i

MEDICAL ASPECTS OF BIOLOGICAL WARFARE 

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The Coat of Arms

1818

Medical Department of the Army

A 1976 etching by Vassil Ekimov of an

original color print that appeared in

The Military Surgeon, Vol XLI, No 2, 1917

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Textbooks of Military Medicine

Published by the

Office of The Surgeon General

Borden Institute

US Army Medical Department Center and School

Health Readiness Center of Excellence

Fort Sam Houston, Texas

Edward A. Lindeke

Colonel (Retired), MSC, US Army

Director, Borden Institute

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v

The TMM Series

Published Textbooks

 Medical Consequences of Nuclear Warfare (1989)

 Conventional Warfare: Ballistic, Blast, and Burn Injuries 

(1991)

 Occupational Health: The Soldier and the Industrial Base 

(1993)

 Military Dermatology (1994)

 Military Psychiatry: Preparing in Peace for War (1994)

 Anesthesia and Perioperative Care of the Combat 

Casualty (1995)

 War Psychiatry (1995)

 Medical Aspects of Chemical and Biological Warfare 

(1997)

 Rehabilitation of the Injured Soldier, Volume 1 (1998)

 Rehabilitation of the Injured Soldier, Volume 2 (1999)

 Medical Aspects of Harsh Environments, Volume 1 (2002)

 Medical Aspects of Harsh Environments, Volume 2 (2002)

 Ophthalmic Care of the Combat Casualty (2003)

 Military Medical Ethics, Volume 1 (2003)

 Military Medical Ethics, Volume 2 (2003)

 Military Preventive Medicine, Volume 1 (2003)

 Military Preventive Medicine, Volume 2 (2005)

 Recruit Medicine (2006)

 Medical Aspects of Biological Warfare (2007)

 Medical Aspects of Chemical Warfare (2008)

 Care of the Combat Amputee (2009)

 Combat and Operational Behavioral Health (2011)

 Military Quantitative Physiology: Problems and Concepts

 in Military Operational Medicine (2012)

 Medical Consequences of Radiological and Nuclear 

Weapons (2013)

 Forensic and Ethical Issues in Military Behavioral Health 

(2014)

 Combat Anesthesia: The First 24 Hours (2015)

 Otolaryngology/Head and Neck Surgery Combat Casualty Care in Operation Iraqi Freedom and Operation 

Enduring Freedom (2015)

 Medical Aspects of Biological Warfare (2018)

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vi

The bacteria that cause anthrax (green) are being enveloped by an immune system phagocytic cell (purple). These bacteria live 

in soil and form dormant spores that can survive for decades. When spores enter humans through the respiratory or gastrointestinal tracts or the skin, they germinate to bacilli and rapidly increase in number. Phagocytic cells of the host immune 

system are essential for ingesting and killing the bacteria, and this is enhanced after vaccination. This is but one example to 

illustrate the important interactions between pathogens and the infected host’s immune system.

Photograph: Courtesy of Sarah Guilman, Camenzind G. Robinson, and Arthur M. Friedlander, US Army Medical Research 

Institute of Infectious Diseases, Fort Detrick, Maryland.

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vii

MEDICAL ASPECTS of

BIOLOGICAL WARFARE

Senior Editors

Joel Bozue, PhD

Division of Bacteriology 

US Army Medical Research Institute of Infectious Diseases

Christopher K. Cote, PhD

Division of Bacteriology 

US Army Medical Research Institute of Infectious Diseases

Pamela J. Glass, PhD

Viral Biology Department, Virology Division

US Army Medical Research Institute of Infectious Diseases

Office of The Surgeon General

Borden Institute

US Army Medical Department Center and School

Health Readiness Center of Excellence

Fort Sam Houston, Texas

2018

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viii

Editorial Staff: Joan Redding

Senior Production Editor

Vivian Mason

Volume Editor

Douglas Wise

Senior Layout Editor

This volume was prepared for military medical educational use. The focus of the information is 

to foster discussion that may form the basis of doctrine and policy. The opinions or assertions 

contained herein are the private views of the authors and are not to be construed as official or as 

reflecting the views of the Department of the Army or the Department of Defense.

Dosage Selection:

The authors and publisher have made every effort to ensure the accuracy of dosages cited herein. 

However, it is the responsibility of every practitioner to consult appropriate information sources 

to ascertain correct dosages for each clinical situation, especially for new or unfamiliar drugs 

and procedures. The authors, editors, publisher, and the Department of Defense cannot be held 

responsible for any errors found in this book.

Use of Trade or Brand Names:

Use of trade or brand names in this publication is for illustrative purposes only and does not 

imply endorsement by the Department of Defense.

Neutral Language:

Unless this publication states otherwise, masculine nouns and pronouns do not refer exclusively 

to men.

certain parts of this publication pertain to copyright restrictions.

all rights reserved.

no copyrighted parts of this publication may be reproduced or 

transmitted in any form or by any means, electronic or mechanical (including photocopy, recording, or any information storage and retrieval 

system), without permission in writing from the publisher or copyright 

owner.

Published by the Office of The Surgeon General 

Borden Institute 

US Army Medical Department Center and School

Health Readiness Center of Excellence

Fort Sam Houston, Texas

Marcia Metzgar

Volume Editor

Ronda Lindsay

Technical Editor

PRINTED IN THE UNITED STATES OF AMERICA

25, 24, 23, 22, 21, 20, 19, 18 5 4 3 2 1

 Library of Congress Cataloging-in-Publication Data

Names: Bozue, Joel, editor. | Cote, Christopher K., editor. | Glass, Pamela 

 J., editor. | Borden Institute (U.S.), issuing body.

Title: Medical aspects of biological warfare / senior editors, Joel Bozue, 

 Christopher K. Cote, Pamela J. Glass.

Other titles: Textbooks of military medicine.

Description: 2. | Fort Sam Houston, Texas : Office of the Surgeon General, 

 Borden Institute, US Army Medical Department Center and School, Health 

 Readiness Center of Excellence, 2018. | Series: Textbooks of military 

 medicine | Includes bibliographical references and index.

Identifiers: LCCN 2017057681 | ISBN 9780160941597

Subjects: | MESH: Bioterrorism--prevention & control | Communicable Disease 

 Control--methods | Military Medicine--methods

Classification: LCC RC971 | NLM WA 295 | DDC 616.9/8023--dc23 LC record available at Caution-https://

lccn.loc.gov/2017057681

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ix

Contents

Contributors xi

Peer Reviewers xvii

Foreword by The Surgeon General xxi

Preface xxiii

1. Historical Overview: From Poisoned Darts to Pan-Hazard Preparedness 1

George W. Christopher, Daniel M. Gerstein, Edward M. Eitzen, and James W. Martin 

2. Epidemiology of Biowarfare and Bioterrorism 37

Zygmunt F. Dembek, Julie A. Pavlin, Martina Siwek, and Mark G. Kortepeter

3. Food, Waterborne, and Agricultural Diseases 71

 Zygmunt F. Dembek, and Edwin L. Anderson

4. Consequence Management: The Local and National Response 93

 Neal E. Woollen, and Gary W. Carter

5. Medical Management of Potential Biological Casualties: A Stepwise Approach 109

 Theodore J. Cieslak 

6. Anthrax 129

 Bret K. Purcell, Christopher K. Cote, Patricia L. Worsham, and Arthur M. Friedlander

7. Brucellosis 159

 Bret K. Purcell, R. Martin Roop II, Arthur M. Friedlander, and David L. Hoover 

8. Glanders 177

 Susan L. Welkos, Bridget Carr Gregory, David M. Waag, and Mary N. Burtnick 

9. Melioidosis 223

Paul J. Brett, David DeShazer, and Nicholas J. Vietri 

10. Plague 247

 Patricia L. Worsham, Thomas W. McGovern, Nicholas J. Vietri, Arthur M. Friedlander, and Joel Bozue 

11. Tularemia 285

Matthew J. Hepburn, Todd M. Kijek, Wendy Sammons-Jackson, Arthur M. Friedlander, and 

 Zygmunt F. Dembek

12. Q Fever 305

 Christina M. Farris and James E. Samuel

13. Multidrug-Resistant Bacterial Infections as a Threat to the US Military Health System: 321

 Acinetobacter Infections as a Case Study 

 Emil Lesho, Luther E. Lindler, Bruno Petruccelli, and Glenn Wortmann

14. Botulinum Toxin 337

Zygmunt F. Dembek, Leonard A. Smith, Frank J. Lebeda, and Janice M. Rusnak

15. Clostridium Perfringens Epsilon Toxin 361

Bradley G. Stiles, Gillian Barth, and Michel R. Popoff

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16. Ricin 373

Virginia I. Roxas-Duncan, Martha L. Hale, Jon M. Davis, John C. Gorbet, Patricia M. Legler, and 

 Leonard M. Smith

17. Staphylococcal Enterotoxin B and Related Toxins Produced by Staphylococcus Aureus and 403

 Streptococcus Pyogenes

 Kamal U. Saikh, Robert G. Ulrich, and Teresa Krakauer 

18. Toxins from Venoms and Poisons 415

	 Scott A. Weinstein and Julian White

19.	 Marine Algal Toxins of Concern as Intentional Contaminants	 461

	 	 Mark A. Poli and Mark R. Withers

	 20.	 Alphavirus Encephalitides	 479

Shelley P. Honnold, Eric C. Mossel, Lesley C. Dupuy, Elaine M. Morazzani, Shannon S. Martin, 

 Mary Kate Hart, George V. Ludwig, Michael D. Parker, Jonathan F. Smith, Douglas S. Reed, and 

 Pamela J. Glass

21. Hemorrhagic Fever-Causing Mammarenaviruses 517

	 	 Sheli R. Radoshitzky, Jens H. Kuhn, Peter B. Jahrling, and Sina Bavari

22. Henipaviruses 547

	 Arnold Park, Sheli R. Radoshitzky, Jens H. Kuhn, and Benhur Lee

23. Filoviruses 569

	 	 Sheli R. Radoshitzky, Sina Bavari, Peter B. Jahrling, and Jens H. Kuhn

24. Smallpox and Related Orthopoxviruses 615

	 Arthur J. Goff, Sara C. Johnston, Kenny L. Lin, Peter B. Jahrling, John W. Huggins, M. Sofi Ibrahim,

	 	 James V. Lawler, and James W. Martin

25. Emerging Infectious Diseases and Future Threats 645

	 	 Chris A. Whitehouse and Brett Beitzel

26.	 Laboratory Identification of Threats	 701

	 	 David A. Norwood, Timothy D. Minogue, Randal J. Schoepp, and Mark J. Wolcott

27. Medical Countermeasures 751

	 	 Phillip R. Pittman, Elizabeth Brown, and Matthew S. Chambers

28.	 Future Prospects of Vaccines and Antibodies in Biodefense	 823

	 	 Jeffrey Froude II, Crystal W. Burke, and Jean-Nicolas Tournier

29.	 Aerobiology: History, Development, and Programs	 855

	 	 Douglas S. Reed, Aysegul Nalca, and Chad J. Roy

30. Biosafety 869

	 	 David E. Harbourt, Catherine L. Wilhelmsen, Kristie M. Yeakle, and Robert J. Hawley

31. Biological Surety 895

	 	 Samuel S. Edwin, Virginia I. Roxas-Duncan, America M. Ceralde, Shelley C. Jorgensen, and Neal E. Woollen

32. Ethical Issues in the Development of Drugs and Vaccines for Biodefense 915

	 	 Arthur O. Anderson, Jeffrey E. Stephenson, and Bret K. Purcell

Abbreviations and Acronyms	 xxv

Index xxx

Biological Warfare 3 - front matter - mark CIP.indd 10 6/21/18 
xi

Contributors

ARTHUR O. ANDERSON, MD

Colonel (Retired), Medical Corps, US Army; Director, Office of 

Human Use and Ethics, US Army Medical Research Institute of 

Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

21702

EDWIN L. ANDERSON, MD

Colonel (Retired), Medical Corps, US Army; Research Professor, 

Department of Internal Medicine, Division of Infectious Diseases, 

Allergy and Immunology, Saint Louis University School of Medicine, 1100 South Grand Boulevard, St. Louis, Missouri 63104

GILLIAN BARTH, BS

Veterinary Technician, Veterinary Department, Wilson College, 

1015 Philadelphia Avenue, Chambersburg, Pennsylvania 17201

SINA BAVARI, PhD

Science Director, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Room 900, Fort Detrick, 

Maryland 21702; formerly, Division Chief, Toxicology Division, 

US Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Room 900, Fort Detrick, Maryland

BRETT BEITZEL, PhD

Principal Investigator, Center for Genomic Sciences, US Army 

Medical Research Institute of Infectious Diseases, 1425 Porter 

Street, Fort Detrick, Maryland 21702

JOEL BOZUE, PhD

Microbiologist, Bacteriology Division, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702

PAUL J. BRETT, PhD

Associate Professor, Department of Microbiology and Immunology, University of South Alabama, 610 Clinic Drive, Laboratory of 

Infectious Diseases Building, Mobile, Alabama 36688

ELIZABETH S. BROWN, PhD

Medical–Technical Writer and Editor, Department of Clinical Research, Division of Medicine, US Army Medical Research Institute 

of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

21702

CRYSTAL W. BURKE, PhD

Research Microbiolgoist, Virology Division, US Army Medical 

Research Institute of Infectious Diseases, 1425 Porter Street, Room 

6, Fort Detrick, Maryland 21702 

MARY N. BURTNICK, PhD

Assistant Professor,Department of Microbiology and Immunology, University of South Alabama, 5851 USA Drive North, Medical 

Science Building, Mobile, Alabama 36688

GARY W. CARTER

Director, Field Operations and Training, National Strategic Research Institute, 3925 Dewey Avenue, Omaha, Nebraska 68198 

AMERICA M. CERALDE, MBA, MT(AMT)

Alternate Surety Officer and Chief, Personnel Reliability Program 

Branch, Biosurety Division, US Army Medical Research Institute 

of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

21702

MATTHEW S. CHAMBERS, MD, MPH

Lieutenant Colonel, Medical Corps, US Army; Chief, Preventive 

Medicine, Fort Wainwright, 4077 Neely Road, Fort Wainwright, 

Alaska 99703; formerly, Chief, Field Studies, Division of Medicine, US Army Medical Research Institute of Infectious Diseases, 

1425 Porter Street, Fort Detrick, Maryland 

GEORGE W. CHRISTOPHER, MD, FACP

Lieutenant Colonel (Retired), Medical Corps, US Air Force; Chief 

Medical Officer, Joint Project Manager-Medical Countermeasure 

Systems (JPM-MCS), 10109 Gridley Road, Building 314, 2nd 

Floor, Fort Belvoir, Virginia 22060-5865

THEODORE J. CIESLAK, MD

Colonel, Medical Corps, US Army; Pediatric Infectious Diseases 

Physician, San Antonio Military Medical Center, Department of 

Pediatrics, 3551 Roger Brooke Drive, Fort Sam Houston, Texas, 

78234

CHRISTOPHER K. COTE, PhD

Principal Investigator, Division of Bacteriology, US Army Medical 

Research Institute of Infectious Diseases, 1425 Porter Street, Fort 

Detrick, Maryland 21702

JON M. DAVIS, PhD

Lieutenant Colonel, Medical Service Corps, US Army; Department of Defense Liaison to the ASPR, Department of Health 

and Human Services, 200 C Street, SW, Washington, DC 20024; 

formerly, Deputy Medical Coordinator, Office of the Assistant 

Secretary of Defense for Nuclear, Chemical, and Biological Defense, 3050 Defense Pentagon, Washington, DC

ZYGMUNT F. DEMBEK, PhD, MS, MPH, LHD

Colonel (Retired), Medical Service Corps, US Army Reserve; Associate Professor, Department of Military and Emergency Medicine, Uniformed Services University of the Health Sciences, 4301 

Jones Bridge Road, Bethesda, Maryland 20814; formerly, Chief 

Biodefense Epidemiology and Education & Training Programs, 

Division of Medicine, US Army Medical Research Institute of 

Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

DAVID DeSHAZER, PhD

Microbiologist, Bacteriology Division, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702

LESLEY C. DUPUY, PhD

Microbiologist, Virology Division, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702

SAMUEL S. EDWIN, PhD

Responsible Official and Surety Officer; Chief, Biosurety Division, 

US Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland 21702

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xii

EDWARD M. EITZEN, MD, MPH

Colonel (Retired), Medical Corps, US Army; Senior Partner, 

Biodefense and Public Health Programs, Martin-Blanck and Associates, 2034 Eisenhower Avenue, Suite 270, Alexandria, Virginia 

22314-4678; formerly, Commander, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland

CHRISTINA M. FARRIS, PhD

Lieutenant, Medical Service Corps, US Navy; Viral and Rickettsial 

Diseases Department, Naval Medical Research Center, 503 Robert 

Grant Avenue, Silver Spring, Maryland 20910

ARTHUR M. FRIEDLANDER, MD

Colonel (Retired), Medical Corps, US Army; Senior Scientist, US 

Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702, and Adjunct Professor of 

Medicine, Uniformed Services University of the Health Sciences, 

4301 Jones Bridge Road, Bethesda, Maryland 20814

JEFFREY FROUDE II, PhD

Clinical Pharmacology Fellow, Experimental Therapeutics Division, Walter Reed Institute of Research, 503 Robert Grant Avenue, 

Silver Spring, Maryland 21702 

DANIEL M. GERSTEIN, PhD

Colonel (Retired), US Army; Adjunct Professor, School of International Studies, American University, 4400 Massachusetts Avenue, 

NW, Washington, DC 20016; formerly, Undersecretary (Acting) 

and Deputy Undersecretary, Science and Technology Directorate, 

Department of Homeland Security, Washington DC

PAMELA J. GLASS, PhD

Chief, Viral Biology Department, Virology Division, US Army 

Medical Research Institute of Infectious Diseases, 1425 Porter 

Street, Fort Detrick, Maryland 21702

ARTHUR J. GOFF, PhD

Research Microbiologist, Department of Virology, US Army 

Medical Research Institute of Infectious Diseases, 1425 Porter 

Street, Fort Detrick, Maryland 21702

JOHN C. GORBET, PhD

Captain, Medical Service Corps, US Army; Chief, Department of 

Aerosol Services, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

BRIDGET CARR GREGORY, DVM, MPH

Colonel, US Air Force, Biomedical Sciences Corps; Deputy Chief 

of Staff, Defense Threat Reduction Agency/Strategic Command 

Center for Combating Weapons of Mass Destruction, 8725 John 

J. Kingman Road, Fort Belvoir, Virginia 22060; formerly, Chief, 

Education and Training, Division of Medicine, US Army Medical 

Research Institute of Infectious Diseases, 1425 Porter Street, Fort 

Detrick, Maryland

MARTHA L. HALE, PhD

Research Microbiologist, Integrated Toxicology Division, US 

Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland 21702

DAVID E. HARBOURT, PhD, SM(NRCM), RBP

Biosafety Officer, Office of Safety, Radiation, and Environment, 

US Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland 21702; formerly, Fellow, National Biosafety and Biocontainment Training Program, National 

Institutes of Health, 13 South Drive, Bethesda, Maryland

MARY KATE HART, PhD

Chief Scientific Officer, Dynport Vaccine Company, 64 Thomas 

Johnson Drive, Frederick, Maryland 21702; formerly, Chief, Virology Division, US Army Medical Research Institute of Infectious 

Diseases, 1425 Porter Street, Fort Detrick, Maryland

ROBERT J. HAWLEY, PhD, RBP, CBSP

Consultant, Biological Safety and Security, Alliance Biosciences, 6810 Deerpath Road, Suite 315, Elkridge, Maryland 21075; 

formerly Senior Advisor, Science, Midwest Research Institute, 

110 Thomas Johnson Drive, Suite 170, Frederick, Maryland; formerly, Chief, Safety and Radiation Protection, US Army Medical 

Research Institute of Infectious Diseases, 1425 Porter Street, Fort 

Detrick, Maryland

MATTHEW J. HEPBURN, MD

Colonel, MC, US Army; Program Manager, Biological Technologies Office, Defense Advanced Research Projects Agency (DARPA), 675 North Randolph Street, Room 08-140, Arlington, Virginia 

22203-2114; formerly, Clinical Research Unit Lead, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, 

Fort Detrick, Maryland

SHELLEY P. HONNOLD, DVM, PhD

Lieutenant Colonel, Veterinary Corps, US Army; Director, Research Support and Chief, Pathology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, 

Fort Detrick, Maryland 21702; formerly, Biodefense Research 

Pathologist, Pathology Division, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland

DAVID L. HOOVER, MD

Colonel (Retired), Medical Corps, US Army; Senior Scientific Advisor, Clinical Research Management, 1265 Ridge Road, Hinckley, 

Ohio 44233; formerly Medical Director, Dynport Vaccine Company LLC, a CSC Company, 64 Thomas Johnson Drive, Frederick, Maryland, and Scientific Coordinator, Brucella Program, 

Department of Bacterial Diseases, Walter Reed Army Institute of 

Research, Silver Spring, Maryland

JOHN W. HUGGINS, PhD

Retired; formerly, Chief, Viral Therapeutics Branch, US Army 

Medical Research Institute of Infectious Diseases, 1425 Porter 

Street, Fort Detrick, Maryland 21702

M. SOFI IBRAHIM, MSC, PhD

Lieutenant Colonel, Medical Service Corps, US Army Reserve; 

Research Microbiologist, Edgewood Chemical Biological Center, 

Ricketts Point Road, Gunpowder, Maryland 21010; formerly, 

Microbiologist, Division of Virology, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland

PETER B. JAHRLING, PhD

Captain (Retired), Medical Service Corps, US Army; Director, National Institutes of Health/Division of Clinical Research/National 

Institute of Allergy and Infectious Diseases/Integrated Research 

Facility, 8200 Research Plaza, Room 1A-111A, Fort Detrick, 

Maryland 21702; formerly, Principal Scientific Advisor (Senior 

Research Scientist), US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

SARA C. JOHNSTON, PhD

Research Microbiologist, Department of Virology, US Army 

Medical Research Institute of Infectious Diseases, 1425 Porter 

Street, Fort Detrick, Maryland 21702

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xiii

SHELLEY C. JORGENSEN, PhD, MT(ASCP)

Lieutenant Colonel, Medical Services, US Army; Chief, Surety 

Branch, US Army Office of The Surgeon General/US Army Medical Command, G-34 Protection Division, DHHQ, 7700 Arlington 

Boulevard, Falls Church, Virginia 22042

TODD M. KIJEK

Major, Medical Service Corps, US Army; PhD candidate and 

Chief, Molecular and Translational Sciences Department, US

Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland 21702; formerly, Research 

Microbiologist, Bacteriology Division, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland

JASON KINDRACHUK, PhD

Staff Scientist, Critical Care Medicine Department, Clinical 

Center, National Institutes of Health, 10 Center Drive, Bethesda, 

Maryland 20814

MARK G. KORTEPETER, MD, MPH

Colonel, Medical Corps, US Army; Director, Infectious Disease 

Clinical Research Program, Department of Preventive Medicine, 

Associate Dean for Research, Associate Professor of Preventive 

Medicine and Medicine, Consultant to the Army Surgeon General 

for Biodefense, Uniformed Services University of the Health 

Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814; formerly, Deputy Commander, US Army Medical Research Institute 

of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

TERESA KRAKAUER, PhD

Microbiologist, Department of Immunology, US Army Medical 

Research Institute of Infectious Diseases, 1425 Porter Street, Fort 

Detrick, Maryland 21702

JENS H. KUHN, MD, PhD, PhD, MS 

Virology Lead, National Institutes of Health/National Institute of 

Allergy and Infectious Diseases/Division of Clinical Research/Integrated Research Facility, B-8200 Research Plaza, Room 1A-132, 

Fort Detrick, Maryland 21702; formerly, Research Scholar, Harvard Medical School, Department of Microbiology and Molecular 

Genetics, New England Primate Research Center, 1 Pine Hill 

Drive, Southborough, Massachusetts

JAMES V. LAWLER, MPH, MD

Commander, Medical Corps, US Navy Reserve; ACESO Director 

and Chief, Clinical Research Department, Biodefense Research Directorate, Naval Medical Research Center, Fort Detrick, Maryland 

21702; formerly, Chief Medical Officer, Integrated Research Facility, Fort Detrick, Maryland

FRANK J. LEBEDA, PhD

Acting Director and Chief Data Officer, Systems Biology Collaboration Center for Environmental Health Research, US Army 

Medical Research and Materiel Command, 568 Doughten Drive, 

Fort Detrick, Maryland 21702; formerly, Civilian Deputy Director, 

Combat Casualty Care Research Program, US Army Medical 

Research and Materiel Command, 504 Scott Street, Fort Detrick, 

Maryland 

BENHUR LEE, MD

Professor, Microbiology Department, Icahn School of Medicine 

at Mount Sinai, One Gustave L. Levy Place, Annenberg Building, 

Room 17-24, New York, New York 10029

PATRICIA M. LEGLER, PhD

Research Biologist, US Naval Research Laboratory, 4555 Overlook 

Avenue, Washington, DC 20375

EMIL LESHO, DO

Director, Multidrug-Resistant Organism Repository and Surveillance Network, Walter Reed Army Institute of Research, 503 

Walter Grant Avenue, 1W102, Silver Spring, Maryland 20910

KENNY L. LIN, MS

GLP Study Coordinator, Nonclinical Development Division, US 

Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland 21702; formerly, Regulatory 

Scientist, Department of Virology, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 

LUTHER E. LINDLER, PhD

Biosurveillance Liaison, Armed Forces Health Surveillance Center, 11800 Tech Road, Silver Spring, Maryland 20904

GEORGE V. LUDWIG, PhD

Deputy Principal Assistant for Research and Technology, US 

Army Medical Research and Materiel Command, 810 Schreider 

Street, Fort Detrick, Maryland 21702; formerly, Science Director, 

US Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland

JAMES W. MARTIN, MD, FACP

Colonel (Retired), Medical Corps, US Army; Chief of Internal 

Medicine, US Army Healthcare Clinic, Vicenza, APO AE 09630-

0040; formerly, Chief, Operational Medicine Department, US 

Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland

SHANNON S. MARTIN, PhD

Vaccine Program Manager, Clinical Research Management, 

Business Plans and Programs Office, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702; formerly, Senior Scientist/Technical Development Manager, Nonclinical Research Department, Dynport Vaccine Company, 64 Thomas Johnson Drive, Frederick, Maryland 

THOMAS W. McGOVERN, MD, FAAD, FACMS, KHS

Major, Medical Corps, US Army (former); Dermatologist/Mohs 

Surgeon, Fort Wayne Dermatology Consultants, 7881 Carnegie 

Boulevard, Fort Wayne, Indiana 46804; formerly, Ward Officer, 

US Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland

TIMOTHY D. MINOGUE, PhD

Chief, Molecular Diagnostics Department, Diagnostic Systems 

Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

ELAINE M. MORAZZANI, PhD

Oak Ridge Institute for Science and Education Fellow, Viral Biology Department, Virology Division, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702; formerly, NRC Postdoctoral Fellow, Virology 

Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

244-949 DLA DS.indb 13 6/4/18 
xiv

ERIC C. MOSSEL, PhD

Major, Medical Service Corps, US Army Reserve; Microbiologist, Division of Virology, US Army Medical Research Institute 

of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

21702; formerly, Science and Technology Advisor, Detachment 

8, Research Development and Engineering Command, 5183 Blackhawk Road, Aberdeen Proving Ground, Maryland 

AYSEGUL NALCA, MD, PhD

Chief, Department of Animal Studies, Center for Aerobiological 

Sciences, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

DAVID A. NORWOOD, PhD

Chief, Diagnostic Systems Division, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702

ARNOLD PARK, PhD

Postdoctoral Fellow, Microbiology Department, Icahn School of 

Medicine at Mount Sinai, One Gustave L. Levy Place, Annenberg 

Building, Room 17-24, New York, New York 10029

MICHAEL D. PARKER, PhD

Retired; formerly, Chief, Viral Biology Department, Virology Division, US Army Medical Research Institute of Infectious Diseases, 

1425 Porter Street, Fort Detrick, Maryland

JULIE A. PAVLIN, MD, PhD, MPH

Colonel (Retired), Medical Corps, US Army; Deputy Director, 

Armed Forces Health Surveillance Center, 11800 Tech Road, 

Silver Spring, Maryland 20904

BRUNO PETRUCCELLI, MD

Colonel (Retired), Medical Corps, US Army; formerly, Director, 

Epidemiology and Disease Surveillance, US Army Center for 

Health Promotion and Preventive Medicine, Aberdeen Proving 

Ground, Maryland; currently, Senior Physician/Epidemiologist, 

US Military HIV Research Program, Henry M. Jackson Foundation for the Advancement of Military Medicine, 3720-A Rockledge 

Drive, Suite 400, Bethesda, Maryland 20817

PHILLIP R. PITTMAN, MD, MPH

Colonel (Retired), US Army; Chief, Department of Clinical Research, Division of Medicine, US Army Medical Research Institute 

of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

21702

MARK A. POLI, PhD, DABT

Research Chemist, Department of Applied Diagnostics, Diagnostic 

Systems Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

MICHEL R. POPOFF, PhD, DVM

Chief, Anaerobic Bacteria and Toxins, Institut Pasteur, 28 Rue du 

Dr Roux, 75724 Paris, France

BRET K. PURCELL, PhD, MD

Colonel, Medical Corps, US Army; Deputy Chief, Bacteriology 

Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

SHELI R. RADOSHITZKY, PhD

Principal Investigator, Molecular and Translational Sciences Division, US Army Medical Research Institute of Infectious Diseases, 

1425 Porter Street, Fort Detrick, Maryland 21702; formerly, Postdoctoral Fellow, Toxicology Division, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland

DOUGLAS S. REED, PhD

Associate Professor, Aerobiological Manager, RBL, Department 

of Immunology, University of Pittsburgh, 3501 Fifth Avenue, 

Pittsburgh, Pennsylvania 15261; formerly, Microbiologist, Center 

for Aerobiological Sciences, US Army Medical Research Institute 

of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

R. MARTIN ROOP II, PhD

Professor, Microbiology and Immunology, Brody School of Medicine, East Carolina University, 600 Moye Boulevard, Room 118, 

Biotechnology Building, Greenville, North Carolina 27834

VIRGINIA I. ROXAS-DUNCAN, PhD

Alternate Responsible Official and Chief, Select Agent Management Branch, Biosurety Division, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702

CHAD J. ROY, PhD

Associate Professor, Department of Microbiology and Immunology, Tulane School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 70112; and Director, Infectious Disease Aerobiology, Division of Microbiology, Tulane National Primate Research 

Center, 18703 Three Rivers Road, Covington, Louisiana 70433

JANICE M. RUSNAK, MD

Lieutenant Colonel (Retired), US Air Force; Medical Director, 

Battelle, 1564 Freedman Drive, Fort Detrick, Maryland 21702; 

formerly, Deputy Director of Special Immunizations Program, 

US Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland 

KAMAL U. SAIKH, PhD

Microbiologist, Department of Immunology, US Army Medical 

Research Institute of Infectious Diseases, 1425 Porter Street, Fort 

Detrick, Maryland 21702

WENDY SAMMONS-JACKSON, PhD

Lieutenant Colonel, Medical Service Corps, US Army; Military 

Deputy, Science and Technology Department, US Army Medical 

Research and Materiel Command, 810 Schreider Street, Suite 103, 

Fort Detrick, Maryland 21702; formerly, Deputy Director, Military 

Infectious Diseases Research Program, Fort Detrick, Maryland

JAMES E. SAMUEL, PhD

Professor and Chair, Department of Microbial Pathogenesis and 

Immunology, College of Medicine, Texas A&M Health Science 

Center, 8447 State Highway 47, Medical Research and Education 

Building, Building 3112, Bryan, Texas 77807

ALAN L. SCHMALJOHN, PhD

Professor, Department of Microbiology and Immunology, University of Maryland School of Medicine, 655 West Baltimore Street, 

HSF-I, 322B, Baltimore, Maryland 21201

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RANDAL J. SCHOEPP, PhD

Chief, Immunodiagnostics and Biologics Department, Diagnostic 

Systems Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

MARTINA SIWEK, PhD

Chief Scientist, Cherokee Nation Technology Solutions in Support 

of Global Emerging Infections Surveillance and Response System, 

Armed Forces Health Surveillance Center, 11800 Tech Road, 

Silver Spring, Maryland 20904; formerly, Science Manager/Liaison, Biosurveillance Management Office, Joint Program Executive 

Office, Aberdeen Proving Ground, Maryland

JONATHAN F. SMITH, PhD

Executive Vice President and Chief Scientific Officer, PaxVax, 

Inc, 3985 Sorrento Valley Boulevard, San Diego, California 92121; 

formerly, Chief, Viral Biology Department, Virology Division, US 

Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland

LEONARD A. SMITH, PhD

Senior Research Scientist (ST) for Medical Countermeasures Technology, Office of the Chief Scientist, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702

JEFFREY E. STEPHENSON, PhD

Regulatory Compliance Specialist, US Army Medical Research 

and Materiel Command, Telemedicine and Advanced Technology Research Center, 1054 Patchel Street, Fort Detrick, Maryland 

21702; formerly, Institutional Review Board Administrator, US 

Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland

BRADLEY G. STILES, PhD

Adjunct Professor of Biology, Biology Department, Wilson College, 1015 Philadelphia Avenue, Chambersburg, Pennsylvania 

17201

JEAN-NICOLAS TOURNIER, MD, PhD

Colonel, French Armed Forces Health Service, Department of 

Infectious Diseases, Unité Biothérapies anti-infectieuses et immunité, Institut de recherche biomédicale des armées, 1 place du 

Général Valérie André, Brétigny-sur-Orge, 91220 France; École du 

Val-de-Grâce, Paris, France; Unité Génomique virale et vaccination, Institut Pasteur, Paris, France

ROBERT G. ULRICH, PhD

Microbiologist, Department of Immunology, US Army Medical 

Research Institute of Infectious Diseases, 1425 Porter Street, Fort 

Detrick, Maryland 21702

NICHOLAS J. VIETRI, MD

Colonel, Medical Corps, US Army; Infectious Diseases Officer, 

Bacteriology Division, US Army Medical Research Institute of 

Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

21702; and Assistant Professor of Medicine, Uniformed Services 

University of the Health Sciences, 4301 Jones Bridge Road, 

Bethesda, Maryland 20814

DAVID M. WAAG, PhD

Microbiologist, Bacteriology Division, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702

SCOTT A. WEINSTEIN, PhD, MD

Physician, Toxinologist, Toxinology Division, Women’s & Children’s Hospital, 72 King William Road, North Adelaide, South 

Australia 5006, Australia

SUSAN L. WELKOS, PhD

Microbiologist, Bacteriology Division, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702

JULIAN WHITE, MD

Head of Toxinology, Toxinology Division, Women’s & Children’s 

Hospital, 72 King William Road, North Adelaide, South Australia 

5006, Australia

CHRIS A. WHITEHOUSE, PhD

Principal Investigator, Molecular and Translational Sciences 

Department, US Army Medical Research Institute of Infectious 

Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702; formerly, Chief, Disease Investigation Branch, US Geological Survey, 

National Wildlife Health Center, 6006 Schroeder Road, Madison, 

Wisconsin

CATHERINE L. WILHELMSEN, DVM, PhD, CBSP

Lieutenant Colonel (Retired), Veterinary Corps, US Army; Veterinary Medical Officer, Division of Pathology; formerly, Biosafety 

Officer, Office of Safety, Radiation Protection, and Environmental 

Health, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland; formerly, Chief, 

Division of Toxicology, US Army Medical Research Institute of 

Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

MARK R. WITHERS, MD, MPH

Colonel, Medical Corps, US Army; Clinical Director, Office of 

Medical Support and Oversight, US Army Research Institute of 

Environmental Medicine, 15 Kansas Street, Natick, Massachusetts 

01760; formerly Deputy Chief, Division of Medicine, US Army 

Medical Research Institute of Infectious Diseases, 1425 Porter 

Street, Fort Detrick, Maryland

MARK J. WOLCOTT, PhD

Chief, Transitional and Operational Diagnostics Department, 

Diagnostic Systems Division, US Army Medical Research Institute 

of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

21702

NEAL E. WOOLLEN, DVM, MSS, PhD

Colonel, Veterinary Corps, US Army; Director, Department of 

Biological Select Agent and Toxin Biosafety Program Office, 1546 

Porter Street, Fort Detrick, Maryland 21702

PATRICIA L. WORSHAM, PhD

Chief, Bacteriology Division, US Army Medical Research Institute 

of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

21702

GLENN WORTMANN, MD

Colonel (Retired), Medical Corps, US Army; formerly, Chief 

of Infectious Diseases, Walter Reed National Military Medical 

Center, Bethesda, Maryland; currently, Assistant Director, Infectious Diseases Section, MedStar Washington Hospital Center, 110 

Irving Street, NW, Washington, DC 20010

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KRISTIE M. YEAKLE, RBP

Alternate Biosafety Officer, Office of Safety, Radiation, and 

Environment, US Army Medical Research Institute of Infectious 

Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

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Peer Reviewers

AMESH A. ADALJA, MD, FACP, FACEP

Senior Associate, UPMC Center for Health Security, 621 East 

Pratt Street, Suite 210, Baltimore, Maryland 21202; and Clinical 

Assistant Professor, Department of Critical Care Medicine Clinical Assistant Professor, and Department of Emergency Medicine 

Adjunct Instructor, Division of Infectious Diseases, Department 

of Medicine, University of Pittsburgh School of Medicine, M240 

Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261

GERARD ANDREWS, PhD

Associate Professor, Department of Veterinary Sciences, University of Wyoming, College of Agriculture and Natural Resources, 

1174 Snowy Range Road, Laramie, Wyoming 82070

ROY BARNEWELL, PhD

Manager and Research Leader, Battelle Memorial Institute, 505 

King Avenue, Columbus, Ohio 43201

HOLGER BARTH, PhD

Professor, Institute of Pharmacology and Toxicology, University 

of Ulm Medical Center, Albert-Einstein-Allee 11, 89081 Ulm, 

Germany

KENNETH BRADLEY, PhD

Associate Professor, Department of Microbiology, Immunology, 

and Molecular Genetics Director, Molecular Screening Shared Resource, University of California, 609 Charles E. Young Drive East,

Los Angeles, California 90095

BARBARA A. BROOKMYER, MD, MPH

Health Officer, Frederick County Health Department, 350 Montevue Lane, Frederick, Maryland 21702

LAURA RUSE BROSCH, RN, PhD

Director, Office of Research Protections and Director, Office of 

Research Protections Human Research Protection Office, Headquarters, US Army Medical Research and Materiel Command, 810 

Schreider Street, Fort Detrick, Maryland 21702

MICHAEL BUCHMEIER, PhD

Professor, Department of Molecular Biology and Biochemistry, 

Division of Infectious Disease, Department of Medicine, Department of Microbiology and Molecular Genetics, and Department of 

Molecular Biology and Biochemistry, University of California–Irvine, 2400 Biological Sciences III, Irvine, California 92697-3900

R. MARK BULLER, PhD

Professor, Department of Molecular Microbiology and Immunology, Saint Louis University, 1100 South Grand Boulevard, St. 

Louis, Missouri 63104

KATHLEEN W. CARR, DVM, PhD, MS

Program Manager, Division of Medicine, US Army Medical 

Research Institute of Infectious Diseases, 1425 Porter Street, Fort 

Detrick, Maryland 21702

BRIAN W. COOPER, MD

Global Medical Affairs Medical Director of Early Viral/Bacterial 

Vaccines, Novartis Vaccines, 350 Massachusetts Avenue, Cambridge, Massachusetts 02139; and Professor of Clinical Medicine, 

University of Connecticut School of Medicine, 263 Farmington 

Avenue, Farmington, Connecticut 06030

PAUL DABISCH, PhD

Senior Principal Investigator and Aerobiology Group Leader, 

National Biodefense Analysis and Countermeasures Center (operated by BNBI for the US Department of Homeland Security), 8300 

Research Plaza, Fort Detrick, Maryland 21702

ROBERT G. DARLING, MD, FACEP

Captain (Retired), Medical Corps, US Navy; Chief Medical Officer, Patronus Medical Corporation, Washington, DC; and Assistant Professor Military and Emergency Medicine, The Uniformed 

Services University of the Health Sciences F. Edward Hébert 

School of Medicine, 4301 Jones Bridge Road, Bethesda, Maryland 

20814 

STACY DeGRASSE, PhD

Science Advisor, Division of Seafood Safety, Office of Food Safety, 

US Food and Drug Administration,

Center for Food Safety and Applied Nutrition, 5100 Paint Branch 

Parkway, College Park, Maryland 20740

JUAN CARLOS De La TORRE, PhD

Professor, Department of Immunology and Microbial Science, 

The Scripps Research Institute, 10550 North Torrey Pines Road, 

La Jolla, California 92037

DAVID DeSHAZER, PhD

Microbiologist, Bacteriology Division, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702

DMITRY DMITROFF, PhD

Head, Protein Interactions Section, Center for Cancer Research, 

National Cancer Institute, Building 567, Room 152, Frederick, 

Maryland 21702

ADAM DRIKS, PhD

Professor, Department of Microbiology and Immunology, Stritch 

School of Medicine, Loyola University Chicago, 2160 South First 

Avenue, Maywood, Illinois 60153

CAROL EISENHAUER, DVM, Dipl ACLAM

Colonel (Retired)

EDWARD EITZEN, MD, MPH

Colonel (Retired), US Army; Senior Partner, Biodefense and 

Public Health Programs, Martin-Blanck and Associates, 2034 

Eisenhower Avenue, Suite 270, Alexandria, Virginia 22314-4678

KENNETH L. GAGE, PhD

Chief, Entomology and Ecology Activity, Bacterial Diseases 

Branch, Division of Vector-Borne Diseases, National Center for 

Emerging and Zoonotic Infectious Diseases, Centers for Disease 

Control and Prevention, 3156 Rampart Road, Fort Collins, Colorado 80521

DAVE HARBOURT, PhD

Biosafety Officer, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702-

5102

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DAN E. HARMS, MS, MT(ASCP)

Program Manager, Operational Laboratory Policy & Programs 

Executive Secretariat, Defense Health Headquarters, 7700 Arlington Boulevard, Suite 5101, Falls Church, Virginia 22042-5190

M. DANA HARRIGER, PhD

Assistant Dean and Professor, Department of Biology, Wilson 

College, 1015 Philadelphia Avenue, Chambersburg, Pennsylvania 

17201

JOHN HUNGERFORD, PhD

Research Chemist, Pacific Laboratory Northwest, US Food and 

Drug Administration, 22201 23rd Drive, SE, Bothell, Washington 

98021

DEBRA L. HUNT, DrPH, CBSP

Assistant Professor and Director, Biological Safety, Occupational, 

and Environmental Safety Office, Duke University/Duke University Health System, Hock Plaza I, 2424 Erwin Road, Durham, 

North Carolina 27705

BRADLEY D. JONES, PhD

Professor, Department of Microbiology, University of Iowa, 

Carver College of Medicine, 375 Newton Road, Iowa City, Iowa 

52242

HEIDI KASSENBORG, DVM, MPH

Director, Dairy and Food Inspection Division, Minnesota Department of Agriculture, 625 Robert Street North, St. Paul, Minnesota 

55155

JEREMIAH J. KELLY, ESQ, MPP

Attorney for Medical Product Development and Regulation, 

Office of the Staff Judge Advocate (JAG), US Army Medical 

Research and Materiel Command, 521 Fraim Street, Fort Detrick, 

Maryland 21701 

ERIC LAFONTAINE, PhD

Associate Professor, Department of Infectious Diseases, College of 

Veterinary Medicine, The University of Georgia, 501 D. W. Brooks 

Drive, Athens, Georgia 30602-7387

DIREK LIMMATHUROTSAKUL, MD, PhD

Assistant Professor, Department of Tropical Hygiene and Mahidol, Oxford Tropical Medicine Research Unit, Faculty of Tropical 

Medicine, Mahidol University, 999 Phuttamonthon 4 Road, 

Salaya 73170, Thailand

MARK LITTLE, MD, MBBS, FACEM, MPH&TM, 

DTM&H, IDHA

Associate Professor, School of Public Health, Tropical Medicine 

and Rehabilitation Sciences, James Cook University, 1 James 

Cook Drive, Townsville City, Queensland 4811, Australia; and 

Emergency Physician and Clinical Toxicologist, Department of 

Emergency Medicine, 165 The Esplanade, Cairns Hospital, Cairns 

City, Queensland 4870, Australia

STEPHEN LUBY, MD

Professor, Department of Medicine-Infectious Diseases and Senior 

Fellow, Stanford Woods Institute for the Environment and Freeman Spogli Institute for International Studies, 473 Via Ortega, 

Stanford, California 94305; and Director of Research, Stanford 

Center for Innovation in Global Health, 473 Via Ortega, Stanford, 

California 94305

NICHOLAS MANTIS, PhD

Associate Professor, Division of Infectious Disease, Wadsworth 

Center, New York State Department of Health, Empire State 

Plaza, PO Box 509, Albany, New York 12201; and Department 

of Biomedical Sciences, School of Public Health, University at 

Albany, One University Place, Albany, New York 12144

GLENN MARSH, PhD

Senior Research Scientist, Molecular Virology, Emerging Zoonotic 

Diseases Stream, CSIRO Biosecurity Flagship, Australian Animal 

Health Laboratory, 5 Portarlington Road, Geelong, Victoria, 

Australia 3220

BRUCE McCLANE, PhD

Professor, Department of Microbiology and Molecular Genetics, 

University of Pittsburgh School of Medicine, 420 Bridgeside Point 

II, 450 Technology Drive, Pittsburgh, Pennsylvania 15219 

CHARLES A. McKAY, Jr, MD, FACMT, FACEP, ABIM 

Director, Division of Medical Toxicology, Department of Emergency Medicine, Hartford Hospital and University of Connecticut 

School of Medicine, 80 Seymour Street, Hartford, Connecticut 

06102

DIETRICH MEBS, PhD

Professor, Goethe-University Frankfurt, Forensic Toxicology, 

Institute of Legal Medicine, Kennedyallee 104, D-60596 Frankfurt, 

Germany

MICHAEL F. MINNICK, PhD

Professor, Division of Biological Sciences, University of Montana, 

32 Campus Drive, HS 104, Missoula, Montana 59812

STEPHEN S. MORSE, PhD

Professor, Department of Epidemiology, Columbia University, 

Mailman School of Public Health, 722 West 168th Street, New 

York, New York 10032

ERIC MOSSEL, PhD

Major, Medical Service Corps, US Army Reserve; Microbiologist, 

Division of Virology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

ROBERT MUNSON, PhD

Professor, Department of Pediatrics, Department of Molecular 

Virology, Immunology, and Molecular Genetics, and Department 

of Microbiology, The Research Institute at Nationwide Children’s 

Hospital and The Ohio State University, 700 Children’s Drive, 

Columbus, Ohio 43205

BILL NAUSCHUETZ, PhD

Lab Biopreparedness Coordinator, US Army Medical Command, 

2748 Worth Road, Suite 10, Fort Sam Houston, Texas 78234-6039

DAVID O’CALLAGHAN, PhD

Director, INSERM U1047, Université Montpellier 1, Place Eugène 

Bataillon, 34090 Montpellier, France 

GENE OLINGER, PhD, MBA

High Containment Coordinator, National Institute of Allergy 

and Infectious Diseases, Integrated Research Facility, Division of 

Clinical Research, 8200 Research Plaza, Fort Detrick, Maryland 

21702

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GEOFFREY K. PHILLIPS, MPH

Safety and Occupational Health Manager, US Army Medical Research and Materiel Command, MCMR-SS, 810 Schreider Street, 

Fort Detrick, Maryland 21702

ANN POWERS, PhD

Chief, Alphavirus Laboratory, Division of Vector-Borne Diseases, 

Centers for Disease Control and Prevention, 3156 Rampart Road, 

Fort Collins, Colorado 80521 

JON ROBERTUS, PhD

Professor Emeritus, The University of Texas at Austin, Molecular 

Biosciences, College of Natural Sciences, 2506 Speedway, Austin, 

Texas 78712

JANICE M. RUSNAK, MD, FACP, FIDSA

Contractor, Battelle, Medical Countermeasure Systems–Joint 

Vaccine Acquisition Program (MCS-JVAP), 1564 Freedman Drive, 

Fort Detrick, Maryland 21702

JOHN M. SCHERER, PhD, MT(ASCP)

Colonel, Medical Corps, US Army; Medical Research and Materiel 

Command, 810 Schreider Street, Fort Detrick, Maryland 21702

LARRY S. SCHLESINGER, MD

Professor and Chair, Department of Microbial Infection and 

Immunity Director, Center for Microbial Interface Biology, The 

Ohio State University, 798 Biomedical Research Tower, 460W 12th 

Avenue, Columbus, Ohio 43210

MICHAEL SCHUIT, MS

Research Scientist, Aerobiology Group, National Biodefense 

Analysis and Countermeasures Center (operated by BNBI for the 

US Department of Homeland Security), 8300 Research Plaza, Fort 

Detrick, Maryland 21702

HARALD SEIFERT, MD

Professor of Medical Microbiology and Hygiene, Institute for 

Medical Microbiology, Immunology, and Hygiene, University of 

Cologne, Goldenfelsstrasse 19-21, 50935 Köln, Germany

EDWARD SHAW, PhD

Associate Professor, Department of Microbiology and Molecular Genetics, Oklahoma State University, 307 Life Sciences East, 

Stillwater, Oklahoma 74078

PETER M. SILVERA, PhD 

Chief, Non-Clinical Development Division, US Army Medical 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702

BAL RAM SINGH, PhD

Professor and Director, Botulinum Research Center, Institute of 

Advanced Sciences, 78-540 Faunce Corner Mall Road, Dartmouth, 

Massachusetts 02747

SHAWN J. SKERRETT, MD

Professor, Division of Pulmonary and Critical Care Medicine, 

University of Washington, Harborview Medical Center, 325 Ninth 

Avenue, Seattle, Washington 98104

BRAD STILES, PhD

Adjunct Professor, Department of Biology, Wilson College, 1015 

Philadelphia Avenue, Chambersburg, Pennsylvania 17201

CHRISTINE TRAVISS, PhD

Microbiologist, Division of Select Agents and Toxins, Office of 

Public Health Preparedness and Response, Centers for Disease 

Control and Prevention, 1600 Clifton Road, NE, MS A-46, Atlanta, 

Georgia 30333

RENÉE M. TSOLIS, PhD

Professor, Department of Medical Microbiology and Immunology, University of California–Davis, 3146 Tupper Hall, 1 Shields 

Avenue, Davis, California 95616

BERNARD VERRIER, PhD

Director, Institut de Biologie et Chimie des Protéines, Fédération de Recherche, 3302 SFR BioSciences (Unité Mixte de Service 

3444/US8), Gerland-Lyon Sud, Université de Lyon 1, 69007 Lyon, 

France; and Laboratoire de Biologie Tissulaire et d’Ingénierie 

Thérapeutique, Centre National de la Recherche Scientifique, 

Unité Mixte de Recherche 5305, 69007 Lyon, France 

NICHOLAS J. VIETRI, MD 

Colonel, Medical Corps, US Army; Chief, Occupational Medicine, 

US Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland 21702; and Assistant Professor of Medicine, Uniformed Services University of the Health 

Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814

SCOTT C. WEAVER, PhD

John Sealy Distinguished University Chair in Human Infections 

and Immunity Director, Institute for Human Infections and 

Immunity Scientific Director, Galveston National Laboratory Professor, Departments of Pathology, Microbiology, and Immunology, Galveston National Laboratory, 301 University Boulevard, 

Galveston, Texas 77555-0610

JUDITH WHITE, PhD

Professor, Department of Cell Biology and Developmental Biology, University of Virginia, 1340 Jefferson Park Avenue, Charlottesville, Virginia 22908-0732

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Foreword

The concept of national defense has been undeniably shaped by the events of September 11, 2001. The 

US anthrax postal attacks immediately following 9/11 forever changed our perspective of biodefense related 

research. More recently, the continued threat of state-sponsored events or individual extremist groups has 

only compounded the severity of this facet of national security. As we focus our medical efforts to succeed at 

the point of injury, and to optimize the success of the operating forces, the identification and preparation for 

biological threats has become a synergistic force multiplier.

The US Department of Defense (DoD) continues to identify potential threats and prepare for possible 

biological attacks by maintaining a knowledge base and by actively developing and testing novel medical 

countermeasures. For example, scientists at the US Army Medical Research Institute of Infectious Diseases 

(Fort Detrick, MD) have developed vaccines against the causative agents of anthrax, plague, ricin intoxication, 

botulinum intoxication, Ebola virus, and encephalitic alphaviruses. Importantly, DoD scientists partner with 

other federal agencies, academic institutions, and pharmaceutical companies to test and evaluate vaccines and 

therapeutics against many other biological threats. There is no better example of this consortium approach 

then the DoD’s efforts during the recent Ebola outbreak in West Africa. DoD scientists and military personnel were on the ground diagnosing samples, sequencing viral genomes, and administering supportive care. 

Concurrently, some of the very products tested and evaluated by the DoD were deployed during the medical 

emergency. If it was not for DoD intervention, this outbreak had the potential to be substantially worse and 

spread further across Africa and around the world. Taken together, these brief examples demonstrate exactly 

why Medical Aspects of Biological Warfare needs to be maintained as an up-to-date information source.

The first edition of Medical Aspects of Chemical and Biological Warfare was published in 1997. A decade later, 

the chemical and biological aspects of this text warranted separate volumes. Thus, in 2007, Medical Aspects of 

Biological Warfare was released as a stand-alone textbook. Because of the fast-paced nature of microbiological 

research, new and emerging threats, and the changing policy, the authors pursued an updated version. In this 

third edition of the Textbooks of Military Medicine’s Medical Aspects of Biological Warfare, the authors have gone 

to great lengths to address many facets of biodefense research, preparedness, and consequence management. 

Individual chapters are devoted to understanding the pathogenesis and disease progression associated with 

bacterial and viral biothreat agents, such as Bacillus anthracis and Ebola virus. Additionally, intoxications by 

toxins such as ricin are also described in detail. These chapters highlight the current state of science for these 

agents and toxins: they clearly underscore the importance of pursuing basic science interests in these arenas, 

and the importance of maintaining a core pool of subject matter experts. Without basic science efforts, our 

continued understanding of these threats would suffer, and knowledge gaps would grow. Accordingly, current clinical treatment protocols and regimens are also discussed throughout the textbook and offer a bridge 

from the basic research to the applied clinical “real-world” applications.

This textbook also examines other less apparent biodefense-related topics. Acinetobacter baumii is used as an 

example of how a drug-resistant bacterium can impact the DoD, and further demonstrates how the institutional 

structure and strategic planning can be used to address such threats. Additional chapters discuss Medical 

Management and Consequence Management, and give current perspectives on patient care and federal and 

local response scenarios in the event of a biological attack. This edition also describes current laboratory biosafety and biosurety philosophies that have tremendous impacts on the execution of biodefense strategies that 

are constantly evolving. Finally, this version of the textbook gives a nod to the history of biodefense research 

while also addressing new and emerging biological threats, be they natural or engineered.

The authors, subject matter expert reviewers, and editors have produced a comprehensive and thoughtful reference source for the DoD, and I am proud of the scientists, physicians, and other professionals who 

contributed their time and efforts to produce the final product.

Lieutenant General Nadja Y. West, MD

The Surgeon General

Commanding General, U.S. Army Medical Command

Washington, DC

March 2017

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Preface

In an ever-changing and complex world, medical defense against biological pathogens must be a central 

pillar of our national defense strategy. Although biological warfare has been a legitimate concern for centuries, 

our current requirements and future operations emphasize the need for a continuing holistic approach to medical biological defense against these threats. From antiquity to the present day, agents such as Bacillus anthracis

(etiological agent of anthrax), Francisella tularensis (etiological agent of tularemia), Burkholderia mallei (etiological 

agent of glanders), Yersinia pestis (etiological agent of plague), and Variola (etiological agent of smallpox) have 

been on the forefront of biowarfare and biodefense. With increased uncertainty associated with terrorist groups, 

rogue nations, and “lone wolf” individuals, the threat of biological weapons is even more relevant today. 

Subject matter experts who wrote and reviewed these chapters focused on the most current data available 

at the time to create the most comprehensive reference source available for the US Department of Defense. 

Revising this textbook is important, not only to highlight the current state-of-the-art application for medical 

countermeasures, but also to discuss myriad current and future threats. Some of these evolving issues include 

the ongoing ramifications of the world’s largest-ever Ebola virus disease outbreak and the impact of emerging 

antibiotic resistance from select bacterial pathogens. Of recent note is the emerging B cereus biovar anthracis

strains isolated in Africa from fatal anthrax-like infections in chimpanzees and western lowland gorillas. These 

strains of B cereus were shown to harbor plasmids highly similar to both B anthracis virulence plasmids and, accordingly, were included on the US Department of Health and Human Services select agent list in 2016. These 

are just a few of the examples that underscore the complexities of biodefense research. Although we must remain 

vigilant in anticipating state-sponsored or terrorist activities, new threats are evolving in the natural world that 

could prove equally catastrophic to our military personnel and national interests. Preparation, cooperation, and 

rehearsal in accordance with the latest methodologies are the key ingredients to success in these current contexts. 

I am deeply grateful for the contributions of the scientists and physicians who collaborated in this endeavor. 

They are nationally and internationally recognized experts in their specialties, and their dedication to updating 

this textbook has been invaluable. I am pleased to introduce the latest edition of Medical Aspects of Biological Warfare. 

Colonel Thomas S. Bundt

Medical Service Corps, US Army

Commander, US Army Medical Research Institute of Infectious Diseases

Fort Detrick, Maryland

October 2016

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1

Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness

Chapter 1 

HISTORICAL OVERVIEW: FROM 

POISONED DARTS TO PAN-HAZARD 

PREPAREDNESS 

GEORGE W. CHRISTOPHER, MD, FACP*; DANIEL M. GERSTEIN, PhD†; EDWARD M. EITZEN, MD, MPH‡; and 

JAMES W. MARTIN, MD, FACP§

INTRODUCTION

EARLY USE

THE WORLD WARS

THE US PROGRAM

THE SOVIET PROGRAM

THE SPECIAL CASE OF IRAQ

OTHER NATIONAL PROGRAMS

BIOCRIMES

BIOLOGICAL TERRORISM

SOLUTIONS: TOWARD PAN-HAZARD PREPAREDNESS

Disarmament: The Biological Weapons Convention

Smallpox Preparedness

Dual Use Research of Concern

Toward Pan-Hazard Preparedness

SUMMARY

*Lieutenant Colonel (Retired), Medical Corps, US Air Force; Chief Medical Officer, Joint Project Manager-Medical Countermeasure Systems (JPMMCS), 10109 Gridley Road, Building 314, 2nd Floor, Fort Belvoir, Virginia 22060-5865 

†

Colonel (Retired), US Army; Adjunct Professor, School of International Studies, American University, 4400 Massachusetts Avenue, NW, Washington, 

DC 20016; formerly, Undersecretary (Acting) and Deputy Undersecretary, Science and Technology Directorate, Department of Homeland Security, 

Washington, DC 

‡

Colonel (Retired), Medical Corps, US Army; Senior Partner, Biodefense and Public Health Programs, Martin-Blanck and Associates, 2034 Eisenhower 

Avenue, Suite 270, Alexandria, Virginia 22314-4678; formerly, Commander, US Army Medical Research Institute of Infectious Diseases, 1425 Porter 

Street, Fort Detrick, Maryland 

§

Colonel (Retired), Medical Corps, US Army; Chief of Internal Medicine, US Army Healthcare Clinic, Vicenza, APO AE 09630-0040; formerly, Chief, 

Operational Medicine Department, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

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Medical Aspects of Biological Warfare 

INTRODUCTION

pathogens may inadvertently provide information 

that could be deliberately misused for biological 

weapons proliferation.3 

Numerous historical examples exist of military 

disasters resulting from failures to adapt policy, strategy, and doctrine to offset the impact of revolutionary 

advances in weapons technology.4 Biological medical 

defense programs, begun as narrowly focused efforts to 

counter a limited number of biological weapons agents, 

are being expanded as versatile capabilities, with a 

shift in emphasis from pathogen-specific approaches to 

capabilities-based programs to enable rapid responses 

to novel, potentially genetically engineered biological 

weapons agents. The response to biological weapons 

has fueled robust enterprises in basic and applied 

medical research, product development, manufacturing, stockpiling, infrastructure, public health policy, 

planning, and response capacities at local, national, 

and international levels.5

 Medical capabilities and biomedical research are being linked to diplomacy, commerce, education, ethics, law enforcement, and other 

activities to enable pan-societal sector responses to both 

biological weapons and the inevitable and dynamic 

challenges of naturally occurring emerging infectious 

diseases.3 Integration of biological defense and public 

health programs and their mutual development must 

be continuous to optimize outcomes and maximize 

efficient utilization of limited resources, because the 

challenges posed by both biological weapons agents 

and naturally emerging pathogens are open-ended.5 

Humans have used technology for destructive as 

well as beneficial purposes since prehistory. Aboriginal 

use of curare and amphibian-derived toxins as arrow 

poisons anticipated modern attempts to weaponize 

biological toxins such as botulinum and ricin. The 

derivation of the modern term “toxin” from the ancient 

Greek term for arrow poison, τωξικον φαρμακον 

(toxicon pharmicon; toxon = bow, arrow)1,2 underscores 

the historical link between weaponry and biological 

agents. 

Multiple factors confound the study of the history of 

biological weapons, including secrecy surrounding 

biological warfare programs, difficulties confirming 

allegations of biological attack, the lack of reliable 

microbiological and epidemiological data regarding alleged or attempted attacks, and the use of 

allegations of biological attack for propaganda and 

hoaxes. A review of historical sources and recent 

events in Iraq, Afghanistan, Great Britain, and the 

United States demonstrates that interest in biological 

weapons by state-sponsored programs, terrorists, 

and criminal elements is likely to continue. Humankind is witnessing a “democratization in the life sciences,” in which the field is becoming industrialized 

and therefore making biotechnology available to an 

ever increasing number of people, some of whom 

will undoubtedly have ill intent. In addition, there 

are growing concerns that well-intentioned life sciences research to advance medical defenses against 

biological weapons agents and other highly virulent 

EARLY USE

The impact of infectious diseases on military forces 

has been recognized since ancient times.6,7 The use of 

disease as a weapon was used long before microbial 

pathogenesis was understood. Military leaders only 

knew that a cause and effect relationship existed 

between certain activities, locations, or exposures to 

victims of disease that resulted in the spread of infections that ultimately provided a military advantage. 

For example, an early tactic was to allow an enemy to 

take sanctuary in locations endemic for infectious diseases in anticipation that its troops would be afflicted, 

thus allowing unimpeded access of opposing armies to 

areas where transmission of malaria was highly likely. 

Numerous anecdotal accounts exist of the attempted 

use of cadavers, animal carcasses, plant-derived 

toxins, and filth to transmit disease during antiquity 

through the Napoleonic era into modern times. Several 

examples illustrate the complex epidemiologic issues 

raised by biological warfare, the difficulty in differentiating epidemics resulting from biological attacks from 

outbreaks of disease that occur due to disruptions of 

war, and the adverse psychological impact of biological 

attacks on military operations. 

During a naval battle against King Eumenes of 

Pergamum in 184 BCE, Hannibal ordered earthen pots 

filled with snakes to be hurled onto the decks of enemy 

ships. The pots shattered on impact, releasing live 

serpents among the enemy sailors. The Carthaginian 

victory is attributed to the ensuing panic rather than 

envenomation8

; this illustrates that the psychological 

contagion of biological weapons may amplify their 

impact beyond their potential to cause organic disease. 

One of the most notorious early biological warfare 

attacks was the hurling of cadavers over the walls 

of the besieged city of Caffa, a Genoese colony in 

the Crimea, in 1346.9,10After war broke out between 

the Genoese and the Mongols in 1343 for control of 

the lucrative caravan trade route between the Black 

Sea and the Orient, the Mongols laid siege to Caffa. 

The plague, later known as the Black Death, was 

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Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness

spreading from the Far East and reached the Crimea in 

1346. The Mongols were severely afflicted and forced 

to abandon their siege. As a parting shot, they hurled 

“mountains of dead” over the city wall, probably with 

the use of a trebuchet, in the hope that “the intolerable stench would kill everyone inside.” An outbreak 

of plague in the city followed. A review by Wheelis10

suggests that the introduction of plague into the city 

by the cadavers—as a result of a tactically successful 

biological attack—may be the most biologically plausible of several competing hypotheses on the source 

of the outbreak. Although the predominant mode of 

plague transmission has been attributed to bites from 

infected fleas (which leave cadavers and carcasses to 

parasitize living hosts), modern experience (United 

States 1970–1995)11 has implicated transmission from 

contact with infected animal carcasses in 20% of 

instances in which the source of the infection could 

be attributed. Contact with tissue and blood would 

have been inevitable during the disposal of hundreds 

or possibly thousands of cadavers. Alternatively, 

plague could have been introduced by imported human cases or infected rodents brought into the city 

through maritime trade, which was maintained during 

the siege. The importation of plague by a rodent-flea 

transmission cycle across the city wall is considered 

less likely because rats are sedentary and rarely venture far from their nests; it is unlikely that they would 

have traversed an open distance of several hundred 

meters between the Mongol encampment and the city 

walls.10 Transmission from sylvatic to urban rodents 

is infrequent, at least under current ecological conditions.12Regardless of the portal of entry, the epidemic 

may have been amplified under siege conditions due 

to deteriorating sanitation and hygiene resulting in 

expansions of rats and fleas.

Smallpox was particularly devastating to Native 

Americans. The unintentional yet catastrophic introduction of smallpox to the Aztec empire during 

1520, and its subsequent spread to Peru in advance of 

Pizarro’s invasion of the Inca empire, played a major 

role in the conquest of both empires.13 During the 

French and Indian Wars (1754–1763), British forces 

provided Native Americans with handkerchiefs and 

blankets contaminated with scabs from smallpox patients to transmit disease.14–18 An epidemic of smallpox 

followed among the Native Americans of the Ohio 

River Valley. It is difficult to evaluate the tactical success of these biological attacks in retrospect because 

smallpox may have been transmitted during other 

contacts with colonists, as had previously occurred 

in New England and the South. Smallpox scabs are 

thought to have low infectivity due to the binding of 

virions in a fibrin matrix, and transmission by fomites 

has been considered less efficient than respiratory 

droplet transmission.13

THE WORLD WARS

The birth of scientific bacteriology during the 19th 

century provided the scientific and technical basis for 

modern biological weapons programs. The Hague Conventions of 1899 and 1904 outlawed the use of “poison 

or poisoned arms,” although bacteriological weapons 

were not specifically addressed.19–20 During World War 

I, German espionage agents reportedly infected draft 

animals intended for military use with Burkholderia 

[Pseudomonas] mallei and Bacillus anthracis.

21–23 Covert 

operations were reportedly conducted in Argentina, 

Norway, Mesopotamia, Romania, Russia, and the 

United States. Unsuccessful attempts were also made to 

cripple grain production in Spain using wheat fungus.21

The German biowarfare program of World War I is 

of special interest because it was the first program with 

a scientific basis; it conducted a large-scale (strategic) 

biological campaign, which targeted neutral nations 

as well as belligerents, and it targeted crops and animals instead of humans. Although German operatives 

thought the program was successful, confirmatory data 

are not available.21

In response to chemical warfare during World War 

I, the 1925 Geneva Protocol, an international protocol 

(for the Prohibition of the Use in War of Asphyxiating, 

Poisonous or Other Gases, and of Bacteriological 

Methods of Warfare), was formulated by the League 

of Nations’ Conference for the Supervision of the 

International Trade in Arms and Ammunition. It had 

no verification mechanism and relied on voluntary 

compliance. Many of the original signatory states 

reserved the right to retaliatory use, making it effectively a no first-use protocol. Signatories that began 

research programs to develop biological weapons between World War I and II included Belgium, Canada, 

France, Great Britain, Italy, The Netherlands, Poland, 

and the Soviet Union.24

After defeating Russia in the 1905 Russo-Japanese 

War, Japan became the dominant foreign power in 

Manchuria, and seized full military control between 

September 1931 and the end of 1932. Major Shiro Ishii, 

a Japanese army physician, established a biological 

weapons laboratory in Harbin, but soon realized that 

his controversial involuntary human research could 

not be conducted freely there. Ishii moved to a secret 

facility at Beiyinhe, 100 km south of Harbin, and began large-scale experimentation. All research study 

subjects died of either experimental infection or live 

vivisection. These studies continued until a prisoner 

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Medical Aspects of Biological Warfare 

riot and escape, which resulted in the closing of the 

facility in 1937. However, larger and more extensive 

facilities were subsequently built.24

In 1936 Ishii built Unit 731, a massive research 

facility 24 km south of Harbin, where a census of 200 

prisoners was kept as expendable subjects of experimentation. Ultimately, more than 3,000 Chinese prisoners were killed during these experiments. Most of 

the evidence was destroyed at the end of the war, and 

in all likelihood the actual number was much greater.24

Additional facilities included Unit 100 at Changchun, 

and Unit Ei 1644 in Nanking. Unit 100 was primarily a veterinary and agricultural biowarfare research 

unit for developing biological weapons for sabotage. 

Although animals and crops were the focus of most 

of the research, numerous human studies were also 

conducted, similar to those conducted by Unit 731. In 

addition to conducting human experimentation, Unit 

Ei 1644 supported Unit 731’s research efforts with 

bacterial agent production and flea cultivation.24

Eleven Chinese cities were allegedly attacked during “field trials” using agents including Yersinia pestis, 

Vibrio cholerae, and Shigella spp. These attacks may 

have backfired because up to 10,000 Japanese soldiers 

reportedly contracted cholera after a biological attack 

on Changde in 1941.25 The field trials were terminated 

in 1943, yet basic research and human experimentation continued until the end of the war.24–26 Despite 

the enormously expensive program (both in terms of 

national treasure and human lives) and the weaponization of many agents, Japan never developed a credible 

biowarfare capability, mainly because of the failure to 

develop an effective delivery system.17

In contrast to Japanese efforts during World War 

II, a German offensive biological weapons program 

never materialized. Hitler reportedly issued orders 

prohibiting biological weapons development. Unethical experimental infections of prisoners were done 

primarily to study pathogenesis and develop vaccines 

and sulfonamides, rather than to develop biological 

weapons. With the support of high-ranking Nazi party 

officials, however, scientists began biological weapons 

research, but their results lagged far behind those of 

other countries.27

Polish physicians used a vaccine and a serologic test 

in a brilliant example of “biological defense.” Knowing 

that inoculation with killed Proteus OX-19 would cause 

false-positive Weil-Felix typhus test results, physicians 

inoculated local populations with formalin-killed 

Proteus OX-19 to create serologic pseudoepidemics of 

typhus. Using serologic surveillance, the German army 

avoided areas with epidemic typhus; consequently, 

residents of these areas were spared deportation to 

concentration camps.28 Unconfirmed allegations indicate that Polish resistance fighters used letters contaminated with B anthracis to cause cutaneous anthrax 

among Gestapo officials21,29 and used typhus against 

German soldiers.21 Czechoslovakian agents reportedly 

used a grenade contaminated with botulinum toxin, 

supplied by British Special Operations, to assassinate 

Reinhard Heydrich, the Nazi governor of occupied 

Czechoslovakia30,31; however, the veracity of this claim 

has been challenged.23 

The perceived threat of biological warfare before 

World War II prompted Great Britain to stockpile 

vaccines and antisera, establish an emergency public 

health laboratory system, and develop biological 

weapons. “Cattle cakes” consisting of cattle feed contaminated with B anthracis spores were designed to 

be dropped from aircraft into Axis-occupied Europe 

to cause epizootic anthrax among livestock,32,33 which 

would in turn induce famine. The cattle cakes were 

intended as a strategic economic weapon rather than 

as a direct cause of human anthrax. In addition, explosive munitions designed to aerosolize and disperse B 

anthracis spores as antipersonnel weapons were tested 

on Gruinard Island near the coast of Scotland in 1942. 

These experiments successfully caused anthrax in 

targeted sheep.34 The antipersonnel weapons were 

not mass produced, and neither the cattle cakes nor 

the explosive munitions were used.21 Great Britain 

continued its offensive biological warfare program 

during the early Cold War era in conjunction with the 

United States and Canada, and it performed secret 

open-air tests using pathogens in off-shore ocean 

sites near the Bahamas and Scotland.21Great Britain’s 

offensive program was terminated between 1955 and 

195635 because of budgetary constraints and reliance 

on nuclear deterrence.32,33Gruinard Island, which had 

been quarantined because of focal soil contamination 

by B anthracis spores following munitions testing, was 

decontaminated in 1986 using 2,000 tons of seawater 

and 280 tons of formaldehyde.36 The United Kingdom 

conducts research to develop medical countermeasures 

at the Defence Science and Technologies Laboratories 

at Porton Down. 

THE US PROGRAM

The US military recognized biological warfare as 

a potential threat after World War I. Major Leon Fox 

of the Army Medical Corps wrote an extensive report 

concluding that improvements in health and sanitation 

made biological weapons ineffective. In 1941, before 

the US entry into World War II, opinions differed 

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Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness

about the threat of biological warfare. Consequently, 

the Secretary of War asked the National Academy of 

Sciences to appoint a committee to study the issue. The 

committee concluded in February 1942 that biowarfare 

was feasible and the United States should reduce its 

vulnerability.

President Franklin D Roosevelt established the 

War Reserve Service (with George W Merck as director) to develop defensive measures against biological weapons. By November 1942 the War Reserve 

Service asked the Army’s Chemical Warfare Service 

to assume responsibility for a secret large-scale 

research and development program, including the 

construction and operation of laboratories and pilot 

plants. The Army selected a small National Guard 

airfield at Camp Detrick in Frederick, Maryland, 

for the new facilities in April 1943. By summer of 

1944 the Army had testing facilities in Horn Island, 

Mississippi (later moved to Dugway, Utah), and 

a production facility in Terre Haute, Indiana. No 

agents were produced at the Terre Haute plant because of safety concerns; simulant tests disclosed 

contamination after trial runs. In the only reported 

US offensive use of a biological weapon, the Office of 

Strategic Services (predecessor of the Central Intelligence Agency) used staphylococcal enterotoxin in 

a food-borne attack to cause an acute but self-limited 

illness in a Nazi party official.37,38 Cattle cakes using 

B anthracis spores were produced at Camp Detrick 

and shipped to Great Britain, but were never used. 

The War Reserve Service was disbanded after the war 

and the Terre Haute plant was leased for commercial 

pharmaceutical production.31 In January 1946 Merck 

reported to the Secretary of War that the United 

States needed a credible capability to retaliate if attacked with biological weapons. Basic research and 

development continued at Camp Detrick.

The United States learned of the extent of Japanese 

biological weapons research after World War II. In 

an action that has become controversial, Ishii and his 

fellow scientists were given amnesty for providing 

information derived from years of biological warfare 

research.24

When war broke out on the Korean peninsula in 

June 1950, concerns about Soviet biological weapons 

development and the possibility that the North Koreans, Chinese, or Soviets might resort to biological 

warfare resulted in an expansion of the US program. 

A large-scale production facility in Pine Bluff, Arkansas, was established. The plant featured advanced 

laboratory safety and engineering measures enabling 

large-scale fermentation, concentration, storage, and 

weaponization of microorganisms. In 1951 the first 

biological weapons, anticrop bombs, were produced. 

The first antipersonnel munitions were produced in 

1954 using Brucella suis. The United States weaponized seven antipersonnel agents and stockpiled three 

anticrop agents (Table 1-1) over 26 years.39 

Field tests using surrogate agents were conducted 

in the United States between 1949 and 1968, in which 

the general public and test subjects were uninformed. 

At least 239 open-air tests were conducted at several 

locations including the Dugway Proving Ground, 

Utah; remote Pacific Ocean sites; and populated areas 

including Minneapolis, St. Louis, New York City, San 

Francisco, and Eglin Air Force Base, Florida. These 

studies tainted the history of the offensive biological 

warfare program. The Special Operations Division 

at Camp Detrick conducted most of the field tests to 

study possible methods of covert attack and to examine 

aerosolization methods, the behavior of aerosols over 

large geographic areas, and the infectivity and rates 

of decay of aerosolized microbes subjected to solar 

irradiation and climatic conditions. Most tests used 

simulants thought to be nonpathogenic, including 

Bacillus globigii, Serratia marcescens, and particulates 

of zinc cadmium sulfide.39,40

In conjunction with the US Department of Agriculture (USDA), several open-air tests were conducted 

using anticrop agents at sites selected for safety. 

TABLE 1-1

BIOLOGICAL AGENTS PRODUCED BY THE US MILITARY (DESTROYED 1971–1973)*

Lethal Agents Incapacitating Agents Anticrop Agents

Bacillus anthracis Brucella suis Rice blast

Francisella tularensis Coxiella burnetii Rye stem rust

Botulinum toxin Venezuelan equine encephalitis virus Wheat stem rust

Staphylococcal enterotoxin B

*Lethal and incapacitating agents were produced and weaponized. Anticrop agents were produced but not weaponized.

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Medical Aspects of Biological Warfare 

Open-air releases of human pathogens (Coxiella burnetii, Francisella [Pasturella] tularensis) were performed 

at the Dugway Proving Ground, Eglin Air Force Base, 

and remote Pacific Ocean sites to study viability 

and infectivity using animal challenge models.21,39,40

Controversial studies included environmental tests 

to determine whether African Americans were more 

susceptible to Aspergillus fumigatus, as had been 

observed with Coccidioides immitis. These studies included the 1951 exposure of uninformed workers at 

Norfolk Supply Center in Norfolk, Virginia, to crates 

contaminated with Aspergillus spores. In 1966 the US 

Army conducted covert experiments in the New York 

City subways. Light bulbs filled with Bacillus subtilis 

var niger were dropped from subway platforms onto 

the tracks to study the distribution of the simulant 

through the subway system.39–41 Similar tests were 

conducted using the ventilation system of the New 

York City subways and the Pentagon.

The first large-scale aerosol vulnerability test 

conducted in San Francisco Bay in September 1950 

using B globigii and S marcescens demonstrated the 

public health issues of such testing.41 An outbreak 

of 11 cases of nosocomial S marcescens (Chromobacterium prodigiosum) urinary tract infection occurred 

at the nearby Stanford University Hospital; one case 

was complicated by fatal endocarditis. Risk factors 

included urinary tract instrumentation and antibiotic 

exposures.42 No similar outbreaks were reported by 

other San Francisco area hospitals. A panel of civilian 

and academic public health experts secretly convened 

by the Army in 1952 failed to reach a conclusion 

regarding the possible link between the Stanford 

outbreak and the testing program, but recommended 

that other microbes be used as simulants.41 Public 

disclosure of the testing program in the Washington 

Post on December 22, 1976, and in US Senate hearings 

in 197743 resulted in harsh criticism of the continued 

use of S marcescens as a simulant after the Stanford 

epidemic. However, a 1977 report from the Centers 

for Disease Control and Prevention (CDC) concluded 

that in 100 outbreaks of S marcescens infection, none 

was caused by the 8UK strain (biotype A6, serotype 

O8:H3, phage type 678) used by the Army testing 

program.44 Other reports from the 1970s postulated 

a link between S marcescens infection and the testing 

program; however, all clinical isolates available for 

strain typing were antigenically distinct from the 

Army test strain. In all likelihood, the 1950 Stanford 

S marcescens epidemic represents an early example 

of a nosocomial outbreak caused by opportunistic 

pathogens of low virulence complicating the use of 

medical devices and surgical procedures in the setting 

of antibiotic selection pressure.44 

The US program developed modern biosafety technologies and procedures including protective equipment, engineering and safety measures, and medical 

countermeasures, including new vaccines. There were 

456 occupational infections and three fatalities (two 

cases of anthrax in 1951 and 1958 and a case of viral 

encephalitis in 1964) reported at Fort Detrick during 

the offensive program (1943–1969).39 The infection rate 

of fewer than 10 infections per million hours of work 

was within the contemporary National Safety Council 

standards; the morbidity and mortality rates were 

lower than those reported by other laboratories. There 

were 48 infections and no fatalities at the production 

and testing sites.39

In 1954 the newly formed Medical Research Unit 

at Fort Detrick began studies to develop vaccines and 

therapy to protect against biological agents. Researchers began using human volunteers in 1956 as part of a 

congressionally approved program called “Operation 

Whitecoat.” This use of volunteers set the standard 

for ethics and human use in research. Active duty 

soldiers with conscientious objector status served as 

research subjects, and participation was voluntary 

with informed consent. The program concluded with 

the end of conscription in 1973.

Numerous unsubstantiated allegations were made 

during the Cold War era. During the Korean War 

(1950–1953), North Korean, Chinese, and Soviet officials made numerous accusations of US biowarfare 

attacks. Many allegations appear to be based on Chinese experiences during World War II field testing 

conducted by the Japanese Unit 731. Polish medical 

personnel were sent to China to support the communist war effort, accompanied by eastern European 

correspondents, who made numerous accusations 

based on anecdotal accounts of patients. These allegations, however, were not supported by scientific 

evidence. Some stories, such as the use of insect vectors 

to spread cholera, had dubious scientific plausibility. 

The North Korean and Chinese governments ignored 

or dismissed offers from the International Committee 

of the Red Cross and the World Health Organization 

(WHO) to conduct impartial investigations. The Soviet 

Union thwarted a proposal from the United States and 

15 other nations to the United Nations (UN) requesting the establishment of a neutral commission for 

investigation. The United States admitted to having 

biological weapons but denied using them. The credibility of the United States may have been undermined 

by the knowledge of its biological weapons program 

and its failure to ratify the 1925 Geneva Protocol until 

1975. Although unsubstantiated, these accusations 

resulted in a loss of international goodwill toward 

the United States and demonstrated the propaganda 

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Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness

value of biological warfare allegations, regardless of 

veracity.43 Reviews of documents from former Soviet 

archives provide evidence that the allegations were 

fictitious propaganda.45–47 

The Soviet Union accused the United States of 

testing biological weapons on Canadian Eskimos, 

resulting in a plague epidemic,48 and of collaborating 

with Colombia in a biological attack on Colombian 

and Bolivian peasants.49 The United States was also 

accused of planning to initiate an epidemic of cholera 

in southeastern China50 and of the covert release of 

dengue in Cuba.51 Similarly, the US allegations that 

Soviet armed forces and their proxies had used “yellow 

rain,” aerosolized trichothecene mycotoxins (inhibitors 

of DNA and protein synthesis derived from fungi of 

the genus Fusarium) in Laos (1975–1981), Kampuchea 

(1979–1981), and Afghanistan (1979–1981), are widely 

regarded as unsubstantiated. The remote locations of 

the alleged attacks made intelligence investigations difficult. Western intelligence operatives never witnessed 

these alleged attacks, and no samples of the aerosols 

were recovered. Confounding factors included:

• contradictory testimonies from survivors of 

alleged attacks; 

• discrepancies in reported symptoms; 

• low disease rates in the allegedly attacked 

populations; 

• the recovery of mycotoxin in fewer than 10% 

of the clinical and environmental samples 

submitted; 

• the presence of Fusarium organisms as environmental commensals; 

• the possible decay of toxin under prevailing 

environmental conditions; 

• conflicting results of toxin assays from different laboratories; 

• the similarity of alleged yellow rain deposits 

recovered from environmental surfaces to bee 

feces in ultrastructural appearance and pollen 

and mold content; and 

• the natural occurrence of showers of bee feces

from swarms of honey bees in the rain forests 

of southeast Asia.52 

The US offensive program resulted in an understanding of the strategic nature of biological weapons. 

By the late 1950s assessments of the potential utility of 

biological weapons were mixed. In a letter from one of 

Dwight D Eisenhower’s President’s Science Advisory 

Council members, George Kistiakowsky, to James 

Killian, the chair of the council, the author made it clear 

that developing highly concentrated formulations of 

biological agents, proper handling of pathogens, and 

appropriate weaponization would result in cases that 

did not act as “normal” disease.53 At high concentrations and in a dried formulation, biological agents had 

the potential for causing high mortality and morbidity. 

Still, questions remained about the potential to successfully use biological weapons in a controlled and 

reliable manner. The follow-on testing authorized by 

President John F Kennedy under the umbrella program 

of Project 112 was designed to fill in these knowledge 

gaps.40,54 In the Bay of Pigs operation of 1961, military 

planners had developed enough interest in biological 

weapons that their use was contemplated. The codenamed “Marshall Plan” called for releasing incapacitating agents to attack defenders on the beach. Ultimately, 

the plan was scrapped and biological weapons were 

not used.55 

By the late 1960s domestic and international pressures were calling for the elimination of the US offensive biological warfare program. At Dugway Proving 

Ground, an incident involving chemical weapons 

testing caused the death of 3,000 sheep. Debates about 

chemical and biological weapons, both for and against 

the development of offensive capabilities, ensued 

between Congress, the administration, industry, and 

even private citizens. In Europe draft texts of what 

would later become the Convention on the Prohibition of the Development, Production and Stockpiling 

of Bacteriological and Toxin Weapons and on their 

Destruction (1972 Biological Weapons Convention 

[BWC]) were being developed by Great Britain, Sweden, and the Soviet Union. 

In May 1969 US President Richard Nixon called for 

an interagency review of chemical-biological warfare 

policies. The review was authorized as part of National Security Study Memorandum 59. The findings 

resulted in recommendations to President Nixon to 

eliminate the US offensive program and retain a defensive program. 

To this end, on November 25, 1969, when visiting 

Fort Detrick, President Nixon announced a new US 

policy on biological warfare, unilaterally renouncing 

the development, production, stockpiling, and use 

of biological weapons. In explaining his decision, 

President Nixon stated, “Biological weapons have 

massive, unpredictable, and potentially uncontrollable 

consequences. They may produce global epidemics 

and impair the health of future generations.”56 Almost 

immediately after the statement, confusion and a potential loophole caused by the ambiguity concerning 

biologically derived toxins that were technically excluded from the renunciation were corrected through 

National Security Study Memorandum–85, “Review 

of Toxins Policy,” which was issued on December 31, 

1969. 

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Medical Aspects of Biological Warfare 

The US Army Medical Unit was closed, and Fort 

Detrick and other installations in the offensive 

weapons program were redirected to solely develop defensive measures such as vaccines, drugs, 

and diagnostics. The US Army Medical Research 

Institute of Infectious Diseases (USAMRIID) was 

created with biosafety level 3 and 4 laboratories 

dedicated to developing medical defensive countermeasures. By May 1972 all antipersonnel agents 

had been destroyed, and the production facility at 

Pine Bluff, Arkansas, was converted into a research 

facility. By February 1973 all agriculture-targeted 

biological agents had been destroyed. Although 

staphylococcal entertoxin was used during World 

War II by Office of Strategic Services’ espionage 

agents,37,38 biological weapons have never been used 

by the US Armed Forces.39 The Central Intelligence 

Agency developed weapons containing cobra venom and saxitoxin for covert operations; all records 

regarding their development and deployment were 

destroyed in 1972; all remaining toxin samples were 

destroyed per presidential orders after a US Senate investigation.37 The United States signed and 

ratified both the 1925 Geneva Convention and the 

1972 BWC, which outlaws all offensive biological 

weapons research, production, and possession, in 

1975 (see Disarmament: The Biological Weapons 

Convention).

Although many welcomed the termination of the US 

offensive program for moral reasons, the decision was 

partly motivated by pragmatic considerations. Biological weapons were unnecessary for national security 

because of a formidable arsenal of conventional, chemical, and nuclear weapons. Although open-air simulant 

studies suggested that biological weapons would be 

effective, the potential effects of aerosols of virulent 

agents on targeted populations were still conjectural 

and could not be empirically validated for ethical and 

public health reasons. Despite evidence to the contrary 

from information obtained through the US offensive 

program, some still considered biological weapons to 

be untried, unpredictable, and potentially hazardous 

for the users. Field commanders and troops were unfamiliar with their use. Most importantly, the United 

States and allied countries had a strategic interest in 

outlawing biological weapons programs to prevent the 

proliferation of relatively low-cost weapons of mass 

destruction. Outlawing biological weapons made the 

arms race for weapons of mass destruction prohibitively expensive, given the cost of nuclear programs.21,57

THE SOVIET PROGRAM

Although a signatory to the 1925 Geneva Convention, the Soviet Union began a weapons development 

program in 192858 under the control of the state security 

apparatus, GPU (the Unified State Political Administration of the Committee of People’s Commissars of the 

USSR). Work was initially done with typhus, reportedly 

with experimentation on political prisoners at Slovetsky 

Island in the Baltic Sea and nearby concentration camps. 

The program subsequently expanded to include work 

with the agents of Q fever, glanders, and melioidosis, 

and possibly tularemia and plague. Outbreaks of Q 

fever and tularemia among German troops are two 

suggested, but unconfirmed, Soviet uses of biological 

warfare during World War II.59 However, the origin of 

epidemic tularemia during the battle of Stalingrad as a 

consequence of biological warfare has been challenged 

and attributed to natural causes and a breakdown of 

public health.60 Similar outbreaks of Q fever in Axis 

troops in Italy, Greece, Bulgaria, and the Ukraine61; in 

Allied troops in the Mediterranean Theater62–64; and 

more recently, among Czech peacekeepers in BosniaHerzegovina65 and tularemia among civilians during the 

Kosovo conflict66 have been attributed to amplification 

of natural transmission cycles during wartime.

Stalin was forced to move his biological warfare 

operations out of the path of advancing German 

forces. Laboratories were moved to Kirov in eastern 

European Russia, and testing facilities were eventually established on Vozrozhdeniya Island on the Aral 

Sea between the Soviet Republics of Kazakhstan and 

Uzbekistan. At the conclusion of the war, Soviet troops 

invading Manchuria captured many Unit 731 Japanese scientists and learned of their extensive human 

experimentation through captured documents and 

prisoner interrogations. Emboldened by the Japanese 

findings, Stalin put KGB (Committee of State Security) 

chief Lavrenty Beria in charge of a new biowarfare 

program. The production facility at Sverdlovsk was 

constructed using Japanese plans. After Stalin died in 

1953, Beria was executed, and Nikita Khrushchev, the 

new Kremlin leader, transferred the biological warfare 

program to the Fifteenth Directorate of the Red Army. 

Colonel General Yefim Smirnov, a strong advocate of 

biological weapons who had been the chief of army 

medical services during the war, became the director.67

In 1956 Defense Minister Marshall Georgy Zhukov 

announced that the Soviet Union would be capable 

of deploying biological and chemical weapons in the 

next war. By 1960 numerous research facilities existed 

in the Soviet Union. Although the Soviet Union signed 

the 1972 BWC, it doubted US compliance, and subsequently expanded its program.58,59,67 Various institutions under different ministries and production facilities were incorporated into an organization known as 

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Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness

Biopreparat to carry out offensive research, development, and production under the label of legitimate 

civil biotechnology research. Biopreparat conducted 

clandestine activities at 52 sites and employed more 

than 50,000 people. Production capacity for weaponized smallpox was 90 to 100 tons annually.59

The Soviet Union was an active participant in 

WHO’s 1964 to 1979 smallpox eradication program. 

Soviet physicians participating in the program sent 

specimens to Soviet research facilities. For the Soviets, 

the program presented an opportunity not only to rid 

the world of naturally occurring smallpox, but also—

reportedly—to obtain virulent strains of smallpox 

virus that could be used to develop biological weapons. 

WHO announced the eradication of smallpox in 1980, 

and the world rejoiced at this public health breakthrough. The bioweapon developers in the former 

Soviet Union had a more cynical reaction. Smallpox 

eradication would result in the termination of vaccination; eventually the world’s population would again 

become vulnerable. It was this vulnerability that would 

inspire the former Soviet Union to develop smallpox 

as part of a strategic weapons system, with production 

of the virus on a massive scale and plans for delivery 

using intercontinental missiles.59

In addition to military biological weapons programs, the Soviets developed toxin weapons for use 

by Warsaw Pact intelligence services. An assassination 

using a biological weapon was executed in September 

1978 when a Bulgarian secret service member attacked 

Georgi Markov, a Bulgarian exile living in London. 

A device concealed in an umbrella discharged a tiny 

pellet into the subcutaneous tissue of his leg. He died 

several days later. The pellet, which had been drilled 

to hold a toxic material, was found at autopsy. No 

toxin was identified, but ricin was postulated as the 

only toxin with the potency to kill with such a small 

dose.68 Vladimir Kostov, a Bulgarian defector living in 

Paris, had been attacked in a similar manner a month 

earlier. He experienced fever and pain and bleeding at 

the wound site, yet had no further complications. After 

learning of Markov’s death, he sought medical evaluation; radiographs disclosed a small metallic pellet in 

subcutaneous fat. The pellet was surgically removed. 

Kostov then tested positive for anti-ricin antibodies, 

supporting the probable use of ricin in these attacks.23

In October 1979 a Russian emigrant newspaper 

published in Frankfurt, Germany, reported a sketchy 

story of a mysterious anthrax epidemic in the Russian 

city of Sverdlovsk (now Yekaterinburg). The military 

reportedly took control of hospitals in Sverdlovsk to 

care for thousands of patients with a highly fatal form 

of anthrax. Soviet officials attributed the epidemic to 

cutaneous and gastrointestinal anthrax contracted from 

contaminated meat. However, US intelligence agencies 

suspected that the outbreak resulted from inhalational 

anthrax following a release of B anthracis spores from 

Compound 17, a Soviet military microbiology facility.69–71

The Central Intelligence Agency sought the opinion of 

Matthew Meselson, a Harvard biologist who had been a 

strong proponent of the Nixon ban of the US biological 

warfare program. He initially doubted the Soviet weapon 

release hypothesis. Other observers reviewing the same 

evidence reached different conclusions, however, and 

satellite imagery from the late spring of 1979 showed 

a flurry of activity at and around the Sverdlovsk installation consistent with a massive decontamination 

effort. The incident generated enough concern within 

the Reagan administration and the Department of 

Defense (DoD) to increase military biopreparedness.

Debate of the incident raged for the next 12 years. 

Meselson testified before the US Senate that the burden of evidence supported the claim that the outbreak 

resulted from the Soviets’ failure to keep B anthracisinfected animals out of the civilian meat supply. In 

1992, after the fall of the Soviet Union, Meselson was 

allowed to take a team of scientists to review autopsy 

material and other evidence from the Sverdlovsk incident. The team’s attempts to review hospital records of 

cases from the outbreak were unsuccessful because the 

KGB had confiscated the records. However, the team 

performed the following: 

• acquired an administrative list of 68 of the 

deceased; 

• obtained information from grave markers in 

a cemetery designated for the anthrax casualties; 

• obtained epidemiological data by interviewing nine survivors and relatives and friends 

of 43 deceased; and 

• determined that the cases occurred among 

people who had either lived or worked in a 

narrow zone southeast of Compound 17 during the first week of April 1979. 

Archived weather reports at the city’s airport 

disclosed that the wind direction on April 2, 1979, 

correlated with the geographic distribution of cases. 

Meselson and his team concluded that the outbreak 

resulted from the escape of aerosolized spores from 

the facility on April 2, 1979, with downwind transmission.69 Furthermore, Russian pathologists who 

had conducted autopsies on 42 fatalities, and had 

courageously preserved tissue specimens and autopsy 

records at great personal risk, shared their findings 

with Meselson’s team and published their results 

confirming inhalational anthrax,72 described the Soviet 

cover-up of the outbreak, and postulated a release of 

spores from Compound 17.71

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Medical Aspects of Biological Warfare 

In 1992 Russian leader Boris Yeltsin admitted in private conversations with President George H Bush that 

the KGB and military had misrepresented the anthrax 

deaths. Subsequently, in a press release, Yeltsin admitted to the offensive program and the origin of the Sverdlovsk biological weapons accident. Additionally, retired 

Soviet general Andrey Mironyuk disclosed that safety 

filters had not been activated on the fateful morning in 

early April 1979, resulting in the escape of aerosolized 

B anthracis and the ensuing epidemic.73 Soviet defectors, 

including Ken Alibek, first deputy chief of Biopreparat 

from 1988 to 1992, confirmed that not only was the 

Sverdlovsk epidemic caused by an accidental release 

of spores from a biological weapons production plant, 

but also that the Soviet biological warfare program 

had been massive.59 In September 1992 Russia entered 

an agreement with the United States and the United 

Kingdom that acknowledged a biological weapons 

program inherited from the Soviet Union, committed 

to its termination, and agreed to onsite inspections. 

The United States assisted the Russian Federation and 

other former Soviet republics through the Nunn-Lugar 

Biological Threat Reduction Program (later called the 

Cooperative Biological Engagement Program) to:

• dismantle biological weapons research, development, and production infrastructure; 

• secure dangerous pathogens into central reference laboratories; 

• upgrade laboratory safety and security; 

• enhance capacities for diagnosis, surveillance, 

and public health response; and 

• engage scientists with biological weapons 

expertise in projects directed to modeling, 

medical countermeasure development, and 

other peaceful purposes.74,75

This led to the dismantlement or conversion 

of three large production facilities and dozens of 

institutes that supported the biological weapons 

program, the destruction of 150 tons of B anthracis weapons agent on Vozrozhdeniya Island, and 

unprecedented transparency at potential dual-use 

facilities that had previously been closed to foreigners.76 However, in 1999 President Vladimir Putin, 

proposed the development of weapons based on 

new genetic technology. Although this directive was 

promptly dropped from publicly available documents, he retracted the 1992 disclosures of President 

Yeltsin.77 The Russian government currently denies 

that the former Soviet offensive program had ever 

existed, claiming that it had only conducted defensive research.58,77 According a 2013 US Department 

of State report, it is still unclear if the Russian Federation has completed the destruction or diversion 

of the offensive program to peaceful purposes, or if 

it continues to conduct activities inconsistent with 

the BWC.78 

THE SPECIAL CASE OF IRAQ

The most ominous biological warfare threat that 

US military forces have faced came during Operations 

Desert Shield and Desert Storm in 1990 and 1991. Intelligence reports suggested that Iraq had developed and 

operated a biological weapons program during the 

1980s. Coalition troops trained in protective gear were 

issued ciprofloxacin in theater for use as postexposure 

prophylaxis against an Iraqi anthrax attack. Before the 

hostilities, approximately 150,000 US troops received 

the Food and Drug Administration–licensed anthrax 

vaccine, and 8,000 received a botulinum toxoid vaccine 

approved by the Food and Drug Administration as an 

investigational new drug. Postwar inspections by the 

multinational UN Special Commission (UNSCOM) on 

Iraq were repeatedly confounded by Iraqi misinformation and obfuscation. After General Hussein Kamal 

defected in 1995, the Iraqi government disclosed that 

it had operated a robust biological weapons program at six major sites since the 1980s, contrary to 

its obligations as a state party to the BWC. The Iraqi 

program conducted basic research on B anthracis, rotavirus, camelpox virus, aflatoxin, botulinum toxins,

mycotoxins, and an anticrop agent (wheat cover 

rust); and it tested several delivery systems including 

aerial spray tanks and drone aircraft. Furthermore, 

the Iraqi government had weaponized 6,000 L of B 

anthracis spores and 12,000 L of botulinum toxin in 

aerial bombs, rockets, and missile warheads before 

the 1991 Persian Gulf War (Table 1-2 and Table 1-3). 

Although these weapons were deployed, they were 

not used.79,80 The reasons behind Saddam Hussein’s 

decision not to use these weapons are unclear; perhaps 

TABLE 1-2

BIOLOGICAL AGENTS PRODUCED BY IRAQ*

Agent Produced (L) Weaponized (L)

Botulinum 19,000 10,000

Bacillus anthracis 8,500 6,500

Aflatoxin 2,200 1,580

*Disclosed by the Iraq government in 1995.

L: liter

Bio Ch 01 - rebuild 4 - pp 1-36 - final d.indd 10 6/21/18 
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Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness

he was concerned about provoking massive retaliation. Alternately, decisive factors may have included 

the possible ineffectiveness of untested delivery and 

dispersal systems, the probable ineffectiveness of liquid slurries resulting from poor aerosolization, and 

the potential hazards to Iraqi troops, who lacked the 

protective equipment and training available to coalition forces.81 The Iraqis claimed to have destroyed 

their biological arsenal immediately after the war 

but were unable to provide confirmatory evidence. A 

covert military research and development program 

continued for another 4 years, with the intent of resuming agent production and weapons manufacture 

after the end of UN sanctions. Infrastructure was 

preserved, and research on producing dried agent 

was conducted under the guise of biopesticide production at the Al Hakam Single Cell Protein Plant 

until its destruction by UNSCOM inspectors in 1996. 

Despite their obvious successes, the UNSCOM inspectors never received full cooperation from the Saddam 

Hussein regime, and were ejected from Iraq in 1998. 

The Iraqi regime continued to promote an air of 

uncertainty after 1998 as to whether it had an active 

ongoing biological weapons program. Amy Smithson, 

in her very detailed account of the Iraqi biological 

weapons program and the UNSCOM inspections, 

suggests three possible reasons why Saddam Hussein 

may have wanted to maintain the perception that his 

biological weapons program was still active82: 

1. To deter attacks by regional rivals, especially 

Iran; 

2. To promote his image internally as a strong 

and unassailable leader and thus preserve his 

own internal stranglehold over Iraq; and 

3. To maintain his own outsized vision of his 

ultimate dream and legacy.

Regardless of his strategic motives, the uncertainty 

about his biological weapons program ultimately 

contributed greatly to the Hussein government’s fall 

and his own demise. The breakdown of the inspections, lack of firsthand information, misinformation 

provided by an informant (Rafid Ahmed Alwan alJanabi, an Iraqi defector code named “Curveball” by 

the Central Intelligence Agency), and the 2001 anthrax 

mailings contributed to growing uncertainties, ambiguities, and apprehension, culminating in the 2002 

US National Intelligence Estimate and assessments 

by the intelligence services of France, Germany, and 

the United Kingdom, that postulated a robust Iraqi 

biological weapons program.83,84 International concern 

led to renewed inspections in 2002 under UN Security 

Council Resolution 1441. The Iraqi government failed 

to cooperate fully, and coalition forces invaded Iraq in 

2003, believing at the time that Iraq’s regime still posed 

a significant biological weapons threat. In 2005 the 

Iraq Survey Group (an international group composed 

of civilian and military members) concluded that the 

Iraqi military biological weapons program had been 

abandoned from 1995 through 1996 because the potential discovery of continued activity would risk severe 

political repercussions including the extension of UN 

sanctions. However, Saddam Hussein had perpetuated 

ambiguity regarding a possible program as a strategic 

deterrent against Iran.85 The Iraqi Intelligence Service 

continued to investigate toxins as tools of assassination, 

concealed its program from UNSCOM inspectors after 

the 1991 Persian Gulf War, and reportedly conducted 

lethal human experimentation until 1994. Small-scale 

covert laboratories were maintained until 2003.86 

TABLE 1-3

DELIVERY SYSTEMS FOR BIOLOGICAL 

AGENTS DEVELOPED BY IRAQ*

Aerial Bombs Missile Warheads

Botulinum 100 Botulinum 13

Bacillus anthracis 50 Bacillus anthracis 10

Aflatoxin 16 Aflatoxin 2

*Disclosed by the Iraq government in 1995.

OTHER NATIONAL PROGRAMS

South Africa is alleged to have operated a small-scale 

biological weapons program between 1981 and 1993, 

after becoming state-party to both the 1925 Geneva 

Convention (1960) and the BWC (1975). The South 

African biological weapons program, code-named 

Operation Coast, reportedly conducted research on B 

anthracis, V cholerae, ricin, botulinum toxin, and other 

agents, and intended to use genetic engineering to 

develop biological agents that would selectively target 

people of black African ancestry. Although Operation 

Coast acquired a collection of pathogens, it was not 

successful in developing large-scale delivery systems. 

V cholerae was reportedly used in 1989, but the attack 

failed because of the targeted water supply’s chlorine 

content. After diplomatic interventions by the United 

States and Great Britain, the program was closed in 1993, 

coincident with the demise of the apartheid regime.87–89 

V cholerae was allegedy used by Rhodesian forces 

with South African assistance during the civil war 

of the 1970s to contaminate rivers used as water 

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Medical Aspects of Biological Warfare 

sources by rebel forces; these attacks are thought to 

have failed because of dilution. Rhodesian forces 

reportedly used B anthracis against livestock; the 

role of these attacks in an anthrax epizootic during 1979–1980 was investigated but could not be 

determined.88 

Libya allegedly launched a clandestine biological 

weapons effort during the 1990s (while a state-party 

to the BWC), and sought assistance from Iraq, North 

Korea, and South Africa. However, in contrast to its 

chemical weapons program, the effort was limited 

to small-scale research, and according to one official 

never progressed beyond initial planning.90 Colonel 

Muammar al-Qaddafi, an authoritarian dictator 

who ruled Libya for 42 years, formally renounced 

all weapons of mass destruction in 2003; inspectors 

from the United States and the United Kingdom 

found no evidence of an offensive biological weapons program.90

An unclassified 2013 US State Department report 

noted that North Korea may still consider the use of 

biological weapons as a military option, and that it is 

unclear if Iran is conducting activities prohibited by 

the BWC.78 

The US Director of National Intelligence reported 

in an open US Senate hearing in 2013 that Syria (a signatory, but not a state-party to the BWC) maintains a 

biological weapons program capable of limited agent 

production; and although Syria is not known to have 

loaded biological agents in effective delivery systems, 

it possesses conventional and chemical weapons 

devices that could be adapted to launch biological 

attacks.91,92 In the context of the ongoing Syrian civil 

war in 2014, there are concerns regarding potential 

deployment93 and that further disintegration of the 

Assad regime could enable Al Qaeda and Hezbollah 

to seize Syrian unconventional weapons.94 

Some 20 nations are thought to have engaged in 

offensive biological weapons efforts. The total number 

of nations and the extent of their efforts are difficult 

to establish because several have engaged in research 

and development, but not taken their efforts to testing, 

deployment, and use. Although the list of states appears to be down from the 20 or so that were thought 

to have biological weapons programs in the assessments in the 1980 and 1990s, several states including 

North Korea, Syria, and Iran are still thought to have 

biological weapons programs.54(p68) 

BIOCRIMES

Biocrime is the malevolent use of biological agents 

when the perpetrator’s motivation is personal, as opposed to a broader ideological, political, or religious 

objective. Although biocrimes constitute only a small 

fraction of criminal assaults and are usually unsuccessful,95 a well-executed attempt may be deadly; the 

resulting disease may pose clinical and forensic challenges. Biocrimes have generally been more successful 

than bioterrorist attacks; 8 of 66 biocrimes reviewed by 

Tucker produced 29 deaths and 31 injuries.96 

Perpetrators with scientific or medical expertise 

or those who have recruited trained accomplices 

typically attempt biocrimes. Criminals without a 

technical background have successfully extracted 

ricin from castor beans but have generally been unable to obtain or produce other agents. In a review 

of 14 episodes in which agent was used, biological 

agents were usually obtained from a legitimate 

source or stolen; the perpetrators produced agent 

in only two cases.21,95 Preferred agents have been 

bacteria and toxins (eg, ricin). Food contamination 

has been preferred over direct injection or topical 

application as a means of attack.

One of the most striking examples of foodborne 

biocrime occurred in Japan between 1964 and 1966. 

Dr Mitsuru Suzuki allegedly contaminated food items, 

medications, barium contrast, and a tongue depressor 

with Salmonella typhi and agents of dysentery on numerous occasions resulting in more than 120 cases and 

four deaths.23 A variation on the Suzuki crime occurred 

in 1996 when Diane Thompson, a hospital microbiologist, deliberately infected 12 coworkers with Shigella 

dysenteriae. She sent an email to her coworkers inviting 

them to eat pastries she had left in the laboratory break 

room. Eight of the 12 casualties and an uneaten muffin 

tested positive for S dysenteriae type 2, identical to the 

laboratory’s stock strain by pulsed-field electrophoresis.97 Police learned that her boyfriend had previously 

suffered similar symptoms and had been hospitalized 

at the same facility, and that Thompson had falsified 

his laboratory test results. Thompson was sentenced 

to 20 years in prison.23

Murders by direct injection included the use of 

diphtheria toxin in Russia in 1910. The director of a 

Norwegian nursing home was convicted in 1983 of 

murdering 22 patients by injecting a curare derivative. There have been at least four murder attempts 

by injecting victims with human immunodeficiency 

virus-infected blood.23

Numerous and highly varied biocrimes have been 

reported; only several representative examples are 

included in this chapter. The works of Carus,23 Leitenberg,21 and Tucker96 provide comprehensive descriptions and analysis.

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Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness

BIOLOGICAL TERRORISM

nization of the Japanese government with “ministries 

and departments.” Seiichi Endo, who headed “health 

and welfare,” had worked in genetic engineering 

at Kyoto University’s viral research center. Hideo 

Murai, who headed “science and technology,” had 

an advanced degree in astrophysics and had worked 

in research and development for Kobe Steel Corporation. Endo attempted to derive botulinum toxin from 

environmental isolates of Clostridium botulinum at the 

cult’s Mount Fuji property. A production facility was 

built and horses were stabled for developing a horse 

serum antitoxin. It is uncertain whether Endo successfully produced potent botulinum toxin.23

In 1993 Aum Shinrikyo built a new research facility 

on the eighth floor of an office building owned by the 

cult in eastern Tokyo. The cult grew B anthracis and 

installed a large industrial sprayer for dissemination. 

The cult is also believed to have worked with C burnetii and poisonous mushrooms, and it sent a team to 

Zaire in the midst of an Ebola epidemic to acquire the 

Ebola virus. According to press accounts from 1990 to 

1995, the cult attempted to use aerosolized biological 

agents against nine targets. Three attacks were attempted with B anthracis and six with botulinum toxin. 

In April 1990 the cult equipped three vehicles with 

sprayers containing botulinum toxin targeting Japan’s 

parliamentary Diet Building in central Tokyo, the 

city of Yokahama, Yosuka US Navy Base, and Nairta 

International Airport. In June 1993 the cult targeted 

the wedding of Japan’s crown prince by spraying 

botulinum toxin from a vehicle in downtown Tokyo. 

Later that month, the cult spread B anthracis using the 

roof-mounted sprayer on its eight-story building. In 

July 1993 the cult targeted the Diet in central Tokyo 

again by using a truck spraying B anthracis, and later 

that month it targeted the Imperial Palace in Tokyo. 

On March 15, 1995, the cult planted three briefcases 

designed to release botulinum toxin in the Tokyo 

subway. Explanations for the cult’s failure include the 

possible use of a nontoxin-producing (or low yield) 

strain of C botulinum, use of a low-virulence veterinary 

vaccine strain of B anthracis, ineffective spraying equipment, and perhaps subversion on the part of some cult 

members who were reluctant to execute the planned 

operation.19 Ultimately, Aum Shinrikyo gave up on its 

biological weapons and released sarin in the Tokyo 

subway on March 20, 1995. 23

Meanwhile in the United States, two members 

of the Minnesota Patriots Council, an antigovernment extremist group, were arrested for producing 

ricin and planning to attack federal agents by contaminating doorknobs. Larry Wayne Harris, a clinical 

Bioterrorism is the use of biological agents by an 

individual or group not acting as official agents of a 

government to achieve a political or ideological objective. Bioterrorist incidents increased markedly after 

1985, with two peaks in 1998 and 2001. The 1998 peak 

followed publicity of the anthrax threat posed by Larry 

Wayne Harris; the 2001 peak followed the September through October anthrax mailings. Successfully 

executed attacks have been few but high in impact; 

the 1984 Rajneeshee Salmonella attack resulted in 751 

cases of infection; the 2001 anthrax mailings resulted 

in 22 cases of infection, five deaths, and approximately 

10,000 individuals being offered postexposure prophylaxis. The vast majority of incidents (at least 98% during 

2000–2002) have been hoaxes, which have nonetheless 

produced considerable social disruption.98,99 

The first large-scale bioterrorism attack in the 

United States occurred in 1984. In the 1960s an Indian 

guru named Bhagwan Shree Rajneesh founded the 

Rajneeshee cult. Rajneesh succeeded in attracting 

followers from the upper middle class and collecting 

significant donations and proceeds from book and 

tape sales. Rajneesh acquired the Big Muddy Ranch 

near The Dalles, Oregon, and built a community for 

his followers named Rajneeshpuram, which became an 

incorporated community. Within a few years, the Rajneeshees came into conflict with the local population 

regarding development and land use. The Rajneeshees 

attempted to gain control of the Wasco County government by bringing in thousands of homeless people 

from cities around the country, counting on their 

votes in the upcoming elections. The Rajneeshees also 

plotted to sicken the local population to prevent them 

from voting.21

Two Wasco County commissioners visiting Rajneeshpuram on August 29, 1984, were given drinking 

water contaminated with Salmonella typhimurium; both 

became ill and one was hospitalized. In trial runs in 

the months leading up to the November 1984 elections, 

several attempts at environmental, public water, and 

supermarket food contamination were unsuccessful. In 

September, Rajneeshees began contaminating food at 

local restaurants by pouring slurries of S typhimurium

into salad bars, salad dressing, and coffee creamers at 

10 restaurants. This attack caused 751 cases of enteritis 

and at least 45 hospitalizations.23,100 

In 1995 in Japan, the Aum Shinrikyo cult released 

sarin gas in the Tokyo subway system, resulting in 

12 deaths and thousands seeking emergency care. 

The cult, founded by Shoko Asahara, had amassed 

approximately 10,000 members and $300 million in 

financial assets. Aum Shinrikyo mimicked the orga244-949 DLA DS.indb 13 6/4/18 
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Medical Aspects of Biological Warfare 

microbiologist with ties to racist groups, was arrested 

in 1995 for using fraudulent information to obtain a 

culture of Y pestis from the American Type Culture 

Collection. He was arrested a second time in 1998 after 

making threatening remarks to US federal officials and 

violating his parole. Harris had constructed a covert 

laboratory in Nevada and was conducting experiments 

with the Sterne strain of B anthracis, a nonencapsulated 

but toxigenic live attenuated veterinary vaccine, and 

he threatened to attack Las Vegas with B anthracis.

68

His case led to the establishment of the Select Agent 

Program (42 CFR Part 73, Possession, Use, and Transfer 

of Select Agents and Toxins) that included the development of stringent regulations for the procurement and 

shipping of select microbes.

On October 4, 2001, just 3 weeks after the September 11th attacks on the World Trade Center and the 

Pentagon had made the nation acutely aware of its 

vulnerability to international terrorism, health officials 

in Florida reported a case of inhalational anthrax. During the first week of September, American Media, Inc, 

received a letter addressed to Jennifer Lopez containing a fan letter and a “powdery substance.” The letter 

was passed among its employees, including Robert 

Stevens. Retrospectively, investigators would consider 

not this letter, but perhaps a subsequent letter, as the 

source of his infection.101

Stevens was admitted to a Palm Beach, Florida, 

hospital with high fever and disorientation on October 

2, 2001. By October 5, he was dead from inhalational 

anthrax, the first such case in the United States in more 

than 20 years. 

Soon afterward anthrax mailings were received at 

civilian news media operations in New York City and 

in the Hart Senate Office Building in Washington, DC. 

At least five (four recovered) letters containing B 

anthracis spores had been mailed on September 18, 

2001, and October 9, 2001. Twenty-two people contracted anthrax, with 11 inhalational cases resulting 

in five deaths. Thirty-five postal facilities and commercial mailrooms were contaminated. Screening and 

postexposure prophylaxis disrupted operations at the 

Hart US Senate Office Building. Decontamination of 

postal facilities cost more than $1.2 billion and resulted 

in the closure of heavily contaminated facilities in 

Washington, DC (October 2001–December 2003), and 

Trenton, New Jersey (October 2001–March 2005).102

More than $27 million was spent on decontaminating 

Capitol Hill facilities.102 Public alarm was compounded 

by numerous “white powder” hoaxes. 

Farsighted emergency planning and training, in addition to the integration of federal and local medical, 

public health, and law enforcement agencies in New 

York City and other cities, enabled an unprecedented 

public health response. The Laboratory Response 

Network and military laboratories such as USAMRIID 

processed more than 125,000 clinical specimens and 1 

million environmental samples. USAMRIID ran more 

than 260,000 assays on more than 30,000 samples in 

9 months. Prophylaxis supplied from the national 

stockpile was offered to nearly 10,000 individuals at 

risk. No cases were found among prophylaxis recipients.103,104 Treatment guidelines advocating multidrug 

antibiotic combinations and aggressive intensive care 

were disseminated,105 and the case fatality rate for 

inhalational anthrax—historically exceeding 90%—

reduced to 45%.106,107 

The attacks provoked an unprecedented criminal 

investigation that coupled traditional law enforcement with the development and validation of novel 

emerging genetic sequencing techniques. The Federal 

Bureau of Investigation (FBI) special agents and US 

Postal Service Inspectors conducted the investigation for 7 years, and 29 government, academic, and 

commercial laboratories supported it. Investigators 

conducted more than 10,000 witness interviews on 

six continents and 80 searches, and they also collected 

more than 6,000 items of potential evidence and 5,730 

environmental samples from 60 locations both within 

the United States and in foreign countries, with the cooperation of the respective host nation governments.102

US Attorney General John Aschroft named Dr 

Steven J Hatfill, a USAMRIID scientist between 1997 

and 1999, a “person of interest” during a television 

interview in 2002. Dr Hatfill vehemently denied 

involvement, and sued the federal government, 

claiming that law enforcement officials had leaked 

information to the media in violation of the Privacy 

Act, and had ruined his reputation and career. The 

FBI exonerated him in 2008, and he received $5.82 

million in restitution.102,108,109 

Forensic analysis was confounded by the highly 

conserved B anthracis genome, which features more 

than 99.99% nucleotide sequence identity among the 

most genetically divergent strains. Investigators went 

beyond the contemporary standard of genetic typing 

by sequencing small DNA segments to advance the 

technique of whole genome sequencing. Comparison 

of the whole genomes of the index case isolate and a 

reference Ames strain disclosed that they were essentially identical, and it could not pinpoint the origin of 

the letter contents. However, a breakthrough followed 

the observation of four phenotypic colony morphology 

variants constituting less than 1% of colonies cultured 

from spore samples taken from three of the anthrax 

letters. Each colony morphology variant was associated with a distinct mutation restricted to four genetic 

loci. These mutations were absent in environmental 

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Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness

isolates taken during the investigation.110 Specimens 

were obtained from every culture of B anthracis Ames 

strain (1,071 samples) from all 15 US and three foreign laboratories known to possess it. One or more 

of the mutants was detected in 71 of 947 samples that 

could be evaluated; all four mutants were present in 

eight samples. The probability of samples to contain 

all four mutants was calculated to be 0.4383 x 10-6 or 

0.0004 samples in the 947 sample collection, if the 

samples were unrelated; these eight samples consisted 

of a specimen from RMR-1029, a flask containing a 

liquid spore preparation in the laboratory of anthrax 

researcher Dr Bruce E Ivins at USAMRIID, and seven 

specimens from another laboratory that were descendents of RMR-1029.111,112 

The FBI concluded that Dr Ivins was the sole perpetrator based on the following: 

• the genetic analysis results; 

• inconsistencies during interviews; 

• erratic conduct that included irregular laboratory hours before each mailing and an unauthorized and unreported decontamination of 

his office and laboratory during the investigation; 

• deteriorating behavior as the investigation 

progressed; and 

• exclusion of other individuals with access to 

RMR-1029 and its descendants. 

The purported motive was to ensure continued support for the anthrax vaccine research in which Dr Ivins 

was personally heavily invested and was under criticism from multiple sectors. The US Attorney’s Office for 

the District of Columbia prepared an indictment charging him with Use of a Weapon of Mass Destruction, in 

violation of Title 18, United States Code, Section 2332a, 

and related charges. Dr Ivins, aware of the indictment, 

took an overdose of over-the-counter medications and 

died on July 29, 2008.102 

Lingering doubts were expressed during a plenary 

session at the 2009 American Society for Microbiology 

Biodefense and Emerging Diseases Research Conference.113 Evidence was considered circumstantial. No 

evidence of B anthracis contamination was found in 

Dr Ivin’s home or vehicles. Unexplained aspects of the 

case included the contamination of the September 18 

mailing with a B subtilis strain that could not be traced 

to USAMRIID and the use of dry spore preparations 

(the production of which is prohibited in the US biodefense program), for which there was no direct evidence 

within USAMRIID. A National Academy of Sciences 

review concluded that the genetic typing results were 

consistent with—but not definitive proof of—the derivation of the letter isolates from RMR-1029. Although 

generally supportive of the FBI’s efforts, the reviewers 

criticized the FBI’s statistical methods and stated that 

an alternative source could not be excluded because of 

possible sharing and mixing of samples among laboratories, and because the possibility of identical mutations 

arising through parallel evolution independently in 

unrelated cultures had not—in their opinion—been 

adequately explored.112 Abnormally high concentrations of silicon114 and tin existed in the spores that were 

absent in spores from RMR-1029; this raised controversies regarding potential production at the Dugway 

Proving Ground or at a civilian contractor laboratory, 

where work with silicon and surrogate spores had 

previously been done.115 Finally, Department of Justice 

lawyers used the argument that Dr Ivin’s lab had no 

equipment to produce dry spore preparations to defend 

the government against a wrongful death lawsuit filed 

by Robert Stevens’ widow.116

However, the investigation spurred the advancement of whole genome sequencing, accelerating the 

time required to sequence a bacterial genome from 4 

months to several days,117 and advanced the emerging 

science of microbial forensics. The investigation raised 

issues regarding laboratory programs for physical security, personal reliability, and mental health screening 

that—while not directly incriminating Dr Ivins—underscored the importance of re-evaluating laboratory 

security measures and the value of robust employee 

occupational health programs to screen and monitor 

the mental health of researchers working with highly 

virulent pathogens. These issues were addressed by 

strengthening the federal regulations that direct CDC 

oversight of research on dangerous pathogens (see 

discussion of the Federal Experts on Security Advisory 

Panel in Toward Pan-hazard Preparedness).118,119 

The threat of bioterrorism did not end with the US 

anthrax experience. Al Qaeda initiated a biological 

weapons program in Afghanistan before the overthrow 

of the Taliban regime. Investigations after the US 

military intervention of 2001 uncovered two Al Qaeda 

laboratories for biological weapons development, supplied with commercially acquired microbiology equipment and staffed by trained personnel. Fortunately, a 

deployable weapon had not been constructed.120 US 

forces operating in northern Iraq in 2003 seized a camp 

linked to Al Qaeda reportedly containing instructions 

and equipment for ricin extraction.121

During the period that followed the US anthrax 

attacks, ricin became the bioweapon of choice for a 

number of misanthropes intent on nefarious use of 

biological agents, perhaps because of its relative ease 

of access. The castor beans (ricin source) are available 

worldwide because the oil is extracted for lubricant in 

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Medical Aspects of Biological Warfare 

many countries. The toxin extraction techniques have 

been published in many forums to include many anarchist and terrorist websites. Examples are provided 

of confirmed cases, but many more incidents have occurred worldwide, and most have proven to be hoaxes. 

In January 2003 British authorities uncovered the 

Wood Green ricin plot. A police raid on a London 

apartment yielded a copy of a protocol for ricin production, toxin source materials (castor beans), and a 

suitable solvent (acetone) for its extraction. Although 

tests for ricin were negative,122 one of the tenants, an Al 

Qaeda-trained operative, was convicted of plotting a 

ricin attack. He had planned to contaminate handrails 

in the railway system connecting London and Heathrow Airport.123 In March 2003 two flasks containing 

ricin were discovered in a railway station in Paris.124

In 2003 US Postal Service employees discovered 

two letters directed to the US Department of Transportation containing vials of ricin. The first letter 

was found on October 15, 2003, at the mail sorting 

center in Greenville, South Carolina.125 The second 

was discovered at the White House mail processing 

facility in Washington, DC. Both letters were from an 

antagonist who identified himself as “Fallen Angel” 

and was angry about the Department of Transportation’s new limitations being placed on truck drivers’ 

daily work hours.126 In February 2004 ricin was found 

in the sorting machine of Senate Majority Leader Bill 

Frist’s office in the Congressional Office Building. No 

evidence was ever found linking the Fallen Angel and 

Frist cases and perpetrators are still at large. On June 

23, 2004, Michael Crooker, a resident of the Boston 

suburb of Agawam, Massachusetts, had his house 

searched by law enforcement officials after attempting 

to mail a firearm. Agents discovered a weapons lab 

that contained castor and abrus seeds (sources of ricin 

and abrin toxins, repectively) as well as the materials 

needed for toxin extraction. Crooker sent a letter to 

the prosecuting attorney threatening to cripple the US 

Postal System by sending toxin-laden letters through 

the mail. He also notified local news journalists that he 

would provide toxins to felons he had met in prison 

who had previously engaged in terrorist activities. He 

pled guilty to possession of ricin and threatening a government official and was sentenced in June 2011.127 In 

February 2008 Roger Bergendorff, an anarchist living 

in an extended stay hotel in Las Vegas, Nevada, developed a mysterious illness that puzzled his healthcare 

providers. He was hospitalized and while investigating 

the cause of his illness, officials discovered evidence 

of a ricin extraction operation in his room. He and 

his cousin were both eventually convicted of charges 

related to ricin production. The specifics of intended 

use—if known—have not been disclosed.128 In March 

2011 four men who were members of a militia organization began having clandestine meetings in which 

they allegedly planned numerous criminal activities 

to include acquisition of illegal weapons, manufacture 

of toxic agents, theft, and assassination. During these 

meetings they allegedly discussed use of weapons to 

include biologic agents to attack government facilities 

and government employees to include law enforcement officials. One of their plans included producing 

10 pounds of ricin and dispersing it from a moving 

vehicle in the Atlanta area. An FBI informant alerted 

authorities and the operation was disrupted without 

incident in November 2011.129

Attacks against government officials resumed after a 

nearly 10-year hiatus with the discovery of an envelope 

testing positive for ricin intercepted at the US Capitol’s 

mail facility in April 2013. The letter was addressed to 

Senator Roger Wicker, and a day later an envelope addressed to President Obama was discovered that also 

contained ricin. A third letter containing ricin was mailed 

to the Lee County Mississippi Court Judge Sadie Holland. 

Within a few weeks the FBI arrested Everett Dutschke 

for producing a toxin weapon and using the mail to 

threaten President Barack Obama, Senator Wicker, and 

Judge Holland. These mailings appear to be acts of 

reprisal in the settlement of personal grudge(s).130 Less 

than 2 months later, in May 2013 three letters intended 

for New York Mayor Michael Bloomberg were intercepted containing a suspicious oily substance that turned 

out to contain ricin. Similar letters were also mailed to 

President Obama, according to a Secret Service press 

release. Gun control opponents purportedly sent the 

letters, and Shannon Richardson notified the FBI that 

her estranged husband was responsible for the mailings. 

When the allegations failed to withstand police scrutiny 

she was arrested, and received an 18-year prison sentence, having falsely implicated her husband.131 Despite 

numerous ricin mailings by many diverse individuals, 

the mail delivery of ricin toxin has been ineffective as 

an instrument of harm or assassination—these mailings 

appear to have little impact beyond their psychological 

“scare” effect. Although ricin is a toxin of very high lethal potency, its effectiveness is limited by the delivery 

method. No illness or significant environmental contamination has resulted from any of the ricin mailings. 

Many of the bioterrorist incidents have been small 

scale, not well perpetrated, and not particularly successful in terms of mortality and morbidity. Still, 

it is clear that several terrorist groups aspire to use 

biological weapons. For example, Al Qaeda radical 

cleric Anwar al-Awlaki in an article stated that “the 

killing of women and children and the use of chemical and biological weapons in addition to bombings 

and gun attacks” is acceptable and even encouraged.54

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Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness

In Inspire, an online Al Qaeda magazine, the authors 

called for “chemists and microbiologists” to develop 

weapons and attack the West. These programs continue to be aspirational, rather than well-established 

developmental efforts. However, with the proliferation and industrialization of biotechnology described 

previously, the threat of bioterrorism continues to 

increase.54(p60) 

SOLUTIONS: TOWARD PAN-HAZARD PREPAREDNESS

Disarmament: The Biological Weapons Convention

In July 1969 Great Britain issued a statement to the 

UN Conference of the Committee on Disarmament calling for the prohibition of the development, production, 

and stockpiling of bacteriological and toxin weapons. 

In September 1969 (the same year) the Soviet Union 

unexpectedly recommended a disarmament convention 

to the UN General Assembly. In November 1969 WHO 

issued a report on biological weapons, after an earlier 

report by the 18-nation Committee on Disarmament, 

describing the unpredictable nature, lack of control, 

and other attendant risks of biological weapons use. 

The United Nations then developed the 1972 Convention on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and 

Toxin Weapons and on their Destruction (1972 BWC), 

which prohibited any malicious research, production, 

or possession of biological agents. Among the 103 initial 

cosignatory nations, agreement was reached to “never 

develop, produce, stockpile, or otherwise acquire or 

retain microbiological agents or toxins, whatever their 

origin or method of production, of types and in quantities that have no justification for prophylactic, protective 

or other peaceful purposes; and weapons, equipment 

or means of delivery designed to use such agents or 

toxins for hostile purposes or in armed conflict.”132 The 

United States ratified both the 1925 Geneva Convention and the BWC in 1975. Signatory states suspecting 

others of treaty violations may file a complaint with 

the UN Security Council, which, in turn, may order an 

investigation. However, mandatory measures for verification and enforcement are lacking; numerous attempts 

to formulate such measures have been unsuccessful 

because of political, security, and proprietary issues.21

Since the BWC entered into force in 1975, seven 

review conferences have taken place; these “RevCons” 

(as they are called) constitute the only decision-making 

forums for the BWC and are held every 5 years in Geneva. RevCons are 3-week international meetings that 

allow member nations to reinforce the norm against 

the prohibition of biological weapons, discuss international collaboration on biotechnological issues, assess 

the continued relevance of the BWC given changes in 

biotechnology, and make proposals for revitalizing 

the BWC. Unfortunately, RevCons have not produced many tangible results and have demonstrated

 an inability to deal with difficult issues. The most 

noteworthy accomplishment was development of 

confidence-building measures for annual reporting by 

member state parties. Only 70 or so of the 170 member nations actually submit annual reports on their 

activities. On questions such as the relationship of the 

Sverdlovsk anthrax epidemic to the Soviet biological 

weapons program, the Iraqi weapons program, and 

the smallpox retention versus destruction issue, the 

BWC has remained unengaged. 

Several RevCons have dealt directly with the 

potential for developing a verification protocol. The 

1991, 1996, and 2001 RevCons saw the establishment 

of the Ad Hoc Group, the progress made in the Review Conference Final Declaration, and the disaster 

of the United States walking out of the RevCon,54(p117)

respectively. After the 2001 RevCon the BWC saw a 

tumultuous period where its future was questioned. 

The “success” of the 2006 RevCon served to reenergize the BWC. The key outcomes were the agreement 

concerning the importance of the BWC forum and the 

development of an intersessional process that would 

include annual member state nations and experts meetings to discuss topical issues. However, neither of these 

two new annual meetings allows for decision-making. 

The lead-up to the 2011 RevCon was anticipated 

by participating nation-states.54(p119) The United 

States had released a national strategy for countering 

biological threats at the 2009 meeting of state parties. 

Several pre-BWC conferences were held in which it 

appeared the international community was moving 

toward tangible outcomes in the 2011 RevCon. The 

president of the 2011 meeting, Paul van den Ijssel from 

The Netherlands, had declared the mantra would be 

“ambitious realism.”54(p122) Unfortunately, it failed to 

live up to expectations. One review of the RevCon 

states, “The December 2011 review conference of the 

Biological Weapons Convention (BWC) demonstrated 

the danger of the bioweapons ban drifting into irrelevance. Standstill was the motto of the meeting. Only 

incremental improvements on some procedural issues 

were achieved.”54(p120) Even modest enhancements, 

such as expanding the implementation support unit’s 

three-person organization, were not approved. 

In examining the BWC’s future, several tensions 

arise because it is a state-to-state treaty, yet many of 

the current biological threats deal with nonstate issues 

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Medical Aspects of Biological Warfare 

such as bioterrorism, biocrimes, and misuse of the 

life sciences. Although member nations allow for 

discussing these issues within the BWC, few have 

demonstrated the desire to make these more topical 

issues the focus of future BWC negotiations, although 

states-parties are obligated under article IV to prohibit 

and prevent proscribed activities within their borders. 

Several other articles of the BWC also create tensions. 

For example, article I establishes the norm against biological weapons, yet provides no ability to enforce the 

convention. Articles III and X call for not transferring, 

assisting, inducing, acquiring, and retaining biological 

weapons, whereas article X encourages the peaceful exchanges of biological science and technology. Although 

the words do not conflict, the interpretation between 

developed and developing nations varies greatly. 

Another area of contention concerns the perennial 

issue of verification. The US position remains as it has 

since 2001 that verification of the BWC is not possible. 

Instead, the United States supports adherence to a policy 

of compliance that begins with national implementation including ensuring all nations have appropriate 

national laws, regulations, and policies that support 

the BWC, as stipulated in article IV. The US position 

on verification also rests on the assertion that articles 

V and VI that call for bilateral and multilateral consultation and the potential for bringing concerns to the 

UN Security Council, respectively, provide sufficient 

opportunities for voicing concerns about compliance. 

Two other issues that feature prominently in the BWC 

debate are continued concerns about its relevance 

given the pace of biotechnological enhancements and 

the lack of universal adherence to it. On the first issue, 

members continue to profess that the BWC remains 

relevant despite exponential changes in biotechnology. With respect to universal adherence, the BWC 

continues to be undersubscribed as compared to other 

treaties dealing with weapons of mass destruction issues, in particular the Nuclear Non-Proliferation Treaty 

and the Chemical Weapons Convention. The BWC 

has 170 member nations, whereas the Nuclear NonProliferation Treaty and the Chemical Weapons Convention have 189 and 188 member nations, respectively. 

Finally, only one allegation has been formally registered under the BWC: in June 1997 Cuba accused 

the United States of a biological attack with a crop 

pest insect, Thrips palmi. The allegations were unsubstantiated in a BWC consultation that concluded in 

December 1997.21 Other attempts at biological arms 

control have been conducted outside of the context 

of the BWC; for example, inspections and sanctions 

against Iraq from 1991 to 1998 and 2002 to 2003 were 

accomplished under separate UN Security Council 

Resolutions, 681 and 1441, respectively.

Smallpox Preparedness

CDC launched a comprehensive smallpox preparedness program in 2002 because of the potential 

use of variola as a biological weapons agent. WHO, the 

United Kingdom, Germany, and other WHO member 

states initiated similar programs including vaccine 

stockpiles. The US program integrated community, 

regional, state, and federal healthcare and public health 

organizations and featured logistical preparation; 

training and education; risk communication; surveillance; and local preparations for mass vaccination, 

isolation, quarantine, active surveillance, and humane 

treatment of patients in designated facilities.A strategy 

was adopted based on preexposure vaccination of carefully screened and trained members of first-response 

teams, epidemiological response teams, clinical teams 

at designated facilities, and military personnel set to 

deploy into the theaters of war.133 More than 400,000 

selected military personnel and 38,000 civilian emergency responders and healthcare workers in designated smallpox response teams were vaccinated. Contracts for the production of a new cell culture-derived 

vaccine were awarded in 2000; the Strategic National 

Stockpile has sufficient cell culture-derived vaccine 

for the entire US population, a replication-deficient 

vaccinia (Modified Vaccinia Ankara) for use in immunocompromised individuals, and vaccinia immune 

globulin to treat vaccine complications. In addition, the 

US government supported the development of new 

smallpox antiviral therapeutic candidates and funded 

animal model development to enable efficacy testing 

of medical countermeasure candidates.

The disposition of the remaining WHO-authorized 

variola virus stocks, held in two secure WHO Collaborating Centers at CDC in the United States and 

at VECTOR in Koltsovo, Novosibirsk, Russia, was 

debated at the WHO 64th World Health Assembly 

in 2011. Two camps emerged, the destructionists and 

retentionists, and each made arguments to support 

their positions. In the end, the World Health Assembly 

remained committed to its previous position calling for 

the destruction of the viral stocks as a long-term goal, 

but agreed to their retention until the completion of 

research leading to two antiviral drugs with different 

mechanisms of action, a safer and effective vaccine, a 

rapid and accurate diagnostic kit, and the refinement 

of nonhuman primate animal models. The issue was 

also revisited at the 67th World Health Assembly in 

2014. The risks posed by recombinant technology 

were also addressed; a private company in the United 

States that had inserted 63 nucleotides from the variola 

genome into an attenuated but transmissible orthopox 

virus to develop a positive control for a diagnostic test 

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Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness

would be asked to destroy its reagent and to report its 

destruction to WHO.134 This underscored the need to 

re-evaluate and publicize WHO guidance regarding 

the use of variola genetic sequences in recombinant 

technology. 

Dual Use Research of Concern

In addition to the threats posed by the deliberate 

release of biological agents, there has been increasing 

recognition of the potential risks posed by legitimate 

scientific research for benevolent medical purposes 

that includes the characterization of, and development of medical countermeasures against, highly 

pathogenic microbes. Risks include laboratory accidents resulting in pathogen release, laboratory 

acquired infections (some of which may be communicable to the community), unanticipated results 

of experiments resulting in increased microbial 

virulence or transmissibility, and the deliberate misuse of knowledge generated by legitimate scientific 

research for biological weapons proliferation. Dual 

use research of concern (DURC) has been identified 

as biological research with legitimate scientific purpose that may be misused to pose a biologic threat 

to public health and/or national security. Examples 

include, but are not limited to, the following: 

• The genetic modification of mousepox virus 

to express both an ovarian protein and the 

immunomodulator interleukin-4 to induce 

sterility in mice for pest control, reported in 

2001. Immunomodulator interleukin-4 was 

intended to enhance immune responses to 

the ovarian protein. However, the vaccine 

candidate was lethal in small-animal testing; 

immunomodulator interleukin-4 had the 

unanticipated effect of immune suppression, 

resulting in a highly virulent mousepox virus.135 

• The in vitro synthesis of wild-strain poliovirus 

type 1 by using synthetic DNA encoding the 

poliovirus genome (with minor mutations 

as genetic markers) in a cell-free extract by 

researchers at the State University of New 

York at Stony Brook in 2002. The researchers 

noted that the knowledge that polioviruses 

can be synthesized using chemical methods 

and reintroduced through bioterrorism may 

inform the closing strategies of WHO’s polio eradication campaign.136,137 It was later 

explained that they had hoped to deliver a 

“wake-up call” regarding the possible misuse 

of viral synthesis for bioterrorism; that WHO’s 

polio eradication campaign may be futile 

because of either possible bioterrorism using 

synthetic virus, laboratory accidents, or live 

attenuated oral polio vaccine and circulating 

oral polio vaccine-derived virus-related disease; and that control may be a more attainable 

outcome.138 Aside from risking an accidental 

reintroduction to the local community (after 

the elimination of circulating wild-strain polioviruses from the western hemisphere), the 

study raised questions regarding its scientific 

value,139 whether demonstrating technical 

capabilities to deliver warnings constitutes a 

legitimate scientific purpose, and whether the 

synthesis of a wild-strain poliovirus, which is 

otherwise available to researchers, served any 

benevolent medical purpose. 

• The reconstruction of the 1918 H1N1 influenza 

A pandemic virus,140 reported in 2005. This 

enabled characterization of a virulent pathogen that—in contrast to poliovirus—was otherwise not available for study. This enabled 

insights into pathogenesis, and potentially the 

identification of virulence factors and drug 

targets that could be relevant to counter future 

pandemic strains.141 Using appropriate biosafety and biosecurity measures minimized 

risks to the public. 

• The generation of a mutant of the highly 

pathogenic avian influenza A virus H5N1 

(HPAI H5N1) with enhanced transmissibility between mammalian hosts (ferrets) that 

was as contagious as seasonal influenza viruses and retained the virulence of the wild 

strain142,143 (55%–60% mortality in humans) 

by researchers at Erasmus University (although it was later reported that the mutant 

was attenuated and not as communicable 

as originally claimed), reported during the 

autumn of 2011. Concurrently, researchers 

at the University of Wisconsin developed 

a recombinant 2009 pandemic H1N1 virus 

expressing H5 hemagglutinin receptor binding proteins that was transmissible between 

ferrets. These announcements stunned many 

in the scientific community and the general 

public as risking a pandemic catastrophe following a laboratory accident or intentional 

release. Policy makers became concerned that 

the publication of these studies would support 

biological weapons proliferation by providing 

information that could be used to produce 

highly communicable and lethal influenza 

viruses. 

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Medical Aspects of Biological Warfare 

Within 4 months of the publication of poliovirus 

synthesis, the Center for Strategic and International 

Studies and the National Academy of Sciences held 

a workshop on scientific openness and national security that involved a wide stakeholder community 

from government, academia, and scientific editorial 

communities that generated voluntary guidelines for 

ensuring the publication of new knowledge while 

safeguarding information that may pose security 

risks. Issues raised by DURC led to the foundation 

of the National Science Advisory Board for Biodefense (NSABB) in 2004. NSABB is a federal advisory 

committee within the Office of Science Policy in the 

National Institutes of Health (NIH) that provides advice, guidance, and leadership regarding biosecurity 

oversight of dual use research, defined as biological research with legitimate scientific purpose that 

may be misused to pose a biological threat to public 

health and/or national security. NSABB is chartered 

to recommend strategies and guidance for enhancing 

personnel reliability among individuals with access 

to biological select agents and toxins; provide recommendations on the development of programs for 

outreach, education, and training in dual use research 

issues for scientists, laboratory workers, students, 

and trainees in relevant disciplines; advise on policies 

governing publication, public communication, and 

dissemination of dual use research methodologies 

and results; recommend strategies for fostering international engagement on dual use biological research 

issues; advise on the development, utilization, and 

promotion of codes of conduct to interdisciplinary 

life scientists and relevant professional groups; advise 

on policies regarding the conduct, communication, 

and oversight of dual use research and results, as requested; advise on the Federal Select Agent Program, 

as requested; and address any other issues as directed 

by the Secretary of Health and Human Services. 

NSABB concerns include knowledge, products, or 

technologies that may: 

• enhance the harmful consequences of a biological agent or toxin; 

• disrupt the immunity or the effectiveness 

of an immunization without clinical and/or 

agricultural justification; 

• confer to a biological agent or toxin, resistance to clinically and/or agriculturally useful prophylactic or therapeutic interventions 

or facilitate their ability to evade detection 

methodologies; 

• increase the stability, transmissibility, or the 

ability to disseminate a biological agent or 

toxin; 

• alter the host range or tropism of a biological 

agent or toxin; 

• enhance the susceptibility of a host population; and

• generate a novel pathogenic agent or toxin or 

reconstitute an eradicated or extinct biological 

agent. 

Examples of initiatives coordinated through NSABB 

include Department of Health and Human Services 

(DHHS) guidelines for synthetic biology144 and guidance for providers of double-stranded DNA to screen 

procurement orders.145 

In December 2011 NSABB reviewed manuscripts 

of the Erasmus University and University of Wisconsin studies on enhanced transmission of HPAI H5N1 

that were being prepared for publication and made 

the unprecedented, nonbinding recommendation to 

redact methods and experimental details.146 In addition, the influenza research community voluntarily 

invoked a moratorium on gain-of-function research 

using HPAI H5N1. 

NSABB members asserted that their recommendation was an exceptional and adaptive response to a 

special case—a situation generated by the life sciences, 

biodefense, and general public communities being 

caught off-guard—and having limited awareness of 

the research until the manuscripts were being prepared 

(even though NIH had funded both projects), they 

reasoned that:

• in the future, the value of conducting and 

supporting specific dual use research projects should be carefully considered a priori 

by a wide stakeholder community including 

experts in life sciences, biosecurity, and members of the general public; and

• decisions to publish results should follow 

the principle of “do no harm,” with the best 

interest of public health in mind.147

However, supporters of the research and its publication argued that:

• medical science must address the most virulent pathogens to be valuable; 

• new knowledge of determinants of transmissibility may be useful to predict the likelihood 

of an epi- or enzootic virus being capable of 

a “species jump” to humans and consequent 

person-to-person transmission; 

• the mutants afforded an opportunity to test 

vaccine and therapeutic candidates against 

potential future emerging viruses;

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Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness

• methods used in the studies are already wellknown in the scientific community; 

• persons with malicious intent could use simpler means to inflict disease and injury; and 

• redacting the manuscripts constituted censorship, thus violating long-standing principles 

of academic freedom.148–151

As the debate raged,152–156 WHO concluded that 

such research and its publication is in public health’s 

best interest and should be continued in the context 

of rigorous biosafety, biosecurity, and risk communication.157 NSABB reconvened in late March 2012 and 

recommended the full publication of the University of 

Wisconsin manuscript and publication of the Erasmus 

University manuscript after appropriate scientific review and revision, with the caveat that the US government should develop a mechanism to control access to 

sensitive scientific information.158 The two manuscripts 

were published later in 2012.159,160

The controversy resulted in an updated US Government Policy for Oversight of Life Sciences DURC, 

which was released in March 2012.161 This policy directed federal departments and agencies that conduct 

or fund life sciences research to do the following:

• review all current or proposed research projects to identify those that could potentially 

provide knowledge, information, products, 

or technologies that could be directly misapplied to pose a significant threat to public 

health and safety, agricultural crops and other 

plants, animals, the environment, materiel, or 

national security; 

• conduct risk assessments and develop risk 

mitigation plans addressing experimental 

design and methods, biosecurity, biosafety, 

and availability of medical countermeasures; 

• review annual progress reports to determine 

whether DURC results have been generated; 

• request voluntary redaction of research publications or communications or classification 

of research findings; and 

• coordinate information regarding DURC 

projects with the Assistant to the President for 

Homeland Security and Counterterrorism. 

In addition, the Office of Science and Technology 

Programs is formulating a complementary policy that 

delineates oversight responsibilities for research institutions receiving federal funds to perform DURC.162 

In December 2012 NIH hosted a meeting of the influenza research community to discuss guidelines for 

funding HPAI H5N1 influenza virus gain-of-function 

research, followed by an opportunity for public comment. The resulting guideline was issued on February 

21, 2013,163,164 and it identified criteria for funding research proposals that may enhance the transmissibility 

of HPAI H5N1 among mammals: 

• the virus anticipated to be generated could 

be produced through a natural evolutionary 

process;

• the research addresses a scientific question 

with high significance for public health;

• there are no feasible alternative methods 

to address the same scientific question in a 

manner that poses less risk than the proposed 

approach;

• biosafety risks to laboratory workers and the 

public can be sufficiently mitigated and managed;

• biosecurity risks can be sufficiently mitigated 

and managed;

• the research information is anticipated to be 

broadly shared to realize its potential benefits 

to global health; and

• the research will be supported through funding mechanisms that facilitate appropriate 

oversight of the conduct and communication 

of the research.

The framework also outlined a review process that 

includes department-level scrutiny of proposals considered for funding by DHHS agencies. 

Five days after the release of the DHHS framework, 

the ethical, societal, scientific, safety, and security 

issues raised by DURC were discussed at the international level at WHO. There was consensus that 

DURC issues are relevant to all nations and multiple 

stakeholders; management of DURC should take place 

during all phases of research; ethical considerations are 

fundamental; and because management of DURC will 

require a diversity of approaches in different member 

states, an internationally binding agreement would 

be difficult, impractical, and not necessarily effective. 

However, the participants remained open to future 

international guidelines and suggested that existing 

international agreements (eg, the BWC, WHO’s International Health Regulations [IHR]) could provide 

a basis for overarching principles. WHO will continue 

to engage member states and other stakeholders to 

explore effective approaches.165 

In the meantime, the influenza research community had already ended its moratorium for scientists 

using biosafety and biosecurity measures in compliance with its respective national regulations.166 The 

subsequent publication of a study completed before 

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Medical Aspects of Biological Warfare 

the moratorium using reverse genetics to generate 127 

hybrids of HPAI H5N1 and 2009 pandemic H1N1 

viruses, of which five were communicable among 

guinea pigs,167 again raised questions regarding the 

medical utility and public health risks of hazardous 

experiments.168 In August 2013 proponents of gain-offunction research publicly announced their intention 

to conduct studies using influenza A H7N9 virus.169

Concurrently, DHHS gave assurances that research 

proposals for H7N9 gain-of-function research would 

undergo rigorous scrutiny by experts in multiple 

disciplines including biosafety and ethics and final 

review at the department level,170 consistent with the 

February DHHS framework. The a priori publication 

of H7N9 research goals was seen as a proactive step 

to enhance transparency and prospective discussion 

and to prevent a recurrence of the 2011–2012 H5N1 

disputes. However, gain-of-function research remains 

a contentious issue171 because no certainty exists that 

laboratory-generated mutants will emerge in nature. 

The issues generated by potential dual use research 

will continue to fuel discourse regarding relationships among stakeholders, and optimal policy and 

technical solutions.172–179

Toward Pan-Hazard Preparedness

During the late 1990s the US government launched 

an ambitious program to enhance biological preparedness at local, state, and federal levels, including 

measures such as the Presidential Decision Directive-39 (1995), Presidential Decision Directive-62 

(1998), and Presidential Decision Directive-63 (1998). 

The Federal Response Plan (now called the National 

Response Plan) coordinates federal agencies responding to disasters. The Select Agent List was created 

to regulate the purchase, shipment, and research of 

designated microbial agents; lead proponents for the 

Select Agent list were DHHS and USDA. DHHS was 

given oversight of health and medical services, and 

its Office of Emergency Preparedness organized local 

medical response teams in 125 jurisdictions. Preparations in New York City and other locations included 

plans and exercises for local incident command; coordinated clinical response; surveillance; and massive 

distribution of postexposure prophylaxis at multiple 

distribution centers designed for efficient screening, 

triage, distribution, and documentation. Federal response teams were organized, staffed, and deployed 

to large official and public gatherings. CDC established a center for bioterrorism response to enhance 

state public health laboratories, improve surveillance 

systems, and improve rapid communication and coordination. The Strategic National Stockpile of key 

pharmaceutical agents and vaccines was prepared. 

The Laboratory Response Network, also managed 

by CDC, provided coordination of testing, sample 

shipment, and communication between designated 

local, regional, and reference laboratories. DoD assets 

integrated into the National Response Plan included 

USAMRIID for emergency medical consultation and 

reference laboratory support; the Naval Medical Research Center for laboratory support; the US Marine 

Corps Chemical and Biological Incident Response 

Force for reconnaissance, initial triage, and the decontamination of casualties; and the Army Technical 

Escort Unit for sampling, transport, and disposal of 

dissemination devices. The Army Medical Department also fielded six regionally based chemical/

biological special medical augmentation response 

teams to deploy within 12 hours to assist local civilian authorities. The National Guard Bureau, under 

legislative direction from Congress, fielded regional 

biological response teams initially called rapid agent 

identification teams, and later renamed civil support 

teams. Many of these new response mechanisms and 

agencies were tested in the autumn of 2001.

After the anthrax mailings of 2001, bioterrorism 

response was strengthened with additional infrastructure and linkages among the emergency response, 

public health, clinical, and laboratory sectors.103,104

The Office of Public Health Emergency Preparedness 

at DHHS was formed to coordinate civilian medical 

countermeasure development by the National Institute 

of Allergy and Infectious Diseases, CDC, and DoD, 

under the leadership of eminent scientists and physicians such as DA Henderson and Philip K Russell. 

In April 2004 President George W Bush signed 

Homeland Security Presidential Decision Directive-10, 

Biodefense for the 21st Century, which outlined a national strategy for combating biological terrorism and 

mandated an interagency approach using strengths of 

various executive branch departments, including the 

Department of Homeland Security, DHHS, and DoD. 

Subsequently, the Homeland Security Council and 

the National Security Council formed an interagency 

steering committee called the Weapons of Mass Destruction Medical Countermeasures Subcommittee, 

whose principals were at the assistant secretary level; 

the group coordinates the various departmental efforts 

to prevent and respond to weapons of mass destruction 

attacks. The Department of Homeland Security took 

the lead on biological threat assessments, and DHHS 

took the lead on medical countermeasures. 

On July 21, 2004, Project Bioshield was initiated as a 

$6 billion, 10-year program for acquiring new medical 

countermeasures for the Strategic National Stockpile. 

This legislation provided a significant funding boost to 

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Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness

the Office of Public Health Emergency Preparedness. 

Medical countermeasures added to the Strategic National Stockpile include significantly increased doses 

of botulinum antitoxins; antibiotics to treat anthrax, 

tularemia, and plague; anthrax adjunctive therapies; 

and ventilators for respiratory support. 

The potential for the malevolent use of genetic 

engineering to develop novel biological threats with 

enhance virulence180 resulted in a shift of technical 

emphasis from pathogen-specific projects to a global 

response capability—a threat-agnostic response 

capacity—to enable responses to outbreaks of any 

known or genetically engineered biological agents, 

or novel emerging pathogens. This capability includes flexible technology platforms to enable rapid 

pathogen identification and characterization, drug 

target identification, and medical countermeasure 

development and mass production. An emphasis 

has been placed on the development of anti-infective 

therapeutics that has a broad spectrum of activity to 

enhance their potential utility against a wide range of 

emerging pathogens. In addition to exploiting highly 

conserved pathogen targets, proposed approaches 

have included host-directed anti-infective therapeutics to upregulate innate immunity, antagonize host 

receptors and processes that are hijacked by pathogens to complete their life cycles, and attenuate sepsis 

and other pathogenesis pathways. 

The National Strategy for Countering Biological Threats3

 proposed an integrated approach to all 

biological threats, whether from intentional releases 

(biological warfare or terrorism) or accidental releases 

(laboratory accidents or unintended consequences of 

legitimate scientific research) or naturally occurring 

emerging diseases. The strategy is based on the concept 

that all of these challenges require a common set of 

responses (pathogen identification and characterization; patient diagnosis; development, mass production, 

and distribution of medical countermeasures; medical 

and public health interventions; risk communication; 

promotion of ethical standards; professional and legal 

codes of conduct; and law enforcement). It proposes a 

pan-sector “all of society” approach that integrates the 

public at large and the scientific, medical, veterinary, 

public health, law enforcement, and diplomatic communities. Initiatives have included reorganization of 

civilian biodefense under the Department of Homeland Security; strengthening of programs under DoD 

and DHHS (NIH, the Biomedical Advanced Research 

and Development Authority, CDC) that have multipurpose utility for biological attacks, naturally occurring 

outbreaks, and other mass casualty disasters; the construction of the Fort Detrick biodefense campus, which 

includes laboratories for the Department of Homeland 

Security and NIH as well as a new USAMRIID facility; 

export controls to regulate exportation of potential 

dual use technologies; the medical countermeasures 

initiative to enhance mass production of medical countermeasures; investments to enhance biosurveillance; 

and federal guidelines for synthetic biology and the 

use of double-stranded DNA. 

The Federal Experts Security Advisory Panel’s interagency working group was initiated in 2010 to update 

42 CFR Part 73, Possession, Use, and Transfer of Select 

Agents and Toxins, to prevent intentional or accidental 

releases of highly virulent pathogens without placing 

counterproductive regulatory burdens on laboratories 

that conduct research on CDC select agents. Topics 

that were considered included revising the list of select 

agents, physical security measures, laboratory safety, 

occupational health, and personal reliability. A simplification of the select agent list was proposed, removing 

or recategorizing agents that are either easy to obtain 

from their natural reservoirs, or that constitute low risk 

due to low virulence, low transmissibility, or the availability of medical countermeasures. The Federal Experts 

Security Advisory Panel developed a comprehensive 

set of recommendations regarding biosecurity—the 

presence of physical security measures such as laboratory access controls, closed circuit visual monitoring, 

etc, and personal reliability—as well as background 

checks of laboratory workers’ law enforcement history, 

substance abuse, and mental health, with continuing 

monitoring and periodic reassessments of suitability 

for continued employment. Robust occupational health 

programs, with mandatory reporting of illnesses requiring medical intervention, were emphasized to prevent 

behaviors that could result in accidental or deliberate 

releases of select agents and to promptly recognize and 

treat laboratory-acquired infections and prevent their 

transmission to the general community. The Final Rule 

(October 5, 2012) included a revised select agent list; 

physical security standards for laboratories possessing 

Tier I Select Agents and Toxins; a requirement to conduct pre-access assessments and ongoing monitoring 

of personnel with access to Tier I agents and toxins; 

and clarifications of regulatory language concerning 

security, training, biosafety, and incident response.118,119

The optimization of biosafety and biosecurity is an iterative process. USDA’s Office of 

the Inspector General noted that while there 

had been enhanced compliance with security 

regulations and inspection processes within the 

USDA Select Agent program between 2005 and 

2012, there had been transfers of B anthracis and 

Y pestis samples to unregistered facilities, and access 

to select agents by a person with an expired security 

clearance. USDA concurred with recommendations 

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Medical Aspects of Biological Warfare 

to clarify restricted access requirements and establish policies and procedures for handling requests 

for transferring select agents under special circumstances to unregistered facilities.181 On March 24, 

2013, a vial of Guanarito virus (a Tier I Select Agent) 

was reported missing from the University of Texas 

Medical Branch at Galveston.182 On the following 

day, the Government Accountability Office issued a 

report concluding that US government interdepartment and interagency biodefense programs using 

high containment laboratories should improve their 

coordination. It also recommended that the Office 

of Science and Technology within the Executive Office of the President conduct periodic assessments 

of the requirements for, and the number, locations, 

and missions of high-containment laboratories, and 

evaluate the need to establish national standards for 

their design, construction, commissioning, operation, and maintenance.183

International efforts include the following:

• outreach by DoD and CDC to enhance surveillance with international partners; 

• DoD’s Cooperative Biological Engagement 

Program that builds partnerships to convert 

former biological weapons programs to 

peaceful purposes and enhance public health 

capacity; 

• collaborations to strengthen biological defense 

capacities of partner nations (eg, through the 

North Atlantic Treaty Organization and the 

Australia–Canada–United Kingdom–US–

New Zealand partnership); 

• US government support of BWC confidencebuilding measures and international public 

health efforts that may also lead to the early 

identification and containment of biological 

attacks (eg, WHO’s IHR); and 

• WHO efforts to enhance implementation of 

the IHR and strengthen ties with the World 

Organization for Animal Health and Interpol. 

SUMMARY

The use of microbes and toxins to intentionally 

cause harm has been attempted repeatedly throughout 

recorded history. However, military use before the 

development of modern microbiology was limited, 

possibly because of the availability of other weapons 

with more rapid and predictable results. 

Following the inception of modern microbiology, 

several nations began offensive biological warfare 

programs. Information regarding the history of 

state-sponsored biological weapons programs is 

obscured by secrecy, propaganda, and a lack of 

rigorous microbiologic or epidemiologic data to 

confirm allegations of use. Disclosures of former 

national programs underscore the ambitious intent 

and potential realization of covert state-sponsored 

programs. However, military deployment has been 

limited, and never decisive in armed conflict. With the 

exceptions of alleged German sabotage during World 

War I, Japanese field trials during World War II, limited deployments by South African and Rhodesian 

forces, and small-scale covert operations, there are no 

well-documented biological attacks by nation-states. 

Deterrents may include poor tactical utility related to 

multiple variables during production, storage, and 

delivery; variable incubations and host susceptibilities; availability of medical countermeasures; nuclear 

deterrence; diplomatic efforts; and political vulnerabilities. The public health disaster at Sverdlovsk, 

the loss of international goodwill toward the United 

States following disclosures during the Cold War, and 

political consequences following the 1996 disclosures 

by Iraq underscore that the attendant liabilities of 

state-sponsored biological weapons programs have 

outweighed potential strategic advantages. 

Non-state groups, lone actors, and even members 

of the medical community have committed bioterrorism and biocrimes. The likelihood of amateurs 

using homemade equipment to successfully develop 

and deploy a biological weapon of mass destruction 

is remote. Terrorists still rely on simple yet effective 

explosives as their weapon of choice. However, the 

Aum Shinrikyo program and Al Qaeda aspirations 

demonstrate intentions to harness modern microbiology for malicious purposes. Although most bioterrorism incidents and biocrimes have had limited results, 

the 1984 Rajneeshee episode and the 2001 anthrax 

mailings illustrate that even relatively small-scale 

attacks can have enormous public health, economic, 

and social consequences. 

Biological weapons have been renounced by 170 

states-parties to the BWC for numerous political 

and strategic considerations. Counterproliferation 

efforts, including verification of compliance of signatory states, remain challenging. According to an 

unclassified 2013 US Department of State report, 

uncertainties exist about activities in Russia, Iran, 

North Korea, and Syria.78 These ambiguities, in addition to the miscalculations of the 2002 National Intelligence Estimate, underscore the difficulty of assessing biological weapons programs even through the 

rigorous efforts of highly dedicated and skilled professionals. These concerns highlight the importance 

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Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness

of strengthening international goodwill and transparency through the BWC and international engagement programs. 

The threats of biological weapons have led to new 

technical strategies: 

• a movement from addressing a static list of a 

limited number of specific pathogens toward 

a threat-agnostic capability-based approach 

using flexible enabling technology platforms 

that can be rapidly adapted to counter novel, 

unanticipated pathogens; 

• broad spectrum therapeutics; and 

• versatile response capacities that can be 

used to counter biological weapons attacks, 

naturally occurring epidemics, or other mass 

casualty disasters. 

The past decade has also seen efforts to integrate 

multidisciplinary societal sectors ranging from research to operational response-surveillance; medical 

care delivery; risk communication; the development, 

mass production, and stockpiling of medical countermeasures; and planning and exercises at local, regional, 

national, and international levels. The enhancement 

of diagnostic platforms, disease surveillance and 

reporting networks, medical countermeasures, and 

health delivery systems that can be rapidly adapted 

as common solution sets to either biological attacks 

or natural epidemics is essential to cost-effective, economically sustainable disease mitigation in an era of 

limited resources. 

Scientific research on highly virulent pathogens is 

essential to biodefense and public health—broadly 

inclusive—to counter biological weapons and novel 

emerging diseases. Such research inevitably carries 

risks, including accidental releases, transmission of 

laboratory-acquired infections to the community, 

unanticipated consequences of well-intended experiments, and the generation of knowledge that could be 

misused to execute biological attacks. Even with effective risk management, risk never reaches zero, but can 

be decreased to an “irreducible minimum” through 

rigorous biosafety and biosecurity. Steps in the right 

direction include the formulation and enforcement of 

standards and regulations for biosafety, biosecurity, 

and handling of select agents. Risks and benefits should 

be carefully considered a priori, with engagement of a 

broad stakeholder community. Risk management must 

preserve opportunities for scientific creativity and academic freedom and also must be open to unanticipated 

experimental results that may serendipitously lead to 

valuable new discoveries, such as the reactogenicity 

of tuberculin purified protein derivative, that led to 

the repurposing of a failed therapeutic to a valuable 

diagnostic reagent, and the fungal contamination of a 

bacterial culture that led to the discovery of penicillin.

Although technical solutions are essential, they are 

not sufficient. An understanding of the history of the 

development and use of biological weapons, as well 

as analyses of risk perception and misperception, and 

appropriate or misguided responses to perceived risks 

requires examination from both technical and sociological points of reference, particularly the sociologies 

of scientific and policy decision-making. Important 

issues include the psycho-social milieus that generate 

biological weapon development and use, and that 

lead either to effective responses to credible threats 

or to misinterpretation and over-reaction to legitimate 

biotechnology.184

The late Joshua Lederberg, the 1958 Nobel laureate for medicine or physiology, a pioneer of bacterial 

genetics and recombinant technology, and an expert 

opinion leader in the fields of emerging infectious 

diseases and biological defense,185 remarked: 

There is no technical solution to the problem of biological weapons. It needs an ethical, human and moral solution if it’s going to happen at all. Don’t ask me 

what the odds are for an ethical solution, but there is 

no other solution.186 

Value-related paradigms of ethical medical research 

directed toward the good of humanity, which underlie 

the preamble of the BWC’s appeal “to the conscience 

of mankind,”187 and the National Strategy for Countering Biological Threats’ emphasis that life sciences 

research should be used “solely for peaceful and 

beneficial purposes,”188 proscribe biological weapons, 

and may also inform approaches to dilemmas posed 

by DURC. Proposals to obtain new data, information, 

and knowledge should be evaluated in the context 

of wisdom and in its relevance to the advancement 

of the common good, and be open to the possibilities that human actions may have intrinsic meaning 

and moral value. History demonstrates that when 

ethics and science are decoupled, potential outcomes 

include biological weapons. Ethical considerations 

are as relevant to basic and applied microbiology as 

the principle of beneficence is to medical research 

involving human subjects. Academic freedom must 

be maximized and ethical constructs must be flexible, 

yet circumstances exist in which it is appropriate to 

take principled stands. 

Moral principles lead to codes of professional conduct based on a commitment that basic and applied 

sciences must be value-related—purposely directed 

toward the benefit of society as their long-term goal 

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Medical Aspects of Biological Warfare 

with a caveat to “do no harm.”179 Professional ethics 

must go deeper than financial disclosures and honest 

reporting of data to address the value and risks of 

proposed experiments. Because an experiment can 

be done—as an achievement outside of a value- and 

goal-related context—does not mean that it should be 

done. It is essential to build a culture of responsibility 

at every level of individual investigators, laboratory 

institutional review boards, funding organizations, 

and national authorities considering the permissibility 

of specific research proposals in the context of purpose, 

methods, potential unintended consequences, and 

value to society. Moral principles underlying the BWC 

and the National Strategy for Countering Biological 

Threats have found expression in the ethical codes of 

the American Society for Microbiology and other professional organizations, US government guidelines for 

synthetic biology and DURC, the Cooperative Biological Engagement Program, support for implementation 

of the IHR, and NSABB’s call for the development and 

dissemination of ethical codes of conduct.189

The use of synthetic biology to produce wild-strain 

poliovirus illustrates the relevance of ethics to biological weapons proliferation and DURC, and the role of 

coordinated multidisciplinary approaches for risk mitigation. An intended outcome was to sound an alarm 

that viruses can be synthetically produced to develop 

biological weapons; a conclusion was that WHO’s goal 

of polio eradication may be unrealistic and should be 

reconsidered in view of issues that include the potential reintroduction of synthetic poliovirus as an act of 

bioterrorism.136,138 Alternatively, the chemical synthesis 

of the oral polio vaccine would have demonstrated 

an innovative cell-free platform for the production of 

attenuated live viruses for vaccines. This would have 

been an unambiguously benevolent action and would 

have supported the investigators’ intention to test 

the hypothesis that live viruses can be synthetically 

produced. The inductive proposition that synthetic 

viruses may pose biological weapons proliferation 

risks would have been obvious. The investigators later 

directed their platform toward novel approaches to 

vaccine development138,190–193; in the context of altruistic medical research, this could have been their stated 

objective and technical approach from the outset.

During the timeframe when the synthesis of wildstrain poliovirus was being conducted and reported, 

WHO was already proposing material and nonmaterial solutions for the contingency of a posteradication 

outbreak resulting from either bioterrorism or an accidental reintroduction.194–198 A 2013 WHO strategic 

plan for the final phase of polio eradication combines 

multidisciplinary pan-sector approaches including 

the global incorporation of inactivated polio vaccine 

into routine immunization programs, coordinated 

withdrawal of oral polio vaccine, biocontainment of all 

wild and vaccine strains, enhanced surveillance, a vaccine stockpile for emergency use, communication, and 

response.199 The potential abuse of synthetic biology 

for biological weapons proliferation has not derailed 

the polio eradication campaign,199–202 just as the risk 

of biological warfare using variola did not obviate the 

goal of smallpox eradication.

Medical capabilities and biomedical research are 

being linked to diplomacy, commerce, education, ethics, law enforcement, and other activities to enable a 

common set of multidisciplinary pan-societal sector 

responses to both biological weapons and the inevitable and dynamic challenges of naturally occurring 

emerging infectious diseases.3 Integration of biological 

defense and public health programs and their mutual 

development must be continuous to optimize outcomes and maximize efficient utilization of limited 

resources and because the challenges posed by both 

biological weapons agents and naturally emerging 

pathogens are open ended. 

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201. World Health Organization. Sixty-fifth World Health Assembly. Poliomyelitis: Intensification of the Global Eradication 

Initiative. Geneva, Switzerland: WHO; 2012. WHA65.5. http://apps.who.int/gb/ebwha/pdf_files/wha65/a65_r5-en.pdf. 

Accessed May 31, 2013.

202. De Quadros C, Tomori O, Thacker N, et al. Scientific Declaration on Poliovirus Eradication. Emory Vaccine Center. 

April 11, 2013. http://vaccines.emory.edu/poliodeclaration/text.pdf. Accessed April 12, 2013.

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Epidemiology of Biowarfare and Bioterrorism

Chapter 2

EPIDEMIOLOGY OF BIOWARFARE 

AND BIOTERRORISM

ZYGMUNT F. DEMBEK, PhD, MS, MPH, LHD*; JULIE A. PAVLIN, MD, PhD, MPH†; MARTINA SIWEK, PhD‡; and 

MARK G. KORTEPETER, MD, MPH§

INTRODUCTION

THE EPIDEMIOLOGY OF EPIDEMICS 

Definition

Recognition

Potential Epidemiological Clues to an Unnatural Event

Outbreak Investigation

EPIDEMIOLOGICAL CASE STUDIES 

Bioterrorism Events

Accidental Release of Biological Agents

Studies of Natural Outbreaks for Potential Bioweapon Use

EPIDEMIOLOGICAL ASSESSMENT TOOL

IMPROVING RECOGNITION AND SURVEILLANCE OF BIOTERRORISM 

POTENTIAL IMPACT OF ADVANCED MOLECULAR TECHNIQUES ON THE 

EPIDEMIOLOGY OF BIOWARFARE AND BIOTERRORISM

SUMMARY

*Colonel (Retired), Medical Service Corps, US Army Reserve; Associate Professor, Department of Military and Emergency Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814; formerly, Chief, Biodefense Epidemiology and Education 

& Training Programs, Division of Medicine, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

†

Colonel (Retired), Medical Corps, US Army; Deputy Director, Armed Forces Health Surveillance Center, 11800 Tech Road, Silver Spring, Maryland 20904

‡

Chief Scientist, Cherokee Nation Technology Solutions in Support of Global Emerging Infections Surveillance and Response System, Armed Forces 

Health Surveillance Center, 11800 Tech Road, Silver Spring, Maryland 20904; formerly, Science Manager/Liaison, Biosurveillance Management Office, 

Joint Program Executive Office, Aberdeen Proving Ground, Maryland

§

Colonel, Medical Corps, US Army; Director, Infectious Disease Clinical Research Program, Department of Preventive Medicine, Associate Dean for 

Research, Associate Professor of Preventive Medicine and Medicine, Consultant to the Army Surgeon General for Biodefense, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814; formerly, Deputy Commander, US Army Medical Research Institute 

of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

A portion of this chapter has previously been published as: Dembek ZF, Kortepeter MG, Pavlin JA. Discernment between deliberate and 

natural infectious disease outbreaks. Epidemiol Infect. 2007;135:353–371.

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Medical Aspects of Biological Warfare 

INTRODUCTION

Preparing for and responding to biological warfare 

(BW) or bioterrorism (BT) is a public health issue and 

falls within the purview of public health professionals, 

because preparation for natural disease outbreaks has 

the dual benefit of BW/BT preparation. An understanding of basic epidemiology is needed before, during, 

and after an event to identify populations at risk, target 

preventive measures such as vaccinations, recognize an 

outbreak, track and limit disease spread, and provide 

postexposure treatment or prophylaxis. Many diseasespecific management needs such as vaccination and 

prophylaxis are discussed elsewhere and are not considered here. Also, agricultural terrorism is discussed 

in chapter 3. This chapter will focus on detection and 

epidemiological investigation including distinguishing between natural and intentional events. Brief case 

studies will be presented to demonstrate important 

indicators and lessons learned from historical outbreaks. 

Finally, traditional methods of surveillance and ways 

to improve surveillance for BW/BT will be discussed.

THE EPIDEMIOLOGY OF EPIDEMICS

Definition

The word epidemic comes from the Greek “epi” and 

“demos,” meaning “upon a mass of people assembled 

in a public place.”1 An epidemic is defined as the occurrence in a community or region of an unusually large or 

unexpected number of disease cases for the given place 

and time.2

 Therefore, a critical foundation is knowing 

baseline rates of disease to determine whether an epidemic is occurring. This information can be at the local, 

regional, national, or global level, and can be seasonal. 

As an example, thousands of influenza cases in January in the United States may not be unusual; however, 

thousands of cases in the summer may be cause for 

concern, similar to what was seen with an early summer wave of cases of H1N1 swine variant influenza in 

2009. Also, even a single case of a rare disease can be 

considered an epidemic. With the absence of a woolen 

mill industry in the United States, any inhalational 

anthrax case should be highly suspect. Many of the 

diseases considered as classic BW agents, such as smallpox (considered to be eradicated), viral hemorrhagic 

fevers, and pneumonic plague are rare, and a single 

case should be investigated. Determining whether an 

outbreak occurs depends, therefore, on the disease, the 

at-risk population, the location, and the time of year.

For an outbreak to occur, three points of the classic 

epidemiological triangle must be present (Figure 2-1). 

There must be a pathogen or agent, typically a virus, 

bacterium, rickettsia, fungus, or toxin, and a host (in 

this case, a human) who is susceptible to that pathogen or agent. The two need to be brought together in 

the right environment to allow infection of the host 

directly by another individual, by a vector, or through 

another vehicle, such as food, water, or contact with 

fomites (inanimate objects). The environment must 

also permit potential transmission to other susceptible 

hosts. Disruption of any of these three points of the 

triangle can limit or disrupt the outbreak; therefore, it is 

important to know and understand the characteristics 

of the three for any specific disease to control an epidemic. For example, if potential hosts are vaccinated, 

disease spread would be significantly limited or if the 

environment is modified, spread may also be limited 

(eg, cleaning up garbage around a home limits rat food 

and harborage, and thus minimizes the risk of contact 

with fleas capable of transmitting plague).3

Recognition

Immediate effects on humans and possibly the environment are evident when an explosion occurs or a 

chemical weapon is released. However, because of the 

incubation periods of infectious pathogens, release of 

a BW/BT agent may be silent and the casualties produced after a release may be dispersed in time and 

space to primary care clinics and hospital emergency 

departments. Even toxins have latent periods prior to 

symptom onset. Therefore, the success in managing a 

biological event hinges directly on whether and when 

the event is recognized.

Host

Agent Environment

Figure 2-1. The epidemiological triangle

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Epidemiology of Biowarfare and Bioterrorism

An example of the ramifications of delayed disease 

outbreak recognition occurred in 1972 in the former 

Yugoslavia. A single unidentified smallpox case led to 

11 secondary cases, also unrecognized. Within a few 

weeks there was an outbreak of 175 smallpox cases and 

35 deaths that led to a massive vaccination effort and 

border closure.4 Early disease recognition may have 

significantly modified the outcome. Modeling studies 

of a BT-caused smallpox outbreak have shown that 

the more rapidly a postrelease intervention occurred, 

including quarantine and vaccination, the greater 

the chances that intervention would halt the spread 

of disease.5–7 When medical professionals identify a 

new case, it is unlikely that a BW/BT event would 

be the first cause suspected, especially if the disease 

presents similar to other diseases that might occur simultaneously, such as influenza. Clinicians generally 

consider the source to be a common endemic disease 

at first. Alternative considerations might include a new 

or emerging disease, or a laboratory accident before 

considering BW/BT.8

 Therefore, care providers should 

be familiar with the diseases of BW/BT that could be 

spread intentionally and maintain a healthy “index of 

suspicion” to recognize an event early enough to significantly modify the outcome.9

 Furthermore, although 

the government has generated lists of potential threat 

agents, public health authorities must be mindful that 

a perpetrator does not necessarily follow any list and 

may choose an organism based on access or some 

other unanticipated reason. Also, a perpetrator might 

listen to government and other media information, and 

respond accordingly, thereby undermining a government terrorism response.

Clinicians, hospital infection control personnel, 

school or healthcare facility nursing staff, laboratory 

personnel, and other public health workers have a 

responsibility to notify public health authorities about 

disease outbreaks. State and local public health officials 

regularly examine and review disease surveillance 

information to detect outbreaks in a timely manner 

and provide information to policymakers on disease 

prevention programs. Time constraints are inherent 

in obtaining case report information because of the 

elapsed time from patient presentation, lab specimen 

collection and submission, and laboratory testing time, 

to final disease or organism reporting. Furthermore, 

the initial BW/BT disease recognition may not come 

from a traditional reporting partner or surveillance 

method. Instead, pharmacists and clinical laboratory 

staff who receive requests or samples from numerous healthcare providers may be the first to note 

an increase in purchases or prescriptions of certain 

medications (eg, antibiotics or antinausea or diarrheal agents) or orders for certain laboratory tests (eg, 

sputum or stool cultures), respectively. Also, because 

many of the category A high-threat diseases are zoonoses (primarily infect animals), with humans serving 

as accidental hosts, veterinarians may be the first to 

recognize the disease in animals prior to the ensuing 

human disease. Media and law enforcement personnel 

and other nontraditional reporters of outbreaks may 

also provide information on a BT event or potential 

cases. Therefore, it is important for all those different 

types of individuals to maintain the same index of 

suspicion as healthcare providers for unusual events 

in their respective fields.

Potential Epidemiological Clues to an Unnatural 

Event

It is often not possible to determine the objectives of a 

BT perpetrator in advance, whether the intent is to kill, 

incapacitate, or obtain visibility. It also may be difficult 

to discern how a biological agent was dispersed, whether through the air, in contaminated food or water, or by 

direct inoculation. In a biological attack, the number of 

casualties may be small and therefore unrecognized as 

intentionally infected, especially if the agent is a common cause of disease in the community. In addition, 

given the agent’s incubation period, individuals may 

seek care from different care providers or travel to different parts of the country before they become ill and seek 

medical care. Despite the potential for these situations 

to occur, it is useful for healthcare providers to be aware 

of potential clues that may be tip-offs or “red flags” of 

something unusual. Although these clues may occur 

with natural outbreaks and do not necessarily signal a 

BW/BT attack, they should at least heighten suspicion 

that something out of the ordinary is occurring. The 

following compilation is an illustrative list; however, 

additional clues may be found elsewhere.10,11

Clue 1: A highly unusual event with large numbers of casualties. Although the mention of BW or 

BT may elicit images of massive casualties, they may 

not actually occur with a real BW/BT event. Numerous examples of naturally spread illness have caused 

massive casualties and some BW/BT events have few or 

no casualties. Nevertheless, the type of large outbreak 

that should receive particular attention is one in which 

no plausible natural explanation for the cause of the 

infection exists.

Clue 2: Higher morbidity or mortality than is 

expected. If clinicians are seeing illnesses that are 

causing a higher morbidity or mortality than what is 

typically seen or reported for a specific disease, this 

may indicate an unusual event. A perpetrator may 

have modified an agent to make it more virulent or 

selected antibiotic resistance in an organism usually 

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40

Medical Aspects of Biological Warfare 

sensitive to antibiotics. Individuals could also be exposed to a higher inoculum than they would normally 

receive with natural spread of the agent, thus causing 

higher morbidity or mortality.

Clue 3: Uncommon disease. Many infectious 

diseases have predictable population and infectivity 

distributions based on environment, host, and vector 

factors; yet unnatural spread may occur if a disease 

outbreak is uncommon for a certain geographical 

area. Concern should be heightened if the naturally 

occurring disease requires a vector for spread and the 

competent vector is missing. For example, if a case 

of yellow fever, which is endemic to parts of South 

America and sub-Saharan Africa, occurred in the 

United States without any known travel, it would be 

a concern. Natural outbreaks have occurred in new 

geographical locations including the West Nile virus 

(WNV) in New York City in 1999.12 It is important to 

consider whether the occurrence of these uncommon 

diseases is natural.

Clue 4: Point source outbreak. For any outbreak, it 

is useful to develop an epidemic curve demonstrating 

the timeline of dates when patients developed illness. 

These curves can have different morphologies depending on whether individuals are exposed at the same 

time from a single source or over time, and whether 

the illness spreads from person to person. In an intentional BT event, a point source outbreak curve would 

most likely be seen13 when individuals are exposed 

at a similar point in time. The typical point source 

outbreak curve has a relatively quick rise in cases, a 

brief plateau, and then an acute drop, as seen in Figure 

2-2. For example, the epidemic curve might be slightly 

compressed after an aerosol release because infected 

individuals were exposed more closely in time (ie, 

within seconds to minutes of each other) compared 

with individuals becoming ill after eating a common 

food over a period of hours. Or the inoculum may be 

greater than what is typically seen with natural spread, 

thus yielding a shorter incubation than expected. It 

should also be considered that the spread of a biological agent capable of being transmitted from person 

to person could result in a propagated (secondary 

transmission) outbreak, with a case distribution more 

similar to that depicted in Figure 2-3.

Clue 5: Multiple epidemics. If a perpetrator can 

obtain and release a single agent, it is also feasible that 

multiple perpetrators could release single or multiple 

agents at different locations. If simultaneous epidemics 

occur at the same or different locations with the same 

or multiple organisms, an unnatural source must be 

considered. It must also be considered that a mixture 

of biological organisms with different disease incubation periods could be released, and thus would cause 

simultaneous or serial outbreaks of different diseases 

in the same population.

Clue 6: Lower attack rates in protected individuals.

This clue is especially important for military personnel. 

If certain military units had some type of respiratory 

protection, such as mission-oriented protective posture 

gear or high-efficiency particulate air-filtered masks, or 

stayed in a high-efficiency particulate air-filtered tent 

and had lower rates of illness than nearby groups that 

were unprotected, this may indicate that a biological 

agent has been released via aerosol.

Clue 7: Dead animals. Historically, animals have 

been used as sentinels of human disease. The storied 

use of canaries in a coal mine to detect the presence 

of noxious gases is one example. This phenomenon 

was observed during the naturally occurring WNV 

outbreak in New York City in 1999, when many of the 

local crows, along with the exotic birds at the Bronx 

Zoo, developed fatal disease.14,15 Because many biological agents that could be used for BW/BT are zoonoses, 

a local animal die-off may also indicate a biological 

agent release that may also infect humans. 

0

5

10

15

20

25

30

35

1 234 567 8 9

Onset by Day of Month

Figure 2-2. Typical point source outbreak epidemic curve

0

2

4

6

8

10

12

14

16

Week 1Week 2Week 3Week 4

Week 5

Week 6

Week 8

Week 9

Week 10

Week 11

Week 0

Week 7

Figure 2-3. Typical continuous common source outbreak 

epidemic curve

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41

Epidemiology of Biowarfare and Bioterrorism

Clue 8: Reverse or simultaneous spread. Zoonotic 

illnesses exhibit a typical pattern: an epizootic first occurs among a susceptible animal population, followed 

by cases of human illness. With anthrax, one would 

expect ill animals to be identified before cutaneous 

disease in workers processing the animals or before 

gastrointestinal disease in people who may have eaten 

meat from the infected animals. After the accidental 

release of anthrax spores in Sverdlovsk (see description 

and case review of the 1979 Sverdlovsk anthrax outbreak), an outbreak occurred simultaneously in people 

and animals downwind of the weapons facility.16 If 

human disease precedes animal disease or human 

and animal disease are simultaneous, then unnatural 

spread should be considered.

Clue 9: Unusual disease manifestation. More than 

95% of worldwide anthrax cases are cutaneous illness. 

Therefore, a single case of inhalational anthrax should 

be considered highly suspicious for BW/BT until 

proven otherwise. The rare exception is an inhalational 

anthrax case in a woolen mill worker or in someone 

handling animal skins from endemic areas, which 

has recently occurred.17 This logic may be applied to 

cases of a disease such as plague, where the majority 

of naturally occurring cases are the bubonic, not the 

pneumonic form.

Clue 10: Downwind plume pattern. The geographic 

locations where cases occur can be charted on a geographic grid or map. If the reported cases appear 

clustered in a downwind pattern, then an aerosol 

release may have occurred. During the investigation 

into the anthrax outbreak in Sverdlovsk in 1979 (as 

examined later in this chapter), mapping out case 

locations helped to determine that the anthrax cases 

were caused by an aerosol release rather than a contaminated food source.16

Clue 11: Direct evidence. The final clue may be the 

most obvious and the most useful. Determining the 

intentional cause of illnesses is easier if a perpetrator 

leaves a “signature” or direct evidence of a biological 

attack. Such a signature could be a letter filled with 

anthrax spores,18 a spray device or another vehicle 

for agent spread, or claims by a person or group of 

a biological attack. It would be useful to compare 

samples from any found device with the clinical 

samples obtained from victims to verify that they are 

the same organism.

Outbreak Investigation

It is important to understand the basic goals of an 

outbreak investigation, as seen in Exhibit 2-1. Any 

outbreak (a greater than expected number of cases in 

a specific location, group of people, or time period) 

should be investigated quickly to find the source of 

the disease. If an outbreak is ongoing, the source of 

infection needs to be identified and eliminated quickly. 

Even if the exposure source has dissipated, all cases 

should be identified expeditiously, so that ameliorative care can be offered and case interviews can be 

conducted. Case identification can assist in preventing 

additional cases, especially with a transmissible infectious disease. Providing information to the public and 

to leaders is also key to ensure the best public health 

policies are enacted and followed. With notification of 

any outbreak, whether natural or intentionally caused, 

there are standard steps to follow in an outbreak investigation (Exhibit 2-2), although these steps may not 

always occur in order.19 The first step is preparation, 

which involves having the necessary response elements (personnel, equipment, laboratory capabilities) 

ready and establishing communications in advance 

with partners who may assist in the investigation. Once 

an event is ongoing, the second step is to investigate, 

verify the diagnosis, and decide whether an outbreak 

exists. Early in an outbreak, its significance and scope 

are often not known. Therefore, existing surveillance 

information and heightened targeted surveillance efforts are used to determine whether reported items 

are cause for concern.

The third step is to define the outbreak and seek a 

definitive diagnosis based on historical, clinical, epidemiological, and laboratory information. A differential 

diagnosis can then be established.

The fourth step is to establish a case definition that 

includes the clinical and laboratory features that the ill 

individuals have in common. It is preferable to use a 

EXHIBIT 2-1

GOALS OF AN OUTBREAK INVESTIGATION

• Find the source of disease.

• Rapidly identify cases.

• Prevent additional cases through implementation of appropriate control measures.

• Identify strategies to prevent further outbreaks.

• Evaluate existing prevention strategies (including control measures immediately put 

into place).

• Address public concerns.

• Provide information to leadership to support 

informed decisions.

• Improve scientific knowledge about the 

disease.

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42

Medical Aspects of Biological Warfare 

broad case definition at first and avoid excluding any 

potential cases too early. Objective clinical features are 

preferred, such as temperature exceeding 100.4°F, or 

diarrhea defined as greater than three watery bowel 

movements per day, as well as laboratory and pathological reports. The case definition enables the investigator to count cases and compare exposures between 

cases and noncases and compare these with other 

investigators and regions using the same case definition. To obtain symptom information, it may not be 

sufficient to look at healthcare facilities only, but also 

necessary to interview the ill persons and their family 

members, as well as coworkers, classmates, or others 

with whom they have social contact. It is important 

to maintain a roster of potential cases while obtaining 

this information. Commonly during an investigation, 

there is a risk of double or even triple counting cases 

because they may be reported more than once through 

different means. Key information needed from each 

ill person, besides identifying information to ensure 

accurate case counting and ability to contact the cases 

again if necessary, includes date of illness onset; signs 

and symptoms; recent travel; ill contacts at work, 

home, or school; animal exposures; and treatments 

received. With this information, an epidemic curve 

can be constructed (see Figure 2-2) that may provide 

information as to when a release may have occurred, 

especially if the disease is known, and an expected 

exposure date based on the typical incubation period, 

known ill contacts, or geographic risk factors.

Different modes of disease spread may have typical 

features that comprise an epidemic curve. If there is 

a common vehicle for disease transmission (such as a 

food or water source) that remains contaminated, it 

might be possible to see a longer illness plateau (a continuous common source curve [Figure 2-3]) than is seen 

with a point source of infection. If the agent is spread 

person to person, successive waves of illness may be 

seen as one group of individuals infects a follow-on 

group, which in turn infects another, and so on (Figure 

2-4). With time and additional cases, the successive 

waves of illness may overlap with each other.

The fifth step is to document potential exposure data. 

Cases need to be identified and counted. Once cases 

have been identified, exposures based on person, place, 

and time can be determined. Obtaining information 

from individuals who would likely have had similar 

exposures but are not ill can also help determine the 

potential cause and method of an agent’s spread. Information can be obtained either informally or formally 

with a case control study. A case control study is a 

type of study where investigators identify individuals 

with and without disease and compare their potential 

exposures or risk factors for disease. With a known 

exposure, one can also identify exposed and nonexposed populations and determine illness rates with a 

retrospective cohort study to help determine whether 

that particular exposure is a risk factor for disease. 

The sixth step is to implement control measures as 

soon as feasible and continuously evaluate them. If necessary, control measures can be quickly implemented 

and then modified as additional case information 

becomes available. The seventh step is to develop a 

hypothesis. Based on the characteristics of the disease, 

the ill persons, and environmental factors, a hypothesis can usually be generated for how the disease occurred, how it is spreading, and the potential risk to 

the uninfected. The eighth step is to test and evaluate 

the hypothesis using analytical studies and refine the 

hypothesis.

EXHIBIT 2-2

TEN STEPS IN AN OUTBREAK 

INVESTIGATION

1. Prepare for fieldwork (identify resources).

2. Verify the diagnosis. Determine whether an 

outbreak exists.

3. Define the outbreak and seek a diagnosis 

(including specimen collection and testing).

4. Develop a case definition and identify and 

count cases.

5. Develop exposure data with respect of person, place, and time.

6. Implement control measures and continually evaluate them.

7. Develop the hypothesis.

8. Test and evaluate the hypothesis with analytical studies and refine the hypothesis.

9. Formulate conclusions.

10. Communicate findings.

0

5

10

15

02610 14 18 22 26 30 34 38

Onset by Day of Month

Figure 2-4. Typical propagated (secondary transmission) 

outbreak epidemic curve

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Epidemiology of Biowarfare and Bioterrorism

Once developed, it is important to test the hypothesis to ensure it fits with the known facts. Does it 

explain how all the cases were exposed? It is possible 

that some outliers may seem as if they should be ill 

but are not, or some who are ill but have no known 

exposure. These outliers can sometimes be the key 

to determining what happened. With preliminary 

control measures implemented, the hypothesis can 

be tested formally with analytical studies. Further 

modifications in control measures might be needed 

and implemented. 

The ninth step is to formulate a conclusion about 

the nature of the disease and exposure route. Findings 

can then be communicated (the tenth and final step) 

through the media or medical literature, depending on 

the urgency of notification to the public and medical 

community.

Experience from the anthrax mailings of 2001 indicates that during any BT event, intense pressure will 

be exerted on public health authorities to provide more 

information than is available.20 As stated earlier, these 

distinct steps may not occur in sequence. It may be necessary to implement control measures with incomplete 

information, especially if an outbreak is fast moving 

or has a high morbidity or mortality rate. Whether the 

control measures appear to limit the disease spread or 

the casualty toll is the ultimate test of the accuracy of 

the original hypothesis. 

Early in an investigation, it will probably not be 

known or suspected that an outbreak was unnaturally 

spread. Therefore, with a few exceptions, the investigation of an unnaturally spread outbreak will not differ 

significantly from the investigation of a naturally occurring outbreak. Public health authorities will work 

on both types of outbreaks. The significant difference 

is that, with a purposeful outbreak, a potential criminal event may have occurred. An additional goal of 

this type of investigation, under the purview of law 

enforcement personnel, is to bring the perpetrator to 

justice. Therefore, law enforcement personnel need to 

partner with public health officials as early as possible 

in any suspected BT case.21

Public health authorities must become familiar 

with the use of chain of custody, the process used 

to maintain and document the chronological history 

of the evidence, so that medical evidence/clinical 

samples or environmental samples obtained in the 

investigation will be admissible in a court of law. 

Environmental and biological samples can be crucial in determining whether a deliberate release of 

a pathogen has occurred (see the case study in this 

chapter about the release of Bacillus anthracis in Tokyo 

by the Aum Shinrikyo). 

Although chain of custody is important, public 

safety should be the primary concern. Public health 

authorities must also have an open mind for unusual 

modes of disease spread, being especially careful to 

ensure their personnel’s safety if a potential exposure 

risk occurs during the investigation. Public health 

authorities conducting a field investigation should 

have personal protective equipment and be trained 

in its proper use, and they should also have access to 

occupational health resources if pre- or postexposure 

prophylaxis or monitoring is needed.

EPIDEMIOLOGICAL CASE STUDIES

The following epidemiological case studies are 

presented to demonstrate the differences between 

naturally occurring and purposefully created epidemics. Biological attacks and some naturally occurring 

epidemics of historical significance are considered in 

the context of BT. Some purposeful BT events have 

not caused illness; however, some naturally occurring 

outbreaks were initially considered as potential BT 

events because of the particular disease or nature of 

clinical case presentation.

Public health authorities could be held accountable to make a determination quickly as to whether 

an infectious disease outbreak has been purposefully 

caused, yet they may lack the necessary information because there may not be clear evidence or 

responsibility claimed for a BT event. A thorough 

understanding of how to investigate suspect outbreak occurrences may better enable public health 

authorities to make difficult public health policy 

decisions.

Bioterrorism Events

The following section describes BT incidents that 

occurred in the United States and Japan. None of these 

events was immediately recognized as having been 

intentional. The 2001 mail-associated anthrax outbreak 

and mail-associated ricin attack were recognized 

within days to weeks. With new sensors installed in 

mail collection facilities, mailings of ricin in 2013 were 

recognized immediately. However, for previous BT 

incidents (anthrax and glanders in 1915, salmonellosis 

in 1984, and anthrax in 1995), intentionality was not 

recognized for a year or longer after the initial event.

Anthrax and Glanders—Maryland; New York, New 

York; and Virginia, 1915–1916

From 1915 through 1918, Germany had a statesponsored offensive BW program to sabotage suppliers 

to the Allies directed at draft, cavalry, and military 

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Medical Aspects of Biological Warfare 

livestock. Human disease was neither intended nor 

recorded from these events, although the program 

could have been expanded to spread zoonotic illness among a target population. Unintended human 

disease may have occurred but was never recorded. 

Countries targeted by Germany included the United 

States, Argentina, Romania, Russia, Norway, and 

Spain. The German army general staff directed and 

implemented the biological sabotage program despite 

official German army doctrine prohibiting such activities. Germany’s plans to spread a wheat fungus and 

contaminate food produced at “meat factories” were 

dropped.22 One 1916 German plan never carried out 

proposed to drop vats of plague cultures from Zeppelins over England.23

In April 1915 German-American physician Anton 

Dilger returned to the United States from Germany 

with cultures of Burkholderia mallei and Bacillus anthracis. His intent was to infect horses and mules being 

shipped from the United States to France and England 

for use in cavalry and transport. These cultures were 

propagated and tested for virulence using guinea pigs 

in the basement of a house (known as “Tony’s Lab”) 

rented by Anton and his brother, Carl, in Chevy Chase, 

Maryland, near Washington, DC.24 From the summer of 

1915 through the fall of 1916, the cultures were used to 

infect horses and mules in holding pens in docks at the 

ports of Baltimore, Maryland; Newport News, Virginia; 

Norfolk, Virginia; and New York, New York. Stevedores 

working for German steamships were recruited and 

given 2-inch, cork-stoppered glass vials containing the 

cultures, in which a hollow steel needle had been placed. 

These stevedores were instructed to wear rubber gloves 

while jabbing the animals with the needle. These cultures were also spread to the animals by pouring them 

into the animal feed and drinking water.25,26

Case Review of 1915–1916 Anthrax and Glanders 

Incidents

Biological Agents: B anthracis, gram-positive bacillus; 

B mallei, gram-negative bacillus

Potential Epidemiological Clues: 2, 7, 8

Review: A full assessment of the success of this BW 

program 90 years later is not possible. German agents 

claimed that epidemics occurred among the animals shipped 

from the US ports. However, disease observed among animals might have originated naturally or from stressful holding 

and shipment conditions. 

Few surveillance systems incorporate comprehensive 

veterinary surveillance. This is an important disease detection vulnerability because many BW agents (ie, B anthracis, 

Brucella suis, B mallei, Burkholderia pseudomallei, Coxiella 

burnetii, Francisella tularensis, Yersinia pestis, encephalitis, 

and hemorrhagic fever viruses) can cause zoonotic illness.

Lessons Learned: Veterinarians discovering glanders 

or anthrax and other US Department of Agriculture (USDA) 

select agricultural agents in livestock should report these 

diseases to state health and federal authorities as possible 

BT indicators.27,28

A comprehensive animal surveillance network would 

include reports from veterinary examinations of farm and 

companion animals, and from wildlife examinations by state 

environmental officials and animal rehabilitators. Current 

animal disease surveillance networks that address these 

deficiencies include the National Animal Health Laboratory 

Network29 and the Centers for Epidemiology and Animal 

Health,30 both part of the USDA. 

Salmonellosis—The Dalles, Oregon, 1984

A large outbreak of Salmonella cases occurred in 

and around The Dalles, Oregon, in 1984. This farming community, with a 1984 population of 10,500, is 

near the Columbia River on the border of Oregon and 

Washington. Salmonellosis is the second most common 

bacterial foodborne illness and is underreported by a 

factor of about 38-fold.31,32 The average onset period for 

salmonellosis is about 12 to 36 hours, and the disease 

manifests as acute gastroenteritis. Fever occurs, anorexia and diarrhea persist for several days, and more 

severe manifestations may at times occur, especially 

in very young or elderly persons. Contaminated food 

(most often poultry) is the principal route of disease 

transmission.33

Given its high incidence in the United States, public health authorities would not normally consider a 

foodborne salmonellosis outbreak as intentional. It 

has been estimated that 1.4 million salmonellosis infections occur annually in the United States, resulting 

in 15,000 hospitalizations and 400 deaths.34 Therefore, 

the index of suspicion for an intentional Salmonella 

outbreak was—and remains—low. However, atypical 

events associated with this outbreak eventually led 

officials to realize that this particular disease occurrence was historically different. Two cohorts of cases 

occurred: (1) from September 9 through 18, 1984, and 

(2) from September 19 through October 10, 1984. Public 

health authorities received initial reports of illness on 

September 17, and local and state health officials interviewed the ill persons. Patronizing two restaurants in 

the city of The Dalles and eating salad bar food items 

were commonly cited in these interviews. Salmonella 

typhimurium isolates were then obtained from clinical 

specimens from the ill persons.35

The source for this outbreak was puzzling. Epidemiological analysis revealed multiple items rather 

than a single suspect item as the cause of the restaurant patrons’ illness. This finding is not uncommon 

either during the initial stages of an investigation of 

a foodborne disease outbreak (until a suspected food 

item is identified), or when an infected food handler 

is identified as the source of the outbreak. Although 

dozens of food handlers became ill, their time of 

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Epidemiology of Biowarfare and Bioterrorism

symptom onset did not precede those of their customers. As gastroenteritis cases occurred in increasing 

numbers, health officials imposed a closure of all salad 

bars in The Dalles on September 25. By the end of the 

outbreak, 751 salmonellosis cases were identified, 

with those affected ranging in age from newborns to 

87 years, and most were associated with dining in 10 

area restaurants. At least 45 persons were hospitalized, 

but no fatalities occurred. 

Bhagwan Shree Rajneesh, a charismatic guru, had 

established a community for his followers in 1981 

at a ranch near The Dalles. These cult members, or 

“Rajneeshees,” attempted to use Oregon’s liberal 

voter registration laws to control zoning and land use 

restrictions to their advantage. Conflict between the 

commune and the neighboring traditional community 

had escalated. To gain political control of the area, 

the Rajneeshees attempted to influence an election 

by making voters too ill to vote.22 Approximately 12 

individuals were involved in the plot, and up to 8 individuals distributed S typhimurium cultures to the salad 

bars. After considering the use of several biological 

agents, including Salmonella typhi (the causative agent 

of typhoid fever) and the human immunodeficiency 

virus, the Rajneeshees legally obtained cultures of 

S typhimurium (American Type Culture Collection 

strain 14028) from a commercial supplier and used 

them to grow bacterial stock cultures. The Rajneeshees 

first spread Salmonella by contaminating the commune members’ hands to greet outsiders, as well as 

the county courthouse’s doorknobs and urinal handles; 

these efforts did not cause illness. The cult also spread 

Salmonella cultures on salad bars in area restaurants.

Public health authorities conducted an extensive 

investigation in response to the salmonellosis outbreak. 

Authorities identified confirmed cases microbiologically by stool culture of S typhimurium, or with the 

clinical criteria of diarrheal illness and at least three 

of the following symptoms: fever, chills, headache, 

nausea, vomiting, abdominal pain, or bloody stools. 

S typhimurium was isolated from 388 patients. In the 4 

years before the outbreak, the local health department 

had collected 16 isolates of Salmonella, 8 of which were 

S typhimurium. No local cases of salmonellosis had been 

reported in 1984 before August.35

The 38 restaurants in The Dalles were grouped according to the number of culture-confirmed customer 

cases with a single restaurant exposure in the week 

before symptom onset. Additional ill customers were 

located through laboratory reporting of clinical specimens or clinician reporting to public health authorities 

(passive disease surveillance). Press releases were 

issued to encourage disease reporting by patients 

and clinicians.35 Public health officials interviewed ill 

persons to obtain their symptoms, risk factors, and 

comprehensive food histories, as well as the names of 

all persons who had eaten with them at the restaurant. 

Restaurant employees with the greatest number of cases 

were interviewed twice and required to submit a stool 

sample as a condition of continued employment. The 

state public health laboratory serotyped the Salmonella 

isolates and performed antibiotic-susceptibility testing on a subset. A representative sample of outbreak 

isolates was sent to the Centers for Disease Control 

and Prevention (CDC) for further characterization, 

during which the outbreak strain was compared with 

national surveys of human and veterinary isolates. 

Sanitarians inspected the restaurants, and tap water 

was collected and analyzed. The local health department and USDA also investigated the food distributors and suppliers used in these restaurants. None was 

found to have contaminated food, nor was a common 

supplier found for all of the implicated restaurants.

Many food items served at the salad bars of the 

restaurants were associated with illness and differed 

among the restaurants. Illness was associated with eating blue cheese dressing at one of the restaurants. The 

consumption of potato salad had the greatest association with illness, followed by lettuce. S typhimurium

was isolated from the blue cheese dressing collected 

at one restaurant, but not from the dry mix used to 

prepare the dressing.

The size and nature of the outbreak eventually 

helped to initiate a criminal investigation. The source 

and cause of the outbreak only became known when 

the Federal Bureau of Investigation (FBI) investigated 

the cult for other criminal violations.36 An Oregon 

public health laboratory official accompanying the 

FBI discovered an open vial containing the original 

culture strain of S typhimurium in the Rajneeshee clinic 

laboratory in October 1985.22,35 This strain was indistinguishable from the outbreak strain as isolated from 

food items and clinical specimens, and records were 

found documenting its purchase before the outbreak.35

Intentional contamination of the salad bars is consistent with the retrospective epidemiology.35 Eventually 

two cult members were arrested and served federal 

prison terms. Despite the Rajneeshees’ success of the 

restaurant-associated BT, the publicity and subsequent 

legal pressure caused them to abandon subsequent 

efforts.22

Case Review of 1984 Salmonellosis Outbreak 

Biological Agents:S typhimurium, gram-negative bacillus 

Potential Epidemiological Clues: 1, 4, 5, 11

Review: Public health authorities found no statistical 

association with any single food item.22 The isolation of S 

typhimurium from the blue cheese dressing, but not from the 

dry mix used in dressing preparation, should have indicated 

to authorities the contamination of the prepared dressing that 

was then served at a salad bar.

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Medical Aspects of Biological Warfare 

The ongoing law enforcement investigation eventually 

revealed purposeful restaurant food contamination by the 

Rajneeshees more than a year after the outbreak occurred. 

Public health and law enforcement authorities lacked cooperative protocols in 1984; however, law enforcement teams 

in Oregon worked together with public health. 

An outbreak of this magnitude now would initiate a joint 

inquiry and investigation by public health and law enforcement, increasing chances that the outbreak cause would be 

identified in a timelier manner.

Lessons Learned: These events illustrate the need to 

have joint public health and law enforcement investigations 

and mutual cooperation. 

This outbreak shows t yohe importance of the mode of 

disease spread in discerning the source. 

Although not occurring in this case, when different geographic locations are affected, there could be a central supplier of a contaminated product shipped to all the locations. 

Since there was not a single supplier in this situation, this 

served as a red flag that multiple contaminations may have 

occurred.

Anthrax—Tokyo, Japan, 1995

Sarin is a chemical (nerve) agent that causes blocking of the postsynaptic enzyme that degrades acetylcholine, thus leading to excessive salivation, lacrimation, respiratory compromise, and seizures. Many may 

be familiar with it as a result of its use in the Syrian 

civil war in 2014. The notorious sarin attacks in a Tokyo 

suburb, Kameido, in 1994 and 1995, culminated with a 

sarin release in the Tokyo subway system.37,38 Less well 

known is that before its efforts with chemical weapons, 

the apocalyptic cult Aum Shinrikyo appears to have 

first invested efforts into producing biological agents 

and had attempted to use them.22

Shoko Asahara, a charismatic guru, built the Aum 

Shinrikyo cult into a membership of approximately 

10,000 individuals with financial assets exceeding 

$300 million. Aum Shinrikyo’s organization mimicked a government entity, with various ministries 

and departments, including a ministry of science and 

technology that included graduate-level researchers 

within modern laboratories interested in developing 

biological and chemical weapons. B anthracis cultures 

were also obtained and grown into a slurry for use as 

a biological weapon. This cult may have also investigated the use of C burnetii (the rickettsial organism 

that causes Q fever) and toxic mushrooms. In 1992 a 

team of 40 cult members, including Asahara, traveled 

to Zaire to attempt to acquire Ebola virus; the success 

of these efforts is unknown.

The Aum Shinrikyo experimented with the release 

of aerosolized biological agents. In June 1993 the 

cult sprayed B anthracis from the roof of one of its 

buildings in downtown Tokyo. In July 1993 the cult 

sprayed B anthracis from a moving truck onto the Diet 

(Japan’s parliament) and also around the Imperial 

Palace in Tokyo.

Information about the anthrax releases became public when, during the arraignment of Asahara on May 

23, 1996, for the Kameido sarin attack, cult members 

testified about their efforts to aerosolize a liquid suspension of B anthracis to cause an inhalational anthrax 

epidemic. Their goal was to have an epidemic trigger 

a world war that would permit Asahara to rule the 

world.39 In 1999 a retrospective case-detection survey 

was conducted to assess the possibility that some anthrax cases may have been unreported. Complaints of 

odors from neighborhood residents were associated 

with the anthrax releases. These complaints were retrospectively mapped to provide the geographic areas 

of the greatest anthrax exposure risk. Physicians at 

39 medical facilities serving this area were surveyed. 

None reported having seen cases of anthrax or relevant syndromes.39 It is not known whether a similar 

retrospective examination of anthrax-caused animal 

deaths was or could have been performed. Danzig and 

colleagues wrote a comprehensive report that analyzed 

the Aum Shinrikyo’s failures and successes in developing biological and chemical weapons.40

Case Review of 1995 Anthrax Releases

Biological Agents: B anthracis, gram-positive bacillus

Potential Epidemiological Clues: 11

Review: Technical errors in either the biological agent 

production or dissemination rendered the attacks harmless. 

In contrast, there were 12 deaths and about 1,000 hospitalizations from the sarin releases by the Aum Shinrikyo.37

Molecular analysis revealed that the B anthracis isolates 

were similar to the Sterne 34F2 strain, the strain of anthrax 

used in animal vaccines. Dispersal of this type of anthrax (regarded as nonpathogenic for immunocompetent individuals) 

had little possibility to cause harm to humans.39

Even if the strain was pathogenic, the concentration of 

spores in the liquid suspension is significantly less (104 bacteria/mL) than that considered optimal for a biological weapon 

(109–1010 bacteria/mL). The viscosity of the suspension was 

also problematic for successful aerosolization.39

The weather on the day of dispersal may have helped 

prevent infection: spore inactivation resulting from solar radiation could have further reduced the anthrax mix’s potency.39

Lessons Learned: These experiences show that it is 

difficult to both create a pathogenic biological weapon and 

deploy it successfully.

Both health and law enforcement officials should be aware 

of the possibility for use of more than one biological agent or 

a combination of agents. 

Environmental sample collection and proper storage are 

important for viability of pathogen cultures. 

The then-emerging discipline of forensic molecular 

biology proved the occurrence of an anthrax release by 

analysis of archived samples 8 years after the incident.41 The 

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Epidemiology of Biowarfare and Bioterrorism

contributions of advanced molecular techniques to the detection of BW and BT is examined in the section, Potential Impact 

of Advanced Molecular Techniques on the Epidemiology of 

Biowarfare and Bioterrorism, at the end of this chapter. 

Shigellosis—Dallas, Texas, 1996

From October 29 through November 1, 1996, 12 

clinical laboratory workers at the St Paul Medical 

Center in Dallas developed severe acute diarrheal 

illness.22 Shigella dysenteriae type 2 was cultured from 

the stool of eight of these cases. This strain of shigella 

is uncommon and, before this outbreak, had last been 

reported as the source of an outbreak in the United 

States in 1983. A 13th individual became ill after eating 

pastries brought home by one of the laboratory workers; this individual also had stool cultures positive for 

S dysenteriae type 2. Five patients were treated in and 

released from hospital emergency departments and 

four were hospitalized, but no deaths resulted.42

During the subsequent epidemiological investigation,43 laboratory employees who had worked during 

the first or third shifts, when the ill employees had 

worked, were interviewed. The employees stated 

that an unsigned email sent from a supervisor’s 

computer invited recipients to take pastries available in the laboratory break room. The supervisor 

was away from the office when the email was sent, 

and the break room could only be accessed using 

a numeric security code. The muffins and pastries 

had been commercially prepared, yet no other cases 

in the community occurred outside of the hospital 

laboratory. The ill persons reported eating a pastry 

between 7:15 am and 1:30 pm on October 29. Diarrhea onset for the ill laboratory workers occurred 

between 9:00 pm that day and 4:00 am on November 1. The mean incubation period until diarrhea 

onset was 25 hours and was preceded by nausea, 

abdominal discomfort, and bloating. All who ate a 

muffin or doughnut became ill (ie, 100% attack rate). 

No increased risk for illness was found from eating 

food from the break room refrigerator or drinking 

any beverage, eating in the hospital cafeteria, or attending social gatherings during the estimated time 

of exposure to the pathogen.

An examination of the hospital laboratory storage 

freezer revealed tampering of reference cultures of S 

dysenteriae type 2. The stored reference cultures had 

each contained 25 porous beads that were impregnated 

with microorganisms. The S dysenteriae type 2 vial 

contained at that time only 19 beads, and laboratory 

records indicated that the vial had not been used. S 

dysenteriae type 2 was isolated in virtually pure culture 

from the muffin specimen, and the same organism was 

isolated from the stools of eight laboratory worker 

patients. Pulsed-field gel electrophoresis revealed 

that the reference culture isolates were indistinguishable from those obtained from a contaminated muffin 

and the collected stool cultures, but differed from two 

nonoutbreak S dysenteriae type 2 isolates obtained from 

other Texas counties during that time.

Case Review of 1996 Shigellosis Food Poisonings

Biological Agents: S dysenteriae type 2, gram-negative 

bacillus

Potential Epidemiological Clues: 3, 4, 11

Review: There was a strong epidemiological link among 

the ill persons, the cultured muffin, and the laboratory’s stock 

culture of S dysenteriae type 2. 

The pathogen provided important clues because it was 

known to be uncommon and no research with this microorganism had been conducted at the hospital; therefore, 

laboratory technicians were not at risk of infection through 

laboratory error. In addition, no concurrent outbreaks of S 

dysenteriae type 2 were reported nationally at the time. 

Pastry contamination during commercial production was 

unlikely. Shigella contamination by a food service worker during food preparation would have had to occur subsequent to 

baking because Shigella bacteria would not have survived 

the heat. 

When the epidemiological report was published,42 it was 

hypothesized that someone had removed the laboratory 

culture of S dysenteriae type 2 from the freezer, cultured the 

microorganism and inoculated the pastries, and had access 

to the supervisor’s computer and the locked break room. 

On August 28, 1997, a laboratory technician who had access to the laboratory culture stocks and a history of purposeful use of biological agents against a boyfriend, was indicted 

on three charges of tampering with a food product, and 

accused of infecting 12 coworkers with S dysenteriae type 

2. She was subsequently sentenced to 20 years in prison. 

Lessons Learned: A match of clinical, food, and laboratory isolates helped to prove an epidemiological link among 

them. The knowledge that only postproduction tampering 

of the baked goods could have resulted in their successful 

contamination assisted with the investigation.

Anthrax—USA, 2001

On October 4, 2001, an inhalational anthrax case 

was reported in a 63-year-old man in Florida.44 Public 

health and government authorities initially misunderstood the nature of inhalational anthrax exposure and 

assumed that he had contracted the illness by outdoor 

hunting activities.45 Two other cases were subsequently 

identified in Florida, and a fourth case of anthrax—via 

cutaneous exposure—was identified in a female employee at NBC News in New York City.43 Investigators 

then realized that the exposures resulted from anthraxcontaining letters placed in the mail. On October 15, 

Senate Majority Leader Tom Daschle’s office received 

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Medical Aspects of Biological Warfare 

a letter that threatened an anthrax attack and also contained anthrax spores. The Hart Senate Office Building 

in Washington, DC, was subsequently closed.46 By the 

end of the year, anthrax-laden letters placed in the 

mail had caused 22 cases of anthrax-related illness (11 

inhalational [all confirmed], and 11 cutaneous anthrax 

[seven confirmed, four suspected]) and five deaths. 

Almost all anthrax cases were among postal workers 

and those who had handled mail.47,48 For two cases, 

it was difficult to determine exact exposure risk. A 

12th cutaneous anthrax case related to these mailings 

occurred in March 2002 in a Texas laboratory where 

anthrax samples had been processed.49,50

Case Review of 2001 Anthrax Mailings

Biological Agents: B anthracis, gram-positive bacillus

Potential Epidemiological Clues: 3, 5, 9, 11

Review: An unprecedented national response occurred 

involving thousands of investigators from federal, state, and 

local agencies. Close collaboration was required of all agencies, and the CDC and FBI formed partnerships to conduct 

public health and criminal investigations.9

 

Public health surveillance to detect previously unreported 

anthrax cases and determine that no new cases were taking place severely strained public health capacity.51,52 This 

outbreak highlighted the importance of containing not only 

the disease but also public panic.

The Laboratory Response Network, a multilevel network 

connecting local and state public health laboratories with 

national public health and military laboratories,53 served as 

a lead resource for both identifying and ruling out a potential biological attack.54 Molecular subtyping of B anthracis

strains played an important role in the differentiation and 

identification of B anthracis. High-resolution molecular 

subtyping determined that the anthrax mail-related isolates were indistinguishable and likely came from a single 

source.55

Postal workers and others handling mail were shown to 

be at risk from the anthrax-containing letters56 and contaminated postal machinery57; therefore, federal and state health 

officials instituted environmental sampling,58 cleaning,59

and protective measures as well as antibiotic prophylaxis.60

Similar protective actions were taken after discovery of the 

anthrax spore-laden envelope opened in the Senate Office 

Building.45 It was later determined that patients frequently 

did not complete the recommended prophylaxis duration.61

As a direct result of the anthrax mailings, on January 31, 

2002, the federal government made $1.1 billion available 

to the states for BT preparedness.62 Disease detection and 

notification efforts, a cornerstone of BT preparedness, have 

changed dramatically since the incident. Continuing efforts to 

strengthen the public health workforce should help to better 

detect, respond, and manage a future BT crisis.63 

Lessons Learned: An enhanced index of suspicion is 

necessary for unusual manifestations of BT diseases. Healthcare providers can learn to heighten their index of suspicion 

and diagnosis early if information is available and they are 

aware of a disease in a community. 

Fine particles of a biological agent can become airborne, 

thereby contaminating areas and placing persons at risk 

without direct exposure to the contaminated vehicle. An 

exposure can occur anywhere along the path of the contaminant, and increased medical surveillance and possibly 

prophylaxis should be instituted for anyone with potential 

pathogen exposure. 

Risk communication and key messages are important to 

contain potential public unrest.

Ricin—South Carolina and Washington, DC, 

2003–2004

After a terrorist plot to use ricin in England in January 2003,64 this plant-based toxin (a ribosome-inactivating protein) was found in a South Carolina postal 

facility in October 2003.65 Ricin was also discovered in 

the office of Senator Bill Frist at the Dirksen Senate Office Building in Washington, DC, on February 3, 2004.66

On October 15, 2003, an envelope containing a note 

threatening to poison water supplies with ricin and a 

sealed container were processed at a mail-processing 

plant and distribution facility in Greenville, South 

Carolina. Laboratory testing at the CDC on October 

21 confirmed the presence of ricin in the container. 

State health authorities interviewed all postal workers at the facility, and statewide surveillance for 

illness consistent with ricin exposure was initiated. 

The postal facility was closed on October 22, and 

epidemiological and environmental investigations 

were conducted. Hospital emergency departments, 

clinicians, health departments, and the postal facility 

were asked to report any cases consistent with ricin 

exposure. State poison control center and intensive 

care unit charts at seven hospitals near the postal 

facility were reviewed daily. A medical toxicologist 

and epidemiologists interviewed all 36 workers at 

the postal facility to determine whether any were ill, 

and no postal employees had illness indicating ricin 

exposure. CDC also conducted environmental testing at the postal facility; all tests were subsequently 

found negative for ricin.65 

In 2013 ricin poisoning again became a newsworthy event when ricin-laced letters were sent to President Barack Obama, New York City Mayor Michael 

Bloomberg, and a gun control lobbyist in Washington, 

DC. A Texas woman, Shannon Guess Richardson, was 

arrested and charged in this case, after her confession 

that she had mailed the letters, and left incriminating evidence that her husband had committed this 

biocrime.67 

Case Review of 2003–2004 Ricin Events 

Biological Agents: Ricin communis toxin 

Potential Epidemiological Clues: 3, 11

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Epidemiology of Biowarfare and Bioterrorism

Review: Ricin is a potent cytotoxin derived from the beans 

of the castor plant (R communis). Ricin will likely continue 

to be a threat agent because castor beans are grown and 

used commercially worldwide, and the toxin can be readily 

extracted. 

Ricin is considered to be a more rapidly acting toxin when 

it is ingested or inhaled than when injected. Treatment for 

ricin toxicity is supportive care because no antidote exists, 

and the toxin cannot be removed by dialysis.

Difficulties inherent in responding to a threat of ricin use 

include the lack of a detection method for locating ricin in 

clinical samples. A mild ricin poisoning may resemble gastroenteritis or respiratory illness. Ingestion of higher ricin 

doses leads to severe gastrointestinal symptoms followed 

by vascular collapse and death; inhalation of a small particle 

aerosol may produce severe respiratory symptoms followed 

by acute hypoxic respiratory failure.68

Lessons Learned: Any ricin threat should be investigated. 

As no cases resulted from the above exposures, it is likely 

that the material used in these incidents was not processed, 

purified, or dispersed in a manner that would cause human 

illness.

Biological agents that are readily available in nature 

remain a threat.

Accidental Release of Biological Agents

The following case studies document the events 

that transpired after what is understood to be the accidental release of BW agents, B anthracis16 and Variola 

major,

69 in the Soviet Union during the 1970s. The 

former Soviet Union had a massive state-sponsored 

biological weapons program, as documented by its 

former deputy director Ken Alibek in his book, Biohazard.

70 This account provides frightening emphasis on 

the dangers to innocent populations from purposeful 

biological weapon development.

Anthrax—Sverdlovsk, Soviet Union, 1979

In April and May 1979, the largest documented 

outbreak of human inhalational anthrax occurred 

in Sverdlovsk in the Soviet Union (now Ekaterinburg, Russia), with at least 77 cases of disease and 

66 deaths. Soviet authorities initially reported the 

occurrence of a gastrointestinal anthrax outbreak. 

Gastrointestinal anthrax is an uncharacteristic clinical manifestation from ingesting B anthracis spores, 

although it occasionally occurs in the republics of 

the former Soviet Union.16,71 When case history and 

autopsy results were reexamined by a joint team 

of Soviet and Western physicians and scientists, it 

became apparent that the Sverdlovsk outbreak and 

subsequent deaths had been caused by inhalational 

anthrax.16 The geographic distribution of human cases 

coupled with the location of animal cases indicated 

that all anthrax disease occurred within a very narrow geographic zone (4 km for the humans, 40 km 

for the animals) from a point of origin in Sverdlovsk. 

Historical meteorological data, when combined with 

this case distribution, demonstrated a point of origin 

at a military microbiological facility, Compound 19.16

These data also indicated that the most likely day on 

which this event occurred was April 2, 1979.16

Public health authorities established an emergency 

commission that directed public health response 

measures on April 10, 1979, which did not include the 

Soviet military. A triage response was established at 

Sverdlovsk city hospital by April 12. Separate areas 

were designated for screening suspected cases and for 

treating nonsystemic cutaneous anthrax cases and for 

intensive care and autopsy. Anthrax illness was not 

believed to be be transmitted from person-to-person. 

Those who had died were placed in coffins containing chlorinated lime and buried in a separate part 

of the city cemetery. Hospital and factory workers 

were recruited into teams that visited homes of both 

suspected and confirmed cases throughout the city 

to conduct medical interviews, dispense tetracycline 

as a prophylactic antibiotic, disinfect kitchens and 

patient sickrooms, and collect meat and environmental samples for microbiological testing. Local 

fire brigades washed trees and building exteriors 

in the section of the city where most cases were located. Some of the control measures that authorities 

enacted likely had little value. Stray dogs were shot, 

and some unpaved streets were paved. Newspaper 

articles were published, and posters were displayed 

that warned residents of the anthrax risk from eating 

uninspected meat or having contact with sick animals. 

Meat shipments entering the city were examined, and 

uninspected meat was embargoed and burned. In 

mid-April a voluntary anthrax vaccination program 

for healthy individuals aged 18 to 55 years was begun in the part of the city where most of the infected 

persons lived. Of the 59,000 people eligible to receive 

anthrax vaccine, about 80% received at least a single 

dose of the vaccine.16,72

Case Review of 1979 Sverdlovsk Anthrax Release 

Biological Agents: B anthracis, gram-positive bacillus 

Potential Epidemiological Clues: 1, 2, 3, 4, 7, 9, 10

Review: In the absence of confirmatory information of 

an aerosol anthrax release, the public health response was 

spectacular. Research has estimated that approximately 14% 

more deaths would have occurred in Sverdlovsk in the absence 

of the public health intervention that included distribution of 

antibiotics and vaccination.72

The Soviet military’s secrecy hid many facts that would 

have helped physicians to diagnose and treat inhalational 

anthrax exposure. It is possible that many more individuals 

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Medical Aspects of Biological Warfare 

than existing medical records indicate may have become ill 

and recovered, or died.73 Ambulance personnel often made 

an initial case diagnosis of pneumonia.74

Government authorities confiscated patient records and 

autopsy reports from the hospital. Some of these records 

could have provided invaluable inhalational anthrax medical 

intervention information from those patients that survived. 

Along with the absence of an epidemiological investigation 

at Sverdlovsk, this was a stunning loss of vital information 

for BW defense purposes.75

Former Soviet physicians released important information 

about anthrax prophylaxis and treatment, some of who took 

tissue samples and records home at their own considerable 

personal risk. This information indicated that the incubation 

period for inhalational anthrax may be as long as 2 months 

and that an antibiotic course of 5 days likely prolonged the 

incubation period for illness.75

Molecular analysis of tissue samples collected from 11 

victims, and retained by Sverdlovsk physicians, indicate that 

these cases had been exposed to a number of different B 

anthracis strains.76

Lessons Learned: Retrospective pathology findings from 

victims, weather patterns, and geographic mapping can help 

to determine the outbreak source and also whether it spread. 

Public health personnel in Sverdlovsk instituted effective 

preventive measures before they knew exactly what the 

exposure was or the cause of the illnesses, and they used information from cases to determine possible exposure routes. 

Once the disease agent was determined, prophylactic 

antibiotics and vaccination and protective environmental 

measures could be provided.

Studies of Natural Outbreaks for Potential 

Bioweapon Use

Although the following accounts are examples 

of naturally occurring outbreaks, some components 

raise suspicion that they were intentionally caused. 

Subsequent to the 1999 WNV outbreak in New York 

City, suggestions were made that Iraqi operatives 

could have covertly released a biological weapon. 

These allegations by Richard Preston in the New 

Yorker magazine were based on documentation 

showing that CDC had provided Iraq with various 

biological agents from 1984 through 1993, including 

Y pestis, dengue, and WNV,77,78 together with the fact 

that the Iraqi government was known to have had a 

covert biological weapons program.79 Although never 

shown to be anything other than an imported disease 

outbreak occurring in an opportunistic manner, this 

claim received a lot of political attention. Similar 

allegations of the covert use of a biological weapon 

could have been made with other outbreaks, including the 2000 Martha’s Vineyard (Massachusetts) 

tularemia outbreak, and they were made during the 

1999 through 2000 Kosovo tularemia outbreak, which 

occurred during wartime.

West Nile Virus, New York, New York, 1999

An outbreak of an unusual encephalitis was first recognized in New York City in late August 1999. On August 23 an infectious disease physician from a Queens 

hospital contacted the New York City Department of 

Hygiene and Mental Health to report two patients with 

encephalitis. The health department then conducted 

a citywide investigation that revealed a cluster of six 

patients with encephalitis in which five had profound 

muscle weakness and four required respiratory support. CDC’s initial clinical tests of these patients’ cerebrospinal fluid and serum samples indicated positive 

results for Saint Louis encephalitis on September 3. 

More cases of encephalitis in New York City ensued, 

and because eight of the earliest cases were residents 

of a 2-square-mile area in Queens, aerial and ground 

applications of mosquito pesticides began in northern 

Queens and South Bronx on September 3.80

Active encephalitis surveillance began in New York 

City on August 30 and in nearby Nassau and Westchester counties on September 3. A clinical case was defined 

as a presumptive diagnosis of viral encephalitis with 

or without muscle weakness or acute flaccid paralysis, 

Guillain-Barré syndrome, aseptic meningitis, or presence 

of the clinical syndrome as identified in earlier cases.80

Before and during this outbreak, an observed increase 

in bird deaths (especially crows) was noted in New 

York City.14 The USDA National Veterinary Services 

Laboratory in Ames, Iowa, analyzed tissue specimens 

taken from dead birds in the Bronx Zoo for common 

avian pathogens and equine encephalitis. When these 

test results were negative, the samples were forwarded 

to CDC, which revealed on September 23 that the virus 

was similar to WNV in genetic composition.81 At that time 

WNV had never been isolated in the western hemisphere.

Concurrently, brain tissue from three New York City 

encephalitis case deaths tested positive for WNV at the 

University of California at Irvine. As of September 28, 

17 confirmed and 20 probable cases had occurred in 

New York City and Nassau and Westchester counties, 

resulting in four deaths. Onset dates were from August 

5 through September 16. The median age of the patients 

was 71 years (range 15–87 years). By October 5 the 

number of laboratory-positive cases had increased to 50 

(27 confirmed and 23 probable). Emergency telephone 

hotlines were established in New York City on September 3, and 130,000 calls were received by September 

28. About 300,000 cans of N,N-diethylmetatoluamide 

(DEET)-based mosquito repellant were distributed 

citywide through local firehouses, and 750,000 public health leaflets were distributed with information 

on protection from mosquito bites. Radio, television, 

and the Internet provided public health messages.80

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A seroprevalence survey later determined that approximately 100 asymptomatic infections and 30 WNV 

fever cases occurred for each WNV encephalitis case 

previously identified in the New York City area.82

Case Review of 1999 West Nile Virus Cases 

Biological Agents: WNV, a flavivirus

Potential Epidemiological Clues: 1, 2, 3, 7

Review: Although some suggestions were made that this 

could have been a bioterrorist attack, the appearance of WNV 

in New York City in 1999 and its subsequent spread to the 

rest of the United States was most likely a natural occurrence.

Saint Louis encephalitis and WNV are antigenically related, and cross reactions can occur with some serologic 

testing.80 Limitations of serologic testing underscore the 

importance of isolation and identification of virus.80

Within its normal geographic area of distribution in Africa, 

West Asia, and the Middle East, birds do not normally show 

symptoms when infected with WNV.83 WNV from this part of 

the world occasionally causes epidemics in Europe that may 

be initiated by migrant birds.84,85 An epizootic that results in the 

deaths of large numbers of crows may be a clue that either 

a new population is susceptible to the virus or a new, more 

virulent strain of a virus has been introduced.80

WNV is transmitted primarily by Culex mosquitoes,86

which contributed to its spread in the United States after the 

1999 outbreak.87

Genetic testing revealed that the virus was 99% identical 

to a virus isolated in 1999 from a goose in Israel.88 Potential 

routes for WNV introduction include importation of WNVinfected birds, mosquitoes, or ill persons. The New York City 

area where WNV was prevalent includes two large international airports.89 

Before this outbreak, death was rarely associated with 

WNV infection.90 In patients with WNV encephalitis, computerassisted tomography often revealed preexisting lesions and 

chronic changes in brain tissue,91 perhaps suggestive of the 

potential for a greater susceptibility to deleterious outcome 

in elderly persons.

Lessons Learned: This outbreak emphasizes the important relationship among veterinarians, physicians, and public 

health authorities in disease surveillance, and the importance 

of considering uncommon pathogens.90 

The incident is an example of a typical zoonotic disease 

epidemic pattern—a natural epidemic occurred first among 

birds, followed by disease in humans. 

 The origin of outbreaks fitting some of the clues for a 

biological attack (a new disease for a geographic region) cannot be immediately determined without further investigation. 

Emerging diseases, whether new for a particular geographic 

area, like WNV, or a totally new disease (eg, severe acute 

respiratory syndrome or Middle East Respiratory Syndrome 

coronavirus), are not uncommon.

Tularemia, Martha’s Vineyard, Massachusetts, 2000

During the summer of 2000, an outbreak of primary 

pneumonic tularemia occurred on Martha’s Vineyard, Massachusetts.92 In July five cases of primary 

pneumonic tularemia were reported, with onset dates 

between May 30 and June 22. The Massachusetts Department of Public Health and CDC initiated active 

surveillance, and 15 confirmed tularemia cases were 

subsequently identified. A confirmed case was defined as occurring in a visitor or resident to Martha’s 

Vineyard who had symptoms suggesting primary 

pneumonic tularemia; was ill between May 15 and 

October 31, 2000; and had test results showing a serum 

titer of anti-F tularensis antibody of at least 1:128 on an 

agglutination assay. Of these cases, 11 had the pneumonic form of the disease, two had ulceroglandular 

disease, and two had fever and malaise. Fourteen of the 

patients were male, and the median age was 43 years 

(range 13–59). One 43-year-old man died of primary 

pneumonic tularemia.92

Control subjects for a case-control study were obtained by random-digit dialing to Martha’s Vineyard 

residents, enrolling 100 control subjects at least 18 

years old that had spent at least 15 days on the island 

between May 15 and their September interviews. 

Both ill persons and control subjects were questioned 

about occupation, landscaping activities, animal and 

arthropod exposures, recreational and outdoor activities, and general health history and status. Information 

was obtained about exposure to risk factors between 

May 15 and the interview, and for 2 weeks before illness for ill persons and 2 weeks before interview for 

control subjects.92

The suspected site of exposure for each patient was 

visited. Activities that may have led to exposure (eg, 

lawn mowing and “weed whacking”) were reproduced, and environmental and personal air samples 

were taken. Samples from soil, water, grass, wild 

mammals, and dogs were also taken. Epidemiological 

analysis revealed that in the 2 weeks before illness, 

using a lawn mower or brush cutter was significantly 

associated with illness. Of all the environmental and 

animal tissue samples taken, only two were positive for 

F tularensis: (1) a striped skunk and (2) a Norway rat.92

Case Review of 2000 Martha’s Vineyard Tularemia 

Outbreak

Biological Agents: F tularensis, a gram-negative bacillus

Potential Epidemiological Clues: 1, 2, 3, 9

Review: Caused by a gram-negative bacillus F tularensis, tularemia is a rare infection in the United States. Between 2001 and 2010, a median number of 126.5 cases per 

year (range: 90–154 cases per year) was reported.91 More 

than half of all cases reported during these 11 years came 

from Arkansas, Missouri, South Dakota, and Oklahoma, 

and most cases were acquired from tick bites or contact 

with infected rabbits. Higher incidences of the disease 

have been noted in persons ages 5 to 9 and older than 75, 

and incidence was greatest among Native Americans and 

Alaskan natives.93

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The only other previously reported pneumonic tularemia 

outbreak in the United States had occurred on Martha’s 

Vineyard during the summer of 1978.94 During a single week 

(July 30–August 6) seven persons stayed in a vacation cottage. By August 12, six of these had a fever, headache, and 

myalgia; and the seventh had a low-grade fever by August 

19. A search for additional cases on the island uncovered six 

other tularemia cases, five of which were pneumonic, and 

one was ulceroglandular. No source for the disease exposure 

was discovered, although two rabbits later found dead were 

culture-positive for F tularensis. 

Tularemia had been reported sporadically since rabbits 

had been introduced to Martha’s Vineyard in the 1930s,93 and 

pneumonic tularemia was first reported in Massachusetts in 

1947.95 Classic research on human tularemia rates showed 

that very high rabbit populations increase the tularemia 

hazard.96

Hospital clinicians on Martha’s Vineyard initially detected 

this outbreak and recognized tularemia caused pneumonic 

summer illness,97 in part based on the experiences with the 

previous outbreak.94

Feldman et al proposed in this outbreak F tularensis was 

shed in animal excreta, persisted in the environment, and 

infected persons after mechanical aerosolization and inhalation. This is a likely exposure scenario, given the principal 

form of primary pneumonic tularemia seen in these cases 

and strong epidemiological association with grass cutting.92

A seroprevalence survey conducted in 2001 in Martha’s 

Vineyard demonstrated that landscapers were more likely to 

have an antibody titer to F tularensis than nonlandscapers, 

revealing an occupational risk for tularemia.92

Lessons Learned: Naturally occurring disease can 

present in the pneumonic form. However, if tularemia were 

used as a biological weapon, an aerosolized release would 

probably result in multiple simultaneous cases presenting 

with the pneumonic form of the disease.97

There may also be disease transmission mechanisms 

(in this example, grass cutting) that are unknown or poorly 

understood.98

Tularemia, Kosovo, 1999–2000

After a decade of political crises and warfare, a 

large outbreak of tularemia occurred in Kosovo from 

1999 through 2000. Tularemia had not been reported 

in Kosovo since 1974.99 By April 2000, 250 suspected 

cases had been identified and spread nationwide, but 

most cases existed in the western area where ethnic 

Albanians resided.100

Unusual outbreaks of zoonoses or vectorborne 

disease may readily occur in war-torn or crisisafflicted regions that have previously been free of 

these diseases. Historically, outbreaks of typhus, 

plague, cholera, dysentery, typhoid fever, and smallpox have long been observed in war-torn regions.101

Among the earliest historic examples is the plague 

of Athens that arose during the second year of the 

Peloponnesian War, as described by Thucydides.102

Speculation may arise that these epidemics were 

purposefully caused. Many biological agents are 

zoonotic pathogens,99 including tularemia, a category A BW pathogen. Purposeful use of this pathogen 

merits consideration when such an outbreak occurs 

with a pathogen having the potential to be a biological weapon.103

Remarks made by the head epidemiologist at the 

Kosovo Institute of Public Health about unidentifiable 

ampoules and white powders discovered near various 

wells could not be verified and added to a perception 

of use of a biological weapon by Serbian forces.99 F tularensis biovar tularensis (type A) is highly pathogenic 

for humans. It is found mostly in North America and 

has been developed for use as a biological weapon. 

Disease progression often follows an acute and severe 

course, with prominent pneumonitis. F tularensis biovar holarctica (type B) is less pathogenic and is found 

throughout the northern hemisphere.104 To further 

complicate matters, a 1998 report documented that 

type A tularemia had been introduced into arthropod populations in the nearby Slovak Republic.105

The United Nations mission in Kosovo requested 

that the World Health Organization assist Kosovar 

health authorities in an epidemiological investigation of the tularemia outbreak. Teams of international 

and Kosovar public health personnel collaborated in 

epidemiological, environmental, and microbiological 

field and laboratory investigations.106

Tularemia cases were discovered by both prospective surveillance and retrospective hospital review of 

a pharyngitis and cervical lymphadenitis syndrome. 

Ill persons were clinically examined and interviewed, 

blood samples were taken from suspected cases, and 

antibiotics were prescribed as appropriate. Rural villagers reported an increase in mice and rats in the 

summer of 1999. A causal association was suspected 

between the increased population density of rodents 

and human tularemia cases. Tularemia is naturally 

transmitted to humans via small lesions in the skin of 

persons handling diseased rabbits, ingestion of contaminated water or food, bites of infectious arthropods, 

or inhalation of infective dusts.99

A matched case-control study was conducted with 

paired households in villages in regions with the 

greatest number of reported cases. Case households 

had one or more family members with a laboratoryconfirmed case of tularemia as of November 1, 1999. 

Control households were the two households closest 

to a suspected case household, having no individuals 

with the disease, and the person who prepared the 

family’s food was serologically negative for tularemia. 

Blood specimens were also drawn from all suspected 

cases. Questionnaires were completed on household 

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Epidemiology of Biowarfare and Bioterrorism

food consumption, water supply, presence of rodents, 

and condition of wells and food preparation and 

storage areas. The study period began a month before 

symptom onset of the first case in the suspected case 

household. Well water sampling and rodent collection 

and analysis were performed.

By June 30, 2000, more than 900 suspected tularemia cases had been discovered. From these, 327 were 

confirmed as serologically positive. The earliest onset 

of reported symptoms in the confirmed cases was 

October 1999, with an epidemic peak in January 2000. 

Confirmed cases were identified in 21 of 29 Kosovo 

municipalities. Cases were equally distributed by 

sex, and all age groups were equally affected. Case 

households were more likely to have nonrodent-proof 

water sources, and members in these households were 

less likely to have eaten fresh vegetables. Risk factors 

for case households included rodent feces in food 

preparation and storage areas and large numbers of 

field mice observed outside the house. Of the field 

samples collected, positive antigen for F tularensis

was detected in striped field mouse and black rat 

fecal specimens.

Case Review of 2000 Kosovo Tularemia Outbreak 

Biological Agents: F tularensis, a gram-negative bacillus 

Potential Epidemiological Clues: 1, 3, 5, 9

Review: Clinical and serologic evidence indicate that a 

tularemia outbreak occurred in Kosovo from October 1999 

through May 2000. The case-control study indicated that 

transmission of tularemia was foodborne based on the 

associations of illness and large numbers of rodents in 

the household environment, rodent contamination of food 

storage and preparation areas, and consumption of certain 

uncooked foods. Unprotected water that was not boiled likely 

contributed to the outbreak. 

Initial field investigations rapidly demonstrated that 

a widespread natural event was occurring and likely resulted from the unusual environmental conditions existing 

in war-torn Kosovo. The principal populations affected 

by the tularemia outbreak were ethnic Albanians in rural 

farming villages with limited economic resources. These 

people had fled during North Atlantic Treaty Organization bombing and Serbian reprisals during the spring 

of 1999. Refugees discovered bombed and ransacked 

homes, unprotected food storage areas, unharvested 

crops, damaged wells, and a rodent population explosion 

when they returned to their cottages. Both ignorance of 

infection and lack of hygienic measures contributed to a 

foodborne infection in the population.99

F tularensis can survive for prolonged periods in cold, 

moist conditions. 

A natural decrease in rodent population resulting from the 

cold winter, food shortages, and the disease itself likely all 

helped to end the zoonoses.99

Although tularemia was not recognized endemically 

or enzootically in Kosovo before the 1999 through 2000 

outbreak, it became well established in a host reservoir. A 

second outbreak occurred there in 2003, causing more than 

300 cases of oropharyngeal tularemia.107

Historically, war in Europe caused tularemia outbreaks. 

During World War II, an outbreak of more than 100,000 cases 

of tularemia occurred in the Soviet Union,108 and outbreaks 

with hundreds of cases following the war occurred in Austria 

and France.107

Lessons Learned: War provides a fertile ground for 

the reemergence of diseases and potential cover for BW 

agent use that is plausible and may go unrecognized as a 

BW event. An extensive epidemiological investigation must 

be conducted to conclude or disprove that a BW event has 

occurred.

Q Fever, Iraq 2005

Q fever is a zoonotic disease caused by C burnetii, a 

bacteria found worldwide. Human cases occur from 

inhalation of aerosols or windborne dust contaminated 

with C burnetii from birth products, milk, urine, and 

feces of infected animals—most frequently cattle, 

camel, goats, and sheep. Infections can also occur 

from ingesting raw milk or eggs as well as tick bites or 

human-to-human transmission.109 Due to the bacteria’s 

ability to survive in harsh environmental climates and 

its high infectivity, there is concern of its use as a biological weapon. The United States developed Q fever 

as a biological weapon before ratifying the Biological 

Weapons Convention. The CDC classifies C burnetii as 

a Category B agent.

From June 18 to July 10, 2005, 22 of 38 Marines 

(58%) from a single platoon in Al Asad, Iraq, experienced a febrile illness.110 All patients had a rapid 

onset of fever and chills, and the majority had headache, respiratory, and gastrointestinal symptoms. 

The patients were diagnosed with upper respiratory 

infection or atypical pneumonia because there was no 

diagnostic capability. Subsequent testing was negative for multiple respiratory pathogens. Follow-up 

serologic testing 6 weeks later on 9 of the affected 

patients revealed positive Q fever immunoglobulin 

for all 9, with 10 unaffected persons from the same 

unit negative for antibody.110

After confirmation of Q fever, the researchers 

distributed follow-up questionnaires to the company 

that included the affected platoon. They found an association between infection and exposure to ticks and a 

trend toward association with exposure to camels and 

the birth of both sheep and dogs. Although the authors 

did not have a sufficient sample size to confirm all risk 

factors, they hypothesize that this particular platoon 

may have sought shelter in an area that was heavily 

infected secondary to recent animal inhabitation and 

birthing or ticks.110

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Before this outbreak, Q fever cases had been reported in US service members deployed to Iraq. An 

evaluation of 62 cases of pneumonia in 2003 found 

eight had seroconverted with Q fever antibody,111

and an additional four diagnosed cases in 2003 and 

2004 were reported.112,113 Three cases of Q fever occurred in US forces in Iraq during the first Persian 

Gulf War (1990–1991).114 Since the 2005 outbreak in 

the Marines, more cases have been reported, and two 

serosurveys have been performed. One serosurvey 

revealed 10% of 909 military personnel hospitalized 

during deployment in 2003–2004 with symptoms 

compatible with Q fever seroconverted,115 and another 

serosurvey studying the same company affected in 

the outbreak in 2005 found seroconversion in 7.2% of 

279 tested.116 The British military has also published 

occurrences of Q fever in deployed forces, including 

26% of “Helmand Fever” cases caused by Q fever in 

Afghanistan.117

Surveillance of deployed military working dogs 

in Iraq revealed no seroconversions in 2007–2008, 

compared to a 5.5% seroconversion in feral dogs.118

This lack of infection is probably secondary to tick 

control and doxycycline prophylaxis for the military 

working dogs.

Case Review of 2005 Q fever cases

Biological Agent: C burnetii, gram-negative, facultative, 

intracellular coccobacillus

Potential Epidemiological Clues: 1, 4

Review: An attack rate of 58% occurred in one platoon. 

Although the research team was unable to determine exact 

movements of the platoon, it is likely they had an exposure 

different from the other platoons.

A relatively short epidemic curve, especially with a long 

and variable incubation period for the pathogen, suggests a 

point source. This outbreak probably resulted from an isolated 

exposure over a short time period.

It is a disease of relatively high severity, had an unknown 

cause at time of outbreak, and can raise concern about 

potential intentional cause.

Q fever is considered a potential bioweapon and a cause 

for concern.

Lessons Learned: All medical personnel should know 

what diseases are endemic in the area and previous history 

in deployed forces.

Cases should be reported immediately to allow dissemination of recommended diagnostics and treatment. In this case, 

the Armed Forces Infectious Disease Society published a set 

of practice guidelines for diagnosis and management of Q 

fever to assist deployed medical personnel.119

Investigate outbreaks of disease, even after resolution. 

Knowledge obtained will assist in preventing, recognizing, 

and rapidly treating future cases. 

EPIDEMIOLOGICAL ASSESSMENT TOOL

It is useful for public health authorities to determine whether an infectious disease outbreak is 

intentional. Grunow and Finke developed an epidemiological assessment tool to rule out biological agent 

use during infectious disease outbreaks.98 This assessment tool’s relevance was demonstrated by analysis 

of the 1999–2000 Kosovo tularemia outbreak.99 In their 

evaluation scheme, each assessment criterion can be 

given a varying number of points dependent on its 

presence and characteristics. There are two types of 

evaluation criteria: (1) nonconclusive and (2) conclusive. The most significant nonconclusive criteria 

include a biological threat or risk, special aspects of 

a biological agent, a high concentration of biological 

agent in the environment, and epidemic characteristics. Conclusive criteria include the unquestionable 

identification of the cause of illness as a BW agent 

(eg, demonstrating modifications that make the agent 

different from its naturally occurring equivalent, such 

as stabilizers or physical modifications) or proof of 

the release of such an agent as a biological weapon. 

With conclusive criteria, additional confirmatory 

information is unnecessary.99

According to Grunow and Finke’s nonconclusive 

criteria, a biological risk may be considered if a political 

or terrorist environment exists from which a biological 

attack could originate:

• Biorisk. Are BW agents available, with the 

means for distribution, and the will to use 

them? Or can an outbreak be explained by 

natural biological hazards, or the changes 

incurred by military conflict? 

• Biothreat. Does a biological threat exist by 

virtue of a group having a BW agent and 

credibly threatening to use it? 

• Special aspects. Is there plausible evidence of 

purposeful manipulation of a pathogen? 

• Geographic distribution. Is the disease’s 

geographic distribution likely given its locale? 

With the advent of a nonendemic pathogen, 

a thorough evaluation should include epidemiological, epizootic, ecological, microbiological, and forensic analysis. 

• Environmental concentration. Is there a high 

environmental concentration of the pathogen? 

• Epidemic intensity. Is the course of illness 

relative to disease intensity and spread in the 

population expected in naturally occurring 

illness?

• Transmission mode. Was the path of disease 

transmission considered naturally occurring? 

The appearance of a naturally occurring epidemic in itself does not rule out the purposeful 

use of a BW agent.

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Epidemiology of Biowarfare and Bioterrorism

• Time. Was the seasonal timing of the epidemic 

unusual? 

• Unusually rapid spread. Was the spread of 

the epidemic unusually rapid? 

• Population limitation. Was the epidemic 

limited to a specific (target) population? If 

certain persons were given prior warning of a 

BW attack, then they may protect themselves, 

as compared to naïve target populations. 

• Clinical. Were the clinical manifestations of 

the disease to be expected? 

The Grunow-Finke epidemiological assessment 

procedure (Table 2-1) was used to evaluate the case 

studies presented in this chapter. To use the assessment tool uniformly for all the events described in this 

chapter, some artificial constraints were placed on the 

analysis. For this exercise, only nonconclusive criteria 

were used because the use of conclusive criteria may 

have excluded many of the case studies with a retrospective assessment. During an outbreak investigation, 

however, epidemiological investigators would also 

initially use the nonconclusive evaluation criteria. With 

TABLE 2-1 

EPIDEMIOLOGICAL ASSESSMENT AND EVALUATION OF CASE STUDY OUTBREAKS

1915 

Assessment Maximum Anthrax 1971 1979 1984 1995 1996

(possible Weighting No. of Eastern Smallpox Anthrax Salmonella Anthrax Shigella

Nonconclusive Criteria points) Factor Points USA Aralsk Sverdlovsk Oregon Tokyo Texas

Biorisk 0–3 2 6 4 4 4 6 6 0

Biothreat 0–3 3 9 0 0 0 0 6 0

Special aspects 0–3 3 9 6 6 6 3 0 6

Geographic distribution 0–3 1 3 3 3 3 2 3 2

Environmental 

concentration 0–3 2 6 6 0 6 0 6 0

Epidemic intensity 0–3 1 3 3 3 3 3 0 3

Transmission mode 0–3 2 6 6 2 6 4 0 0

Time 0–3 1 3 3 3 3 1 0 1

Unusually rapid spread 0–3 1 3 3 1 3 3 0 3

Population limitation 0–3 1 3 1 0 1 0 0 3

Clinical 0–3 1 3 3 3 3 0 0 1

Score 54 38 25 38 22 21 19

 2000

1999 1999 Tularemia 2001 2003

WNV Tularemia Martha’s Anthrax Ricin 2005

Nonconclusive Criteria NYC Kosovo Vineyard USA USA Q Fever

Biorisk 6 2 0 6 6 2

Biothreat 6 3 0 6 9 6

Special aspects 0 0 0 9 0 0

Geographic distribution 3 3 3 3 3 0

Environmental 

concentration 4 4 4 6 6 0

Epidemic intensity 3 3 3 3 0 1

Transmission mode 2 2 6 6 0 0

Time 1 0 3 3 0 0

Unusually rapid spread 3 1 3 3 0 1

Population limitation 0 0 2 3 0 3

Clinical 1 1 3 3 0 0

Score 29 19 27 51 24 13

NYC: New York City 

USA: United States of America

WNV: West Nile Virus

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IMPROVING RECOGNITION AND SURVEILLANCE OF BIOTERRORISM

Existing disease surveillance systems may not 

be sensitive enough to detect a few cases of illness, 

unless they are legally reportable diseases that have 

confirmed laboratory diagnoses. However, even before 

confirmed diagnoses, disease reporting can be initiated upon patient presentation to healthcare providers with initial diagnoses, laboratory testing, and the 

reason provided by the patient for the hospital visit. 

Clinicians, laboratories, hospitals, ancillary healthcare 

professionals, veterinarians, medical examiners, morticians, and others may be partners in reporting diseases 

to public health authorities.

If a medical surveillance system first detects a biological attack, there may already be a significant number of cases, and the available time to prevent further 

illness is short or perhaps already over. The point of 

release is the earliest detection point of a biological 

event. Some disease exposures could be prevented 

through publicized avoidance of the area at risk, 

prophylactic medication use, or vaccination of those 

exposed, coupled with immediate disease recognition 

and patient treatment. The Department of Homeland 

Security’s BioWatch program has deployed biological 

detectors in major urban centers nationwide to detect 

trace amounts of airborne biological materials120,121 to 

help determine the presence and geographic extent of 

a biological release to focus emergency public health 

response and consequence management. Such detectors could be of great utility when pre-positioned 

at large well-publicized gatherings or in cities that 

may be the greatest targets for terrorist activity.

Although deployed sensors may detect an agent’s 

release, the infinite number of venues coupled with 

limited resources to position sensors and analyze air 

samples minimizes the chances that an agent release 

will be detected. In most instances, the earliest opportunity to detect an attack will be by recognizing 

ill patients. Depending on the agent, the mode of dissemination, and the number exposed, initial cases will 

present in different ways. If the disease is severe, such 

as is possible with category A biological agents, one 

properly diagnosed case will launch an investigation, 

as seen during the 2001 anthrax attacks.47

Even if the cause is initially unknown, extremely 

severe or rapidly fatal cases of illness in previously 

healthy individuals should be reported to public 

health authorities. If many people are exposed, as 

would be expected with a large aerosol release of a 

biological agent, an overwhelming number of people 

may eventually visit hospital emergency departments 

and outpatient clinics. Even with less severe disease, 

such cases should be recognized and quickly reported.

However, in the absence of confirmed laboratory diagnoses or high attack rates, infectious disease 

outbreaks are often not reported. If the disease is not 

rapidly fatal or cases are distributed among a variety 

of healthcare practitioners, it may not be readily apparent that a disease outbreak is under way. Therefore, 

there is a need for better awareness of the health of 

communities—a way to quickly detect shifts in potentially infectious diseases, whether of bioterrorist origin 

or not. This need has been recognized and has resulted 

in the proliferation of what are commonly known as 

syndromic surveillance systems.

Syndromic surveillance has been defined as the 

ongoing, systematic collection, analysis, and interpretation of data that precede diagnosis and can indicate 

a potential disease outbreak earlier than when public 

the exception of the 2001 anthrax and 2003 ricin events, 

none of the outbreaks described had been positively 

identified as having been caused by a biological agent 

until sometime after the events had occurred.

Grunow and Finke provide the following cut-off 

scores for nonconclusive criteria with respect to the 

likelihood of biological weapon use:

• unlikely (0%–33% confidence): 0 to 17 points;

• doubtful (18%–35% confidence): 18 to 35 

points;

• likely (67%–94% confidence): 36 to 50 points; and

• highly likely (95%–100% confidence): 51 to 54 

points.

Based on this scoring, only the 2001 anthrax mailings would be considered as highly likely to have been 

caused by a BW agent. The 1915 and 1979 anthrax 

events qualify as likely to have been caused by a BW 

agent. All of the other case study scenarios are either 

doubtful or unlikely to have been caused by a BW agent.

The authors conducted this evaluative exercise by 

consensus of opinion. Although subjective, the exercise 

underscores the challenges facing epidemiologists to 

determine whether a BT/BW event has occurred, unless direct evidence indicates a purposeful event, or 

someone credibly claims responsibility. The basic epidemiological principles described earlier in this chapter (including those needed for disease recognition) to 

determine the occurrence of an unnatural event, and 

for basic outbreak investigation, are the foundation of 

infectious disease response and control. Public health 

authorities must remain vigilant to quickly and appropriately respond to any infectious disease event.

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Epidemiology of Biowarfare and Bioterrorism

health authorities would usually be notified.122 The 

data used in syndromic surveillance systems are usually nonspecific potential signs and symptoms of an 

illness spectrum indicating that disease may be higher 

than expected in a community. These data can be from 

new or existing sources.123 For syndrome surveillance 

of BT, the emphasis is on timeliness, with automated 

analysis and visualization tools such as Web-based 

graphs and maps. These tools provide information 

that initiates a public health investigation as soon as 

possible.124

Numerous regional and national syndromic surveillance systems have been developed, including programs 

that rely on data collected specifically for the surveillance system and those that use existing medical data 

(eg, diagnostic codes, chief complaints, nurse advice 

calls, ambulance runs) and other information (eg, pharmacy sales, absenteeism, calls to poison control centers, 

Internet searches for specific symptoms or pathogens, 

participatory epidemiology where people voluntarily 

provide information to a system like Flu Near You125 or 

even scanning Twitter feeds and other social media sites 

for the use of terms related to illness) to detect changes 

in population health. Systems that use active data collection can be “drop-in” (those instituted for a specific 

high-threat time) such as those performed immediately 

after September 11, 2001,126–128 or during large gatherings for sports (eg, the Olympics) or other events,129 or 

they can be sustained systems for continuous surveillance.69,130 Systems that require new data entry benefit 

from greater specificity in the type of syndromes and 

illnesses reported, but they require extra work and are 

difficult to maintain. Systems that use existing data 

can be less specific, especially with information taken 

from behaviors early in the disease, such as over-thecounter pharmacy sales, absenteeism, Internet searches, 

and social media use. However, these programs have 

the large advantage of continuous data streams that 

are not dependent on provider input or influenced by 

news reports of disease rates. Such systems (examples 

of which are described below) have become standard 

in many health departments, the military, and CDC.

In the US Department of Defense, the Electronic 

Surveillance System for the Early Notification of 

Community-based Epidemics uses outpatient diagnostic International Classification of Diseases, Ninth Revision

codes, chief complaints, radiology and laboratory tests, 

and pharmacy prescriptions to track disease groups 

in military beneficiaries. Temporal and spatial data 

are presented through a Web-based interface, and 

statistical algorithms are run to detect any aberrations that could indicate a disease outbreak.131 This 

system is available for all permanent US military 

treatment facilities worldwide. Some local and state 

health departments use civilian versions of the Electronic Surveillance System for the Early Notification of 

Community-based Epidemics. Other civilian systems, 

such as the North Carolina Disease Event Tracking and 

Epidemiologic Collection Tool132 and various software 

packages made available by the Real-time Outbreak 

and Disease Surveillance Laboratory at the Department of Biomedical Informatics at the University of 

Pittsburgh,133 and the EpiCenter application134 also use 

syndromic information from emergency departments, 

911 calls, ambulance runs, and poison control center 

calls to monitor the health of populations. 

CDC has developed the BioSense 2.0 program using 

national data sources such as the US Department of 

Defense and Department of Veterans Affairs outpatient 

diagnostic codes, state and local emergency department visits, and laboratory test orders from commercial vendors to track disease patterns nationwide. 

The information is provided in a Web-based format to 

health departments.135 Algorithms are run on the data 

and send out an alert when levels of medical visits or 

laboratory test orders exceed those expected. The information is presented in temporal and spatial format, 

allowing the health department to track disease based 

on the patient’s home zip code. The BioSense 2.0 goal 

is to facilitate sharing of automated detection and visualization algorithms and promote national standards.

Despite the proliferation of systems, there are definite limitations in the ability to detect bioterrorist attacks using syndromic surveillance. Some have argued 

that even if syndromic surveillance could detect an 

outbreak faster than traditional methods, the advanced 

warning may not assist with disease mitigation.73 The 

warning may not be early enough or effective countermeasures may not be available. In addition, although 

nonspecific data such as absenteeism and social media 

may provide some early warning, it is very difficult to 

institute preventive measures without more specific 

information. However, nonspecific data can still serve 

as an early indicator, prompting authorities to monitor 

specific data sources more carefully. 

Most importantly, because a BT attack can present 

in a variety of ways depending on the agent, population, 

method of dispersal, and environment, it is impossible 

to predict how any individual surveillance system will 

perform. It is generally agreed that most syndromic 

surveillance systems will not detect a few cases of disease, but they can assist in detecting more widespread 

disease increases and assessing the population impact, 

an outbreak’s spread, and the success of mitigation 

efforts. The coverage area of the surveillance system is 

crucial in determining outbreak detection sensitivity 

in any part of a community. In the future, syndromic 

surveillance will probably be based on national models 

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Medical Aspects of Biological Warfare 

POTENTIAL IMPACT OF ADVANCED MOLECULAR TECHNIQUES ON THE

EPIDEMIOLOGY OF BIOWARFARE AND BIOTERRORISM

In addition to the use and application of syndromic 

surveillance for the detection of shifts in potentially 

infectious diseases, advances in technologies used for 

both disease diagnosis and surveillance are helping 

scientists and healthcare and public health professionals more quickly determine what is causing or 

has the potential to cause illness.136–139 These technological advances, which include multiplex polymerase 

chain reaction, immunoassays, arrays, and even 

next-generation sequencing, allow a more accurate 

determination of not only the pathogen,138,139–142 but 

also the presence of mutations or other factors that 

distinguish the organism(s) from previous outbreaks 

or near neighbors143 and have the potential to result in 

more severe disease. These techniques have identified 

several emerging infectious diseases.144–146 

Many of the technologies listed have been available for 30 years or more147,148; however, the increased 

speed and multiplex capability, lower cost, and 

greater application of the technologies as surveillance tools, combined with enhanced surveillance 

reporting systems, create a more likely environment 

for the detection of a possible natural or intentional 

biological event.149–150 Specifically, the more routine 

use of sequencing has significantly affected biological 

sciences and has the potential to be influential in the 

arena of the epidemiology of biowarfare. Ten years 

ago the cost and sample-to-result time of sequencing 

were prohibitive for routine use. However, the cost 

and processing time continues to decrease, making 

the accessibility to sequencing more universal and 

easily adaptable for inclusion in pathogen identification and characterization.151 In 1990 the National 

Institutes of Health and Department of Energy initiated the human genome project, which required 10 

years to publish a working draft and cost millions 

of dollars.152,153 A viral or bacterial genome can be 

sequenced in a few hours and can cost as little as 

$100 per isolate.151,153–157 The use of sequence technology has been instrumental in not only pathogen 

detection and characterization, including mutations 

that increase morbidity and mortality, but also in 

the development of detection and diagnostic assays 

and therapeutic and prophylactic solutions and/or 

countermeasures.155

Most recently, sequencing was used in the Middle 

East Respiratory Syndrome coronavirus outbreak 

to identify the source of the disease, determine the 

distinction from severe acute respiratory syndrome 

corona virus,143 and develop polymerase chain reaction detection and diagnostic capabilities.23 Sequencing was also used in the H7N9 and H1N1 influenza 

outbreaks,158,159 and in the Escherichia coli O104:H4 

in Germany in 2011160–162 to assist with identifying 

the causative agent and developing possible countermeasures. Although it appears as though these 

events have all been naturally occurring, the addition of characterization information in the form of 

sequence has allowed researchers to go back and look 

for possible index cases and the source or reservoir 

for the outbreak in humans. Rapid sequencing may 

also facilitate a more rapid vaccine development, 

as demonstrated in the use of novel techniques for 

influenza vaccine production.163 The use of sequencing will continue to assist scientists and public health 

professionals in their search for not only the reservoir, 

point of exposure, possible nefarious intention, and 

comparison with currently known and well characterized diseases, but will also assist in limiting the 

spread of the disease and possible prevention of 

future outbreaks by identifying potential zoonotic 

crossover before it even occurs.164–167

Many organizations are conducting surveillance 

globally with the goal of predicting and preventing 

the next outbreak or pandemic, often in zoonotic 

sources.164,165 The US Agency for International Development,168 the US Department of Defense, and both forprofit and nonprofit, nongovernmental organizations 

are all engaged in surveillance efforts using some of 

these technological advancements to identify the next 

potential source of an outbreak and develop detection 

and prophylactic or therapeutic solutions and other 

nonmedical countermeasures to prevent such an event, 

or at the very least, to be well prepared to respond 

robustly and quickly. 

However, not all uses of advanced technologies 

have been without controversy. One recent example 

of the use of sequencing in the creation of a potential 

BW agent came in late 2011 and continues today.169–171

Flu researchers Ron Fouchier, of the Erasmus Medical 

such as BioSense 2.0 and use readily available electronic 

databases. Local health departments could then build 

on a national system using local data that can improve 

population coverage. Future disease monitoring and 

reporting systems need to be seamlessly integrated 

with other traditional disease surveillance systems. 

Ideally, these systems should also help to educate 

clinicians on the importance of maintaining a high 

index of suspicion and to promptly report unusual 

diseases or disease clusters to public health authorities.

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Center in The Netherlands, and Yoshihiro Kawaoka, 

of the University of Wisconsin-Madison, engineered 

more transmissible strains of H5N1, and, more recently, have focused on H7N9.172,173 They believe genetic engineering can be used to determine which—if 

any—mutations accelerate the spread of influenza 

between mammals.173–175 Additionally, scientists 

claim genetically modifying the H7N9 virus in the 

lab will help drive efforts to develop pandemic drugs 

and vaccines, and result in better preparedness and 

response.175 However, not all scientists agree with the 

type of research being conducted, including infectious 

disease specialist Adel A F Mahmoud, of Princeton 

University.171 Some scientists worry that these strains 

could escape the laboratory and possibly kill millions, 

or get in the hands of the wrong people.173 Even the 

US National Science Advisory Board for Biosecurity 

became involved in this debate and has issued several 

rulings and restrictions on publication of information 

from this type of research. Dual use research considerations are also being carefully evaluated in some of 

these instances to ensure that the global populace is 

protected from potential harm. 

There are other limitations with this type of information gathering and sharing. As seen during 

the E coli outbreak in Germany, when an initial error is made in the suspected source of the outbreak 

(in this case erroneously stated to be from Spanish 

cucumbers)176 the information can seriously and 

detrimentally affect a nation, manufacturing or processing group, or product identified as the source.177

Although the initial source of the outbreak was 

suspected based on epidemiological investigation 

and early molecular testing, the desire to release the 

information superseded molecular validation of the 

suspected outbreak source information178; it was not 

until the results obtained using advanced molecular 

techniques160,179,180 combined with further epidemiological investigation identified the more likely outbreak source.175,181 Additionally, some nations may 

not approve the release of information regarding an 

outbreak or may not allow other scientists to continue 

surveillance or investigations into the source if they 

feel their economy or other factors such as national 

security may be threatened. The existence, or lack 

thereof, of surveillance efforts, systems, and software 

solutions may also hinder the transfer of information 

regarding a potential outbreak or emerging infectious 

disease.150 

The use of high throughput screening and sequencing technologies can also be instrumental in detection 

of anomalies indicative of not only natural mutation 

and resistance, but also engineered and intentional 

activities.180,181 The addition of virulence factors such 

as plasmids that are not typical to given organisms, 

but convey greater morbidity, communicability, and 

so forth can be a potential sign of human manipulation. 

Phylogenetic comparison with known pathogens can 

not only narrow prevention and treatment options, but 

also can highlight a possible unnatural combination 

of strains. Sequence information can even be used to 

generate a pathogen of interest de-novo, without the 

pathogenic element, allowing for possible manipulation of once pathogenic organisms in a lower class 

safety environment and additional options for assay 

and countermeasure development.182–184 However, 

this capability also allows for generation of dangerous 

pathogens with the proper authorization.185 Although 

the knowledge obtained from sequencing can be very 

beneficial, it has the potential to cause harm if it falls 

into the wrong hands or is not accurate and does not get 

reported to the appropriate public health professionals.

However, as evidenced in the last few years when 

several anthrax and plague cases were detected in patients in the United States, advanced technologies can 

rapidly assist an epidemiologic investigation. Public 

health and laboratory officials moved quickly to investigate and determine the source of these infections; 

and using a combination of molecular techniques and 

epidemiological outbreak investigation, they found 

none were suspected to be intentionally caused.186–193

The addition of advanced molecular techniques can 

lead to faster diagnosis, treatment, and determination 

of intent or origin of infection(s).

SUMMARY

Because management of BT and BW events depends 

on the disease surveillance, laboratory, and outbreak 

investigation capabilities of public health authorities, 

the science of epidemiology will always be the foundation for a response to these events. An enhanced 

index of suspicion, awareness of potential red flags, 

open lines of communication between local healthcare 

providers and law enforcement authorities, knowledge 

of historical outbreak investigation information, robust 

disease surveillance systems, and the use of advanced 

molecular techniques will improve the ability to respond to any future BT or BW event.

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Food, Waterborne, and Agricultural Diseases

Chapter 3

FOOD, WATERBORNE, AND 

AGRICULTURAL DISEASES

ZYGMUNT F. DEMBEK, PhD, MS, MPH, LHD,* and EDWIN L. ANDERSON, MD†

INTRODUCTION

FOODBORNE AND WATERBORNE PATHOGENS AND DISEASES 

Bacillus anthracis

Clostridium botulinum

Campylobacter jejuni

Salmonella

Listeria monocytogenes 

Escherichia coli

Shigella

Cryptosporidium

Hepatitis A

Mycotoxins

Parasites

Threat Potential Summary

WATER SUPPLY CONCERNS 

AGRICULTURAL TERRORISM 

SMUGGLING AND INVASIVE SPECIES

FOOD AND WATER SECURITY

SUMMARY

*Colonel (Retired), Medical Service Corps, US Army Reserve; Associate Professor, Department of Military and Emergency Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814; formerly, Chief, Biodefense Epidemiology and Education 

& Training Programs, Division of Medicine, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland. 

†

Colonel (Retired), Medical Corps, US Army; Research Professor, Department of Internal Medicine, Division of Infectious Diseases, Allergy and Immunology, Saint Louis University School of Medicine, 1100 South Grand Boulevard, St Louis, Missouri 63104

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INTRODUCTION

Food and waterborne pathogens cause a considerable amount of disease in the United States. The economic impact from foodborne diseases is estimated at 

about $78 billion per year.1 The top five pathogens that 

contribute to domestic foodborne illness are, Novovirus, Salmonella species, Clostridium perfringens, Campylobacter species, and Staphylococcus aureus.

2,3 Many of 

the common foodborne pathogens, whether bacteria, 

viruses, parasites, or toxins, can cause disease if purposefully introduced into water or food sources. These 

pathogens characteristically have the potential to cause 

significant morbidity or mortality, have low infective 

dose and high virulence, are universally available, 

and can be stable in food products or potable water. 

These agents include Clostridium botulinum toxin, the 

hepatitis A virus, Salmonella, Shigella, enterohemorrhagic Escherichia coli species, Cryptosporidium parvum, 

Campylobacter jejuni, Listeria monocytogenes, and Vibrio 

cholerae, among others. Pathogens in the Centers for 

Disease Control and Prevention (CDC) list of biological 

threat agents that also may cause food or waterborne 

disease are Bacillus anthracis, Brucella species, staphylococcal enterotoxin B, and ricin.4

 The potential for 

nonlisted biological agents such as mycotoxins and 

parasites (eg, Taenia species) to be used in a bioterrorist 

event should also be considered.

This chapter provides an introduction to the farreaching subjects of food and waterborne diseases, 

the potential for terrorist attacks on the food and 

water supply, and terrorism directed at sources of 

the nation’s farm-to-food continuum (agricultural terrorism). For a more extensive review of these topics, 

readers may consult more specialized texts on food5

and waterborne6 diseases and agricultural terrorism.7,8

FOODBORNE AND WATERBORNE PATHOGENS AND DISEASES

Bacillus anthracis

B anthracis is the causative agent of two forms of 

foodborne anthrax: oropharyngeal and gastrointestinal. Although spores of B anthracis would cause the 

most potential harm via an aerosol release, anthrax 

disease is not normally perceived as having bioterrorism potential as a foodborne bacterial contaminant 

because the infective dose required for such an attack 

would be high.9 However, given that the early diagnosis of gastrointestinal anthrax is difficult for clinicians 

who have never treated cases of this disease, a higher 

mortality rate than expected may result from a natural 

or purposeful outbreak. Anthrax spores are resistant to 

disinfection by contact chlorination as used by water 

treatment facilities, although higher levels of chlorination (≥ 100 ppm) for longer contact times (5 minutes) 

will kill Bacillus spores.10

Clostridium botulinum

C botulinum is the causative agent of botulism intoxication, of which there are three natural manifestations: classic, wound, and infant botulism. Bioterrorism 

use of botulinum toxin could possibly occur through 

inhalational intoxication, as was considered by the 

Aum Shinrikyo cult in Japan in 1990.11,12 C botulinum 

produces the most potent natural toxin known; the 

human lethal dose of type A toxin is approximately 

1.0 μg/kg.13 There are seven antigenic types of botulinum toxin, denoted by the letters A through G. 

Most human disease is caused by types A, B, and E. 

Botulinum toxins A and B are often associated with 

home food preparation14 and home canning15 and 

pickling.16 Botulism-contaminated food cannot be 

distinguished by visual examination, and the cook is 

often the first to show the toxin’s effects (via sampling 

the food during cooking). A 12- to 36-hour incubation 

period is common. The incubation period is followed 

by blurred vision, speech and swallowing difficulties, 

and descending flaccid paralysis.17

The current mortality rate associated with botulism 

intoxication is less than 10%. Foodborne botulism mortality during the 1950s (before the advent of modern 

clinical therapies) was approximately 25%.18 Little 

evidence of acquired immunity from botulinum intoxication exists, even after a severe infection. Successful 

treatment consists of aggressive trivalent (A, B, E) 

botulinum antitoxin therapy and ventilatory support. 

Early diagnosis is critical for patient survival. Toxin 

can be found in food, stool, and serum samples, which 

may all be used in the standard mouse model assay to 

test for the presence of botulism toxin.19

A controversial paper published in 200520 explored 

the potential for botulinum toxin contamination of the 

milk supply. A nine-stage dairy cows-to-consumer 

supply chain was examined, which accurately modeled a single milk-processing facility. The release of 

botulinum toxin was assumed to have occurred either 

at a holding tank at the dairy farm, in a tanker truck 

transporting milk from the farm to the processing 

plant, or at a raw milk silo at the plant. By the use of this 

model, it was predicted that 100,000 individuals could 

be poisoned with over 1 gram of toxin, and 10 grams 

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would affect about 568,000 milk consumers.20 The 

National Academy of Sciences published this information to foster further discussion and alert authorities to 

dangers to the milk supply from purposeful contamination.21 The paper also describes interventions that 

the government and the dairy industry could take to 

prevent this scenario. Officials at the US Department 

of Health and Human Services requested that the 

paper not be published, but the National Academy of 

Sciences published it anyway, convinced that the information would not enable bioterrorists to conduct an 

attack, and that the paper would stimulate biodefense 

efforts. Whether the paper’s information presents a 

”roadmap for terrorists” by exposing vulnerabilities 

in food processing remains to be determined22; however, the hypothetical use of botulinum toxin placed 

at various points into the food supply was proposed 

in a fictional novel over 35 years ago.23

Campylobacter jejuni

Campylobacter, Salmonella, Listeria, and E coli 

O157:H7 can be transmitted zoonotically from contaminated animal food sources. These bacteria species 

are ubiquitous and cannot be completely eliminated 

from the food supply. C jejuni is the most commonly 

reported bacterial cause of foodborne infection in the 

United States.24 Chronic sequelae associated with C 

jejuni infections include Guillain-Barre syndrome25 and 

arthritis.26 Infants have the highest age-specific isolation rate for this pathogen in the United States, which 

is attributed to a greater susceptibility upon initial 

exposure and a lower threshold of seeking medical 

treatment for infants.24 Reservoirs for C jejuni include 

wild fowl and rodents.27 The intestines of poultry are 

easily colonized with C jejuni,

28 and it is a commensal 

inhabitant of the intestinal tract of cattle.29 Antibiotic 

resistance of Campylobacter is a growing concern for 

poultry farmers.30 The infective dose for Campylobacter

is 100 to 1,000 cells, with poultry the primary source of 

infection in the United States.31 Insect transmission by 

several fly species has also been documented.32 There 

is a 3- to 5-day illness onset for campylobacteriosis and 

a 1-week recovery time. Immunity is conferred upon 

recovery, which accounts for a significantly higher 

incidence rate among individuals younger than 2 years 

of age in developing countries.33

Salmonella

Salmonellosis is the second most common foodborne illness,34 and contaminated food is the principal 

route of disease transmission.35 There are over 2,400 

Salmonella serotypes, many of which can cause gastroenteritis, manifested as diarrhea, abdominal pain, 

vomiting, fever, chills, headache, and dehydration.34,35

Other diseases from Salmonella infections include enteric fever, septicemia, and localized infections. Poultry is 

a principal reservoir of the salmonellae.35 Water, shellfish, raw salads, and milk are also commonly implicated as vehicles for this pathogen. In humans, the most 

highly pathogenic Salmonella species is S typhi.

35 This 

bacterium is the causative agent of typhoid fever, which 

comprises about 2.5% of salmonellosis in the United 

States.35 The symptoms of typhoid include septicemia, 

high fever, headache, and gastrointestinal illness.35

During World War II, the Japanese developed 

biological weapons, poisoning prisoners with S typhimurium and many other bacteria and viruses during 

their experimentation, and contaminating wells with S 

typhimurium along the Russian border of Mongolia.36 In 

September and October 1984, two large groups of salmonellosis cases occurred in The Dalles, Oregon. Case 

interviews by health officials associated patronage of 

two restaurants in The Dalles with illness, especially 

with food items eaten from salad bars. S typhimurium 

isolates were then obtained from clinical specimens.37

The size and nature of this outbreak helped to initiate 

a criminal investigation, which was rarely done in 

conjunction with a foodborne disease outbreak. The 

cause of the epidemic became known when the Federal Bureau of Investigation investigated a nearby cult 

(the Rajneeshees) for additional criminal violations.38

In October 1985 authorities found an opened vial that 

contained the original culture type of S typhimurium

inside a refrigerator within the Rajneeshee clinic 

laboratory.

A large multistate outbreak of milk-borne salmonellosis from Salmonella enteritica serovar typhimurium 

occurred in northern Illinois in 1985, with more than 

14,000 people reported ill and five deaths.39,40 The 

cause of the outbreak was the accidental comingling 

of raw milk into the pasteurized product in a milk 

plant.41 The contaminated milk was distributed via 

supermarket distribution systems, and cases were also 

reported in the neighboring states of Indiana, Iowa, 

and Michigan.42 Medical treatment was complicated 

because the strain of S typhimurium involved was 

found to be resistant to antibiotics. Such inadvertent 

milk-borne contamination reinforces the potential for a 

ready-made vehicle for transmission of disease among 

a population by deliberate means.20

Listeriamonocytogenes

L monocytogenes is ubiquitous in the environment 

and often found in silage, water, and the environs of 

animal fodder.43 Soft cheeses,44 raw or contaminated 

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milk,45 and contaminated refrigerated foods46 are often sources of this organism. Listeriosis can result in 

meningo-encephalitis and septicemia in neonates and 

adults, and fever and abortion in pregnant women.47

Fetuses, newborns,48 the elderly,49 and immunocompromised persons50 are at greatest risk for serious illness. Listeriosis case investigations can be problematic 

because of the variable incubation period for illness (3 

to >90 days). Large outbreaks of foodborne listeriosis 

have occurred, including a 1983 Massachusetts epidemic where improperly pasteurized milk was the 

source of the infection.51 Of the 49 infections associated 

with this outbreak, 14 patients died.51

Escherichia coli

E coli O157:H7 infections often originate from contamination due to a bovine reservoir.52 This organism 

produces two verotoxins and is a significant cause 

of serious pediatric illness.53 It can result in bloody 

diarrhea and hemolytic uremic syndrome, which is 

defined as the demonstration of three clinical conditions: (1) microangiopathic hemolytic anemia, (2) acute 

renal failure, and (3) thrombocytopenia.53 Children 

younger than 5 are at greatest risk for hemolytic uremic syndrome when infected with E coli O157:H7 or 

other enterohemorrhagic E coli (EHEC) species, and 

deaths from these infections occur most often in the 

age ranges of 1 to 4 years and 61 to 91 years.52 A major 

source of EHEC exposure is from consumption of and 

contact with beef cattle.54 About 20% of the ground beef 

consumed in the United States is derived from culled 

dairy cattle, which may be an important contributor 

to this bacterial contamination of the food supply.55

For example, during July 2002, the Colorado Department of Public Health and Environment identified 

an outbreak of E coli O157:H7 infections that linked 

28 illnesses in Colorado and six other states to the 

consumption of contaminated ground beef products. 

Seven patients were hospitalized, and five developed 

hemolytic uremic syndrome.56

E coli-contaminated food items commonly result 

from use of cattle waste as fertilizer or other contact 

with cattle products. Outbreaks have occurred from 

exposure to various E coli-tainted food items, including alfalfa57 and radish58 sprouts, parsley,59 lettuce,60,61

hazlenuts,62 apple cider,63 unpasteurized gouda 

cheese,64,65 raw milk,66 recontaminated pasteurized 

milk,67 prepackaged cookie dough,68 and salami,69

as well as through petting zoos70 and environmental transmission.71,72 Waterborne outbreaks of E coli 

O157:H7 have also occurred. From mid-December 1989 

to mid-January 1990, 243 cases of gastrointestinal illness from antibiotic-resistant E coli O157:H7 occurred 

in a rural Missouri township as a result of an unchlorinated water supply.73 Swimming-associated outbreaks 

of E coli O157:H7 have also occurred.74,75

Other enterohemorrhagic E coli strains containing 

Shiga toxins (Shiga toxin-producing E coli [STEC] 

infections) have appeared as public health concerns, 

including STEC O121,76 STEC O26,77 STEC O145,78 

and STEC O104:H4.79 Novel STEC strains can easily 

develop due to opportunistic microbial growth and 

spread in the food supply chain and distribution systems. The CDC E coli investigation page80 lists current 

and past identified outbreaks. A recent 10-year study 

in Connecticut of 663 reported STEC infections demonstrated that both O157 and non-O157 STEC infection 

incidence decreased from 2000 through 2009, and also 

that O157 was the most common and clinically severe 

type of STEC infection.81 However, in this and other 

studies, non-E coli O157 accounted for a minority of all 

clinically significant STEC infections.82,83 Importantly, 

STEC O104 and O157:H7 infections are more likely 

to lead to hospitalization and death than other STEC 

serogroups, as shown by a recent 8-year retrospective 

cohort study of 8,400 patients in Germany.84 Another 

recent German study demonstrated that cattle density 

is a risk for exposure to E coli O157:H7 and other STEC 

strains, including all major disease-causing groups 

(O26, O103, O111, O128, O145), but not O91.85 STEC 

strains therefore appear to be a diverse group of organisms that demonstrate differences as well as many 

commonalities in exposure and epidemiology.

Shigella

Humans are the major reservoir for Shigella and 

the primary source of subsequent infections.86 It is 

thought that worldwide Shigella-associated illness 

causes about 165 million cases per year, of which 

fewer than 1% occur in industrialized nations.86 Shigella 

dysenteriae produces severe disease, may be associated 

with life-threatening complications, and causes about 

25,000 cases of illness each year in the United States.86

Although not an environmentally hardy organism, 

Shigella is highly infectious and can be very persistent 

in a close community environment.87 Four serogroups 

(A through D) cause approximately 80% of shigellosis 

cases in the United States. Immunity is serotype-specific.88 Vaccine development has been problematic,89 and 

the species can easily become resistant to antibiotics.90

Infants and young children are most susceptible to 

shigellosis, attributable in part to toiletry behaviors 

and child care practices. The infectious dose for Shigella 

is 10 to 100 organisms, and Shigella contamination can 

cause outbreaks associated with food, water, and milk. 

Shigellosis has also been associated with recreational 

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Food, Waterborne, and Agricultural Diseases

swimming.91 Shigellosis is readily transferred from 

person-to-person contact and through fomites92; it can 

also be transmitted by insect vectors (primarily flies).93

There is a 1- to 3-day incubation period for shigellosis. Shigella organisms are shed for 3 to 5 weeks after 

symptoms cease, ultimately contributing to a greater 

person-to-person spread than with other enteric pathogens such as Salmonella and V cholerae.

Cryptosporidium

Cryptosporidium, a protozoan and an obligate intracellular parasite, can cause food and waterborne illness and 

can also be acquired from exposure to contaminated 

recreational water.94–98 Seroprevalence surveys indicate 

that about 20% of the US population have been infected 

with Cryptosporidium by adulthood.99 The severity and 

course of infection can vary considerably, dependent 

upon the immune status of the individual. Intestinal 

cryptosporidiosis is often characterized by severe watery 

diarrhea but may also be asymptomatic. Pulmonary 

and tracheal cryptosporidiosis in humans is associated 

with coughing and low-grade fever; these symptoms 

are often accompanied by severe intestinal distress. The 

duration of illness in one study of 50 healthy individuals 

varied from 2 to 26 days, with a mean of 12 days.100

The precise infectious dose is unknown; research 

indicates that a range of 9 to 1,024 oocysts will initiate 

infection.101 The pathobiology is not completely known 

either; however, the intracellular stages of the parasite 

can cause severe tissue alteration. Infected food handlers are a major contributor to disease transmission. 

Consequently, cryptosporidiosis incidence is higher in 

facilities that serve uncooked foods, such as restaurants 

with salad bars. Child care centers can be a problematic 

source of cryptosporidium infection because diarrhea 

in children in diapers can be difficult to contain.102

A significant reservoir worldwide for Cryptosporidium 

parvum is domestic livestock, predominately cattle.103

Drinking water outbreaks have affected as many as 

403,000 individuals (in a 1993 outbreak in Milwaukee).94 In the Milwaukee incident, the water was both 

filtered and chlorinated.104 The organism’s resistance to 

chlorine treatment ensures that it will remain a concern 

in treated potable water,105 and therefore a risk to immunocompromised individuals, in whom it causes severe 

and chronic life-threatening gastroenteritis.106

Hepatitis A

Humans are the source of the Hepatovirus hepatitis 

A virus.35 Illness caused by hepatitis A is characterized 

by sudden onset of fever, malaise, nausea, anorexia, 

and abdominal discomfort, followed by jaundice.35 The 

infectious dose is not precisely known but is thought 

to be 10 to 100 virus particles.35 The virus is hardy, 

and it survives on hands and fomites. Because viral 

particles are excreted in the feces during clinical illness, 

stringent personal hygiene is crucial to prevent disease 

transmission. Hepatitis A is commonly transmitted via 

personal contact, and fewer than 5% of all hepatitis A 

cases are demonstrated to have been caused by food 

or waterborne transmission.107 Permanent immunity 

to hepatitis A is assumed subsequent to infection108 or 

immunization completion.109 The advent of nationwide 

hepatitis A vaccination programs is gradually causing 

a decrease in disease incidence and the susceptible 

population.110 As a result, hepatitis A may in time cease 

to be a public health concern.111

The potential for hepatitis A virus transmission 

in drinking water was demonstrated in an outbreak 

among members of the varsity football team at the College of the Holy Cross in Worcester, Massachusetts, in 

1969. The same water supply was used for both irrigation and potable water. Water used by firefighters to 

battle a blaze nearby caused a drop in water pressure, 

and back-siphonage brought groundwater into the 

football practice field’s irrigation system. The groundwater had been contaminated by children infected with 

hepatitis A in a building immediately adjacent to the 

playing field. The football team members became ill 

after consuming the water from a faucet hooked up to 

this contaminated water source.112,113 Although 90 of 97 

players and coaches on the team became ill (93% attack 

rate), serologic testing performed years later revealed 

that only 33 had IgM anti-hepatitis A virus in serum 

(34% attack rate).114 Because of this discrepancy, the 

illness may have been caused by another pathogen 

also present in the water. 

Mycotoxins

Fungi are plant pathogens that can cause both 

mycoses (infections) and mycotoxicoses (exposures to 

toxic fungal metabolites that may be dietary, dermal, 

or respiratory). Mycotoxins are ubiquitous worldwide 

toxic fungal metabolites and contaminants of stored 

cereal grains.115,116 Although mycotoxins are not on the 

CDC threat list, individuals with chronic exposure to 

mycotoxins (including aflatoxin B1, ochratoxin, T-2 

toxin, deoxynivalenol [DON], nivalenol [NIV], and 

others), often exhibit oncogenic symptoms, including liver damage, liver cancer, hemorrhaging, mental 

impairment, abdominal pain, vomiting, convulsions, 

and edema. The fact that these toxins are found naturally in commercially available cereal-based foods, 

including bread and related products, noodles, breakfast cereals, baby and infant foods, and rice, indicate 

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Medical Aspects of Biological Warfare 

that a ready substrate for growth is available, and 

deliberate contamination of these foodstuffs may be 

possible. Mycotoxicoses are often undiagnosed and 

hence unrecognized by public health authorities, 

except when large numbers of people are affected.117

The symptoms of mycotoxicosis depend on the type 

of mycotoxin; the amount and duration of exposure; 

the age, health, and sex of the exposed individual; 

and many unknown synergistic effects including 

genetics, dietary status, and interactions with other 

toxic insults.118

Large naturally occurring outbreaks of trichothecene intoxications have occurred, including a large 

exposure of trichothecene mycotoxin from moldy 

grain and bread in Orenburg, Russia, in 1944, which 

caused alimentary toxic aleukia and subsequent 

mortality in at least 10% of the population.119 A 1991 

outbreak caused by moldy wheat and barley affected 

130,000 people in the Anhui province in China.115

Fusarium mycotoxins including DON and NIV have 

also been discovered in corn samples in Linxian, 

China, in positive correlation with the incidence 

of esophageal cancer.119,120 Although outbreaks of 

mycotoxicoses have decreased greatly as a result 

of increases in hygiene measures, they still occur 

in developing countries,121 are considered a serious 

international health problem,122 and also pose a risk 

to domestic animals.122–126

The history of mycotoxin use as a biological weapon 

includes efforts by Iraq to develop and use aflatoxins 

during the 1980s.127,128 Iraq’s biological weapons program 

cultured strains of Aspergillus flavus and A parasiticus

and extracted 2,300 liters of concentrated toxin.127,128 This 

aflatoxin was used mostly to fill missile warheads, and 

the remainder was kept stockpiled. 127,128 The Soviet Union 

is suspected of deploying trichothecene toxins (NIV, 

DON, and T-2) in the “yellow rain” incidents in Laos and 

Cambodia during the 1980s. Whether the toxin exposures 

that occurred at that time were the result of purposeful129 or natural130 events has never been completely 

resolved. These events indicate the potential for mycotoxin use as a biological weapon or bioterrorism agent.

Parasites

Parasites such as tapeworms (eg, Taenia species) 

may have potential for use as bioterrorism agents. It 

is conceivable, for example, that a culture of Taenia 

solium eggs could be poured onto a salad bar or into 

water, be ingested, and cause illness. Symptoms of 

TABLE 3-1

PROPERTIES RELATED TO THREAT POTENTIAL OF COMMON FOOD AND WATERBORNE 

DISEASE PATHOGENS 

Pathogen Incubation Period Infective or Toxic 

Dose*

Mortality in United 

States

Bloody Diarrhea

Enterohemorrhagic Escherichia coli3–4 d 10–102 rare yes

Salmonella typhi 8–14 d 10–102 low yes

Salmonella species 6–72 h 102–103 low yes

Shigella dysenteriae 1–7 d 10–102 rare yes

Campylobacter jejuni 2–5 d ≥ 5 × 102 rare no

Clostridium botulinum 

toxin

12–72 h 70 μg† 5%–10% no

Vibrio cholera 2–3 d 106 rare no

Cryptosporidium species 7 d 9–1,024 rare no

Listeria monocytogenes 3 ≥ 90 d unknown high no

Hepatovirus hepatitis A 30 d 10–102 low no

Norovirus 1–2 d < 102 rare no

Mycotoxins Minutes to months‡ 4 mg/kg§ rare yes

*The number of organisms unless otherwise noted.

†

Oral lactate dehydrogenase for a 70-kg human.

‡

Dose-dependent.

§

Oral lactate dehydrogenase for laboratory rat.

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taeniasis from ingestion of the eggs would include 

cysticercosis (parasite tissue infection), which would 

not appear for weeks to years following infection. 

However, this infection timeline should not eliminate 

parasites from consideration as potential bioterrorism 

agents; such a scenario has been proposed.131 T solium 

can be transmitted person to person by food handlers 

with poor personal hygiene, adding to the spread of 

the outbreak.132 Such an outbreak may go undiagnosed 

for an additional period, during which ill persons are 

seen by healthcare providers unfamiliar with tapeworm infections. A purposeful outbreak of giardiasis 

that occurred in Edinburgh, Scotland, in 1990 demonstrates that parasites can be used for bioterrorism. 

Nine individuals living in the same apartment complex 

developed giardiasis subsequent to the purposeful 

fecal contamination of an unsecured water supply.133

Threat Potential Summary

Table 3-1 provides information about various pathogens related to their potential threat as purposeful

food contaminants. Both bacterial and viral enteric 

pathogens were considered for this compilation. This 

taxonomic approach may prove useful in stimulating 

further discussion of pathogenicity and potential for 

misuse. For example, Salmonella was not considered 

a threat agent before its use in the salad bar contamination in 1984. The prior view may have been based 

on factors inherent in Salmonella infection—a high 

dose of Salmonella is required to cause illness. If the 

infectious or toxic dose required for illness from an 

organism is the sole consideration for its classification 

as a bioweapon, then salmonellae should not even be 

considered as a threat agent. However, the use of S 

typhimurium to sicken many hundreds of people in 

the Dalles incident demonstrated a reality of biological agents: those that can be cultured and dispersed to 

cause illness will prove effective. Although no deaths 

occurred, the incident involved a rapid-onset illness 

with gastrointestinal effects that spread through 10 

restaurants, causing widespread fear of food poisoning and long-lasting economic consequences in the 

community.134 Given suitable circumstances, almost 

any pathogen could be used to make a target population ill. The severity of illness, including symptoms 

such as bloody diarrhea, also should be considered. 

For example, an outbreak of bloody diarrhea could 

have strong psychological effects upon those directly 

affected and perhaps lead to widespread psychological 

effects in the general public135 if exacerbated by media 

coverage of the outbreak.136

WATER SUPPLY CONCERNS

Poisoning water supplies is one of the oldest methods of biological warfare.137 The earliest documented 

poisoned drinking water occurred in Greece in 590 

bce, when the Amphictyonic League used hellebore 

to poison the city of Kirrha’s water source, causing 

the inhabitants to become violently sick and unable 

to move.138 In current developed countries, it is more 

difficult for a terrorist to contaminate water because of 

the large volumes of water and the extensive purification processes used in modern water treatment facilities, including aeration, coagulation and flocculation, 

clarification, filtration, and chlorination.138 All of these 

methods remove contaminants and pathogens in the 

water, whether purposefully added or not.

However, the risk to the US water supply has been 

known for some time. Federal Bureau of Investigation 

Director J Edgar Hoover noted in 1941, “It has long 

been recognized that among public utilities, water 

supply facilities offer a particularly vulnerable point of 

attack to the foreign agent.”139 A terrorist might bypass 

the purification process and introduce a pathogen later 

in the distribution system. A private well water supply system with a smaller volume of water and a less 

extensive purification system may be more vulnerable. 

Another potential avenue for deliberate waterborne 

contamination is the addition of a pathogen to a building’s water supply (ie, an enclosed system), with likely 

little or no subsequent water treatment processes and 

a specific target community.

Waterborne pathogens included on the CDC threat 

list are V cholerae and C parvum. The Milwaukee 

outbreak of C parvum demonstrates the potential of 

public water supply contamination to affect great 

numbers of people. Another example of an extensive 

waterborne disease outbreak resulting from contaminated well water was the 1999 E coli O157:H7 

and Campylobacter outbreak involving more than 900 

illnesses and 2 deaths among attendees of a New York 

county fair.140 According to a comprehensive review 

of potable water threats by Burrows and Renner, 

potential water threat agents also include B anthracis, 

Brucella, V cholera, C perfringens, Yersinia pestis, Chlamydia psittaci, Coxiella burnetii, Salmonella, Shigella, 

Francisella tularensis, enteric viruses, smallpox virus, 

aflatoxin, C botulinum toxin, microcystins, ricin, saxitoxin, staphylococcal enterotoxins, T-2 mycotoxin, 

and tetradotoxin.141 The 1969 hepatitis A outbreak 

at the College of the Holy Cross demonstrates the 

potential for this pathogen to cause illness when 

distributed in a water supply.

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Medical Aspects of Biological Warfare 

Communitywide outbreaks of gastroenteritis, 

caused by Giardia lamblia, Cryptosporidium, various E 

coli serotypes, Torovirus, and other infectious agents, 

have occurred from recreational water use, including 

swimming pools, water slides, and wave pools.142

Nongastroenteritis recreational water outbreaks often include those caused by Pseudomonas aeruginosa, 

Naegleria fowleri, and Legionella.

142 A recent naturally 

occurring outbreak of gastroenteritis associated with a 

contaminated recreational water fountain at a Florida 

beachside park demonstrates the potential for disease 

transmission.143 In this incident, 44% of the interviewed 

park visitors who used an interactive water fountain 

became ill. Both C parvum and Shigella sonnei were 

subsequently isolated from clinical specimens obtained from these ill persons. The median age of the 

ill persons was 8 years. One can imagine the effect of 

a powerful biological agent such as C botulinum toxin 

covertly added to a recreational public water fountain 

in similar circumstances.144

The water utility industry and federal public health 

agencies have carried out plans to improve the ability 

to prevent as well as detect deliberate contamination 

of water systems.145 An example of a new program 

to detect purposeful contamination of the water supply is the Water Sentinel program.146 However, much 

work remains to attain full biosecurity of the US water 

supply.147,148

AGRICULTURAL TERRORISM

Agricultural terrorism (agroterrorism) may be 

directed at stored or processed food, but some of the 

greatest vulnerabilities may exist close to the farm end 

of the farm-to-food continuum (Figure 3-1). Many of the 

potential bioterrorist agents are endemic, and therefore 

cannot easily be controlled. As with processed food and 

water terrorism, agroterrorism concerns are not recent 

developments. The historical use of biological agents to 

affect livestock includes the attempt to interrupt supply 

lines by infecting cavalry and transport animals with 

anthrax and glanders during World War I. In April 

1915, German-American physician Anton Dilger (who 

had served in the German Army) returned to the United 

States from Germany along with cultures of Burkholderia 

mallei and B anthracis. His intent was to infect animals 

(horses and mules) that were shipped from the United 

States to France and England for use in cavalry and 

transport to support their war with Germany. Dilger 

propagated the bacterial cultures and tested them for 

virulence using guinea pigs in the basement of a house 

(known as “Tony’s Lab”) he and his brother Carl 

rented in Chevy Chase, Maryland, near Washington, 

DC.149 Over the next 2 years, Dilger’s bacterial cultures 

were used to infect horses and mules in holding pens 

in docks at the ports of Baltimore, Maryland; Newport 

News, Virginia; Norfolk, Virginia; and in New York 

City. Stevedores working for German steamships were 

recruited and provided with cork-stoppered glass vials 

containing the bacterial cultures, in which a hollow steel 

needle had been placed. The stevedores were instructed 

to wear rubber gloves while jabbing the animals with 

the needles. These cultures were also spread among the 

animals by pouring them directly into the animal feed 

and drinking water.150,151

The significance of foot and mouth disease (FMD) 

as a biological weapon has been known for some time, 

and it is perhaps the greatest agroterrorism threat for 

Figure 3-1. Some of the greatest vulnerabilities from agricultural terrorism may exist at the farm end of the farm-to-food 

continuum.

Photograph courtesy of the US Department of Agriculture, 

Washington, DC.

livestock. Field trials of FMD virus dissemination were 

conducted in Nazi Germany’s offensive biological warfare program. FMD is thought to be inherently spread 

through airborne virus transmission, a problematic 

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Food, Waterborne, and Agricultural Diseases

issue for outbreak containment,152 and Germany considered aerial dissemination and dispersal of the FMD 

virus through contaminated hay and grass.153(p114) 

Another attack on livestock occurred during the 

Mau-Mau uprising in British-controlled Kenya (1952–

1960), when the Mau-Mau used the indigenous poisonous African milk bush (Synadenium compactum) to kill 

33 cows at a mission station in 1952.154,155 This use of 

locally obtained poisonous plants could be replicated 

anywhere lacking constant monitoring of animal feed.

Anticrop terrorism has also been suspected on numerous occasions. The Colorado potato beetle (Leptinotarsa decemlineata) is a crop pest of plants of the genus 

Solanum, which includes potatoes, tomatoes, and eggplants. During World War II, Germany initiated largescale breeding and field trial dispersals of the insects 

in Germany, when Dr Martin Schwartz conducted an 

offensive research program at the Kruft Potato Beetle 

Research Station near Koblenz.153(p110) This program 

may have backfired by initiating local crop infestations; however, outbreaks of the pest also occurred in 

England and the United States, which were suspected 

to be caused by a German release of the insects.156,157

Perhaps because of this research conducted in Nazi 

Germany, in 1950 Soviet-occupied East Germany accused the United States of releasing the beetle during 

in infestation.158 Herbicides have also been used for 

wartime missions, such as the large-scale use of the 

defoliant Agent Orange by the United States to both 

defoliate and destroy crops used by North Vietnamese 

forces.159 In 1989 a group known as “the Breeders” announced that it had released Mediterranean fruit flies 

in southern California to protest the use of pesticides 

in that region.160

State-sponsored agricultural terrorism remains a 

global concern today, given vulnerabilities inherent in 

modern farming practices. In the United States, livestock may be susceptible to agroterrorism (Figure 3-2). 

Because US disease eradication efforts among livestock 

herds have been so successful, much of the nation’s 

livestock is either vaccinated or monitored for disease 

by farmers and veterinarians. However, the risk of harm 

from agroterrorism to large numbers of livestock has 

been increased through the widespread use of modern 

livestock farming, such as concentrated animal feeding operations (CAFOs). The pervasiveness of CAFOs 

in the US agriculture industry is all-encompassing. 

For example, in the US between 1997 and 2007:161

• hog factory farms added 4,600 hogs every day;

• factory farm dairies added nearly 650 cows 

every day;

• factory farms added 5,800 broiler chickens 

every hour;

• factory farmed broiler chickens doubled to 1.1 

billion;

• hog factory farms grew by 42%, to 5,144;

• the average size of egg factory farms increased 

by half to 614,000 hens;

• the number of cows on factory-farm dairies 

nearly doubled, to 4.9 million;

• the number of hogs on factory farms grew by 

more than a third, to 62.9 million;

• the number of factory farm egg-laying hens 

increased by 24%, to 266.5 million;

• the number of US beef cattle on industrial 

feedlots grew by 17%, to 13.5 million; and

• nearly half of factory-farm egg-laying hens 

were located in just five states: Iowa, Ohio, 

Indiana, California, and Pennsylvania.

Altogether, CAFO aggregation has had the greatest effect on livestock operations with the greatest 

numbers of animals. From 1982 to 1997, livestock 

operations with 1,000 or more animals increased by 

47%. In comparison, farms with less than 25 animals 

(a family farm) decreased by 28%.162 While such aggregation has enabled economic viability and success 

for the farming industry, it also can provide a singlesource opportunity for foodborne contamination or 

adulteration for the would-be bioterrorist desiring to 

affect the food supply.

Upon infection, livestock may become a vector163

or reservoir164 for disease transmission. This potential 

was plainly demonstrated in the 2001 outbreak of 

Figure 3-2. Livestock may be more susceptible to agroterrorism than crops.

Photograph courtesy of the US Department of Agriculture, 

Washington, DC.

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Medical Aspects of Biological Warfare 

FMD in the United Kingdom.165 This outbreak was the 

single largest FMD epidemic ever experienced in the 

world.166 Agricultural and food losses to the United 

Kingdom exceeded $4.6 billion,167 and psychological 

effects in residents of the worst affected areas were 

extensive and long-lasting.168 The United States has 

not had an outbreak of this disease since 1929,169

and the US Department of Agriculture (USDA) has 

developed national protective measures to prevent a 

reintroduction.170

Perhaps the greatest national risks from agroterrorism involve the potential for widespread economic 

consequences. Not only would immediate loss to a 

crop occur from such an event, but incidental costs 

would also result from lost production, the destruction of potentially diseased products, and containment 

(including quarantine, drugs, and diagnostic and veterinary services). The costs of these programs would be 

borne by farmers as well as federal and state governments.171 Export markets would be rapidly lost as other 

nations close their borders to imports from a country 

with diseased livestock. As an example, a single case 

of mad cow disease (bovine spongiform encephalopathy) was found in Washington state on December 23, 

2003; by December 26, Japan had banned all US beef 

imports, and beef prices dropped by as much as 20% 

in the following week.172 Additionally, multiplier 

economic effects would occur from decreased sales 

by agriculturally dependent businesses and tourism. 

Other animal pathogens besides FMD and bovine 

spongiform encephalopathy that could have severe 

economic consequences if uncontrolled include highly 

pathogenic avian influenza,173 rinderpest,174 and African175 and classical swine fever.176

The USDA’s Animal and Plant Health Inspection 

Service has developed a select agent and toxin list of 

pathogens and toxins that endanger agriculture in the 

United States177 (some of these zoonotic pathogens also 

endanger humans and appear on the CDC Category 

A list4; these pathogens are listed separately by the 

USDA as overlap agents and toxins). Another USDA 

list enumerates harmful plant pathogens.177

SMUGGLING AND INVASIVE SPECIES

The problem of smuggling and the unintentional 

introduction of invasive species has grown in recent years. Increased and more rapid international 

trade, increased trade in fresh commodities, new 

travel and trading routes, and increased difficulties 

in enforcing quarantines have all contributed to this 

problem. The potential consequences of smuggling 

and unintentional introduction of invasive species 

may go far beyond the direct damages or costs of 

control. The economic costs of all invasive species in 

the United States is estimated at between $120 billion 

and $138 billion per year.178,179 These invasive species 

consist of microbes (30.1%), mammals (27.2%), plants 

(25.0%), and arthropods (15.4%). The full range of 

economic costs of biological species invasions reaches 

far beyond the immediate impacts on the affected 

producers and often include consequences to local, 

national and global markets. In 1995, member nations of the World Trade Organization signed the 

Uruguay Round Agreement on the Application of 

Sanitary and Phytosanitary Measures, which set out 

basic rules for food safety and animal and plant health 

standards and increased the transparency of sanitary 

and phytosanitary measures.180 The agreement covers all measures to protect human or animal health 

from foodborne risks, human health from animal- or 

plant-carried diseases, and animals and plants from 

pests or diseases.180

FOOD AND WATER SECURITY

On December 3, 2004, the former secretary of the 

Department of Health and Human Services, Tommy 

Thompson, warned of a possible terrorist attack on 

the nation’s food supply: “For the life of me, I cannot 

understand why the terrorists have not attacked our 

food supply, because it is so easy to do . . . We are 

importing a lot of food from the Middle East, and it 

would be easy to tamper with that.”182 In American 

society, the farm-to-food continuum, which includes 

production, processing, distribution, and preparation, 

has myriad potential vulnerabilities for natural and 

intentional contamination.182 Centralized food production and widened product distribution systems 

present increased opportunities for the intentional 

contamination of food.183 As covered in the discussions above, many opportunities exist along the food 

and water production continuum to accidentally or 

intentionally introduce various pathogens, many 

of which are not categorized as threat agents.184

Strategies to counter these threats should focus on 

enhancing knowledge of all raw material inputs to 

the system; identifying and addressing the most likely 

points of vulnerability; disposing of end products 

after they leave the systems; and accounting for employees, visitors, computers, and physical security 

throughout the continuum.

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Food, Waterborne, and Agricultural Diseases

Knowledge of the various processes involved in 

food production will help to determine potential vulnerabilities for agricultural terrorism. The typical food 

distribution system includes agricultural production 

and harvesting, storage and transport of raw commodities, processing and manufacture, storage and 

transport of processed and manufactured products, 

wholesale and retail distribution, and the food service 

sector.185 The responsibility for food safety and security 

throughout the food distribution network is shared by 

the producers and suppliers as well as many different 

state and federal agencies. Typically, a state’s health 

and agricultural agencies ensure that the food comes 

from safe sources and is served with safeguards to 

prevent foodborne disease transmission. Equivalent 

federal agencies share these responsibilities, including 

the US Food and Drug Administration, USDA, Department of Health and Human Services, US Public Health 

Service, CDC, and other partner agencies now part of 

the Department of Homeland Security, including the 

Federal Bureau of Investigation and the US Customs 

Service.

One prevention strategy is to anticipate intentions or motivations that could result in an attack 

using a particular product or organization. These 

motivations could include religion or ideology; 

personal grievances (real or perceived); and contentious issues such as animal rights, environmental 

protection, and abortion. Research facilities, food 

processors, and food retailers could be targets of terrorism and should take extra preventive measures. 

Knowledge of terrorism trends can be an indicator 

for the need to change security measures to meet 

the threat. However, because the US food industry 

is highly competitive on a price basis, additional 

preventive measures may only be an option if they 

are government subsidized.

From an attacker’s standpoint, the choice of methods and weapons is determined by the target and the 

delivery medium. It is rare that someone would attempt to cause harm without consideration of whom 

or how many people are affected. The target population may then define the vulnerabilities. For example, 

animal feed could be contaminated if the goal was to 

affect a CAFO.

Strategies also can be implemented to address specific vulnerabilities. The first task is to define production processes in terms of the inputs and outputs at all 

potential nodes of vulnerability. For example, foods 

that are either eaten uncooked or that can be contaminated after cooking should receive special quality control attention. Also, knowledge of where raw materials 

including water are obtained can help identify needs 

for enhanced security and accountability. 

A thorough knowledge of the existing hazard 

analysis critical control points (HACCPs) for each food 

item considered to be a potential vehicle for foodborne 

disease is essential to understanding and preventing 

illness. A comprehensive HACCP analysis will provide 

a systematic method of documenting that food safety 

hazards have been addressed.186 Hazard analysis involves food safety issues only, including storage and 

holding temperatures, pH, sanitary conditions, physical storage security, and any other factor that could 

impact the safety and integrity of a food item during 

manufacture, storage, delivery, or food preparation.186

General guidance to conducting an HACCP program 

would necessarily include:186

• Hazard analysis: what are the food safety 

hazards that can be controlled?

• Establish critical control points (CCP): where 

can things go wrong, and how can they be 

controlled?

• Establish critical limits: what physical values 

(temperature, pH, etc) indicate that the process is in control?

• Establish monitoring procedures: how will 

the CCPs be monitored?

• Establish corrective actions: what happens if 

a critical limit is exceeded?

• Establish a record keeping system: “If it isn’t 

written down, it didn’t occur.”

• Establish verification procedures: how can 

you know if the system works?

HACCP principles have been successfully applied 

to the production, storage, and serving of many types 

of food items.187–189 However, there remain many challenges to providing a safe and wholesome food supply, 

and resolution of issues through the use of HACCP may 

also provide solutions that could prevent bioterrorism. 

For example, food items that are common sources of 

foodborne infections may also present opportunities 

to a potential bioterrorist, by virtue of a lack of proper 

temperature use and monitoring. It has been demonstrated that salsa and guacamole are frequent vehicles 

of foodborne disease outbreaks in the United States.190

Unsurprisingly, fresh serrano and jalapeno peppers 

used in these food items have caused huge multistate 

Salmonella outbreaks.191,192 Fresh salsa and guacamole 

require careful preparation and storage, and food prevention strategies based upon the HACCP principles can 

greatly help to reduce the incidence of foodborne disease, 

as well as to maintain monitoring of these food items.

Focus is often targeted on the inputs to food, 

water, or agricultural production, and when a 

product leaves the plant, that attention may be dis244-949 DLA DS.indb 81 6/4/18 
82

Medical Aspects of Biological Warfare 

continued. The time and route of delivery, as well as 

the security of the transportation, may be the most 

vulnerable points in the continuum and should not 

be overlooked when planning security. Studying 

incidents of nonpurposeful foodborne pathogen 

contamination, such as the 1985 Minnesota salmonellosis outbreak,193 may reveal potential avenues for 

purposeful outbreak scenarios. This outbreak and 

many others demonstrate that foodborne bioterrorism might have greater chances of success when 

pathogens are introduced after processing and as 

close to consumption as possible, thus circumventing opportunities for dilution and destruction by 

cooking or pasteurization.

Implementing rational employee hiring and accountability procedures may also effectively mitigate 

food, water, or agricultural vulnerabilities.186 Additional strategies include implementing procedures 

for laboratory testing and monitoring, reporting and 

investigating inspection discrepancies, and ensuring 

computer and information security.186 

Various disease surveillance systems (covered in 

greater details in other chapters) are in place, including 

local, state health agency, and CDC programs to track 

and identify trends in foodborne illness, including 

FoodNet,194 PulseNet,195 CalciNet,196 WBDOSS,197,198

and syndromic surveillance systems such as RODS199

and BioSense.200 Additional methods to inspect and 

protect food and water supply chains, and rapidly 

integrate disease surveillance, are being actively examined and implemented.201

Furthermore, under the Food Safety and Modernization Act of 2010 the Food and Drug Administration 

has proposed a rule that would require the largest 

food businesses in the United States and overseas to 

take measures to prevent food facilities from being 

targeted by intentional attempts to contaminate the 

food supply.202 Under the proposed rule, food facilities would be required to have a written food defense 

plan addressing significant vulnerabilities in their 

food production process, and to take measures to 

address these vulnerabilities, establish monitoring 

measures and corrective actions, confirm that the 

system is working, and ensure that workers assigned 

to vulnerable areas receive suitable training and 

maintain records.202

SUMMARY

Any biological pathogen, whether bacteria, virus, 

toxin, or parasite, has the potential to be used in a terrorism context. Historical examination of both purposeful and inadvertent food and waterborne disease 

outbreaks can greatly assist in understanding how 

such events occur and how they may be prevented. 

A comprehensive understanding of animal production and crop farming, as well as food production 

and distribution, is required to ensure protection 

for the agricultural industry from terrorism events. 

Absolute safety of the food supply is perhaps an 

unattainable goal, but should be the benchmark for 

which all food protection and agricultural efforts 

are directed.

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Consequence Management: The Local and National Response

Chapter 4

CONSEQUENCE MANAGEMENT: 

THE LOCAL AND NATIONAL 

RESPONSE 

NEAL E. WOOLLEN, DVM, MSS, PhD,* and GARY W. CARTER†

INTRODUCTION

CONSEQUENCE MANAGEMENT

CONSEQUENCES OF A BIOLOGICAL INCIDENT

LOCAL AND NATIONAL RESPONSE

RECOVERY

SUMMARY

*Colonel, Veterinary Corps, US Army; Director, Department of Defense Biological Select Agent and Toxin Biosafety Program Office, 1546 Porter Street, Fort 

Detrick, Maryland 21702

†

Director, Field Operations and Training, National Strategic Research Institute, 3925 Dewey Avenue, Omaha, Nebraska 68198 

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INTRODUCTION

regional, state, and federal response capabilities and 

functions and outline a mechanism for requesting assistance. Effective consequence management starts at 

the local level, but can rapidly escalate with the need 

to coordinate higher-level supportive response with 

ongoing local response and recovery efforts.

Federal response and recovery key planning considerations and responsibilities are identified in the 

National Response Framework (NRF) and the National 

Disaster Recovery Framework (NDRF), available for 

download from the Department of Homeland Security (DHS) and the Federal Emergency Management 

Agency (FEMA) digital libraries. They identify essential support functions and recovery support functions that guide local, state, and interagency federal 

all-hazards planning for response and recovery. The 

Biological Incident Annex to the NRF outlines the 

actions, roles, and responsibilities associated with a 

human disease outbreak of known or unknown origin 

requiring federal assistance. These documents emphasize a common theme: response and recovery will start 

at the local level and local involvement throughout is 

critical to success. Robust local planning to reduce an 

entity’s vulnerability to the threats of biological incidents, with a well-thought plan for disaster response 

and recovery that meshes well with state and federal 

assistance plans, can save lives, minimize impact, and 

defeat terrorist objectives.

Consequence management is critical to minimizing 

the long-term impact from any natural or manmade 

disaster. A common aim of organizations that seek 

to induce terror is to cause maximum disruption to 

societies with no regard for the impact on human life. 

Effective consequence management will minimize 

a disaster’s impact on a society, its people, its infrastructure, and its economy. It will deliver the right 

solutions at the right time to the right locations in a coordinated and controlled response. Many, but not all, 

consequences of a catastrophic event are predictable, 

and many catastrophic events share common types of 

consequences. An effective consequence management 

plan focuses on the critical functions and capabilities 

necessary to span multiple types of disasters that effectively minimize the disaster’s impact. The plan must 

be comprehensive, flexible, and scalable. It must be 

adaptable to variable outcomes of varying magnitude 

and potential cascading effects of catastrophic events 

that may require a rapid transition from the initial plan 

to a more comprehensive one. 

Constructing an effective consequence management 

plan starts with understanding the threats to a populace, 

geographic location, or specific entity and the potential 

impact of those threats on all facets of the affected area. 

It must identify local response capabilities and functions 

and develop an activation strategy for timely implementation. It should also demonstrate an understanding of 

CONSEQUENCE MANAGEMENT

The definition of consequence management has 

evolved over time. At one time a clear separation 

existed between crisis management and consequence 

management. In a November 20, 2003 hearing before the House of Representatives, Subcommittee 

on Crime, Terrorism, and Homeland Security, Mr. 

Howard Coble defined crisis management as actions 

taken to anticipate, prevent, or resolve a threat, with 

consequence management fulfilling the cleanup and 

restoration functions after an attack.1

 To truly mitigate 

the short-term and long-term impacts of catastrophic 

events, one must think and plan over a continuum 

of prevention, protection, and mitigation through 

response and recovery. 

The NDRF emphasizes that community recovery 

can be accelerated through a community’s efforts in 

predisaster preparedness, mitigation, and recovery 

capacity building.2

 Recovery efforts must not interfere 

with immediate response efforts to preserve life and 

health and maintain critical infrastructure. However, 

immediate response efforts can mitigate long-term 

effects and the overall impact of the disaster with significant impact on postdisaster recovery—positively 

or negatively—and the two should be planned and 

executed in harmony. 

The NDRF emphasizes that recovery encompasses 

far more than the restoration of a community’s physical 

structures; it must also provide a continuum of care to 

meet the needs of the affected community members 

who have experienced financial, emotional, or physical 

impacts.2

 Good communication and coordination early 

and throughout a disaster response will help inform 

long-term recovery planning as well as prevent selection of short-term solutions that may result in negative 

long-term impact. Integration among the frameworks 

is considered one of the key themes of the National 

Preparedness Goal (NPG).3

Planning must be risk based and risk assessments 

must be comprehensive and standardized. Planners, 

regardless of their affiliation, must understand what 

threats have the greatest potential impact on their area 

of responsibility. Threat and hazard identification and 

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Consequence Management: The Local and National Response

risk assessment guidance is provided in the Comprehensive Preparedness Guide 201.4

 The guide describes 

a five-step process for threat and hazard identification 

and risk assessment: 

1. identifying threats and hazards of concern; 

2. giving them context; 

3. examining core capabilities; 

4. establishing capability targets; and 

5. applying results. 

The Army Techniques Publication No. 5-19 outlines 

a risk management approach to analyzing and mitigating hazardous operations. Core components of risk 

management are conducting a hazard analysis based 

on source, mechanism, and outcome of each hazard; 

an assessment of risk based on the probability the 

hazard will be experienced; and the severity of the 

outcome, followed by development of a strategy to 

mitigate risk that focuses on lowering the probability 

or lessening the severity resulting in an acceptable level 

of residual risk. Risk assessment must be continuous 

and updated throughout the course of a response to 

hazardous conditions, and it must be specific to the 

environment, local infrastructure, and population. 

Severity of outcomes will likely vary significantly between heavily populated areas and sparsely populated 

areas and could vary considerably within the same 

city depending on what part of the city is affected, 

time of day, or whether the incident occurs during a 

workweek, weekend, or special event.

Both risk management systems stress the importance of identifying hazards through lessons learned 

from past events and subject matter expert opinion, 

understanding how they may affect an entity’s operations, identifying capabilities that can mitigate the 

impact of the hazard, and proper resourcing to implement the necessary control measures. These systems 

can assist planners with selecting controls to mitigate 

outcomes, inform predisaster resourcing to enhance 

preparedness (eg, establishment of memoranda of 

agreement), decrease the residual risk, and assist with 

comprehensive consequence management planning. 

An example of where this can assist consequence 

management following a population’s exposure to a 

biological agent or toxin is to factor in the mechanism 

by which the resultant disease can spread. 

Some aspects of consequence management following exposure to a biological agent that causes contagious disease could significantly differ from one that 

is not. An overreaction for a disease-causing agent that 

is not contagious could cause unnecessary negative 

impact to the local infrastructure and economy and 

further complicate long-term recovery. Conversely, 

a lack of planning or inability to control the spread 

of a contagious disease could result in uncontrolled 

spread beyond the contamination zone with severe to 

catastrophic impact on life, health, infrastructure, and 

economy over a broad geographic region. A contagion 

will likely require implementation of quarantine and/

or isolation as a control measure, but quarantine and/

or isolation may not be appropriate for an agent that 

does not cause a contagious disease. The mechanism 

by which a hazard produces negative outcomes is 

important. When local planners do not have subject 

matter experts available to assist with planning, they 

should seek assistance from county, state, and federal 

public health professionals. 

The general population does not understand 

the unique differences among the various potential 

pathogens, their mechanisms of transmission, and the 

differences in risk created by each. Misinformation disseminated through rumors or poorly informed news 

outlets can create additional challenges to the response 

and recovery effort. It can lead to confusion as well 

as a loss of confidence in those leading the response 

and recovery effort. Timely accurate information dissemination to the community is important, whenever 

a threat to public safety occurs. 

An analysis of the human response to the catastrophic events of September 11, 2001 reveals that 

fear during a crisis situation does not automatically 

result in panic, and the negative impact of fear may 

manifest more significantly during the consequence 

management period when considerable uncertainty 

exists.5

 It revealed a tremendous spirit of cooperation 

and compassion among the affected population during 

the crisis period. Timely release of accurate information can mitigate the effects of misinformation and 

facilitate a spirit of teamwork throughout response 

and recovery. Close coordination of press releases 

with public affairs professionals is critical to accurate 

information dissemination.

One intending to induce terror will rely on a hazard’s natural and intended consequences but will 

also benefit from unintended consequences that are 

a product of poor or narrowly focused planning, that 

is not flexible and scalable, resulting in an inefficient 

response with poor communication. Similarly, the 

magnitude of impact from a naturally occurring 

outbreak of infectious disease can be minimized or 

magnified by the quality of response planning and 

efficiency of execution. 

Selecting the appropriate medical countermeasures 

and understanding the potential effects will rely on accurate agent identification at the time of the incident, 

but detailed planning for specified agents would be an 

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Medical Aspects of Biological Warfare 

ment and would risk not having an actionable plan in 

place when needed. Local, state, and federal response 

and recovery planning can leverage common mechanisms and common potential outcomes to be prepared 

for many potential threats rather than constructing 

plans for every possible disease causing agent.

CONSEQUENCES OF A BIOLOGICAL INCIDENT

Biological incidents may be one of the most challenging threat conditions for planning consequence management. Response and recovery for most disasters follow 

a major catastrophic event that is relatively rapidly definable in scope and magnitude of impact. Conversely, 

it is unlikely that a biological incident will present as 

a well defined major catastrophic event. However, it 

carries the potential for significant casualties and major 

disruption to a community, region, or nation’s infrastructure and economy, with a significant fear factor 

and potential for panic among a nation’s population. If 

it involves a contagion, it also has the potential to spread 

well beyond the original target area in a short period 

of time. If it involves a zoonotic disease-causing agent, 

or is equally infectious to a local animal population, 

secondary spread from human–animal contact may 

persist and human health efforts must be synchronized 

with that of the animal health and feral animal control 

industries. With the exception of an emerging infectious disease with pandemic potential, where initial 

cases have already been identified, no warning or opportunity will likely occur for leaders to proactively 

surge or position resources. A scalable response must be 

part of a community’s and nation’s normal framework. 

A series of framework documents can guide planners through consequence management planning and 

execution: 

• National Prevention Framework, 

• National Mitigation Framework, 

• NRF, and 

• NDRF. 

All disasters have the potential for cascading effects, 

especially for a biological incident. Those intending to 

induce terror will attempt to leverage this potential to 

achieve their objectives and the response and recovery 

efforts must control them to minimize the impact on 

a society. Whether a biological incident is intentional, 

accidental, or results from a natural disease outbreak, 

it is important to rapidly identify the potentially affected area and population and control movement. 

Without these controls rapidly implemented, the zone 

of potential contamination will grow. Concurrent with 

controlling movement into and out of the affected area, 

the agent or agents involved must be rapidly identified. 

Differing biological agents have differing incubation 

periods, environmental survivability, and modes of 

transmission. Appropriate medical countermeasure 

and decontamination procedure identification and 

implementation timelines will rely on accurate agent 

identification. If animals and insect vectors can be 

potential sources of residual infection and sources for 

spreading the disease causing agents, the appropriate 

subject matter experts must be included in response 

and recovery planning.

Sick people with relatively generic conditions show 

up in emergency departments, urgent care clinics, and 

primary care clinics on a daily basis. It is likely that 

a disaster will not be declared until their condition is 

identified as part of a natural disease with pandemic 

potential or tied to an act of bioterrorism. By this time 

the consequences of the causative event are being 

experienced, and the challenge of determining where 

it started and what caused it exists. The immediate 

consequences are obvious and rely on availability of 

the appropriate diagnostic assays and medical countermeasures. Some critical questions must be immediately 

answered: 

• Is this disease typical of the human population 

or geographic location? 

• Is it contagious?

• Are animals also affected? 

• Is it zoonotic? 

• Does the pattern of disease in the population 

suggest a natural outbreak or an intentional 

release? 

Answers to these questions will inform immediate 

response and identify potential cascading effects and 

consequences that must be managed. If the incident is 

determined to be suspicious for an intentional release, 

a criminal investigation must immediately accompany 

the public health and epidemiologic investigations. 

These efforts must be synchronized to ensure no disruption to response efforts intended to preserve life 

and health while also preserving evidence to facilitate 

the criminal investigation.

A biological incident will present a significant 

amount of uncertainty for professionals responding 

to the incident and this will be magnified throughout 

the community. Timely accurate dissemination of 

information can mitigate a public reaction that may 

compound the problem if not appropriately managed.

The National Incident Management System, which 

calls for a unified approach to multiagency coordination during response and recovery operations, 

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emphasizes the unified approach concept based on 

chain of command, unity of command, unity of effort, 

and when implemented, unified command.6 Unity of 

effort is critical to disseminating consistent informative messages at the right time to assist with managing public fear and reaction. Conflicting information 

from multiple agencies creates confusion, a lack of 

confidence in the response and recovery effort, and 

potential panic. 

The anthrax (Bacillus anthracis) attacks of 2001 

provide lessons on the value of effective risk communications and public reaction. Confusion following the initial reports was widespread on a national 

scale. Examples of communication failures include 

the following: 

• 46% of the population thought anthrax was a 

contagious disease.

• 70% of the New Jersey population were concerned that they or someone close to them had 

been exposed. 

• Agencies were overwhelmed by requests for 

information. 

• Reports of discontent with both quality and 

timeliness of information were heard.7 

An example of risk communication success came 

from a multiagency task force in New Jersey that 

was reported as beneficial for enhancing cooperation 

between law enforcement and health organizations 

and reducing tensions. Law enforcement and health 

organizations approach communications differently, 

with law enforcement leaning toward secrecy and 

public health valuing openness.7 By managing disparate philosophies through a task force or unified 

command element, messaging can be less confusing 

for the recipients, with greater efficiency in providing 

informative messages in a timely manner that do not 

compromise criminal investigations and serve to palliate public fear and reaction.

Preserving the life and health of an affected population must be the primary concern during response and 

recovery, but consequence management planning and 

execution for a biological incident cannot be narrowly 

focused on individual patient care. If a biological agent 

is intentionally dispersed, the dispersion method must 

be rapidly characterized to determine the extent of 

contamination and identify the affected human and/or 

animal population(s). If agent dispersion is covert and 

the first indication of an act of bioterrorism or criminal 

act involving a biological agent is one or more patients 

presenting with clinical disease, determining the extent 

of contamination and the affected population will be 

challenging but critical to effectively managing the 

consequences. Once the affected area is determined, 

access to and egress from the area must be controlled, 

but controls must be tailored to the type of agent(s) 

identified. 

A detailed assessment of the affected area that 

includes personnel and equipment movement, wild 

and domestic animal populations and movement, 

and surface water flow patterns must be conducted 

to determine the potential for contamination spread 

and other potentially affected populations. If the affected area involves significant community services, 

planners will need to determine how to continue 

providing those services to the unaffected community 

to minimize the incident’s overall impact, especially 

if it involved any services critical to the response and 

recovery effort. The initial assessment of causative 

agent, method of dispersion, and extent of contamination will also assist health providers and planners on 

selection of medical countermeasures and the potential 

number of expected human casualties. If the extent of 

contamination is large or multifocal, or if the causative 

agent is a contagion, patient care facilities and medical 

countermeasures could be rapidly overwhelmed, necessitating the need for rapid coordination of external 

support. Plans and support agreements to manage this 

potential consequence must be in place before an incident. These agreements are an area where neighboring 

communities and neighboring states can seek mutual 

aid and support when one or the other is affected by 

a catastrophic event.

The impact of a biological incident will likely extend 

well beyond the primary concern of human health 

considerations. Whether secondary hazards from a 

biological incident are real or perceived, they will have 

significant impact on the affected entity and surrounding population. Biological incidents have the potential 

to overwhelm local healthcare resources and render 

any business or public service provider inoperable. 

If a community’s healthcare facility and emergency 

response capabilities are contaminated, it is imperative 

that a backup plan for care and emergency response is 

in place. How effectively a community conducts waste 

management will either create or mitigate secondary 

concerns and the impact of accumulating waste. Waste 

may also require special treatment that is not part of 

a community’s standard operating procedures, and 

special assistance may be required. 

Communities should develop continuity of operations plans to identify backup resourcing for critical 

services. Businesses should also develop continuity 

of operations plans to protect their livelihood in case 

they are directly or indirectly affected by an emergent 

situation and unable to occupy their normal place of 

business. The former $3.8 million American Media Inc. 

building in Boca Raton, Florida, was quarantined on 

October 10, 2001, sold for $40,000 in April 2003, and 

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was not reported to be clear of Bacillus anthracis until 

February 7, 2007.8 The Hart Senate Office Building 

reopened after it cost $27 million to decontaminate 

it. Economic impact on an affected business can be 

catastrophic and a protracted disruption to services 

can occur when a public service facility is affected. 

LOCAL AND NATIONAL RESPONSE

The NPG suggests that successful consequence 

management of a biological incident will result from 

capabilities being available across the whole community to prevent, protect against, mitigate, respond to, 

and recover from the threats and hazards that pose 

the greatest risk.9 The NPG and supporting framework documents emphasize that an effective response 

starts at the local level with individuals, community 

organizations, the private and nonprofit sector, faithbased organizations, and local governments all having 

a critical role with support from the state and federal 

governments. 

The NPG identifies core capabilities for five mission 

areas: (1) prevention, (2) protection, (3) mitigation, 

(4) response, and (5) recovery. It recognizes that the 

core capabilities listed are ambitious and will require 

a national effort involving the whole community to 

be effective, with three core capabilities spanning all 

five mission areas: (1) planning, (2) public information and warning, and (3) operational coordination.9

The NPG lists five key findings from the Strategic 

National Risk Assessment; two key risk areas correlate 

with the subject of this chapter: a virulent strain of 

pandemic influenza and terrorist use of weapons of 

mass destruction.9

Planners at all levels should assess their level of 

readiness to manage consequences associated with 

these risks, but not limit their planning to just these 

risks. Every community and state may have differing 

priority lists for planning. Military planners routinely 

assess two types of risks to operations: most likely 

and most dangerous. This approach can also prove 

valuable for planners as they develop local and state 

core capabilities for effectively managing risks posed 

by various biological threats.

Although planners should be familiar with several 

framework documents, the remainder of this chapter 

will primarily focus on guidance from the NRF, the 

NRF Biological Incident Annex, the NDRF, and other 

supportive documents. Framework documents emphasize the significance of local readiness and response 

and provide federal agency level guidance that can be 

tailored to all levels of government planning. With a 

few exceptions, federal assets will not be mobilized 

until local and state capabilities have been or likely 

will be exceeded and a state’s governor requests federal 

assistance under the Stafford Act. These documents 

provide valuable guidance for planning at all levels. 

The NRF lists 14 core capabilities, 15 emergency 

support functions, and four priorities for the response 

mission area that local and state planners can use as a 

template for their planning activities. Core capabilities 

include the following: 

1. planning;

2. public information and warning;

3. operational coordination;

4. critical transportation; 

5. environmental response/health and safety; 

6. fatality management services;

7. infrastructure systems; 

8. mass care services; 

9. mass search and rescue operations; 

10. on-scene security and protection; 

11. operational communications; 

12. public and private services and resources; 

13. public health and medical services; and 

14. situational assessment.10

Emergency support functions (ESF) include the 

following: 

1. ESF1 transportation; 

2. ESF2 communications; 

3. ESF3 public works and engineering; 

4. ESF4 firefighting; 

5. ESF5 information and planning; 

6. ESF6 mass care, emergency assistance, temporary housing and human services; 

7. ESF7 logistics; 

8. ESF8 public health and medical services; 

9. ESF9 search and rescue; 

10. ESF10 oil and hazardous materials response; 

11. ESF11 agriculture and natural resources; 

12. ESF12 energy; 

13. ESF13 public safety and security; 

14. ESF14 (replaced by the NDRF); and 

15. ESF15 external affairs. 

Response mission priorities include the following: 

1. save lives; 

2. protect property and the environment; 

3. stabilize the incident; and 

4. provide for basic human needs.10 

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TABLE 4-1 

CORE CAPABILITIES AND OBJECTIVES

Capability Objectives

Planning Conduct a systematic process engaging the whole community in the development of executable strategic, operational, and/or community-based approaches to meet defined objectives.

Public Information and Warning Deliver coordinated, prompt, reliable, and actionable information to the whole community 

through the use of clear, consistent, accessible, and culturally and linguistically appropriate methods to effectively relay information regarding any threat or hazard and the actions 

being taken and the assistance being made available.

Operational Coordination Establish and maintain a unified and coordinated operational structure and process that appropriately integrates all critical stakeholders and supports the execution of core capabilities.

Critical Transportation Provide transportation (including infrastructure access and accessible transportation services) 

for response priority objectives, including the evacuation of people and animals, and the 

delivery of vital response personnel, equipment, and services to the affected areas.

Environmental Response/Health and Safety Ensure the availability of guidance and resources to address all hazards, including hazardous 

materials, acts of terrorism, and natural disasters, in support of the responder operations 

and the affected communities.

Fatality Management Services Provide fatality management services, including body recovery and victim identification to 

provide temporary mortuary solutions, sharing information with Mass Care Services for 

the purpose of reunifying family members and caregivers with missing persons/remains, 

and providing counseling to the bereaved.

Infrastructure Systems Stabilize critical infrastructure functions, minimize health and safety threats, and efficiently 

restore and revitalize systems and services to support a viable resilient community.

Mass Care Services Provide life-sustaining services to the affected population with a focus on hydration, feeding, 

and sheltering to those with the most need, as well as support for reunifying families.

Mass Search and Rescue Operations Deliver traditional and atypical search and rescue capabilities, including personnel services, 

animals, and assets to survivors in need, with the goal of saving the greatest number of 

endangered lives in the shortest time possible.

On-scene Security and Protection Ensure a safe and secure environment through law enforcement and related security and 

protection operations for people and communities located within affected areas and for 

all traditional and atypical response personnel engaged in lifesaving and life-sustaining 

operations.

Operational Communications Ensure the capacity for timely communications in support of security, situational awareness, 

and operations by any and all means available between affected communities in the impact 

area and all response forces.

Public and Private Services and Resources Provide essential public and private services and resources to the affected population and 

surrounding communities to include emergency power to critical facilities, fuel support for 

emergency responders, and access to community staples (eg, grocery stores, pharmacies, 

and banks) and fire and other first response services.

Public Health and Medical Services Provide lifesaving medical treatment via emergency medical services and related operations, 

and avoid additional disease and injury by providing targeted public health and medical 

support and products to all people in need within the affected area.

Situational Assessment Provide all decision makers with decision-relevant information regarding the nature and 

extent of the hazard, any cascading effects, and the status of the response.

Data source: Department of Homeland Security. National Response Framework. 2nd ed. Washington, DC: DHS; May 2013.

Coordinating agencies are identified for each of the 

emergency support functions at the federal level; local 

and state planners should identify assets for planning 

at their respective levels. Objectives for each of the 

core capabilities listed in the NRF are summarized in 

Table 4-1. Local planners will likely not have resources 

available to meet all of these objectives, but the list can 

assist them with determining what local assets need 

to be factored into a local response plan and what 

support requirements are needed to coordinate with 

neighboring communities or the private sector through 

support agreements, or requests from county, state, or 

federal partners.

The effective response to a catastrophic event will 

require meeting many of the core capability objectives 

through local assets, at least for initial response and 

later to augment state or federal response efforts. Local assets may include individuals, the private sector, 

nongovernmental organizations, and neighboring 

communities. 

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Individuals within a community possess talents 

and experience that can be organized for response 

through community organizations. Individuals 

should participate in community preparedness planning activities and develop household emergency 

plans.10 Individuals can also participate in FEMA’s 

Community Emergency Response Team Program, 

which educates people on how to prepare for hazards that may affect their area and trains them in 

basic disaster response skills, such as fire safety, light 

search and rescue, team organization, and disaster 

medical operations.11 Community Emergency Response Team Program trained individuals can play 

a critical role in assisting with local planning and 

response. Private sector entities can support local 

emergency management and should also participate 

in community preparedness planning activities.10

Private sector entities should also conduct continuity 

of operations planning within their own organization to establish a plan that will foster their continued support or service to the community while also 

preserving their livelihood. 

Nongovernmental organizations may factor into 

any level: local, state, or federal response. They manage 

volunteers and resources to support incident response 

through collaboration with responders, all levels of 

government, and other agencies and organizations.10

Neighboring communities can play a critical role 

in consequence management for a biological incident. 

Community dynamics can be significantly disrupted 

within the contamination zone of a biological incident, 

but life outside the contamination zone will continue 

with no change in requirement for services and support. 

If a community’s critical services are located within 

the contamination zone and are rendered inoperable 

because of real or perceived contamination, then mutual 

aid agreements among communities can fill critical gaps 

in the response effort as well as continuation of services 

to the unaffected parts of the community. 

When local resources are exhausted or prove to 

be inadequate, local authorities may seek county or 

state assistance; in some situations, local authorities 

may seek assistance directly from the federal government for non-Stafford Act incidents.10 Some federal 

departments or agencies, using funding sources other 

than the President’s Disaster Relief Fund, can conduct 

or lead federal response actions under their own 

authorities.10 Examples include immediate lifesaving assistance, wild-land firefighting, response to an 

agricultural disease, cybersecurity incidents, and oil 

and hazardous substance response operations. 10 The 

Secretary of the Department of Health and Human 

Services (DHHS) has the authority to take actions to 

protect the public health and welfare and declare a 

public health emergency.10

State governors are responsible for the public safety 

and welfare of their state’s residents. Their responsibilities and authorities include making, amending, 

or suspending orders or regulations associated with 

response; communicating to the public; coordinating 

with tribal governments; commanding the state military force (National Guard personnel not in federal 

service); coordinating assistance from other states; and 

requesting federal assistance.10 A state’s response to 

emergency situations is coordinated through the state 

emergency management agency. 

Numerous state departments and agencies have a 

role in response and recovery, but the National Guard 

is one of the governor’s key assets for a biological 

incident. National Guard members can be valuable to 

consequence management because of their expertise 

in emergency medical response; communications; 

logistics; search and rescue; civil engineering; and 

chemical, biological, radiological, and nuclear response, planning, and decontamination.10 Weapons 

of mass destruction/civil support teams are highly 

specialized National Guard units designed to provide 

unique capabilities for response to chemical, biological, 

radiological, or nuclear incidents, primarily in a Title 32 

operational status within Washington, DC, the United 

States, its territories, and its possessions.12

Federal financial aid or other support to response, 

recovery, and mitigation efforts are authorized following a Stafford Act emergency or major disaster 

declaration by the president. An emergency declaration is more limited in scope, provides fewer federal 

programs, and is not normally associated with recovery programs, but it may be used before an incident 

to mitigate the threat of a potential catastrophe.10 Most 

of the president’s authority under the Stafford Act has 

been delegated to the FEMA administrator through the 

Secretary of Homeland Security.10 A state’s governor 

may request federal assistance through the FEMA regional administrator when the situation is considered 

beyond the capabilities of the state and affected local 

government.10

The DHHS is the coordinating agency for the biological incident annex to the NRF. Federal government 

objectives for response to a biological incident—naturally occurring or as an act of terrorism—are as follows:

• detect the event through disease surveillance 

and environmental monitoring; 

• identify and protect the population(s) at risk; 

• determine the source of the disease; 

• assess the public health, law enforcement, and 

international implications; 

• control and contain any possible epidemic; 

• augment and surge public health and medical 

services; 

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• identify the cause and prevent the recurrence 

of any potential resurgence, additional outbreaks, or further spread of disease; and 

• assess the extent of residual biological contamination and conduct response, restoration, 

and recovery actions.13

Detection of a biological incident may be by the 

presentation of disease in humans or animals or environmental surveillance systems, or by acts of bioterrorism detected though the normal operations of other 

cooperating departments and agencies.13 The National 

Biosurveillance Integration System is a tool that supports detecting disease outbreaks by leveraging data 

from multiple surveillance systems that monitor human 

health, animal health, plant health, and food and water.13

Monitoring for dangerous pathogens in some heavily 

populated places is accomplished through the BioWatch 

program, which serves as an early detection and warning 

system.14 This program has been criticized for creating 

false alarms, but those criticisms probably come from 

individuals who do not fully understand the technologies 

used, intent of the program, and confirmatory process 

that follows an alert. DHS partners with public health 

laboratories through the Laboratory Response Network 

to rapidly confirm any alerts from BioWatch systems. 

Detection technologies need to be rapid and sensitive; they need to ensure that no false negatives occur. 

Specificity is ensured through the laboratory confirmatory process that follows. People will live with a few 

false alarms, but they will become sick and potentially 

die from false negative results at the detection level. It 

is unlikely that samples will be forwarded for confirmatory analysis if results are negative at the detection 

level, so these systems should be judged more on their 

potential to prevent false negatives than false positives. 

These systems are value added if they are not over interpreted before confirmatory analysis. Claims of false 

positives have been characterized as unsubstantiated, 

with more than 7 million tests performed by public 

health laboratories and no false positives.14

DHHS convenes a meeting of ESF #8 partners, after 

notification of a credible threat or disease outbreak, to 

assess the situation and determine appropriate public 

health and medical actions.13 If the threat or disease 

outbreak is suspected to be tied to a criminal or terrorist act, the Federal Bureau of Investigation will 

lead a concurrent criminal investigation and possibly 

establish a joint operations center.13 Joint operations 

centers are valuable to establishing unity of effort. 

Agencies with disparate primary objectives will be 

working simultaneously toward a common outcome, 

but do not always fully understand each other’s mission priorities; synchronization of efforts is critical to 

mission success. 

It is important for first responders to understand 

that they may be working within a crime scene 

and that all materials may have evidentiary value, 

but this fact cannot compromise mitigating the 

immediate threats to life and health. If a criminal 

or terrorist incident initially presents as disease in 

humans or animals, criminal intent may not be apparent for some time and evidence may already be 

compromised. The Laboratory Response Network 

is used to test samples whenever a credible threat 

of a biological crime or act of terrorism exists.13 If 

contamination of food is suspected the Food Emergency Response Network, a complementary system 

to the Laboratory Response Network, may be used 

for food sample analysis.13

Other federal agencies will support DHHS during 

a biological incident response. The DHS will serve 

as the incident coordinator. The Environmental 

Protection Agency will develop and implement 

sampling strategies when a potential for environmental contamination exists. The Department of 

Agriculture will provide support for an outbreak 

of an agriculturally significant zoonotic disease 

or human foodborne pathogen. Federal public announcements, statements, or press releases will be 

coordinated with the DHS Office of Public Affairs, 

consistent with ESF #15.13

An epidemic resulting from the introduction of a 

contagious biological agent into a population is one 

of the most significant—and likely the most dangerous—potential consequences of a biological incident. 

Effectively managing this potential consequence relies 

on the following:

• rapid detection, and identification and confirmation of the biological agent; 

• identification of the population at risk; 

• determination of how the agent is transmitted; 

• determination of appropriate medical countermeasures;

• administration of countermeasures; 

• rapid dissemination of safety information to 

the public; and 

• control and containment strategies. 

Planning must include worst-case scenario branches 

for mass casualties if early control measures are not 

effective and containment is not achieved, requiring 

augmentation and surging of health and medical 

resources in order to track and prevent additional 

disease outbreaks. 

DHHS assists partner public health and medical authorities with epidemic surveillance and coordination, 

and it will assess the need for increased surveillance. 

DHS, with partner organizations, coordinates timely, 

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consistent, accurate, and actionable information dissemination. The public health system, starting at the 

local level, initiates appropriate protective measures 

for the affected population, including all workers 

involved in incident response. DHHS, with partner organizations involved, evaluates the need for isolation, 

quarantine, or shelter-in-place measures to prevent 

spread of disease. If isolation and/or social distancing 

are recommended, the affected state’s governor may 

implement these measures under state or local legal 

authorities. Tribal leaders also possess this authority 

under tribal legal authority. 

DHHS may take appropriate federal actions to 

prevent the import or interstate spread of disease. 

If the source of the disease outbreak is identified as 

originating outside the United States, DHHS works 

with DHS and other agencies to identify and isolate 

persons, cargo, mail, or conveyances that may be 

contaminated. If it is determined that food, animals, 

and other agricultural products need to be quarantined, livestock or poultry need to be vaccinated 

or depopulated, and/or movement of animals and 

equipment need to be restricted, DHHS will work 

with the Department of Agriculture and other partner 

organizations. DHHS works through the Department of State to notify affected foreign governments 

if foreign nationals are subjected to isolation and/or 

quarantine.13

The ability to care for sick and/or potentially 

exposed people is one of the most critical response 

requirements that must be incorporated into predisaster response planning. The Strategic National 

Stockpile is a national repository of medical countermeasures, vaccines, and medical supplies stored 

in strategic locations.15 Division of Strategic National 

Stockpile personnel, from the Centers for Disease 

Control and Prevention’s Office of Public Health 

Preparedness and Response, will assist as local and 

state health departments prepare for receipt, distribution, and dispensing of medical countermeasures 

from the Strategic National Stockpile.15 These medical countermeasures, vaccines, and medical supplies 

are free to the public, and states have plans to receive 

and distribute them once federal and local authorities agree that they are needed.16 Strategic National 

Stockpile supplies include 12-hour push packages, 

CHEMPACKs (program that provides antidotes to 

nerve agents [three countermeasures used concomitantly] for prepositioning by state, local, and tribal 

officials), and federal medical stations. The 12-hour 

push packages contain 50 tons of a broad spectrum 

of medical assets and can be delivered to any state in 

the continental United States within 12 hours from 

the decision to deploy; if the incident requires additional or different supplies, they can be delivered 

within 24 to 36 hours.15 Federal medical stations 

are rapidly deployable and modular, stocked with 

beds and supplies to care for up to 250 patients for 

up to 3 days.15

As with other aspects of the integrated local, state, 

and federal response effort, local technical expertise 

and local planning will be critical to efficient and successful delivery of medical care to those who need it. 

Centers for Disease Control and Prevention developed 

the public health preparedness capabilities/national 

standards for state and local planning that can assist 

state and local public health officials with planning 

for this and other critical public health planning considerations. It identifies 15 public health preparedness 

capabilities under six domains17: 

1. biosurveillance; 

2. community resilience; 

3. countermeasures and mitigation; 

4. incident management; 

5. information management; and 

6. surge management. 

This downloadable document is an excellent planning guide that links planning and execution activities 

back to NRF emergency support functions and provides links to additional resources.

Beyond the challenge of medical countermeasure 

availability and distribution, a biological incident 

may challenge the ability of healthcare systems to 

adequately care for large numbers of patients that 

exceed local capabilities and capacities, and it will 

likely affect their ability to continue providing a 

standard of care to the local community for routine 

health issues. Maintaining medical system resiliency 

may require regional, state, or federal coordination 

and medical surge capacity and capability. The 

medical surge capacity and capability management 

system was developed to provide a systems-based 

approach for managing the complexity of mass casualty or complex incidents.18 Surge capacity is the 

ability to respond to a markedly increased number 

of patients. Surge capability is the ability to address 

unusual or very specialized medical needs. The 

medical surge capacity and capability management 

system is consistent with the National Incident Management System and guides public health and medical response through a six-tier approach, escalating 

from management of individual healthcare assets 

to federal support to state, tribal, and jurisdictional 

management. 

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RECOVERY

will remain important to identify additional cases of 

human or animal disease and potential contamination 

spread that will need to be included in decontamination efforts. Decontamination can and will likely be 

challenging. Its effectiveness will depend on accurate 

identification of the contaminating infectious agent 

or toxin; assessment of primary and secondary areas 

of contamination; and selection of suitable decontamination reagents, equipment, and methods that 

factor in effectiveness for the contaminating agent 

and the environment. Appropriate subject matter 

experts should be included in planning and executing 

decontamination. 

Personnel involved in recovery operations will not 

have the benefit of established clearance strategies for 

reoccupation of contaminated facilities or resumed 

use of contaminated equipment for all potential 

biological agents. Members of the Environmental 

Protection Agency and the Centers for Disease Control and Prevention published an interim clearance 

strategy for a building or an outdoor environment 

after an incident involving Bacillus anthracis in July 

2012. It was determined that no detection of viable 

spores is the best practicable clearance goal,19 which 

is a sound goal for B anthracis as well as many other 

potential biological agents. It infers that the agent 

identification technology used will identify viability 

as well as continued presence of the pathogen on or 

in the sampled item. 

Some of the more sensitive agent identification 

technologies (nucleic acid amplification and antigen detection) will not demonstrate agent viability. 

Agents killed or neutralized during decontamination may still be detected by these technologies and 

not properly inform clearance decisions. Cleanup 

procedures could be unnecessarily prolonged with 

no added benefit if decisions are being made based 

on technologies that do not aid the risk assessment 

procedure by demonstrating agent viability. Local 

or state public health officials or property owners 

will likely make the final decision on clearance.19

However, the lack of established standards and 

complexity of this decision process will likely 

necessitate the support of external subject matter 

experts.

Establishing transportation routes becomes critical during both response and recovery to facilitate 

response and recovery mitigation activities as well 

as continue providing critical services and support 

inside the contaminated area. Once a biological incident has occurred, containment becomes important 

Recovery operations focus on returning the affected 

region or entity, as closely as possible, to predisaster 

conditions. Initiation of recovery efforts does not require full completion of response operations, but the 

transition process must be well synchronized with any 

continuing response efforts. Many components of the 

response effort will also influence recovery activities. 

As healthcare transitions from emergent and temporary medical care, activities must ensure continuity of 

care and reestablishment of any disrupted healthcare 

capabilities. Continuity of care may need to be established through temporary facilities until services are 

fully restored; candidate facilities should be identified 

during consequence management planning activities. 

Surveillance should be initiated during response, as discussed in the previous section, and continued through 

recovery until health officials determine that the discovery of new cases has met criteria for discontinuation.

Effective messaging by public health professionals 

can serve to mitigate public fear and prevent panic. 

Several people will be identified, throughout response 

and recovery, who may benefit from counseling and 

behavioral health services. These services should be 

restored or made available as soon as possible. Lessons 

learned from previous disasters suggest that during 

the transition and recovery period, public fear can 

increase. During this period people will have gained 

awareness of the morbidity and mortality associated 

with the infectious agent or toxin and will have continued—possibly escalating—fear about exposure to 

this invisible threat. 

Ineffective messaging may have contributed to 

public fear and panic during the 2001 Amerithrax 

incident. Many people thought anthrax was a contagious disease, and because the infectious agent was 

delivered in a powdered form there was widespread 

fear of powders in general. People were more aware 

of powders and powder-appearing residues after the 

incident. Powders associated with many normal activities that went unnoticed or created no concern before 

the incident suddenly created concern, fear, and panic. 

The US Army Medical Research Institute of Infectious 

Diseases and many other laboratories involved in the 

recovery effort received thousands of samples for 

analysis that normally have an innocuous powder associated with them. Effective accurate communications 

may have mitigated some of these concerns and will 

remain important throughout any recovery. 

Exposed populations and contaminated buildings, 

equipment, and environments will likely be identified 

during the response effort, but continued surveillance 

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Medical Aspects of Biological Warfare 

to all aspects of the management strategy. Factoring containment considerations into all subsequent 

planning will prevent the incident from growing in 

scale and magnitude, minimizing impact on human 

health, infrastructure, and economy. Strategies will 

vary depending on the situation and conditions, but 

some basic principles can be applied to all. Whenever 

one is dealing with biological contamination, it is 

beneficial to establish at least three zones: 

1. known or high probability to be contaminated 

(hot zone); 

2. not expected or low probability to be contaminated (warm zone); and 

3. expected to be clean (cold zone). 

Operational procedures should be established 

for each zone that facilitate the movement of necessary supplies, personnel, and equipment to sustain 

operations and facilitate recovery without spreading 

contamination. If a clean corridor cannot be established through the warm zone to the hot zone, handoff 

procedures will need to be established for cross-zone 

movement. Decontamination procedures at each handoff point will need to be established for any movement 

from hot zone to warm zone and from the warm zone 

to the cold zone. One strategy may be to have dedicated 

equipment in each zone that will facilitate the movement of personnel and supplies from the cold zone to 

the hot zone and sustain operations in the warm zone 

and hot zone. Personal protective equipment requirements must also be established for each zone to prevent 

secondary contamination to workers. 

The NDRF promotes nine core principles for recovery success: 

1. individual and family empowerment; 

2. leadership and local primacy; 

3. predisaster recovery planning; 

4. partnerships and inclusiveness; 

5. public information; 

6. unity of effort; 

7. timeliness and flexibility; 

8. resilience and sustainability; and 

9. psychological and emotional recovery.2

It promotes a concept of all-community involvement in recovery efforts to ensure that no groups 

of people and their unique interests are excluded 

during the recovery effort and that services are made 

equally available to everyone as all affected members 

of a community attempt to rebound from their losses. 

It affirms that local leaders and local governments 

maintain a primary role even when their response 

capabilities have been overwhelmed and state or 

federal assistance is required. It recognizes that 

partnerships and collaborations with unity of effort 

are essential to successful recovery and emphasizes 

that compliance with the principles of equal opportunity and civil rights must be upheld. It further 

emphasizes the importance of clear, consistent, culturally appropriate, and frequent communications 

to the affected public. Timeliness and flexibility are 

emphasized to minimize missed opportunities and 

foster the ability to adapt to changing conditions. 

It recognizes that recovery can be negatively affected by cascading effects and additional hazards, 

emphasizing the significance of risk management 

to enhance resilience and sustainability practices 

to reconstruct the environment and revitalize the 

economic, social, and natural environments. Psychological and emotional recovery is recognized 

as vital to individuals, families, and communities. 

Local governments are responsible for planning and 

managing a community’s recovery from all disasters.2

They shoulder the burden of preparing hazard mitigation and recovery plans, raising hazard awareness, 

and educating their people on resources available to 

enhance resilience. Even though state and federal 

standards exist, the local government decides whether 

to adopt, codify, and enforce mitigation measures. 

Individuals, families, and businesses will look to local 

leaders for support during disasters, and local governments should establish continuity of government 

and continuity of operations plans. They are at risk of 

becoming overwhelmed and will likely need assistance 

from state and federal offices for critical staffing and 

recovery expertise. A critical local asset during any 

biological incident is the local or county public health 

agency, which will have established contingency plans 

and can assist with coordinating medical surge when 

needed.

States lead, manage, and drive the overall recovery process; they coordinate recovery activities that 

include providing financial and technical support.2

They serve as a conduit to local and tribal governments for federal recovery assistance programs, and 

they may develop programs or secure funding to 

finance or implement recovery projects. States can 

also reassign existing resources to facilitate recovery, 

and they play a critical role in strategic messaging to enhance public awareness. The state public 

health agency will play a critical role in messaging 

and coordinating medical assistance to the affected 

community. It is critical that state offices remain mission capable during a disaster, and they should also 

develop and maintain continuity of government and 

continuity of operations plans.

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Consequence Management: The Local and National Response

The federal government may use the NDRF to engage necessary and available department and agency 

capabilities to support local recovery efforts when a 

disaster exceeds the capacity of the state and tribal 

resources or affects federal property or national security interests.2

 Federal support is important when 

local and state resources are overwhelmed, especially 

during the early weeks following a large-scale disaster or catastrophic incident; the duration and extent 

of federal support will be partially determined by 

scale and enduring impact of the disaster. The federal 

government also plays a critical role in messaging to 

enhance public awareness about the threat and to 

inform stakeholders about federal grants and loans 

that can assist recovery efforts. The lead federal agency 

for coordination of health and social services during 

recovery is the DHHS. 

Similar to ESFs in the NRF outlining federal assistance for disaster response, the NDRF identifies 

multiagency coordinated recovery support functions 

in the following areas: 

• community planning and capacity building; 

• economics; 

• health and social services; 

• housing; 

• infrastructure systems; and 

• natural and cultural resources. 

Each annex outlines pre- and postdisaster activities 

as well as a list of objectives. The recovery support 

functions develop guidance and standard operating 

procedures for rapid activation to support community 

recovery.2

SUMMARY

Consequence management has historically 

received the least amount of planning emphasis 

and has not been adequately tested through robust exercises. By the nature of the problem it is 

complex, involves multiple agencies, and spans a 

considerable amount of time to exercise through 

response and recovery operations. The national 

framework documents offer a template for core 

capability development that can lead to readiness 

across a broad spectrum of potential disasters, 

and they can facilitate robust planning at the local 

level. These documents are already being used to 

develop plans at the state and federal levels. Unity 

of effort, which is critical to both effective response 

and recovery, can be developed through multiagency exercises. Historically, response without 

continuation through full recovery has been exercised. Emergency response exercises can be time 

compressed, but should span the full spectrum of 

response and recovery, so agencies will be prepared 

to work together when a disaster strikes. 

Biological incidents are unique challenges. It is unlikely to know when a biological agent is dispersed or 

when the index case of a pandemic crosses a nation’s 

border. Buildings will not be flattened, but they may 

be unsuitable for human occupation for an extended 

time, compromising critical services to the community, 

state, or nation. Economic impact could be significant 

and devastating to individuals and industries. A small 

focal dispersion of a biological agent could lead to 

broad impact with significant morbidity, mortality, 

and public fear if response and recovery efforts are 

not efficiently implemented to identify, contain, treat, 

protect, and clean. 

Consequence management is critical to mitigating 

the magnitude of impact a disaster has on a community, state, and nation. History has proven that 

all disasters or terrorist acts cannot be prevented, but 

through effective consequence management the impact 

of both can be minimized and the terrorist’s aim of 

maximal disruption can be defeated. Most importantly, 

lives can be saved.

REFERENCES

1. Serial No. 65: Homeland Security: The Balance Between Crisis and Consequence Management Through Training and Assistance: 

Hearing before the Subcommittee on Crime, Terrorism, and Homeland Security of the Committee on the Judiciary House of Representatives, One Hundred Eighth Congress, First Session on HR 2512, HR 3266, and HR 3158. Washington, DC: Government 

Printing Office; November 20, 2003. https://www.hsdl.org/?view&did=446489. Accessed October 30, 2015.

2. Department of Homeland Security. National Disaster Recovery Framework: Strengthening Disaster Recovery for the Nation. 

Washington, DC: DHS; September 2011. https://www.hsdl.org/?view&did=687785. Accessed October 30, 2015.

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Medical Aspects of Biological Warfare 

3. Department of Homeland Security. Overview of the National Planning Frameworks. Washington, DC: DHS; May 2013:1. 

https://www.hsdl.org/?view&did=736071. Accessed October 30, 2015. 

4. Department of Homeland Security. Threat and Hazard Identification and Risk Assessment Guide: Comprehensive Preparedness Guide (CPG) 201, Second Edition. Washington, DC: DHS; August 2013. https://www.fema.gov/medialibrary/assets/

documents/26335. Accessed October 30, 2015. 

5. Department of Health and Human Services. Terrorism and Other Public Health Emergencies: A Reference Guide for Media. 

Washington, DC: DHHS; September 2005.http://permanent.access.gpo.gov/lps65017/HHSMedisReferenceGuideFinal.

pdf. Accessed October 30, 2015.

6. Department of Homeland Security. National Incident Management System. Washington, DC: DHS; December 2008:9. 

https://www.hsdl.org/?view&did=232899. Accessed October 30, 2015.

7. Chess C, Clarke L. Facilitation of risk communication during the anthrax attacks of 2001: the organizational backstory. 

Am J Public Health. 2007;97:1578–1583.

8. Sarmiento G. Former AMI building declared free of anthrax contamination. Palm Beach Post. February 8, 2007. http://

www.ph.ucla.edu/epi/bioter/formeramibldganthraxfree.html. Accessed October 30, 2015.

9. Department of Homeland Security. National Preparedness Goal. 1st ed. Washington, DC: DHS; September 2011:1. https://

www.hsdl.org/?view&did=689207. Accessed October 30, 2015.

10. Department of Homeland Security. National Response Framework. 2nd ed. Washington, DC: DHS; May 2013. http://

www.dhsem.state.co.us/sites/default/files/2013%20National%20Response%20Framework.pdf. Accessed October 30, 

2015.

11. Department of Homeland Security. Community Emergency Response Teams (CERT). Washington, DC: DHS; January 16, 

2014. http://www.fema.gov/community-emergency-response-teams. Accessed October 30, 2015.

12. Weapons of Mass Destruction Civil Support Team Management. Arlington, VA: Departments of the Army and the Air Force, 

National Guard Bureau; January 12, 2006. NGR 500-3/ANGI 10-2503. http://www.ngbpdc.ngb.army.mil/pubs/10/

angi10_2503old.pdf. Accessed October 30, 2015.

13. Department of Health and Human Services. Biological Incident Annex. Washington, DC: DHHS; 2008. https://www.

hsdl.org/?view&did=236117. Accessed October 30, 2015.

14. Garza A. The Truth About BioWatch: The Importance of Early Detection of a Potential Biological Attack. Washington, DC: 

Department of Homeland Security, July 12, 2012. http://www.dhs.gov/blog/2012/07/12/truth-about-biowatch. Accessed 

October 30, 2015.

15. Centers for Disease Control and Prevention. Division of Strategic National Stockpile Fact Sheet. Atlanta, GA: CDC, 

Office of Public Health Preparedness and Response; June 2014. www.cdc.gov/phpr/documents/DSNS_fact_sheet.pdf. 

Accessed October 30, 2015.

16. Centers for Disease Control and Prevention. Strategic National Stockpile. Atlanta, GA: CDC, Office of Public Health 

Preparedness and Response; July 10, 2014. http://cdc.gov/phpr/stockpile/stockpile.htm. Accessed October 30, 2015.

17. Centers for Disease Control and Prevention, Department of Health and Human Services. Public Health Preparedness 

Capabilities: National Standards for State and Local Planning. Washington, DC: CDC, DHHS; March 2011:2. http://www.

cdc.gov/phpr/capabilities/DSLR_capabilities_July.pdf. Accessed October 30, 2015.

18. Department of Health and Human Services. Medical Surge Capacity and Capability: A Management System for Integrating Medical and Health Resources During Large-Scale Emergencies, Second Edition. Washington, DC: DHHS; September 

2007:1–3. http://www.phe.gov/Preparedness/planning/mscc/handbook/Documents/mscc080626.pdf. Accessed October 

30, 2015.

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Consequence Management: The Local and National Response

19. Centers for Disease Control and Prevention and Environmental Protection Agency. Interim Clearance Strategy for 

Environments Contaminated with Bacillus anthracis. Washington, DC: CDC and EPA; July 2012:4. http://www.epa.gov/

osweroe1/docs/misc/cdc-epa-interim-clearance-strategy.pdf. Accessed October 30, 2015.

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Medical Management of Potential Biological Casualties: A Stepwise Approach

Chapter 5

MEDICAL MANAGEMENT OF 

POTENTIAL BIOLOGICAL CASUALTIES: 

A STEPWISE APPROACH

THEODORE J. CIESLAK, MD*

INTRODUCTION

10-STEP APPROACH TO CASUALTY MANAGEMENT

Step 1: Maintain a Healthy Index of Suspicion

Step 2: Protect Yourself

Step 3: Save the Patient’s Life (Primary Assessment)

Step 4. Disinfect or Decontaminate as Appropriate

Step 5: Establish a Diagnosis (Secondary Assessment)

Step 6: Provide Prompt Therapy

Step 7: Institute Proper Infection Control Measures

Step 8: Alert the Proper Authorities

Step 9: Conduct an Epidemiological Investigation and Manage the Psychological 

Aftermath of a Biological Attack 

Step 10: Maintain a Level of Proficiency

SUMMARY

*Colonel, Medical Corps, US Army; Pediatric Infectious Diseases Physician, San Antonio Military Medical Center, Department of Pediatrics, 3551 Roger 

Brooke Drive, Fort Sam Houston, Texas, 78234

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INTRODUCTION

Response to a biological attack is relatively straightforward when the etiologic agent employed is known. 

A larger problem arises, however, in the context of 

diagnostic uncertainty. In some cases, an attack may 

be threatened or suspected, but whether such an attack 

has, in fact, occurred can remain unclear. Moreover, 

it may be uncertain whether casualties in certain 

situations arise from exposure to a biological agent, a 

chemical or radiological agent, a naturally occurring 

infectious disease process, or toxic industrial exposure, 

or may simply reflect a heightened awareness of background disease within a community or population. Experience with West Nile virus,1

 severe acute respiratory 

syndrome,2 pneumonic tularemia,3,4 and monkeypox5

highlight this dilemma. In each of these cases, the possibility of bioterrorism was properly raised, although 

each outbreak ultimately proved to have a natural 

origin. In some instances, proof of such an origin may 

be difficult or impossible to attain, providing “plausible 

deniability,” precisely the reason some belligerents may 

opt to employ biological agents. This chapter provides 

a structured framework for dealing with outbreaks 

of unknown origin and etiology on the battlefield, as 

well as in a potential bioterrorism scenario involving 

military support installations or the civilian populace.

In responding to the unknown, it is helpful in many 

situations to employ a standardized, stepwise approach. This is especially true in the setting of a medical 

mass casualty event (MASCAL), where the use of such 

an approach (as advocated by the Advanced Trauma 

Life Support [ATLS] model sponsored by the American 

College of Surgeons6

) is already well accepted and 

practiced. It is also especially true under austere or 

battlefield conditions. Although major theater-level 

and continental United States-based military medical centers (and research institutions, such as the US 

Army Medical Research Institute of Infectious Diseases 

[USAMRIID] and US Army Medical Research Institute of Chemical Defense) may possess sophisticated 

diagnostic and response capabilities, providers on the 

battlefield and at lower-role medical treatment facilities are typically required to make rapid therapeutic 

decisions based on incomplete information and with 

little immediate support. Civilian clinicians, first responders, and public health personnel practicing in 

rural or remote areas during a terrorist attack would 

face similar decision-making challenges. In the setting 

of a biological (or chemical or radiological) attack, 

similar to the setting of a MASCAL trauma event, 

such decisions may have life-and-death implications. 

In such situations, a stepwise or algorithmic approach 

becomes invaluable.

USAMRIID has developed a 10-step approach to 

managing casualties that might result from biological 

warfare or terrorism. Many facets of this approach 

may be helpful in dealing with potential chemical 

or radiological casualties as well. In today’s complex 

world, it is no longer adequate for most clinicians 

and medical personnel to simply understand disease 

processes. Rather, these personnel, whether military or 

civilian, must have tactical, operational, and strategic 

knowledge of threat response—and, in fact, of disaster 

response in general—as it applies to weapons of mass 

destruction. Tactical response concerns those elements 

of diagnosis and treatment of specific diseases that 

traditionally have been the realm of the individual 

practitioner. Operational response can be thought of 

as involving the mechanisms by which the provider 

interacts with his or her institution (hospital, clinic, 

medical unit) to provide mass care during a disaster. 

Strategic response involves system-wide disaster 

preparedness and response. In a civilian setting, this 

includes mechanisms by which state and federal 

disaster response elements might become involved. 

Medical personnel today need to have at least a basic 

understanding of operational and strategic response 

in addition to a firm grounding in tactical medical and 

public health intervention. The first 7 steps of this 10-

step approach deal predominately with tactical issues 

(ie, at the level of the individual provider). Steps 8 and 

9 transition into operational and strategic response 

(ie, at the level of the institution and of the system, 

as a whole). The derivation of the 10-step approach is 

reported elsewhere,7–10 and a condensed version appears in recent editions of USAMRIID’s Blue Book.

11 It 

is expanded upon here.

10-STEP APPROACH TO CASUALTY MANAGEMENT

Step 1: Maintain a Healthy Index of Suspicion 

In the case of chemical warfare or terrorism, the 

intentional nature of an attack is often evident. In this 

case, victims would likely be tightly clustered in time 

and space; they would succumb in close proximity 

(both temporally and geographically) to a dispersal 

device. Complicating the discovery of the intentional 

nature of a biological attack, however, is the fact that 

biological agents possess inherent incubation periods, 

while conventional, chemical, and nuclear weapons do 

not. These incubation periods, typically of several days 

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Medical Management of Potential Biological Casualties: A Stepwise Approach

(but up to several weeks in the case of agents such as 

Coxiella burnetii and the Brucellae), allow for the wide 

dispersion of victims in time and space. Additionally, 

they make it likely that the first responder to a biological attack would not be the firefighter, police officer, 

paramedic, or other traditional first responder, but 

rather primary care providers, hospital emergency 

departments, and public health officials. In such circumstances, maintaining a healthy index of suspicion 

is imperative. 

In some instances, maintaining an index of suspicion might be simplified by the fact that diseases 

caused by biological agents may present with specific 

characteristic clinical findings, which allow for a very 

limited differential diagnosis. The hallmark of inhalational anthrax is a widened mediastinum, a clinical 

finding seen in few naturally occurring conditions. 

With botulism, the hallmark presentation is that of a 

descending, symmetric, flaccid paralysis. Whereas an 

individual patient with flaccid paralysis might prompt 

consideration of disorders such as Guillain-Barré syndrome, Eaton-Lambert syndrome, poliomyelitis, and 

myasthenia gravis, the near-simultaneous presentation of multiple patients with flaccid paralysis should 

quickly lead one to a diagnosis of botulism. Similarly, 

patients with plague and melioidosis may exhibit hemoptysis in the later stages of illness. Such a finding 

is uncommon among previously healthy individuals, 

but can be caused by tuberculosis, staphylococcal and 

Klebsiella pneumonia, carcinoma, and trauma. Multiple 

patients with hemoptysis, however, should prompt 

consideration of a plague or melioidosis diagnosis. 

Smallpox is characterized by a very unique exanthem, 

perhaps evocative of Varicella or syphilis in its earliest 

stages, but readily distinguishable from these entities 

as it progresses. 

Yet, by the time each of these characteristic findings 

develops, treatment is less likely to be effective. Therapy is thus best instituted during the incubation or prodromal phases of these diseases if it is to be beneficial. 

Unfortunately, during their prodromes, these diseases 

are likely to appear as undifferentiated febrile illnesses, 

difficult, if not impossible, to distinguish from myriad 

other common infectious diseases. Similarly, many 

other diseases potentially arising from a biological 

attack (such as tularemia, brucellosis, melioidosis, Q 

fever, and Venezuelan equine encephalitis) may appear 

simply as undifferentiated febrile illnesses throughout 

their course. Prompt diagnosis and targeted therapy is 

thus possible only with a very high index of suspicion.

Epidemiological clues can lead a clinician to suspect 

that a disease outbreak may have been intentional (Exhibit 5-1).12 Large numbers of victims tightly clustered 

in time and space, or limited to a discrete population, 

should raise suspicion. Similarly, unexpected deaths 

and cases of unexpectedly severe illness merit concern. 

An outbreak of a disease not typically seen in a specific 

geographic location, in a given age group, or during a 

certain season, likewise warrants further investigation. 

Simultaneous outbreaks of a disease in noncontiguous 

areas should prompt one to consider an intentional 

release, as should simultaneous or sequential outbreaks of different diseases in the same locale. Even a 

single case of rare disorders, such as anthrax or certain 

viral hemorrhagic fevers (Ebola, Marburg, Lassa, and 

many others) would be suspicious, and a single case of 

smallpox, because it no longer occurs naturally, would 

almost certainly represent an intentional release. The 

presence of dying animals (or the simultaneous occurrence of zoonotic disease outbreaks among humans 

and animals) might provide evidence of an unnatural 

aerosol release. Evidence of a disparate attack rate 

between those known to be indoors and outdoors at 

a given time should also be sought and evaluated. 

EXHIBIT 5-1

EPIDEMIOLOGICAL CLUES TO A 

BIOTERRORIST ATTACK

• Presence of an unusually large epidemic

• High infection rate

• Disease limited to a discrete population

• Unexpected severity of disease

• Evidence of an unusual route of exposure

• Disease in an atypical geographic locale

• Disease occurring outside normal transmission seasons

• Disease occurring in the absence of usual 

vector

• Simultaneous outbreaks of multiple 

diseases

• Simultaneous occurrence of human and 

zoonotic disease

• Unusual organism strains

• Unusual antimicrobial sensitivity patterns

• Disparity in attack rates among persons 

indoors and outdoors

• Terrorist claims

• Intelligence reports

• Discovery of unusual munitions

Data source: Pavlin JA. Epidemiology of bioterrorism. 

Emerg Infect Dis. 1999;5:528–30.

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Medical Aspects of Biological Warfare 

Finally, intelligence reports, terrorist claims, and the 

discovery of aerosol spray devices would obviously 

lend credence to the theory that a disease outbreak 

was of sinister origin. 

On the modern battlefield, an array of developing 

technology is available to assist clinicians, preventive 

medicine and chemical corps personnel, operators, 

and commanders in maintaining their index of suspicion through early “stand-off” detection of biological threats. The Portal Shield is the Department 

of Defense’s (DoD’s) first automated biological detection system, and was designed to provide fixed-site 

protection to air and port facilities. Portal Shield is 

equipped with modular sensors capable of simultaneously assaying for eight different agents and providing 

presumptive identification within about 25 minutes. 

The Biological Integrated Detection System, a system 

mounted on a high-mobility multipurpose wheeled 

vehicle, is equipped with samplers, an aerodynamic 

particle sizer, a flow cytometer, and a chemical biological mass spectrometer. The Joint Biological Point 

Detection System integrates into the M31A2 Biological 

Integrated Detection System platform (Figure 5-1) to 

permit rapid, real-time detection of 10 separate biological threat agents on the battlefield; the system is 

capable of definitively identifying biowarfare threat 

agents within 18 minutes. The Joint Biological Agent 

Identification and Diagnostic Systems (JBAIDS) is a 

reusable, portable, and modifiable biological agent 

identification and diagnostic system capable of rapid, 

reliable, and simultaneous identification of multiple 

biological agents and other pathogens of operational 

concern. The JBAIDS anthrax, tularemia, plague, and 

Q fever detection systems are cleared by the Food and 

Drug Administration (FDA) for diagnostic use. Until 

these technologies are refined, validated, and made 

widely available, though, those tasked with responding to an attack must rely on clinical, epidemiological, and intelligence clues to maintain their index of 

suspicion.

Step 2: Protect Yourself 

Providers who themselves become casualties are of 

little use to their patients. Before approaching casualties of biological or chemical warfare or victims of a 

potential terrorist attack, clinicians should be familiar 

with basic means of self-protection. Such protective 

measures generally fall into one of three categories: 

(1) physical protection, (2) chemical protection, and 

(3) immunologic protection. Under a given set of circumstances, clinicians and laboratory personnel might 

appropriately avail themselves of one or more of these 

forms of protection.

Physical Protection

Since the beginning of modern gas warfare on the 

battlefields near Ypres, Belgium, in 1915, physical 

protection during military operations has involved 

gas masks and, more recently, charcoal-impregnated 

chemical protective overgarments. Although militarystyle protective clothing and masks were designed with 

chemical agent protection in mind, they are capable of 

offering protection against biological agents as well. 

Although some countries have advocated the issuance 

of military-style protective masks and ensembles to 

civilians (eg, the Israeli government has issued masks 

to its general populace), such items, even if offered, 

would likely be unavailable to civilians at the precise 

moment of agent release; the unannounced release of 

odorless and colorless biological agents by belligerents or terrorists would afford no opportunity to don 

protective gear, even if it were available. Furthermore, 

misuse of protective equipment in the past has led to 

fatalities, including cases of infants and adults suffocating in protective ensembles.13,14 Although military masks such as the M40/42, M45, and M50 series 

provide ample protection against inhalation hazards 

posed by chemical and biological weapons as well as 

against radioactive dust particles, they add heat stress 

and are potentially mission-degrading. Moreover, a 

simple surgical mask will usually afford adequate 

protection against inhalation of infectious aerosols 

of virtually any of the biological agents typically mentioned in a terrorism context. An important exception 

might be smallpox, in which case a high-efficiency 

Figure 5-1. The Biological Integrated Detection System 

(BIDS) is a semi-automated biological agent detection/

identification suite mounted on a dedicated heavy high 

mobility multipurpose wheeled vehicle. The system uses 

multicomplimentary bio-detection technologies.

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particulate air (HEPA) filter mask would be ideal. With 

the exception of smallpox, pneumonic plague, and certain viral hemorrhagic fevers, the agents in the Centers 

for Disease Control and Prevention’s (CDC’s) categories 

A and B (Exhibit 5-2) are not contagious via the respiratory route. Respiratory protection is thus necessary 

when operating in an area of primary release, but would 

not be required in most patient-care settings (see step 7). 

Chemical Protection

During Operations Desert Shield and Desert Storm, 

tens of thousands of US troops were given pyridostigmine under an emergency-use authorization, and in 

early 2003, the FDA gave its final approval for the use 

of pyridostigmine bromide as preexposure prophylaxis 

against intoxication with soman, an organophosphatebased chemical nerve agent. It is conceivable, given 

credible and specific intelligence, that similar strategies might be employed against biological weapons. 

For example, if a specific terrorist group possessing a 

specific weaponized agent were known to be operating 

in a given locale, public health authorities might conceivably contemplate the widespread distribution of 

an appropriate prophylactic antibiotic. Obviously, the 

opportunities to employ such a strategy are likely to 

remain few and far between, and the logistics of doing 

so would be exceedingly difficult in a civilian setting.

Immunologic Protection

For the near future, active vaccination is likely 

to provide one of the most practical methods for 

administering preexposure prophylaxis against 

biological attack. In the military, decisions regarding vaccination policy are typically made through 

the office of the Assistant Secretary of Defense for 

Health Affairs, with input from high-level military 

medical, public health, and intelligence sources. 

The decision to offer a specific vaccine in a specific 

circumstance is a complex one that must take into 

account a careful risk-benefit calculation. During 

Operations Desert Shield and Desert Storm, some 

150,000 service members received at least one dose 

of anthrax vaccine, while about 8,000 received a 

botulinum toxoid vaccine. Since 1998, the US military has intermittently employed force-wide anthrax vaccination, and since 2003 has administered 

smallpox vaccine to deploying troops and certain 

medical response teams. 

In a civilian counter-terrorism context, the decision to employ a specific vaccine is even more difficult and complex. Factors that would influence a 

decision by public health officials to recommend 

vaccination include intelligence (eg, how likely or 

plausible is an attack? How imminent is the threat? 

How specific is the threat?), vaccine safety, vaccine 

EXHIBIT 5-2

CRITICAL AGENTS FOR HEALTH PREPAREDNESS

Category A* Category B† Category C‡

• Variola virus

• Bacillus anthracis

• Yersinia pestis

• Botulinum toxin

• Francisella tularensis

• Filoviruses and arenaviruses

• Coxiella burnetii

• Brucellae

• Burkholderia mallei

• Burkholderia pseudomallei

• Alphaviruses

• Certain toxins (ricin, staphylococcal enterotoxin B, trichothecenes)

• Food safety threat agents 

(Salmonellae, Escherichia coli

O157:H7)

• Water safety threat agents (Vibrio cholerae, etc)

Other biological agents that may 

emerge as future threats to public 

health, such as:

• Nipah virus

• Hantaviruses

• Yellow fever virus

• Drug-resistant tuberculosis

• Tick-borne encephalitis

*

Agents with high public health impact requiring intensive public health preparedness and intervention.

†

Agents with a somewhat lesser need for public health preparedness.

‡

Other biological agents that may emerge as future threats to public health.

Data source: Centers for Disease Control and Prevention. Biological and chemical terrorism: strategic plan for preparedness and 

response. MMWR. 2000;49(RR-04):1–14.

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availability, disease consequences (ie, is the threat 

from a lethal agent or from an incapacitant?), and the 

availability of postexposure prophylaxis or therapy. 

Recently, civilian public health and policy planners 

have given extensive consideration to the widespread 

distribution of anthrax and smallpox vaccines.

Anthrax. Anthrax Vaccine, Adsorbed (AVA, BioThrax; Emergent BioSolutions, Lansing MI) is a fully 

licensed product, approved by the FDA in 1970. The 

vaccine consists of a purified preparation of protective antigen, a potent immunogen necessary for entry 

of key anthrax toxin components (lethal and edema 

factors) into mammalian cells. Administered alone, 

protective antigen is nontoxic. In a large controlled 

trial, AVA was effective in preventing cutaneous anthrax among textile workers.15 Based on an increasing 

amount of animal data, there is every reason to believe that this vaccine is quite effective at preventing 

inhalational anthrax as well.16 Moreover, well over 

20 clinical studies, surveys, and reports now attest to 

the safety of AVA,17,18 and the FDA has reaffirmed the 

vaccine as being safe and effective in light of those 

studies.19 Nonetheless, although widespread use of 

AVA has occurred within the US military (as of January 2014, more than 12.1 million doses of AVA had 

been given to more than 2.4 million service members), 

logistical and other considerations make large-scale 

civilian vaccination impractical at present. The vaccine is licensed as a five-dose series, given at 0 and 4 

weeks, and at 6, 12, and 18 months. Yearly boosters are 

recommended for those at ongoing risk of exposure. 

Further complicating any potential civilian anthrax 

vaccination strategy is the fact that AVA is approved 

by the FDA only for individuals 18 to 65 years old. 

Although a large-scale preexposure offering of AVA 

to the general public might thus be problematic, some 

have recommended that a three-dose series of AVA 

(given at time zero and at 2 and 4 weeks after the 

initial dose), combined with 60 days of antibiotics 

under an investigational new drug (IND) protocol or 

emergency use authorization, might be an acceptable 

alternative to longer (60–100 days) antibiotic courses 

alone for postexposure prophylaxis against inhalational anthrax.20 This recommendation was based 

on nonhuman primate challenge studies; no human 

studies currently exist to support such a strategy, 

and AVA is not licensed by the FDA for postexposure 

prophylaxis or therapy.

Smallpox. Widespread vaccination against smallpox is equally controversial and problematic. Nonetheless, in 2002, President George W. Bush announced 

a plan to vaccinate selected American healthcare 

workers and military personnel. Within the DoD, 

service members deploying to locations thought at 

risk for biological attack and members of designated 

smallpox epidemiological and clinical response teams 

were selected for vaccination. The program includes 

prevaccination screening to exclude members with 

vaccine contraindications or household contacts at 

risk, instruction on vaccine site care and potential 

complications, and mandatory follow-up. As of January 10, 2014, over 2.4 million military response team 

members, hospital workers, and operational forces 

had been vaccinated, with one death occurring due to 

a lupus-like illness. Although the emergence of myopericarditis (there were 161 confirmed, suspected, 

or probable cases among 1.4 million vaccinees as of 

January 2008) as a complication of vaccination21 led 

to a revision of prevaccine screening (candidates with 

multiple cardiac risk factors are now excluded), rates 

of other adverse reactions were low. Cases of autoinoculation or transmission to household and other 

contacts have been rare.22–24 One case of progressive 

vaccinia occurred in a primary vaccine recipient,25 and 

three cases of eczema vaccinatum occurred among 

contacts of vaccinees.26,27 No cases of fetal vaccinia 

have been reported. Vaccinia immune globulin was 

required on only seven occasions, to treat ocular 

vaccinia,28 progressive vaccinia,26 eczema vaccinatum,27,28 and as prophylaxis for a vaccinated patient 

who sustained large burn wounds. The success of 

this smallpox immunization program suggests that 

mass vaccination can be accomplished with greater 

safety than previously thought possible.29 

Although universal civilian vaccination was not 

recommended under President Bush’s plan, the possibility of a future strategy calling for such recommendations was allowed for, and provisions were 

made to provide smallpox vaccine to those members 

of the general public who specifically requested it. The 

wisdom of widespread civilian vaccination is difficult 

to assess. Most medical decisions involve a risk-benefit 

analysis on the part of the responsible clinician. In the 

case of smallpox vaccination, the risks are well known, 

and they are significant.30,31 The benefits, however, 

are far less certain; although the global eradication of 

smallpox surely ranks among the greatest public health 

accomplishments of recent history and the wisdom 

of vaccination with live vaccines went unquestioned 

during the era of endemic smallpox, the likelihood 

of contracting smallpox today via a terrorist attack is 

unknown and likely miniscule for the average civilian. 

In this regard, the risk-benefit calculation is not based 

on medical considerations, but rather on intelligence 

estimates to which few are privy.

Despite these concerns, a prerelease mass vaccination program for the general population may be the 

most effective countermeasure to the terror threat 

244-949 DLA DS.indb 114 6/4/18 
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Medical Management of Potential Biological Casualties: A Stepwise Approach

posed by smallpox. By inducing individual and herd 

immunity and by obviating the extreme difficulty of 

conducting postrelease vaccine and quarantine efforts, 

a program involving the resumption of universal 

smallpox vaccination possesses distinct advantages 

over other response plans. However, such an approach 

is hampered not only by the unknown risk of a smallpox release, but also by safety and logistics issues.32,33

A large number of persons are at risk for severe 

vaccine reactions today compared to the previous era 

of routine civilian smallpox vaccination, which ended 

in 1972. This increase in risk is due to the presence 

in the population of a large number of persons with 

compromised immunity associated with human immunodeficiency virus and with advances in immunosuppressive therapy and bone marrow and solid 

organ transplantation. This phenomenon raises concern 

about the safety and risk-benefit ratio of any preexposure vaccination program.34 Similarly, the occurrence 

of rare but severe smallpox vaccine complications in 

otherwise healthy recipients could result in morbidity 

and mortality that would be unacceptable in times of 

low risk. Risk analysis favors prerelease mass vaccination of the general population only if the probability of 

a large-scale attack is high. Prerelease mass vaccination 

of healthcare workers might again be contemplated in 

the future, owing to the risk of exposure while caring for 

patients, and the value of keeping healthcare workers 

healthy and functioning in the setting of an epidemic.35 

The smallpox vaccine currently employed in the 

United States is ACAM2000 (Acambis Inc, Cambridge, 

MA), which uses modern cell-culture-based production of vaccinia, an orthopoxvirus closely related to 

variola. ACAM2000 was licensed by the FDA in 2007, 

and replaced Dryvax (Wyeth Laboratories, Marietta, 

PA), a preparation derived from the harvested lymph 

of inoculated calves, in 2008. It is unlikely that this will 

significantly diminish the risk of adverse reactions, however, as the new vaccine employs the same live strain of 

vaccinia virus. The vast majority of adverse reactions to 

current vaccinia-containing vaccines derive from the live 

nature of the virus rather than the method of preparation.

The CDC controls release of civilian ACAM2000 

stocks and conditions for release have been established.36 The current CDC smallpox response strategy is based on preexposure vaccination of carefully 

screened first responders and members of epidemiological and clinical response teams. CDC plans also 

provide for a program to treat certain severe complications of vaccination using vaccinia immune globulin 

under an IND protocol, as well as for compensation of 

persons experiencing such complications, through the 

establishment of a smallpox vaccine injury compensation program.37 

The CDC’s response plan calls for “ring vaccination” after a smallpox release: identification and isolation of cases, with vaccination and active surveillance 

of contacts. Mass vaccination would be reserved for 

those instances when the number or location of cases 

renders the ring strategy inefficient, or when the risk 

of additional virus release is high.38 Although ring 

vaccination was successful historically (in the setting 

of herd immunity), mathematical models predict that 

this strategy may be problematic when applied to 

large or multifocal epidemics today.39 Furthermore, 

there is controversy among experts regarding the predicted benefit of postrelease mass vaccination due to 

lack of herd immunity, a highly mobile population, a 

relatively long incubation period, and the difficulties 

associated with prompt implementation of quarantine 

and mass vaccination.40,41 Finally, it should be kept 

in mind that vaccination is but one component of a 

multifaceted response, which should also include 

farsighted planning and logistical preparation, risk 

communication, surveillance, treatment, isolation, 

and quarantine.

Other Agents. Few authorities, either military or 

civilian, have advocated widespread vaccination 

against potential agents of bioterrorism other than 

anthrax and smallpox, and the implementation of any 

such strategy would currently be problematic. A vaccine against plague, previously licensed in the United 

States, is currently out of production. It required a 

three-dose primary series followed by annual boosters. Moreover, it was licensed only for persons 18 to 

61 years old. Finally, although reasonably effective 

against bubonic plague and widely employed by the 

DoD to protect against endemic disease, it probably 

afforded little protection against pneumonic plague, 

the form of disease likely to be associated with warfare or terrorism. A vaccine against one specific viral 

hemorrhagic fever, namely yellow fever, is widely 

available, although its causative virus is not regarded 

as a significant weaponization threat by most policymakers and health officials. Again, while the US 

military has administered yellow fever vaccine to 

large numbers of troops, it does so to guard against 

endemic disease, rather than a bioweapon threat. Additionally, a vaccine against Q fever (Q Vax, CSL Ltd, 

Victoria, Australia) is licensed in Australia. Although 

this vaccine might conceivably prove a useful addition to the military biodefense armamentarium, the 

self-limited nature of Q fever makes it unlikely that 

widespread use of this vaccine would be contemplated for the general public. Numerous research efforts 

are aimed at developing improved next-generation 

vaccines against anthrax, smallpox, and plague. 

Similarly, vaccines effective against tularemia, 

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Medical Aspects of Biological Warfare 

brucellosis, botulism, the equine encephalitides, 

staphylococcal enterotoxins, ricin, and several viral 

hemorrhagic fevers, as well as other potential agents 

of bioterrorism, are in various stages of development.42

Investigational vaccines against tularemia, botulism, the 

equine encephalitides (especially Venezuelan equine 

encephalitis), staphylococcal enterotoxin B, Q fever, 

and other agents, have been used under IND protocols 

to protect scientists studying these agents.

Step 3: Save the Patient’s Life (Primary Assessment)

Once self-protective measures are implemented, 

the clinician can approach the MASCAL scenario and 

begin assessing patients (the “primary survey,” in 

keeping with ATLS guidelines6). This initial assessment 

is intended to be brief and its purpose limited to the 

discovery and treatment of those conditions presenting an immediate threat to life or limb. Biological (or 

chemical) warfare victims may also have conventional 

injuries; attention should thus be focused at this point on 

maintaining a patent airway and providing for adequate 

breathing and circulation. The need for decontamination and administration of antidotes for rapid-acting 

chemical agents (nerve agents and cyanide) should be 

determined at this time. An “ABCDE” algorithm aids 

the clinician in recalling the specifics of the primary 

assessment. “A” stands for airway, which should be 

evaluated for the presence of conventional injury, but 

should also be examined because exposure to certain 

chemical agents (such as mustard, lewisite, or phosgene) can damage the airway. “B” denotes breathing; 

many agents of biological (and chemical) terrorism may 

cause the patient to experience respiratory difficulty. 

Examples include anthrax, plague, tularemia, botulism, 

Q fever, the staphylococcal enterotoxins, and ricin, as 

well as cyanide, nerve agents, and phosgene. “C” denotes circulation, which may be compromised due to 

conventional or traumatic injuries sustained during a 

MASCAL event, but may also be involved in the septic 

shock associated with plague and in the circulatory 

collapse associated with the viral hemorrhagic fevers. 

“D” refers to disability, specifically, neuromuscular disability. Note that botulism and nerve agent exposures 

are likely to present with a preponderance of neuromuscular symptomatology. Finally, “E” refers to exposure. 

In a MASCAL setting, this serves as a reminder to remove the victim’s clothing to perform a more thorough 

secondary assessment. It is here that one considers the 

need for decontamination and disinfection.

Step 4: Disinfect or Decontaminate as Appropriate

Once patients have been stabilized, decontamination can be accomplished, where appropriate. 

On the battlefield, considerable mature military 

doctrine drives decontamination efforts, which are 

carried out by unit personnel, guided or assisted by 

specific, highly trained Chemical Corps decontamination units. It should be pointed out, however, that 

decontamination, in the classical sense, may not be 

necessary after a biological attack (the same cannot 

always be said after a chemical attack). This is due, 

again, to the inherent incubation periods of biological agents. Because victims will not typically become 

symptomatic until several days after exposure to such 

agents, they are likely to have bathed and changed 

clothing several times before presenting for medical 

care, thus effectively accomplishing self-decontamination. Exceptions might include personnel directly 

exposed to an observed attack or persons encountering a substance in a threatening letter, where common 

sense might dictate topical disinfection. Even in these 

situations, bathing with soap and water and conventional laundry measures would likely be adequate. 

Moreover, it should be kept in mind that situations 

such as the case of the threatening letter represent 

crime scenes. Any medical interest in disinfection 

must be weighed against law enforcement concerns 

regarding preservation of vital evidence, which can be 

destroyed through hasty and ill-considered attempts 

at decontamination. Furthermore, significant psychological stress has been caused by unnecessary, costly, 

and resource-intensive attempts at decontamination 

in the past.43 Some of these attempts have involved 

forced disrobing and showering in public streets; 

to avoid such problems, the following measured 

responses should be considered.44

The Announced Threat (or Presumed Hoax). The 

need to preserve evidence, and maintain a chain-ofcustody when handling that evidence, is an important 

consideration at any crime scene. Although human and 

environmental health protection concerns take precedence over law enforcement procedures, threat and 

hoax scenarios nonetheless require the early involvement of law enforcement personnel and a respect for the 

need to maintain an uncompromised crime scene. Decontamination or disinfection is not typically necessary. 

The Telephoned Threat or the “Empty Letter.” In 

the majority of cases involving a telephoned threat, 

no delivery device or package is located. If a device 

is found or a threat is subsequently deemed credible, 

public health authorities should contact potentially 

exposed individuals, obtain appropriate information, 

and consider instituting prophylaxis or therapy. An 

envelope containing nothing other than a written 

threat poses little risk and should be handled in the 

same manner as a telephoned threat. Because the 

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Medical Management of Potential Biological Casualties: A Stepwise Approach

envelope constitutes evidence in a crime, however, 

further handling should be left to law enforcement 

professionals. In these cases, no decontamination is 

typically necessary, pending results of the legal and 

public health investigation. 

The Suspicious Package. When a package is discovered and found to contain powder, liquid, or other 

physical material, response should be individualized. 

In most cases, the package should not be disturbed further, the room should be vacated, additional untrained 

persons should be prohibited from approaching the 

scene and from handling the package or its contents, 

and law enforcement and public health officials should 

again be promptly notified. Persons who have come 

in contact with contents should remove clothing as 

soon as practical and seal it in a plastic bag. Victims 

should then wash with soap and water45 and, in most 

cases, may be sent home after adequate instructions 

for follow-up are provided and contact information 

obtained. In general, antibiotic prophylaxis would 

not be necessary before the preliminary identification of package contents by a competent laboratory, 

although decisions to provide or withhold postexposure prophylaxis are best made after consultation with 

public health authorities. Floors, walls, and furniture 

would not require decontamination before laboratory analysis is completed. Nonporous contaminated 

personal items, such as eyeglasses and jewelry, may 

be washed with soap and water or immersed in 0.5% 

hypochlorite (household bleach diluted tenfold) if a 

foreign substance has contacted the items.

The Delivery Device. If an aerosol delivery device or other evidence of a credible aerosol threat is 

discovered, the room (and potentially the building) 

should be evacuated. Law enforcement and public 

health personnel should be notified immediately 

and further handling of the device left to personnel 

with highly specialized training, such as the Army’s 

22nd and 110th Chemical Battalions (Technical Escort 

Units), the Marine Corps Chemical-Biological Incident 

Response Force (CBIRF), or the Federal Bureau of 

Investigation’s Hazardous Materials Response Unit. 

Contact information should be obtained from potential 

victims and detailed instructions provided. Clothing 

removal, soap and water showering, and decontamination of personal effects should be accomplished as 

above (the CBIRF brings with it extensive decontamination capabilities). Decisions regarding institution of 

empiric postexposure prophylaxis pending determination of the nature of the threat and identification of 

the involved biological agents should again be left to 

local and state public health authorities. In providing 

a reasoned and measured response to each situation, 

public health and law enforcement personnel can assist in minimizing the disruption and cost associated 

with large-scale decontamination, costly hazardous 

materials unit involvement, and broad institution of 

therapeutic interventions, and can help avoid widespread public panic.

Step 5: Establish a Diagnosis (Secondary Assessment)

Once decontamination has been considered, and 

accomplished as warranted, the clinician may perform 

a more thorough and targeted assessment aimed at 

establishing a diagnosis (the ATLS “secondary survey”). The thoroughness and accuracy with which one 

establishes this diagnosis will vary depending upon 

the circumstances the clinician finds him- or herself in. 

At robust roles of care (Role 4), the clinician may well 

have access to infectious disease and microbiology 

professionals, as well as to sophisticated diagnostic 

assays. Under such circumstances, it may be possible 

to arrive at a definitive microbiologic diagnosis fairly 

promptly. On the other hand, it is equally conceivable 

that the primary care provider, practicing at lower 

roles of care (Roles 1 to 3) or in more austere circumstances, may need to intervene promptly based on 

limited information and without immediate access to 

subspecialty consultation. Even in such cases, however, 

reasonable care can be instituted based simply on a 

syndromic diagnosis. An “AMPLE” (A: allergies, arthropod exposures; M: medications [as well as military 

occupational specialty and mission-oriented protective 

posture status]; P: past illnesses and vaccinations; L: 

last meal; E: environment) history may aid in establishing this diagnosis. A brief but focused physical 

examination, even one performed by inexperienced 

practitioners, can, at a minimum, reveal whether a 

victim of a biological or chemical attack exhibits primarily respiratory, neuromuscular, or dermatologic 

signs, or suffers simply from an undifferentiated febrile 

illness. By placing patients into one of these broad 

syndromic categories, empiric therapy can be initiated 

(see step 6); such empiric therapy can be refined and 

tailored once more information becomes available.46,47

When the situation permits, laboratory studies 

should be obtained to aid in later definitive diagnosis 

(Exhibit 5-3). On the battlefield, samples obtained at 

lower echelons would normally be submitted to the local clinical laboratory and, from there, through clinical 

laboratory channels to the 1st Area Medical Laboratory 

(AML). The AML is a theater-level tactical laboratory 

with very robust scientific capabilities, including the 

ability to rapidly identify biological, chemical, and 

radiological threat agents, as well as endemic, occupational, and environmental health hazards. The 

AML also has “reach-back” ability and works closely 

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Medical Aspects of Biological Warfare 

with national laboratories at USAMRIID and the US 

Army Medical Research Institute of Chemical Defense 

in Maryland. 

Step 6: Provide Prompt Therapy

Once a diagnosis (whether definitive or syndromic) 

is established, prompt therapy must be provided. 

In the cases of anthrax and plague, in particular, 

survival is directly linked to the speed with which 

appropriate therapy is instituted. A delay of more 

than 24 hours in the treatment of either disease leads 

to a uniformly grim prognosis. When the identity of 

a bioterrorist agent is known, the provision of proper 

therapy is straightforward (Table 5-1). When a clinician is faced with multiple victims and the nature of

the illness is not known, however, empiric therapy 

must be instituted. Guidelines for providing empiric 

therapy in such situations have been published, and 

an algorithmic approach to syndromic diagnosis and 

empiric therapy has been developed (Figure 5-2). 

Doxycycline, ciprofloxacin, or levofloxacin should be 

administered empirically to patients with significant 

respiratory symptoms when exposure to a biological 

attack is considered a possibility. 

Step 7: Institute Proper Infection Control Measures

The clinician must practice proper infection control 

procedures to ensure that contagious diseases are not 

propagated among patients. The majority of biological 

threat agents are not contagious. Among these are the 

causative agents of anthrax, tularemia, brucellosis, Q 

fever, the alphaviral equine encephalitides, glanders, 

melioidosis, and many others, including all of the 

toxins. Standard precautions alone suffice, in most 

cases, when caring for victims of such diseases.48 More 

stringent transmission-based precautions should be 

applied in certain circumstances. Three subcategories 

of transmission-based precautions exist. Droplet precautions are required to manage victims of pneumonic 

plague. Ordinary surgical masks are a component of 

proper droplet precautions and constitute adequate 

protection against acquisition of plague bacilli by 

the aerosol route. Contact precautions should be 

employed when managing certain viral hemorrhagic 

fever patients. In theory, these would be adequate for 

managing even Ebola victims given the transmission 

of this disease through infected blood and body fluids. 

Recent experience with Ebola in West Africa, however, 

illustrates the ease with which such precautions might 

be compromised. Given the prodigious amounts of 

body fluids (emesis and diarrhea) produced by these 

patients, the very low infectious inoculum of Ebola, and 

the propensity for hemorrhagic sputum to be aerosolized during coughing, the CDC now recommends that 

both contact and droplet precautions be employed when 

managing Ebola victims. Airborne precautions, ideally 

including an N-95 HEPA-filter mask, should be used 

when caring for smallpox victims. A summary of hospital infection control precautions as they apply to victims of biological terrorism is presented in Exhibit 5-4. 

Step 8: Alert the Proper Authorities

As soon as it is suspected that a case of disease might 

be the result of exposure to biological or chemical 

agents, the proper authorities must be alerted so that 

appropriate warnings may be issued and outbreakcontrol measures implemented. On the battlefield 

and in other military settings, the command must be 

notified immediately. It is similarly important to notify preventive medicine officials, as well as chemical 

corps and laboratory personnel. Early involvement 

of preventive medicine personnel ensures that an 

epidemiological investigation is begun promptly (see 

step 9) and that potential victims (beyond the index 

cases) are identified and treated early, when such 

treatment is most likely to be beneficial. Similarly, 

early notification of Army chemical corps personnel 

allows for battlefield surveillance, detection, and 

EXHIBIT 5-3

SAMPLES TO CONSIDER OBTAINING 

FROM POTENTIAL BIOWARFARE OR 

BIOTERRORISM VICTIMS*

• Complete blood count

• Arterial blood gas

• Nasal swabs for culture and PCR

• Blood for bacterial culture and PCR

• Serum for serologic studies

• Sputum for bacterial culture

• Blood and urine for toxin assay

• Throat swab for viral culture, PCR, and 

ELISA

• Environmental samples

*This list is not all-inclusive, nor is it meant to imply 

that every sample should be obtained from every 

patient. In general, laboratory sampling should be 

guided by clinical judgment and the specifics of the 

situation. This is a list of samples to consider obtaining 

in situations where the nature of an incident is unclear 

and empiric therapy must be started before definitive 

diagnosis.

ELISA: enzyme-linked immunosorbent assay

PCR: polymerase chain reaction

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Medical Management of Potential Biological Casualties: A Stepwise Approach

TABLE 5-1

RECOMMENDED THERAPY OF AND PROPHYLAXIS AGAINST DISEASES CAUSED BY 

CATEGORY A BIOTHREAT AGENTS

Condition Adults Children

Anthrax, inhalational, 

therapy* (patients who are 

clinically stable after 14 days 

can be switched to a single 

oral agent [ciprofloxacin or 

doxycycline] to complete a 

60-day course†

) 

Ciprofloxacin 400 mg IV q12h OR

Levofloxacin 500 mg IV q24h OR

Doxycycline 100 mg IV q12h

AND

Clindamycin‡ 900 mg IV q8h

AND

Penicillin G§ 4 mil U IV q4h 

AND CONSIDER

Raxibacumab 40 mg/kg IV

Ciprofloxacin 10–15 mg/kg IV q12h OR

Levofloxacin 8 mg/kg IV q12h OR

Doxycycline 2.2 mg/kg IV q12h 

AND

Clindamycin‡ 10–15 mg/kg IV q8h

AND

Penicillin G§ 400–600 k U/kg/d IV × q4h 

AND CONSIDER

Raxibacumab IV (> 50 kg: 40 mg/kg; 15–50 kg: 60 

mg/kg; < 15 kg: 80 mg/kg)

Anthrax, inhalational, postexposure prophylaxis (60-day 

course†

)

Ciprofloxacin 500 mg PO q12h OR

Levofloxacin 500 mg PO q24h OR

Doxycycline 100 mg PO q12h

Ciprofloxacin 10–15 mg/kg PO q12h OR

Levofloxacin 8 mg/kg PO q12h OR

Doxycycline 2.2 mg/kg PO q12h

Anthrax, cutaneous in setting 

of terrorism, therapy¥

Ciprofloxacin 500 mg PO q12h OR

Levofloxacin 500 mg PO q24h OR

Doxycycline 100 mg PO q12h

Ciprofloxacin 10–15 mg/kg PO q12h OR

Levofloxacin 8 mg/kg PO q12h OR

Doxycycline 2.2 mg/kg PO q12h

Plague, therapy Gentamicin 5 mg/kg IV qd OR

Doxycycline 100 mg IV q12h OR

Ciprofloxacin 400 mg IV q12h OR

Levofloxacin 500 mg IV q24h

Gentamicin 2.5 mg/kg IV q8h OR

Doxycycline 2.2 mg/kg IV q12h OR

Ciprofloxacin 15 mg/kg IV q12h OR

Levofloxacin 8 mg/kg IV q12h

Plague, prophylaxis Doxycycline 100 mg PO q12h OR

Ciprofloxacin 500 mg PO q12h OR

Levofloxacin 500 mg PO q24h

Doxycycline 2.2 mg/kg PO q12h OR Ciprofloxacin 

20 mg/kg PO q12h OR

Levofloxacin 8 mg/kg PO q12h

Tularemia, therapy Gentamicin 5 mg/kg IV qd OR

Doxycycline 100 mg IV q12h OR

Ciprofloxacin 400 mg IV q12h

Gentamicin 2.5 mg/kg IV q8h OR

Doxycycline 2.2 mg/kg IV q12h OR

Ciprofloxacin 15 mg/kg IV q12h

Tularemia, prophylaxis Doxycycline 100 mg PO q12h OR

Ciprofloxacin 500 mg PO q12h 

Doxycycline 2.2 mg/kg PO q12h OR Ciprofloxacin 

20 mg/kg PO q12h

Smallpox, therapy Supportive care Supportive care

Smallpox, prophylaxis Vaccination may be effective if given 

within the first several days after 

exposure.

Vaccination may be effective if given within the first 

several days after exposure.

Botulism, therapy Supportive care; antitoxin may halt 

the progression of symptoms but is 

unlikely to reverse them.

Supportive care; antitoxin may halt the progression 

of symptoms but is unlikely to reverse them.

Viral hemorrhagic fevers, 

therapy

Supportive care; ribavirin may be beneficial in select cases.Supportive care; ribavirin may be beneficial in 

select cases.

*In a mass casualty setting, where resources are severely constrained, oral therapy may need to be substituted for the preferred parenteral option.

†

Assuming the organism is sensitive, children may be switched to oral amoxicillin (80 mg/kg/d× q8h) to complete a 60-day course. We recommend that the first 14 days of therapy or postexposure prophylaxis, however, include ciprofloxacin, levofloxacin, or doxycycline regardless 

of age. A three-dose series of Anthrax Vaccine Adsorbed may permit shortening of the antibiotic course to 30 days.

‡

Rifampin or clarithromycin may be acceptable alternatives to clindamycin as drugs that target bacterial protein synthesis. If ciprofloxacin 

or another quinolone is employed, doxycycline may be used as a second agent, as it also targets protein synthesis.

§

Ampicillin, imipenem, meropenem, or chloramphenicol may be acceptable alternatives to penicillin as drugs with good central nervous 

system penetration.

¥

10 days of therapy may be adequate for endemic cutaneous disease. A full 60-day course is recommended in the setting of terrorism, however, because of the possibility of a concomitant inhalational exposure.

IV: intravenous; PO: per os (by mouth)

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Medical Aspects of Biological Warfare 

Figure 5-2. An empiric and algorithmic approach to the diagnosis and management of potential biological casualties. 

cipro: ciprofloxacin; CXR: chest X-ray; doxy: doxycycline; JE: Japanese encephalitis; nl: normal limits; prn: as needed; Rx: 

prescription; VEE: Venezuelan equine encephalitis; VHF: viral hemorrhagic fever; +: positive finding; ++: strongly positive 

finding; +/–: with or without finding

Adapted with permission from Henretig FM, Cieslak TJ, Kortepeter MG, Fleisher GR. Medical management of the suspected 

victim of bioterrorism: an algorithmic approach to the undifferentiated patient. Emerg Med Clin North Am. 2002;20:351–364.

Clinical evaluation with emphasis on vital signs and neurological, respiratory, and dermatological examination. Note many

diseases evolve rapidly to clinical sepsis with shock and acute respiratory failure. Resuscitate prn as per primary survey. 

YES NO

Encephalopathy, seizures, meningeal signs?

Neurological syndrome predominance?

Likely VEE, JE, etc;

Rx: supportive care

(consider bacterial

meningitis)

Likely botulism

(without fever); Rx: botulinum

antitoxin, ventilator?

Skin findings predominance?

Likely smallpox; Rx:

isolate patient, vaccinate

contacts

Likely VHFs or septicemic

plague; Rx: isolation, cipro

or doxy for plague (see

text)

Likely cutaneous anthrax

Likely anthrax; Rx: cipro

or doxy (see text)

Likely tularemia, brucellosis,

Q fever; Rx: doxy (see

text), aminoglycosides

Likely plague pneumonia;

Rx: isolate patient, cipro or

doxy (see text)

Respiratory syndrome predominance?

Bulbar palsies, muscle weakness,

intact sensation?

Possible early anthrax,

plague; Rx: cipro or doxy

(see text)

Possible early smallpox;

isolate and vaccinate

contacts

Undifferentiated febrile

syndrome, in context of

any of the above in

earlier presenting

patients?

Further clinical evaluation; CXR

Centrifugal,

synchronous,

pustulovesicular

rash?

YES NO YES NO

YES NO

YES NO

Hemorrhagic rash?

YES NO

Vesicle progressing

to ulcer to black

eschar with marked

edema

Severe respiratory distress;

CXR: wide mediastinum,

mediastinal adenopathy, effusion;

+/– pulmonary infiltrates

YES NO

YES NO

Pneumonia syndrome, bloody

sputum; CXR: nl mediastinum

with ++ parenchymal disease?

Pneumonia syndrome; CXR: +

parenchymal infiltrates, hilar

adenopathy?

Figure 5-3. The M93 “Fox” nuclear, biological, and chemical 

reconnaissance vehicle.

delineation of the limits of contamination. Using M93 

“Fox” or M1135 Stryker (Figure 5-3) nuclear, biological, 

chemical reconnaissance vehicles, these personnel can 

collect soil, water, and vegetation samples, mark areas 

of contamination, and transmit data to commanders in 

real time. Finally, notifying laboratory personnel not 

only permits them to focus their efforts at diagnosis, 

but also allows them to take necessary precautions. 

In a civilian terrorism response scenario, notification of a suspected biological, chemical, or radiological attack would typically be made through local or 

regional health department channels. In the United 

States, a few larger cities have their own health departments. In most areas, though, the county represents 

the lowest jurisdiction at which an independent health 

department exists. In some rural areas lacking county 

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Medical Management of Potential Biological Casualties: A Stepwise Approach

EXHIBIT 5-4

CONVENTIONAL INFECTIOUS DISEASES AND DISEASES POTENTIALLY RESULTING FROM 

AN ACT OF BIOTERRORISM: REQUIRED HOSPITAL INFECTION CONTROL PRECAUTIONS*

Standard (handwashing) Contact (gloves and 

gown†)

Droplet (private room‡ and 

surgical mask§)

Airborne (private room,‡ 

negative pressure room, 

HEPA filter mask)

All patients

Anthrax

Botulism

Tularemia

Brucellosis

Q Fever

Glanders

Melioidosis

Ricin intoxication

SEB intoxication

T-2 intoxication

VEE, EEE, WEE

MRSA, VRE

Enteric infections

Skin infections

Lice

Scabies

Clostridium difficile disease

RSV, parainfluenza

Certain VHFs

• Ebola¥

• Marburg¥

• Lassa Fever

Smallpox

Melioidosis (with cutaneous lesions)

Meningococcal disease

Resistant pneumococci

Pertussis

Group A streptococci

Mycoplasma

Adenovirus

Influenza

Pneumonic plague

Pulmonary TB

Measles

Varicella

Smallpox

*

Thorough guidelines for hospital infection control can be found in: Cole LA. Bioterrorism threats: learning from inappropriate 

responses. J Publ Hlth Manage Pract. 2000;6:8–18. †

Gloves and/or gown should also be worn as a part of standard precautions (and other forms of precaution) when contact with blood, 

body fluids, and other contaminated substances is likely.

‡

Mixing patients with the same disease is an acceptable alternative to a private room.

§

Surgical masks should also be employed as a part of standard and contact precautions (along with eye protection and a face shield) 

if procedures are likely to generate splashes or sprays of infectious material.

¥

While Ebola is transmitted primarily via infected blood and body fluids, the voluminous emesis and diarrhea produced by Ebola 

patients, the very low infectious inoculum of the virus, and the ease with which hemorrhagic respiratory secretions can be aerosolized during coughing, the CDC now recommends that both contact and droplet precautions be employed when managing Ebola 

victims; similar caution would likely apply to Marburg (and perhaps other VHF) patients as well.

EEE: eastern equine encephalomyelitis; HEPA: high-efficiency particulate air; MRSA: methicillin-resistant Staphylococcus aureus; RSV: 

respiratory syntactical virus; SEB: staphylococcal enterotoxin B; TB: tuberculosis; VEE: Venezuelan equine encephalitis; VHF: viral 

hemorrhagic fever; VRE: vancomycin-resistant enterococci; WEE: western equine encephalomyelitis

health departments, practitioners would access the 

state health department directly. Once alerted, local 

and regional health authorities know how to request 

additional support from health officials at higher 

jurisdictions. Each practitioner should have a point of 

contact with such agencies and should be familiar with 

mechanisms for contacting them before a crisis arises. 

If an outbreak proves to be the result of terrorism, or if the scope of the outbreak overwhelms local 

resources, a regional or national response becomes 

imperative. Under such circumstances, an extensive 

panoply of supporting assets and capabilities may 

be summoned. The National Incident Management 

System and its component Incident Command System 

(ICS) provide a standardized approach to command 

and control at an incident scene.49 Local officials use 

the ICS when responding to both natural and humanmade disasters, and ICS would be equally applicable 

in responding to a biological attack. Under the ICS, a 

designated official, typically the fire chief or the chief 

of police, serves as local incident commander. The 

incident commander may be able to summon groups of 

volunteer medical personnel through the Metropolitan 

Medical Response System, which includes medical 

strike teams in 124 local jurisdictions. These teams, 

under contract with mayors of the 124 municipalities, 

are organized under the Department of Homeland 

Security’s Office of Domestic Preparedness. 

In any incident or disaster, whether natural or 

human-made, the local incident commander may 

request assistance from the state through the state 

coordinating officer if it appears that local resources 

or capabilities will be exceeded. The state coordinating 

officer works with the governor and other state of244-949 DLA DS.indb 121 6/4/18 
122

Medical Aspects of Biological Warfare 

EXHIBIT 5-5

THE LABORATORY RESPONSE NETWORK

Sentinel laboratories. These laboratories, found in many hospitals and local public health facilities, have the ability 

to “rule-out” specific bioterrorism threat agents, to handle specimens safely, and to forward specimens on to higher 

echelon laboratories within the network. 

Reference laboratories. These laboratories, typically found at state health departments, and at military, veterinary, 

agricultural, and water-testing facilities, can employ BSL-3 practices, and can often conduct nucleic acid amplification and molecular typing studies. The more than 100 reference laboratories can confirm (“rule-in”) the presence of 

the various biological threat agents.

National laboratories. These laboratories, including those at the CDC and USAMRIID, can employ BSL-4 practices, 

and serve as the final authority in the work-up of bioterrorism specimens. These laboratories provide specialized 

reagents to lower level laboratories and have the ability to bank specimens, perform serotyping, and detect genetic 

recombinants and chimeras.

BSL: biosafety level

CDC: Centers for Disease Control and Prevention

USAMRIID: US Army Medical Research Institute of Infectious Disease

EXHIBIT 5-6

BIOSAFETY LEVELS

Biosafety Level 1: includes practices employed by a microbiology laboratory that deals only with well-characterized organisms that do not typically produce disease in humans. Work is conducted on open bench tops using 

standard microbiologic practices. Example: high school biology laboratory

Biosafety Level 2: includes practices employed by laboratories that deal with most human pathogens of moderate 

potential hazard. Laboratory coats and gloves are typically worn, access to the laboratory is restricted to trained 

personnel, and safety cabinets are often employed. Example: clinical hospital laboratory

Biosafety Level 3: Includes practices employed by laboratories that work with agents with the potential to cause 

serious and lethal disease by the inhalational route of exposure. Work is generally conducted in safety cabinets, 

workers are often vaccinated against the agents in question, and respiratory protection is worn. Clothing (such 

as scrub suits) is exchanged upon exiting the laboratory. Laboratories are negatively pressurized. Example: state 

health department laboratory 

Biosafety Level 4: Also includes practices employed by laboratories working with highly hazardous human pathogens infectious via the inhalational route. BSL-4 organisms differ from those requiring BSL-3 precautions in that 

no vaccine or antibiotic therapy is available. Personnel may only enter the laboratory through a series of changing 

and shower rooms. Equipment and supplies enter via a double-door autoclave. Strict and sophisticated engineering 

controls are employed and personnel wear sealed positive-pressure space suits with supplied air. Laboratories are 

negatively pressurized. Examples: laboratories at the CDC, USAMRIID, the Canadian Science Center for Human 

and Animal Health, and a few other research facilities

BSL: biosafety level

CDC: Centers for Disease Control and Prevention

USAMRIID: US Army Medical Research Institute of Infectious Disease

ficials to make state-level assets (such as state health 

departments, state public health laboratories, and state 

police assets) available. Most state public health laboratories participate as “reference” laboratories in the 

Association of Public Health Laboratories and CDC’s 

Laboratory Response Network. These facilities support 

hundreds of “sentinel” laboratories in local hospitals 

throughout the nation, and can provide sophisticated confirmatory diagnosis and typing of biological 

agents50 (an overview of public health laboratory capa244-949 DLA DS.indb 122 6/4/18 
123

Medical Management of Potential Biological Casualties: A Stepwise Approach

bilities is provided in Exhibit 5-5; the biosafety-level51

precautions they employ are outlined in Exhibit 5-6). 

State police can provide law enforcement assistance 

and state police laboratories can assist with forensic 

analysis. Finally, governors can access military assets 

at the state level through National Guard units under their direct control. These units can provide law 

enforcement, public works assistance, mobile field 

hospital bed capacity, and other support. Every state 

governor now has, at his or her disposal, one of some 57 

military Weapons of Mass Destruction–Civil Support 

Teams (WMD-CSTs). These 22-person advisory teams 

can offer expertise and provide liaison to additional 

military assets at the federal level. 

When state capabilities are overwhelmed or insufficient, the state coordinating officer may alert the 

federal coordinating officer, who can, in turn, assist 

in activating the national response framework. The 

national response framework guides delivery of federal assets and provides for a coordinated multiagency 

federal response. Federal response and support to state 

and local jurisdictions, according to the framework, is 

organized into 15 emergency support functions (ESFs). 

ESF 8 provides for health and medical services. While 

a specific agency is assigned primary responsibility 

for each of the 15 ESFs, more than two dozen federal 

agencies, as well as the American Red Cross, can, 

under federal law, be tasked to provide assistance. 

Federal disaster medical support is primarily the responsibility of the Department of Health and Human 

Services which, through its Office of Emergency Response, oversees the National Disaster Medical System 

(NDMS).52 A principal component of the NDMS is its 

network of disaster medical assistance teams, each 

of which consists of trained medical volunteers with 

the ability to arrive at a disaster site within 8 to 16 

hours. Another important component of the NDMS 

is its excess hospital bed capacity, held at numerous 

Department of Veterans Affairs, military, and civilian 

hospitals throughout the nation.

Finally, several other federal agencies may play an 

important role in the response to disasters, including, in particular, those resulting from a biological 

attack. The CDC and USAMRIID provide national 

laboratories, which support the reference labs at the 

state level and are capable of dealing with virtually 

all potential biological threat agents.53 Expert consultation and epidemiological investigative assistance 

is also available through the CDC, and bioweapons 

threat evaluation and medical consultation is likewise available through USAMRIID. Additionally, the 

military can provide expert advice and assistance to 

civilian authorities through Army National Guard’s 

CBRNE Enhanced Response Force Package Teams, 

which can arrive at a disaster site within a few hours 

of notification, as well as through the aforementioned 

CBIRF, which is capable of providing reconnaissance, 

decontamination, and field treatment. Military support, when provided, would be subordinate to civilian 

authorities and would be provided and tailored by the 

Joint Task Force for Civil Support, a component of US 

Army Northern Command that provides a commandand-control element for all military assets involved 

in disaster response missions and other contingencies within the United States. Finally, the CDC has 

developed the Strategic National Stockpile, whereby 

critical drugs and vaccines necessary to combat a large 

disaster or terrorist attack are stockpiled at several 

locations throughout the country, available for rapid 

deployment to an affected area.54 Release of stockpile 

components is currently controlled by the Department 

of Health and Human Services. 

Step 9: Conduct an Epidemiological Investigation 

and Manage the Psychological Aftermath of a 

Biological Attack 

Clinicians must be versed in the basic principles 

of epidemiology and be prepared to assist in the 

epidemiological investigation, which will be of paramount importance after a suspected terrorist attack. 

Although preventive medicine officers, environmental 

science officers, veterinarians, preventive medicine 

technicians (68S in US Army organizations), and 

field sanitation personnel may be invaluable in the 

course of such an investigation, the clinician should, 

nonetheless, have a working knowledge of the steps, 

known as the epidemiological sequence,55 involved 

in the conduct of an epidemiological investigation 

EXHIBIT 5-7

THE EPIDEMIOLOGICAL SEQUENCE

1. Make an observation

2. Count cases

3. Relate cases to population

4. Make comparisons

5. Develop the hypothesis

6. Test the hypothesis

7. Make scientific inferences

8. Conduct studies

9. Intervene and evaluate

Data source: Centers for Disease Control and Prevention. 

Investigating an outbreak. In: Principles of Epidemiology: Self 

Study Course SS3030. 2nd ed. Atlanta, GA: CDC; 1998: 347–424.

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Medical Aspects of Biological Warfare 

(Exhibit 5-7). Although the well-prepared clinician 

may positively impact the health and well-being of 

individual patients, it is only through the rapid conduct of a competent epidemiological investigation 

that large numbers of exposed persons are likely to 

be reached, and successful medical and psychological prophylaxis implemented, before the widespread 

outbreak of disease or panic. 

In addition to the instigation of an epidemiological 

investigation and the institution of specific medical 

countermeasures against biological agent exposures, 

the clinician should be prepared to address the psychological effects of known, suspected, or feared exposure 

to threat agents.56 An announced or threatened biological attack can provoke fear, uncertainty, and anxiety 

in the population, and can result in an overwhelming 

number of patients seeking evaluation and demanding 

therapy for feared exposure. Such a scenario might also 

follow the covert release of an agent once the resulting 

epidemic is characterized as being the consequence of 

a biological (or chemical or radiological) attack. Symptoms due to anxiety and autonomic arousal, as well 

as side effects from postexposure prophylactic drugs, 

may mimic prodromal disease due to biological agent 

exposure and pose dilemmas in differential diagnosis. 

Persons with symptoms arising from naturally occurring infectious diseases may likewise pose significant 

challenges to healthcare providers and public health 

officials. 

Public panic and behavioral contagion are best prevented by timely, accurate, well-coordinated, and realistic risk communication from health and government 

authorities. Such communication should include an 

assessment of the risk of exposure, information regarding the resulting disease, and a recommended course 

of action for suspected exposure. As the epidemic subsides and public knowledge increases, public anxiety 

will decrease to realistic and manageable levels. This 

cycle of uncertainty, panic, response, and resolution 

occurred during the October 2001 anthrax bioterror 

event.57 Readily accessible, biological, chemical, and 

radiological agent-specific information packages for 

local public health authorities and the general public 

are available through the CDC website, and can be of 

valuable assistance in risk communication. 

Effective risk communication is possible only in 

the presence of well-conceived risk communication plans and tactics, worked out well in advance 

of an actual event. Similar advanced planning must 

take into account the need to rapidly establish local 

centers for the initial evaluation and administration of 

postexposure prophylaxis. Finally, the development 

of patient and contact tracing mechanisms and vaccine screening tools, the mechanisms for accession of 

stockpiled vaccines and medications, and the means 

by which to identify and prepare local facilities and 

healthcare teams for the care of mass casualties must 

be clearly elucidated in advance. The CDC’s smallpox 

response plan40 provides a useful template for such a 

coordinated, multifaceted approach, and the wisdom 

of farsighted planning and coordination was amply 

demonstrated by the efficient mass prophylaxis of over 

10,000 individuals in New York City during the events 

surrounding the discovery of anthrax-contaminated 

mail in 2001.58 

Step 10: Maintain a Level of Proficiency 

Once response plans have been developed, they 

must be exercised. Military commanders and their 

units are typically well versed in planning and executing conventional field-training and command-post 

exercises. In the future, such exercises must account 

for the real possibility that military units may encounter biological weapons on the battlefield. Similarly, 

planning and exercises must account for the tandem 

threat posed by bioterrorist attacks against garrison 

activities. Local civilian exercises (which can often 

include military participants) are likewise a necessary 

component of disaster preparation. Such exercises 

should be designed so as to test incident command 

and control, communications, logistics, laboratory 

coordination, and clinical capabilities. These exercises 

may involve only the leadership of an organization 

and focus on planning and decision making (the 

command-post exercise), they may involve notional 

play around a tabletop exercise, or they may involve 

actual hands-on training and evaluation in a disaster 

drill or field-training exercise. In fact, the CDC expended considerable effort prior to the 2009 H1N1 

influenza pandemic preparing for just such an event, 

conducting numerous tabletop and full-scale exercises 

involving CDC personnel as well as state public health 

participants. The Joint Commission requires hospitals 

to conduct a hazard vulnerability analysis, develop 

an emergency operations plan, and evaluate this plan 

twice yearly; one of these evaluations must include 

a community-wide drill.59 Moreover, the Joint Commission specifically mandates that hospitals provide 

facilities (and training in the use of such facilities) for 

radioactive, biological, and chemical isolation and 

decontamination.

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SUMMARY

Many resources, including this text, are now available to assist both military and civilian clinicians 

and public health professionals in planning for, and 

maintaining proficiency in, the management of real 

or threatened terror attacks. Finally, as discussed 

under step 8 above, numerous governmental, military, and civilian organizations have now been organized, trained, and equipped to provide assistance 

and consultation to clinicians, first responders, and 

public health officials faced with planning for and 

treating the victims of a potential terrorist attack. It 

is assistance that, if incorporated into thorough planning efforts, will hopefully never be needed for actual 

patient care purposes.

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17. National Research Council. The Anthrax Vaccine: Is It Safe? Does It Work? Washington, DC: The National Academies 

Press; 2002.

18. US Army Medical Command, Military Vaccine Agency. Detailed Safety Review of Anthrax Vaccine Adsorbed, January 

2012. US Army Medical Command: Falls Church, VA.

19. Food and Drug Administration, Department of Health and Human Services. Biological products; bacterial vaccines 

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Advisory Committee on Immunization Practices (ACIP), 2009. MMWR. 2010;59(RR-6):1–36.

21. Halsell JS, Riddle JR, Atwood JE, et al. Myopericarditis following smallpox vaccination among vaccinia-naive US 

military personnel. JAMA. 2003;289:3283–3289.

22. Centers for Disease Control and Prevention. Secondary and tertiary transfer of vaccinia virus among US military 

personnel—United States and worldwide, 2002–2004. MMWR. 2004;53:103–105.

23. Hughes CM, Blythe D, Yu L, et al. Vaccinia virus infections in martial arts gym, Maryland, USA, 2008. Emerg Infect 

Dis. 2011;17. http://dx.doi.org/10.3201/eid1704.101010. Accessed July 30, 2014.

24. Young G, Hidalgo C, Sullivan-Frohm A, et al. Secondary and tertiary transmission of vaccinia virus from US service 

member. Emerg Infect Dis. 2011:17. http://dx.doi.org/10.3201/eid1704.101316. Accessed July 30, 2014.

25. Lederman ER, Davidson W, Groff HL, et al. Progressive vaccinia: case description and laboratory-guided therapy 

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26. Vora S, Damon I, Fulginiti V, et al. Severe eczema vaccinatum in a household contact of a smallpox vaccinee. Clin 

Infect Dis. 2008;46:1555–1561.

27. Centers for Disease Control and Prevention. Secondary and tertiary transmission of vaccinia virus after sexual contact 

with a smallpox vaccinee—San Diego, California, 2012. MMWR. 2013;62;145–147.

28. Centers for Disease Control and Prevention. Update: adverse events following civilian smallpox vaccination—United 

States, 2003. MMWR. 2003;52:819–820.

29. Grabenstein JD, Winkenwerder W Jr. US military smallpox vaccination experience. JAMA. 2003;289;3278–3282.

30. Centers for Disease Control and Prevention. Smallpox vaccination and adverse reactions: guidance for clinicians. 

MMWR. 2003;52(Dispatch):1–29.

31. Centers for Disease Control and Prevention. Update: cardiac and other adverse events following civilian smallpox 

vaccination—United States, 2003. MMWR. 2003;52;27:639–642.

32. Fauci AS. Smallpox vaccination policy—the need for dialogue. N Engl J Med. 2002;346:1319–1320.

33. Morosa VK, Isaacs SN. Separate worlds set to collide: smallpox, vaccinia virus vaccination, and human immunodeficiency virus and acquired immunodeficiency syndrome. Clin Infect Dis. 2003;37:426–432.

34. Kemper AR, Davis MM, Freed GL. Expected adverse events in a mass smallpox vaccination campaign. Eff Clin Pract. 

2002;5:84–90.

35. Bozzette SA, Boer R, Bhatnagar V, et al. A model for smallpox-vaccination policy. N Engl J Med. 2003;348:416–425.

36. Centers for Disease Control and Prevention. Recommendations for using smallpox vaccine in a pre-event vaccination 

program. MMWR. 2003;52(RR-07):1–16.

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37. Health Resources and Services Administration, Department of Health and Human Services. Smallpox vaccine injury 

compensation program: smallpox (vaccinia) vaccine injury table. Fed Reg. 2003;68:51492–51499.

38. Centers for Disease Control and Prevention. Smallpox response plan and guidelines (version 3.0), 2003. http://www.

bt.cdc.gov/agent/smallpox/response-plan/index.asp. Accessed July 30, 2014.

39. Kaplan EH, Craft DL, Wein LM. Emergency response to a smallpox attack: the case for mass vaccination. Proc Natl 

Acad Sci. 2002;99:10935–10940.

40. Mortimer PP. Can postexposure vaccination against smallpox succeed? Clin Infect Dis. 2003;36:622–629.

41. Mack T. A different view of smallpox and vaccination. N Engl J Med. 2003;348:460–463.

42. Cieslak TJ, Christopher GW, Kortepeter MG, et al. Immunization against potential biological warfare agents. Clin 

Infect Dis. 2000;30:843–850.

43. Cole LA. Bioterrorism threats: learning from inappropriate responses. J Public Health Manag Pract. 2000;6:8–18.

44. Kortepeter MG, Cieslak TJ. Bioterrorism: plague, anthrax, and smallpox. In: Baddour L, Gorbach SL, eds. Therapy of 

Infectious Diseases. Philadelphia, PA: WB Saunders; 2003.

45. Centers for Disease Control and Prevention. Bioterrorism alleging use of anthrax and interim guidelines for management—United States, 1998. MMWR. 1999;48:69–74.

46. Henretig FM, Cieslak TJ, Kortepeter MG, Fleisher GR. Medical management of the suspected victim of bioterrorism: 

an algorithmic approach to the undifferentiated patient. Emerg Med Clin N Am. 2002;20:351–364.

47. Cieslak TJ, Henretig FM. Biological and chemical terrorism. In: Kliegman RM, Stanton BMD, St. Geme J, Schor NF, 

and Behrman RE, eds. Nelson Textbook of Pediatrics. 19th ed. Philadelphia, PA: WB Saunders; 2011. Chap 704.

48. Siegel JD, Rhinehart E, Jackson M, Chiarello L, and the Healthcare Infection Control Practices Advisory Committee. 

2007 Guideline for Isolation Precautions: Preventing Transmission of Infectious Agents in Healthcare Settings. http://

www.cdc.gov/ncidod/dhqp/pdf/isolation2007.pdf. Accessed July 30, 2014.

49. US Department of Homeland Security. National Incident Management System, December 2008. Washington, DC: DHS; 

2008. https://s3-us-gov-west-1.amazonaws.com/dam-production/uploads/20130726-1824-25045-1942/national_incident_management_system_2008.pdf. Accessed July 30, 2014.

50. Morse SA, Kellogg RB, Perry S, et al. Detecting biothreat agents: the laboratory response network. ASM News. 

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51. US Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories. 5th ed. Washington, DC: HHS; 2009.

52. Knouss RF. National disaster medical system. Public Health Rep. 2001;116(suppl 2):49–52.

53. Centers for Disease Control and Prevention. Biological and chemical terrorism: strategic plan for preparedness and 

response. MMWR. 2000;49(RR-04):1–14.

54. Esbitt D. The Strategic National Stockpile: roles and responsibilities of health care professionals for receiving the 

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55. Centers for Disease Control and Prevention. Investigating an outbreak. In: Principles of Epidemiology: Self-Study Course 

SS3030. 2nd ed. Atlanta, GA: CDC; 1998: 347–424.

56. Holloway HL, Norwood AE, Fullerton CS, Engel CC, Ursano RJ. The threat of biological weapons: prophylaxis and 

mitigation of psychological and social consequences. JAMA. 1997;278:425–427.

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57. Rundell JR, Christopher GW. Individual and group responses to bioterrorism agent exposure: differentiating manifestations of infection from psychiatric disorder and fears of having been exposed. In: Ursano RJ, Fullerton AE, Norwood 

CS, eds. Bioterrorism. Psychological and Public Health Interventions. Cambridge, UK: Cambridge University Press; 2004: 

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58. Blank S, Moskin LC, Zucker JR. An ounce of prevention is a ton of work: mass antibiotic prophylaxis for anthrax, New 

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EM 1-24.

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Anthrax

Chapter 6

ANTHRAX

BRET K. PURCELL, PhD, MD*; CHRISTOPHER K. COTE, PhD†; PATRICIA L. WORSHAM, PhD‡; and 

ARTHUR M. FRIEDLANDER, MD§

INTRODUCTION AND HISTORY 

THE ORGANISM 

EPIDEMIOLOGY

PATHOGENESIS

CLINICAL DISEASE 

Cutaneous Anthrax 

Inhalational Anthrax

Meningitis

Oropharyngeal and Gastrointestinal Anthrax

DIAGNOSIS 

TREATMENT 

PROPHYLAXIS

Prophylactic Treatment After Exposure

Active Immunization

Side Effects

SUMMARY

*Colonel, Medical Corps, US Army; Deputy Chief, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter 

Street, Fort Detrick, Maryland 21702

†

Principal Investigator, Division of Bacteriology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

21702

‡

Chief, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

§

Colonel (Retired), Medical Corps, US Army; Senior Scientist, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort 

Detrick, Maryland 21702, and Adjunct Professor of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, 

Maryland 20814

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INTRODUCTION AND HISTORY

household interviews, grave markers, pathologists’ 

notes, various hospital lists, and clinical case histories 

of five survivors yielded evidence of 77 anthrax cases, 

with 66 deaths and 11 survivors.15 Cases were also 

reported in animals located more than 50 km from 

the site.16,17 Polymerase chain reaction examination of 

tissue samples collected from 11 of the victims demonstrated that virulent B anthracis DNA was present 

in all these patients, and at least five different strains 

of virulent B anthracis were detected based on variable 

number tandem repeat analysis.18

The retrospective data associated with the Sverdlovsk incident as well as studies performed for the 

Department of Homeland Security have been used 

by several computer modeling efforts to better understand the human infectious dose.19,20 Under the 

direction of the Department of Health and Human 

Services, the Office of the Assistant Secretary of 

Preparedness and Response, Public Health Emergency Medical Countermeasures Enterprise, and 

Biomedical Advanced Research and Development, 

these agencies have developed a variety of computer 

dissemination models for a wide variety of potential 

scenarios.

Although the Sverdlovsk incident is not well 

known among US civilians, most people are familiar 

with the 2001 bioterrorist attack in the United States 

in letters containing dried B anthracis spores. The 

spore powder, which was sealed in letters addressed 

to members of Congress and the press, was mailed 

through the US Postal Service.21–24 According to the 

Centers for Disease Control and Prevention, 22 people 

contracted anthrax from the letters.21,25–29 Of the 11 

individuals who developed inhalational anthrax, five 

died and six survived after intensive antimicrobial 

therapy. Eleven other people contracted cutaneous 

anthrax; all survived after treatment. Thousands of 

other persons received prophylaxis with antibiotics 

and, in some cases, postexposure vaccination.30–33

Considerable research has been devoted to biodefense research and modeling since this event.34–45 It has 

been estimated that the 2001 anthrax attacks cost the 

United States more than $1 billion in medical planning, 

response, and remediation costs.46–49 Additionally, this 

incident profoundly affected the law enforcement, 

scientific, and medical communities within the United 

States and throughout the world. Although the source 

of these letters has never been definitively identified, 

the impact on biodefense research establishments has 

been a transformational event for researchers and 

institutes. 

Anthrax, a zoonotic disease caused by Bacillus 

anthracis, occurs in domesticated and wild animals, 

primarily herbivores, including goats, sheep, cattle, 

horses, and swine.1–5 Humans usually become infected 

by contact with infected animals or contaminated 

animal products, most commonly via the cutaneous 

route and only rarely via the respiratory or gastrointestinal routes.6,7 Anthrax has a long association with 

human history. The fifth and sixth plagues described 

in Exodus may have been anthrax in domesticated 

animals followed by cutaneous anthrax in humans. 

Virgil described anthrax in domestic and wild animals 

in his Georgics, and anthrax was an economically important agricultural disease during the 16th through 

18th centuries in Europe.8,9

Anthrax, which is intimately associated with 

the origins of microbiology and immunology, was 

the first disease for which a microbial origin was 

definitively established. Robert Koch established 

the microbial origin for anthrax in 1876.10,11 Anthrax 

also was the first disease for which an effective live 

bacterial vaccine was developed; Louis Pasteur developed that vaccine in 1881.12 Additionally, anthrax 

represents the first described occupational respiratory infectious disease. During the latter half of the 

19th century, inhalational anthrax,13 a previously 

unrecognized form, occurred among wool-sorters 

in England as a result of the generation of infectious 

aerosols of anthrax spores under industrial conditions from the processing of contaminated goat hair 

and alpaca wool.14

The military has long been concerned about B 

anthracis as a potential biological weapon because 

anthrax spores are infectious by the aerosol route, 

and a high mortality rate is associated with untreated 

inhalational anthrax. In 1979 the largest inhalational 

anthrax epidemic of the 20th century occurred in 

Sverdlovsk, Russia. B anthracis spores were accidentally released from a military research facility located 

upwind from where the cases occurred. According 

to the accounts provided by two Soviet physicians, 

96 human anthrax cases were reported, of which 79 

were gastrointestinal and 17 cutaneous. The 79 gastrointestinal cases resulted in 64 deaths.15 Although the 

initial report of this event attributed the infections to 

a gastrointestinal source, later evidence indicated that 

an aerosol release of weaponized anthrax spores from 

a military production facility had occurred, and thus, 

inhalational anthrax was the predominant cause of 

these civilian casualties. Retrospective analysis using 

administrative name lists of compensated families, 

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Anthrax

THE ORGANISM

tion of capsule by fluorescent antibody, and virulence 

for mice and guinea pigs.54,55 Additional confirmatory 

tests to identify toxin and capsule genes by polymerase 

chain reaction, developed as research tools, have been 

Figure 6-1. (a) Gram stain of a blood smear from an infected guinea pig demonstrating intracellular bacilli chains within a 

polymorphonuclear leukocyte. (b) Gram stain of peripheral blood smear from a nonhuman primate infected with Bacillus 

anthracis, Ames strain.

Photographs: (a) Courtesy of Susan Welkos, PhD, Division of Bacteriology, US Army Medical Research Institute of Infectious 

Diseases, Fort Detrick, Maryland. (b) Courtesy of John Ezzell, PhD, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland.

a b

Figure 6-2. Scanning electron micrograph of a preparation 

of Bacillus anthracis spores. Two elongated bacilli are also 

presented among the oval-shaped spores. Original magnification × 2,620. 

Photograph: Courtesy of John Ezzell, PhD, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, 

Maryland.

B anthracis is a large, gram-positive, spore-forming, 

nonmotile bacillus (1–1.5 μm × 3–10 μm) that is closely 

related to Bacillus cereus and Bacillus thuringiensis. The 

organism grows readily on sheep blood agar aerobically and is nonhemolytic under these conditions. 

The colonies are large, rough, and grayish white, with 

irregular, curving outgrowths from the margin. The 

organism forms a prominent capsule both in vitro in 

the presence of bicarbonate in the culture media and 

elevated levels of carbon dioxide in the bacterial plate 

incubator and in tissue in vivo. In tissue, the encapsulated bacteria occur singly or in chains of two or 

three bacilli (Figure 6-1). The organism does not form 

spores in living tissue; sporulation occurs only after 

the infected carcass tissues are exposed to oxygen. The 

spores, which cause no swelling of the bacilli, are oval 

and they occur centrally or paracentrally (Figure 6-2). B 

anthracis spores are composed of dozens of spore coat 

proteins that—in part—protect the genomic material 

housed in the core.50,51 The spores are surrounded by 

a loose fitting membrane referred to as the exosporium. The exosporium has been shown to impact how 

the spore interacts with certain types of mammalian 

cells.52,53 The spores, which are resistant to environmental stressors, may survive for decades in certain 

soil conditions. Bacterial identification is confirmed 

by demonstration of the protective antigen (PA) toxin 

component, lysis by a specific bacteriophage, detec244-949 DLA DS.indb 131 6/4/18 
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Medical Aspects of Biological Warfare 

incorporated into the Laboratory Response Network 

established by the Centers for Disease Control and 

Prevention.56–59

The diagnosis of anthrax has been complicated by 

the identification of strains of B cereus, which produce 

anthrax-like disease. Because B cereus is hemolytic and 

resistant to the anthrax-specific gamma bacteriophage, 

such isolates would not typically be tested for the 

presence of genes encoding anthrax toxin, especially 

because B cereus is often regarded as an environmental 

contaminant.60 Continued reports of bacterial strains 

harboring anthrax toxin genes have demonstrated 

not only the importance of appropriate detection 

strategies, but also the possibility of emerging risks 

associated with the possible transfer of B anthracis 

characteristics to other organisms.61,62

EPIDEMIOLOGY

B anthracis, an organism that exists in the soil as a 

spore, occurs worldwide. Whether its persistence in 

the soil results from significant multiplication of the 

organism, or from cycles of bacterial amplification in 

infected animals whose carcasses then contaminate the 

soil, remains unsettled.63–67 The form of the organism 

in infected animals is the bacillus. 

Domestic or wild animals become infected when 

they ingest spores while grazing on contaminated 

land or eating contaminated feed. Pasteur originally reported that environmental conditions such as 

drought, which may promote trauma in the oral cavity on grazing, may increase the chances of acquiring 

anthrax.68 Spread from animal to animal by mechanical 

means—by biting flies and from one environmental 

site to another by nonbiting flies and by vultures—has 

been suggested to occur.64,69

Anthrax in humans is associated with agricultural, horticultural, or industrial exposure to infected 

animals or contaminated animal products. In less 

developed countries, primarily Africa, Asia, and the 

Middle East, disease occurs from contact with infected 

domesticated animals or contaminated animal products. Contact may include handling contaminated 

carcasses, hides, wool, hair, and bones or ingesting 

contaminated meat. Cases associated with industrial 

exposure—rarely seen—occur in workers processing 

contaminated hair, wool, hides, and bones. Direct 

contact with contaminated material leads to cutaneous disease, and ingestion of infected meat leads to 

oropharyngeal or gastrointestinal forms of anthrax. 

It has been well documented that intravenous drug 

users can become infected with B anthracis, resulting 

in a septicemic form of anthrax.70–78 Inhalation of a 

sufficient quantity of spores, usually seen only during 

generation of aerosols in an enclosed space associated 

with processing contaminated wool or hair, leads to 

inhalational anthrax. Military research facilities have 

played a major role in studying and defining anthrax, 

as well as many other zoonotic diseases in wild and 

domestic animals and the subsequent infections in 

humans.79

Unreliable reporting makes it difficult to estimate 

with accuracy the true incidence of human anthrax. It 

was estimated in 1958 that between 20,000 and 100,000 

cases occurred annually worldwide.80 In more recent 

years, anthrax in animals has been reported in 82 

countries, and human cases continue to be reported 

from Africa, Asia, Europe, and the Americas.81–85 In the 

1996–1997 global anthrax report, a general decrease appeared in anthrax cases worldwide; however, anthrax 

remains underdiagnosed and underreported.86

In the United States the annual incidence of human 

anthrax has steadily declined from about 127 cases in 

the early part of the 20th century to about 1 per year for 

the past 10 years.87 The vast majority of these cases have 

been cutaneous. Under natural conditions, inhalational 

anthrax is rare; before the anthrax bioterrorism event 

in 2001, only 18 cases had been reported in the United 

States in the 20th century.88,89 In the early part of the 

20th century, inhalational anthrax cases were reported 

in rural villagers in Russia who worked with contaminated sheep wool inside their homes.90 However, in 

recent years a significant decrease occurred in anthrax 

cases in domestic animals in east Russia. Five inhalational anthrax cases occurred in woolen mill workers 

in New Hampshire in the 1950s.91 During economic 

hardship and disruption of veterinary and human 

public health practices (eg, during wartime), large 

anthrax epidemics have occurred. The largest reported 

human anthrax epidemic occurred in Zimbabwe from 

1978 through 1980, with an estimated 10,000 cases.92

Essentially all cases were cutaneous, including rare 

gastrointestinal disease cases and eight inhalational 

anthrax cases, although no autopsy confirmation was 

reported.93

PATHOGENESIS

B anthracis produces two protein exotoxins, known 

as the lethal toxin (LT) and the edema toxin (ET); an 

antiphagocytic capsule; and other known and putative virulence factors.94 The role of the capsule in 

pathogenesis was demonstrated in the early 1900s, 

when anthrax strains lacking a capsule were shown 

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Anthrax

to be attenuated.95 In more recent years, the genes 

encoding synthesis of the capsule were identified on 

the 96-kilobase plasmid known as pXO2. Molecular 

analysis revealed that strains cured of this plasmid no 

longer produced the capsule and were attenuated, thus 

confirming the critical role of the capsule in virulence.96

The capsule is composed of a polymer of D-glutamic 

acid, which confers resistance to phagocytosis and 

may contribute to the resistance of anthrax to lysis 

by serum cationic proteins.97–102 Capsule production 

is necessary for dissemination to the spleen in a murine inhalational anthrax model.103 The capsule has 

also been the focus of several efforts to develop new 

generation anthrax vaccines.104–106 Evidence indicates 

that the capsule may enhance the protection afforded 

by PA-based vaccines against anthrax if opsonizing 

antibodies are produced.106

Koch first suggested the importance of toxins 

in his initial studies on anthrax. In 1954 Smith and 

Keppie107 demonstrated a toxic factor in the serum of 

infected animals that was lethal when injected into 

other animals. The role of toxins in virulence and immunity was firmly established by many researchers in 

the ensuing years.108,109 Advances in molecular biology 

have produced a more complete understanding of the 

biochemical mechanisms of action of the toxins, and 

they have begun to provide a more definitive picture 

of their role in the pathogenesis of the disease.

Two protein exotoxins, known as the LT and the 

ET, are encoded on a 182-kb plasmid (pXO1), distinct 

from that coding for the capsule. In an environment 

of increased bicarbonate in the growth media, atmospheric carbon dioxide within the plate incubation chamber, and increased temperature, such as is 

found in the infected host, transcription of the genes 

encoding these and other virulence-associated gene 

products is enhanced.94,110–113 A complex regulatory 

cascade controlled in large part by the atxA and acpA

genes encoded on the toxin plasmid pXO1 and pXO2, 

respectively, directs the production of virulence factors 

in response to these environmental signals.114,115 The 

anthrax toxins, like many bacterial and plant toxins, 

possess two components: (1) a cell binding, poreforming, or B, domain; and (2) an active, or A, domain 

that has the toxic and—usually—the enzymatic activity 

(Figure 6-3). The B and A anthrax toxin components, 

which are synthesized from different genes, are secreted as noncovalently linked proteins. The anthrax 

toxins are unusual because both toxins share the B 

protein, PA. Thus, the LT is composed of the PA63 

(MW [molecular weight] 63,000 after cleavage from a 

MW 83,000 protein) heptamer or octamer combined 

with a second protein, which is known as lethal factor (LF [MW 90,000]), and the ET is composed of PA 

complexed with the edema factor (EF [MW 89,000]). 

Each of these three toxin proteins—the B protein and 

both A proteins—individually is without biological 

activity. The critical role of the toxins in pathogenesis 

was established when it was shown that deletion 

of the toxin-encoding plasmid pXO196,116 or the PA 

gene alone117 attenuates the organism. Crude toxin 

preparations have been shown to impair neutrophil 

chemotaxis118,119 and phagocytosis.97

The ET, which causes edema when injected into the 

skin of experimental animals, is likely responsible for 

the marked edema often present at bacterial replication 

sites.120,121 This toxin is a calmodulin-dependent adenylate cyclase that impairs phagocytosis and priming for 

the respiratory burst in neutrophils; it also inhibits 

the production of interleukin-6 and tumor necrosis 

factor by monocytes, which may further weaken host 

resistance.122–124 ET also impairs dendritic cell function 

and appears to act with LT to suppress the innate immune response.125

Figure 6-3. Composition of anthrax lethal protein toxin. 

Molecular models of the protective antigen (PA)63 heptamer 

and the PA63 heptamer-lethal factor (LF) complex. (a, b) Side 

and top views of PA63 heptamer (green) bound to three LF 

molecules (yellow). (c, d) The surface renderings are colored 

according to the negative (red) and positive (blue) electrostatic 

surface potential. (c) Top view of the PA63 heptamer. The 

yellow box highlights the protomer-protomer interface and 

where LF binds to heptameric PA. (d) A hypothetical PA63

heptamer–LF interface. 

Photographs: Courtesy of Kelly Halverson, PhD, US Army 

Medical Research Institute of Infectious Diseases, Fort Detrick, 

Maryland.

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Medical Aspects of Biological Warfare 

The LT is a zinc metalloprotease that is lethal for 

experimental animals120,121,126 and is directly cytolytic 

for rodent macrophages, causing release of the potentially toxic cytokines interleukin-1 and tumor necrosis 

factor.127 In in vitro models, LT cleaves members of the 

mitogen-activated protein kinase (MAPK) kinase family, which are an integral part of a phosphorelay system 

that links surface receptors to transcription of specific 

genes within the nucleus. Thus, LT interferes with 

the MAPK signaling pathways necessary for many 

normal cell functions.128 In macrophage and dendritic 

cell models, LT leads to inhibition of proinflammatory 

cytokines, downregulation of costimulatory molecules, 

and ineffective T-cell priming.128–131 In vitro it also 

appears to promote apoptosis of endothelial cells lining the vascular system, leading to speculation that 

LT-induced barrier dysfunction leads to the vascular 

permeability changes accompanying systemic anthrax 

infection.132 Effects on hormone receptors, including 

glucocorticoids, have also been reported. Although 

much of the information regarding LT activity has been 

obtained from animal-derived cell culture models, 

Fang et al reported that—in vitro—LT inhibits MAPK 

kinase dependent interleukin-2 production and proliferative responses in human CD4+ T cells.133 Studies 

using tissue-specific CMG2 knockout mice strongly 

indicate that LTs/ETs target myeloid-derived cells to 

promote bacterial survival early in infection.134 In addition, the data suggest that elevated levels of toxin 

specifically target host organs and are responsible 

for the significant morbidity and mortality caused by 

anthrax infection.135

Studies in cell culture models have provided a 

clearer understanding of the molecular interactions 

of the toxin proteins.128 PA first binds, most likely by 

a domain at its carboxy-terminus, to a specific cell receptor.136–138 Two proteins have been proposed as the 

PA receptor: (1) Tumor endothelial marker 8 TEM8, 

(ANTX1); and (2) capillary morphogenesis protein, 

CMG2 (ANTX2).139–141 Both receptors have a von Willibrand factor type A domain that appears to interact 

with PA. Once bound, PA is cleaved by a furin-like 

protease, resulting in retention of a 63-kilodalton 

fragment of PA on the cell surface.142,143 This cleavage 

promotes formation of PA heptamers and creates a 

binding site on PA to which up to three molecules 

of the LF and the EF can bind with high affinity.129

Heptamerization144 and octamerization141–144 stimulates 

endocytosis of PA (or PA EF or PA LF complexes), 

which are then delivered into early endosomes. The 

mildly acidic pH of the endosome is hypothesized to 

trigger membrane insertion of the heptameric PA into 

intraluminal vesicles.145 EF and LF are translocated into 

the lumen of the vesicle and are thereby protected from 

lysosomal proteases.145 The toxins are then translocated 

via endosomal carrier vesicles to the cell cytosol, where 

they express their toxic activity.145 In addition, studies 

have also suggested that the formation of octamers 

provides stability to these toxin products and permits 

active LT to travel freely in the circulatory system.146

The processes leading to toxin activity in the infected animal may be more complicated because the 

toxin proteins appear to exist in the serum as a complex 

of PA and EF/LF.147 The proteolytic activation of PA 

necessary to form LT or ET may occur in interstitial 

fluid or serum rather than on the cell surface.147 The 

LT or ET may then bind to target cells and be internalized. This theory was bolstered by Panchal et al who 

demonstrated that purified LF complexed with the PA 

heptamer cleaved both a synthetic peptide substrate 

and endogenous MAPK kinase substrates and killed 

susceptible macrophage cells.148 In addition, complexes of the heptameric PA-LF found in the plasma 

of infected animals showed functional activity.148 Terminally, toxin is present in very high concentrations 

in the blood, which probably accounts for the sudden 

death observations in infected experimental animals. 

Although these toxins were once thought to be 

exclusively found in B anthracis, recent cases of inhalational disease have been identified that possess the 

hallmarks of anthrax disease; however, the bacteria 

recovered were not B anthracis but did possess anthrax 

toxin genes.149–152 Studies have identified isolates of B 

cereus that carried a plasmid homologous to the anthrax 

toxin plasmid pXO1. The polyglutamate capsule was 

not produced by this B cereus isolate. However, gene 

sequences encoding a polysaccharide capsule were 

present on a smaller plasmid.149 Capsule-producing 

strains of B cereus have caused severe pneumonia.150

Consequently, a possibility of false positives exists in 

diagnostic tests that rely on toxin-based identification 

of genes or gene products. Subsequent investigations 

of these strains determined that the virulence of these 

strains in mice, guinea pigs, and rabbits was significantly attenuated when compared to fully virulent B 

anthracis.

153,154 It was also shown that vaccines that are 

effective against fully virulent B anthracis can protect 

mice and guinea pigs from infection with the anthraxlike B cereus strain.154

Infection begins when the spores are introduced 

through the skin or mucosa. Spores are then ingested 

at the local site by macrophages. Phagocytosed spores 

can have multiple fates depending on the stage of 

infection and the spore burden of individual phagocytes.155,156 Within the lungs, spores are translocated by 

pulmonary macrophages and dendritic cells. Phagocytes have a dual role; they can transport spores to the 

lymphatic system157–160 but also are bactericidal toward 

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Anthrax

germinating spores under certain conditions.158,161–163

Another hypothesis has been proposed that may explain the toxins’ effects early in the infectious process. 

Banks, Ward, and Bradley164 have hypothesized that 

intoxication may occur after spores have been engulfed 

by phagocytic cells. The anthrax toxin receptors have 

been located on the inside of the phagolysosome, and 

the germinating spore may secrete toxins that interact 

with these receptors within the phagolysosome. The 

effector molecules (EF and/or LF) can then be translocated into the cytoplasm.155,164

Once a spore becomes vegetative, it can produce a 

robust capsule and large amounts of toxins. At these 

sites, the bacteria proliferate and produce the ETs and 

LTs that impair host leukocyte function and lead to the 

distinctive pathological findings: edema, hemorrhage, 

tissue necrosis, and a relative lack of leukocytes. Once 

the vegetative cells emerge from the phagolysome, 

they replicate within the cell and finally exit through 

the host cell plasma membrane.160 In inhalational anthrax, the spores are ingested by alveolar phagocytes, 

which transport them to the regional tracheobronchial 

lymph nodes, where germination occurs.165

Anthrolysin O (ALO) and phospholipases may also 

play critical roles as virulence factors for B anthracis166

and mediate the toxicity of B anthracis to lung epithelial cells under microaerobic conditions.167 ALO has 

been found to cause lysis of human phagocytes and 

epithelial cells. The mechanism of action appears to 

be from ALO pore-forming alterations of the cellular 

membrane, resulting in acute primary membrane permeabilization followed by a burst of reactive radicals 

released from the mitochondria.

The evidence reported from animal studies overwhelmingly suggests that the alveolar spaces are not 

permissive for significant levels of spore germination. 

Rather, spores begin to germinate once phagocytosed 

during translocation to and upon deposition within 

lymph nodes.165,168–171 However, several studies have 

suggested that small amounts of germination may occur within the alveolar spaces.171,172 Additionally, the 

nasal-associated lymphoid tissue has been explored 

as another area from which infection may be initiated.171,173,174 These data, largely collected through invivo imaging technologies, suggest that other scenarios 

may lead to spore germination after inhalation.159

Once in the tracheobronchial lymph nodes, the local 

production of toxins by extracellular bacilli generates 

the characteristic pathology picture: massive hemorrhagic, edematous, and necrotizing lymphadenitis; 

and mediastinitis (the latter is almost pathognomonic 

of this disease).175

These findings in human disease have been replicated in various animal disease models.176,177 The bacilli 

can then spread to the blood, leading to septicemia 

with seeding of other organs and frequently causing hemorrhagic meningitis. Death is most likely the 

result of systemic inflammatory response syndrome 

triggered by the release of endogenous cellular contents from damaged or dying cells, termed damageassociated molecular patterns and in combination with 

exogenous microbial exposure or pathogen-associated 

molecular patterns,178 resulting in respiratory failure 

associated with pulmonary edema, direct cardiac tissue damage, overwhelming bacteremia, accompanied 

frequently with meningitis. 

CLINICAL DISEASE

The military seeks to defend against anthrax used 

as an inhalational biological weapon. However, other 

anthrax forms are more likely to be seen by medical 

officers—particularly when deployed to third world 

countries—and are therefore included for completeness.

Cutaneous Anthrax

More than 95% of anthrax cases are cutaneous.179–181

After inoculation, the incubation period is 1 to 5 days. 

The disease first appears as a small papule that progresses over a day or two to a vesicle containing serosanguineous fluid with many organisms and a paucity of leukocytes. Histopathology findings consist of 

varying degrees of ulceration, vasculitis, perivascular 

inflammation, coagulative necrosis, hemorrhage, and 

edema.182 The vesicle—which may be 1 to 2 cm in diameter—ruptures, leaving a necrotic ulcer (Figure 6-4). 

Satellite vesicles may also be present. The lesion is 

usually painless, and varying degrees of edema may 

be present around it.183 The edema may occasionally 

be massive, encompassing the entire face or limb, 

which is described as “malignant edema.” Patients 

usually have fever, malaise, and headache, which may 

be severe in those with extensive edema. There may 

also be local lymphadenitis. The ulcer base develops a 

characteristic black eschar, and after 2 to 3 weeks the 

eschar separates, often leaving a scar and sometimes 

requiring surgical reconstruction.184,185 Debridement 

has been shown to improve survival rates in a mouse 

model of subcutaneous anthrax159; however, no clinical 

studies have been conducted to validate this procedure 

in human clinical disease. Septicemia is rare, and with 

treatment, mortality should be less than 1%.184,186–188 In 

addition, no age-related risk factor appears to be associated with cutaneous human anthrax.189

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Medical Aspects of Biological Warfare 

Of recent interest has been the identification of anthrax cases among intravenous drug users in western 

Europe.70–75 In 2000 a case of cutaneous anthrax was 

identified in a Norwegian patient who participated in 

subdermal drug injection, commonly known as “skin 

popper.”76 The first reported case of intravenous drug 

user-associated anthrax was in Scotland with subsequent 47 confirmed cases and 13 fatalities. These numbers increased to a total of 119 cases from December 

2009 to December 2010.74 This disease is thought to be 

initiated by direct injection of spore-contaminated heroin, which led to clinical presentations ranging from 

subcutaneous disease to septicemic anthrax.70–78,190–192

Inhalational Anthrax

Inhalational anthrax begins after an incubation period 

of 1 to 6 days with nonspecific symptoms of malaise, 

fatigue, myalgia, and fever.193–195 A nonproductive cough 

and mild chest discomfort may also occur. These symptoms usually persist for 2 or 3 days, and in some cases 

there may be a short period of improvement. Then a sudden onset of increasing respiratory distress with dyspnea, 

stridor, cyanosis, increased chest pain, and diaphoresis 

occurs. Associated edema of the chest and neck may also 

be present. Chest radiograph examination usually shows 

the characteristic widening of the mediastinum from 

necrosis and hemorrhage of the lymph nodes and surrounding tissues, often with associated pleural effusions 

(Figure 6-5). In the 2001 bioterrorist event, the pleural 

effusions were initially small but rapidly progressed 

and persisted despite effective antibiotic therapy.195,196

The effusions were predominantly serosanguineous, 

and immunohistochemistry revealed the presence of 

B anthracis cell wall and capsule antigens. Effusion 

fluid from deceased patients who had received fewer 

than 55 hours of antibiotic therapy revealed bacilli.197

Polymerase chain reaction analysis of the pleural 

fluid was also positive for B anthracis DNA.198 Pneumonia has not been a consistent finding but can occur 

in some patients and may be attributed to vascular 

permeability, intra-alveolar edema, and hyaline membrane formation.197 Although inhalational anthrax cases have been rare in this century, except for the 11 cases 

arising from the anthrax letters in 2001, several cases 

have occurred in patients with underlying pulmonary 

disease, suggesting that this condition may increase 

susceptibility to the disease.68 Meningitis is present 

in up to 50% of cases, and some patients may present 

with seizures. The onset of respiratory distress is fola b

c

Figure 6-4. Cutaneous lesions of anthrax. (a) Ulcer with 

vesicle ring. (b) Black eschar with surrounding erythema. 

(c) Marked edema of extremity secondary to anthrax edema 

toxin with multiple black eschar. 

Photographs: Courtesy of the Centers for Disease Control 

and Prevention, Atlanta, Georgia. www.bt.cdc.gov/agent/

anthrax/anthrax-images/cutaneous.asp.

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Anthrax

lowed by the rapid onset of shock and death within 

24 to 36 hours. Mortality had been essentially 100% in 

the absence of appropriate treatment; however, during 

2001 the mortality rate was 45%.195,196

An inhalational pulmonary disease thought initially 

to be anthrax has been identified to be caused by B 

cereus strains.152,199 These cases were found in metal 

welders, and susceptibility of these patients to this 

unusual pathogen may be related to inhalation of 

heavy metals during welding. Heavy metal exposure 

produces immunosuppression and an increased susceptibility to infection. 

Meningitis

Meningitis may occur after bacteremia as a complication of any of the disease’s clinical forms.190–192 Meningitis 

may also occur—rarely—without a clinically apparent 

primary focus, and it is often hemorrhagic, which is important diagnostically, and almost always fatal (Figure 

6-6). Studies have suggested that LF, EF, and protease 

InhA inhibit neutrophil signaling pathways in brain 

endothelium, thus promoting anthrax meningitis.193–195

Figure 6-5. (a) Frontal chest radiograph reveals mediastinal 

and hilar widening, bilateral pleural effusions, and decreased 

lung volumes. (b) Chest axial computed tomography (CT) 

(mediastinal window) shows enlarged, hyperdense subcarinal (arrow) and left hilar (arrowhead) lymph nodes, 

compatible with intranodal hemorrhage. (c) On lung window CT, peribronchial consolidation (curved arrow) reflects 

lymphatic spread of anthrax infection.

Radiologic Images: Courtesy of JR Galvin, MD and AA 

Frazier, MD, Department of Radiologic Pathology, Armed 

Forces Institute of Pathology, Washington, DC.

a b

c

Figure 6-6. Meningitis with subarachnoid hemorrhage in 

a man from Thailand who died 5 days after eating undercooked carabao (water buffalo). 

Reproduced from: Binford CH, Connor DH, eds. Pathology 

of Tropical and Extraordinary Diseases. Vol 1. Washington, 

DC: Armed Forces Institute of Pathology; 1976: 121. AFIP 

Negative 75-12374-3.

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Medical Aspects of Biological Warfare 

Oropharyngeal and Gastrointestinal Anthrax

Oropharyngeal and gastrointestinal anthrax result 

from ingesting infected meat that has not been sufficiently cooked or by ingesting anthrax spores either 

directly or from swallowing contaminated respiratory 

secretions.178,200,201 After an incubation period of 2 to 

5 days, patients with oropharyngeal disease present 

with severe sore throat or a local oral or tonsillar ulcer, 

usually associated with fever, toxicity, and swelling 

of the neck resulting from cervical or submandibular 

lymphadenitis and edema. Dysphagia and respiratory 

distress may also be present. Gastrointestinal anthrax 

begins with nonspecific symptoms of nausea, vomiting, and fever; in most cases severe abdominal pain 

follows. The presenting sign may be an acute abdomen, 

which may be associated with hematemesis, massive 

ascites, and bloody diarrhea. Mortality in both forms 

may be as high as 50%, especially in the gastrointestinal form.

DIAGNOSIS

The most critical aspect in making an anthrax diagnosis is a high index of suspicion associated with a compatible history of exposure. Cutaneous anthrax should be 

considered after a painless pruritic papule, vesicle, or 

ulcer develops—often with surrounding edema—and 

then becomes a black eschar. With extensive or massive edema, such a lesion is almost pathognomonic. 

Gram stain or culture of the lesion usually confirms 

the diagnosis. Bacterial culture tests include colony 

morphology on sheep blood agar plates incubated at 

35°C to 37°C for 15 to 24 hours. B anthracis colonies are 2 

to 5 mm in diameter, flat or slightly convex, irregularly 

round with possible comma-shaped (“Medusa-head”) 

projections with a ground-glass appearance (Figure 6-7). 

The colonies tend to have tenacious consistency when 

moved with a bacterial loop and are not β-hemolytic. 

The bacteria appear as gram-positive, 1 to 8 μm long 

and 1 to 1.5 μm wide bacilli. India ink staining reveals capsulated bacteria. A motility test should be 

performed either by wet mount or motility media; B 

anthracis is nonmotile. Gamma bacteriophage lysis 

and direct fluorescent antibody tests are performed at 

Level D laboratories as confirmatory tests (Figures 6-7 

and 6-8). Commercial polymerase chain reaction kits

specific for the B anthracis pXO1 and pXO2 plasmids 

are also available to assist in identifying this organism. 

The differential diagnosis should include tularemia, 

staphylococcal or streptococcal disease, and orf (a viral 

disease of sheep and goats transmissible to humans).

The diagnosis of inhalational anthrax is difficult, 

but the disease should be suspected with a history of 

exposure to a B anthracis–containing aerosol. The early 

Figure 6-7. (a) Isolated colonies of Bacillus anthracis on sheep blood agar plate. (b) Detection of B anthracis using specific 

gamma-phage mediated cell-lysis. 

Photographs: Courtesy of Bret K Purcell, PhD, MD, Division of Bacteriology, US Army Medical Research Institute of Infectious Diseases and the Defense Threat Reduction Agency/Threat Agent Detection and Response Program, National Center 

for Disease Control, Tbilisi, Georgia, 2005.

a b

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Anthrax

symptoms are nonspecific194,202–204 and include fever, 

chills, dyspnea, cough, headache, vomiting, weakness, myalgias, abdominal pain, and chest or pleuritic 

pain. This stage of the disease may last from hours to 

a few days. However, the development of respiratory 

distress in association with radiographic evidence of 

a widened mediastinum resulting from hemorrhagic 

mediastinitis and the presence of hemorrhagic pleural 

effusion or hemorrhagic meningitis should suggest the 

diagnosis. Contrast-enhanced computer tomography 

images reveal diffuse hemorrhagic mediastinal and 

hilar adenopathy with edema, perihilar infiltrates, 

bronchial mucosal thickening, and hemorrhagic 

pleural, and pericardial effusions.205 During the later 

stages of the disease patients develop sudden fever, 

dyspnea, diaphoresis, cyanosis, hypotension, shock, 

and death.202 Blood culture should demonstrate growth 

in 6 to 24 hours if the patient has not received antibiotics before collection, and a Gram stain of peripheral 

blood smears often reveals large bacilli in later disease 

stages. Sputum examination is not helpful in making 

the diagnosis because pneumonia is usually not a 

feature of inhalational anthrax.

Gastrointestinal anthrax is difficult to diagnose 

because of its rarity and nonspecific symptoms including nausea, vomiting, anorexia, and fever. As the 

disease progresses, patients often develop acute, severe 

abdominal pain, hematemesis, and bloody diarrhea. 

Diagnosis is usually considered only with a history 

of ingesting contaminated meat in the setting of an 

outbreak. Microbiological cultures do not help confirm 

the diagnosis. The diagnosis of oropharyngeal anthrax 

can be made from the clinical and physical findings 

in a patient with the appropriate epidemiological 

history. Sore throat, dysphagia, hoarseness, cervical 

lymphadenopathy, and edema as well as fever are 

often presenting symptoms.194,206,207

Meningitis resulting from anthrax is clinically indistinguishable from meningitis attributable to other 

etiologies. An important distinguishing feature is that 

the cerebral spinal fluid is hemorrhagic in as many 

as 50% of cases. The diagnosis can be confirmed by 

identifying the organism in cerebral spinal fluid by 

microscopy, culture, or both.

Serology is generally only useful in making a retrospective diagnosis. Antibody to PA or the capsule 

develops in 68% to 93%208–211 of reported cutaneous 

anthrax cases and 67% to 94%210,211 of reported oropharyngeal anthrax cases. A positive skin test to anthraxin 

(an undefined antigen derived from acid hydrolysis 

of the bacillus that was developed and evaluated in 

the former Soviet Union) has also been reported212 to 

help with the retrospective anthrax diagnosis. Western 

countries have limited experience with this test.213 The 

Food and Drug Administration (FDA) has recently 

approved two tests: (1) the QuickELISA Anthrax-PA 

Kit (Immunetics, Boston, MA) for identification of PA 

toxin in blood from infected human casualties, and (2) 

the PCR Joint Biological Agent Identification and Diagnostic System (Idaho Technology Inc, Salt Lake City, 

UT) anthrax test for rapid identification of bacteria in 

blood and blood culture samples.214

Figure 6-8. Direct fluorescent antibody stain of Bacillus 

anthracis capsule. 

Photograph: Courtesy of David Heath, PhD, Division of Bacteriology, US Army Medical Research Institute of Infectious 

Diseases and the Defense Threat Reduction Agency/Threat 

Agent Detection and Response Program, National Center 

for Disease Control, Tbilisi, Georgia, 2005.

TREATMENT

Cutaneous anthrax without toxicity or systemic 

symptoms may be treated with oral penicillin if the 

infection did not originate with a potential aerosol exposure. However, if an inhalational exposure is also suspected, ciprofloxacin or doxycycline is recommended 

as first-line therapy.202,203,215 Effective therapy reduces 

edema and systemic symptoms but does not change 

the evolution of the skin lesion. Treatment should be 

continued for 7 to 10 days, unless inhalational exposure 

is suspected; then treatment should be continued for 60 

days. However, recent studies of the 2001 bioterrorism 

event have identified problems associated with pro244-949 DLA DS.indb 139 6/4/18 
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Medical Aspects of Biological Warfare 

longed treatment, mass prophylaxis, and medication 

compliance.216–221 Amoxicillin is recommended for patients who cannot take fluoroquinolones or tetracyclineclass drugs; however, increasing evidence shows that B 

anthracis possesses β-lactamase genes that may reduce 

the efficacy of this treatment.222–227 In addition, if a bioterrorism event occurs, the bacterial strains used may be 

naturally antibiotic resistant or genetically modified to 

confer resistance to one or more antibiotics.

Tetracycline, erythromycin, and chloramphenicol 

have also been used successfully228 for treating rare 

cases caused by naturally occurring penicillin-resistant 

organisms. Additional antibiotics shown to be active 

in vitro include gentamicin, cefazolin, cephalothin, 

vancomycin, clindamycin, and imipenem.229–231 These 

drugs should be effective in vivo, but no reported 

clinical experience exists. Experimental infections using the inhalational mouse model have demonstrated 

significant efficacy using these additional antibiotics.

 Inhalational, oropharyngeal, and gastrointestinal 

anthrax should be treated with intravenous therapy 

using two or more antibiotics. The therapy should 

initially include a fluoroquinolone or doxycycline with 

one or more of the following antibiotics: clindamycin, rifampin, penicillin, ampicillin, vancomycin, 

amino-glycosides, chloramphenicol, imipenem, and/

or clarithromycin.202,215 Tactical Combat Casualty Care 

guidelines have been established for medical management of patients in chemical, biological, radiological, 

nuclear, or high-yield explosives environments.232 The 

Centers for Disease Control and Prevention issued 

guidelines for the treatment and management of human anthrax disease.205,206 New guidelines published 

in 2014 recommend linezolid over clindamycin—when 

appropriate—to prevent toxin formation and the use 

of adjunctive corticosteroids when indicated.233–236 The 

World Health Organization has also issued guidelines 

for the surveillance and control of anthrax in humans 

and animals and can be accessed at the following website: http://www.who.int/csr/resources/publications/

anthrax/WHO_EMC_ZDI_98_6/en/. 

Patients often require intensive care unit support, 

including appropriate vasopressors, oxygen, and 

other supportive therapy, because of the disease’s 

severity and rapid onset. Recommendations for treatment during pregnancy and for pediatric populations 

follow similar guidelines.234–236 The development of 

severe bacterial sepsis has been well documented for 

anthrax in both human clinical disease and experimental animal models. The expression of LT and ET as 

well as other virulence factors such as ALO promote 

the development of systemic inflammatory response 

syndrome by both damage-associated molecular patterns and pathogen-associated molecular patterns.178

This immunologic stimulation, if unregulated or limited, results in the formation of a cytokine cascade and 

eventual storm resulting in multiorgan system failure 

and rapid death of humans exposed to inhalational 

anthrax as well as other select agents. This immunologic over-response has prompted the evaluation 

of various augmentation therapies to mitigate these 

events. One such therapy that received FDA approval 

in 2012 is raxibacumab (GlaxoSmithKline, Brentford, 

Middlesex, United Kingdom), a human IgG1 monoclonal antibody directed against the PA antigen of B 

anthracis.

237,238 This product was the first monoclonal 

antibody approved for use in the treatment of severe inhalational anthrax under the FDA’s Animal 

Efficacy Rule.237–239 The study found that 64% of 

Cynomolgus macaque monkeys and 44% of rabbits with 

inhalational anthrax survived, whereas all placebo 

control animals died from both groups.239 An additional study comparing antibiotics and raxibacumab 

against antibiotics demonstrated a 82% survival for 

combination therapy versus 65% for antibiotics only. 

When rabbits were treated with levofloxacin plus 

raxibacumab verses levofloxacin alone, the absolute 

difference in survival rates between the groups was 

not statistically significant; however, clinically there 

was only an 18% death rate in the levofloxacin plus 

raxibacumab group and a 35% death rate in the levofloxacin only group.240,241

PROPHYLAXIS

Prophylactic Treatment After Exposure

Experimental evidence242 has demonstrated that 

treatment with antibiotics (including ciprofloxacin, 

doxycycline, and penicillin) beginning 1 day after 

exposure to a lethal aerosol challenge with anthrax 

spores can significantly protect against death. Combining antibiotics with active vaccination provides 

the optimal protection. Recent analysis has suggested 

postexposure vaccination may shorten the duration of 

antibiotic prophylaxis, providing the least expensive 

and most effective strategy to counter a bioterrorism 

event.243–245

Active Immunization

Emergent BioSolutions (Rockville, MD) produces 

the only licensed human vaccine against anthrax, 

anthrax vaccine adsorbed (BioThrax). This vaccine is 

made from sterile filtrates of microaerophilic cultures 

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of an attenuated, unencapsulated, nonproteolytic 

strain (V770-NP1-R) of B anthracis. The filtrate, containing predominantly 83-kDa PA, is adsorbed to 1.2 mg/

mL of aluminum hydroxide in 0.85% sodium chloride. 

The final product also contains 100 μg/mL of formaldehyde and 25 μg/mL of benzethonium chloride as 

preservatives. Some vaccine lots contain small amounts 

of LF and lesser amounts of EF, as determined by antibody responses in vaccinated animals.246,247 Low levels 

of antibody to LF and EF by Western blot have been 

reported in some vaccines, but these did not contribute 

significantly during toxin neutralization assays.248 The 

vaccine is stored at 2°C to 8°C. The vaccine should be 

given to industrial workers exposed to potentially 

contaminated animal products imported from countries in which animal anthrax remains uncontrolled. 

These products include wool, goat hair, hides, and 

bones. People in direct contact with potentially infected 

animals and laboratory workers should also be vaccinated. Vaccination is also indicated for protection 

against anthrax use in biological warfare. 

Recommendations have been made for anthrax vaccine use in the United States.249,250 The current guidelines recommend the anthrax vaccine adsorbed vaccine 

should be administered to prime the immune system 

to prevent infection as either a preexposure vaccine 

or after exposure to aerosolized B anthracis pores. For 

preexposure protection the Advisory Committee on 

Immunization Practices recommends intramuscular 

injections starting on day 0 followed by week 4, and 

every 6 months (6, 12, and 18 months) for a total of 

5 doses as the initial vaccination series. Since no in 

vitro correlate of immunity exists for humans, annual 

boosters are recommended if the potential for exposure 

continues. For postexposure to anthrax, those persons 

who have been previously unvaccinated should receive 

the vaccine as a three dose, subcutaneous series (at 0, 2, 

and 4 weeks) in addition to the administration of a 60-

day course of an appropriate antimicrobial therapeutic. 

More than 2.6 million US military personnel have 

received the licensed anthrax vaccine adsorbed vaccine, and no unusual rates of serious adverse events 

have been noted.251 Additional studies also support the 

safety of the anthrax vaccine.252–260 The next generation vaccine, recombinant PA, may afford equivalent 

protection with a decrease in reactogenicity. A live 

attenuated, unencapsulated spore vaccine is used 

for humans in the former Soviet Union. The vaccine is given by scarification or subcutaneously. Its 

developers claim that it is reasonably well tolerated 

and shows some degree of protective efficacy against 

cutaneous anthrax in clinical field trials.212 New attenuated vaccines developed in the United States are 

being evaluated for efficacy in inhalational anthrax 

animal models.261 Recent studies have demonstrated 

a fourfold rise in anti-PA immunoglobulin G (IgG) 

titers of 85% and 100% in adults receiving two and 

three doses, respectively, of either subcutaneous or 

intramuscular AVA.262–265

One hundred percent of the vaccinees developed 

a rise in titer in response to the yearly booster dose. 

When tested by an enzyme-linked immunosorbent assay, the current serologic test of choice, more than 95% 

of vaccinees seroconvert after the initial three doses.248

A rough correlation exists between antibody titer 

to PA and protection of experimental animals from 

infection after vaccination with the human vaccine. 

However, the exact relationship between antibody 

to PA as measured in these assays and immunity to 

infection remains obscure because the live attenuated 

Sterne veterinary vaccine (made from an unencapsulated, toxin-producing strain) protects animals better 

than the human vaccine, yet it induces lower levels of 

antibody to PA.246–248

A recent study evaluating the response of mice to 

recombinant PA revealed significant variation of fine 

specificity of humoral response to the antigen even 

among genetically identical mice using the same immunogen and environment.266,267 The authors demonstrated a heterogeneity of response to the PA antigen 

and identified specific epitopes that correlated to 

seroconversion and LeTx neutralization. Then they 

speculated that this observed stochastic variation in 

humoral immunity was likely a major contributing factor to the heterogeneity of vaccine response. Although 

these data suggest enhancing immunologic recognition 

of specific epitopes can improve vaccine protective 

response, the current anthrax vaccine adsorbed vaccine 

has demonstrated significant protection to nonhuman 

primates when exposed to inhalational challenge with 

large doses of anthrax spores.268–274

The protective efficacy of experimental PA-based 

vaccines produced from sterile culture filtrates of B 

anthracis was clearly demonstrated by various animal 

models and routes of challenge.275 A placebo-controlled 

clinical trial was conducted with a vaccine similar to 

the currently licensed US vaccine.276

This field-tested vaccine was composed of the sterile, cell-free culture supernatant from an attenuated, 

unencapsulated strain of B anthracis, different from that 

used to produce the licensed vaccine and grown under 

aerobic, rather than microaerophilic, conditions.277

This vaccine was precipitated with alum rather 

than adsorbed to aluminum hydroxide. The study 

population worked in four mills in the northeastern 

United States where B anthracis–contaminated imported goat hair was used. The vaccinated group, 

compared to a placebo-inoculated control group, 

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was afforded 92.5% protection against cutaneous 

anthrax, with a lower 95% confidence limit of 65% 

effectiveness. There were insufficient inhalational 

anthrax cases to determine whether the vaccine was 

effective. This same vaccine was previously shown 

to protect rhesus monkeys and other animal models 

against an aerosol exposure to anthrax spores.277–282

No controlled clinical trials in humans of the efficacy 

of the currently licensed US vaccine have been conducted. This vaccine has been extensively tested in 

animals and has protected guinea pigs against both an 

intramuscular247,248,280 and an aerosol challenge.246 The 

licensed vaccine has also been shown to protect rhesus 

monkeys against an aerosol challenge.242,270,278,282 The 

Centers for Disease Control and Prevention issued 

recommendations on the use of the anthrax vaccine 

in 2009.273

Recombinant PA is undergoing clinical trials and is 

considered the next-generation anthrax vaccine. Additionally, other nontoxin based vaccine approaches 

are being explored. These approaches include using 

the B anthracis capsule270,279,281,283–285 and spore-specific 

proteins.286–289 Although these novel antigens have 

been promising, it is generally agreed that PA will 

continue to have a prominent role in licensed anthrax 

vaccines.

Side Effects

In two different studies, the incidence of significant 

local and systemic reactions to the vaccine used in the 

placebo-controlled field trial was 2.4% to 2.8%82 and 

0.2% to 1.3%.277 The vaccine licensed in the United States 

is reported to have a similar incidence of reactions.290

Local reactions considered significant include induration, erythema in an area larger than 5 cm in diameter, 

edema, pruritus, warmth, and tenderness. These reactions peak at 1 to 2 days and usually resolve within 2 to 3 

days afterward. Rare reactions include edema extending 

from the local site to the elbow or forearm, and a small, 

painless nodule that may persist for weeks. A recent 

study indicated that administering the vaccine over the 

deltoid muscle instead of the triceps can significantly 

reduce the frequency of local reactions.251

People who have recovered from a cutaneous 

infection with anthrax may have severe local reactions from being vaccinated.276 Systemic reactions are 

characterized by flu-like symptoms, mild myalgia, 

arthralgia, headache, and mild-to-moderate malaise 

that last for 1 to 2 days. No long-term sequelae of local or systemic reactions exist and no suggestion of a 

high frequency or unusual pattern of serious adverse 

events exists.251,256,257,291,292

SUMMARY

Anthrax is a zoonotic disease that occurs in domesticated and wild animals. Humans become infected 

by contact with infected animals or contaminated 

products. Under natural circumstances, infection occurs by the cutaneous route and only rarely by the 

inhalational or gastrointestinal routes. An aerosol 

exposure to spores causes inhalational anthrax, which 

is of military concern because of its potential for use 

as a biological warfare agent. Aerosol exposure begins 

with nonspecific symptoms followed in 2 to 3 days by 

the sudden onset of respiratory distress with dyspnea, 

cyanosis, and stridor; it is rapidly fatal. Radiography 

of the chest often reveals characteristic mediastinal 

widening, indicating hemorrhagic mediastinitis. 

Hemorrhagic meningitis frequently coexists. Given 

the rarity of the disease and its rapid progression, it is 

difficult to diagnose inhalational anthrax. Treatment 

consists of massive doses of antibiotics and supportive 

care. Postexposure antibiotic prophylaxis is effective 

in laboratory animals and should be instituted as soon 

as possible after exposure. A licensed, antigen-based, 

nonviable vaccine is available for human use.

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237. Mazumdar S. Raxibacumab. mAbs. 2009;1:531–538.

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251. Sever JL, Brenner AI, Gale AD, et al. Safety of anthrax vaccine: an expanded review and evaluation of adverse events 

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255. Pittman PR. Aluminum-containing vaccine associated adverse events: role of route of administration and gender. 

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anthrax vaccine adsorbed, US Armed Forces, 1998–2000. Vaccine. 2003;21:1620–1628.

257. Wasserman GM, Grabenstein JD, Pittman PR, et al. Analysis of adverse events after anthrax immunization in US Army 

medical personnel. J Occup Environ Med. 2003;45:222–233.

258. Tierney BC, Martin SW, Franzke LH, et al. Serious adverse events among participants in the Centers for Disease Control 

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259. Grabenstein JD. Anthrax vaccine: a review. Immunol Allergy Clin North Am. 2003;23:713–730.

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protective antigen (PA) of Bacillus anthracis induced by anthrax vaccine adsorbed (AVA) among US military personnel. 

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263. Pittman PR, Norris SL, Barrera Oro JG, Bedwell D, Cannon TL, McKee Jr KT. Patterns of antibody response in humans 

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264. Marano N, Plikaytis BD, Martin SW, et al. Effects of a reduced dose schedule and intramuscular administration of 

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267. Dumas EK, Nguyen ML, Cox PM, et al. Stochastic humoral immunity to Bacillus anthracis protective antigen: identification of anti-peptide IgG correlating with seroconversion to lethal toxin neutralization. Vaccine. 2013;31:1856–1863. 

268. Ivins BE, Fellows PF, Pitt ML, et al. Efficacy of a standard human anthrax vaccine against Bacillus anthracis aerosol 

spore challenge in rhesus monkeys. Salisbury Med Bull. 1996;87:125-126.

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270. Ivins BE, Pitt ML, Fellow PF, et al. Comparative efficacy of experimental anthrax vaccine candidates against inhalation 

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Am J Public Health. 1962;52:632–645.

277. Wright GG, Green TW, Kanode RG Jr. Studies on immunity in anthrax. V. Immunizing activity of alum-precipitated 

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279. Ivins BE, Fellows PF, Nelson GO. Efficacy of a standard human anthrax vaccine against Bacillus anthracis spore challenge in guinea-pigs. Vaccine. 1994;12:872–874.

280. Ivins BE, Fellows PF, Pitt ML, et al. Efficacy of a standard human anthrax vaccine against Bacillus anthracis aerosol 

spore challenge in rhesus monkeys. Salisbury Med Bull. 1996;87:125–126.

281. Pitt ML, Little S, Ivins BE, et al. In vitro correlate of immunity in an animal model of inhalational anthrax. J Appl Microbiol. 1999;87:304.

282. Fellows PF, Linscott MK, Ivins BE, et al. Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin. Vaccine. 2001;19:3241–3247.

283. Chabot DJ, Scorpio A, Tobery SA, Little SF, Norris SL, Friedlander AM. Anthrax capsule vaccine protects against 

experimental infection. Vaccine. 2004;23:43–47.

284. Joyce J, Cook J, Chabot D, et al. Immunogenicity and protective efficacy of Bacillus anthracis poly-gamma-D-glutamic 

acid capsule covalently coupled to a protein carrier using a novel triazine-based conjugation strategy. J Biol Chem. 

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285. Chabot DJ, Joyce J, Caulfield M, et al. Efficacy of a capsule conjugate vaccine against inhalational anthrax in rabbits 

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286. Brossier F, Weber-Levy M, Mock M, Sirard JC. Protective antigen-mediated antibody response against a heterologous 

protein produced in vivo by Bacillus anthracis. Infect Immun. 2000;68:5731–5734.

287. Gauthier YP, Tournier JN, Paucod JC, et al. Efficacy of a vaccine based on protective antigen and killed spores against 

experimental inhalational anthrax. Infect Immun. 2009;77:1197–1207. 

288. Cote CK, Kaatz L, Reinhardt J, et al. Characterization of a multi-component anthrax vaccine designed to target the 

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289. Vergis JM, Cote CK, Bozue J, et al. Immunization of mice with formalin-inactivated spores from avirulent Bacillus cereus

strains provides significant protection from challenge with Bacillus anthracis Ames. Clin Vaccine Immunol. 2013;20:56–65. 

290. Puziss M, Wright GG. Studies on immunity in anthrax. X. Gel-adsorbed protective antigen for immunization of man. 

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291. Sever JL, Brenner AI, Gale AD, et al. Safety of anthrax vaccine: a review by the Anthrax Vaccine Expert Committee 

(AVEC) of adverse events reported to the Vaccine Adverse Event Reporting System (VAERS). Pharmacoepidemiol Drug 

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292. Sulsky SI, Luippold R, Garman P, et al. Disability among US Army veterans vaccinated against anthrax. Vaccine. 

2012;30:6150–6156.

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Brucellosis

Chapter 7

BRUCELLOSIS

BRET K. PURCELL, PhD, MD*; R. MARTIN ROOP II, PhD†; ARTHUR M. FRIEDLANDER, MD‡; and 

DAVID L. HOOVER, MD§

INTRODUCTION

THE INFECTIOUS AGENT

EPIDEMIOLOGY

PATHOGENESIS

CLINICAL MANIFESTATIONS

DIAGNOSIS

TREATMENT

PROPHYLAXIS

SUMMARY

*Colonel, Medical Corps, US Army; Deputy Chief, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter 

Street, Fort Detrick, Maryland 21702 

†

Professor, Microbiology and Immunology, Brody School of Medicine, East Carolina University, 600 Moye Boulevard, Room 118, Biotechnology Building, Greenville, North Carolina 27834

‡

Colonel (Retired), Medical Corps, US Army; Senior Scientist, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort 

Detrick, Maryland 21702

§

Colonel (Retired), Medical Corps, US Army; Senior Scientific Advisor, Clinical Research Management, 1265 Ridge Road, Hinckley, Ohio 44233; formerly Medical Director, Dynport Vaccine Company LLC, a CSC Company, 64 Thomas Johnson Drive, Frederick, Maryland, and Scientific Coordinator, 

Brucella Program, Department of Bacterial Diseases, Walter Reed Army Institute of Research, Silver Spring, Maryland

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INTRODUCTION

deployment of US military and coalition forces into 

Iraq, Afghanistan, Libya, and other Middle Eastern 

countries has posed particular risk from environmental 

and food source animals.11–13 The disease frequently 

becomes chronic and may relapse, even with treatment. 

Laboratory-acquired infections have been documented 

as awareness of this disease has increased,14–17 and 

as biodefense research expands in the academic and 

biotechnology industries, laboratory accidents may 

unfortunately become more frequent and significant.18

Strict adherence to proper engineering controls, good 

laboratory and microbiology techniques, and the 

use of personal protective equipment significantly 

reduces the incidence of laboratory-acquired infections.19,20 No vaccine is available that can safely be 

used to prevent laboratory-acquired brucellosis. 

The ease of transmission by aerosol underscores 

the concern that Brucella might be used as a biological warfare agent. The United States began developing Brucella suis as a biological weapon in 1942. The 

agent was formulated to maintain long-term viability, 

placed into bombs, and tested in field trials during 

1944–1945 with animal targets. By 1969 the United 

States terminated its offensive program for development and deployment of Brucella as a weapon and 

destroyed all of its biological weapon munitions. 

Although the munitions developed were never used 

in combat, studies conducted under the offensive 

program reinforced the concern that Brucella might be 

used against US troops as a biological warfare agent.21

Even before the post–September 11, 2001 attacks, 

civilian populations were recognized as potential 

high yield targets. In 1997 a model of aerosol attack 

with Brucella on an urban population estimated an 

economic impact of $477.7 million per 100,000 persons 

exposed.22 Brucella represents one of many biological 

agents of zoonotic disease that could pose threats 

as terrorist weapons against human or agricultural 

targets.23 Several reviews that focus on the potential 

use of the brucellae as agents of bioterrorism or biowarfare have been published.24–26

Brucellosis is a zoonotic infection of domesticated 

and wild animals caused by bacteria of the genus 

Brucella. Humans become infected by ingesting animal 

food products directly contacting infected animals or 

inhaling infectious aerosols either inadvertently or by 

intentional means through bioterrorism. Brucellosis is 

currently considered to be one of the world’s leading 

zoonoses.1

Military medicine has played a large role in discovering and defining brucellosis in humans.2

 In 1751 

G Cleghorn, a British army surgeon stationed on the 

Mediterranean island of Minorca, described cases of 

chronic, relapsing febrile illness and cited Hippocrates’ 

description of a similar disease more than 2,000 years 

earlier.3 Three additional British army surgeons working on the island of Malta during the 1800s were responsible for important descriptions of the disease. JA 

Marston described clinical characteristics of his own 

infection in 1861.4

 In 1887 David Bruce, for whom the 

genus Brucella is named, isolated the causative organism from the spleens of five fatal cases and placed this 

bacterium within the genus Micrococcus.

5

 Ten years 

later, ML Hughes, who had coined the name “undulant 

fever,” published a monograph that detailed clinical 

and pathological findings in 844 patients.6

In that same year, Bernhard Bang, a Danish investigator, identified a bacterium, which he called the 

“bacillus of abortion,” in placentas and fetuses of cattle 

suffering from contagious abortion.7 In 1917 Alice C 

Evans recognized that Bang’s organism was identical to that described by Bruce as the causative agent 

of human brucellosis. The bacterium infects mainly 

cattle, sheep, goats, and other ruminants, in which it 

causes abortion, fetal death, and genital infections.8,9

Humans, who are usually infected incidentally by 

contact with infected animals or ingestion of dairy 

foods, may develop numerous symptoms in addition 

to the usual ones of fever, malaise, and muscle pain. 

With the worldwide distribution of brucellosis, international travel and military deployments increase 

the risk of exposure to this disease.10 In particular, the 

THE INFECTIOUS AGENT

Brucellae are small, nonmotile, nonsporulating, 

nontoxigenic, nonfermenting, facultatively intracellular, gram-negative bacteria that represent a single 

“genospecies” from a phylogenetic perspective.27

However, for epidemiologic purposes and ease and 

accuracy of communication, Brucella strains are classified as separate “nomenspecies” based on readily 

distinguished phenotypic characteristics that include 

host specificity.28 There are presently 10 of these recognized “nomenspecies” (Table 7-1). Brucella melitensis, B 

suis, Brucella abortus, and Brucella canis are the classic 

causative agents of disease in humans. Human infections with the marine mammal strain Brucella ceti29,30

and a strain (Brucella inopinata) of unknown origin31–33 

have also recently been described, but prevalence of 

such infections is unclear.

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TABLE 7-1

TYPICAL HOST SPECIFICITY OF BRUCELLA

SPECIES

Brucella Species Animal Host Human Pathogenicity

B melitensis Sheep, goats High

B suis Swine High

B abortus Cattle, bison Intermediate

B canis Dogs Low

B ceti Dolphins, Unknown*

porpoises

B inopinata Humans Unknown*

B pinnipedialis Seals Not reported

B ovis Sheep Not reported

B neotomae Rodents Not reported

B microti Rodents Not reported

*B ceti and B inopinata strains have been isolated from human disease, but the importance of these strains as human pathogens is 

presently unknown. 

Human infections with Brucella ovis, Brucella neotomae, Brucella pinnipedialis, and Brucella microti have not 

been described. Brucellae grow best on trypticase soybased media or other enriched media with a typical 

doubling time of 2 hours in liquid culture. Although 

B melitensis bacteremia can be detected within 1 week 

by using automated culture systems,34 cultures should 

be maintained for at least 4 weeks, with weekly subculture, for diagnostic purposes. Most biovars of B 

abortus require incubation in an atmosphere of 5% 

to 10% carbon dioxide for growth. Brucellae may 

produce urease and may oxidize nitrite to nitrate; 

they are oxidase- and catalase-positive. Species and 

biovars are differentiated by their carbon dioxide 

requirements; ability to use glutamic acid, ornithine, 

lysine, and ribose; hydrogen sulfide production; 

growth in the presence of thionine or basic fuchsin 

dyes; agglutination by antisera directed against 

certain lipopolysaccharide (LPS) epitopes; and by 

susceptibility to lysis by bacteriophage. Brucella can 

grow on blood agar plates and does not require X or 

V factors for growth. 

Serological agglutinating antibodies have been 

used worldwide as the definitive diagnostic test for 

brucellosis infection. The standard tube agglutination test is the modified Brucella microagglutination 

test.35 This test uses direct agglutination of bacterial 

antigens by specific antibodies of the immunoglobulin 

(Ig), IgG, and IgA classes. Acute infection is indicated 

by the presence of antigen-specific IgM antibodies, 

but these antibodies decline rapidly within weeks of 

the onset of infection. Chronic or relapsing disease is 

characterized by elevated or increasing levels of IgG 

and IgA classes.36 A four-fold or greater rise in Brucella

agglutination titers demonstrated between acute and 

convalescent serum specimens collected at least 2 

weeks apart in conjunction with clinically compatible 

illness is considered a confirmatory test for brucellosis 

infection. Additional confirmatory tests for infection 

include the isolation of Brucella from clinical specimens or the identification of Brucella bacteria in tissue 

cultures by specific immunohistochemical staining.37

Although highly sensitive and specific, occasionally 

false positive tests and cross reactions do occur using 

Brucella antibody tests. The cell wall lipopolysaccharide of the Brucella organism is antigenically similar to 

other gram-negative bacteria. Antibodies to Moraxella 

phenylpyruvica, Yersinia enterocolitica, Escherichia coli

O157, and specific Salmonella strains are known to 

provide false positive reactions.38,39

Analysis of fragment lengths of DNA cut by 

various restriction enzymes has also been used to 

differentiate brucellae groupings.33 Single nucleotide 

polymorphism analyses using real time polymerase 

chain reaction (PCR) have been used to rapidly 

identify Brucella isolates to the species level.40 Both 

the multiple loci variable number of tandem repeat 

analysis and the Bruce-ladder multiplex PCR assays 

have been recently used to type a variety of marine 

Brucella isolates and differentiate by biovar typing of 

B suis and B canis.

41,42 Recent studies using proteomics, 

complete genomic sequencing, and multi-locus analysis of variable number tandem repeats have rapidly 

expanded the information on virulence determinants, 

identification of pathogenicity islands, and evolutionary relatedness among the Brucella species.43–47 Microarrays have now been developed to phylogenetically 

classify and forensically identify unknown pathogens 

as well as genotype Brucella species.48,49 The LPS component of the outer cell membranes of the brucellae is 

different—both structurally and functionally—from 

that of other gram-negative organisms.31,32 For instance, in addition to its capacity to provide resistance 

to complement and potentially serve as a ligand for 

binding to host cells, experimental evidence indicates 

that the O-chain of LPS of “smooth” (fully expressed 

O-chain versus “rough” strains with substantially 

reduced or absent O-chain) Brucella strains directly 

interferes with the capacity of host macrophages to 

process antigens via the major histocompatibility 

complex class II pathway50 and influences in the 

intracellular trafficking of the Brucella containing 

vacuoles in host macrophages preventing their fusion 

with lysosomes.51 The chemical compositions of the 

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lipid A and core moieties of the Brucella LPS are also 

distinct from those found in the enteric and many 

other gram-negative bacteria, and these differences 

greatly reduce the “recognition” of the brucellae by 

the Toll-like receptors on host macrophages, which 

allows these bacteria to induce a dampened inflammatory response and use a “stealthy” approach for 

establishing infections.52 

One of the unique features of Brucella strains is 

that unlike most pathogenic bacteria, these bacteria 

produce relatively few “classical” virulence factors.53

Probably the most widely studied virulence determinants in the Brucella strains are the LPS and the Type 

IV secretion system.54 The brucellae use this transport 

system to secrete effector proteins into the cytoplasm 

of infected mammalian cells. These effector proteins 

interfere with the activity of the host cell proteins 

that control the intracellular membrane trafficking. 

The net result is that the phagosomes within which 

the brucellae reside in host macrophages avoid 

extensive interactions with lysosomes and eventually fuse with the host cell endoplasmic reticulum. 

The formation of these so-called replicative Brucella

containing vacuoles (or rBCVs) is essential for the 

virulence of the naturally occurring smooth Brucella

strains such as B melitensis, B suis, and B abortus. The 

capacity of Brucella strains to survive and replicate in 

host macrophages is critical for their virulence. Accordingly, in addition to gene products such as these 

that overtly interfere with biology of the host cell, the 

brucellae also produce numerous proteins that allow 

them to successfully resist the environmental stresses 

they encounter during their intracellular residence in 

host macrophages. These stresses include exposure to 

acidic pH, reactive oxygen species and antimicrobial 

peptides, and nutrient deprivation.55

EPIDEMIOLOGY

Animals may transmit Brucella organisms during 

septic abortion, at the time of slaughter, and in their 

milk. For infected patients, no conclusive evidence 

indicates that brucellosis can be transmitted from 

person to person. The incidence of human disease is 

thus closely tied to the prevalence of infection in sheep, 

goats, pigs, and cattle, and to practices that allow 

exposure of humans to potentially infected animals 

or their products. In the United States, where all 50 

states are considered to be “free” of bovine brucellosis 

and dairy products are routinely pasteurized, illness 

occurs primarily in individuals such as veterinarians, 

shepherds, cattlemen, and slaughterhouse workers 

who have occupational exposure to infected animals. 

In many other countries, humans more commonly 

acquire infection by ingesting unpasteurized dairy 

products, especially cheese.

Less obvious exposures can also lead to infection. In 

the United States and Australia, for example, hunters 

have acquired B suis infection from feral swine.56,57 It 

was also not uncommon for veterinarians to develop 

brucellosis after accidental exposure to B abortus Strain 

19 in the United States when this strain was being 

used as a live vaccine in cattle.58 Another bovine vaccine strain, Brucella Abortus Vaccine, Strain RB-51, 

has been used to eradicate brucellosis from the US 

livestock herds.59 Accidental human infections with 

this vaccine cannot be identified using the standard 

LPS-based diagnostics assay. Brucellae are also highly 

infectious in laboratory settings; numerous laboratory 

workers who culture the organism become infected. 

Disease with a relatively high proportion of respiratory 

complaints has also been reported in individuals who 

have camped in the desert during the spring lambing 

season.46 B canis, a naturally rough strain that typically causes genital infection in dogs, can also infect 

humans.60 Although B canis infections were once considered rare, it has become apparent that in some areas 

of the world these infections were probably unrecognized.60 In the United States the total number of cases 

of brucellosis remains very low (0.02 to 0.09 cases per 

100,000 person-years).61,62 A major contributing factor 

to this low incidence of brucellosis can be attributed 

to a national eradication campaign to eliminate brucellosis in domestic cattle herds. When implemented 

the human incidence of disease dropped from a high 

of 6,321 cases in 1947 to 136 cases in 2001 (0.48 cases 

per million). These few cases are primarily caused by 

infections with B melitensis and now most human cases 

are distributed in Hispanic populations residing on 

either side of the Mexico border.61 The endemic regions 

located in Latin America, Europe, Africa, and Asia account for most of the human cases of brucellosis with 

the highest incidences occurring in the former Yugoslav Republic of Macedonia, Algeria, Peru, Iraq, Iran, 

Syria, Turkey, Kyrgyzstan, and Mongolia.61,62 With the 

improvement of diagnostic methods, ever increasing 

international tourism, and establishment of new eradication programs, the epidemiology of brucellosis will 

continue to shift and evolve requiring constant vigilance for new foci of disease. Unfortunately, with the 

rapidly changing political, international, and financial 

environments, worldwide eradication of this zoonotic 

disease will be extremely difficult. 

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PATHOGENESIS

Brucellae can enter mammalian hosts through 

skin abrasions or cuts, the conjunctiva, and the 

respiratory tract, and, unlike enteric pathogens 

such as Salmonella or Shigella species that infect the 

lower gastrointestinal tract, the most likely site of 

bacterial entry is the mucosae of the upper gastrointestinal tract.63,64 Organisms are rapidly ingested 

by polymorphonuclear leukocytes, which generally fail to kill them,65,66 and are also phagocytosed 

by macrophages (Figure 7-1). Bacteria transported 

in macrophages, which traffic to lymphoid tissue 

draining the upper gastrointestinal mucosa, may 

eventually disseminate to lymph nodes, liver, spleen, 

mammary glands, joints, kidneys, and bone marrow. 

As noted previously, the brucellae are resistant to the 

microbicidal activity of macrophages, and it is their 

capacity to survive and replicate for prolonged periods in these phagocytes that underlies their ability 

to produce chronic infections.55 Histopathologically, 

the host cellular response may range from abscess 

formation to lymphocytic infiltration to granuloma 

formation with caseous necrosis.58

Studies in experimental models have provided 

important insights into host defenses that eventually control infection with Brucella organisms. 

Serum complement effectively lyses some rough 

strains (ie, those that lack O-polysaccharide side 

chains on their LPS), but has little effect on smooth 

strains (ie, bacteria with a long O-polysaccharide 

side chain); B melitensis may be less susceptible 

than B abortus to complement-mediated killing.67,68

Administration of antibody to mice before challenge with rough or smooth strains of brucellae 

reduces the number of organisms that appear in 

liver and spleen. This effect is caused mainly by 

antibodies directed against LPS, with little or no 

contribution of antibodies directed against other 

cellular components.69

The intensity of an infection in mice can be reduced 

by transferring from immune to nonimmune animals 

differentiated CD4+and CD8+ T cells70 or by the Ig 

fractions of serum. In particular, the T-cell response 

to Brucella appears to play a key role in the development of immunity and protection against chronic 

disease.71,72 Neutralization of B abortus-induced 

host interferon gamma (IFN-g) during infection in 

pregnant mice prevents abortion.73 Moreover, macrophages treated with IFN-g in vitro inhibit intracellular 

bacterial replication.74 Studies in humans support a 

role for IFN-g in protection; homozygosity for the 

IFN-g +874A allele is associated with about a two-fold 

increase in incidence of brucellosis.75 In ruminants, 

vaccination with live vaccines is required in order to 

provide protection.76–78

These observations suggest that brucellae, like 

other facultative or obligate intramacrophage 

pathogens, are primarily controlled by macrophages 

activated to enhanced microbicidal activity by IFN-g

and other cytokines produced by immune T lymphocytes. It is likely that antibody, complement, 

and macrophage-activating cytokines produced by 

natural killer cells play supportive roles in early 

infection or in controlling growth of extracellular 

bacteria.

In ruminants, Brucella organisms bypass the most 

effective host defenses by targeting embryonic and 

trophoblastic tissue. In cells of these tissues, the bacteria grow not only in the phagosome but also in the 

cytoplasm and the rough endoplasmic reticulum.79

In the absence of effective intracellular microbicidal 

mechanisms, these tissues permit exuberant bacterial 

growth, which leads to fetal death and abortion. In 

ruminants, the presence of erythritol in the placenta 

may further enhance growth of brucellae. Products 

of conception at the time of abortion may contain 

up to 1010 bacteria per gram of tissue.80 When septic 

abortion occurs, the intense concentration of bacteria 

and aerosolization of infected body fluids during 

parturition often results in infection of other animals 

and people.

Figure 7-1. Impression tissue smear from a bovine aborted 

fetus infected with Brucella abortus. The bacteria appear as 

lightly stained, gram-negative cells. 

Photograph: Courtesy of John Ezzell, PhD, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, 

Maryland.

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Medical Aspects of Biological Warfare 

CLINICAL MANIFESTATIONS

purpura, and erythema nodosum may occur during 

brucellosis infections.84–86 Disease manifestations 

cannot be strictly related to the infecting species. 

Infection with B melitensis leads to bone or joint 

disease in about 30% of patients; sacroiliitis develops 

in 6% to 15%, particularly in young adults.87–89 Arthritis 

of large joints occurs with about the same frequency as 

sacroiliitis. In contrast to septic arthritis caused by pyogenic organisms, joint inflammation seen in patients 

with B melitensis is mild, and erythema of overlying 

skin is uncommon. Synovial fluid is exudative, but cell 

counts are in the low thousands with predominantly 

mononuclear cells. In both sacroiliitis and peripheral 

joint infections, destruction of bone is unusual. Organisms can be cultured from fluid in about 20% of cases; 

culture of the synovium may increase the yield. Spondylitis, another important osteoarticular manifestation 

of brucellosis, tends to affect middle-aged or elderly 

patients, causing back (usually lumbar) pain, local 

tenderness, and occasionally radicular symptoms.90

Radiographic findings, similar to those of tuberculous infection, typically include disk space narrowing 

and epiphysitis, particularly of the antero-superior 

quadrant of the vertebrae, and presence of bridging 

syndesmophytes as repair occurs. Bone scan of spondylitic areas is often negative or only weakly positive. 

Paravertebral abscess occurs rarely. In contrast with 

frequent infection of the axial skeleton, osteomyelitis 

of long bones is rare.91 

Infection of the genitourinary tract, an important 

target in ruminant animals, also may lead to signs 

and symptoms of disease in humans.92–94 Pyelonephritis, cystitis, Bartholin’s gland abscess and, in males, 

epididymo-orchitis, may occur. Both diseases may 

mimic their tuberculous counterparts, with “sterile” 

pyuria on routine bacteriologic culture. With bladder and kidney infection, Brucella organisms can be 

cultured from the urine. Brucellosis in pregnancy can 

lead to placental and fetal infection.95 Whether abortion 

is more common in brucellosis than in other severe 

bacterial infections, however, is unknown.

Lung infections have also been described, particularly before the advent of effective antibiotics. 

Although up to one-quarter of patients may complain 

of respiratory symptoms, mostly cough, dyspnea, 

or pleuritic pain, chest radiograph examinations are 

usually normal.96 Diffuse or focal infiltrates, pleural 

effusion, abscess, and granulomas may be noted.

Hepatitis and, rarely, liver abscess also occur. Mild 

elevations of serum lactate dehydrogenase and alkaline 

phosphatase are common. Serum transaminases are 

frequently elevated.97 Biopsy may show well-formed 

TABLE 7-2 

SYMPTOMS AND SIGNS OF BRUCELLOSIS

Symptom or Sign Patients Affected (%)

Fever 90–95

Malaise 80–95

Body aches 40–70

Sweats 40–90

Arthralgia 20–40

Splenomegaly 10–30

Hepatomegaly 10–70

Data sources: (1) Mousa AR, Elhag KM, Khogali M, Marafie AA. 

The nature of human brucellosis in Kuwait: study of 379 cases. Rev 

Infect Dis. 1988;10:211–217. (2) Buchanan TM, Faber LC, Feldman 

RA. Brucellosis in the United States, 1960–1972: an abattoir-associated disease, I: clinical features and therapy. Medicine (Baltimore).

1974;53:403–413. (3) Gotuzzo E, Alarcon GS, Bocanegra TS, et al. 

Articular involvement in human brucellosis: a retrospective analysis 

of 304 cases. Semin Arthritis Rheum. 1982;12:245–255.

Clinical manifestations of brucellosis are diverse 

and the course of the disease is variable.81 Patients 

with brucellosis may present with an acute, systemic 

febrile illness; an insidious chronic infection; or a 

localized inflammatory process. However, in the 

absence of suspicion for brucellosis, many cases 

seen in the United States are not diagnosed in the 

early stage of disease, but they are discovered once 

a focal complication has developed, such as a joint 

infection. Disease may be abrupt or insidious in 

onset, with an incubation period of 3 days to several 

weeks. Patients usually complain of nonspecific 

symptoms such as fever, sweats, fatigue, anorexia, 

and muscle or joint aches (Table 7-2). Neuropsychiatric symptoms, notably depression, headache, 

and irritability, occur frequently. In addition, focal 

infection of bone, joints, or genitourinary tract may 

cause local pain. Cough, pleuritic chest pain, and 

dyspepsia may also be noted. Symptoms of patients 

infected by aerosol are indistinguishable from those 

of patients infected by other routes. Chronically 

infected patients frequently lose weight. Symptoms often last for 3 to 6 months and occasionally 

for a year or more. Physical examination is usually 

normal, although hepatomegaly, splenomegaly, or 

lymphadenopathy may occur. Brucellosis does not 

usually cause leukocytosis, and some patients may 

be moderately neutropenic82; however, cases of 

pancytopenia have been noted.83 In addition, bone 

marrow hypoplasia, immune thrombocytopenic 

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granulomas or nonspecific hepatitis with collections of 

mononuclear cells.81 Spontaneous bacterial peritonitis 

has also been reported.98,99

Other sites of infection include the heart, central 

nervous system, and skin. Brucella endocarditis, a 

rare, but most feared complication, accounts for 

80% of deaths from brucellosis.100,101 Central nervous 

system infection usually manifests itself as chronic 

meningoencephalitis, but subarachnoid hemorrhage 

and myelitis also occur. Guillain-Barré syndrome has 

been associated with acute neurobrucellosis and involvement of spinal roots has been noted on magnetic 

resonance imaging.102,103 A few cases of skin abscesses 

have been reported. 

DIAGNOSIS

A thorough history that describes details of appropriate exposure (eg, laboratories, animals, animal 

products, or environmental exposure to locations 

inhabited by potentially infected animals) is the most 

important diagnostic tool. The differential diagnosis for 

brucellosis is broad and includes noninfectious causes 

such as vasculitis, sacroiliitis, lumbar disk disorders, 

thrombotic thrombocytopenic purpura, ankylosing 

spondylitis, abortion complications, depression/suicide, collagen-vascular disease, erythema nodosum, 

pediatric chronic fatigue syndrome, and malignancy. 

The infectious disease differential includes fever of 

unknown origin, rickettsial diseases, bacterial and viral 

pneumonia, bronchitis, cat scratch fever, cryptococcosis, 

acute epididymitis, cystitis in females, gastroenteritis, 

hepatitis, histoplasmosis, infectious mononucleosis, 

infective endocarditis, influenza, leptospirosis, malaria, 

meningitis, osteomyelitis, Epstein-Barr virus infection, spontaneous bacterial peritonitis, tuberculosis, 

tularemia, typhoid fever, and urinary tract infections 

in men. Brucellosis should also be strongly considered 

in differential diagnosis of febrile illness if troops have 

been exposed to a presumed biological attack. PCR and 

antibody-based, antigen-detection systems may demonstrate the presence of the organism in environmental 

samples collected from the attack area.

When the disease is considered, diagnosis is based 

on clinical history, bacterial isolation from clinical 

samples, biochemical identification of the organism, 

and by serology. The Centers for Disease Control and 

Prevention’s clinical description of brucellosis is “an 

illness characterized by acute or insidious onset of 

fever, night sweats, undue fatigue, anorexia, weight 

loss, headache and arthalgia.”104 Cultivation of Brucella

poses a significant hazard to clinical laboratory personnel.105–108 Rapid detection of the organism in clinical 

samples using PCR-enzyme-linked immunosorbent 

assays (ELISAs) or real-time PCR assays can be used 

to detect Brucella DNA in clinical specimens as well 

as cultivated bacteria and may eventually prove to be 

the optimal method for identification of these infections.109–112 Although PCR may have many advantages, 

a positive PCR is not proof of viable Brucella. Many of 

the assays used are not standardized and have led to 

false “outbreak” investigations in the United States 

and, therefore, these assays require proper validation 

and standardization by the testing laboratory. Typically, the most reliable and simple PCR identification 

uses a single pair of primers directed against the 16S23S rRNA operon containing the IS711 or BCSP31 

genes.111 To identify four of the major Brucella species, 

combination primers directed against the BCSP31, 

OMP3B, OMP2A, and OMP31 external membrane 

protein genes are used.111 Multiplex PCR provides 

a method to identify all known species of Brucella. 

Despite these technical advances, PCR has sensitivity 

and specificity limitations that depend heavily on the 

quality of DNA isolated and potential inhibitors present within the clinical samples.109–111 

According to the Centers for Disease Control and 

Prevention case definition for brucellosis, the infection 

may be diagnosed if any of the following laboratory 

criteria is met: 

• isolation of the organism from a clinical 

specimen; 

• fourfold or greater rise in Brucella agglutination titer between acute and convalescentphase serum obtained greater than 2 weeks 

apart; and 

• demonstration by immunofluorescence of 

Brucella in a clinical specimen.104,112

Although several serologic techniques such as the 

Coombs test have been developed and tested, the tube 

agglutination test remains the standard method.113 This 

test, which measures the ability of serum to agglutinate killed organisms, reflects the presence of anti–Opolysaccharide antibody. Use of the tube agglutination 

test after treating serum with 2-mercaptoethanol or 

dithiothreitol to dissociate IgM immunoglobulin into 

monomers makes these antibodies inactive and permits agglutination by immunoglobulin G antibodies 

that are resistant to dissolution by chemical agents. A 

titer of 1:160 or higher is considered diagnostic. Most 

patients already have high titers at the time of clinical 

presentation, so a fourfold rise in titer may not occur. 

Immunoglobulin M rises early in disease and may 

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Medical Aspects of Biological Warfare 

persist at low levels (eg, 1:20) for months or years 

after successful treatment. Persistence or increase of 

2-mercaptoethanol–resistant (essentially immunoglobulin G) antibody titers has been associated with 

persistent disease or relapse.114 Serum testing should 

always include dilution to at least 1:320, as inhibition 

of agglutination at lower dilutions may occur. The 

tube agglutination test does not detect antibodies to 

B canis because this rough organism does not have 

O-polysaccharide on its surface. Unfortunately, given 

the need for trained personnel and standardization of 

the test reagents and control sera, only some references 

laboratories, such as the Centers for Disease Control 

and Prevention in Atlanta, Georgia, and the ARUP 

National Reference Laboratory in Utah, perform the 

tube agglutination test. ELISAs have been developed 

for use with B canis, but are not well standardized. 

Although ELISAs developed for other brucellae 

similarly suffer from lack of standardization, recent 

improvements have resulted in greater sensitivity and 

specificity. ELISAs will probably replace the serum agglutination and Coombs tests, thus allowing for screening and confirmation of brucellosis in one test.115,116

In addition to serologic testing, diagnosis should 

be pursued by microbiologic culture of blood or body 

fluid samples. If unautomated systems are used, blood 

cultures should be incubated for 21 days, with blind 

subculturing every 7 days and terminal subculturing 

of negative blood cultures. For automated systems, 

incubation of cultures for 10 days with blind culture 

at 7 days is recommended.117 Because it is extremely 

infectious for laboratory workers, the organism should 

be subcultured only in a biohazard hood. Appropriate personal protective equipment such as a powered 

air purifying respirator with hood, gown, and gloves 

should be used when working with cultures or preparing and manipulating bacteria for studies. The reported 

frequency of isolation from blood varies widely, from 

less than 10% to 90%; B melitensis is said to be more 

readily cultured than B abortus. A recent study indicated that BACTECTM Myco/F lytic medium (Becton 

Dickinson Diagnostic Instrument Systems, Sparks, 

MD), pediatric Peds Plus/F or adult Plus Aerobic/F 

medium in conjunction with BACTECTM 9240 blood 

culture system yielded detection rates of 80% and 

100%, respectively.34 Culture of bone marrow may increase the yield and is considered superior to blood.118

In addition, direct fluorescent antibody tests under 

development may offer a method of rapidly identifying 

these organisms in clinical specimens (Figure 7-2). The 

case classification of “probable” is defined as a clinically compatible case that is epidemiologically linked 

to a confirmed case or that has supportive serology 

(ie, Brucella agglutination titer greater than or equal 

to 160 in one or more serum specimens obtained after 

the onset of symptoms), and a “confirmed” is a clinically compatible case that is laboratory confirmed.104,119

Future trends on rapid identification may use 

sophisticated protein microarrays to rapidly screen 

clinical samples or bacterial isolates.111 However, many 

of these state-of-the-art identification methods will remain out of reach for resource and fiscally constrained, 

endemic countries, and thus for many of these areas the 

primary methods of identification of Brucella infections 

will remain the clinical presentation and traditional 

diagnostic methods.

Figure 7-2. Direct fluorescent antibody staining of Brucella 

abortus. 

Photograph: Courtesy of Dr John W Ezzell and Terry G 

Abshire, US Army Medical Research Institute of Infectious 

Diseases, Fort Detrick, Maryland. 

TREATMENT

Brucellae are sensitive in vitro to a number of oral 

antibiotics and to aminoglycosides. In June 2005 at the 

Clinical Laboratory Standards Institute (CLSI formally 

known as National Committee for Clinical Laboratory 

Standards or NCCLS) meeting, the minimum inhibitory 

concentration breakpoints were established (Table 7-3) 

for Brucella along with the standard procedures for in 

vitro testing.120 Therapy with a single drug has resulted 

in a high relapse rate, so combined regimens should 

be used whenever possible.104,121–125 A 6-week regimen 

of doxycycline (200 mg/day administered orally) and 

streptomycin (1 g/day administered intramuscularly for 

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Brucellosis

the first 2 to 3 weeks) is effective therapy for adults with 

most forms of brucellosis.125,126 However, a randomized, 

double-blind study using doxycycline plus rifampin or 

doxycycline plus streptomycin demonstrated that 100 

mg twice daily oral doxycycline plus 15 mg/kg body 

weight of oral rifampin once a day for 45 days was as 

effective as the classical doxycycline plus streptomycin 

combination, provided these patients did not have evidence of spondylitis.127 A 6-week oral regimen of both 

rifampin (900 mg/day) and doxycycline (200 mg/day) 

is an effective therapeutic treatment with a relapse rate 

lower than 10%.128 Several studies, however, suggest 

that treatment with a combination of streptomycin 

and doxycycline is more successful and may result in 

less frequent relapse than treatment with the combination of rifampin and doxycycline.126–130 Although it is a 

highly effective component of therapy for complicated 

infections, streptomycin has disadvantages of limited 

availability and requirement for intramuscular injection. 

Other aminoglycosides (netilmicin and gentamicin), 

which can be given intravenously and may be more 

readily available, have been substituted for streptomycin with success in a limited number of studies.97 Fluoroquinolones in combination with rifampin have demonstrated efficacy similar to the doxycycline-rifampin 

regimen and may replace doxycycline plus rifampin due 

to potential doxycycline–rifampin interactions.125,131–134

Endocarditis may best be treated with rifampin, 

streptomycin, and doxycycline for 6 weeks; infected 

valves may need to be replaced early in therapy.125,135

However, if patients do not demonstrate congestive 

heart failure, valvular destruction, abscess formation, 

or a prosthetic valve, conservative therapy with three 

antibiotics—(1) doxycycline, fluoroquinolone and 

trimethoprim/sulfamethoxazole, (2) tetracycline or 

doxycycline plus rifampin, and (3) aminoglycoside 

or trimethoprim/sulfamethoxazole—may be effective 

therapy.136 Patients with spondylitis may require treatment for 3 months or longer. Central nervous system 

disease responds to a combination of rifampin and trimethoprim/sulfamethoxazole, but patients may need 

prolonged therapy. The latter antibiotic combination is 

also effective for children younger than 8 years old.137

The Joint Food and Agriculture Organization–World 

Health Organization Expert Committee recommends 

treating pregnant women with rifampin.128

Organisms used in a biological attack may be resistant to these first-line antimicrobial agents. Medical 

officers should make every effort to obtain tissue and 

environmental samples for bacteriological culture, so 

that the antibiotic susceptibility profile of the infecting 

brucellae may be determined and the therapy adjusted 

accordingly.

TABLE 7-3

BRUCELLOSIS MINIMUM INHIBITORY 

CONCENTRATION BREAKPOINT RANGES

Minimum Inhibitory Concentration

Antimicrobial range (mg/mL)

Azithromycin 0.25 – > 64

Chloramphenicol 0.5 – 4

Ciprofloxacin 0.25 – 8

Streptomycin* ≤ 8

Tetracycline 0.03 – 0.5

Doxycycline < 1

Gentamicin 0.5 – 4

Rifampin < 0.12 – 2

Levofloxacin < 0.06 – 4

Trimethoprim –

Sulfamethoxazole ≤ 2/38

*The streptomycin-susceptible breakpoint is > 16 mg/mL when the 

test is incubated in CO2 and > 8 mg/mL when incubated in room air. 

Data sources: (1) Jorgensen JH. CLSI M45-A2: Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently 

Isolated or Fastidious Bacteria; Approved Guideline-Second Edition, M45A2. 2010, Clinical and Laboratory Standards Institute, 

ISBN(s):1562387324. (2) Patel J, Heine H, oral personal communication between these principal investigators at the Clinical and Laboratory Standards Institute Guideline Meeting, June 2005.

PROPHYLAXIS

To prevent brucellosis, animal handlers should 

wear appropriate protective clothing when working 

with infected animals. Meat should be well cooked; 

milk should be pasteurized. Laboratory workers 

should culture the organism only with appropriate 

biosafety level 2 or 3 containment, depending on the 

stage of bacterial identification (diagnostic sample 

verses isolated culture).138 Chemoprophylaxis is not 

generally recommended for possible exposure to 

endemic disease.

In the event of a biological attack, the M40 mask 

(3M, St Paul, MN) should adequately protect personnel from airborne brucellae, as the organisms 

are probably unable to penetrate intact skin. After 

personnel have been evacuated from the attack area, 

clothing, skin, and other surfaces can be decontaminated with standard disinfectants to minimize risk 

of infection by accidental ingestion, or by conjunctival inoculation of viable organisms. A 3- to 6-week 

course of therapy with one of the treatments listed 

above should be considered after a confirmed biological attack or an accidental exposure in a research 

laboratory.138,139 There is no safe and effective vaccine 

currently available to use in humans.

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SUMMARY

Brucellosis is a naturally occurring disease in a 

wide variety of wild and domestic mammals. Although humans are not natural hosts for Brucella

strains, they can be infected by ingesting contaminated foods (oral route) or slaughtering infected 

animals (percutaneous route). The brucellae are 

highly infectious by the airborne route, and this is 

the route of infection that is presumed to be of the 

biggest threat to military personnel. Laboratory 

workers can easily become infected when Brucella

cultures are handled outside of a biosafety cabinet. 

Individuals presumably infected by aerosol have 

symptoms indistinguishable from patients infected 

by other routes: fever, chills, and myalgia are most 

common. 

Because the brucellae disseminate throughout the 

reticuloendothelial system, they may cause disease 

in virtually any organ system. Large joints and the 

axial skeleton are favored targets; arthritis appears in 

approximately one-third of patients. Fatalities occur 

rarely, usually in association with central nervous 

system or endocardial infection.

Serologic diagnosis uses an agglutination test that 

detects antibodies to LPS. This test, however, is not useful to diagnose infection caused by B canis, a naturally 

O-polysaccharide-deficient strain. Although ELISAs 

can more easily be standardized and performed in 

most clinical laboratories, these tests tend to have a 

higher degree of false-positive results,139 and therefore the Rose Bengal (slide-type) agglutination test140

or Brucella microagglutination test141 continue to be 

considered the gold standards for diagnosis. Infection 

can be most reliably confirmed by culture of blood, 

bone marrow, or other infected body fluids, but the 

sensitivity of culture varies widely.

Nearly all patients respond to a 6-week course 

of oral therapy with a combination of rifampin and 

doxycycline; fewer than 10% of patients relapse. Alternatively, doxycycline plus fluoroquinolone may 

be as effective for treating this disease. Six weeks of 

doxycycline plus streptomycin for the first 3 weeks is 

also effective therapy; the limited availability of streptomycin may be overcome by substitution of netilmicin 

or gentamicin. No vaccine is available for humans.

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Glanders

Chapter 8

GLANDERS

SUSAN L. WELKOS, PhD*; BRIDGET CARR GREGORY, DVM, MPH†; DAVID M. WAAG, PhD‡; and 

MARY N. BURTNICK, PhD§

INTRODUCTION

MILITARY RELEVANCE

HISTORY

INFECTIOUS AGENT

DISEASE

Epidemiology

Transmission

Pathogenesis

Clinical Disease in Animals

Clinical Disease in Humans

Laboratory Diagnosis

Treatment

Prophylaxis

SUMMARY

*Microbiologist, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

†

Colonel, US Air Force, Biomedical Sciences Corps; Deputy Chief of Staff, Defense Threat Reduction Agency/Strategic Commmand Center for Combating 

Weapons of Mass Destruction, 8725 John J. Kingman Road, Fort Belvoir, Virginia 22060; formerly, Chief, Education and Training, Division of Medicine, 

US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

‡

Microbiologist, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702 

§

Assistant Professor, Department of Microbiology and Immunology, University of South Alabama, 5851 USA Drive North, Medical Science Building, 

Mobile, Alabama 36688

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INTRODUCTION

ing equinia, malleus, droes, and farcy. Farcy is an 

ancient term given to a particular cutaneous manifestation of glanders that at the time (before 1882) 

was believed to be a completely separate disease 

in horses. With this cutaneous manifestation of 

glanders, nodular abscesses (farcy buds) became ulcerated, and regional cutaneous lymphatic vessels 

became thickened and indurated (farcy pipes) and 

oozed a glanders-typical yellow-green gelatinous 

pus (farcy oil).6 Pure farcy without ulceration of the 

mucous membranes is rare, if not just a temporary 

stage, as is vice versa.3 Humans, goats, dogs, cats, 

rabbits, and carnivorous predators living in close 

proximity to infected equids or carcasses have 

been naturally infected.1,7 Camels have also been 

infected and are associated with human disease.7

Naturally occurring glanders has been eradicated in 

most countries, but is still found in parts of Africa, 

the Middle East, Eastern Europe, Asia, and South 

America. Glanders has drawn interest as a possible 

warfare agent in the biological weapons programs 

of several countries. 

Glanders is a debilitating and often fatal zoonotic 

disease of solipeds including horses, mules, and 

donkeys caused by infection with the bacterium 

Burkholderia mallei. It is characterized by ulcerating 

granulomatous lesions of the skin and mucus membranes. Disease progression and pathology in humans 

and horses are similar, yet the clinical presentation 

of any two cases in the same species—even if related 

by direct transmission—may vary significantly.1–5

Generalized symptoms include fever, myalgia, headache, fatigue, diarrhea, and weight loss. After infection, the organism travels through lymph channels 

first to regional lymph nodes often causing irritation 

(lymphangitis, lymphadenitis) en route. Unchecked, 

organisms may enter the bloodstream and be carried 

throughout the body. Without effective treatment, the 

course of disease may range from one that is acute and 

rapidly fatal to one that is very slow and protracted 

with alternating remissions and exacerbations. 

Glanders is an old disease, having been described toward the beginning of recorded history. 

It is less commonly known by other names, includMILITARY RELEVANCE

B mallei was one of the first biological warfare 

agents used in the 20th century. Germany used an 

ambitious biological sabotage campaign in several 

countries, including the United States, Russia, Romania, France, and Mesopotamia, on both the western 

and eastern fronts during World War I. Additionally, 

cattle, horses, mules, and other livestock being shipped 

from the United States to the Allies were beleaguered 

and inoculated with cultures of B mallei.

8

 In 1914, a 

member of the German army named Anton Dilger, 

an American-educated surgeon, was sent home to live 

with his parents in Virginia after a nervous breakdown. 

He brought strains of Bacillus anthracis and B mallei and 

set up a laboratory with his brother’s help to grow the 

organisms in a private home in Chevy Chase, Maryland. Organisms were delivered to another contact 

from Germany waiting in Baltimore, who then inoculated horses awaiting shipment to the Allies in Europe. 

German agents also infected 4,500 mules in Mesopotamia with glanders, a German agent was arrested 

in Russia with similar intentions in 1916, and French 

cavalry horses were also targets for intentional glanders infection.9

 Germany and its allies infected many 

mules and horses on Russia’s eastern front, and this 

action successfully impaired artillery movement and 

troop and supply convoys. Concurrent with this rise 

in animal cases during and after the war, human cases 

increased in Russia. Attempts to contaminate animal 

feeds also occurred in the United States. Between 1932 

and 1945 the Japanese used B mallei to deliberately infect horses, civilians, and prisoners of war at the Ping 

Fan Institute, also known as Unit 731, in occupied Manchuria. Two laboratory workers accidentally exposed 

to B mallei died at the institute in 1937.10

In response to perceived biological warfare 

threats from Japan and Germany, the United States 

began work on biological warfare agents at Camp 

Detrick, Maryland (now Fort Detrick) in 1942. B 

mallei was studied for potential use but was not 

weaponized. Between November 1944 and September 1953, seven laboratory-acquired human infections from Malleomyces mallei (the taxonomic name 

of glanders at that time) occurred in Camp Detrick 

employees. Howe and Miller reported the first six 

of these infections in a case series, which remains 

the largest reported human case series in US medical literature.5

 Information on the seventh case was 

not published before 2005. All seven original case 

files were thoroughly reviewed for this chapter. 

An eighth laboratory-acquired infection occurred 

in March 2000 during US defensive research on B 

mallei.

11 Also, the Soviets were alleged to have used 

weaponized B mallei against opposition forces in 

Afghanistan between 1982 and 1984.12

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Glanders

The United States signed the Convention on the 

Prohibition of the Development, Production and 

Stockpiling of Bacteriological and Toxin Weapons, 

which banned development, production, stockpiling, 

acquisition, and retention of biological agents, toxins, 

and the weapons to deliver them in 1972.9 All offensive 

biological warfare work at Fort Detrick had ceased 

by this time; any remaining biological weapons were 

destroyed by 1973. Biodefense related research aimed 

toward the development of countermeasures to combat 

B mallei infections, however, continues to be conducted 

in the United States. A report by the Monterey Institute 

of International Studies states that between 1931 and 

1945 Japan developed B mallei as a biowarfare agent. 

There are no known current attempts for acquisition 

and use by terrorists.13

B mallei was considered a potential biothreat agent 

in 1947 because of its high infectivity, high degree 

of incapacitation among those infected, and agent 

availability.14 It could be a more significant threat 

if weaponized. As exemplified by past clusters of 

laboratory-acquired infections, B mallei is particularly 

infectious by the respiratory route. It is not considered 

to be highly contagious among humans, and reports of 

person-to-person transmission are rare. If a determined 

bioterrorist gained access to the agent, whether from 

an infected animal, laboratory culture, or commercial 

culture, the consequences could be severe. Because 

the clinical symptoms of glanders are protean and 

nonspecific, and most physicians in the west are not 

familiar with the disease, diagnosis and treatment 

may be delayed postattack, even in regions with the 

most advanced medical facilities. Delayed diagnosis 

and treatment would likely result in significant morbidity and mortality. In addition, treatment may be 

complicated by the relative scarcity of knowledge 

and experience in therapy. As equids and some other 

animals are susceptible, further spread from animals 

to humans may continue long after an initial attack. 

Fortunately, glanders is curable and postexposure 

prophylaxis may be an option if an attack was rapidly 

confirmed. As with other agents, genetic engineering 

could be used to produce a strain with unpredictable 

virulence and atypical antibiotic resistance. Thus, if B 

mallei was cultured, concentrated, and delivered as an 

infectious aerosol, significant casualties could result.15

HISTORY

Glanders is one of the oldest documented infectious 

diseases with symptoms being recorded by Hippocrates 

as early as 425 bce. Aristotle described the disease in 

horses in 330 bce and named it “malleus,” meaning hammer or mallet. It was associated with clustered horses 

around the globe, particularly army horses and mules. 

The occurrence of glanders in domesticated equids was 

so familiar that horses and their glanders commonly 

appeared together in early literature. By about the 4th 

century, Apsyrtus and Vegetius recognized the contagious nature of the disease and recommended isolation 

of affected animals. Glanders was not studied in a systematic matter until centuries later. The first veterinary 

school was established in Lyon, France, in the mid-1700s 

to deal with the serious problems of rinderpest and glanders.16 Many researchers at the school became infected 

and died of glanders during their studies. The first 

account of humans glanders was not published until 

1821.3

 In 1837, Rayer proved the transmissibility of the 

disease by successfully infecting a horse using material 

taken from a pustule of a human glanders patient.17,18

In 1882, Loeffler and Schutz isolated the causative 

agent, now called Burkholderia mallei, in pure culture 

from the liver and spleen of a glanderous horse.1,2

Up until the industrial revolution, horses and mules 

were the primary modes of transportation in all developing economies. Particularly in urban locations, these 

animals were housed under crowded conditions, and 

glanders was passed from the infected to the uninfected. 

Horses and mules were in high demand during the 

American Civil War. Thousands of animals passed 

through remount stations where glanders was found 

in epidemic proportions. The problem was exacerbated 

after the American Civil War when infected military 

stock was sold to civilians, which facilitated spread 

of the disease to communities. Heavy losses of horses 

and the infrequent but deadly transmission to humans 

in the late 19th century led several countries to consider glanders control and eradication programs. Early 

programs in some countries involved destroying only 

clinically ill equids, with compensation, and meticulous 

disinfection of the premises of such cases. Despite these 

tactics, glanders would reemerge in new or remaining 

animals in stables and barns that once housed infected 

animals and the number of countrywide cases increased. 

The notion of a carrier-state began to be accepted. 

In spite of epidemic disease in equine populations, 

there were no simultaneous epidemics in humans.

Vaccines and therapeutic agents were developed but 

they did not reduce the glanders burden. By 1890, the 

mallein diagnostic skin test was developed. Control 

and eradication programs would soon incorporate 

the testing of all contact equids, followed by quarantine and a recommendation for slaughter of all skin 

test-positive animals. This program failed in some 

locales at first because of lack of enforcement and lack 

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Medical Aspects of Biological Warfare 

of incentive to owners for killing their nonclinically 

ill animals. Some horse owners would deliberately 

hide contact animals to avoid testing, or they would 

sell these and asymptomatic test-positive animals to 

unsuspecting individuals to salvage economic loss.4

Inexpensive steam transportation helped the disease 

spread by shipping B mallei-infected animals to other 

regions and countries. The United States was blamed 

for the import of glanderous horses to Cuba in 1872 and 

for the great increase of glanders cases in Canada near 

the turn of the 20th century, where tens of thousands 

of US horses were shipped annually.3,4

Once control programs offered indemnity to testpositive and contact animals, and popular belief 

accepted the existence of a carrier-state, glanders 

eradication progressed more rapidly. Eliminating 

glanders in livestock effectively also eradicated the 

disease in humans in countries with such programs. 

Great Britain’s experience with the rise and fall of glanders outbreaks in equids typifies many countries, and 

is shown in Figure 8-1.19 Eradication of glanders was 

achieved in Great Britain by 1928 and in Canada by 

1938, about 30 years after eradication programs were 

initiated.20 Glanders was successfully eradicated in 

the United States by 1942; the last naturally occurring 

human case was recorded in 1934.21,22

Glanders is a zoonotic disease of concern internationally and is notifiable to the 164-member Office 

International des Epizooties in accordance with the 

International Animal Health Code.23 Eradication 

programs still exist for several countries attempting 

to eliminate the disease. In more than 500,000 equids 

tested in Turkey between 2000 and 2001, for example, 

less than 2% tested positive and were destroyed. Only 

one of these, a mule, showed clinical signs of infection. 

Over the past two decades, glanders in livestock was 

reported in Afghanistan, Bahrain, Belarus, Bolivia, 

Brazil, China, Eritrea, Ethiopia, India, Iran, Iraq, Kuwait, Latvia, Lebanon, Mongolia, Myanmar, Pakistan, 

Russia, Turkey, and the United Arab Emirates.21,24–35

Between 1996 and 2003 glanders in humans was reported in Cameroon, Curacao, Sri Lanka, Turkey, and 

the United States (laboratory acquired).21 Exhibit 8-1 

depicts the year equine glanders was last reported to 

Equine Glanders (and Farcy) in Great Britain: 1877–1928

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

1877

1879

1881

1883

1885

1887

1889

1891

1893

1895

1897

1899

1901

1903

1905

1907

1909

1911

1913

1915

1917

1919

1921

1923

1925

Year

Observations

Outbreaks

Cases

Eradicated in 1928

1927

Figure 8-1. Glanders cases and outbreaks reported to the Department for Environment, Food, and Rural Affairs in Great 

Britain, 1877–1928. Glanders was eradicated in Great Britain in 1928. 

Data source: http://www.defra.gov.uk/animalh/diseases/notifiable/glanders/index.htm. 

244-949 DLA DS.indb 180 6/4/18 
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Glanders

the Office International des Epizooties among countries and territories that have been without glanders 

activity (by Office International des Epizooties report) 

since 1996. Given the recent outbreaks in horses, 

donkeys, and dromedaries in some regions of India, 

Bahrain, Brazil, Lebanon, Pakistan, and the United 

Arab Emirates, glanders is currently considered a reemerging infectious disease in these areas.24–26,28,30–32,34–37

Bioterrorism should be considered as a possible source 

in the event that confirmed human glanders occurs, in 

the absence of infected animals, in the countries and 

territories listed in Exhibit 8-1.

EXHIBIT 8-1 

YEAR EQUINE GLANDERS LAST REPORTED TO OIE BEFORE 1996* 

Country or Territory Year Country or Territory Year

Australia 1891 Moldavia 1957

Austria 1952 Nambia 1925

Bulgaria 1954 Netherlands 1957

Canada 1938 Northern Ireland 1910

Croatia 1959 Norway 1889

Denmark 1928 Poland 1957

Egypt 1928 Portugal 1952

Estonia 1945 Romania 1960

Finland 1943 Serbia and Montenegro 1959

France 1965 Slovakia 1954

Georgia 1960 South Africa 1945

Germany 1955 Spain 1956

Great Britain 1928 Sudan 1989

Greece 1965 Sweden 1943

Hungary 1956 Switzerland 1937

Ireland 1920 United States of America 1942

Israel 1951 Yug Rep of Macedonia (former) 1957

Japan 1935 Zimbabwe 1911

*The most recent year evidence of equine glanders was reported to the OIE among countries and territories free of equine glanders for at 

least 5 years (between 1996 and 2013). Included only are territories for which data exist on the reporting of equine glanders to the OIE.

OIE: Office International des Epizooties

INFECTIOUS AGENT

Glanders is caused by B mallei, a gram-negative 

bacillus that is a close relative to Burkholderia pseudomallei, the etiologic agent of melioidosis. Wholegenome comparisons of B pseudomallei and B mallei

in combination with multilocus sequence typing 

(MLST) analyses suggest that B mallei is a clonal descendant of B pseudomallei that has evolved through 

genome downsizing.38 Unlike B pseudomallei, which 

can be isolated from tropical soil, B mallei is an 

obligate animal pathogen and has not been found 

free-living in the environment.39 The lack of flagellarbased motility is a primary means by which B mallei

can be differentiated from B pseudomallei. Growth 

requirements are not complex and B mallei can be 

cultivated on basic nutrient medium. However, 

glycerol or glucose can be added to the medium to 

enhance growth. When stained, the cells typically 

exhibit bipolar staining. 

B mallei is well traveled taxonomically. Since its 

discovery, this microorganism has been placed in 

several genera, including Bacillus, Corynebacterium, 

Mycobacterium, Loefflerella, Pfeifferella, Malleomyces, 

Actinobacillus, and Pseudomonas, and was finally 

assigned to the genus Burkholderia in 1992.40,41 This 

microorganism is not particularly hardy in the environment.40 B mallei is susceptible to drying, heat, 

and sunlight. In warm and moist environments, the 

organism may survive a few months and can survive 

in room temperature water as long as 1 month.1,19,42

Experimentally and under the most favorable temperature and moisture conditions, Loeffler was able to 

extend the viability of B mallei to 100 days. Survival 

of B mallei in distilled water in the laboratory was 

determined to be less than 30 days.43 In nature, viability of the organism is unlikely after 90 days, and 

most infectivity is lost within 3 weeks.

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Medical Aspects of Biological Warfare 

Particularly in culture, B mallei is easily aerosolized 

as demonstrated by at least seven of the eight laboratory-acquired infections in the United States since 

1944. Given its high infectivity by aerosol, laboratory 

studies on this Centers for Disease Control and Prevention Tier 1 select agent are performed at biosafety 

level 3 (BSL-3) facilities. Varying degrees of virulence 

among strains have been shown in the laboratory and 

in nature.4,5,7 The infectious dose is considered to be 

low, depending on the route of infection, susceptibility, and strain virulence. One to 10 organisms of some 

strains by aerosol are lethal to hamsters.5 Inhaling only 

a very few organisms may cause disease in humans, 

equids, and other susceptible species.44–46

DISEASE

Epidemiology

Naturally acquired cases of glanders in humans or 

equines are sporadic and rare; most countries have 

eradicated glanders. Glanders is still infrequently 

reported in northern Africa, the Middle East, South 

America, Asia, and eastern Europe.21 Serologic crossreactivity with B pseudomallei precludes the accurate 

distribution and prevalence of B mallei by serologic 

means alone. However, new reagents and assays 

potentially leading to improved serodiagnosis of human glanders have been described, as detailed below. 

Although human outbreaks have been reported in 

Austria and Turkey, no human epidemic has been 

recorded.47

In nature, chronically infected horses are considered 

to be the reservoir of B mallei, and they may also serve 

as amplifying hosts. A disease of primarily solipeds, 

donkeys are considered most prone to develop acute 

forms of glanders, whereas horses are more prone to 

develop chronic and latent disease. Mules, a crossbred 

animal resulting from the mating of horse and donkey, 

are susceptible to both acute and chronic disease as 

well as latent infections.40,48,49 A recent report indicates 

that Old World camels (dromedaries) can acquire 

glanders naturally when kept in close proximity to 

infected horses.35 Clinical disease in dromedaries 

closely resembled that seen in equids. Humans are an 

accidental host. 

Zoonotic transmission of B mallei from equid to 

human is uncommon, even with close and frequent 

contact with infected animals, which may be explained 

by low concentrations of organisms from infection sites 

and a species-specific difference in susceptibility to 

virulent strains. During World War II human glanders 

was rare despite a 30% prevalence in horses in China.50

Between 5% and 25% of tested animals in Mongolia 

were reactive, yet no human cases were reported. 

With successful transmission, however, humans are 

susceptible to infection. 

Humans exposed to infected equids have contracted 

glanders in occupational, hobby, and lifestyle settings. 

Naturally infected humans have included veterinarians and veterinary students, farriers, flayers (hide 

workers), transport workers, soldiers, slaughterhouse 

personnel, farmers, horse-fanciers and caretakers, and 

stable hands. Subclinical or inapparent infections in 

horses and mules have posed a hidden risk to humans. 

Infection by ingesting contaminated food and water 

has occurred; however, it does not appear to be a significant route of entry for infections in humans.1,7,51

Laboratory workers have also been rarely and sporadically infected. In contrast to zoonotic transmission, 

culture aerosols are highly infectious to laboratory 

workers. The six infected workers in the Howe and 

Miller case series represented 46% of the personnel 

actually working in the laboratories during the year 

of occurrence.5

Different strains of B mallei can now be discriminated by multiple-locus variable number tandem 

repeat analysis (MLVA), MLST, random amplified 

polymorphic DNA, and other fingerprinting methods. 

Such procedures have been shown to be useful in tracking the source and spread of an outbreak strain and in 

geographic/clonal relationship studies, and they have 

been described and summarized elsewhere.24,52 For 

example, Godoy et al used MLST with a set of seven 

loci in epidemiologic studies analyzing many isolates 

of B mallei, B pseudomallei, and Burkholderia thailandensis

(a closely related but nonpathogenic environmental 

species) from diverse geographical locations that represented 71 sequence types; specific clones isolated from 

animals that were associated with disease in humans 

were identified.35,53 MLST was most useful for distinguishing strains of B pseudomallei and B thailandensis, 

which were clearly distinguished by the divergence 

between the alleles of seven loci. However, all the 

geographically diverse isolates of B mallei analyzed 

had identical allelic profiles that clustered within the 

B pseudomallei group of isolates; alleles at six of the loci 

in B mallei were also present in B pseudomallei isolates, 

and the allele at the seventh locus in B mallei differed 

at only a single nucleotide site from B pseudomallei. B 

mallei was considered to cluster within and to be a clone 

of B pseudomallei instead of a separate species. However, one recent analysis of camel-associated glanders 

used one B mallei-specific sequence type to confirm the 

laboratory identification of glanders.35

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Glanders

MLVA has been found to be more useful for subtyping different strains of B mallei. For example, an MLVA 

analysis of a B mallei strain isolated from a diseased 

camel in Bahrain revealed close genetic proximity to 

a specific strain, which caused an earlier outbreak 

of glanders in horses in the United Arab Emirates in 

2004.35 The MLVA was based on 23 different loci, as 

reported previously.28 Similar analyses focused on B 

mallei isolates from the Punjab region of Pakistan demonstrated that these strains were genetically distinct 

from isolates from other countries.28 In the event of 

a deliberate release of B mallei or a focal outbreak of 

glanders in humans or animals, these types of analyses 

would be critical tools for facilitating investigations 

aimed at determining the source of the organism.

Transmission

Transmission is direct by bacterial invasion of the 

nasal, oral, and conjunctival mucous membranes; by 

inhalation into the lungs; and by invasion of abraded 

or lacerated skin. Areas of the body most often exposed 

include the arms, hands, and face. Considering the 

affinity for warm and moist conditions, B mallei may 

survive longest in stable bedding, manure, feed and 

water troughs (particularly if heated), wastewater, and 

in enclosed equine transporters.1

 Transmission has 

occurred via handling contaminated fomites such as 

grooming tools, hoof trimming equipment, harnesses, 

tack, feeding and husbandry equipment, bedding, and 

veterinary equipment. Such equipment stored away 

from any contact with equids for at least 3 months, even 

without disinfection, is not likely to be an infection source.

Reports of the circumstances surrounding zoonotic 

transmission are diverse. Here are a few examples: 

• equids snorting in the vicinity of humans or 

human food; 

• the wiping of equine nasal exudate off a human arm with a blade of grass (local infection 

occurred at wipe site); 

• sleeping in the same barn or stall as apparently 

healthy equids; 

• accidental puncture with contaminated 

equipment; 

• wiping an eye or nostril after contact with an 

equid; 

• being licked by a glandered horse; and 

• stall cleaning without any direct equine 

contact.3,54,55

The nature of much of the work in horse handling is 

physical, often producing skin abrasions under normal 

circumstances. Although absorption through intact 

skin is believed to be unlikely, patients may insist their 

skin was intact at the time of exposure. Among 105 

chronic human cases associated with equid exposure 

described by Robins, only 40 (38%) reported a wound 

present.3

 In 27 cases (17%) the absence of a wound was 

specifically noted.3

Laboratory infections have followed procedures 

that involved washing and aeration of cultures. Air 

samples and swabs from equipment, tables, and 

benches failed to detect residual contamination in 

laboratories after the six US laboratory-acquired events 

that occurred between 1944 and 1945. Seven of the 

eight Fort Detrick laboratory-acquired infections also 

occurred at a time when mouth-pipetting was common 

practice. The first six patients acknowledged using this 

technique to clear blocked pipettes and to blow contents out of pipettes that were calibrated to the tip. The 

eighth case patient involved a microbiologist who had 

2 years of experience of working with B mallei in BSL-3 

containment but did not always wear latex gloves.11,56

Based on the clinical manifestation of unilateral axillary lymphadenopathy, transmission in this case was 

believed to be percutaneous, yet a break in the skin or 

a specific exposure-associated laboratory incident was 

not recalled. This is not surprising as most laboratoryacquired infections are not associated with injury, or 

a recollection of injury.57 This patient had a 13-year 

history of diabetes, however, and collected blood via 

finger-stick morning and evening. It is possible that 

a recent finger-stick site may have been a potential 

entry point. Bacterial surveys of the laboratory found 

no contamination, and all engineering controls were 

validated as functional.

Human-to-human transmission is rare, but it has 

been reported. The majority of documented events 

were in medical practice, at autopsy, in the diagnostic 

laboratory, and in patient care settings before clearer 

understanding of universal precautions existed.1,3,11

Transmission also occurred in home settings. Close 

contact while caring for glanders-infected individuals 

at home led to infecting other family members.3 At least 

one entire family became infected. In this case, two 

children and the wife of a chronically infected stable 

hand contracted glanders. The wife was presumably 

infected sexually; the 4-year-old child was likely infected by close contact with a 2-year-old sibling who 

was presumably infected by one of the parents. Robins 

found that among the 156 chronic infections he studied, 

10% were directly caused by another human case.3

Human infection by ingestion has not been definitively reported. Stomach contents were found to 

inactivate B mallei experimentally in 30 minutes.47 In 

his detailed 1886 report on the etiology of glanders, 

Loeffler describes several accounts of feeding meat 

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Medical Aspects of Biological Warfare 

from glanderous horses to humans without causing 

disease.1 In one account, more than 100 glanderous 

horses were slaughtered and fed to soldiers without 

incident. Although not clear in his report, it is most 

likely that in these cases the meat was cooked just as 

was customary for a military mess at that time. In another case, consumption of raw glanderous meat by a 

veterinarian seeking to answer the ingestion question 

did not produce disease. An 1866 veterinary journal 

report, however, describes two persons who contracted 

glanders after consuming milk from a glanderous mare. 

Because these individuals were also exposed to the 

mare, infection by ingestion could not be determined.1

Monogastric animals, including the lion, tiger, 

domestic cat, dog, and bear, became infected with 

B mallei after ingesting raw meat.1 Regarding wild 

animals, Loeffler posited that crunching bones might 

cause enough oral trauma to introduce the organism 

through defects in the oral mucosa rather than by entry 

through the healthy digestive tract.1 This explanation, 

however, does not explain infections in dogs, domestic 

cats, and captive wildlife that were fed only boneless 

meat from glanderous horses. In 2010, four lions and 

one tiger at a zoo in Tehran died from glanders.29 Although not definitively proven, this outbreak was attributed to ingestion of soliped meat that had not been 

screened for B mallei. Based on this limited collection of 

testimonies and the current understanding of glanders 

pathogenesis, one may infer that ingestion of the live 

organism by humans is unlikely to cause disease. 

These features of transmission exemplify the requirement for BSL-3 containment and safety practices 

when working with B mallei. Adherence to safety 

procedures and universal barrier precautions is also 

prudent. In the presence of potentially infected equids, 

transmission risk is also reduced by universal precautions as well as procedures that reduce inhalation risk 

of potentially contaminated aerosols. Advances in 

medicine, infection control, and therapeutics make it 

less likely today than 100 years ago for human-to-human transmission to occur even in the event of a human 

outbreak, whether related to bioterrorism or not. It is 

also highly unlikely that an equid reservoir would become established. Acute disease is expected to manifest 

in a significant proportion of exposed equids, which 

would allow emergency response, quarantine, traceback, and eradication procedures. Long-term exposure 

to asymptomatic chronically infected equids that evade 

detection and are handled without precautions could 

become a sporadic but perilous risk to humans, and 

less caution may be used around them.3

Among equids, transmission is primarily by oronasal mucous membrane exposure, inhalation, and 

mastication (possibly ingestion) of skin exudates and 

respiratory secretions of infected animals, including 

those with latent and subclinical infection. The sharing 

of feed and water troughs facilitates this, as do common equid behaviors that include grooming and 

snorting.40,48,49 Since equids cannot breathe through 

their mouths, simple exhalation and snorting to clear 

nasal passages serve to finely aerosolize infectious 

nasal efflux from an infected equid, which poses a 

transmission risk to susceptible hosts (including humans) in the vicinity.

Transmission through ocular mucous membranes 

and abrasions in the skin is also possible. Vertical 

transmission from mare to foal has occurred naturally 

in horses. In utero transmission from sow guinea pig to 

pup has also occurred in housed laboratory animals.1

Sexual transmission from stallion or jack to mare or 

jenny has also been observed. The breeding of asymptomatic stallions was responsible for some glanders 

spread near the turn of the 20th century.4

Carnivores can become infected after eating contaminated carcasses and meat.29,58 Reported outbreaks 

in captive wild felids suggest that they appear to be 

more susceptible than canids.40,48,58,59 Glanders has also 

been transmitted to goats housed with infected horses.1

Laboratory animals are also susceptible, including mice, 

hamsters, guinea pigs, rabbits, and monkeys.1 Cattle, 

swine, and chickens appear to be resistant, even after 

experimental injection.1,59,60 Pigeons were infected experimentally.1

 A review of experiments on glanders in 

animals led Loeffler to suggest that the field mouse, donkey, mule, horse, goat, cat, and guinea pig were more 

susceptible to glanders infection and clinical disease 

than humans.1 Among other susceptible host species, 

the rabbit and dog appeared to be less susceptible to 

disease than humans. Recent reports have described the 

use of invertebrate species including wax moth larvae, 

cockroaches, and nematodes as viable experimental 

models of glanders that are primarily useful for identifying virulence factors.61–63

Pathogenesis

Overview of Pathogenesis

Although glanders has been largely eradicated 

in humans, and for the most part in animal populations, B mallei is considered a significant potential 

biothreat agent.64 Both the acute and chronic forms of 

glanders were described in detail long before effective treatments were available and when the disease 

was still prevalent. In the 1906 review of 156 chronic 

human glanders cases, Robins stated that distinguishing between chronic and acute disease was difficult 

because chronic disease was often interrupted with 

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Glanders

acute symptoms and acute onset disease may run a 

chronic course.3 For convenience purposes he defined 

chronic cases as those lasting longer than 6 months. 

Most historical literature attempting to differentiate 

the two classifies a more fulminant and rapidly fatal 

clinical course (within 2 to 4 weeks) as an acute form 

of glanders. An acute course is found more often with 

untreated acute pneumonic and frank septicemic infection, whether primary or recurrent.5,47,65 Chronic infections are most common in horses where they comprise 

the majority of cases, whereas acute disease is more 

common in humans and donkeys.7

B mallei most often enters the human body through 

abrasions or openings in the skin, particularly where 

occupationally exposed on the hands and forearms, 

face, and neck. An abrasion is not always present, 

however, at least grossly. Normal, intact skin resists 

penetration of the organism; however, in several human infections, the affected persons insisted no wound 

or penetration occurred during the likely exposure 

interval. Thus, a patient history in which there is no 

recollection of exposure to horses or of abrasion should 

not preclude glanders as a differential diagnosis. 

Organisms may also enter through oral, nasal, and 

ocular mucous membranes, as well as via inhalation. 

The latter has occurred in several laboratory-acquired 

infections; however, at least one laboratory-acquired 

case most likely occurred through cutaneous exposure. 

When present, the most characteristic feature of the 

disease is glanders nodes—small papular to egg-sized 

abscesses—that are very slow to heal if they open. 

The incubation period for glanders is variable, ranging from less than a day to several weeks. Cutaneous 

and mucous membrane exposure generally leads to 

symptoms in 3 to 5 days; without direct inoculation of 

the organism, however, the duration may be longer.3

Inhalational exposure may incur a slightly longer 

range of about 7 to 21 days.3,5

Figure 8-2. Interactions of Burkholderia mallei with host cells. (a) Proposed model of the intracellular lifestyle of B mallei in 

phagocytic cells. Following entry in host cells, B mallei rapidly escapes from the phagosome and enters into the cytosol 

where it can grow, polymerize host cell actin (red), spread cell to cell, and induce host cell fusion resulting in the formation 

of multinucleated giant cells. B mallei interacts with various pattern recognition receptors and evades host cell autophagy. 

Genes, proteins, or systems that are known to be important at various points are indicated in blue text. VirAG senses a signal within the phagosome that activates T6SS-1 gene expression; T3SSAP is required for escape from the phagosome; BimA 

is necessary for actin-based motility and actin tail formation; T6SS-1 is critical for multinucleated giant cell formation. (b) 

Fluorescent micrograph of B mallei infected RAW 264.7 murine macrophages. B mallei was added to RAW 264.7 cells at a 

multiplicity of infection of 10; at 12 hours postinfection cells were fixed and stained for actin and nuclei. B mallei expressing 

green fluorescent protein is shown in green; actin stained with Alexa Fluor568 phalloidin is shown in red; nuclei stained with 

DRAQ5 are shown in blue. MNGC: multinucleated giant cell

Photograph: Courtesy of Mary N Burtnick, University of South Alabama, Mobile, Alabama.

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Intracellular Lifestyle

B mallei is a host-adapted deletion derivative of B 

pseudomallei and is genetically more uniform and less 

diverse than the latter.66,67 The severity, clinical course, 

and frequent chronicity of glanders are likely related 

to the capacity of B mallei to survive and persist within 

host cells and thus evade destruction by the immune 

system. As an intracellular pathogen, survival of B mallei involves binding to and invading eukaryotic cells, 

successfully escaping phagosomal compartments, and 

growing in the cytosol. Relatively little is known concerning how the organism binds to cells or the surface 

receptor involved. B mallei adheres poorly to and does 

not invade A549 and LA-4 respiratory epithelial cell 

lines, but readily invades phagocytic cell lines such 

as J774.2 and RAW 264.7 murine macrophages.68–70 As 

shown in Figure 8-2 and as discussed below, B mallei

uses a type III secretion system to escape from the 

phagosome into the cytosol, where it can multiply and 

use actin-based motility to move about the cell.68,69,71,72

In addition, B mallei can induce cell-to-cell fusion resulting in the formation of multinucleated giant cells 

(MNGCs), which are thought to provide a protective 

niche and metabolic resources for the bacteria, but 

little is known about the bacterial or host factors that 

are required for MNGC formation. Recent evidence 

points to expression of a type VI secretion system as a 

requirement for efficient intra- and intercellular spread 

and host cell fusion.73,74 B mallei can also evade innate 

host immune responses when present extracellularly 

due in part to its surface structures, which will be 

described below. However, little is known about the 

specific molecular mechanisms of B mallei virulence, 

and more study is urgently needed.

Animal Models

Research on pathogenicity requires the availability 

of relevant, well-characterized animal models. Various 

animal models of glanders have been reported for use 

in studies on pathogenesis and countermeasure evaluation, including guinea pigs, mice, hamsters, nonhuman primates (NHPs), and several invertebrates. Major 

models will be described and are summarized in Table 

8-1; several recent reviews are available that provide 

further details of the development of in vivo models 

for glanders.44,75,76

Guinea Pigs and Hamsters

Guinea pigs and hamsters are the laboratory animals exhibiting the greatest susceptibility to B mallei, 

and guinea pigs were initially used most extensively. 

However, these animals varied in their individual susceptibility to infection. Syrian hamsters proved to be 

uniformly susceptible to infection and have been used 

more extensively in recent studies on B mallei.

44,60,75,77 In 

1999, Fritz et al characterized disease in these animals 

bacteriologically and pathologically to include gross, 

histological, immunologic, and electron microscopic 

pathology.78 The hamster was shown to be much more 

susceptible to B mallei than is presumed for humans, 

with an LD50 of less than 10 colony-forming units 

(cfu) by the intraperitoneal (IP) route, with mortality/morbidity monitored for 5 days.77,78 Nevertheless, 

the course of disease and extensive development of 

glanders-associated pathology in a broad range of 

organs, especially in reticuloendothelial organs (such 

as spleen, lymph nodes, liver, and bone marrow) but 

also in other organs such as lung and brain, are similar 

to those in humans. The changes observed consist of 

infiltrates with an equal mixture of macrophage and 

polymorphonuclear leukocyte inflammatory cells; 

these can become organized into discrete, often bacteria-filled, nodules referred to pyogranulomas. More 

extensive information on the pathology of glanders 

in these susceptible species is described elsewhere.44

Mice 

Mice vary by strain in their susceptibility to B mallei, 

a finding that mirrors that observed in studies with B 

pseudomallei.

79–81 However, mice are all considered to 

be moderately resistant to infection, similar to humans. 

Depending on the strain, route, and dose of infection 

selected, they have been used to model a range of 

disease manifestations and states ranging from latent 

to acute or chronic. Since genetic constructs and reagents specific to mice are widely available, they are 

common tools for studies on pathogenesis and protection. In most studies, the C57Bl/6 strain was more 

resistant than BALB/c strain mice by the pulmonary 

route.44,76,82 Studies by Goodyear et al have shown 

that when B mallei was administered intranasally to 

C57Bl/6 mice, high dose inocula (5,000 cfu) resulted 

in acute infections that were lethal within 3 to 4 days, 

whereas low dose inocula (500 cfu) resulted in chronic 

infections.82 In recent as well as previous studies, the 

relatively more sensitive BALB/c strain has been used 

most often.44,76,83,84 In acute infection studies with 

BALB/c mice, a lethal IP dose often results in splenomegaly with multiple splenic white foci consisting 

of pyogranulomatous inflammation. This pathology 

occurs also in other reticuloendothelial organs, as 

typically described for hamsters; but mice usually 

do not exhibit this pathology in other organs, such as 

the lung, as occurs later in infection of hamsters.44,83

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Glanders

TABLE 8-1 

EXPERIMENTAL ANIMAL MODELS OF GLANDERS

Model Route of Infection Features

Horses IT Physiologically relevant model that mimics the natural development of chronic 

glanders in its reservoir host. Horses typically exhibit a long disease course 

with periods of improvement and relapse.

Rhesus monkeys SC and aerosol Development of SC lesions that healed after 3 weeks. No evidence of chronic or 

acute infection. Evidence of potential nonlethal chronic infection after aerosol 

exposure.

African green Aerosol Development of acute glanders resembling the human infection.

monkey

Syrian golden IP Extremely susceptible infection model (LD50 <10 cfu) with development of acute 

hamsters infection resulting in a rapid disease course. Bacteria are transported to the 

(Mesocricetus mediastinal lymph nodes and seeded to other tissues, forming lesions in the 

auratus) spleen as early as 1 day postinoculation and death occurring around 6 days 

later. 

Guinea pigs SC and IP Development of acute glanders. Bacteria are transported to inguinal and axillary lymph nodes (SC), or mediastinal and mesenteric lymph nodes (IP), at 

early time points postinoculation. 

BALB/c mice Aerosol, IN, and IP Good model for acute and potentially chronic glanders. Commonly used model 

due to low cost, susceptibility to B mallei infection, and a well-documented 

disease pathology. Mice are more resistant to B mallei when delivered IP. 

C57Bl/6 mice Aerosol, IN, and IP More resistant to B mallei infection than BALB/c mice by pulmonary routes. 

High doses delivered IN resulted in acute disease, whereas low doses produced chronic infections.

Wax moth larvae Injection into Used to screen putative virulence mutants; between 3 to 200 cfu of wild type 

(Galleria hemocoel B mallei leads to >90% killing within 6 days.

mellonella)

Madagascar Injection into dorsal Used to screen putative virulence mutants; highly susceptible infection model 

hissing abdomen (LD50 <10 cfu) resulting in death within 5 days.

cockroaches

Nematodes Feeding Used to screen putative virulence mutants; limited sensitivity resulting from 

(Caenorhabditis high infectious dose.

elegans)

cfu: colony forming unit

IN: intranasal

IP: intraperitoneal

IT: intratracheal

LD: lethal dose

SC: subcutaneous 

Aerosol exposure to a lethal dose of B mallei produces 

gross and microscopic pathologic changes similar to 

those after IP injection, except that there is more lung 

involvement, that is, focal inflammation and necrosis 

early in infection develop into extensive consolidation 

with chronic inflammatory cell infiltration (David L 

Fritz, David DeShazer, David M Waag, USAMRIID, 

Fort Detrick, MD, unpublished data, 2012).84 It has 

been observed that aerosol-exposed BALB/c mice 

develop acute inflammation of the nasal passages, 

which later extends to the nasal sinuses and ultimately 

into the brain, especially the olfactory lobe (David M 

Waag, USAMRIID, Fort Detrick, MD, unpublished 

data, 2013).84 Unlike humans, mice are obligate nose 

breathers, an anatomical difference that may explain 

these findings. Recent models using an intratracheal 

(IT) route of infection address this potentially confounding issue.85 However, the brain infection in mice 

might serve as a potential protected site for bacterial 

survival and provide a model for studying recrudescence of disease.

Acute infection virulence data obtained with murine 

models often used laboratory strains such as the type 

strain B mallei ATCC 23344 (aka China 7) or the most 

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Medical Aspects of Biological Warfare 

recent human clinical isolate of B mallei, referred to as 

the FMH 23344 strain of B mallei ATCC 23344.56 The 50% 

lethal doses (LD50s) for BALB/c mice have varied by 

route, and the mice were more susceptible by pulmonary routes than by the IP route. The range of LD50s, as 

determined 10 to 35 days postchallenge (most 21 or 28 

days), is exemplified as follows for BALB/c mice: IP (7 

x 105 cfu), intranasal (IN; 680 cfu), IT (818 cfu), wholebody aerosol (913 cfu), and nose-only aerosol (1,859 

cfu).76,77,83–88 Recently, the virulence of B mallei strain 

FMH 23344 was reevaluated in C57Bl/6 and BALB/c 

mice challenged by the whole-body aerosol route. As 

expected, C57Bl/6 mice were significantly more resistant than BALB/c mice; the respective LD50s (after 21 

days) were 7,665 cfu and 395 cfu, respectively (David 

M Waag, USAMRIID, Fort Detrick, MD, unpublished 

data, 2013). 

No well-defined murine models for long-term 

chronic or latent glanders exist. However, B mallei

clearly establishes protracted persistent infections 

in mice as it does in horses and humans, as shown 

especially in BALB/c mice. In recent aerosol and IP 

challenge studies, spleen cultures revealed the presence of long-term residual infection with B mallei in 

surviving mice. Such infections were more consistently detected in BALB/c than in C57Bl/6 survivors, 

that is, BALB/c mice surviving greater than or equal 

to 60 days after aerosol challenge with the FMH 

derivative of strain B mallei ATCC 23344 had positive spleens; whereas C57Bl/6 survivors had usually 

cleared the infection (Christopher K Cote and David 

M Waag, USAMRIID, Fort Detrick, MD, unpublished 

data, 2012). Fritz et al conducted a natural history 

study of B mallei ATCC 23344 in mice infected lethally 

and sublethally by the IP route.83 Pyogranulomatous 

inflammation was observed histologically in multiple reticuloendothelial organs, and the incidence 

and severity of the changes did not decrease in the 

sublethally infected mice. Barnes et al treated mice 

that had been infected by aerosol with B mallei with a 

postexposure prophylactic regimen of trimethoprimsulfamethoxazole that effectively prevented acute infection; two-thirds of the mice survived to study end 

at day 74.89 However, the treatment did not eradicate 

the bacteria and a clinical relapse of infection occurred 

by day 30 postchallenge. B mallei was detected in the 

organs of all surviving mice. Similar data have been 

reported, and the results overall suggest that BALB/c 

mice could serve as a useful model for both acute and 

chronic glanders in a relatively resistant host such 

as humans.89,90 Further research to develop defined 

murine models for persistent forms of B mallei infection are needed to identify the factors involved in the 

evolution of infection.

Nonhuman Primates

Although NHPs are phylogenetically the closest 

animal to humans, few studies have described their 

use as laboratory models. In one study, rhesus monkeys were given different doses of a virulent strain 

by the subcutaneous route, and the monkey receiving 

the highest dose developed a cutaneous abscess that 

resolved completely after 3 weeks.44,60,76 Russian investigators reported studies with baboons but few details 

are available.44 Different species of NHPs appear to 

vary significantly in susceptibility but overall there is 

a lack of substantial data characterizing experimental 

infection in these animals. The challenge is to develop a 

model(s) that best mimics the human acute and chronic 

responses to B mallei. Recent studies have shown that 

rhesus macaques are resistant to acute infection (John 

J Yeager and M Louise Pitt, USAMRIID, Fort Detrick, MD, unpublished data, 2012); however, Yingst 

et al showed that rhesus monkeys given subclinical 

doses of B mallei exhibited clinical presentations and 

pathological lesions that correlated well with those 

described for human cases of glanders.91 They suggested that the rhesus macaque is a potentially viable 

model for human disease albeit not acute lethal illness. 

A longer-term study is being done to include LD50 determinations with extensive clinical, pathological, and 

laboratory evaluations to further analyze three NHPs 

as models of human glanders (Patricia L Worsham, 

David M Waag, and Taylor B Chance, USAMRIID, Fort 

Detrick, MD, unpublished data, 2013). African Green 

monkeys (Chlorocebus aethiops) were much more sensitive to infection than cynomolgus macaques or rhesus 

macaques and appear to be a potential model for 

acute infection (Patricia L Worsham, David M Waag, 

and Taylor B Chance, USAMRIID, Fort Detrick, MD, 

unpublished data, 2013). 

Invertebrate Models 

Several nonmammalian surrogate models have 

recently been used to study virulence mechanisms 

and host-pathogen interactions of the human pathogenic species of Burkholderia, including insect larvae, 

cockroaches, a phagocytic amoeba, and soil-dwelling 

nematode. For B mallei, models using wax moth larvae 

(Galleria mellonella) and the Madagascar hissing (MH) 

cockroach have been reported.62,63 Insect models are 

useful mammalian surrogates for several reasons. Significant similarities exist between the innate immune 

systems. Both hosts harbor Toll receptors (insects) or 

Toll-like receptors (TLRs, mammals) that recognize 

pathogen markers and produce protective responses 

such as antimicrobial peptides; also insects possess 

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Glanders

phagocytic hemocytes that can take up and kill microbes, in a manner similar to that of neutrophils. Other 

advantages of using insects as models involve ready 

availability, reduced costs and facile housing/maintenance, and exemption from regulatory oversight and 

expense. B mallei was shown to be as virulent for the 

larvae as it was for hamsters and mice, whereas Burkholderia that are nonpathogenic in mammals were not 

pathogenic for the insects. Notably, in tests with mutants 

of B mallei harboring known virulence-associated gene 

defects (eg, in capsule production or in the type three 

secretion system [T3SS]AP), lethality for wax moth larvae 

corresponded to the extent of reduced virulence of the 

mutants in hamsters and mice.63 Fisher et al found the 

MH cockroach to be easier to handle than wax moth 

larvae and their ability to grow at 37°C made them 

more amenable than other insects to mutant analysis 

with B mallei, a mammalian host-adapted pathogen.62

Thus, MH cockroaches appear to be a valid surrogate 

and alternative to mammals as a model for virulence 

mechanisms of B mallei important in host interactions. 

Virulence Mechanisms

Surface Polysaccharides. Since B mallei appears to 

be genetically derived from the environmental saprophyte B pseudomallei, it shares many virulence factors 

with the latter.75,92 However, some of the B pseudomallei

factors required for its independent lifestyle appear 

to have been lost in B mallei as a consequence to its 

adaptation to the equine hosts. In addition, differences 

in the presence or role of some virulence factors in B 

mallei compared to B pseudomallei have been described 

(possibly related to the increased presence in B mallei

of insertion sequence elements and genetic rearrangements), as will be illustrated. The factors and activities 

identified as being essential for B mallei virulence and 

host persistence include the following: 

• a capsular polysaccharide (CPS);

• lipopolysaccharide (LPS); 

• animal pathogen-like T3SS (T3SSAP);

• the cluster 1 T6SS (T6SS-1); and 

• the VirAG two-component regulatory 

system.38,72,74,77,93

Other putative virulence factors, including various 

autotransporter proteins, adhesins, quorum sensing, 

and iron-binding compounds, have been identified but 

their roles in virulence are unconfirmed (as described 

below). 

Two major polysaccharide (PS) antigens that are 

present on the surface of B mallei, a CPS and LPS, play 

important roles in the pathogenesis of glanders and 

in host responses to the infection. The presence of a 

CPS on the surface of B mallei was shown by immunoelectron microscopy.77 The structure of the CPS antigen 

has recently been characterized and shown to be identical to the CPS expressed by B pseudomallei, which is a 

homopolymer of -3)-2-O-acetyl-6-deoxy-ß-D-mannoheptopyranose-(1-.77,94 Consistent with this finding, 

anti-CPS monoclonal antibodies (mAbs) that have been 

characterized recognize the CPS of both pathogens.95–97

The surface-expressed nature of the LPS is evidenced 

by its availability to the host immune system and its 

ability to activate TLR4 complexes.97–99 The structure of 

B mallei O-polysaccharide (OPS) has been determined 

to be a repeating unit of -3)-β-D-glucopyranose-(1,3)-6-

deoxy-α-l-talopyranose-(1- where the talose residues 

contain 2-O-methyl or 2-O-acetyl side groups.93,100 In 

comparison to B pseudomallei, B mallei OPS lacks 4-Oacetyl modifications on the talose residues.93,100,101 This 

structural difference explains why mAbs specific for 

either B mallei LPS or B pseudomallei can be isolated.101,102

The virulence roles of these PS antigens were demonstrated by the construction of mutant strains lacking 

either CPS or OPS, which proved to be avirulent in 

animal models.77,93,103 The precise functions of CPS 

and LPS in pathogenesis are not fully characterized; 

however, the CPS may contribute to survival in serum 

by inhibiting complement deposition, opsonization, 

and phagocytosis, as well as possibly conferring resistance to the harsh environment of the phagosome 

until the bacteria are able to escape.75 OPS is known 

to be critical for serum resistance since B mallei strains 

lacking OPS moieties are rapidly killed by 30% normal 

human serum.93 mAbs to both B mallei CPS and LPS 

have been identified that are either bactericidal for the 

organism or have strong opsonic activities.97 The roles 

of these two PS moieties in pathogenesis were further 

confirmed by demonstrating that passive administration of LPS- or CPS-specific mAbs effectively protected 

mice against lethal pulmonary challenge.97,104 

Secretion Systems and Secreted Proteins. B mallei, 

a highly successful facultative intracellular pathogen 

that can survive in many eukaryotic cell lines, possesses a variety of mechanisms to adapt to and alter the 

host environment.68,69,71 The organism harbors an array 

of specialized secretory systems that are essential to 

this process. Little is known about these systems or the 

specific roles of their components, although a number 

of the genes identified appear to be homologous to 

the more extensively studied species B pseudomallei. 

B mallei-specific studies have focused primarily on 

characterization of T3SSAP and T6SS-1.69,72–75,105,106 Genes 

encoding other secretion systems, including the type 

II and type V systems, are also present in B mallei.

75

The effector proteins delivered by these systems are 

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Medical Aspects of Biological Warfare 

predicted to disable or modulate critical host proteins 

and pathways involved in cell signaling, cytoskeleton 

and ubiquitin function, and cell death pathways, 

thus facilitating pathogen survival and propagation 

in the host.70,75,107–109 In addition, both B mallei and B 

pseudomallei exhibit actin-based motility and the distinct ability to induce formation of MNGCs in tissue 

culture models, potential mechanisms that allow the 

pathogen to spread in the host via direct cell-to-cell 

passage (Figure 8-2).73,75,110 

After B mallei is phagocytosed by the host cell, it 

escapes from the endocytic vacuoles into the host cell 

cytoplasm where it uses actin-based motility to spread 

intra- and intercellularly. Virulence-associated T3SSAP

and T6SS-1, as well as other secreted proteins, appear to 

be essential for these various functions.68,69,71–73,75,103,111,112

Many gram-negative bacterial pathogens harbor such 

secretion systems and use them to synchronize the 

secretion and delivery of effector proteins directly into 

target host cells via needle-like injection apparatuses. 

B mallei, T3SSAP is required for virulence in animal 

models of infection, as well as for phagosomal escape 

and survival in macrophage tissue culture models of 

infection.69,72,75 Once free in the cytosol, the microbe 

activates processes for evading host cell killing and 

for polymerization of host cell actin.75 BimA, a type V 

secreted (T5S) protein, plays a major role in facilitating 

actin-based motility in B mallei.

113,114

Although the T3SSAP is important for phagosomal 

escape and survival within host cells, the exact roles 

of specific effector proteins delivered by this system 

have yet to be clearly identified. Many proteins are 

predicted to be part of the B mallei T3SSs by in silico 

annotation; some have been partially characterized, 

and the potential roles of a few in virulence have been 

studied.75,92 These proteins include BopA and BopE; 

Bip B, C, and D; and BapB, as described in detail in previously published reports.70,75,92 In B mallei a mutation 

in the T3SS-encoded BopA effector protein resulted in 

a slower growth rate in macrophages and an apparent 

reduced ability to escape the cells. This mutation also 

attenuated infection by B mallei in BALB/c mice, suggesting BopA may contribute to survival within—and 

possibly escape from—host alveolar macrophages.70,115

In B pseudomallei, BopA appears to enhance survival 

by helping the microbe evade autophagy-induced 

phagocytic vacuole degradation.116 Bip B, C, and D 

proteins appear to be structural components of the 

T3SS injector apparatus that are involved in contact 

of the tip with host cells.75

T6SS-1, which is important during the intracellular 

lifestyle of the B mallei, is essential for B mallei virulence 

in a hamster model of glanders.63,74 In a RAW 264.7 

macrophage model, T6SS-1 expression was shown to 

occur following internalization of B mallei, but before 

escape of the organism from the phagosomal environment.73 Once in the cytosol of host cells, T6SS-1 

mutants displayed defects in actin polymerization and 

an inability to induce MNGC formation.73,105 T6SSs are 

proposed to resemble inverted bacteriophage tail-like 

structures involved in delivering effector molecules directly into target cells. These systems are tightly regulated at the genetic level so that they are only expressed 

at the appropriate time and place. Two key components 

of T6SS-1 are the Hcp1 and VgrG1 proteins, which are 

both secreted and structural components of the T6SS 

apparatus and are considered reliable indicators of 

T6SS function.74,112,117,118 Various components of T6SS1 are being characterized because these proteins may 

represent potential diagnostic, therapeutic, or vaccine 

targets.73,74,105,111,112 

The VirAG two-component system is an important 

regulator of virulence gene expression in B mallei and 

is required for virulence in hamsters.38 Approximately 

60 genes are under the regulatory control of VirAG, 

including the T6SS-1 gene cluster and genes involved 

in actin-based intracellular motility (bimBCADE).74

When the VirAG system is overexpressed in vitro, 

Hcp1 and VgrG1 are secreted into culture supernatant by T6SS-1. VirAG also controls the expression 

of tssM, which encodes a putative ubiquitin-specific 

protease (ie, deubiquitinase) that is expressed shortly 

after intracellular uptake and provides the bacteria 

with an enzymatic tool that can potentially regulate 

multiple eukaryotic cell processes.74,108 Shanks et al 

characterized the expression and regulation of B 

mallei TssM, and demonstrated that it was a potent 

ubiquitin-specific protease.108 Ubiquitin is a host protein that attaches to other proteins so as to direct their 

intracellular fate. Bacterial deubiquitinases remove 

the ubiquitin residues, disrupting this process and 

promoting bacterial evasion of host immune responses 

and survival. Although the TssM protease may provide 

B mallei a selective advantage within the cell during 

infection, a role in virulence for hamsters was not 

shown. Interestingly, even though tssM is coregulated 

with and physically linked to the T6SS-1 gene cluster, 

TssM is not secreted by either T6SS-1 or T3SSAP.

108 More 

research is needed to determine the signal sensed by 

VirAG in vivo that ultimately results in the expression 

of this important regulon.

Autotransported and ATP-binding Cassette Proteins. Several autotransporter (AT) and ATP-binding 

cassette (ABC) transporters that may have roles in the 

infectious process have been described for B mallei. 

ATs are large families of outer membrane proteins 

in gram-negative bacteria that are secreted by the 

T5S pathway, and they include virulence-associated 

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invasins, adhesins, proteases, and actin-nucleating factors.119,120 AT proteins have three common features: (1) 

an N-terminal signal sequence for periplasmic translocation, (2) a central functional domain(s), and (3) an 

outer membrane channel-forming C-terminus needed 

for surface export of the central domain.119 Eight of 

the 11 AT analogs of B pseudomallei are shared by B 

mallei and include BimA, an AT involved in actin tail 

formation and actin-based motility, and BoaA, an AT 

with a potential role in bacterial adhesion to epithelial 

cells.74,76,114,121 BimA is expressed by both B pseudomallei

and B mallei, although their sequences vary. In both 

species BimA is required for actin-based motility and 

MNGC formation in infected tissue culture monolayers.74,76,114,122 Interestingly, in B mallei, the bimBCADE 

genes were found to be dispensable for virulence in 

hamsters.74

A group of immunodominant Burkholderia antigens, 

designated Hep-Hag autotransporter (BuHA) proteins, 

shares structural similarities with hemagglutinins and 

invasins.123 These proteins were present in 53% of a B 

mallei expression library examined and only 3% of a 

B pseudomallei library. They appear to function as surface proteins that modulate interactions of the bacterial cell with the host and environment; homologs in 

other bacteria have significant roles in virulence, but 

their possible roles in B mallei virulence and immune 

modulation require further study. Finally, several ABC 

protein systems with established roles in the virulence 

and pathogenicity of various gram-negative pathogens 

have been identified in the Burkholderia.

124,125 Although 

their contribution to B mallei pathogenicity has not been 

evaluated, some components (eg, the ABC transporter 

protein LolC) have been shown to be immunogenic 

and to elicit significant partial protection against both 

B mallei and B pseudomallei. These proteins deserve 

further analyses as both putative virulence factors and 

vaccine targets.

Quorum Sensing Systems. Quorum sensing (QS) 

permits bacteria to monitor their population density 

and modify gene transcription at critical population 

levels.126 Many host-associated bacteria use small 

amphipathic acyl-homoserine lactone signals for QS; 

and Duerkop et al identified octanoyl-homoserine 

lactone as the predominant BmaI1 synthase-produced 

acyl-homoserine lactone signal and activator for the 

B mallei LuxR QS system.126 Nevertheless, numerous 

animal pathogens lack such systems, yet are virulent. Using mutants in bmaI genes of the B mallei QS 

systems, Ulrich et al showed that QS was critical for 

virulence of B mallei in aerosol-exposed BALB/c mice.127

However, using constructs with similar mutations in 

bmaI, Majerczyk et al recently determined that QS 

was not required for lethal infection of mice exposed 

by aerosol to B mallei.

128 These studies do not exclude 

a role for QS in glanders in other animal models or the 

natural equine host. 

Other Potential Virulence Factors. A role for pilin/

fimbriae structures in the pathogenesis of glanders 

is poorly defined.75,129 For instance, type IV pili are 

required for virulence of B pseudomallei, and although 

they are expressed by B mallei in vivo and are highly 

immunogenic, a role for them in adherence and virulence of B mallei has yet to be shown. Neither active 

nor passive immunization with pilin or anti-pilin 

antibodies protected mice against subcutaneous or 

aerosol challenge. Its protective role in a natural host 

or incidentally infected human or after exposure by 

different means remains to be determined.129 Several 

other virulence mechanisms are being investigated, 

and innovative techniques and combination approaches (in silico, in vitro, and in vivo) are being used to 

identify putative novel virulence factors with possible 

roles in pathogenesis of B mallei or in protective immunity.67,92 One of these virulence mechanisms is the 

carboxy-terminal processing protease of B mallei and 

compounds potentially involved in iron acquisition, 

such as the siderophore malleobactin.130–132

Clinical Disease in Animals

B mallei naturally infects horses, donkeys, and 

mules, but other species have occasionally become 

infected.41,45,58,133 If glanders is suspected as a differential diagnosis, local and regional animal and public 

health authorities must be immediately notified. The 

incubation period for glanders in equids ranges from 

a few days to many months, with most between 2 to 

6 weeks. The infectious process, disease progression, 

and pathology in equids are similar to those in humans. 

Donkeys are most likely to succumb to acute disease 

and die in a week to 10 days.1,4 Horses are more likely 

to incur a slowly progressive, chronic disease. Recurring clinical disease and even death in horses may 

manifest months to years after dormancy, particularly 

after any stress that causes a rise in temperature such 

as infectious disease, roundup, transport, overwork, 

poor diet, exercise, immunization, and even mallein 

testing.1,4,134 Changes in season, from winter to spring 

and from summer to fall, have also been associated 

with recurrent disease.4

The primary route of infection in the natural host 

is oral, by chewing or contacting contaminated food 

and water, feeding and husbandry equipment, and 

by direct close contact with infected animals.135 Tooth 

eruption, irregular tooth wear, coarse feeds, and bridling contribute to oral trauma, a common finding that 

leaves the mucosa and mucocutaneous junctions more 

244-949 DLA DS.indb 191 6/4/18 
192

Medical Aspects of Biological Warfare 

vulnerable to infection. Equids are also gregarious and 

prefer to be in close contact with at least one other. 

Grooming and nibbling behavior also exacerbate the 

potential for exposure from direct contact. Contaminated aerosols, such as those produced by snorting or 

coughing, may also easily find their way into the eyes, 

mouth, or skin abrasions of other equids in the vicinity. Tack, such as a harness, can cause skin irritation 

that—if contaminated—may allow easy entry of the organism. Despite the oral route of infection, significant 

pathology is usually seen in the airways and lungs.40

With early infection or resurgence, constitutional 

signs are often the first to manifest. These signs may 

include thirst, fever (low grade to high), shivering, 

head drooping, tachycardia, tachypnea, weight 

loss, rough hair coat, indolence, prostration, and a 

reluctance to move.136 Swelling of the limbs and joints 

may be seen. The lungs, mucosa of the respiratory tract, 

and lymphatic system are most frequently involved 

wherever the infection originates. Horses experimentally infected by cutaneous flank injection of infectious 

material developed a respiratory tract infection within 

a few weeks.1

 In some cases (or at various stages of 

disease) the lungs may appear to be the only organ 

involved. Regional or diffuse pneumonia and pleuritis 

are common. The lungs and upper respiratory tract are 

also the organs and tissues that show the oldest signs 

of chronic disease. Lung pathology is typically more 

marked and extensive in donkeys than in horses.

The nasal form of glanders classically described in 

equids is a somewhat local infection of the nasal cavity 

at least characterized by yellowish-green unilateral or 

bilateral nasal discharge, with or without nodules or 

ulcers on the nasal mucosa. Regional lymphadenopathy and lymphangitis most often accompany nasal 

signs. Laryngeal, tracheal, and lower respiratory tract 

pathology is often present, however, even if microscopically supporting the concept that a local infection 

is more likely just early infection, or rare. Nasal signs 

are common with recurrence of chronic infection. Although the nasal form has been associated with equids, 

similar pathology has been described in humans.3,54

With clinical expression of upper respiratory infection, a highly infectious, sticky, yellow-gray to greenish 

viscid unilateral or bilateral nasal exudate is produced. 

The glottis may be edematous and nasal discharge may 

be so thick as to obstruct nasal passages. The margins 

of the external nares are often swollen and crusted. The 

exudate may be periodically blood-tinged. The muzzle 

and distal forelimbs may be covered with this exudate; 

the latter is due to wiping the nose. The nasal mucosa 

may be nodular and ulcerous with ulcers often rapidly 

spreading. Ulcers may be deep and coalesce forming 

larger ulcers. Mucosal abscesses of the septum and 

nasal conchae may have swollen edges and display 

small yellow and gray nodules. These abscesses may 

invade the turbinates and cartilaginous structures, 

leading to perforation and erosion of the nasal septum. 

Particularly where the larger ulcers heal, white stellate 

or radial scars are left on the mucosa. These scars may 

be seen with an endoscopy, and they are near-hallmark 

signs of prior infection. Visible or palpable regional 

lymphadenopathy (particularly submandibular) and 

lymphangitis are present.

The equid will frequently snort to clear nasal passages, effectively showering the immediate area with 

the infectious exudate. The animal may cough, or a 

cough may be easily elicited by placing pressure on 

the throat over the larynx when there is laryngeal 

involvement. The air-exchange produced by a cough 

may exacerbate nasal discharge as equids breathe 

through their nose and not their mouths. Dyspnea, 

particularly inspiratory, may result from swelling in 

the nasal cavity or larynx. Expiratory dyspnea is also 

not uncommon, particularly with chronic involvement 

of the upper and lower respiratory tract.55 Auscultation 

and diagnostic imaging findings may support localized 

or diffuse lung disease and pleurisy. Clinical signs may 

be mild and transient, or severe and progressive. Death 

may occur within a few days.

At necropsy, glanders nodes will likely be found 

in the lungs, even if incidentally. Their consistency 

may be caseous to calcified depending on lesion age. 

These nodes may be of any size and occur as just a few 

or as hundreds in a diffuse miliary pattern. Pleuritis 

may also be found at necropsy. The microorganism is 

relatively abundant in the affected tissues. Animals 

may die within 3 to 4 weeks from bronchopneumonia 

and septicemia.

The progression of cutaneous and mucous membrane infection in the equid is similar to infections in 

humans. An entry wound may not be found. Lymphatic 

involvement may be more visible, however. Subsequent 

to cutaneous or mucosal infection, regional lymphangitis develops within 7 to 10 days. Typically, the lymphatics undergo a visible or palpable “string of pearls” stage 

within 10 days and then turn to more solid, fingerlike 

cords that can be traced to regional lymph nodes. Nodules along the lymphatic vessels may erupt, exuding 

gelatinous pus. Lymph nodes may be enlarged and 

indurated, and less frequently they may rupture and 

suppurate. With disease progression, more eruptions, 

enlargement of eruptions, and coalescence of lesions are 

expected. As a rule these are very slow to heal. Thick 

crusts of wound secretions, hair, bedding, and dirt 

may mat around the lesions. With ocular involvement, 

photophobia, excessive lacrimation, mucopurulent 

ocular discharge, conjunctivitis, and apparent partial 

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193

Glanders

blindness may occur, and this may result in behavioral 

changes such as avoidance or fear. With disseminated 

disease, cutaneous and mucous membrane lesions 

may appear anywhere, particularly the respiratory 

tract, as previously mentioned, and the limbs. The hind 

limb is more commonly affected than the forelimb.42,48

Acute septicemia may occur at any stage of infection. A septicemic course is typically progressive 

with signs leading to multiple organ failure including 

watery diarrhea, colic, marked dyspnea, prostration, 

cardiovascular collapse, and death. Donkeys are 

particularly susceptible to B mallei septicemia; this 

form manifests in most of those naturally and experimentally infected. Disseminated disease in horses is 

typically more protracted, however. Clinical signs 

vary widely and may include any of those previously 

mentioned. Horses may be asymptomatic. They may 

appear slightly thin, unthrifty, or have an occasional 

or persistent nasal discharge. There may be a transient mild to moderate fever. Mucous membrane and 

cutaneous lesions as well as lymphadenopathy and 

lymphangitis may also be transient or chronic. Visceral 

abscess is common, and the spleen and the liver are 

frequently involved. Intact males may have orchitis, 

which may not be evident without a reproductive 

examination.40,137 Remission is unlikely with disseminated disease particularly if it involves visceral organs. 

In the event an equid presents with clinical or necropsy signs consistent with glanders, the premises 

should be immediately quarantined and local and 

regional animal health authorities should be notified. 

Treatment should not be attempted. Although a clinical prognosis for various forms of glanders infection 

may be surmised, it is less relevant now because of the 

global interest in eradicating (by test-and-slaughter) 

the disease.

Chronically infected horses may display cycles of 

worsening disease followed by apparent recovery 

where few symptoms are displayed. Clinical signs 

include intermittent cough, lethargy, and lesions in 

the nasal region, lungs, and skin, just as with acute 

disease.136 Lungs may develop lesions similar to tubercules. Nodules may appear in the submucosa of 

the nasal cavity, particularly the nasal septum and 

turbinates. Nodules found in the liver and spleen may 

be up to 1 cm in diameter and have a purulent center 

surrounded by epithelioid and giant cells.138 Attempts 

to isolate B mallei from chronically infected animals are 

usually unsuccessful. Thrombosis can be found in the 

large venous vessels of nasal mucous membranes.25

Nodules in the skin along lymphatics may be seen in 

chronically infected animals as they thicken. Nodules 

may ulcerate and rupture, spewing a thick exudate 

that may be a source of infection. 

Clinical Disease in Humans 

Even during its peak near the turn of the 20th 

century, human glanders was uncommon but well 

documented. The clinical course of glanders is based 

on reports of hundreds of cases published before antibiotics were developed and from a small series of cases 

that occurred in the United States since the discovery 

of sulfonamides. The earlier reports describe a nearly 

always fatal disease of short (a few days to weeks) 

to long (months to years) duration that was usually 

acquired from close contact with infected equids. The 

most recent cases were laboratory acquired, and all 

patients survived.

Glanders manifestations can vary. At least five forms 

of infection have been described, including localized, 

pulmonary, septicemic, disseminated, and the aforementioned chronic infection, but none is exclusive. The 

most important distinction is whether the infection is 

localized, which is unusual except early in the infectious process. The variety of forms is largely explained 

by route of infection, regional lymphatic inflammation 

and drainage, and loci of dissemination and embolism 

via hematogenous or lymphatic spread. With disease 

progression and chronicity, all forms may manifest. 

Localized infections are regionally confined and 

typically characterized by pus-forming nodules and 

abscesses that ulcerate and drain for long periods of 

time. Lymphangitis or regional lymphadenopathy may 

develop in the lymphatic vessels that drain the entry or 

infection site. Increased mucus production from affected 

ocular, nasal, and respiratory mucosa is often present. 

Localized infections typically disseminate, leading 

to pulmonary, septicemic, or disseminated infection.

Constitutional signs and symptoms typically occur 

early in the disease and some may persist through 

treatment. These signs and symptoms may be severe, 

leaving the patient extremely prostrate. Common 

signs and symptoms include fever or low grade fever 

in the afternoon to evening, chills with or without 

rigors, severe headache, malaise, generalized myalgias (particularly of the limbs, joints, neck, and back), 

dizziness, nausea, vomiting, diarrhea, tachypnea, 

diaphoresis (includes night sweats), altered mental 

status, and fatigue. Other nonspecific signs may be 

tender lymph nodes, sore throat, chest pain, blurred 

vision, splenomegaly, abdominal pain, photophobia, 

and marked lacrimation. Any or many of these signs 

may be present. Following constitutional signs, clinical courses are discussed in greater detail as they are 

associated with route of entry and disease spread.

Cutaneous manifestations include multiple papular 

or pustular lesions that may erupt anywhere on the 

body. Cutaneous or mucosal infections may spread, 

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Medical Aspects of Biological Warfare 

leading to disseminated infections. Dissemination to 

internal organs produces abscesses in virtually any 

organ, most commonly the spleen, liver, and lungs. 

Disseminated infections are associated with septic 

shock and high mortality, yet they may also produce 

a more chronic, indolent course of infection.

With cutaneous entry through an abrasion, an 

inflammatory response of varying degrees (virulence 

dependent) occurs with accompanying pain and swelling. A glanders node may appear usually as a single 

blister, gradually developing into an ulcer that may 

be hemorrhagic.7,55 Localized infection develops at the 

entry site with a mucopurulent discharge. Inflammation may extend along regional lymphatics and cause 

lymphangitis perhaps with numerous foci of suppuration along their course. This irritation is caused by 

endotoxins present in some B mallei strains affecting 

the smooth muscle of the lymphatics. Lymphatic 

vessels may be easily palpable as firm, ropy cords. 

Regional lymph nodes become involved and similarly 

inflamed. Infection may remain localized, but more often spreads, particularly without adequate treatment. 

Further spread occurs via the lymphatics and through 

hematogenous dissemination as thrombi and emboli 

are formed. Local endothelial tissue inflammation and 

suppuration can occur at any place along the route of 

spread, producing abscesses that may drain through 

the skin. Superficially, these abscesses may appear as 

papules or diffuse abscesses in inflamed skin, or larger 

(egg-sized) swellings deeper in the subcutaneous tissue 

and superficial musculature. Published case descriptions have described glanders nodes anywhere including the face, neck, shoulders, lumbosacral region, arms, 

and legs.7,55 When the legs are affected, glanderous 

nodes occur more often on the medial aspect than the 

lateral. At first these glanderous nodes may be hard 

and painful, but eventually they ulcerate and slough. 

These nodes may exude relatively tenacious pus that 

varies in consistency from mucopurulent to gelatinous 

to oily, depending somewhat on chronicity. The nodes 

heal slowly and recur without adequate treatment. At 

any time the patient may become acutely ill and septicemic. Other organs and tissues may also be showered 

with infectious emboli. 

The infectious process through the oral, nasal, or 

ocular mucus membrane is similar to the cutaneous 

process. Weakened or abraded membranes are more 

vulnerable to entry than are intact membranes. Potential entry may be associated with contaminated hands, 

fingers, objects, and aerosols contacting the eye, nose, 

and mouth. A localized infection typically follows. 

Within 1 to 5 days the affected membranes become 

injected, swell, and weep a serosanguinous to mucopurulent discharge. Papular and ulcerative lesions similar 

in character to those in the skin may appear. Single or 

multiple oral blisters and sores may also appear. Hyperemia may be diffuse (entire pharynx, conjunctiva, 

etc) or localized. With ocular involvement, excessive 

lacrimation and photophobia are common. With nasal 

involvement, the nose may become greatly swollen 

and inflamed and copious nasal discharge may occur. 

Infection may invade the nasal septum and bony tissues, causing fistulae and tissue destruction. The entire 

face can become swollen, and regional lymph glands 

may inflame and suppurate. Infection may also extend 

lower in the respiratory tract resulting in tracheitis and 

bronchitis, which can be accompanied by cough and 

the production of mucopurulent sputum. If mucous 

membrane involvement is extensive, constitutional 

signs are also usually severe including high fever, 

severe headache, fatigue, prostration, earache, and 

various neurologic signs.

Infection of the respiratory tract may be anticipated 

after aerosol exposure or secondarily as a consequence of 

disseminated infection. A pulmonary infection typically 

produces pneumonia, pulmonary abscess, pleuritis, and 

pleural effusion, with associated signs and symptoms 

such as cough, dyspnea, chest pain, and mucopurulent 

sputum. Nasal exudate and cervical lymphadenopathy 

may also be present if the upper respiratory tract is 

involved. Nonspecific signs and symptoms often accompany respiratory infections, such as fatigue, fever, 

chills, headache, myalgias, and gastrointestinal signs. 

Pulmonary abscess and pleuritis are common sequelae. 

Symptoms including tender cervical lymph nodes, 

fatigue, lymphangitis, sore throat, pleuritic chest pain, 

cough, fever (often exceeding 102°F), chills, tachypnea, 

dyspnea, and mucopurulent discharge may take 2 to 3 

weeks to develop. Nonspecific signs are also usually 

present including night sweats, rigors, myalgia, severe 

headache, tachycardia, nausea, weight loss, dizziness, 

and mucosal eruptions. Some of the latter symptoms 

may indicate disseminated infection. Imaging studies 

may show diffuse or localized infiltration depending on 

the stage of infection. Miliary to necrotizing nodules or a 

localized (lobar to bilateral) bronchopneumonia are other potential radiographic signs. Developing abscesses 

may be well circumscribed and circular, later becoming 

cavitated with evidence of central necrosis. Pleural irritation may also be visible on imaging studies. Acute 

bronchopulmonic or pneumonic disease untreated 

tends to have a rapid onset of symptoms and was once 

said to be almost uniformly fatal within 10 to 30 days.5

Most laboratory-acquired infections have resulted from 

inhalational exposure resulting in pulmonary infection.

Clinical features of eight laboratory-acquired infections from Camp (Fort) Detrick are summarized in 

Table 8-2. These infections include the six-case series 

244-949 DLA DS.indb 194 6/4/18 
195

Glanders

published by Howe and Miller in 1945, a previously 

unpublished case that occurred in 1953, and the 2000 

case first presented by the Centers for Disease Control 

and Prevention.11 The most common symptoms experienced by at least four of the eight include—in order 

of most common occurrence—afternoon to evening 

low-grade fever, malaise, fatigue, headache, myalgias 

including backache, lymphadenopathy, and chest pain 

(Table 8-2). Shaded elements in the table represent the 

first signs and symptoms according to the medical 

records of the first seven patients, and according to 

the published case description of the eighth patient. 

An important clinical feature that is not reflected in the 

table is that at least half of the patients not only “felt 

better” but also were clinically better for a time after 

the first wave of disease symptoms. This period lasted 

from a few days for patient 7, to 2 months for patient 2. 

Inhalation is suspected as the route of exposure for the 

first seven patients, whereas percutaneous exposure 

probably led to the eighth case.

Septicemic glanders results from the seeding of 

B mallei into the bloodstream, whether as a primary 

event, secondary to a local or pulmonary infection, or 

as a relapse to chronic or latent infection. Septicemia 

may be passing and lead to protracted disseminated 

infection or be fulminant and rapidly fatal. Septicemic 

glanders may produce numerous signs consistent 

with a highly pathogenic bacterial septicemia. Without aggressive treatment, B mallei septicemia runs an 

acute course and may lead to death in 7 to 10 days. 

The thromboembolic process of glanders was well 

described by the early 1900s.1,3 B mallei causes damage 

and subsequent death of the endothelial cells lining 

the vessels. As the cells detach, the endothelial lining 

is predisposed to thrombosis. Thrombi serve as an 

excellent culture medium and seed the bloodstream 

with bacteria. The embolic process may be realized by 

the patient as sharp stinging pain in the receiving part 

or tissue of the body. Robins describes one protracted 

chronic infection in which the patient was always aware 

of pain before multiple impending dissemination sites.3

Bacteremia is transient; however, the more acute or 

sudden the onset of a septicemic course, the more likely 

B mallei may be isolated from the blood. Bacteremia is 

also more likely shortly before and during the appearance of multiple eruptions and pustules, if they occur. 

Century-old accounts of acute septicemic glanders 

suggest that virulent organisms and toxins may be so 

rapidly absorbed that systemic disease is actually primary, preceding the more patent ulcerative and lymphoglandular manifestations. Death may occur before 

these develop, however. Clinical signs and symptoms 

of the septicemic process may develop immediately or 

up to 2 weeks after initial infection or resurgence. These 

signs and symptoms include any severe constitutional 

sign and any of the cutaneous, mucous membrane, 

nervous, and respiratory signs previously discussed. 

Multiple organs may be involved. Erythroderma, 

jaundice, severe gastrointestinal distress, abdominal 

spasm, and severe respiratory signs may develop. 

Tachycardia, blurred vision, photophobia, excessive 

lacrimation, altered mental status, hepatomegaly, 

splenomegaly, granulomatous or necrotizing lesions, 

and lymphadenopathy may also be present. Death 

usually occurs in 7 to 30 days without adequate treatment. The prognosis for acute B mallei septicemia is 

guarded regardless of treatment. 

Dissemination can also occur in a more benign 

process resulting in a chronic course, which may be 

interrupted with latent periods of up to 10 years.6

Dissemination typically occurs without adequate treatment 1 to 4 weeks after B mallei infection of the lymph 

nodes. The organs most often involved in disseminated 

infection are the spleen, liver, and lungs, although 

any can be affected. Other sites include the skeleton, 

brain, meninges, musculature, and any cutaneous or 

mucous membrane locations. The kidneys are rarely 

affected, however. Clinical signs may be absent, limited 

simply to weight loss, or be highly severe and variable 

and include any of the aforementioned. Cutaneous 

eruptions may appear anywhere on the body and 

often originate from deep pockets of infection in the 

musculature. The extremities are often affected. Generalized lymphadenopathy with induration, enlargement, and nodularity of regional lymphatic vessels 

may be found on the extremities and in other affected 

areas. Miliary abscesses of organs and tissues may 

resemble tuberculosis. Robins described several cases 

of disseminated chronic infections in which no clinical symptoms were apparent, yet at autopsy patients 

were riddled with abscesses, including in the lungs.3

Robins chronicles a patient with the longest known 

infection (15 years, only 5 of which were latent) who 

finally died of disseminated disease. Symptoms of this 

particular disseminated infection included nasal and 

aural discharge, submaxillary adenitis, phlegmon of 

the nose, perforation of the nasal septum, jaundice, 

diarrhea, and amyloid disease.3

The amount of infection and pathology in a surviving patient can be particularly alarming when compared to a usually more rapidly fulminant disease such 

as septicemic anthrax. Protracted disseminated infections are associated with septic shock and a guarded 

prognosis. Diagnostic imaging studies are indicated 

to identify potential infection. Before antibiotics, disseminated infection was ultimately fatal either by 

resurgence of acute disease or from exhaustion of the 

patient. Based on the few cases treated with antibiotics, 

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Medical Aspects of Biological Warfare 

TABLE 8-2

CLINICAL FEATURES OF EIGHT US LABORATORY-ACQUIRED BURKHOLDERIA MALLEI

INFECTIONS 

Signs and Symptoms* Patient 1† Patient 2† Patient 3† Patient 4† Patient 5† Patient 6† Patient 7† Patient 8†

November November February April August August July March

1944 1944 1945 1945 1945 1945 1953 2000

Fever, pm rise‡99.0–99.4 99.0–101.2 101.0–103.4 99.0–103.8 99.0–102.8 - 99.0–101.4 99–104.5

Rigors, chills + + +

Night sweats + + +

Pain in chest + + + +

Myalgia + +

Malaise + + + + + + +

Headache + + + + +

Backache + + + 

Stiff or sore neck + 

Dehydration + +

Earache + 

Cough + - +

Mucopurulent sputum +

Oropharyngeal Postnasal Blister Sore

drip under throat

tongue;

nasal

obstruction

Pharynx injected + + + 

Lymphadenopathy Cervical Cervical - Cervical L axilla

Neurologic signs Stupor Carpo-

 pedal

spasm

Drowsy + +

Apprehension + +

Dizziness +

Fatigue + + + + + +

Weight loss + + +

Anorexia + +

Blurred vision + 

Lacrimation + 

Photophobia + + 

Abdominal signs - Pain L- Diarrhea Indigestion, Epigastric

 upper flatulence, tenderness

quadrant; belching

 spasm

Nausea, vomiting + 

Enlarged spleen + +

Chest radiographs R-upper; R-lower; R-upper, Clear L-middle, L-lower, L-hilum Clear

~Abscess ~Abscess ~Abscess ~Abscess pneumo- ~Abscess

 nitis

(Table 8-2 continues)

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197

Glanders

survival is likely if early and long-term effective 

therapy is instituted. Even with treatment, clinical 

symptoms may continue several months before complete resolution, particularly if treatment is delayed. 

Radiographic imaging is useful to monitor pulmonary infection. Early radiographic signs are typically infiltrative or support early abscess formation. 

WBC Normal-low; Normal High; High to Normal Normal Normal, Normal

neutropenia neutro- normal to to high- L-shift; late in

philia low; normal; atyp mono, disease

 Neutro- Neutro- lymph

 phils phils

Primary site Pulmonary Pulmonary Pulmonary Unknown Pulmonary Pulmonary Pulmonary Cutaneous

Disseminated Possible Likely Possible +

 spleen

Secondary sites Unknown Liver,

 spleen

Likely route of entry Inhalation Inhalation Inhalation Inhalation Inhalation Inhalation Inhalation Percutaneous

Sputum/throat culture - - - + NA

Blood culture - - - - - - - + at 2 mos

Isolation of organism - - - - - - + +

CFT positive§ Day 50 Day 50 Day 12 Day 40 - - - NA

Agglutinin positive¥ Day 50 Day 50 Day 5 Day 23 Day 22 Day 23 Day 19 NA

Mallein test positive Day 58 Day 58 Day 21 Day 18 Day 72 - - NA

Successful treatment Sulfa- Sulfa- Sulfa- Sulfa- Sulfa- Sulfa- Aureo- Doxycydiazine diazine diazine diazine diazine diazine mycin cline

10 days 10 days 36 days 20 days 20 days 20 days 28 days 6.5 mos

Onset of antibiotic¶ Day 60 Day 60 Days 2, Day 18 Day 16 Day 9 Day 21 ~ 5 wks

15, 115

Recovery time post trx 21 days Immediate 188 days 12 days 15 days Immediate Immediate > 6.5 mos

*Shaded elements in the table represent the first signs and symptoms according to the medical records of the first seven patients and according to the eighth patient’s published case description.

†Patients 1 through 7: Data from original case files. WBC deviations involved only neutrophils. Absolute lymphocyte counts were all normal.

Patients 1 and 2: Glanders as a differential diagnosis was delayed. CFTs positive > 10 months, agglutinin titers positive > 10 months, mallein 

positive > 16 months.

Patient 3: First sulfadiazine treatment was halted because of falling sedimentation rate; two more treatments followed at onset days indicated.

Patient 4: See “Patients 1 through 7” note above. 

Patient 5: Eleven normal complete blood counts except occasional slight relative lymphocytosis; lymphadenopathy also at axillary, epitrochlear, and inguinal. 

Patient 6: Patient did not take temperature but felt feverish. Agglutinin test considered positive due to titers rising from zero to 1:320.

Patient 7: Previously unpublished case. Early WBC cytology showed transient atypical monocytes and lymphocytes (atyp mono, lymph).

Patient 8: Initial blood culture was negative; data from Srinivasan A, Kraus CN, DeShazer D, et al. Glanders in a military research microbiologist. N Engl J Med. 2001;345:256–258. 

‡Temperature ranges represent the span of recordings that exceeded normal.

§

CFTs were considered positive if >/= 1:20.

¥

Agglutinin titers were positive if >/= 1:640 because of background titers in healthy patients of up to 1:320.

¶

Onset of antibiotic refers to the day of disease that the successful antibiotics were started; Patient 8 received two prior unsuccessful courses.

+: positive or present 

–: negative or not present 

[blank]: not reported or no mention 

CFT: complement fixation test

mos: months

Table 8-2 continued

Segmental or lobar infiltrates are common. With 

time, pulmonary abscesses tend to undergo central 

degeneration and necrosis, which radiographically 

resembles cavitation, and these may be single or multiple. Unilateral or bilateral bronchopneumonia may 

be seen, as well as a smattering of miliary nodules. 

Because of the potential for disseminated disease, 

NA: not applicable or not done

post trx: posttreatment

WBC: white blood cell

wks: weeks

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Medical Aspects of Biological Warfare 

computed tomography scan is useful for monitoring 

deep tissues and visceral organs. 

Complete blood count and chemistry studies for 

glanders patients vary depending on the disease’s 

location of infection and duration, and the degree of 

dissemination or septicemia. Complete blood count 

may be normal early and throughout the pretreatment 

disease course. Based on the laboratory-acquired cases, 

deviations in the white blood cell count typically involve 

only the absolute neutrophil count rather than other cell 

lines (Table 8-2). Neutropenia or neutrophilia, with or 

without a left shift, may be transient findings. Leukcopenia with mild to moderate relative lymphocytosis was 

seen in three of the six laboratory-acquired infections 

reported by Howe and Miller,5 which may be attributed 

to a low absolute neutrophil count. Absolute lymphocyte counts were consistently within normal limits.

Historically, mortality rates have been reported to 

be 95% without treatment and up to 50% with treatment. A more recent analysis estimates the mortality 

rate for localized disease is 20% when treated, and 

the overall mortality rate is 40%.65 Since the near 

eradication of glanders and the development of effective antibiotics, even these may be high estimates. 

Successful cure was achieved in 100% of the eight US 

laboratory-acquired cases, despite three of the eight 

cases (37%) experiencing a delay in effective treatment of 2 months.5,11,56 A brief period of “apparent 

recovery” is a common clinical feature that can easily 

lead to delayed treatment and complications. Four 

of the eight patients were successfully treated with 

sulfadiazine for at least 20 days. The first two who 

received delayed treatment still recovered with only 

10 days of sulfadiazine, yet recovery was protracted. 

The most recent patient (patient 8) had disseminated 

disease, which included abscesses of the spleen and 

liver, and required ventilatory assistance before improving on a prolonged course of several antibiotics. 

These recent cases imply that prognoses range from 

good with localized infection and prompt treatment 

to guarded with septicemic infection. 

Laboratory Diagnosis

Morphology and Growth Characteristics

A definitive glanders diagnosis in humans occurs 

when the organism is isolated in culture and correctly 

identified. In endemic regions, phenotypic characteristics such as colony and cell morphology in combination with biochemical assays may still be a practical 

means to definitively diagnose glanders. These methods may take 2 to 7 days to confirm a diagnosis.139

Gram stains alone of pus from lesions may be productive, but microorganisms are generally difficult to 

find or isolate. B mallei can be cultured and identified 

with standard bacteriological media.46 In potentially 

contaminated samples, supplements to inhibit the 

growth of gram-positive organisms (eg, crystal violet, 

bacitracin, penicillin) and some gram-negatives (eg, 

polymyxin B) and facilitate selective isolation of B 

mallei can be useful.140,141 The optimum growth temperature is approximately 37°C. Growth is typically 

slow on nutrient agar, but is more rapid (2 days) when 

enhanced with 1% to 5% glucose and/or glycerol, and 

on most meat infusion nutrient media.140,142 B mallei

colonies typically are smooth and about 1 mm in 

width, white (turning yellow with age), semitranslucent and viscid on Loeffler’s serum agar and blood 

agar. After incubating for 3 days on sterile potato 

slices, growth appears as a shiny, moist, yellowish 

transparent film.24,143 Selective differentiation from the 

related organisms B pseudomallei and Pseudomonas aeruginosa may be achieved by examining the following 

phenotypes. Whereas B mallei does not grow at 42°C 

or at 21°C or in the presence of 2% sodium chloride, 

B pseudomallei and P aeruginosa do. Also, it has been 

reported that B mallei does not grow on MacConkey 

agar, whereas both B pseudomallei and P aeruginosa

grow.7,24 However, others found that B mallei strains 

grew on this agar as nonlactose fermenting colonies.24

B mallei is a small, nonsporulating, aerobic gramnegative bacillus approximately 2 to 4 μm long and 

0.5 to 1 μm wide. It is nonmotile, a characteristic 

that differentiates it from related organisms such 

as B pseudomallei. The presence of a thick polysaccharide capsule can be demonstrated on the surface 

by immuno-electron microscopy.77 In the presence 

of nitrogen, the organism can grow as aerobic and 

facultative anaerobe.22,24,144 Size may vary by strain 

and by environmental factors including temperature, 

growth medium, and age of culture. Organisms from 

young cultures and fresh exudate or tissue samples 

typically stain in a bipolar fashion with Wright stain 

and methylene blue. Organisms from older cultures 

may be pleomorphic.140 In vivo B mallei is found most 

often to be extracellular. Since the disease is rare, 

samples should be designated as “glanders suspect” 

until confirmed by more extensive testing. Sample 

security to include appropriate chain-of-custody 

documentation is also prudent for all samples. 

Isolation 

The isolation of B mallei in culture is the gold standard for a glanders diagnosis. However, it can be 

difficult to obtain clinical specimens harboring viable 

bacteria; and invasive techniques, such as aspiration 

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Glanders

and biopsy, may be required. Even then, B mallei bacteria are often difficult to find, even in acute abscesses. 

Although isolation from blood has sometimes been 

successful in acute human cases, blood cultures are 

frequently negative until the disease’s terminal stages 

and do not generally appear to be a reliable indicator 

of infection, at least in animals such as NHP (Patricia 

L Worsham, David M Waag, and Taylor B Chance, 

USAMRIID, Fort Detrick, MD, unpublished data, 2013; 

Samuel L Yingst and Mark J Wolcott, USAMRIID, 

Fort Detrick, MD, unpublished data, 2013).24,44,56 To 

amplify the presence of low numbers of bacteria in 

normally sterile sites, animal inoculation methods 

were often used previously. However, such studies are 

impractical and inadvisable now for several reasons: 

• the time required for disease to manifest; 

• logistical requirement for special containment 

facilities; 

• stringent current animal regulatory requirements; and 

• adverse public reception of such animal 

work.

Isolation of the agent in nonendemic regions or 

from potentially contaminated samples may require 

use of selective media. Several media are commercially available for isolation of human Burkholderia

pathogens, such as BCA (Burkholderia cepacia agar 

also referred to as PC [Pseudomonas cepacia] agar), 

OFPBL (Oxidative-Fermentative-Polymyxin BBacitracin-Lactose agar), and Burkholderia Cepacia 

Selective Agar. These and other media have been 

described previously.145–147 For example, BCA was 

originally developed as a selective medium to isolate Burkholderia cepacia complex from the sputum 

of individuals with cystic fibrosis. Commercial 

preparations typically contain crystal violet, bile 

salts, ticarcillin, and polymyxin B (Remel, Lenexa, 

KS). It was found to be sensitive and selective for 

both B pseudomallei and B mallei.

146,147 OFPBL agar is 

another commercially available selective medium 

used to isolate B cepacia from cystic fibrosis patients. 

It permitted growth of 80% of strains tested, even 

though the colonies are very small and translucent; 

the growth often causes the agar to change from 

green to yellow due to lactose fermentation.146 OFPBL can be used in conjunction with (but not in place 

of) BCA agar for selective isolation of B mallei or B 

pseudomallei. Although both media are significantly 

discriminating for B mallei and B pseudomallei, they 

are not totally selective and strains of B cepacia, other 

Burkholderia, and some non-Burkholderia organisms 

can be expected to be isolated with them.146

Identification

Biochemical Identification. Automated biochemical kits are available commercially, such as API 20NE 

(bioMerieux, Durham, NC), RapID NF (Remel, 

Lenexa, KS), VITEK (bioMerieux, Durham, NC), and 

Biolog Inc phenotype microarray systems; however, 

they have often misidentified the Burkholderia. In 

2012, USAMRIID Supervisory Research Microbiologist Mark Wolcott relayed in several written and 

oral communications that B mallei and B pseudomallei

have been misidentified as nonpathogenic bacteria 

or other pathogens such as the B cepacia complex or 

Pseudomonas.

24,56,148,149 This situation was exemplified 

with the most recent case of human glanders in which 

the infecting B mallei strain was identified by an automated bacterial identification system as Pseudomonas 

fluorescens or Pseudomonas putida.

56 Another drawback 

is that many of these methods require the organism 

to be cultured in vitro before testing, resulting in a 

delay in the diagnosis.

The MIDI Sherlock Microbial Identification System 

(Microbial Identification System, version 4; MIDI Inc, 

Newark, DE) can identify isolates of the Burkholderia

by gas–liquid chromatography of cellular fatty acids. 

Inglis obtained good results using MIDI with B pseudomallei; and gas–liquid chromatography was used 

to correctly classify the B mallei from the most recent 

case of human glanders in the Burkholderia.

56,150 However, the bacteria must first be cultured under specific 

standardized conditions, and sample preparation is 

laborious and time-consuming. It does not usually 

allow speciation of the Burkholderia because of, for 

example, the highly similar cellular fatty acid compositions.53 Yet specific fatty acids and derivatives, such 

as methyl esters, are being identified that appear to be 

Burkholderia species-specific.148,151

Nucleic Acid-based Identification 

A major obstacle to isolating the pathogenic Burkholderia directly from samples is their low concentration 

in tissues and biological fluids of infected hosts. The 

development of methods for the reliable detection of 

glanders that does not rely on isolation of the organism is especially important for the diagnosis of chronic 

glanders. Therefore, many attempts have been made to 

develop indirect assays, such as nucleic acid analysis. 

The genomes of at least nine strains of B mallei have been 

sequenced,152 and the data are being used to enhance 

the ability to specifically identify this microorganism 

and increase understanding of how B mallei interacts 

with its host.38 Several nucleic acid-based diagnostic 

methods can confirm specific identification of B mallei, 

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often within several hours. Whereas some of the DNAbased procedures reported could be performed directly 

on clinical samples, others required preliminary culturing to isolate the bacteria. Many of the methods include 

polymerase chain reaction (PCR)-based and DNA gene 

sequencing-based assays.153–155 The latter have included 

the 16S and 23S rRNA-encoding genes, S21 ribosomal 

protein gene loci sequences, and MLST procedures. 

For instance, Frickmann et al showed that the sequence 

comparison of a 120 base pair ribosomal protein S21 

gene fragment was useful as a diagnostic procedure 

for the discrimination of B mallei and B pseudomallei

from the nonpathogenic B thailandensis and several 

other environmental species of Burkholderia, but it did 

not differentiate between B mallei and B pseudomallei.

156

Gene fragments and a single nucleotide polymorphism 

in the 16S rRNA gene have been reported to differentiate B mallei from B pseudomallei.

155,157 Analysis of the 

16S ribosomal RNA gene sequence analysis identified 

B mallei from other Burkholderia species in the 2000 US 

laboratory-acquired infection.56 However, it will be 

necessary to analyze many different species of B mallei and B pseudomallei (and other species) to establish 

the specificity of these and similar single-locus typing 

procedures. For discrimination of the closely related 

species B mallei and B pseudomallei, sequencing of the 

entire 16S rRNA gene, sequencing of the 16S-23S rRNA 

intergenic spacer, or the addition of a specific PCR or 

other genotyping method to a 16S rRNA gene fragment 

test has been recommended.158

PCR-based techniques and DNA gene sequencing 

are increasingly used in clinical settings and public 

health laboratories for bacterial identification.159 Automation of sequencing and improved reagents have 

also reduced the cost per test and the time required 

for identification. Furthermore, because killed bacteria 

or their templates may be used, these techniques also 

have the advantage of reducing the risk of exposure 

and infection to laboratory personnel compared 

to conventional methods.153 The current interest in 

biowarfare defense research prompted an increased 

capability based on recent publications.153–155,157,160–165

Numerous PCR assays have been described, some of 

which were recently described in detail in a review 

on PCR methods.166 They target various genetic elements, such as specific insertion sequences or single 

nucleotide polymorphisms, secretion system genes, 

and flagellar biosynthetic genes.157,162–165 Most of these 

assays require evaluation using many more diverse 

strains of the pathogenic Burkholderia and related/

unrelated species and testing for diagnostic applicability in a controlled infection study, such as an animal 

natural history study, for full validation. For instance, 

a real-time PCR assay, BurkDiff, was designed to 

target a unique conserved region in the B mallei and B 

pseudomallei genomes containing a single nucleotide 

polymorphism that differentiates the two species.157

Assay sensitivity and specificity were assessed and 

confirmed by screening BurkDiff across 469 isolates of 

B pseudomallei, 49 isolates of B mallei, and 390 nontarget isolates. The agreement of results with traditional 

identification methods and lack of cross-reactivity 

prompted the suggestion that BurkDiff may be a robust 

and specific assay for the detection and differentiation 

of B mallei and B pseudomallei; however, test results may 

be difficult to interpret and the assay must be assessed 

for its diagnostic applicability in controlled models for 

B mallei infection.167

Several PCR assays have targeted genes of the T3SS 

or other secretion systems; these multigenic systems 

have been shown to be important in the pathogenesis of 

B mallei and B pseudomallei.

165,166,168–171 Two assays based 

on the bimA (Burkholderia intracellular motility A) gene 

of the type V secretion system were developed.162,163

It was demonstrated that the N-terminal nucleotide 

sequence of bimA contains a unique B mallei region 

not present in the B pseudomallei and B thailandensis

bimA genes, as verified in tests with numerous strains 

of these three species.162,163 The value of the assays for 

early, rapid, and specific diagnosis of glanders in mice 

infected by the aerosol route was shown.162 However, 

it was reported later that the bimA of B pseudomallei

strains from Australia contain an N-terminus identical 

to that in B mallei bimA and a B thailandensis-like strain 

was detected in a bimA-based PCR assay.109,166,172 Finally, other PCR assays based on flagellar biosynthesis 

proteins (such as fliP) have been shown to be highly 

sensitive in tests with B mallei.

29,166,173,174

Because of potential problems intrinsic to PCR assays such as false positives, gene mutation, and PCR 

inhibitors, some have recommended the use of two 

DNA targets (two PCR assays) in combination with 

sequencing of the amplicons.52 Several multiplex PCR 

assays that target several and partially alleviate these 

issues have been developed. For example, Lee, Wang, 

and Yap described a sensitive and specific multiplex 

PCR using a short variable copy repetitive sequence, a 

metalloprotease gene fragment, and a sequence unique 

to B thailandensis that could distinguish B mallei, B 

pseudomallei, and B thailandensis.

175 Koh et al separated 

the species B mallei, B pseudomallei, B thailandensis, and 

the Burkholderia cepacia complex by using four specific 

primers: (1) a putative sugar binding protein, (2) a 

hypothetical protein, (3) a putative outer membrane 

protein, and (4) 16S rDNA.176

Other DNA-based techniques using pulsed-field 

gel electrophoresis and ribotyping have been used 

to identify strains of B pseudomallei and differentiate 

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Glanders

their virulence; these methods have not been tested 

with B mallei and would likely be more time and labor 

intensive than gene sequencing.177,178 More recently, 

improved in silico probe design based on short unique 

regions of the target genome has aided in the potential 

use of microarray procedures to selectively distinguish 

B mallei and B pseudomallei genetically; more microarray tests of these genetic targets with many bacterial 

strains are needed.179

Immunological Detection

Immunoassays that detect the presence of specific 

microorganisms can be useful in disease diagnosis. 

No such tests specifically for B mallei are established 

despite advancements in immunodetection of B pseudomallei.

180,181 Similar efforts to develop B mallei-specific 

immunodetection methods are important because 

antibiotics that are efficacious for one disease might 

not be effective for the other. mAbs elicited by B mallei whole cells and that targeted the LPS have been 

described.104 They appeared to be specific for B mallei, 

failing to recognize B pseudomallei, and might be useful reagents for a direct immunoassay for B mallei. In 

related studies, two groups developed large panels of 

mAbs to capsule polysaccharide and LPS that were 

specific for B mallei, B pseudomallei, or both and demonstrated strong binding to the bacteria.99,165 The study 

by Zou et al also revealed additional mAbs of possible 

diagnostic value, specifically, a pathogenicity-linked 

antigen epitope(s) on capsule-like polysaccharides 

found only in the pathogenic species of Burkholderia

(both B mallei and B pseudomallei), and several B mallei LPS-specific mAbs.99 It is possible that by using a 

combination of mAbs from different antigen groups, 

different strains of B mallei and B pseudomallei can be 

effectively differentiated from each other and from 

other nonpathogenic Burkholderia species. 

Serologic Diagnosis 

Although serological tests have been developed 

for diagnostic use in equines, no such tests exist to 

identify glanders specifically in humans. The mallein 

skin test has been primarily used to detect glanders 

in horses.24,182 A human version of the skin test was 

of little diagnostic value because of the multiweek 

delay to obtain a positive result. However, modified 

tests yielded somewhat improved results. In eight 

laboratory-acquired, confirmed cases of human glanders in the United States, the test was negative in two, 

not completed in one, and first positive in five on days 

18 to 72 postinfection.177 Overall, it appears that this 

diagnostic test for human glanders is minimally useful.

In vitro tests to include the indirect hemagglutination assay (IHA) and complement fixation test (CFT) 

have been used for serologic glanders detection. The 

IHA, which is the most frequently used serological test 

for human melioidosis, can also be used to identify 

glanders cases.183–185 In melioidosis testing, the failure of the IHA to detect antibody responses despite 

culture-confirmed disease has been observed.183 The 

CFT is still used universally in veterinary medicine 

as a reasonably reliable and low-cost procedure for 

animal glanders diagnosis.44,186–188 However, the 

CFT can be nonspecific and may not detect all cases 

or stages of glanders. In addition to occasional false 

negative results, it has also produced frequent false 

positive results (low specificity) and been hampered 

by inhibitory effects on complement of sera.24,183,186,189

The major problem leading to the low sensitivity and 

specificity of the CFT and other in vitro procedures 

has been linked to the test antigens currently used, 

that is, crude preparations of whole cells.44,190 Use of 

such antigens has led to frequent false positive results 

resulting from cross-reactive antigens. To address 

these shortcomings, numerous new tests have been 

reported for animal diagnosis (described below); 

however, improved assays are also clearly needed for 

human glanders serodiagnosis. 

Several assays and reagents have been described recently for the improved serodiagnosis of human glanders. Waag et al developed a whole cell enzyme-linked 

immunosorbent assay (ELISA) using irradiation-killed 

B mallei.

44,191 The test identified patients that have melioidosis or glanders (and excluded other differential 

diagnosis candidates, ie, anthrax, brucellosis, tularemia, Q fever). Similarly, Parthasarathy et al developed 

a polysaccharide microarray using extracted CPS and 

LPS to facilitate specific detection of B pseudomallei

and B mallei antibodies in animal and human sera.192

However, neither assay can discriminate between the 

two Burkholderia diseases because of serological crossreactivities.56,191,192 By using bioinformatic or similar 

state-of-the art approaches, others have identified B 

mallei-specific proteins that have potentially improved 

prospects for glanders-specific serodiagnosis.190,193 For 

example, Varga et al used pre-and postexposure sera 

from 2,000 cases of human glanders and a protein array platform made for studying melioidosis patients 

to characterize the human immunological response 

to B mallei.

56,191,193–195 Significantly increased antibody 

responses to 17 of 156 peptides were detected and 

antibodies to only two (a pilus biosynthetic protein 

and 50S ribosomal protein) were shared between 

the two diseases, implying that the human antibody 

response to B mallei is markedly distinct from that 

to B pseudomallei. The results of these recent studies 

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Medical Aspects of Biological Warfare 

suggest that these antigens may be useful for an improved glanders diagnosis; however, additional studies to include nonspecific and glanders-specific sera 

from human (and animal model) sources are needed.

Other Identification Methods

New methods to verify identification of isolated 

organisms as exemplified by phage-based identification have been described. Bacteriophages have been 

isolated which exhibit infectivity and high specificity 

for B mallei. For instance, φE125 and φ1026b infect B 

mallei, but not B pseudomallei.

196,197 Efforts to develop 

phage variants with species-specific receptors offer 

the potential for convenient phage-based diagnostics. 

Mass spectrometry methods, such as matrix-assisted 

laser desorption/ionization mass spectrometry and 

Raman spectroscopy, are being exploited for the 

identification of B mallei and B pseudomallei.

198,199 For 

example, signatures specific to pathogens at the species 

level were developed to evaluate a Raman chemical 

imaging spectroscopy method for reagentless detection and identification.198 Raman chemical imaging 

spectroscopy combines Raman and fluorescence 

spectroscopy and digital imaging to allow detection 

of low levels of biothreat organisms in the presence of 

complex environmental backgrounds without prior 

amplification methods. Raman spectra for viable select 

agents and toxins including B mallei and B pseudomallei were reported; however, most of the studies were 

conducted to distinguish Burkholderia from other 

pathogens (eg, B anthracis) and more efforts to distinguish near-neighbors of B mallei are needed. Other new 

assays for direct detection of the organism in culture 

and tissues and that use probes for surface or secreted 

antigens of the pathogenic Burkholderia are also being 

evaluated.133,200 One example is a fluorescence in situ 

hybridization assay that correctly identified all of the 

B mallei, B pseudomallei, and B thailandensis strains.133

However, the assay requires a relatively high bacterial 

concentration; thus, fluorescence in situ hybridization 

analysis may be more useful for evaluating tissue 

sections in pathogenesis studies than for diagnosis of 

direct clinical samples. 

New combination methods are being developed. 

An assay coupling biothreat group-specific PCR with 

electrospray ionization mass spectrometry and using 

DNA extracts from killed bacteria correctly identified 

seven bacterial biothreat bacterial species (including 

B mallei and B pseudomallei) to the genus if not species 

level.201 A gas chromatography-mass spectrometry 

method was used to identify several cellular fatty acid 

methyl ester fragments that could differentiate the 

species B mallei, B pseudomallei, and B thailandensis.

148

Diagnosis in Equids

Several diagnostic tests have been developed and 

used extensively for glanders diagnosis in equines.24,53

Khan et al provides a comprehensive review and 

evaluation of diagnostic tests developed previously 

as well as an overview of recent assays aimed at 

improving the sensitivity and specificity of glanders 

diagnosis.24 Tests used currently consist primarily 

of serodiagnostic assays, that is, the CFT and the 

mallein skin test. Serum anti-B mallei antibodies are 

detected by the CFT within a week of infection, and 

the CFT can detect carriers and animals chronically 

infected with B mallei24,189; it is the only mandatory 

serological test for international trade of equids, 

but it can be hampered by the problems detailed in 

the previous section.22,187 The mallein test is another 

well-known and established test used for glanders 

diagnosis in animals and involves injecting a purified 

protein derivative of the Burkholderia glycoprotein 

mallein intradermally. An immune cellular response 

as manifested by a delayed type hypersensitivity 

reaction is observed and considered diagnostic for 

glanders.24,177,182

More recently developed assays include competitive 

and indirect ELISAs using B mallei-specific monoclonal 

Abs, ELISAs with purified recombinant proteins used 

as test antigens, and Western blot techniques.189,202,203

Using sera from horses in an endemic region of 

South East Asia, Elschner and Khan et al reported the 

significantly improved specificity and sensitivity of 

the CFT when it was combined with a complement 

independent immunoblot technique, an approach 

that increased both the detection rate and specificity 

of the test for glanders serodiagnosis.189,202 Kumar et al 

cloned a novel recombinant Burkholderia intracellular 

motility A (rBimA) protein and used it with many 

positive and potentially negative serum samples in an 

indirect ELISA to detect equine glanders.203 The results 

revealed 100% sensitivity and 98.9% specificity. Also, 

rBimA protein did not react with melioidosis patient 

and normal healthy human serum samples, and thus 

showed high specificity.

Treatment

Because human cases of glanders are rare, limited 

information exists regarding the use of modern antibiotic treatment for humans. B mallei infection responds 

to antibiotic therapy; however, recovery may be slow 

after a delayed diagnosis or with disseminated disease. 

Reports in the scientific literature indicate that most 

strains of B mallei are susceptible to the following 

antibiotics in vitro: 

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Glanders

• amikacin, 

• netilmicin, 

• gentamicin, 

• streptomycin, 

• tobramycin, 

• azithromycin, 

• novobiocin, 

• piperacillin, 

• imipenem, 

• ceftazidime, 

• tetracycline, 

• oxytetracycline, 

• minocycline, 

• doxycycline, 

• ciprofloxacin, 

• norfloxacin, 

• ofloxacin, 

• enrofloxacin, 

• erythromycin, 

• sulfadiazine, 

• trimethoprim/sulfadiazine, 

• trimethoprim/sulfmethoxazole (co-trimoxazole), and 

• amoxicillin-clavulanate (co-amoxiclav).89,204–212

Aminoglycosides and other antibiotics incapable 

of penetrating host cells probably will not be useful 

in vivo because B mallei is a facultative intracellular 

pathogen.204,205,211 Susceptibility to streptomycin 

and chloramphenicol in vitro has been inconsistent, 

with some reporting sensitivity and others reporting resistance.7,205,209,212 B mallei is susceptible to the 

lytic action of human granulysin, a broad-spectrum 

antimicrobial peptide, and silver containing compounds.213,214

Most B mallei strains exhibit resistance to the following antibiotics:

• amoxicillin, 

• ampicillin, 

• penicillin G, 

• bacitracin, 

• chloromycetin, 

• carbenicillin, 

• oxacillin, 

• cephalothin, 

• cephalexin, 

• cefotetan, 

• cefuroxime, 

• cefazolin, 

• ceftriaxone, 

• metronidazole, and 

• polymyxin B.7,11,47

In addition, a study focused on 41 isolates of B mallei

obtained from various outbreaks of equine glanders 

occurring between 1999 and 2006 in Punjab, Pakistan, 

reported that less than 50% of the isolates were resistant to oxytetracycline, roxithromycin, and norfloxacin, 

and less than 40% were resistant to ciprofloxacin.212

Consistent with the published literature, all of the 

Pakistani isolates were susceptible to co-amoxiclav, 

chloramphenicol, doxycycline, gentamicin, and trimethoprim-sulfamethoxazole, and approximately 95% 

were susceptible to both enrofloxacin and ofloxacin.

Antibiotics have been tested against glanders 

in hamsters, mice, equids, guinea pigs, and monkeys.207,208,211,215–217 Sodium sulfadiazine—but not 

penicillin or streptomycin—was effective for treating 

acute glanders in hamsters.208 Doxycycline and ciprofloxacin were also examined in the hamster model 

of glanders.211 Doxycycline therapy was superior to 

ciprofloxacin therapy, but relapse did occur in some 

of the treated animals 4 to 5 weeks after challenge. 

Hamsters were also infected subcutaneously or by 

aerosol with B mallei and were treated with ofloxacin, 

biseptol, doxycycline, and minocycline.215 All of the antibiotics exhibited some activity in animals challenged 

subcutaneously, but ofloxacin was superior. None of 

the antimicrobials demonstrated appreciable activity 

against a high dose of B mallei delivered by aerosol, 

but doxycycline provided 70% protection against a 

low dose delivered by this route.214

Ceftazidime and levofloxacin were examined as 

treatments for glanders in BALB/c mice infected intranasally with B mallei ATCC 23344.218 Despite good in 

vitro activity against B mallei, intraperitoneal delivery 

of the antibiotics failed to eradicate the organism and 

resulted in the development of nonlethal, chronic 

glanders. More recent studies using a BALB/c model of 

inhalational glanders reported that oral administration 

of co-trimoxazole twice daily for 14 days prevented the 

development of acute disease, but was not able to completely eliminate B mallei and ultimately resulted in the 

establishment of a chronic infection.89 Co-trimoxazole 

is recommended for postexposure prophylaxis in humans; however, an extended course of therapy of up 

to 21 days is likely to be indicated.89,219,220

In most countries, strict regulations mandate that 

animals testing positive for glanders are destroyed 

rather than treated. Given the difficulties in implementation of such regulations in some countries along 

with the high monetary value of horses in equestrian 

sport and breeding, the usefulness of modern antibiotics for treatment or postexposure prophylaxis of 

horses has recently been reevaluated. In a 2012 report 

Saqib et al described the effectiveness of antibacterial 

therapy on 23 culture positive horses involved in a 

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Medical Aspects of Biological Warfare 

confined glanders outbreak at the Lahore Polo Club 

in Pakistan.37 A treatment protocol was implemented 

consisting of once daily parenteral administration of 

enrofloxacin and trimethoprim/sulfadiazine followed 

by twice daily oral administration of doxycycline 

for 12 weeks (84 days). All horses showed a marked 

improvement during the initial week of treatment. 

Abatement of fever, renewal of appetite, and healing 

of ulcerated nasal septa were reported by the end 

of week 1. Nodules and ulcers were cleared in most 

horses by the end of week 3. No clinical signs were observed in any horse following week 12. To confirm the 

absence of disease, immunosuppression was induced 

(by daily corticosteroid injection for 10 days) in six 

randomly selected horses starting on day 90 following 

completion of therapy. No recrudescence of disease as 

determined by a lack of clinical signs was observed. 

By 8 months posttreatment, the IHA titers of all of the 

horses were considered to be negative (≤1:320). At the 

time of the report (September 2012), nine of the horses 

in the study were still at the Lahore Polo Club and remained healthy; the status of the remaining eight was 

unknown. Whereas the findings of the Saqib et al are 

promising, the widespread treatment of glanderous 

horses is unlikely to replace the practice of “testing and 

culling” because the latter is a more cost-effective and 

efficient means of containing the disease spread. This 

study does, however, provide important information 

that may be applicable in certain situations where 

treatment is desirable.

The majority of human glanders cases occurred 

before the antibiotic era, and more than 90% of cases 

resulted in death.221 Several human glanders cases have 

been recorded since the 1940s, primarily in laboratory 

workers, and these have been successfully treated with 

antibiotics.5,56,222,223 Sulfadiazine was used successfully 

in the first six US laboratory-acquired infections.5 The 

seventh was successfully treated with the tetracycline 

compound, aureomycin. Two additional cases were 

successfully treated with sulfadiazine in 1949 and 

1950.222 Disseminated glanders in a stablehand who 

had only indirect contact with horses was also treated 

successfully with aureomycin in Austria in 1951.55

Streptomycin was used to treat a patient infected with 

B mallei and Mycobacterium tuberculosis.

223 Treatment 

with streptomycin reportedly cured the glanders, but 

had little effect on the bone’s tuberculosis in this patient. In a recent case of laboratory-acquired glanders, 

the patient received imipenem and doxycycline intravenously for 1 month followed by oral azithromycin 

and doxycycline for 6 months.56 Susceptibility testing 

of the B mallei isolate in this case demonstrated sensitivity to the former two drugs, although retrospective 

susceptibility testing found that the organism was 

resistant to azithromycin.51 Diagnostic imaging of the 

patient’s splenic and hepatic abscesses through the 

6-month course showed their near complete resolution.

No Food and Drug Administration-approved 

therapy for glanders exists. Recommendations for 

antibiotic therapy depend on the infection site and 

severity. Localized disease should be treated with at 

least a 2-month—and preferably a 6-month—course of 

antibiotics based on sensitivity. Without susceptibility 

test results and for mild disease, oral doxycycline (100 

mg twice/day) plus trimethoprim-sulfamethoxazole 

(4 mg/kg/day in two divided doses) for 20 weeks is 

recommended.224 Historically, oral chloramphenicol 

has been added to this regimen for the first 8 weeks; 

however, based on recent recommendations for melioidosis treatments, this may no longer be necessary. 

Amoxicillin/clavulanate is the recommended alternative for pregnant women and children or individuals 

that cannot tolerate trimethoprim-sulfamethoxazole. 

For severe disease ceftazidime at 40 mg/kg intravenously (IV) every 8 hours or imipenem IV at 15 mg/

kg every 6 hours (maximum 4 g/day) or meropenem 

at 25 mg/kg IV every 8 hours (maximum 6 g/day) plus 

trimethoprim-sulfamethoxazole at 8 mg trimethoprim/

kg per day IV in four divided doses is recommended. 

Intravenous therapy should be continued for at least 

14 days and until the patient is clinically improved. 

Oral maintenance therapy as for mild disease can be 

continued from that point.224 Combined therapy for at 

least the first month should be considered for patients 

even with the mildest of systemic symptoms. For visceral and severe disease, prolonged treatment up to 

a year is recommended. Abscesses may be surgically 

drained, depending on their location.65 Because of the 

intractable nature of glanders, long-term follow-up 

and possibly prolonged, tailored therapy is indicated 

for infections that are slow to clear. Patients should be 

followed at regular intervals for at least 5 years after 

recovery. Diagnostic imaging is useful to follow the 

reduction and resurgence of abscesses, serology may 

help to monitor the clearing of antibody, and inflammatory markers may also suggest resurgence of a latent infection. Patients should be advised of the lifelong 

risk of relapse and to alert their healthcare providers 

of their previous history, particularly if they develop 

a febrile illness. This situation becomes even more 

important when potentially dealing with a genetically 

engineered strain of B mallei. Current postexposure 

prophylaxis recommendations for laboratory workers 

consist of oral trimethoprim-sulfamethoxazole for 21 

days.219,220,224 If the patient is allergic to or intolerant of 

this, or the organism is known to be resistant to the 

first choice, the second-line choice is oral amoxicillin/

clavulanic acid (co-amoxiclav) for 21 days.219

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Prophylaxis

Host Immunity

B mallei can establish chronic or acute infections 

in multiple species, which suggests that native host 

immune responses are unable to eradicate wild type 

organisms upon initial challenge. In addition, no 

evidence indicates previous infection provides immunity against glanders.7,225 Infections in horses that 

appeared to symptomatically recover from glanders 

would recrudesce when the animals were challenged 

with B mallei. Inoculation of B mallei into chronically 

infected horses produced at least local infections most 

of the time, and occasionally a manifestation of classic 

glanders. Similar to equines, protective immunity in 

humans after recovery from glanders is not believed 

to occur. In an 1869 human case report from Poland as 

told by Loeffler, one attempt at autoinoculation with 

the fluid from a pustule produced more pustules.1

Thus, patients who recover may still be susceptible, 

making reuse of the agent in biowarfare necessary to 

consider. 

Immune responses to glanders appear to be complex 

requiring both humoral and cell-mediated immune 

(CMI) responses. The role of antibodies in immune 

protection has been investigated experimentally in 

animals as well as retrospectively in a recent human 

case. Experiments on horses regarding protective 

immunity have given ambiguous results.1,7 Passive 

immunity experimentation using equine sera has 

also failed.7 Conversely, passive immunization of 

BALB/c mice using B mallei OPS- or CPS-specific 

mAbs conferred nonsterilizing protection against a 

lethal bacterial challenge delivered either intranasal 

or by aerosol.97,104 Consistent with a role for antibodies in protection against glanders, investigations by 

Whitlock et al involving the depletion of B cells in 

BALB/c mice demonstrated a significantly decreased 

survival time following B mallei infection compared 

to control mice.226 In contrast, Rowland et al reported 

that when B cell knockout mice (μMT C57Bl/6 mice) 

were infected with B mallei, survival of B cell deficient 

animals did not differ from wild type mice.227 The 

reason for these contrasting results is unclear, but it is 

possible that these differences may be a consequence 

of the manner in which the B cells were depleted (ie, 

antibody depletion versus genetic deletion) or the 

specific mouse models used.

Analysis of humoral immune responses from a 

laboratory-acquired case of glanders in 2000 indicated 

that B mallei specific immunoglobulin A, immunoglobulin G, and immunoglobulin M levels were highly 

elevated (8-, 16- and 4-fold, respectively) by 2 to 4 

months postinfection and then began to decline.191 By 

14 months postinfection antibody titers returned to 

near baseline levels. More extensive characterization 

of the antibody responses in this case demonstrated 

reactivity of highly increased (≥2-fold compared to preexposure serum) antibodies with a variety of B mallei

and/or B pseudomallei proteins including T3SS and T6SS 

components, type IV pili, outer membrane proteins, 

chaperones (eg, GroEL and GroES), and hypothetical proteins.193 Screening of equine glanders serum 

against a bacteriophage expression library revealed 

the presence of antibodies to a Burkholderia Hep_Hag 

autotransporter (BuHA) proteins, a family of immunodominant antigens predicted to be hemagglutinins and 

invasins.123 Such proteins represent potential candidate 

antigens to develop glanders diagnostics, therapeutics, 

and vaccines.

Since B mallei is a facultative intracellular pathogen, 

CMI mechanisms as well as cytokine and chemokine 

expression, significantly contribute to the clearance of 

the organism from infected hosts. B mallei is capable 

of infecting and surviving in many cell types including professional phagocytes. In a BALB/c model of 

glanders, neutrophils and macrophages infiltrated the 

spleen 5 hours postinfection and an increase in activated macrophages, neutrophils, and T cells occurred 

by 24 hours postinfection.227 When neutrophils were 

depleted, mice became acutely susceptible to B mallei

infection and succumbed within 5 days. In contrast, if 

mice were depleted of both CD4+ and CD8+ T cells, they 

did not succumb until 14 days postinfection. In wild 

type C57Bl/6 mice, macrophages have been shown 

to be important for reducing the susceptibility of the 

animals to pneumonic B mallei infection.82,228 Monocyte 

chemoattractant protein-1 (MCP-1), a chemokine involved in the chemoattraction of macrophages to sites 

of infection, plays an important role in protective immunity to B mallei infection. Mice lacking either MCP-1 

or the MCP-1 receptor were more susceptible to disease 

following IN challenge and exhibited higher bacterial 

burdens in organs at 3 days postinfection compared 

to wild type mice.82 Monocyte and inflammatory 

dendritic cell recruitment was defective in the MCP-1 

knockout mice and increased numbers of neutrophils 

were observed in the lungs. These data support a critical role for phagocytic cells (neutrophils, monocytes 

and macrophages, and dendritic cells) in controlling 

the early, innate responses to B mallei infection while 

T cells appear to be important later in infection.227

Interferon-gamma (IFN-γ) plays an important role 

in macrophage activation and clearance of intracellular organisms. Examination of cytokine responses 

24 hours following IP injection of female BALB/c mice 

with B mallei exhibited a strong IFN-γ response and 

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Medical Aspects of Biological Warfare 

elevated levels of IL-18, IL-12, IL-27, IL-6, and MCP1.229 IFN-γ knockout mice infected with B mallei died 

within 2 to 3 days after infection, and uncontrolled 

bacterial replication in several organs confirmed a 

critical role for this cytokine during innate immune 

responses to B mallei. Similar findings were reported 

when specific antibodies were used to deplete IFN-γ 

in mice.226 The proinflammatory cytokines IL-12 and 

IL-18 were shown to be critical for IFN-γ production 

at early time points postinfection.229 Neutralization of 

IL-12 in vivo led to increased susceptibility of mice to 

lethal infection, possibly from its role in promoting 

IFN-γ production by natural killer cells and T cells.230

Consistent with this notion, in vitro assays have 

confirmed that natural killer cells and CD8+ T cells 

were the main cellular sources of IFN-γ generated in 

response to B mallei.

229 Furthermore, MyD88 knockout 

mice (which cannot produce IFN-γ) were also highly 

susceptible to pulmonary challenge with B mallei

compared to wild type mice.228 Treatment of MyD88 

knockout mice with exogenous recombinant IFN-γ 

helped to restore effective immunity and significantly 

increased survival.82,228 Thus, B mallei appears to be 

susceptible to CMI responses promoting the expression 

of type 1 cytokines (eg, IFN-γ and IL-12). Although the 

initial burst of IFN-γ was able to control bacterial replication, clearance was not achieved.229 These studies 

highlight the importance of cytokine and chemokine 

production during B mallei infection and suggest that 

strategies targeting the production of IFN-γ should 

be considered when attempting to achieve effective 

vaccine induced immunity.

LPS and various microbial products can activate 

macrophages through TLRs and other patternrecognition receptors. Pattern-recognition receptors 

signaling leads to transcription of genes encoding 

cytokines, chemokines, and enzymes, such as inducible nitric oxide synthase that aids in the clearance of 

intracellular bacteria. Purified B mallei LPS is a strong 

activator of human TLR4 complexes and stimulates 

production of tumor necrosis factor-alpha (TNF-α), 

IL-6, and regulated upon activation normal T-cell 

expressed, and secreted (RANTES) protein in human 

macrophages and dendritic cells.98 The expression of 

these cytokines and chemokines by antigen presenting 

cells reflects signaling through both MyD88-dependent 

(TNF-α and IL-6) and -independent (RANTES) pathways. Recent studies using LPS-activated macrophages 

and iNOS-2 knockout mice suggest that iNOS activity 

and the production of reactive nitric oxide species 

contribute to the killing of intracellular B mallei.

98,227

These studies illustrate the importance of TLR4 signaling and reactive nitric oxide species in macrophage 

activation and clearance of intracellular B mallei. The 

role of other pattern-recognition receptors (eg, TLR2 

and NOD-like receptors) in B mallei infection remains 

to be investigated. 

Relatively little is understood regarding the role 

of T-cell subsets in controlling B mallei infections. 

Recent studies suggest that strong T-cell helper (Th)1-

polarized responses will likely be required for protection against B mallei infection.231,232 More research is 

needed in this area. Maximizing the effectiveness of 

CMI responses is predicted to limit the duration of 

B mallei infections and to reduce disease pathology, 

and understanding how innate and adaptive immune 

responses fail to result in complete bacterial clearance 

may provide clues for the design of effective vaccination strategies. 

Vaccine Candidates

No human or veterinary glanders vaccine exists. 

From 1895 to 1928 numerous attempts to vaccinate 

horses and laboratory animals against glanders were 

unsuccessful. Vaccines were initially prepared by treating bacterial cells with urea or glycerin7

 or by drying 

the glanders bacilli.225 For most chronically infected 

horses, experimental vaccination did not change the 

course of their illness. Experiments on protective immunity in horses have given ambiguous results.1,7 Mendelson reported guarded postvaccination success in a 

youth with severe ocular and oro-nasal involvement.54

Although attempts (with limited success) to develop 

a glanders vaccine were initiated more than 100 years 

ago, using modern approaches to identify virulence 

factors and studying the ways putative vaccines 

modulate the immune system may aid in developing 

an efficacious glanders vaccine. 

Interest in glanders vaccine development has increased in recent years, mostly resulting from B mallei’s biothreat potential. Several up-to-date reviews 

provide further details of glanders vaccine development.233–235 The most desirable glanders vaccine will 

be a recombinant protein or a biochemically purified 

preparation that gives long-term sterile immunity 

when administered. As previously mentioned, since 

B mallei is an intracellular pathogen it is likely that 

both CMI and humoral responses will be critical in 

developing protective immunity. Activating both arms 

of adaptive immunity and ultimately achieving sterile 

immunity will be significant challenges in B mallei vaccine design. A better understanding of the correlates 

of vaccine-induced immunity is needed. 

Killed Whole Cells

The initial attempts to protect mice against an 

aerosol-acquired infection with B mallei using either an 

irradiation-killed B mallei or heat-killed B pseudomallei 

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Glanders

cellular vaccine resulted in an increased time to death, 

compared to controls, but spleens of survivors were not 

sterile.236 A nonviable B mallei cellular vaccine failed to 

protect mice from a parenteral live challenge.237 This 

vaccine stimulated a weak mixed Th1- and Th2-like 

immune response. This study suggested that nonviable B mallei cell preparations may not protect mice 

because of the failure inducing a strong Th1-like immune response. In a subsequent study, adding IL-12 to 

an irradiated B mallei vaccine preparation stimulated a 

Th1-like antibody response and induced an increase in 

splenocyte proliferation and IFN-γ production in comparison to mice vaccinated with killed B mallei alone.231

Following an IP challenge with B mallei, increased 

survival was observed in mice vaccinated with both 

IL-12 and killed bacteria in comparison to control mice. 

Sterile immunity was not achieved, and the spleens of 

the vaccinated survivors were enlarged and heavily 

infected with B mallei. More recent studies by SarkarTyson et al and Whitlock et al using heat killed B mallei

and/or B pseudomallei are consistent with the findings 

of Amemiya et al, and indicate that TNF-α, IFN-γ, and 

B cells are necessary for an effective immune response 

against the organism.226,231,238

Live Attenuated Vaccines

Three attenuated strains of B mallei, a CPS (wcbB) 

mutant, a branched-chain amino acid auxotroph 

(ilvI) mutant, and a carboxy-terminal protease (ctpA) 

mutant have been evaluated as vaccines in a BALB/c 

mouse model of glanders.77,90,131 The CPS and ilvI vaccine strains were delivered aerogenically, and mice 

were subsequently exposed to wild type B mallei via 

whole-body aerosols. Immunization with the CPS 

mutant resulted in a Th2-like antibody response that 

failed to protect mice (all animals died within 5 days 

of infection). In contrast, immunization with the ilvI

mutant resulted in a Th1-biased immune response that 

conferred significant protection against lethal aerosol 

challenges. At 1 month postchallenge, 25% and 50% 

of the mice had survived high- and low-dose aerosol 

challenges, respectively. Analysis of bacterial loads in 

the organs of the surviving mice revealed high numbers (>105

 cfu) of B mallei in the spleens of all of the ilvI

mutant vaccinated mice. B mallei was also present in 

the livers and lungs of most of the surviving animals, 

suggesting the development of a chronic infection. A 

B mallei ctpA mutant, which was evaluated as a live 

attenuated vaccine in CD1 mice, provided partial 

protection against an IP challenge of wild type B mallei.

131 Whereas 75% of the ctpA mutant-vaccinated mice 

survived the 15-day postchallenge, all survivors displayed splenomegaly and high splenic loads of B mallei. 

A mixed Th1/Th2-like antibody response was noted. 

Based on these studies, it appears that live attenuated 

strains promoting Th1-like antibody responses may 

be useful as glanders vaccine candidates.

Protein Subunit Vaccines

Two studies by Whitlock et al describe the identification and testing of purified B mallei protein antigens 

as potential vaccine candidates.125,239 Immunogenic 

antigens were identified from genomic screens of B 

mallei expressed proteins. In initial studies, the candidate protein antigens included LolC (ABC transporter 

protein), BimA (autotransporter protein), BopA (T3SS 

effector protein), and Hcp1 (T6SS-1 component).239 IN 

immunization with purified recombinant LolC, BimA, 

BopA, and Hcp1 alone or as a quadrivalent mixture, 

administered with CpG 2395 and ISCOM adjuvants, 

provided BALB/c mice significant protection against 

an IN challenge of B mallei. While the recombinant proteins protected the mice from the initial acute infection, 

sterile immunity was not achieved. In a subsequent 

study, recombinant forms of five additional B mallei

proteins and GroEL were evaluated for their protective 

capacity in BALB/c mice.125,194 Three of these proteins 

and GroEL provided partial protein against an IN 

challenge of B mallei. Several of the highly immunogenic proteins (eg, LolC and Hcp1) evaluated in these 

studies are also considered promising components for 

melioidosis vaccine development.105,124,239

Polysaccharide-based Subunit Vaccines

B mallei isolates appear to be capable of expressing 

only a limited repertoire of structurally diverse CPS 

and LPS antigens.36,93,97 The protective efficacy of LPS 

and CPS as vaccines against B pseudomallei infection 

was tested in BALB/c mice and partial protection was 

observed.240,241 Active immunization studies using CPS 

or LPS for protection against B mallei have not been 

reported; however, it has been shown that CPS- and 

LPS-specific mAbs passively protect animals from 

challenge with B mallei.

97,104 In addition, the bactericidal 

or opsonophagocytic activities of various anti-LPS or 

anti-CPS mAbs appeared to correlate with their ability 

to passively protect mice.97,104 Such findings confirm 

the protective capacity of these surface exposed antigens and support the rationale for developing CPS 

and OPS-based glanders vaccines. However, since 

carbohydrates such as CPS and OPS are T-independent 

antigens that would be poorly immunogenic if administered in purified form, methodologies for the preparation of CPS- and OPS-protein conjugates have been 

developed.242–245 Immunization of mice with CPS- or 

OPS-based glycoconjugates resulted in the generation 

of high titer carbohydrate-specific antibody responses. 

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Medical Aspects of Biological Warfare 

Depending on the adjuvant system used, Th1- or 

Th2-polarized antibody responses could be achieved. 

The protective capacity of these glycoconjugates in 

animal models of glanders has not been reported. 

Further development of these antigenically defined 

CPS- and OPS-based vaccine candidates is an active 

area of research.

Immunotherapies

In addition to passive immunization studies with 

PS-specific antibodies, activators of innate immune 

responses have been experimentally evaluated as 

potential immunotherapies for pre- or postexposure 

prophylaxis. BALB/c mice pre-treated with CpGcontaining oligodeoxynucleotides (CpG 7909) that 

signal through TLR9 exhibited elevated levels of IL-6, 

IL-12, and IFN-γ following an aerosol challenge with 

B mallei, resulting in lower numbers of bacteria in 

lungs and spleen, and prolonged survival.232 Similarly, 

mucosal administration of cationic liposome DNA 

complexes (CLDC), potent activators of innate immunity, to BALB/c mice before or shortly after bacterial 

challenge generated nearly complete protection from 

inhalational challenge with 100% lethal doses of B 

mallei.

86 Substantially reduced acute organ pathology 

was observed in CLDC-treated mice in comparison to 

controls. Protection was dependent on CLDC-mediated induction of IFN-γ responses in lung tissues and 

was partially dependent on the activation of natural 

killer cells. These findings suggest that preexposure 

or timely postexposure therapy with CpG or CLDC 

may help to protect individuals exposed to aerosolized B mallei.

Control and Decontamination

Historically, no vaccines were successful in protecting animals from glanders. Control and eradication of 

the disease has been dependent on eliminating infected 

horses and preventing infected horses from entering 

glanders-free stables. The greatest risk for glanders exposure to humans—outside of a biowarfare attack—is 

infected equids, particularly the asymptomatic horse. 

When glanders infection is considered as a differential diagnosis in countries with ongoing or completed 

eradication programs, local and state public health and 

veterinary authorities should be contacted immediately. Where human infection has occurred, potential 

exposure to infected equids should be investigated 

by a team approach involving patient care personnel, 

public health officials, and local veterinarians. Equids 

suspected as a possible human exposure source should 

be tested and, if positive, humanely destroyed in accordance with the local regulatory animal health authority. Facilities and transporters traced back to positive 

equine cases should be quarantined and disinfected in 

accordance with the local animal health authority. Stall 

bedding, feed, and manure in the vicinity of infected 

livestock should be burned.

In the event of deliberate release of B mallei, emergency response personnel entering a potentially heavily contaminated area should wear personal protective 

equipment, which includes Tyvek coveralls (DuPont 

USA, Wilmington, DE), gloves, and powered air purifying respirators. Decontamination procedures for 

the patient include the removal and containment of 

outer clothing. Such clothing should be regarded as 

contaminated or high risk and handled in accordance 

with local protocol. All waste should be managed in 

accordance with BSL-3 containment protocols. Patient 

showers are indicated, preferably in a facility for which 

decontamination and containment can be managed. 

The risk of acquiring infection from contaminated 

persons and their clothing is low.246 Personal protective 

equipment may prevent infection in those potentially 

exposed, including emergency responders. 

Environmental contamination will decline over time 

as a result of sunlight exposure and drying. Monitoring 

highly contaminated areas is indicated, however, and 

the advice of foreign animal disease experts should be 

sought. B mallei can remain viable in tap water for at 

least 1 month.40 B mallei can be destroyed by heating to 

at least 55°C for 10 minutes, and by ultraviolet irradiation. It is susceptible to several disinfectants including 

1% sodium hypochlorite, chlorine dioxide, 5% calcium 

hypochlorite, 70% ethanol, 5% Micro-Chem Plus 

(National Chemical Laboratories Inc, Philadelphia, 

PA), 2% glutaraldehyde, 1% formaldehyde, iodine, 

benzalkonium chloride, 1% potassium permanganate, 

3% solution of alkali, and 3% sulfur-carbolic solution. 

Phenolic and mercuric chloride disinfectants are not 

recommended.7,42,247,248 

Because human-to-human transmission has occurred nosocomially and with close personal contact, 

standard precautions are recommended. These precautions include use of disposable gloves, face shields, 

surgical masks, and—when appropriate—surgical 

gowns to protect mucous membranes and skin. Personnel, microbiological, and containment procedures 

for BSL-3 are advised in the laboratory. Appropriate 

barriers to direct skin contact with the organisms are 

mandatory at all times.249,250 Family contacts should 

be advised of blood and body fluid precautions for 

patients recovering at home. Barriers protecting mucus 

membranes, cuts and sores, and potential skin abrasions from genital, oral, nasal, and other body fluids 

are recommended. 

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Glanders

Many countries have import restrictions for equids. 

Veterinary health authorities may require testing 

within a few weeks of shipment and again at the place 

of disembarkation, as well as documentation of the 

animal’s location in the exporting country for the 6 

months before shipment.23 Restrictions vary by country and glanders-free status under the International 

Animal Health Code. The most current information 

regarding import and export should be sought from 

the regional animal health authority.

SUMMARY

B mallei is designated as a Tier 1 select agent by 

the Centers for Disease Control and Prevention, since 

the organism is considered to present a high risk of 

deliberate misuse and could pose a severe threat to 

public health and safety. B mallei also is believed to be 

moderately easy to disseminate, and enhancements 

to current diagnostic capabilities and disease surveillance are required to diagnose the disease rapidly and 

accurately. Given the biothreat potential associated 

with B mallei, raising the clinical index of suspicion 

for glanders in humans is crucial. The rarity of recent 

human cases may make glanders a difficult diagnosis 

even in regions with exceptional medical facilities. 

As with many rare diseases, final diagnosis and appropriate treatment is often delayed, with sometimes 

disastrous results. Without a higher index of suspicion, 

diagnostic laboratories may not conduct tests appropriate to detect B mallei, as what happened in the eighth 

US laboratory-acquired infection in 2000.56

The genetic homology between B mallei and B 

pseudomallei may cause confusion in identifying the 

infectious agent, especially in endemic areas for B 

pseudomallei. However, once they are thoroughly assessed, new developments in nucleic acid-based PCR 

and DNA sequencing assays may improve the accuracy and speed of B mallei diagnosis. These developments include PCR assays targeting B mallei-specific 

sequences or single nucleotide polymorphisms in the 

bimA gene or in a unique conserved region of the B 

mallei and B pseudomallei genomes, and DNA sequence 

analyses that identify gene fragments and single polymorphisms in the 16S ribosomal RNA gene specific for 

B mallei. Effective treatments for glanders are available; 

however, due in part to the development of chronic 

infections, the disease remains difficult to treat. More 

research on treatments for B mallei is warranted. Considering an aerosol threat from a virulent strain, studies 

to distinguish the effectiveness of therapeutic agents 

for treating septicemic and pulmonary infections are 

indicated. The potential for prophylactic treatment 

regimens should be further investigated.

Significant progress has been made toward a better understanding of host immunity to B mallei infection. Effective innate immune responses are essential 

for controlling the early phase of the infection, and 

monocytes and macrophages are crucial for limiting 

dissemination of the organism. The role of adaptive 

immune responses in controlling B mallei infection 

requires more investigation, although it is evident that 

T cell responses will be important for vaccine-induced 

immunity. Efforts aimed at developing effective vaccine candidates for prevention of glanders are critical, 

and research is active in this area. Overcoming chronic 

infections and achieving the ultimate goal of sterile immunity will be challenging. Investigations focusing on 

determining correlates of vaccine-induced protection 

in both acute and chronic infection are needed.

Aerosol dissemination of B mallei would likely cause 

disease in humans, equids, goats, and possibly cats in 

the vicinity. Unintentional infection may first manifest 

in equids or humans. Thus, public health workers 

should team with animal health officials in a suspected 

outbreak to expedite identification and control of 

an event. Although a formal surveillance system for 

glanders does not exist in the United States, local and 

state veterinary and public health authorities would 

be among the first to recognize a potential outbreak 

regardless of intent. These agencies would then work 

with the US Department of Agriculture, the Centers 

for Disease Control and Prevention, the Department 

of Health and Human Services, and the Department 

of Defense to control and eradicate the disease. 

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195. Felgner PL, Kayala MA, Vigil A, et al. A Burkholderia pseudomallei protein microarray reveals serodiagnostic and crossreactive antigens. Proc Natl Acad Sci U S A. 2009;106:13499–13504.

196. DeShazer D. Genomic diversity of Burkholderia pseudomallei clinical isolates: subtractive hybridization reveals a Burkholderia mallei-specific prophage in B pseudomallei 1026b. J Bacteriol. 2004;186:3938–3950.

197. Woods DE, Jeddeloh JA, Fritz DL, DeShazer D. Burkholderia thailandensis E125 harbors a temperate bacteriophage 

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203. Kumar S, Malik P, Verma SK, et al. Use of a recombinant Burkholderia intracellular motility a protein for immunodiagnosis of glanders. Clin Vaccine Immunol. 2011;18:1456–1461.

204. Heine HS, England MJ, Waag DM, Byrne WR. In vitro antibiotic susceptibilities of Burkholderia mallei (causative agent 

of glanders) determined by broth microdilution and E-test. Antimicrob Agents Chemother. 2001;45:2119–2121.

205. Kenny DJ, Russell P, Rogers D, Eley SM, Titball RW. In vitro susceptibilities of Burkholderia mallei in comparison to 

those of other pathogenic Burkholderia spp. Antimicrob Agents Chemother. 1999;43:2773–2775.

206. Batmanov VP. Sensitivity of Pseudomonas mallei to fluoroquinolones and their efficacy in experimental glanders [in 

Russian]. Antibiot Khimioter. 1991;36:31–34.

207. Batmanov VP. Sensitivity of Pseudomonas mallei to tetracyclines and their effectiveness in experimental glanders [in 

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208. Miller WR, Pannell L, Ingalls MS. Experimental chemotherapy in glanders and melioidosis. Am J Hyg. 1948;47:205–213.

209. Batmanov VP. Treatment of experimental glanders with combinations of sulfazine or sulfamonomethoxine with 

trimethoprim [in Russian]. Antibiot Khimioter. 1993;38:18–22.

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211. Russell P, Eley SM, Ellis J, et al. Comparison of efficacy of ciprofloxacin and doxycycline against experimental melioidosis and glanders. J Antimicrob Chemother. 2000;45:813–818.

212. Naureen A, Saqib M, Muhammad F, et al. Antimicrobial susceptibility of 41 Burkholderia mallei isolates from spontaneous outbreaks of equine glanders in Punjab, Pakistan. J Equine Vet Sci. 2010;30:131–141.

213. Panzner MJ, Deeraksa A, Smith A, et al. Synthesis and in vitro efficacy studies of silver carbene complexes on biosafety 

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217. Manzeniuk IN, Manzeniuk O, Filonov AV, Stepanshin IuG, Svetoch EA. Resistance of Pseudomonas mallei to tetracyclines: assessment of the feasibility of chemotherapy [in German]. Antibiot Khimioter. 1995;40:40–44.

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218. Judy BM, Whitlock GC, Torres AG, Estes DM. Comparison of the in vitro and in vivo susceptibilities of Burkholderia 

mallei to Ceftazidime and Levofloxacin. BMC Microbiol. 2009;9:88.

219. Lipsitz R, Garges S, Aurigemma R, et al. Workshop on treatment of and postexposure prophylaxis for Burkholderia 

pseudomallei and B. mallei infection, 2010. Emerg Infect Dis. 2012;18:e2.

220. Peacock SJ, Schweizer HP, Dance DA, et al. Management of accidental laboratory exposure to Burkholderia pseudomallei

and B mallei. Emerg Infect Dis. 2008;14:e2.

221. Howe C. Glanders. In: Christian HA, ed. The Oxford Medicine. New York, NY: Oxford University Press; 1950:185-202.

222. Ansabi M, Minou M. Two cases of chronic human glanders treated with sulfamides (in French). Ann Inst Pasteur. 

1951;81:98–102.

223. Womack CR, Wells EB. Co-existent chronic glanders and multiple cystic osseous tuberculosis treated with streptomycin. Am J Med. 1949;6:267–271.

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225. Mohler JR, Eichhorn A. Immunization tests with glanders vaccine. J Comp Path. 1914;27:183–185.

226. Whitlock GC, Lukaszewski RA, Judy BM, Paessler S, Torres AG, Estes DM. Host immunity in the protective response 

to vaccination with heat-killed Burkholderia mallei. BMC Immunol. 2008;9:55.

227. Rowland CA, Lever MS, Griffin KF, Bancroft GJ, Lukaszewski RA. Protective cellular responses to Burkholderia mallei

infection. Microbes Infect. 2010;12:846–853.

228. Goodyear A, Troyer R, Bielefeldt-Ohmann H, Dow S. MyD88-dependent recruitment of monocytes and dendritic cells 

required for protection from pulmonary Burkholderia mallei infection. Infect Immun. 2012;80:110–120.

229. Rowland CA, Lertmemongkolchai G, Bancroft A, et al. Critical role of type 1 cytokines in controlling initial infection 

with Burkholderia mallei. Infect Immun. 2006;74:5333–5340.

230. Santanirand P, Harley VS, Dance DA, Drasar BS, Bancroft GJ. Obligatory role of gamma interferon for host survival 

in a murine model of infection with Burkholderia pseudomallei. Infect Immun. 1999;67:3593–3600.

231. Amemiya K, Meyers JL, Trevino SR, Chanh TC, Norris SL, Waag DM. Interleukin-12 induces a Th1-like response to 

Burkholderia mallei and limited protection in BALB/c mice. Vaccine. 2006;24:1413–1420.

232. Waag DM, McCluskie MJ, Zhang N, Krieg AM. A CpG oligonucleotide can protect mice from a low aerosol challenge 

dose of Burkholderia mallei. Infect Immun. 2006;74:1944–1948.

233. Bondi SK, Goldberg JB. Strategies toward vaccines against Burkholderia mallei and Burkholderia pseudomallei. Expert Rev 

Vaccines. 2008;7:1357–1365.

234. Choh LC, Ong GH, Vellasamy KM, et al. Burkholderia vaccines: are we moving forward? Front Cell Infect Microbiol. 

2013;3:5.

235. Silva EB, Dow SW. Development of Burkholderia mallei and pseudomallei vaccines. Front Cell Infect Microbiol. 2013;3:10.

236. Waag DM. Efficacy of postexposure therapy against glanders in mice. Antimicrob Agents Chemother. 2015;59:2236–2241. 

237. Amemiya K, Bush GV, DeShazer D, Waag DM. Nonviable Burkholderia mallei induces a mixed Th1- and Th2-like cytokine response in BALB/c mice. Infect Immun. 2002;70:2319–2325.

238. Sarkar-Tyson M, Smither SJ, Harding SV, Atkins TP, Titball RW. Protective efficacy of heat-inactivated B thailandensis, 

B mallei or B pseudomallei against experimental melioidosis and glanders. Vaccine. 2009;27:4447–4451.

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239. Whitlock GC, Deeraksa A, Qazi O, et al. Protective response to subunit vaccination against intranasal Burkholderia 

mallei and B pseudomallei challenge. Procedia Vaccinol. 2010;2:73–77.

240. Nelson M, Prior JL, Lever MS, Jones HE, Atkins TP, Titball RW. Evaluation of lipopolysaccharide and capsular polysaccharide as subunit vaccines against experimental melioidosis. J Med Microbiol. 2004;53:1177–1182.

241. Ngugi SA, Ventura VV, Qazi O, et al. Lipopolysaccharide from Burkholderia thailandensis E264 provides protection in 

a murine model of melioidosis. Vaccine. 2010;28:7551–7555.

242. Brett PJ, Burtnick MN, Heiss C, et al. Burkholderia thailandensis oacA mutants facilitate the expression of Burkholderia 

mallei-like O-polysaccharides. Infect Immun. 2010;79:961–969.

243. Brett PJ, Woods DE. Structural and immunological characterization of Burkholderia pseudomallei O-polysaccharideflagellin protein conjugates. Infect Immun. 1996;64:2824–2828.

244. Burtnick MN, Heiss C, Roberts RA, Schweizer HP, Azadi P, Brett PJ. Development of capsular polysaccharide-based 

glycoconjugates for immunization against melioidosis and glanders. Front Cell Infect Microbiol. 2012;2:108.

245. Burtnick MN, Heiss C, Schuler AM, Azadi P, Brett PJ. Development of novel O-polysaccharide based glycoconjugates 

for immunization against glanders. Front Cell Infect Microbiol. 2012;2:148.

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London, England: Health Protection Agency Centre for Infections; 2006.

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Microbiol. 2011;53:225–230.

248. Waag DM, DeShazer D. Glanders: new insights into an old disease. In: Linder LE, Lebeda FJ, Korch G, eds. Biological 

Weapons Defense: Infectious Diseases and Counterbioterrorism. Totowa, NJ: Humana Press; 2004:209–237.

249. Centers for Disease Control and Prevention, National Institutes of Health, and US Department of Health and Human Services. Biosafety in Microbiological and Biomedical Laboratories (BMBL). 5th ed. Washington, DC: US Government 

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250. Centers for Disease Control and Prevention. Guidelines for isolation precautions in hospitals. Part II. Recommendations 

for isolation precautions in hospitals. Hospital Infection Control Practices Advisory Committee. Am J Infect Control. 

1996;24:32–52.

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Chapter 9

MELIOIDOSIS

PAUL J. BRETT, PhD*; DAVID DeSHAZER, PhD†; and NICHOLAS J. VIETRI, MD‡

INTRODUCTION

THE INFECTIOUS AGENT

MILITARY RELEVANCE

THE DISEASE

Epidemiology

Clinical Disease

Diagnosis

Treatment

Prevention

SUMMARY

*Associate Professor, Department of Microbiology and Immunology, University of South Alabama, 610 Clinic Drive, Laboratory of Infectious Diseases 

Building, Mobile, Alabama 36688

†

Microbiologist, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

‡Colonel, Medical Corps, US Army; Infectious Diseases Officer, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland 21702; and Assistant Professor of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones 

Bridge Road, Bethesda, Maryland 20814

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INTRODUCTION

In 1911, Captain Alfred Whitmore and Dr CS 

Krishnaswami described a previously unrecognized 

disease that was prevalent among the ill-nourished 

and neglected inhabitants of Rangoon, Burma.1 The 

new disease resembled glanders, a zoonotic disease 

of equines,2 and the gram-negative bacillus they isolated post-mortem from tissue samples resembled the 

glanders bacillus, Bacillus mallei.3 However, the new 

bacillus could be differentiated from B mallei by its motility, luxuriant growth on peptone agar, and wrinkled 

colony morphology, and was subsequently named 

Bacillus pseudomallei.3,4 Whitmore’s detailed account of 

the first 38 human cases of this disease demonstrated 

most were morphine injectors that died of septicemia 

with abscesses in multiple organs.4 As a result, the 

disease became known as “Whitmore’s disease” or 

“morphine injector’s septicemia.”5,6 In 1921, Stanton 

and Fletcher reported an outbreak of a septicemic disease in a guinea pig colony at the Institute for Medical 

Research in Kuala Lumpur.7

 The infectious agent they 

isolated from diseased animals was indistinguishable 

from Whitmore’s bacillus, and they coined the term 

“melioidosis” (a Greek term meaning glanders-like 

illness) to describe this new disease of the tropics.7

Stanton and Fletcher subsequently published a classic monograph in 1932 describing their observations 

of melioidosis in humans and animals occurring in 

Burma, Malaya, French Indochina, and Ceylon over 

a number of years.8

Today, melioidosis is regarded as an emerging infectious disease and a potential bioterrorism threat.9–11

The etiologic agent of melioidosis is present in water 

and soil in tropical and subtropical regions and is 

spread to humans through direct contact with the 

contaminated source. Clinical manifestations range 

from subclinical infection to overwhelming septicemia 

that resembles disseminated or localized, suppurative 

infection due to a variety of pathogens, resulting in the 

nickname “the remarkable imitator.”12 The majority 

of melioidosis cases have one or more identified risk 

factors, including diabetes, alcoholism, chronic renal 

disease, cystic fibrosis, and steroid abuse.13 Interestingly, acquired immunodeficiency syndrome does not 

seem to be a major risk factor for melioidosis. Healthy 

individuals can also get the disease, especially if they 

work in muddy soil without good hand and foot 

protection.14 Many animal species are susceptible to 

melioidosis, including sheep, goats, horses, swine, 

cattle, dogs, and cats.15 Numerous review articles on 

melioidosis have been published since 1990.11,13–30

THE INFECTIOUS AGENT

The bacterium that causes melioidosis, now designated Burkholderia pseudomallei,

31 has undergone 

numerous name changes since its original classification 

as Bacillus pseudomallei, including Bacterium whitmori, 

Bacillus whitmori, Pfeifferella whitmori, Pfeifferella pseudomallei, Actinobacillus pseudomallei, Lofflerella whitmori, 

Flavobacterium pseudomallei, Malleomyces pseudomallei, 

and Pseudomonas pseudomallei. The non-sporulating, 

gram-negative bacillus is an environmental saprophyte found in surface waters and wet soils in endemic 

regions.32–39 Individual cells are approximately 0.8 × 1.5 

μm with a polar tuft of two to four flagella and may 

exhibit bipolar staining with a “safety pin” appearance.40,41 B pseudomallei is metabolically versatile and 

can grow on numerous carbon sources.31,42 Anaerobic 

growth is possible, but only in the presence of nitrate 

or arginine.11 The microbe accumulates intracellular 

stores of poly-b-hydroxybutyric acid and can survive 

in distilled water for years.10,43,44 The optimal survival 

temperature for B pseudomallei is between 24°C and 

32°C, but it can grow at temperatures up to 42°C.45,46

B pseudomallei demonstrate considerable interstrain 

and medium-dependent colony morphology.47–49 The 

oxidase-positive organism can grow on a variety of 

microbial media, but Ashdown’s selective medium is 

often used for isolating B pseudomallei from environmental and clinical specimens.50 Two distinct colony 

phenotypes are commonly observed on this medium 

(Figure 9-1a), presumably due to the differential uptake of crystal violet and neutral red or to the differential production of ammonia and oxalic acid.50,51 Most 

strains appear lavender after 2 to 3 days of incubation 

at 37°C, but some isolates appear deep purple (see 

Figure 9-1a). After 3 days at 37°C, the colonies often 

become dull and wrinkled (Figure 9-1b) and emit a 

distinctive sweet, earthy smell. Other selective media 

have also been used to isolate B pseudomallei from 

contaminated specimens.52,53

The complete genome sequence of B pseudomallei

K96243, a strain isolated in 1996 from a 34-year-old diabetic patient in Khon Kaen, Thailand, was published 

in 2004.54 The 7.25-megabase pair (Mb) genome was 

comprised of two circular replicons, termed chromosome 1 (4.07 Mb) and chromosome 2 (3.17 Mb). The G 

+ C content of the genome is 68% and is predicted to 

encode 5,855 proteins. Chromosome 1 encoded a high 

proportion of core housekeeping functions (DNA replication, transcription, translation, amino acid and nucleotide metabolism, basic carbohydrate metabolism, 

and cofactor synthesis), while chromosome 2 encoded 

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Melioidosis

Figure 9-1. Burkholderia pseudomallei colony morphologies as demonstrated on Ashdown’s selective medium50 supplemented 

with 100 μg/mL streptomycin. Plates were incubated for 3 days at 37°C (a) and 5 days at 37°C (b).

a

b

a high proportion of accessory functions (adaptation 

to atypical conditions, osmotic protection, and secondary metabolism).54 Plasmid-like replication genes 

and accessory genes on chromosome 2 suggest it may 

have been derived from a plasmid (or megaplasmid) 

that became an indispensable replicon by acquiring 

essential functions such as transfer ribonucleic acid 

genes, amino acid biosynthesis genes, and energy metabolism genes. There are 16 “genomic islands” in the B 

pseudomallei K96243 genome that appear to have been 

acquired through horizontal gene transfer.54 Analysis 

of 11 additional B pseudomallei strains has led to the 

identification of at least 128 different genomic islands 

that are variably present among these strains.55,56 Mobile genetic elements, such as prophages, insertion 

sequences, and integrated plasmids, account for most 

of the laterally acquired genomic sequences. Recent 

studies have shown that B pseudomallei strains exhibit 

significant genomic diversity and that much of the 

genetic heterogeneity is due to laterally acquired mobile genetic elements.54,57–61 These genomic islands may 

provide strains that give them a metabolic or virulence 

advantage over strains that do not contain such sequences. Similarly, autonomously replicating plasmids 

are variably present in B pseudomallei isolates, but little 

is known about their biological significance.30,62–64 Complete genome sequences of 13 B pseudomallei isolates 

(K96243, 1026b, 1106a, 1710b, 668, BPC006, MSHR146, 

MSHR305, MSHR511, NAU20B-16, NCTC13178, 

NCTC13179, MSHR520) and draft genome sequences 

of an additional 54 B pseudomallei isolates are available 

in GenBank, dramatically enhancing the amount and 

diversity of genome sequence data available for study 

of B pseudomallei.

65

MILITARY RELEVANCE

Throughout the 20th century, melioidosis had an 

impact on the health of soldiers serving in Asia during 

times of war and peace.66 Sporadic melioidosis infections occurred in US and Japanese soldiers during 

World War II (WWII),41,67,68 and cases of recrudescent 

melioidosis in WWII veterans were also reported.69,70

During the French Indochina War (1946–1954), there 

were at least 100 cases of melioidosis in French forces 

during their fight against the resistance movement led 

by the Viet Minh.22,66 There were more than 300 cases 

of melioidosis in US soldiers during the Vietnam War22

and additional cases that did not surface until years 

after the war’s end, leading to the nickname “Vietnam 

Time Bomb.”71–73 Twenty-three melioidosis cases were 

reported in the Singapore Armed Forces from 1987 to 

1994.74 The infection rate in these relatively healthy 

Singapore Armed Forces was approximately fourfold 

the rate in the general population of Singapore, suggesting that close contact with the soil during military 

training may lead to an increased risk for melioidosis.

B pseudomallei is a Centers for Disease Control and 

Prevention Tier 1 select agent that must be handled in 

biosafety level 3 laboratories.9

 Biosafety level 3 facilities 

incorporate specialized negative-air pressure ventilation systems and well-defined biosafety containment 

equipment and protocols to study agents that can 

be transmitted through the air and cause potentially 

lethal infection. Tier 1 agents present the greatest risk 

of deliberate misuse and pose a severe threat to public 

health and safety. B pseudomallei was studied by the 

United States, the former Soviet Union, and possibly 

Egypt as a potential biological warfare agent, but was 

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never used in this capacity.75–77 On the other hand, B 

mallei was used as a biological warfare agent several 

times in the past, including during the American Civil 

War, World War I, WWII, and in Afghanistan between 

1982 and 1984.2,76,78,79 The usefulness of B pseudomallei as 

a biological warfare agent is currently unknown, but 

the ease of acquiring strains from the environment, 

the ability to genetically manipulate the agent to be 

multiply antibiotic resistant, and the lack of a melioidosis vaccine make this possibility a serious concern.

THE DISEASE

Epidemiology

Melioidosis cases are most commonly reported 

from countries located between 20°N and 20°S in 

latitude, with the greatest concentration in Vietnam, 

Cambodia, Laos, Thailand, Malaysia, Singapore, and 

northern Australia.11,13,23 The disease has also been 

observed in the South Pacific, Africa, India, and the 

Middle East.80–83 In addition, sporadic cases of melioidosis have occurred in the Western Hemisphere in 

Aruba, Brazil, Mexico, Panama, Ecuador, Haiti, Peru, 

and Guyana.11,13,23,84,85 In endemic regions, the disease 

occurs in humans, sheep, goats, horses, swine, cattle, 

dogs, cats, deer, camels, monkeys, zebras, kangaroos, 

koalas, birds, and crocodiles.15,27,86–88 Melioidosis cases 

that occur in temperate regions are typically acquired 

in endemic areas. Human cases can often be attributed 

to recent travel to such areas.21,89–92 For example, the 

first case report of cerebral melioidosis in the United 

States was recently described in a individual that had 

immigrated from Cambodia and frequently visited 

his home country.93 A case of neurological melioidosis 

was reported in a pigtail macaque that was imported 

from Indonesia to the United States for research purposes.94 In addition, B pseudomallei infections have 

been described in pet green iguanas in California 

and, based on the multilocus sequence typing of the 

isolates in these cases, are believed to have originated 

in Central America.95 With the exception of a single 

case of melioidosis that occurred in Arizona in 2008, 

no B pseudomallei infections have been documented 

in US patients without a history of prior travel to an 

endemic region.96,97 While the source of the Arizona 

isolate could not be determined despite extensive 

investigation, molecular analysis indicated that it was 

consistent with Southeast Asian origin.97,98

Pathogenesis

Several animal models of melioidosis have been 

developed to study pathogenesis, virulence factors, 

and efficacy of antibiotics and vaccines.99–107 In general, hamsters and ferrets are highly susceptible to 

experimental melioidosis, while rats, pigs, and rhesus 

monkeys are relatively resistant. Infant rats can be 

made more susceptible to infection by intraperitoneal 

injection of streptozotocin, a compound that induces 

diabetes.103,108 The LD50 (amount necessary to kill 50% 

of the subject population) of B pseudomallei for nondiabetic infant rats is greater than 108

 bacteria, while 

the LD50 in streptozotocin-induced diabetic infant rats 

is approximately 104 bacteria. Mice and guinea pigs 

exhibit intermediate susceptibility to experimental 

infection with B pseudomallei, but the LD50 for mice varies widely depending on the route of infection, mouse 

strain, and bacterial strain.101,102,105,109 Recent reviews 

describe in detail the various mouse models of melioidosis that have been developed and the advantages 

and disadvantages of each.110,111

Due to fears that B pseudomallei might be used as 

a biological weapon, basic research on this pathogen 

has progressed rapidly over the past 10 years. The 

identification of virulence factors has been facilitated 

by the availability of genomic sequence data54 and the 

existence of a nonpathogenic B pseudomallei-like species 

designated B thailandensis.112–114 B pseudomallei and B

thailandensis strains are genetically and immunologically similar to one another, but B thailandensis is less 

virulent in animal models of infection and has only 

rarely been reported to cause disease in humans.115

Genetic determinants that confer enhanced virulence 

in B pseudomallei relative to B thailandensis have been 

identified by comparative analysis of genomic DNA 

from these species.58,116,117 Table 9-1 provides a brief description of B pseudomallei virulence factors identified 

to date, their mechanisms of action, and their relative 

importance in animal models of melioidosis.

B pseudomallei is a facultative intracellular pathogen 

whose ability to survive and replicate in phagocytic 

and nonphagocytic cell lines has been well documented.118–123 The organism possesses a variety of mechanisms to adapt to the host environment and harbors 

an array of specialized secretory systems, which are 

required within this niche.124–128 Although relatively 

little is known about the initial interactions of B pseudomallei with host cells, recent studies have identified 

a number of adhesins that mediate adherence of the 

organism to eukaryotic cells.129–133 Following internalization, B pseudomallei rapidly escapes from endocytic 

vacuoles and enters into the host cell cytosol, where it 

can replicate to high numbers, polymerize actin, and 

induce host cell fusion. These processes are believed 

244-949 DLA DS.indb 226 6/4/18 
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Melioidosis

to facilitate intracellular spread and multinucleated 

giant cell formation. Type III and type VI secretions 

systems have been shown to play key roles in endosomal escape and multinucleated giant cell formation, 

respectively.124,134 The effector proteins delivered by 

these systems are predicted to modulate critical host 

proteins and pathways involved in cytoskeleton rearrangement, cell signaling, and cell death, thereby 

enabling pathogen survival and proliferation within a 

host.17 It has been postulated that after the initial phase 

of infection, B pseudomallei can persist in a dormant 

stage in macrophages for months or years.123 Melioidosis has the potential for a long latency period and B 

pseudomallei’s intracellular persistence could provide a 

mechanism by which this occurs. Intracellular survival 

and cell-to-cell spread may also provide B pseudomallei 

protection from the humoral immune response. 

Clinical Disease

Melioidosis is a tropical bacterial disease with 

primary endemic foci in southeast Asia, northern 

Australia, south Asia, and China. Hyperendemic 

areas for melioidosis include northern Australia and 

northeast Thailand, where the disease incidence peaks 

in the rainy season. The routes of infection include 

percutaneous inoculation, inhalation, or ingestion of 

contaminated food or water. Although percutaneous 

inoculation is the most common route of infection, 

heavy rainfall is associated with pneumonia and more 

severe disease and may represent a shift from percutaneous inoculation to inhalation as the primary mode 

of infection.135 In hyperendemic areas, B pseudomallei 

causes a substantial burden of infectious disease. For 

example, at the Sappasit Prasong Hospital in Ubon 

Ratchatani, northeast Thailand, which serves a rural 

community of rice farmers and their families, nearly 

20% of all community-acquired bacteremia is due to 

B pseudomallei.136 Likewise, melioidosis is the most 

common cause of fatal community-acquired bacteremic pneumonia at the Royal Darwin Hospital in the 

Northern Territory of Australia.137

Cases of human-to-human transmission of B pseudomallei, although very rare, have been documented.138,139

The incubation period (time between exposure and 

appearance of clinical symptoms) varies, as infectious dose, route of infection, B pseudomallei strain 

characteristics, and host risk factors are all believed 

to play an important role. One study that looked at 

the incubation period after inoculation exposures in 

Darwin, Australia, revealed a mean incubation period 

of 9 days, with a range of 1 to 21 days.140 Although 

serologic studies suggest that most infections with B 

pseudomallei are asymptomatic,141 individuals with risk 

factors such as diabetes mellitus, alcoholism, cirrhosis, 

thalassemia, or other immunosuppressed states are 

at an increased risk of developing symptomatic infection. Other melioidosis-associated risk factors include 

chronic lung disease, kava consumption, and cystic 

fibrosis. Diabetes appears to be the most important 

of all the known risk factors, as up to 50% of patients 

with melioidosis have diabetes mellitus.27

Melioidosis, which presents as a febrile illness, has 

an unusually broad range of clinical presentations. 

The diversity of infectious presentations includes 

acute localized suppurative soft tissue infections, acute 

pulmonary infections, acute fulminant septicemia, 

and chronic localized infections. Clinical disease with 

B pseudomallei is generally caused by hematogenous 

spread of bacteria and seeding to various organs within 

the host.27 The Infectious Disease Association of Thailand, the country with the largest number of reported 

cases (2,000–3,000 per year), divided 345 cases into the 

following categories: (a) disseminated septicemia, 45% 

of the cases with 87% mortality; (b) nondisseminated 

septicemia, 12% of the cases with 17% mortality; (c) 

localized septicemia, 42% of the cases with 9% mortality; and (d) transient bacteremia, 0.3% of cases.142,143

Melioidosis is characterized by abscess formation, 

and the majority of patients with melioidosis are bacteremic. The most commonly involved organ is the lung. 

The nidus of infection is either a primary pneumonia 

or lung abscess, or the infection results from hematogenous seeding of the lung from bacteremia (Figures 9-2 

and 9-3). For example, of the 540 cases of melioidosis 

analyzed in the 20-year Darwin Prospective Melioidosis Study, pneumonia was the most common primary 

Figure 9-2. Chest radiograph demonstrating a severe multilobar pneumonia. 

Photograph courtesy of Bart Currie, MD, Royal Darwin 

Hospital, Australia.

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Medical Aspects of Biological Warfare 

TABLE 9-1

CANDIDATE VIRULENCE FACTORS OF BURKHOLDERIA PSEUDOMALLEI

Factor Description

Capsular polysaccharide CPS is a 200-kDa 1,3-linked 2-O-acetyl-6-deoxy-β-d-manno-heptan.1 CPS mutants are highly attenuated in 

hamsters and mice.2,3 The CPS may contribute to survival in serum by reducing complement factor C3b 

deposition.4,5

Type III secretion system B pseudomallei harbors three distinct TTSS loci, termed TTSS1, TTSS2, and TTSS3.6

 The TTSS1 and TTSS2 

loci are similar to TTSS genes of the plant pathogen Ralstonia solanacearum and are not necessary for 

virulence in hamsters.6 The TTSS3 locus is similar to the TTSS in Salmonella and Shigella7 and is required 

for full virulence of B pseudomallei in both hamsters and mice.6,8 The effector proteins of TTSS3 facilitate 

the invasion of epithelial cells and escape from endocytic vesicles.7,9

Type VI secretion system B pseudomallei harbors six distinct T6SS loci designated T6SS-1, T6SS-2, T6SS-3, T6SS-4, T6SS-5, and T6SS6.10 Of the six systems, only T6SS-1 has been shown to be necessary for virulence in animal models of 

melioidosis. This system is expressed following uptake by murine macrophages (RAW264.7 cells) 

and is required for optimal intracellular growth, actin-based motility and multinucleated giant cell 

formation.11,12,13 

Transcriptional 

regulators and 

two-component regulatory systems

B pseudomallei harbors numerous transcriptional regulators and two component regulatory systems. 

BspR (TetR family regulator), bsaN, and bprC (AraC family regulators) are involved in a complex regulatory cascade that ultimately controls TTSS3 and T6SS-1 gene expression.14,15 B pseudomallei bspR, bsaN,

and bprC mutants exhibited significantly attenuated virulence in mice.12,14,15 The VirAG two-component 

regulatory system is known to positively regulate T6SS-1 gene expression, and mutations in VirAG 

render the organism avirulent in mice. 11,12,14

Quorum sensingB pseuβdomallei encodes three luxI homologues that produce at least three quorum-sensing molecules, 

including N-octanoyl-homoserine lactone (C8-HSL),16,17 N-decanoyl-homoserine lactone (C10-HSL),16,18

and N-(3-hydroxyoctanoyl)-L-homoserine lactone (3-hydroxy-C8-HSL).16 It also has five luxR homologs 

to sense these signals. Mutations in all of the luxI and luxR homologues result in strains with decreased 

virulence in hamsters and mice,16,18 but the virulence-associated genes regulated by this complex 

quorum-sensing system are still under investigation.

Lipopolysaccharide OantigenAn unbranched heteropolymer consisting of disaccharide repeats having the structure -3)-β-dglucopyranose-(1-3)-6-deoxy-α-l-talopyranose-(1-, in which the 6-deoxy-α-l-talopyranose residues 

posses 2-O-acetyl or 2-O-methyl and 4-O-acetyl modifications.19,20,21 LPS O-antigen mutants are attenuated in hamsters, guinea pigs, and infant diabetic rats and are killed by serum.22 This factor promotes 

survival in serum by preventing killing by the alternative pathway of complement. Levels of anti-LPS 

O-antigen antibodies are significantly higher in patients who survive than in those who die.23 

Flagellin A surface-associated 43-kDa protein that is required for motility.24,25 Flagellin mutants are attenuated in 

mice,26 but not in hamsters or infant diabetic rats.25 Passive exposure studies demonstrated that flagellinspecific antiserum was capable of protecting infant diabetic rats from challenge with B pseudomallei.

24

Type II secretionRequired for the secretion of several exoproducts, including protease, lipase, and phospholipase C.27 The 

products secreted by this pathway appear to play a minor role in B pseudomallei pathogenesis.28

Type IV pili B pseudomallei K96243 encodes four complete type IV pilin clusters.29 A mutation in pilA, a gene encoding 

a type IVA pilin subunit, resulted in a strain exhibiting decreased attachment to cultured respiratory cell 

lines relative to wild type. The pilA mutant was not attenuated in mice by the intraperitoneal challenge 

route, but was slightly attenuated by the intranasal challenge route.30

Biofilm formationThe extracellular slime matrix produced by B pseudomallei appears to be polysaccharide in nature, but 

the exact structure is unknown.31 Biofilm mutants were not attenuated in the mouse model of melioidosis, suggesting that the biofilm plays a relatively minor role, if any, in virulence.31

Malleobactin A water-soluble siderophore of the hydroxamate class.32 The compound is capable of scavenging iron 

from both lactoferrin and transferrin in vitro.33 The genes encoding malleobactin biosynthesis and 

transport were recently identified, but malleobactin mutants were not tested in animal models of melioidosis.34

Rhamnolipid A 762-Da glycolipid with the structure 2-O-α-l-rhamnopyranosyl-α-l-rhamnopyranosyl-βhydroxytetradecanoyl-β-hydroxytetradecanoate (Rha-Rha-C14-C14).35 Rhamnolipid-treated cell lines exhibit profound morphological alterations, but the role of this glycolipid in virulence remains unknown.36

(Table 9-1 continues)

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Melioidosis

Exopolysaccharide A linear, unbranched polymer of repeating tetrasaccharide units composed of d-galactose and 3-deoxyd-manno-octulosonicacid (KDO), with the following structure: -3)-2-O-Ac-β-d-Galp-(1-4)-α-d-Galp-(1-

3)-β-D-Galp-(1-5)-β-D-KDOp-(2-.37–39 EPS is not produced by the closely related nonpathogenic species 

B thailandensis, suggesting that it may be a virulence determinant of B pseudomallei. EPS is probably 

produced during infection because sera from melioidosis patients contain IgG and IgM antibodies to 

EPS.38,40

Endotoxin The lipid A portion of B pseudomallei LPS contains amide-linked 3-hydroxyhexadecanoic acids, which 

are longer than the fatty acid chains of enterobacterial LPS.41 The endotoxic activity of B pseudomallei

LPS was 10- to 100-fold weaker than enterobacterial LPS in pyrogenic activity in rabbits, lethal toxicity in GalN-sensitized mice, and macrophage activation assays. However, the mitogenic activity of B 

pseudomallei LPS was much higher than enterobacterial LPS.41 The LD50 of purified B pseudomallei LPS in 

hamsters was 1,000 mg.42

Actin-based 

motility

Once B pseudomallei gains access to the host cell cytoplasm, it can replicate and exploit actin-based motility for cell-to-cell spread and evasion of the humoral immune response.43–45 The autotransported protein 

bimA is located at the pole of the bacterial cell and is responsible for the formation of actin tails.44 It is 

currently unknown if actin-based motility is required for virulence in animal models of melioidosis.

Autotransporters and 

adhesins

B pseudomallei harbors 11 autotransporter analogs, including bimA, and two ATs (boaA and boaB) with 

roles in bacterial adhesion to epithelial cells, eight ATs (BpaA-F; BcaA-B) that contribute to adherence to 

and efficient invasion of A549 cells.46–48 BpaC and bcaA have been implicated in virulence in BALB/c mice, 

since mutants in these ATs displayed defects in dissemination to the liver or spleen, respectively.47 A B 

pseudomallei bbfA (also known as bpaF) mutant demonstrated a moderate attenuation in a murine model 

of melioidosis.48

Exotoxins There have been several reports in the literature about B pseudomallei exotoxins,49–53 but the genes encoding these exotoxins have not been identified and no defined exotoxin mutants have been constructed. 

The role of exotoxins as B pseudomallei virulence factors is highly controversial and there appears to 

be no correlation between in vitro cytotoxicity and in vivo virulence.42,54 In fact, the K96243 genome 

sequence does not encode any homologues of known major toxins produced by other pathogenic bacteria.29

Intracellular 

toxin

Burkholderia lethal factor 1 (BLF1; BPSL1549) is structurally related to cytotoxic necrotizing factor 1 and 

has been shown to specifically deamidate Gln339 of eukaryotic initiation factor 4A (a translation initiation factor) leading to inhibition of protein synthesis.55,56 Purified recombinant BLF1 was toxic to mice 

(via intraperitoneal injection), J774 macrophages, but not 3T3 cells.55,56 A B pseudomallei bfl1 mutant was 

significantly attenuated in mice and exhibited a 100-fold higher median lethal dose in comparison to the 

wild type strain.55,56

CPS: capsular polysaccharide; EPS: exopolysaccharide; Gln: glutamine; IgG: immunoglobulin G; IgM: immunoglobulin M; LPS: lipopolysaccharide; T6SS: type VI secretion system; TTSS: type III secretion system

(1) Isshiki Y, Matsuura M, Dejsirilert S, Ezaki T, Kawahara K. Separation of 6-deoxy-heptan from a smooth-type lipopolysaccharide preparation of Burkholderia pseudomallei. FEMS Microbiol Lett. 2001;199:21–25. (2)Reckseidler SL, DeShazer D, Sokol PA, Woods DE. Detection of 

bacterial virulence genes by subtractive hybridization: identification of capsular polysaccharide of Burkholderia pseudomallei as a major 

virulence determinant. Infect Immun. 2001;69:34–44. (3) Atkins T, Prior R, Mack K, et al. Characterization of an acapsular mutant of Burkholderia pseudomallei identified by signature tagged mutagenesis. J Med Microbiol. 2002;51:539–547. (4) Reckseidler-Zenteno SL, DeVinney R, 

Woods DE. The capsular polysaccharide of Burkholderia pseudomallei contributes to survival in serum by reducing complement factor C3b 

deposition. Infect Immun. 2005;73:1106–1115. (5) Woodman ME, Worth RG, Wooten RM. Capsule influences the deposition of critical 

complement C3 levels required for the killing of Burkholderia pseudomallei via NADPH-oxidase induction by human neutrophils. PLoS One. 

2012;7:e52276. (6) Warawa J, Woods DE. Type III secretion system cluster 3 is required for maximal virulence of Burkholderia pseudomallei in 

a hamster infection model. FEMS Microbiol Lett. 2005;242:101–108. (7) Stevens MP, Wood MW, Taylor LA, et al. An Inv/Mxi-Spa-like type 

III protein secretion system in Burkholderia pseudomallei modulates intracellular behaviour of the pathogen. Mol Microbiol. 2002;46:649–659. 

(8) Stevens MP, Haque A, Atkins T, et al. Attenuated virulence and protective efficacy of a Burkholderia pseudomallei bsa type III secretion 

mutant in murine models of melioidosis. Microbiology. 2004;150:2669–2676.(9) Stevens MP, Friebel A, Taylor LA, et al. A Burkholderia pseudomallei type III secreted protein, BopE, facilitates bacterial invasion of epithelial cells and exhibits guanine nucleotide exchange factor activity. J Bacteriol. 2003;185:4992–4996. (10) Schell MA, Ulrich RL, Ribot WJ, et al. Type VI secretion is a major virulence determinant in Burkholderia mallei. Mol Microbiol. 2007;64:1466–1485. (11) Burtnick MN, Brett PJ, Harding SV, et al. The cluster 1 type VI secretion system is a 

major virulence determinant in Burkholderia pseudomallei. Infect Immun. 2011;79:1512–1525. (12)Chen Y, Wong J, Sun GW, Liu Y, Tan GY, Gan 

YH. Regulation of type VI secretion system during Burkholderia pseudomallei infection. Infect Immun. 2011;79:3064–3073. (13) Shalom G, Shaw 

JG, Thomas MS. In vivo expression technology identifies a type VI secretion system locus in Burkholderia pseudomallei that is induced upon 

invasion of macrophages. Microbiology. 2007;153:2689–2699. (14) Sun GW, Chen Y, Liu Y, et al. Identification of a regulatory cascade controlling type III secretion system 3 gene expression in Burkholderia pseudomallei. Mol Microbiol. 2010;76:677–689. (15) Sun GW, Gan YH. Unravel-

(Table 9-1 continues)

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ing type III secretion systems in the highly versatile Burkholderia pseudomallei. Trends Microbiol. 2010;18:561–568. (16) Ulrich RL, DeShazer 

D, Brueggemann EE, Hines HB, Oyston PC, Jeddeloh JA. Role of quorum sensing in the pathogenicity of Burkholderia pseudomallei. J Med 

Microbiol. 2004;53:1053–1064. (17) Song Y, Xie C, Ong YM, Gan YH, Chua KL. The BpsIR quorum-sensing system of Burkholderia pseudomallei. J Bacteriol. 2005;187:785–790. (18) Valade E, Thibault FM, Gauthier YP, Palencia M, Popoff MY, Vidal DR. The PmlI-PmlR quorumsensing system in Burkholderia pseudomallei plays a key role in virulence and modulates production of the MprA protease. J Bacteriol. 

2004;186:2288–2294. (19) Ulett GC, Currie BJ, Clair TW, et al. Burkholderia pseudomallei virulence: definition, stability and association with 

clonality. Microbes Infect. 2001;3:621–631. (20) Perry MB, MacLean LL, Schollaardt T, Bryan LE, Ho M. Structural characterization of the lipopolysaccharide O antigens of Burkholderia pseudomallei. Infect Immun. 1995;63:3348–3352. (21) Knirel YA, Paramonov NA, Shashkov AS, 

et al. Structure of the polysaccharide chains of Pseudomonas pseudomallei lipopolysaccharides. Carbohydrate Res. 1992;233:185–193. (22) DeShazer D, Brett PJ, Woods DE. The type II O-antigenic polysaccharide moiety of Burkholderia pseudomallei lipopolysaccharide is required for 

serum resistance and virulence. Mol Microbiol. 1998;30:1081–1100. (23) Charuchaimontri C, Suputtamongkol Y, Nilakul C, et al. Antilipopolysaccharide II: an antibody protective against fatal melioidosis. Clin Infect Dis. 1999;29:813–818. (24) Brett PJ, Mah DC, Woods DE. Isolation 

and characterization of Pseudomonas pseudomallei flagellin proteins. Infect Immun. 1994;62:1914–1919. (25) DeShazer D, Brett PJ, Carlyon R, 

Woods DE. Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: Isolation of motility mutants and molecular characterization of the 

flagellin structural gene. J Bacteriol. 1997;179:2116–2125. (26) Chua KL, Chan YY, Gan YH. Flagella are virulence determinants of Burkholderia pseudomallei. Infect Immun. 2003;71:1622–1629. (27) Ashdown LR, Koehler JM. Production of hemolysin and other extracellular enzymes 

by clinical isolates of Pseudomonas pseudomallei. J Clin Microbiol. 1990;28:2331–2334. (28) DeShazer D, Brett PJ, Burtnick MN, Woods DE. 

Molecular characterization of genetic loci required for secretion of exoproducts in Burkholderia pseudomallei. J Bacteriol. 1999;181:4661–4664. 

(29) Holden MT, Titball RW, Peacock SJ, et al. Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei. Proc Natl 

Acad Sci U S A. 2004;101:14240–14245. (30) Essex-Lopresti AE, Boddey JA, Thomas R, et al. A type IV pilin, PilA, Contributes to adherence 

of Burkholderia pseudomallei and virulence in vivo. Infect Immun. 2005;73:1260–1264. (31) Taweechaisupapong S, Kaewpa C, Arunyanart C, et 

al. Virulence of Burkholderia pseudomallei does not correlate with biofilm formation. Microb Pathogen. 2005;39:77–75. (32) Yang HM, Chaowagul 

W, Sokol PA. Siderophore production by Pseudomonas pseudomallei. Infection & Immunity. 1991;59:776–780. (33) Yang H, Kooi CD, Sokol PA. 

Ability of Pseudomonas pseudomallei malleobactin to acquire transferrin-bound, lactoferrin-bound, and cell-derived iron. Infect Immun. 

1993;61:656–662. (34) Alice AF, Lopez CS, Lowe CA, Ledesma MA, Crosa JH. Genetic and transcriptional analysis of the siderophore malleobactin biosynthesis and transport genes in the human pathogen Burkholderia pseudomallei K96243. J Bacteriol. 2006;188:1551–1566. (35) 

Haussler S, Nimtz M, Domke T, Wray V, Steinmetz I. Purification and characterization of a cytotoxic exolipid of Burkholderia pseudomallei. 

Infect. Immun. 1998;66:1588-1593. (36) Haussler S, Rohde M, von Neuhoff N, Nimtz M, Steinmetz I. Structural and functional cellular 

changes induced by Burkholderia pseudomallei rhamnolipid. Infect Immun. 2003;71:2970–2975. (37) Kawahara K, Dejsirilert S, Ezaki T. Characterization of three capsular polysaccharides produced by Burkholderia pseudomallei. FEMS Microbiol Lett. 1998;169:283–287. (38) Masoud 

H, Ho M, Schollaardt T, Perry MB. Characterization of the capsular polysaccharide of Burkholderia pseudomallei 304b. J Bacteriol. 1997;179:5663–

5669. (39) Nimtz M, Wray V, Domke T, Brenneke B, Haussler S, Steinmetz I. Structure of an acidic exopolysaccharide of Burkholderia pseudomallei. Eur J Biochem. 1997;250:608–616. (40) Steinmetz I, Nimtz M, Wray V, Haussler S, Reganzerowski A, Brenneke B. Exopolysaccharides 

of Burkholderia pseudomallei. Acta Trop. 2000;74:211–214. (41) Matsuura M, Kawahara K, Ezaki T, Nakano M. Biological activities of lipopolysaccharide of Burkholderia (Pseudomonas) pseudomallei. FEMS Microbiol Lett. 1996;137:79–83. (42) Brett PJ, DeShazer D, Woods DE. Characterization of Burkholderia pseudomallei and Burkholderia pseudomallei-like strains. Epidemiol Infect. 1997;118:137–148. (43) Kespichayawattana W, 

Rattanachetkul S, Wanun T, Utaisincharoen P, Sirisinha S. Burkholderia pseudomallei induces cell fusion and actin-associated membrane 

protrusion: a possible mechanism for cell-to-cell spreading. Infect Immun. 2000;68:5377–5384. (44) Stevens MP, Stevens JM, Jeng RL, et al. 

Identification of a bacterial factor required for actin-based motility of Burkholderia pseudomallei. Mol Microbiol. 2005;56:40–53. (45) Breitbach 

K, Rottner K, Klocke S, et al. Actin-based motility of Burkholderia pseudomallei involves the Arp 2/3 complex, but not N-WASP and Ena/VASP 

proteins. Cell Microbiol. 2003;5:385–393. (46) Balder R, Lipski S, Lazarus JJ, et al. Identification of Burkholderia mallei and Burkholderia pseudomallei adhesins for human respiratory epithelial cells. BMC Microbiol. 2010;10:250. (47) Campos CG, Borst L, Cotter PA. Characterization of 

BcaA, a putative classical autotransporter protein in Burkholderia pseudomallei. Infect Immun. 2013;81:1121–1128. (48) Lazar Adler NR, Dean 

RE, Saint RJ, et al. Identification of a predicted trimeric autotransporter adhesin required for biofilm formation of Burkholderia pseudomallei. 

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Burkholderia pseudomallei toxin, connects inhibition of host protein synthesis with melioidosis. Biochem Soc Trans. 2012;40:842–845.

Table 9-1 continued

clinical presentation, occurring in 278 (51%) of the 

patients.144 Patients with acute pulmonary melioidosis 

present with cough, fever, sputum production, and 

respiratory distress, and can present with or without 

shock. Chronic pulmonary melioidosis mimics tuberculosis, with side effects including purulent sputum 

production, cough, hemoptysis, and night sweats. 

Patients with the acute septic form of melioidosis 

present characteristically with a short history of 

fever and no clinical evidence of focal infection. 

Most patients are profoundly ill with signs of 

sepsis. Septic shock may appear on presentation. 

In the Darwin Prospective Melioidosis Study, 298 

(55%) of patients were bacteremic on presentation 

to the hospital. Septic shock, usually occurring on, 

or within 24 hours of, admission to the hospital, 

was associated with 50% mortality, while bacteremia without septic shock was associated with a 7% 

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Figure 9-3. Autopsy specimen demonstrating extensive 

pulmonary involvement with abscess formation due to B 

pseudomallei.

Photograph courtesy of Bart Currie, MD, Royal Darwin 

Hospital, Australia.

mortality.144 Hematogenous seeding and abscess 

formation can occur in any organ (Figure 9-4); 

however, liver, spleen, skeletal muscle, prostate, 

and kidney are the most common abscess sites 

(Figures 9-5 and 9-6).27

Less common presentations of melioidosis include 

uncomplicated infections of the skin (Figure 9-7), 

subcutaneous tissues, or the eye. Corneal ulcerations 

resulting from trauma, which become secondarily 

infected with B pseudomallei, are rapidly destructive.145

Septic arthritis and osteomyelitis (Figure 9-8) have 

also been described, but cellulitis appears to be rare. 

In a prospective study of more than 2,000 patients 

with melioidosis in Thailand, primary meningitis 

Figure 9-4. Pustules with an erythematous base due to septicemic melioidosis. 

Photograph courtesy of Bart Currie, MD, Royal Darwin 

Hospital, Australia.

or endocarditis was not observed, but meningitis 

secondary to cerebral abscess rupture and mycotic 

aneurysms was seen.27 Other unusual melioidosis 

presentations include mediastinal masses, pericardial 

fluid collections, and adrenal abscesses. The clinical 

presentation of melioidosis also varies among different regions. In Thailand, 30% of the melioidosis cases 

in children present as acute suppurative parotitis.136

These children present with fever, pain, and swelling over the parotid (salivary) gland without other 

evidence of underlying predisposing conditions. In 

10% of the cases, the swelling is bilateral.27 Although 

acute suppurative parotitis is unusual in Australia, 

approximately 4% of the melioidosis cases there present as brainstem encephalitis with peripheral motor 

Figure 9-5. Computed tomography scan showing multiloculated liver abscess. 

Photograph courtesy of Bart Currie, MD, Royal Darwin 

Hospital, Australia.

Figure 9-6. Computer tomography scan showing prostatic 

abscess. 

Photograph courtesy of Bart Currie, MD, Royal Darwin 

Hospital, Australia.

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weakness or flaccid paraparesis. Features associated with this presentation include limb weakness, 

cerebellar signs, and cranial nerve palsies. Patients 

with this syndrome usually have an initial normal 

state of consciousness. Multiple focal B pseudomallei 

micro abscesses in the brainstem and spinal cord are 

probably responsible for this syndrome.27

Although acute infections in individuals with predisposing risk factors are the most common, latent 

infection with reactivation, resulting in an illness that 

can resemble tuberculosis, also occurs with melioidosis. During the Vietnam War, large numbers of Western 

soldiers were exposed to B pseudomallei through inhalation, contaminated wounds, or burns. A serologic survey of US military personnel demonstrated that mild 

or unapparent infection was common, and estimated 

that 225,000 people with subclinical infection were 

potentially at risk for reactivation.146 Fortunately, the 

number of cases of reactivation melioidosis in these 

individuals has remained rare compared to the number 

of individuals exposed. Long latency periods between 

exposure and development of melioidosis in nonendemic regions have been reported.70 Recently, a case 

of cutaneous melioidosis in a man taken prisoner by 

the Japanese during World War II was described. This 

man is presumed to have had reactivated melioidosis 

62 years after exposure, as he had not returned to an 

area of melioidosis endemicity after being imprisoned 

in northwest Thailand.69

Diagnosis

Because of its protean clinical manifestations, the 

diagnosis of melioidosis depends on the isolation 

and identification of B pseudomallei from clinical 

specimens. Melioidosis should be suspected in any 

severely ill, febrile patient with an associated risk 

factor who has been in an endemic area. B pseudomallei can grow on most routine laboratory media 

and can be isolated from normally sterile sites, such 

as blood, by standard techniques.23 The organism is 

usually detected in blood culture within 48 hours. 

Isolator centrifugation blood culture systems result 

in quicker detection times, but are less sensitive compared to conventional broth-based blood culture.147

Ashdown’s medium, a crystal violet and gentamicincontaining medium that permits selective growth 

of B pseudomallei (see Figure 9-1), has been used to 

significantly increase the frequency of recovery of B 

pseudomallei from the rectum, wounds, and sputum 

compared to recovery on blood and MacConkey 

agars.50 Patients with suspected melioidosis should 

submit blood, sputum, urine, and abscess fluid, as 

well as throat wound and rectal swabs for culture.

B pseudomallei is intrinsically resistant to aminoglycosides and polymyxins.148,149 This unusual antibiotic 

profile (gentamicin and colistin resistance, but amoxicillin-clavulanate susceptibility) in an oxidase-positive, 

gram-negative bacillus is helpful for identifying B 

pseudomallei in the microbiology laboratory. Commercially available kits for bacterial identification, such 

as the API 20NE (bioMérieux, Marcy l’Etoile, France), 

have been reported to reliably confirm the identity 

of B pseudomallei,

49 although other investigators have 

reported mixed results.150 The Vitek 1 (bioMérieux) has 

also been found to be highly sensitive, having identified 99% of the 103 B pseudomallei isolates tested.151

Figure 9-7. Skin lesions associated with melioidosis on the 

lower extremity. 

Photograph courtesy of Bart Currie, MD, Royal Darwin 

Hospital, Australia.

Figure 9-8. Chronic osteomyelitis of the lower extremity due 

to melioidosis. 

Photograph courtesy of Bart Currie, MD, Royal Darwin 

Hospital, Australia.

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Melioidosis

However, in this same study, the Vitek 2 (bioMérieux) 

identified only 19% of these same isolates.151 Other 

studies continue to document difficulties in identifying B pseudomallei with the Vitek 2.152,153 It has also 

been recently reported that B pseudomallei specimens 

from infections that occurred in exotic locations, such 

as Malaysian Borneo, were misidentified as B cepacia

by the Vitek 2.154

Serologic testing alone is not a reliable method 

of diagnosis. An indirect hemagglutination test and 

other serologic tests may produce false negatives in 

patients with sepsis, as well as false positives due to 

a high prevalence of antibodies to B pseudomallei in 

healthy individuals from endemic areas.143 A recently 

published paper from Australia proposed a highly 

sensitive B pseudomallei identification algorithm that 

makes use of screening tests (Gram stain, oxidase test, 

gentamicin, and polymyxin susceptibility testing) combined with monoclonal antibody agglutination testing 

and gas-liquid chromatography analysis of bacterial 

fatty acid methyl esters.155 Various polymerase-chainreaction–based identification techniques have also 

been developed to aid in the identification of B pseudomallei.

156,157 A recent comparison of the sensitivities and 

specificities of seven different real-time TaqMan (Life 

Technologies, Grand Island, New York) polymerase 

chain reaction assays for detecting B pseudomallei demonstrated that an assay targeting the type III secretion 

system (TTS1-ofr2) performed the best at detecting B 

pseudomallei directly from clinical samples.158

Treatment

Asymptomatic carriage of B pseudomallei appears 

to be a very rare event159; therefore, the isolation of B 

pseudomallei from a clinical specimen indicates that treatment is required. Melioidosis requires prolonged antibiotic therapy to cure the infection and prevent relapse. 

Melioidosis cases should be treated with initial intensive 

therapy (at least 2 weeks of intravenous [IV] therapy) 

followed by oral eradication therapy for a minimum of 

3 months. Cases presenting with localized mild disease 

can be treated with oral eradication therapy without 

initial parenteral treatment. The choice of therapy for 

treating melioidosis is complicated because B pseudomallei is resistant to many antibiotics, including penicillin, 

ampicillin, aminoglycosides, first- and second-generation cephalosporins, and colistin.160,161 B pseudomallei 

is also relatively insensitive to quinolones and macrolides162; therefore, therapeutic options are limited. 

The first study demonstrating the effectiveness of 

ceftazidime for severe melioidosis was published in 

1989. In this study, ceftazidime treatment (120 mg/

kg/day) was associated with a reduction of overall 

mortality from 74% to 37% (P = 0.009) when compared 

to “conventional therapy” with chloramphenicol (100 

mg/kg/day), doxycycline (4 mg/kg/day), trimethoprim 

(TMP; 10 mg/kg/day), and sulfamethoxazole (SMX; 50 

mg/kg/day).163 In 1992, a second randomized clinical 

trial of treatment of severe melioidosis conducted in 

Thailand also demonstrated a substantial reduction in 

mortality when ceftazidime plus TMP-SMX was used, 

as compared to the four-drug conventional therapy.164

In 1999, a comparative treatment trial in Thailand 

found that imipenem/cilastatin was as effective as 

ceftazidime for the treatment of severe melioidosis. 

Although there was no difference in mortality, fewer 

treatment failures were observed in the patients given 

imipenem/cilastatin as compared to the ceftazidime 

group.165 Therefore, initial intensive therapy for melioidosis should consist of high doses of ceftazidime (50 

mg/kg, up to 2 g IV every 6 hours), imipenem/cilastatin 

(25 mg/kg, up to 1 g IV every 6 hours), or meropenem 

(25 mg/kg, up to 1 g IV every 8 hours) combined with 

TMP-SMX (320 mg/1,600 mg IV or by mouth every 12 

hours) for patients with severe infection involving the 

brain, prostate, or other privileged site, for at least 14 

days.166 Critically ill patients with extensive pulmonary 

disease, organ abscesses, osteomyelitis, septic arthritis, 

or neurological melioidosis require longer intensive IV 

therapy (4 weeks or even longer).

The benefit of adding TMP-SMX to the initial 

antimicrobial regimen is supported by animal data 

and expert opinion.26 However, a 2005 paper from 

Thailand, which described two randomized controlled 

trials comparing ceftazidime alone versus ceftazidime 

combined with TMP-SMX for severe melioidosis, 

failed to demonstrate a mortality benefit associated 

with the addition of TMP-SMX, although the dose 

of TMP-SMX used in this study appears to be lower 

than that used in Australia.167 Nonetheless, patients 

in the Royal Darwin Hospital in the Northern Territory of Australia with severe melioidosis involving 

the brain, bone, prostate or other sequestered site, or 

severe pulmonary disease are treated with meropenem 

and TMP-SMX (Bart Currie, MD, Royal Darwin Hospital, Australia, written communication, April 2013). 

Meropenem is used rather than imipenem/cilastatin 

because it has fewer neurological side effects.162

The median time to resolution of fever is 9 days, but 

patients with large abscesses or empyema often have 

fluctuating fevers lasting a month or more. In a 10-year 

prospective review of 252 melioidosis cases in Australia, internal organ abscesses were common, with the 

largest majority found in the prostate. Although other 

internal collections frequently resolve with medical 

therapy, prostatic abscesses usually require drainage 

to prevent treatment failures.137

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Adjunctive therapy with recombinant granulocyte 

colony-stimulating factor (G-CSF) is routinely used for 

melioidosis patients with septic shock in the Northern 

Territory of Australia. A retrospective review of mortality rates before and after the addition of G-CSF therapy 

at the Royal Darwin Hospital was recently published. 

In this study, the introduction of G-CSF as adjunctive 

therapy for patients with septic shock was associated 

with a decrease in mortality from 95% to 10%.168 However, a randomized controlled clinical trial of G-CSF 

for treating melioidosis sepsis in Thailand failed to 

demonstrate a significant mortality benefit to the group 

that received it in addition to ceftazidime. The authors 

pointed out that the resource-constrained environment 

where the trial took place (limited ventilator and inotropic support, no invasive monitoring, no dialysis) 

may have introduced confounding variables.169

After initial intensive therapy, oral maintenance 

therapy is given for another 12 to 20 weeks to prevent 

disease relapse.13 Oral maintenance therapy traditionally consists of chloramphenicol (40 mg/kg/day), 

doxycycline (4 mg/kg/day), and TMP-SMX (10 mg/50 

mg/kg/day).170 However, this combination frequently 

causes side effects that result in problems with compliance. Some experts recommend high-dose TMP-SMX 

(8 mg/40 mg/kg, up to 320/1,600 mg, by mouth twice 

daily) combined with doxycycline.142 The combination of TMP-SMX with doxycycline was recently 

shown to be as effective and better tolerated than the 

conventional four-drug regimen (chloramphenicol, 

doxycycline, and TMP-SMX) for maintenance therapy 

in an open-labeled randomized trial conducted in 

Thailand.171 In this study, failure to complete at least 12 

weeks of maintenance therapy was the most important 

determinate of relapse. In the Northern Territory of 

Australia, TMP-SMX (8 mg/40 mg/kg) given every 12 

hours is used as monotherapy for maintenance for at 

least 3 to 6 months, with a low relapse rate (1 failure 

in fewer than 60 patients).13,137 It should be noted, 

however, that improvements in rates of relapse, from 

6.4% (prior to September 2003) to 1.2% (after September 2003) in the Darwin Prospective Melioidosis 

Study have been attributed to the improved used of 

efficacious antimicrobials as well as a lengthened IV 

treatment phase for complex cases.172 A recent, multicenter, double-blind, randomized placebo-controlled 

trial comparing the efficacy of 20 weeks of TMP-SMX 

plus placebo to TMP-SMX plus doxycycline for oral 

maintenance therapy was recently published. This 

trial, which enrolled 626 patients from five hospitals 

in northeast Thailand, demonstrated that TMP-SMX 

was noninferior to TMP-SMX plus doxycycline for the 

oral phase of melioidosis treatment. Adverse drug reactions were less common in the TMP-STX group than 

in the TMP-SMX plus doxycycline group, suggesting 

that TMP-STX monotherapy is preferred on the basis 

of safety and patient tolerance.173

Although evidence suggests that it is associated 

with a higher rate of relapse, amoxicillin-clavulanate 

can be used for oral maintenance therapy in individuals with a sulfonamide allergy or in pregnant 

patients. The recommended dose of oral amoxicillinclavulanate is 20 mg/5 mg/kg, three times a day—a 

dose of amoxicillin-clavulanate that is higher than 

usually prescribed.174

Quinolone antibiotics are not recommended as 

therapy for eradicating B pseudomallei. Ciprofloxacin 

and ofloxacin were found inferior, with a failure rate 

of 29%, when compared to a 20-week course of maintenance therapy consisting of amoxicillin-clavulanate 

or the combination of chloramphenicol, doxycycline, 

and TMP-SMX.175 Another study also found that the 

combination of ciprofloxacin plus azithromycin was 

associated with an unacceptably high rate of relapse.176

Prevention

Several experimental melioidosis vaccines have 

been tested in rodent models of infection, including 

live attenuated vaccines, heterologous vaccines, acellular vaccines, and subunit vaccines.177–180 Variability 

in vaccination protocols, routes of challenge, and animal models make it difficult to directly compare the 

experimental melioidosis vaccine studies published. In 

general, most vaccine candidates provided significant 

protection compared to unvaccinated controls, but 

none resulted in 100% protection and sterilizing immunity. Several recent comprehensive reviews thoroughly 

describe the melioidosis vaccine candidates that have 

been developed to date.178–180

Live attenuated vaccines have been shown to be immunogenic and protective against a variety of facultative intracellular pathogens, including Mycobacterium 

tuberculosis, Shigella, Salmonella, Yersinia, Listeria monocytogenes, Francisella tularensis, and Brucella melitensis.181–185

B pseudomallei purine auxotrophic mutants generated 

by ultraviolet and chemical mutagenesis were highly 

attenuated in mice and provided significant protection 

against subsequent challenge with virulent strains.186,187

Unfortunately, the molecular nature of the purinedependent mutations in these strains was unknown, 

and the possibility of reversion to wild-type could not be 

eliminated. A B pseudomallei temperature-sensitive mutant (chemically induced) and a branched-chain amino 

acid auxotroph (transposon mutant) were also tested as 

live attenuated vaccines and provided significant protection in mice against challenge with virulent strains.186,188

Vaccination of mice with an attenuated strain harboring 

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Melioidosis

a suicide plasmid disruption of bipD, a gene encoding a 

type III secretion system translocation protein, resulted 

in partial protection against challenge with wild-type 

B pseudomallei.189 In contrast, vaccination with purified 

bipD protein did not significantly protect this animal 

model.189 More recently, a highly attenuated B pseudomallei purM mutant (strain Bp82) was evaluated as 

a live attenuated vaccine and shown to provide mice 

significant protection against an intranasal challenge 

with wild-type B pseudomallei.

190 These studies suggest 

that live attenuated vaccines are promising candidates 

for melioidosis vaccines; however, strains with defined 

deletion mutations would be preferred to prevent the 

possibility of reversion to wild-type. 

Iliukhin et al vaccinated guinea pigs with live B 

thailandensis strains and protected less than 50% of the 

animals challenged with 200 times the LD50of wildtype B pseudomallei.191 B thailandensis and B pseudomallei 

produce identical lipopolysaccharide (LPS) O-antigens 

and contain immunologically related secreted and cellassociated antigens,112,113,192,193 which probably account 

for the protection that B thailandensis affords. The B 

pseudomallei exopolysaccharide and capsular polysaccharide (CPS; see Table 9-1) are not produced by most 

B thailandensis strains, and both polysaccharides may 

be necessary for full protection against challenge with 

B pseudomallei. Recently, B thailandensis strains (eg, 

E555) that express the CPS have been identified and 

tested as vaccine candidates.194,195 A study by Scott et 

al showed that immunization with E555 conferred 

significant protection against a lethal intraperitoneal 

challenge of B pseudomallei in mice.195 Live attenuated 

F tularensis strains were also tested as heterologous 

vaccine candidates against melioidosis in rodents.186,196

Attenuated F tularensis strains did afford some protection against challenge with virulent B pseudomallei. 

A crude acellular melioidosis vaccine was produced 

to protect captive cetaceans at Ocean Park in Hong 

Kong.197 The vaccine consisted of a protein-polysaccharide mixture (1:3), and it significantly protected 

hamsters against experimental challenge with virulent 

B pseudomallei. The acellular vaccine reduced melioidosis mortality in cetaceans from 45% to less than 1%.197

Unfortunately, the exact chemical components of the 

vaccine were not well characterized, leaving a high 

probability of lot-to-lot variation. A naturally derived 

outer membrane vesicle vaccine has been developed 

and tested in BALB/c mice and shown to provide significant protection against a lethal B pseudomallei aerosol challenge.198 In addition, studies have described the 

testing of purified protein antigens, including LolC 

(ABC transporter protein), PotF (periplasmic binding protein), OppA (oligopeptide-binding protein), 

BimA (autotransporter protein), BopA (T3SS effector 

protein), and Hcp1 (T6SS-1 component) as potential 

vaccine candidates.124,199,200 Significant protection was 

conferred by several of these protein antigens; sterilizing immunity was not.

In a study by Nelson et al, mice were actively immunized with purified B pseudomallei CPS or LPS and 

challenged with virulent B pseudomallei by the intraperitoneal or aerosol route.201 The LPS-vaccinated mice 

exhibited an increased mean time to death relative to 

controls, and 50% of the mice survived for 35 days 

after intraperitoneal challenge. By comparison, mice 

vaccinated with the purified CPS had an increased 

mean time to death, but 100% of the vaccinated mice 

were dead by day 28.201 Neither of the subunit vaccines provided substantial protection against a lethal 

aerosol challenge, probably because B pseudomallei 

appears to be more virulent by this route of infection.102,135 Improved subunit vaccines that generate 

both humoral and cell-mediated immune responses 

are probably necessary to protect against infection 

with B pseudomallei.202

Several studies have shown that CPS- and LPSspecific monoclonal antibodies (mAbs) can passively 

protect animals against challenge with B pseudomallei.

203–205 The bactericidal and opsonophagocytic activities associated with various anti-LPS or anti-CPS 

mAbs correlated with their protective capacity in 

mice.203 CPS and LPS have also been shown to be major components of an outer membrane vesicle vaccine 

that provided significant protection in mice.198 Taken 

together, such findings indicate that these surfaceexposed carbohydrates are protective antigens and 

support the rationale for developing LPS- and CPSbased vaccines for immunization against melioidosis. 

Both CPS- and OPS-based glycoconjugates have been 

produced and shown to be capable of eliciting hightiter, carbohydrate-specific antibody responses; however, the protective capacity of these subunit vaccines 

remains to be reported.206,207

There is no licensed vaccine available to prevent 

human melioidosis and no definitive evidence that infection with B pseudomallei confers immunity, because 

reinfection with a different strain of B pseudomallei has 

occurred after successful melioidosis treatment.21 The 

only proven method of disease prevention for individuals with known risk factors is avoiding B pseudomallei 

in the environment. Recommendations for postexposure prophylaxis (PEP) following a laboratory exposure or bioterrorism event involving B pseudomallei are 

complicated by the lack of efficacy data from either 

clinical studies or animal experiments. Recommendations are therefore based on expert consensus opinion 

from physicians who frequently treat melioidosis cases 

and with data extrapolated from clinical studies on oral 

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SUMMARY

 Melioidosis, a disease caused by the saprophytic 

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biosynthetic pathway, is attenuated and protective in a murine model of melioidosis. Infect Immun. 2002;70:5290–5294.

189. Stevens MP, Haque A, Atkins T, et al. Attenuated virulence and protective efficacy of a Burkholderia pseudomallei bsa

type III secretion mutant in murine models of melioidosis. Microbiology. 2004;150:2669–2676.

190. Silva EB, Goodyear A, Sutherland MD, et al. Correlates of immune protection following cutaneous immunization with 

an attenuated Burkholderia pseudomallei vaccine. Infect Immun. 2013;81:4626–4634.

191. Iliukhin VI, Senina TV, Plekhanova NG, Antonov VA, Merinova LK, Seimova IK. Burkholderia thailandensis: biological 

properties, identification and taxonomy (in Russian). Mol Gen Mikrobiol Virusol. 2002;1:7–11.

192. Heiss C, Burtnick MN, Black I, Azadi P, Brett PJ. Detailed structural analysis of the O-polysaccharide expressed by 

Burkholderia thailandensis E264. Carbohydr Res. 2012;363:23–28.

193. Heiss C, Burtnick MN, Roberts RA, Black I, Azadi P, Brett PJ. Revised structures for the predominant O-polysaccharides 

expressed by Burkholderia pseudomallei and Burkholderia mallei. Carbohydr Res. 2013;381:6–11.

194. Sim BM, Chantratita N, Ooi WF, et al. Genomic acquisition of a capsular polysaccharide virulence cluster by nonpathogenic Burkholderia isolates. Genome Biol. 2010;11:R89.

195. Scott AE, Laws TR, D’Elia RV, et al. Protection against experimental melioidosis following immunization with live 

Burkholderia thailandensis expressing a manno-heptose capsule. Clin Vaccine Immunol. 2013;20:1041–1047.

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196. Iliukhin VI, Plekhanova NG, Senina TV, Stanovaia OV, Kislichkin NN. Experimental study on the possibility of using 

live tularemia vaccine to increase resistance to heterologous infection disease (in Russian). Zh Mikrobiol Epidemio Immunobiol. 2004;2:38–42.

197. Vedros NA, Chow D, Liong E. Experimental vaccine against Pseudomonas pseudomallei infections in captive cetaceans. 

Dis Aquat Org. 1988;5:157–161.

198. Nieves W, Asakrah S, Qazi O, et al. A naturally derived outer-membrane vesicle vaccine protects against lethal pulmonary Burkholderia pseudomallei infection. Vaccine. 2011;29:8381–8389.

199. Harland DN, Chu K, Haque A, et al. Identification of a LolC homologue in Burkholderia pseudomallei, a novel protective antigen for melioidosis. Infect Immun. 2007;75:4173–4180.

200. Whitlock GC, Deeraksa A, Qazi O, et al. Protective response to subunit vaccination against intranasal Burkholderia 

mallei and B. pseudomallei challenge. Procedia Vaccinol. 2010;2:73–77.

201. Nelson M, Prior JL, Lever MS, Jones HE, Atkins TP, Titball RW. Evaluation of lipopolysaccharide and capsular polysaccharide as subunit vaccines against experimental melioidosis. J Med Microbiol. 2004;53:1177–1182.

202. Healey GD, Elvin SJ, Morton M, Williamson ED. Humoral and cell-mediated adaptive immune responses are required for protection against Burkholderia pseudomallei challenge and bacterial clearance postinfection. Infect Immun. 

2005;73:5945–5951.

203. Zhang S, Feng SH, Li B, et al. In vitro and in vivo studies of monoclonal antibodies with prominent bactericidal activity against Burkholderia pseudomallei and Burkholderia mallei. Clin Vaccine Immunol. 2011;18:825–834.

204. AuCoin DP, Reed DE, Marlenee NL, et al. Polysaccharide specific monoclonal antibodies provide passive protection 

against intranasal challenge with Burkholderia pseudomallei. PLoS One. 2012;7:e35386.

205. Bryan LE, Wong S, Woods DE, Dance DA, Chaowagul W. Passive protection of diabetic rats with antisera specific 

for the polysaccharide portion of the lipopolysaccharide isolated from Pseudomonas pseudomallei. Can J Infect Dis. Jul 

1994;5:170–178.

206. Burtnick MN, Heiss C, Schuler AM, Azadi P, Brett PJ. Development of novel O-polysaccharide based glycoconjugates 

for immunization against glanders. Front Cell Infect Microbiol. 2012;2:148.

207. Burtnick MN, Heiss C, Roberts RA, Schweizer HP, Azadi P, Brett PJ. Development of capsular polysaccharide-based 

glycoconjugates for immunization against melioidosis and glanders. Front Cell Infect Microbiol. 2012;2:108.

208. Peacock SJ, Schweizer HP, Dance DA, et al. Management of accidental laboratory exposure to Burkholderia pseudomallei

and B. mallei. Emerg Infect Dis. 2008;14:e2.

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Plague

Chapter 10

PLAGUE

PATRICIA L. WORSHAM, PhD*; THOMAS W. McGOVERN, MD, FAAD, FACMS, KHS†; NICHOLAS J. VIETRI, MD‡; 

ARTHUR M. FRIEDLANDER, MD§; and JOEL BOZUE, PhD¥

INTRODUCTION

HISTORY

The Justinian Plague (The First Pandemic)

The Black Death (The Second Pandemic)

The Third Pandemic

PLAGUE AND WARFARE

Endemic Disease

Plague as a Biological Warfare Agent

THE INFECTIOUS AGENT

Taxonomy

Morphology

Growth Characteristics

Biochemistry

EPIDEMIOLOGY

INCIDENCE

VIRULENCE DETERMINANTS

Type III Secretion System

F1 Antigen

Other Virulence Factors in the Mammalian Host

Virulence and Transmission Factors in the Flea

PATHOGENESIS

CLINICAL MANIFESTATIONS

Bubonic Plague

Septicemic Plague

Pneumonic Plague

Plague Meningitis

Pharyngeal and Gastrointestinal Plague

Cutaneous Manifestations

DIAGNOSIS

Signs and Symptoms

Laboratory Confirmation

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Medical Aspects of Biological Warfare 

TREATMENT

Isolation

Antibiotics

Prevention

Postexposure Prophylaxis

Vaccination

SUMMARY

*Chief, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

†

Major, Medical Corps,US Army (former); Dermatologist/Mohs Surgeon, Fort Wayne Dermatology Consultants, 7881 Carnegie Boulevard, Fort Wayne, 

Indiana 46804; formerly, Ward Officer, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

‡

Colonel, Medical Corps, US Army; Infectious Diseases Officer, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 

1425 Porter Street, Fort Detrick, Maryland 21702; Assistant Professor of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones 

Bridge Road, Bethesda, Maryland 20814

§

Colonel (Retired), Medical Corps, US Army; Senior Scientist, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort 

Detrick, Maryland 21702, and Adjunct Professor of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, 

Maryland 20814

¥

Microbiologist, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

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249

Plague

INTRODUCTION

plague was endemic among the native population, but 

US soldiers were relatively unaffected. The protection 

of troops was largely attributable to the US military’s 

understanding of the rodent reservoirs and flea vectors of disease, the widespread use of a plague vaccine during the war, and prompt treatment of plague 

victims with effective antibiotics. Mortality from 

endemic plague continues at low rates throughout the 

world despite the availability of effective antibiotics. 

Deaths resulting from plague occur not because the 

bacilli have become resistant but, most often, because 

physicians do not include plague in their differential 

diagnosis, or because treatment is absent or delayed. 

To be best prepared to treat plague in soldiers who 

are affected by endemic disease or a biological agent 

attack, military healthcare providers must understand 

the natural mechanisms by which plague spreads 

between species, the pathophysiology of disease in 

humans, and the diagnostic information necessary to 

begin treatment with effective antibiotics. No vaccine 

is available for plague in the United States, although 

candidates are undergoing clinical trials.

The US military’s concern with plague is both as 

an endemic disease and as a biological warfare threat. 

A better understanding of the preventive medicine 

aspects of the disease will aid in the prompt diagnosis 

and effective treatment necessary to survive an enemy 

attack of plague and protect military and civilian 

personnel in plague endemic areas where military 

operations are underway.

Key terms in this chapter include enzootic and 

epizootic. Enzootic refers to when plague is present 

in a small number of animals; the host, vector, and 

bacterium live in an apparent equilibrium in which 

mortality among rodent hosts is difficult to detect and 

not obviously resulting from plague. During an epizootic, which typically follows a longer period of enzootic 

maintenance, widespread plague infections frequently 

lead to death among susceptible host populations (ie, 

equivalent to an epidemic in a human population), an 

event that is often noticeable to residents in affected 

areas. The death of a rodent causes the living fleas to 

leave that host and seek other mammals, and when 

those other mammals die in large numbers, they may 

seek humans. Knowledge of these two concepts of 

enzootic and epizootic will help to clarify how and 

when humans may be infected, in endemic or biological warfare scenarios.

Plague, a severe febrile illness caused by the gramnegative bacterium Yersinia pestis, is a zoonosis usually transmitted by fleabites. It is foremost a disease 

of rodents; more than 200 species of mammals have 

been reported to be infected with Y pestis, but maintenance of plague in nature relies almost exclusively on 

a smaller number of rodent species and their fleas.1,2

Humans most often become infected by fleabites during an epizootic event; less frequently they are exposed 

to blood or tissues of infected animals (including ingestion of raw or undercooked meat) or aerosol droplets 

containing the organism.1,3 Humans or animals with 

plague pneumonia, particularly cats, can generate infectious aerosols.4,5 The resulting primary pneumonic 

plague is the most severe and frequently fatal form of 

the disease. Pneumonic plague is of particular concern 

to the military because it can also be acquired from 

artificially generated aerosols.

In the 6th, 14th, and 20th centuries Y pestis was the 

cause of three great pandemics of human disease. The 

bubonic form of the disease in humans is characterized 

by the abrupt onset of high fever, painful local lymphadenopathy draining the exposure site (ie, a bubo, the 

inflammatory swelling of one or more lymph nodes, 

usually in the groin, axillary, or cervical regions; the 

confluent mass of nodes, if untreated, may suppurate), 

and bacteremia. Septicemic plague can ensue from 

untreated bubonic plague or by passage of bacteria directly into the bloodstream bypassing the local lymph 

nodes. Patients with the bubonic and septicemic forms 

of the disease may develop secondary pneumonic 

plague, which can lead to human-to-human spread by 

the respiratory route. Cervical lymphadenitis has been 

noted in several human plague cases, including many 

fatal cases, and is often associated with the septicemic 

form of the disease. However, it is possible that these 

patients were exposed by the oral/aerosol route and 

developed pharyngeal plague that progressed into 

a systemic infection and cervical lymphadenitis.1,6–8

According to Dr Kenneth Gage with the Centers for 

Disease Control and Prevention (CDC) laboratory in 

Fort Collins, Colorado, in an email in 2006, cervical 

lymphadenopathy, which is more common in patients 

from developing countries, may result from fleabites 

on the neck or face while sleeping on the dirt floors of 

heavily flea-infested buildings.

During the past 4 millennia, plague has played a role 

in many military campaigns. During the Vietnam War, 

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Medical Aspects of Biological Warfare 

HISTORY

The Justinian Plague (The First Pandemic)

Procopius gave us the first identifiable description 

of epidemic plague in his account of the plague of 

the Byzantine Empire during the reign of Justinian I 

(541–542 ce [the common era]), which we now consider to be the first great pandemic of the Common 

Era.9 At the height of the epidemic, more than 10,000 

people died each day. As many as one hundred million 

Europeans died, including 40% of Constantinople’s 

population.10,11 Repeated, smaller epidemics followed 

this plague into the 8th century.12 Recently, Y pestis was 

definitively determined to have caused this pandemic 

based on extraction of plague-specific DNA from the 

dental pulp of plague victim skeletons in Germany. 

DNA fingerprinting strongly suggests China as the 

source of this First Pandemic.13

The Black Death (The Second Pandemic)

The second plague pandemic, known as the Black 

Death, brought the disease into the collective memory 

of Western civilization.12 Plague bacilli probably entered Europe via the trans-Asian Silk Road during 

the early 14th century in fleas on the fur of marmots 

(a rodent of the genus Marmota). When bales of these 

furs were opened in Astrakhan and Saray, hungry fleas 

jumped from the fur seeking the first available blood 

meal, often a human leg.12,14,15 In 1346, plague arrived 

in Caffa (modern Feodosiya, Ukraine) on the Black Sea. 

Caffa’s large rat population helped spread the disease 

as they were carried on ships bound for major European ports such as Pera, a suburb of Constantinople, 

and Messina, in Sicily. By 1348, plague had already 

entered Great Britain at Weymouth.9

The Black Death probably killed 24 million people 

between the years 1346 and 1352 and perhaps another 

20 million by the end of the 14th century.10 However, 

some believe that the plague persisted through 1750, 

following a final foray into Marseilles in 1720. During 

the 15th through the 18th centuries, 30% to 60% of 

the populations of major cities, such as Genoa, Milan, 

Padua, Lyons, and Venice, died of plague.15 Debate 

has raged for decades about the cause of the Black 

Death. Some believed that a viral hemorrhagic fever 

or unknown agent caused the Black Death instead of Y 

pestis. Y pestis was only recently proven to have caused 

the Black Death based on finding Y pestis DNA and F1 

capsular antigen in the dental pulp of plague victims 

buried in mass graves in England, France, Germany, 

Italy, and the Netherlands, and later characterization 

of the genome of a Y pestis strain in DNA extracted 

from the skeletal material in a cemetery where Black 

Death victims had been buried in England.16

Failing to understand the plague’s epidemiology 

and etiology, physicians could offer no effective treatment. Physicians at the University of Paris theorized 

that a conjunction of the planets Saturn, Mars, and 

Jupiter at 1:00 pm on March 20, 1345, corrupted the 

surrounding atmosphere, which led to the plague.10

Physicians recommended a simple diet; avoidance of 

excessive sleep, exercise, and emotion; regular enemas; 

and abstinence from sexual intercourse.17 Although 

some people killed cats and dogs because they were 

thought to carry disease, rats seemed to escape attention.10 Christians blamed plague on Muslims, Muslims 

blamed it on Christians, and both Christians and Muslims blamed it on Jews or witches.12

In 1666, a church rector in Eyam, Derbyshire, England, persuaded the whole community to quarantine 

itself when plague erupted, but this was the worst possible solution because the people then remained close 

to the infected rats and/or fleas. The city experienced 

virtually a 100% attack rate with 72% mortality. The 

average mortality for the Black Death was consistently 

70% to 80%.12,18

Contemporary observers such as Giovanni Boccaccio (1313–1375), who wrote in his Decameron, provided 

accurate clinical descriptions of the Black Death:

The symptoms were not the same as in the East, where 

a gush of blood from the nose was a plain sign of inevitable death, but it began both in men and women 

with certain swellings [buboes] in the groin or under 

the armpit. They grew to the size of a small apple or 

an egg, more or less, and were vulgarly called tumours. In a short space of time these tumours spread 

from the two parts named all over the body. Soon after this, the symptoms changed and black or purple 

spots appeared on the arms or thighs or any other 

part of the body, sometimes a few large ones, sometimes many little ones.19

Marchione di Coppo Stefano Buonaiuti (1327–

1385) wrote in his memoir about the Black Death in 

Florence: 

In the year of our Lord 1348 there occurred in the city 

and contado of Florence a great pestilence and such 

was its fury and violence that in whatever household 

it took hold, whosoever took care of the sick, all the 

carers died of the same illness, and almost nobody 

survived beyond the fourth day, neither doctors nor 

medicine proving to any avail . . . those symptoms 

were as follows: either between the thigh and the 

body, in the groin region, or under the armpit, there 

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Plague

appeared a lump, and a sudden fever, and when the 

victim spat, he spat blood mixed with saliva, and 

none of those who spat blood survived. Such was 

the terror this caused that seeing it take hold in a 

household, as soon as it started, nobody remained: 

everybody abandoned the dwelling in fear, and fled 

to another; some fled into the city and others into the 

countryside . . . sons abandoned fathers, husbands 

wives, wives husbands, one brother the other, one 

sister the other. The city was reduced to bearing the 

dead to burial . . ..20

Some writers described bizarre neurological disorders (which led to the term “dance of death”), followed 

by anxiety and terror, resignation, blackening of the 

skin, and death. The sick emitted a terrible stench: 

“Their sweat, excrement, spittle, breath, [were] so 

foetid as to be overpowering” [in addition, their urine 

was] “turbid, thick, black, or red.”10

The second great pandemic slowly subsided in 

Europe by 1750. The pandemic’s decline has been 

attributed to the replacement of the black rat (Rattus 

rattus) in the area by the Norwegian rat (Rattus norvegicus), which is a less efficient host; natural vaccination 

of animals and/or humans by other Yersinia species or 

by less virulent Y pestis strains; and other less plausible 

hypotheses. The theories are all flawed to some extent, 

and the disappearance of plague from Europe remains 

one of the great epidemiological mysteries.3,8,21

It is not known why plague spread so easily during the First and Second Pandemics and had such a 

high case-fatality rate. Based on an analysis of Y pestis

genomes from Black Death victims, no significant differences existed between 14th century and 21st century 

strains of the bacterium.22 The environment, vector 

dynamics, and host susceptibility likely contributed 

to the disease spread. Although there are legitimate 

reasons to question the Oriental rat flea’s role in parts 

of northern Europe where it and black rats appeared 

absent, the human flea’s role in transmitting plague 

to humans in these regions probably has been underemphasized, and could explain the rapid humanto-human spread during the Black Death.23–25 It also 

is possible that human lice became vectors during 

pandemics. Even though lice are not normal vectors, 

they are capable of transmitting Y pestis infection in 

rabbits, and presumably humans.26

The Third Pandemic

The third, or modern, plague pandemic arose in 

1894 in China and spread throughout the world as rats 

and their fleas traveled via modern transportation.12,18

In 1894, both Shibasaburo Kitasato and Alexandre JE 

Yersin independently discovered the plague bacillus. 

However, it was Yersin who was able to satisfy Koch’s 

postulates for the bubonic plague and his plague bacillus fits the characterization of Y pestis.

6

 The reservoir 

of plague bacilli in the fleas of the Siberian marmot 

was likely responsible for the Manchurian pneumonic 

plague epidemic of 1910 through 1911, which caused 

50,000 deaths.27 The modern pandemic arrived in Bombay in 1898, and during the next 50 years, more than 

13 million Indians died of rat-associated plague.27–29 

The disease officially arrived in the United States in 

March 1900, when a plague-infected Chinese laborer’s 

lifeless body was discovered in a hotel basement in 

San Francisco, California. The disease subsequently 

appeared in New York City and Washington state the 

same year.30,31 The disease appeared in New Orleans, 

Louisiana, in 1924 and 1926.31 The Texas Gulf Coast 

and Pensacola, Florida, also saw the influx of plague. 

Among these numerous events, only the outbreaks 

in California appear to have spread inland, leading 

to the establishment of permanent plague foci in native rodent and flea populations in the interior of the 

western United States.32,33 Human plague in the United 

States was initially a result of urban rat and ground 

squirrel epizootics until 1925. After general rat control 

and hygiene measures were instituted in various port 

cities, urban plague vanished—only to spread into rural areas, where virtually all cases in the United States 

have been acquired since 1925.34 Rodents throughout 

the western United States were probably infected from 

the San Francisco focus.

PLAGUE AND WARFARE

It is an axiom of warfare that battle casualties are 

much fewer than casualties caused by disease and 

nonbattle injuries.35 Y pestis can initiate disease both 

through exposure to natural sources of infection, such 

as fleabites, and to a biological warfare agent. Medical 

officers need to distinguish cases likely acquired from 

natural sources in a plague-endemic region from those 

occurring following exposure to a biological warfare 

agent. 

Endemic Disease

Plague has also afflicted armies in more recent times. 

In 1745, Frederick the Great’s troops were devastated 

by plague. Catherine the Great’s troops returned from 

the Balkans with plague in 1769 through 1771. French 

military operations in Egypt were significantly impeded by plague in 1798, which caused them to abandon 

their attack on Alexandria. The modern pandemic 

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252

Medical Aspects of Biological Warfare 

began in China when its troops were deployed in an 

epidemic plague area to suppress a Muslim rebellion. 

Military traffic is responsible for the rapid plague 

spread to nearly every country in Asia.27

Endemic plague has not been a source of disease 

and nonbattle injuries for the US military since the mid 

20th century. During World War II and the Vietnam 

War, US military forces were almost free of plague, 

although civilian populations in certain areas were 

subjected to plague outbreaks in both of these wars. 

However, plague remains on and near military bases 

in the southwestern United States because the local 

mammal populations are reservoirs of infection, and it 

may be found in many areas around the world where 

US military forces are deployed.

World War II

Endemic plague became established in Hawaii (on 

the islands of Hawaii and Maui) in December 1899. 

However, no evidence of the disease in either rodents 

or humans has been found on Oahu or Kauai since the 

first decade of the 20th century. A “small outbreak” 

occurred during World War II on the island of Hawaii 

(in 1943), but it was contained by strict rat control 

measures, which prevented any plague spread to military personnel during the war in the Pacific.36 Official 

policy during World War II was to vaccinate US troops 

with the whole-cell killed plague vaccine. No troops 

contracted plague despite serving in known endemic 

areas.36,37 Plague has since disappeared from Hawaii.

Vietnam War

Plague entered Vietnam in Nha Trang in 1898 and 

several pneumonic epidemics have occurred since 

then.27,38,39 Cases were reported in Vietnam every year 

from 1898 to 2002, except during the Japanese occupation during World War II.27,40 When French forces 

departed Vietnam after the Indochina War, public 

health conditions deteriorated and plague flourished. 

The reported plague incidence increased from eight 

cases in 1961 to 110 cases in 1963, and to an average 

of 4,500 cases from 1965 through 1969.34,41–44 The mortality in clinically diagnosed cases was between 1% 

and 5%. In untreated individuals, it was much higher 

(60%–90%).27,42 However, only eight American troops 

were affected (one case per 1 million human-years) 

during the Vietnam War.44 The low infection rate in the 

US troops was attributed to insecticide use, vaccination 

of virtually all troops, and a thorough understanding of plague’s epidemiology, which led to the insect 

repellent use, protective clothing, and rat-proofed 

dwellings.27,42 During this period, knowledge of plague 

grew dramatically, in large part due to the work of 

two officers of the US Army Medical Service Corps, 

Lieutenant Colonel Dan C Cavanaugh and Lieutenant Colonel John D Marshall. These scientists studied 

plague ecology, related plague epidemics to weather, 

described the effects of high temperatures (>28°C) on 

the abilities of fleas to transmit plague, developed 

serologic tests for plague infection, and significantly 

contributed to the field of plague vaccinology.27,45 

Disease Threat on US Military Installations

Human exposure to plague on military installations 

may occur when pets bring home infected rodents or 

fleas, at recreation areas with sick or dead rodents and 

their infected fleas, or at field training and bivouac 

sites. The consequences of plague at a military installation include morbidity and mortality of both humans 

and pets; loss of training and bivouac sites; large 

expenditures of money, personnel, and equipment to 

eliminate the plague risk; and the loss of recreation 

areas.34 Plague risk has been identified on and near 

several US military installations (Exhibit 10-1). For a 

description of relevant rodent/flea complexes found 

in the United States, see the Epidemiology section.

Plague as a Biological Warfare Agent

The first known attempt at what is now called 

“biological warfare” is purported to have occurred at 

the Crimean port city of Caffa on the Black Sea in 1346 

and 1347.10,27 During the conflict between Christian 

Genoese sailors and Muslim Tatars, the Tatar army 

was struck with plague. The Tatar leader catapulted 

corpses of Tatar plague victims at the Genoese sailors. 

The Genoese became infected with plague and fled to 

Italy. However, the disease was most likely spread by 

the local population of infected rats, not by the corpses, 

because an infected flea leaves its host as soon as the 

corpse cools.10

The 21st century use of plague as a potential biological warfare weapon is the immediate concern of 

this chapter. Medical officers need to consider this 

use of plague, particularly if the disease appears in 

an unlikely setting.

World War II

During World War II, Japan established a secret 

biological warfare research unit (Unit 731) in Manchuria, where pneumonic plague epidemics occurred 

from 1910 through 1911, 1920 through 1921, and 1927; 

a cholera epidemic also spread in 1919. General Shiro 

Ishii, the physician leader of Unit 731, was fascinated 

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253

Plague

by plague because it could create casualties disproportionate to the number of bacteria disseminated, 

the most dangerous strains could be used to make a 

lethal weapon, and its origins could be concealed to 

appear as a natural occurrence. Early experiments, 

however, demonstrated that aerial bomb dropping of 

bacteria had little effect because air pressure and high 

temperatures created by the exploding bombs killed 

nearly 100% of the bacteria.46

One of Ishii’s more frightening experiments was his 

use of the human flea, Pulex irritans, as a stratagem to 

simultaneously protect the bacteria and target humans. 

This flea is resistant to air drag, naturally targets humans, and can infect a local rat population to prolong 

an epidemic. Spraying fleas from compressed-air containers was not successful because high-altitude release 

resulted in too much dispersion and aircraft had to 

fly low for safety. However, clay bombs solved these 

technical difficulties and resulted in an 80% survival 

rate of fleas.46

At 5:00 am on a November morning in 1941, a lone 

Japanese plane made three low passes over the business center of Changteh, a city in the Hunan province. 

This area of China was not a plague endemic area. 

Although no bombs were dropped, a strange mixture 

of wheat and rice grains, pieces of paper, cotton wadding, and other unidentified particles were observed 

falling from the plane. Within 2 weeks, individuals in 

this same area began dying of plague. No individual 

who contracted plague had recently traveled outside 

Changteh. Unlike the zoonotic form of the disease that 

is typically observed, rat mortalities were not noted 

until months after the human cases. It was also observed that plague usually spreads with rice shipments 

(because rats infest the grain) along shipping routes, 

but the nearest plague epizootic was 2,000 km away by 

land or river. Furthermore, Changteh exported—rather 

than imported—rice. These unusual circumstances 

surrounding the plague outbreak suggest that it may 

have been of deliberate human origin.46

EXHIBIT 10-1

PLAGUE RISKS AT US MILITARY INSTALLATIONS*

Plague-infected animals on the installation; human case 

reported on post:

Fort Hunter Liggett, California

US Air Force Academy, Colorado†

Human case reported in the same county:

Edwards Air Force Base, Colorado‡

FE Warren Air Force Base, Wyoming

Kirtland Air Force Base, New Mexico§

Peterson Air Force Base, Colorado

Plague-infected animals on the installation:

Dugway Proving Ground, Utah

Fort Carson, Colorado

Fort Ord, California

Fort Wingate Army Depot Activity, New Mexico

Marine Corps Mountain Warfare Training Center, 

Bridgeport, California

Navajo Army Depot Activity, Arizona

Pueblo Army Depot Activity, Colorado

Rocky Mountain Arsenal, Colorado

Vandenberg Air Force Base, California

White Sands Missile Range, New Mexico

Plague-infected animals or fleas in the same county but 

not on the installation:

Bridgeport Naval Facility, California

Camp Roberts, California

Dyess Air Force Base, Texas

Fort Bliss, Texas

Fort Lewis, Washington

Sierra Army Depot, California

Tooele Army Depot, Utah

Umatilla Army Depot Activity, Oregon

Nellis Air Force Base, Nevada

No plague-infected animals or fleas on the installation or 

in the county, but susceptible animals present:

Fort Huachuca, Arizona

*Does not include military installations near Los Angeles and San Francisco, California, where urban plague cases and deaths were 

common in the first quarter of the 20th century; no plague cases have occurred in these urban areas since the mid 1920s.

†

Fatality: 18-month-old child died of pneumonic plague; rock squirrels and their fleas had taken up residence in the ducts of the 

child’s on-base house.

‡

Two human cases in the same county in 1995; animal surveillance on base began in 1996.

§

Plague-infected animals in the county in 1995; last human case in the county in 1993; no animal surveillance on base since 1986.

Data sources: (1) Harrison FJ. Prevention and Control of Plague. Aurora, CO: US Army Center for Health Promotion and Preventive 

Medicine, Fitzsimons Army Medical Center; September 1995: 3–8. Technical Guide 103. (2) Data collected from Preventive Medicine 

Officers on 30 military bases in the United States, March 1996.

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In another incident, on October 4, 1940, a Japanese 

plane dropped rice and wheat grains mixed with 

fleas over the city of Chuhsien, in Chekiang province. 

In November, bubonic plague appeared for the first 

time in the area where the particles had been dropped. 

Plague caused 21 deaths in 24 days. On October 27, 

1940, a Japanese plane was seen releasing similar particles over the city of Ningpo, in Chekiang province. 

Two days later, bubonic plague occurred for the first 

time in that city, resulting in 99 deaths in 34 days. No 

epizootic disease or excessive mortality was found in 

the rat population.46

Since World War II 

During the Korean War, allied forces were accused 

of dropping on North Korea insects that were capable 

of spreading plague, typhus, malaria, Japanese B encephalitis, and other diseases. However, no evidence 

exists to support such claims.47

In 1999, Dr Ken Alibek (Kanatjan Alibekov), a 

former Soviet army colonel and scientist, published 

a book titled Biohazard that illuminates the former 

Soviet Union’s extensive biological weapons program.48 Alibek describes the weaponization of Y pestis

(including a powdered form) and the development 

of genetically engineered organisms, one of which 

was a Yersinia strain producing “myelin toxin” that 

induced both disease and paralysis in animal models. 

Alibek states that “In the city of Kirov, we maintained 

a quota of twenty tons of plague in our arsenal every 

year.”48 Although the accuracy of details presented 

in the memoir has been debated in some circles, the 

former Soviet Union had entire institutes devoted to 

the study of Y pestis. 

Other state-sponsored or extremist group efforts to 

obtain Y pestis will likely occur. For example, in 1995, 

a white supremacist and microbiologist fraudulently 

purchased vials of lyophilized Y pestis from the American Type Culture Collection.49,50 The intended use of 

these organisms was never determined but it caused 

alarm and led to legislation requiring that the transfer 

of disease causing pathogens be reported (CDC Select 

Agent Program).49,50 

THE INFECTIOUS AGENT

Taxonomy

Y pestis, the causative agent of plague, is a gramnegative coccobacillus belonging to the family Enterobacteriaceae. The genus was named in honor of Alexandre Yersin, the scientist who originally isolated Y pestis

during a plague outbreak in Hong Kong in 1894; the 

species name pestis is derived from the Latin for plague 

or pestilence. Previous designations for this species 

have included Bacterium pestis, Bacillus pestis, Pasteurella pestis, and Pesticella pestis.

51 This species is closely 

related to two other pathogens of the genus Yersinia: 

Y pseudotuberculosis and Y enterocolitica. The extensive 

genetic similarity (>90%) between Y pseudotuberculosis

and Y pestis led to a recommendation that Y pestis be 

reclassified as a subspecies of Y pseudotuberculosis.

52

This proposal was not well received, primarily because 

of the fear that this change in nomenclature would 

increase the potential for laboratory-acquired plague 

infections. The most recent molecular fingerprinting 

analysis of Y pestis suggests that this pathogen arose 

from Y pseudotuberculosis through microevolution 

over the past few millennia, during which the enzootic “pestoides” isolates evolved (see Biochemistry 

on next page). The pestoides strains appear to have 

split from Y pseudotuberculosis more than 10,000 years 

ago, followed by a binary split approximately 3,500 

years later that led to the populations of Y pestis more 

frequently associated with human disease. The isolation of Y pestis “pestoides” from both Africa and Asia 

suggests that Y pestis spread globally long before the 

first documented plague (Justinian) in 784 ce.

53 Recent 

phylogenetic analyses suggest that among the Enterobacteriaceae, Y pestis is more closely related to insect and 

invertebrate-associated genera (such as Photorhabdus, 

Serratia, and Sodalis) than to vertebrate-associated 

genera like Escherichia and Salmonella.

54

Morphology

The characteristic “safety pin” bipolar staining of 

this short bacillus (0.5–0.8 μm by 1.0–3.0 μm) is best 

seen with Wayson’s or Giemsa stain (Figure 10-1). 

Depending on growth conditions, Y pestis can exhibit 

marked pleomorphism with rods, ovoid cells, and 

short chains present. A gelatinous capsule, known 

as the F1 antigen, is produced by the vast majority of 

strains at 37°C. Y pestis is nonmotile, unlike the other 

mammalian pathogens of the genus that produce 

peritrichous flagella at growth temperatures lower 

than 30°C.

51,55

Growth Characteristics

Y pestis can grow at a broad range of temperatures (4°C–40°C) in the laboratory, with an optimal 

growth temperature of 28°C. Although Y pestis

grows well on standard laboratory media, such as 

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Plague

and the loss of one or more virulence plasmids.21,51,55

Strains to be archived should be grown at a low 

temperature and frozen promptly at –70°C.

Biochemistry

Y pestis is a facultative anaerobe, fermenting glucose with the production of acid. It is incapable of a 

long-term saprophytic existence, partly because of 

complex nutritional requirements, including a number 

of amino acids and vitamins. Y pestis also lacks certain 

enzymes of intermediary metabolism that are functional in the closely related but more rapidly growing 

species such as Y enterocolitica or Y pseudotuberculosis. 

Y pestis strains have traditionally been separated into 

three biovars, based on the ability to reduce nitrate 

(Nit+) and ferment glycerol (Gly+).21 Some molecular 

methods of typing, such as ribotyping and restriction 

fragment-length polymorphisms of insertion sequence 

locations, support this division of strains.56,57 Biovar 

orientalis (Gly-

, Nit+), which is distributed worldwide 

and is responsible for the third (modern) plague pandemic, is the only biovar present in North and South 

America. Biovar antiqua (Gly+, Nit+), which is found 

in Central Asia and Africa, may represent the most ancient of the biovars.21,53 Biovar mediaevalis (Gly+

/Nit-) 

is geographically limited to the region surrounding the 

Figure 10-1. (a) This Wright-Giemsa stain of a peripheral 

blood smear from a patient with septicemic plague demonstrates the bipolar, safety-pin staining of Yersinia pestis. 

Gram’s and Wayson’s stains can also demonstrate this pattern. (b) Micrograph of the CO92 strain of Y pestis stained 

with Wayson’s stain and examined by microscopy (original 

magnification × 100). 

Photographs: (a) Courtesy of Kenneth L Gage, PhD, Centers 

for Disease Control and Prevention Laboratory, Fort Collins, 

Colorado. (b) Courtesy of Joel Bozue, PhD, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, 

Maryland. 

a

b

sheep blood agar, MacConkey agar, or heart infusion agar, growth is slower than that of Y pseudotuberculosis or Y enterocolitica; more than 24 hours of 

incubation are required to visualize even pinpoint 

colonies. Appearance of colonies can be hastened 

by growth in an environment containing 5% carbon 

dioxide. The round, moist, translucent, or opaque 

colonies are nonhemolytic on sheep blood agar and 

exhibit an irregular edge (Figure 10-2). A fried egg 

appearance is common in older colonies and is more 

pronounced in certain strains. Long-term laboratory 

passage of Y pestis or short-term growth under less 

than optimal conditions is associated with irreversible genetic changes leading to attenuation. These 

changes include the deletion of a large chromosomal 

pathogenicity island that encodes factors necessary 

for growth in both the flea and the mammalian host 

Figure 10-2. Growth of the CO92 strain of Yersinia pestis

grown on a 5% sheep blood agar plate following 2 days of 

incubation at 28°C. 

Photograph: Courtesy of Joel Bozue, PhD, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, 

Maryland. 

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Medical Aspects of Biological Warfare 

Caspian Sea. No apparent differences in pathogenicity 

exist among the biovars.21,58 Recently, three different 

multilocus molecular methods were used to investigate 

the microevolution of Y pestis.

56 Eight populations were 

recognized. An evolutionary tree for these populations 

rooted on Y pseudotuberculosis was proposed. The 

eight population groups do not correspond directly to 

the biovars; thus, it was suggested that future strain 

groupings be rooted in molecular typing. Four of the 

groups were made up of transitional strains of Y pestis, 

“pestoides,” which exhibit biochemical characteristics 

of both Y pestis and Y pseudotuberculosis.

59 These isolates represent the most ancient of the Y pestis strains 

characterized to date.53

Figure 10-3. The oriental rat flea (Xenopsylla cheopis) has 

historically been most responsible for plague spread to 

humans. This flea has a blocked proventriculus (indicated 

at the arrow), the equivalent to a human’s gastroesophageal 

region. In nature, this flea would develop a ravenous hunger 

because of its inability to digest the fibrinoid mass of blood 

and bacteria. The ensuing biting of the nearest mammal will 

clear the proventriculus through regurgitation of thousands 

of bacteria into the bite wound, thereby inoculating the mammal with the plague bacillus. 

Photograph: Courtesy of Kenneth L Gage, PhD, Centers for 

Disease Control and Prevention Laboratory, Fort Collins, 

Colorado. 

EPIDEMIOLOGY

During the modern pandemic, WG Liston, a member of the Indian Plague Commission (1898–1914), 

associated plague with rats and identified the rat flea 

as a vector.27 Subsequently, more than 200 species of 

mammals and 150 species of fleas have been implicated 

in maintaining Y pestis endemic foci throughout the 

world, although only a relatively few species play a 

significant role in disease transmission and maintaining plague in nature.34,60,61 Y pestis is not capable of 

establishing lasting infections in all flea species, and 

there appears to be variability in the ability of various 

flea species to transmit the organism.58

The oriental rat flea (Xenopsylla cheopis) has been 

largely responsible for spreading Y pestis during bubonic plague epidemics. Some researchers think it is 

the most efficient flea for transmitting plague.9 However, this long-held belief has recently been challenged. 

After the flea ingests a blood meal from a bacteremic 

animal, bacilli can multiply and eventually block the 

flea’s foregut, or proventriculus, with a biofilm mass 

containing bacteria as shown in Figure 10-3. When 

feeding, the flea ingests approximately 0.1 μl to 0.3 μl 

of blood during a blood meal. High-level bacteremia 

is a hallmark of Y pestis infection in susceptible hosts. 

This bacteremia provides a sizeable inoculum for the 

flea and promotes the subsequent infection, and in 

the case of X cheopis it likely leads to blockage of the 

proventriculus. This blockage limits feeding, resulting in repeated desperate attempts by the flea to feed. 

Because of the blockage, blood carrying Y pestis is 

regurgitated into the bite wounds, thus spreading the 

disease to new hosts. The blocked flea, also a victim 

of the disease, eventually starves to death.2

 As many 

as 24,000 organisms may be inoculated into the mammalian host.27 This flea species desiccates rapidly in 

hot and dry weather when away from its hosts, but 

flourishes at humidity just above 65% and temperatures between 20°C and 26°C; in these conditions it can 

survive 6 months without a feeding.27,34

Interestingly, the belief that feeding by infectious 

blocked fleas represents the only means by which Y 

pestis can be transmitted efficiently has recently been 

challenged as a result of experiments with unblocked 

Oropsylla fleas.24 These fleas rarely become blocked and 

were shown to transmit Y pestis better. The infected 

Oropyslla montana fleas became infectious within a 

day after feeding and remained infectious for at least 

4 days. This timeframe is much shorter than the 2 

weeks required for flea blockage to occur and allows 

O montana and certain other fleas to support the rapid 

plague spread during epizootics, even in the absence 

of proventricular blockage. 

Although the largest plague outbreaks have been 

associated with X cheopis, all fleas should be considered dangerous in plague-endemic areas.2,60 During 

the Black Death, the human flea, Pulex irritans, may 

have aided in human-to-human plague spread; during other epidemics, bedbugs (Cimex lectularius), lice, 

and flies were found to contain Y pestis.

9,62 However, 

the presence of plague bacilli in these latter insects is 

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Plague

associated with ingestion of contaminated blood from 

plague victims, and they apparently had little or no 

role as vectors for the disease, although some have 

recently suggested lice may be important in some 

situations.63 In one laboratory study, bedbugs were 

able to infect guinea pigs after feeding on a moribund 

Y pestis-infected mouse.64 The most important vector of 

human plague in the United States is O montana, often 

the most common flea on rock squirrels and California 

ground squirrels,34 although cases have been linked to 

infectious bites of other flea species, including those 

found on other ground squirrels, prairie dogs, chipmunks, and wood rats. 

Throughout history, the black rat, Rattus rattus, 

has been most responsible worldwide for plague’s 

persistence and spread in urban and most rural village 

epidemics. R rattus is a nocturnal, climbing animal that 

does not burrow, but instead nests overhead and lives 

close to humans.9 In the United Kingdom and much 

of Europe, the brown rat, R norvegicus, has replaced 

R rattus as the dominant city rat.65 Unlike R rattus, R 

norvegicus is essentially a burrowing animal that lives 

under farm buildings and in ditches. Although often 

considered less important than R rattus as a source 

of Y pestis infection, R norvegicus may be involved in 

both rural and urban plague outbreaks.9 Most carnivores, except wild and domestic cats, are resistant to 

plague infection, but animals such as domestic dogs, 

all rodents, and burrowing owls may transport infected fleas into homes. However, in some instances 

individuals of normally resistant species, such as dogs, 

can experience serious illness.66 For example, in 2009 

a pneumonic plague outbreak occurred in China. The 

index case, which was a herdsman who contracted the 

disease from a sick dog,67 was unique because it was 

the first known pneumonic plague case attributed to 

an infected dog (generally they are considered naturally resistant). Mammals that are partially resistant to 

plague infection (ie, consist of a mixture of individuals that are either resistant or susceptible to plagueinduced mortality) are continuous plague reservoirs. 

Some epidemiologists propose that the true plague 

hosts are rodent species with populations consisting of 

both sensitive and resistant individuals, while others 

have questioned the need for resistant individuals to 

maintain plague foci.68 In the United States, Cynomous 

species (prairie dogs) and Spermophilus species (rock 

squirrels and ground squirrels) are most often associated with plague activity because of the high mortality 

they often experience during epizootics. A variety of 

susceptible mammals, such as chipmunks, tree squirrels, cottontail rabbits, ferrets, and domestic cats, are 

occasionally infected. Epizootic spread among tree 

squirrels in Denver, Colorado, in the 1960s resulted in 

the first urban plague case since the 1920s.60 A more 

recent epizootic in Denver, Colorado, also involving 

tree squirrels, occurred in the summer of 2007.69

Although not associated with any human plague 

cases, the appearance of two infected fox squirrels in 

Dallas, Texas, in 1993 also caused considerable concern.69,70 An increasing number of human infections has 

been associated with domestic cats, usually through 

bites, contact with tissues, suppurating buboes, or 

aerosol rather than by flea transmission.4,5 Cats appear 

to be particularly efficient at transmitting disease to 

humans.4,5

Highly susceptible animals amplify both flea populations and bacilli within their bloodstreams and often 

support the spread of epizootics, especially when these 

animals occur at high densities.71 In many developing 

countries, these epizootics often involve commensal 

rat species (Rattus) and potential human exposure to 

infectious rat fleas. In the United States, such epizootics occur in chipmunks, ground squirrels, and wood 

rats, but especially in prairie dogs, rock squirrels 

(Spermophilus variegatus), and California ground squirrels (Spermophilus beechyi). Although prairie dog fleas 

rarely bite humans, they have been sources of infection 

for humans, who can acquire the disease by handling 

infected prairie dogs. Rock squirrels and California 

ground squirrels both infect humans via direct contact 

and fleas.34,72,73 Many other mammals in the United 

States harbor plague, and a few, including wild carnivores, have served as infection sources for humans 

(Exhibit 10-2). In 2007, a National Park Service wildlife 

biologist died of primary pneumonic plague resulting from Y pestis, likely contracted from a necropsy 

EXHIBIT 10-2

MAMMALS KNOWN TO HARBOR 

PLAGUE IN THE UNITED STATES

Carnivores Black bears, cats (including bobcats 

and mountain lions), coyotes, dogs, 

foxes, martens, raccoons, skunks, 

weasels, wolverines, wolves

Rodents Chipmunks, gophers, marmots, mice, 

prairie dogs, rats, squirrels, voles

Lagomorphs Hares, rabbits

Hooved Stock Pigs, mule deer, pronghorn antelope

Adapted from Harrison FJ. Prevention and Control of Plague. 

Aurora, CO: US Army Center for Health Promotion and 

Preventive Medicine, Fitzsimons Army Medical Center; 

September 1995: 25–28. Technical Guide 103.

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Medical Aspects of Biological Warfare 

performed on a dead mountain lion found within 

Grand Canyon National Park.74 Thinking that the 

mountain lion died from trauma, the biologist did 

not protect himself while removing the lion’s skin and 

skull while performing the necropsy in his garage.

In the United States, where human plague cases are 

likely to be associated with exposures to native rodents 

and their fleas rather than rats and rat fleas, knowledge of local host species is critical because certain 

mammal-flea complexes are particularly dangerous: 

these complexes consist of both a susceptible mammal genus or species and one or more associated fleas 

known to bite humans. More than one host-epizootic 

complex can occur in a given area. These pairings can 

include the following34,61:

• the rock squirrel (S variegatus) of the Southwest and the California ground squirrel (S 

beechyi) of California and bordering regions 

that both are host to the flea O montana, which 

is known to readily bite humans;

• the antelope ground squirrel (Ammospermophilus leucurus) and the flea Thrassus bacchi of the 

Southwest;

• the prairie dogs (Cynomys species) of the Great 

Plains, Rocky Mountains, and Colorado plateau region and the flea Opisochrostis hirsutus;

• the Wyoming ground squirrel (Spermophilus 

elegans, formerly known as S richardsoni) 

of Colorado and Wyoming or the goldenmantled ground squirrel (S lateralis) of the 

Rocky Mountains and Sierras, and the fleas 

Opisochrostis labis, Opisochrostis idahoensis, or 

Thrassus bacchi; and

• various wood rat species (Neotoma sp) found 

across the West and the fleas Orchopeas sexdentatus and Orchopeas neotomae.

Plague exists in one of two states in nature: enzootic 

or epizootic. An enzootic cycle is a stable rodent–flea 

infection cycle presumably occurring in a relatively 

resistant host population that experiences low mortality. Enzootic maintenance cycles also appear to occur 

in some areas in more susceptible populations when 

the animals occur in separate patches or colonies and 

transmission among them is delayed to some extent 

by geographical barriers, seasonal changes, or other 

means.68,75 In an enzootic cycle, rodent mortality is 

limited and the fleas have less need to seek alternative 

hosts, such as humans. During an epizootic, however, plague bacilli also infect moderately or highly 

susceptible mammals, and infections spread quickly 

causing high mortality. High mortality occurs, most 

conspicuously in larger colonial rodents, such as 

prairie dogs, but it can occur among animals of the 

relatively resistant rodent populations presumed to 

be involved in the enzootic cycle, although this tends 

to be less noticeable.1

 These epizootics are most common when host populations are dense. Evidence has 

been presented that epizootics and the frequency of 

human cases are influenced not only by host density 

but also by climatic variables.75 Humans are accidental 

hosts in the plague cycle and are not necessary for the 

persistence of the organism in nature (Figure 10-4). 

Humans typically acquire plague via infectious

bites of fleas whose natural host is another mammal, 

usually a rodent. Infection via flea feces inoculated 

into skin with bites may also occur, but this mode of 

transmission is not considered important compared to 

direct inoculation of the plague bacilli into the feeding 

site through the flea’s mouthparts. Less common infection sources include infectious human fleas, contact 

with tissues or body fluids from an infected animal, 

consumption of infected tissues, handling of contaminated pelts, and respiratory droplet transmission from 

animals with pneumonic disease.1,3,27,60,61 Fleas removed 

by humans during the grooming behavior practiced in 

some cultures are sometimes killed when the person 

doing the grooming bites the flea, which can squirt the 

flea’s gut contents and viable Y pestis into the mouth 

and pharynx, an act that has been implicated in some 

cases of plague.62 The greatest risk to humans occurs 

when large concentrations of people live under unsanitary conditions in close proximity to large commensal 

or wild rodent populations that are infested with fleas 

that bite both humans and rodents.27 

Figure 10-4. This drawing shows the usual, occasional, and 

rare routes by which plague has spread between various 

mammals and humans. 

Courtesy of William Discher, US Army Medical Research 

Institute of Infectious Diseases, Visual Information Office, 

Fort Detrick, Maryland.

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Plague

Human-to-human plague transmission can occur 

from patients with pulmonary infection and cough. 

However, the understanding of pneumonic plague 

is incomplete. Most large pneumonic epidemics have 

occurred in cool climates with moderate humidity 

and close contact between susceptible individuals. 

Pneumonic plague outbreaks have been rare in tropical climates even during bubonic disease. The role of 

particle size in efficiency of transmission is unknown, 

although it may occur more efficiently via larger 

respiratory droplets or fomites rather than via smallparticle aerosols.76

Only the pneumonic form of plague can spread 

between humans. The risk of person-to-person plague 

transmission via infectious respiratory droplets is 

lower than once believed. A pneumonic plague outbreak in Madagascar resulting from an index case with 

secondary pneumonic plague infected 18 individuals 

and killed 8 of them.77 However, once the outbreak’s 

cause was determined and appropriate measures were 

taken, including avoidance of severely ill persons with 

cough, no person developed infection. Of 154 contacts 

of these patients who understood the risk, only 8.4% 

(13/154) developed antibodies to F1 antigen; few were 

symptomatic and then only had pharyngitis.77 

Pneumonic plague patients typically transmit 

disease only several hours before death when they 

cough up copious amounts of bloody sputum full of 

bacteria, and then only to individuals who approach 

them within 1 to 2 meters.78 The initial pneumonic 

plague cough is dry. During the Manchurian pneumonic plague epidemics in the first half of the 20th 

century, prolonged and close contact with end-stage 

patients were necessary to transmit disease; layered 

cotton and gauze masks were effective transmission 

barriers.78 

A physician with 20 years of experience who cared 

for 400 to 500 patients with pneumonic plague reportedly has never seen a healthcare worker develop 

plague from these patients.78 This record has been 

attributed to maintaining well-ventilated wards, having patients cough away from healthcare workers 

during examinations, and limiting time spent close to 

patients. Most workers in this situation did not have 

protective masks. 

No human-to-human plague transmission cases 

have been documented after exposure to droplet nuclei 

(particles <10 microns), which linger for minutes to 

hours after coughing. All person-to-person transmission seems to be caused by airborne droplets (>10 

microns) released immediately during a cough; these 

droplets rapidly fall to the ground.77–79 High concentrations of aerosolized droplet nuclei that can transmit 

plague are used in the laboratory to infect experimental 

animals.80 Such small particle aerosols are of particular 

concern from a biological defense perspective. 

INCIDENCE

Under the 2007 revised International Health Regulations, the World Health Organization (WHO) member 

states are required to report human plague cases in 

suspected cases in areas not known to be endemic.81

The cases are then verified, which involves consulting 

an expert committee to confirm plague based on evidence and additional laboratory testing. Plague may 

be significantly underreported for several reasons, 

including the reluctance of some endemic countries 

to admit to public health problems, difficulties in diagnosis, and the absence of laboratory confirmation. 

Generally, the distribution of human plague coincides 

with the geographical distribution of its natural foci.82,83

In the first decade of the 21st century, 21,725 cases were 

reported worldwide; 1,612 patients died (7.4%), and 

97% of cases were in Africa.40,84

Plague is endemic in many countries in Africa, the 

former Soviet Union, the Americas, and Asia. From recent reports the Democratic Republic of the Congo had 

10,581 human plague cases followed by Madagascar 

(7,182), Zambia (1,309), and Uganda (972). The United 

States placed 11th with 57 cases, but at least one was 

reported every year of the decade.40,82,84 

Since the early 1990s, there have been increasing 

reports of plague in Africa. It is not clear whether this 

represents an increase of disease or an improvement of 

notification to WHO. However, for the Congo, the increase in human plague cases is attributed to civil wars, 

breakdown in health services, and a greater association 

of humans with rats.85–87 Recent plague resurgence in 

India, Indonesia, and Algeria during the past decade 

occurred after “silent” periods of years.82,84 Worldwide 

distribution of plague and its epidemiology can be 

found in the WHO’s Plague Manual (http://www.who.

int/csr/resources/publications/plague/). Recent reports 

of plague activity and occasional summaries of plague 

activity can be found at the websites for WHO’s Weekly 

Epidemiological Record (http://www.who.int/wer/en/) 

and the CDC Morbidity and Mortality Weekly Report 

(http://www.cdc.gov/mmwr/). Known foci of plague are 

shown in Figure 10-5. 

WHO reported 57 deaths among 130 suspected 

plague cases in the Democratic Republic of the Congo 

based on a retrospective analysis of cases since December 2004. The victims were employed as miners in a diamond mine at the time of the outbreak. All cases, except 

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Medical Aspects of Biological Warfare 

for two cases of the septicemic form, were reported as 

pneumonic plague. No evidence of bubonic disease was 

observed. WHO sent multidisciplinary health teams 

to investigate the potential outbreak, but no report 

has been issued since March 2005.88 The prevalence 

of pneumonic disease in this group of cases (assuming that this was plague) has not yet been explained.

Plague has been endemic in the continental United 

States since at least 1900 and now is permanently established from the High Plains on the eastern slope of the 

Rocky Mountains westward—especially in pine–oak or 

piñon–juniper woodland habitats at altitudes of 5,000 

to 9,000 feet, or on lower, dry grassland or desert scrub 

areas.2,30,34,61 In the first quarter of the 20th century, 

virtually all 432 cases and 284 deaths (65.7% mortality) in the United States occurred in urban port cities.34

Epidemics occurred in San Francisco, California, from 

1900 through 1904 (118 deaths) and from 1907 through 

1908 (78 deaths).34 The last time plague was transmitted 

between humans in the United States was during the 

1924–1925 pneumonic plague epidemic in Los Angeles, 

California. Eighty percent of cases since 1925 have been 

sylvatic, involving contact with wild-rodent habitats.31

Most cases (58%) are in men and occur within a 1-mile 

radius of home, and half of the US victims have been 

younger than 20 years old.31,34

Between 1926 and 1960, the United States averaged 

only one plague case per year. This number steadily 

rose to 3 per year during the 1960s, 11 during the 1970s, 

and 18 during the 1980s; then it decreased to 9 per year 

since 1990.72,73 Between 2000 and 2010, 57 cases were 

reported; the most active year was 2006 (17 cases).40,89

The number of states reporting human plague cases 

has steadily increased over the past 5 decades, most 

likely because increasing encroachment of humans on 

previously wild areas brings people closer to infected 

animals and their fleas.34 Most human plague cases 

are reported from New Mexico, Arizona, Colorado, 

and California.70,90 In 2002, a couple from New Mexico 

travelled to New York City and subsequently became 

ill with plague. However, the couple most likely 

acquired the infection in an endemic area because Y 

pestis was identified in the dead wood rats and fleas 

on their property. The bacterial strains recovered from 

the rats and fleas were indistinguishable from those of 

the infected couple.91 In addition, in 2010 two bubonic 

plague cases from the same household were reported 

in Oregon, the first in this state since 1995. Most likely, 

a pet dog’s fleas infected the patients. The dog was 

found to be seropositive.92

Epizootic cycles occur approximately every 5 

years; the last extremely widespread epizootic 

with a large die-off of rodents over multiple states 

(1982–1984) was accompanied by the highest number of humans infected with plague since the urban 

epidemics of the first quarter of the century.72,73 In 

2009, a fatal laboratory-acquired plague infection 

occurred by an unknown route with an attenuated 

Figure 10-5. Known worldwide foci of human plague infection as indicated by yellow. 

Data source: Epidemic Readiness and Interventions, Department of Epidemic and Pandemic Alert and Response, World 

Health Organization, Geneva, Switzerland.

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Plague

strain of Y pestis (KIM D27), which contains defects 

in the ability to acquire iron.93,94 However, the scientist was diagnosed portmortem with hemochromatosis. The possibility exists that the excess iron 

resulting from the condition may have compensated 

for the iron limitations of the attenuated strain and 

led to the septicemic infection.95 This hypothesis 

was further supported by a recent study that demonstrated virulence can be restored to a pgm-strain 

of Y pestis in a mouse model of hereditary hemochromatosis.95 Before the 2009 case, the last known 

laboratory-acquired infection in the United States 

was a pneumonic plague case that occurred in 1959 

with a virulent strain of Y pestis.

96

VIRULENCE DETERMINANTS

The persistence of plague in endemic areas requires 

cyclic transmission between rodents and fleas; thus, Y 

pestis has evolved to survive and replicate in two very 

different hosts. To maintain the transmission cycle, Y 

pestis must either be transmitted within the few days of 

the early phase period or multiply within the flea sufficiently to cause blockage and promote the infection of 

a new mammalian host. Equally critical is the ability to 

establish an infection and induce a sufficient bacteremia 

in the mammal to infect fleas during the blood meal. 

The milieu of the mammalian host is radically different 

from the flea’s midgut, yet, clearly, the organism successfully adapts to each host to complete its life cycle. 

The adaptation occurs through environmental regulation of virulence factors. For example, gene products 

necessary for growth in the flea are expressed most 

efficiently at the flea’s body temperature. Likewise, 

genes required for replication in the mammalian host 

are expressed at highest levels at 37°C, the internal body 

temperature of these animals; and the synthesis of some 

proteins, thought to be induced in the phagolysosome, 

is also regulated by pH. In the laboratory, the synthesis 

and secretion of certain essential virulence factors are 

controlled by both growth temperature and calcium

concentration; the induction of these proteins has been 

termed the low calcium response.2,21,97,98

Recent genetic analyses of Y pestis and the other 

pathogenic Yersiniae have begun to unravel the unique 

qualities that make Y pestis a successful pathogen in 

both the flea and the mammalian host. Most strains of 

Y pestis carry three plasmids, two of which are unique 

to this species: 

• pMT (or pFra), which encodes the F1 antigen 

“capsule”; and

• pPCP, which carries the gene for the virulence 

factor plasminogen activator. 

The third plasmid is common to the human pathogenic Yersiniae and is known as pCD (calcium dependence), pYV (Yersinia virulence), or pLcr (low calcium 

response). This plasmid, which is responsible for the 

synthesis of many antihost factors, is an absolute requirement for virulence.21

Type III Secretion System

Like a number of other gram-negative pathogens, 

the human pathogenic Yersinae possess a type III 

secretion system that enables an organism in close 

contact to host cells to deliver toxic proteins directly 

into the eukaryotic cell cytosol.97,99 In the case of 

the Yersinia species, this system is encoded on the 

pYV plasmid, which encodes the components of 

the low calcium response. Toxic activities of the 

low calcium response effector proteins, designated 

Yops (Yersinia outer protein), include disruption 

of the cytoskeleton, interference with phagocytic 

activity, prevention of proinflammatory cytokine 

synthesis, inhibition of the oxidative burst, and 

induction of programmed cell death (apoptosis). 

Yop delivery is necessary for growth of Y pestis in 

the liver and spleen.100 Specifically, YopM appears 

to induce a global depletion of natural killer cells. 

YopH, a protein tyrosine phosphatase, inhibits host 

cell phagocytosis by dephosphorylating several focal 

adhesive proteins and inhibiting calcium signaling 

in neutrophils. YopE, YpkA, and YopT are also antiphagocytic; these toxins inhibit cytoskeletal mobilization. YopJ plays an immunosuppressive role by 

inhibiting inflammatory cytokine production and 

inducing apoptosis in macrophages.97,101,102 Overall, 

the effect is that of paralyzing professional phagocytes. It is clear why the pathogen-host interaction 

mediated by the type III secretion system has been 

designated the “Yersinia Deadly Kiss.”102 

LcrV (historically known as V [or “virulence”] 

antigen), another virulence factor associated with the 

type III secretion system, is an important protective 

immunogen in new-generation plague vaccines. This 

protein serves many roles for the pathogen: 

• as regulator of Yop transcription; 

• for translocation of Yops into the host cell; and 

• as a virulence factor in its own right.21,101

LcrV appears to stimulate production of the antiinflammatory cytokine interleukin 10 through interactions with Toll-like receptors 2 and 6 as well as CD14 

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signaling. These effects appear to be mediated by the 

N-terminal portion of LcrV.102,103 Repression of proinflammatory cytokines is presumed to be a result of the 

interleukin 10 induction. In addition, LcrV released 

from the cell appears to interact directly with IFN-g

and may contribute to immunosuppression through 

this binding.104

The secretion mechanism includes an “injectisome” 

that can be visualized as a needle-like structure using 

electron microscopy. The type III secretion injectisome 

consists of a cylindrical basal structure spanning the 

two bacterial membranes and the peptidoglycan, connected to a hollow “needle.”105,106 The needle is tipped 

by a structure that allows formation of pores in the host 

cell membrane, and the length of the needle is governed 

by a protein deemed the “molecular ruler.” At body 

temperature, the secretion apparatus is synthesized 

on the outer surface of the bacterial cell. Contact with 

the host cell induces transcription of the Yops and 

opens this secretion channel that allows the Yops to be 

translocated through the membrane and into the host 

cell.105,106 YopK (also called YopQ) controls the rate of 

Yop injection from within the host cell.107 Under certain 

environmental conditions, proteins with adhesin activity (Ail, Pla, Psa) appear to facilitate Yop delivery.108

F1 Antigen

The F1 antigen, encoded by the largest plasmid of 

Y pestis (pMT), is produced in large quantities by Y 

pestis in vivo and when cultured in the laboratory at 

37°C. The F1 antigen structure has been described as 

both capsular- and fimbrial-like because it is composed 

of fibers that can be shed from the bacteria.109–112 This 

capsule-like polymer is generally thought to protect 

the organism from host phagocytic cells by interfering 

at the level of receptor interaction in the phagocytosis 

process.113 It likely acts in concert with the type III 

secretion system to provide Y pestis with protection 

from phagocytes. Although the vast majority of natural 

isolates produce the antigen, F1-negative strains have 

been isolated from rodent hosts and reportedly from 

one human case.110,114–116 In the laboratory, spontaneous mutants defective in F1 production have been 

obtained from immune animals, cultures treated with 

antiserum containing F1 antibody, and chronically 

infected rodents.114–116 Examination of isogenic F1-

positive/-negative strain pairs revealed that F1 is not 

an absolute requirement for virulence in the mouse 

and the African green monkey models, including 

aerosol models, although mutations leading to loss 

of the capsular antigen increase time to death in the 

mouse.114,117 However, a recent study demonstrated a 

F1 mutant in Y pestis was attenuated by bubonic and 

pneumonic (intranasal) models of infection depending 

on the strain of mouse.118 Older studies suggesting a 

role of F1 in the infection of guinea pigs and rats used 

F1-negative strains that were not genetically defined 

and, thus, are more difficult to interpret. However, 

these studies suggest that the importance of F1 in 

pathogenesis may vary with the species of the host. 

The fact that F1-negative strains are relatively rare 

among natural isolates suggests that the capsular antigen, or other gene products encoded by this plasmid, 

may play an important role in the maintenance of the 

disease in animal reservoirs. Historically, F1 has been 

important as a diagnostic reagent because it is specific 

to Y pestis.

110,119 It is the major antigen recognized in 

convalescent sera of humans and rodents,120,121 and also 

a highly effective protective immunogen.

Other Virulence Factors in the Mammalian Host

Plasminogen Activator

The virulence factor plasminogen activator (Pla) 

is encoded on a 9.5 kb plasmid, pPCP1, unique to Y 

pestis. Inactivation of the pla gene leads to a significant 

attenuation of virulence from a subcutaneous but not 

an intraperitoneal or intravenous route of infection in 

mice, suggesting that Pla promotes dissemination of 

the organism from peripheral sites of infection, and 

plasminogen-deficient mice are 100-fold more resistant to Y pestis than normal mice.21,121,122 Although Pla 

is necessary for full virulence in some Y pestis strains, 

a few strains that are Pla- and appear to be fully virulent have been identified among natural isolates or 

generated in the laboratory.61,122 Presumably, these 

isolates synthesize other proteins that substitute for 

Pla function. 

Fimbrae

 The so-called pH 6 antigen is a fimbrial structure on 

the surface of Y pestis that is necessary for full virulence 

in the mouse model. Researchers have proposed that 

pH 6 antigen mediates attachment of the organism 

to host cells via binding to glycosphingolipids. The 

temperature and pH of the environment tightly control 

the biosynthesis of these fimbriae; the expression of 

pH 6 antigen is most efficient in vitro with a growth 

temperature between 35°C and 41°C and a pH range 

of 5.0 to 6.7. This situation suggests that, in vivo, the 

adhesin activity is likely to be expressed only in specific microenvironments, such as the phagolysosome, 

necrotic tissue, or an abscess. Intracellular association 

with macrophages in the laboratory induces synthesis 

of the fimbriae.123 More recent data, however, suggest 

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that the pH 6 antigen does not enhance adhesion to 

mouse macrophages but rather promotes resistance 

to phagocytosis.124 Additional data suggest that this 

protein is not an essential virulence factor in wild 

type Y pestis; the use of laboratory-passaged strains 

may have influenced the results of previous studies.125

Alternatively, there may be redundancy of some functions in Y pestis as implied by the work of Felek et al.108

Iron and Manganese Sequestration 

Acquisition of nutrients in the host is an essential 

part of pathogenesis. In the mammalian host, iron is 

sequestered from invading pathogens; therefore, the 

level of free iron in the extracellular milieu is less than 

that necessary for bacterial growth. Like most bacterial 

pathogens, Y pestis possesses a high-affinity iron uptake system that is capable of procuring this essential 

nutrient from the host. Strains that do not produce 

the low-molecular-weight iron chelator, known as 

yersiniabactin, or those unable to transport yersiniabactin are highly attenuated by the subcutaneous route 

of infection and somewhat affected in pneumonic 

models. Such strains are capable, however, of infecting via the intravenous route (septicemic model). The 

genes encoding this iron transport system are situated 

on a chromosomal pathogenicity island known as the 

pigmentation locus (pgm).126 Manganese transport is 

also important for full virulence.127

Phage Shock Protein Response 

The phage shock protein (PSP) response is almost 

ubiquitous among microbes; homologues are found 

in numerous gram-positive and gram-negative bacteria, as well as archaebacteria and even chloroplasts. 

This regulon appears to respond to environmental 

stressors, including disturbances in the cell envelope 

and changes in the proton motive force that are induced by impaired inner membrane integrity.128,129

For pathogens, environmental stressors triggering 

the PSP regulon likely include environments within 

the host, and the PSP response is associated with 

virulence in Salmonella enterica, Yersinia enterocolitica, 

Yersinia pseudotuberculosis, and Y pestis.

128,130,131 For Y 

pestis, it is required for virulence by both the aerosol 

and subcutaneous routes of infection.131

Twin Arginine Transport 

Gram-negative bacteria have numerous ways to 

transport molecules across their membranes. One of 

these mechanisms is the twin arginine transport (Tat) 

pathway. The Tat pathway secretes folded proteins that 

are identified by an N-terminal signal peptide containing a twin arginine motif across the inner membrane. 

The TatA gene product mediates the actual translocation event,132 and it is an important virulence factor 

of Y pestis in both bubonic and pneumonic models 

of infection.133 The tatA mutation in Y pestis leads to 

many phenotypic changes, including a defect in the 

secretion/assembly of the F1 antigen on the cell surface. 

However, the attenuation of a Y pestis tatA mutant 

cannot be explained by the defect in F1 synthesis; the 

tatA mutant is more attenuated than mutants affected 

in the capsular synthetic genes per se.133 

Surface Structures

Bacterial surface structures, such as porins and 

phage receptors, have been implicated in virulence. 

OmpA, a major outer membrane porin, was identified as an in vivo-expressed protein and subsequently 

proven to be essential for virulence.134 Receptors for 

Y pestis-specific bacteriophage also play an important 

role in virulence; these tend to be associated with 

various portions of the lipopolysaccharide inner and 

outer core.135

Small RNAs 

Posttranscriptional control of virulence determinant 

expression by small RNAs was recently documented 

in Y pestis.

136 Expression of the majority of small RNAs 

in Y pestis is dependent on the RNA-binding regulatory protein Hfq. Hfq is necessary for growth of the 

organism at 37oC and for virulence.136 Identification of 

regulons governed by small RNAs may lead to identification of virulence factors previously unknown.137

Yersinia Autotransporter Proteins 

Bacterial autotransporters proteins are secreted via 

type V secretion pathway and have three conserved 

domains necessary for mediating secretion. An aminoterminal signal peptide targets the autotransporter to 

the general secretory pathway for secretion across the 

inner membrane. From the periplasm, the proteins 

are translocated to the outer membrane for tethering 

to the bacterial surface for release following proteolytic cleavage. Numerous autotransporters have been 

established to be virulence factors in many bacterial 

pathogens.138–140 The genome of Y pestis encodes for 

numerous functional Yersinia autotransporter proteins

(Yaps).141–143 Many of these Yap genes have increased 

expression during infection of mammals,141 and the 

proteins are necessary for efficient adherence to host 

cells and colonization of the mammalian host.142,144,145 

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Medical Aspects of Biological Warfare 

 Virulence and Transmission Factors in the Flea

Researchers have identified many factors that allow Y pestis to block the flea and promote vectorborne 

transmission.146 Y pestis has a natural resistance to antimicrobial peptides at growth temperatures similar to 

that of the flea gut. Such peptides are an integral part 

of the insect immune system. 

Blockage of the proventriculus occurs as a result of 

bacterial aggregates embedded in a biofilm synthesized 

by the bacterium at temperatures lower than 28°C. This 

allows the organism to persist in the proventriculus 

despite the shearing forces that flush nonaggregating 

cells into the midgut.147 The two-component regulatory 

system, phoP-phoQ, contributes to stable biofilm production in the flea.148 The chromosomal hmsHFRS operon, part of the Yersinia pathogenicity island (“pgm”), 

encodes the polysaccharide extracellular matrix that 

is essential to biofilm formation. The temperaturedependent biofilm synthesis is posttranscriptionally 

regulated.149 Although Hms-

 mutants are capable of 

colonizing the flea midgut, they are unable to colonize 

the proventriculus and, therefore, do not block the flea. 

The hms operon mediates storage of hemin or Congo 

red in the outer membrane of Y pestis on agar medium 

containing these compounds. This “pigmentation” 

phenotype, or Pgm, has been associated with virulence 

of Y pestis in animal models; however, Hms per se does 

not appear to play a role in mammalian plague other 

than promoting flea transmission. The spontaneous 

loss of pigmentation in the laboratory usually results 

from a large chromosomal deletion affecting not only 

the genes necessary for the Hms phenotype, but also 

the genetically linked yersiniabactin uptake system. 

The absence of the high affinity iron transport system 

in Pgm strains, rather than the loss of Hms, is responsible for attenuation in animal models.126

Studies examining the role of the Y pestis plasmids 

in the flea host indicated that one or more genes on 

the plasmid pMT are necessary for colonizing the 

midgut.150 The so-called murine toxin encoded by 

this plasmid appears to be one of these colonization 

factors. Murine toxin has phospholipase D activity, 

and although toxic to mice and rats in pure form, it is 

not important for virulence in rodent models.150 This 

may be explained by the regulation of toxin synthesis. 

Like Hms, it is produced more efficiently at 28°C than 

at mammalian body temperatures. Ymt, the Yersinia

murine toxin, appears to protect the bacterium from an 

unidentified antibacterial substance in the midgut.146 

PATHOGENESIS

As few as 1 to 10 Y pestis organisms are sufficient 

to cause infection by the oral, intradermal, subcutaneous, or intravenous routes.21 Estimates of infectivity 

by the respiratory route for nonhuman primates vary 

from 100 to 20,000 organisms.110,151,152 After being introduced into the mammalian host by a flea, where 

it had been at ambient temperature, the organism is 

thought to be initially susceptible to phagocytosis and 

killing by neutrophils. In rodent models of bubonic 

plague, it has been shown that neutrophils are quickly 

recruited to the area associated with the bacteria. 

Furthermore, the ability to evade and neutralize 

neutrophils was necessary for infection.153 However, 

some of the bacteria may grow and proliferate within 

tissue macrophages.154 A vigorous debate has raged 

for many years regarding the relative importance 

of intracellular versus extracellular replication in 

plague. Although most of the bacterial multiplication 

in the mammalian host is extracellular, evidence indicates that Y pestis can survive and multiply in macrophages. As reviewed by Pujol and Bliska, growth 

inside host cells is likely to be of greatest importance 

at the early stages of colonization.155 They suggest 

that, although considerable attention has focused on 

how Y pestis subverts the functions of phagocytes 

from the outside, there is less understanding of how 

these bacteria affect macrophage functions from the 

inside.155 Once the antiphagocytic gene products are 

expressed, the bacteria are resistant to phagocytosis 

and multiply extracellularly. However, another recent study demonstrated that a Y pestis mutant strain 

severely defective for intracellular recovery within 

macrophages was still fully virulent in a murine 

pneumonic plague challenge.156 The ability of Y pestis

to reside and replicate in an intracellular environment may be dependent upon the route of infection 

(parenteral versus respiratory).

During the incubation phase, the bacilli most commonly spread to regional lymph nodes, where lymphadenitis develops, producing the characteristic bubo. 

Dissemination from this local site leads to septicemia 

and seeding of other organs, including the liver, spleen, 

lung, and (less often) the meninges. The endotoxin of 

Y pestis probably contributes to the development of 

septic shock, which is similar to the shock state seen in 

gram-negative sepsis from other causes. The endotoxin 

may also contribute to the resistance of the organism 

to the bactericidal activity of serum.151

Primary pneumonic plague, the most severe form of 

disease, arises from inhalation of infectious respiratory 

droplets or an aerosol. Primary pneumonic plague is 

more rapidly fatal than secondary.1

 During primary 

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pneumonic plague, the disease can be divided into 

two host response phases. During the initial preinflammatory phase, the bacteria are actively replicating in 

the absence of host innate immune responses. Next, 

a proinflammatory phase occurs with a neutrophil 

influx, proinflammatory cytokine storm, and tissue 

destruction within the lung. Evidence suggests that 

Y pestis suppresses the host immune cells in the lung 

early during infection. As discussed above, the type 

III secretion system Yop effectors act upon numerous 

mammalian cells to elicit antiinflammatory and antiphagocytic effects. During pneumonic infection, the 

type III secretion system initially targets macrophages 

and neutrophils.157 

Primary septicemic plague can occur from direct 

inoculation of bacilli into the bloodstream, bypassing 

initial multiplication in the lymph nodes. Asymptomatic pharyngeal carriage of plague has occurred in 

contacts of patients with either bubonic or pneumonic 

plague.77,158,159

CLINICAL MANIFESTATIONS

From 1947 through 1996, 390 cases of plague were 

reported in the United States, resulting in 60 (15.4%) 

deaths.70,91 Of these deaths, bubonic plague accounted 

for 327 cases (83.9%) and 44 deaths (13.5%); primary 

septicemic plague accounted for 49 cases (12.6%) and 

11 deaths (22.4%); and primary pneumonic plague 

accounted for 7 cases (1.8%) and 4 deaths (57.1%).70,91

Seven cases (1.8%) were unclassified, including 1 death 

(14.3%).70,160 If Y pestis was used as a biological warfare 

agent, the clinical manifestations of plague would be 

(a) rapidly progressive, highly fatal epidemic pneumonia if aerosolized bacteria were used, or (b) bubonic 

and septicemic plague if fleas were used as carriers. 

Infections via ingestion could also occur.1

Bubonic Plague

Human symptoms of bubonic plague typically develop 2 to 8 days after being bitten by an infected flea. 

Presenting symptoms include prostration or severe 

malaise (75% of cases), headache (20%–85% of cases), 

vomiting (25%–49% of cases), chills (40% of cases), 

altered mentation (26%–38% of cases), cough (25% of 

cases), abdominal pain (18% of cases), and chest pain 

(13% of cases).27 In the United States, fleabites typically 

occur in the lower extremities; therefore, buboes are 

most common in the femoral and inguinal regions. As 

noted previously, the proportion of bubonic cases with 

cervical buboes is often higher in poverty-stricken areas 

of developing countries because these cases involve 

persons that frequently sleep on the dirt floors of huts 

where fleas are likely to bite them on the head and neck. 

Infection arising from skinning plague-infected animals 

typically produces axillary buboes due to inoculation 

of the upper extremities. Six to 8 hours after onset of 

symptoms, buboes, heralded by severe pain, appear 

(Figure 10-6). Buboes may drain spontaneously and 

rarely require incision and drainage because of pronounced necrosis. Within 2 days, a warm, erythematous 

bubo can grow to the size of an egg and eventually 

reach 10 cm in diameter as a cluster of infected lymph 

nodes. Buboes are so intensely painful that even nearly 

comatose patients attempt to shield them from trauma 

and will abduct their extremities to decrease pressure. 

Buboes are often associated with considerable surrounding edema, but lymphangitis is rare. Occasionally, 

buboes become fluctuant and suppurate. Histologically 

buboes demonstrate hemorrhagic necrosis, numerous 

neutrophils, a plethora of extracellular bacteria, and 

completely destroyed lymph node architecture.40

A small minority of patients bitten by plagueinfected fleas develop Y pestis septicemia without a 

discernable bubo. Other manifestations of bubonic 

plague include bladder distention, apathy, confusion, 

fright, anxiety, oliguria, and anuria. Tachycardia, hypotension, leukocytosis, thrombocytopenia, and fever 

are frequently encountered. In about half of untreated 

cases of bubonic plague, septicemia ensues 2 to 6 days 

later, which is virtually 100% fatal if untreated.40,161

In the United States, approximately 10% to 15% of 

bubonic plague patients have developed secondary 

pneumonic plague with the potential for airborne 

transmission of the organism.162

Septicemic Plague

Septicemic plague may occur primarily—if the bacteria is inoculated by a fleabite or other means, such as 

a puncture wound caused by a knife used while skinning an animal or bypass regional lymph nodes—or 

secondarily as a complication of hematogenous dissemination of bubonic plague.40 Presenting signs and 

symptoms of primary septicemic plague are essentially 

the same as those for any gram-negative septicemia: 

fever, chills, nausea, vomiting, and diarrhea. Purpura 

(Figure 10-7), disseminated intravascular coagulation, and acral cyanosis and necrosis (Figure 10-8) 

may be seen later. In New Mexico between 1980 and 

1984, plague was suspected in 69% of patients who 

had bubonic plague, but in only 17% of patients who 

had the septicemic form. The mortality was 33.3% for 

septicemic plague versus 11.5% for bubonic, which 

indicates the difficulty of diagnosing septicemic 

plague.162 Diagnosis of septicemic plague took longer 

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Medical Aspects of Biological Warfare 

Figure 10-6. A femoral bubo (a), the most common site of 

an erythematous, tender, swollen, lymph node in patients 

with plague. This painful lesion may be aspirated in a sterile 

fashion to relieve pain and pressure; it should not be incised 

and drained. The next most common lymph node regions 

involved are the inguinal, axillary (b), and cervical areas. 

Bubo location is a function of the region of the body in which 

an infected flea inoculates the plague bacilli. 

Photographs: Courtesy of Kenneth L Gage, PhD, Centers 

for Disease Control and Prevention Laboratory, Fort Collins, Colorado.

a b

Figure 10-7. Purpuric lesions can 

be seen on the upper chest of this 

girl with plague. The bandage on 

her neck indicates that a bubo has 

been aspirated. 

Photograph: Courtesy of Kenneth 

L Gage, PhD, Centers for Disease 

Control and Prevention Laboratory, 

Fort Collins, Colorado. 

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Plague

Figure 10-8. This patient is recovering from bubonic plague that disseminated to the blood (septicemic form) and the lungs 

(pneumonic form). Note the dressing over the tracheostomy site. At one point, the patient’s entire body was purpuric. Note 

the acral necrosis of (a) the patient’s nose and fingers and (b) the toes. 

Photographs: Courtesy of Kenneth L Gage, PhD, Centers for Disease Control and Prevention Laboratory, Fort Collins, 

Colorado.

a b

(5 vs 4 days) after onset, although patients sought care 

earlier (1.7 vs 2.1 days) and were hospitalized sooner 

(5.3 vs 6.0 days) than patients with bubonic plague. The 

only symptom present significantly more frequently 

in septicemic than in bubonic plague was abdominal 

pain (40% vs <10%), which was probably caused by 

hepatosplenomegaly.162

Pneumonic Plague

Pneumonic plague may occur primarily, from inhaling infectious respiratory droplets or aerosols, or 

secondarily, from hematogenous dissemination. It is the 

only form of plague that can be transmitted from one 

person to another.78 Patients with pneumonic plague 

rapidly develop symptoms of a severe bronchopneumonia, severe headache, chills, malaise, tachypnea, 

tachycardia, dyspnea, cough, chest pain, and hemoptysis.78,163,164 Patients initially exhibit a dry cough that 

progressively becomes productive as sputum concentration of blood and bacilli increases to “almost pure 

culture” levels at the end.78 The findings on a chest x-ray 

may be variable, but bilateral alveolar infiltrates appear 

to be the most common finding (Figure 10-9).78,165,166 A 

chest x-ray with bilateral alveolar infiltrates in a bubonic 

plague patient sometimes represents adult respiratory distress syndrome and disseminated intravascular 

coagulation in the absence of plague pneumonia.164

Depending on the stage of infection, the sputum may 

be clear, purulent, or hemorrhagic, and contain gramnegative rods. Unless appropriate antimicrobial therapy 

is begun during the first day of symptoms, pneumonic 

plague is rapidly fatal.5,40 The time from respiratory 

exposure to death in humans is reported to have been 

between 2 to 6 days (and from symptoms to death 1–3 

days) in epidemics during the preantibiotic era.78,163,167

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Medical Aspects of Biological Warfare 

Plague Meningitis

Plague meningitis is seen in 6% to 7% of cases. The 

condition manifests itself most often in children after 9 to 

14 days of ineffective treatment. Symptoms are similar 

to those of other forms of acute bacterial meningitis.165

Pharyngeal and Gastrointestinal Plague

In late December 2007, the first known cases of 

plague in Afghanistan developed among 83 persons 

who developed acute gastroenteritis after eating the 

meat of a slaughtered, sick camel, and 17 of those 

patients died.168 

Transient asymptomatic pharyngeal carriage 

may occur in healthy contacts of bubonic plague 

patients.77,158,159,169 Symptomatic pharyngeal plague 

presents with pharyngitis, fever, and cervical lymphadenopathy after inhalation of plague bacteria or 

ingestion of meat from infected camels or goats.1,3,6,161

For example, in early 1997, 12 individuals in Jordan 

ate raw or cooked meat from the same (infected) camel 

and all developed pharyngeal plague. One developed 

pneumonic plague. However, all survived because 

they were serendipitously treated with gentamicin for 

the suspected diagnosis of tularemia.170

A plague syndrome of cervical buboes, peritonsillar 

abscesses, and fulminant pneumonia has been reported 

in Vietnam161 and among Indians of Ecuador, who are 

known to catch and kill fleas and lice with their teeth.62

Endobronchial aspiration from peritonsillar abscesses 

may lead to fulminant pneumonia. 

Cutaneous Manifestations

While most plague patients have normal appearing skin (apart from buboes), approximately 4% to 

10% of patients develop an inoculation-site pustule, 

ulcer, eschar, or carbuncle (Figure 10-10).40,154,165,171–173

A sample from a plague patient eschar (ecthyma gangrenosum lesion) grew Y pestis, which suggests that 

local skin lesions are the result of septicemic seeding 

of the organism.173

Petechiae and ecchymoses may develop during 

hematogenous spread of bacteria when patients 

develop disseminated intravascular coagulation 

secondary to the Y pestis endotoxin. When purpura 

and acral gangrene occur, possibly exacerbated by 

the tissue plasminogen activator, the prognosis is 

Figure 10-9. This chest roentgenogram shows right middleand lower-lobe involvement in a patient with pneumonic 

plague.

Photograph: Courtesy of Kenneth L Gage, PhD, Centers for 

Disease Control and Prevention Laboratory, Fort Collins, 

Colorado.

Figure 10-10. This child has left axillary bubonic plague. The 

erythematous, eroded, crusting, necrotic ulcer on the child’s 

left upper quadrant is located at the presumed primary 

inoculation site. 

Photograph: Courtesy of Kenneth L Gage, PhD, Centers for 

Disease Control and Prevention Laboratory, Fort Collins, 

Colorado.

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poor.27,40,173 Recently, an American man contracted 

bubonic plague that progressed to septic shock; he 

developed ischemic necrosis of his feet requiring 

bilateral foot amputation.174 Patients in the terminal 

stages of pneumonic and septicemic plague often 

develop large ecchymoses on their backs. Lesions like 

these are likely to have led to the medieval epithet 

“the Black Death.”

DIAGNOSIS

Signs and Symptoms

The early diagnosis of plague requires a high index 

of suspicion. Presence of a painful bubo in the setting 

of fever, prostration, and possible exposure to rodents 

or fleas in an endemic area should readily suggest the 

diagnosis of bubonic plague. However, if the healthcare provider is not familiar with the disease or does 

not ask the patient for a travel or exposure history, or 

if the patient presents in a nonendemic area or without 

a bubo, then the diagnosis will be difficult to make. 

For example, in the United States in 1996, fatal plague 

cases occurred in two young adults who presented 

for treatment without obvious buboes.175 The first, 

an 18-year-old male, presented with left groin swelling and tenderness that was misdiagnosed as a groin 

muscle strain attributed to a fall 2 days earlier. The 

second, a 16-year-old female, presented with left arm 

numbness and left axillary pain that was misdiagnosed 

as a possible brachial plexus injury related to a fall from 

a trampoline 3 days earlier. In both cases, the patients 

were sent home with a pain reliever, and they both 

experienced rapid progression of their illness within 

the next day and died.90

The wildlife biologist who died of pneumonic 

plague after necropsy of an infected mountain lion did 

present to a health clinic with fever, chills, nausea, myalgias, and a cough producing blood-tinged sputum.74

No chest x-ray was performed. No exposure history to 

wildlife during his job was elicited.74 Either the chest xray or a job exposure history could have saved his life. 

The laboratory plague researcher who died in Chicago of septicemic plague presented to an outpatient 

clinic 3 days before death.94 He complained of fever, 

body aches, and a 3-day history of nonproductive 

cough. Influenza or other acute respiratory infection 

was suspected, and he was referred to an emergency 

department, but the patient did not follow through. 

Neither at that clinic visit nor at his hospital admission 12 hours before death was his occupation noted.94

When a bubo is present, the differential diagnosis 

should include tularemia, cat scratch disease, lymphogranuloma venereum, chancroid, tuberculosis, 

streptococcal adenitis, and scrub typhus (Figure 10-11). 

In both tularemia and cat scratch disease, the inoculation site is typically more evident and the patient will 

usually not be septic. In chancroid and scrofula, the 

patient has less local pain, the course is more indolent, 

and there is no sepsis. Patients with chancroid and 

lymphogranuloma venereum will have a recent history 

of sexual contact and genital lesions. Those with the 

latter disease may be as sick as patients with plague. 

Streptococcal adenitis may be difficult to distinguish 

initially, but the patient is usually not septic, and the 

node is more tender when plague is present.

The implications of the absence of a bubo were 

demonstrated in a review of 27 plague cases seen in 

New Mexico.166 In this study, there were eight cases 

of septicemic plague and 19 cases of bubonic plague, 

with six fatalities. Of the patients who died, three had 

septicemic plague and three had bubonic plague, but 

all six presented with nonspecific febrile symptoms 

or symptoms of an upper respiratory tract infection. 

The authors concluded that the lack of a bubo was associated with a delay in the diagnosis of plague and 

increased mortality.166

The differential diagnosis of septicemic plague also 

includes meningococcemia, gram-negative sepsis, and 

the rickettsioses. The patient with pneumonic plague 

who presents with systemic toxicity, a productive 

cough, and bloody sputum suggests a large differential 

diagnosis. However, demonstration of gram-negative 

rods in the sputum should readily suggest the correct 

diagnosis, because Y pestis is perhaps the only gramnegative bacterium that can cause extensive, fulminant 

pneumonia with bloody sputum in an otherwise 

healthy, immunocompetent host.

Laboratory Confirmation

Procedures for the isolation and presumptive 

identification of Y pestis by Level A laboratories can 

be downloaded from the CDC website (http://www.

bt.cdc.gov/agent/plague/index.asp).

172 The World 

Health Organization offers its Plague Manual online 

(http://www.who.int/emc-documents/plague/whocdscsredc992c.html). A recent review of the methodology for isolating and identifying Y pestis from clinical 

samples and animals is available.55 Standard bacterial methodologies include staining and microscopic 

analysis of the organism, isolation on culture medium, 

and biochemical tests. Misidentification of Y pestis by 

automated systems used for bacterial identification 

resulting in the delayed diagnosis of human plague 

has been reported.175 Therefore, if plague is suspected, 

immediate appropriate treatment should be started 

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and isolates should be sent to the appropriate laboratories experienced in the identification of Y pestis. 

Care should be taken to avoid aerosols; in this regard, 

fixing slides with methanol rather than heat fixing is 

preferred. CDC summarizes diagnosis of plague at 

its website (http://www.cdc.gov/plague/healthcare/

clinicians.html).

Reference laboratories, such as those found in major 

county or state health departments, have additional 

tests to confirm the diagnosis of Y pestis. These tests 

include direct fluorescent antibody tests to detect 

the F1 capsular antigen and polymerase chain reaction based assays, which can be used on isolates or 

direct clinical samples. Confirmatory testing includes 

lysis by a species-specific bacteriophage.1

 Serological 

testing such as passive hemagglutination antibody 

detection in acute or convalescent-phase plasma or 

enzyme-linked immunosorbent assay are found at 

national laboratories such as the CDC at Fort Collins, 

Colorado, and the US Army Medical Research Institute 

of Infectious Diseases at Fort Detrick, Maryland.176

Serological assays measuring the immune response to 

plague infection are mainly of value, retrospectively, 

because patients present clinically before they develop 

a significant antibody response. A four-fold change 

in antibody between acute and convalescent serum 

samples is considered confirmatory. 

When using the fluorescent antibody test to detect 

the plague-specific capsular antigen, it is important to 

recall that F1 antigen is produced only at temperatures 

Figure 10-11. (a) Small femoral bubo and presumed inoculation site (on the inferior thigh) in a patient with tularemia. This 

gram-negative bacterial infection (with Francisella tularensis) may closely mimic bubonic plague and is successfully treated with 

the same antibiotics. (b) Axillary bubo seen in child with cat scratch disease. (c) Greenblatt’s sign of ipsilateral femoral and 

inguinal buboes with intervening depression over the inguinal ligament seen in a patient with lymphogranuloma venereum 

caused by Chlamydia trachomatis. (d) Large inguinal bubo seen in a patient with chancroid caused by Haemophilus ducreyi. 

Photographs: Courtesy of Dermatology Service, Fitzsimons Army Medical Center, Aurora, Colorado.

c

b

a

d

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greater than 33°C. Thus, this method requires a relatively fresh sample from the patient/animal or from a 

laboratory culture incubated at the appropriate temperature. Therefore, flea samples, as well as samples 

refrigerated for more than 30 hours, are F1 antigen 

negative.55 For diagnosing plague in the field, a new 

rapid diagnostic test with monoclonal antibodies to 

the F1 antigen has been developed and field tested in 

Madagascar. The rapid diagnostic test detected concentrations of F1 antigen as low as 0.5 ng/mL in as little as 

15 minutes and had a shelf life of 21 days at 60°C. This 

test had 100% sensitivity and specificity against laboratory isolates of Y pestis, and the agreement between 

field testing and reference laboratory testing was 89.9%. 

This test demonstrated positive and negative predictive 

values of 90.6% and 86.7%, respectively.176 A rapid and 

reliable test such as the rapid diagnostic test, which 

healthcare workers can easily perform at the patient’s 

bedside, holds considerable promise for rapid plague 

diagnosis in endemic countries, but further testing is 

needed. A polymerase chain reaction test using primers for the plasminogen activator gene (pla) can detect 

as few as 10 Y pestis organisms, even from flea tissue. 

This test may be useful in surveillance of rats and 

can be adapted to help diagnose human infection.177

More recently, the use of Pla primers for simulated 

detection of Y pestis in sputum was reported to have a 

sensitivity of 104 CFU/mL and a 5-hour turnaround.178

In cases where use of Y pestis as a biological weapon is 

a possibility, it should be kept in mind that F1 or Pla 

are not necessary for virulence in animal models.59,110,179

Strains lacking these important diagnostic targets may 

still be threats.

Cultures of blood, bubo aspirate, sputum, and 

bronchial/tracheal washings, and/or cerebrospinal 

fluid (if indicated) should be performed based on the 

clinical presentation. Tiny 1- to 3-mm “beaten-copper” 

colonies will appear on blood agar by 48 hours, but Y 

pestis is slow growing and cultures may appear negative at 24 hours. In one study, 24 of 25 blood cultures 

(96%) of patients with bubonic plague were positive 

on standard supplemented peptone broth.6

 In patients 

with lymphadenopathy, a bubo aspirate should be 

obtained by inserting a 20-gauge needle attached to 

a 10-mL syringe containing 1 mL of sterile saline. Saline is injected and withdrawn several times until it is 

tinged with blood. Repeated, sterile bubo aspiration 

may also be done to decompress buboes and relieve 

pain. Drops of the aspirate should be air-dried on a 

slide and methanol-fixed for staining. When evaluating 

stained material, it should be considered that the characteristic bipolar staining is not specific for Y pestis nor 

is it always observed. If available, a direct fluorescent 

antibody stain of bubo aspirate for the presence of Y 

pestis capsular antigen should be performed; a positive 

direct fluorescent antibody result is more specific for Y 

pestis than are the other listed stains (Figure 10-12).180

In patients with plague, complete blood counts 

often reveal leukocytosis with a left shift. Leukemoid 

reactions with up to 100,000 white blood cells/μL may 

be seen, especially in children. Platelet counts may be 

normal or low, and partial thromboplastin times are 

often increased. Leukocytosis with thrombocytopenia 

is common in plague, but rare in other conditions.181

When disseminated intravascular coagulation is present, fibrin degradation products will be elevated. Because of liver involvement, alanine aminotransferase, 

aspartate aminotransferase, and bilirubin levels are 

often increased.

Figure 10-12. These Yersinia pestis fluorescent cells are from 

an infected mouse spleen. Notice how the outlines of the 

coccobacilli “light up” in this direct fluorescent antibody 

(DFA) test. The DFA test is specific and therefore better than 

the other stains discussed in this chapter (original magnification × 1,000). 

Photograph: Courtesy of MC Chu, Centers for Disease 

Control and Prevention Laboratory, Fort Collins, Colorado.

TREATMENT

Isolation

Previous editions of this chapter recommended 

strict isolation until patients had received treatment 

for 48 hours. However, only standard infection precautions are necessary when caring for bubonic and 

septicemic plague patients, whereas droplet precautions are still recommended until pneumonic plague 

patients have received 48 hours of antibiotic therapy. 

However, such droplet precautions are strictly only 

necessary when patients are coughing up of bloody 

sputum. Simply wearing masks, practicing good 

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hand hygiene, and avoiding close contact (within 2 

m) will almost always prevent pneumonic plague 

transmission.77,182

Microbiology laboratory personnel must be alerted 

when Y pestis is suspected because laboratory-acquired plague cases have been reported in the United 

States.92,96 Treatment of plague is summarized on the 

CDC website (http://www.cdc.gov/plague/healthcare/

clinicians.html). 

Antibiotics

Both because of difficulty acquiring streptomycin 

and its unfavorable side effect profile, the Working 

Group on Civilian Biodefense and the CDC now 

recommend gentamicin as a first-line alternative to 

streptomycin.79,183 Gentamicin is given 5 mg/kg intramuscularly (IM) or intravenously (IV) once daily, 

or 2 mg/kg loading dose followed by 1.7 mg/kg IM 

or IV three times daily.79 A recent review of 75 cases 

of human plague in New Mexico demonstrated that 

gentamicin alone or in combination with a tetracycline 

was as efficacious as streptomycin for treating human 

plague cases.184 Alternate regimens recommended by 

the Working Group on Civilian Biodefense include 

doxycycline (100 mg IV twice daily or 200 mg IV once 

daily), ciprofloxacin (400 mg IV twice daily) or chloramphenicol (25 mg/kg IV as a loading dose, followed 

by 60 mg/kg/d in four divided doses).79

Recently, a randomized, comparative, open label 

clinical trial comparing monotherapy with gentamicin or doxycycline for treating plague was conducted 

in Tanzania.185 Thirty-five plague patients received 

gentamicin (2.5 mg/kg IM every 12 hours for 7 days) 

and 30 patients received doxycycline (100 mg [adults] 

and 2.2 mg/kg [children] orally every 12 hours for 7 

days). Both gentamicin and doxycycline were found 

to be effective therapies for adult and pediatric 

plague, resulting in favorable response rates of 94% 

and 97% for gentamicin and doxycycline, respectively. 185 The three deaths occurred on the first or second day of treatment when disease was thought to be 

too advanced to respond to antibiotics. However, in 

this study a paucity of pneumonic plague cases existed, and the authors also recommended the use of a 

loading dose when using doxycycline (200 mg every 

12 hours for 72 hours before completing the therapy 

with the conventional dose of 100 mg every 12 hours 

[or 200 mg every 24 hours]) to ensure early attainment 

of high-tissue concentrations of doxycycline in the face 

of a life-threatening infection.186

Chloramphenicol is indicated for conditions in 

which high tissue penetration is important, such as 

plague meningitis, pleuritis, or myocarditis, but it 

can cause bone marrow suppression. It can be used 

separately or combined with an aminoglycoside. In 

pregnant women, the preferred choice is gentamicin 

with doxycycline or ciprofloxacin as alternatives, and 

streptomycin should be avoided if possible.79 The 

treatment of choice for plague in children is streptomycin or gentamicin. The Working Group on Civilian 

Biodefense recommended doxycycline, ciprofloxacin, 

or chloramphenicol as alternatives.79 Chloramphenicol 

should not given to children younger than 2 years old 

because of the risk of grey baby syndrome. 

In 2013, the Food and Drug Administration approved the fluoroquinolone antibiotic levofloxacin for 

treating patients with plague. This approval was based 

on the agency’s Animal Efficacy Rule, which allows 

studies from animals to be used in situations where 

it is not feasible or ethical to conduct clinical trials in 

humans. In this study, 24 African green monkeys who 

had been challenged with aerosolized Y pestis of the 

CO92 strain received either placebo or levofloxacin at 

a dose equivalent to 500 mg IV every 24 hours for 10 

days. Treatment was begun after the fever developed. 

Sixteen of 17 levofloxacin treated animals survived 

as compared with none of the seven control animals, 

which all died with 5 days of exposure. The one treated 

animal that died was euthanized because of vomiting 

and inability to retain food; blood cultures 2 to 4 days 

before death were negative.80

The Working Group on Civilian Biodefense has 

also proposed recommendations for antibiotic therapy 

in a mass casualty setting and for postexposure prophylaxis. Because IV or IM therapy may not be possible in these situations, oral therapy preferably with 

doxycycline or ciprofloxacin is recommended.79 Levofloxacin, 500 mg once a day for 10 days, is also Food 

and Drug Administration- approved for postexposure 

prophylaxis187 based on the Animal Efficacy Rule. If 

treated with antibiotics, buboes typically recede in 

10 to 14 days and do not require drainage. Patients 

are unlikely to survive primary pneumonic plague if 

antibiotic therapy is not initiated within 18 hours of 

symptom onset. Without treatment, mortality is 60% 

for bubonic plague and 100% for the pneumonic and 

septicemic forms.169 

Prevention

All plague-control measures must include insecticide use, public health education, environmental sanitation to reduce sources of food and shelter for rodents, 

and perhaps reduction of rodent populations with 

chemicals such as cholecalciferol.27,40 Fleas must always 

be targeted before rodents, because killing rodents 

may release massive amounts of infected fleas.161 The 

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use of insecticides in rodent areas is effective because 

rodents pick up dust on their feet and carry it back to 

their nests, where they distribute it over their bodies 

via constant preening.27

Postexposure Prophylaxis

Asymptomatic individuals such as family members, 

healthcare providers, or other close contacts with 

persons with untreated pneumonic plague should 

receive antibiotic prophylaxis for 7 days. Close contact is defined as contact with a patient at distance 

less than 2 m.79 Prophylaxis is also recommended for 

laboratory workers who have had an exposure to Y 

pestis. Doxycycline is the preferred antibiotic, given 

as 100 mg twice daily for 7 days. Levofloxacin or 

ciprofloxacin are alternatives. The Working Group 

for Civilian Biodefense recommends that people who 

develop fever or cough while receiving prophylaxis 

should seek prompt medical attention and begin parenteral antibiotic treatment.79 Hospital personnel who 

are observing recommended isolation procedures do 

not require prophylactic therapy, nor do contacts of 

bubonic plague patients. However, people who were 

in the same environment and were potentially exposed 

to the same source of infection as the plague patients 

should be given prophylactic antibiotics. The CDC also 

recommends that prophylactic antibiotics be given to 

persons potentially exposed to the bites of infected 

fleas (during a plague outbreak, for example) or who 

have handled animals infected with the plague bacterium. In addition, previously vaccinated individuals 

should receive prophylactic antibiotics if they have 

been exposed to plague aerosols. 

Natural antibiotic resistance is rare in Y pestis; 

however, a chilling report appeared in 1997 of a human isolate in Madagascar resistant to streptomycin, 

tetracycline, chloramphenicol, ampicillin, kanamycin, 

and sulfonamide. A transmissible plasmid, pIP1202, 

was responsible for the multidrug-resistant phenotype of this isolate, suggesting a potential for transfer 

to other Y pestis strains in nature.188 This plasmid is 

closely related to transmissible multidrug-resistant 

plasmids of Yersinia ruckeri and Salmonella enterica

serotype Newport SL254 identified in the United 

States.189 More recently, a multidrug-resistant strain 

of Y pestis was isolated from a rodent in Mongolia.190

Russian scientists have published descriptions of 

multidrug-resistant plague vaccine strains produced 

in the laboratory; these techniques could conceivably 

be used on virulent strains.191 Ciprofloxacin-resistant 

isolates have been obtained in the laboratory from 

attenuated strains.192 If Y pestis is used as a biological 

weapon, then antibiotic resistance is a possibility; the 

stability and transmissibility of the multidrug-resistant 

pIP1202 plasmid in Y pestis suggests that such a strain 

could be engineered in the laboratory via conjugation 

without modern molecular technologies. 

Vaccination

While working in India in 1897, Ukrainian microbiologist Waldemar MW Haffkine developed the first 

plague vaccine consisting of killed whole cells. In 1942, 

Karl F Meyer, DVM, began developing an immunogenic 

and less reactogenic vaccine for the US Army from 

an agar-grown, formalin-killed, suspension of virulent plague bacilli. This same procedure (with minor 

modifications) was used to prepare the licensed vaccine, Plague Vaccine USP, which was routinely given 

to military personnel stationed in Vietnam and other 

individuals such as field personnel working in plagueendemic areas with exposure to rats and fleas and 

laboratory personnel working with Y pestis. However, 

this vaccine was discontinued by its manufacturers 

in 1999 and is no longer available. Although Plague 

Vaccine USP was effective in preventing or ameliorating bubonic disease, as seen by the low incidence of 

plague in US military personnel serving in Vietnam, 

data from animal studies suggest that this vaccine 

does not protect against pneumonic plague.151,152,193–195

The former Soviet Union and many other nations 

have traditionally focused on live attenuated vaccines, with tens of millions of humans receiving the 

live plague vaccine. Many investigators continue to 

believe that live attenuated vaccines are preferable to 

subunit vaccines. Live plague vaccines, usually derived 

from the attenuated EV76 strain, have been used alone 

and also successfully in a prime-boost strategy with 

subunit vaccines. Even the most recent reviews on 

plague vaccination continue to revisit the appeal of 

live attenuated vaccines.196,197

Two new plague vaccine candidates that use the 

F1 and V antigens of Y pestis have been developed. 

F1, a capsular antigen of Y pestis, appears to prevent 

phagocytosis of plague bacilli, whereas V antigen has 

a key role in the translocation of the cytotoxic Yops 

into host cells, as well as stimulating the production 

of immunosuppressive cytokines. US Army Medical 

Research Institute of Infectious Diseases scientists 

developed the first vaccine, F1-V, which consists 

of a recombinant fusion protein expressing F1 and 

V antigens (F1-V).198 Porton Down, the biodefense 

laboratory in the United Kingdom, developed a 

similar candidate that is a recombinant protein-based 

vaccine consisting of two separate proteins, F1 and 

V.199 The separate proteins are then combined, two 

parts F1 to one part V, to form a subunit vaccine. 

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The F1-V vaccine, which has been shown to protect 

African green monkeys from pneumonic plague,198 is 

currently in advanced development by the Department of Defense’s Joint Project Manager Medical 

Countermeasures, a component of the Joint Program 

Executive Office for Chemical and Biological Defense. 

The Joint Project Manager Medical Countermeasures 

facilitates the advanced development and acquisition 

of medical countermeasures and systems to enhance 

the US biodefense response capability. 

SUMMARY

Plague is a zoonotic infection caused by the gramnegative bacillus Y pestis. Plague is maintained in 

nature, predominately in urban and sylvatic rodents 

and flea vectors. Humans are not necessary for the 

persistence of the organism, and they acquire the disease from animal fleas, contact with infected animals, 

or, rarely, from other humans via aerosol or direct 

contact with infected secretions. Healthcare providers 

must understand the typical way in which humans 

contract plague in nature to differentiate endemic 

disease from plague used in biological warfare. First, 

a die-off of the mammalian reservoir that harbors 

bacteria-infected fleas will occur. Second, troops 

who have been in close proximity to such infected 

mammals will become infected and typically develop 

the bubonic form of the disease. By contrast, in the 

most likely biological warfare scenario, plague would 

spread via aerosol and result in primary pneumonic 

plague cases. Person-to-person spread of fulminant 

pneumonia, characterized by blood-tinged sputum, 

would then ensue. If, however, an enemy force released fleas infected with Y pestis, the soldiers would 

present with classic bubonic plague before a die-off 

in the local mammalian reservoir occurred, although 

such a die-off may be possible later if the introduced 

strain of Y pestis succeeded in proliferating among 

local rodent populations.

The most common form of the disease is bubonic 

plague, characterized by painful lymphadenopathy 

and severe constitutional symptoms of fever, chills, 

and headache. Septicemic plague without localized 

lymphadenopathy occurs less commonly and is difficult 

to diagnose. Secondary pneumonia may follow either 

the bubonic or the septicemic form. Primary pneumonic 

plague is spread by airborne transmission, when infectious respiratory droplets from an infected human or 

animal are inhaled or a person inhales an aerosol released as the result of biological weapon attack.

Diagnosis is established by isolating the organism 

from blood or other tissues. Rapid diagnosis may be 

made with fluorescent antibody stains of sputum or 

tissue specimens or detection of F1 antigen in serum. 

Patients should be isolated and treated with aminoglycosides. Chloramphenicol should be added when 

meningitis is suspected or shock is present. Although 

the licensed, killed, whole-cell vaccine is no longer 

available, a new vaccine that appears to protect against 

pneumonic plague is in advanced development. 

Acknowledgments

We thank Andrea Stossel and Laura Kalinyak for assistance with collecting references for this chapter and 

the reviewers for their thoughtful comments. In addition, Dr Kenneth Gage provided valuable insight into 

the ecology of plague. This chapter is dedicated to long-time plague researchers Lieutenant Colonel Dan C 

Cavanaugh and Lieutenant Colonel John D Marshall, who made outstanding and enduring contributions 

to public health and military medicine in their careers as both Army officers and as civil servants. 

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Kerala, India: Research Signpost; 2011:175–234.

197. Wang X, Zhang X, Zhou D, Yang R. Live-attenuated Yersinia pestis vaccines. Expert Rev Vaccines. 2013;12:677–686.

198. Heath DG, Anderson GW Jr, Mauro JM, et al. Protection against experimental bubonic and pneumonic plague by a 

recombinant capsular F1-V antigen fusion protein vaccine. Vaccine. 1998;16:1131–1137.

199. Williamson ED. Plague vaccine research and development. J Appl Microbiol. 2001;91:606–608.

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Tularemia

Chapter 11

TULAREMIA

MATTHEW J. HEPBURN, MD*; TODD M. KIJEK†; WENDY SAMMONS-JACKSON, PhD

‡

; ARTHUR M. FRIEDLANDER, MD§; 

and ZYGMUNT F. DEMBEK, PhD, MS, MPH, LHD¥

INTRODUCTION 

INFECTIOUS AGENT 

THE CLINICAL DISEASE

Epidemiology

Pathogenesis

Clinical Manifestations

Diagnosis

Treatment

PROPHYLAXIS 

Postexposure Prophylaxis 

Vaccination With Live Vaccine Strain 

ISSUES FOR LABORATORY WORKERS 

USE OF TULAREMIA AS A BIOLOGICAL WEAPON

SUMMARY 

*Colonel, MC, US Army; Program Manager, Biological Technologies Office, Defense Advanced Research Projects Agency (DARPA), 675 North Randolph 

Street, Room 08-140, Arlington, Virginia 22203-2114; formerly, Clinical Research Unit Lead, US Army Medical Research Institute of Infectious Diseases, 

1425 Porter Street, Fort Detrick, Maryland

†

Major, Medical Service Corps, US Army; PhD candidate and Chief, Molecular and Translational Sciences Department, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702; formerly, Research Microbiologist, Bacteriology Division, US Army 

Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

‡

Lieutenant Colonel, Medical Service Corps, US Army; Military Deputy, Science and Technology Department, US Army Medical Research and Materiel 

Command, 810 Schreider Street, Suite 103, Fort Detrick, Maryland 21702; formerly, Deputy Director, Military Infectious Diseases Research Program, 

Fort Detrick, Maryland

§

Colonel (Retired), Medical Corps, US Army; Senior Scientist, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland 21702, and Adjunct Professor of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones 

Bridge Road, Bethesda, Maryland 20814

¥

Colonel (Retired), Medical Service Corps, US Army Reserve; Associate Professor, Department of Military and Emergency Medicine, Uniformed Services 

University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814; formerly, Chief Biodefense Epidemiology and Education & 

Training Programs, Division of Medicine, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

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Medical Aspects of Biological Warfare 

INTRODUCTION

Kosovo conflict, although subsequent epidemiological 

investigations suggest that the observed cases were not 

caused by an intentional release.5,6

Given its highly pathogenic nature, low infectious dose, 

and ability to infect via aerosol, F tularensis is classified by 

the US Department of Health and Human Services as 

a tier 1 select agent. This classification is reserved for 

those pathogens deemed to pose the highest risk for 

intentional misuse. The nonspecific disease presentation of tularemia, the high morbidity, the significant 

mortality if untreated, and the limited ability to obtain 

a rapid diagnosis are other characteristics that contribute to the effectiveness of F tularensis as a potential 

biological weapon. Although tularemia responds to 

antibiotic therapy, the intentional use of a genetically 

modified antibiotic-resistant strain could make these 

countermeasures ineffective.

Tularemia is a life-threatening, debilitating disease 

caused by infection with the bacterium Francisella tularensis. This bacterium is one of the most infectious microorganisms known and poses a substantial threat as a potential biological weapon.1

 Both the United States and the 

former Soviet Union developed weaponized F tularensis

during the Cold War. 1,2 The Japanese experimented with F 

tularensis as a biological weapon,3 but no documentation 

shows that it was deliberately used as a biological weapon. 

There is also speculation that the former Soviet Union 

used F tularensis as a biological weapon against German 

troops in the Battle of Stalingrad during World War II.2

However, other authors suggest that natural causes, as opposed to an intentional release, were responsible for the 

tularemia epidemic that occurred in both armies during 

this battle.4 There was similar speculation that F tularensis

was used as a biological weapon by Serbia during the 

INFECTIOUS AGENT

Infection associated with F tularensis was first 

identified in Tulare County, California, where an epidemic disease outbreak resembling plague occurred 

in ground squirrels in 1911. McCoy and Chapin successfully cultured the causative agent and named it 

Bacterium tularense.

7

 Subsequently, Wherry and Lamb 

identified this pathogen as the cause of conjunctival 

ulcers in a 22-year-old man.8 Edward Francis’ pioneering work significantly increased the understanding of 

human disease associated with this pathogen in the 

early 20th century. He described the clinical syndromes 

associated with Francisella infection and named it “tularemia.”9 F tularensis was formerly included in both 

the Pasteurella and the Brucella genera. However, as 

mounting scientific data supported the creation of 

a new genus for this remarkable pathogen, this bacterium was assigned to its own genus and the name 

Francisella was proposed in tribute to Edward Francis.10

F tularensis is an aerobic, nonmotile bacterium and 

member of the Gammaproteobacteria. By Gram stain, 

it appears as a small (approximately 0.2–0.5 μm by 

0.7–1.0 μm),

11 faintly staining coccobacillus (Figure 

11-1). F tularensis is considered to have four subspecies: (1) tularensis, (2) holarctica, (3) mediasiatica, and (4) 

novicida.

12 F tularensis subspecies tularensis, also known 

as type A (or biovar A), occurs predominantly in North 

America and is the most virulent subspecies in both 

animals and humans. This subspecies was recently 

divided into A.I. and A.II. subpopulations based on 

extensive genotyping of isolates. Subpopulation A.I. 

causes disease in the central United States, and subpopulation A.II. is found mostly in the western United 

States.13 F tularensis subspecies holarctica (formerly described as palearctica), also known as type B (or biovar 

B), is found throughout the Northern Hemisphere. F 

tularensis subspecies holarctica typically causes a less 

clinically severe disease than subspecies tularensis, 

but has been documented to cause bacteremia in immunocompetent individuals.14,15 Before antibiotics, F 

tularensis subspecies tularensis resulted in 5% to 57% 

mortality, yet F tularensis subspecies holarctica was 

rarely fatal.15 F tularensis subspecies mediasiatica has 

Figure 11-1. Gram’s stain of Francisella tularensis. 

Photograph: Courtesy of Dr Larry Stauffer, Oregon State 

Public Health Laboratories, Centers for Disease Control and 

Prevention, Atlanta, Georgia, Public Health Image Library, 

Image 1904.

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Tularemia

been isolated in the central Asian republics of the 

former Soviet Union, and it appears to be substantially 

less virulent in a rabbit model compared to F tularensis

subspecies tularensis.

16,17

The clinical significance of this subspecies is not 

known. F tularensis subspecies novicida, also referred 

to as F novicida, is believed to be avirulent in healthy 

humans. Reported cases associated with this subspecies 

usually involve patients with other underlying health 

conditions.18 The four subspecies can be distinguished 

using biochemical tests and genetic analysis. Another 

closely related species, Francisella philomiragia, has also 

been described as a human pathogen.19,20 However, 

similar to F tularensis subspecies novicida, infections 

attributed to F philomiragia were typically found in 

patients with underlying health conditions.19,20

THE CLINICAL DISEASE

Tularemia is an infection with protean clinical manifestations. Healthcare providers need to understand 

the range of possible presentations of tularemia to use 

diagnostic testing and antibiotic therapy appropriately 

for these infections. Most cases of naturally occurring 

tularemia are ulceroglandular disease, involving an ulcer at the inoculation site and regional lymphadenopathy. Variations of ulceroglandular disease associated 

with different inoculation sites include ocular (oculoglandular) and oropharyngeal disease. Occasionally 

patients with tularemia present with a nonspecific 

febrile systemic illness (typhoidal tularemia) without 

evidence of a primary inoculation site. Pulmonary 

disease from F tularensis can occur naturally (pneumonic tularemia), but is uncommon and should raise 

suspicion of a biological attack, particularly if the cause 

is not readily discernable and significant numbers of 

cases are diagnosed. Because of the threat of this microorganism as a biological weapon, clusters of cases 

in a population or geographic area not accustomed to 

tularemia outbreaks should trigger consideration for 

further investigation.21 Rotz et al provide criteria for 

determining the likelihood that a tularemia outbreak is 

caused by intentional use of F tularensis as a biological 

weapon.21 A tularemia outbreak in US military personnel deployed to a nonendemic environment would be 

one example of an incident that should be investigated. 

An investigation should yield the likely cause of the 

outbreak, which could be varied (exposure to infected 

animals, arthropod borne, etc). By determining the 

source of the outbreak, it may be possible to implement 

control measures, such as water treatment or use of an 

alternative water supply if the outbreak is traced to a 

waterborne source.

Epidemiology

F tularensis subspecies tularensis(type A) is the most 

virulent subspecies and found predominantly in 

North America. This subspecies has recently been 

genetically subdivided into two subpopulations, A.I. 

and A.II. The subpopulations are distinct in mortality 

rates, geographic distribution, transmission vectors, 

and hosts.22–27 F tularensis subspecies holarctica (type 

B), which is found throughout the Northern Hemisphere, is associated with a milder form of disease. In 

the United States, 90 to 154 cases of tularemia have 

been reported yearly from 2001 to 2010, according to 

the Centers for Disease Control and Prevention.28 More 

than half of all cases reported came from Arkansas, 

Missouri, South Dakota, and Oklahoma, where the 

foci of infection are well established. Every state except 

Hawaii has reported cases of tularemia. 

Human outbreaks, which are often preceded by 

animal outbreaks, are seasonal, with the highest 

incidence in late spring, summer, and autumn.28,29 F 

tularensis has been detected in more than 100 mammalian species and several arthropods.30 F tularensis

can be transmitted to humans by direct contact with 

infected animals or their tissues, ingestion of undercooked infected meat or contaminated water, animal 

bites or scratches, arthropod bites, and inhalation of 

an aerosol or contaminated dust. However, humanto-human transmission has not been described. F 

tularensis is unique in its ability to adapt to a wide 

range of environmental, host, and vector conditions, 

and it can be categorized into two distinct transmission 

cycles involving different hosts and arthropod vectors. 

The cycle of disease is commonly associated with a 

subspecies, with type A commonly associated with the 

terrestrial cycle and type B commonly associated with 

the aquatic cycle.23,27,31,32 The human clinical syndromes 

can be classified by the portal of entry. 

Direct Contact

In 1914, a meat cutter with oculoglandular disease, 

manifested by conjunctival ulcers and preauricular 

lymphadenopathy, had the first microbiologically 

proven human tularemia case reported.8 An early review of tularemia established that a majority of human 

cases (368 of 488, or 75%) in North America resulted 

from dressing and eating wild rabbits.9 Other wild 

mammals may potentially serve as sources for tularemia transmission from direct contact, such as wild 

prairie dogs that are captured and sold as pets.33

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Medical Aspects of Biological Warfare 

Food and Water Ingestion

Tularemia can also be contracted by eating meat 

from infected animals9 or food contaminated by infected animals.34 Water can also become contaminated 

from animals infected with tularemia and cause human infection. From March through April 1982, 49 

cases of oropharyngeal tularemia were identified in 

Sansepolcro, Italy.3 The case distribution in this city 

suggested that a water system was the source. The 

infected individuals had consumed unchlorinated 

water, and a dead rabbit from which F tularensis was 

isolated was found nearby. 35 Waterborne transmission 

of ulceroglandular tularemia also occurred during a 

Spanish outbreak among 19 persons who had contact 

with river-caught crayfish.36 The crayfish appear to 

have served as mechanical vectors, but some evidence 

suggests a potential role as hosts.23,36 Contaminated 

water may have also contributed to recent outbreaks of 

oropharyngeal tularemia in Turkey37 and Bulgaria.34 It 

is unclear how F tularensis survives in water, but it may 

be linked to its ability to survive in certain protozoa 

species, such as Acanthamoeba castellanii.

38

Mammalian Bites and Arthropod Vectors

Mammalian bites are another source of F tularensis

transmission to humans. Instances of transmission from 

the bites or scratch of a cat, coyote, ground squirrel, and 

a hog to humans were documented more than 80 years 

ago.9 In April 2004, a 3-year-old boy from Denver, Colorado, contracted tularemia from a hamster bite, providing evidence of disease transmission from these pets.39

Transmission of F tularensis by the bites of ticks and 

flies is also well documented.11 Dermacentor species 

ticks (dog ticks) are important vectors in areas where 

enzootic transmission occurs in North America40 and 

Europe.41 Ixodes species ticks may also contribute to F 

tularensis transmission.42 In Utah during the summer 

of 1971, 28 of 39 tularemia cases were contracted from 

deerfly (Chrysops discalis) bites.43 An epidemic of 121 

tularemia cases (115 ulceroglandular) in Siberia from 

July through August 1941 may have resulted from 

transmission of F tularensis by mosquitoes, midges 

(Chironomidae), and small flies (Similia).44

Aerosol Transmission

The largest recorded pneumonic tularemia outbreak 

occurred in Sweden during the winter of 1966 through 

1967, when 676 cases were reported.45 Most of the cases 

occurred among the farming population, 71% among 

adults older than 45 years and 63% among men. The 

hundreds of pneumonic cases likely resulted from 

contact with hay and dust contaminated by voles 

infected with tularemia. F tularensis was later isolated 

from the dead rodents found in barns, as well as from 

vole feces and hay.

In the summer of 2000, an outbreak of primary 

pneumonic tularemia occurred in Martha’s Vineyard, 

Massachusetts.46 Fifteen confirmed tularemia cases were 

identified, 11 of which were the pneumonic form of tularemia. One 43-year-old man died of primary pneumonic 

tularemia. Epidemiological analysis revealed that using a 

lawn mower or brush cutter was significantly associated 

with illness in the 2 weeks before presentation of this 

case.47 Feldman et al proposed that in Martha’s Vineyard, 

F tularensis was shed in animal excreta, persisted in the 

environment, and was transmitted to humans after 

mechanical aerosolization by mower or brush cutter 

and subsequent inhalation.47 The strong epidemiological 

link with grass cutting adds plausibility to this explanation.48 A seroprevalence survey conducted in 2001 

in Martha’s Vineyard demonstrated that landscapers 

were more likely to have antibodies to F tularensis than 

nonlandscapers, suggesting an increased occupational 

risk for tularemia.47

The only other previously reported outbreak of 

pneumonic tularemia in the United States occurred in 

Martha’s Vineyard during the summer of 1978.49 In a 

single week, seven persons who stayed together in a 

vacation cottage eventually developed typhoidal tularemia. Asearch for additional cases on the island uncovered six other tularemia cases (five typhoidal and one 

ulceroglandular). No confirmed source for the disease 

exposure was discovered. Tularemia had been reported 

sporadically since the introduction of rabbits to Martha’s 

Vineyard in the 1930s,49 and pneumonic tularemia was 

initially reported in Massachusetts in 1947.50

Tularemia in an Unusual Setting

Some tularemia cases have occurred in geographic 

areas where the disease has never been reported. An 

orienteering contest on an isolated Swedish island in 

2000 resulted in two cases of ulceroglandular tularemia.51 These cases were theorized to have occurred 

from contact with migratory birds carrying the microorganism. 

The social disruption caused by war also has been 

linked to tularemia outbreaks. During World War II, 

an outbreak of more than 100,000 tularemia cases occurred in the former Soviet Union,4 and outbreaks with 

hundreds of cases after the war occurred in Austria 

and France.52 Outbreaks of zoonoses during war since 

that time have led to speculation that these epidemics 

were purposefully caused. For example, no tularemia 

cases had been reported from Kosovo between 1974 

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Tularemia

and 1999, and tularemia was not previously recognized 

endemically or enzootically in the Balkan countries.5

However, after a decade of warfare, an outbreak of 

more than 900 suspected tularemia cases occurred in 

Kosovo during 1999 and 2000, leading researchers 

to investigate claims of use of this agent as a biological 

weapon by the Serbs against the Albanian inhabitants 

of the country. 5,6 The Kosovo outbreak and subsequent investigation are described in detail in chapter 

2, Epidemiology of Biowarfare and Bioterrorism.

Laboratory-acquired Tularemia

Soon after the discovery of F tularensis as a pathogen, 

cases of laboratory-acquired infection were recognized. 

Edward Francis observed that many laboratory personnel working with the pathogen, including himself, 

became infected.9 Six tularemia cases occurred during 

US Public Health Service laboratory investigations of 

tularemia outbreaks from 1919 through 1921.53 Tularemia is the third most commonly acquired laboratory 

infection,54 and recent laboratory-acquired infections 

of tularemia emphasize the laboratory hazard that this 

organism presents.55 Because of the extreme infectivity 

of this microorganism, investigators of a 2000 outbreak 

in Kosovo chose not to culture the organisms from patients, but instead relied on empirical clinical evidence 

of tularemia cases.

Pathogenesis

One of the remarkable attributes of F tularensis is 

its low infectious dose. As few as 10 organisms can 

produce clinical disease in healthy human volunteers 

when administered by either subcutaneous injection or 

by aerosol exposure.56,57 Research aimed at elucidating 

the unique characteristics that permit this organism 

to cause disease at such low numbers revealed that F 

tularensis boasts a variety of mechanisms to not only 

evade host defenses, but also to modulate them to 

survive and proliferate within its host. 

F tularensis, which is an intracellular pathogen, is 

known to survive and replicate within a wide variety 

of cells including professional phagocytic cells, such 

as macrophages. To gain entry into these cells, F tularensis can efficiently use multiple receptors including 

the mannose receptor, FcγR, and complement receptor 

3. Interestingly, a recent study using a fully virulent 

type A strain showed that entry of opsonized bacteria 

into human macrophages via complement receptor 3 

suppressed the Toll-like receptor 2-dependent proinflammatory responses.58 Bacterial entry through the 

mannose receptor resulted in rapid phagosomal escape 

and prolific cytosolic replication.59 These findings 

indicate that Francisella has evolved to use multiple 

entry pathways to enhance its ability to replicate in 

the intracellular environment.

Once inside the macrophage, Francisella can avoid 

the bactericidal activity of reactive oxygen species 

and nitrogen species through expression of enzymes 

including bacterial acid phosphatases (Acp), super oxide dismutases (Sod), and catalase enzymes (Kat).60–65

Inhibition of these host defense mechanisms promotes 

bacterial virulence, as F tularensis live vaccine strain 

(LVS) mutants deficient in expression of SodB, SodC, 

or KatG are highly attenuated in mouse models of 

tularemia.60,61,63 Phagosomal acidification is another 

host defense mechanism designed to restrict growth 

of bacterial pathogens. However, both F tularensis type 

A and B stains can inhibit acidification of the phagosome and subsequently escape from the phagosome, 

and reside in the macrophage cytoplasm.66,67 The 

ability of Francisella to escape into the cytosol is in 

part dependent on proteins encoded on the Francisella

pathogenicity island (FPI). Nano et al first described 

the FPI in 2004 and subsequently most genes contained 

within the FPI have been linked to virulence.68 The FPI 

also contains genes that encode for a putative type 

VI secretion system that is required for phagosomal 

escape and virulence.69,70 IglC, a 23-kDa protein, is 

believed to be both a core component and secreted 

effector of the T6SS. IglC has been implicated not only 

in phagosomal escape but also in influencing Toll-like 

receptor-4 signal transduction.71–75 Regulation of the 

FPI is controlled by the MglA transcriptional regulator, 

which responds to various cues and in turn influences 

expression of more than 100 genes, including several 

other virulence factors.76 

Once Francisella reaches the cytoplasm, replication 

begins slowly, but eventually large numbers of organisms can be found within a single macrophage.73,77,78

Although F tularensis may initially delay apoptosis 

(programmed cell death) of the macrophage, the organism eventually induces apoptosis through mechanisms similar to intrinsic cellular signals.79 This strategy 

to escape the macrophage may help shield Francisella

from host immune surveillance mechanisms. 

Another survival mechanism of F tularensis is the 

inhibition of Toll-like receptor signaling and cytokine secretion, as demonstrated in experiments with 

murine macrophages and the LVS of F tularensis.

75

Avoidance of Toll-like receptor signaling inhibits the 

typical robust innate immune response that is active 

in eliminating typical bacterial pathogens. The early 

innate immune response to F tularensis involves intracellular killing of the pathogen by the macrophages 

and proinflammatory cytokine secretion. Murine 

experiments have demonstrated the importance 

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Medical Aspects of Biological Warfare 

of an effective early cytokine response. Interferonγ-deficient mice die from sublethal doses of LVS,80

and tumor necrosis factor-α is at least as important 

as interferon-γ for control of F tularensis infection.81,82

The host defense within macrophages appears to be 

crucial at controlling infection by F tularensis. In human monocytes/macrophages, the LVS strain and F 

novicida induced the processing and release of interleukin (IL)-1β, an essential component of the inflammatory immune response.83 However, killed bacteria 

did not induce this response, but did induce the early 

phases required for IL-1β, such as mRNA transcription. This suggests that only live Francisella can escape 

from the phagosome, and thus trigger the function of 

caspase-1, which converts the precursor of IL-1β to its 

active form. In mice deficient in caspase-1 as well as 

ASC, an adaptor protein involved in the assembly of 

inflammasome complexes, substantially higher bacterial loads were observed, as well as early mortality, 

compared to normal mice.84 Neutrophils perform an 

important function in limiting the spread of F tularensis

after inoculation. Experiments have demonstrated that 

neutrophils can kill F tularensis,

85 and mice depleted of 

neutrophils appear more susceptible to infection with 

F tularensis LVS.86

The late adaptive immune response to F tularensis

requires an intact cell-mediated immune system, 

particularly in resolving the initial infection and in 

producing long-term immunity.87 There is no clear 

immunodominant epitope on any one F tularensis virulence protein that stimulates the required cell-mediated 

response; however, studies have demonstrated that 

multiple protein/peptides may be required to produce 

a sufficient response.88 Vaccination with F tularensis

LVS appears to produce a long-term memory T-cell 

response (as measured by lymphocyte stimulation),89

but it is unclear what degree of long-term protection is conferred by this response. Both CD4+ and 

CD8+ lymphocytes are required for an effective cellmediated response to F tularensis.

80 The protective 

memory response is dependent on a robust proinflammatory cellular response, because administration 

of anti-interferon-γ and antitumor necrosis factor-α

antibodies to previously vaccinated mice dramatically lowers the lethal infective intradermal dose of F 

tularensis.

82 This memory response initially appears 2 

to 4 weeks after initial infection,90–92 and it can remain 

detectable for many years.89,93

The importance of humoral immunity in the defense 

against tularemia is not completely understood, but it 

appears that the humoral response by itself provides 

little or no value in protecting the host.94 When laboratory workers received a formalin-killed whole-cell 

vaccine developed by Foshay et al,95 a strong humoral 

response was elicited but was not protective against 

cutaneous58 or respiratory57 challenge. The failure of 

this vaccine suggested that the formalin inactivation 

procedures destroyed some of the essential protective antigens or that these protective antigens were 

not expressed in vitro. A persistent humoral response 

does develop during human infection and after vaccination. Waag et al reported that sera from five of nine 

vaccinees resulted in Western blot banding profiles 

that were identical to F tularensis lipopolysaccharide.90

Investigations focused on identifying protective antigens are ongoing, particularly in animal models.12

Unfortunately, the antigens that induce humoral immunity appear to be different than antigens inducing 

cell-mediated immunity, making determinations of 

the most immunogenic antigen challenging.94 The 

ultimate goal of these investigations is to optimize 

the cell-mediated immune response to F tularensis, 

thereby suggesting improvements to prophylactic and 

therapeutic strategies.

The lipopolysaccharide structure of many gramnegative pathogens elicits a profound proinflammatory immune response, which can lead to the clinical 

manifestations of septic shock.96 However, although 

F tularensis lipopolysaccharide can elicit a strong humoral response, it does not induce significant tumor 

necrosis factor-α and nitric oxide production in macrophages or IL-1 from polymorphonuclear cells,97 in contrast to lipopolysaccharide from other gram-negative 

pathogens. Additionally, the lipopolysaccharide of F 

tularensis is structurally different in composition than 

typical gram-negative pathogens, which is believed 

to result in the poor Toll-like receptor 4 stimulation 

observed in type A and type B strains.98 The O-antigen 

of Francisella has also been shown to be required for 

virulence. The ability of Francisella to avoid complement mediated killing is dependent on the presence 

of O-antigen as F tularensis mutants deficient in Oantigen expression are more sensitive to complement.98

O-antigen was required for virulence of F tularensis in 

mice99 and also played a role in cytosolic survival by 

avoiding autophagy.100 

Clinical Manifestations

Tularemia has a diversity of clinical presentations, 

and it is likely that many cases are unrecognized, 

especially because of the diagnostic challenges associated with this infection.101 The disease manifestations 

of tularemia have been ascribed to at least 10 different 

clinical categories (ulceroglandular, glandular, oculoglandular, oropharyngeal, enteric, gastrointestinal, 

typhoidal, respiratory, pneumonic, and septic). Symptoms overlap among these categories.102 Alternatively, 

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Tularemia

Evans’ review of 88 tularemia cases more than 30 years 

ago describes two syndromes based on clinical signs 

(ulceroglandular or typhoidal), portal of entry, and 

disease prognosis. This categorization simplifies this 

often confusing nomenclature, while emphasizing the 

obscure but potentially fatal typhoidal presentation, 

and may also reflect differences in host response to F 

tularensis infection.103 With ulceroglandular tularemia, 

there is a vigorous inflammatory reaction, pneumonia 

is uncommon, and the patient rarely succumbs from 

infection. Typhoidal tularemia presents with few localizing manifestations, pneumonia is common, and 

mortality is higher in the absence of antimicrobial 

therapy.11,104

Typhoidal tularemia (15%–25% of cases) primarily 

occurs after infectious aerosol inhalation, but may 

also result from an intradermal or gastrointestinal 

infection.11,104 The disease presents as a nonspecific 

syndrome with an abrupt onset of fever (38°C to 

40°C), headache, malaise, myalgias, and prostration, 

but without lymphadenopathy.11 Lymph nodes are 

less than 1 cm in diameter, and no skin or mucous 

membrane lesions are present. Nausea, vomiting, diarrhea, and abdominal pain may also occur. Mortality 

is greater with pneumonia.11 Case fatality rates are 

approximately 35% in untreated naturally acquired 

typhoidal tularemia.102 Untreated tularemia survivors 

may have persistent symptoms for weeks or months 

with progressive debilitation.102

Ulceroglandular tularemia (75%–85% of naturally 

occurring disease) most often occurs through mucous 

membrane or skin inoculation with blood or tissue 

fluids of infected animals.104 Clinical symptoms in 

cases of ulceroglandular tularemia typically appear 

after an incubation period of 3 to 6 days. The lesions 

present on the skin or mucous membranes (including conjunctiva, oropharynx, etc) and lymph nodes 

are greater than 1 cm in diameter.11 This form of the 

disease is characterized by sudden onset of fever 

(85% of cases), chills (52% of cases), headache (45% 

of cases), cough (38% of cases), and myalgias (31% of 

cases), along with the emergence of a painful papule 

at the inoculation site.104 The fever may be associated 

with pulse-temperature disassociation (42% of cases 

in one series)104 where the pulse increases fewer than 

10 beats per minute per 1°F increase in temperature 

above normal. However, this finding is not specific for 

tularemia. Other nonspecific complaints include chest 

pain, vomiting, arthralgia, sore throat, abdominal pain, 

diarrhea, dysuria, back pain, and nuchal rigidity.102,104

A rapid progression occurs at the site of inoculation in 

the untreated patient, with pustule formation leading 

to a painful ulcer, accompanied by regional painful 

lymphadenopathy. A persistent ulcer is the hallmark 

of ulceroglandular tularemia. Ulcers generally range 

in size from 0.4 cm to 3.0 cm and occasionally have 

raised borders. 

The location of the lesion may provide an indirect 

clue as to the route of exposure: inoculation from an 

arthropod vector, such as a tick, is more likely on the 

lower extremities, and exposure to a mammal with 

tularemia tends to cause lesions on the upper extremities.104 Lesions are typically associated with regional 

lymphadenopathy, and a lack of lymphadenopathy 

may suggest another etiologic agent.104 Enlarged 

lymph nodes can occur singly, in groups, or enlarged 

in a sequential fashion along the lymphatic tracts 

(sporotrichoid pattern). The lymph node is typically painful and may precede, occur simultaneously, 

or follow the appearance of the cutaneous ulcer in 

ulceroglandular disease.102 Enlarged lymph nodes 

may become fluctuant and spontaneously drain. If 

untreated, these fluctuant lymph nodes may persist 

for months or years.102 The ulceroglandular form in the 

eye (oculoglandular) presents with ocular erythema 

and exudative conjunctivitis as key distinguishing 

features. The mechanism of exposure is usually from 

contact with infected mammals. 

One case report describes infection after tick 

removal; the tick contents were inadvertently inoculated into the eye.105 Food and water contamination 

can also lead to oculoglandular infection.34 In one 

series pharyngitis was observed in 24% of patients 

with tularemia.104 Possible findings on examination 

include erythema, exudates, petechiae, hemorrhage, 

or ulceration. Other findings may include retropharyngeal abscess or suppuration of the regional lymph 

nodes. Severe exudative pharyngitis suggests ingestion of contaminated food or water as the likely source 

of infection. The appearance of pharyngitis may be 

linked to lower respiratory tract disease, or possibly 

to ingestion as the route of exposure. Oropharyngeal 

signs and symptoms and cervical adenitis have been 

the primary manifestation of recent outbreaks in 

Turkey (83% of cases)37 and Bulgaria (89% of cases),34

and these outbreaks appear to be associated with a 

contaminated water source.

The overall incidence of symptoms of lower respiratory tract disease in patients with tularemia is 

high, ranging from 47% to 94%.52,104 These percentages are influenced by the route of exposure and the 

diagnostic approach to a patient with tularemia. The 

routine use of chest radiographs increases the likelihood of detecting mild or asymptomatic respiratory 

infections. Additionally, case series may only involve 

patients who are hospitalized, or receive a thorough 

evaluation, and may not include milder case presentations. Pneumonic tularemia can result from cases 

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Medical Aspects of Biological Warfare 

of ulceroglandular tularemia through hematogenous 

spread, with an onset ranging from a few days to 

months after the appearance of initial nonpulmonary 

symptoms.52 Approximately 30% of patients with 

ulceroglandular disease and 80% of patients with 

typhoidal tularemia also have pulmonary signs and

symptoms consistent with pneumonia.104 Pneumonic 

tularemia can also occur from direct inhalation of the 

organism, which has been demonstrated in human 

experimental models.56,106 In experimental infections 

of humans, cases were characterized by abrupt onset 

of fever, headache, sore throat, malaise, myalgias, 

coryza, and cough, which was typically nonproductive.106 Chest radiographic findings in pneumonic tularemia are highly variable and nonspecific107 because 

they can mimic findings associated with other clinical 

syndromes such as bacterial pneumonias, tuberculosis, lymphoma, or lung carcinoma.108 Patients can 

have infiltrates consistent with pneumonia and hilar 

adenopathy. In patients with pneumonia, 15% have 

an associated pleural effusion. Other less common 

findings include interstitial infiltrates, cavitary lesions, 

and bronchopleural fistulas.

A pneumonic tularemia outbreak in Martha’s 

Vineyard, Massachusetts, provides an instructive 

example of tularemia’s diagnostic challenges. The 

index case was a Connecticut resident with a second 

home at Martha’s Vineyard. His family physician in 

Connecticut empirically treated this case of “summer 

pneumonia.” Hospital clinicians in Martha’s Vineyard 

noticed the outbreak more than a month later while 

searching for the cause of another pneumonic summer 

illness.46,109 After seeing news accounts of the Martha’s 

Vineyard tularemia outbreak, the Connecticut man 

reported to Connecticut health authorities with a history of symptoms, exposure risk, and laboratory tests 

compatible with tularemia. 

Other examples of pneumonic tularemia have 

presented as diagnostic challenges. In 1994, a California case of community-acquired pneumonia was 

recognized as typhoidal tularemia in a 78-year-old 

with an absence of any epidemiological association 

for the illness.110 A decade earlier, of the 96 patients 

with tularemia presenting to a Veteran’s Hospital in 

Arkansas, five had pneumonic tularemia.111

The clinical manifestations of typhoidal and septic 

forms of tularemia overlap. Septic tularemia can be 

considered the result of clinical progression of any of 

the other forms of tularemia to a state of septic shock. 

Typhoidal tularemia presents as a nonspecific febrile 

syndrome, with or without lymphadenopathy, that can 

lead to death if untreated.108 This presentation mimics 

an extensive number of other disease entities, making 

the diagnosis challenging. A wide range of additional 

clinical manifestations has been described with all 

forms of tularemia, including pericarditis, enteritis, 

appendicitis, peritonitis, erythema nodosum, and 

meningitis.101,104,112

The laboratory findings with tularemia are nonspecific. Hemoglobin and platelet counts are typically normal, but anemia has been associated with 

disease. White blood cell counts are usually only 

mildly elevated, with no alteration in the normal cell 

differential.104 Microscopic pyuria may be observed104

and nonspecific inflammatory markers such as erythrocyte sedimentation rate and C-reactive protein can 

be elevated. One case series described tularemia associated with skeletal muscle abscesses, elevated creatine 

kinase, and rhabdomyolysis.113 Nonspecific elevations 

of liver transaminases and alkaline phosphatase may 

be observed with tularemia. The cerebrospinal fluid 

is usually normal, but may have mildly abnormal 

glucose, protein, and cell counts.104

Untreated tularemia patients usually have a prolonged illness lasting for months. The disease can be 

fatal, although rarely in ulceroglandular tularemia 

with antibiotic intervention. Before the use of streptomycin for therapy, tularemia—particularly the typhoidal form—had a mortality rate of 33%.102 No specific 

infection control practices are recommended for tularemia, other than universal precautions, because no 

documented cases of human-to-human transmission 

exist.1 However, special precautions are needed for 

clinical microbiology laboratory workers because of 

the high incidence of laboratory-acquired infections114

(see Issues for Laboratory Workers). 

Diagnosis

The diagnosis of tularemia is difficult because 

the clinical presentations of the various forms are 

not specific and are consistent with several other 

syndromes. This nonspecific presentation combined 

with a low incidence rate may have the unintended 

consequence of excluding tularemia as a differential 

diagnosis. This situation is exemplified by a review 

of cases in Missouri, a known focal point of infection in the United States, where more than half of 

the documented tularemia infections were misdiagnosed as other infectious diseases.115 Additionally, 

the diagnostic modalities available for isolation and 

identification of F tularensis have limitations. In a 

scenario in which F tularensis is used as a biological 

weapon, a rapid increase in pneumonic cases may be 

the initial clue implicating a biological weapon attack. 

In this scenario, either astute clinical judgment116 or 

epidemiological syndromic surveillance117 would be 

useful in detecting the attack.

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Bacterial Culture Techniques

The diagnosis of tularemia by culture can be 

challenging because the organism grows poorly on 

routine culture medium. Although positive cultures 

have been obtained from the blood,118–120 blood 

cultures are typically negative, even in cases of severe disease.15 Similarly, cultures from ulcer sites, 

sputum, gastric washings, and pharyngeal and 

conjunctival exudates are also u s u a l l y negative.11

Occasionally, positive blood cultures have been observed in immunocompromised persons (infected 

with the less virulent subspecies holarctica), and 

recovery may be improved when blind subculture 

is conducted.120

F tularensis is difficult to grow using standard media and requires media supplemented with cysteine 

or other sources of sulfhydryl groups.121 Chocolate 

agar, charcoal yeast extract agar, and Thayer-Martin 

agar support the growth of F tularensis. F tularensis

colonies appear gray-white on chocolate or ThayerMartin agar (Figure 11-2). The organism is optimally grown in a CO2 incubator and tends to grow 

more slowly than bacteria routinely encountered 

in clinical practice typically taking 48 to 72 hours to 

discern. The fastidious growth characteristics of F 

tularensis can often make the diagnosis of tularemia 

difficult, particularly when only routine culture 

techniques are used. However, some strains of F 

tularensis (ie, novicida subspecies) do not have these 

fastidious growth requirements.122

When recovered from clinical specimens, the organism may be presumptively identified with traditional 

microbiology techniques and biochemical testing. 

Automated identification systems in microbiology 

laboratories should be avoided because they may 

create aerosols and often misidentify the pathogen. 

Presumptive or suspected F tularensis isolates should 

be referred to a specialized laboratory for definitive 

testing. 

Serology

Traditionally, tularemia diagnosis has been based 

on serology, with a 4-fold rise in antibody titer as an 

acceptable diagnostic criterion. When using a microagglutination test, levels of antibody may be measurable 

within 1 week after infection, although significant 

levels usually appear in 2 weeks. An agglutination titer 

of greater than 1:160 tends to be specific for F tularensis

infection. These criteria are used in a major case series 

on tularemia.104

The limitations of serologic diagnosis are as pertinent to tularemia as they are to other infections. This 

technique depends on obtaining acute and convalescent sera, which may not be practical, especially if the 

suspicion of tularemia is delayed because of a nonspecific presentation.123 Antibodies to F tularensis may 

cross-react with other bacteria, such as Brucella, Proteus, 

and Yersinia species, which decreases the specificity of 

serology-based assays. Antibiotic therapy can blunt the 

serologic response, which could mask the convalescent 

rise in titer needed to confirm the diagnosis. Finally, 

antibody levels against F tularensis can persist for years, 

so distinguishing between acute and remote infection 

may be difficult. For all of these reasons, the development of better diagnostic capabilities for tularemia has 

become imperative.1

Rapid Diagnostic Methods

The most promising recent development in tularemia diagnosis has been the application of polymerase

chain reaction (PCR) technology. F tularensis can be 

detected by standard PCR of the 16S rRNA gene124,125

and the genus-specific tul4 gene encoding a 17-kd 

membrane lipoprotein.125–128 Other PCR assays have 

been designed to target fopA, a locus encoding an 

outer membrane protein.126,129,130 PCR testing of tissue 

specimens has been performed with mouse models,131

rabbit tissue,132 and humans with ulceroglandular 

tularemia.125,133 However, PCR as a diagnostic test has 

some limitations. The limit of detection of F tularensis

in blood samples may be suboptimal because of the 

presence of PCR inhibitors11 or other unknown conFigure 11-2. Chocolate agar plate of Francisella tularensis. 

Photograph: Courtesy of Dr Larry Stauffer, Oregon State 

Public Health Laboratories, Centers for Disease Control and 

Prevention, Atlanta, Georgia, Public Health Image Library, 

Image 1912.

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Medical Aspects of Biological Warfare 

founding factors. Antigen-detection techniques have 

also been developed for F tularensis,

132,134 although 

extensive data on the specificity and sensitivity of 

these techniques have not been published. These 

techniques offer the potential of rapid detection, but 

have not been extensively used in human clinical case 

scenarios. Other assays to detect F tularensis have 

been studied, including capture enzyme-linked 

immunosorbent assays based on monoclonal antibodies specific for lipopolysaccharide and fluorescent antibody tests for detection in pathological 

samples.31,132

Treatment

Antibiotics usually provide curative therapy for 

tularemia, with resulting mortality rates of only 1% to 

2.5%.1,104 Mortality varies, depending on type of infection (ulceroglandular vs typhoidal), overall health of 

the infected individual, and rapidity after infection 

that antimicrobial therapy was initiated. Streptomycin 

has traditionally been used to treat tularemia, with 

individuals often demonstrating a clinical response 

within 48 hours of administration.1,11,135 Relapses with 

streptomycin rarely occur. Gentamicin or other aminoglycosides are thought to be as effective as streptomycin,136, 137 but no controlled trials have been reported. 

Beta-lactam antibiotics such as ceftriaxone136 are 

typically ineffective. 

Antibiotics other than the aminoglycosides have 

been proposed for treating tularemia. Tetracycline 

and doxycycline are effective, but are associated with 

a higher relapse rate than the aminoglycosides.1,123,136

Chloramphenicol is another alternative,1 but it is rarely 

used in the United States. The fluoroquinolones offer 

an additional treatment option,138–140 especially with 

the high bioavailability of oral preparations. Although 

extensive clinical data are lacking for the fluoroquinolones, one report of a tularemia outbreak resulting 

from F tularensis subspecies holoarctica in Spain noted 

a 5% failure rate for ciprofloxacin, compared to a 23% 

failure rate for streptomycin and 43% failure rate for 

doxycycline.123 However, the number of patients treated with streptomycin in this study was 94, compared to 

only 22 being treated with ciprofloxacin. Although the 

clinical effectiveness with fluoroquinolones has been 

demonstrated in mild to moderate cases resulting from 

F tularensis subspecies holoarctica, in severe cases a combination with gentamicin has been recommended.141

However, there is limited experience using fluoroquinolones to treat tularemia disease due to the 

more virulent F tularensis subspecies tularensis, but 

TABLE 11-1

ANTIBIOTICS FOR THE TREATMENT OF TULAREMIA*

Patient Group Preferred Antibiotic Dose Alternate Dose

Adults Streptomycin 1 g IM twice daily Doxycycline 100 mg IV twice daily

Gentamicin* 5 mg/kg IM or IV Ciprofloxacin* 400 mg IV twice daily

once daily

Chloramphenicol* 15 mg/kg IV four times a day

Children Streptomycin 15 mg/kg IM Doxycycline If weight is >45 kg, 100 mg IV twice 

twice daily daily; if weight is <45 kg, 2.2 mg/

kg IV twice daily

Gentamicin* 2.5 mg/kg IM or IV Ciprofloxacin* 15 mg/kg IV twice daily

three times daily

 Chloramphenicol* 15 mg/kg IV four times daily

Pregnant Women Gentamicin* 5 mg/kg IM or IV Doxycycline 100 mg IV twice daily

once daily

Streptomycin 1 g IM twice daily Ciprofloxacin† 400 mg IV twice daily

*Recommendations are from the Working Group on Civilian Biodefense, and assume a contained casualty setting. Recommendations would 

differ in a mass casualty scenario.

†Usage is not approved by the Food and Drug Administration.

IM: intramuscular.

IV: intravenous.

Data source: Dennis DT, Inglesby TV, Henderson DA, et al. Tularemia as a biological weapon: medical and public health management. 

JAMA. 2001;285:2763–2773.

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Tularemia

it has been used successfully in a case that relapsed 

after doxycycline.142 The use of combination antibiotic 

therapy has not been studied for severe tularemia 

cases, nor has the antimicrobial susceptibility of 

antibiotic-resistant strains been extensively studied. 

The treatment options are summarized in Table 11-1. 

The general recommendations for length of therapy depend on the antibiotic used. Aminoglycosides 

and ciprofloxacin are thought to have a low incidence 

of relapse and, therefore, a course of 10 days is recommended.1 For doxycycline and chloramphenicol, a 

longer course of 14 to 21 days is indicated.1

PROPHYLAXIS

Postexposure Prophylaxis

Recent consensus recommendations have addressed 

the issue of postexposure prophylaxis after the use of F 

tularensis in a biological attack.1 These recommendations 

have suggested that antibiotics are indicated, especially 

if the exposure is thought to be recent. Data from human challenge models have suggested that tetracycline 

can be used to prevent infection after exposure.143 In an 

experiment in which volunteers received tetracycline 

within 24 hours after airborne exposure to F tularensis, 

no tularemia symptoms were detected in 8 volunteers 

receiving 2 g per day for 14 days, or in 8 volunteers 

receiving 1 g per day for 28 days. In a group in the 

same experiment receiving 1 g per day for 15 days, 2 of 

10 volunteers developed symptoms after therapy was 

discontinued. Therefore, if patients can be treated in the 

early incubation period, oral therapy with either ciprofloxacin or doxycycline (a compound closely related 

to tetracycline) for 14 days is suggested. However, if 

the exposure is not detected immediately and it is suspected that individuals were exposed more than a few 

days ago, a ”fever watch” is recommended, involving 

self-monitoring for constitutional symptoms such as a 

fever or flu-like illness.1 Individuals who develop these 

symptoms should be presumptively treated as if they 

had tularemia. Consensus statements for postexposure 

prophylaxis are described in Table 11-2. 

Vaccination With Live Vaccine Strain

A live vaccine for F tularensis was first developed in 

the former Soviet Union in the 1930s and reportedly 

used to safely vaccinate millions of individuals.144 This 

vaccine, developed from a type B strain, was transferred in 1956 to the United States,145 where researchers 

Eigelsbach and Downs further characterized the strain 

designating it as the LVS of F tularensis. It is the only 

tularemia vaccine available in the United States 

and is currently in Food and Drug Administration 

Investigational New Drug status. This vaccine has 

been administered to thousands of recipients since 

the 1950s at the US Army Medical Research Institute 

of Infectious Diseases (USAMRIID). The vaccine is 

administered by a scarification process (similar to 

smallpox vaccination) to the volar surface of the 

forearm. A small papule forms initially, developing 

occasionally into a pustule and ulcer. Most vaccine recipients develop a minor scab, and few have systemic 

side effects. In human challenge studies, the vaccine 

protected against low to moderate-dose respiratory 

challenge and partially protected against high-dose 

respiratory challenge with virulent type A strains.56,106

Alternative vaccine strategies have been the focus of 

considerable research, but none of these candidate 

vaccines are ready for human use.

F tularensis LVS has been studied extensively in 

mice, but significant differences exist in the immune 

response of mice to this type B strain and the immune 

response of humans to type A strains. LVS can be fatal 

in mice when administered as an intraperitoneal injection, yet it can confer protective immunity if given as an 

intradermal injection.81 Intradermal administration of 

LVS can also protect mice from a lethal challenge dose 

TABLE 11-2

ANTIBIOTICS FOR POSTEXPOSURE 

PROPHYLAXIS*

Type of Preferred

Patient Antibiotic Therapy

Adult Doxycycline 100 mg orally twice daily

Ciprofloxacin† 500 mg orally twice daily

Children Doxycycline If weight is >45 kg, 100 mg 

 orally twice daily; if weight is 

 <45 kg, 2.2 mg/kg orally twice 

 daily

Ciprofloxacin† 15 mg/kg orally twice daily

Pregnant Ciprofloxacin† 500 mg orally twice daily

women

Doxycycline 100 mg orally twice daily

*Recommendations are from the Working Group on Civilian Biodefense.

†Usage is not approved by the Food and Drug Administration.

Data source: Dennis DT, Inglesby TV, Henderson DA, et al. Tularemia as a biological weapon: medical and public health management. 

JAMA. 2001;285:2763–2773.

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Medical Aspects of Biological Warfare 

of virulent strains of F tularensis. Mice can be protected 

from the virulent form of F tularensis as early as 2 to 

3 days after intradermal injection of LVS.146 Injections 

of bacterial DNA (as unmethylated CpG motifs) can 

also confer a similar early protective response.147 The 

prompt development of immunity after vaccination 

in mice suggests that the protective mechanisms are 

attributable to innate immunity80 because an adaptive 

response requires more time to develop. It is unknown 

whether the vaccine in humans induces an early immune response that is protective. This type of early 

protection after vaccination would be useful in the 

military environment because unexposed soldiers may 

be rapidly protected from further intentional use of F 

tularensis as a weapon.

The correlates of immune response to vaccination 

have been suggested by prior investigations, but are 

not definitively established. Before the use of LVS, a 

killed F tularensis vaccine was used.95 This vaccine was 

documented to elicit a serologic response, but was not 

protective. Markers of cell-mediated immunity, such as 

delayed-type hypersensitivity testing, have also been 

correlated with protection after vaccination.94

The LVS tularemia vaccine is offered at the special 

immunizations clinic at USAMRIID for laboratory 

workers at risk for exposure to F tularensis. This vaccine is efficacious, as documented in a human challenge model; however, this protection is not 100%, 

particularly at high-dose aerosol challenges.56,106 In 

addition, an epidemiological study showed that the 

incidence of typhoidal tularemia in laboratory workers 

decreased significantly after the introduction of vaccination with LVS.148 The primary disadvantages are the 

potential hazards associated with a live vaccine (such 

as severe infection in immunocompromised individuals) and the lack of effectiveness against high-dose 

respiratory challenge. For these reasons, there is much 

interest in the development of a subunit F tularensis

vaccine.11,12,149 Promising vaccine candidates are being 

explored.12,149,150

ISSUES FOR LABORATORY WORKERS

Tularemia is considered a significant hazard for 

laboratory workers.114 All experiments that involve 

using F tularensis subspecies tularensis and fully virulent F tularensis subspecies holarctica strains should 

be conducted under biosafety level 3 conditions. 

Additionally, vaccination of at-risk personnel may 

diminish clinical manifestations of laboratory-acquired infections. A retrospective review of tularemia 

cases at USAMRIID was conducted. This study documented that typhoidal tularemia incidence dropped 

substantially after the LVS was offered to at-risk 

laboratory workers. Incidence rates decreased from 

5.70 to 0.27 cases per 1,000 at-risk employee-years.148

The occurrence of ulceroglandular tularemia did 

not decline significantly (from 0.76 to 0.54 cases per 

1,000 at-risk employee-years), but milder symptoms 

were observed in the recipients of the LVS vaccine.148

Another review of occupational exposures at USAMRIID suggested that the incidence of tularemia (15 

cases/year) did not decrease with the introduction 

of biosafety cabinets, but did decline after LVS vaccination was introduced.151

USE OF TULAREMIA AS A BIOLOGICAL WEAPON

F tularensis could be used as a biological weapon 

in many scenarios, causing varying degrees of casualties. The most dangerous scenario involves an aerosol 

release with large numbers of persons exposed. Additional complications would result if an antibioticresistant strain—as is claimed to have been developed 

in the former Soviet Union—was used.2

Researchers have estimated that a large-scale aerosol release of 50 kg over a large metropolitan area 

could cause 250,000 incapacitating casualties.29 Most 

of those affected could present with a nonspecific 

febrile illness 3 to 5 days after exposure (range: 1–14 

days, depending on the inoculum of exposure), and 

would subsequently develop pulmonary symptoms 

consistent with pneumonic tularemia.1 However, because of the aforementioned difficulties in tularemia 

diagnosis and the nonspecific clinical presentation, 

the determination of F tularensis as the causative agent 

may be delayed. The initial presentation of cases may 

be difficult to distinguish from a natural influenza 

outbreak or other respiratory pathogens.1

F tularensis may also be confused with another biological weapon. Epidemiological clues to distinguish 

tularemia from plague or anthrax are the clinical course 

of disease (slower with tularemia), case fatality rate 

(higher with plague152 or anthrax153), and possibly the 

pattern of pulmonary manifestations observed on 

chest radiograph, such as the large pleural effusions 

and mediastinal widening characteristic of inhalational 

anthrax.154 Pulmonary tularemia may be difficult to 

distinguish from Q fever, another potential biological 

weapon agent.

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SUMMARY

care professionals to minimize the impact of its use. 

Although the current LVS vaccine provides some 

protection against clinical disease associated with F 

tularensis, much interest remains in the development 

of a more effective vaccine. Further research will likely 

continue to elucidate the pathogenesis of this organism and yield improved preventive, diagnostic, and 

therapeutic options.

F tularensis constitutes a substantial threat as a 

biological weapon. The variety of clinical manifestations of F tularensis infection and the benefits of early 

antibiotic intervention necessitate a high degree of 

suspicion from healthcare providers. Familiarization with the variety of epidemiological and clinical 

manifestations of this disease, along with available 

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108. Tarnvik A, Berglund L. Tularaemia. Eur Respir J. 2003;21:361–373.

109. Dembek ZF, Buckman RL, Fowler SK, Hadler JL. Missed sentinel case of naturally occurring pneumonic tularemia 

outbreak: lessons for detection of bioterrorism. J Am Board Fam Pract. 2003;16:339–342.

110. Fredricks DN, Remington JS. Tularemia presenting as community-acquired pneumonia: implications in the era of 

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114. Evans ME. Francisella tularensis. Infect Control. 1985;6:381–383.

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Q Fever

Chapter 12

Q FEVER

CHRISTINA M. FARRIS, PhD,* and JAMES E. SAMUEL, PhD†

INTRODUCTION AND HISTORICAL PERSPECTIVES

MILITARY RELEVANCE

World War II

Gulf War

Operation Enduring Freedom and Operation Iraqi Freedom

INFECTIOUS AGENT

Pathogenesis

Epidemiology

DISEASE

Humans (Q Fever)

Animals (Coxiellosis)

DIAGNOSIS

TREATMENT

VACCINE

M-44

Chloroform:Methanol Residue

Q-Vax

SUMMARY

*Lieutenant, Medical Service Corps, US Navy; Viral and Rickettsial Diseases Department, Naval Medical Research Center, 503 Robert Grant Avenue, 

Silver Spring, Maryland 20910

†

Professor and Chair, Department of Microbial Pathogenesis and Immunology, College of Medicine, Texas A&M Health Science Center, 8447 State 

Highway 47, Medical Research and Education Building, Building 3112, Bryan, Texas 77807

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INTRODUCTION AND HISTORICAL PERSPECTIVES

Q fever is a zoonotic infection with almost worldwide distribution, characterized by sudden fever, 

headache, and atypical pneumonia, and which, in 

some cases, results in chronic disease. The causative 

agent of Q fever is the gram-negative bacterium Coxiella 

burnetii. In two separate but concurrent instances, a 

new disease and the causative agent were discovered 

on different continents. An illness of unknown etiology 

struck slaughterhouse workers in Queensland, Australia, in 1933.1,2 Symptoms included fever, headache, 

and malaise, but serological tests for all suspected 

infectious agents were negative. This led to the designation as query, or “Q,” fever. Attempts to culture the 

agent on bacterial medium proved fruitless; however, 

scientists were able to demonstrate the transmissibility 

of the agent by inoculating guinea pigs with blood and 

urine from infected patients. It seemed likely that the 

causative agent was viral.

Around the same time in the United States, scientists 

in Montana were investigating a filter-passing agent 

isolated from ticks collected in the Nine Mile Creek 

area near Missoula, Montana. Initially looking for the 

agent of Rocky Mountain Spotted Fever, they found 

something new that did not cross-react with sera from 

patients infected with Rickettsia rickettsii: the causative 

agent of Rocky Mountain Spotted Fever, or any other 

known rickettsial agents.3,4 When placed on guinea 

pigs, the ticks infected with this unknown agent caused 

febrile illness, which was transmissible to other guinea 

pigs by injection of blood from the infected animals. 

Examination of the inflammatory cells revealed that 

the agent was rickettsia-like.4 Although the researchers in Montana were also unable to culture their agent 

on traditional bacterial media, they discovered that 

they were able to culture it in chicken embryos.5 This 

discovery was a significant breakthrough not only for 

studying the Nine Mile agent, but also for the study 

of all rickettsial organisms. The scientists in Montana 

now had a Rickettsia-like microorganism that they 

had demonstrated to be infectious, but whether it 

caused disease in humans remained unknown until 

a researcher from the National Institutes of Health 

visited the public health laboratory in Hamilton, 

Montana, and became ill after working with the isolate 

from Nine Mile Creek. When blood from the researcher 

was injected into guinea pigs, the same febrile illness 

that resulted from the infected ticks was produced. 

After considerable effort by researchers in Montana 

and Washington, DC, a correlation was made between 

the agent found in the Nine Mile ticks and the disease 

that manifested in the visiting scientist. Serum from the 

visiting scientist was found to protect against infection 

by Nine Mile ticks in guinea pigs.2

The similarities between Q fever and the disease 

caused by the filter-passing agent found in the ticks 

from Nine Mile did not go unnoticed. In 1938, Dr Frank 

Macfarlane Burnet, who, along with Doctors Edward 

Holbrook Derrick and Mavis Freeman, published on 

Q fever in Australia, sent mouse spleens infected with 

Q fever agent to the National Institutes of Health in 

Washington, DC. The spleens were used to pass the 

infection to guinea pigs; the guinea pigs infected with 

Q fever were subsequently immune to the Nine Mile 

agent, but not the agents of Rocky Mountain Spotted Fever.2

 After issues arose with its source, Burnet’s Q fever 

sample was lost before any further comparisons could 

be made; however, the similarities between the infections and immunity observed against the Nine Mile in 

guinea pigs that had recovered from Q fever infection 

indicated these two agents were one and the same.

Due to the similarities between the Q fever agent 

and members of the Rickettsia genus, it was originally 

classified as Rickettsia diaporica,

6

 and then later Rickettsia burnetii,7

 in honor of Dr Burnet. In 1948, R burnetii

was reclassified to its own genus named in honor of Dr 

Cox and became known as Coxiella burnetii.

8

 Q fever’s 

prevalence in the world was soon discovered; to date, it 

has been found in every country except New Zealand.

MILITARY RELEVANCE

Fevers of unknown origin and atypical pneumonia 

have plagued troops throughout history. Even as late 

as World War II (WWII), it was common for infectious 

disease to sideline significant numbers of troops and 

impact the outcome of battles. Because Q fever was 

identified just prior to WWII, it was during this war 

that the impact of the disease was first noted, though 

likely not understood to the full extent because of the 

nonspecific nature of the symptoms. While Q fever 

does not have a high mortality rate, it has the potential to debilitate large numbers of troops for extended 

periods of time. 

World War II

Service members from both the Allied and the Axis 

forces sustained Q fever outbreaks during WWII. German troops were plagued with outbreaks of an atypical 

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pneumonia they called “balkengrippe” in northern 

Yugoslavia, Serbia, Bulgaria, Italy, Crimea, Greece, 

Ukraine, and Corsica in 1941.9 The outbreaks were not 

well contained and a single consulting physician for the 

German army saw over 1,000 cases of balkengrippe. 

Although the mortality rate of Q fever is not high, the 

impact of these outbreaks was not insignificant; the 

minimum absence from duty for infected individuals 

was more than 6 weeks. Similar outbreaks occurred in 

1942, 1943, and 1944, including a very severe outbreak 

in Swiss troops during which half of the soldiers in 

two battalion subunits developed pneumonia. As the 

balance of power shifted and Germany was pushed 

out of its previously conquered territories, the Allied 

troops now occupying these areas began to experience outbreaks of the same atypical pneumonia that 

had been sidelining German troops in previous years. 

From February to April 1945, there were 511 cases of 

“primary atypical pneumonia” admitted to British and 

New Zealand military hospitals near Naples, Italy. 

From winter 1944 to spring 1945, there were nine significant outbreaks of atypical pneumonia reported in 

Allied troops in Greece, Italy, and Corsica.9 Although 

most of the outbreaks of atypical pneumonia were 

never confirmed to be Q fever during the acute stage 

of infection, serological analysis of select individuals 

up to 2 years after infection revealed high antibody 

titers against C burnetii.

9

In the years following WWII, there were several 

small outbreaks of Q fever in military personnel. Between 1951 and 1958, there were three outbreaks of Q 

fever in Libya, Algeria, and the Isle of Man that impacted US, French, and British units, respectively. There 

were no further reports until an outbreak among British 

soldiers in Cyprus in 1974 that affected 78 soldiers.10,11

Gulf War

There were very few confirmed cases of Q fever in 

American service members during the Persian Gulf 

War. Only one case of acute illness was attributed to 

Q fever during this campaign: a severe case of meningoencephalitis associated with acute Q fever in a 

soldier following deployment.12,13 Three additional 

soldiers within the same battalion as the afflicted 

soldier seroconverted during the same period, as 

evidenced by subsequent testing.14 This small number of confirmed cases should not be taken as an 

indication that Q fever did not affect other military 

personnel or that it was absent in the environment. 

The nondescript nature of the symptoms associated 

with Q fever generally lead to gross under estimations of the disease.

Operation Enduring Freedom and Operation Iraqi 

Freedom

The impact of Q fever on military personnel in 

Iraq was realized starting in 2003.15 To date, there 

have been over 150 cases of Q fever in US military 

personnel in Iraq and Afghanistan.16–23 An evaluation of pre- and post-deployment sera of US military 

personnel hospitalized with symptoms consistent 

with acute Q fever between April 2003 and December 2004 revealed a seroconversion rate of 10%.24 In 

addition to pneumonia, the infected personnel also 

presented with hepatitis, high fever, cholecytitis, and 

meningoencephalitis. In May of 2010, the Centers for 

Disease Control and Prevention issued an official 

health advisory detailing the potential for Q fever in 

travelers returning from Iraq.25

INFECTIOUS AGENT

C burnetii is classified phylogenetically within the 

gamma subdivision of proteobacterium in the Legionellales order, with several unique properties.26 It is a 

pleomorphic coccobacillus with a gram-negative cell 

wall and replicates within phagolysosome-like parasitophorous vacuoles (PVs) of eukaryotic cells. Replication depends on trafficking to a PV with low pH; this 

“biochemical stratagem” as an acidophile27 involves 

developmental cycle forms with metabolically active 

large cell variants and metabolically quiescent small 

cell variants.28–30 The type isolate, Nine Mile (RSA493), 

was found to contain few highly degraded genes but a 

large accumulation of point mutations, leading to the 

hypothesis that C burnetii is in an early stage of reductive evolution.31 This is in contrast to other obligate 

intracellular pathogens, like Rickettsia and Chlamydia, 

that appear to have undergone massive genome fragmentation and reduction since their separation from 

free-living organisms. Coxiella genomes are composed 

of one chromosome and one large plasmid or plasmidrelated sequences integrated into the chromosome.32–34

Sequencing genomes of four isolates identified approximately 2,150 to 2,300 open reading frames per 

genome and a high degree of homology among phylogenetically distinct isolate groups, with most major 

genetic variation resulting from transposon-mediated 

rearrangements.35

C burnetii undergoes a phase variation population 

shift where virulent phase I converts to avirulent 

phase II upon serial passage in a nonimmunologically 

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competent host.36 Phase I organisms have “smooth” 

lipopolysaccharides (LPS) with complete O antigen, 

while phase II have a “rough” LPS, missing, at a 

minimum, O-antigen sugars. Two clonal isolates of 

the Nine-Mile-type strain—phase I, RSA493 (clone 7) 

and phase II, RSA439 (clone 4)—have been used for 

comparative analysis to demonstrate the requirement 

for O-antigen expression for virulence. The phase II 

isolate has a well-defined genetic deletion (~ 20 Kbp),37

which encodes O-antigen LPS biosynthesis and will 

not revert to wild-type. This clone 4 has been exempted 

from Centers for Disease Control and Prevention’s 

select agent regulation and requires only biosafety 

level 2 biocontainment. 

C burnetii are primarily found in cells of the reticuloendothelial lineage, especially monocytes, 

macrophages, and polymorphonuclear cells during 

infection. Intracellular interactions have been modeled 

in vitro in a variety of continuous and primary cell 

lines, including L929 mouse fibroblasts, Vero (African 

green monkey kidney), J774 mouse macrophages, 

THP-1 human monocytes/macrophages, primary human peripheral blood mononuclear cells, and mouse 

bone-marrow-derived macrophages. The following 

models of the essential stages of uptake and survival 

have emerged from these studies.38 C burnetii is taken 

up via complement receptor 3 and alpha V beta 3 

integrin-mediated mechanisms into human macrophages.39 The adhesins recognized by the host receptors are uncharacterized, but complement receptor 3 

uptake appears dependent on loss of LPS O antigen.39

An actin-dependent, endocytic mechanism of uptake 

leads to trafficking through an early endosome, progressing to a late endosome/phagolysosome PV.40 The 

trafficking appears slightly delayed compared to latex 

bead uptake41 and membrane markers for Rab5, Rab7, 

Rab24, LC3, lysosomal-associated membrane protein 

(LAMP)-1, LAMP-2, and LAMP-3, and flotillin 1 and 

2 progressively decorate the PV.42–45 Connection to the 

autophagosome compartment appears essential to 

support a productive replication compartment, and 

PV membrane development to a spacious vacuole requires access to continual cholesterol biosynthesis.46

The PV biogenesis process requires de novo synthesis 

of C burnetii proteins, suggesting actively expressed 

bacterial factors in the modulation of host processes.44

Infection of activated macrophages appears to favor 

pro-survival stimulation through Akt and extracellular 

signal-regulated kinases (Erks) 1 and 2 and restriction of proapoptotic events in a caspase-dependent 

manner.47 Alternatively, the interaction with pro- and 

antiapoptotic host factors appears to be additionally 

complicated based on the observations that infected 

monocytic THP1 cells undergo a caspase-independent 

apoptosis soon after infection that may be mediated, 

in part, by tumor necrosis factor and require protein 

synthesis by C burnetii.

48,49 Infection of monocytes or 

macrophages does not result in subsequent activation, 

and several pathogen-associated common molecules 

appear modified to avoid serving as agonists for tolllike receptor recognition, including the lipid A of LPS 

that contains a tetraacylated structure with antagonistic activity for toll-like receptor 4.50 When taken up by 

polymorphonuclear leukocytes, an incompletely defined, secreted acid phosphatase prevents release of reactive oxygen intermediates through the nicotinamide 

adenine dinucleotide phosphate-oxidase pathway.51,52

This avoidance of activating macrophages appears to 

be a key pathogenic strategy, and hypersensitivity to 

oxidative stress suggests avoidance of reactive oxygen 

intermediates as well as detoxification as evolutionary 

pressures.53,54 Through reductive evolution, several 

virulent isolates have lost expression of a functional 

catalase or secreted superoxide dismutase without 

a loss of virulence, and the organism requires very 

low iron levels, in part, as a strategy to avoid Fenton 

chemistry.53,55

The model of pathogen–host interaction suggests 

that C burnetii actively remodels the host cell to establish a productive intracellular niche, and two secretion 

systems are likely key to the effector molecule release 

that mediates this remodeling. Virulent isolates with 

acute disease-causing potential appear to encode a 

functional type IV pilus-structured, type II secretion 

system to release several enzymes, including acid 

phosphatase, phospholipases D and A1, copper/zinc 

superoxide dismutase, chitin, and a family of enhanced 

entry proteins.55 C burnetii also encode and express 

a type IVB secretion system with striking similarity 

to the defective in organelle trafficking/intracellular 

multiplication (dot/icm) system (type IV secretion 

system [T4SS]) of L pneumophila.

31,56 It encodes 23 of the 

26 dot/icm proteins, lacking homologs of the chaperone IcmR and the inner membrane proteins DotJ and 

DotV.31,56,57 C burnetii dotB, icmS, icmW, and icmT are 

able to complement these mutations in Legionella,

58,59

while icmX, icmQ, dotM, dotL, dotN, and dotO do not 

complement.59 These results suggest strong functional 

similarities as well as unique properties associated 

with each system. The Legionella dot/icm system has 

identified over 300 substrate effector proteins using 

a variety of approaches, and most of these effectors 

have been shown to subvert some step in the host 

cell process connected with replication in its unique 

niche.60 Interestingly, although not surprising given 

the PV of Legionella and Coxiella are so dramatically 

distinct, C burnetii encodes relatively few homologs 

of the Legionella effectors. This suggests that although 

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C burnetii has maintained a functional dot/icm-related 

T4SS, the effectors, which are substrates for this secretion system, are almost entirely unique to C burnetii. 

Pursuit of molecular pathogenesis studies for C 

burnetii has dramatically advanced with the development of the extracellular growth media acidified 

citrate cysteine media under microaerophilic conditions and the advent of genetic tools to randomly and 

site-specifically mutagenize and rescue mutant phenotypes using complementation methods.61–66 Using 

these techniques, the isolation of T4SS mutants verified 

that, like Legionella, the dot/icm system of Coxiella is 

essential for intracellular replication but not growth 

in artificial media.67,68 The identification of specific 

effectors released via T4SS has begun to determine 

which effectors are essential for this replication and 

potentially many additional Coxiella-unique virulence 

properties. Among potential type IV secreted effector 

molecules are a diverse family of pathotype-specific 

ankyrin repeat-containing proteins.57,69 Ankyrin repeat 

domains (Anks) are commonly found in eukaryotic 

systems to mediate protein-to-protein and proteinto-DNA interactions and may be involved in host 

modulation events. Like Legionella, some redundancy 

appears to exist among Coxiella secretion substrates, 

as three substrates, ankyrin repeat-containing protein 

G (AnkG), C burnetii anti-apoptotic effector A protein 

(CaeA), and C burnetii anti-apoptotic effector B protein 

(CaeB), appear to act to promote host cell viability by 

modulating apoptosis. Recent studies suggest that 

AnkG binds the proapoptotic protein p32 to inhibit 

apoptosis.70 Either redundantly or in support of AnkG, 

CaeB inhibits apoptosis through a mitochondrial 

pathway, whereas the nuclear effector CaeA was also 

noted to block apoptosis.71 A variety of essential host 

manipulations via T4SS substrate/effectors released 

into the host cytoplasm will likely be identified to 

elucidate the intimate relationship between host and 

pathogen in this exquisitely adapted agent.

Pathogenesis

The pathogenesis of Q fever in humans is not well 

studied, and knowledge of acute C burnetii infection 

has been elucidated primarily in animal models. 

Upon inoculation, C burnetii are engulfed by resident 

macrophages and transported systemically. Alveolar 

macrophages have been identified as the resident 

cells that are primarily infected upon aerosol infection.38,72,73 C burnetii grows and replicates within these 

macrophages and then bursts the cell, resulting in 

release and the subsequent infection of other phagocytic cells. In mice and guinea pigs, the spleen and 

liver are the most heavily burdened organs, and it is 

assumed that this is the same in human infection.74

Chronic Q fever is far more complex. In chronic infection, reactivation of the microorganisms is possible 

years after the initial infection. Studies in guinea 

pigs and mice have demonstrated that these animals 

remain infected throughout their lives, but growth 

is uncontrolled during parturition and other periods 

of immunosuppression. Immunosuppressed animals 

have also been used to model chronic infection,75 as 

have mice that are altered genetically to over produce 

the cytokine interleukin 10.76 The early prediction that 

phylogenetic groups are uniquely virulent to cause 

either acute or chronic disease in humans is supported by studies that demonstrate in acute disease 

animal models that pathotypes are distinct in their 

ability to cause acute inflammatory disease.77 Yet, 

there remains much to learn about chronic infection, 

including where the microorganisms persist during 

periods of latency and if the microorganisms are ever 

cleared from the body.

Epidemiology

Q fever is a worldwide zoonotic infection found in 

every country, with the exception of New Zealand. C 

burnetii is able to infect a wide range of species, but 

symptomatic infection is only found in humans. Occupational exposure is the primary source of human 

infection, with the vast majority of cases occurring in 

abattoir (slaughter house) workers, farmers/ranchers, 

and veterinarians. Although Q fever is primarily a 

problem in rural areas with domestic animals such as 

cattle, sheep, and goats as the primary sources, domestic 

pets can spread infection in urban areas, though at a 

much lower rate.78,79 Infected animals shed C burnetii in 

urine, feces, milk, and, in highest concentration, in the 

placenta and other materials that are released during 

birth. C burnetii is incredibly stable in the environment 

and can persist as dried infectious particles for months 

or even years, perpetuating the infectious cycle.80,81 This 

can also result in infections in individuals indirectly associated with the infected animals, such as those living 

in the area, because infectious particles can be carried 

by the wind. Although infectious aerosols are the most 

common source of infection in humans, bites by infected 

ticks and consumption of contaminated milk are also 

associated with infection82 (though it is possible that 

long-term consumption of contaminated milk may 

result in seroconversion without causing disease).

C burnetii is one of the most infectious organisms 

known, with an infectious dose of fewer than 10 microorganisms, and possibly as low as 1 microorganism.83,84

Routes of infection include aerosol, ingestion, and, 

rarely, human to human. The route of infection impacts 

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the manifestation of the disease; ingestion generally 

results in granulomatous hepatitis, while pneumonia is 

more common with aerosol transmission.85 C burnetii is 

ubiquitous in the environment, yet there has been little 

work done to estimate its seroprevalence around the 

world. In the United States, the estimated seroprevalence 

is around 3% but the number of reported cases is very 

low, indicating that the majority of cases do not produce 

clinical disease.19 Extensive studies were completed 

in Nova Scotia before 1990 that revealed a 14% seropositivity rate, but significantly fewer reported cases.86,87

Between 2007 and 2010, the Netherlands experienced one of the most significant Q fever outbreaks 

known. Approximately 4,000 cases of human Q 

fever were reported during this 3-year period, an 

exponential increase from the 1 to 32 cases per year 

that the country experienced in the years prior to the 

outbreak.88,89 The outbreak resulted in the culling of 

millions of domestic animals, which was a devastating 

loss to the farming industry. This outbreak highlights 

the potential for large-scale Q fever outbreaks even in 

highly developed countries.

DISEASE

Humans (Q Fever)

Q fever is a zoonotic infection that produces symptomatic infection only in humans. Even in humans, 

only approximately 50% of infected individuals develop clinical disease, and the mortality rate is less 

than 1%.1

 Typically, Q fever presents as an acute, selflimited, systemic infection after an incubation period 

ranging from a few days to several weeks; there is 

evidence that disease severity is directly related to the 

infectious dose.83,90,91 Symptoms, although acute, are 

typically nonspecific and may include fever, chills, severe headache warranting lumbar puncture,19 fatigue, 

pneumonia, myalgia, nausea, vomiting, diarrhea, chest 

pain, weight loss, and abdominal pain. Of infected 

individuals, 30% to 50% develop pneumonia, and it is 

not uncommon for patients to develop hepatitis. Less 

common clinical manifestations include acalculous 

cholecystitis, acute respiratory distress syndrome, 

gastroenteritis, myelitis, orchitis, epididymitis, pericarditis and myocarditis, and rhabdomyolysis, among 

others.12,17–20 In many cases, patients clear the infection 

without antibiotic intervention. 

In addition to acute infection, Q fever is also capable 

of inducing chronic infection. Chronic Q fever has the 

potential to manifest in excess of 20 years after the 

initial exposure and can persist for 6 or more months 

at a time. In its chronic form, Q fever is especially 

dangerous in patients with heart disease, as endocarditis is known to develop in conjunction with chronic 

disease. Immunocompromised patients, such as those 

undergoing immunosuppressive therapy, suffering 

from diseases such as AIDS, or receiving antirejection 

therapy after organ transplant are particularly at risk 

for developing chronic Q fever.92,93 The most common 

disease associated with chronic Q fever is endocarditis of the aortic and mitral valves, though it is not 

uncommon to see chronic hepatitis.94–97 A preexisting 

heart disease is a major risk factor for chronic Q fever. 

It is estimated that of those individuals that develop 

Q fever endocarditis, close to 90% have preexisting 

valvular disease96; as many as one third of all chronic 

Q fever patients with cardiac valve abnormalities go 

on to develop Q fever endocarditis.97 In patients with 

chronic Q fever, the immune responses necessary 

to fight the infection, such as T-cell responses, are 

not present. Patients have been observed to produce 

increased immunosuppressive cytokines, including 

tumor necrosis factor98 and interleukin 10.76,99 Immune 

response suppression is responsible for the persistence 

of the microorganism. 

Animals (Coxiellosis)

As a zoonosis, infection with C burnetii is termed 

“coxiellosis” and infects both wild and domestic 

animals. Q fever is especially common in domestic 

ruminants such as cattle, sheep, and goats. Unlike infection in humans, coxiellosis does not produce overt 

symptoms, and pathological changes are generally 

limited to the genital tract, manifesting as spontaneous abortion and fertility issues.100 Sheep appear 

to have only transient infection while the infection 

persists in other mammals.101 Infection reactivates 

in female mammals during pregnancy. As with infection in humans, animals are infected by aerosol 

transmission, tick bites, and milk (nursing). A 2005 

study demonstrated that over 90% of dairy cows in 

the northeastern United States are infected with C 

burnetii,

102 but pasteurization prevents human infection via consumption of contaminated milk. Although 

exposure via contaminated milk can be prevented by 

simply pasteurizing milk, infection by the aerosol 

route (ie, inhalation of dried infectious particles shed 

by infected animals) cannot be prevented. Without 

obvious symptoms, it is difficult to identify infected 

animals and therefore next to impossible to eradicate 

C burnetii from domestic animals. 

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Q Fever

DIAGNOSIS

Q fever cannot be diagnosed based on clinical 

symptoms because of the nonspecific presentation 

of symptoms; instead, diagnosis is made based on 

the combination of clinical signs and serological 

testing. History of exposure to animals or time 

spent on farms bolsters what is usually a presumptive diagnosis. Humoral responses are more consistently activated during Q fever infection than 

cellular responses and, as such, serological testing 

is considered the more reliable immunoassay for 

Q fever. The most commonly used assays are indirect immunofluorescence assay (IFA), complement 

fixation, microagglutination, and enzyme-linked 

immunofluorescence assay (ELISA).19,103

The gold standard for Q fever serological diagnosis is the IFA because it is highly sensitive and very 

specific, and does not require purified antigen.103,104

This method is very convenient for laboratories with 

limited space for equipment, but it is not feasible for 

testing large numbers of samples. Complement fixation is one of the most specific assays for diagnosing 

Q fever, but it lacks sensitivity and cannot detect 

specific antibody early in the course of an infection.105

Microagglutination is sensitive, but its use is hindered 

by the requirement for large amounts of antigen.103,106

ELISA rivals IFA in specificity and sensitivity, and is 

a platform conducive for analyzing large numbers 

of samples. Unlike IFA, however, ELISA requires 

highly purified antigen to achieve sensitivity and 

specificity.103,104,107

Phase variation plays an important role in 

serodiagnosis of Q fever. Acute and chronic Q 

fever produce characteristic, yet distinct, antibody 

profiles.108–111 During acute infection, antibody to 

the phase II, nonpathogenic organism is detectable before antibody to the phase I, pathogenic 

organism. Phase II antibody titers peak much 

higher than phase I antibody and remain elevated 

for years after infection, while phase I antibody 

titers wane shortly after infection. Individuals 

with chronic infection have an antibody profile 

exactly opposite the profile of individuals with 

acute infection. Chronic infection produces and 

sustains high titer phase I antibody, but much 

lower phase II antibody titers.

Although it is possible to diagnose Q fever based 

on bacterial culture, this method is not widely used. 

C burnetii is highly infectious and must be grown 

under biosafety level 3 conditions, a requirement 

that is not feasible at most medical treatment facilities. Culturing C burnetii from patient samples 

can be done by using the sample to infect research 

animals such as mice, or by infecting a monolayer of 

cells and subsequently staining and visualizing the 

cocci. These methods are very time consuming and 

are not conducive for large numbers of samples.103

The advent of the use of acidified citrate cysteine 

media65 for axenic culture of C burnetii will likely 

make culturing patient samples easier, but it seems 

unlikely that this will become the primary method 

for diagnosing Q fever. Detection by quantitative 

polymerase chain reaction is also a viable method 

and can be used to detect the bacteria in the infected 

individual much earlier than methods that rely on 

the production of antibody.112,113 Most of the information on the use of PCR for diagnosis have come 

from animal studies, but there has also been success 

detecting C burnetii in the buffy coat of citrated or 

ethylenediaminetetraacetic acid (EDTA)-treated 

blood.103 In the spring of 2011, the US Food and Drug 

Administration cleared the first test to diagnose Q 

fever in military personnel serving overseas. The 

nucleic acid amplification test by Idaho Technologies 

produces results within 4 hours.114

TREATMENT

Acute Q fever is easily treated. The treatment 

of choice is doxycycline, 100 mg twice daily for 14 

days.115 This treatment is not effective for chronic Q 

fever; instead, drug combinations such as doxycycline plus hydroxychloroquine are considered the 

most efficacious treatment in chronic cases. In cases 

with endocarditis, an 18-month regimen of 100 mg of 

doxycycline twice per day and 200 mg of chloroquine 

three times per day is effective.117 When chloroquine 

is not an option for a particular patient, a combination of 100 mg doxycycline twice per day and 200 mg 

ofloxacin three times per day is recommended for a 

period of 3 years.116 There is evidence of reactivation 

of disease when the drug regimens are shortened or 

not completed. Doxycycline is not bactericidal, but 

is an effective treatment for intracellular bacteria 

such as C burnetii and Chlamydia. The effectiveness 

of doxycycline is improved when used in combination with hydroxychloroquine and it is hypothesized 

that the hydroxychloroquine increases the pH of the 

phagolysosome, which would decrease the metabolic 

activity of C burnetii.

117

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Medical Aspects of Biological Warfare 

VACCINE

with great success, but studies in guinea pigs revealed 

that the organisms persisted in the animals and led to 

lesions in the heart, spleen, and liver, suggesting either 

reactivation or low-level contamination with phase I 

organisms.124

Chloroform:Methanol Residue

Another attempt to prevent the side effects of vaccination in the previously exposed individuals was to 

vaccinate with an extraction from phase I organisms, 

rather than the WCV. Scientists at the United States 

Army Medical Research Institute of Infectious Diseases 

vaccinated with the residue from chloroform and 

methanol extracted from phase I organisms that had 

been formalin inactivated. They have demonstrated 

that the vaccine is protective in animals, nontoxic, 

and immunogenic in humans.125,126 However, at high 

doses the residue is reactive, though to a lesser extent 

than whole cell vaccine. Studies using a low-dose 

prime-boost scheme have shown success in animals 

and induce protection without reaction.127

Q-Vax

Q-Vax (short for Q fever vaccine) is a WCV that is 

licensed for use in Australia from CSL Limited, a company based in Victoria, Australia; it is the most widely 

studied Q fever vaccine.128 Studies have demonstrated 

that this vaccine is 100% effective for more than 5 

years in individuals who are considered extremely 

at risk due to their occupation.128 Q-Vax, however, 

is hampered by the need for pretesting to determine 

prior immunity. It is currently not licensed for use in 

the United States.

SUMMARY

Q fever is a worldwide zoonotic disease with 

one of the most highly infectious causative agents 

known, C burnetii. Q fever significantly impacted 

wartime efforts as recently as the past few years, 

and as evidenced by the recent outbreak in the Netherlands, can produce prolific disease even in areas 

with sufficient preparation. The ubiquitous nature of 

C burnetii in the environment indicates that Q fever 

will be a concern for years to come; not only does it 

have the potential to produce disease in humans, it 

can also result in devastating losses to domestic animals. Extremely resistant to environmental stresses, 

C burnetii can persist for long periods of time in the 

environment.

Q fever is easily treated by antibiotics of the tetracycline class, though diagnosis can be difficult based 

on the nonspecific symptoms observed in Q fever 

patients. Physicians must rely on a combination of 

clinical presentation and serological testing to diagnose 

Q fever. Although the vaccine used in Australia is not 

available for use in the United States, efforts continue 

to find a safe and efficacious alternative.

The ability to create an efficacious vaccine has never 

been a problem in the field of Q fever research. The 

failure of most of these vaccines has been the inability to 

uncouple the protection and the accompanying adverse 

reactions. Within a few years of identifying C burnetii,

researchers had successfully developed an effective vaccine.118 The composition of the vaccine was crude and 

consisted of formalin-inactivated C burnetii extracted 

with ether from egg culture and was contaminated 

with 10% yolk sack. Knowledge of the phase variation 

that occurs in C burnetii came after this early vaccine 

was developed. It was later determined that vaccines 

consisting of phase I antigen are 100- to 300-fold more 

protective than those consisting of phase II antigen in 

guinea pigs.119 Whole cell vaccines (WCVs) consisting 

of formalin inactivated phase I C burnetii have been 

studied extensively. There is no question that these vaccines are highly protective against Q fever, but the side 

effects that occur in a specific population of people has 

prevented their wide-spread use. Individuals that have 

been exposed to C burnetii prior to vaccination, such as 

those who have had Q fever or have spent significant 

time in close proximity to livestock, are very likely to 

have adverse reactions at the site of injection.120,121 The 

reactions can range from mild redness and swelling to 

painful granulomas and sores. The same can be seen 

after multiple vaccinations. To circumvent the adverse 

reactions to the WCV, alternative antigen sources have 

been, and continue to be, investigated.

M-44

The Soviet Union developed a live attenuated 

oral vaccine using phase II organisms from the M-44 

strain.122,123 The vaccine was tested in Soviet volunteers 

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Multidrug-Resistant Bacterial Infections as a Threat to the US Military Health System: Acinetobacter Infections

Chapter 13

MULTIDRUG-RESISTANT BACTERIAL 

INFECTIONS AS A THREAT TO THE 

US MILITARY HEALTH SYSTEM: 

ACINETOBACTER INFECTIONS 

AS A CASE STUDY

EMIL LESHO, DO*; LUTHER E. LINDLER, PhD†; BRUNO PETRUCCELLI, MD‡; and

GLENN WORTMANN, MD§

INTRODUCTION

EPIDEMIOLOGICAL CONSULTATION 

Mission

First Reservoirs in the Combat Zone

Nosocomial Infection as the Dominant Problem

MOLECULAR ANALYSIS

Strain Collection and Sampling

Genetic Analysis, Characterization, and Identification of the Source

Antibiotic Resistance

MILITARY HEALTH SYSTEM RESPONSE TO ACINETOBACTER AND OTHER 

“ESKAPE” PATHOGENS

The Way Forward

SUMMARY

*Director, Multidrug-Resistant Organism Repository and Surveillance Network, Walter Reed Army Institute of Research, 503 Walter Grant Avenue, 

1W102, Silver Spring, Maryland 20910

†

Biosurveillance Liaison, Armed Forces Health Surveillance Center, 11800 Tech Road, Silver Spring, Maryland 20904 

‡

Colonel (Retired), Medical Corps, US Army; formerly, Director, Epidemiology and Disease Surveillance, US Army Center for Health Promotion and 

Preventive Medicine, Aberdeen Proving Ground, Maryland; currently, Senior Physician/Epidemiologist, US Military HIV Research Program, Henry 

M. Jackson Foundation for the Advancement of Military Medicine, 3720-A Rockledge Drive, Suite 400, Bethesda, Maryland 20817 

§

Colonel (Retired), Medical Corps, US Army; formerly, Chief of Infectious Diseases, Walter Reed National Military Medical Center, Bethesda, Maryland; 

currently, Assistant Director, Infectious Diseases Section, MedStar Washington Hospital Center, 110 Irving Street, NW, Washington, DC 20010

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INTRODUCTION

Although not new to the civilian critical care community, infections with multidrug-resistant (MDR) 

Acinetobacter species were rare in the US Military 

Health System (MHS) before the wars in Afghanistan 

and Iraq. In fact, the marked increase in such infections immediately following these conflicts led some 

to postulate that MDR Acinetobacter species may be a 

result of human engineering with malicious intent, 

leading some news reports to refer to it as “Iraqibacter.” However, there is no evidence supporting this 

contention. Acinetobacter species are environmentally 

hardy and difficult to eradicate from inanimate healthcare surfaces, but their relatively low virulence makes 

them poor candidates for weaponizing. In fact, even 

in the most severely war-wounded patients, Acinetobacter species infections are rarely fatal.1,2 Nonetheless, 

MDR Acinetobacter species pose an equally concerning 

risk to global public health as bacteria engineered for 

weapons use. 

Infection has always been a complication of war 

trauma. Treatment of traumatic wound infections 

has evolved over time, as a belief in “laudable pus” 

yielded to surgical debridement, and the emergence 

of penicillin in 1942 ushered in a period when recovery from serious infections became possible, if not 

expected. However, the 21st century has witnessed 

the expansion of bacteria that are resistant to multiple 

antibiotics, and a dearth in new drug development has 

resulted in infections from bacteria that are resistant to 

virtually all available antibiotics. These MDR bacteria 

have become well recognized in hospitals around 

the world and are especially problematic in locations 

where antibiotic use is frequent, such as in intensivecare units and long-term acute care facilities. During 

Operation Enduring Freedom (OEF) and Operation 

Iraqi Freedom (OIF), forward-deployed US medical 

facilities often provided acute care to service members 

who were rapidly medically evacuated, as well as to 

local national patients who required sustained care. 

Antibiotic use following traumatic injury to prevent or 

treat infection was the standard of care, and thus the 

stage was set for the selection of MDR bacteria, which 

subsequently spread through the evacuation chain. 

Although several different species of MDR bacteria 

emerged to complicate war trauma care, Acinetobacter 

calcoaceticus baumannii (ACB) complex first heralded 

the problem, which led to the investigations discussed 

in this chapter. 

Bacteria of the genus Acinetobacter are glucose 

nonfermentative, nonfastidious, catalase-positive, 

oxidase-negative, strictly aerobic, gram-negative, 

coccobacilli (or pleomorphic) and commonly occur in 

diploid formation or in chains of variable length. However, different genospecies cannot be easily identified 

using traditional methods. Members of the genus have 

been classified in various ways; therefore it is difficult 

to understand the true status of the epidemiology and 

clinical importance of these organisms. Since 1986, the 

taxonomy of the genus Acinetobacter has undergone 

extensive revision. The original single species named 

Acinetobacter calcoaceticus has been abandoned, and 

at least 32 genospecies have now been proposed, 17 

of which have been correlated with species’ names. 

Identifying the members of the genus Acinetobacter to 

the species level by traditional methods is problematic. 

Acinetobacter baumannii (genospecies 2), Acinetobacter 

pittii (formerly known as Acinetobacter genospecies 

3), and Acinetobacter nosocomialis (formerly known as 

Acinetobacter genospecies 13TU) are genetically and 

phenotypically similar to A calcoaceticus (Acinetobacter

genospecies 1) and hence are grouped in the so-called 

ACB complex. Molecular methods are needed to 

identify members of the complex to the species level 

because each member has a distinct antimicrobial susceptibility profile and shows different clinical characteristics. In this chapter, we use the term Acinetobacter 

or ACB interchangeably and to indicate the overall 

phenotype that includes the four most clinically relevant species mentioned above. These have gained 

notoriety for a predilection to cause nosocomial infections and to develop resistance to multiple antibiotics.3

The importance of ACB has been recognized with 

its inclusion in the ESKAPE pathogens (Enterococcus 

faecium, Staphylococcus aureus, Klebsiella pneumoniae, 

ACB, Pseudomonas aeruginosa, and Enterobacter species), 

a group of bacteria identified by the Infectious Diseases 

Society of America that risk becoming resistant to all 

available antibiotics.4 ACB is predominantly associated 

with medical technology, as most infections occur in 

the setting of artificial ventilation (ventilator-associated 

pneumonia), intravenous access (line-associated bacteremia) or urinary catheterization (urinary tract infections).5

 Although spontaneous, invasive infection of 

immune-competent patients appears to be uncommon, 

infections associated with trauma have been reported. 

Following an earthquake in Turkey in 1999, a local 

hospital experienced an 18.6% rate of hospital-acquired 

infection (HAI; predominantly wound infections) 

with MDR ACB being the most commonly isolated 

pathogen.6 In 2002, 62% of trauma victims following 

a bombing in Bali had ACB infections, and in 2004 

MDR ACB was discovered in 18% of cultures among 

injured tsunami victims evacuated from Thailand to 

Germany.7,8 

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Multidrug-Resistant Bacterial Infections as a Threat to the US Military Health System: Acinetobacter Infections

The presence or relative absence of ACB as a “militarily relevant” pathogen prior to 1970 is unknown. 

Among casualties of the Korean War, at least one blood 

culture was reported to grow Achromobacter species,9

and during the Vietnam conflict, the predominant 

gram-negative pathogen recovered from a series of 

30 Marines was in the Mimeae-Herellea-BacteriumAlcaligenes group, which is postulated as being ACB. 

However, it is unclear if any of these bacteria would 

be classified as ACB using current taxonomy. Furthermore, the organism was not prevalent in other studies 

of combat wounds during either war.10–14 The numbers 

of US casualties in Operations Just Cause (Panama, 

1989–1990), Desert Storm/Shield (Iraq, 1990–1991) and 

Restore Hope (Somalia, 1992–1993) were relatively low, 

and ACB infection was not reported.15 

US forces entered Afghanistan in 2001 in support of 

OEF and Iraq in 2003 in support of OIF, and military 

healthcare providers shortly thereafter began noting 

an increase in the number of patients infected with 

ACB. A collaborative report from military physicians 

and the Centers for Disease Control and Prevention 

was published in 2004, highlighting 102 injured service members whose blood cultures grew ACB. Most 

of these cases were reported from Landstuhl Regional 

Medical Center (LRMC) and Walter Reed Army Medical 

Center (WRAMC), with 32 OIF and 29 OEF bacteremic 

patients. The number of patients with ACB bloodstream 

infections in 2003 and 2004 exceeded those reported in 

previous years (one case during 2000–2002 at LRMC, 

and two cases during 2001–2002 at WRAMC).16 A review of 211 trauma casualties evacuated from Iraq to 

the United States Naval Ship (USNS) Comfort during the 

first month of OIF) in 2003 revealed 44 cultures positive 

for Acinetobacter species, representing 33% of all isolates. 

Specifically, 36% of wound isolates and 41% of bloodstream isolates were of Acinetobacter species.17 A review 

of MDR bacteria at WRAMC demonstrated a marked 

increase in the incidence of ACB infections, peaking in 

2004 (Figure 13-1), and a study using multilocus polymerase chain reaction (PCR) and mass spectrometry to

genotype isolates demonstrated that some of the strains 

belonged to an atypical and evolving group of isolates, 

distinct from those found at nonmilitary hospitals in 

the United States.18,19 Additionally, molecular typing of 

isolates from one of the worst outbreaks of MDR ACB 

in the MHS occurred at WRAMC and revealed eight 

major clone types, 60% of which were related to the 

three International Clonal Complex (ICC) types, suggesting multiple independent origins.20, 21

The clinical impact of the ACB outbreak associated 

with OIF/OEF was acute, with lingering downstream 

effects. A retrospective study of 93 war-related trauma 

patients at WRAMC with ACB bacteremia determined 

that there was a median of 1 day between admission 

and detection of infection. Of these isolates, 86% were 

resistant to multiple antibiotics. The 30-day mortality 

rate in this group of patients was only 2%, and those 

2 patients died from massive pulmonary emboli believed to be unrelated to ACB bacteremia. The authors 

concluded that the absence of severe comorbidities 

(as measured by the Acute Physiology and Chronic 

Health Evaluation II score and Charlson comorbidity 

index), compared to cohorts of patients in other published reports of ACB bacteremia, contributed to the 

low mortality rate.1 

Along with bacteremia, military providers began 

noting the development of ACB wound and burn 

infections, as well as osteomyelitis. Davis et al reported 23 patients with MDR ACB, 18 of whom had 

osteomyelitis, 2 with burn infections, and 3 with deep 

wound infections. Within a mean follow-up period of 9 

months, all patients were cured of infection.22 ACB was 

reported to be the most prevalent organism recovered 

from military burn patients injured during operations 

in Iraq and Afghanistan, with the percentage of ACB 

resistant to four classes of antimicrobial agents increasing from 17% in 2003 to 2005 to 49% in 2006 to 2008.23,24

Trauma-related skin and soft-tissue infections were 

reported by Sebeny et al, who reported their findings 

0

50

100

150

200

250

300

350

400

VRE

ESBL

Acinetobacter Species

MRSA

2000 2001 2002 2003 2004 2005 2006

Year

Number of Isolates

Figure 13-1. Annual incidence of drug-resistant organisms 

isolated from clinical specimens at Walter Reed Army Medical Center (2000–2006). 

ESBL: extended-spectrum β-lactamase-producing organisms

MRSA: methicillin-resistant Staphylococcus aureus

VRE: vancomycin-resistant enterococci

© 2008 by The Society for Healthcare Epidemiology of 

America. Originally published in Infecton Control and Hospital 

Epidemiology 2008;29:661-63. Used with permission.

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Medical Aspects of Biological Warfare 

in eight patients with infection due to ACB.25 Osteomyelitis was noted in several cohorts, often in association with orthopedic fixation devices.26,27ACB central 

nervous system infection associated with trauma was 

reported in several cases.28,29

In addition to causing infectious complications in 

traumatically injured patients, ACB developed resistance to multiple classes of antibiotics, posing treatment challenges to clinicians. At both WRAMC and 

Brooke Army Medical Center (BAMC), the prevalence 

of MDR ACB rose dramatically during the course of 

OIF/OEF. At BAMC, the percentage of MDR ACB 

rose from 4% to 55% between 2001 and 2008, while at 

WRAMC the percentage of ACB that was susceptible 

to imipenem dropped from 100% in 2002 to 61% in 

2006.19,30 Faced with the loss of traditional antibiotics, 

physicians turned to other agents, including tigecycline, minocycline, and colistimethate sodium.31–33 The 

switch to second-line agents came at a cost, however, 

as the incidence of acute renal failure (defined as the 

first three criteria in the RIFLE acronym [risk, injury, 

failure, loss, and end-stage renal disease]) in 66 patients 

who received colistimethate sodium was 45%; and 21% 

of patients stopped therapy because of nephrotoxicity. 

Fortunately, renal function returned to normal once 

colistimethate sodium was stopped. 

In summary, prior to OIF/OEF, ACB had been 

recognized as a pathogen among hospitalized patients, and scattered reports had noted an association 

with civilian trauma. However, its emergence as a 

pathogen associated with war trauma was unexpected, as was the breadth of its clinical presentation 

(including bacteremia, skin and soft-tissue infection, meningitis, and osteomyelitis). The organism’s 

ability to develop resistance to multiple antibiotics 

complicated treatment decisions, leading to the use 

of more toxic agents. Healthcare on the modern 

battlefield now extends from the point of injury to 

US-based, Echelon V (Level V) facilities, separated 

by a transit time of only a few days. Traumatically 

wounded patients are cloistered in an intensivecare environment, supported by invasive medical 

devices (ventilators, chest tubes, urinary catheters 

and intravenous lines), and administered antibiotics that invariably select for resistant pathogens. In 

retrospect, the emergence of a nosocomial pathogen 

was perhaps inevitable, and ACB was well positioned to exploit the opportunity. Trauma-induced 

disruption of anatomic barriers to infection and 

antibiotic use appear to select for ACB, and thus 

the military healthcare system will likely face this 

challenge again in future conflicts. 

 EPIDEMIOLOGICAL CONSULTATION 

Mission

Lieutenant General James B Peake, who was Surgeon General of the Army and Commander, US Army 

Medical Command, ordered an epidemiological consultation (EPICON) on August 27, 2004. The charge to 

the US Army Center for Health Promotion and Preventive Medicine (now the Army Public Health Command) included objectives that had typified military 

epidemiological investigations for decades and now 

addressed ACB. Descriptive and risk-factor analysis, 

identification of sources of infection, and recommendations for control, prevention, and future surveillance 

were all expected. However, the scope and complexity 

of the ACB problem prompted an approach that differed substantially from previous outbreak responses. 

A multicenter effort was organized that included the 

Walter Reed Army Institute of Research (WRAIR) and 

the Department of Defense Global Emerging Infections Surveillance and Response System (DoD-GEIS), 

in addition to four hospitals in the chain of medical 

evacuation from Southwest and Central Asia: the 31st 

Combat Support Hospital (31st CSH in Iraq, Level 

III), LRMC (Level IV), WRAMC (Level V), and BAMC 

(Level V). Twenty principal personnel, including four 

civilians, provided data or conducted local studies to 

support the investigation; these included clinicians, 

epidemiologists, infection control practitioners, microbiologists, an environmental scientist, and a statistical programmer. Numerous other individuals from 

various military organizations served as consultants or 

collaborators, including Navy and Air Force personnel, 

and advice from medical and laboratory contacts with 

ACB experience in other countries, particularly those 

in Southwest Asia, was also obtained.34

The three-way networking of public health, research, and clinical assets was not a new endeavor; 

neither was applying tools like genetic fingerprinting, 

web-based data collection, and multipoint conferencing. Nevertheless, bringing these together in response 

to the ACB problem seriously threatening hospitalized 

military beneficiaries set a new precedent for the MHS; 

just as the coordinated, international response to severe acute respiratory syndrome, on a broader scale, 

had set a new precedent for global public health the 

previous year. 

Acinetobacter infections had been rare at LRMC and 

WRAMC; but cases began to emerge in 2002 during the 

first year of OEF in Afghanistan and their frequency accelerated immediately after OIF began in March 2003. 

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Since microbiology and clinical data within military 

hospitals were archived, confirmation of an outbreak 

at LRMC and WRAMC was rather straightforward. 

Even after adjusting for the number of admissions, 

intensive-care bed days, and the frequency of culture 

specimen collection, rates of all types of ACB infection 

exceeded historical counts at these hospitals. At LRMC, 

36 bloodstream infections were observed in 2003 and 

2004, compared to only 4 in the preceding 3 years. At 

LRMC and WRAMC combined, the average number of 

ACB isolates had increased from 1 per month during 

2002 to 1 per day during 2003 and 2004.34

Among the aims of data analysis and interpretation by EPICON investigators were: (a) weighing the 

relative importance of wound contamination at the 

time of injury with that of nosocomial infection; (b) 

determining the role of Southwest or Central Asia as 

a geographic source, versus a primary role of intensive 

hospital care regardless of geographic location; and (c) 

distinguishing the importance of fomite versus personto-person transmission as an underlying propagation 

mechanism. It was not assumed a priori that these 

were mutually exclusive dichotomies; and much was 

already well known about ACB as a species complex, 

such as the ability of the organisms to colonize humans 

in addition to the inanimate, or built environment of 

the hospital, and soil and water.35 The newly accumulated evidence was mixed with respect to each of these 

aims when considered individually; however, taken 

together, the findings empirically supported a set of 

practical countermeasures and ongoing surveillance 

procedures.34

First Reservoirs in the Combat Zone

A baumannii strain distribution across international 

borders has been described, with evidence of drug 

resistance transfering between globally distant hospitals.36,37 Evidence of drug-resistant A baumannii as 

a growing problem in Southwest Asia was revealed 

during the outbreak in US military hospitals, both 

through investigators’ conversations with hospital 

infections experts in the region and through published 

reports.38,39 Also, surveillance at the 31st Combat Support Hospital in Baghdad had shown that non-US 

patients (coalition and local national) were colonized 

with ACB at a proportion that was more than five-fold 

that of US patients. Among hospital staff, 15 pairs of 

hands had been screened in one series, and none of the 

specimens grew ACB. During a 2-month period after 

the EPICON was initiated, 102 screening specimens 

from the US field hospital in Baghdad were obtained 

from patients receiving care in the emergency treatment area or being admitted to intensive care. One (2%) 

of 64 US patients and 4 (11%) of 38 Iraqi patients were 

found to be colonized. Furthermore, the hospital ship 

providing Level III care in the Persian Gulf during the 

early weeks of OIF (USNS Comfort, discussed earlier) 

was receiving primarily Iraqi nationals when Acinetobacter isolates had become relatively common.17,25

Screening of 96 ambulatory patients evacuated 

from Iraq to Landstuhl from its usual catchment area 

revealed no ACB skin colonization. However, among 

472 inpatients admitted to LRMC during the same 

period, 19 (3.9%) had skin cultures that were positive 

for ACB. Patients admitted to intensive care had a 

colonization prevalence of 10% (relative risk [RR] = 2.8 

compared with regular ward admissions, P < 0.0001). 

Patients with ACB bloodstream infection were found 

to have the infection within 48 hours of arrival (over 

50% on the day of admission), suggesting acquisition 

before admission to Level IV in the most seriously affected ACB patients.34

Analysis of locations in Iraq and Afghanistan from 

which patients were evacuated to Landstuhl over time 

revealed that the US hospital at Ibn Sina (Baghdad) 

contributed patients with the highest proportion colonized or infected with ACB, and that originating from 

field hospitals in Iraq presented a higher risk than from 

Afghanistan. When the results of colonization studies 

at three echelons (Baghdad, LRMC, and WRAMC, 2003 

to 2004) were examined together, including stratification between admission and discharge screening at 

LRMC and WRAMC, the proportions of patients with 

positive ACB cultures showed a clear, progressive 

increase with each level of care.34

At least one culture of the inanimate hospital environment from each of seven field hospitals (five in 

Iraq, two in Kuwait) was positive for ACB. Specific 

sampling locations were documented for 37 isolates. 

All of these were subjected to 16S rDNA (ribosomal 

deoxyribonucleic acid) sequencing, and 34 also underwent pulsed field gel electrophoresis (PFGE). In 

addition, there were 170 isolates available from 145 

individuals tested in 2003 while they were inpatients 

at Baghdad, the USNS Comfort, LRMC, or WRAMC. All 

of these underwent PFGE, and 164 of them underwent 

16S rDNA sequencing. The results are in the Molecular 

Analysis section, but key to the issue of strain importation was the demonstration that 43 patients treated at 4 

different military hospitals were infected with related 

strains from a single cluster group which, in turn, was 

genetically related to an isolate derived from environmental sampling of an operating room in the Baghdad 

field hospital. This group included both US and nonUS patients, and both those who had and those who 

had not deployed to the Central Command area of 

operations. No cultures were positive for ACB organ244-949 DLA DS.indb 325 6/4/18 
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Medical Aspects of Biological Warfare 

isms when 31 archived soil samples from the general 

environment of Iraq and Kuwait were tested (collected 

March 2003 to December 2004 from various locations, 

not in the vicinity of hospitals).21 A separate report, also 

published in 2007 but focusing on Canadian soldiers 

with A baumannii ventilator-associated pneumonia, 

similarly described genetic linkage between a field 

hospital environmental isolate (from a ventilator intake 

filter in Afghanistan) and isolates from four soldiers 

whose hospital treatment continued in Canada. Linkage to clinical specimens at Level IV (LRMC in these 

cases as well) was also found for three of the patients.40

Observations based on colonization and environmental studies of ACB may be confounded, biased, or diluted 

in significance when low-yield skin sampling sites are 

used for surveillance and colonization studies. This can 

also occur when reliance on routine cultures fails to 

distinguish A baumannii, the species most often causing 

opportunistic infection, from clinically less-significant 

species.41,42 During the EPICON, investigators found poor 

agreement between different body sites (eg, axilla versus 

groin, hands, feet, or forehead) at LRMC and BAMC 

when skin cultures were observed for ACB growth; 

and most isolates from environmental sampling were 

ACB other than A baumannii. Thus, a separate focus on 

clinical specimens, and the subjecting of both clinical and 

environmental isolates to species identification and molecular typing, provided critical data for the investigation. 

In addition to the EPICON, separate endeavors by 

Air Force personnel contributed data regarding ACB. 

In 2005, 83 environmental samples were taken from 

two C-141 aircraft used for aeromedical evacuation 

from Iraq to Germany, and from a deployed hospital 

of the Expeditionary Medical Dental Group (332nd 

EMDG, Balad Air Base, Iraq). The source locations 

included the walls, seats and floors from the front, 

middle, and back sections of the aircraft; the operating 

rooms (ORs) and wards of the hospital; and a variety 

of equipment (litters, litter straps, life pack monitor 

covers, and outer surface of endotracheal tubes). 

Also sampled were personnel working directly with 

patients while receiving, flying with, or transferring 

them. Three samples were taken from personnel working with patients from both the gloves and hands of 

caregivers. Finally, 16 of 58 patients who were transported during the observed flights were screened for 

ACB at LRMC. All of the environmental, equipment, 

personnel, and patient specimens were negative for 

ACB, except for one sample taken from a patient air 

warmer in the Balad hospital, which produced an 

imipenem-sensitive isolate. Of course, the possibility 

of colonization or contamination during transport of 

known, ACB-infected patients could not be ruled out.34

At the 332nd EMDG hospital, the surgical staff 

observed a significant reduction in ACB infections 

after implementing very aggressive infection control 

procedures, which indirectly supported the conclusion that the outbreak at higher echelons of care was 

likely preceded by nosocomial transmission in field 

hospitals. Countermeasures applied in Balad included 

strict enforcement of contact precautions as well as 

standardized intraoperative wound management, 

imposed conservative use of antimicrobials, and initiated special interventions (ie, plastic draping for all 

OR entrances, opening an additional OR for the most 

contaminated wounds, opening an additional ward 

to reduce patient crowding, greater use of heat and 

bleaching for linen cleaning, and thorough cleaning 

of ORs and wards, including weekly filter and duct 

cleaning in environmental control units). 

Nosocomial Infection as the Dominant Problem

When the EPICON report was submitted in mid2005, nosocomial transmission had been clearly 

documented in at least 39 A baumannii infection cases 

at WRAMC, and three quarters of these were cross infections in patients who were not evacuated from a deployed setting, including four civilians who died with 

bloodstream infection as an underlying cause. There 

had also been a fatal case at LRMC in a non-OEF/OIF 

patient: a 63-year-old woman who had been admitted 

to the ward with exacerbation of chronic obstructive 

lung disease, and who initially improved. Unfortunately the case went on to illustrate the serious impact 

ACB was having on persons completely unassociated 

with military operations. On hospital day 11, there was 

a sudden clinical deterioration, requiring the patient 

to be transferred to intensive care and mechanically 

ventilated. She expired the next day. Culture results 

supported the diagnosis of A baumannii pneumonia 

and bacteremia. During the patient’s hospital stay, at 

least five patients colonized with A baumannii had been 

admitted to the same ward and one of them stayed in 

the same room, but PFGE analysis distinguished the 

colonized patient’s strain from that of the deceased. 

However, an isolate completely matching that of the 

deceased was obtained from a patient staying in a different room on the same ward. This favored transmission by healthcare personnel over fomites in the room 

as an explanatory mechanism in this case.34

An analysis of nosocomial ACB transmission at 

WRAMC and BAMC revealed that the resulting 

infections primarily involved the respiratory tract, 

and that ACB acquisitions were primarily among 

civilian beneficiaries. Comparing 2004 with 2003 at 

LRMC and WRAMC combined, ACB wound specimens accounted for a diminishing fraction of positive 

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cultures, while bloodstream infections increased as a 

proportion of ACB infections. At LRMC the respiratory tract was considered a likely portal of entry for 

many of the nosocomial infections and, after excluding specimens taken from the skin and wounds, the 

respiratory tract was also the most common site of 

infection. The urinary tract was not a major site of 

infection, but infection there was more common at 

WRAMC than at LRMC. 

The EPICON report concluded that 

While all stages of the military healthcare system can 

propagate or sustain the presence of A baumannii on 

patients, the initial source of the current outbreak appears to be the (Level III facilities) in Iraq and Afghanistan. Nosocomial transmission accounts not only for 

some of the infections at (higher echelons) but also 

for the initial infection of US troops who acquire 

the infection before or during strategic MEDEVAC 

[medical evacuation]. Patients with relatively long 

inpatient stays in these hospitals (especially non-US 

patients) represent a likely reservoir for transmission 

of the organism. Pre-hospital, primary wound infections in theater are not likely to have a significant role 

in transmission.34 

With respect to environmental surface contamination versus colonized people, both were linked sufficiently to transmission to warrant both enhanced 

sanitation (room, equipment) and strict personal hygiene. Despite such measures, control of nosocomial 

transmission and of further progression toward drug 

resistance continued to prove extremely challenging 

for the MHS in the years following recognition of the 

A baumannii outbreak.43 Nevertheless, it is highly likely 

that morbidity and mortality would have continued 

to increase without strict preventive interventions, as 

now promulgated in national guidelines.44

MOLECULAR ANALYSIS

Strain Collection and Sampling

Around April 2003, physicians in the MHS noticed 

a marked increase in the number of Acinetobacter infections within Level IV and Level V medical treatment 

facilities (MTFs). The formal investigation described 

in the previous section was launched the following 

year. A critical part of the EPICON was to, “identify 

the cause(s) or source(s) of infection.” More than 200 

clinical and environmental isolates were collected from 

148 different patients and 37 environmental isolates 

collected in and around 7 deployed field hospitals in 

the Central Command area of responsibility. These 

isolates were referred to WRAIR for genetic analysis. 

The results of this EPICON were published in the 

journal Clinical Infectious Diseases.

21 

Genetic Analysis, Characterization, and Identification of the Source

Molecular epidemiology was performed on the 

clinical and environmental Acinetobacter isolates using 

PFGE and 16S rDNA sequencing in a single-blinded 

study.21,45 A total of 201 of the 207 isolates were identified using 16S rDNA sequencing. The clinical isolates 

were almost evenly split between A baumannii and 

other ACB organisms. In contrast, only 19% of the 

environmental isolates were A baumannii, and 70% 

were ACB organisms other than A baumannii. PFGE 

was able to establish 66 clinical isolate clusters and 25 

different environmental isolate clusters when clusters 

were defined as greater than 90% identical. Three different PFGE clusters contained isolates from clinical 

samples and the environmental isolates that were 100% 

identical. The matching environmental and clinical 

isolates were obtained from Camp Dogwood and 

WRAMC; LRMC, WRAMC, USNS Comfort, and Field 

Hospital Baghdad; and Mosul, Camp Dogwood, and 

LRMC. Additionally, two isolates from LRMC were 

100% identical to an ACB isolate from WRAMC.21

Taken together, these results strongly indicated that the 

outbreak of MDR Acinetobacter infections seen in the 

larger military medical centers began as nosocomial 

infection that originated in CSHs in Iraq. In essence, 

the MHS had become “infected” with Acinetobacter

because patients who were not involved in Iraqi military operations became infected with the organisms.

Military and civilian casualties from OIF were evacuated to the United States and the United Kingdom. 

Hospitals in the United Kingdom reported an outbreak 

of Acinetobacter infections soon after OIF commenced. 

Initially, there was concern that the infections caused 

by Acinetobacter may have been due to an intentional 

release of the organism. To determine if the isolates 

obtained in the United Kingdom were similar to those 

found in US casualties; the two countries initiated a 

collaboration and analyzed representative strains.46

The laboratory at WRAIR chose representative isolates 

from all of the major clusters that had been identified 

by PFGE and supplied them to the UK investigators. 

The UK Laboratory of HealthCare Associated Infections compared their Acinetobacter isolates to the ones 

supplied by the WRAIR laboratory. PFGE revealed 

that three of the US isolates were similar to the UK 

isolates with greater than 90% similarity. The antibiotic 

susceptibility profiles among these three isolates were 

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Medical Aspects of Biological Warfare 

also similar. DNA sequence analysis of the integron 

region associated with antibiotic resistance in Acinetobacter revealed that two of these common isolates 

had identical sequences. The third common isolate 

revealed a duplication of one gene in the US isolate 

but was otherwise identical to the UK isolate. These 

results suggested that there was a common origin for 

the Acinetobacter isolates causing wound infections in 

both the United States and the United Kingdom.

Research, epidemiological investigation, and 

molecular typing indicated that the Acinetobacter

infections were nosocomial such that casualties were 

becoming infected in theater, then the organism was 

becoming disseminated through the medical evacuation chain. A study utilizing multilocus PCR and mass 

spectroscopy (MS) was undertaken to analyze how 

the genotype of the organism might change over time 

in an MTF. A total of 267 Acinetobacter isolates were 

analyzed; 216 of the isolates were isolated from 2002 

through 2004 and were part of the original EPICON 

previously analyzed by PFGE.21,47 The additional 

isolates in this study were obtained from the American Type Culture Collection and from European 

hospitals. A baumannii accounted for 83% of the total 

isolates and could be divided into 46 unique sequence 

types (STs). This study also showed a strong correlation between isolates obtained from US OIF casualties and isolates obtained from European hospitals. 

Although the resolution of the PCR MS technique was 

lower than PFGE, there was good correlation between 

the two molecular methods. Additionally, this group 

analyzed the change in genotype of the Acinetobacter

over time at WRAMC using PCR MS.18 This study 

compared the ST of isolates obtained from 2002 to 

2004 with the ST of strains isolated from 2006 to 2007 

at WRAMC. The STs were relatively constant; a few 

minor STs either disappeared or increased with time, 

yet the major STs remained constant. Comparison of 

STs with nonmilitary hospital isolates revealed the 

distribution of STs was markedly different between 

the two groups. The antibiotic susceptibility profile 

generally correlated with ST as well. The study suggested that the Acinetobacter population in WRAMC 

had become less diverse and more stable with time. 

This was possibly due to effective countermeasures, 

such as sanitation and specific early therapy that 

resulted in reduction of less fit Acinetobacter strains 

both in patients and in the environment.

Antibiotic Resistance

MDR in Acinetobacter increased with time, making 

it more difficult for the MHS to respond to the threat. 

The genus is known as an opportunistic pathogen that 

resides in the environment and is naturally resistant 

to many antibiotics35,48; however, the organism also 

responds to antibiotic treatment by acquiring antibiotic-resistant genes. Interestingly, isolates obtained at 

BAMC from deployed service members were generally 

more resistant than those from nondeployed personnel.49 Resistance to the drug of choice, imipenem, 

increased as casualties from OIF continued. Several 

studies have looked at which genes are involved in the 

MDR phenotype of Acinetobacter involved in military 

wound infections. 

The study by Hujer et al used antibiotic-resistant, 

gene-specific PCR to analyze selected isolates from 

WRAMC to determine which MDR-associated genes 

the strains harbored.50 Approximately 20% of the 75 

isolates they analyzed were resistant to imipenem. 

Many isolates encoded multiple genes that were responsible for resistance to a class of antibiotic. Almost 

all of the isolates were resistant to ciprofloxacin through 

chromosomal housekeeping gene mutation. The strains 

were also highly resistant to cephalosporins by the 

production of beta-lactamase belonging to seven different classes of genes. Ninety percent of the imipenemresistant isolates encoded the blaOXA-23beta-lactamase 

allele. In another study, based on microarray analysis of 

antibiotic resistance genes found in 102 Acinetobacter isolates obtained from the National Naval Medical Center 

(NNMC) in 2006, 93% of the imipenem-resistant isolates 

were found to encode the blaOXA-23 beta-lactamase.51

All of the imipenem-resistant isolates belonged to one 

of two PFGE clusters. Accordingly, the increase in 

imipenem resistance in Acinetobacter-colonizing OIF 

casualties was mostly due to acquisition of a single allele 

of beta-lactamase. The blaOXA-23gene has been shown 

to be associated with bacterial mobile genetic elements 

allowing for rapid resistance acquisition and spread.

MILITARY HEALTH SYSTEM RESPONSE TO ACINETOBACTER AND OTHER “ESKAPE” PATHOGENS

Following the EPICON described above, many clinicians, scientists, and microbiologists recognized the 

use and value of establishing a centralized laboratory 

for receiving and archiving multidrug-resistant organisms (MDROs). Some of the major referral centers, 

such as NNMC, BAMC, and WRAMC, had already 

been preserving some MDR isolates, especially Acinetobacter, recognizing their inherent scientific and epidemiologic value. However, there was no central and 

standardized repository. Additionally, the institutions 

lacked the necessary human and financial resources to 

fully characterize these isolates. Centralized collection, 

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Multidrug-Resistant Bacterial Infections as a Threat to the US Military Health System: Acinetobacter Infections

comprehensive characterization, and long-term storage of MDROs is essential to understanding the healthcare challenges and informing future approaches. The 

Agency for Healthcare Research and Quality estimates 

that 5% to 10% of all inpatients acquire one or more 

HAIs during their stay, at an annual cost of $28 to $33 

billion. Twenty percent of HAIs are considered preventable through surveillance programs; however, as 

of late 2008, no agency in the Department of Defense 

was performing what would become the mission of the 

MDR Organism Repository and Surveillance Network 

(MRSN): to conduct enterprise-wide epidemiologic 

surveillance of MDROs to inform clinical practice and 

healthcare policy and enhance infection control.

The idea for a repository (“Joint Bacterial Repository”) was presented to the leadership of the US 

Army Medical Research and Materiel Command in 

2008; however, given the heightened sensitivity to 

biosurety amid the ongoing anthrax investigation 

involving scientists in the command, senior leaders 

were reluctant to authorize and establish what might 

be misperceived as another “freezer farm.” A few 

months before, DoD-GEIS (now part of the Armed 

Forces Health Surveillance Center) had funded two 

small surveillance studies in Eastern Iraq during the 

troop surge of 2007.52,53 These became the proof of 

concepts for a less static repository and more dynamic 

surveillance network. These studies demonstrated 

that regardless of location or environment, a distant 

facility could submit samples to a central laboratory 

and could receive useful and actionable information 

relating to infection control. The concept of a central 

repository laboratory at the nexus of a multifacility, 

bidirectional surveillance network (Figure 13-2) was 

successfully proffered to the US Army Medical Command (MEDCOM) in early 2009. 

In June 2009, the bacterial diseases branch of 

WRAIR in Silver Spring, Maryland, launched the 

MRSN. Under a performance improvement mandate 

from MEDCOM, Army hospitals, including those in 

Iraq and Afghanistan, submit MDROs isolated from 

clinical infections and active surveillance efforts. 

Isolates undergo integrated phenotypic, clonal, and 

phylogenetic analyses, including high-resolution 

ordered whole genome restriction optical mapping, 

followed by archival cryopreservation. The MRSN 

works closely with the Centers for Disease Control 

and Prevention to ensure genotyping methods are 

the same. Repository personnel provide epidemiologic reports and infection control information to 

hospitals and policy makers, conduct site assistance 

visits, and post site-specific and global antibiograms 

on a secure website.54,55 Other US military services are 

encouraged, but not required, to participate. Synergy 

is achieved through interagency collaboration and 

information sharing with the other military services, 

especially the Army’s Pharmacovigilance Center and 

the Navy and Marine Corps Public Health EpiData 

center, and with international military and civilian 

colleagues.

Substantial evidence supports the MRSN’s role in 

enhancing infection prevention and control efforts and 

reducing associated healthcare costs.56–59 Authors of 

one outbreak investigation concluded that if an aggressive surveillance program had not been in place, the 

source of an outbreak of severe ventilator-associated 

pneumonia in Canadian soldiers caused by MDR A 

baumannii would have been missed.60 The Association 

of Professionals in Infection Control and Epidemiology manual lists over 70 references demonstrating the 

usefulness of surveillance data in reducing infection 

occurrence and supporting the use of surveillance data 

to improve the quality of healthcare outcomes and 

processes.61 McQuillen et al cited more than 20 studies 

quantifying the financial impact of HAIs and the cost 

savings associated with surveillance.62 Infection control 

component monitoring and feedback, the approach 

taken by the MRSN, has been key in reducing the rate 

of HAIs (see Figure 13-2).63,64 Choosing the correct 

therapy requires knowledge of the underlying disease, 

the previous patient colonization, and the microbial 

trends in the community, as well as in the specific 

healthcare facility. Submitting isolates to the MRSN 

makes the latter two possible by enabling the MRSN 

to produce facility-specific and regional antibiograms. 

Using that modus, the MRSN has achieved notable 

firsts in the MHS and, in 2010, won first place in the 

Army Surgeon General’s Excalibur Award for health 

innovation practices. In 2012, the MRSN received accreditation by the College of American Pathologists 

(CAP), and in 2013 it won the Military Health System 

Award for Healthcare Innovation.

Currently more than 30 hospitals, including some 

in unstable areas and war zones, request molecular 

assistance with outbreak investigation or submitted 

isolates (an average of 375 per month). Isolates are 

identified and their susceptibility tested with the three 

most commonly used commercial automated susceptibility testing systems (VITEK 2 [bioMérieux, Inc, 

Durham, NC]; MicroScan [Siemens Medical Solutions, 

Malvern, PA]; and Phoenix [Becton, Dickinson, and 

Company, Franklin Lakes, NJ]). Discordant results of 

susceptibility testing are resolved by use of microbroth 

dilution panels. These data enable direct comparison 

of the three systems across multiple antibiotics and 

organisms, ranging from 8 drugs for A baumannii to 

14 drugs for Escherichia coli, Klebsiella pneumoniae, and 

methicillin-resistant Staphylococcus aureus. 

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Medical Aspects of Biological Warfare 

Unique to MRSN, associated clinical and demographic information, including securely maintained 

personal identifiers, is also submitted, helping to avoid 

isolate duplication when producing antibiograms and 

enabling MDRO tracking across regions. Isolates without this information are collected, but their ability to 

inform infection control efforts is severely degraded.

The MRSN developed and validated multiplex 

real-time PCR platforms to test isolates for the presence of clinically important antimicrobial resistance 

genes, such as mupA, qacA/B, PVL, cfr, and etA in 

Staphlococcus species; all variants of blaNDM and 

blaKPC, and the most common variants of blaVIM 

and blaIMP, and the most relevant alleles of blaOXAs 

in carbapenem-resistant gram-negatives. These are 

usually plasmid-borne genes that encode for highlevel mupirocin resistance (mupA), chlorhexidine tolerance or resistance (qacA/B), or carbapenemase production (blaKPC, blaNDM, blaVIM, blaIMP, and certain blaOXAs). They contribute to the dissemination 

Continental United States, Alaska & Hawaii Conflict areas

Military medical 

policy makers

Military hospitals

MDRO lab & 

repository AFHSC

Research investigators, 

clinicians, consultants

Isolates

IPCP

JTTR

EDC-ARO

Theater policy 

makers

VA, DHHS, civilian 

agencies

24-7 database 

queries

Figure 13-2. The purple-shaded square represents medical treatment facilities (Roles 3, 4, and above), which submit isolates and 

associated clinical data (red arrow) to the repository at Walter Reed Army Institute of Research (WRAIR; yellow oval) and in 

turn receive reports and assistance from WRAIR (black arrow). Reports and guidance will also be provided to theater medical 

leaders and combatant commanders (green box via purple arrow). Bidirectional information sharing and technical assistance 

can occur between the repository and other agencies, such as the Infection Prevention and Control Panel, the Joint Theater 

Trauma Registry, and the Navy’s Public Health EpiData Center Antibiotic Resistance Organization (EDC-ARO; blue box via 

yellow arrow) to enhance surveillance and reports. Data and reports from the repository will also be forwarded to the Armed 

Forces Health Surveillance Center (AFHSC; purple arrow) for broad dissemination (via blue arrows); the AFHSC can also assist 

with epidemiologic analysis or generation of the final reports (green arrow). Reports and information can also be provided to 

consultants for microbiology, infectious diseases, surgery, trauma, the surgeons general and others (gray box via purple and 

blue arrows). Consultants, policy makers, clinicians, and research investigators will be able to query certain portions of the 

database through web-enabled architecture (olive-tan arrow). Investigators can request de-identified isolates and specimens 

for approved research protocols (pink arrows).

AFHSC: Armed Forces Health Surveillance Center 

DHHS: US Department of Health and Human Services

EDC-ARO: EpiData Center Antibiotic Resistance Organization

IPCP: Infection Prevention and Control Panel

JTTR: Joint Theater Trauma Registry

VA: US Department of Veterans Affairs

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Multidrug-Resistant Bacterial Infections as a Threat to the US Military Health System: Acinetobacter Infections

of biocide or antibiotic resistance. BlaNDM1 carried 

on a novel plasmid was detected in Providencia stuartii

from Afghanistan.65 The MRSN was first to report 

qacA/B in methicillin-resistant Staphylococcus aureus

in the United States, which had higher tolerance of 

chlorhexidine gluconate than those that did not have 

the gene.66 A cluster of colistin-resistant Acinetobacter

emerged during therapy with colistin. This clone 

or strain, MLST 94, appeared to have a tendency to 

develop colistin resistance rapidly upon exposure to 

colistin, so the MRSN developed an assay capable of 

detecting the genetic element associated with that 

strain. It combined optical mapping and sequencing 

to identify gene copy number changes in sequential 

Acinetobacter isolates from the same patient.67 The 

MRSN also improved the speed and reduced the cost 

of optical genome mapping by successfully mapping 

multiple genomes on the standard map card.68 

The MRSN provided the foundation for enhanced 

de novo assembly of high-throughput pyrosequencing data using optical genome mapping, and demonstrated the first clinically relevant application of 

next-generation sequencing in the MHS.69 It also 

allowed identification of a heretofore notoriously 

difficult-to-characterize species of Acinetobacter that 

is typically misidentified by commercially available 

identification systems.70 The intent is that the MRSN 

become a reference laboratory for DoD-GEIS for 

high-resolution characterization of the MDR ESKAPE 

pathogens, with the ultimate goal of becoming the first 

CAP-accredited laboratory for optical mapping and 

sequencing in the DoD. Aligned with the One Health 

philosophy, the MRSN also supports or performs 

canine and environmental surveillance. Currently 

the MRSN has collaborators in 32 hospitals from 12 

countries in Central and South America, Europe, Asia 

(including Iraq and Afghanistan), and North America. 

The demonstrable power of surveillance increases in 

proportion to the geographic area surveyed and the 

degree of sharing between those who need to know 

and those who can act on findings to improve patient 

safety.61 To that end, the MRSN continually invites 

more international civilian and military colleagues to 

collaborate and submit isolates. 

The Way Forward

Current typing technologies have been useful in 

revealing relationships between isolates of ACB, but 

they are unable to resolve differences between closely 

related isolates from small-scale outbreaks, where 

chains of transmission are often unclear. Increasingly, genome scale epidemiology is required to detect and respond to outbreaks of highly resistant and 

virulent bacterial “superbugs.”71–73 Recall the recent 

fatal outbreak at the National Institutes of Health and 

the amount of sequencing and computing power that 

was needed to determine the origination and pattern 

of spread.72 Another study investigated a polyclonal 

outbreak of MDR A baumannii using whole genome 

sequencing. Comparison of the complete genome 

sequences of three dominant outbreak strain types 

showed that these strains diverged before their arrival at the authors’ institution despite all belonging 

to the same epidemic lineage (International Clonal 

Complex II).72 The simultaneous presence of three 

divergent strains of the same lineage is in accordance 

with its increasing prevalence in international hospitals, further supporting the ongoing adaption of 

clonal complex II to the hospital environment.72,74

Finally, a recent study found that nearly every strain 

of ACB from one integrated hospital system in the 

United States was unique despite being indistinguishable by conventional sequence typing methods, 

and in some cases by core single nucleotide variation 

typing.73 

The recent availability of rapid and inexpensive 

whole genome sequencing permits detailed investigation of genetic differences between bacterial isolates belonging to a single species and gives insight 

into the nature of genetic changes between isolates 

under antibiotic selection pressure. This is the approach now taken by the MRSN, and has been used 

to elucidate the evolutionary origin of an outbreak 

of colistin-resistant A baumannii containing a novel 

operon, the genomic characterization of a separate 

clone of Acinetobacter responsible for a group of fatal 

infections, and a new strain type in Honduras.2,75

Overall, as whole genome sequencing technologies 

continue to improve with respect to price and speed, 

these approaches should become the gold standard 

for rigorous epidemiological analysis in variable circumstances ranging from single-hospital outbreaks 

to worldwide epidemics, and from retrospective 

analysis to real-time monitoring. 

Applying basic and translational research methods to improve surveillance conducted for quality 

improvement and infection control resulted in the 

evolution of the MRSN into the Antimicrobial Resistance Monitoring and Research (ARMoR) Program. 

It is an enterprise-wide collaboration to aid in infection prevention and control. This approach consists 

of a network of epidemiologists, bioinformaticists, 

researchers, policy makers, and hospital-based infection preventionists who collaborate to collect relevant 

antimicrobial resistance monitoring (ARM) data, 

conduct centralized molecular characterization, and 

use ARM characterization feedback to implement ap244-949 DLA DS.indb 331 6/4/18 
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Medical Aspects of Biological Warfare 

propriate infection prevention and control measures. 

Policy compliance and enforcement further distinguish 

the ARMoR Program from the MRSN (Figure 13-3). 

A particularly concerning type of ARM, carbapenemresistant Enterobacteriaceae, peaked immediately after 

the program was launched, then significantly declined. 

Similarly, there have been no further reports or outbreaks of another concerning type of ARM, colistin 

resistance in Acinetobacter, in the DoD since the program was initiated. 

Figure 13-3. The Department of Defense Antibiotic Resistance Monitoring and Research Program (ARMoR) isolate and data 

flow.

SUMMARY

The MHS response to Acinetobacter exemplifies 

how different parts of the MHS coming together to 

investigate and respond to a major threat to wounded 

warriors and all MHS beneficiaries. Infectious disease 

physicians recognized the problem early and engaged 

DoD medical leadership to task public health professionals with identifying the problem so actions could 

be taken to stop these costly infections. Public health 

professionals took advantage of laboratory expertise 

that had been initially developed for the DoD chemical 

and biological defense program to perform molecular 

analysis to determine genetic relationships among the 

Acinetobacter isolates. This fact underscores the need 

for collaboration between the Chemical Biological Defense Program and that part of the MHS that conducts 

“routine” infectious disease and public health investigations. Such collaboration supports service members and 

enhances the MHS’s ability to respond during crises.

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Botulinum Toxin

Chapter 14

BOTULINUM TOXIN

ZYGMUNT F. DEMBEK, PhD, MS, MPH, LHD*; LEONARD A. SMITH, PhD†; FRANK J. LEBEDA, PhD‡; and 

JANICE M. RUSNAK, MD§

INTRODUCTION

HISTORY

DESCRIPTION OF THE AGENT

Botulinum Neurotoxin Production

PATHOGENESIS

CLINICAL DISEASE

DIAGNOSIS

Diagnostic Assays in Botulism

Foodborne Botulism

Inhalation-acquired Botulism

Other Forms of Botulism

TREATMENT

Antitoxin

Clinically Relevant Signs of Bioterrorist Attack

Preexposure and Postexposure Prophylaxis

New Vaccine Research

New Therapeutic Drug Research 

SUMMARY

*Colonel (Retired), Medical Service Corps, US Army Reserve; Associate Professor, Department of Military and Emergency Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814; formerly, Chief, Biodefense Epidemiology and Education 

& Training Programs, Division of Medicine, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

†

Senior Research Scientist (ST) for Medical Countermeasures Technology, Office of the Chief Scientist, US Army Medical Research Institute of Infectious 

Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

‡

Acting Director and Chief Data Officer, Systems Biology Collaboration Center, US Army Center for Environmental Health Research, US Army Medical Research and Materiel Command, 568 Doughten Drive, Fort Detrick, Maryland 21702; formerly,Civilian Deputy Director, Combat Casualty Care 

Research Program, US Army Medical Research and Materiel Command, 504 Scott Street, Fort Detrick, Maryland

§

Lieutenant Colonel (Retired), US Air Force; Medical Director, Battelle, 1564 Freedman Drive, Fort Detrick, Maryland 21702; formerly, Deputy Director 

of Special Immunizations Program, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

A portion of this chapter has previously been published as: Dembek ZF, Smith LA, Rusnak JM. Botulism: cause, effects, diagnosis, clinical 

and laboratory identification, and treatment modalities. Disaster Med Public Health Prep. 2007;1(2):122–134.

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INTRODUCTION

The seven neurotoxin serotypes (A-G) produced 

by Clostridia species are among the most potent toxins 

known. These structurally similar neurotoxins are 

immunologically distinct because neutralizing antibodies for one serotype does not protect against the 

other six serotypes.1 Because of their extreme toxicity, 

neurotoxin from Clostridia botulinum was one of the 

first agents to be considered as a biological weapons 

agent. Botulinum neurotoxin has been developed as 

a biological weapon by many countries, including 

Japan, Germany, the United States, Russia, and Iraq 

(Figure 14-1). 

Botulism in its various forms (foodborne, wound, 

infant and adult intestinal, and iatrogenic) is a 

potentially fatal neuroparalytic disease that most 

Figure 14-1. Representations of the structure of BoNT/A. 

Left panel: the protein is composed of a ~50 kDa light chain 

(LC, blue-green) and a ~100 kDa heavy chain. The heavy 

chain is composed of three distinct structural domains: 

two C-terminal ganglioside binding domains are used in 

recombinant vaccines, HCc

 (red) and HCn (yellow), and an 

N-terminal translocation domain (HN, green). The LC functions as a zinc-dependent endopeptidase. Right panel: in this 

rendering of the molecule, the belt-like portion of the heavy 

chain (red) is more clearly seen to wrap around the LC (blue).

Data sources: (1) Lacy DB, Tepp W, Cohen AC, et al. Crystal 

structure of botulinum neurotoxin type A and implications 

for toxicity. Nat Struct Biol. 1998;5:898–902. (2) Swaminathan 

S, Eswaramoorthy S. Structural analysis of the catalytic and 

binding sites of Clostridium botulinum neurotoxin B. Nat Struct 

Biol. 2000;7:693–699.

HISTORY

In the early 1930s during its occupation of Manchuria, Japan formed a biological warfare research 

program. The largest facility in this program, which 

was located in Pingfang, was known as Unit 731.5

General Shiro Ishii, the Japanese military medical 

commander of Unit 731, admitted to feeding lethal 

cultures of C botulinum to prisoners.6 US researchers 

began working on weaponization of botulinum toxin 

in the 1940s, and Allied intelligence indicated that 

Germany attempted to develop botulinum toxin as a 

weapon to be used against invasion forces.7 At the time, 

neither the composition of the toxic agent produced 

by C botulinum nor its mechanism of injury were fully 

known. Therefore, the earliest research goals were to 

isolate and purify the toxin and determine its pathogenesis,8,9 with the latter work conducted at Camp 

Detrick. The potential of botulinum neurotoxin as an 

offensive biological weapon was also investigated.10–12

The US code name given to botulinum neurotoxin at 

that time was “agent X.”

Following President Richard M Nixon’s executive 

orders in 1969–1970, as explicitly stated in National 

Security Decision Memoranda 35 and 44,13 all biological 

agent stockpiles in the US offensive biological program, 

including botulinum neurotoxin, were destroyed.14 The 

1975 Convention on the Prohibition of the Development, 

Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction prohibited the production of offensive toxins.15 Although 

the Soviet Union signed and ratified this convention, its 

biological warfare program, which included botulinum 

neurotoxin research, biological weapons development, 

and production, continued and was expanded in the 

post-Soviet era.16,17 The Soviet Union reportedly tested 

botulinum-filled weapons at the Soviet site Aralsk-716 on 

Vozrozhdeniye (Renaissance) Island in the Aral Sea17,18

and attempted to use genetic engineering technology 

to transfer complete toxin genes into other bacteria.19 In 

April 1992, President Boris Yeltsin publicly declared that 

his country had covertly continued a massive offensive 

often presents as a descending, symmetric flaccid 

paralysis,2 which is typically associated with neurotoxin types A, B, and E. Foodborne outbreaks receive 

considerable public health attention, as the risk of 

widespread food dissemination of botulinum toxin 

constitutes a public health emergency and is often 

fatal if untreated. However, the most common form 

of botulinum intoxication in the United States is infant 

botulism from the intestinal colonization with toxinproducing Clostridium in infants younger than 1 year 

of age.3 For a recent 10-year period (2002–2011), of 

1,379 reported cases of botulism in the United States, 

the greatest number was infant botulism (68%), followed by wound botulism (18%), and foodborne 

botulism (12%).4

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Botulinum Toxin

biological warfare buildup, which included developing 

botulinum toxin as a weapon. Yeltsin’s assertions gave 

credence to the claims of Biopreparat laboratory director Vladimir Pasechnik, who defected to the United 

Kingdom in 1989.20 Also in 1992, Colonel Kanatjan Alibekov (Kenneth Alibek),19 the former deputy director 

of Biopreparat (a Soviet agency whose primary function was to develop and produce biological weapons 

of mass casualties), defected to the United States and 

eventually described in considerable detail the Soviet 

biological weapons program in his book Biohazard.

19

Iraq, which also signed the 1975 convention, expanded its biowarfare program in 1985. Ten years 

later, it admitted to the United Nations Special Commission inspection team to having produced 19,000 

liters of concentrated botulinum neurotoxin for use in 

specially designed missiles, bombs, and tank sprayers 

in 1989 and 1990.16,21 Of this preparation, 10,000 liters 

were used to fill 13 SCUD missiles with a 600-km 

range and 100 181-kg (R-400) bombs (each bomb could 

hold 83 liters of toxin solution). However, Iraq did not 

use biological agents during the Persian Gulf War or 

Operation Iraqi Freedom, and it has maintained that 

its biological weapon stockpiles were destroyed.22 No 

biological weapons were discovered in Iraq in wartime 

from 2003 through 2011.23

The Aum Shinrikyo, a Japanese cult formed in 1987 

by Shoko Asahara, attempted to develop biological 

weapons after its political party was defeated in the 

1990 election campaign. Known for its deadly 1995 

sarin attack in the Tokyo subway, Aum Shinrikyo 

also attempted to produce botulinum neurotoxin. 

Five days before the sarin attack, three briefcases 

containing portable disseminating devices generating 

water vapor were found in the Kasumigaseki subway 

station.24 At his 1996 trial, Asahara said he believed 

the cases contained botulinum neurotoxin, although 

the toxin was not detected in the devices. With 50,000 

followers worldwide and an estimated $1 billion in financial resources, the cult had the capability to develop 

biological toxins for use as weapons, and the intent to 

do so.25 Although no cult members were specialists 

in biological weapons development, microbiologists, 

medical doctors, and other scientists were among 

the followers. In their thorough analysis of the Aum 

Shinrikyo’s efforts to develop biological and chemical 

weapons, Richard Danzig and his colleagues postulate 

that the group specifically failed to develop a viable 

botulinum toxin because of the following reasons24:

• inability to acquire an appropriate strain of 

C botulinum capable of producing sufficient 

concentrations of active botulinum toxin (five 

different strains were isolated by the cult);

• inability to achieve specific culture conditions 

(eg, appropriate fermentation broth, appropriate anaerobic environment) required for toxin 

production;

• presence of bacterial contamination, as Bacillus subtilis natto was identified in the cult’s C 

botulinum product (as an aerobic bacteria, also 

indicates inappropriate anaerobic cultural 

conditions); 

• toxin degradation that may have occurred 

during postfermentation recovery or storage; 

and

• suboptimal concentrations of toxin (if present 

in any amount) that were disseminated that 

did not cause harm.

A successful bioterrorist attack on large numbers of 

people with botulinum neurotoxin would overwhelm 

the public health system. The medical intervention 

required to assist patients with botulism includes mechanical ventilation and urgent attendant healthcare. 

If the Rajneeshee cult (the followers of the Bhagwan 

Shree Rajneesh who had carried out a biological attack 

to influence a local election) had used botulinum toxin 

instead of Salmonella typhimurium on salad bars in its 

1984 attack in The Dalles, Oregon,26–28 many of the 751 

persons who contracted Salmonella gastroenteritis 

would likely have died; the neurological sequelae of 

hundreds of patients with botulinum toxin poisoning 

would have quickly overwhelmed community medical resources.29

The potential consequences of a botulinum toxininduced mass casualty disaster can be estimated by 

reviewing the March 2006 foodborne botulism outbreak in Thailand.30 This event signifies a profound 

national public health mass casualty event, and successful patient recovery is not trivial. In this outbreak, 

home-canned bamboo shoots were consumed in a 

village, where 209 individuals consumed a common 

meal. Of 163 individuals examined in a hospital, 140 

were hospitalized and 42 developed respiratory failure 

and required mechanical ventilation.30 Sufficient antitoxin was donated to Thailand by various international 

health organizations to treat 90 patients. This antitoxin 

was administered to patients with the most severe 

symptoms, but treatment was delayed 5 to 9 days from 

exposure. The median duration of hospital admission 

was 6 days for patients without mechanical ventilation 

and 25 days for patients with mechanical ventilation.31

A long incubation time was associated with a better prognosis.32 The massive public health response 

undertaken by the Thai healthcare system, the global 

health community, and others outside of healthcare, 

including many embassies, airlines, and commercial 

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Medical Aspects of Biological Warfare 

partners, is credited for no mortalities.30 Besides the 

need for adequate ventilators and healthcare support, 

successful treatment of such large numbers of severely 

affected patients required intervention from neurologists, pulmonologists, intensivists, cardiologists, infectious disease specialists, and rehabilitation and referral 

services.31 Other supportive personnel required for a 

mass casualty botulism event included pharmacists, 

respiratory care officials, and psychological services 

personnel.33,34

In 2005, Wein and Liu35 described in detail how 

a bioterrorism attack using botulinum neurotoxin 

could be perpetrated on the nations’ milk supply. 

They describe a mathematical model representative of 

California’s dairy industry with milk traveling from 

cows to consumer in a supply chain: milk is processed 

from cows; picked up by tanker truck; piped through 

milk silos; processed via separation, pasteurization, 

homogenization, and vitamin fortification; and eventually distributed to the public.35 Naturally occurring 

salmonellosis outbreaks from milk and milk products 

affecting more than 200,000 persons have occurred, 

leading to a realistic assessment of such vulnerability 

in the national milk distribution system.36,37 The ability 

to spread botulinum neurotoxin via a liquid media, if 

present in sufficient concentration, makes this agent a 

logical choice for such a scenario. Modeling of botulinum in a liquid dispersal medium is not new and has 

been posited for terrorist use in a water fountain,38

based on microbiological contamination at a recreational facility.39 However, Wein and Liu’s modeling 

goes further than tocsin generation, pinpointing critical 

entry points of neurotoxin into the milk supply, estimating the amount of toxin required, and identifying 

weaknesses in current detection technology.35,39 The 

Wein and Liu paper generated considerable debate40

by the possible security risk it exposed.41 Stewart Simonson, former assistant secretary for public health 

emergency preparedness at the US Department of 

Health and Human Services, regrets the publication 

decision.42 It has been demonstrated that the milk supply is likely not at a risk for botulism contamination 

during the presterilization process because standard 

pasteurization at 72°C for 15 seconds inactivates at 

least 99.99% of BoNT/A and BoNT/B and at least 99.5% 

of their respective complexes.43 

DESCRIPTION OF THE AGENT

Clostridium species bacteria are sporulating, obligate anaerobic, gram-positive bacilli. The spores of C 

botulinum are ubiquitous, distributed widely in soil 

and marine sediments worldwide, and often found in 

domestic grazing animals’ intestinal tracts.44–48 Under 

appropriate environmental or laboratory conditions, 

spores can germinate into vegetative cells that will 

produce toxin. C botulinum grows and produces neurotoxin in the anaerobic conditions frequently encountered in food canning or preservation. The spores are 

hardy, and special efforts in sterilization are required 

to ensure that the spores are inactivated.49 Modern 

commercial procedures have virtually eliminated food 

poisoning by botulinum toxin. However, the leading 

cause of foodborne botulism is attributed to homecanned foods (particularly vegetables such as beans, 

garlic, peppers, carrots, and corn that are pH >4.6) or 

food items improperly prepared by restaurants.50–52

C botulinum produces eight antigenic types of neurotoxins denoted by the letters A through H. Seven neurotoxins (A-G) are structurally similar (approximately 

150 kD in mass) but immunologically distinct.53,54

However, there is some serum cross-reactivity among 

the serotypes because they share some sequence 

homology with one another as well as with tetanus 

toxin.55,56 The unique strain Clostridium baratii produces 

only serotype F,57 and the Clostridium butyricum strain, 

serotype E.58

Botulism is a neuroparalytic disease. Human 

botulism cases are caused primarily by neurotoxin 

types A, B, and E,50 and rarely by type F.59 Clostridium 

argentinense produces type G, which has been associated with sudden death, but not neuroparalytic 

illness, in a few patients in Switzerland.60 Neurotoxin 

types C and D cause disease in animals. All seven 

toxins are known to cause inhalational botulism in 

primates,61 and therefore could potentially cause 

disease in humans. Clostridial C2 cytotoxin is a 

nonhomologous enterotoxin, and not a selective 

neurotoxin.62 It affects multiorgan vascular permeability via cellular damage from its action on actin 

polymerization in the cellular cytoskeleton, and has 

been implicated in a fatal enteric disease in waterfowl.63,64 Although the newly described botulinum 

neurotoxin (H) is structurally related to serotypes 

A and F, it is not a distinct serotype because it has 

been demonstrated to be neutralized with serotype 

A antitoxin.65 A gene sequence of two botulinum 

toxin gene clusters differed substantially from the 

sequences of BoNT genes for toxins A-G.55

Botulinum Neurotoxin Production

Spore germination and subsequent growth of toxinproducing bacteria occur in improperly preserved 

foods,66–72 decaying animal carcasses and vegetable 

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matter,73–77 and microbiology laboratories.78–82 A terrorist with the proper expertise and resources could 

obtain a toxin-producing strain of C botulinum. Various 

scientific journals, textbooks, and Internet sites provide 

information on how to isolate and culture anaerobic 

bacteria and, specifically, how to produce botulinum 

toxin. A major cause of botulism is the ingestion of 

foods contaminated with C botulinum and preformed 

toxin. The food supply remains vulnerable to a botulinum toxin attack.

PATHOGENESIS

The seven neurotoxins have different specific toxicities83–85 and durations of persistence in nerve cells.86,87

All botulinum toxin serotypes inhibit acetylcholine 

release, but they act through different intracellular 

protein targets, exhibit different durations of effect, 

and have different potencies.88 All seven toxins may 

potentially cause botulism in humans given a large 

enough exposure. Botulinum neurotoxin can enter 

the body via the pulmonary tract (inhalational botulism), the gastrointestinal tract (foodborne and infant 

botulism, adult intestinal [adult infectious] botulism), 

direct injection (iatrogenic botulism), and from infected 

wounds (wound botulism). Infant, adult intestinal, 

and wound botulism are also referred to as infective 

botulism, as toxin produced by C botulinum either 

colonizes the lumen of the intestinal tract or is produced in a wound.89 Upon absorption, the circulatory 

system transports the toxin to peripheral cholinergic 

synapses, primarily targeting neuromuscular junctions.90 The toxin binds to high-affinity presynaptic 

receptors and is transported into the nerve cell through 

receptor-mediated endocytosis. In the nerve cell, it 

functionally blocks neurotransmitter (acetylcholine) 

release, thereby causing neuromuscular paralysis. 

Other neurotransmitters co-located with acetylcholine 

may also be inhibited,91,92 and noncholinergic cells may 

also be affected.93 The estimated human dose (assuming a weight of 70 kg) of type A toxin lethal to 50% of 

an exposed population (the LD50) is estimated, based 

on animal studies, to be approximately 0.09 to 0.15 μg 

by intravenous administration, 0.7 to 0.9 μg by inhalation, and 70 μg by oral administration.94–97

CLINICAL DISEASE

Untreated botulism is frequently fatal. The rapidity 

of the onset of symptoms, as well as the severity and 

duration of the illness, is dependent on the amount 

and serotype of toxin.50,98 In foodborne botulism, 

symptoms appear several hours to within a few days 

(range 2 hours to 10 days) after contaminated food is 

consumed.50,99 In most cases the onset of symptoms occurs within 12 to 72 hours postexposure. In one study, 

the median incubation period for the onset of symptoms from all toxin serotypes was 1 day.98 However, 

the median time to onset of symptoms for serotype 

E was shorter (range 0–2 days) compared to toxin 

serotypes A (range 0–7 days) and B (range 0–5 days); 

most individuals with toxin serotype E had symptoms within 24 hours of ingestion. Symptoms from 

foodborne botulism from toxin serotype A generally 

are more severe than from toxin serotypes B and E.98

As a neuroparalytic illness, botulism presents as an 

acute, symmetrical, descending, and flaccid paralysis. 

However, early symptoms may be nonspecific and 

difficult to associate with botulinum intoxication. 

Individuals with foodborne botulism often present initially with gastrointestinal symptoms such as nausea, 

vomiting, abdominal cramps, and diarrhea. Initial neurologic symptoms usually involve the cranial nerves, 

with symptoms of blurred vision, diplopia, ptosis, and 

photophobia, followed by signs of bulbar nerve dysfunction such as dysarthria, dysphonia, and dysphagia. Onset of muscle weakness ensues in the following 

order: muscles involving head control, muscles of the 

upper extremities, respiratory muscles, and muscles 

of the lower extremities. Weakness of the extremities 

generally occurs in a proximal-to-distal pattern, and is 

generally symmetric.51 However, asymmetric extremity weakness may occasionally be observed, occurring 

in 9 of 55 botulism cases in one review.100

Respiratory muscle weakness can result in respiratory failure, which may be abrupt in onset. In one study, 

the median time between the onset of intoxication 

symptoms and intubation was 1 day.98 Other commonly 

reported symptoms included fatigue, sore throat, dry 

mouth, constipation, and dizziness.100 Botulism is not 

associated with sensory nerve deficits. However, one 

review of botulism from toxin serotype A or B showed 

that 8 of 55 cases reported symptoms of paresthesias.100

Death is usually the result of respiratory failure or secondary infection associated with prolonged mechanical 

ventilation. In general, intoxication with toxin serotype 

A results in a more severe disease, often with bulbar 

and skeletal muscle impairment, and thus the need for 

mechanical ventilation.98,100,101 Intoxication with toxin 

serotype B or E is more often associated with symptoms 

of autonomic dysfunction, such as internal ophthalmoplegia, nonreactive dilated pupils, and dry mouth. 

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Medical Aspects of Biological Warfare 

Paralysis from botulism can be long lasting. Mechanical ventilation may be required for 2 to 8 weeks 

with foodborne botulism, with paralysis lasting as long 

as 7 months.100 Symptoms of cranial nerve dysfunction 

and mild autonomic dysfunction may persist for more 

than a year.102–104

The following symptom triad should suggest a botulism diagnosis: (1) an acute, symmetric, descending, 

flaccid paralysis with prominent bulbar palsies in (2) 

an afebrile patient with (3) a normal sensorium. The 

bulbar palsies of botulism consist of the “four Ds”: 

diplopia, dysarthria, dysphonia, and dysphagia. Five 

classic symptoms have also been used to diagnose 

botulism: (1) nausea and vomiting, (2) dysphagia, (3) 

diplopia, (4) dry mouth, and (5) fixed dilated pupils.100

However, individuals may not exhibit all five symptoms; a review from the country of Georgia reported 

that only 2% of patients (13/481) presented with all 

five criteria.72

Epidemiological history of injection of black tar 

heroin (wound botulism), laboratory work with 

botulinum toxins, or therapeutic use (eg, for cervical dystonia or cosmetic purposes) of botulinum 

neurotoxin preparations not approved by the Food 

and Drug Association (FDA) may also support the 

diagnosis of botulism.105–107 Patients with botulism 

may have a delay in diagnosis because of vague 

complaints or symptoms only present early in the 

illness or from misdiagnosis of paralytic symptoms.108 In one study, less than half of the patients 

with botulism requiring hospitalization were diagnosed and admitted during the initial physician 

contact.100 Alternate diagnoses may include drug 

overdose or intoxication (particularly with injection 

drug users having wound botulism) malingering, 

conversion disorder, stroke, myasthenia gravis, viral 

syndromes, and others.108,109 Clinicians should be 

aware of early symptoms of botulism in adults (ie, 

blurred vision, diploplia, and/or ptosis) and infants. 

In addition, delays to diagnosis are associated with 

more prolonged hospital course and an increased 

need for ventilatory support.108,110

Although foodborne botulism is the most likely 

route of exposure for botulism from either natural 

causes or a bioterrorist event, botulism acquired on 

the battlefield is most likely to occur from botulinum 

toxin inhalation, a route of exposure that does not 

naturally occur. The duration from exposure to the 

onset of symptoms for inhalational botulism is similar 

to that observed with ingestion of botulinum toxin, 

generally ranging from 24 to 36 hours to several days 

postexposure.98,111 Clinical symptoms resulting from 

inhalational intoxication are similar to botulism acquired from toxin ingestion.

The first reported inhalation-acquired botulism 

in humans occurred in 1962 in a German research 

laboratory.112 Three laboratory workers experienced 

symptoms of botulinum intoxication after conducting 

a postmortem examination of laboratory animals that 

had been exposed to botulinum toxin type A. Hospitalized 3 days after their exposure, the workers were 

described as having (a) a “mucous plug in the throat,” 

(b) difficulty in swallowing solid food, and (c) “the 

beginning of a cold without fever.” The symptoms 

had progressed on the 4th day, and the patients complained of “mental numbness,” extreme weakness, and 

retarded ocular motions. Their pupils were moderately 

dilated with slight rotary nystagmus, and their speech 

became indistinct and their gait uncertain. The patients 

were given antibotulinum serum on the 4th and 5th 

days. Between the 6th and 10th days after exposure, the 

patients experienced steady reductions in their visual 

disturbances, numbness, and difficulties in swallowing. They were discharged from the hospital less than 2 

weeks after the exposure, with a mild general weakness 

as their only remaining symptom.112 Botulism acquired 

through illicit drug (cocaine) inhalation has also been 

reported, and the incubation period was difficult to 

calculate because of frequent drug use.113

Other forms of botulism (wound, infant, and iatrogenic botulism) have similar symptoms as foodborne 

botulism, but they may have some variation in clinical 

presentation or clinical findings. Wound botulism is 

associated with an infected wound. However, the 

wound botulism associated with injecting drug use 

(usually an abscess) may not be grossly infected (15% 

to 50% cases), the wound site (usually at injecting 

sites) may be at an unusual location (ie, base of the 

tongue), and fever is generally not present.108 The 

onset of symptoms in wound botulism associated 

with injecting drug use (mainly black tar heroin) has 

ranged from 2 to 14 days, and in wound botulism 

not associated with injecting drug use (ie, traumatic 

wounds) from 2 to 18 days (mean of 5 to 7 days).105,108

Early symptoms in infant botulism are constipation or 

change in stool pattern, irritability, poor feeding, lethargy, and cranial nerve findings.110 Cranial findings in 

infant botulism may initially be mild and overlooked, 

and generally manifest as a weak cry, poor suck or gag 

reflex, sluggish and incomplete pupillary response, 

ophthalmoplegia, and ptosis. Mortality from infant 

botulism with supportive intensive care (even before 

availability of antitoxin for infants) is generally less 

than 1%.114 In iatrogenic botulism associated with 

therapeutic BoNT products (but not reported with 

lower doses given for cosmetic purposes), onset of 

symptoms generally occurred within hours to 3 weeks 

after the last injection.108 

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DIAGNOSIS

sensory deficits) with a normal sensorium. Any occurrence of botulism requires notification of public health 

officials and an epidemiological evaluation. Electrophysiological studies are helpful in distinguishing 

botulism from other causes of acute flaccid paralysis 

and support a presumptive diagnosis of botulism.116–118

An electromyogram with repetitive nerve stimulation 

at 20 Hz to 50 Hz showing facilitation (an incremental 

response to repetitive stimulation), usually occurring only at 50 Hz, may be helpful in distinguishing 

botulism from Guillain-Barré syndrome or myasthenia 

gravis, but not from Lambert-Eaton syndrome.51 Botulism patients with neuromuscular respiratory failure 

showed mostly incremental responses to high-rate 

repetitive nerve stimulation testing of the extensor 

muscles of the fifth digit in 17 patients, especially in 

response to >20 Hz stimulation.119 Electrophysiological testing in botulism may also demonstrate a small 

evoked muscle action potential response to a single 

supramaximal nerve stimulus, with normal sensory 

nerve function and nerve conduction velocity test results. However, electrophysiological tests may be normal in botulism. Approximately 15% of patients with 

botulism may have normal muscle action potential 

amplitudes, and as many as 38% of patients may not 

exhibit facilitation.100 CSF findings are usually normal 

in botulism, and abnormal findings should suggest 

another diagnosis. However, mild elevation of CSF 

protein (between 50 and 60 mg/dL) has been reported 

in 3 of 14 patients (21%) who had spinal fluid analysis 

performed.98 Laboratory findings, such as complete 

blood count, chemistries, liver and renal function tests, 

and electrocardiogram, are normal in botulism, unless 

a complication (eg, secondary infection, respiratory 

failure) has occurred. 

Diagnostic Assays in Botulism

A confirmatory diagnosis of botulism can often be 

made by demonstrating the presence of toxin in patient 

specimens, such as the serum, stool, gastric aspirate, 

vomitus, or wound, using mouse bioassays. Mouse 

bioassays, which are highly sensitive to botulinum 

toxin (0.01 ng/mL detection limit), are performed by 

injecting mice intraperitoneally with the specimen 

sample suspected to contain toxin (with and without 

various antitoxins). If toxin is present in the specimen, mice injected with the clinical specimen alone 

(without the specific antitoxin) will usually die from 

botulism within 6 to 96 hours, but mice injected with 

the specimen treated with the specific antitoxin will 

survive. Specimens for mouse bioassays may be sent 

The differential diagnosis of botulism includes other 

diseases with paralysis symptoms100:

• Diphtheria (may cause paralysis but also 

exhibits fever, typical pharyngeal or nasal 

mucosal lesions, cervical adenopathy, cardiac 

conduction abnormalities).

• Guillain-Barré syndrome (usually ascending 

paralysis, paresthesias common, elevated cerebrospinal fluid [CSF] protein [may be normal 

early in illness], electromyogram findings). The 

CSF findings are usually normal in botulism, 

but mild elevation of CSF protein between 

50 and 60 mg/dL has been noted in a minority of botulism patients. Most patients (90%) 

with the Miller Fisher syndrome variant of 

Guillain-Barré syndrome also have serum antibodies to the gangliosides GQ1b and GT1a.115

• Myasthenia gravis (dramatic improvement 

with edrophonium chloride, autoantibodies 

present, electromyogram findings). Botulism 

cases may have a positive response to edrophonium chloride (26%), but the response is 

generally not dramatic.

• Tick paralysis (ascending paralysis, paresthesias common, usually does not involve cranial 

nerves; detailed exam often shows presence 

of tick).

• Lambert-Eaton syndrome (commonly associated with carcinoma, particularly lung 

carcinomas; deep tendon reflexes absent; 

usually does not involve cranial nerves; electromyogram findings similar to botulism).

• Stroke or central nervous system mass lesion 

(paralysis usually asymmetric, brain imaging 

abnormal).

• Paralytic shellfish poisoning (history of shellfish ingestion; paresthesias of mouth, face, 

lips, and extremities common).

• Belladonna toxicity, such as atropine (history 

of exposure, tachycardia, and fever).

• Aminoglycoside toxicity (drug history of 

aminoglycoside therapy).

• Other neurotoxins, such as snake toxin (history of snake bite, presence of fang punctures).

• Chemical nerve agent poisoning (often associated with ataxia, slurred speech, areflexia, 

Cheyne-Stokes respiration, and convulsions).

A botulism diagnosis may be suggested by the clinical presentation of an afebrile patient with an acute, 

symmetric, descending, flaccid paralysis (without 

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Medical Aspects of Biological Warfare 

to the Centers for Disease Control and Prevention 

(CDC) or other designated state or municipal public 

health laboratories.120

Diagnosis can also be achieved by anaerobic culture 

and isolation of Clostridium species toxigenic strains 

from clinical specimens, including fecal specimens, 

gastric aspirates, vomitus, or infected wounds. The 

organism or toxin can also be isolated from the suspect 

food to help support the diagnosis.

In recent years the CDC has also used real-time 

polymerase chain reaction (PCR), Endopep-MS 

(mass spectrometry), and/or an enzyme-linked 

immunosorbent assay (A, B, E, F) to optimize its 

evaluation of botulism cases and outbreaks. However, real-time PCR detects DNA in fragments of 

C botulinum (not active BoNT), and PCR results 

require confirmation with the mouse bioassay or 

other validated test.108 PCR has the advantage of 

providing results within 24 hours (including serotype of botulism) versus up to 4 days (range 6 to 96 

hours) for results from mouse bioassays and 7 to 10 

days for cultures. Endopep-MS has a high sensitivity and specificity for detecting botulinum toxin, 

but generally is used as a secondary assay (ie, if 

mouse bioassay is negative or cannot be done due 

to inadequate sample). Other tests in various stages 

of development for detecting botulinum toxin (ie, 

lateral flow, endopeptidases, enzyme-linked immunosorbent assays, and electrochemiluminesence 

assays) have been developed.108

Foodborne Botulism

Toxin assays of specimens from foodborne botulism cases from 1975 to 1988 showed the presence 

of toxin in specimens from various sites as follows: 

sera, 53% (126/240); stool, 23% (65/288); and gastric 

aspirate, 5% (3/63). Specimens were more likely to 

be positive if obtained soon after toxin ingestion. 

Toxin assays of sera were positive in more than 

60% of specimens obtained within 2 days after toxin 

ingestion, in 44% of specimens obtained within 3 

days of toxin ingestion, but in only 23% of specimens 

obtained at day 4 or later.98 Toxin assays of sera were 

more likely to be positive in intoxications from toxin 

serotype A than from toxin serotypes B and E. Toxin 

assays of the stool were positive in 50% of specimens 

obtained within 1 day following toxin ingestion, in 

39% of specimens obtained within 3 days of ingestion, but in less than 20% of specimens obtained at 

day 5 or later.99

Stool and gastric aspirate cultures for C botulinum

resulted in a higher yield of diagnosis than toxin 

assays.99 Gastric aspirates were positive in 45% of 

specimens (35/78). Nearly 80% of stool cultures 

were positive at day 2 postingestion of toxin, with 

nearly 40% of specimens remaining positive at 7 to 

9 days after ingestion. However, in this cohort of 

patients, laboratory confirmation of botulism could 

not be obtained in 32% of patients, which reflects 

the insensitivity of the diagnostic testing, especially 

when specimens are obtained more than 3 days after toxin ingestion. In these patients, the diagnosis 

must be based on clinical history, physical examination, electromyography results, epidemiological 

history (including food consumption), and tests 

on ingested food samples from epidemiologically 

linked food. 

Inhalation-acquired Botulism

Laboratory confirmation of botulism acquired 

by inhalation may be difficult, because toxin acquired by inhalational exposure is not generally 

identifiable in the serum or stool, as in foodborne 

botulism.121,122 Although not validated, an enzymelinked immunosorbent assay or PCR test has been 

reported to detect botulinum toxin (using nasal 

mucosal swabs) from the nares for up to 24 hours 

after exposure.122,123 Antibody titers also have 

limited use in the diagnosis of botulism, because 

individuals may not develop an antibody response 

to the small quantity of toxin protein required to 

cause symptoms. Additionally, cultures of C botulinum are not helpful for definitive confirmation of 

inhalation of toxin preparations that do not contain 

spores of the organism.

Other Forms of Botulism

In wound botulism, serum toxin assays have been 

reported in one US cohort to be positive in 95% of cases 

associated with injecting drug use (mainly subcutaneous injection of black tar heroin) and in 83% of cases 

not associated with injecting drug use, but reported 

in other cohorts to be lower (range 38% to 68%).108

Toxin assays from the wound were reported positive 

in a third of cases, and C botulinum was cultured from 

the wound in 65% of cases (61% of cases in injecting 

drug use-associated and 83% of noninjecting drug 

use-associated botulism).108 Laboratory confirmation 

of infant botulism is often by toxin assay and culture 

of stool (positive in nearly all cases); toxin in the serum is less sensitive (only 13% [9/67] positive in one 

US cohort).108,124 Laboratory confirmation of iatrogenic botulism associated with injection of therapeutic 

preparations of botulinum toxin is by toxin detection 

in serum.125

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Botulinum Toxin

TREATMENT

usually required for treatment of botulism. However, a 

second or subsequent doses may be required if symptoms should continue to progress or relapse, as may 

occur after exposure to extremely high toxin doses, 

incomplete wound debridement in wound botulism, 

or persistent colonization in adult intestinal botulism. 

In such cases, a serum toxin assay is recommended 

24 hours after antitoxin administration.108 Antitoxin 

levels in four botulism patients given trivalent equine 

botulism antitoxin had previously demonstrated peak 

serum levels of antitoxin 10 to 1,000 times higher than 

the amount required to neutralize serum toxin levels.133

In 2010, equine antitoxins available at CDC were 

replaced with an investigational new drug (IND) 

despeciated equine heptavalent (A-G) antitoxin product (Cangene Corporation, Winnepeg, Canada) then 

known as HBAT. It was subsequently FDA approved 

in 2013 and renamed as BAT (Emergent Biosolutions, 

Gaithersburg, MD).134,135 Despeciated equine antitoxins 

are made by cleaving the Fc fragments from the horse 

immunoglobulin G molecules, resulting in only F(ab’)2

and Fab’ fragments that contain less than 2% horse 

protein. The decrease in the amount of horse protein 

may potentially reduce the risk of serum sickness and 

hypersensitivity reactions as observed with nondespeciated equine botulinum antitoxin products (1% 

risk with a single vial and 9% risk with 2 to 4 vials).136 

Symptoms in clinical trials in 16 healthy subjects were 

headache (9%), pruritus (5%), nausea (5%), and urticaria (5%). Safety data from 213/216 adult and 13/15 

pediatric subjects who received BAT for suspected or 

confirmed botulism (5 subjects receiving 2 doses) noted 

10% of subjects reported adverse reactions.137 Most 

common reactions reported were fever (4%), rash (2%), 

chills (1%), nausea (1%), and edema (1%). No subject 

experienced anaphylaxis, but one subject experienced 

mild serum sickness and one subject experienced hemodynamic instability characterized by bradycardia, 

tachycardia, and asystole during BAT administration. 

Rebound intoxication was observed in a case of adult 

intestinal botulism resulting from toxin serotype F 

and occurred 10 to 12 days after receiving the BAT.2

The rebound was attributed to the more rapid clearance of BAT from the circulation due to BAT’s shorter 

estimated serum half-life, and also due to intestinal 

colonization and toxin production from Clostridia.

2,108

BAT is the only botulism antitoxin available for noninfant patients. In emergencies it may be obtained from 

the CDC (contact the state or county health department 

or, alternatively, the CDC Emergency Operations Center at 770-488-7100 or 800-CDC-INFO [800-232-4636], 

or http://www.cdc.gov/phpr).134,135

The current recommended treatment for botulism, 

although limited, includes antitoxin therapy and supportive care as needed, including mechanical ventilation. Because respiratory failure may begin suddenly, 

individuals with suspected botulism should be closely 

monitored, with frequent assessment of the vital capacity and maximal inspiratory force.126 If ingestion of 

the implicated food has been recent, removal of unabsorbed toxins may be hastened with cathartic agents 

or enemas, provided ileus is not present. Decrease in 

gastric motility may require parenteral nutritional 

support. In wound botulism, antibiotic therapy and 

surgical debridement are recommended to remove 

the source of toxin production. Wound manipulation 

preferably should be done after antitoxin therapy administration because it may result in release of toxin 

into the bloodstream.105 Aminoglycosides, clindamycin, and magnesium containing medications should 

be avoided, if possible, as they may potentiate neuromuscular blockade.127–129

Antitoxin

Mortality from foodborne botulism before 1950 was 

approximately 60%,51 and has been reduced to less 

than 10% by the use of antitoxin therapy coupled with 

supportive care (often mechanical ventilation).31,101 As 

botulism antitoxins can only neutralize circulating 

antitoxin and have no effect on toxin already bound to 

nerve terminals (antitoxins do not reverse paralysis), 

antitoxin therapy should be administered as soon as 

possible.31,101 Early antitoxin administration has been 

associated with a decrease in the duration of illness, 

number of days of mechanical ventilation, requirement 

for mechanical ventilation, and duration of hospitalization.130 Early treatment, especially within 24 hours, 

is most effective in preventing paralysis progression. 

Therefore, it is recommended that antitoxin treatment commence with clinical suspicion, before the 

availability of definitive laboratory test results, and 

especially when a case is epidemiologically linked to 

a botulism outbreak.122,120 One retrospective analysis 

of 134 cases demonstrated a 10% mortality rate in 

patients who received antitoxin within 24 hours of 

symptom onset versus 15% among those who received 

late treatment.131

Because antitoxin cannot neutralize toxin once it 

has bound to the nerve receptors, the antitoxin cannot reverse paralysis; it can only prevent paralysis 

progression. Symptoms may often progress for 12 to 

24 hours after antitoxin administration before an effect 

is observed.132 Only one dose of antitoxin treatment is 

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Medical Aspects of Biological Warfare 

For treatment of infants with botulism resulting 

from toxin serotypes A and B, an FDA-approved bivalent (A/B) human botulism immune globulin product 

(BabyBIG/BIG-IV),138 is available at the California 

Department of Health.1,114,138 BabyBIG was derived 

from pooled plasma of adults immunized with an 

investigational botulinum toxoid. Because it is derived 

from humans, BabyBIG does not have the high risk of 

anaphylaxis observed with equine products, nor the 

risk of lifelong hypersensitivity to equine antigens. 

A placebo-controlled trial with BabyBIG in treating 

infant botulism (associated with a mortality less than 

2% even with supportive care without antitoxin) demonstrated efficacy by decreased duration of hospital 

stay, intensive care unit stay, mechanical ventilation, 

tube feedings, and a $55,000 cost savings per case.114 

A single infusion of BabyBIG has been estimated to 

neutralize toxin for up to 5 months, based on its prolonged half-life of approximately 28 days.139 BabyBIG 

may interfere with the response to live viral vaccines if 

given shortly before or within 5 months after BabyBIG 

administration. When botulism in infants is not due to 

toxin serotypes A or B (or if BabyBIG is not available), 

HBAT may be considered, as it has been successfully 

administered to one infant in the United States.140 Also, 

a retrospective review of 31 cases of infant botulism 

in Argentina treated with equine botulinum antitoxin 

was well tolerated (no serious hypersensitivity reactions) and was associated with a reduction in hospital 

stay and tube feeding by 24 days and mechanical 

ventilation by 11 days and a 47% decrease in sepsis.141

Animal studies with a heptavalent despeciated 

antitoxin (IND product developed at the US Army 

Medical Research Institute of Infectious Diseases that 

was a basis for BAT development) demonstrated efficacy in preventing and treating botulism in both 

mice and nonhuman primates against aerosol toxin 

challenge.97,134,142 The F(ab’)2 heptavalent, despeciated 

equine antitoxin toxin (known as Hfab-BAT) given to 

asymptomatic mice within a few hours after aerosol 

challenge with approximately 10 LD50 of serotype A, 

was protective, even with a dose as low as one-tenth 

of one human dose. This dose resulted in low levels 

of antitoxin titers, 0.02 IU/mL or lower.97 The product 

was also protective against aerosol challenge to toxin 

serotype A at a dose of approximately 2,000 mouse 

intraperitoneal LD50/kg, when given to nonhuman primates immediately before exposure (protection in 5/5 

animals), and when given 48 hours after inhalational 

exposure (protective in 3/5 monkeys).97

However, if antitoxin was given at the onset 

of respiratory failure, the Hfab-BAT product was 

not protective in the mouse model against aerosol 

exposure or intraperitoneal exposure, even with a 

dose that was 3-fold greater than the recommended 

human-equivalent dose. The ineffectiveness of delayed 

antitoxin administration in mice may be because the 

majority of toxin is no longer present in the circulation at the time of the antitoxin administration (ie, 

it is already bound to nerve terminals). Respiratory 

failure in mice occurred within 1 to 3 hours, and death 

occurred within 2.8 to 11 hours postexposure, which 

is earlier than observed in humans and nonhuman 

primates whose death generally does not occur until 

2 to 3 days postexposure. In one review of human 

foodborne botulism, shortness of breath at presentation was also identified as a poor prognostic factor for 

survival, even with antitoxin therapy; it was noted in 

94% (50 of 55) of the deaths.72 The Hfab-BAT and the 

HE-BAT despeciated equine antitoxin (also developed 

at the US Army Medical Research Institute of Infectious 

Diseases) are no longer available as IND products, and 

they have been replaced with the FDA-approved BAT 

despeciated antitoxin product.

In a successful Phase I clinical trial, a product made 

from three IgG1 monoclonal antibodies (Xoma 3AB) 

that target different regions of botulinum neurotoxin 

A and were engineered to neutralize toxin serotype 

subclasses A1, A2, A3, and A4 was well tolerated when 

given as a single intravenous injection infused over 

1 hour.143 The product has demonstrated a reduced 

mortality in mice when given prior to or up to 23 hours 

after toxin exposure. Although all three monoclonal 

antibodies were detected for a minimum of 4 weeks 

after infusion, the protective level of the monoclonal 

antibodies in humans is currently unknown. Further 

testing of this product may provide an antitoxin 

therapy for botulism that offers potential advantages 

of (1) a longer serum half-life, (2) decreased risk of allergic reactions (contains no residual equine proteins), 

and (3) mass production potential.144,145

Clinically Relevant Signs of Bioterrorist Attack

The first evidence of a bioterrorist attack with 

botulinum toxin would likely be reports from hospitals and urgent care medical facilities as they begin to 

receive victims with symptoms suggestive of botulism. 

Because antitoxin therapy given early has a greater 

beneficial effect, the initial diagnosis of botulism is 

based on clinical presentation with epidemiological associations, with subsequent confirmation by laboratory 

findings.34 Neurological signs and symptoms resulting 

from a toxin-induced blockade of neurotransmission 

at voluntary motor and cholinergic junctions dominate 

the clinical manifestation of botulism.98,146,147 A diagnosis of botulism is suggested in individuals presenting 

with an acute onset of cranial nerve weakness (ie, dip244-949 DLA DS.indb 346 6/4/18 
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Botulinum Toxin

lopia, ptosis, dysphonia, dysphagia, and dysarthria). In 

mild cases, no further symptoms may develop. In more 

severe cases, individuals may progress and develop 

descending symmetrical weakness and flaccid paralysis. Because mechanical ventilation may be required 

for individuals with respiratory failure resulting from 

paralysis of the respiratory muscles, hospital bioterrorism plans should include contingency plans for 

additional ventilatory and intensive care unit support 

for mass intoxication. Antitoxin therapy is indicated 

in cases of suspected botulism to inactivate and clear 

toxin from the circulatory system before it can enter 

peripheral cholinergic nerve cells.

An outbreak of botulism in 2004 illustrates the 

vulnerability of readily accessible bulk botulinum 

toxin. Four cases of botulism resulted from the use of 

toxin serotype A for cosmetic purposes. A vial of raw 

bulk botulinum toxin (a non-FDA approved formulation) containing between 20,000 and 10 million units 

of botulinum toxin (a vial of FDA-approved BOTOX 

[Allergan, Inc, Irvine, CA] contains only 100 units of 

toxin) was used by an unlicensed physician for cosmetic injections into three patients and himself.148,149

The four individuals were subsequently admitted 

to medical facilities with symptoms of botulism and 

faced a long-term recovery.150 One of these patients 

was iatrogenically injected with as much as 8 million 

mLD50 (2,857 times the human lethal dose by injection) 

and has survived with intensive medical care, including long-term mechanical ventilation, although with 

chronic sequelae.106,150 This patient began improving 

4 weeks after the event, and achieved a remarkable 

recovery 14 weeks from injection.107

Preexposure and Postexposure Prophylaxis

Although passive antitoxin prophylaxis has been 

effective in protecting laboratory animals from toxin 

exposure, the limited availability and short-lived 

protection of antitoxin preparations make preexposure or postexposure prophylaxis with these agents 

impractical for large numbers of persons.121,151 Administration of equine antitoxin is not recommended 

for preexposure prophylaxis because of the risk of 

anaphylaxis from the foreign equine proteins, particularly with repeated doses. These products are 

not generally recommended for use in asymptomatic persons. In asymptomatic persons with known 

exposure to botulinum toxin, the risk of anaphylaxis 

from the equine antitoxin must be weighed against 

the risk of disease from botulinum toxin. However, 

botulinum immune globulin is most effective when 

administered within 24 hours of a high dose aerosol 

exposure to botulinum toxin. 

No FDA-approved vaccines exist to prevent botulism. Of historical note, a bivalent botulinum toxoid 

(serotypes A and B) had been given to at-risk laboratory workers in the US offensive biological warfare 

program at Fort Detrick beginning in 1945.152 Between 

1945 and 1969, 50 accidental exposures to botulinum 

toxins (24 percutaneous, 22 aerosol, and 4 ingestion) 

were reported, but no cases of laboratory-acquired 

botulism occurred, possibly because of the toxoid immunizations. An IND product, the pentavalent botulinum toxoid (PBT) against botulinum toxin serotypes 

A through E, had been used since 1959 for persons at 

risk for botulism (ie, laboratory workers)152–154 but is 

no longer available as an investigational product on 

protocol through the CDC. In 2012, due to declining 

immunogenicity and increased local reactogenicity 

observed with the requirement for annual booster 

doses to maintain immunity,155–157 the CDC discontinued its sponsorship of IND that provided PBT to 

at-risk laboratory workers. 158–160 The declining immunogenicity of the PBT was not unexpected, given 

that the PBT was manufactured more than 30 years 

earlier. PBT is a toxoid (toxin that has been inactivated) derived from formalin-inactivated, partially 

purified toxin serotypes A, B, C, D, and E, which 

was developed by the Department of Defense at Fort 

Detrick and originally manufactured by Parke-Davis 

and Company (Detroit, MI). PBT was found to be 

protective in animal models against challenge with 

botulinum toxin serotypes A through E,161 including 

protection in nonhuman primates against aerosol 

challenge to toxin serotype A.162

PBT was originally given as a primary series of 

three subcutaneous injections (0.5 mL at 0, 2, and 

12 weeks), a booster dose at 12 months, and annual 

booster doses thereafter.163 PBT was administered to 

thousands of at-risk persons, and clinical experience 

has shown the toxoid to be safe and immunogenic. 

The vaccine has mainly been used for laboratory 

workers who work directly with botulinum toxin. 

Approximately 8,000 service members also received 

the toxoid between January 23 and February 28, 

1991, as part of the US force deployed to the Persian 

Gulf War.164 The main adverse event was local reactions. Adverse events passively reported to the CDC 

between 1970 and 2002 for more than 20,000 vaccinations included moderate local reactions (edema 

or induration between 30 mm to 120 mm) in 7% of 

vaccinees, and severe local reactions (reaction size 

>120 mm, marked limitation of arm movement, or 

marked axillary node tenderness) in less than 1%.158

To allow recent vaccinees to complete the primary 

series of PBT, the investigational new drug protocol 

remained in effect through May 31, 2012.158

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Medical Aspects of Biological Warfare 

New Vaccine Research

Future vaccine candidates for FDA approval will 

probably not include formalin-inactivated toxoids 

similar to the formalin-inactivated PBT for several 

reasons.165,166 Production of formalin-inactivated toxoids requires partially purified culture supernatants 

containing botulinum toxin to be treated with formaldehyde, which must be performed by highly trained 

staff using a dedicated high-containment laboratory 

space.167 Furthermore, the relative impurity of the toxoid (PBT contained only 10% neurotoxoid and 90% is 

irrelevant material) likely contributed significantly to 

the need for multiple injections to achieve and sustain 

protective titers, as well as increased local reactogenicity associated with multiple injections.

The use of pure and concentrated antigen in 

recombinant vaccines offers the advantages of 

increased immunogenicity and a decrease in reactogenicity (local reactions at the injection site) 

over formalin-inactivated toxoids.168 Recombinant 

techniques use an immunogenic toxin fragment, 

which does not have the capability of blocking 

cholinergic neurotransmitters. Both Escherichia coli

and yeast expression systems have been used in 

recombinant fragment production, primarily the 

carboxy-terminal fragment (Hc) of the toxin’s heavy 

chain. Vaccine candidates using recombinant fragments of botulinum toxins against serotypes A, B, 

C, E, and F were protective in mice.169–177 A vaccine 

recombinant candidate for serotype A was protective in mice against intraperitoneal challenge and 

produced immunity levels similar to that attained 

with PBT, but with an increase in safety and decrease 

in cost per dose.167 Recombinant vaccines given by 

inhalational route are also being investigated.178,179

The only botulinum vaccine candidate currently in 

advanced development is a bivalent recombinant botulinum vaccine (rBV A/B [P pastoris] for toxin serotype 

A and B) developed by the US Army Medical Research 

Institute of Infectious Diseases for protection against 

botulinum toxin serotype A (subtype 1) and botulinum 

toxin serotype B (subtype B1).180 Animal studies have 

demonstrated protection against toxin challenge by 

aerosol and intramuscular challenge with the two 

toxin serotypes.181 The safety data and immunological 

responses from Phase 1 and Phase 2 clinical trials and 

animal data support the continued investigation of the 

rBV A/B for FDA vaccine licensure.179,181–184 

Another vaccine candidate evaluated involved the 

insertion of a synthetic carboxy-terminal fragment 

(Hc) gene of the heavy chain of toxin serotype A into 

the vector system of the Venezuelan equine encephalitis virus.173 This vaccine induced a strong antibody 

response in the mouse model and remained protective 

in mice against intraperitoneal challenge at 12 months. 

However, the presence of BoNT variants as subtypes 

or chimeras challenges the development of any comprehensive “pan-botulinum neurotoxin” vaccines and 

therapies.185 BoNT subtypes are natural variants of the 

prototype BoNT serotype that vary in primary amino 

acid sequence from 3% to 26% depending on the serotype, whereas BoNT chimeras are natural variants 

that appear to have derived from recombinant events 

between two BoNT serotypes.186 BoNT subtypes have 

unique activities relative to the prototypical BoNT 

serotype.186 One novel approach to vaccine design 

uses structure-based knowledge to produce a single 

molecule containing the immunodominant epitopes 

of multiple, antigenic distinct variants to develop a 

meningococcal pan-vaccine.187 A similar research effort 

may eventually allow the development of a similar 

pan-vaccine strategy for all seven variants of botulinum neurotoxins.188

New Therapeutic Drug Research

In addition to the vaccine research for prophylaxis, 

efforts are underway to find postexposure, pharmacologic treatments. The development of low molecular 

weight inhibitors is related to work that was designed 

to pharmacologically “deduce commonalities” among 

the serotypes.189 Different molecular targets for inhibitor candidates include those that prevent toxin binding to nerve terminals and internalization or those 

that inhibit the neurotoxin’s proteolytic activity. One 

limitation for this approach to be useful is that when 

the neurotoxin molecule is internalized within the presynaptic endings, the neurotoxin is—as with neutralizing antibodies—no longer susceptible to circulating 

inhibitors. Thus, effective therapeutic inhibitors must 

also be internally delivered to nerve termini without 

creating additional adverse central effects. Another 

problem concerns the diversity of the substrate binding sites (exosites)190 and active site structures among 

the serotypes.191 This diversity implies that a single 

inhibitor will not antagonize the substrate binding and 

subsequent proteolytic activity of all the neurotoxin 

serotypes.

Active site inhibitor candidates have been extensively reviewed192,193 with new candidates appearing 

frequently in the literature.194–197 Within the BotDB 

resource (http://botdb.abcc.ncifcrf.gov),198 the BotDBI 

section199 has information on more than 60 inhibitor 

candidates that lists peptides, synthetic and natural 

compounds, and antibodies.200

A steady rise over the past 30 years has occurred in 

the clinical/therapeutic uses of this neurotoxin for vari244-949 DLA DS.indb 348 6/4/18 
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Botulinum Toxin

ous disorders. The FDA-approved uses include treatment of blepharospasm, strabismus, cervical dystonia, 

upper limb spasticity, axillary hyperhidrosis, detrusor 

overactivity associated with a neurologic condition, 

and chronic migraine.200 Other potential uses are still 

under investigation including those for wound healing,201 treatment of cerebral palsy,202,203 treatment of 

lower urinary tract disorders,204,205 controlling multiple 

sclerosis spasticity,206 and various modalities of pain 

management.207–209 Because of these clinical conditions, 

very small doses of the neurotoxin may be used for a 

measurable beneficial effect. One novel clinical application involves the neurotoxin-induced reduction 

of hyperhidrosis.210

SUMMARY

The neurotoxins produced by Clostridia species 

are among the most potent toxins known. Botulinum 

toxin has been studied and developed as a biological 

weapon by many countries, and it should be considered as a bioterrorism threat agent. A mass casualty 

event caused by botulinum toxin, which has been depicted by a mathematical model, has the potential to 

cause great harm. Botulism is most commonly caused 

by neurotoxin types A, B, and E, is a neuroparalytic 

disease, and often fatal if untreated. Paralysis from 

botulism can be long-lasting, with concomitant and 

demanding supportive care requirements. Clinicians 

should be able to recognize the early signs and symptoms of botulinum intoxication, as early initiation of 

antitoxin therapy has been associated with decreased 

duration of mechanical ventilator support and days 

spent in intensive care, and increased survival. Antitoxin therapy should be initiated based on clinical 

presentation consistent with botulism and epidemiological history, as results of laboratory confirmatory 

assays may not be available for days.120

For infants with botulism resulting from toxin serotypes A and B, an FDA-approved human antitoxin 

known as BabyBIG is available at the California Department of Public Health.143 An FDA-approved despeciated equine heptavalent antitoxin product known 

as BAT (serotypes A-G) is the only antitoxin for adults 

(and for infants if BabyBIG is not available or botulism 

not due to serotypes A or B), and is available through 

the CDC.120 No FDA-approved vaccine for botulism 

currently exists. The IND PBT product is no longer 

available for protection of at-risk laboratory workers 

because of declining immunogenicity and increased 

local reactogenicity associated with required annual 

booster doses.122 Future vaccine research may lead to 

a new class of recombinant vaccines to protect against 

botulism while pharmacologic approaches may lead 

to viable postexposure drug treatments. 

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161. Cardella MA, Fiock MA, Wright GG. Immunologic Response of Animals to Purified Pentavalent ABCDE Botulinum Toxoid 

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163. Fiock MA, Cardella MA, Gearinger NF. Studies on immunity to toxins of Clostridium botulinum. IX. immunologic 

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166. Torii Y, Tokumaru Y, Kawaguchi S, et al. Production and immunogenic efficacy of botulinum tetravalent (A, B, E, F) 

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167. Middlebrook JL. Protection strategies against botulinum toxin. Adv Exp Med Biol. 1995;383:93–98.

168. Holley JL, Elmore M, Mauchline M, Minton N, Titball RW. Cloning, expression and evaluation of a recombinant subunit vaccine against Clostridium botulinum type F toxin. Vaccine. 2000;19:288–297. 

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172. Shyu RH, Shaio MF, Tang SS, et al. DNA vaccination using the fragment C of botulinum neurotoxin type A provided 

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174. Potter KJ, Bevins MA, Vassilieva EV, et al. Production and purification of the heavy-chain fragment C of botulinum 

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205. Mangera A, Andersson KE, Apostolidis A, et al. Contemporary management of lower urinary tract disease with 

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207. Fu J, Ngo A, Shin K, Bruera E. Botulinum toxin injection and phenol nerve block for reduction of end-of-life pain. J 

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208. Varma G. Role of botulinum toxin type-A (BTX-A) in the management of trigeminal neuralgia. Pain Res Treat. 2013; 

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Chapter 15

CLOSTRIDIUM PERFRINGENS EPSILON 

TOXIN

BRADLEY G. STILES, PhD*; GILLIAN BARTH, BS†; and MICHEL R. POPOFF, PhD, DVM‡

INTRODUCTION

HISTORY

DESCRIPTION OF THE EPSILON TOXIN

Natural Occurrence

Chemical and Physical Properties 

Mechanism of Action

CLINICAL SIGNS AND SYMPTOMS

MEDICAL MANAGEMENT

SUMMARY

*Adjunct Professor of Biology, Biology Department, Wilson College, 1015 Philadelphia Avenue, Chambersburg, Pennsylvania 17201

†

Veterinary Technician, Veterinary Department, Wilson College, 1015 Philadelphia Avenue, Chambersburg, Pennsylvania 17201

‡

Chief, Anaerobic Bacteria and Toxins, Institut Pasteur, 28 Rue du Dr Roux, 75724 Paris, France

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INTRODUCTION

Clostridium perfringens is a gram-positive, sporeforming anaerobe commonly found throughout nature 

(ie, soil, water, gastrointestinal tracts of humans and 

animals, etc). This bacillus is one of the most “toxic” 

bacteria described to date, producing at least seventeen different “major” and “minor” protein toxins.1,2

Other pathogenic species of Clostridium synthesize 

the most potent protein toxins known, such as tetanus 

and botulinum neurotoxins. Unlike a number of other 

bacterial pathogens (ie, Listeria, Rickettsia, Salmonella, 

Shigella, and Yersinia species), current understanding 

of C perfringens pathogenesis during various diseases 

does not include invasion, and subsequent replication, 

in eukaryotic cells. 

C perfringens was first isolated in 1892 by William 

Welch and George Nuttall at Johns Hopkins University 

in Baltimore following an autopsy of a cancer patient. 

Of note was a rather profuse, unusually explosive 

formation of gas bubbles within the cadaver’s blood 

vessels and organs only 8 hours after death. Gas is a 

common byproduct of anaerobic growth by clostridial 

species, explaining the term “gas gangrene” during 

severe myonecrosis induced by C perfringens. Over 

time and throughout the literature, C perfringens has 

also been known as Bacillus aerogenes capsulatus, Bacillus 

welchii, and Clostridium welchii. Many ill-effects induced 

by C perfringens in humans and animals are linked to 

protein toxins. Below are succinct descriptions of the 

classically defined major (typing) toxins with a particular emphasis on epsilon, which has been targeted 

recently as a select agent by various agencies within 

the United States and other countries.

HISTORY

Protein toxins are considered important virulence 

factors for C perfringens, and have thus received much 

attention by various laboratories throughout the world. 

For many bacterial pathogens, toxins possessing diverse modes of action play critical roles in survival 

that include nutrient gathering and thwarting the 

host’s immune system. There are two primary modes 

of action described for the four major (typing) toxins 

produced by C perfringens: 1) increasing permeability 

of cell membranes (ie, alpha, beta, and epsilon toxins) 

resulting in ion imbalances and general leakiness; and 

2) destroying the actin cytoskeleton (ie, iota toxin).2

 In 

either scenario, the end result elicited by any of these 

toxins is the same: cell dysfunction and death. Multiple 

studies by many groups reveal that C perfringens possesses highly evolved tactics, involving offensive (ie, 

secreted protein toxins plus enzymes) and defensive 

(ie, protein toxins plus spores) tools for surviving, 

and then thriving, in harshly diverse environments. 

C perfringens consists of five toxin types: A, B, C, D, 

and E (Table 15-1), based on the production of one or 

more protein toxins.1,2

Each of these toxins is lethal, dermonecrotic, and 

associated with a wide range of diseases that include 

a rapid, life-threatening myonecrosis (gas gangrene) 

and various enteric illnesses in both animals as well 

as humans (Table 15-2). Historically, for diagnostic 

purposes these typing toxins would be neutralized in 

the laboratory by type-specific antisera in mouse-lethal 

and guinea-pig dermonecrotic assays. The toxin source 

would consist of culture filtrate from C perfringens

isolated from a patient.3 Today, rapid genetic methods involving multiplex polymerase chain reactions 

TABLE 15-1

TOXIN TYPES OF CLOSTRIDIUM PERFRINGENS

Major (typing) 

Toxins

Toxin Type

A B C D E

Alpha √ √ √ √ √

Beta √ √

Epsilon √ √

Iota √

are more commonly used by diagnostic laboratories 

around the world for typing C perfringens.

4,5 This technique, although rapid and accurate, suggests the presence of a toxin gene but indicates neither production 

nor relative quantities of a biologically-active protein. 

Rapid quantitation of epsilon toxin protein in complex 

matrices (eg, milk and serum) is also possible using 

a novel, mass spectrometry technique; however, this 

does not determine whether the toxin is biologically 

active.6 

Unlike the other typing toxins, alpha is, by definition, produced by all C perfringens and has played a 

significant role in military casualties over time. Type A 

strains are most commonly found in the environment 

and cause gas gangrene.1,7–11 Alpha toxin facilitates gas 

gangrene due to C perfringens infection, an omnipresent threat to soldiers wounded on the battlefield.7,8

Deep penetrative wounds, soiled by dirt that contains 

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Clostridium Perfringens Epsilon Toxin

TABLE 15-2

CLOSTRIDIUM PERFRINGENS TOXIN TYPES 

AND DISEASES

Toxin Type Disease/Intoxication

A Myonecrosis (gas gangrene), necrotic enteritis of fowl and piglets, human food poisoning, antibiotic-associated diarrhea

B Dysentery in lambs; hemorrhagic enteritis in 

calves, foals, and sheep

C Necrotizing enteritis in humans (known 

as pigbel, darmbrand, or fire-belly), pigs, 

calves, goats, and foals; enterotoxemia in 

sheep (struck)

D Enterotoxemia in lambs (pulpy kidney or 

overeating disease) and calves; enterocolitis 

in goats and cattle

E Calf and lamb enterotoxemia

various clostridial species (especially C perfringens), 

are often to blame for quickly advancing disease in 

the buttocks, thigh, shoulder, upper extremity, and 

leg (in order of decreasing prevalence).7,10,11 The fatality rate from gas gangrene was 50% during World 

War II; it was especially high when fighting occurred 

in cultivated land (commonly fertilized with animal 

feces), as opposed to desert (eg, Tunisia).10,11 The threat 

of gangrene from C perfringens or other clostridial 

species11 due to wound contamination in the field 

or nonsterile operating conditions was particularly 

prevalent before 1900 and resulted in many amputations and deaths that can be avoided in modern 

times. If administered promptly after disease onset, 

medical countermeasures, such as extensive surgical 

debridement, various antibiotics (eg, beta-lactams, 

clindamycin, metronidazole), and hyperbaric oxygen provide effective treatment for most cases of C 

perfringens-induced gangrene. Antitoxin (historically, 

polyclonal antibodies of equine origin) administration 

is also another possible therapy that targets alpha toxin 

and mitigates myonecrosis.7,10,12 Vaccine studies from 

various groups using the carboxy-terminal (cell binding) domain of alpha toxin show prophylactic protection against either toxin-induced lethality or bacterial 

challenge in a mouse gangrene model.13,14 

Biochemically, alpha toxin is a zinc-containing 

phospholipase C (43 kDa) composed of two structural 

domains that destroys eukaryotic cell membranes.15,16

The amino-terminal domain contains a catalytic site 

and ganglioside (GM1a) binding motif, the latter being similar to that found on another clostridial toxin 

studied by the biodefense community: Clostridium 

botulinum neurotoxin.17 Interaction of GM1a by alpha 

toxin promotes clustering and activation of tyrosine kinase A involved in signal transduction.17 The carboxyterminal domain of alpha toxin binds to phospholipids 

on cell membranes. 

In comparison to the alpha toxin and due to recent national and international biodefense concerns, C perfringens epsilon toxin has received much more government 

attention (ie, funding and regulated oversight) over the 

past 15 years as a potential agent used in biowarfare and 

bioterrorism.18 Epsilon is the most potent of all C perfringens toxins as determined by a very low LD50 (toxin 

amount necessary to kill 50% of the subject population; 

murine intravenous assay), ranking behind only the C 

botulinum and C tetani neurotoxins among all clostridial 

toxins. In the very recent past, the Centers for Disease 

Control and Prevention placed epsilon toxin on the 

Category B list of select agents, along with bacterial diseases (eg, brucellosis, glanders, and typhus) plus other 

protein toxins (eg, ricin and staphylococcal enterotoxin 

B). Additionally, epsilon toxin represented a potential 

agroterrorism threat and was deemed a select agent by 

the United States Department of Agriculture. However, 

when the select agents list was modified in December 2012, C perfringens epsilon toxin was removed.19 

In France, but not throughout Europe, epsilon toxin 

is still classified as a potential biological weapon and 

requires special authorization from a federal agency 

(Agence Nationale de Securite du Medicament) before 

being approved for laboratory work. There are varying opinions around the world regarding C perfringens

epsilon toxin, its potential nefarious applications, 

and imposed level of government regulations. Such 

nonconsensus among allies affects sustained funding 

and effective collaborations between investigators, 

laboratories, and nations. 

DESCRIPTION OF THE EPSILON TOXIN

Natural Occurrence

The epsilon toxin is naturally produced by types B 

and D C perfringens that are involved in animal (cattle, 

goat, and sheep) enterotoxemias that often prove widespread, rapidly fatal, and economically damaging for 

the agriculture industry.1,20 Although C perfringens is 

considered normal intestinal flora in ruminants, types 

B and especially D can cause severe, life-threatening 

illness in a “naïve” digestive system shortly after 

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Medical Aspects of Biological Warfare 

birth, or following a diet change involving higher 

carbohydrate levels (particularly starch) among older 

animals. If there is little microbial competition within 

the gastrointestinal tract or an overly nutrient-rich diet 

is suddenly consumed, resident C perfringens types 

B and D can rapidly proliferate in the intestines and 

concomitantly produce life-threatening levels of toxins 

that include epsilon. Those who study C perfringens

and disease naturally associate the epsilon toxin with 

veterinary issues. In fact, neither the epsilon toxin nor 

C perfringens types B and D infections are commonly 

linked to human disease, which may make the toxin 

ideal for nefarious use as a biological weapon against 

humans: a bioterrorist event employing epsilon toxin 

against humans could be very difficult to diagnose 

and treat because there is no “natural” precedent and 

classically trained physicians do not anticipate human 

illness linked to C perfringens epsilon toxin. 

Chemical and Physical Properties 

C perfringens epsilon toxin is a plasmid-borne, 311 

amino acid (32.9 kDa) protein secreted as a protoxin, 

activated subsequently by extracellular proteases 

(trypsin and chymotrypsin) that remove amino-terminal (14 amino acids) and carboxy-terminal (29 amino 

acids) peptides.20 The nascent protoxin contains a 

typical leader sequence (32 amino-terminal residues) 

that normally facilitates protein secretion from the 

bacterium into the environment. The toxin is resistant 

to inactivation by serine-type proteases commonly 

found throughout nature, including those in the gastrointestinal tracts of various mammals. 

The crystal structure of epsilon toxin (Figure 15-1) 

reveals an elongated beta-sheet (100 Å × 20 Å × 20 Å) 

containing three domains, which shares conformation 

with other bacterial (aerolysin family) pore-forming 

toxins, including Aeromonas hydrophila aerolysin, 

C perfringens enterotoxin, and Clostridium septicum

alpha toxin.21,22 The putative roles for each domain 

of epsilon toxin include receptor binding (domain 

I: amino terminus), channel formation (domain II: 

central region), and monomer-monomer interaction 

(domain III: carboxy terminus).2,21,23 Loss of a carboxyterminal peptide from epsilon toxin seems primarily 

responsible for monomer-monomer interactions and 

subsequent homoheptamer formation.23 

Proteolysis is a common method of activating many 

bacterial toxins, and for epsilon this process induces 

conformational changes that facilitate homooligomerization on the external surface of a eukaryotic cell. 

Essentially, proteolytic activation causes “protein 

priming” that enables the toxin to quickly act after 

binding to diverse target cells of neuronal, renal, 

and endothelial origins (described in detail below). 

Additionally, proteolysis of the amino- and carboxytermini on the epsilon protoxin leads to a more acidic 

isoelectric point (5.4 versus 8.0), which may play a 

role in receptor interactions.20,24 For enteric-produced 

toxins requiring proteolysis, the proteases synthesized 

by resident bacteria (including C perfringens lambda 

toxin)25 and host23 are bountiful. Recent evidence 

suggests that epsilon toxin can be activated intracellularly (in select strains), remains in C perfringens until 

stationary or death phase, and is finally released into 

the environment following autolysis.26 This unique 

protease and a further understanding of its novel 

activation mode in clostridia (and possibly in other 

secreted toxin-producing pathogens) remains elusive.

Mechanism of Action

The mode of action for epsilon toxin involves pore 

formation in eukaryotic cell membranes facilitated by 

detergent-resistant membrane fractions, also known 

Figure 15-1. Crystal structure of Clostridium perfringens epsilon toxin. Three domains exist and are putatively involved 

in (I) receptor binding (amino terminus), (II) channel formation, and (III) monomer-monomer interactions (carboxy 

terminus). Designated amino acids (tyrosines 29, 30, 36, and 

196) facilitate receptor (hepatitis A virus cellular receptor 1) 

binding, and when individually replaced with glutamic acid, 

yield nontoxic variants of epsilon toxin.

Data sources: (1) Cole AR, Gibert M, Popoff MR, Moss 

DS, Titball RW, Basak AK. Clostridium perfringens epsilontoxin shows structural similarity to the pore-forming toxin 

aerolysin. Nat Struct Mol Biol. 2004;11:797–798. (2) Ivie SE, 

McClain MS. Identification of amino acids important for 

binding of Clostridium perfringens epsilon toxin to host cells 

and to HAVCR1. Biochemistry. 2012;51:7588–7595. (3) Madej T, 

Addess KJ, Fong JH, et al. MMDB: 3D structures and macromolecular interactions. Nucleic Acids Res. 2012;40:D461–464.

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Clostridium Perfringens Epsilon Toxin

as lipid rafts, that concentrate toxin monomers into 

homoheptamers.27,28 These cholesterol-rich membrane 

domains play important roles in many diseases elicited 

by bacteria, associated toxins, and viruses.29 Furthermore, caveolins 1 and 2 found in these domains are 

bound directly by epsilon toxin and necessary for toxin 

oligomerization.30 Epsilon toxin oligomers, critical for 

biological activity: 

• form within 15 minutes at 37°C on MadinDarby canine kidney cells (MDCK); 

• are more stable at 37°C versus 4°C; 

• promote potassium efflux; and 

• are internalized from the cell surface, forming vacuoles in the late endosomes and lysosomes.31,32 

Sialidases, also known as neuraminidases, are 

produced by C perfringens and enhance binding of 

the bacterium as well as epsilon toxicity to select 

cultured cells.33 For example, in the presence of 

bacterial-derived sialidase, there is increased adherence of C perfringens to a human colorectal epithelial 

line (Caco-2), but not to MDCK or Vero (monkey 

kidney) cells. Contact between the bacterium and 

certain cell lines like Caco-2 leads to gene upregulation and increased activity for C perfringens-derived 

sialidase. Increased toxin binding to target cells following sialidase activity has been described previously for another enteric toxin-producing pathogen, 

Vibrio cholerae.

34 

In concert with disrupted cell membranes facilitating free passage of 1 kDa-sized molecules,35 secondary 

effects of epsilon toxin involve cytoskeletal dysfunction that becomes lethal for an intoxicated cell.36 Additionally, the integrity of a cell monolayer is readily 

disrupted by epsilon toxin,27 providing another clue 

toward understanding subsequent dysfunction of 

the vascular endothelium, edema, and crossing of 

the blood-brain barrier.37 The ill effects induced by 

epsilon toxin upon the circulatory system are quite 

substantial, with albumin-sized molecules (~ 65 kDa) 

subsequently transiting from the blood stream into 

the brain.38

CLINICAL SIGNS AND SYMPTOMS

Although epsilon toxin can be found in the heart, 

lungs, liver, and stomach following intoxication, it 

noticeably accumulates in kidneys, causing what 

veterinarians classically refer to as “pulpy kidney 

disease.”1,2,20,39–41 Another indicator that kidneys are 

a primary target of epsilon toxin is that the few susceptible cell lines discovered to date are of kidney 

descent from dog, mouse, and human.20 Postmortem 

results from lambs and mice given epsilon toxin show 

similar results in the kidneys that include congestion, 

interstitial hemorrhage, and degenerated epithelium 

within the proximal tubules. Toxin accumulating in the 

kidney may represent a natural defense mechanism by 

the host to prevent lethal concentrations in the brain.41

During lamb enterotoxemia, glucose excretion into the 

urine following epsilon toxin exposure may be a result 

of liver-released glycogen.42 

The ability of C perfringens epsilon toxin to rapidly disrupt the blood-brain barrier, bind neuronal 

cells, and cause lethality is of obvious concern.20,37,39

Among neuronal cell populations (sheep), neurons 

are most susceptible, followed by oligodendrocytes 

and astrocytes.43 In the brain, there are signs of swelling, vacuolation, and necrosis. Edema in the rat brain 

following an intraperitoneal injection of epsilon toxin 

causes increased levels of aquaporin-4 in astrocytes, 

which may be the body’s attempt to reduce osmotic 

pressure surrounding sensitive neurons.44 Aquaporins 

are membrane proteins that regulate water flow in 

various cell types and could be a target for therapies 

against epsilon toxin. Necrosis of the brain following 

epsilon intoxication is likely due to multiple factors 

that include reduced blood flow, sustained hypoxic 

state, and direct toxicity on various cell types. 

Clinical signs attributed to epsilon toxin given intravenously to calves, lambs, and young goats occur 

rapidly (approximately 30 minutes, depending on 

dose).45,46 These animals can experience labored breathing, excited or exaggerated movements, intermittent 

convulsions, loss of consciousness, and ultimately 

death. Further signs of epsilon intoxication include 

elevated blood pressure and vascular permeability, 

lung edema, and brain congestion with edema.20 Results from another laboratory reveal that an intravenous injection of epsilon toxin (2–4 LD50, in which an 

LD50 is ~ 70 ng/kg) into mice yields seizures within 60 

minutes.47 More naturally, duodenal inoculation of 

goats with whole culture or supernatant of C perfringens type D leads to diarrhea, respiratory distress, and 

central nervous system dysfunction (ie, recumbency 

and convulsions).48 Similar symptoms are also evident in lambs, minus the diarrhea.49 Furthermore, in 

ovines (namely lambs) there can be sudden death or 

acute disease involving neurological manifestations 

that include struggling, opisthotonos, convulsions, 

lateral recumbency, and violent paddling. The mode 

of action for epsilon toxin in vivo seemingly involves 

ion imbalance, endothelial disruption, and edema. 

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A vicious cycle is established by C perfringens epsilon 

toxin in the intestinal tract via increased permeability, 

leading to higher circulating levels of toxin.43 

Further studies with a wild-type D culture (sheep 

isolate, CN1020) given intraduodenally to sheep, goats, 

and mice reveal that a genetic knockout of epsilon 

toxin does not cause disease compared to controls.50

Complementing this mutant with the wild-type toxin 

gene generates the original phenotype that causes 

epsilon toxin-based disease. In this thorough study, 

clinical signs of epsilon intoxication in mice included 

depression, ataxia, circling, and dyspnea. Overall, these 

results show that the epsilon toxin from a type D strain 

is critical for disease in these diverse animal models. 

Different animal models show that the toxin is quite 

active when given intravenously or intraduodenally. 

However, from a biodefense perspective, critical data 

are lacking in the literature for epsilon toxin and effects 

following aerosol delivery. It is unclear what happens 

to nonhuman primates following epsilon intoxication. 

To establish a basal level of knowledge for further 

study and develop medical management strategies 

for humans, it is necessary to carry out and publish 

nonhuman primate studies. 

MEDICAL MANAGEMENT

From a therapeutic perspective, very little has been 

done with C perfringens epsilon toxin, partly because 

of its natural association with animal (and not human) 

disease. An effective vaccine against epsilon toxin 

(described below) is readily available for animal use, 

thus obviating the need for a therapeutic in susceptible 

populations. There is no therapeutic agent or vaccine 

against epsilon toxin approved for human use at this 

time. Findings from different laboratories and various in vivo and in vitro studies suggest that therapy 

is possible. Perhaps a proteomics-based approach 

following epsilon toxin exposure can reveal unique, 

host-based targets for therapeutic intervention. This 

approach has recently been taken in mice given epsilon toxin intravenously, with subsequent analysis of 

select organs (brain plus kidney), plasma, and urine 

for differentially-expressed proteins.51 However, there 

is much more work to be done on this front. 

Miyamoto et al47 show that riluzole, a benzothiazole 

(234 Da) used for treating human amyotrophic lateral 

sclerosis by preventing presynaptic glutamate release, 

can minimize murine seizures induced by epsilon toxin. These results were manifest after riluzole injection 

(16 mg/kg, intraperitoneal) given 30 minutes before 

toxin (140 or 280 ng/kg, intravenous), but the drug 

was evidently not used in subsequent experiments as a 

therapeutic administered after toxin. Another murinebased study shows that epsilon toxin binds preferentially to the cerebellum, particularly oligodendrocytes 

plus granule cells.52 Incubation of primary-cultured 

granule cells with epsilon toxin causes a rapid increase 

in intracellular calcium levels and glutamate release. 

This study provides a brain cell-specific target (and 

assay) for therapeutic intervention (and screening of 

potential therapeutic molecules).52

Small molecule libraries have also been screened 

using an MDCK cell assay for therapeutic effects that 

counter epsilon toxin.53 Three structurally unique 

inhibitors were discovered that do not prevent toxin 

binding or oligomerization on cells, but likely affect 

pore function or an unidentified cofactor important 

in epsilon intoxication. Two of these compounds 

(N-cycloalkylbenzamide and furo[2,3-b]quinoline) 

protected cells when added 10 minutes after toxin 

exposure, thus providing therapeutic potential in an 

in-vitro scenario. Such results logically lead to efficacy 

studies in animals, though none have been published 

to date. 

Another therapeutic approach against epsilon toxin 

includes dominant-negative inhibitors, which have 

been successfully employed as experimental therapeutics for other oligomer-forming bacterial toxins 

produced by Bacillus anthracis, Escherichia coli, and 

Helicobacter pylori.

54–57 This concept involves a recombinantly modified toxin that is no longer toxic after 

deleting a peptide region or substituting specific amino 

acids. Upon integrating a dominant-negative protein 

into a complex with wild-type toxin monomers in solution or on a cell surface, a nonfunctional toxin oligomer 

is generated. Two dominant-negative inhibitors for 

epsilon toxin were created via cysteine substitutions of 

four amino acids (isoleucine 51 plus alanine 114, and 

valine 56 plus phenylalanine 118) that constrain the 

membrane insertion domain. These paired mutations 

facilitate an intramolecular cystine bond, oligomer 

dysfunction (decreased heat and detergent stability 

plus poor prepore-to-pore transition), and ultimately 

toxin inactivation in vitro.57 When used in vitro with 

MDCK cells, these inhibitors dose-dependently inhibit 

epsilon-induced cytotoxicity at a 1-, 2-, 4-, or 8 (wildtype toxin)-to-1 (dominant-negative protein) mole 

mixture. Furthermore, dominant-negative molecules 

like those presented in this study represent potential 

vaccine candidates worthy of future study. 

Additional therapy and prophylaxis studies show 

that the epsilon protoxin protects mice (ie, delays time 

to death) when given intravenously before activated 

toxin. This protective effect presumably occurs via 

competitive occupation of the cell surface receptor by 

the protoxin, primarily localized within the brain.39

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Clostridium Perfringens Epsilon Toxin

Such data suggest that a receptor-targeted approach 

for prophylaxis and therapy is possible; Buxton discovered that a formalin toxoid of the protoxin (100 

mg/mouse, intravenous) affords protection up to 100 

minutes after intravenous exposure to epsilon toxin 

(0.5 mg/mouse).58 The readout in this study was extravasation of horseradish peroxidase from the blood 

stream into the brain. Recent studies by Dorca-Arévalo 

et al report a similar binding dissociation constant 

(Kd ~ 4–6 nM) for the epsilon protoxin and activated 

toxin to MDCK cells.59 These results evidently depend 

on plasma membrane integrity plus an unidentified 

glycoprotein. Existing literature suggests that further 

work with receptor antagonists as potential therapeutics against epsilon toxin has not been readily pursued 

by various laboratories.

Knowledge of cell surface receptors for epsilon 

toxin and intimate molecular interactions can be useful in formulating effective receptor-based therapies. 

Early studies by Nagahama and Sakurai reveal that 

the receptor is perhaps a heat-labile sialoglycoprotein; a pretreatment of rat synaptosome membranes 

with heat (70°–80°C for 10 minutes), neuraminidase, 

or pronase effectively reduces the binding of epsilon 

toxin.60 Furthermore, this same study reveals that a 

snake presynaptic neurotoxin (beta-bungarotoxin) 

decreases epsilon toxin binding in a dose-dependent 

fashion, suggesting a common receptor for these very 

different toxins. In contrast, the presynaptic neurotoxin produced by C botulinum type A had no effect 

on epsilon toxin binding. It seems that kidney cells 

and synaptosomes have different receptors for epsilon toxin, as evidenced by varying results following 

sialidase pretreatment of cells.33,60 

Recent studies by Ivie et al show that hepatitis A 

virus cellular receptor 1 (HAVCR1) acts as a receptor, or coreceptor, for epsilon toxin.61,62 The natural 

role of HAVCR1 appears linked to regulatory T cells 

and maintaining immunological balance. This class 

I, integral-membrane glycoprotein contains multiple 

isoforms varying within a mucin-like domain that possesses multiple glycosylation sites. Domain I tyrosines 

(29, 30, 36, 196) found on one end of epsilon toxin (see 

Figure 15-1) are surface-accessible and important for 

binding to HAVCR1.62 Although these data advance 

our understanding of epsilon toxin–receptor interactions and are potentially useful for therapeutic agent 

and vaccine development, additional studies are 

needed to clarify in further detail these cell-surface 

interactions. 

Epsilon toxin is naturally found in the veterinary 

arena and efficacious vaccines are commonly used in 

the field, as explained earlier.63–65 Hyperimmune sera 

can also afford temporary, passive protection for 3 to 

4 weeks in weaned lambs; however, animals showing 

clinical signs of epsilon intoxication are not typically 

afforded protection by antitoxin immunoglobulins.66

It is also possible that a monoclonal antibody targeting 

a critical epitope, like the membrane insertion region 

of epsilon toxin, could be a more useful therapeutic, 

better characterized and purified, than polyclonal antibodies.67 Immunoglobulins derived by either active 

or passive immunization are effective tools against C 

perfringens epsilon toxin when present before or after 

toxin exposure. 

Historically, vaccines for humans and animals have 

proven remarkably effective against myriad diseases 

throughout the world. However, as with many veterinary vaccines, those for C perfringens and associated 

toxins are often formaldehyde toxoids consisting of 

various antigens from culture filtrates or whole cells. 

These vaccines are efficacious and cost effective for 

animals, but considered too crude for humans.68 Furthermore, current veterinary vaccines containing epsilon toxoid can vary in immunogenicity and ultimately 

protective efficacy.64 The vaccination regimen also 

varies depending on the animal species.65 Typically, 

two doses are given within 2 to 6 weeks of each other 

using aluminum hydroxide adjuvant, followed by an 

annual (sheep) or quarterly (goat) boost. In animals, 

and perhaps in a very heterogeneous population like 

humans, an epsilon toxin vaccine is clearly not a “one 

and done” scenario for lasting, protective immunity. 

Recombinantly produced epsilon toxin that is 

subsequently inactivated by 0.5% formaldehyde can 

be used as a superior cost-effective vaccine (eliciting 

a sustained, higher antitoxin titer) at much lower 

protein doses than standard epsilon toxoids.64 Use 

of a more defined (purified) vaccine, as opposed to a 

crude culture filtrate, also affords easier quality control. The gene for epsilon toxin was first successfully 

cloned, sequenced, and expressed in 1992, making 

subsequent recombinant work possible.69 Any human 

vaccine against epsilon toxin will likely be chemically 

or recombinantly mutated critical amino acids for receptor binding, oligomerization, or channel formation 

detoxified versions of the purified protein. In terms 

of recombinant protein, replacing histidine 106 with 

proline results in a nontoxic form of epsilon toxin70 

that provides vaccine-based protection against 1,000 

LD50 of wild-type toxin given intravenously to mice. 

Further recombinant work could be done using data 

derived from earlier chemical modifications of epsilon toxin.71 X-ray crystallography of a toxin-receptor 

complex would likely yield definitively useful data, 

leading to an even better recombinant vaccine or novel 

therapeutics. Medical management of epsilon intoxication, particularly in humans, is currently a wide-open 

field, not only for physicians but also microbiologists, 

biochemists, and immunologists. 

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SUMMARY

C perfringens is a very “toxic” bacterium, employing 

various proteins to promote life in (and out of) various 

mammals. Myriad proteins are toxins intimately linked 

to many C perfringens diseases in humans and animals; 

in particular, the epsilon toxin has been studied by 

various groups around the world and is primarily a 

veterinary concern. Vaccines are available for veterinary use, but an equivalent has not been generated 

for human use in biodefense. From a therapeutic or 

short-term prophylactic perspective, toxin-specific 

immunoglobulins should be of logical interest for human use. However, critical experiments employing 

characterized immunoglobulins (monoclonal and especially those humanized) are lacking in the literature.

Simply put, countermeasures for epsilon intoxication 

in humans are currently highly experimental. 

Finally, military and civilian physicians throughout 

time have been concerned with soiled penetrating 

wounds involving muscle tissue for fear of gas gangrene due to C perfringens (and other anaerobes). More 

recently, biodefense in the 21st century has targeted 

a C perfringens toxin, epsilon, as a potential nefarious agent. As the discovery of C perfringens over 120 

years ago suggests, along with subsequent work on 

the various virulence factors of this extraordinarily 

toxic bacterium, a collaborative international effort 

propelled by scientific endeavor is key to discovering 

knowledge-based medical interventions. 

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Chapter 16

RICIN

VIRGINIA I. ROXAS-DUNCAN, PhD*; MARTHA L. HALE, PhD†; JON M. DAVIS, PhD‡; JOHN C. GORBET, PhD§; 

PATRICIA M. LEGLER, PhD¥; and LEONARD A. SMITH, PhD¶

INTRODUCTION

HISTORY, BIOLOGICAL WARFARE, AND TERRORISM

History 

Ricin as a Biological Weapon

Ricin as a Terrorist Weapon and Use in Biocrimes

DESCRIPTION OF THE AGENT

Biochemistry 

Pathogenesis

CLINICAL SYMPTOMS, SIGNS, AND PATHOLOGY

Oral Intoxication

Injection

Inhalation

Cause of Death

DETECTION AND DIAGNOSIS

Enzyme-Linked Immunosorbent Assay 

Handheld Assay Detection Devices

Sample Verification Platforms

Liquid Chromatography/Mass Spectrometry

Ricin Activity Assay

Diagnosis

MEDICAL MANAGEMENT

Ricin Vaccines

Antibody Treatment

Supportive and Specific Therapy

Development of Ricin Small Molecule Inhibitors

Ricin A Chain Inhibitors

Transport Inhibitors

Cellular Stress Response Inhibitors 

SUMMARY

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*Alternate Responsible Official and Chief, Select Agent Management Branch, Biosurety Division, US Army Medical Research Institute of Infectious 

Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

†

Research Microbiologist, Integrated Toxicology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702

‡

Lieutenant Colonel, Medical Service Corps, US Army; Department of Defense Liaison to the ASPR, Department of Health and Human Services, 200 

C Street, SW, Washington, DC 20024; formerly, Deputy Medical Coordinator, Office of the Assistant Secretary of Defense for Nuclear, Chemical, and 

Biological Defense, 3050 Defense Pentagon, Washington, DC 

§

Captain, Medical Service Corps, US Army; Chief, Department of Aerosol Services, US Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland 21702

¥

Research Biologist, US Naval Research Laboratory, 4555 Overlook Avenue, Washington, DC 20375

¶

Senior Research Scientist (ST) for Medical Countermeasures Technology, Office of the Chief Scientist, US Army Medical Research Institute of Infectious 

Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

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Ricin

INTRODUCTION

and media coverage fortify the appeal of ricin for those 

in quest of retribution and public attention.

In the United States, the possession or transfer of ricin and genes encoding its functional form is regulated 

by the Centers for Disease Control and Prevention 

(CDC) Select Agents and Toxins Program. CDC has 

classified ricin as a category B threat agent. Category 

B agents, which are the second highest priority agents, 

are moderately easy to disseminate, result in moderate 

morbidity and low mortality rates, and require specific enhancements of CDC’s diagnostic capacity and 

enhanced disease surveillance.11,12 Investigators must 

register with the CDC before using nonexempt quantities of ricin in their research. No federal regulations 

restrict the possession of castor plants; however, some 

states or cities (eg, Hayward, CA) prohibit possession 

of castor plants or seeds. 

Ricin is listed as a schedule 1 toxic chemical under 

both the 1972 Convention on the Prohibition of the 

Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on 

their Destruction, usually referred to as the Biological 

Weapons Convention, or Biological and Toxin Weapons Convention, and the 1997 Chemical Weapons 

Convention.13,14

Ricin is a potent toxin derived from the ornamental 

and widely cultivated castor plant, Ricinus communis 

L (Euphorbiaceae). Ricin is mostly concentrated in the 

seed of the plant—popularly known as the castor 

bean—that, despite its name, is not a true bean. The 

purified ricin toxin is a white powder that is water 

soluble; it inhibits protein synthesis leading to cell 

death. Ricin is stable under normal conditions, but 

can be inactivated by heat exceeding 80°C. After oil 

extraction and inactivation of ricin, the defatted mash 

and seed husks are used as animal feed supplement 

and fertilizer, respectively.1

In 1978, the lethality of ricin was overtly established 

after the high-profile assassination of Bulgarian dissident Georgi Markov.2

 Numerous incidents involving 

ricin or castor seeds for nefarious purposes have been 

reported since 1978.3-9 More recently, various extremists and terrorist groups have also experimented with 

ricin; some involved mailing ricin-tainted letters to the 

offices of US politicians. These events have heightened 

concerns regarding ricin’s potential for urban bioterrorism, and thus prompted its constant inclusion in 

weapons of mass destruction investigations.10 The 

wide availability of the castor plants; the relative ease 

of toxin production; and the toxin’s lethality, stability, 

HISTORY, BIOLOGICAL WARFARE, AND TERRORISM

History

The castor plant, also known as the Palm of Christ, 

was initially indigenous to the southeastern Mediterranean region, eastern Africa, and India, but is now 

widespread throughout temperate and subtropical 

regions.15–17 Ricinus is a Latin word for “tick” to describe the castor seed’s appearance that resembles a 

tick (Ixodes ricinus), and communis meaning “common” 

to describe its worldwide distribution. For centuries, 

the castor plant has been cultivated for numerous 

economically important products, primarily castor 

oil.17 In ancient Egypt, Europe, India, and China, castor 

oil was used for lighting and body ointments, and as 

purgative or cathartic, and other ethnomedical uses. 

Castor oil was also reportedly used as an instrument 

of coercion by the Italian Squadristi, the Fascist armed 

squads of Benito Mussolini.18 Political dissidents and 

regime opponents were forced to ingest large amounts 

of castor oil, triggering severe diarrhea and dehydration that often led to death.19 Presently, castor oil has 

abundant commercial applications including medicinal and industrial purposes.20–24 Castor seeds are being produced in more than 30 countries in the world 

because of their economic benefits and myriad uses. 

In 2013, world castor oil seed production totaled 1.86 

metric tons, and the leading producers include India, 

China (mainland), and Mozambique.25

In 1888, Peter Hermann Stillmark, a student at 

the Dorpat University in Estonia (Stillmark 1888, as 

cited in Franz and Jaax5), discovered ricin. During 

Stillmark’s extensive research, he observed that ricin 

caused agglutination of erythrocytes and precipitation of serum proteins.17 In 1891, Paul Ehrlich studied 

ricin and abrin in pioneering research that is now 

recognized as the foundation of immunology.17 Ehrlich found that animals vaccinated with small oral 

doses of castor beans were protected against a lethal 

dose of the toxin. Additional experiments using abrin 

and ricin showed that the immunity was specific, was 

associated with serum proteins, and could be transferred to the offspring through milk. Further research 

on ricin showed that the toxin described by Stillmark 

was actually two proteins, one with an agglutinin 

with a molecular weight of 120 kDa (R communis agglutinin I) possessing little toxicity and the other, R 

communis agglutinin II, a smaller molecular weight 

protein (60 kDa) with little agglutinating capacity 

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Medical Aspects of Biological Warfare 

but extremely toxic. Nearly a century after Stillmark’s 

original discovery, Olsnes and Pihl26 demonstrated 

that the 60 kDa toxic protein (ricin) inhibited protein 

synthesis and that the 60S ribosomal subunit is the 

toxin’s molecular target.27

Although ricin is considered a possible biological 

threat (see below), its potential medical applications 

have been also explored. During the past decade, ricin 

has been used extensively in the design of therapeutic 

immunotoxins, often called “magic bullets.” Specifically, ricin, ricin A chain (RTA), or a related toxin is 

chemically or genetically linked to a binding ligand 

such as an antibody or used in other conjugates to 

specifically target and destroy cancer cells, and also as 

alternative therapies for AIDS and other illnesses.28–30

Ricin-based immunotoxins conjugated to either the 

anti-CD22 antibody RFB431,32 or its Fab fragment33 have 

been reported to provide enhanced therapeutic efficacy 

and improved antitumor activity.34–36 However, the 

US Food and Drug Administration has placed a hold 

on the clinical testing of RTA-based immunotoxins 

because they caused vascular leak syndrome (VLS) in 

humans, a life-threatening condition in which fluids 

leak from blood vessels leading to hypoalbumina, 

weight gain, and pulmonary edema.37 Although progress has been made in understanding the mechanisms 

of immunotoxin-mediated VLS, significant effort is still 

required to understand VLS and generate RTA-derived 

immunotoxins that do not cause VLS but also maintain 

RTA’s potent antitumor activity.38

Ricin as a Biological Weapon

During World War I, the United States, which was 

aware of the German biological warfare program, examined ricin for retaliatory intentions.39 Two methods 

of ricin dissemination were described in a 1918 technical report: (1) adhering ricin to shrapnel bullets for 

containment in an artillery shell, and (2) production of 

a ricin dust cloud (Hunt et al, 1918, as cited in Smart39). 

The thermal instability of ricin constrained its initial 

use in exploding shells, and ethical and treaty issues 

limited its use as a poison or blinding agents. World 

War I ended before the toxin could be weaponized 

and tested. During World War II, ricin was evidently 

never used in battle despite its mass production and 

being armed into ricin-containing bombs (also known 

as W bombs), because its toxicity was surpassed by 

the even more potent biological agents of the time.5

Interest in ricin diminished with the production and 

weaponization of other chemical agents, for example, 

sarin. During the Cold War, the Soviet Union studied 

ricin as a possible biological weapons agent. A former top Russian official who defected to the United 

States in 1991 asserted that Russia developed ricin as a 

weapon, and that the toxin used against the Bulgarian 

dissidents Georgi Markov and Vladimir Kostov was 

formulated in Russian laboratories.40 Iraq reportedly 

manufactured and tested ricin in animals and used it 

as payload in artillery shells.41–44 Syria and Iran were 

believed to have produced unknown quantities of the 

toxin.45 Ricin was also found in Afghanistan in 2001 

after the collapse of the Taliban government.46,47

Although ricin’s potential use as a military weapon 

was investigated, its utility over conventional weaponry remains ambiguous. Despite its toxicity, ricin is 

less potent than other agents such as botulinum neurotoxin or anthrax. It has been estimated that eight metric 

tons of ricin would have to be aerosolized over a 100 

km2 area to achieve about 50% casualty, whereas only 

kilogram quantities of anthrax spores would cause 

the same effect.48 Furthermore, wide-scale dispersal 

of ricin is logistically impractical. Thus, while ricin is 

relatively easy to produce, it is not as likely to cause 

as many casualties as other agents.49

Ricin as a Terrorist Weapon and Use in Biocrimes

The well-publicized “umbrella murder” of the Bulgarian writer and journalist Georgi Markov in 19782

represents the first documented case of a modern 

assassination using a biological agent,50 although this 

remained unproven. Markov defected to the West in 

1969 and was a vocal critic of the Bulgarian communist 

regime. The Bulgarian secret police had previously 

attempted to kill Markov twice, but failed. However, 

on September 7, 1978, Markov was assaulted with an 

umbrella tip while waiting at a bus stop in London. 

He subsequently developed severe gastroenteritis 

and a high fever, and died on September 11, 1978. 

The autopsy revealed a small platinum pellet with 

an X-shaped cavity. Further examination of the pellet 

revealed ricin.2,51 Prosecutors have failed to identify, 

arrest, or charge anyone for the crime. On September 

11, 2013, a news report disclosed that Bulgaria was 

abandoning its investigations of the notorious case 

35 years after the cessation of the absolute statute of 

limitations.52

Days before Markov’s assassination, an attempt was 

made to kill another Bulgarian defector, Vladimir Kostov.2

 However, the pellet lodged in the fatty tissue in 

Kostov’s back prevented the toxin from being released 

from the sugar-coated pellet; he survived the incident. 

Several cases involving the possession, experimentation, or planned misuse of ricin by bioterrorists and 

extremist groups have been investigated or prosecuted 

by law enforcement agencies worldwide.5-7 Recent 

related incidents include the following:

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• In April 2013, letters containing ricin were 

mailed to Republican Senator Roger Wicker 

(of Mississippi) and the White House.53,54 A 

Mississippi martial arts instructor, Everett 

Dutschke, was charged with sending those 

ricin-tainted letters.55

• In May 2013, five letters, three of which that 

tested positive for ricin, were mailed from Spokane, Washington, to a local judge, downtown 

Spokane post office, President Barack Obama, 

Fairchild Air Force Base, and the Central 

Intelligence Agency in McLean, Virginia.56

• On June 7, 2013, actress Shannon Richardson 

was arrested for allegedly sending ricintainted letters to New York Mayor Michael 

Bloomberg and President Barack Obama.57

These reports further substantiate ricin’s image as 

an attractive lethal poison, and ostensibly, a biological 

weapon of choice by extremist groups and individuals. 

DESCRIPTION OF THE AGENT 

Biochemistry 

While ricin is a well-known toxin that can be extracted from castor bean mash, most do not realize 

that it is related in structure and function to the bacterial Shiga toxins and Shiga-like toxin (also known as 

Verotoxin) of Shigella dysenteriae and Escherichia coli. 

Antibiotic-resistant Shiga toxin-producing E coli was 

responsible for 54 deaths in Germany in 201158; the 

Shiga toxin gene encoding the toxin was carried by this 

infectious pathogen. Ricin is noninfectious; however, 

both the structure and enzymatic activities of ricin and 

Shiga toxins are similar (Figure 16-1). These protein 

toxins belong to a family of toxins known as ribosome

inactivating proteins (RIPs). More than 60 different 

plant and bacterial species produce RIPs.59–61 Type I and 

II RIPs include the plant toxins ricin, abrin, mistletoe 

lectins, volkensin, modeccin, saporin, trichosanthin, 

luffin, and the bacterial Shiga toxin and Shiga-like toxin. 

Ricin, a type II RIP, consists of two glycoprotein 

subunits: a catalytic A-chain (RTA) and a lectin B-chain 

(RTB), which binds cell surface oligosaccharides containing galactose.59,62–64 The RTA and the RTB, which 

are of approximately equal molecular mass (~32 kDa), 

are covalently linked by a single disulfide bond. The 

protein-coding region of ricin consists of a 24 amino 

acid N-terminal signal sequence preceding a 267 amino 

acid RTA. The RTB has 262 amino acids. It consists 

of two major domains with identical folding topologies,62 each of which comprises three homologous 

subdomains (α, β, γ) that probably arose by gene duplication from a primordial carbohydrate recognition 

domain.65 RTB binds terminal β 1,4, linked galactose 

and N-acetyl galactosamine (Gal/GalNac)66 that are 

on the surface of most mammalian cells. A 12-amino 

acid linker in the pre-protein joins the two chains. The 

carboxyl-terminal end of the RTA folds into a domain 

that interacts with the two domains of the B chain.62

Figure 16-1. Structural and functional similarities among RIPs. The A-chains of plant RIPs such as ricin (PDB 3HIO) and 

abrin (PDB 1ABR) are structurally and functionally related to the bacterial Shiga toxin A-chains (PDB 1R4Q and 1R4P). The 

A-chains catalyze the same reaction to inactivate ribosomes and halt protein synthesis.

Data sources: (1) Ho MC, Sturm MB, Almo SC, Schramm VL. Transition state analogues in structures of ricin and saporin 

ribosome-inactivating proteins. Proc Natl Acad Sci U S A. 2009;106:20276–20281. (2) Tahirov TH, Lu TH, Liaw YC, Chen YL, 

Lin JY. Crystal structure of abrin-a at 2.14 A. J Mol Biol. 1995;250:354–367. (3) Fraser ME, Fujinaga M, Cherney MM, et al. 

Structure of shiga toxin type 2 (Stx2) from Escherichia coli O157:H7. J Biol Chem. 2004;279:27511–27517. 

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Medical Aspects of Biological Warfare 

A disulfide bond is formed between amino acid 259 

of the RTA and amino acid 4 of the RTB.59,63,64 Thirty 

percent of the RTA protein is helical. The RTA folds 

into three somewhat arbitrary domains. The active site 

cleft of the RTA is located at the interface between all 

three domains. 

Pathogenesis

Pathogenesis resulting from ricin intoxication is a 

two-step process. The first phase occurs at the cellular 

level in which the toxin kills cells in a cell-cycle independent fashion; the second phase is primarily caused 

by systemic reactions that develop in response to cell 

death and tissue damage.

The cell binding component of the toxin (RTB) 

binds to cell surface lipids and proteins with exposed 

terminal β-1,4-linked galactose molecules that are 

found on most mammalian cells, permitting ricin to 

bind indiscriminately to most cells in the body.17,66,67

In addition to binding to surface glycoproteins, ricin 

contains three mannose oligosaccharide chains, two in 

RTB and one in RTA, that provide another route for 

ricin binding to the cell via mannose receptors located 

primarily on macrophages and dendritic cells.68 

Once RTB binds to the cell, it is endocytosed.69 At 

this point, three possible fates exist for ricin: 

1. entry into endosomes and recycling to the 

cell surface; 

2. degradation via the late endosomes; or 

3. entry to the trans-Golgi network and entry 

to the endoplasmic reticulum (ER) via retrograde trafficking. 

In the ER, a protein disulfide isomerase reduces 

the toxin into RTA and RTB components.70 The low 

lysine content of RTA probably enables the molecule 

to evade the ER-associated protein degradation pathway and chaperone proteins, such as calreticulin, 

and to transport RTA from the Golgi apparatus to the 

ER; dislocation of RTA from the ER may involve the 

translocon component Sec61p.71–73 Postdislocation of 

RTA in the cytosol probably involves Hsp70, which 

may also aid the protein in binding to its ribosomal 

substrate.74 Additionally, the ribosome itself may act 

as a suicidal chaperone by facilitating proper refolding 

of RTA, which is required for the catalytic activity of 

the enzyme.73 

Extensive investigations on reactions controlling 

RTA’s binding to ribosomes provide detailed information on RTA’s enzymatic functions.17,75 RTA catalyzes 

the hydrolysis of a specific adenine in the ricin-sarcin 

loop of the 28S ribosomal RNA (rRNA) (Figure 16-2). 

The ricin-sarcin loop interacts with eukaryotic elongation factor EF-2. The binding of EF-2 to the ricin-sarcin 

loop is required for the translocation of the peptidyltRNA from the A-site to the P-site on the ribosome 

during protein synthesis. The depurinated ricin-sarcin 

loop fails to bind EF-2 and the ribosome stalls with the 

peptidyl-tRNA stuck in the A-site.76,77 The overall effect 

is a halt in protein translation and cell death. 

Analysis of reactions resulting from mixing purified 

rat ribosomes with RTA shows that the RTA reaction 

follows classical Michaelis-Menten enzyme kinetics, 

and the enzymatic action has been calculated to be 

0.1 mol/L.78 Furthermore, these studies predict that 

one RTA molecule would depurinate 1,500 ribosomes 

per minute, thus making one ricin molecule sufficient 

to kill the cell. Site-directed mutagenesis and the 

development of transition state mimics have yielded 

mechanistic information. The hydrolysis reaction catalyzed by RTA is thought to proceed via a dissociative 

mechanism with an oxocarbenium transition state.79

Glu-177 in the active site stabilizes the developing 

positive charge on the ribosyl ring while Tyr-80 and 

Tyr-123 have been proposed to activate the leaving 

group by pi-stacking with the adenine80 (Figure 16-3). 

The enzymatic activity of RTA is the primary source of 

toxicity and therefore must be attenuated in RTA subunit vaccines by incorporating the Y80A mutation81 or 

removing the C-terminal residues (residues 199–267).82

The mutations interfere with rRNA binding. 

Activation of apoptotic processes is one method 

by which RTA kills cells, but the apoptotic pathways 

are somewhat cell dependent.81–83 Evidence indicates 

that some cells have novel ricin-specific pathways 

for activating apoptosis. Wu and colleagues84 found 

that RTA binds to a novel binding protein (BAT3) 

that is found in the cytoplasm and nucleus of many 

cells. BAT3 possesses a canonical caspase-3 cleavage 

site that appears to be exposed when RTA binds to 

BAT3; apoptosis is then activated with caspase-3 

cleavage. The finding that BAT3 may play a role in 

ricin-induced apoptosis could identify new targets 

for preventing ricin toxicity.

Ricin intoxication has been shown to activate 

numerous signaling pathways including mitogenactivated protein (MAP) kinases and subsequent 

secondary signaling pathways, such as the stress 

activated protein kinase family.85 MAP kinases regulate activation of cytokines such as interleukin (IL)-8, 

IL-1β, and tumor necrosis factor-α that, in turn, cause 

inflammatory reactions and tissue damage. Although 

inflammatory responses caused by ricin have been 

described previously, pathways and resulting cellular 

responses were only recently examined.86 Korcheva 

et al87 demonstrated that intravenous administration 

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Ricin

Figure 16-2. The ricin A chain catalyzes the hydrolysis of an adenine in the ricin-sarcin loop. The depurinated rRNA is shown 

as a dotted line. The aminoacyl-tRNA is delivered to the A-site by eukaryotic elongation factor eEF1A and peptidyl transfer 

follows. The binding of eukaryotic elongation factor eEF-2 carrying GTP is required for the peptidyl-tRNA to translocation 

from the A-site to the P-site; this movement requires eEF-2. The depurinated loop fails to bind eEF-2, and the ribosome stalls 

with the peptidyl-tRNA in the A-site. GTP: guanosine triphosphate 

Data source: Figure adapted from Mansouri S, Nourollahzadeh E, Hudak KA. Pokeweed antiviral protein depurinates the 

sarcin/ricin loop of the rRNA prior to binding of aminoacyl-tRNA to the ribosomal A-site. RNA. 2006;12:1683–1692. 

of ricin in mice resulted in cellular signaling pathway 

activation and a significant increase in serum proinflammatory cytokine levels. Additional research in 

which ricin was instilled by an intratracheal route 

showed similar signaling pathway activation as well 

as an increase in proinflammatory cytokine levels, 

although more inflammatory reactions and tissue 

damage were observed in the lungs.88 Although these 

studies have initiated the systemic pathogenesis characterization of ricin intoxication, further efforts aimed 

to determine the cellular responses induced by ricin 

will lead to a greater understanding of its pathogenesis 

and may also enable the development of new treatment 

strategies to combat the effects of intoxication.

CLINICAL SYMPTOMS, SIGNS, AND PATHOLOGY

Experimental animal studies reveal that clinical signs and pathological manifestations of ricin 

toxicity depend on the dose as well as the route of 

exposure.5,27,89 The common routes of entry are oral 

intoxication (ingestion), injection, and inhalation. 

The differences observed in pathology among various routes likely result from the fact that RTB binds 

to a wide array of cell surface carbohydrates.90 Once 

bound, RTA is internalized and results in the death of 

intoxicated cells. Although symptoms may vary, in 

most cases, there is a time-to-death delay of approximately 10 hours, even with a high dose of toxin.91 Additionally, in animals and humans intoxicated either 

by injection or oral ingestion, a transient leukocytosis is 

commonly observed, with leukocyte counts rising two 

to five times above their normal values. The LD50 and 

time to death for animals by various routes have been 

reported, and the values for humans were estimated 

based on animal experiments and accidental human 

exposures.5,7,40

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Medical Aspects of Biological Warfare 

Oral Intoxication

Oral or intragastric delivery is the least effective 

and least toxic route, reportedly 1,000 times less toxic 

than parenteral routes. The reduction in toxicity may 

result from poor absorption of the toxin across the 

epithelium and slight enzymatic degradation of the 

toxin as it traverses the gastrointestinal tract. Ingestion of castor beans is the most common route of poisoning for humans and domestic animals. Worbs and 

Figure 16-3. RTA catalyzed depurination reaction. (a) Structure of a cyclic G(9-DA)GA 2’-OMe transition state mimic 

determined by Ho et al. (PDB 3HIO). A methylene carbon between the nitrogen of the aza-sugar and the adenine mimics 

the increased riobsyl-adenine distance in the dissociative transition state. (b) Proposed mechanism of the RTA catalyzed 

depurination reaction. The hydrolysis reaction is thought to proceed via a dissociative mechanism with an oxocarbenium 

transition state. Arg-180 protonates the leaving group (adenine) and the N-glycosidic bond is broken. Glu-177 deprotonates 

the hydrolytic water that attacks at carbon to complete the depurination reaction.

Data sources: (1) Ho MC, Sturm MB, Almo SC, Schramm VL. Transition state analogues in structures of ricin and saporin 

ribosome-inactivating proteins. Proc Natl Acad Sci U S A. 2009;106:20276–20281. (2) Roday S, Amukele T, Evans GB, Tyler PC, 

Furneaux RH, Schramm VL. Inhibition of ricin A-chain with pyrrolidine mimics of the oxacarbenium ion transition state. 

Biochemistry. 2004;43:4923–4933.

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colleagues92 provide an updated list of ricin intoxication in humans worldwide. Since the late 1880s, 

875 cases of accidental poisoning and 13 fatalities 

were reported in the literature (1.5% death rate); 

there were 11 intentional poisonings, five of which 

were fatal (45.5% death rate). In recent years, ricin 

intoxication resulting from intentional poisoning 

using mashed seeds or crude preparations of ricin 

has become a major bioterror weapon as well as a 

method of suicide.8,9 

Great variability exists in the effects from seed 

ingestion, which is probably related to the number of 

seeds, the degree of mastication that releases ricin from 

the seeds, the age of the individual, and—to a lesser 

extent—the cultivar of the castor bean plant.92,93 In addition, an accurate description of ricin intoxication in 

humans is complicated by other factors including the 

presence of other somewhat toxic components, such as 

the ricin agglutinin protein and the alkaloid ricinine, 

that is found in castor seeds and crude preparations of 

ricin.92 These substances can also cause tissue damage 

and contribute to pathological manifestations. 

Despite numerous differences that may play a 

role in oral toxicity, all fatal or serious cases appear 

to have a similar clinical history; a recent case study 

is presented in this reference (Exhibit 16-1).94 Within 

a few hours, an onset of nausea, vomiting, and abdominal pain occurs, which is followed by diarrhea, 

EXHIBIT 16-1

CASE REPORT: RICIN POISONING CAUSING DEATH AFTER INGESTION OF 

HERBAL MEDICINE

A 42-year-old male Saudi patient presented to the emergency department with a 12-hour history of epigastric pain, 

nausea, repeated attacks of vomiting, chest tightness, and mild nonproductive cough.

These symptoms were preceded by a 5-day history of constipation for which the patient ingested a large amount of a 

mixture of herbal medicine preparation 2 days before his admission. A review of systems was unremarkable. He had 

no history of any medical illnesses and medication use except for the herbal medicine. Initial examination showed 

a mild elevation of temperature (38o

C), with generalized abdominal tenderness and hyperactive bowel sounds. His 

respiratory system examination showed equal bilateral air entry and no added sounds. The rest of his systemic examinations were unremarkable. 

Laboratory investigations on admission showed mild leukocytosis of 14 x 109

/L, a normal platelet count of 200 x 109/L, 

and normal hemoglobin level of 15.8 g/dL. Liver enzymes initially showed mild to moderate elevation of alanine 

transaminase (ALT) 86 U/L (normal range up to 37 U/L), aspartate transaminase (AST) 252 U/L (normal range up to 

40 U/L), and serum lactate dehydrogenase 281 U/L (normal range 72–182 U/L), and the renal function was normal. 

The initial coagulation profile was impaired as documented by a prolonged prothrombin time (19 seconds, control 12 

seconds) and a prolonged activated partial thromboplastin time (56 seconds, control 32 seconds). Electrocardiogram 

showed a right bundle branch block, and a chest radiograph was normal. 

After 4 hours of admission, the abdominal pain became worse, and the patient started showing subcutaneous bleeding 

at the intravenous sites and upper gastrointestinal bleeding, manifested as hematamesis. The patient was managed by 

intravenous fluid therapy, fresh frozen plasma and platelet transfusion, and gastric decontamination with activated 

charcoal. A gastrointestinal consultation was requested in which endoscopy was planned after stabilization of the 

patient, but was not performed because of the patient’s rapid deterioration.

In the second day after admission, his liver enzymes increased to a level of 5980 U/L for ALT and 7010 U/L for AST. 

Serum albumin was 31 g/L (normal range 38–50 g/L), total protein was 59 g/L (normal range 66–87 g/L), and the platelet 

count dropped to 85 x 109

/L. His renal function also deteriorated, elevating the creatinine level to 150 Umol/L (normal 

range up to 123 Umol/L), and urea to 110 mmol/L (normal range 1.7–83 mmol/L). His blood and sputum cultures and 

sensitivity were negative for bacterial pathogens, and an abdominal computerized tomography scan was normal. The 

patient was managed conservatively with supportive measures as maintained earlier; however, he remained persistently hypotensive necessitating inotropic support. On the third day, he developed cardiopulmonary arrest and was 

resuscitated; however, he could not be revived. The sample of the herbal medicine powder was sent to the university 

lab. The chemical contents were extracted by the liquid chromatography/mass spectrometry technique, revealing the 

presence mainly of ricin powder that was further identified by the immuno-polymerase chain reaction assay that 

confirmed the presence mainly of ricin with no other significant contaminants. This finding could be implicated as 

the cause for the patient’s fulminant clinical course.

Data source: Assiri AS. Ricin poisoning causing death after ingestion of herbal medicine. Ann Saudi Med. 2012;32:315–317.

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hemorrhage from the anus, anuria, cramps, and pupil 

dilation.95 Fever develops, followed by thirst, sore 

throat, and headache, leading to vascular collapse and 

shock. Death usually occurs by day three or thereafter. 

Common histopathological findings during autopsy 

are multifocal ulcerations and hemorrhages in the 

stomach and small intestinal mucosa. Significant lymphoid necrosis occurred in intestinal associated lymph 

nodes, lymphoid tissue, and spleen. Necrosis was also 

observed in cells of the reticuloendothelial system 

resulting in liver damage and nephritis. Macrophages 

and macrophage-derived cells appear to be very susceptible, probably because of the large numbers of 

mannose receptors present in the cell membrane.68,96

Injection

Pathological damage caused by injection of ricin depends largely upon the dose. Results of a clinical trial in 

which 18 to 20 μg/m2 of ricin were given intravenously 

to cancer patients indicated that the low dose was fairly 

well tolerated, with flu-like symptoms, fatigue, and 

muscular pain as the main side effects.97 Some patients 

experienced nausea and vomiting, but after 2 days, they 

had recovered and experienced no more side effects. At 

low doses, intramuscular or subcutaneous injections 

may result in necrosis at the injection site possibly resulting in secondary infections.98 High doses by either 

route cause severe local lymphoid necrosis, gastrointestinal hemorrhage, diffuse nephritis, and splenitis. 

Targosz and colleagues99 describe a suicide case in 

which an individual injected himself subcutaneously 

with a large dose of ricin extracted from castor beans. 

The 20-year-old man was admitted to the hospital 36 

hours after injection. He experienced severe weakness, 

nausea, dizziness, headache, and chest pain. Clinical 

exams showed hypotension, anuria, metabolic acidosis, and hematochezia. The patient was observed with 

hemorrhagic diathesis and liver, kidney, cardiovascular, 

and respiratory systems failure requiring endotracheal 

intubation and artificial ventilation. Although given 

maximal doses of pressor amines and treated for hemorrhagic diathesis, treatments were ineffective and the 

patient developed symptoms of multiorgan failure 

followed by asystolic cardiac arrest. Resuscitation was 

not effective, and the patient died shortly thereafter. A 

postmortem examination revealed hemorrhagic foci in 

the brain, myocardium, and pleura.

In the case of Georgi Markov,2

 the lethal injected 

dose was estimated to be 500 μg. Markov experienced 

severe local pain after the injection, which was followed by a general weakness 5 hours later. Fifteen 

to 24 hours later, he had an elevated temperature, 

felt nauseated, and vomited. He was admitted to the 

hospital with a high fever and signs of tachycardia. 

While his blood pressure remained normal, lymph 

nodes in the affected groin were swollen and sore, 

and a 6-cm diameter area of induration was observed 

at the injection site. Just over 2 days after the attack, he 

suddenly became hypotensive and tachycardic with a 

pulse rate of 160 beats/minute and a white blood cell 

count of 26,300/mm.3 He became anuric developing 

gastrointestinal hemorrhaging and complete atrioventricular conduction block. Shortly thereafter, Markov 

died from cardiac failure complicated by pulmonary 

edema; the time of death was 3 days after he was initially poisoned.2

 

Inhalation

No reports exist in which humans have been subjected to ricin by accidental inhalation or premeditated 

aerosolized exposure. Most of the human data comes 

from descriptions of workers being exposed to castor 

bean dust in or around castor bean processing plants.100

Allergic manifestations induced by ricin dust were first 

described in 1914.101 Symptoms and clinical signs of 

intoxication were later differentiated from the allergic 

syndrome and further investigations showed that the 

allergens and toxin were two different molecules.102–104

Because no data exist for human exposure, it is 

important to determine whether a consistency exists 

between rodents and nonhuman primates (and other 

animal models) that can be used to extrapolate an accurate representation of inhalational ricin in humans. 

Unlike other routes of intoxication, damage caused by 

an aerosol exposure is greatly dependent on particle 

size, and to a lesser extent on the dose and cultivar 

from which ricin was obtained.86 Ricin extracted from 

R communis var. zanzibariensis was twice as lethal as 

ricin extracted from the Hale Queen variety.86 The 

differences are more than likely related to variations 

in the isotoxins of ricin found in the seeds from different cultivars.86

For ricin to reach the lung, the particles would need 

to be a size that could move around the nasal turbinates 

and flow with the airstream to the lung. Roy and colleagues105 compared the outcome of mice receiving 1 

μm versus 5 μm particle size by an aerosol challenge. 

With the 1 μm particles, the majority of ricin was found 

in the lung and by 48 hours, lung tissue show significant lesions with alveolar edema, fibrin, and hemorrhage. Seventy-two hours postexposure, all of the mice 

had died. Conversely, no deaths were observed when 

mice were exposed to ricin with a 5 μm mass median 

diameter. Most of the toxin was found in the trachea, 

and little lung damage was observed in histological 

sections of lung tissue taken 48 hours postexposure. 

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When rats were exposed to a sublethal dose (LCt30) of 

ricin with particle sizes less than 1 μm, damage was limited to the lung and no histological changes were noted 

before 8 hours postchallenge.86,106 By 48 hours, pathological changes observed included necrosis and apoptosis in 

bronchial epithelium and macrophages present in the 

alveolae septae. Photographs of tissue sections from CD1 

mouse lungs 48 hours after exposure show perivascular 

edema and pulmonary epithelial cell necrosis (Figure 

16-4). Three days post-exposure, there was significant 

diffuse alveolar edema, and severe capillary congestion 

and macrophage infiltration of the alveolar interstitium. 

By day four, there was a rapidly resolving pulmonary 

edema and renewal of the bronchial epithelium, even 

though severe pas-sive venous congestion existed in all 

solid peripheral organs. Fourteen days postexposure, 

all animals survived. Examination of tissue sections 

from sacrificed animals were similar to control tissues, 

except for focal areas of intraalveolar macrophage infiltration.106 Additionally, when rats and mice were given 

lethal doses of ricin by aerosol, no indication of lung 

damage was observed during the first 4 to 6 hours.14,106,107

By 12 hours, there was an increase in total protein and 

polymorphonuclear cells in the bronchial lavage, indicating damage to the epithelial cell barrier. Thirty hours 

after challenge, alveolar flooding was apparent, along 

with arterial hypoxemia and acidosis. Histopathology 

showed lesions throughout the respiratory tract, spleen, 

and thymus. A median lethal dose of ricin by inhalation 

was determined to be 1 μg/kg body weight for both 

Sprague Dawley rats and BALB/c mice.107 Further characterization of inhaled ricin exposure was performed by 

examining lung tissue sections for the presence of ricin.5

Immunohistochemical studies showed that ricin binds 

to the ciliated bronchiolar lin-ing, alveolar macrophages, 

and alveolar lining cells.14 This finding further substantiates the importance of the lung epithelium and alveolar 

macrophages in the inhaled ricin intoxication process. 

As with other laboratory animal models, investigations in which nonhuman primates were challenged 

with an aerosolized dose of ricin indicate that disease 

progression is proportional to particle size.108 Inhalational challenge with a particle size of 8 μm was not 

lethal and did not cause lung damage, suggesting that 

the upper airways can effectively remove the toxin 

before it reaches the lung. Inhalational challenge with 

a particle size of 1 μm presented an entirely different 

picture with histopathologic changes beginning as early 

as 4 to 6 hours postexposure.108,109 By 8 hours, pulmonary 

changes included alveolar edema, perivascular interstitial edema, lymphangiectasis, alveolar septal necrosis, 

and hemorrhage. At 16 hours, progression of pulmonary 

tissue damage continued, and by 24 hours, there was 

edema, pulmonary congestion, necrotic alveolar septa, 

and necrotic bronchiolar epithelium (Figure 16-5). Thirty-two hours later, there was marked perivascular and 

peribronchiolar interstitial edema and alveoli contained 

fluid (edema) mixed with fibrin and viable or degenerate neutrophils and macrophages. The bronchiolar 

epithelium was necrotic and often sloughed into the lumen, whereas lymphatics surrounding the airways were 

moderately dilated and the endothelium of many small 

vessels had atrophied. In the tracheal mucosa, there was 

epithelial degeneration with scattered areas of necrosis 

and subacute inflammation. The cortex of adrenal glands 

showed mild degeneration and necrosis, and there 

Figure 16-4. Histological sections of lungs from CD1 mice exposed to ricin by aerosol showing (a) perivascular edema and 

pulmonary epithelial necrosis. Hematoxylin and eosin stain at original magnification × 25; (b) pulmonary epithelial cell 

necrosis, hematoxylin and eosin stain at original magnification × 100. 

Photographs: Courtesy of Lieutenant Colonel (Retired) Catherine L. Wilhelmsen, Pathology Division, US Army Medical 

Research Institute of Infectious Diseases, Fort Detrick, Maryland.

a b

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Medical Aspects of Biological Warfare 

was lymphoid depletion and lymphocytolysis in the 

mediastinal lymph nodes. A similar course of disease 

was observed in an earlier study in which nonhuman 

primates were challenged with ricin (~1 μm particle 

size), but the preclinical period varied between 8 and 

24 hours in relation to the size of the original challenge 

dose.109 This stage was followed by anorexia and decrease in physical activity. The time of death was also 

dose dependent and occurred between 36 and 48 hours. 

Cause of Death

Although the exact cause of death from ricin toxicity is not known, clinical symptoms of individuals 

exposed to lethal doses of the toxin suggest that death 

results from a severe inflammatory response and 

multiorgan failure.94,95,99 A lethal dose of ricin given to 

mice by intraveneous injection or intratracheal instillation results in a systemic inflammatory response, 

thrombocytopenia, hemolytic anemia, renal failure, 

and microvascular thrombosis, pathologies that are 

similar to those observed in humans.87, 88 Initially, the 

fact that macrophages are extremely sensitive to ricin 

led investigators to believe that macrophages might 

play a significant role in ricin intoxication.110 Recent 

findings demonstrated that pulmonary inflammation 

caused by ricin required the presence of both macrophages and interleukin-1 signaling pathways.111 Also, 

studies using bone marrow derived macrophages 

showed that ricin inhibition of protein translation 

led to activation of IL-1β-dependent inflammation 

by activating innate immune signaling through the 

Figure 16-5. Lungs from a nonhuman primate exposed to 

ricin by aerosol exposure. (a) Goss picture of lungs removed 

from thorax. The lungs are edematous with hemorrhage and 

necrosis. (b) Histologically, microscopic changes show severe 

perivascular edema; hematoxylin and eosin stain at original 

a b

c

magnification × 10.(c) Alveolar fibrinopurulent exudate is observed hematoxylin and eosin stain at original magnification × 100.

Photographs courtesy of Lieutenant Colonel (Retired) Catherine L Wilhelmsen, DVM, PhD, Pathology Division, US Army 

Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland.

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nod-like receptor (NLR) family member, NLRP3.112

NLRP3 is an innate immune pattern recognition 

receptor found in the cytosol that is activated by molecular patterns found on many pathogens or other 

danger-associated proteins. Activation of NLRP3 

stimulates IL-1β processing via a multiprotein complex, the inflammasome. More investigations are 

necessary to understand how ricin activates severe 

inflammatory responses that lead to multiorgan 

failure, shock, and death. 

DETECTION AND DIAGNOSIS

Early clinical symptoms of ricin intoxication may 

resemble symptoms caused by other biothreat agents, 

and therefore it is essential to identify the etiological 

agent to provide the best treatment for exposed patients. The cellular uptake of ricin is rapid and thus 

limits the diagnosis of ricin in blood and other fluid 

samples. Additionally, the ricin concentration may be 

below the current levels of detection, making diagnosis 

more difficult.113 Because of the inability to detect ricin 

in patients, identifying the toxin in environmental or 

forensic samples associated with the exposure remains 

the most reliable method for determining the presence 

of ricin and the possibility of intoxication. Ricin does 

not replicate, so detection relies on the ability to identify physical attributes of the toxin within the sample. 

The most common method for toxin identification 

uses antiricin antibodies to which ricin would bind. 

In recent years, several variations of antigen (toxin)-

antibody assays have been developed.114 Physical 

characterization using liquid chromatography and 

mass spectroscopy complements the antibody-based 

methods and permits development of signatures of 

the toxin preparation.115 

Enzyme-linked Immunosorbent Assay 

An enzyme-linked immunosorbent assay (ELISA) 

provides an economical and straightforward method 

for detecting the presence of ricin in environmental 

and forensic samples. A capture antibody ELISA is a 

common method of detection. Ricin is initially “captured” onto the matrix via an antiricin monoclonal 

antibody (Mab) recognizing RTB. A second anti-ricin 

Mab, usually recognizing RTA, binds to the immobilized ricin, and the second Mab is then detected by 

an anti-mouse immunoglobulin 3 conjugated to an 

enzyme such as horse radish peroxidase that forms a 

colometric reaction upon the addition of its substrate 

solution.86 The limit of detection (LOD) for these assays has been greatly improved by using methods that 

amplify the detection signal or use a more sensitive 

signal such as those generated by electrochemiluminescence (ECL). Using slight modifications of these 

assays, Poli and colleagues116 reported LODs of 100 

pg/100 μL in human serum and urine that had been 

spiked with various concentrations of ricin. Other 

studies, such as those by Roy et al,105 detected ricin 

in lungs, stomach, trachea, and nares using an ELISA 

based on time-resolved fluorescence. Although these 

colorimetric and ECL methods permit detection of high 

pg or low ng concentrations, sensitivity issues still exist, 

particularly when assessing foods or biological tissues. 

Recently, an immuno-polymerase chain reaction assay 

that uses a polymerase chain reaction to amplify a 

DNA-labeled reporter system bound to the anti-mouse 

immunoglobulin G (IgG) permits accurate detection of 

ricin in these biologic samples ranging from 1 pg/mL to 

100 pg/mL.117 The immuno-polymerase chain reaction 

may not only offer a method that greatly enhances the 

ability to detect ricin in environmental samples, but also 

and more importantly it provides a technique that will 

accurately determine ricin in tissues from individuals 

exposed to ricin. 

Handheld Assay Detection Devices 

Although routine capture ELISAs provide accurate 

diagnostic tools, these assays require a laboratory setting and instruments to measure the signal. Antibodybased handheld assay (HHA) devices were developed 

to enable first responders to assess the situation in the 

field.118 HHAs were initially developed to detect the 

anthrax in the letters sent through the mail to Senator 

Tom Daschle’s office in 2001. The success of anthrax 

spore identification initiated development of HHAs 

for ricin and other biothreat agents. In 2004, HHAs 

identified ricin in letters sent to the Dirksen Senate 

Office building.118 For ricin, HHAs have an antiricin 

Mab bound as a single line on the matrix bed. The 

sample is added to one end of the bed, and capillary 

action causes the sample to flow across the matrix. 

The toxin binds to the antibody and then another 

detection antibody is added. If ricin is present, then 

the detecting antibody causes color development at 

the line. If samples are positive using a HHA, samples 

are sent to a laboratory for confirmation and further 

analysis.

Sample Verification Platforms 

Laboratories, such as the US Army Medical Research Institute of Infectious Diseases (USAMRIID) 

and the National Biodefense Analysis and Countermeasure Center, need capabilities that will accurately 

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identify ricin. Multiple instrumental platforms with 

ELISA-based formats have been developed, including 

the following: 

• ECL-based ricin immunoassay (LOD, 0.05 ng/

mL); 

• Luminex MAGPIX multiplex (LOD, 0.001 ng/

mL); and 

• MesoScale Discovery (MSD) PR2 Model 1900 

ECL (LOD, 0.2 ng/mL). 

Both the M1M ECL-based ricin immunoassay and 

the Luminex MAGPIX use magnetic beads that are 

labeled with antiricin antibodies.119 Once ricin binds to 

the magnetic beads via the antibody, the sample is sent 

through the instrument where the magnetic beads are 

captured by an internal magnet. The magnet is set on 

an electrode that delivers the proper amount of electrical potential resulting in the emission of light identifying that the sample contains ricin. The MSD PR2, which 

is a highly sensitive ELISA, has the advantage of using 

less sample amount (25 μL). The detection antibody is 

conjugated to a chemiluminescent label that allows for 

ricin detection by ECL. 

On May 30, 2013, a multiplexed rapid ricin detection assay was launched by Radix BioSolutions Ltd 

(Austin, TX) through the CDC’s Laboratory Response 

Network.120 This assay was developed using Luminex 

xMAP technology that permits concurrent detection of several agents in an adaptable, multiplexed 

assay architecture. Following this report, Tetracore 

Inc (Rockville, MD) publicized the successful study 

completion and validation of its BioThreat Alert Lateral 

Flow Assay and BioThreat Alert Lateral Flow Assay 

Reader by the Department of Homeland Security for 

ricin detection.121

Liquid Chromatography/Mass Spectrometry 

Another method for ricin detection includes identification by liquid chromatography/mass spectrometry. 

The combination of liquid chromatography and mass 

spectrometry allows for the separation of mixtures in a 

sample while being able to identify specific substances 

based on their molecular mass via their mass to charge 

ratio (m/z).122 The ionization of the molecules in the 

sample can either be protonated or deprotonated 

depending on the characteristics of the analyte and 

the mode of detection.123,124 The advantage of using 

this technique allows for ricin detection when very 

little sample is available. Picogram amounts of ricin 

can be detected within a 5-hour timeframe allowing 

for fast, reliable detection.125 Liquid chromatography/

mass spectrometry can also be used to characterize 

other components within the sample because they 

may provide “signatures” that suggest the origin of 

the agent. For example, a highly pure form of the toxin 

might indicate that an organized terrorist group, such 

as Al Qaeda, produced the ricin while a less pure form 

may indicate fewer organized groups or individuals 

acting alone.86 

Ricin Activity Assay

When ricin is detected using an ELISA or other 

physical types of assays, the ability to determine 

whether the toxin is active becomes important for 

forensic evidence. The assay itself needs to accurately 

detect ricin’s biological activity in samples of limited 

size (about 50 mL) and low toxin concentration (about 

10 ng/mL), and preferably, with an assay time less 

than 6 hours. To meet these criteria, a cell-free translation (CFT) assay was developed at USAMRIID.126

The CFT assay measures luminescence generated by 

the enzyme luciferase produced from the translation 

of luciferase m-RNA in a rabbit reticulocyte lysate 

system. The amount of luminescence, produced when 

the luciferin substrate is added to luciferase, is proportional to the amount of luciferase produced in the 

in vitro translation system. When ricin is added to the 

mixture, translation of luciferase mRNA is reduced, 

which decreases the amount of luciferase produced. 

Since the amount of luminescence developed is proportional to the amount of luciferase present in the 

CFT mixture, a reduction in luminescence, as compared to a ricin standard control, provides a quantitative assessment of active ricin in the sample.126 Table 

16-1 summarizes the most commonly used techniques 

for ricin detection and their sensitivity limits. 

Diagnosis

Diagnosis of ricin intoxication is challenging 

because the cellular uptake of ricin is extremely 

rapid and limits the availability of ricin for diagnosis in blood and other fluid samples to 24 hours 

postintoxication.113 Experimental data suggest that 

the plasma half-life of ricin is biphasic with the early 

α phase half-life lasting approximately 4 minutes; 

the longer β phase half-life was determined to last 

approximately 83 minutes. The biphasic half-life 

suggests rapid distribution and uptake of the toxin 

followed by the slow clearance of excess toxin.113

Additional liquid chromatography/mass spectrometry assessment of urine samples for metabolites, 

particularly alkaloids such as ricinine that are 

commonly found in ricin preparations, indicates 

ricin intoxication if the individual has symptoms 

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associated with it.86 Individuals who survive ricin intoxication develop circulating antibodies in their blood 

that can be used to confirm intoxication. However, 

these antibodies are not present until approximately 

2 weeks postintoxication and, therefore, could not be 

used in the initial diagnosis. 

MEDICAL MANAGEMENT

TABLE 16-1 

BIOCHEMICAL METHODS FOR RICIN DETECTION AND THEIR LIMITS OF SENSITIVITY

Method* LOD† (ng/mL) Time‡ Detection§ Reference

ELISA-based 0.01–10 5–7 h Ricin 1–4

Handheld 10–50 90 min Ricin 5

ECL-based ELISA 0.001–10 4–7 h Ricin 6–8

Immuno-PCR 0.01–0.l 3–5 Ricin 3, 9

LC/MS 0.1–8 5 h Ricin/ricinine 10–13

CFT 10–50 4–5 h Biological activity 14

*

Each method may include several different assays using similar principles and formats

†

The limit of detection is the lowest amount of ricin detected

‡

The time required to perform the assay 

§

The assays detect either the physical form of ricin or determine the biological activity

CFT: cell-free translation

ELISA: enzyme-linked immunosorbent assay

LC/MS: liquid chromatography/mass spectrometry

LOD: limit of detection

PCR: polymerase chain reaction

Data sources: (1) Griffiths GD. Understanding ricin from a defensive viewpoint. Toxins (Basel). 2011;3:1373–1392. (2) Roy CJ, Hale M, 

Hartings JM, Pitt L, Duniho S. Impact of inhalation exposure modality and particle size on the respiratory deposition of ricin in BALB/c 

mice. Inhal Toxicol. 2003;15:619–638. (3) Bozza WP, Tolleson WH, Rosado LA, Zhang B. Ricin detection: tracking active toxin. Biotechnol 

Adv. 2015;33:117–123. (4) Poli MA, Rivera VR, Hewetson JF, Merrill GA. Detection of ricin by colorimetric and chemiluminescence ELISA. 

Toxicon. 1994;32:1371–1377. (5) Wade MM, Biggs TD, Insalaco JM, et al. Evaluation of handheld assays for the detection of ricin and staphylococcal enterotoxin B in disinfected waters. Int J Microbiol. 2011;2011:132627. (6) DHS fund ricin detection. Homeland Security News Wire. 

October 20, 2011. http://www.homelandsecuritynewswire.com/dhs-funds-ricin-detection. Accessed May 29, 2015. (7) Radix BioSolutions 

News, 2013. Accessed May 29, 2015. (8) GlobalBiodefense.com. Tetracore Completes DHS Validation of Ricin Detector. http://globalbiodefense.com/2013/06/12/tetracore-completes-validation-of-ricin-detector-for-dhs/. Accessed March 15, 2016. (9) He X, McMahon S, Henderson TD II, Griffey SM, Cheng LW. Ricin toxicokinetics and its sensitive detection in mouse sera or feces using immuno-PCR. PLoS One. 

2010;55:e12858. (10) Thompson M. High-performance liquid chromatography/mass spectrometry (LC/MS). AMC Technical Brief. Analytical 

Methods Committee AMC TB 34. London, England: Royal Society of Chemistry; 2008. (11) Fredriksson SA, Hulst AG, Artursson E, de Jong 

AL, Nilsson C, van Baar BL. Forensic identification of neat ricin and of ricin from crude castor bean extracts by mass spectrometry. Anal 

Chem. 2005;15;77:1545–1555. (12) Becher F, Duriez E, Volland H, Tabet JC, Ezan E. Detection of functional ricin by immunoaffinity and liquid 

chromatography-tandem mass spectrometry. Anal Chem. 2007;79:659–665. (13) Kanamori-Kataoka M, Kato H, Uzawa H, et al. Determination of ricin by nano liquid chromatography/mass spectrometry after extraction using lactose-immobilized monolithic silica spin column. J 

Mass Spectrom. 2011;46:821–829. (14) Hale ML. Microtiter-based assay for evaluating the biological activity of ribosome-inactivating proteins. 

Pharmacol Toxicol. 2001;88:255–260.

Despite the history of ricin’s use as a weapon, and unlike other toxin-mediated illnesses such as botulism, no 

Food and Drug Administration-approved therapeutic 

for ricin exposure exists. Given that ricin does not have 

cell specific selectivity, treatment of ricin intoxication 

is dependent on the site or route of entry, is largely 

symptomatic, and basically supportive to minimize the 

poisoning effects of the toxin. Medical countermeasures 

that have demonstrated capability to disrupt the ricin 

intoxication process include vaccines and antibody 

therapy. Both rely on the ability of antibody to prevent the binding of ricin to cell receptors. To ensure 

maximum protection, the vaccine must be given before 

exposure, and sufficient antibody must be produced.

Ricin Vaccines

Development of a ricin vaccine has previously 

focused on either a deglycosylated ricin A chain 

(dgRTA) or formalin-inactivated toxoid.127 Although both preparations conferred protection 

against aerosolized ricin, the proteins aggregated 

and precipitated over time. Additionally, ricin is 

not completely inactivated by formalin and may 

retain some of its enzymatic activity (albeit approximately 1,000-fold lower than native ricin). 

Thus, other approaches to vaccine development 

have been investigated to develop a safe and efficacious candidate. 

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Recent research has focused on developing recombinant RTA subunit vaccines to eliminate cytotoxicity and improve the stability of the vaccine13

(Figure 16-6). Researchers at the University of Texas 

developed RiVax that contains the Y80A mutation 

to inactivate catalysis, and the V76M mutation to 

ensure the removal of any trace of VLS activity from 

the immunogen.128,129 RiVax is at least 10,000-fold less 

active than wild type RTA but has also been shown to 

protect rodents against aerosol challenge.129 In 2006, 

RiVax was tested in phase I clinical trials. Results of 

these studies showed that RiVax appeared to be immunogenic and well tolerated in humans.130,131 However, while such findings were encouraging, vaccine 

formulation and stability remain problematic. Hence, 

a lyophilized formulation that retained immunogenicity when stored at 4°C was developed.132,133 RiVax 

has been out-licensed to Soligenix (Princeton, NJ) for 

more advanced clinical trials.134,135

To overcome both safety and stability issues 

simultaneously, researchers at USAMRIID structurally modified the RIP-protein fold of RTA to create a 

nonfunctional scaffold for presentation of a specific 

protective epitope.82 The engineered RTA 1–33/44–198 

(RVEc) was produced in E coli and lacks the Cterminal residues 199–276 as well as a loop between 

residues 34–43 (Figure 16-6). RVEc contains a number 

of well-characterized protective B-cell epitopes, but 

is more stable and less prone to aggregation. Based 

on preclinical studies, this product was determined 

to have a reasonable safety profile for use in human 

studies; it demonstrated no detectable RIP activity 

or evidence of VLS.136–139 In April 2011, USAMRIID 

launched a phase I escalating, multiple-dose study 

to evaluate the safety and immunogenicity of RVEc 

in healthy adults, and it was completed in November 

2012.138 The vaccine was well tolerated and immunogenic.138,139 In June 2013, a phase 1a (Version 2.0) 

protocol was implemented as a single-dose, singlecenter clinical study to allow for the administration 

and evaluation of a fourth boost vaccination.139 The 

ELISA and TNA anti-ricin IgG endpoint titers for the 

four boosted subjects indicated a robust response 

very soon after a boost vaccine. In conjunction with 

this study, another protocol was also started in June 

2013 for the collection of plasma from previously 

RVEc vaccinated subjects for passive transfer studies 

in animal models to demonstrate IgG as a surrogate 

marker for clinical efficacy. No adverse events have 

been reported on this study.139 

The RVEc final drug product passed stability testing 

through the 48 months.139 In addition, the potency assay 

results confirmed the vaccine elicited protective immunity in mice against 5 times the lethal ricin toxin dose, 

and it was capable of inducing anti-ricin neutralizing 

antibodies. An end of clinical use stability testing to include the 54-month time point was initiated in October 

2013 for both the final drug product and the diluent.139

A comparative immunogenicity and efficacy 

study between RVEc and RiVax has been conducted 

in mice.140 Both candidate RTA vaccines were found 

equally effective in eliciting protective immunity; 

however, quantitative differences were observed at the 

serologic level. RVEc was slightly more effective than 

RiVax in eliciting ricin-neutralizing antibodies. Furthermore, the antisera elicited by RVEc were toward an 

immunodominant neutralizing linear epitope on RTA 

(Y91 to F108), whereas those of RiVax were confined 

to residues 1–198.140

Antibody Treatment

Passive protection with aerosolized antiricin 

IgG has been evaluated as prophylaxis before aerosol challenge. In mice, pretreatment of nebulized 

antiricin IgG protected against aerosol exposure 

to ricin.141 Preclinical studies also have shown the 

protection afforded by neutralizing monoclonal antibodies against a lethal dose challenge of ricin.142–144

Researchers at Defence Science and Technology 

Laboratory in Porton Down, United Kingdom, have 

developed polyclonal antiricin antibodies that were 

raised in sheep immunized with ricin toxoid plus 

incomplete Freund’s adjuvant.145 The protective efficacy of both IgG and F(ab’)2 were demonstrated in 

mice against ricin intoxication when administered 

2 hours following either systemic or inhalational 

ricin challenge, while the smaller Fab′ fragment 

did not prevent death from ricin intoxication.145,146

This demonstrates the feasibility of producing an 

effective ovine antiricin antibody for use following 

ricin intoxication. In a recent study, four chimeric 

toxin-neutralizing monoclonal antibodies were produced and evaluated for their ability to passively 

protect mice from a lethal-dose ricin challenge.147

The most effective antibody, c-PB10, had the lowest 

IC50 (half-maximal inhibitory concentration) in a cellbased toxin-neutralizing assay and was sufficient to 

passively protect mice against systemic and aerosol 

toxin challenge.147

The use of antitoxins as therapies for toxin exposure 

has limitations including the following: 

• anaphylactoid or anaphylactic reactions;

• requirement of timely detection of exposure; 

and 

• the therapeutic window is dependent on the 

toxin and the dose received.145

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Supportive and Specific Therapy

The route of exposure for any agent is an important consideration in determining prophylaxis and 

therapy. For oral intoxication, supportive therapy includes intravenous fluid and electrolyte replacement 

and monitoring of liver and renal functions. Standard 

intoxication principles should be followed. Because of 

the necrotizing action of ricin, gastric lavage or induced 

emesis should be used cautiously. An aerosol-exposed 

patient may require the use of positive-pressure ventilator therapy, fluid and electrolyte replacement, antiinflammatory agents, and analgesics.148 Percutaneous 

exposures necessitate judicious use of intravenous fluids 

and monitoring for symptoms associated with VLS.

Development of Ricin Small Molecule Inhibitors

Reaching intracellular space with a ricin inhibitor 

provides an ideal pre- and postexposure therapeutic. 

At a minimum, small molecule inhibitors must possess sufficient safety and efficacy to enable a pathway 

to licensure. A strong safety profile is critical since no 

diagnostic capability exists to identify personnel who 

have received a clinically significant dose of ricin. 

Ideally, the inhibitor is also self-administered, which 

would greatly reduce the burden on the healthcare 

system and allow the provider to focus on patients who 

require more intensive care and medical resources.

A variety of approaches have been used to identify 

suitable small molecule ricin therapeutics. Potential 

compounds fall into three broad mechanisms of action: 

1. those that target RTA; 

2. those that target the retrograde transport 

pathway used by ricin to gain access to the 

cytosol; and 

3. a group that alters the cellular stress response 

following ricin intoxication. 

A notable absence among published work includes 

molecules directed against the RTB that might prevent 

ricin from entering cells. However, the molecular 

Figure 16-6. Ricin vaccines have been derived from the A-chain of the toxin. (a) Ricin consists of an A-chain and a B-chain. 

The A-chain is shown in ribbon, and B-chain in worm. (b) The ricin A-chain can be produced recombinantly in Escherichia 

coli apart from the B-chain. The structure of RiVax (PDB 3SRP) is similar to the structure of the A-chain of the toxin. (c) 

Truncation of the hydrophobic C-terminal residues of the A-chain and the loop increased the thermal stability of the protein 

and reduced its propensity to aggregate. The incorporation of disulfide bonds further enhanced the thermal stability of the 

immunogens (PDB 3MK9, 3LC9, and 4IMV). The protective epitopes are colored on each protein. The UNIVAX R70 epitope 

is shown in magenta; the B-cell epitope recognized by human neutralizing antibodies between Leu161-Ile175 was identified 

by Castelletti et al (2004) and is shown in green. The T-cell epitope between Ile175-Tyr183 is shown in blue. 

Data sources: (1) Legler PM, Brey RN, Smallshaw JE, Vitetta ES, Millard CB. Structure of RiVax: a recombinant ricin vaccine. 

Acta Crystallogr D Biol Crystallogr. 2011;67(Pt 9):826–830. (2) Compton JR, Legler PM, Clingan BV, Olson MA, Millard CB. 

Introduction of a disulfide bond leads to stabilization and crystallization of a ricin immunogen. Proteins. 2011;7:1048–1060. 

(3) Janosi L, Compton JR, Legler PM, et al. Disruption of the putative vascular leak peptide sequence in the stabilized ricin 

vaccine candidate RTA1-33/44-198. Toxins (Basel). 2013;5:224–248. (4) Lemley PV, Amanatides P, Wright DC. Identification and 

characterization of a monoclonal antibody that neutralizes ricin toxicity in vitro and in vivo. Hybridoma. 1994;13:417–421. (5) 

Castelletti D, Fracaso G, Righetti S, et al. A dominant linear B-cell epitope of ricin A-chain is the target of a neutralizing antibody response in Hodgkin’s lymphoma patients treated with an anti-CD25 immunotoxin. Clin Exp Immunol. 2004;136:365–372.

(6) Tommasi M, Castelletti D, Pasti M, et al. Identification of ricin A-chain HLA class II-restricted epitopes by human T-cell 

clones. Clin Exp Immunol. 2001;125:391–400. 

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structure of RTB makes it an extremely difficult drug 

target. X-ray studies show that RTB is composed of 

two domains with each domain possessing three subdomains that bind to sugars.62,149 Selected mutations of 

RTB suggest that three of the six sites must be inactivated to prevent cellular intoxication.62 Since these three 

sites are widely separated on RTB, it would present a 

formidable challenge in the design of inhibitors that 

still possess drug-like characteristics. Furthermore, 

because the RTB carbohydrate binding regions are 

small and shallow, these features present yet another 

hurdle for the design of drug-like molecules.150

In contrast, RTA presents a more tractable drug 

target. Although the large, open, and polar nature 

of the active site makes it a difficult drug target,151,152

high-resolution X-ray structures of the active site can 

help in the design of inhibitors.153 Furthermore, the 

mechanism of action for ricin is well described and 

provides additional criteria for the design of drugs 

that target the active site. Drug discovery approaches 

for ricin therapeutics have typically relied on virtual 

screening (VS), or high throughput cell-based assays. 

Virtual screening uses computational methods to 

evaluate large numbers of compounds for possible 

activity against ricin but requires careful consideration 

of molecular parameters to ensure optimal results, 

access to libraries of appropriate chemicals,154,155 and 

structural data, such as high resolution crystal structures of the target molecule.156 No single VS software 

is ideal as each produces different results.160 Several 

excellent recent in-depth reviews provide additional 

background on VS.157–159 Although it allows for evaluation of a large number of chemicals, VS has not 

always identified appropriate candidates for drug 

development.152,156,160 Regions of proteins, such as the 

active site of ricin, that have large and polar pockets 

are difficult drug targets,151 but successes have been 

seen in discovering RTA inhibitors.161,162,163

Another screening technique, cell-based high 

throughput screening (HTS), requires an appropriate 

cellular model of intoxication and a method to identify 

compounds that have activity against ricin. For cellbased assays the tested compounds should be soluble 

in cell culture media or with an excipient compatible 

with cellular growth. The solubility requirement 

significantly reduces the number of compounds that 

can be tested in cell-based assays. Furthermore, poor 

solubility may mask an otherwise useful molecule 

because it cannot be delivered to the cells in a high 

enough concentration to have an observable effect. 

More comprehensive discussion on cell-based HTS can 

be found in several recent reviews.164–167 Despite these 

limitations, the complementary methods of VS and 

cell-based HTS assays have identified a variety of active compounds. Similar approaches have been used to 

identify small molecule inhibitors of shigatoxin, a prokaryotic enzyme with related enzymatic activity but 

limited structural homology to ricin, suggesting pharmacophore discovery is broadly applicable.151–161,168–170 

Some of these VS and HTS successes for RTA inhibitor 

design will be highlighted in the next sections.

Ricin A Chain Inhibitors

Several research groups used RTA as a target to 

identify potential lead compounds from chemical 

libraries. One of the most potent active site inhibitors 

was developed by a rational drug design process171 and 

built on an earlier observation that pteroic acid (PTA) 

bound to the ricin active site with a modest IC50 of 600 

μm.163 Although PTA is not a suitable candidate because 

of its limited solubility, it served as a platform for designing derivatives. Several derivatives of PTA were 

found to have increased solubility, and when a linker 

was included that enabled additional contacts between 

RTA and the inhibitor, RTA inhibition was enhanced.172

Saito et al171 built on this observation by adding di- and 

tri-peptide linkers to PTA that allowed binding both 

the specificity pocket and a distant secondary pocket 

within the ricin active site. The addition of these linkers and the resulting interaction with the secondary 

pocket provided a 100-fold improvement in the IC50.

171

Additional successes in identifying RTA inhibitors through virtual screening have been reported by 

Pang et al161 and Bai et al.162 Although both groups’ 

compounds target RTA, Pang’s161 molecules targeted 

a site distant from the active cleft yielding inhibition 

of ricin enzymatic activity, whereas Bai’s162 are active 

site inhibitors. Pang’s161 deliberate choice was based on 

the recognition that the large size and polar features as 

well as the multiple electrostatic interactions between 

rRNA and the active site made it an unattractive and 

difficult drug target. Pang et al161 also took advantage 

of a structural change that occurs in the ricin active 

site upon binding of the toxin to the α-sarcin-ricin loop 

in the 28S ribosome, which causes Tyr80 in the active 

cleft to move to a new position where it participates 

in the depurination of the ribosome by packing with 

the bases of rRNA.153,173,174 Thus, if the movement of 

Tyr80 is blocked ricin is rendered inactive. Preventing the movement of the Tyr80 is a novel approach to 

developing RTA inhibitors by avoiding the complication of designing drugs for the ricin active site. This 

approach to inhibitor design was designated as the 

“door-stop” approach because it prevents Tyr80 from 

undergoing the necessary conformational change for 

enzymatic activity. Pang et al161 screened more than 

200,000 molecules with molecular weights lower than 

300 Da and 226 were predicted to block the movement 

of Tyr80. When evaluated in a CFT assay using firefly 

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luciferase, several of these compounds inhibited ricin. 

Unexpectedly, several compounds enhanced the firefly 

luciferase assay, but were the result of the compounds 

directly interacting with the firefly luciferase and not 

RTA. This interaction precluded the determination of 

the IC50 of the compounds and serves to underscore 

that appropriate controls need to be present when 

screening chemicals for activity in this reporter assay.175–178 However, functional studies revealed that 

the Pang compounds151 protected cells exposed to ricin, 

suggesting that ricin inhibition using the “door-stop” 

approach is a validated model. Furthermore, these 

results demonstrated that direct competition with the 

ricin active site, a difficult target, was not essential to 

achieve inhibition of the ricin catalytic activity.

The VS approach conducted by Bai et al162 identified 

several new classes of inhibitors. Bai162 used two different VS programs, one to identify molecules that could 

bind to RTA in which the Tyr80 has been displaced 

and a separate program that identified candidate compounds that bound to the RTA form in which Tyr80 

was not displaced. Compounds ranked highly by both 

programs were selected for further study, and they 

revealed a variety of new chemical entities for further 

development.162 In vitro kinetic studies showed that 

these compounds possess a potency similar to PTA. 

Although many of the compounds were cytotoxic, 

two were identified that protected vero cells exposed 

to ricin. The best performing compound showed little 

cytotoxicity and protected about 90% of cells exposed 

to ricin.162 Nevertheless, the cytotoxic compounds can 

still serve as starting points to improve their binding 

to RTA while reducing their toxicity.

Transport Inhibitors 

The second category of inhibitors, transport inhibitors, blocks the retrograde movement of ricin through 

the cell and may have its greatest utility as preexposure 

treatments. Compounds that inhibit the retrograde 

transport of ricin have substantial efficacy in animal 

models when used in a preexposure setting.179 However, because of the retrograde pathway taken by ricin 

to arrive at its cellular target, inhibitors of this normal 

cellular process also have a potential to exhibit significant toxicity. For example, ilimaquinone (IQ), a marine 

sponge metabolite, inhibited ricin in a dose-dependent 

manner in a vero cell assay.180 However, IQ also caused 

the Golgi apparatus to fragment into smaller vesicles; 

yet this effect was reversible when IQ was removed.181

Additional molecules have been identified that alter retrograde transport and protect cells from ricin challenge; 

however, the utility of these molecules for continued 

development is questionable because they also disrupt 

the Golgi architecture.182 In spite of the potential toxicity of retrograde transport inhibitors, several groups 

identified inhibitors of ricin transport, some of which 

show limited toxicity in cellular and animal based assays of efficacy. Stechmann and colleagues179 used a 

protein synthesis cell-based HTS assay to identify compounds that restored normal levels of protein synthesis 

after ricin exposure. Of more than 16,000 compounds, 

they identified two that were inhibitors of retrograde 

transport. Despite functionally blocking retrograde 

movement, these compounds exhibited no effect on the 

architecture of the Golgi complex or on cellular transport pathways such as endocytosis, vesicle recycling, 

degradation, or secretion.179 These two compounds 

were further examined in an animal model of intranasal 

ricin challenge. The compounds completely protected 

challenged animals when treatment was given 1 hour 

before ricin exposure; no acute toxicity was observed in 

animals that received only the test compounds.179 However, these compounds may not be ideal candidates for 

further development because of instability.183 

Cellular Stress Response Inhibitors 

Rather than targeting the ricin molecule or the retrograde transport pathway described previously, another target is the cellular response to ricin. When ricin 

depurinates ribosomes in target cells, these cells enter a 

condition known as ribotoxic stress response.184,185 The 

ribotoxic stress response leads to activation of stress associated protein kinases and other cellular changes.184 

Activation of stress associated protein kinases including p38 mitogen activated protein kinase (p38MAPK) can 

lead to the release of proinflammatory cytokines and 

the induction of apoptosis in cells.186–188 A screen of molecules that protected cells from ricin challenge but did 

not act on ricin or the retrograde pathway identified 

two molecules for further analysis.189 One compound 

reduced the activation of the SAPK p38MAPK by acting 

upstream of p38MAPK activation. The other compound 

acted as an inhibitor of caspase 3 and 7 activation, thus 

blocking a critical step in the induction of apoptosis.189

SUMMARY

Ricin is a potent toxin derived from the castor plant, 

R communis L, which has been cultivated worldwide 

for its oil since ancient times. Because of its potency, 

stability, wide availability of its source plants, and 

popularity on the Internet, ricin is considered a significant biological warfare or terrorism threat. Ricin 

was developed as an aerosol biological weapon during World War II, but was not used in combat nor in 

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mass casualty attacks. As a biological weapon, ricin 

has not been considered as useful in comparison with 

other biological agents such as anthrax or botulinum 

neurotoxin. Nevertheless, its popularity and its track 

record in actually being exploited by extremist groups 

and individuals accentuate the need to be vigilant of 

its surreptitious misuse. 

Despite ricin’s notoriety as a potential biological 

agent, its medical applications have been also explored. Ricin has contributed to early immunology; 

the understanding of both immunological and cell 

biological processes; and the treatment of cancer, 

AIDS, and other illnesses. Clinical manifestations 

of ricin poisoning vary depending on the routes of 

exposure. Aerosol exposure represents the greatest 

threat posed by ricin and can lead to death via hypoxia. Diagnosis of ricin exposure is based on both 

epidemiological and clinical parameters. No Food 

and Drug Administration-approved drug or vaccine 

against ricin intoxication exists; treatment is mainly 

symptomatic and supportive. Since vaccination offers a practical prophylactic strategy against ricin 

exposure, considerable efforts have been devoted to 

develop a safe and effective ricin vaccine to protect 

humans, in particular soldiers and first responders. 

Recombinant candidate ricin vaccines are currently 

in advanced development in clinical trials. Efforts are 

also underway to develop small molecule inhibitors 

for the treatment of ricin intoxication. Recent findings 

suggest that refinement of the newly identified ricin 

inhibitors will yield improved compounds suitable 

for continued evaluation in clinical trials. 

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184. Iordanov MS, Pribnow D, Magun JL, et al. Ribotoxic stress response: activation of the stress-activated protein kinase 

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185. Tesh VL. Activation of cell stress response pathways by Shiga toxins. Cell Microbiol. 2012;14:1–9.

186. Hui L, Bakiri L, Stepniak E, Wagner EF. p38alpha: a suppressor of cell proliferation and tumorigenesis. Cell Cycle. 

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187. Higuchi S, Tamura T, Oda T. Cross-talk between the pathways leading to the induction of apoptosis and the secretion 

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188. Coulthard LR, White DE, Jones DL, McDermott MF, Burchill SA. p38(MAPK): stress responses from molecular mechanisms to therapeutics. Trends Mol Med. 2009;15:369–379.

189. Wahome PG, Ahlawat S, Mantis NJ. Identification of small molecules that suppress ricin-induced stress-activated 

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Chapter 17

STAPHYLOCOCCAL ENTEROTOXIN 

B AND RELATED TOXINS PRODUCED 

BY STAPHYLOCOCCUS AUREUS AND 

STREPTOCOCCUS PYOGENES

KAMAL U. SAIKH, PhD*; ROBERT G. ULRICH, PhD†; and TERESA KRAKAUER, PhD‡

INTRODUCTION

CHARACTERIZATION OF TOXINS

HOST RESPONSE AND ANIMAL MODELS

CLINICAL DISEASE

Fever

Respiratory Symptoms

Headache

Nausea and Vomiting

Other Signs and Symptoms

DETECTION AND DIAGNOSIS

MEDICAL MANAGEMENT

VACCINES

DEVELOPMENT OF THERAPEUTICS

SUMMARY

*Microbiologist, Department of Immunology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

†

Microbiologist, Department of Immunology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

‡

Microbiologist, Department of Immunology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

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INTRODUCTION

Staphylococcus aureus and Streptococcus pyogenes are 

ubiquitous, gram-positive cocci that play an important 

role in numerous human illnesses such as food poisoning, pharyngitis, toxic shock, autoimmune diseases, 

and skin and soft tissue infections. These common bacteria readily colonize humans via numerous virulence 

factors that facilitate their survival and dissemination. 

Among these factors, staphylococcal enterotoxins 

(SEs), toxic shock syndrome toxin-1 (TSST-1), and 

streptococcal pyrogenic exotoxins (SPEs) share a common three-dimensional protein fold characteristic of 

these bacterial exotoxins called “superantigens” due 

to their potency in activating cells of the immune 

system.1,2 Picomolar concentrations of these bacterial 

superantigens activate specific Vβ-bearing T cells by 

binding to and cross-linking the major histocompatibility complex (MHC) class II molecules on antigenpresenting cells (APC) and the T-cell receptor (TCR). 

Activated T cells proliferate and, together with APC, 

produce proinflammatory mediators that, in elevated 

quantities, can induce fever, hypotension, and lethal 

shock. Most strains of S aureus and S pyogenes examined harbor genes for superantigens and are likely to 

produce at least one of these toxins. Strains that lack the 

ability to produce superantigens are usually attenuated 

in virulence. The staphylococcal enterotoxins are most 

frequently associated with food poisoning, yet not all 

superantigens are enterotoxins. Life-threatening toxic 

shock syndrome (TSS) may result from exposure to any 

of the superantigens through a nonenteric route. High 

dose, microgram-level exposures to staphylococcal enterotoxin B (SEB) will result in fatalities, and inhalation 

exposure to nanogram or lower levels may be severely 

incapacitating as well as fatal.3 In addition, the severe 

perturbation of the immune system caused by superantigen exposure may lower the infectious or lethal 

dose of replicating agents, such as influenza virus.4

SEB is a prototype enterotoxin and potential biological threat agent produced by many isolates of S aureus. 

During the 1960s, SEB was studied extensively as a 

biological incapacitant in the US offensive program. 

Recent studies on countermeasures and diagnostics 

have focused on SEB because of its effectiveness as 

a biological weapon, especially by inhalation. However, SEB represents many related biologically active 

superantigens that are readily isolated and manipulated by recombinant DNA (deoxyribonucleic acid) 

techniques. Moreover, the coadministration of SEB or 

related toxins with replicating pathogens or pathogenassociated molecules can lower the lethal dose of toxin 

by thousands fold. Pathogen-associated molecules 

such as endotoxins bind to toll-like receptors (TLRs) 

present on many cell types and activate similar intracellular signaling pathways as SEB, accounting for the 

synergy between these molecules and SEB in inducing 

pathophysiological effects.5

CHARACTERIZATION OF TOXINS

Genes encoding superantigens of S aureus and S 

pyogenes arise from a common ancestral gene. Most of 

the streptococcal superantigens are encoded by mobile 

genetic elements. SPE-A, SPE-C, SEA, and SEE are all 

phage-borne, while SED is plasmid-encoded. A chromosomal cluster of SE and SE-like genes are present in 

strains of S aureus.6 Transcriptional control of TSST-1, 

SEB, SEC, and SED is mediated through the accessory 

gene regulator (agr) locus,7 whereas SEA expression 

appears to be independent of agr. Strains that are agrnegative generally produce less toxin; however, there 

are also considerable differences in production levels 

among agr-positive isolates. These toxins are synthesized during the late logarithmic to stationary phases 

of growth, and production of many SEs is dependent 

on glucose concentration and environmental pH. The 

great diversity of superantigens and the highly mobile 

nature of their genetic elements suggest an accelerated 

rate of evolution. Staphylococcal and streptococcal 

strains that colonize domestic animals are potential genetic reservoirs for new toxin genes,8

 and the transfer of 

these sequences may contribute to hybrid polypeptides. 

The bacterial superantigens are 19- to 30-kD singlechain proteins with two major domains, containing 

β-sheet and α-helix structures, separated by a shallow 

groove.1,9 Based on amino acid sequences and structural homology, superantigens can be compiled into 

five different groups.10 TSST-1 is the most distantly 

related and lacks a “disulfide loop” commonly found 

in SEs, whereas SEs with emetic properties such as 

SEA, SEB, SEC, SED, and SEE all possess this loop 

structure. Despite significant sequence divergence, 

with similarities as low as 14%, overall protein folds 

are similar among staphylococcal and streptococcal 

superantigens. Cross-reactivities of polyclonal and 

monoclonal antibodies to SEs, TSST-1, and SPEs indicate common epitopes among these superantigens.11 

The toxin genes have evolved by strong selective 

pressures to maintain receptor-binding surfaces by 

preserving three-dimensional protein structure. The 

contact surfaces with MHC class II molecules involve 

variations of conserved structural elements,12,13 which 

include a ubiquitous hydrophobic surface loop, a 

polar-binding pocket present in most superantigens, 

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and one or more zinc-binding sites found in some 

toxins. Comparison of antibody recognition among 

superantigens11 suggests that antigenic variation is 

maximized while three-dimensional structures, and 

hence receptor-binding surfaces, are conserved. From 

a practical standpoint, this observation indicates that 

a large panel of antibody probes will be required for 

proper sample identification. 

Molecular details of the receptor interaction and 

biological actions of bacterial superantigens are 

well established. Superantigens target cells that 

mediate innate and adaptive immunity, resulting 

in an intense activation and subsequent pathology 

associated with aberrant host-immune responses. In 

contrast to “conventional” antigens, bacterial superantigens bind on the outside of the peptide-binding 

groove of MHC class II molecules and exert their 

biological effects without being “processed.” Most 

superantigens share a common mode for binding 

MHC class II molecules, with additional stabilizing interactions that are unique to each toxin.14 A 

second, zinc-dependent molecular binding mode 

for some superantigens increases T cell signaling 

and may impart greater toxicities in some cases. 

In normal T-cell responses to peptide antigens, the 

CD4 molecule stabilizes interactions between TCR 

and MHC class II molecules on APC (Figure 17-1). 

Superantigens also cross-link TCR and MHC class

II molecules, mimicking the CD4 molecule,15 and 

hence stimulate large numbers of T cells. Recognition of a superantigen by TCR is dependent on the 

variable region of the β chain (Vβ) of the TCR. Each 

toxin binds to a distinct repertoire of TCR Vβ, thus 

revealing the unique Vβ specificities of an individual 

superantigen.16 An intense and rapid release of cytokines, such as interferon-g, interleukin-1 (IL-1), 

interleukin-2 (IL-2), interleukin-6 (IL-6), and tumor 

necrosis factor-α (TNFα) is responsible for the systemic effects of the toxins.17–19 Although SEB has enterotoxic effects, the interaction of toxin with specific 

cells and receptors of the gastrointestinal tract is less 

well-defined. A specific region in SEB is involved in 

transcytosis of toxin.20 Other studies suggest various 

binding regions of SEB to epithelial cell membrane 

proteins.21,22 The release of histamine and cysteinyl 

leukotriene from mast cells likely accounts for the 

emetic effects of staphylococcal enterotoxins.23

Figure 17-1. Molecular model of receptor binding. Staphylococcal enterotoxins and other bacterial superantigens 

target the multireceptor communication between T cells 

and antigen-presenting cells that is fundamental to initiating pathogen-specific immune clearance. The superantigen 

inserts itself between the antigen receptor of T cells and the 

major histocompatibility complex class II molecule displaying peptides from potential pathogens. Toxin exposure 

results in hyperactivation of the immune system, and the pathology is mediated by tumor necrosis factor-α, interferon-γ, 

and other cytokines. 

HLA-DR: Human Leukocyte Antigen DR; SEB: staphylococcal enterotoxin B; TCR: T-cell receptor

 HOST RESPONSE AND ANIMAL MODELS 

Individuals may respond differently to superantigen 

exposure as a result of MHC polymorphisms, age, and 

many physiological factors. Each toxin exhibits varying 

affinities toward the HLA-DR, HLA-DQ, and HLA-DP 

isotypes and distinct alleles of class II MHC molecules, 

as observed by differences in T-cell responses in vitro. 

Generally, SE and TSST-1 bind HLA-DR better than 

HLA-DP or -DQ, whereas SPEA preferentially binds 

HLA-DQ better than HLA-DR. Primates, including 

humans, are most sensitive to superantigens when 

compared to other mammals.24 Lethal or incapacitating doses of toxin may be lowered by coexposure to 

endotoxin from gram-negative bacteria17 or hepatotoxins,25 or by infection with replicating agents.4

At the cellular level, the interaction of superantigens 

with receptors on APC and T cells leads to intracellular 

signaling.26 As with conventional antigens, costimulatory receptors are also required for cell activation by 

superantigens. The best-characterized costimulatory receptors are CD80/CD86 on APC and CD28 on T cells.27–29 

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The expression of intercellular adhesion molecule-1 

(ICAM-1) on APC promotes stable cell conjugate with 

T cells and provides costimulatory activation signals.27

The interactions of LFA-1 (lymphocyte function-associated antigen)/ICAM-1 and CD28/CD80 have both 

been implicated in SEA (staphylococcal enterotoxin 

A)-mediated T-cell activation.30 High concentrations 

of SEB elicit induction of phosphatidylinositol and the 

activation of protein kinase C (PKC) and protein tyrosine kinase (PTK) pathways,31,32 similar to mitogenic 

activation of T cells. PKC and PTK activation affect 

many intracellular signaling pathways, ultimately activating the transcription factors NF-κB (nuclear factor 

kappa-light-chain-enhancer of activated B cells), NF-AT 

(nuclear factor of activated T cells), and AP-1 (activator 

protein 1), resulting in the expression of proinflammatory cytokines, chemokines, and adhesion molecules.33–35 

Both IL-1 and TNFα can directly activate the transcription factor NF-κB in many cell types, including epithelial 

cells and endothelial cells, perpetuating the inflammatory response. Another mediator, IFNγ (interferon 

gamma), produced by activated T cells and natural 

killer cells, synergizes with TNFα and IL-1 to enhance 

immune reactions and promote tissue injury. PTKs and 

T-cell cytokines also activate phosphoinositide 3 kinase 

(PI3K), affecting many intracellular processes and 

pathways, ultimately activating the mammalian target 

of rapamycin (mTOR).36 SEB and other superantigens 

also directly induce chemotactic mediators, interleukin-8, monocyte chemoattractant protein-1 (MCP-1), 

macrophage inflammatory protein 1α, and macrophage inflammatory protein-1β, which can selectively 

chemoattract and activate leukocytes.37–39 Thus, cellular 

activation by SEB and other superantigens leads to severe inflammation, hypotension, and shock. Additional 

mediators contributing to SEB-induced shock include 

prostanoids, leukotrienes, and tissue factor from monocytes; superoxide and proteolytic enzymes from neutrophils; and chemokines from epithelial and endothelial 

cells. Activation of coagulation via tissue factor leads to 

disseminated intravascular coagulation, tissue injury, 

and multiorgan failure. SE-induced TSS thus presents a 

spectrum and progression of clinical symptoms, including fever, tachycardia, hypotension, multiorgan failure, 

disseminated intravascular coagulation, and shock.40,41

In humans and nonhuman primates (NHP), SEs 

induce an emetic response and toxic shock when ingested.42,43 Typically, the SEB-intoxicated NHPs developed anorexia, vomiting, and diarrhea within 6 to 24 

hours postexposure, followed by depression, dyspnea, 

and shock 24 to 72 hours later.43,44 Specific cells and 

receptors in the intestinal tract have not been identified 

for emesis, but some studies suggest the interaction of 

a dodecapeptide binding region of superantigen with 

epithelial cells.21,22 Pulmonary edema and lung lesions 

with infiltrated leukocytes and macrophages appeared 

in NHP exposed to SEB.44 

Although the SE studies in NHP are considered 

a “gold standard” for in vivo investigations, many 

rodent models have been developed as alternatives to 

study the toxic shock and acute lung injury aspects of 

superantigens.4,17,25,37,39,45 The lower cost associated with 

maintaining mice, the availability of immunological 

reagents, and certain similarities to NHP models are 

obvious advantages for their use in the development 

of therapeutics and vaccines. 

Mice are naturally less susceptible to SEs, TSST-1, 

and SPEs because of the lower toxin affinity to murine 

MHC class II molecules.17 Potentiating agents, such as 

d-galactosamine,25 actinomycin D,

46 lipopolysaccharide 

(LPS),17 and viruses4 have often been used to amplify 

the toxic effects of superantigens so that lower, practical 

amounts of toxins can be used for in-vivo studies. Many 

vaccine studies with SEB have been accomplished with 

an LPS-potentiated mouse model, as a natural synergy 

exists between these bacterial exotoxins and LPS.17,47 

Results of these studies show a correlation between increased serum levels of IL-1, IL-2, TNFα, and IFNγ with 

bacterial superantigen-induced shock. Pulmonary lesions 

with severe interstitial and alveolar edema, as well as 

perivascular leukocytic infiltrates in mouse models were 

similar to those in NHP exposed to SEB.45,46 Transgenic 

mice with inserted human HLA class II molecules have 

also been developed to study SEB-induced shock.48–50 In 

some cases, high doses of SEB and d-galactosamine were 

still required to induce toxic shock with transgenics.48,50 

Nevertheless, there is a correlation of proinflammatory cytokine induction and pulmonary lesions in the 

various transgenic models of SEB-induced toxic shock. 

A “double-hit” low-dose SEB model was developed in 

C3H/HeJ mice, an LPS-resistant mouse strain, to simulate 

human SEB-induced toxic shock.39 This model mimics 

human TSS closely as intranasal delivery of SEB triggers lung inflammation, systemic release of cytokines, 

and hypothermia that culminate in death at later time 

points similar to human toxic shock.45 All of these murine 

models have various drawbacks but they are useful as 

tools for the development of therapeutics and vaccines. 

CLINICAL DISEASE

The clinical documentation of TSS provides the 

most comprehensive source of information on the 

pathology of superantigen (eg, SEB) exposure. To 

meet strict criteria of the Centers for Disease Control 

and Prevention for TSS,51 negative blood (except 

for S aureus or S pyogenes), throat, or cerebrospinal 

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fluid cultures, as well as negative serological tests 

for Rocky Mountain spotted fever, leptospirosis, and 

measles should be obtained. TSS disease symptoms 

are characterized by a rapid drop in blood pressure, 

elevated temperature, and multiple organ failure. 

The profound hypotension and desquamation of the 

palms and soles of the feet characteristic of TSS is 

not observed in exposure by inhalation, and respiratory involvement is rapid, unlike other forms of 

TSS. Furthermore, the fever prominent after aerosol 

exposure is generally not observed in cases of SEB 

ingestion. 

An accidental laboratory inhalation exposure of 

nine laboratory workers to SEB best exemplifies the 

clinical disease as reported below. The following description illustrates a severely incapacitating illness of 

rapid onset (3–4 hours) and modest acute duration (3–4 

days) upon exposure to SEB.43 Details of the disease 

and signs and symptoms are described below.

Fever

Fever was prominent in all nine of those exposed. 

Eight of the individuals experienced at least one 

shaking chill that heralded the onset of illness. Using 

the morning peak level of SEB aerosol generation in 

the laboratory as the most likely time of exposure, 

onset of fever occurred from 8 to 20 hours post initial 

exposure, with a mean time of onset of 12.4 ± 3.9 (SD) 

hours. Duration of fever was from 12 to 76 hours after 

onset, with a mean duration of 50 ± 22.3 hours. Fever 

ranged as high as 106° acutely. Myalgias were often 

associated with the initial fever. Onset of myalgia was 

between 8 and 20 hours, with a mean onset of 13 ± 5 

hours. Duration was from 4 to 44 hours, and the mean 

duration was 16 ± 15 hours. 

Respiratory Symptoms

All nine patients were admitted to the hospital 

with a generally nonproductive cough. Onset was at 

10.4 ± 5.4 hours, and duration was 92 ± 41 hours. Five 

had inspiratory rales with dyspnea. The three most 

seriously compromised patients had dyspnea, moist 

inspiratory and expiratory rales, and orthopnea that 

gradually cleared. One individual had profound dyspnea for the first 12 hours that moderated to exertional 

dyspnea and rales, which persisted for 10 days. Chest 

radiographs on admission showed densities compatible with “patches of pulmonary edema” and Kerley 

lines suggesting interstitial edema. During recovery, 

discoid atelectasis was noted. Moderate compromise 

of the respiratory system was often accompanied by 

radiographic evidence of peribronchial accentuation, or “cuffing.” The mildly ill patients had normal 

radiographs. One of the three severely ill patients had 

severe pulmonary compromise and profound dyspnea 

and received only slight relief when treated with an 

aminophylline suppository. Moderately intense chest 

pain, of a substernal pleuritic type, occurred in seven 

individuals. Onset of chest pain was at 12 ± 6.5 hours 

and lasted for 4 to 84 hours, with a mean duration of 

23 ± 27 hours.

Headache 

Eight of the nine patients experienced headache 

with onset ranging from 4 to 36 hours, and the mean 

time of onset was at 13.3 ± 10 hours. Duration ranged 

from 8 to 60 hours, with a mean duration of 30.6 ± 19 

hours. The headaches ranged from severe to mild, but 

were usually mild by the second day of hospitalization. Five individuals’ headaches responded to Darvon 

(propoxyphene hydrochloride; Eli Lilly & Company, 

Indianapolis, IN) or codeine.

Nausea and Vomiting 

Gastrointestinal symptoms occurred in more than 

half of the individuals, nausea and anorexia in six, 

and vomiting in four. The onset of nausea ranged 

from 8 to 24 hours, with a mean onset of 17 ± 6.3 hours. 

Duration ranged from 4 to 20 hours, with a mean of 9 

± 5.5 hours. The time to onset of anorexia ranged from 

8 to 24 hours, with a mean onset of 18.5 ± 5.6 hours. 

Duration of anorexia ranged from 4 to 136 hours, and 

the mean duration was 44.5 ± 45 hours. Vomiting occurred in four patients, sometimes after prolonged 

paroxysms of coughing. The range of onset of vomiting was 8 to 20 hours, with a mean time to onset of 14 

± 5.1 hours. Duration was not prolonged and usually 

consisted of one episode. The patients were successfully treated with Compazine (prochlorperazine; 

SmithKline Beecham Pharmaceuticals, Philadelphia, 

PA) and Benadryl (diphenhydramine hydrochloride; 

Pfizer Pharmaceuticals Company, New York, NY). 

Only one individual demonstrated hepatomegaly 

and bile in the urine, although another patient also 

demonstrated mildly elevated liver-function tests. 

No diarrhea was reported in any of the exposed 

individuals. 

Other Signs and Symptoms

Cardiovascular

All patients who experienced chest pain had normal 

electrocardiograms. Throughout the illness, all patients 

were normotensive. Vomiting was of brief duration, and no one, including those vomiting, required 

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intravenous fluid administration. The patients’ pulse 

rates, when elevated, paralleled temperature elevation.

Hematology

Leukocytosis was observed in most of the patients 

12 to 24 hours after exposure to the toxin.

Ocular Effects

None of the patients experienced conjunctivitis, 

although one individual later stated he remembered 

that his eyes had “burned” during the believed time 

of exposure. This contrasts with reports of conjunctivitis resulting from separate accidental laboratory 

exposures.52

DETECTION AND DIAGNOSIS

The staphylococcal enterotoxins are moderately 

stable proteins; therefore, immunological evaluation should be possible in field or clinical samples. A 

variety of rapid and sensitive detection methods are 

available.53,54 Immunoassays are very sensitive and can 

detect picogram quantities of toxins in environmental 

and serum samples. Plasma concentrations of superantigens were measured in septic patients of an intensive 

care unit using an enzyme-linked immunosorbent assay.55 In one study, 56 the mean concentration of TSST-1 

in human sera from TSS patients was reported to be 

440 pg/mL. In contrast, anti-TSST-1 antibody titers are 

often low in TSS patients57,58 and only recover during 

convalescence. Furthermore, most normal human 

serum samples contain detectable levels of antibody 

reacting with several different toxins, including SEB. 

Therefore, serum antibody titers are of little diagnostic 

value. If bacterial sepsis is suspected and cultures can 

be obtained, detecting minute quantities of potentially 

toxigenic strains is possible using polymerase chain 

reaction (PCR) amplification and toxin gene-specific 

oligonucleotide primers. The results from both PCR 

and immunoassays are rapid, allowing quantitative 

or qualitative measurements in less than 24 hours. 

Finally, as the best approach to early diagnosis on the 

battlefield, toxins may be identifiable in nasal swabs 

from individuals exposed to aerosols for at least 12 to 

24 hours postexposure.

MEDICAL MANAGEMENT

No specific therapy has been identified or described.3,41,43 Supportive therapy in the nine mild accidental exposure cases described earlier seemed to 

provide adequate care. Symptoms of fever, muscle 

aches, and arthralgias may respond to cool compresses, 

fluids, rest, and judicious use of acetaminophen or 

aspirin. For nausea, vomiting, and anorexia, symptomatic therapy should be considered.

Antihistamines (eg, diphenhydramine) and phenothiazine derivatives (eg, prochlorperazine) have been 

used parenterally or as suppositories. The success 

of these drugs in controlling nausea may have been 

augmented by the relatively short duration of nausea 

and vomiting induced by aerosolized SEB. Because of 

the brevity of vomiting episodes, fluid replacement 

was not considered or required in the series discussed. 

However, replacement may be necessary in the event 

of prolonged vomiting resulting in fluid and electrolyte 

depletion. Although diarrhea was not observed in human accidental exposure cases, deposition of toxin on 

foodstuffs could produce the syndrome, which should 

be treated symptomatically.

Initial symptomatic therapy with cough suppressants containing dextromethorphan or codeine 

should be routinely employed. Prolonged coughing 

unrelieved by codeine might benefit from a semisynthetic, centrally acting narcotic antitussive containing 

hydrocodone (dihydrocodeinone). 

Pulmonary status should be monitored by pulse 

oximetry, and when respiratory status is compromised, 

prompt evacuation to a site with capacity for intensive 

respiratory care by mechanical ventilation should be 

considered. 

Infusion of intravenous immunoglobulin has been 

successfully used59,60 to treat episodes of Kawasaki’s 

syndrome linked to SE and TSST-1. An anecdotal case 

of TSS with elevated TSST-1 and SEA levels, complicated by life-threatening multiorgan dysfunction, was 

successfully treated by early introduction of plasma 

exchanges.61 Prior exposure to SEB by inhalation does 

not appear to protect against a subsequent episode; 

however, increased antibody titers to SEB are protective, and efforts to devise both passive and active immunotherapy show promise. Because of the rapidity 

of receptor binding by these toxins (apparent saturation less than 5 minutes), active immunity should be 

considered the best defense. 

The treatment of toxic shock with S aureus-secreting 

superantigens such as SEB and TSST-1 is much more 

complex in a clinical setting. Both S aureus and S 

pyogenes produce multiple virulence factors that aid 

in bacterial survival and dissemination in the host. 

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Furthermore, the emergence of methicillin-resistant S 

aureus strains poses constraint in treatment options and 

clinical guidelines were revised and updated recently.62

A recent study in a rabbit model of S aureus pneumonia 

suggests that vaccination against superantigens and 

secreted cytolysins provides protection against S aureus, whereas vaccination against bacterial cell-surface 

antigens increases disease severity.63

VACCINES

A formalin-treated SEB toxoid demonstrated some 

degree of efficacy in animal trials, but is not approved 

for human use. Vaccines produced by site-specific mutagenesis of the toxins, delivered by intramuscular or 

intradermal routes, have also shown promising results 

in animal and human trials. These recombinant subunit vaccines were produced by substitution of active 

receptor-binding amino acid side chains that reduced 

affinities and consequential T-cell activation13,14,47,64

without altering the three-dimensional structure of the 

antigen. Though promising, these engineered vaccines 

are neither licensed nor available for human use. 

DEVELOPMENT OF THERAPEUTICS

An understanding of the cellular receptors, signaling pathways used by staphylococcal superantigens, 

and the biological mediators induced has provided 

insights to selecting appropriate therapeutic targets. 

Potential targets to prevent the toxic effects of SEs 

include (a) blocking the interaction of SEs with the 

MHC, TCRs,26 or other costimulatory molecules27,28; 

(b) inhibition of signal transduction pathways used by 

SEs26; (c) inhibition of cytokine and chemokine production36; and (d) inhibition of the downstream signaling 

pathways used by proinflammatory cytokines and 

chemokines. 

Limited therapeutics for treating superantigeninduced toxic shock are currently available. Intravenous immunoglobulin was effective as a treatment in 

humans after the onset of toxic shock syndrome.41,59

Antibody-based therapy targeting direct neutralization of SEB or other superantigens is most suitable during the early stages of exposure before cell activation 

and the release of proinflammatory cytokines.64 Because some neutralizing antibodies cross-react among 

different superantigens,11 a relatively small mixture of 

antibodies might be effective in treating exposures to a 

greater variety of superantigens. Vaccines of SEB and 

SEA with altered critical residues involved in binding 

class II MHC molecules were also used successfully 

to vaccinate mice and monkeys against SEB-induced 

disease.47,65

Most therapeutic strategies in animal models of 

SEB-induced shock have targeted proinflammatory 

mediators. Therapeutic regimens include corticosteroids and inhibitors of cytokines, caspases, or phosphodiesterases.45,66,67 Several in vivo murine models have 

been used to study potential therapies that prevent 

superantigen-induced shock. Therapeutic agents, such 

as nitric oxide inhibitors, decrease SEA and SEB effects 

by inhibiting the production of IL-1, -2, -6, TNFα, and 

IFNγ in the LPS-potentiated model.68 Blockade of the 

CD28 costimulatory receptor by its synthetic ligand, 

CTLA4-Ig, prevented TSST-1-induced proliferation 

of T cells and lethal TSS.69 Decreased mortality rates 

accompanied by an attenuation in liver apoptosis

and hemorrhagic necrosis were seen in mice given dgalactosamine plus SEB along with a cell-permeable 

cyclic peptide targeting NFκB.70 Dexamethasone, a 

well-known FDA-approved immunosuppressant 

and NFκB inhibitor, prevented toxic shock in the 

LPS-potentiated mouse model and the “double-hit” 

SEB-induced shock model.45,71 Rapamycin, another 

FDA-approved drug currently used to prevent kidney 

graft rejection, was efficacious even when given 24 

hours after SEB in the “double-hit” SEB-induced shock 

model.72 Recently, myeloid differentiation primary 

response protein (MyD88)–mediated proinflammatory 

signaling has been shown to be activated after SEB 

binding to MHC class II73 and that MyD88-/- mice are 

resistant to SEB and SEA intoxication.74,75 Administration of a synthetic small molecule mimetic (EM-163) to 

the conserved BB loop in the toll/IL-1 receptor (TIR) 

domain of MyD88, reduced multiple cytokines and 

protected mice from lethal shock in the LPS-sensitized 

model. 76,77

SUMMARY

SEB is representative of a group of bacterial proteins 

that exert profound toxic effects upon the immune 

system. Many sensitive immunoassays have been 

developed for laboratory detection of most of the 

staphylococcal and streptococcal superantigen toxins, 

but the limit of field detection is unknown. Inhalation 

exposure to agents such as SEB may result in severe but 

temporary incapacitation, while high-dose exposures 

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will result in fatalities. Supportive symptomatic therapy is the only known method of treatment. Vaccines 

currently under development may afford protection 

to individuals but are not yet licensed for human use. 

Therapeutics tested in murine models may provide 

insights to future development in treating toxic shock. 

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cells by staphylococcal toxin superantigens. J Clin Invest. 1993;91:602–607.

60. Leung DY, Meissner HC, Fulton DR, Murray DL, Kotzin BL, Schlievert PM. Toxic shock syndrome toxin-secreting 

Staphylococcus aureus in Kawasaki syndrome. Lancet. 1993;342:1385–1388.

61. Kohro S, Imaizumi H, Yamakage M, Masuda Y, Namiki A, Asai Y. Reductions in levels of bacterial superantigens/cannabinoids by plasma exchange in a patient with severe toxic shock syndrome. Anaesth Intensive Care. 2004;32:588–591.

62. Liu C, Bayer A, Cosgrove SE, Daum RS, Fridin SK, et al. Clinical practice guidelines by the Infectious Diseases Society 

of America for the treatment of methicillin-resistant Staphylococcus aureus infections in adults and children. Clin Infect 

Dis. 2011; 52:e18-55. 

63. Spaulding AR, Salgado-Pabon W, Merriman JA, Stach CS, Ji Y. et al. Vaccination against Staphylococcus aureus. J Infect 

Dis. 2014; 209:1955-1962.

64. Larkin EA, Stiles BG, Ulrich RG. Inhibition of toxic shock by human monoclonal antibodies against staphylococcal 

enterotoxin B. Plos One. 2010; 5(10):e13253.

65. Ulrich RG, Olson M, Bavari S. Bacterial superantigen vaccines. In: Brown F, Norrby E, Burton D, Mekalanos J, eds. 

Vaccines 96: Molecular Approaches to the Control of Infectious Diseases. Cold Spring Harbor, NY: Cold Spring Harbor 

Laboratory Press; 1996: 135–141.

66. Krakauer T, Stiles BG. Pentoxifylline inhibits staphylococcal superantigen induced toxic shock and cytokine release. 

Clin Diagn Lab Immunol. 1999; 6:594–598.

67. Annane D, Sebille V, Charpentier C, et al. Effect of treatment with low doses of hydrocortisone and fludrocortisone 

on mortality in patients with septic shock. JAMA. 2002;288:862–871.

68. LeClaire RD, Kell W, Bavari S, Smith T, Hunt RE. Protective effects of niacinamide in staphylococcal enterotoxin B 

induced toxicity. Toxicology. 1996;107:69–81.

69. Saha B, Jaklic B, Harlan DM, Gray GS, June CH, Abe R. Toxic shock syndrome toxin-1-induced death is prevented by 

CTLA4Ig. J Immunol. 1996;157:3869–3875.

70. Liu D, Zienkiewicz J, DiGiandomenico A, Hawiger J. Suppression of acute lung inflammation by intracellular peptide 

delivery of a nuclear import inhibitor. Molec Ther. 2009;17:796–802.

71. Krakauer T, Buckley M. Dexamethasone attenuates staphylococcal enterotoxin B-induced hypothermic response and 

protects mice from superantigen-induced toxic shock. Antimicrob Agents Chemother. 2006; 50:391–395.

72. Krakauer T, Buckley M, Issaq HJ, Fox SD. Rapamycin protects mice from staphylococcal enterotoxin B-induced toxic 

shock and blocks cytokine release in vitro and in vivo. Antimicrob Agents Chemother. 2010;54:1125–1131.

73. Kissner TL, Ruthel G, Ulrich RG, Fernandez S, Saikh KU. Activation of MyD88 signaling upon staphylococcal enterotoxin binding to MHC class II molecules. Plos One. 2011;6(1):e15985.

74. Kissner TL, Ruthel G, Cisney ED, Ulrich RG, Fernandez S, Saikh KU. MyD88-Dependent pro-inflammatory cytokine 

response contributes to lethal toxicity of staphylococcal enterotoxin B in mice. Innate Immunity. 2011;17:451–462. 

75. Kissner TL, Cisney ED, Ulrich RG, Fernandez S, Saikh KU. Staphylococcal enterotoxin A induction of pro-inflammatory

cytokines and lethality in mice is primarily dependent on MyD88. Immunology. 2010;130:516–526.

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76. Kissner TL, Moisan L, Mann E, et al. A small molecule that mimics the BB-loop in the toll interleukin-1 (IL-1) receptor domain of MyD88 attenuates staphylococcal enterotoxin B-induced pro-inflammatory cytokine production and 

toxicity in mice. J Biol Chem. 2011;286:31385–31396.

77. Kissner TL, Ruthel G, Alam S, et al. Therapeutic inhibition of pro-inflammatory signaling and toxicity to staphylocooccal enterotoxin B by a synthetic dimeric BB-loop mimetic of MyD88. PLoS One. 2012;7:e40773.

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Chapter 18

TOXINS FROM VENOMS AND POISONS

SCOTT A. WEINSTEIN, PhD, MD,* and JULIAN WHITE, MD†

INTRODUCTION

SOME DEFINITIONS: VENOMS, TOXINS, AND POISONS

NONWARFARE EPIDEMIOLOGY OF VENOM-INDUCED DISEASES AND 

RELATED TOXINS

Venomous Bites and Stings

Venomous Snakes

Scorpions, Spiders, and Other Arachnids

Insects

Marine Envenoming: Sea Snakes, Cnidarians, and Venomous Fish

Poisoning by Animals, Plants, and Mushrooms

Relevance to Biological Warfare

MAJOR TOXIN CLASSES AND THEIR CLINICAL EFFECTS

Paralytic Neurotoxins

Excitatory Neurotoxins

Myotoxins

Hemostasis-Active Toxins

Hemorrhagic and Hemolytic Toxins

Cardiotoxins

Nephrotoxins

Nectrotoxins

Other Toxins

CONCLUSIONS AND DIRECTIONS FOR RESEARCH

*Physician, Toxinologist, Toxinology Division, Women’s & Children’s Hospital, 72 King William Road, North Adelaide, South Australia 5006, Australia

†

Head of Toxinology, Toxinology Division, Women’s & Children’s Hospital, 72 King William Road, North Adelaide, South Australia 5006, Australia

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INTRODUCTION

venom-derived toxins have not generally been considered suitable for use as a mass offensive weapon; 

however, primarily fungal- or plant-derived toxins 

certainly have been weaponized (eg, ricin; see chapter 

16). Using venoms as a weapon is also obviously distinct from the weaponization of toxins from bacteria.

However, many toxins found in animals, plants, 

and mushrooms are highly toxic—even lethal—to 

humans. These toxins, which can form the basis for 

developing tailored toxin derivatives for specific functions, are the subject of current intense research in the 

pharmaceutical industry. A well-known example is 

paclitaxel, a taxane initially derived from bark extract 

of the Western yew, Taxus brevifolia. Taxanes, such as 

paclitaxel, are potent cytotoxins that stabilize microThis chapter considers toxins that might be exploited as offensive biological weapons, or that may 

have medical relevance to deployed military personnel. The major characteristics of important toxin 

classes are summarized, and their medical effects are 

covered. Venom toxins are emphasized because little 

information is available about venomous animals in 

relation to military medicine. This chapter highlights 

selected plant, fungal, and animal toxins as examples 

of potent agents that target essential physiological 

processes, and it provides information that may facilitate recognition of types of envenoming or poisoning 

in an affected patient. This chapter also supports the 

perception that animal toxins are generally of low 

relevance to military applications, and contrarily, the 

relevant—but limited—nontactical importance of some 

plant and fungal toxins. This information is intended 

to increase awareness of the potential hazards posed 

by animal toxins that can be used for offensive applications on a small scale and also provide some important 

considerations about possible exposures to venomous 

and poisonous animals, plants, and mushrooms that 

might occur during military deployments. 

The use in warfare of diverse animal-derived venoms, as well as the venomous animals themselves, has 

probably been contemplated for most of human history. The well-known mythical account of the second 

labor of Hercules slaying the malevolent nine-headed 

serpent, the Lernaean Hydra (Figure 18-1), featured 

him using venom-coated arrowheads to accomplish 

the deed. Some folklore scholars consider this to be 

the first description of the use of a bioweapon.1

 The 

practice may have been used in any of the ancient 

Greek Wars, and, as has been noted by numerous authors, the word toxic is derived from toxikon, Greek 

for poison arrow. This is one of the reasons why Findlay E Russell (1919–2012), in consultation with other 

founders of the International Society on Toxinology 

(IST), named the IST journal Toxicon. Circa 200 bce, 

Hannibal reportedly used pottery containing venomous snakes to “bombard” opposing maritime vessels. 

The Roman Legion’s assault on Hatra in 199 ce resulted 

in retaliation by civil forces that included the hurling of 

clay pots filled with scorpions over the walls.1

 Several 

Native American tribes (eg, Wishram, Yuma) used 

venom- or venom gland extract-coated arrowheads in 

warfare, and some such as the Achomawi and Karok 

used arrowheads dipped in rattlesnake organs or their 

extracts that they believed to be toxic.2

Historically, the offensive military use of venoms 

and their component toxins (all of natural origin) has 

been a rare and small-scaled occurrence. Venoms or 

Figure 18-1. Hercules slaying the Lernaean Hydra. 

Illustration: Antonio del Pollaiolo, Ercole e l’Idra e Ercole e 

Anteo, Google Art Project. Wikimedia Commons, public 

domain. https://it.wikipedia.org/wiki/File:Antonio_del_

Pollaiolo_-_Ercole_e_l%27Idra_e_Ercole_e_Anteo_-_

Google_Art_Project.jpg. 

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Toxins From Venoms and Poisons

tubule assembly, thereby disrupting physiological 

assembly/disassembly of microtubules in a guanosine 

triphosphate-independent manner.3 These taxanes, 

which have a proven pharmacotherapeutic efficiency 

against a wide array of solid neoplasms, are a significant part of the chemotherapeutic armamentarium. 

Several pharmacotherapeutics are derived from 

venom components, and some are in various stages 

of clinical trials. Two prominent examples are the 

entire class of antihypertensives: (1) the angiotensinconverting enzyme inhibitors (ACEIs) and (2) the 

parenteral insulin secretagogue exendin-4. Exendin-4 

is a 39-amino acid peptide isolated from venom of the 

helodermatid lizard, the Gila monster (Heloderma suspectum), that has greater than 50% structural homology 

with glucagon-like peptide 1. Exendin-4 exhibits functional similarity with glucagon-like peptide 1, but has a 

longer half-life and biological stability. This peptide increases insulin secretion, accelerates gut emptying, and 

stimulates β-islet cell proliferation and survival, as well 

as other actions. It was tested as an antidiabetic agent 

and introduced as Byetta (Amylin Pharmaceuticals, 

San Diego, CA) in 2005.4 Similarly, the ACEI arose from 

the study of bradykinin-potentiating oligopeptides 

present in several South American lance head pit vipers 

(eg, the jararaca, Bothrops jararaca, and others) in conjunction with study of the complex renin-angiotensin 

and kallikrein-kinin systems. Extensive investigation 

eventually resulted in teprotide, an early, parenterally administered ACEI, and eventually the first oral 

ACEI, Captopril (Par Pharmaceutical, Woodcliff Lake, 

NJ), was developed.5 A broad variety of ACEIs have 

become among the three most frequently prescribed 

classes of antihypertensive medications in the United 

States and most of the world. 

The potential threat posed by these toxins and their 

derivatives (as discussed further in the section on 

Relevance to Biological Warfare) relates mostly to their 

practicality for weaponization and delivery, rather 

than their inherent toxicity.

SOME DEFINITIONS: VENOMS, TOXINS, AND POISONS

Toxins are substances produced by living organisms (animals, plants, mushrooms, bacteria) that 

cause significant adverse effects when administered 

to another living organism, particularly those that 

offer the producing organism some advantage, either 

offensive or defensive.

Venoms are mixtures—often complex mixtures—of 

toxins produced in defined organs (usually venom 

glands) or organelles (eg, nematocysts/cnidocysts located in specialized cells and nematocytes/cnidocytes 

in jellyfish) that are delivered to the target organism 

usually using an evolved delivery system such as fangs 

or a stinging apparatus. Therefore, venom is delivered 

as an active process, and if sufficient (this may be minute in some cases) amounts are introduced into the 

target organism, it causes envenomation (also known 

as “envenoming”). Venom may be used defensively 

against predators (eg, stings from bees or venomous 

fish), but more commonly it is used offensively to assist 

in acquiring prey (eg, as by venomous snakes). Venom 

used offensively may be used to either kill or immobilize 

the prey, possibly aid digestion of the prey, or combine 

these functions, which may also be useful when venom 

is used defensively. The evolution of venom has been 

positively selected among a wide range of taxa, suggesting that it provides diverse organisms with selective 

advantages and fitness. The definition of venom from 

evolutionary, phylogenetic, and functional perspectives 

is actively debated.6–10 The criteria defining the words 

venom and venomous and the related terminology may 

be subjected to interdisciplinary consensus in the future.11

Poisons are technically differentiated from venoms 

because they need to be ingested rather than injected 

(as venom is delivered) to induce their toxic effects. 

Animal, plant, or fungal toxins consist of individual or 

mixtures of toxins that are produced by the organism or, 

in some cases, by symbiotic bacteria (eg, the synthesis 

of tetrodotoxin by at least 18 microbial taxa, including 

Vibrio spp12,13 and Shewanella putrefaciens14) that colonize the poisonous animal. These toxins are generally 

delivered in a passive and, in most cases, a defensive 

way to an organism attacking or trying to consume the 

toxin producer. Examples include tetrodotoxic fish that 

cause poisoning when eaten, some toads (eg, common 

African toad, Amietophrynus regularis) that exude toxins 

from parotid skin glands when mouthed by predators, 

and both poisonous plants and mushrooms when ingested. However, other animals such as several species 

of hedgehogs (eg, four-toed, spiny, or Cape hedgehog, 

Atelerix albiventris pruneri15) anoint their spines with toxic 

toad parotid secretions, and thereby can be considered 

to actively deter predators by exposing them to toad 

toxins via their spines. Therefore, in nature, poisoning by toxins is most often a passive process because 

the poison is introduced by the aggressor organism’s 

actions. In terms of natural selection, it is often better 

to deter rather than kill a predator. When delivered at 

the typically delivered dose, the toxins in poisoning 

can often cause unpleasant but nonlethal effects. Clear 

exceptions exist when ingestion of only a tiny quantity of some of these poisonous organisms, because 

of their high lethal potency, can be fatal for humans.

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NONWARFARE EPIDEMIOLOGY OF VENOM-INDUCED DISEASES AND RELATED TOXINS

Venomous Bites and Stings

Venomous animals include a vast array of organisms found in many phyla, from primitive to highly 

advanced, but certain groups have a particularly important impact on human health.

Venomous Snakes

Snakebite has the most significant impact on human health. Most regions contain some venomous 

species, but the rural tropics have a particularly high 

Toxin-induced disease affects millions of humans 

every year. Detailed epidemiology is unavailable 

for any toxin-induced disease (other than selected 

microbial toxin diseases, and this information is 

often incomplete) at the global level, but more 

epidemiologic data may emerge for some key disease types, as a result of increasing international 

efforts directed at the improved management of 

regionally important venom diseases (eg, several 

snakebite initiatives). An approximate estimate of 

epidemiology for some principal groups is provided in Table 18-1. 

TABLE 18-1

ESTIMATED HUMAN IMPACT OF ENVENOMING AND POISONING BY SOME PRINCIPAL GROUPS 

OF TOXIN-PRODUCING FAUNA AND FLORA*

Estimated Annual Global Impact

Organism Group Number of Cases Number of Fatalities

Venomous snakes >2.5 million >100,000

Scorpions >1 million >3,000

Spiders >100,000 <100

Paralysis ticks >1,000 <10

Insects† >1 million >1,000

Spiny venomous fish >100,000 <10, likely close to zero‡

Stingrays >100,000 <10§

Cone snails <1,000 <10

Octopus (blue-ringed octopuses) <100 <10

Jellyfish and related coelenterates >1 million <10¥

Fugu poisoning (tetrodotoxic fish) Unknown Unknown, likely >100

Ciguatera (ciguatoxic fish) >20,000 Unknown, but few

Shellfish poisoning (several types) Unknown Unknown

Poisonous mushrooms Unknown Unknown, likely >100

Poisonous plants Unknown Unknown, possibly >1,000

*Data are based on most reliable published data, extrapolations of that data, or best guess estimates based on fragmented published data. 

The most reliable data are for snakebite and the annual global fatalities are probably underestimated. Most authorities consider published 

epidemiology data for envenoming as underestimates, and it is possible the estimated incidence given here is similarly an underestimate. 

†

Figures for insects include severe and fatal allergic reactions to venomous stings, which are responsible for the vast majority of medically 

significant cases.

‡

No well-documented fatalities exist, and insufficient evidence for any approximations of possibly reliably reported semianecdotal cases.

§

The handful of rare fatalities almost always results from intraperitoneal penetrative envenoming.

¥

Almost all of the uncommon fatalities occur after envenoming by Chironex fleckeri, or one of several other chirodropid or charybdeid taxa 

that cause Irukandji syndrome (see Marine Envenoming in text).

Data sources: (1) Williams D, Gutiérrez JM, Harrison R, et al. The global snake bite initiative: an antidote for snake bite. Lancet. 2010;375:89–91. 

(2) Skinner MP, Brewer TD, Johnstone R, Fleming LE, Lewis RJ. Ciguatera fish poisoning in the Pacific Islands (1998 to 2008). PLoS Negl 

Trop Dis. 2011;5:e1416. doi:10.1371/journal.pntd.0001416. (3) Mebs D. Venomous and Poisonous Animals: A Handbook for Biologists, Toxicologists 

and Toxinologists, Physicians and Pharmacists. Boca Raton, FL: CRC Press; 2002: 360. (4) Meier J, White J. (eds). Handbook of Clinical Toxicology 

of Animal Venoms and Poisons. Boca Raton, FL: CRC Press; 1995. (5) Wang DZ. Neurotoxins from marine dinoflagellates: a brief review. Mar 

Drugs. 2008;11:349–371. (6) Noguchi T, Arawaka O. Tetrodotoxin-distribution and accumulation in aquatic organisms, and cases of human 

intoxication. Mar Drugs. 2008;6:220–242. (7) Warrell DA. Venomous bites, stings and poisoning. Infect Dis Clin North Am. 2012;26:207–223. 

(8) Personal observations of the authors.

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burden of envenoming. The estimated global toll from 

snakebite remains a mixture of some quality evidence 

and projected speculation. Recent estimates suggest 

more than 2.5 million cases per year, with more than 

1 million of these resulting in significant morbidity, 

approximately 400,000 cases requiring amputations, 

and more than 100,000 fatalities.16 The economic impact 

likely is correspondingly enormous, but it has yet to 

be reliably quantified. Despite this impact, snakebite 

has generally been relegated to minor status in medical planning. The World Health Organization (WHO) 

briefly classified snakebite as one of three globally 

important “other neglected conditions” included under the recognized grouping of “neglected tropical 

diseases” that contains 17 infectious diseases responsible for a large proportion of morbidity and mortality 

in rural Third World regions.17 However, in 2015, the 

WHO removed it from the list, and thus the considerable global impact of snakebite (especially among the 

world’s most medically underserved communities) is 

no longer recognized (see http://www.who.int/gho/

neglected_diseases/en/).

Scorpions, Spiders, and Other Arachnids

Scorpion stings are second, after snakebite in regards to medically significant occurrences, and probably affect more than 1 million humans each year, but 

with a low fatality rate (see Chippaux and Goyffon, 

2010). 

Spiderbite is also common, but with a few notable 

exceptions (widow spiders, Latrodectus spp, family 

Theridiidae [Figure 18-2]; recluse spiders, Loxosceles 

spp, family Sicariidae [Figure 18-3]; banana spiders, 

Phoneutria spp, family Ctenidae; Australian funnelweb spiders, Atrax spp [Figure 18-4]; and Hadronyche

spp, family Hexathelidae), these are most commonly 

of minor medical significance (see www.toxinology.

com). Tick envenoming causing paralysis is a problem 

in Australia, North America, and southern Africa, 

and possibly elsewhere, but reported cases are few, 

although rare fatalities have occurred (see Meier and 

White, 1995). The ticks involved are often members of 

Figure 18-2. A medically important widow spider (Latrodectus spp) and comparison of the vertically deployed venom delivery 

apparatus of a mygalomorph spider and the horizontally deployed venom delivery apparatus of an araenomorph spider. 

(A) Red back widow spider (Latrodectus hasselti, Theridiidae). (B) Fangs of the mygalomorph, Aganippe subtristis (four-spotted 

trapdoor spider, female specimen, family Idiopidae). Note the vertical direction of the fang-bearing chelicerae traditionally 

termed paraxial, with the spider in an adopted defensive posture. (C) Fangs of the mygalomorph, Aganippe subtristis. The 

figure again illustrates the vertical direction of the fang-bearing chelicerae. (D) Fangs of the araenomorph, Pediana spp (a 

taxon of huntsman spider, family Sparassidae). The figure illustrates the horizontal deployment of the chelicerae-bearing 

fangs traditionally termed diaxial.

Photographs: Copyright © Julian White. In: White J. A Clinician’s Guide to Australian Venomous Bites and Stings. Melbourne, 

Australia: Commonwealth Serum Laboratories; 2013: 300+ pp.

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the family Ixodidae (hard-bodied ticks), particularly 

including members of the genera Ixodes, Amblyomma, 

Dermacentor, and the less common family Argasidae, 

notably the genus Argas (soft-bodied ticks, which so 

far have caused paralysis in animals only, not humans). 

Insects

Insect sting envenoming causing toxin-induced 

disease of medical significance is uncommon, but 

allergic reactions to hymenopteran insect stings (eg, 

anaphylaxis from ants, bees, wasps, and hornets) are 

common and sometimes fatal. Retrospective studies 

have suggested that typically 40 to 100 deaths occur 

each year from hymenopteran sting anaphylaxis in the 

United States.18 This amount is a significantly higher 

annual patient fatality rate than that of snakebite 

envenoming in the United States (typically between 

5 and 8 patients). 

Some bee venoms (eg, bumblebee, Megabombus 

pennsylvanicus; honey bee, Apis mellifera; Figure 18-5) 

contain mast cell degranulating peptide, a 22-amino 

acid cationic peptide that can directly trigger release of 

proanaphylactic mediators without prior sensitization. 

One unusual example of a medically important 

insect venom is the Lepidopteran larvae (caterpillar) 

of the giant silkworm moth, Lonomia obliqua (family 

Saturniidae; Figure 18-6), whose spines contain several 

direct and indirect prothrombin activators, as well 

as several other toxins.19 The sting of this caterpillar 

can cause a hemorrhagic diathesis, and fatalities have 

been documented.34 Other Lepidopteran larvae have 

been implicated in human disease, at least some of 

which may involve local envenoming. Other terrestrial venomous animals cause few cases of human 

disease.

Marine Envenoming: Sea Snakes, Cnidarians, and 

Venomous Fish

Marine envenoming, such as jellyfish stings, are 

common, but few are medically significant. Significant types of marine envenoming include box 

jellyfish (eg, Chironex fleckeri, family Chirodropidae) 

stings (sometimes lethal); Irukandji jellyfish stings 

(resulting in a syndrome caused by several taxa of 

cnidarians, rarely lethal; see below under Excitatory Neurotoxins); and blue bottle (Physalia spp, 

family Physaliidae) stings (nonlethal envenoming, 

but occasional cases of potentially lethal allergic 

reactions). Sea snake (family Elapidae) bites can 

cause lethal envenoming, but are increasingly 

uncommon because of changes in fishing methods 

(eg, decreased manual removal of snakes from 

purse nets). Painful stings from venomous fish from 

several different families (eg, Scorpaenidae, Trachinidae, and Tetrarogidae), including many popular 

food and aquarium fishes as well as marine and 

fresh or brackish water stingrays, are common but 

generally unlikely to be lethal. 

Some 200 species of stingrays, which belong to 

seven of nine families, can deliver venomous stings, 

or more accurately termed, penetrative envenoming. 

The most medically important stingrays belong to the 

following families: 

Figure 18-3. Brown recluse spider (Loxosceles reclusa). One of 

two genera (Sicarius and Loxosceles) belonging to the family 

Sicaridae, there are approximately 113 recognized taxa of 

Loxosceles. Several of these, including L reclusa, have inflicted 

medically significant bites that may occasionally cause a 

recurrent ulcer. In some parts of South America, several 

species may cause systemic envenoming (viscerocutaneous 

loxoscelism), an uncommon but potentially life threatening venom disease. There is antivenom available in several 

Latin American countries (eg, Brazil, Chile, Peru, Mexico), 

but treatment in the United States remains somewhat controversial with no quality evidence supporting the previous 

management (eg, surgical debridement, etc) of verified L 

reclusa bites. Meticulous wound management and possibly 

bariatric oxygen treatments are the most appropriate management methods. Recluse spider bites are among the most 

misdiagnosed presentations to emergency departments 

and outpatient/urgent care facilities, and diagnosis must be 

founded on verified identification of a spider or, in lieu of a 

specimen, with a well-supported history of a bite occurring 

within the range of recluse spiders (many suspected bites 

have occurred well outside any natural range or region in 

which the species has been accidentally introduced).

Photograph: Copyright © Julian White. In: White J. Venomous animals: clinical toxinology. EXS. 2010;100:233–291. In:

Luch A (ed). Molecular, Clinical and Environmental Toxicology. 

Vol 2: Clinical Toxicology. Basel, Switzerland: Birkhäuser; 

2010. 

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Toxins From Venoms and Poisons

• Urolophidae (stingarees), 

• Dasyatidae (whiptail stingrays), 

• Hexatrygonidae (sixgill stingrays),

• Potamotrygonidae (river stingrays), and 

• Plesiobatidae (giant stingrays). 

Stingrays, which are cartilaginous relatives of sharks (all 

in the class Elasmobranchii) and do not possess venom 

glands, deliver their stings using a serrated spine that is 

covered with mucosal secretions and venom-secreting 

cells (Figure 18-7A, B). The cells release the venom into 

the wound produced by the spine penetration (penetrative envenoming). The venom contains cytotoxic and 

vasculotoxic (including probably cardiotoxic) properties, and secondary infection from the wounds is common. Laceration of the lower extremities (especially the 

foot and ankle) is common (Figure 18-7C) with later 

clinical evolution of edema, cellulitis, and occasionally

necrosis. Rare deep penetrative envenoming from giant 

species such as the Australian smooth stingray (Dasyatis 

brevicauda, family Dasyatidae) can be fatal if the thoracic 

cavity is pierced. In these uncommon cases, fatal effects usually result from physical trauma rather than 

envenoming, although, as mentioned previously, some 

experimental data have demonstrated cardiotoxicity of 

some stingray venoms (see Mebs, 2002 and Meier and 

White, 1995).34,35 The well-known television personality Steve Irwin succumbed rapidly to the intracardiac 

Figure 18-4. Sydney funnel-web spider (Atrax robusta). 

Approximately 13 fatalities have resulted from A robusta

envenoming that clinically presents as a catecholamine storm 

produced by potent neuroexcitatory venom toxins. 

Data source: White J. A Clinician’s Guide to Australian Venomous Bites and Stings. Melbourne, Australia: Commonwealth 

Serum Laboratories; 2013.

Photograph: Copyright © Julian White. In: White J. A 

Clinician’s Guide to Australian Venomous Bites and Stings. 

Melbourne, Australia: Commonwealth Serum Laboratories; 

2013: 300+ pp.

Figure 18-5. Honeybee (Apis mellifera). Stings from hymenopterans (especially bees and wasps) can cause lifethreatening anaphylaxis in susceptible individuals. There 

are significantly more annual fatalities in the United States 

from hymenopteran sting-induced anaphylaxis than from 

snakebite envenoming. 

Data sources: (1) Weinstein SA, Dart RC, Staples A, White J. 

Envenomations: an overview of clinical toxinology for the 

primary care physician. Am Fam Physician. 2009;80:793–802. 

(2) Weinstein SA, Warrell DA, White J, Keyler DE. “Venomous” Bites from Non-venomous Snakes. A Critical Analysis of 

Risk and Management of “Colubrid” Snake Bites. 1st ed. New 

York, NY: Elsevier; 2011.

Photograph: Copyright © Julian White. In: White J. A 

Clinician’s Guide to Australian Venomous Bites and Stings. 

Melbourne, Australia: Commonwealth Serum Laboratories; 

2013: 300+ pp. 

Figure 18-6. Larvae of the giant silkworm moth (Lonomia 

obliqua). The larvae of this moth can inflict a life-threatening 

envenoming that features coagulopathy.

Photograph: Centro de Informações Toxicológicas de Santa 

Catarina, Brazil. Wikipedia Commons, public domain. http://

www.cit.sc.gov.br.

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penetration from a giant Australian stingray, and his 

death most likely resulted from cardiac tamponade, 

not envenoming. 

Poisoning by Animals, Plants, and Mushrooms

Poisonings from ingestion of poisonous animals, 

especially marine animals, are common. These animals 

include the fugu puffer fish (eg, Takifugu spp, family 

Tetraodontidae); most species of fish that belong to the 

order Tetraodontiformes, which contain tetrodotoxin; 

ciguatoxic fish (numerous species, including many 

colorful diverse reef species and apex predators such 

as the great barracuda, Sphyraena barracuda); and several types of shellfish. In some Pacific island populations, ciguatera poisoning affects up to 1 in 5 people 

each year, and from 1973 to 2008 an estimated 500,000 

people were affected by ciguatera poisoning.20 Some 

types of marine poisoning carry a significant fatality 

rate, particularly the ingestion of sushi made with 

fugu (especially when including visceral organ meats), 

which equates with tetrodotoxin poisoning (see Mebs, 

2002 and Meier and White, 1995).34,35 Some types of 

shellfish poisoning also have a substantial risk of death 

(see Mebs, 2002).34

Poisoning from ingestion of poisonous plants and 

mushrooms, which is similarly common, is particularly 

frequent or important in some regions, notably parts 

of the tropics (poisonous plants) and in Europe and 

parts of North America (poisonous mushrooms). It 

can occur as a consequence of accidental circumstances 

(misidentification of the plant/mushroom) or as a deliberate act (eg, use of oleander [eg, Thevetia peruviana, 

family Apocynaceae] ingestion in suicides in the Indian 

subcontinent).21,22 Some types of plant or mushroom 

poisoning carry a relatively high fatality rate, especially in delayed or late presentations; however, this 

may reflect regional trends because in some Western 

countries (eg, the United States), the case fatality rate 

for mushroom poisoning remains low.23

Figure 18-7. South American freshwater or river stingray (Potamotrygon motoro), venom apparatus and stingray penetrative 

envenoming. (A) Ocellate or peacock-eye river stingray (Potamotrygon motoro). This increasingly rare species is popular among 

home aquarists. It is capable of inflicting a sting that can cause moderate to severe local effects, including severe pain, and 

systemic effects including shock. (B) Stingray tail spine. Stingrays do not possess a venom gland; rather, their serrated tail 

spines have venom-containing cells that release their contents when physically disrupted. The spine also is coated with a 

mucous layer that may be colonized with several taxa of marine microorganisms and can predispose to serious local infection in an envenomed victim. (C) Stingray spine (species unidentified)-inflicted wound on foot. Wounds may result from 

directly stepping on a ray covered with sand, or from a glancing, slash-like wound. These wounds often require medical 

imaging in order to determine if any spine fragments remain imbedded in the wound, as well as meticulous wound care 

and prophylactic antibiotics.

Photographs: Copyright © Julian White. In: White J. A Clinician’s Guide to Australian Venomous Bites and Stings. Melbourne, 

Australia: Commonwealth Serum Laboratories; 2013: 300+ pp.

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Toxins From Venoms and Poisons

Relevance to Biological Warfare

Venomous animals generally evolved to target prey 

or predators on an individual basis—not en masse—so 

they are not readily adapted to act as ideal weapons 

in human warfare. However, either using the native 

toxins or modifying those toxins to enhance a particular action and developing an artificial weaponization 

and delivery strategy is possible, but presents logistical 

challenges that would likely outweigh practicality in 

most cases.

Venoms contain some highly potent agents that 

can kill humans in small doses. In most cases the 

lethal outcome will not be instantaneous, but likely 

prolonged over several hours. Venoms are not ideal 

overall for biological warfare because of these delivery 

problems as well as the absorption/direct administration required in most examples to optimize their actions. Even for potent neurotoxins, such as paralytic or 

neuroexcitatory toxins, other equally or more potent 

chemicals are available that allow mass delivery. Also, 

most venom-derived toxins are susceptible to thermolability and varying degrees of denaturation through 

other environmental influences that can affect their potency. Some of them also exhibit nonmammalian prey 

target specificity, for example, some antagonize the 

acetylcholine receptors of lizards and birds. Thermostable toxins are found in a few venoms such as those 

of the venomous helodermatid lizards (Heloderma spp; 

see below) and the unusual peptide neurotoxins from 

temple or Wagler’s pit viper (Tropidolaemus wagleri) 

venom.24,25 Some of the specific factors influencing the 

use of biological toxins in biowarfare and preparedness against such a threat have been discussed by 

Osterbauer and Dobbs.26

Research into specific toxins, including their molecular modification, and possible xenogenic incorporation of toxin-encoding genes into potentially infectious 

microorganisms may allow substances to be developed 

with biological warfare potential. Several countries 

have explored the potential uses for such recombinant 

products. The ethics and realities of such research are 

beyond the scope of this chapter.

Similarly, toxins from poisonous animals, plants, 

and fungi may—in general—be unattractive as 

biological warfare agents, although ricin (from 

the castor bean plant, Ricinus communis [family 

Euphorbiaceae]) and the aflatoxins (from the molds, 

Aspergillus spp, family Trichocomaceae) are exceptions. Specifically excluded from this discussion is 

the casual/accidental interaction between combatants and venomous fauna on the battlefield or in 

otherwise deployed locations. 

However, the risk posed by accidental envenoming, especially snakebite and scorpion sting, should 

not be overlooked in developing risk-mitigation strategies for any potential combat zone. Some authors 

have described the impact that venomous animals 

may have on field troops. Maretic27 reported several 

accounts of mass envenoming of large numbers of 

troops by widow spiders, Latrodectus spp. These 

troops include the troops of Ludwig in Calabria in 866 

ce who were “decimated” by spiders. Also, during the 

eve of the Battle of Loncomilla that occurred during 

the Chilean Revolution on the 8th of December 1851, 

soldiers bitten by Latrodectus spp were chloroformed 

so as not to “betray with their screams” the position 

of the army.27

However, the general concerns about risks posed 

by venomous animals to modern troops deployed in 

locales with several medically important venomous 

species have appeared to be disproportionate to the 

small number that are seriously or fatally envenomed. 

For example, Ellis28 reported only three recorded 

snakebite-related deaths among British troops during 

World War II, and Minton and Minton29 reported only 

one well-documented fatal snakebite inflicted on an 

American soldier during the Vietnam War. It is likely 

that such cases were underreported and the actual 

numbers of those less seriously envenomed are unknown. Although few figures account for envenomings 

among coalition troops in Operations Desert Shield 

or Desert Storm, the Persian Gulf War, or Operation 

Enduring Freedom in Afghanistan, there are a handful 

of documented cases. Two enlisted American military 

service personnel were among 17 snakebite victims 

treated at three US medical facilities in Afghanistan. 

Most of the patients in this series were local Afghans, 

and the identity of the envenoming snake species was 

unknown in 11 of 17 cases (65%).30 There were no fatalities, and 10 of 17 patients (58%) received antivenom.30

However, in some circumstances, natural disasters may share some features with the effects of 

warfare on civilian populations. Envenoming can 

become a significant risk in certain disasters. The 

effects of massive flooding in Bangladesh are well 

studied because it is a frequent disaster event. Although drowning is the single most common reason 

for fatalities in floods by a substantial margin, snakebite is the second most common reason for fatalities 

and causes as many or more deaths than all other 

causes (except drowning) combined.31 The diagnosis, 

first aid, medical treatment, and prevention of such 

accidental envenoming are major subjects beyond 

the scope of this chapter, although some basic recommendations are presented.

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Medical Aspects of Biological Warfare 

MAJOR TOXIN CLASSES AND THEIR CLINICAL EFFECTS

18-9) allows safe subjugation and consumption of 

prey (eg, respectively, crabs, mammals) that otherwise 

might injure the octopus/snake. 

For poisonous animals, the effects of neurotoxins 

may either permanently deter potential predators or 

allow escape by the poisonous animal. The theoretical 

evolutionary value for poisonous plants and mushrooms is less clear, although it can be speculated that 

excessive foraging of these species may have occurred 

and endangered their survival. Perhaps these plants 

produce noxious and toxic substances to discourage 

their consumption. Also, these toxins may serve (or 

have served) other functions important to the physiological functions of the plant that are unrecognized. 

The costs of evolving such a biosynthetic capability 

may be a factor contributing to the less common occurrence of such toxins in these taxa.

The mechanism that causes paralysis is variable, 

but in most cases it involves direct toxin activity 

in the peripheral nervous system, rather than a 

central effect. The molecular variability in these 

toxins is considerable, but within particular venomous animal groups tends to be more homogenous 

and conserved. The best described examples are 

snake venom neurotoxins, many of which have 

been duplicated among different ophidian clades 

(Table 18-2).32–35

There are essentially four principal snake paralytic 

neurotoxin types of clinical significance (see Table 

18-2). Most are polypeptides, some have multimeric 

structures that include one or more basic phospholipases A2 (PLA2) subunits, and a few unusual neurotoxins are small peptides. Within each type, some 

molecular variability exists, particularly among presynaptic neurotoxins, which are generally the most 

potent though slightly slower acting paralytic toxins. 

Postsynaptic neurotoxins generally fall within one 

of two classes that are commonly termed short and 

long chain, but these are now collectively classed as 

“three-finger-fold” toxins in reference to the three 

β-stranded loops extending from their central core 

that contain all four conserved disulfide bonds. These 

toxins most commonly have molecular masses ranging from 6 to 8 kDa (see Table 18-2). The other wellcharacterized toxins such as the fasciculins (a group 

of anticholinesterases that have the three-finger fold 

conformation) and dendrotoxins (neuronal voltagegated potassium channel inhibitors; see Table 18-2) 

from mamba venoms act differently from each other, 

but are synergistic. All act at the neuromuscular junction (Figure 18-10).32–35 The cited references offer more 

detailed reviews of these essential toxin classes. 

Figure 18-8. Textile cone snail (Conus textilis). These gastropods are highly coveted by amateur conchologists for their 

beautiful shells. There are rare human deaths from stings 

delivered by these snails when they are handled. A modified 

tooth, the radula that closely resembles a miniature harpoon, 

delivers the venom into prey or an unfortunate human 

victim. This adaptation allows this slow-moving snail to 

capture fast moving fish. Various Conus species (>650 currently recognized) often favor specific prey such as fish or 

other gastropods (including other Conus spp). 

Photograph: By Jan Delsing. Wikipedia Commons, public 

domain. https://commons.wikimedia.org/wiki/File:Conus_

textile_010.jpg.

There are many ways of classifying toxins, including by taxonomic origin, chemical structure, molecular 

targets, and biological activity. For this chapter, a 

pathophysiologically based scheme is most relevant 

in considering the primary actions of venom toxins 

and possible clinical presentations that may occur as 

a result of their action.

Paralytic Neurotoxins

For venomous animals, paralysis is a biologically 

useful state to induce in either prey or predator. For 

some arthropods, paralyzing prey allows them to 

both overcome larger prey and provide a food store 

for leisurely later feeding, or for their offspring to 

feed on during their larval stage. For cone snails 

(Conus spp, family Conidae; Figure 18-8), the use of 

paralytic neurotoxic peptides delivered by ejection 

of a harpoon-like modified radula tooth allows this 

slow moving predator to capture and ingest fast moving prey (fish). For most cephalopods (eg, octopuses) 

and some squamate reptiles (eg, venomous snakes), 

the use of neurotoxins delivered respectively by beak 

or canaliculated fang (containing an internal lumen)/

externally grooved modified maxillary teeth (Figure

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Toxins From Venoms and Poisons

Other neurotoxins, such as tetrodotoxin and saxitoxin, are nonproteinaceous and low molecular mass 

(often between 300 and 1,000 Da) toxins (the former is 

a guanidinium class toxin, the latter a polyether), and 

thus have different structures as well as mechanisms 

of action. These are mostly ion channel toxins that 

are very potent with notably low minimal concentrations required to affect biological activity.36,37 Small 

amounts ingested (eg, through eating fugu fish [tetrodotoxin] or contaminated shellfish [eg, saxitoxins, 

yessotoxin]) can result in rapid complete paralysis, 

but the paralysis is usually of shorter duration, in 

comparison with snake venom neurotoxins.33,38–40

Figure 18-9. Representative venom delivery apparatuses 

found among some snakes. (A) Distensible fangs of a 

representative viperid, Western diamondback rattlesnake 

(Crotalus atrox). This dentitional arrangement has often been 

termed solenoglyphous. Elapids, viperids, and front-fanged 

lamprophiid snakes are collectively termed front-fanged 

colubroids. (B) Fixed fangs of a representative elapid, Eastern 

brown snake (Pseudonaja textilis). This dentitional arrangement has often been termed proteroglyphous. (C) The posterior grooved maxillary teeth of a non–front-fanged colubroid, 

the Mangrove or ringed cat eye snake (Boiga dendrophila). The 

maxillary is placed upside down to better illustrate the characteristics of the teeth. The deep external grooves conduct 

the venom from the low-pressure glands associated with 

the delivery apparatus. This dentitional arrangement that 

can include mid- or notably posterior maxillary teeth that 

may be enlarged and may also be grooved has been termed 

opisthoglyphous, or rear fanged with aglyphous referring 

to those non–front-fanged colubroids that have mid or posterior teeth that lack grooves and in some instances are also 

associated with a low pressure gland. These terms are not 

precisely accurate because the modified dentition may occur 

midway in the maxillary. 

Photographs: (A) Copyright © Julian White. In: Brent J, 

Wallace KL, Burkhart KK, et al (eds). Critical Care Toxicology: 

Diagnosis and Management of the Critically Poisoned Patient. 

1st ed. Philadelphia, PA: Mosby; 2005. (B) Copyright ©

Julian White. In: Covacebich J, Davie P, Pearn J (eds). Toxic 

Plants and Animals: A Guide for Australia. Brisbane, Australia: 

The Queensland Museum; 1987. (C) Copyright © Scott A 

Weinstein. In: Weinstein SA, Warrell DA, White J, Keyler 

DK. “Venomous” Bites from Non-Venomous Snakes: A Critical 

Analysis of Risk and Management of “Colubrid” Snake Bites. 

New York: Elsevier; 2011: 141. 

The biothreat potential of tetrodotoxin has been long 

recognized. It is included on the US Department of 

Health and Human Services’ regulated select agent 

list and considered an agent “determined to have the 

potential to pose a severe threat to both human and 

animal health.”41,42

Clinically, these paralytic neurotoxins cause grossly 

similar presentations, with progressive development of 

flaccid paralysis. In most cases a descending paralysis 

affecting cranial nerves occurs first (however, envenoming by paralytic tick species causes an ascending 

paralysis commencing with ataxia).40,43–45 In a classic 

presentation after snakebite, the patient develops bilateral ptosis after one to several hours, which may progress to complete ophthalmoplegia and fixed dilated 

pupils if untreated, although even timely treatment 

does not always prevent this progression. Dysarthria, 

dysphagia, drooling, and loss of upper airway protection may occur, followed by limb muscle weakness, 

loss of deep tendon reflexes, diaphragmatic paralysis, 

and complete respiratory paralysis. Without intubation 

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TABLE 18-2 SOME PRINCIPAL PARALYTIC NEUROTOXIN TYPES Toxin Class Structure Site of Action Mode of Action Source 

Presynaptic neu- rotoxins PLA2-based, mono- or multi- meric NMJ Bind to surface membrane of terminal axon, modify SNAP proteins, enter the axon via synaptosomes, and then dam- age mitochondria and other cell structures, thus disrupting syn- aptosome production, thereby 

causing complete paralysis

Many elapid snakes (Australia

elapids, coral snakes, kraitsa few vipers (South Amercan rattlesnakes, a few NortAmerican rattlesnakes, som“old world” viperids, such aRussell’s vipers)

Postsynaptic 

neurotoxins

Polypeptide, with 

variable number of disulfide bonds; variably termed, long-chain, shortchain, or threefinger-fold neurotoxins

NMJ Bind to acetylcholine receptor 

on muscle end plate and cause reversible or irreversible block preventing activation of receptor

Many elapid snakes (Australia

elapids, sea snakes, coral snakecobras, kraits) and some nonfront-fanged colubroid snake(NFFCs); studied NFFC toxinhave so far been largely preyspecific

Dendrotoxins Polypeptides that 

are structurally homologous with Kunitz-type proteinase inhibitors

NMJ Cause massive release of acetylcholine from terminal axons 

through activation of potassium channels, flooding the junctional space, and receptors

African mambas (Dendroaspis spp

Fasciculins Three-finger-fold 

polypeptides 

NMJ Prevent regulated removal of 

acetylcholine from the junctional space, thereby overstimulating and inactivating receptors causing muscle fasciculation

African mambas (Dendroaspis spp

Tetrodotoxins Steroidal alkaloid 

(Guanidinium class)

Na+ channels 

in excitable nerve and muscle cells

Causes blockade of the voltagegated sodium channels (NaV) 

in nerve and muscle cell membranes by binding to site 1 of the NaV α-subunit, thus blocking 

ion conduction and preventing the cells from activation, as well as inhibiting release of neurotransmitter

Blue-ringed octopus, puffer (fugu

fish, selected newts, toads, flaworms, and a diverse series other animals; the toxin with thbroadest phylogenetic distribution; probably produced in somspecies by symbiotic bacteria (epresent in the beak-associatevenom/salivary glands of bluringed octopuses and relatives

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427

Batrachotoxins, 

homobatrachotoxins

Steroidal alkaloid 

with oxazepine ring

Axolemma Binds to site 2 of the NaV 

α-subunit, thereby increasing the permeability of the voltagedependent sodium channel by prolonging the open state; this causes persistent activation and shifted voltage dependence; the toxin has approximately 10- to 12-fold greater experimental lethal potency than tetrodotoxin 

Poison dart frogs (Dendrobatidae

pitohui birds (PNG); the dendrbatid frogs obtain these toxinfrom insect food sources (ecoleopteran, hymenoptera, anothers), and the toxin becomeabsent in specimens maintaineon nonindigenous insects icaptivity

Saxitoxins, 

gonyautoxins; others

Purine derivatives (polyethers: 

the molecular structures classify into groups based on potency [most potent to least]; carbamates, decarbameyl, N-sulfacarbamyl, hydroxybenzoate)

Excitable cell 

membranes

Bind adjacent to the sodium channel, blocking the channel and 

preventing action potentials; like tetrodotoxin, saxitoxin exerts its activity by binding to site 1 of the NaV 

α-subunit

Selected shellfish (paralytic shel

fish poisoning); toxins are prduced by a wide variety of dinflagellates and bioconcentratein filter-feeding shellfish; somtoxins may be produced in cynobacteria and algal spp

Holocyclotoxins 

(HT-1)

Probably several isotoxins (HT-1, HT-2, 

HT-3, and possibly others); HT-1 is a basic polypeptide with a calculated molecular mass of 5.9 kDa

NMJ Similar to snake venom presynaptic neurotoxins Paralysis ticks in Australia, Nort

America, and Southern Africa 

Conopeptides Broad array of 

peptides

A variety of mechanisms, depending on toxin; as an example, the 

μ-conotoxins bind to site 1 of the NaV 

α-subunit

Selected Conus spp cone snail

an indeterminate number othe >650 species produce thestoxins; of those tested, many pr

duce toxins that are prey-speciffor either fish or invertebrateincluding other molluscs; only handful produce toxins that amedically significant in human

NMJ: neuromuscular junction; PLA2: phospholipase A2; PNG: Papua New Guinea; SNAP: synaptosomal-associated proteinData sources: (1) Weinstein SA, Warrell DA, White J, Keyler DE. ‘Venomous’ Bites from Non-venomous Snakes. A Critical Analysis of Risk and MaBites. 1st ed. New York, NY: Elsevier; 2011. (2) Mebs D. Venomous and Poisonous Animals: A Handbook for Biologists, Toxicologists and ToxinologistFL: CRC Press; 2002: 360. (3) Meier J, White J. (eds). Handbook of Clinical Toxicology of Animal Venoms and Poisons. Boca Raton, FL: CRC Press; 199tion materials (eg, lectures) of the authors.Table 18-2 continued

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Medical Aspects of Biological Warfare 

Figure 18-10. Sites of action of principal types of snake venom neurotoxins. Simplified overview of the general actions of 

phospholipase A2-multimeric presynaptic neurotoxins and postsynaptic neurotoxins. The numbered steps included in the 

figure indicate the following: (1) shows the initiation of translation of the electrical impulse into a biochemical release; (2) 

synaptic vesicles fuse with the axonal membrane thereby leading to exocytic discharge of acetylcholine (ACh); (3) discharged 

ACh enters the synaptic cleft; (4) the discharged ACh binds to motor end-plate receptors of the myocyte membrane leading 

to stimulation of contraction; and (5) the ACh at the motor end plate is then hydrolyzed by acetylcholinesterases, thus terminating the contractile stimulus. Note the site of respective site of actions of the presynaptic and postsynaptic neurotoxins. 

The investigation of presynaptic neurotoxin pharmacology has largely focused on the actions of several potent snake venom 

neurotoxins isolated from Australian or Asian elapids, as well as a few viperids (several species of rattlesnakes and viperine 

viperids). Some of the well-studied toxins—such as taipoxin, β-bungarotoxin, and crotoxin—ultimately inhibit ACh release 

at the neuromuscular junction, and in nerve-muscle preparations accomplish this in three phases: (1) an initial transient 

decrease/inhibition of evoked transmitter release that is promoted by Ca2+ (this phase has been absent in studies of some 

toxins, eg, notexin), (2) a facilitated transmitter release phase, and (3) the final phase that features a progressive fall in evoked 

(Figure 18-10 continues)

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Toxins From Venoms and Poisons

and mechanical ventilation, this condition is fatal. It 

may take many hours to reach this final, potentially 

fatal stage. With external respiratory support, most 

affected patients can be expected to survive.

If the paralysis is caused by presynaptic snake 

venom neurotoxins, then complete paralysis may 

extend to days and weeks (but rarely months) until 

the damaged terminal axons at the neuromuscular 

junction regenerate. Antivenom cannot reverse this 

process and is only effective if given early enough to 

neutralize the neurotoxins before they bind and enter 

the terminal axon. 

If the paralysis is caused by only postsynaptic 

snake neurotoxins, then the blockade may sometimes 

be reversed by giving antivenom, or it can be moderated by increasing the supply of neurotransmitter 

release resulting in transmission failure and paralysis. Miniature end-plate potential frequency is similarly affected, although 

spontaneous release tends to occur at a low frequency after the failure of evoked neuromuscular transmission. However, 

spontaneous release amplitude does not change significantly, suggesting that synaptic vesicles do not fuse inside the axonal 

terminal and that the ACh-synaptosomal packaging mechanism is not impaired by the action of the studied species of these 

toxins. The phospholipase A2 subunit(s) in some of these toxins (eg, Mojave toxin) also have a degenerative effect on the motor 

end plate. There is still controversy about the specific enzymatic influence of phospholipase A2 subunits on the neurotoxicity 

and the initial phospholipid hydrolysis role in initiating the three-phase mechanism that ultimately results in paralysis, but 

the lysophospholipids produced by hydrolysis do alter the active zones of neuroexocytosis thereby making them less prone 

to membrane fusion with synaptic vesicles. Unlike the actions of several botulinum toxins (eg, botulinum toxins A and E) 

from Clostridium botulinum and tetanus toxin from C tetani that function as endopeptidases by cleaving integral proteins 

(eg, SNAP-25; other botulinum toxins cleave VAMP or SNAP-25 and syntaxin) of the presynaptic membrane, snake venom 

phospholipase A2-containing neurotoxins do not directly hydrolyze these SNARE proteins. Although other presynaptically 

acting neurotoxins, such as α-latrotoxin from the widow spider (Latrodectus spp) venoms function with a mechanism different from that of snake venom phospholipase A2 toxins, these different toxins still alter the axolemmal permeability and 

cause Ca2+ overload within the terminals and subsequent neuronal degeneration, a process that likely causes activation of the 

calcium-activated proteolytic activities of calpains. The increased Ca2+ permeability induced by α-latrotoxin occurs via toxin 

binding to its receptor(s) (eg, latrophilin) subsequently forming pores in the presynaptic membrane. The terminal is thus 

essentially flooded with Ca2+ and vesicle fusion is over-stimulated resulting in a massive release of neurotransmitter. Most 

snake venom postsynaptic neurotoxins bind to subunit interfaces in either muscle type (α-1) or neuronal type (α-7) nicotinic 

Ach receptors (AChR), thereby antagonizing neurotransmitter binding with resultant paralysis. The length of their primary 

sequence, long-chain and short-chain, has been commonly used to classify these toxins but recently all of these toxins have 

been renamed as three-finger-fold neurotoxins. The concave aspect of the three-dimensional structure of these toxins contains 

several amino acids that function as active sites for binding to respective AChR subunits. These toxins are widespread among 

elapid venoms and some are found in venoms of other snakes such as some colubrids and lamprophiids, as well as a few 

viperids (eg, Daboia russelii). Snake venom neurotoxicity should not be viewed as a result of the isolated action of discrete 

toxins such as those outlined above. For example, fasciculins and dendrotoxins from mamba (Dendroaspis spp) venoms can 

facilitate the subjugation of prey animals, as well as compound the clinical manifestations in envenomed patients. Fasciculins 

are reversible, selective acetylcholinesterase antagonists that cause an accumulation of acetylcholine at the neuromuscular 

junction thereby causing marked and prolonged fasciculations. Pita et al considered the potential use of fasciculins, as well 

as other anticholinesterase toxins, as biological warfare agents. Originally isolated from venom of the Eastern green mamba 

(D angusticeps), dendrotoxin is a 7-kDa polypeptide homologue of Kunitz-type serine protease inhibitors. However, unlike 

mammalian Kunitz-type inhibitors, the dendrotoxins are potent, selective blockers of voltage-dependent potassium channels. These toxins induce repetitive and sustained terminal neuronal firing, and have increasingly been used as molecular 

tools in ion channel neuropharmacology. 

ChEsterase: cholinesterase; SNARE: soluble N-ethylmaleimide-sensitive factor activating protein receptor; VAMP: vesicleassociated membrane protein

Data sources: (1) Pungerčar J, Križaj I. Understanding the molecular mechanism underlying the presynaptic toxicity of secreted phospholipases A2. Toxicon. 2007;50:871–892. (2) Rossetto O, Montecucco C. Presynaptic neurotoxins with enzymatic 

activities. Handb Exp Pharmacol. 2008;184:129–170. (3) Duregotti E, Tedesco E, Montecucco C, Rigoni M. Calpains participate 

in nerve terminal degeneration induced by spider and snake presynaptic neurotoxins. Toxicon. 2013;64:20–28. (4) Davletov 

B, Ferrari E, Ushkaryov Y. Presynaptic neurotoxins: an expanding array of natural and modified molecules. Cell Calcium. 

2012;52:234–240. (5) Teixeira-Clerc F, Ménez A, Kessler P. How do short neurotoxins bind to a muscular-type nicotinic acetylcholine receptor? J Biol Chem. 2002;277:25741–25747. (6) Harvey AL, Robertson B. Dendrotoxins: structure–activity relationships 

and effects on potassium ion channels. Curr Med Chem. 2004;11:3065–3072. (7) Pita R, Anadón A, Martínez-Larrañaga MR. 

Neurotoxins with anticholinesterase activity and their possible use as warfare agents. Med Clin (Barcelona). 2003;121:511–517.

Illustration: Copyright © Julian White. In: White J. Venomous animals: clinical toxinology. EXS. 2010;100:233–291. In: Luch 

A (ed). Molecular, Clinical and Environmental Toxicology. Vol 2: Clinical Toxicology. Basel, Switzerland: Birkhäuser; 2010. 

Figure 18-10 continued

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Medical Aspects of Biological Warfare 

(acetylcholine) from the terminal axons. This can 

sometimes be accomplished by administering anticholinesterases (usually intravenous neostigmine) to 

partially overcome the receptor blockade.

For poisoning from the nonprotein neurotoxins such 

as tetrodotoxin, the patient is most often wholly reliant on mechanical ventilation that constitutes the only 

treatment during major paralysis. However, complete 

paralysis and the concomitant artificial ventilation may 

often last only a few hours. These toxins act rapidly 

and can cause complete respiratory paralysis within 

20 to 60 minutes of toxin exposure; thus the onset of 

symptoms and signs is precipitous.

Excitatory Neurotoxins

Excitatory neurotoxins cause nonparalytic stimulation of the nervous system and may exert their effect 

on many or all parts of the peripheral nervous system, 

including the autonomic nervous system. These are the 

classic arthropod toxins found in selected scorpion and 

spider venoms. They also occur in some other venoms, 

such as Irukandji jellyfish venoms. 

Most are potent and highly selective ion channel 

toxins, variously affecting sodium, potassium, and 

calcium channels and either activating or blocking 

these channels. For example, the δ-atracotoxins from 

Atrax robusta (Sydney funnel-web spider) venom interact with a specific voltage sensor transmembrane 

segment (S4) of α-subunit domain IV. The interaction 

of the δ-atracotoxins with S4 prevents the normal outward ionic movement, and associated conformational 

changes that are required for channel inactivation. 

This results in prolonged action potentials at autonomic or somatic synapses, which induces massive 

transmitter release.46 Many excitatory toxins have 

become vital tools in unlocking the secrets of nerve 

signaling at the molecular level because of their specific mechanisms, and they are the subject of ongoing 

intensive research. Most are low molecular weight 

peptides (eg, the aforementioned δ-atracotoxins 

consist of 42 amino acids), and they have proved 

amenable to molecular manipulation/modification to 

enhance specific activities. These peptides are ideal 

as structural scaffolds for the production of unique 

new biologically active molecular species with highly 

specific targets or actions. Although of resultant great 

interest to the pharmaceutical industry, the problem 

of successful delivery of peptide therapeutics (or, 

potentially, offensive agents) remains an issue. This 

problem applies equally to the peptide toxins with 

different sites and modes of action, which also is the 

subject of pharmaceutical discovery research. For 

example, the diverse conopeptides are typically low 

molecular mass (<5 kDa) peptides that have complex 

pharmacology. More than a dozen classes of conotoxins exist, including: 

• inhibitors or activators of voltage-gated sodium channels,

• nicotinic acetylcholine receptor inhibitors, and

• serotonergic 5-HT3 antagonists and many 

others.47,48

Some of these already have proven uses as pharmacotherapeutics. One conopeptide, ziconotide (Prialt, Jazz 

Pharmaceuticals, Dublin, Ireland), is already used as 

an alternative analgesic to treat moderate to severe 

refractory pain. Ziconotide, which is the only intrathecal nonopioid analgesic approved by the US Food 

and Drug Administration, binds to N-type calcium 

channels on primary nociceptive afferent neurons in 

the dorsal horn of the spinal cord.49

Some excitatory neurotoxins are larger polypeptides, and most of these, like the small pepide atracotoxins, stimulate neuroexocytosis. For example, 

the 110- to 140-kDa α-latrotoxins from widow spider 

(Latrodectus spp) venoms bind to several target proteins (eg, soluble N-ethylmaleimide-sensitive factor 

activating protein receptor [SNARE] proteins) in the 

neurolemma, and they cause calcium and sodium influx that results in massive neurotransmitter release.50 

Clinically, these toxins cause symptoms and signs 

that reflect hyperexcitation of the nervous system, 

manifesting clinical effects, such as muscle fasciculations and spasms, tachycardia, paraesthesia, and 

more.34,35,43,51 The pharmacology of these toxins may 

have broad similarities across many taxa of venomous 

animals, although with sometimes distinctive specific 

effects in particular groups.

Of the approximately 1,800 species of scorpions, 

only about 30 to 35 taxa are known to be dangerously venomous to humans. Most of these medically 

important species belong to the family Buthidae, and 

are widely distributed globally. The venom delivery 

apparatus (sting, the aculeus) is located at the end of 

the telson and delivers a bolus of venom. Many scorpions typically insert the sting into prey with protracted 

penetration. Stings delivered to potential predators 

or unfortunate humans can be relatively brief, but 

still may cause life-threatening effects, especially in 

children. 

Medically dangerous buthid scorpions (Figure 18-

11) often have numerous neurotoxic isotoxins present 

in their venoms. Some scorpion venoms contain longchain polypeptide neurotoxins that act by stabilizing 

the open conformation of voltage-dependent sodium 

channels, thereby causing continuous and prolonged 

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Toxins From Venoms and Poisons

firing of the somatic, sympathetic, and parasympathetic neurons. The repetitive firing results in autonomic 

and neuromuscular hyperexcitation. Many of these 

toxins also stimulate neuroexocytosis. Well over 100 

α-neurotoxins have been characterized from various 

scorpion venoms and have often been classified as toxic 

to mammals, insects, or both.52 Interestingly, computational analyses of structure–function have suggested 

that scorpion α-toxins possess modular organization, 

and individual modules interact with different parts 

of their target sodium channels.53 Most of these toxins 

have molecular mass around 65 kDa, share a common 

βαββ organization, contain four disulfide bridges, and 

primarily bind to site 3 of the voltage-gated sodium 

channel, thereby delaying inactivation; whereas other 

scorpion venom toxins (β-toxins) shift the membrane 

potential dependence of channel activation by binding to site 4.54

Some of the short polypeptide neurotoxins present in the same venoms simultaneously antagonize 

potassium channels. Most of these consist of 23 to 

64 amino acids with molecular mass less than 4 

kDa, and these structurally constrained polypeptides adhere to either the inhibitor cysteine knot or 

disulfide-directed β-hairpin folding motif.55,56 The 

first structurally elucidated bound toxin-potassium 

channel complex contains the well-studied potassium channel-binding toxin charybdotoxin, isolated 

from venom of the Israeli yellow scorpion, Leiurus 

quinquestriatus hebraeus.

57 This imaginatively named 

distinctive toxin has specificity for a single site on 

the external end of big potassium channels, a form 

of Ca2+-dependent, voltage-dependent K+ channel 

that facilitates the passive flow of a relatively large 

current of potassium ions.58,59 (The toxin was named 

after Charybdis, the daughter of Poseidon, who was 

transformed into a marine behemoth by Zeus. The 

whirlpools produced by the mythic Charybdis were 

analogized with the figuratively turbulent external 

face of the big potassium channel.58,59 Other medically 

important scorpions often have been named with 

similar mythological flair. The toxic South American 

scorpion, Tityus serrulatus [Brazilian yellow scorpion, 

family Buthidae] derives its genus name from the giant, Tityus, who, according to Greek mythology, was 

banished to Hades by Zeus because of the former’s 

attempt to rape his bride, the goddess Leto, a female 

Titan.) Charybdotoxin essentially “plugs” the channel 

closed, thereby antagonizing the flow of potassium 

current. Na+

 channel and K+ channel toxins in scorpion 

venoms function synergistically, causing persistent 

depolarization of autonomic nerves and resulting in 

the massive release of autonomic neurotransmitters.60

Although the actual frequency and case morbidity/mortality rates of scorpion stings are unknown, 

at least several thousand fatalities occur per year. A 

recent global estimate suggested 1.2 million scorpion 

stings occur annually, with about 3,250 deaths (case 

fatality rate around 0.27%).61 The hallmark features 

of envenoming are instant and severe local pain at 

the sting site, followed by rapid onset (usually within 

15 to 60 minutes) of systemic envenoming that may 

include generalized pain, cardiac dysfunction, profuse 

sweating, labile blood pressure (hypertension or hypotension), and pulmonary edema. After envenoming by 

some members of the genus Centruroides spp (Central 

America, southwestern United States, and Mexico), 

bizarre signs such as rotational nystagmus may be seen 

in children. The cardiac dysfunction or pulmonary 

edema may prove fatal, especially in children. 

Envenoming by Australian funnel-web spiders 

(Atrax spp and Hadronyche spp) produces a similar 

pattern of rapid hyperexcitation, starting with perioral 

parasthesiae and tongue fasciculation. These initial 

effects can progress in minutes to include hypertension, excessive sweating, salivation, lacrimation, 

tachycardia, pulmonary edema, hypoxia, coma, and 

death. If the envenomed victim survives this stage, 

they may develop progressive muscle fasciculation, 

bradycardia, hypotension, and terminal cardiac collapse. The envenoming syndrome has been likened 

to a catecholamine storm.

Figure 18-11. The medically important scorpion, Androctonus 

australis (thick or fat-tailed scorpion, family Buthidae), is one 

of the most toxic species studied. This species is extensively 

distributed from Asia through the Middle East and northern 

Africa.

Photograph: Copyright © Julian White. In: White J. Venomous animals: clinical toxinology. EXS. 2010;100:233–291. In:

Luch A (ed). Molecular, Clinical and Environmental Toxicology. 

Vol 2: Clinical Toxicology. Basel, Switzerland: Birkhäuser; 

2010. 

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Medical Aspects of Biological Warfare 

Widow spiders (family Theridiidae, genus Latrodectus; approximately 31 species, including well-known 

species such as black widows [Latrodectus mactans] 

and red-backed spiders [Latrodectus hasselti; see Figure 

18-2]) cause less rapid or severe envenoming, with 

initial bite site pain, and sometimes with local sweating and piloerection. Regional pain and sweating that 

progresses in severe cases to generalized pain, sweating, hypertension, nausea, and malaise may follow. 

This syndrome (latrodectism) is unpleasant and may 

last many hours to days, but is rarely fatal. Brazilian 

banana spiders (family Ctenidae, genus Phoneutria; approximately eight species; Figure 18-12) cause similar 

clinical effects (phoneutrism), but with more prominent local pain; additionally, envenomation in young 

males may result in priapism. 

Australian Irukandji jellyfish (several genera of the 

class Cubozoa) may also cause a catecholamine stormlike envenoming. Irukandji syndrome is named for 

the Aboriginal clan in Cairns, Queensland, Australia, 

where this type of envenoming was first noted. The 

agent responsible for these cases remained elusive for 

years and eventually was determined to be a single 

cubozoan species, the chirodropid, Carukia barnesi, 

named for Dr Jack Barnes, who in 1964 first associated 

this jellyfish species with the syndrome. It is now clear 

that at least two species of chirodropid [C barnesi, 

Malo kingi], one or more species of cubozoan carybdeid [Carybdea spp], and probably several others are 

responsible for these serious envenomings. The victim 

initially experiences minor local sting effects and then 

a delayed (usually 20 to 40 minutes) onset of systemic 

envenoming with severe muscle spasm pain (especially 

in the back), sweating, nausea, and hypertension that 

can fluctuate and recur over hours. In severe cases, 

cardiac dysfunction and pulmonary edema can occur, 

and although deaths have occurred, they are rare (see 

Table 18-2). Still, any patient presenting with a significant Irukandji envenoming should have continuous 

cardiac monitoring.

Antivenoms are available for some medically important scorpion species and, despite some controversy, 

the majority of evidence indicates that these are effective and lifesaving if administered intravenously at the 

earliest opportunity. Some information has suggested 

that glucocorticoids might improve outcomes in some 

severe scorpion envenoming cases. However, these 

suggestions have largely been based on low quality 

evidence, and a recent Tunisian case-control study 

found no benefit.62,63

Antivenom against Australian funnel-web spiders 

is highly effective and can be lifesaving. Antivenom 

for widow spider bites is used routinely in Australia, 

where most clinical practice suggests it is effective, 

or at least more effective than alternative therapies. 

Elsewhere, its use is often reserved only for the most 

severe cases, mostly due to largely overrated fears of 

side effects that are actually uncommon. 

A similar situation exists for Phoneutria bites, where 

local pain relief, including local anesthesia, is used in 

preference to antivenom, except in children and the 

most severely envenomed. No antivenom for Irukandji stings exists, and treatment is supportive and 

includes use of opioid analgesia to help alleviate the 

severe systemic pain associated with serious Irukandji 

envenoming.

Myotoxins

Myotoxins are mostly systemic in action and commonly are PLA2, but some myotoxins, such as the 42 

amino acid basic polypeptide crotamine, appear to 

have focused action on muscle groups in the lower 

extremities. Crotamine, which is found in selected 

North American rattlesnake venoms, probably aids 

prey capture by causing rear limb dysfunction, that 

inhibits locomotor ability (eg, prevention of lengthy 

prey travel post-envenoming). Hypothetically, it may 

decrease the metabolic cost of trailing envenomed 

prey (eg, following bite-release, an envenomed prey 

animal may not expire for a few minutes and have 

time to flee from the site of the encounter with the 

snake). Locally acting myotoxins cause cellular damage around the bite site, but the systemically acting 

myotoxins, particularly PLA2, selectively target 

Figure 18-12. Brazilian banana spider (Phoneutria nigriventer). A medically important South American species of 

aeranomorph. 

Photograph: Wikipedia Commons, public domain. 

https://commons.wikimedia.org/wiki/File:Wandering_

spider.jpg.

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Toxins From Venoms and Poisons

skeletal muscle and can cause extensive and severe 

muscle damage. Although binding may occur early, 

once venom has reached and then exited the circulation, a delay occurs before onset of clinical detection 

of pathology. Therefore, significant myolytic effects 

may occur before clinical indicators for treating 

myolysis, which has led some researchers to suggest 

that early intervention with antivenom is justified to 

prevent myolysis in some cases of bites inflicted by 

species known to produce serious myolytic effects.64

This remains to be studied, as no current evidence 

supports this approach.

The systemic myotoxins bind to the skeletal muscle 

cells and cause progressive and severe damage to the 

cells. Experimental animals injected with purified 

myotoxic PLA2 often exhibit skeletal muscle changes 

characterized by dissolution of actin and myosin 

filaments, disruption of Z-band material, dilation of 

the sarcoplasmic reticulum, and swelling and disruption of mitochondria, as well as disorganization of 

the T-tubule system.65–68 However, if the basement 

membrane is preserved, some muscle regeneration 

can occur that may commence 24+ hours postbite, 

but may require weeks to complete. It is thought that 

the PLA2 enzymatic action is a crucial component in 

their toxicity, but chemical modification of specific 

residues (eg, Asp49, Lys49, and others) in the primary 

sequence of some of these enzymes has suggested that 

some PLA2 species may have a pharmacologically 

active domain discrete from the catalytic functional 

site.69 Clearly, cellular binding to the target cells is an 

Figure 18-14. Australian eastern brown snake (Pseudonaja 

textilis). The most medically important snake in Australia; 

its range encompasses a large proportion of Australia and 

southeastern Papua New Guinea. Its venom contains potent 

procoagulants and a presynaptic neurotoxin (textilotoxin) 

with the highest experimental lethal potency of any snake 

venom toxin isolated to date. Fortunately, many bites inflicted on human victims are dry, meaning no venom is injected. 

Photograph: Copyright © Julian White. In: Brent J, Wallace 

KL, Burkhart KK, et al (eds). Critical Care Toxicology: Diagnosis and Management of the Critically Poisoned Patient. 1st ed. 

Philadelphia, PA: Mosby; 2005.

Figure 18-13. Australian common tiger snake (Notechis scutatus). One of the world’s most venomous snakes, its venom 

contains potent presynaptic neurotoxins, myotoxins, and 

procoagulants. 

Photograph: Copyright © Julian White. In: White J. A 

Clinician’s Guide to Australian Venomous Bites and Stings. 

Melbourne, Australia: Commonwealth Serum Laboratories; 

2013: 300 pp.

essential first step, as the myotoxic PLA2 species do 

not cause widespread cellular injury and specifically 

target muscle cells.

PLA2 myotoxins or multimeric toxins containing 

PLA2 subunits (many with myolytic activity; see Table 

18-2 for representative examples) are found principally 

in snake venoms, notably selected Australian elapid 

venoms (eg, notexin [common tiger snake, Notechis 

scutatus; Figure 18-13], textilotoxin [Eastern brown 

snake, Pseudonaja textilis; Figure 18-14], taipoxin [coastal 

taipan (Oxyuranus scutellatus)]), sea snake venoms [eg, 

myotoxin VI5 (beaked sea snake, Hydrophis [Enhydrina]

schistosus70], PLA2-H1 [blue-banded or annulated sea 

snake, Hydrophis cyanocinctus]), and some krait venoms (eg, β-bungarotoxin found in several species [eg, 

Bungarus candidus; Figure 18-15]). They are also found 

in several rattlesnake (family Viperidae, subfamily 

Crotalinae) venoms (eg, crotoxin, found in venoms of 

several taxa of tropical rattlesnakes, including C durissus

spp, and others such as Mojave toxin and its isotoxins 

in venom of the Mojave rattlesnake, C scutulatus, tiger 

rattlesnake, C tigris [Figure 18-16], timber or canebrake 

rattlesnake, C horridus and others), as well as some Russell’s viper (family Viperidae, subfamily Viperinae) venoms (eg, possibly, VRV-PL4 [D russelii; Figure 18-17]). 

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Medical Aspects of Biological Warfare 

Clinically, systemic myotoxicity presents several to 

many hours postbite as muscle pain, muscle weakness, 

myoglobinuria, and gross elevation of plasma creatine 

phosphokinase and often-raised hepatic enzymes (eg, 

alanine transaminase, alkaline phosphatase, aspartate 

aminotransferase). In some cases, a notable creatine 

phosphokinase elevation may be observed before 

muscle discomfort. 

It is important to note that some snake species often 

considered as purely neurotoxic may inflict bites that 

can cause mixed neurotoxicity and rhabdomyolysis 

akin to that seen in some Australian elapid envenoming by coastal taipans, tiger snakes, and others. For 

example, bites from greater black kraits (Bungarus niger) and Malayan kraits (Bungarus candidus; see Figure 

18-15) have respectively caused mixed neurotoxicity 

and myotoxicity in Bangladesh71 and myotoxicity, 

cardiovascular instability, neurotoxicity, and hyponaetremia in southern Vietnam.72 Thus, due to unpredictable venom variability, as well as clinical response to 

variable venom components, it is essential not to view 

a given species as solely “neurotoxic” or “hemotoxic,” 

because the venom-induced disease may notably vary. 

Treatment is with early intravenous administration 

of antivenom and supportive treatment, especially 

to ensure good renal output with aggressive fluid 

resuscitation (as in any recommendation for clinical 

Figure 18-16. Tiger rattlesnake (Crotalus tigris). This distinctive rattlesnake has venom that contains the crotoxin homologue, Mojave toxin, a potent presynaptic heterodimeric 

neurotoxin. There are only a few documented bites by this 

taxon, all of which were medically insignificant. However, 

any rattlesnake species with venom that contains Mojave 

toxin, crotoxin or related isotoxins (eg, horridus toxin, or 

canebrake toxin found in venom of some geographic populations of the timber rattlesnake, C horridus) must be considered 

capable of delivering a potentially fatal envenomation.

Photograph: Copyright © Julian White. In: White J, Dart RC. 

Snakebite: A Brief Medical Guide. Denver, CO: Rocky Mountain 

Poison & Drug Center; 2008.

Figure 18-17. Russell’s viper (Daboia russelii), Bannerghatta, 

India. Along with the saw-scaled vipers (Echis spp) and 

several species of cobras (Naja spp), D russelii and D siamensis (Eastern Russell’s viper) are the species most important 

in the global envenoming burden. The snakes have a wide 

distribution and are plentiful; they constitute a public health 

problem particularly among rural communities in the Indian 

subcontinent, as well as parts of Southeast Asia. Envenoming from D russelii from different geographic populations 

can result in several differing clinical syndromes including 

hypogonadism, one of the consequences of pituitary hemorrhagic infarct (Sheehan’s syndrome) resulting in panhypopituitarism. Another member of the genus, D palaestinae

(Palestine viper) is medically significant in the Middle East. 

Its venom has been studied as a source of several classes of 

pharmacotherapeutics, including analgesics. 

Photograph: Wikipedia Commons, public domain. https://

commons.wikimedia.org/wiki/Daboia_russelii#/media/

File:Russellsviper_sal.jpg.

Figure 18-15. Malayan or blue krait (Bungarus candidus), 

Thailand. A semi-fossorial species that ranges in Malaysia, 

parts of Southeast Asia, and Indonesia. Studied Bungarus spp 

have venoms containing highly potent presynaptic neurotoxins (bungarotoxins), postsynaptic neurotoxins, and other 

components, including some with cardiotoxic properties. 

Photograph: Copyright © Julian White. In: Brent J, Wallace 

KL, Burkhart KK, et al (eds). Critical Care Toxicology: Diagnosis and Management of the Critically Poisoned Patient. 1st ed. 

Philadelphia, PA: Mosby; 2005.

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Toxins From Venoms and Poisons

management, approaches such as aggressive fluid resuscitation must be applied in the setting of risk/benefit with 

consideration for the patient’s possible preexisting 

comorbidities, such as congestive heart failure or other 

volume overload states).

With major myolysis, secondary renal failure is a 

risk and can contribute to severe and sometimes fatal 

hyperkalemic cardiac toxicity. Alkalinization of urine 

is sometimes recommended, but is unproven to provide any added benefit in snakebite myolysis, as well 

as in most other presentations (eg, serotonergic syndrome, crush injuries) featuring acute myoglobinuria 

capable of producing nephropathy.

Myolysis can also follow some other envenomings, 

including massive bee stings and ingestion of certain 

mushrooms (eg, family Tricholomataceae, Tricholoma 

[flavovirens] equestre, yellow knight mushroom).73 Studies of some venomous fish have reported myotoxicity 

in the murine model,74 but, to date, there are no welldocumented clinical cases of fish stings having caused 

myotoxicity.

Hemostasis-Active Toxins

The complex human hemostatic system is a common 

target of venoms, particularly snake venoms, that may 

cause a wide variety of clinical effects most commonly 

associated with an increased bleeding tendency. When 

combined with the action of proteolytic hemorrhagins, 

some cause severe hemorrhagic diathesis, a pathological state that could hypothetically be viewed as 

inducing terror in some individuals or populations. 

Thus, although impractical as tactically deployed bioweapon agents, some of these toxins could conceivably 

be used offensively on a small scale. An overview of 

the major toxin groups involved is shown in Table 18-3.

Venoms cause activation or inhibition of the clotting 

system by several different mechanisms and at many 

possible target points (Figure 18-18). Some venoms 

contain multiple toxins affecting hemostasis that may 

be synergistic, independent, or counteracting (see 

Figure 18-18). 

In most cases, this type of envenoming (most 

accurately termed coagulopathic, not hemotoxic) 

causes an increased bleeding tendency, often by either hydrolyzing clotting protein (fibrinogenases) or 

by activating normal systems of clot formation and 

dissolution (procoagulants). These activities are most 

widely represented in viperid snake venoms, and a 

prominent example is that of ecarin, the prothrombinconverting metalloprotease (and closely related toxins) 

found in saw-scaled viper (Echis spp; Figure 18-19) 

venoms (a group A prothrombin activator). Ecarin 

plays an important role in the clinical laboratory by 

providing a meizothrombin generation test allowing 

for the precise quantification of direct thrombin inhibitors. Russell’s viper (Daboia russelii) venom is a pivotal 

reagent in laboratory medicine because it contains a 

phospholipid and Ca2+-dependent potent activator of 

factor X that forms a complex with prothrombin and 

thereby converts fibrinogen to fibrin. This mechanism 

facilitates the diagnosis of phospholipid antibodies 

including lupus anticoagulant, because in these states 

the antibodies bind to the essential venom co-factor 

phospholipid and thus inhibit the venom-induced 

factor X activation and prolong the clotting time. 

Some Australian elapids also have potent procoagulant venoms. A number of the procoagulants present 

in these venoms, such as those found in the common 

tiger snake and Eastern brown snake, have structures 

homologous to human clotting factors Xa (group D 

prothrombin activator) or Va and Xa combined (group 

C prothrombin activator). Other animals, such as the 

venomous lizards Heloderma suspectum (Gila monster) 

and Heloderma horridum (beaded lizard), have venoms 

that contain procoagulant toxins and other hemostasisactive toxins, such as the H horridum venom acidic 

PLA2 that inhibits thromboxane-induced platelet aggregation.75 As mentioned previously, caterpillars of 

the Brazilian saturniid moth, Lonomia, have irritating 

hairs that contain toxins with powerful procoagulant 

effects. Envenoming by a caterpillar may seem farfetched, but it is not to be taken lightly because Lonomia

stings can produce fatal disseminated intravascular 

coagulopathy in humans. A few animals, such as the 

Martinique lancehead viper or fer-de-lance Bothrops 

lanceolatus, have prothrombotic venom, and envenoming can lead to deep venous thrombosis, pulmonary 

emboli, cerebral infarction, and related thrombotic 

events.

Clinically, toxins promoting increased bleeding 

tendency may cause few initial or early symptoms. 

In many cases, almost all circulating fibrinogen can 

be consumed without apparent bleeding until or unless bleeding is induced through injury or a medical 

procedure such as venipuncture. In the former case, 

a fall with relatively mild cranial trauma can result 

in catastrophic intracranial bleeding that may occur 

minutes to hours later. 

To detect abnormalities at the earliest opportunity 

after a potentially coagulopathic bite or sting, serial 

laboratory assessment of clotting function is essential. 

Careful serial laboratory testing is a cornerstone of 

management because early provision of antivenom 

and other potential treatments (eg, replacement therapy; see next paragraph) can limit the possible cardiovascular effects induced by the venom disease. There 

is ongoing controversy over the role of antivenom in 

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Medical Aspects of Biological Warfare 

TABLE 18-3

MAJOR VENOM TOXIN GROUPS AFFECTING HUMAN HEMOSTASIS

Toxin Type Effect Examples

Procoagulants Factor V activating

Factor X activating

Factor IX activating

Factor II (prothrombin) 

activating:

 Group A

 Group B

 Group C

 Group D

Fibrinogen clotting

RVV-V (Factor V-activating serine protease from venom of the Russell’s viper, Daboia russelii)

Contortrixobin (Factor V activating serine protease from venom of 

the copperhead, Agkistrodon contortrix)

RVV-X (metalloproteinase disintegrin activator of Factor X from 

venom of the Russell’s viper)

TSV-FIX-BP (C-type lection-activating Factor IX from venom of 

Stejneger’s green tree viper, Trimeresurus stejnegeri)

Ecarin (cofactor-independent metalloproteinase Group A prothrombin activator from venom of the saw-scaled viper, Echis carinatus)

Carinactivase (Ca2+-dependent metalloproteinase Group B 

prothrombin activator from venom of the saw-scaled viper)

Oscutarin (Ca2+- and phospholipid-dependent serine protease 

Group C prothrombin activator from venom of the coastal taipan, 

Oxyuranus scutellatus)

Notecarin (Ca2+-, phospholipid, and Factor Va-dependent serine 

protease Group D prothrombin activator from venom of the common tiger snake, Notechis scutatus)

Anticoagulants Protein C activating

Factor IX/X activating protein

Thrombin inhibitor

Phospholipase A2

ACC-C (protein C-activating serine protease from venom of the 

copperhead, Agkistrodon controtrix)

Bothrojaracin (C-type lectin thrombin inhibitor from venom of the 

jararaca, Bothrops jararaca)

CM-IV (anticoagulant PLA2 from venom of the African black-necked 

spitting cobra, Naja nigricollis)

Fibrinolytic Fibrin(ogen) degradation

Plasminogen activation

Ancrod (fibrinogenolytic enzyme from venom of Malayan pit viper, 

Calloselasma rhodostoma)

Neuwiedase (α-chain fibrinogenase [metalloproteinase] from venom 

of the jararaca pintada, Bothrops neuwiedi) 

Brevinase (β-chain fibrinogenase (serine protease) from venom of 

the mamushi, Gloydius blomhoffi) 

TSV-PA (plasminogen-activating serine protease from venom of 

Stejnegers green tree viper, Trimeresurus stejnegeri)

Vessel wall interactive Hemorrhagins Echistatin (RGD disintegrin from venom of the saw-scaled viper, 

Echis sochureki)

Platelet activity Platelet aggregation inducers

Platelet activation inhibitors

Botrocetin (platelet agglutination with VWF from venom of the 

jararaca)

Convulxin (C-type lectin platelet aggregation, from venom of the 

South American rattlesnake, Crotalus durissus terrificus)

Jararhagin (RGD disintegrin snake venom metalloproteinase that 

causes inhibition of platelet aggregation, from venom of the 

jararaca)

Echicetin (C-type lectin platelet aggregation inhibitor from venom 

of the saw-scaled viper)

Plasma protein 

activators

Serine protease inhibitors Proteinase I and II (inhibition of serine protease inhibitors from venom of the eastern diamondback rattlesnake, Crotalus adamanteus)

RGD: arginine-glycine-aspartic acid, a peptide motif found in this group of snake venom metalloproteinases; vWF: Von Willebrand Factor

Data sources: (1) Mebs D. Venomous and Poisonous Animals: A Handbook for Biologists, Toxicologists and Toxinologists, Physicians and Pharmacists. 

Boca Raton, FL: CRC Press; 2002: 360. (2) Meier J, White J (eds). Handbook of Clinical Toxicology of Animal Venoms and Poisons. Boca Raton, FL: 

CRC Press; 1995. (3) Mackessy SP (ed). Handbook of Venoms and Toxins of Reptiles. Boca Raton, FL: CRC Press, Taylor & Francis; 2010: 528 pp. 

(4) Synthesized information included in lectures and presentations of the authors.

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treating snakebite-induced coagulopathy in Australia, 

but most authorities consider timely administration of 

antivenom as the optimal treatment. Outside Australia, 

antivenom remains the evidence-based treatment for 

snakebite coagulopathy, either for increased bleeding 

(eg, from bites by the saw-scaled vipers, Echis spp) or 

for increased clotting (eg, from bites by the Martinique 

lancehead viper or fer-de-lance B lanceolatus). 

The role of blood clotting products (eg, fresh frozen 

plasma and cryoprecipitate) replacement remains controversial, and when antivenom is available, it should 

be used in preference to and before such blood products 

if the clinical circumstances (eg, severe depletion with 

notable bleeding risk) suggest the need for replacement. 

For patients with major bleeding, despite adequate antivenom, blood products may have a place as adjunctive 

treatment depending on clinical need. Anticoagulant 

drugs such as heparin and warfarin are not useful in 

treating snakebite coagulopathy, probably intensify 

bleeding, and are positively contraindicated.

Hemorrhagic and Hemolytic Toxins

Some snakes have venom toxins that actively 

damage blood vessels and other tissues, thereby promoting bleeding. When combined with pro- and/or 

anticoagulant toxins, the actions are synergistic and 

potentially cause extensive bleeding or tissue injury, 

particularly around the bite site. These toxins are 

often also called hemotoxins, but are more accurately 

termed vasculotoxins because of their direct effects on 

the microvasculature.

Most hemorrhagic toxins are metalloproteinase enzymes, usually with a zinc moiety. However, some are 

comprised of peptide complexes, such as the synergistically hemorrhagic PLA2-peptide complex (DR-HC-1), 

characterized from D russelii venom.76 The larger venom 

metalloproteinases have additional domains carboxy 

to the zinc-binding domain. Some metalloproteinases, 

termed class P-II by many investigators, contain domains that are further processed and give rise to free 

domains such as disintegrins.77–79 Class P-III metalloproteinases have disintegrin-like and cysteine-rich 

domains, whereas class P-IV is similar to P-III, but its 

metalloproteinases have additional lectin-like domains. 

Homologs of the venom P-III structures (ADAMs: A 

Disintegrin-Like And Metalloproteinase-containing 

protein) have been identified in a variety of mammalian 

sources and tissues77 and possess myriad activities, 

including participation in essential cellular functions 

such as angiogenesis regulation, inflammation, matrix 

protein processing, and many others.80 However, particular focus has been directed toward those that occur 

in reptile venoms and mammalian reproductive tissues 

(the reprolysins, or M12 metalloproteinase subfamily79). 

The angiogenic inhibitor and cell adhesion molecule 

regulation functions have attracted scrutiny of some 

of the venom disintegrins as potential antineoplastic 

agents.81 In addition, tissue inhibitors of metalloproteinases not only regulate proteinases in mammalian 

systems, but also have signal-transduction roles that 

continue to be characterized.82 This is a fertile and promising area of research, and full consideration of their 

structure-function is beyond the scope of this chapter. 

The proteolytic actions of these toxins damage the 

endothelium/basement membrane of cells comprising 

the microvasculature. Some crotaline viperids such as 

the Western diamondback rattlesnake have venoms 

that contain several different isoforms (eg, atrolysins) 

of these toxins. Some hemorrhagins have a shared 

disintegrin domain (a 13-amino acid loop containing 

specific sequences of arginine, glycine, and aspartic 

acid, or RGD in abbreviated nomenclature) that facilitates binding to platelet receptor gIIb/IIIa. These 

toxins have attracted more attention than most snake 

venom hemorrhagins because of their use in integrin 

function studies83 and for their roles as structurefunction scaffolds for pharmacotherapeutics (especially for the development of antithrombotics and as 

antineoplastics). One of these peptides, barbourin, has 

been characterized from venom of the dusky pygmy 

rattlesnake (Sistrurus miliarius barbouri).

Clinically, these hemorrhagins contribute to rapid 

and potentially severe local tissue effects such as 

blistering, bleeding, and necrosis, which are mostly 

associated with significant local pain and sometimes 

major fluid shifts from the circulation into local tissues. 

This latter effect can result in profound hypovolemic 

shock. Although some characterized hemorrhagins, 

such as the 50 kDa nonproteolytic hemorrhagin from 

venom of Atractaspis engaddensis (Israeli or oasis mole 

viper or burrowing asp), exhibit dose-related hemorrhagic activity, others such as the atrolysins mentioned 

previously may induce dose-independent hemorrhage 

in vivo as a result of the combined activities of this 

rhexic hemorrhagin and other venom components, 

such as the 30 kDa antagonist that binds to platelet 

glycoprotein receptor Ib. In this instance, the inhibition of platelet adhesion probably acts synergistically 

with the hemorrhagins and other anticoagulant toxins, 

thereby increasing the hemorrhagic effect to a greater 

extent than that accomplished by any of the single 

components alone.

The role of antivenom in treating effects of hemorrhagins is controversial, although most authorities 

recommend using antivenom therapy for prohemorrhagic envenomings. However, the molecular size 

and steric hindrance posed by antibodies present in 

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Figure 18-18. Diagrammatic representation of the human hemostasis system, with examples of where venoms may interact to 

activate or inhibit the clotting process. The diagram only represents some key elements of hemostasis, not every component 

currently known and similarly, only some common examples of toxin effects, not an exhaustive listing of all known toxin 

interaction sites. The diagram concentrates particularly on the coagulation cascade elements, whereby various triggers in 

normal hemostasis activate top levels of the cascade, resulting in progressive activation downstream until common pathways 

are reached. Principal among the latter are activation of procoagulant enzymes (mostly serine proteases), such as factor VIII 

to FVIIIa, factor X to FXa, factor V to FVa, with FXa and FVa forming the prothrombinase complex that then activates the final 

enzymatic step, prothrombin (factor II) to thrombin (FIIa). It is thrombin that then converts the clotting protein, fibrinogen, 

to fibrin, so that fibrin molecules can be cross-linked by factor XIIIa, to form a stable fibrin clot. Normally this clot formation 

occurs inside a protective platelet plug environment, at the site of blood leakage from a damaged blood vessel. The damaged 

edges (endothelial cells) of the blood vessel are a potent stimulus to activation of the hemostasis system. The fibrin blood clot 

then provides a semipermanent plug to prevent further blood leakage from the damaged blood vessel, allowing time (6–10 

days) for the endothelium to repair and effect a permanent repair of the damage, at which time the fibrin clot is no longer 

required and is broken down by plasmin (activated form of plasminogen). There are a number of promoters and inhibitors 

of each step in this complex multistep process, each designed to ensure clots only form where needed and are dissolved once 

no longer needed. Each and every step in the process, including promoters and inhibitors, is a potential target for venom 

toxins. In addition, if the process is activated freely in blood, rather than in a protected platelet plug environment, some 

normal controls are bypassed, allowing rapid progression from procoagulant activation, through conversion of fibrinogen 

to fibrin, and then degradation of fibrin strands and cross-linked fibrin by plasmin. This process can potentially cause rapid 

consumption of all circulating or available fibrinogen, rendering the patient unable to form blood clots, so at risk of major 

bleeding. In one sense this is a profound form of anticoagulation, but it is achieved by aggressive stimulation of clotting, 

hence is usually termed a procoagulant defibrination coagulopathy (referred to as VICC by a few authors). This consumptive 

coagulopathy can be initiated by a variety of venom toxins, particularly the calcium-independent procoagulants (Groups A 

and B) found in saw-scaled viper (Echis spp) venoms, and the calcium-dependent procoagulants (Groups C and D) found 

in many Australian Elapid snake venoms (eg, Pseudonaja spp, Oxyuranus spp, Notechis spp, Tropidechis carinatus, Hoplocephalus spp). These latter procoagulants mimic closely the normal clotting factors, thus have extensive homology with either 

FXa, or the FVa–FXa (prothrombinase) complex. It can be speculated that these toxins were developed from recruitment of 

normal clotting factor genes. Once this process is activated there can be massive consumption of clotting factors, especially 

fibrinogen, but also FII, FV, FVIII, and FX among others, and while antivenom may, at least for some venoms, switch off the 

process, it cannot instantly restore depleted fibrinogen and other factor levels, so several hours may pass before protective 

levels are reached. Although giving factor replacement therapy (fresh frozen plasma, cryoprecipitate, or whole blood) may 

speed return to normal levels, if neutralization of venom toxins is incomplete, such replacement therapy can instead add 

fuel to the coagulopathy fire, potentially worsening the clinical picture. In contrast, phospholipase A2-based anticoagulant 

toxins (eg, from Pseudechis spp) merely inhibit portions of the hemostasis pathways without causing consumption of clotting 

factors, so that antivenom can almost instantly reverse this effect. 

PL: phospholipid; TAFI: thrombin-activatable fibrinolysis inhibitor; VICC: venom-induced consumption coagulopathy 

Photograph: Copyright © Julian White. In: White J. Venomous animals: clinical toxinology. EXS. 2010;100:233–291. In: Luch 

A (ed). Molecular, Clinical and Environmental Toxicology. Vol 2: Clinical Toxicology. Basel, Switzerland: Birkhäuser; 2010. 

antivenom (usually whole IgG or the F(ab’)2 portion of 

IgG) may not easily leave the circulation to penetrate 

the areas being damaged by hemorrhagins. Drugs to 

directly target the enzymatic action of these toxins 

have been explored as adjunctive therapy and continue to attract study to identify possible alternative 

therapeutic agents. 

In cases of hemorrhagic snakebite, it is essential to 

monitor for shock and ensure adequate intravenous 

hydration. The damaged bite area may appear markedly swollen and with poor vascular return, with 

concomitant pain, possibly suggesting an underlying 

compartment syndrome. Although snakebites may 

rarely cause compartment syndrome, most snakebite 

patients do not have definitive evidence of compartment syndrome. In addition, the clinical criteria defining compartment syndrome varies among surgical 

specialties (eg, orthopedics and vascular surgery), 

facilities, and individual physicians and surgeons. In 

facilities lacking intracompartmental measurement 

catheter systems, clinically experienced use of basic 

Doppler ultrasound may help clinical interpretation. Conversely, hospitals equipped with magnetic 

resonance imaging or multidetector computed tomographic arteriography could provide additional 

means to assess complicated cases, but the latter 

method would be positively contraindicated in any 

patient with coagulopathy. 

Fasciotomy is the standard treatment for compartment syndrome, but when used injudiciously for 

snakebite it can accelerate blood loss and shock, and 

cause severe permanent tissue injury. Fasciotomy 

should only be performed in snakebite if there is direct 

pressure measurement confirmation of compartment 

syndrome with clinical correlation and then only after 

any coagulopathy has been treated. Several patients of 

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snakebite-induced, verified compartment syndrome 

have also been treated successfully by purely medical 

methods (eg, hyperbaric oxygen therapy and cautious 

use of mannitol, the latter contraindicated in cases 

featuring hypovolemia).

Cardiotoxins

True cardiotoxins that cause direct cardiac effects 

in humans are uncommon. Some examples include 

the following: 

• the 2.5 kDa endothelin homologues, the sarafotoxins from some venoms of the mole vipers 

or burrowing asps (Atractaspis spp; these are 

not vipers at all, but instead they are frontfanged members of the subfamily Atractaspinae of the African family Lamprophiidae), 

• bufotoxins from toad (eg, the marine toad, 

Rhinella marina Linnaeus 1758, formerly Bufo 

marinus) parotid gland secretions, and

• the oleandrins from oleander plants (Nerium 

oleander [oleander; Figure 18-20] and Cascabela 

(Thevetia) peruviana [yellow oleander or lucky 

nut; Figure 18-21]). 

The cardiac glycosides of both of the latter plants 

produce effects similar to digoxin by binding to and inactivating the Na+

/K+-ATPase pump on the cytoplasmic 

Figure 18-19. Northeast African saw-scaled or carpet viper 

(Echis pyrimidum). There are approximately 11 current 

recognized species of Echis. Several Echis spp cause a large 

proportion of the world’s human envenoming burden, and 

are among the three most medically important venomous 

snakes. Conversely, E carinatus venom has yielded components important in laboratory medicine (eg, the metalloprotease prothrombin activator, ecarin), as well as a platelet 

aggregation inhibitor (tirofiban, marketed as Aggrastat) that 

reversibly binds to platelet GPIIb/IIIa receptors. 

GP: glycoprotein

Photograph: Copyright © Julian White. In: Brent J, Wallace 

KL, Burkhart KK, et al (eds). Critical Care Toxicology: Diagnosis and Management of the Critically Poisoned Patient. 1st ed. 

Philadelphia, PA: Mosby; 2005.

Figure 18-20. Oleander (Nerium oleander) and yellow oleander or lucky nut (Cascabella [Thevetia] peruviana). These attractive 

and popular ornamental plants contain digoxin-like toxins that inactivate the Na+/K+-ATPase pump on the cytoplasmic 

membrane of myocardiocytes. Concentrations of the toxins present in the plant vary according to plant component (eg, 

seeds, leaves, etc), but ingestion of small amounts is potentially fatal. (A) Oleander (Nerium oleander). (B) Yellow oleander or 

lucky nut (Cascabella [Thevetia] peruviana).

Photographs: Courtesy of Julian White.

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membrane of cardiac cells.22 As a result, intracellular 

Na+ concentration increases, which affects the Na+/

Ca2+ exchange channels, resulting in an increase in 

intracellular Ca2+ that leads to a positive inotropic effect. The increase of intracellular Ca2+ ions also raises 

the resting membrane potential of the cell, leading to 

increasing rates of spontaneous cellular depolarization 

and myocardial automaticity.84 Inhibition of the Na+

/

K+-ATPase pump affects the intracellular movement 

of K+ leading to hyperkalemia.84,85

A few reports show dysrhythmias/cardiotoxicity after cobra bites (especially the monocellate or monocled 

cobra, Naja kaouthia), and some of these probably are 

caused by the direct effects of cardiotoxin (also known 

as direct lytic factor due to its weakly hemolytic and 

other cytotoxic properties). Similar effects may be 

caused by secondary complications that might be more 

likely in victims with cardiovascular comorbidities (see 

Figure 18-21).86,87

Secondary cardiac effects are more common, 

such as the severe and sometimes lethal hyperkalaemia, secondary to severe myolysis that can follow secondary renal failure, which in some cases 

probably results from myoglobinuric nephropathy. 

The myolysis causes massive release of intracellular 

potassium into the circulation, which is controlled by 

kidney excretion, but with renal failure this normal 

kidney function is impaired or ceases, and potassium levels in blood subsequently rise to toxic levels. 

Some scorpion venom toxins cause neuroexcitation 

that can result in secondary cardiac dysfunction, 

sometimes with output failure, arrhythmias, and 

cardiogenic pulmonary edema. Cardiac arrhythmias 

and arrest may also be indirectly caused by hyperkalemia resulting from the cytotoxicity caused by high 

doses of toxins from box jellyfish (eg, the Australian 

box jellyfish or sea wasp, Chironex fleckeri) and the 

closely related multiple taxa (eg, C barnesi and M 

kingi) that cause Irukandji syndrome. The clinical 

effects of cardiotoxins depend on the toxin and its 

mechanisms of action. 

Antidotes may be available for some “true,” or 

more accurately, directly acting cardiotoxins. For 

digoxin-like toxins (such as bufonid toad poisoning or ingestion of oleander plants), cardiac effects 

can sometimes be reversed using anti-digoxin Fab’ 

(digoxin fragment antibodies, or antigen-binding 

fragments). In one case of possible cobra envenominginduced cardiotoxicity, antivenom administration 

appeared to correct ventricular bigeminy, possibly 

a result of the neutralization of toxin effects, or an 

improvement of other venom effects, thereby alleviating the secondary effects of envenoming.87 For 

box jellyfish, although antivenom can neutralize 

the toxins, the indirect cardiovascular effects can 

develop so rapidly that, under experimental conditions using rodents, even giving antivenom together 

with the toxin does not prevent death. Nevertheless, 

prevailing opinion currently supports provision of 

high dose antivenom in cardiac collapse caused by 

box jellyfish stings, combined with aggressive cardiac 

resuscitation. 

Figure 18-21. Indo-Chinese spitting cobra (Naja siamensis). 

This cobra is medically important throughout its range 

in Southeast Asia (Thailand, Lao, southern Vietnam and 

Cambodia), but its congener, N kaouthia (the monocellate, 

or monocled cobra) is responsible for a large proportion of 

snakebite mortality and morbidity throughout its extensive 

range in Southeast Asia and the Indian subcontinent. Envenoming commonly features serious local effects including desquamation, cellulitis and necrosis, and can include 

systemic neurotoxicity, as well as rare direct cardiotoxicity. 

Predominance of local envenoming or neurotoxicity is variable due to marked population venom variance, and some 

populations can inflict envenoming with mixed clinical 

presentation of local effects and neurotoxicity. In addition, 

N siamensis and probably several populations of N kaouthia

can forcibly eject (“spit”) venom through modified fangs that 

contain an anteriorly oriented orifice. The venom is targeted 

at the head of the recipient, and often enters the eyes causing 

venom ophthalmia. Agrarian-based rural communities may 

be seriously impacted by these snakes, as well as by Echis spp 

and Daboia spp, because farmers working sustenance crops 

often are the victims of envenomings by these species and, 

when not fatal, the common disabling sequelae can threaten 

their livelihood. 

Data source: Chu ER, Weinstein SA, White J, Warrell DA. 

Venom ophthalmia caused by venoms of spitting elapids and 

other snakes: report of ten cases with review of epidemiology, clinical features, pathophysiology, and management. 

Toxicon. 2010;56:259–272.

Photograph: Copyright © Julian White. In: Brent J, Wallace 

KL, Burkhart KK, et al (eds). Critical Care Toxicology: Diagnosis and Management of the Critically Poisoned Patient. 1st ed. 

Philadelphia, PA: Mosby; 2005.

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Nephrotoxins

Few direct animal venom or plant-derived nephrotoxins are currently known. Examples include 

toxins from Russell’s viper venom and from several 

mushroom taxa: Cortinarius spp mushrooms (sometimes called cortinars or webcaps), Amanita smithiana

(Smith’s amanita or Lepidella), Amanita proxima (near 

neighbor amidella), and others.88 The former contains 

toxins (eg, a 7 kDa basic cytotoxin) that damage the tubular epithelium, while the latter contains orellanines 

(eg, 3,3’-4,4’-tetrahydroxy-2,2’-bipyridyl-N,N’-dioxide 

from C orellanus) that induce delayed renal tubular 

necrosis, which leads to renal failure.

Most renal pathology associated with venom, plant, 

or fungal toxins is secondary. In snakebite, it may follow coagulopathy and bleeding, myolysis, hemolytic 

anemia, or a period of hypotension/shock. Venoms 

causing major hemolysis are associated with renal 

failure, as seen after massive multiple hymenopteran 

stings such as those inflicted by bees or vespid wasps, 

as well as after some snakebites. Recluse spider bites 

may also cause hemolysis as part of widespread tissue 

injury (viscerocutaneous loxoscelism), with associated 

multiple organ involvement, including renal failure.

The nature of the kidney injury depends on the toxin 

mechanism or that of the secondary effects. In snakebites, the injury is often due to acute tubular necrosis, 

from which full recovery after a period of hemodialysis 

is possible. However, cases of bilateral renal cortical 

necrosis are reported, where renal recovery occurs 

far less frequently.35 Renal dysfunction secondary to 

hemolysis is similarly likely to be transient, but renal 

pathology following orellanine mushroom poisoning 

is permanent in some cases.

Treatment for possible impending nephrotoxicity 

is firstly preventative, by ensuring adequate cardiovascular function and hydration. Renal output must 

be carefully monitored, and hydration appropriately 

adjusted, sometimes with the addition of diuretics. 

For envenoming where antivenom is available, early 

neutralization of venom may help prevent or moderate 

renal damage, but in some snakebites a microangiopathic hemolytic anemia (with secondary renal damage) can occur despite early and adequate antivenom 

treatment. The biomedical basis of this phenomenon 

is not well characterized.

Once significant renal failure has developed, dialysis—preferably hemodialysis—is the key to management. In many developing countries renal dialysis 

facilities are uncommon and because of their unavailability, some envenomed patients who may have otherwise survived die from renal failure. However, some 

severe patients may be poorly responsive to dialysis 

and have fatal outcomes despite aggressive treatment.

Necrotoxins

Necrotoxins are major components in some venoms, 

such as those of the recluse spiders (family Sicariidae, 

Loxosceles spp) and the Iranian scorpion, Hemiscorpius 

lepturus (family Hemiscorpiidae; this species does not 

have a generally used common name, but the genus 

is sometimes collectively called the Asian thin-tailed 

scorpions); while cytotoxic and hemorrhagic toxins in 

some snake venoms may cause similarly direct injury 

and necrosis around the bite site. Although several 

species of Hemiscorpius are found in Iran, Pakistan, 

Yemen, and Iraq, H lepturus occurs only in Iran and 

is the only known medically important taxon in the 

genus. There are no available data regarding the possible medical significance of other species, and these 

should be treated as dangerous until proven otherwise.

Recluse spider (Loxosceles spp) venoms contain 

sphingomyelinase D that initiates neutrophil recruitment and thereby ultimately stimulates neutrophilinduced cell lysis at the bite site. This destructive 

neutrophil response has been linked to locally 

elevated levels of complement such as C5a. Interestingly, sphingomyelinase D from Loxosceles spp venom 

also activates target cellular matrix proteases that 

cleave the C5a receptor, thereby initiating a protective mechanism against the elevated levels of C5a.89

The sphingomyelinases also alter the morphology 

of target cell membranes by transforming sphingomyelin into ceramide-1-phosphate. However, there 

are many gaps in the characterization of the precise 

pathophysiological mechanisms of some necrotoxins 

such as sphingomyelinase. Further elucidation of these 

mechanisms could provide information important to 

characterization of regulatory mechanisms governing 

local complement levels and autoimmune cellular 

responses in a variety of pathological states.

Clinically, local necrosis is often painful and obvious from an early stage, but recluse spider bites may 

be painless with few visible clinical manifestations in 

the first 12 to 24 hours, followed by the development 

of a classic “target” lesion (necrosing blue-black central skin, with surrounding pallor and an outer ring 

of erythematous reaction). This lesion can evolve over 

several days into epidermal necrosis that may be painful, and local blistering occasionally occurs. Necrosis 

following selected snakebites is usually painful and 

often accompanied by blistering, either centrally or at 

the margins of the evolving demarcated necrotic focus.

H lepturus is a particularly medically important 

species in Iran.90 It is the only scorpion species known 

to commonly cause hemoglobinuric nephropathy 

and subsequent renal failure. In a series of H lepturus

envenomings involving children younger than 10 

years, Afzali and Pezeshki91 reported that 8% had a 

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Figure 18-22. The venom delivery apparatus of a scolopendrid centipede. The fang-like structures located on each side 

of the head are modified legs (maxillipedes or forcipules) 

that deliver venom into grasped prey or a potential predator. Thus, centipedes deliver venom through stinging, rather 

than via a bite. 

Photograph: By Fritz Geller-Grim. Wikipedia Commons, 

public domain. https://commons.wikimedia.org/wiki/

File:Scolopendra_fg02.JPG.

fatal outcome and 2.2% succumbed to renal failure. 

Stings from this species also cause local edema and 

hemorrhagic effects. 

Treatment of toxin-induced necrosis is controversial. 

Early debridement of necrotic tissue may sometimes 

help, but for recluse spider bite it may actually increase 

the necrotic area. For this reason, current opinion 

discourages debridement, but delayed debridement 

may be necessary.92 Some patients have been treated 

with hyperbaric oxygen and other modalities, but have 

not resulted in consistent benefit, and thus cannot be 

generally endorsed without further supporting evidence.92 Secondary infection can occur and may either 

accelerate the necrosis, or even be the more important 

contributor to the necrotic process. Therefore, careful 

infection control is important, including appropriate 

antibiotics, as determined by anaerobic and aerobic 

culture, sensitivity determination, and regional trends 

in antibiotic resistance. 

Other Toxins

There are many lesser toxins that are either of minor 

clinical importance in humans, poorly understood, 

or clinically significant but uncommonly to rarely 

encountered. For example, the giant tropical and desert centipedes, particularly of the genus Scolopendra

(class Chilopoda, family Scolopendridae), are semifossorial and often nocturnal, and are not frequently 

encountered. These fast-moving active predators can 

inflict a painful sting with a pair of modified front 

legs often called maxillipedes, or forcipules (with the 

tarsungulum being the fang-like business end93) that 

are associated with pressurized venom glands (Figure 

18-22). Although of generally minor importance, stings 

from several species have caused mild to moderate 

local effects (eg, pain [sometimes severe], edema, 

secondary infection and necrosis, pruritis), as well as 

uncommon systemic effects (eg, nausea, headache), 

some of which may also be due to autonomic responses 

to pain or anxiety. There are a few anecdotal reports of 

fatalities, and Scolopendra subspinipes has been viewed 

as a species of potentially greater medical importance 

in some rural Southeast Asian communities. An S 

subspinipes reportedly inflicted a fatal sting in a child 

in the Philippines.94 These isolated reports require 

careful assessment and further documentation before 

assigning a defined hazard index for the species. 

However, the possible medical importance for giant 

centipedes may be underestimated in some regions, 

and rare deaths result from secondary complications 

of centipede stings (eg, necrotizing fasciitis).95

Several taxa and strains of the mold Aspergillus spp 

(family Trichocomaceae) produce difuranocoumarin 

derivatives, the aflatoxins, which are synthesized 

by a polyketide pathway. Interestingly, this fungal 

genus can be subjectively viewed as one that possibly contains the greatest contrasting mix of species 

because some provide respective benefits to humans 

(eg, utilization of A niger in the fermentation industry 

for citric acid synthesis96), yet others (eg, A flavus) are 

among the most medically hazardous, especially due 

to their toxins’ carcinogenicity. The most studied aflatoxins (B1, B2, G1, G2) were named as a result of their 

blue or green fluorescence under ultraviolet light and 

their thin-layer chromatographic mobility properties.96

These toxins are acutely toxic in sufficient doses and 

are also potent carcinogens. 

An accumulating body of interdisciplinary information suggests that interspecies variability in response 

to aflatoxins probably results from multifactorial influences including variability in the cytochrome P450 

system that converts aflatoxins to the reactive, DNA 

adduct-forming, and protein-binding 8,9-epoxide 

forms.97 Clinical signs of acute aflatoxicosis include 

hypolipidemia, hepatic steatosis, and necrosis.98 Etzel99

estimated the human LD50 (derived from murine data) 

of ingested aflatoxin B1 in liquid medium as 0.15 to 0.30 

mg/kg. The longer term, carcinogenic consequences of 

aflatoxin exposure are a result of toxin binding to both 

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TABLE 18-4

PRINCIPAL TYPES OF MUSHROOM POISONING

Mushroom Clinical Group* Principal Toxin(s) Major Effect

Group 1 — Amatoxic mushrooms, 

notably Amanita phalloides (death cap) 

and related spp, as well as several other 

taxa (eg, Lepiota spp; Figure T1) 

Amatoxins Delayed-onset cytotoxicity via inhibition of 

RNA polymerases II that arrest 

transcription resulting in cellular 

(especially hepatocellular) destruction

Figure T1. Lepiota spp (Agaricaceae), Outer Banks, Nags Hags 

Head Woods, North Carolina. Some taxa of these gilled 

mushrooms (eg, L helveola) contain amatoxins and ingestion 

of several species including L helveola has caused fatalities. 

Photograph: By Jason Hollinger. Wikipedia Commons, 

public domain. https://commons.wikimedia.org/wiki/

File:Lepiota_(4503849093).jpg. 

Figure T2. Fly agaric (Amanita muscaria), Ulm, southern Germany. Typically not fatal, ingestion of these mushrooms can 

produce agitation, elation, disorientation, depersonalization, 

manic excitement, visual misperceptions (rather than true 

hallucinations), confusion; the syndrome can be mistaken for 

alcohol intoxication. There can be ataxia, incoordination, dizziness, mydriasis, myoclonus, muscle fasciculation/tremors, 

hyporeflexia, coma, and in severe poisoning, especially in 

children, convulsions. Ingestion of the congener, A phalloides, can be fatal, and there are a substantial number of well 

documented life-threatening and fatal cases. 

Photograph: By Holger Krisp. Wikipedia Commons, public 

domain. https://commons.wikimedia.org/wiki/File:Amanitamuscaria-fliegenpilz-b.jpg. 

Group 2 — Gyrometrin-containing 

mushrooms, notably Gyromitra spp

Gyrometrin (hydrolyzed to 

monomethyl hydrazine)

Delayed-onset cytotoxicity; cellular 

destruction

Group 3 – Coprine-containing 

mushrooms (various)

Coprine (N-5,1-hydroxycyclopropyl-l-glutamine)Rapid autonomic effects, only in 

association with co-ingestion of alcohol 

(disulfarim-mimetic)

Group 4 – Muscarinic mushrooms, notably Inocybe spp, Clitocybe spp, Mycena

spp

Muscarine (2,5-anhydro-1,4,6-trideoxy6-(trimethylammonio)-dribo-hexitol)Rapid-onset neuroexcitatory effects 

(parasympathetic stimulation; classically 

sweating, salivation, and lachrymation)

Group 5 — Ibotenic mushrooms, notably 

Amanita muscaria (Figure T2) and 

Amanita pantherina 

Ibotenic acid, muscimol Rapid onset of central nervous system 

effects, including agitation, elation, 

disorientation, mania, and visual 

misperceptions

(Table 18-4 continues)

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Toxins From Venoms and Poisons

DNA and RNA, which cause inhibition of replication, 

transcription, and protein synthesis; protein binding 

can also cause enzyme inactivation.100

Although it may seem unlikely that carcinogenic 

toxins might be used as biological warfare agents, 

significant evidence indicates that aflatoxins from A 

flavus and A parasiticus were a significant part of Iraqi 

bioweapons development during the 1980s100 and were 

loaded into warheads and stockpiled.101–103 Several 

possible reasons for their selection as potential offensive agents have been considered including obvious 

stimulation of fear and terror, and simple exploitation 

of favored or available resources.97 However, consideration must be given to the possibility of intended 

infliction of longer term serious health effects on a 

given population (eg, as the initial Iraqi intent may 

have been directed against the Kurds in northern Iraq), 

a potential form of generational genocide. Thus, these 

toxins remain of concern on a relatively small scale 

because they are impractical as tactical weapons.104

Several filamentous fungi, particularly of the genus Fusarium (family Nectriaceae), produce tricothecenes such as T-2 and deoxynivalenol (vomitoxin), 

which are potent eukaryotic protein synthesis inhibitors and may disrupt initiation, elongation, and/or 

termination. There are several groups of these toxins 

including the nonmacrocyclic group (which contains 

an ester-ether bridge between C-4 and C-15 instead 

of a macrocyclic ester), which is further subclassified 

into structurally based groups, A and B.96 T-2 is a potent immunotoxin, which binds to the 60S ribosomal 

subunit and subsequently inhibits elongation; it also 

inhibits protein synthesis induction.105,106 Ingestion 

of T-2 toxin causes severe gastrointestinal distress 

including nausea, profuse diarrhea, vomiting, distention, and pain; those affected may also present 

with dizziness, chills, and other flu-like symptoms. 

T-2 toxins damage labile cells such as those in the 

gastrointestinal tract; they cause degeneration and 

necrosis of the lymphoid tissues and the surface and 

crypt epithelium of the gastrointestinal mucosa,104 as 

well as induction of thymic lymphocytic apoptosis.106

In humans T-2 toxin can induce apoptosis in megakaryocyte progenitors, and affected animals exhibit 

a notable loss of cell-mediated immunity.104,107 Due 

to the acute effects caused by T-2 and its natural 

Group 6 — Hallucinogenic mushrooms, 

notably spp of Psilocybe, Conocybe, 

Gymnopilus, Panaeolus, and others

Psilocybin (4-phosphoryloxyN,N-dimethyltryptamine; 

note: purchased hallucinogenic mushrooms may be 

laced with LSD and related 

substances)

Rapid onset of central nervous system 

effects including illusions (visual, 

auditory, or tactile), most commonly 

euphoric (but potentially sinister)

Group 7 — Gastrointestinal irritants, 

many species

Diverse array of toxins Rapid onset (1–3 h) of gastrointestinal 

effects (vomiting, diarrhea, or abdominal 

pain)

Group 8 — Orellanine (nephrotoxic) 

mushrooms, notably Cortinarius spp

Orellanine (2,2’-bipyridine3,3’,4,4’-tetrol-1,1’-dioxide)Delayed-onset cytotoxicity; renal failure 

often occurs days after ingestion

Group 9 — Nephrotoxic mushrooms, 

notably Amanita smithiana and 

A pseudoporphyria

Aminohexadienoic acid Delayed-onset cytotoxicity; renal failure 

with prerenal symptoms occurring 

0.5–12 h later

Group 10 — Primary myolytic 

mushrooms, notably Tricholoma equestre

Unknown (note: these are 

normally edible mushrooms)

Delayed-onset myolysis due to unknown 

mechanism or association

Group 11 — Myolytic mushrooms, 

notably Russula subnigricans

Unknown Rapid-onset myolysis 

Group 12 — Pain-inducing mushrooms, 

notably Clitocybe acromelalga

Acromelic acid (several toxins; 

eg, acromelic acids A and B; 

all are heteroaromatics)

Rapid onset of rash, edema, and digit-tip 

pain (allodynia)

*The groupings listed here are based on the type of clinical presentation, rather than toxin type or mushroom species. Some groups may 

have many mushrooms, while others may have only a single species known to cause the specific clinical syndrome. From a clinical perspective, patients frequently ingest mushrooms of multiple species and potentially from multiple groups; thus, the clinical picture in any given 

patient may be a mixture of effects from a variety of mushroom groups. Clinical cognizance is especially important in relation to potentially 

lethal groups, particularly Group 1 amatoxic mushrooms, when coingested with other mushrooms causing gastrointestinal disturbance, as 

this may lead to a mixed clinical picture initially suggestive of poisoning by nonlethal Group 7 species.

Data source: (1) White J, Weinstein SA, De Haro L, et al. Mushroom poisoning: a proposed new clinical classification. Clin Toxicol. 2017 

(under review). 

Table 18-4 continued

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Figure T3. (A) Castor bean or castor oil plant (Ricinus communis). This common member of the Euphorbiaceae contains the 

potent heterodimeric, type 2 ribosome inactivating cytotoxin, 

ricin. This toxin has limited offensive tactical potential, but 

does pose a danger for possible small-scale offensive use. 

Several well-publicized incidents have occurred in which 

intercepted ricin-containing letters were mailed to prominent 

A B

public figures, including the former Mayor of New York City, Michael Bloomberg, and President Barack Obama. This toxin 

has been used in several well-known perpetrated or attempted assassinations. (B) Castor bean (Ricinus communis) seeds.

Photographs: By H Zell. Wikipedia Commons, public domain. (A) https://commons.wikimedia.org/wiki/Ricinus_communis#/media/File:Ricinus_communis_005.JPG. (B) https://commons.wikimedia.org/wiki/File:Ricinus_communis_008.JPG. 

TABLE 18-5

SELECTED PRINCIPAL TYPES OF PLANT POISONING

Plant Clinical Group Principal Toxin(s) Major Effects

CYTOTOXIC PLANTS

Ricinus communis (castor 

bean, castor oil plant; 

Figure T3) 

Ricin is a heterodimeric subunit 

protein that belongs to the ribosome inactivating protein family. 

One subunit (B chain) is a lectin 

and the other (A chain) is responsible for most of the toxicity.

Cytotoxic, inhibits protein synthesis by catalytic (via 

N-glycosidase) depurination of the 28S RNA, thereby 

arresting translation. The toxin can potentially cause 

lethal multiorgan failure, but most cases of simple ingestion (eg, raw ingestion of intact or slightly disrupted 

seeds) cause nonlethal gastrointestinal effects only.

Colchicum autumnale (autumn crocus, meadow 

saffron; Figure T4)

Colchicine (antimitotic; mitotic 

spindle poison)

Concentrates in polymorphonuclear leukocytes and 

inhibits microtubule assembly. Abdominal pain, vomiting, diarrhea, paralysis, convulsions, hypovolemic 

shock, respiratory failure, bone marrow failure.

Abrus precatorius (jequirty pea)Abrin (also a ribosome inactivating 

protein)

Concentrates in polymorphonuclear leukocytes and 

inhibits microtubule assembly. Abdominal pain, vomiting, diarrhea, paralysis, convulsions, hypovolemic 

shock, respiratory failure, and bone marrow failure.

(Table 18-5 continues)

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Toxins From Venoms and Poisons

Symphytum officinale

(comfrey) and others

Pyrrolizidine alkaloids Cyotoxic, toxic mechanisms uncertain, causes multiorgan 

damage, especially jaundice secondary to fibrosing 

hepatic venoocclusive disease.

Mentha pelugium

(pennyroyal)

Pelugone Cytotoxic, causing potentially fatal hepatotoxicity.

(Table 18-5 continues)

Table 18-5 continued

Figure T5. Purple foxglove (Digitalis purpurea). The genus 

Digitalis contains approximately 20 species of perennials, 

shrubs and biennials. The leaves of some of these such as D 

purpurea contains the steroid, digoxin, the extract of which 

is used to produce digitalis and other related medications 

that are centrally important in the management of congestive 

heart failure and several dysrhythmias. Its mechanism of 

action targets the Na+

/K+-ATPase pump on the cytoplasmic 

membrane of myocardiocytes (see Table 18-5), and acts as a 

positive inotrope. Its discovery is attributed to the Scottish 

physician, Dr William Withering, who reportedly obtained 

an early herbal mixture containing Digitalis spp from a 

gypsy, which led to his noting its effective therapeutic uses. 

Although it has no tactical potential, it has been used as a 

poison targeting individuals.

Photograph: Courtesy of Julian White.

Figure T4. Autumn crocus or meadow saffron (Colchicum autumnale). This genus Colchicaceae contains more than 160 taxa. 

Colchicum autumnale is one of the species that contains the 

cytotoxic alkaloid, cochicine, an important pharmacotherapeutic especially used in the treatment of gout. Colchicine is 

a potent mitotic spindle poison, and has been suspected in 

several cases of intentional poisoning, but has no significant 

tactical potential.

Photograph: By Meneerke Bloem. Wikipedia Commons, 

public domain. https://commons.wikimedia.org/wiki/

File:Colchicum_autumnale_clump_02.jpg. 

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Table 18-5 continued

(Table 18-5 continues)

CYANOGENIC PLANTS

Prunus spp (apricot, 

almond, peach, plum, 

apple, etc); hydrangea; 

cassava and others

Amygdaline Metabolized to form hydrocyanic acid, causes metabolic 

failure, potentially lethal.

CARDIOTOXIC PLANTS

Nerium oleander, Thevetia 

peruviana (oleanders; 

see Figure 18-20)

Cardiac glycosides (digitalis-like; 

oleandrin, nerium, thevetin, etc)

Cause digoxin-type cardiotoxicity by binding to and inactivating the Na+

/K+-ATPase pump on the cytoplasmic 

membrane of myocardiocytes. As a result, the intracellular Na+

 concentration increases, and this affects the 

Na+/Ca2+ exchange channels, thereby resulting in an increased intracellular Ca2+. This causes a positive inotropic effect and also raises the resting membrane potential 

of the cell, leading to increasing rates of spontaneous 

cellular depolarization and myocardial automaticity. 

These effects can cause potentially lethal dysrhythmias, 

conduction anomalies, hyperkalemia, and death.

Carissa spectabilis

(wintersweet)

Digoxin-like toxin Cause digoxin-type cardiotoxicity by binding to and inactivating the Na+

/K+-ATPase pump on the cytoplasmic 

membrane of myocardiocytes. As a result, the intracellular Na+

 concentration increases, and this affects the 

Na+

/Ca2+ exchange channels, thereby resulting in an increased intracellular Ca2+. This causes a positive inotropic effect and also raises the resting membrane potential 

of the cell, leading to increasing rates of spontaneous 

cellular depolarization and myocardial automaticity. 

These effects can cause potentially lethal dysrhythmias, 

conduction anomalies, hyperkalemia, and death.

Digitalis purpurea, D lantana (foxglove) (Figure 

T5)

Digoxin Cause digoxin-type cardiotoxicity by binding to and inactivating the Na+

/K+-ATPase pump on the cytoplasmic 

membrane of myocardiocytes. As a result, the intracellular Na+

 concentration increases, and this affects the 

Na+

/Ca2+ exchange channels, thereby resulting in an increased intracellular Ca2+. This causes a positive inotropic effect and also raises the resting membrane potential 

of the cell, leading to increasing rates of spontaneous 

cellular depolarization and myocardial automaticity. 

These effects can cause potentially lethal dysrhythmias, 

conduction anomalies, hyperkalemia, and death.

Convallaria majalis (lily of 

the valley)

Convallatoxin (cardiac glycoside) As for other cardiac glycosides, but less potent.

Taxus breviffolia, T baccata

(yew tree) (Figure T6)

Taxines (alkaloids derived from 

esterified diterpenes)

Cardiotoxins affecting cardiomyocytes by antagonizing 

sodium and calcium channels. These can potentially 

cause cardiac failure, but raw ingestions of plants are 

rarely lethal. The effects of isolated toxin ingestion are 

poorly documented, but probably carry a high risk of 

fatal outcome.

Veratrum spp (hellebore) Veratridines (steroid alkaloids) Act as agonists of the NaV and have neurotoxic and 

cardiotoxic effects. These toxins most commonly cause 

hypotension and bradycardia.

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Table 18-5 continued

(Table 18-5 continues)

Figure T8. Belladonna, deadly nightshade (Atropa belladonna). 

Another genus in the family Solanaceae, and the source for 

several very important tropane alkaloids used as pharmacotherapeutics: atropine, scopolamine, and hyoscyamine. The 

genus is named for Atropos, one of the three female deities 

who supervised fate, the Three Fates, and was the one who 

cut the thread of life. This is a worthy appellation, because 

these plants also can cause a potentially fatal classic anticholinergic syndrome as noted for Brugsmansia (see Figure T7 

caption and Table 18-5). 

Photograph: By Rüdiger Kratz. Wikipedia Commons, public 

domain. https://commons.wikimedia.org/wiki/File:Atropabella-donna_Staude_102_b.jpg.

Figure T7. Angel’s trumpet (Brugsmansia spp). The genus 

Brugsmansia consists of about seven Solanaceae taxa that 

feature large flowers with notably strong aroma. Ingestion of parts of these plants causes a classic anticholinergic 

syndrome, including delirium, hallucinations, tachycardia, 

blurred vision, dry mucosa, and seizures. Serious poisoning 

can result in death. 

Photograph: Courtesy of Julian White.

Figure T6. Pacific Yew tree (Taxus brevifolia), Wenatchee 

Mountains, Wenatchee National Forest, Washington. The 

genus Taxus contains approximately seven species of monoecious or dioecious trees and shrubs. Taxus extracts have 

been long recognized as poisons, and the component alkaloids, the taxines, are potent cardiotoxins that antagonize 

cardiomyocyte ion channels (see Table 18-5). The yew tree 

also contains the diterpenes, the taxanes, which function as 

mitotic inhibitors. The very important chemotherapeutic 

agents, paclitaxel and docetaxel, are particularly used to 

treat a wide variety of solid neoplasms.

Photograph: By Walter Siegmund. Wikipedia Commons, 

public domain. https://commons.wikimedia.org/wiki/

File:PacificYew_7790.jpg. 

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ANTICHOLINERGIC PLANTS

Brugmansia spp (angel’s 

trumpet) (Figure T7), 

Datura spp (jimsonweed), Atropa belladonna (deadly nightshade) 

(Figure T8), Hyoscamus niger (henbane), 

Mandragora officinarum

(mandrake), and 

others

Anticholinergics (scopolamine, 

atropine, hyoscyamine, etc)

Classic anticholinergic toxidrome (delirium, hallucinations, pupillary dilatation, blurred vision, dry skin/

mucosa, hyperthermia, flushed skin, tachycardia, 

hypertension, potentially urinary retention, coma, convulsions, and death).

NEUROTOXIC PLANTS

Nicotiana spp (tobacco 

tree)

Nicotine (bicyclic alkaloid; structurally resembles acetylcholine, 

the ligand for the receptor to 

which it binds)

Binds with the nicotinic acetylcholine receptor and causes 

excitation with tremor, tachycardia, sweating, convulsions, gastrointestinal effects, then depressant effects 

including coma, paralysis, bradycardia, and cardiovascular collapse; potentially fatal.

Conium maculatum (poison hemlock)Coniine (piperidine alkaloid) Causes nicotine-like effects (see nicotine, above).

Aconitum spp (aconite) Aconitine (aconitum alkaloid) Acts as an agonist of tetrodotoxin-sensitive NaV; causes 

excitation, widespread paresthesia, muscle weakness, 

hypotension, cardiac arrhythmias, gastrointestinal effects (vomiting, diarrhea, abdominal pain), sweating, 

lacrimation, confusion, headache, and death.

Cicuta spp (water hemlock)Cicutoxin (a C17-conjugated polyacetylene)Noncompetitively antagonizes γ-aminobutyric acid 

(GABA) receptors thereby cause unregulated central 

nervous system neuronal depolarization. Delayed onset 

of oral mucosal pain (oral ingestion), abdominal pain, 

vomiting, coma, frothing at the mouth, convulsions, 

and death. 

Laburnum anagyroides

(Laburnum; Faboideae)

Cytisine (tricyclic quinolizidine 

alkaloid)

Acts as a partial agonist of nicotinic acetylcholine receptors containing specific combinations of the α4 and β2 

subunits. Delayed-onset salivation, sweating, vomiting, 

delirium, excitation, convulsions, respiratory paralysis, 

and death. 

Solanum spp (nightshades, bittersweet, 

Jerusalem cherry, 

potato; Solanaceae)

Solanine and related glycoalkaloidsFunction as reversible inhibitors of human plasma cholinesterase, and may also be cytotoxic. Poisoning may 

include vomiting, diarrhea, dilated pupils, drowsiness, 

cholinesterase inhibition, respiratory failure, and death.

Cannabis sativa (marijuana)Cannabinoids (tetrahydrocannabinol)Depression or excitation, tremors, hallucinations, and 

aberrant behavior.

Cycads (number of taxa 

belonging primarily 

to Cycadaceae and 

Zamiaceae)

Cycasin (a nitrogen-containing 

methylazoglucoside)

In mammals, cycasin undergoes modification (cleavage) 

in vivo and forms methylazoxymethanol resulting in 

acute intoxication. It is probably teratogenic and is a 

tumor initiator in experimental rodents. It has been 

strongly implicated as a cause of Pacific parkinsonism 

dementia/amyotrophic lateral sclerosis complex.

NEPHROTOXIC PLANTS

Rheum rhaponticum (rhubarb; Polygonaceae)Oxalates Soluble oxalates cause local irritation/corrosion and 

potential renal damage related to the excretion of the 

oxalate crystals.

Table 18-5 continued

(Table 18-5 continues)

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Table 18-5 continued

GASTROINTESTINAL IRRITANTS

Juniperus sabina (juniper; Cupresaceae)Diterpenics (communic acids; J 

communis also contains isocupressic acid, an abortificant)

Ingestion of oils, berries, and other parts of the plant can 

cause vomiting and diarrhea.

Wisteria spp (wisteria; 

Fabaceae)

Wisterin (glycoside most commonly reported from seeds and 

pods)

Reports suggest that ingestion causes gastroenteritis that 

may be severe, particularly in children.

Melia azedarach (white 

cedar; Meliaceae)

Limonoids (oxygenated, modified 

triterpenes) and other 

triterpenoids

Ingestion of the fruits can cause gastrointestinal effects, 

coma, and convulsions; potentially fatal in severe cases.

LOCAL IRRITANTS

Rheum rhaponticum

(rhubarb, Polygonaceae), Dieffenbachia spp 

(dumbcane, Araceae), 

Zantedeschia aethiopica (arum lily, Araceae), Philodendron spp 

(Araceae), and other 

oxalate-containing 

plants

Oxalate salts Oxalate crystals released when plant (especially parts 

that contain high concentrations such as Rheum leaves) 

is chewed, causing local pain and edema; may cause 

dermal irritation in some individuals.

Euphorbia spp (poinsettia, candelabra cactus, 

etc, Euphorbiaceae)

Phorbol esters (tigliane diterpenes) Contact with sap causes intense local irritation to 

mucosal membranes and the eye; some authors have 

reported potentially blinding effects. These compounds 

mimic the action of diacylglycerol; thereby may activate 

protein kinase C and function as tumor promoters. 

Toxicodendron spp (poison ivy, poison sumac, 

and poison oak; Anacardiaceae), Metopium

spp (poisonwood, 

Anacardiaceae)

Urushiols (3-substituted catechols) Contact with the plant, including the intact leaves causes 

local skin irritation. Some species (eg, T verniciflua, 

Melanorrhea usitata) containing urushiol or related 

irritants are sometimes used in the preparation of 

furniture varnish and may present an occupational 

irritant hazard.

LSD: lysergic acid diethylamide; NaV: voltage-gated sodium channel

Data sources: (1) Brent J, Wallace KL, Burkhart KK, et al (eds). Critical Care Toxicology: Diagnosis and Management of the Critically Poisoned 

Patient. 1st ed. Philadelphia, PA: Elsevier-Mosby; 2005: 1,690 pp. (2) Riet-Correa F, Psister J, Schild AL, Wierenga TL (eds). Poisoning by Plants, 

Mycotoxins and Related Toxins. Oxfordshire, England: CABI; 2011: 660 pp. (3) Information synthesized from lectures presented by the authors.

occurrence in rice, it remains an agent that attracts 

concern about possible offensive use. However, as 

noted in regard to aflatoxins, such reasonable concern should be balanced with their impracticality for 

tactical applications.

The toxins in cytotoxic mushrooms, particularly the 

potent transcription inhibitors, the bycyclic heterogeneous octapeptide amatoxins, are clinically important 

because they regularly cause fatal poisoning after 

accidental ingestion. Cytotoxic mushrooms include 

the death cap (Amanita phalloides) and other gilled 

mushrooms that may closely resemble edible species 

such as some Lepiota spp (Table 18-4 and Figure T1). 

Amatoxin acts by inhibiting DNA-dependent RNA

polymerase II, thereby stimulating hepatocellular 

acute death and apoptosis. Amanita poisoning is 

characterized by delayed—typically 8 to 12 hours—

severe gastrointestinal effects, followed by apparent 

recovery; 2 to 4 days later, progressive onset of liver 

failure occurs, which can be fatal. Early recognition 

of the risk, timely charcoal decontamination when 

possible, and treatment with silibinin, penicillin G, or 

d-penicillamine may reduce the severity of liver damage, but after closure of the early treatment window, 

patients with major liver failure can only be managed 

with liver transplant. However, transplants can be 

either medically impractical because the patient is 

already too ill or a transplantable liver is unavailable.

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The hallucinogens (eg, alkaloids such as mescaline, 

indole alkaloids such as psilocybin) and other nonlethal toxins found in some mushrooms (eg, Psilocybe

spp, “magic mushrooms”) or cactus (eg, the spineless 

cactus, Lophophora williamsii, peyote) cause characteristic clinical effects (see Table 18-4). Treatment varies 

with the toxin type, but is most commonly symptomatic and supportive. Some Psilocybe spp are sold for 

recreational purposes in Amsterdam, and thus are 

legally available in quantity.

There is a vast array of poisonous plants. A particularly well-studied example of a potent plant-derived 

cytotoxin is ricin, the seed-derived heterodimeric, 

type 2 ribosome-inactivation protein from Ricinus communis (castor bean or oil plant, Euphorbiaceae; Table 

18-5 and Figures T3A, B)108. Ricin contains A and B 

polypeptide chains with molecular masses of 32 and 

34 kDa, respectively, that are covalently linked via a 

single disulphide bond. The lectin-binding properties 

associated with the B chain aids entry into the target 

cell, which leads to endocytic vesicle internalization, 

thereby facilitating its retrograde transport through 

the Golgi and endoplasmic reticulum. It enters the 

cytosol, where the A-chain re-natures and then inactivates ribosomes.107 The ribosomal inactivation is effective because one molecule of A chain can inactivate 

1,777 ribosomes per minute,109 and is enzymatically 

accomplished because the A chain is a highly active Nglycosidase responsible for selectively deadenylating 

the first adenine in a GAGA sequence in the α-sarcin/

ricin loop of 28S rRNA.107,110 Removal of this adenine 

prevents mammalian elongation factor-2 from binding to the ribosome,111 thus blocking protein synthesis 

and activating apoptotic pathways.112 Ricin poisoning 

can lead to fulminant multiorgan failure and death. 

Bozza et al (2013) list at least seven incidents involving 

potential terrorist actions or assassinations (planned 

or completed) involving ricin.109 Therefore, although 

having limited offensive military potential, ricin still 

poses a significant danger for possible small-scale offensive use. 

Other plant toxins could conceivably be used in a 

limited offensive manner like ricin. Selected principal 

types of plant poisoning are shown in Table 18-5. 

CONCLUSIONS AND DIRECTIONS FOR RESEARCH

Only a relatively small minority of natural toxins 

(especially animal-derived) have been thoroughly 

characterized, and the clinical effects and management 

of this limited group are similarly well understood. 

Most venomous and poisonous organisms have yet 

to be subjected to any toxinologic research, so their 

possible risk to humans is unknown. Unfortunately, 

limited public and private funding is available for most 

toxinological investigations.

There is limited potential for offensive military applicability of venom toxins because of their physical 

and biochemical characteristics (eg, unpredictable 

stability of some purified toxins), as well as limitations on practical use per routes of administration, and 

even marked species-specificity of some toxins. The 

experimental lethal potencies of some venom toxins 

are notable and far greater than that of cyanide (eg, 

the presynaptic neurotoxin textilotoxin, from venom 

of the Australian Eastern brown snake [murine ip LD50

1 μg/kg] is approximately 3,000-fold more potent than 

that for oral ingestion of sodium cyanide by nonfasted 

rats [3 mg/kg]).113 However, bacterial toxins such as 

botulinum toxins a–g (murine LD50 1–2 ng/kg; toxin d 

does not have affinity for human tissues)114,115 or readily 

dispersible organophosphate nerve agents such as the 

cholinesterase inhibitor sarin (methylphosphonofluoridic acid l-methylethyl ester; acute toxicity [LCt50] in 

resting humans estimated to occur with inhalation 

of 70 mg–min/m3

 aerosolized agent)116 are far more 

concerning as offensive biological weapons. This concern is largely because of their toxicity and potential 

practical application per mass delivery or population 

exposure. Furthermore, although textilotoxin is the 

snake venom toxin with the highest experimental 

(murine) lethal potency, few patients envenomed by 

Pseudonaja spp whose venoms contain this toxin present with—or develop—paralytic features. In contrast, 

very low doses of either botulinum toxins or sarin 

produce severe neurotoxicity in humans with a high 

risk of lethal outcome. 

However, some plant toxins (eg, ricin), as well as 

fungal toxins (eg, aflatoxins such as aflatoxin B1, and 

the trichothecenes T-2 toxin and deoxynivalenol), have 

either been offensively used in a small scale (in political assassinations), or have been suspected of being 

deployed against small populations (eg, suspected 

yellow rain in Southeast Asia and Afghanistan).104

They remain a threat as a biological weapon, as does 

the possible small scale offensive application potential 

of tetrodotoxin. 

Even for well-known causes of envenoming or poisoning, major gaps in knowledge exist, and optimized 

(or unambiguous) evidence-based treatment strategies 

have not been developed. Where antidotes such as 

antivenom are available, they may not be optimal in 

design and function, and no viable antivenoms can 

counter the effects of the venoms from many medically 

important venomous animals.

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An important starting point for toxinology research 

is to understand the epidemiology of envenoming and 

poisoning so realistic risk profiles can be generated. 

Such data are often scant and fragmentary, or of 

questionable validity. Major methodological hurdles 

exist in collecting valid and useful epidemiologic data 

on toxin-induced diseases, not least because even the 

taxonomy of some of the fauna and flora of concern 

remains incomplete or uncertain. These problems 

are compounded by the tendency in developing nations—particularly in rural populations—for patients 

to be treated outside the health system, mostly by 

native healers, witch doctors, or shamans. Thus, even 

where government data on presentations for envenoming and poisoning are available, they may represent only a small minority of the actual percentage of 

the population affected by venomous or poisonous 

animals, fungi, and plants. Therefore, it is necessary 

to conduct detailed community-based surveys such 

as the Million Death Study of snakebite mortality in 

India,117 in addition to garnering prospective hospitalbased statistics.

Once important risk organisms are identified, a 

targeted toxin identification process is required if the 

toxins have not been previously elucidated. This process may require complex fractionation of the venom or 

poison to locate relevant toxins, including test systems 

to identify important potential activities. Sometimes 

a global “fishing” approach may be required, as used 

in the discovery of the huge diversity of action of cone 

snail toxins, where systematic injection of each individual toxin into test mice revealed a complex array 

of precisely acting toxins. This facilitated a detailed 

examination of individual toxins to understand how 

they exerted their often highly specific action in the 

central nervous system. Both in vitro and in vivo 

experimentation are usually required to understand 

the potential clinical effects of a given toxin and the 

mechanistic basis of its pathophysiological effects.

Recognizing the risks associated with a venomous 

or poisonous species and then identifying the medically important toxins opens the door to development 

of specific treatments. These treatments may be either 

pharmacologic antidotes to specific toxin actions, or 

antibody-based antidotes such as antiserums, developed to neutralize either specific toxins (eg, cardiotoxins such as oleandrin that are neutralized by antidigoxin antibodies) or antivenoms developed against 

mixtures of toxins present in venoms. Efforts to standardize and optimize production of antivenoms have 

recently been discussed in detail in a World Health 

Organization publication, which is essential reading 

for those undertaking antivenom production.118

Biomedical research identifying animal and plantderived toxins of medical importance and their biological activities should be elevated to a significantly 

greater level of priority. It is also essential that the 

evidence-based assessment of medical management 

of envenoming and other toxin-based diseases and 

emergencies should be recognized as a significant 

public health problem among a large proportion of 

the world’s population.119-121 The World Health Organization recently removed snakebite from its previous 

inclusion as an “other neglected tropical disease.” 

Thus, the unknown and undoubtedly substantial, as 

well as underestimated, human cost of envenoming 

in many economically disadvantaged nations remains 

very much “neglected.” The likelihood of military 

deployments in some of these regions adds to the importance of carefully assessing and addressing the risks 

of venom diseases and poisoning syndromes, and this 

is where the military importance of venomous animals 

and their venoms, as well as poisonous organisms and 

plants, are most relevant. 

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Chapter 19

MARINE ALGAL TOXINS OF 

CONCERN AS INTENTIONAL 

CONTAMINANTS

MARK A. POLI, PhD, DABT,* and MARK R. WITHERS, MD, MPH†

INTRODUCTION

Paralytic Shellfish Poisoning 

Neurotoxic Shellfish Poisoning 

Amnesic Shellfish Poisoning

Palytoxin

SUMMARY

*Research Chemist, Department of Applied Diagnostics, Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter 

Street, Fort Detrick, Maryland 21702

†

Colonel, Medical Corps, US Army; Clinical Director, Office of Medical Support and Oversight, US Army Research Institute of Environmental Medicine, 

15 Kansas Street, Natick, Massachusetts 01760; formerly, Deputy Chief, Division of Medicine, US Army Medical Research Institute of Infectious Diseases, 

1425 Porter Street, Fort Detrick, Maryland 

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INTRODUCTION

be harvested from laboratory cultures of the toxic 

organism, yields are insufficient to supply the large 

amounts required for deployment in traditional biological warfare aerosols or munitions.

Targeting food supplies as an act of biological terrorism is a more likely scenario. The toxins occur naturally in seafood products in concentrations sufficient 

to cause incapacitation or death. The contaminated 

foodstuffs appear fresh and wholesome, and cannot 

be differentiated from nontoxic material except by 

chemical analysis, which obviates the need for isolation 

of large quantities of pure toxins and subsequent adulteration of the food supply. In theory, the toxic seafood 

needs only to be harvested and then introduced into 

the food supply at the desired location. Regulatory 

testing, if any, is typically done only at the harvester 

and distributor levels. The natural occurrence of these 

toxins in seafood may provide cover for an act of intentional bioterrorism.

In some cases, harvesting toxic seafood is difficult. 

In the case of ciguatoxin, contaminated fish are typically a small percentage of the catch, and levels of toxin 

within toxic fish tissues are extremely low. In other 

cases, harvesting could be easy. The United States and 

other countries maintain monitoring programs at the 

state and local levels to ensure consumer safety. On 

the US Gulf Coast, concentrations of toxin-producing 

dinoflagellate Karenia brevis in the water column are 

closely monitored. When cell numbers increase to 

levels suggestive of an imminent bloom, harvesting 

of shellfish is officially halted. The shellfish are then 

monitored by chemical analysis or mouse bioassay 

until toxin concentrations in the edible tissues fall to 

safe levels, at which point harvesting is allowed to 

resume. During the period when shellfish are toxic, 

information is made available through the news media 

and regulatory agencies to discourage recreational 

harvesting, and anyone could conduct surreptitious 

harvesting during such a time.

Of the six marine toxin syndromes, three—ciguatera fish poisoning, diarrhetic shellfish poisoning, and 

azaspiracid poisoning—are unlikely to be significant 

bioterrorism threats. Diarrhetic shellfish and azaspiracid 

poisoning cause mild to moderate intoxications that 

are self-limiting and likely to be mistaken for common gastroenteritis or bacterial food poisoning; the 

syndromes are unlikely to cause the sorts of disruptions sought by terrorists. Ciguatera fish poisoning can 

present a more serious intoxication, but toxic fish are 

difficult to procure. Acquiring sufficient toxin to launch 

a food-related bioterrorist attack of any magnitude is 

nearly impossible.

Marine biotoxins are a problem of global distribution, estimated to cause more than 60,000 foodborne 

intoxications annually. In addition to human morbidity, some marine toxins may cause massive fish kills, 

such as those occurring during the Florida red tides. 

Others have been implicated in mass mortalities of birds 

and marine mammals. However, their presence in the 

environment is more often “silent,” unless detected 

by monitoring programs or when contaminated foodstuffs are ingested.

The long-term environmental and public health effects of chronic exposure in humans have not been extensively studied, although questions are beginning 

to arise about whether chronic exposures to some 

marine toxins, such as okadaic acid, may increase the 

risk of cancer through their action as tumor promoters. Intoxication syndromes from ingestion of marine 

toxins have long been known, primarily associated with 

molluscan shellfish. These syndromes include paralytic 

shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), diarrhetic shellfish poisoning, amnesic 

shellfish poisoning (ASP), and azaspiracid poisoning. 

Another important marine intoxication is ciguatera fish 

poisoning, which is caused by ingesting contaminated 

finfish. Although for the most part, molluscan shellfish 

are the primary vectors to humans, filter feeding and 

other trophic transfers can result in occurrence in other 

seafood such as crustaceans and finfish.

With the exception of ASP, which is of diatom origin, 

the causative toxins all originate from marine dinoflagellates. More recently, palytoxin—a marine toxin 

originally described from zoanthid soft corals of the 

genus Palythoa in the Pacific Ocean, but now known 

to be produced by the dinoflagellate Ostreopsis spp—

has become a potential human health problem in the 

Adriatic and Mediterranean seas. The toxin-producing 

algal species are a small fraction of the thousands of 

known phytoplankton species. However, under the 

proper environmental conditions, they can proliferate 

to high cell densities known as blooms. During these 

blooms, they may be ingested in large quantities by 

zooplankton, filter-feeding shellfish, and grazing or 

filter-feeding fishes. Through these intermediates, 

toxins can be vectored to humans who consume the 

seafood.

In general, marine algal toxins are not viewed as 

important biological warfare threat agents for several 

reasons. Marine toxins occur naturally at low concentrations in wild resources, and extraction of large 

quantities is difficult. Most are nonproteinaceous and, 

therefore, not amenable to simple cloning and expression in microbial vectors. Although some toxins can 

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The three marine algal toxin syndromes with bioterrorism potential and their causative toxins (Table 19-1) 

are described in the following sections. In addition, 

a brief description of palytoxin and its physiological 

effects is presented. Some of these effects are of greater 

concern for homeland security than others. Issues that 

may impact or limit their potential use as weapons of 

bioterror will be discussed, followed by clinical aspects 

and treatment.

Paralytic Shellfish Poisoning

Description of the Toxin

PSP results from exposure to a family of toxins called 

paralytic shellfish poisons, or saxitoxins. Saxitoxin (STX) 

was the first known member of this family, named for 

the giant butter clam, Saxidoma giganteus, from which it 

was first isolated.1 Later it was learned that STX is the 

parent compound of more than 20 derivatives of varying potency produced by marine dinoflagellates of the 

genera Alexandrium (previously Gonyaulax), Pyrodinium, 

and Gymnodinium, as well as several species of freshwater cyanobacteria. In the 1990s, STX was isolated from 

bacterial species associated with dinoflagellate cells, 

suggesting the possibility of a bacterial origin in at least 

some dinoflagellates.2

 STX also occurs in other benthic 

marine organisms, such as octopi and crabs, from which 

the ultimate source of toxin is unknown but assumed 

to be the food web.3

In humans, the greatest risk is associated with 

consumption of filter-feeding mollusks such as clams, 

mussels, and scallops that ingest dinoflagellate cells 

during bloom conditions or resting cysts from the 

sediment. The original toxin profiles in the dinoflagellate cells may be metabolically altered by the shellfish. 

Ingestion by humans results in signs and symptoms 

characteristic of PSP. Approximately 2,000 cases occur 

annually across regions of North and South America, 

Central and South America, Europe, Japan, Australia, Southeast Asia, and India. PSP-related fatalities 

have been reported in South Africa, Canada, Chile, 

Guatemala, and Mexico. Because of this, numerous 

monitoring programs are now in place worldwide, 

which have minimized risks and drastically reduced 

fatalities.4 The overall mortality rate has been estimated 

at 15%,5 although mortality is highly dependent on the 

quality of medical care received. Etheridge provides a 

review of PSP toxins from a human health perspective.4

Mechanism of Action

STX and its derivatives elicit their toxic effects by 

interacting with the voltage-dependent sodium channels in electrically excitable cells of heart, muscle, 

and neural tissue. High-affinity binding to a specific 

binding site (denoted neurotoxin binding site 1) on 

sodium channels blocks ionic conductance across the 

membranes, thereby inhibiting nerve polarization. Although voltage-dependent sodium channels in many 

TABLE 19-1

COMPARISON OF SELECTED MARINE ALGAL TOXINS

Paralytic Shellfish Poisoning Neurotoxic Shellfish Poisoning Amnesic Shellfish Poisoning

Toxin Gonyautoxins (saxitoxin) Brevetoxins Domoic acid

Source Marine dinoflagellates Karenia brevis Pseudo-nitzschia multiseries

Mechanism of action Binds to site 1 of voltage- Binds to site 5 of voltage- Binds to kainate and AMPA

dependent sodium channels, dependent sodium channels subtypes of glutamate recepleading to inhibition of nerve and prevents channel tors in the central nervous

polarization. inactivation. system, leading to excitotoxic

effects and cell death.

Clinical manifestations Circumoral parasthesias that Symptoms similar to paralytic Vomiting, diarrhea, and abmay rapidly progress to the shellfish poisoning, but usually dominal cramps, which may

extremities. May result in milder. Nausea, diarrhea, and be followed by confusion,

diplopia, dysarthria, and abdominal pain. Neurological disorientation, and memory

dysphagia. Progression may symptoms include oral loss. Severe intoxications

lead to paralysis of extremities parasthesias, ataxia, myalgia, may result in seizures, coma,

and respiratory musculature. and fatigue. or death. 

AMPA: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

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tissues are susceptible to these toxins, pharmacokinetic 

considerations make the peripheral nervous system the 

primary target in seafood intoxications.

Clinical Signs and Symptoms

Ingestion. Ingestion of PSP toxins results in a rapid 

onset (minutes to hours) complex of paresthesias, 

including a circumoral prickling, burning, or tingling 

sensation that rapidly progresses to the extremities. At 

low doses, these sensations may disappear in a matter of 

hours with no sequelae. At higher doses, numbness can 

spread to the trunk, and weakness, ataxia, hypertension, 

loss of coordination, and impaired speech may follow. 

Death has been known to occur in as little as 3 to 4 

hours.6 

A 20-year retrospective analysis of PSP documented 

by the Alaska Division of Public Health from 1973 to 

1992 revealed 54 outbreaks involving 117 symptomatic 

patients. 7 The most common symptom in these outbreaks was paresthesia, and 73% of patients had at least 

one other neurological symptom. Other documented 

symptoms in descending order of occurrence included 

perioral numbness, perioral tingling, nausea, extremity numbness, extremity tingling, vomiting, weakness, ataxia, shortness of breath, dizziness, floating 

sensation, dry mouth, diplopia, dysarthria, diarrhea, 

dysphagia, and limb paralysis.7

Approximately 10 outbreak-associated PSP cases 

are reported to the Centers for Disease Control and 

Prevention each year. Thirteen cases of neurological 

illness associated with consumption of pufferfish containing STX caught near Titusville, Florida, occurred 

in 2002.8 All 13 symptomatic patients reported tingling 

or numbness in the mouth or lips. Additionally, eight 

patients reported numbness or tingling of the face, 10 

patients reported these symptoms in the arms, seven 

patients reported these symptoms in the legs, and one 

patient reported these symptoms in the fingertips. 

Six of the 13 patients experienced nausea, and four 

reported vomiting. Symptoms began between 30 

minutes and 8 hours after ingestion, with a median 

of 2 hours. The illness lasted from 10 hours to 45 days, 

with a median of 24 hours. All of these cases resolved.

At lethal doses, paralysis of the respiratory musculature results in respiratory failure. Intoxication 

of a 65-year-old female in the Titusville case series is 

illustrative. The patient experienced perioral tingling 

within minutes of meal ingestion. Her symptoms 

worsened over the next 2 hours, and she experienced 

vomiting and chest pain. Emergency department 

evaluation noted mild tachycardia and hypertension. 

Over the next 4 hours, she developed an ascending 

paralysis, carbon dioxide retention, and a decrease in 

vital capacity to less than 20% predicted for her age, 

which led to intubation and mechanical ventilation. 

She regained her reflexes and voluntary movement 

within 24 hours and was extubated in 72 hours.9

Children appear to be more susceptible than adults. 

The lethal dose for small children may be as low as 

25 μg of STX equivalents, whereas that for adults may 

be 5 to 10 mg of STX equivalents.10 In adults, clinical 

symptoms probably occur upon ingestion of 1- to 3-mg 

equivalents. Because shellfish can contain up to 10 to 20 

mg equivalents per 100 grams of meat, ingestion of only 

a few shellfish can cause serious illness or death.10,11

Fortunately, clearance of toxin from the body is 

rapid. In one series of PSP outbreaks in Alaska resulting from the ingestion of mussels, serum half-life 

was estimated at less than 10 hours. In these victims, 

respiratory failure and hypertension resolved in 4 to 

10 hours, and toxin was no longer detectable in the 

urine 20 hours postingestion.11 A paper describing a 

2007 outbreak in Maine evaluated the clearance of 

individual PSP toxin congeners from the urine. 12 Halflives were shorter for the sulfated derivatives GTX 

(gonyautoxin) 1/4 and GTX 2/3 (5–6 h) than for the 

parent toxins STX and neoSTX (16–20 h), suggesting 

that the toxin profile in the ingested shellfish may be 

an important factor in recovery time.12

Inhalation. In mice, STX is considerably more toxic 

by inhalation (LD50 of 2 μg/kg) or by intraperitoneal 

injection (LD50 of 10 μg/kg) than by oral administration 

(LD50 of 400 μg/kg).13 Unlike PSP in humans, which is 

an oral intoxication and has a lag time to toxicity resulting from adsorption through the gastrointestinal tract, 

inhalation of STX can cause death in animals within 

minutes. At sublethal doses, symptoms in animals 

appear to parallel those of PSP, albeit with a more 

rapid onset reflective of rapid absorption through the 

pulmonary tissues.

Cause of Death

The cause of death in human cases of STX ingestion, as well as in experiments with animal models, is 

respiratory failure. Postmortem examination of STX 

victims reveals that the most notable effects are on the 

respiratory system, including pulmonary congestion 

and edema, without abnormalities of the heart, coronary 

arteries, or brain.6,14 In vitro, STX does not directly affect 

the smooth muscle of airways or large blood vessels, but 

in vivo axonal blockade may lead to respiratory failure 

and hypotension.15 Intoxication with large doses of STX 

may lead to metabolic acidosis, cardiac dysrhythmias, 

and cardiogenic shock, even with correction of ventilatory failure.16 For patients that survive 24 hours, the 

prognosis is good, regardless of respiratory support.

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Diagnosis

Clinicians should consider PSP patients who present with rapid onset of neurological symptoms that 

are sensory, cerebellar, and motor in nature and occur 

shortly after the consumption of seafood. Confirmatory diagnosis should rely on an analysis of body fluid 

samples, as well as an analysis of gastric contents or 

uneaten portions of recent meals. Urine, which is the 

primary mechanism of elimination, is used for analysis; 

the residence time of PSP toxins in the serum is short.12

Animal studies and human intoxications both indicate 

toxins present in the urine for several days postingestion, although this time frame is dose dependent. 

Urine samples should be collected as soon as possible 

to ensure accurate analysis.

Postmortem examinations of fatally intoxicated 

humans have identified STX in gastric contents, body 

fluids (serum, urine, bile, and cerebrospinal fluid), and 

tissues (liver, kidney, lungs, stomach, spleen, heart, 

brain, adrenal glands, pancreas, and thyroid).6,14 The 

largest concentrations of toxin existed in the gastric 

contents and urine.

Food or clinical samples can be evaluated by several 

methods. The traditional “gold standard” method is 

the mouse bioassay, which is an official method of 

the Association of Official Analytical Chemists International. High performance liquid chromatography, 

which is also an official method, can detect individual 

toxin congeners, but requires either precolumn or 

postcolumn derivatization of toxin mixtures for optimal detection.12,17,18 Receptor binding assays based on 

either rat brain membranes19 or purified STX binding 

proteins from frogs or snakes20 measure total toxicity 

rather than individual toxin profiles. All of these have 

been used to detect PSP in urine and serum of intoxicated victims.11 Immunoassays can detect individual 

toxins, but cross-reactivity among different congeners 

is highly variable; although useful in some situations, 

good correlation with analytical methods has so far 

been problematic. Rapid test kits are commercially 

available.

Medical Management 

Treatment of STX intoxication consists exclusively 

of supportive care. Patients may benefit from gastric 

lavage if ingestion is recent. Patients need to be monitored closely for 24 hours, and if signs of respiratory 

compromise occur, aggressive respiratory management should be instituted. Intravenous fluids should 

be used judiciously to maintain urine output and blood 

pressure. Large doses of STX or intoxication in patients 

with underlying medical conditions may lead to cardiovascular abnormalities including hypotension, Twave inversions, dysrhythmias, and cardiogenic shock. 

Sodium bicarbonate may be required for correction of 

metabolic acidosis. Vasopressor agents may be used to 

maintain blood pressure and perfusion of vital organs. 

Dobutamine may be the preferred agent; in experiments with high doses of STX given intravenously to 

cats, dobutamine improved recovery over dopamine.16

Research into specific treatments has examined heterologous antibody therapy and pharmacologic agents 

to overcome inhibition of the voltage-dependent 

sodium channel. However, at this time, no specific 

chemotherapeutic or immunotherapeutic agents exist 

for STX intoxication. 

Because of its high potency and relative stability, 

STX must be considered a potential bioterrorist threat 

agent. Toxins are easily isolated from laboratory cultures, but production constraints would limit the scope 

of an aerosol attack. The more likely threat is through 

the food supply, with the vector being naturally 

contaminated fresh shellfish. Blooms of the causative 

organism occur annually along both the Atlantic and 

Pacific coasts of the United States and Canada, as well 

as elsewhere around the world, often in underdeveloped nations with poor screening programs. Toxins 

can easily reach lethal levels in filter-feeding shellfish. 

Depuration is slow enough that in some areas, such 

as George’s Bank, some shellfisheries are permanently 

closed to harvesting. Threats to the water supply are 

minimal. Small-scale contamination (eg, of water coolers) is feasible, but large-scale contamination of reservoirs or even water towers is unlikely to be successful 

because of dilution effects coupled with the reduced 

potency of the oral route.

Neurotoxic Shellfish Poisoning

Description of the Toxin

NSP results from exposure to brevetoxins, a group 

of cyclic polyether toxins produced by the marine 

dinoflagellate K brevis (formerly Ptychodiscus brevis or 

Gymnodinium breve). Blooms of K brevis, with the associated discolored water and mass mortalities of inshore 

fish, have been described in the Gulf of Mexico since 

1844.21 As are paralytic shellfish poisons, brevetoxins 

are typically vectored to humans through shellfish; 

although in the case of NSP, the proximal agents are 

actually molluscan metabolites of the parent brevetoxins.22 In addition to causing NSP, annual blooms 

of K brevis in the Gulf of Mexico can cause significant 

revenue losses in the tourism and seafood industries. 

Beachgoers can be especially affected because the unarmored dinoflagellates are easily broken up by rough 

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wave action, and the toxins become aerosolized into 

airborne water droplets, causing respiratory irritation 

and potentially severe bronchoconstriction in people 

with asthma.

Historically, NSP has been virtually nonexistent 

outside the Gulf of Mexico. However, an outbreak was 

reported in New Zealand in 1993. Blooms of another 

dinoflagellate, Chattonella verruculosa, occurred in Rehoboth Beach, Delaware, in 2000 and caused a series 

of localized fish kills.23 Although no cases of NSP were 

reported, these events suggest a possible NSP range 

extension.

Mechanism of Action

Brevetoxins exert their physiological effects by binding 

with high affinity and specificity to neurotoxin receptor 

site 5 on the voltage-dependent sodium channel.24Unlike 

STX, which inhibits the sodium channel by binding to site 

1, binding of brevetoxins to site 5 prevents channel inactivation. This shifting of the voltage-dependence of channel 

activation leads to channel opening at lower membrane 

potentials25 and inappropriate ionic flux. Clinical effects 

are typically more centrally mediated than peripherally 

mediated. Brevetoxins can cross the blood–brain barrier, 

and it hypothetically leads to injury and death of cerebellar neurons by stimulation of glutamate and aspartate 

release, activation of the N-methyl-d-aspartate (NMDA) 

receptor, and excitotoxic cell death.26 A detailed review 

of the molecular pharmacology and toxicokinetics of brevetoxin can be found in Poli’s Recent Advances in Marine 

Biotechnology. Vol 7: Seafood Safety and Human Health.

27

Clinical Signs and Symptoms

Ingestion. Symptoms of NSP are similar to that 

of PSP, but are usually milder. Manifesting within 

hours after ingestion of contaminated seafood, symptoms include nausea, diarrhea, and abdominal pain. 

Typical neurological symptoms are oral paresthesia, 

ataxia, myalgia, and fatigue. In more severe cases, 

tachycardia, seizures, loss of consciousness, and respiratory failure can occur. During a 1987 outbreak, 

48 cases of NSP occurred in the United States. Acute 

symptoms documented in the outbreak included 

gastrointestinal (23% of cases) and neurological (39% 

of cases) symptoms. Symptoms occurred quickly, 

with a median of 3 hours to onset, and lasted up 

to 72 hours. Most of the victims (94%) experienced 

multiple symptoms, and 71% reported more than one 

neurological symptom.28

Although a fatal case of NSP has never been reported, children may be more susceptible, and a fatal dose 

must be considered a possibility.22 The toxic dose of 

brevetoxins in humans has not been established. However, important information has been gleaned from a 

clinical outbreak. A father and two small children became ill after ingesting shellfish harvested in Sarasota 

Bay, Florida, in 1996. Both children were hospitalized 

with severe symptoms, including seizures. Brevetoxin 

metabolites were detected in urine collected 3 hours 

postingestion. With supportive care, symptoms resolved in 48 to 72 hours, and no brevetoxin was detectable in the urine 4 days later. 22 Mass chromatography 

of serum samples taken immediately after the family 

checked into the hospital demonstrated ion masses 

suggestive of brevetoxin metabolites, although these 

compounds were never isolated. The amount of toxin 

ingested was not determined, although the father, 

who had milder symptoms and was released from 

the hospital after treatment, reported eating “several” 

shellfish. The number eaten by the children (ages 2 and 

3) was unknown. 

The toxicity of brevetoxins in mice is well established. LD50 values range from 100 to 200 μg/kg after 

intravenous or intraperitoneal administration for 

PbTx-2 and PbTx-3, the two most common congeners. Oral toxicity is lower: 500 and 6,600 μg/kg for 

PbTx-3 and PbTx-2, respectively.29 Animal models 

indicate brevetoxin is excreted primarily in the bile, 

although urinary elimination is also significant. Toxin 

elimination is largely complete after 72 hours, although 

residues may remain in lipid-rich tissues for extended 

periods.30

Inhalation. Respiratory exposure may occur with 

brevetoxins associated with harmful algal blooms or 

“red tides.” As the bloom progresses, the toxins are 

excreted and released by disruption of the dinoflagellate cells. Bubble-mediated transport of these toxins 

leads to accumulation on the sea surface; the toxins 

are released into the air by the bursting bubbles. The 

toxins are then incorporated into the marine aerosol 

by on-shore winds and breaking surf, leading to respiratory symptoms in humans and other animals. Sea 

foam may also serve as a source of toxin and result in 

symptoms if it is ingested or inhaled. During harmful 

algal blooms, the on-shore concentration of aerosolized 

toxins varies along beach locations by wind speed and 

direction, surf conditions, and exposure locations on 

the beach. Concentrations of the toxin are highest near 

the surf zone.31

Systemic toxicity from inhalation is a possibility. 

Distribution studies of intratracheal instillation of 

brevetoxin in rats have shown that the toxin is rapidly 

cleared from the lung, and more than 80% is distributed 

throughout the body. Twenty percent of the initial 

toxin concentration was present in several organs for 

7 days.32

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Diagnosis

Brevetoxin intoxication should be suspected 

clinically when patients present with gastrointestinal 

symptoms and neurological symptoms occurring 

shortly after ingesting shellfish. Although these symptoms may be similar to those of STX intoxication, they 

do not progress to paralysis. Epidemiological evaluation of cases may identify additional cases during 

an outbreak and allow for public health measures, 

including surveillance, to be put into place.

Human cases are typically self-limiting, with improvement in 1 to 3 days, but symptoms may be more 

severe in young persons, elderly persons, or those with 

underlying medical conditions. Evaluation of biological 

samples should include urine as well as any uneaten 

shellfish from the meal. Toxins in clinical samples 

can be detected by liquid chromatography mass spectrometry receptor-binding assays, or immunoassay. 

Because metabolic conversion of parent toxins occurs 

in shellfish and the metabolites are apparently less 

active at the sodium channel, it appears that immunoassays are better screening tools. However, secondary 

metabolism in humans has yet to be fully investigated.

Medical Management

No specific therapy exists for NSP. If the ingestion is 

recent, treatment may include removal of unabsorbed material from the gastrointestinal tract or binding of residual 

unabsorbed toxin with activated charcoal. Supportive 

care, consisting of intravenous fluids, is the mainstay of 

therapy. Although brevetoxin has not been implicated 

in human fatalities, symptoms of NSP may overlap with 

symptoms of STX and thus warrant observation for developing paralysis and respiratory failure. Aggressive 

respiratory management may be required in severe cases.

Pulmonary symptoms resulting from inhalation of 

marine aerosols typically resolve upon removal from 

the environment, but may require treatment for reactive 

airway disease, including nebulized albuterol and anticholinergics to reverse bronchoconstriction. Mast cell 

release of histamine may be countered with the use of 

antihistamines. Mast cell stabilizers, such as cromolyn, 

may be used prophylactically in susceptible persons 

exposed to marine aerosols during red tide events.

No antitoxins for NSP are available. However, experiments with an anti-brevetoxin immunoglobulin G 

showed that treatment before exposure blocked nearly 

all neurological symptoms.33 Additional research 

into pharmacologic agents should be pursued. Two 

brevetoxin derivatives that function as brevetoxin antagonists but do not exhibit pharmacologic properties 

have been identified. Other agents that compete with 

brevetoxin binding for the sodium channel include 

gambierol, gambieric acid, and brevenal.34,35 Future 

research with these agents may assist in developing 

adequate therapeutics.

Brevetoxins are likely to have only moderate potential as agents of bioterror. Although unlikely to cause 

mortality in adults, oral intoxication can be severe and 

require hospitalization. Disruption of a local event, 

inundation of medical facilities by the “worried well,” 

and societal overreaction possibly leading to economic 

disruption of local industry are the most likely repercussions. K brevis is easily cultured and produces toxins well in culture. Unpublished animal experiments 

suggest brevetoxins may be 10-fold to 100-fold more 

potent by aerosol—versus oral—exposure. Thus, smallscale aerosol attacks are technically feasible, although 

isolation and dissemination of toxins would be difficult 

for nonexperts.

Amnesic Shellfish Poisoning

Description of the Toxin

ASP was defined after an outbreak of mussel poisoning in Prince Edward Island, Canada, in 1987. More 

than 100 people became ill with an odd cluster of symptoms, and three died. Canadian researchers quickly 

isolated the causative agent and identified it as domoic 

acid.36 Domoic acid, which was previously known as 

a compound tested and rejected as a potential insecticide, is a common ingredient in Japanese rural folk 

medicine. Domoic acid was originally isolated from the 

marine red algae Chondria spp, and researchers were 

surprised to discover that the diatom Pseudo-nitzschia 

pungens f multiseries (now Pseudo-nitzschia multiseries) 

was the causative organism. ASP remains the first and 

only known seafood toxin produced by a diatom.

Since the 1987 outbreak, several toxic species of diatoms have been found around the world and are now 

the subject of many regional monitoring programs. 

Domoic acid is seasonally widespread along the US 

Pacific Coast and the Gulf of Mexico. It has also been 

found in New Zealand, Mexico, Denmark, Spain, Portugal, Scotland, Japan, and Korea. Although amounts 

of domoic acid in shellfish occasionally reach levels 

sufficient to stimulate harvesting bans, no further human cases have been reported, reflecting the efficacy of 

monitoring programs. However, the toxicity of domoic 

acid remains evident in biotic events.

In 1991, numerous cormorants and pelicans died 

after feeding on anchovies (a filter-feeding fish) during 

a bloom of Pseudo-nitzschia australis in Monterey Bay, 

California. High levels of domoic acid were detected in 

the gut contents of the anchovies. Later that year, after 

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Medical Aspects of Biological Warfare 

the bloom moved northward along the coast, razor clams 

and Dungeness crabs became toxic off the Washington 

and Oregon coasts. Several cases of human intoxication 

apparently followed ingestion of razor clams, although 

a definitive link was not found.37 More than 400 sea lions 

died and numerous others became ill in 1998 after ingesting anchovies feeding in a bloom of P australis, again in 

Monterey Bay.38 Domoic acid was detected in both the 

anchovies and feces from the sea lions.39 These events 

suggest that periodic blooms of domoic acid–producing Pseudo-nitzschia on the western coast of the United 

States may cause significant toxicity in seafood items. 

Mechanism of Action

Domoic acid is a neuroexcitatory amino acid structurally related to kainic acid. As such, it binds to the 

kainate and AMPA (alpha-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid) subtypes of the glutamate 

receptor in the central nervous system, which subsequently elicits nonsensitizing or very slowly sensitizing 

currents,40 causes a protracted influx of cations into the 

neurons, and stimulates a variety of intracellular events 

leading to cell death.41 This effect may be potentiated 

by synergism with the excitotoxic effects from high glutamate and aspartate levels found naturally in mussel 

tissue.42 The kainate and AMPA receptors are present 

in high densities in the hippocampus, a portion of the 

brain associated with learning and memory processing. Mice injected with domoic acid develop working 

memory deficits.43 Neuropathological studies of four 

human fatalities revealed neuronal necrosis or loss 

with astrocytosis, mainly affecting the hippocampus 

and the amygdaloid nucleus.44 Developing and aging 

brains show higher susceptibility to domoic acid, and 

thus define susceptible subpopulations. 

Clinical Signs and Symptoms

Ingestion. The 1987 Prince Edward Island outbreak 

provided information on the clinical effects of domoic 

acid ingestion in humans.45 The outbreak occurred 

during November and December, with 250 reports 

of illness related to mussel consumption (107 of these 

reports met the classic case definition). All but seven of 

the patients reported gastrointestinal symptoms ranging from mild abdominal discomfort to severe emesis 

requiring intravenous hydration. Forty-three percent 

of patients reported headache, frequently characterized as incapacitating, and 25% reported memory loss, 

primarily affecting short-term memory.

At higher doses, confusion, disorientation, and 

memory loss can occur. Severe intoxications can 

produce seizures, coma, and death. Nineteen of the 

patients required hospitalization for between 4 and 

101 days, with a median hospital stay of 37.5 days. 

Twelve patients required care in an intensive care 

unit. The intensive care patients displayed severe 

neurological dysfunction, including coma, mutism, 

seizures, and purposeless chewing and facial grimacing.45 Severe neurological manifestations, which were 

more common in elderly persons, included confusion, 

disorientation, altered states of arousal ranging from 

agitation to somnolence or coma, anterograde memory 

disorder, seizures, and myoclonus. Although mean 

verbal and performance IQ scores were average and 

language tests did not reveal abnormalities, severe 

memory deficits included difficulty with initial learning of verbal and visuospatial material, with extremely 

poor recall. Some of the more severely affected patients 

also had retrograde amnesia that extended to several 

years before ingestion of the contaminated mussels.44

Nine of the intensive care patients required intubation 

for airway control resulting from profuse secretions, 

and seven of them suffered unstable blood pressures 

or cardiac dysrhythmias. Three patients died during 

their hospitalization.45

Symptoms of intoxication occur after a latency 

period of a few hours. In mild cases, the gastrointestinal symptoms of vomiting, diarrhea, and abdominal 

cramps occurred within 24 hours. The time from ingestion of the mussels to symptom onset ranged from 15 

minutes to 38 hours, with a median of 5.5 hours.45 In 

a study of 14 patients who developed severe neurological manifestations, 13 developed gastrointestinal 

symptoms between 1 and 10 hours after ingestion, and 

all of the patients became confused and disoriented 

1.5 to 48 hours postingestion. Maximal neurological 

deficits were seen 4 hours after mussel ingestion in the 

least affected patients and up to 72 hours postingestion 

in those patients who became unresponsive.44 All the 

patients who developed severe neurological symptoms 

were older than 65 or had preexisting medical conditions such as diabetes or renal failure that altered their 

renal clearance.

Inhalation. No natural cases of domoic acid inhalation exist, and no experimental models have evaluated 

an aerosol exposure to this toxin. It may be assumed 

that the toxin would be absorbed through the pulmonary tissues leading to systemic symptoms comparable 

to other exposure routes, although no data confirm 

this theory.

Diagnosis

Diagnosis should be suspected by the clinical presentation after ingestion of filter feeding seafood. Patients 

may have mild symptoms that resolve spontaneously 

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or may present with more severe signs of neurotoxicity, 

including confusion, altered mental status, or seizures. 

Symptomatic patients typically are older than 65 or 

have underlying medical conditions that affect renal 

clearance.45 Initial evaluation of these patients should 

include standard protocols for patients with altered 

mental status, including toxicological screens to rule out 

more common intoxicants, especially illicit substances. 

Other diagnostic tests can be used to rule out other 

clinical causes of the symptoms including imaging with 

computed tomography scans, which does not show abnormality related to domoic acid intoxication, and monitoring of brain activity with electroencephalography. Of 

the 12 patients that were admitted to the intensive care 

unit during the 1987 outbreak, electroencephalograms 

showed that nine had generalized slow-wave activity 

and two had localized epileptogenic activity.45 Positron 

emission tomography scanning of four patients with 

varying degrees of illness revealed a correlation between 

glucose metabolism in the hippocampus and amygdala 

with memory scores.44

Based primarily on levels measured in Canadian 

shellfish after the 1987 outbreak, mild symptoms in 

humans may appear after ingestion of approximately 

1 mg/kg of domoic acid, and severe symptoms may 

follow ingestion of 2 to 4 mg/kg. The official regulatory testing method uses analytical high performance 

liquid chromatography, although both immunological 

methods and a simple, inexpensive thin-layer chromatography method are available.46–48 No evidence of 

domoic acid metabolism by rodents or primates exists, 

as shown by recovery in an unchanged form from the 

urine or feces.49 Samples to be included for definitive 

testing include serum, feces, urine, and any uneaten 

portions of the suspected meal.

Medical Management

Treatment for intoxication with domoic acid is 

supportive care. For patients who present early after 

ingesting the meal, gastric lavage or cathartics may 

decrease toxin amounts absorbed systemically. A key 

issue with this intoxication is the maintenance of renal clearance; hydration or other measures may also 

be required. Additionally, severe intoxications may 

cause alterations in hemodynamic functions, requiring 

pharmacologic interventions to maintain perfusion. In 

the 1987 outbreak, some severely intoxicated patients 

developed substantial respiratory secretions requiring 

intubation. Patients should be monitored for seizure 

activity that may require anticonvulsants. Studies in 

mice have shown that sodium valproate, nimodipine, 

and pyridoxine suppress domoic acid-induced spike 

and wave activity on an electroencephalogram.50

No specific therapy exists for domoic acid intoxications. Research has revealed that competitive and 

noncompetitive NMDA receptor antagonists reduce 

the excitable amino acid cascade that leads to brain 

lesions.51 Additionally, NMDA receptor antagonists 

have also been shown to antagonize domoic acid 

toxicity.51

Domoic acid should be considered a legitimate—if 

moderate—bioterrorist threat agent. Toxic shellfish 

are available, and ingestion elicits symptoms that can 

be life threatening. Although mass casualties are not 

likely, mortality can occur, and the frightening nature 

of the symptoms in survivors may cause the disruption 

sought by an aggressor.

Palytoxin

Description of the Toxin

Palytoxins are a group of complex marine natural 

products originally isolated from zoanthid soft corals of the genus Palythoa. The structure of the first 

palytoxin was elucidated in 198152,53 when first described by Moore and Scheuer in 1971.54 Palytoxins 

are complex hemiketals with molecular weights of 

approximately 2,600 to 2,700 g/mol, and containing 

cyclic ethers, 64 chiral centers, and multiple hydroxyl 

groups. The original palytoxin from Palythoa toxica

contains a continuous chain of 115 carbon atoms, 

making it unique among known natural products.55

Since that discovery, a family of palytoxins has 

emerged, including congeners from Palythoa tuberculosa and Palythoa margaritae in Japan, and Palythoa 

caribaeorum from Puerto Rico. In addition, a palytoxinlike compound was isolated from the sea anemone 

Radianthus macrodactylus from the Seychelles,56 and 

two analogs have been isolated from the red alga 

Chondria armata, which also produces domoic acid.57

More recently, dinoflagellates of the genus Ostreopsis

have been shown to produce a variety of palytoxin 

analogs, including ostreocins and ovatoxins (from Ostreopsis ovata and Ostreopsis siamensis) and macarenotoxins from Ostreopsis mascarenensis.

55 Finally, both 

palytoxin and one of the newly described palytoxin 

analogs, 42-hydroxy-palytoxin, have been identified 

in a marine cyanobacterium of the genus Trichodesmium in New Caledonia, raising the possibility of 

additional food–web sources.58 

In this chapter, all congeners, including ovatoxinsa–f, ostreocins, and palytoxins are collectively referred 

to as “palytoxins.” In many of the references cited, the 

compounds referred to as “palytoxin” were not highly 

purified, or only compared to a chromatographically 

derived palytoxin standard. It is very likely that many 

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were actually mixtures of palytoxin analogs, which 

have only recently been described and are difficult 

to separate.

Palytoxins are some of the most potent nonproteinaceous toxins known. Intravenous LD50 values (in 

μg/kg) range from 0.025 (rabbits), 0.033 (dogs), 0.078 

(monkeys), 0.089 (rats), and 0.11 (guinea pigs). Of the 

animal models tested, mice are the least susceptible at 

0.15 μg /kg to 0.45 μg /kg, depending on the investigator and the origin of the toxin.59,60 Potency is somewhat 

less by other routes. The lowest potency seems to be 

by the oral route, with LD50 values for palytoxin and 

ostreocin-D in the range of 500 μg/kg to 1,000 μg/kg 

in mice.61–64

In spite of the rather unimpressive oral toxicity values in mice, fatal human intoxications from palytoxins 

have been reported since the 1700s, primarily from 

grazing or filter feeding species of fish in the Atlantic 

and Pacific oceans and the Caribbean Sea.65 Palytoxins 

are now thought to be the causative agent in the often 

fatal syndrome formerly known as clupeotoxism, 

which results from the ingestion of filter feeding fish 

of the family Clupeidae (herrings and sardines) and 

Engraulidae (anchovies) among others.66 They have also 

been implicated in several fatal cases of poisoning from 

various species of xanthid crabs in the Philippines.66

Palytoxins are known to bioaccumulate in important 

seafood species such as mussels, sea urchins, and 

cephalopods (octopus and squid), suggesting a real 

possibility of human intoxication from the ingestion of 

seafood.67,68 Deeds and Schwartz55 provide an excellent 

review of human intoxication from seafood.

More recently, palytoxins have begun to cause problems in the Mediterranean and Adriatic seas, probably 

as a result of the introduction of O ovata into these 

waters. Several instances of respiratory symptoms 

associated with blooms of Ostreopsis were reported 

in the early years of the 21st century along the coasts 

of Italy, Spain, and France (reviewed in Del Favero 

et al69). The most striking of these occurred in 2005, 

when more than 200 beachgoers near Genoa, Italy, 

experienced respiratory symptoms of bronchoconstriction, dyspnea, cough, and rhinorrhea. Of these 200, 20 

beachgoers experienced symptoms severe enough to 

warrant extended hospitalization and intensive care. 

These symptoms occurred during a bloom of O ovata

near the coast.70,71 Symptoms peaked during the peak of 

the bloom and dissipated with the bloom. Cell samples 

of O ovata collected during the bloom tested positive 

for palytoxins by high performance liquid chromatography and mass spectrometry.72 Similar testing from 

subsequent events has resulted in the identification of 

new palytoxin analogs, including ovatoxin-a, which 

may predominate in these blooms.73 Blooms of O 

ovata now regularly occur in the Mediterranean and 

Adriatic seas,69 raising important questions regarding 

public health, seafood safety, and the potential for 

the accumulation of large amounts of toxins through 

dinoflagellate culture. More importantly, the intoxication of beachgoers during periods of little wind from 

dinoflagellate blooms in the water column suggests 

high aerosol potency. Thus, aerosol toxicology studies 

of palytoxin are clearly warranted.

Mechanism of Action

The well-accepted, oft-cited mechanism of action of 

palytoxin is through interference of the Na+/K+-ATPase 

ionic pump, converting it to a nonspecific ion pore and 

altering the internal ionic concentration of susceptible 

cells.60,61 Binding of palytoxin to the pump causes an 

immediate influx of Na+, which triggers an influx of 

K+ and Ca++. This interference with intracellular ionic 

homeostasis, especially the increase in intracellular 

Ca++, entrains a variety of toxic cell responses. Although 

inhibition of many of the toxic effects by ouabain supports this as the primary source of the pathophysiology 

of palytoxins, significant evidence from both in vivo 

and in vitro models indicate ancillary mechanisms 

as well (see the review by Munday60). Tubaro et al62

provides a review of the in vitro and in vivo biological 

effects of palytoxin. 

Clinical Signs and Symptoms

Ingestion. Manifestations of palytoxin intoxication by ingestion typically involve malaise, nausea, 

vomiting and diarrhea, a bitter or metallic taste in the 

mouth, myalgia and cramps, numbness or tingling in 

the extremities, bradycardia, and dyspnea. Renal failure has occurred in severe cases, probably secondary 

to the myoglobinemia associated with skeletal muscle 

damage. Human oral intoxications by palytoxins 

are reviewed in Yasumoto and Murata,57 Deeds and 

Schwartz,55 and Tubaro et al.74 However, the example 

of a fatal intoxication occurring after the ingestion 

of a crab, Demania reynaudii, in the Philippines is illustrative. 

In November 1984, at around noon, a man cooked 

a crab caught in a net off Tanjay Town in the province 

of Negros Oriental, Philippines.66 Within minutes of 

partially ingesting the crab, he experienced dizziness, 

fatigue, nausea, and a metallic taste in his mouth. 

Later he experienced diarrhea. When a dog died 

that had eaten the remainder of his crab, the man requested transport to a local hospital. During the trip, 

he experienced fatigue, numbness in the extremities, 

restlessness, and vomiting. Upon admission at about 

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5:45, he complained of restlessness, muscle cramps, 

and vomiting. He died at 3:14 the next morning, approximately 15 hours postingestion.

Clinical records revealed alternating periods of normal heart rate and severe bradycardia (30 beats/min), 

rapid and shallow respiration, cyanosis around the 

mouth and in the hands, and renal failure (anuria).66

Administration of atropine, diphenhydramine, pethidine, and adrenaline was ineffective. The causative 

agent was determined to be palytoxin based on the 

dose/survival time relationship in the mouse bioassay and the chromatographic characteristics of the 

extracted toxin, both of which were identical to those 

of a palytoxin standard.

Inhalation. Although O siamensis is well known 

around Japan, Australia, New Zealand, and the Mediterranean, O ovata has only recently become established 

in the Mediterranean/Adriatic. The first blooms occurred in 2003, and now have become regular events. 

Blooms associated with respiratory effects in humans 

occurred in 2003 and 2004 in Italy, and they have been 

reported in Spain, France, Croatia, Tunisia, Greece, and 

Algeria since that time.75

The most complete description of aerosol exposure 

to palytoxin is a Genoa event in 2005. In late July, during a period of warm weather with little wind and 

calm seas, a large bloom of O ovata occurred along 

the coastline. Concomitantly, a total of 209 beachgoers were afflicted with a complex of symptoms that 

included fever (64%), sore throat (50%), cough (40%), 

dyspnea (39%), headache (32%), nausea (24%), rhinorrhea (21%), lacrimation (16%), vomiting (10%), and 

dermatitis (5%).71 Although no deaths occurred, 20 

people were hospitalized, some for as long as 3 days. 

Mean onset of symptoms was 4.5 hours (range 30 

min–23 h). Laboratory analyses were available for 82 

patients, including all those hospitalized. Of these, 46% 

had leukocytosis (mean white cell count was 13,900/

mm2) and 40% had neutrophilia (mean 82%); transaminases, gamma-glutamyl transpeptidase, creatinine, 

and sedimentation rate values were normal. Chest 

X-rays and electrocardiogram values were normal. 

Dermal. Dermal exposure to palytoxins can occur 

with exposure to water containing blooms of Ostreopsis, 

although this seems to be a minor hazard. For example, 

in the Genoa bloom event of 2005, only 5% of patients 

reported a mild dermatitis.71 However, in describing 

bloom occurrences along the French Mediterranean 

coast, Tichadou et al reported skin irritation as the most 

common sign in people exposed to cells in the water 

column.75 At low cell numbers, erythema resolved 

rapidly after exposure. At higher cell numbers, clinical 

findings included pruritis of exposed skin, conjunctivitis, rhinorrhea, and oral irritation. 

Exposure Through Home Aquaria. A more serious 

dermal issue can occur through the home aquarium 

trade. Colonies of zoanthid corals are popular aquarium decorations. They readily propagate and provide 

a colorful marine backdrop to other species. Home 

aquarists are usually cognizant of the potential toxicity 

of zoanthid corals and handle them with gloves when 

cleaning aquaria or dividing colonies. Occasionally, 

however, intoxications occur. In one case, a 25-yearold woman handled a zoanthid coral without gloves 

in her home aquarium.76 She noted a metallic taste in 

her mouth within minutes, followed by perioral paresthesias and hives on her torso and extremities. The 

next day she noted edema of the upper lip without 

airway compromise. By the second day, the paresthesia 

had resolved, but she experienced increasing edema, 

erythema, and pruritis in both hands. In addition, a 

bilateral urticarial rash was noted on her upper arms, 

thighs, abdomen, upper chest, and back. Symptoms 

improved after treatment with intravenous diphenhydramine, methylprednisolone, and lorazepam. 

In another instance of aquarium dermal intoxication, a 32-year-old man was admitted to the emergency 

department 20 hours after cutting three fingers on his 

right hand on a zoanthid colony while cleaning his 

aquarium.77 Within 2 hours, he exhibited shivering, 

myalgia, and general weakness of the extremities. 

After 16 hours, he collapsed with dizziness, speech 

disturbances, and glassy eyes. Upon hospital admission, swelling and erythema around the cuts were 

noted, which spread over his whole arm over the next 

20 hours. An electrocardiogram detected incomplete 

left bundle block. Bloodwork was normal, with the 

exception of slightly elevated levels of creatine kinase, lactate dehydrogenase, and C-reactive protein. 

He was treated with infusion of physiological fluids. 

Electrocardiogram changes receded in 24 hours, but 

paresthesias, weakness, and myalgia persisted for 48 

hours until discharge.

Several cases of pulmonary exposure to palytoxin 

have occurred during the aquaria cleaning using boiling water78 or otherwise handling Palythoa.

79 Bernaconi 

et al79 reported on three individuals handling Palythoa

in a new aquarium to which sea salt had been added, 

which produced some foam and mist and resulted in 

pulmonary effects consisting of a restrictive ventilatory 

pattern with significant hypoxia. Bernasconi et al79

also reported on an entire family that became ill after 

a professional aquarium cleaner poured boiling water 

on a Palytoxin-encrusted coral fragment. The cleaner, 

along with four members of the family elsewhere 

in the room, reported to the emergency department 

complaining of respiratory symptoms. All patients 

developed a low-grade fever, increased white cell 

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Medical Aspects of Biological Warfare 

count, and elevated creatine phosphokinase during 

their hospital stay. The intoxication of other people 

not directly involved with the aquarium cleaning 

process strongly suggests extremely high potency of 

the aerosolized toxic material.

Diagnosis

Context is key to the clinical diagnosis of the known 

palytoxin intoxication syndromes. Palytoxin poisoning should be suspected in patients who present with 

rapid onset of respiratory distress and tonic muscle 

contractions soon after eating grazing fish species 

(especially sardines, but also other fish such as herring, anchovies, etc), and especially during warm 

summer months.80 Essentially the same high fatality 

syndrome is described in cases of xanthid crab ingestion. Cases have been described only in Caribbean, 

African coastal, and Indo-Pacific waters. The complication of rhabdomyolysis, with creatine kinase levels 

peaking at about 24 to 36 hours after symptom onset, 

would also be an important later clue if the diagnosis 

has not yet been made.55,81 Paresthesias and numbness, 

as well as gastrointestinal symptoms, also occur in the 

other seafood toxidromes, but absence of frank paresis 

or paralysis—which has not been described with palytoxins—may be a relative discriminator from PSP. 

Other palytoxin intoxication syndromes differ from 

fish poisoning in varying degrees according to the 

route of exposure: dermal exposure, as with marine 

aquarium hobbyists, can be expected to produce local 

and systemic skin manifestations as described above; 

inhalational exposure near an algal bloom is known 

to produce respiratory distress and a mild dermatitis, 

but also fever (in a majority of cases) and conjunctivitis (in a minority). Gastrointestinal symptoms were 

also reported in a significant minority of inhalational 

cases, and low-grade fever was reported in most of the 

aquarium hobbyists.

Definitive laboratory identification of palytoxins in 

seafood can be accomplished with rapid and sensitive 

hemolysis neutralization assays,82 but the relative rarity of these syndromes does not make the licensing or 

widespread commercial availability of these tests likely 

in the foreseeable future. A number of other investigational methods of detection (including immunoassays 

and a fluorescence polarization) have been described 

in the literature.62

Medical Management

No specific therapy exists for palytoxin intoxications. For exposures restricted to contact with intact 

skin/mucosa only (eg, beachgoers exposed to algal 

blooms), experience indicates that nonsteroidal antiinflammatory drugs are effective and that the condition is self-limited.75 For recent ingestions, however, 

gastric emptying procedures, implemented as early as 

possible, are highly desirable and may be undertaken 

even with uncertainty as to the specific seafood-related 

toxin involved. Supportive care and the amelioration 

of symptoms are then the basis of treatment, with aggressive hydration via infusion of intravenous fluids 

being the primacy focus in significant intoxications 

where avoidance of rhabdomyolysis-induced renal 

failure is the goal. Palytoxin(s) have been implicated 

in a number of human fatalities involving ingestion 

of tropical fishes, with one author estimating a case 

fatality rate—based on an admittedly limited number 

of cases—of 45%.80 Cardiac conduction disorders and 

heart failure (with consequent hypotension), as well 

as renal failure, appear to be important mechanisms 

in these patients.80,83

As with other toxic marine aerosols, the respiratory symptoms associated with inhalation would 

be expected to rapidly diminish upon vacating the 

contaminated site, but severe intoxications by this 

route may require standard treatment for reactive 

airway disease (nebulized albuterol, anticholinergics, 

antihistamines).78 Aggressive treatment in an intensive 

care facility may prove necessary, and—although not 

validated—administration of steroids,79 antihistamines, or benzodiazepines76 have proved helpful and 

may be tried.

Given the apparent potency of exposures to aerosols, as well as the lethality associated with ingestions, 

palytoxins seem to have potential as bioterrorism 

agents. The large numbers sickened (more than 200 

individuals) in the 2005 Genoa event (20% of whom 

were hospitalized) indicate these possibilities. 

SUMMARY

Exposure to marine algal toxins may occur via 

ingestion or delivery as an aerosol at the tactical 

level. Although the toxins may be highly lethal, 

extracting and weaponizing them is relatively 

difficult because of the relatively small amounts 

of toxins typically produced by the source organisms. Such toxins may be more suitable for causing 

incapacitation or death among small groups or for 

assassinations. The toxins presented in this chapter 

are diverse in structure and mode of action. Proper 

diagnosis and care represent a daunting challenge 

for physicians.

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STX, brevetoxins, and domoic acid are marine 

algal toxins associated with human illness in natural outbreaks related to harmful algal blooms. STX 

blocks ionic conductance of the voltage-dependent 

sodium channels, leading to neurological symptoms 

(parasthesias and paralysis) as well as respiratory 

distress and cardiovascular instability. Treatment 

includes respiratory support and intensive cardiovascular management. Anti-STX serum and antibodies have shown promise in animal models, but such 

reagents are unavailable for human use. Brevetoxins 

inhibit sodium channel inactivation, leading to depolarization of membranes. Brevetoxin symptoms 

are similar to those of STX but are usually milder 

and lack paralysis. Although naturally acquired 

cases typically resolve spontaneously in 1 to 3 days, 

patients should be carefully observed and may 

require aggressive airway management. Domoic 

acid is a neuroexcitatory amino acid that kills cells 

within the central nervous system, particularly in 

the hippocampus, which is associated with learning 

and memory. Patients with domoic acid intoxication 

develop gastrointestinal symptoms and neurological symptoms, including anterograde memory loss 

and myoclonus. Severe intoxications may lead to 

convulsions and death. Medical management of 

domoic acid intoxications includes monitoring of 

hemodynamic status and pharmacological treatment 

of seizures.

Palytoxins derive from both dinoflagellate blooms 

or from the polyps of soft corals of the genus Palythoa. Their primary mechanism of action is to bind 

to the Na+/K+-ATPase, converting it to a nonselective 

cationic pore and interfering with intracellular ionic 

homeostasis. Oral intoxication can lead to malaise, 

vomiting and diarrhea, a bitter or metallic taste in 

the mouth, muscle aches and cramps, numbness or 

tingling in the extremities, bradycardia, and dyspnea. Renal failure resulting from myoglobinemia 

secondary to rhabdomyolysis can occur. Inhalation 

exposure can lead to respiratory symptoms such as 

fever, sore throat, cough, dyspnea, headache, nausea/

vomiting, rhinorrhea, lacrimation, and dermatitis. An 

underappreciated exposure mechanism is through the 

home aquarium trade, whereby several incidences 

of inhalational or dermal intoxication have occurred 

through the handling or disinfection of Palythoa polyps. 

Treatment of palytoxin intoxication, which is nonspecific, is based on the route of exposure. The apparent 

extreme aerosol toxicity of palytoxins, as suggested 

by several incidences of beachgoers being intoxicated 

during near-shore blooms of O ovata, makes it critical 

for further investigation of the aerosol toxicology of 

these compounds. 

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(Revised Edition). Princeton, NJ: Darwin Press; 1978:325–402.

66. Alcala AC, Alcala LC, Garth JS, Yasumura D, Yasumoto T. Human fatality due to ingestion of the crab Demania reynaudii that contained a palytoxin-like toxin. Toxicon. 1988;26:105–107.

67. Amzil Z, Sibal M, Chomerat N, et al. Ovatoxin-a and palytoxin accumulation in seafood in relation to Ostreopsis cf. 

ovata blooms on the French Mediterranean coast. Mar Drugs. 2012;10:477–496.

68. Lopes VM, Lopes AR, Costa P, Rosa R. Cephalopods as vectors of harmful algal bloom toxins in marine food webs. 

Mar Drugs. 2013;11:3381–3409.

69. Del Favero G, Sosa S, Pelin M, et al. Sanitary problems related to the presence of Ostreopsis spp. in the Mediterranean 

Sea: a multidisciplinary scientific approach. Ann Ist Super Sanita. 2012;48:407–414.

70. Gallitelli M, Ungaro N, Addante N, Procacci V, Silveri NG, Sabba C. Respiratory illness as a reaction to tropical algal 

blooms occurring in a temperate climate. JAMA. 2005;293:2599–2600.

71. Durando P, Ansaldi F, Oreste P, et al. Ostreopsis ovata and human health: epidemiological and clinical features of 

respiratory syndrome outbreaks from a two-year syndromic surveillance, 2005-06, in north-west Italy. Euro Surveill. 

2007;12:3212–3217.

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72. Ciminiello P, Dell’Aversano C, Fattorusso E, et al. The Genoa 2005 outbreak: determination of putative palytoxin 

in Mediterranean Ostreopsis ovata by a new liquid chromatography tandem mass spectrometry method. Anal Chem. 

2006;78:6153-6159.

73. Ciminiello P, Dell’Aversano C, Fattorusso E, et al. Putative palytoxin and its new analog, ovatoxin-a, in Ostreopsis 

ovata collected along the Ligurian coasts during the 2006 toxic outbreak. J Am Soc Mass Spectrom. 2008;19:111–120.

74. Tubaro A, Durando P, Del Favero G, et al. Case definitions for human poisonings postulated to palytoxins exposure. 

Toxicon. 2011;57:478–495.

75. Tichadou L, Glaizal M, Armengaud A, et al. Health impact of unicellular algae of the Ostreopsis genus blooms in the 

Mediterranean Sea: experience of the French Mediterranean coast surveillance network from 2006 to 2009. Clin Toxicol 

(Phila). 2010;48:839–844.

76. Nordt PS, Wu J, Zahller S, Clark RF, Cantrell FL. Palytoxin poisoning after dermal contact with zoanthid coral. J Emerg 

Med. 2011;40:397–399.

77. Hoffmann K, Hermann-Clausen M, Buhl C, et al. A case of palytoxin poisoning due to contact with zoanthid corals 

through a skin injury. Toxicon. 2008;51:1535–1537.

78. Sud P, Su MK, Greller HA, Majlesi N, Gupta A. Case series: inhaled coral vapor – toxicity in a tank. J Med Toxicol. 

2013;9:282–286.

79. Bernasconi M, Berger D, Tamm M, Stolz D. Aquarism: an innocent leisure activity? Palytoxin-induced acute pneumonitis. Respiration. 2012;84:436-439.

80. Minns AB, Matteucci MJ, Ly BT, et al. Seafood toxidromes. In: Auerbach PA, ed. Wilderness Medicine. 6th ed. Maryland 

Heights, MO: Elsevier Mosby; 2012:1454–1455. 

81. Okano H, Masuoka H, Kamei S, et al. Rhabdomyolysis and myocardial damage induced by palytoxin, a toxin of blue 

humphead parrotfish. Intern Med. 1998;37:330–333.

82. Bignami GS. A rapid and sensitive hemolysis neutralization assay for palytoxin. Toxicon. 1993;31:817–820.

83. Melton RJ, Randall JE, Fusetani N, Weiner RS, Couch RD, Sims JK. Fatal sardine poisoning: a fatal case of fish poisoning in Hawaii associated with the Marquesan sardine. Hawaii Med J. 1984;43:114.

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Chapter 20

ALPHAVIRUS ENCEPHALITIDES

SHELLEY P. HONNOLD, DVM, PhD*; ERIC C. MOSSEL, PhD†; LESLEY C. DUPUY, PhD‡; ELAINE M. MORAZZANI, 

PhD§; SHANNON S. MARTIN, PhD¥; MARY KATE HART, PhD¶; GEORGE V. LUDWIG, PhD**; MICHAEL D. PARKER, 

PhD††; JONATHAN F. SMITH, PhD‡‡; DOUGLAS S. REED, PhD§§; and PAMELA J. GLASS, PhD¥¥

INTRODUCTION

HISTORY AND SIGNIFICANCE 

ANTIGENICITY AND EPIDEMIOLOGY

Antigenic and Genetic Relationships

Epidemiology and Ecology

STRUCTURE AND REPLICATION OF ALPHAVIRUSES 

Virion Structure

PATHOGENESIS

CLINICAL DISEASE AND DIAGNOSIS 

Venezuelan Equine Encephalitis 

Eastern Equine Encephalitis

Western Equine Encephalitis

Differential Diagnosis of Alphavirus Encephalitis

Medical Management and Prevention

IMMUNOPROPHYLAXIS

Relevant Immune Effector Mechanisms

Passive Immunization

Active Immunization

THERAPEUTICS

SUMMARY

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*Lieutenant Colonel, Veterinary Corps, US Army; Director, Research Support and Chief, Pathology Division, US Army Medical Research Institute 

of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702; formerly, Biodefense Research Pathologist, Pathology Division, US Army 

Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

†Major, Medical Service Corps, US Army Reserve; Microbiologist, Division of Virology, US Army Medical Research Institute of Infectious Diseases, 

1425 Porter Street, Fort Detrick, Maryland 21702; formerly, Science and Technology Advisor, Detachment 8, Research Development and Engineering 

Command, 5183 Blackhawk Road, Aberdeen Proving Ground, Maryland 

‡

Microbiologist, Virology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

§

Oak Ridge Institute for Science and Education Fellow, Viral Biology Department, Virology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702; formerly, NRC Postdoctoral Fellow, Virology Division, US Army Medical Research 

Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

¥

Vaccine Program Manager, Clinical Research Management, Business Plans and Programs Office, US Army Medical Research Institute of Infectious 

Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702; formerly, Senior Scientist/Technical Development Manager, Nonclinical Research Department, Dynport Vaccine Company, 64 Thomas Johnson Drive, Frederick, Maryland

¶

Chief Scientific Officer, Dynport Vaccine Company, 64 Thomas Johnson Drive, Frederick, Maryland 21702; formerly, Chief, Virology Division, US Army 

Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

**Deputy Principal Assistant for Research and Technology, US Army Medical Research and Materiel Command, 810 Schreider Street, Fort Detrick, 

Maryland 21702; formerly, Science Director, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland ††Retired; formerly, Chief, Viral Biology Department, Virology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter 

Street, Fort Detrick, Maryland

‡‡Executive Vice President and Chief Scientific Officer, PaxVax, Inc, 3985 Sorrento Valley Boulevard, San Diego, California 92121; formerly, Chief, Viral 

Biology Department, Virology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

§§Associate Professor, Aerobiological Manager, RBL, Department of Immunology, University of Pittsburgh, 3501 Fifth Avenue, Pittsburgh, Pennsylvania 

15261; formerly, Microbiologist, Center for Aerobiological Sciences, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, 

Fort Detrick, Maryland 

¥¥Chief, Viral Biology Department, Virology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702

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INTRODUCTION

most of which were known or thought to be aerosol 

infections.10 Before vaccines were developed, most 

laboratories working with VEEV reported disease 

among their personnel. The ability of aerosolized EEEV 

and WEEV to infect humans is less certain, relying 

on anecdotal evidence and animal studies. EEEV and 

WEEV are less commonly studied in the laboratory 

than VEEV, which may explain the lower incidence 

of laboratory-acquired infections. Therefore, fewer 

human exposures have occurred or the infectious dose 

is higher resulting in fewer incidences. 

Perhaps as a consequence of their adaptation to 

dissimilar hosts in nature, the alphaviruses replicate 

readily and generally to very high titers in a wide 

range of cell types and culture conditions. Virus titers 

of 1 billion infectious units per milliliter of culture 

medium are not unusual, and the viruses are stable in 

storage and in various laboratory procedures. Because 

they can be easily manipulated in the laboratory, 

these viruses have long served as model systems by 

which to study various aspects of virus replication, 

pathogenesis, induction of immune responses, and 

virus–vector relationships. As a result, the alphaviruses are well described and their characteristics well 

defined.9,11,12

The designers of offensive biological warfare 

programs initiated before or during World War II13

recognized that the collective in vitro and in vivo 

characteristics of alphaviruses, especially the equine 

encephalitis viruses, lend themselves well to weaponization. Although other encephalitic viruses could 

be considered as potential weapons (eg, the tickborne 

encephalitis viruses), few possess as many of the required characteristics for strategic or tactical weapon 

development as the alphaviruses:

• These viruses can be produced in large 

amounts in inexpensive and unsophisticated 

systems.

• They are relatively stable and highly infectious 

for humans as aerosols.

• Strains are available that produce either incapacitating or lethal infections.

• The existence of multiple serotypes of VEEV, 

as well as the inherent difficulties of inducing 

efficient mucosal immunity, confound defensive vaccine development.

The equine encephalitis viruses remain as highly 

credible threats, and intentional release as a small-particle aerosol from a single airplane could be expected to 

infect a high percentage of individuals within an area 

During the 1930s, three distinct but antigenically 

related viruses recovered from moribund horses were 

shown to be previously unrecognized agents of severe 

equine encephalitis. Western equine encephalitis 

virus (WEEV) was isolated in the San Joaquin Valley 

in California in 19301; Eastern equine encephalitis 

virus (EEEV) was isolated in Virginia and New Jersey in 19332,3; and Venezuelan equine encephalitis 

virus (VEEV) was isolated in the Guajira peninsula 

of Venezuela in 1938.4 By 1938, it was clear that EEEV 

and WEEV were also natural causes of encephalitis in 

humans,5–7 and naturally acquired human infections 

with VEEV occurred in Colombia in 1952 in association 

with an equine epizootic.8 

Although these viruses cause similar clinical syndromes in horses, the consequences of the infections 

they cause in humans differ. Eastern equine encephalitis (EEE) is the most severe of the arboviral encephalitides, with case fatality rates of 30% to 70%, and 

neurological sequelae common in survivors.9

 WEEV 

appears to be less neuroinvasive but has pathology 

similar to that of EEE in patients with encephalitis. In 

contrast, severe encephalitis resulting from VEEV is 

rare in humans except for children. In adults, VEEV 

usually causes an acute, febrile, and incapacitating 

disease with prolonged convalescence.

The three viruses are members of the Alphavirus

genus of the family Togaviridae. As with most of the 

alphaviruses, VEEV, EEEV, and WEEV are transmitted by mosquitoes, and are maintained in enzootic 

cycles with various vertebrate hosts. Thus, the natural 

epidemiology of these viruses is controlled by environmental factors that affect the interactions of the 

relevant mosquito and reservoir host populations. Of 

the 31 viruses currently classified within this group, 

VEEV, EEEV, and WEEV are the only viruses regularly 

associated with encephalitis. Although these encephalitic viruses are restricted to the Americas, as a group, 

alphaviruses have worldwide distribution and include 

other epidemic human pathogens. Among those 

pathogens, chikungunya virus (Asia, Africa, and the 

Americas), Mayaro virus (South America), o’nyongnyong virus (Africa), Ross River virus (Australia and 

Oceania), and Sindbis virus (SINV; Africa, Europe, 

and Asia) can cause an acute febrile syndrome often 

associated with debilitating polyarthritic symptoms.

Although natural infections with the encephalitic 

alphaviruses are acquired by mosquito bite, these 

viruses are also highly infectious by aerosol. VEEV 

has caused more laboratory-acquired disease than any 

other arbovirus. Since its initial isolation, at least 150 

symptomatic laboratory infections have been reported, 

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Medical Aspects of Biological Warfare 

of at least 10,000 km2. Furthermore, these viruses are 

readily amenable to genetic manipulation by modern 

recombinant DNA technology. This characteristic is 

being used to develop safer and more effective vaccines,14,15 but, in theory, it could also be used to increase 

the weaponization potential of these viruses.

HISTORY AND SIGNIFICANCE

Descriptions of encephalitis epizootics in horses 

thought to have been caused by EEEV were recorded 

as early as 1831 in Massachusetts.16 However, it was 

not until the outbreaks of EEE in Delaware, Maryland, and Virginia in 1933 and 1934 that the virus was 

isolated, and not until a similar outbreak in North 

Carolina in 1935 that birds were suspected as the 

natural reservoir.17 The initial isolation of EEEV from 

a bird18 and from Culiseta melanura mosquitoes,18 the 

two major hosts of the EEEV natural cycle, were both 

reported in 1951. Outbreaks of EEEV have occurred 

in most eastern states and in southeastern Canada, 

but they have been concentrated along the eastern 

and Gulf coasts. Although only 270 cases of EEE 

in humans were reported between 1964 and 2010 

(http://www.cdc.gov/easternequineencephalitis/

tech/epi.html), the social and economic impact of 

this disease has been larger than expected because 

of the high case fatality rate, significant long-term 

sequelae among survivors, equine losses, extreme 

concern among individuals living in endemic 

areas during outbreaks, and the surveillance and 

mosquito-control measures required. Isolation of 

EEEV from Aedes albopictus mosquitoes, which are 

prevalent in EEE endemic areas in the United States, 

has heightened concern because the opportunistic 

feeding behavior of these mosquitoes and their apparent high vector competence for EEEV suggest 

that they may be efficient bridge vectors for spillover 

infections of humans.19

The initial isolation in 1930 of WEEV from the brain 

tissues of a horse with encephalitis was made in the 

midst of a large epizootic in California, which involved 

at least 6,000 horses and with an approximate mortality of 50%.1

 Cases of human encephalitis in California 

were not linked to WEEV until 1938, when the virus 

was isolated from the brain of a child. During the 1930s 

and 1940s, several other extensive epizootics occurred 

in western and north-central states, as well as in Saskatchewan and Manitoba in Canada, which affected 

large numbers of equids and humans. For example, it 

has been estimated that during 1937 and 1938, more 

than 300,000 equids were infected in the United States, 

and in Saskatchewan, 52,500 horse infections resulted 

in 15,000 deaths.20,21 Unusually high numbers of human 

cases were reported in 1941: 1,094 in Canada and 2,242 

in the United States. The attack rate in these epidemics 

ranged from 22.9 to 171.5 per 100,000, with case fatality 

rates of 8% to 15%.21 

In the early 1940s, workers isolated WEEV from Culex tarsalis mosquitoes22 and demonstrated the presence 

of specific antibodies to WEEV in birds,23 suggesting 

that birds are the reservoirs of the virus in nature. 

The annual incidence of disease in both equids and 

humans continues to vary widely, which is expected of 

an arthropodborne disease, and significant epidemics 

occurred in 1952, 1958, 1965, and 1975.21

VEEV was initially isolated during investigations 

of an epizootic occurring in horses in Venezuela in 

1936, and the isolate was shown to be antigenically 

different from the EEEV and WEEV isolated previously in the United States.4,24 Over the following 30 

years, many VEEV outbreaks were reported among 

horses, and humans became infected in large numbers 

in association with these epizootics.25 Most of those 

infected recovered after suffering an acute, febrile 

episode, but abortions and stillbirths were observed in 

pregnant women and severe disease with encephalitis 

and death also occurred, mostly in children and older 

individuals. In the 1960s, major epizootics occurred in 

Venezuela, Colombia, Peru, and Ecuador, and spread 

to Central America in 1969.26 These epizootics and 

previous ones were associated with significant human 

suffering, especially among rural people, who suffered 

not only from disease, but also from the loss of their 

equids, which were essential for transportation and 

agriculture. Between 1969 and 1971, epizootics were 

reported in essentially all of Central America and subsequently continued north to Mexico and into Texas. 

The most recent major epizootic occurred in Venezuela 

and Colombia in 1995.27

Between active epizootics it was not possible to 

isolate the equine virulent viruses. During the 1950s 

and 1960s, however, several other attenuated, antigenically different VEEV strains were isolated from 

different geographical areas. These enzootic strains 

could be differentiated antigenically not only among 

themselves but also from the epizootic strains.28 Enzootic strains used different mosquito vectors than the 

epizootic strains,29 and most used rodents as reservoir 

hosts.30 However, despite apparent avirulence for 

equids, at least some of the enzootic strains caused 

human disease.31

Laboratory studies with EEEV, WEEV, and VEEV 

quickly and often inadvertently demonstrated how easily these viruses could cause disease when inhaled. In 

1943, eight cases of VEE in laboratory personnel resulted 

from aerosolization of the virus from contaminated 

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animal caging.32 In 1959, two reports from the former 

Soviet Union detailed an incident in which nine vials of 

VEEV were dropped in a stairwell infecting 24 people, 

including people who worked on adjacent floors.33,34

Before the vaccine and more sophisticated personal 

protective measures were developed, VEEV was among 

the most common laboratory-acquired infections.35 It 

was primarily because of laboratory-acquired infections 

that it was realized that the current investigational new 

drug (IND) vaccines may not protect well against aerosol exposure to enzootic strains of VEEV.36,37 Two fatal 

laboratory accidents involving WEEV were reported in 

the late 1930s, one involving a centrifuge accident and 

another in which the route of infection was unknown.38,39

Other reports of laboratory-acquired infection of WEEV 

were not fatal.40,41 Although EEEV is considered the most 

virulent of the encephalitic alphaviruses, before 1967 

only two cases of laboratory-acquired EEE occurred, 

neither of which was fatal.35 Experimental studies in 

mice, hamsters, rats, and nonhuman primates (NHPs) 

have all corroborated the disease potential of VEEV, 

WEEV, and EEEV when aerosolized and inhaled.42

Therefore, within 30 years of the initial isolation 

of EEEV, WEEV, and VEEV, an essentially accurate 

picture had emerged with respect to their endemic 

and epidemic behavior, arthropod vectors, reservoir 

hosts, potential for infection via the respiratory tract, 

and the diseases produced. Although not then understood at the molecular level, these three viruses were 

well described as agents of disease, and the basic 

methods for their manipulation and production were 

known. The development of this knowledge occurred 

during the same period of war and political instability 

that fostered the establishment of biological warfare 

programs in the United States43 and elsewhere, and it 

was evident that the equine encephalitis viruses were 

optimal candidates for weaponization. The viruses 

were incorporated into these programs for both potential offensive and defensive reasons. The offensive 

biological warfare program in the United States was 

disestablished in 1969 and all stockpiles were destroyed13 by executive order, which stated:

The United States shall renounce the use of lethal biological agents and weapons and all other methods of 

biological warfare. The United States shall confine its 

biological research to defensive measures such as immunization and safety measures.44

Continuing efforts within the US defensive program 

in the 1960s and 1970s produced four vaccines for 

the encephalitis viruses: live-attenuated (TC-83) and 

formalin-inactivated (C84) vaccines for VEEV, and 

formalin-inactivated vaccines for EEEV and WEEV. 

These vaccines are used under US Food and Drug 

Administration IND status for at-risk individuals, 

distributed under the provisions of the IND, and recommended for use by any laboratory working with 

these viruses.10 Although these vaccines are useful, 

they have certain disadvantages (which are discussed 

later in this chapter), and next-generation vaccines are 

being developed.14

ANTIGENICITY AND EPIDEMIOLOGY

Antigenic and Genetic Relationships

The three American equine encephalitides antigenic 

complexes, VEE, EEE, and WEE, have been grouped 

with eight additional virus complexes into the Alphavirus genus based on their serologic cross-reactivity 

(Table 20-1).9,45 Analysis of structural gene sequences 

obtained from members of the VEEV and EEEV complexes confirms the antigenic classification for the 

most part and serves as another tool for classifying 

these viruses. Viruses of the WEE complex, including Highlands J, Fort Morgan, and WEEV, have been 

identified as recombinant viruses originating from 

ancestral precursors of EEEV and Sindbis virus and 

fall into a unique genetic grouping of alphaviruses.46–49

Venezuelan Equine Encephalitis Complex

The VEE complex consists of eight closely related 

viruses that manifest different characteristics with 

respect to ecology, epidemiology, and virulence for 

humans and equids (Table 20-2). The IA/B and IC varieties are commonly referred to as epizootic strains. 

These strains, which have been responsible for extensive epidemics in North, Central, and South America, 

are highly pathogenic for humans and equids. All epizootic strains are exotic to the United States and have 

been isolated only twice since 1973.50–52 Enzootic strains 

include Everglades (formerly subtype II), Mucambo 

(formerly subtype IIIA), Pixuna (formerly subtype IV), 

Cabassou (formerly subtype V), Rio Negro (formerly 

subtype VI), and varieties ID, IE, and Mosso das Pedras 

(formerly subtype IF).53–59 Like the epizootic strains, 

the enzootic strains may cause disease in humans, but 

they differ from the epizootic strains in their lack of 

virulence for equines. Infection of equids with some 

enzootic subtypes leads to an immune response capable of protecting the animals from challenge with 

epizootic strains.60 Limited data, acquired following 

laboratory exposures, suggest that cross-protection 

between epizootic and enzootic strains may be much 

less pronounced in humans.37,61,62 

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Medical Aspects of Biological Warfare 

TABLE 20-1

ANTIGENIC CLASSIFICATION OF ALPHAVIRUSES

Virus

Antigenic Complex Species Subtype Variety

Western Equine Encephalitis (WEE) WEE virus

Highlands J virus

Fort Morgan virus Buggy Creek

Aura virus

Whataroa virus

Sindbis virus Ockelbo

 Babanki

Kyzylagach

Venezuelan Equine Encephalitis (VEE) VEE virus I A-B

 I C

I D

I E

Mosso das Pedras virus

Everglades virus

Mucambo virus Mucambo (IIIA)

Tonate virus Tonate (IIIB)

Bijou Bridge (IIIB)

71D-1252 (IIIC)

Pixuna virus

Cabassou virus

Rio Negro virus

Eastern Equine Encephalitis (EEE) EEE virus

Madariaga virus Madariaga II

Madariaga III

Madariaga IV

Semliki Forest Semliki Forest virus

Bebaru virus

Chikungunya virus Chikungunya Several

O’nyong-nyong virus Igbo Ora

Getah virus Getah

Ross River virus Sagiyama

Mayaro virus Mayaro

Una virus

Middelburg Middelburg virus

Nduma Nduma virus

Barmah Forest Barmah Forest virus

Trocara Trocara virus

Southern elephant seal Southern elephant seal virus

Eilat Eilat virus

Salmon pancreas disease Salmon pancreas disease virus 1-6

Sleeping disease

Data sources: (1) Nasar F, Palacios G, Gorchakov RV, et al. Eilat virus, a unique alphavirus with host range restricted to insects by RNA 

replication. Proc Natl Acad Sci U S A. 2012;109:14622–14627. (2) King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ, eds. Virus Taxonomy: 

Classification and Nomenclature of Viruses: Ninth Report of the International Committee on Taxonomy of Viruses. San Diego, CA: Elsevier; 2012.

Eastern Equine Encephalitis Virus

The EEEV complex previously consisted of 

viruses in two antigenically distinct forms: (1) 

the North American and Caribbean (NA EEEV), 

and (2) the South American (SA EEEV). A recent 

proposal accepted by the International Committee 

on Taxonomy of Viruses resulted in the reclas244-949 DLA DS.indb 484 6/4/18 
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Alphavirus Encephalitides

TABLE 20-2

THE VENEZUELAN EQUINE ENCEPHALOMYELITIS COMPLEX

Disease in

Subtype Variety Prototype Strain Origin Cycle Horse Man

VEEV IA/B Trinidad donkey Donkey (Trinidad)1 Epizootic + +

IC P-676 Horse (Venezuela)2 Epizootic + +

ID 3880 Human (Panama)3 Enzootic – +

IE Mena II Human (Panama)1 Enzootic – +

Mosso das Pedras

virus 78V-3531 Mosquito (Brazil)4 Enzootic – ?

Everglades virus Fe3-7c Mosquito (Florida)5 Enzootic – +

Mucambo virus Mucambo (BeAn8) Monkey (Brazil)6 Enzootic – +

Tonate virus IIIB Tonate (CaAn410-D) Bird (French Guiana)7 Enzootic – +

IIIC 71D-1252 Mosquito (Peru)8 Enzootic – ?

Pixuna virus Pixuna (BeAn356445) Mosquito (Brazil)6 Enzootic – ?

Cabassou virus Cabassou Mosquito (French Guiana)7 Enzootic – ?

Rio Negro virus AG80-663 Mosquito (Argentina)9 Enzootic – +

Sources that contain original descriptions of or additional information about this strain: (1) Young NA, Johnson KM. Antigenic variants 

of Venezuelan equine encephalitis virus: Their geographic distribution and epidemiologic significance. Am J Epidemiol.1969;89:286. (2) 

Walton TE. Virulence properties of Venezuelan equine encephalitis virus serotypes in horses. In: Venezuelan Encephalitis: Proceedings of 

the Workshop-Symposium on Venezuelan Encephalitis Virus, Washington, DC, 14–17 Sep 1971. Washington, DC: Pan American Health 

Organization; 1972:134. PAHO Scientific Publication 243. (3) Johnson KM, Shelokov A, Peralta PH, Dammin GJ, Young NA. Recovery of 

Venezuelan equine encephalomyelitis virus in Panama: a fatal case in man. Am J Trop Med Hyg. 1968;17:432–440. (4) Walton TE, Grayson MA. 

Venezuelan equine encephalitis. In: Monath TP, ed. The Arboviruses: Epidemiology and Ecology. Vol 4. Boca Raton, FL: CRC Press; 1988:203–231. 

(5) Chamberlain RW, Sudia WD, Coleman PH, Work TH. Venezuelan equine encephalitis virus from South Florida. Science. 1964;145:272. 

(6) Shope RE, Causey OR, de Andrade AHP, Theiler M. The Venezuelan equine encephalitis complex of group A arthropod-borne viruses, 

including Mucambo and Pixuna from the Amazon region of Brazil. Am J Trop Med Hyg. 1964;13:723. (7) Karabatsos N. International Catalogue 

of Arboviruses Including Certain Other Viruses of Vertebrates. 3rd ed. San Antonio, TX: American Society for Tropical Medicine and Hygiene; 

1985. (8) Scherer WF, Anderson K. Antigenic and biological characteristics of Venezuelan encephalitis virus strains including a possible 

new subtype isolated from the Amazon region of Peru in 1971. Am J Epidemiol. 1975;101:356. (9) Contigiani MS, De Basualdo M, Camara A, 

et al. Presencia de anticuerpos contra el virus de la encefalitis equina Venezolana subtipo VI en pacientes con enfermedad aguda febril [in 

Spanish]. Revista Argentina de Microbiologia. 1993;25:212–220.

Adapted with permission from Walton TE, Grayson MA. Venezuelan equine encephalitis. In: Monath TP, ed. The Arboviruses: Epidemiology 

and Ecology. Vol 4. Boca Raton, FL: CRC Press; 1989: 206.

sification of the South American variants as a 

distinct species, Madariaga virus (MADV), within 

the Alphavirus genus.63,64 Thus, EEEV will refer to 

the former NA EEEV, whereas the new species 

designation, MADV, will refer to the former SA 

EEEV. The two species can be distinguished readily by hemagglutination-inhibition (HI) assays and 

plaque-reduction neutralization tests.63,65 All EEEV 

isolates show a high degree of genetic and antigenic 

homogeneity. However, they are distinct from the 

MADV isolates, which are more heterogeneous 

and form three genetic subtypes (II, III, IV).66,67

EEEV strains are extremely virulent for humans 

and horses. Although MADV subtypes infect both 

horses and humans, infections are rarely associated with significant clinical disease.68,69 However, 

a MADV subtype III virus (ArgM) resulted in up 

to 75% lethality, depending on the dose, in rodent 

models infected by aerosol.70

Western Equine Encephalitis Complex

Six virus species, including WEE, Sindbis, Aura, 

Fort Morgan, Highlands J, and Whataroa, comprise 

the WEEV complex.9,64 Several antigenic subtypes of 

WEEV have been identified, but their geographical 

distributions overlap.52 Most of the members of the 

WEE complex are distributed throughout the Americas, although Whataroa and subtypes of SINV have 

strictly Old World distributions.9,12 The New World 

WEE complex viruses can be distinguished readily 

by neutralization tests. In addition, WEE complex 

viruses isolated in the western United States (eg, 

WEEV) are genetically distinct from those commonly 

found in the eastern United States (eg, Highlands 

J).67,71 SINV is considered a member of the WEEV 

complex based on antigenic relationships. However, 

sequence comparisons show that WEEV, Highlands 

J, and Fort Morgan are actually derived from a 

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Medical Aspects of Biological Warfare 

recombination event between ancestral SINV and 

EEEV (or MADV). The structural domains of the 

recombinant viruses were derived from the SINV 

ancestor, whereas the nonstructural domains were 

derived from the EEEV ancestor.67,72

Epidemiology and Ecology

The evolution of the equine encephalitides is 

closely tied to the ecology of these viruses in naturally 

occurring endemic foci. Evidence indicates that the 

relative genetic homogeneity of the EEE and WEE 

complex viruses may result from the mixing of virus 

subpopulations as a result of the movement of the virus from one location to another by the avian hosts.73

In general, these viruses are maintained in a consistently virulent state, capable of initiating epizootics 

without development of any significant mutations. In 

contrast, diversity within the VEEV complex results 

from local evolution of these viruses in mammalian 

hosts that live in defined habitats. Initiation of epizootic and epidemic activity is almost always associated with appearance of specific genetic change.73

Most commonly, human involvement in the form 

of endemic and epidemic activity, occurs following 

intrusion into geographical regions where natural 

transmission cycles are occurring or following perturbation of these cycles by environmental changes or the 

addition of other vectors.74 The dramatic exception to 

this is epizootic VEEV, in which the spreading waves 

of the epizootic among equines can move rapidly 

over large distances, and humans become infected 

by mosquitoes that have fed on viremic equines. 

The high levels of viremia in equines infected with 

epizootic VEEV make them efficient amplifying 

hosts, with the result that equine infections normally 

precede human infections by days to weeks.75 Recent 

evidence suggests that it is the adaptation of these 

enzootic subtype ID viruses for efficient replication in 

horses that leads to emergence and efficient epidemic 

spread of disease.19,76 Medical personnel should view 

with some suspicion evidence of widespread human 

VEEV infections outside of endemic areas, in the absence of mosquito vectors or in the absence of equine 

disease, because this combination of circumstances 

may indicate an unnatural release of virus into the 

environment.

Enzootic VEEV subtypes, as described above, 

are maintained efficiently in transmission cycles 

involving primarily rodents and Culex mosquitoes 

belonging to the subgenus Melanoconion.

77–79 These 

mosquitoes live in humid locales with abundant open 

spaces such as sunny, swampy pastures cut by slowly 

flowing streams. The mosquitoes are ground feeders, 

seldom found higher than 8 meters above ground, and 

some prefer feeding on mammals rather than birds.80

Ground-dwelling rodents, partly because their ecologies are similar to that of the mosquito vectors, are 

the primary vertebrate hosts for the enzootic forms 

of VEEV. Following infection, these animals develop 

viremia of sufficient magnitude and duration to infect 

mosquitoes feeding on their blood.81 Other animals, 

such as bats and certain birds, may play a secondary 

role.82 Seroprevalence rates among human populations living in or near endemic VEEV areas vary 

but can approach 100%, suggesting that continuous 

transmission occurs presumably in the absence of 

significant human disease.75 However, virus activity 

within endemic zones can also be highly focal. In 

one incident at the Fort Sherman Jungle Operations 

Training Center in the Panama Canal Zone in December 1967, 7 of 12 US soldiers camped in one area 

developed VEE disease within 2 days, but another 

group that camped only a few yards away showed 

no disease.83,84 The incidence of human disease during 

epizootics also varies, but it is often high. During an 

outbreak in Venezuela, attack rates of 119 per 1,000 

inhabitants per month were reported.85 Following 

an epizootic in Guatemala and El Salvador, overall 

seroprevalence was estimated at 20%.86 

Unlike the enzootic strains, the fate of the epizootic 

strains during interepidemic periods is unclear. The 

most appealing theory on how epizootic strains arise 

suggests that they evolve by mutation and equine 

selection from enzootic strains. Results from oligonucleotide fingerprinting and sequence analysis of ID 

isolates from Colombia and Venezuela reveal a close 

similarity to the epizootic strains, suggesting that the 

equine virulent epizootic strains arise naturally from 

variants present in populations of ID virus.87,88 

Although the genetic evidence indicates that mutation of enzootic strains may lead to the development 

of epizootic strains, ecological data suggest a strong 

selective pressure to maintain the enzootic genotype in 

certain habitats. The enzootic VEEV vector Culex (Melanoconion) taeniopus is fully susceptible to both IAB and 

IE strains following intrathoracic inoculation. Orally 

exposed mosquitoes are fully competent vectors of the 

enzootic strain; however, they fail to develop disseminated infection or transmit epizootic virus.29,89 In the 

absence of genetic change, this virus–host interaction 

appears to be relatively stable. Mosquito resistance 

to epizootic strains of VEEV is rare. Epizootic strains 

have been isolated from a large number of mosquito 

species, and many have been shown to be efficient vectors.90 Thus, host switching from enzootic to epizootic 

vectors may be an important factor in the evolution of 

epizootic VEEV strains. Researchers suggest that emer244-949 DLA DS.indb 486 6/4/18 
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Alphavirus Encephalitides

gence of epizootic strains may result from acquisition 

of mutations that allow for transmission by abundant, 

equiphilic mosquitoes. More specifically, adaptation 

to Aedes (Ochlerotatus) taeniorhynchus mosquitoes has 

been a determinant of some recent emergence events, 

providing further evidence that the ability to switch 

hosts is critical for emergence of epizootic strains.76 The 

introduction of mosquito species into previously unoccupied geographical ranges (eg, Aedes albopictus into 

North America) may, therefore, offer the opportunity 

for epizootic strains to reemerge.

A major outbreak of epizootic VEEV occurred in 

the late 1960s and early 1970s. Epizootic virus first 

reached North America in 1969,25 but did not reach 

the United States until 1971. Studies of this epizootic 

demonstrated that the virus easily invaded territories 

in which it was formerly unknown,85 presumably as 

a result of (a) the availability of large numbers of susceptible equine amplifying hosts and (b) the presence 

of competent mosquito vectors. The initial outbreak 

in North America, attributed to enzootic strain IE, 

occurred in 1966 in Tampico, Mexico, involving approximately 1,000 equids.91

By the end of 1969 and the beginning of 1970, the 

expansion of the outbreak prompted the Mexican 

government to request the TC-83 vaccine from the 

US Army through the US Department of Agriculture.92 Despite the immunization of nearly 1 million equids, the epizootic continued to spread and 

reached the United States in June 1971. The nature 

of the virus and the number of human and equine 

cases prompted the US Secretary of Agriculture to 

declare a national emergency on July 16, 1971.93

Subsequent immunization of more than 2 million 

horses and unprecedented mosquito abatement 

efforts eventually stopped the epizootic before it 

was able to spread from Texas. Epizootic VEEV 

has not been isolated in the United States since the 

1971 outbreak.

The first large outbreak since the 1969–1971 

epizootic occurred in 1995 (Figures 20-1 and 20-2). 

The epizootic began in northwestern Venezuela 

and spread across the Guajira peninsula into northeastern Colombia. An estimated 75,000 to 100,000 

humans were infected, with more than 20 deaths 

reported. This outbreak was caused by a VEEV IC 

strain. By sequence analysis, this strain proved to 

be essentially identical to a virus that caused an 

outbreak in Venezuela in 1962–1964.27 Outbreaks 

of traditionally enzootic strains of VEEV also have 

Figure 20-1. This photograph was taken in 1995 near Buena 

Vista, Colombia. During large Venezuelan equine encephalitis (VEE) epizootics, typical morbidity rates among unvaccinated equines are 40% to 60%, with at least half of the 

affected animals progressing to lethal encephalitis. Note the 

disruption of the ground surface, which is caused by the 

characteristic flailing or swimming syndromes of moribund 

animals. Although clinically indistinguishable from the syndromes produced by eastern equine encephalitis and western 

equine encephalitis viruses, the capability of VEE to initiate 

explosive and rapidly expanding epizootics makes reliable 

diagnostic tests essential for the initiation of appropriate 

veterinary and public health measures. 

Figure 20-2. This photograph was taken in 1995 near 

Maicao, Colombia. Equine vaccination is the most effective 

means available to prevent Venezuelan equine encephalitis 

(VEE) epizootics as well as to control emerging outbreaks. 

Equines are the major amplifying hosts, and maintaining a 

high rate of immunity in the equine population will largely 

prevent human infection with the epizootic strains of VEE. 

Both inactivated and live attenuated vaccines are available 

for veterinary use, but the ability of the live attenuated 

vaccine to induce immunity in 7 to 10 days with a single 

inoculation makes it the only practical vaccination strategy 

in the face of an outbreak. Other measures used to control 

outbreaks include using insecticides to reduce mosquito 

populations and prohibiting the transportation of equines 

from affected areas.

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Medical Aspects of Biological Warfare 

occurred in Mexico and Central America. Genetic 

analysis confirmed acquisition of mutations and provided further evidence that emergence of epizootic 

strains may result from the accumulation of genotypic 

changes in enzootic strains.94,95

EEEV is endemic to focal habitats ranging from 

southern Canada to Central America. The virus, 

which has been isolated as far west as Michigan, is 

most common along the eastern coast of the United 

States between New England and Florida. Enzootic 

EEEV transmission occurs almost exclusively between 

passerine birds (eg, the perching songbirds) and the 

mosquito Culiseta melanura. Because of the strict ornithophilic feeding behavior of this mosquito, human 

and equine disease requires the involvement of more 

general feeders, such as members of the genera Culex

and Coquillettidia. Recent evidence suggests EEEV may 

overwinter in the southeastern United States in reptiles 

or amphibians, further necessitating the participation 

of more general feeding vectors.96,97 Mosquito vectors 

belonging to Culex species, subgenus Melanconion, may 

play a role in maintaining and transmitting MADV 

subtypes.98

WEEV is the best studied member of the WEE 

complex in terms of its epidemiology. The virus is 

maintained in cycles involving passerine birds and the 

mosquito Culex tarsalis. Humans and equids become 

infected only tangentially and are considered to be 

dead-end hosts,99 indicating that they do not normally 

contribute to further spread of the virus in nature. 

STRUCTURE AND REPLICATION OF ALPHAVIRUSES

Virion Structure

The alphavirus virion, a spherical particle approximately 65 nm to 70 nm in diameter, is typically composed 

of three structural proteins enclosing a single molecule 

of single-stranded RNA. The RNA genome is packaged 

within an icosahedral nucleocapsid, which is constructed 

from multiple copies of the capsid (C or CP) protein 

(Figure 20-3). The nucleocapsid is, in turn, surrounded 

by a lipid envelope derived from areas of the host cell 

plasma membrane that had previously been modified by 

the insertion of two viral glycoproteins. These envelope 

glycoproteins, E1 and E2, form heterodimers that associate further into trimers100,101 to form the short spikes on 

the surface of the virion. Although a third glycoprotein, 

E3, was thought to be absent in the mature virion of most 

alphaviruses, three-dimensional reconstruction of VEEV 

virions (TC-83 strain) from cryoelectron microscopic 

images revealed that E3 is associated with the E1–E2 

dimers on the virion surface, but at a lower stoichiometry.102 Evidence of E3 on the surface of Semliki Forest 

virus virions has also been reported.103 However, E1 and 

E2 dimers—but not E3—are known to be targets of the 

neutralizing antibody response and are among the determinants of tropism and virulence.104,105 Although nonneutralizing, a monoclonal antibody directed against the 

VEEV E3 protein provided complete protection against 

an intraperitoneal challenge when administered before 

exposure.106 It is possible that this monoclonal antibody 

protects by binding to the E3 protein at the surface of 

infected cells blocking the ability of the virus to bud.

Viral Infection

The infection cycle is initiated when the glycoprotein spikes on the virion bind to receptors on the cell 

Figure 20-3. 3D structure of Venezuelan equine encephalitis 

virus (VEEV). (a) A typical CCD image of VEEV TC-83 embedded in vitreous ice. Scale bar: 50nm. (b) Radially colored 

3D reconstruction of VEEV, showing the E1 basal triangle 

(green) and the E2 central protrusion (blue) for each spike. 

Scale bar: 10nm. (c) A slice through the 3D density map 20 

pixels from the origin. The inset is the 1D radial density 

profile of the map and is aligned to the slice image. (d) One 

asymmetric unit of the virus containing four unique copies 

of E1 (magenta), E2 (cyan), E3 (orange), and CP (blue). The 

cryo-EM densities for the viral membrane (yellow) and 

genomic RNA (green) are also displayed at a slightly lower 

isosurface threshold. Scale bar: 2nm. 

Data source: Zhang R, Hryc CF, Cong Y, et al. 4.4 Å cryo-EM 

structure of an enveloped alphavirus Venezuelan equine encephalitis virus. EMBO J. 2011;30(18):3854–3863. Reproduced 

with permission from EMBO.

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surface. The virus is localized initially to clathrincoated pits, where it is engulfed in a coated vesicle 

and transported to the endosomal compartment 

within the interior of the cell. A decrease in the pH 

in the interior of the vesicle induces a conformational 

change in the glycoprotein spikes, and rearrangement 

of the E1 glycoprotein mediates fusion of the virion 

envelope with the endosomal membrane.107 This fusion results in the release of the nucleocapsid into the 

cytoplasm, where disassembly of the nucleocapsid 

releases the viral RNA genome to the synthetic apparatus of the cell.108

Genomic RNA 

The viral genome, a positive-sense RNA of approximately 11,700 nucleotides, has the structural features of messenger RNA (ie, mRNA, a 5’ methylated 

cap [m7GpppA] and a poly-A tract at the 3’ end).109

As a complete and functional mRNA, genomic RNA 

purified from virions is fully infectious when artificially introduced (ie, transfected) into susceptible 

cells. Similarly, RNA transcribed from a full-length 

complementary DNA clone of an alphavirus is also 

infectious, which allows relatively easy genetic manipulation of these viruses. Mutations introduced 

into a complementary DNA clone by site-directed 

mutagenesis are reflected in the RNA transcribed 

from the altered clone and in the virus produced in 

transfected cells. These procedures are being used to 

develop improved vaccines,14 but they could also be 

used to enhance specific characteristics required for 

weaponization.

Structural Protein Synthesis

 The alphavirus genome contains two protein 

coding regions or open reading frames. The 5’ 

7,500 nucleotides encode a 220,000-dalton precursor polypeptide, which is proteolytically processed 

to produce the four components of the viral RNA 

polymerase. The polymerase genes are followed by a 

second coding region of approximately 3,800 nucleotides, which contains the information that directs the 

synthesis of the viral structural proteins. Soon after 

release of the viral genome from the nucleocapsid, 

the 5’ 7,500 nucleotides of the genome RNA are 

translated to produce the viral RNA polymerase. 

Early in infection, the incoming viral genome is also 

used as a template for the synthesis of a negativesense RNA, identical in length to the genome RNA, 

but of opposite polarity. The negative-sense RNA 

subsequently serves as a template for the synthesis 

of additional genomic RNA. The negative-sense 

RNA is also used as a template for transcription of 

a capped and polyadenylated subgenomic mRNA, 

which is identical to the 3’ third of the genome. The 

subgenomic mRNA is translated to yield a precursor polypeptide that is proteolytically processed 

by cotranslational and posttranslational cleavages 

to produce the viral structural proteins. The order 

of the structural proteins within the precursor is 

C-E3-E2-6K/TF-E1.

As the subgenomic mRNA is translated, the 

C protein is produced first and catalyzes its own 

cleavage from the nascent polypeptide soon after 

the ribosome transits into the sequences that encode 

E3. After release of the C protein, the free amino 

terminus of E3 is bound to the membranes of the 

rough endoplasmic reticulum. As the synthesis of 

nascent E3 and E2 (precursor E2 or pE2) continues, 

the polypeptide is translocated into the lumen of the 

endoplasmic reticulum, where oligosaccharides and 

fatty acids are added.110 A domain of hydrophobic 

amino acids near the carboxyl terminus of E2 inhibits 

further transmembranal movement so that the last 

30 to 40 amino acids of the E2 polypeptide remain 

exposed on the cytoplasmic side of the membrane. 

The 6K polypeptide serves as a signal for membrane insertion of the second glycoprotein, E1, and 

is subsequently cleaved from both E2 and E1 by 

signal peptidase.111 A hydrophobic anchor sequence 

present near the carboxyl terminus of E1 secures the 

protein in the membrane.

Budding and Release of Progeny Virus Particles

Soon after synthesis, the precursor of PE2 and 

E1 interact to form multimeric complexes,112 which 

are then transported through the Golgi apparatus, 

where the final modifications of the oligosaccharide 

are made. The pE2 protein is cleaved to generate 

the mature E2 and E3 glycoproteins soon after the 

glycoproteins leave the Golgi apparatus,113 and the 

mature viral spikes assume an orientation in the 

plasma membrane with the bulk of the E2 and E1 

polypeptides exposed on the exterior surface of the 

cell. In vertebrate cells, final assembly of progeny 

virus particles happens by budding exclusively at 

the plasma membrane114; whereas in cultured arthropod cells, budding also occurs at intracellular 

membranes.115

In vertebrate cells, budding is initiated when intracellular nucleocapsids bind to the 30 to 40 amino 

acid cytoplasmic domain of the E2 glycoprotein,116–118

inducing the formation of a locally ordered array 

of glycoprotein spikes, which excludes most of 

the cellular membrane proteins from the region. 

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Medical Aspects of Biological Warfare 

PATHOGENESIS

result, in the guinea pig and hamster models, death 

occurs before serious CNS disease develops.124,125 The 

host species and the route of administration of VEEV 

greatly affect CNS disease development. Mice uniformly exhibit a severe paralytic episode before death 

from diffuse encephalomyelitis following peripheral 

or aerosol administration of TrD or V3000.122,126,132,133

NHPs, however, exhibit few if any clinical signs of 

encephalitis following peripheral inoculation with 

TrD, and only modest perivascular cuffing and gliosis, 

mainly in the thalamus, hypothalamus, and olfactory 

areas of the brain.122,123 In one study, NHPs inoculated 

by the intraperitoneal route developed transient viremia and biphasic fever but otherwise exhibited no 

evidence of clinical disease.122 NHPs in this study developed brain lesions as early as 6 days postinfection, 

which typically included lymphocytic perivascular 

cuffs and gliosis, with the thalamus being the site of 

the most intense inflammation.122 NHPs infected by 

intranasal inoculation had more moderate inflammation, especially in the cortex and hypothalamus,134

whereas a Colombian epizootic strain of VEEV given 

by aerosol caused severe clinical and pathological CNS 

signs and resulted in death in approximately 35% of 

rhesus macaques.123 In another study, cynomolgus 

macaques infected with the VEEV IE or Mucambo 

virus (IIIA) developed fever, viremia, and mild clinical signs of encephalitis (tremors, loss of coordination) 

but recovered.135 Both mice and cynomolgus macaques 

challenged intracerebrally with TrD or related VEEV 

strains developed severe and lethal neurological signs 

with moderate to severe brain histopathology.134,136

The mechanisms of neuroinvasion by VEEV represent an important issue, particularly regarding 

immunoprophylaxis. The specific mechanism of 

neuroinvasion in the case of peripheral inoculation of 

virus is not completely understood; however, important features of the process have been elucidated by 

animal studies. In mice inoculated peripherally and 

subsequent to the development of viremia, virulent 

VEEV is detectable in the brain, initially in the olfactory 

bulbs, and usually within 48 hours of infection.133,137,138

In humans, the pathogenesis of VEEV, EEEV, and 

WEEV infections acquired by aerosol, which is the 

route of greatest biological defense concern, is unknown. Little is known of the pathogenesis following 

natural vectorborne infections of humans, mainly 

because of the limited autopsy material. Much of the 

information on VEEV pathogenesis in humans is based 

on a histological review of 21 human fatalities from the 

1962–1963 VEEV epidemic in Zulia, Venezuela.120 With 

few exceptions, the histopathological lesions in these 

cases, all among children or young adults, were comparable to those observed in experimentally infected 

animals. Tissues commonly affected in both humans 

and animals121–129 include those of the lymphoid and reticuloendothelial systems as well as the central nervous 

system (CNS). Widespread hepatocellular degeneration and interstitial pneumonia, not ordinarily seen in 

experimental animals, were frequent histological findings in these cases of severe human disease. Much of 

the understanding of the pathogenesis of VEEV, EEEV, 

and WEEV has relied on animal studies. 

The clinical and pathological responses of the host 

to VEEV, EEEV, and WEEV infection are highly dependent on a number of host and viral factors, including:

• the species, immune status, and age of the 

host;

• the route of infection; and

• the strain and dose of virus.

Most of the existing experimental data are from 

studies using rodent models challenged with the 

virulent Trinidad donkey (TrD) strain of VEEV, an 

epizootic IAB serotype virus, or its genetic clone V3000. 

A few NHP studies have also been reported.130,131 In 

animal models, as in humans, the lymphatic system 

and the CNS are consistent target organs. However, 

the relative degree of injury caused to these tissues 

varies. Virulent VEEV causes limited and reversible 

lesions to the lymphoid organs of mice and NHPs,122,126

but in guinea pigs and hamsters, it causes extreme 

and irreversible damage to those organs.127,128 As a 

Additional lateral associations between the individual 

spikes stabilize the lattice and promote additional 

E2–C protein interactions. The growing lattice may 

draw the membrane around the nucleocapsid, 

completing the envelopment with the release of the 

spherical virus particle. Maximal amounts of virus are 

typically produced from mammalian cells within 8 to 

10 hours after infection, and disintegration of the infected cell is likely caused by programmed cell death 

(apoptosis) rather than direct effects of the virus on 

cellular function.119 In contrast, alphaviruses initially 

replicate to high titer in arthropod cells with little or 

no evidence of cytopathology. The surviving cells 

continue to produce lesser amounts of virus, often for 

weeks or months. The ability of the virus to replicate 

without causing cell death in arthropod cells may be 

critical for maintenance of the virus in the mosquito 

vector in nature.

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It appears that virus in the blood escapes from fenestrated capillaries supplying the olfactory lining of the 

nasal tract. Virus may then invade olfactory neuron 

cell bodies or their axons and may be carried via the 

olfactory nerves into the olfactory bulbs of the brain. 

However, surgical or chemical ablation of the olfactory lining did not significantly affect the mortality 

rate or average survival time of infected mice. In this 

case, neuroinvasion was suspected to occur via the 

trigeminal nerves.137 However, direct invasion of the 

brain across the blood–brain barrier125,139 seems less 

compelling than the olfactory route. 

The understanding of the mechanism of neuroinvasion following respiratory infection is more clear. 

In mice, an early and strong target of virulent VEEV 

administered by aerosol has been shown to be the 

olfactory neuron.133 This cell type, a so-called “bipolar neuron,” is in direct contact with inspired air at 

one pole and synapses with resident neurons in the 

olfactory bulb at the opposite pole, offering a direct 

connection to the brain independent of viremia development. Both the nasal olfactory epithelium and 

the olfactory nerve axon bundles in the underlying 

connective tissue exhibit VEEV antigen within 24 

hours of aerosol infection (Figure 20-4), and the olfactory bulbs also show viral infection shortly thereafter 

(Figure 20-5). In a study of rhesus macaques inoculated intranasally with VEEV, the virus gains access 

to the olfactory bulb within 24 hours after infection 

and before the onset of viremia, suggesting direct 

neuroinvasion via olfactory neurons similar to neuroinvasion in the mouse.140

However, in inoculated macaques whose olfactory 

nerves had been surgically removed, VEEV was still 

able to reach the olfactory bulb by 36 hours after infection, presumably by the vascular route. Although the 

olfactory bulb and olfactory tract were sites of early 

viral replication, the virus did not appear to spread 

to the rest of the brain along the neural tracts in these 

monkeys, as it does in mice. In a more recent study in 

which cynomolgus macaques were exposed to VEEV 

by the aerosol route, virus was not detected in the brain 

until 4 days postinfection and was only detected in the 

region of the olfactory tubercle.132,141 However, the dose 

delivered to the cynomolgus macaques in this study 

was not reported, which could influence the resulting 

pathology. The teeth are another early target of VEEV 

administered peripherally or by aerosol,132,133,137 and the 

trigeminal nerves appear to carry VEEV from the teeth 

into the brains as an alternate, although it is probably 

a less significant route of neuroinvasion. 

The pathogenesis of EEEV has not been as thoroughly evaluated. In contrast to VEEV, EEEV replication in lymphoid tissues is limited by tissue-specific 

microRNA142; primary EEEV replication after subcutaneous inoculation occurs in fibroblasts, skeletal muscle, 

and osteoblasts.133 In a recent study, it was shown that 

when mice are infected with EEEV strain FL93-939 

by either the aerosol or intranasal route that the virus 

specifically targets the olfactory epithelium and enters 

the brain via the olfactory tract.143,144 In mice exposed 

by the aerosol route, virus was detected in the brain as 

early as 6 hours postinfection. In mice inoculated by the 

subcutaneous route, the mechanism of neuroinvasion 

Figure 20-4. Nasal tissue, BALB/c mouse, 2 days after exposure to aerosolized Venezuelan equine encephalitis (VEE) virus. Note immunoreactive olfactory epithelium and olfactory 

nerves. Alkaline phosphatase-labeled streptavidin method 

using rabbit antiserum to VEE virus (Mayer’s hematoxylin 

counterstain, original magnification x 300).

Figure 20-5. Olfactory bulb, BALB/c mouse, 2 days after 

exposure to aerosolized Venezuelan equine encephalitis 

(VEE) virus. Note immunoreactive cells. Alkaline phosphatase-labeled streptavidin method using rabbit antiserum 

to VEE virus (Mayer’s hematoxylin counterstain, original 

magnification x 150).

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is less clear and the virus may enter the brain either by 

the olfactory tract or the vascular route. In all cases, 

once in the brain, the neuron is the main viral target 

and animals exhibited varying degrees of neuronal cell 

death and meningoencephalitis.143,144

In a study in which guinea pigs were infected with 

EEEV by aerosol, animals developed clinical signs within 

24 hours of infection and rapidly progressed to include 

circling, recumbency, coma, and death.71 No difference 

in virulence or time to death was seen whether virus 

was targeted to the lower respiratory tract or upper 

respiratory tract by manipulating particle size. In these 

animals, virus was found in the olfactory epithelium 

and olfactory bulbs followed by spread to all parts of 

the brain by 4 days postinfection. Again, the neuron 

was the main viral target and brain lesions included 

neuronal necrosis, perivascular cuffs, and encephalitis 

with vasculitis noted in few animals in late-stage cases. 71

EEEV natural history and pathogenesis studies in 

NHPs are limited. However, in a recent natural history 

study cynomolgus macaques were challenged with 

aerosolized EEEV. In this study, 66% of the animals 

exhibited fever, leukocytosis, and neurological signs 

and succumbed 5 to 9 days postchallenge.145 The major pathological changes in the brain included severe 

meningoencephalomyelitis characterized by neuronal 

necrosis, perivascular cuffs, gliosis, satellitosis, edema, 

hemorrhage, and vasculitis in some animals.141

Since there has been a dramatic decline in the incidence of WEEV infection in humans and horses since 

the middle of the 20th century, few animal studies with 

this virus have been conducted in recent years. In one 

study, mice were infected with several strains of WEEV 

by various routes.146 The McMillian strain of WEEV 

was 100% lethal by the intracranial, subcutaneous, and 

aerosol routes and 90% lethal by the intravenous and 

intranasal routes. Histopathological lesions occurred 

in the brains of all mice and were characterized by 

neuronal necrosis, edema, lymphocytic meningitis, 

and occasional fibrin thrombi. Two studies in hamsters using various strains of WEEV147,148 showed 

lethality by various routes of infection with the major 

histopathological lesions in the brain being neuronal 

necrosis, lymphocytic meningitis and perivascular 

cuffs, astrocytosis, microgliosis, and hemorrhage. In 

a recent study, cynomolgus macaques were infected 

with the CBA-87 strain of WEEV by aerosol.149 Affected 

animals developed fever, inappetence, lethargy, and 

tremors, as well as leukocytosis and hyperglycemia. 

The histopathological lesions were characterized as 

nonsuppurative meningoencephalomyelitis. In particular, infection was noted in Purkinje cells in the 

cerebellum and hypothalamus, the region of the brain 

that controls body temperature.

The specific viral and host mechanisms that contribute to neuroinvasion and neurovirulence for each of 

these viruses have yet to be elucidated. The importance 

of these mechanisms and the differences observed between peripheral and aerosol administration are of significant practical concern because the immunological 

mechanisms of virus neutralization respective to each 

route can vary greatly, as studies have shown.130,150,151

The efficiency and rapidity of neuroinvasion following 

aerosol infection also place very high demands on the 

vaccines used for immunoprophylaxis (vaccines are 

discussed later in this chapter).

CLINICAL DISEASE AND DIAGNOSIS

VEEV, EEEV, and WEEV are also recognized for 

their potential for neuroinvasion and encephalitis in 

humans, sometimes in epidemic proportions. However, many of the infections caused by these viruses 

are manifested as systemic viral febrile syndromes, 

and infections by EEEV and WEEV may remain subclinical. Furthermore, these alphaviruses vary markedly in both their neurotropism and the severity of 

their neurological sequelae. Depending on the virus, 

patients presenting with the general syndrome of 

alphavirus encephalitis have a varying combination 

of fever, headache, confusion, dysphasia, seizures, 

paresis, ataxia, myoclonus, and cranial nerve palsies.

Venezuelan Equine Encephalitis

The IAB and IC subtypes of VEEV, which are 

pathogenic for equines, have the capacity for explosive epizootics with epidemic human disease. 

Epidemics of VEE affecting 20,000 to 75,000 people 

have been documented in Colombia, Venezuela, and 

Ecuador.25,75,152 In contrast to the other alphavirus 

encephalitides (EEEV and WEEV), epizootic strains 

of VEEV are mainly amplified in equids, rather than 

birds, so that equine disease normally occurs before 

reports of human disease. Enzootic VEEV strains 

(subtypes ID and IE, as well as Mosso das Pedras, 

Everglades, Mucambo, Tonate, Pixuna, Cabassou, 

and Rio Negro viruses, previously known as VEEV 

subtypes IF, II, IIIA, IIIB, IV, V, and VI, respectively) 

are not recognized as virulent for equines, but disease 

has been documented with most of these variants in 

humans who reside in or move into enzootic foci, 

or after laboratory infections (see Table 20-2). The 

resulting syndromes appear to be similar—if not 

indistinguishable—from the syndrome produced by 

epizootic variants, which ranges from undifferentiated febrile illness to fatal encephalitis. In NHPs, 

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Alphavirus Encephalitides

aerosol exposure to enzootic strains results in a febrile 

illness with indications of encephalitis virtually indistinguishable from that seen with epizootic strains 

in terms of onset, severity, and duration.153

Following an incubation period that can be as short 

as 28 hours but is usually 2 to 6 days, patients typically 

develop a prostrating syndrome of chills, high fever 

(38°C–40.5°C), headache, and malaise.154 Photophobia, 

sore throat, myalgias, and vomiting are also common 

symptoms. Frequent signs noted on physical examination include conjunctival injection, erythematous 

pharynx, and muscle tenderness. Although essentially 

all human infections with VEEV are symptomatic,83,84

only a small percentage manifest neurological involvement.155 In one epidemic, the ratio of encephalitis to 

infections was estimated at less than 0.5% in adults, although possibly as high as 4% in children.156 Mild CNS 

involvement is evidenced by lethargy, somnolence, 

or mild confusion, with or without nuchal rigidity.8

Seizures, ataxia, paralysis, or coma indicate more severe CNS involvement. In children with overt encephalitis, case fatalities may be as high as 35% compared 

with 10% for adults.157 However, for those adults who 

survive encephalitic involvement, neurological recovery is usually complete,158 although one report documented motor disorders and an increased incidence 

of seizures in children following VEE outbreaks.158

Abortions and increased fetal deaths have also been 

attributed to VEEV infection.27,152 School-aged children 

are believed to be more susceptible to a fulminant 

form of disease, which follows a lethal course over 48 

to 72 hours in which depletion of lymphoid tissues is 

prominent.120,159,160

In the first 3 days of illness, leukopenia and elevated 

aspartate aminotransaminase are common. For those 

with CNS involvement, a lymphocytic pleocytosis of 

up to 500 cells per microliter can be observed in the 

cerebrospinal fluid (CSF). The CSF pleocytosis may 

be acutely polymorphonuclear but soon becomes 

predominantly lymphocytic.

Specific diagnosis of VEEV infection can be accomplished by virus isolation, serologic testing, identification of virus-specific nucleic acid, or all three.59,161–165

Identification of the VEEV subtype of an isolate 

involved can usually be determined by cross-neutralization tests or more reliably by genetic sequence 

analysis. During the first to third days of symptoms 

of nonspecific febrile illness, VEEV may be recovered 

from either the serum or the nasopharynx.166 Despite 

the theoretical possibility of person-to-person transmission of virus present in the nasopharynx, no such 

occurrences have been reported. In NHPs, the virus is 

found in the blood for the first 2 to 3 days after aerosol 

exposure but levels are low compared to what has 

been reported for natural infection and may not be 

detectable after fever onset for enzootic strains.131,153

VEEV can be isolated from the nasopharynx of 

NHPs for up to 5 days postexposure after aerosol 

exposure of naïve animals. Antibodies detectable by 

an HI assay, enzyme-linked immunosorbent assay, 

or plaque-reduction neutralization tests appear as 

viremia diminishes. Complement-fixing antibodies 

make their appearance later during convalescence. 

VEEV immunoglobulin M (IgM) antibodies are present in acute phase sera,84 and VEEV IgM tests do not 

react with sera from patients with EEEV or WEEV.167

Since patients with encephalitis typically come to 

evaluation later in the course of clinical illness, virus 

is recovered less often from them,131 and they usually 

have virus-specific serum antibody by the time of 

clinical presentation.168 Immunity after infection is 

probably lifelong to the homologous serotype, but 

cross-immunity may be weak or nonexistent to heterologous serotypes.37,61,62 Thus, when viewed either as 

an endemic disease threat or as a potential biological 

warfare threat, adequate immunization may require 

polyvalent vaccines.

Eastern Equine Encephalitis

In North America, EEEV is maintained in a natural 

transmission cycle between Culiseta melanura mosquitoes and passerine birds in freshwater hardwood 

swamps and forested areas, primarily in the Atlantic 

and Gulf Coast states. EEEV outbreaks are typically 

recognized when severe equine or human encephalitis occurs near such areas.169 In the southeastern 

United States, Culex erraticus may play an important 

role in the transmission of the virus to humans and 

horses.170–172 During vectorborne EEEV epidemics, 

the incidence of human infection is low (<3% of the 

population at risk), and the neurological attack rate in 

one outbreak was estimated at 1 in every 23 cases of 

human infection.173 However, the effect on morbidity 

and mortality of aerosol-acquired (the expected route 

of infection in a biological warfare offensive) EEEV 

infection in humans is unknown, although animal 

studies suggest that EEEV by aerosol is lethal.145 The 

incubation period in humans varies from 5 to 15 days. 

Adults typically exhibit a febrile prodrome for up to 

11 days before the onset of neurological disease.174

However, illness in children exhibits a more sudden 

onset.175 In natural outbreaks, viremia occurs during 

the febrile prodrome,176 but is usually undetectable by 

the time clinical encephalitis develops, when HI and 

neutralizing antibodies become evident.173 Despite the 

development of a prompt and neutralizing humoral 

response, the virus is not eliminated from the CNS, 

and progressive neuronal destruction and inflammation continue.

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Medical Aspects of Biological Warfare 

EEE is the most severe of the arboviral encephalitides, with high mortality and severe neurological sequelae.177 During EEEV outbreaks, the attack, morbidity, and fatality rates are highest in young children178

and elderly people.179 Overall, approximately 4% to 5% 

of human EEEV infections result in clinically apparent 

EEE in the United States, with an average of six human 

cases of EEE reported annually.180 Case fatality rates 

are estimated to be from 30% to 70%, but asymptomatic infections and milder clinical illness are underreported. The illness is characterized by a nonspecific 

prodrome followed by severe headache, high fevers, 

lethargy, and seizures, often with rapid progression 

to coma and death.181–183 Motor involvement with paresis is common during the acute phase of the illness. 

Major disturbances of autonomic function, such as 

impaired respiratory regulation or excess salivation, 

may dominate the clinical picture. Between 30% and 

70% of survivors are left with long-term neurological sequelae such as seizures, spastic paralysis, and 

cranial neuropathies. Cognitive impairment ranges 

from minimal brain dysfunction to severe dementia. 

In a recent retrospective study of 15 cases of EEE in 

children, fever, headache, and seizures were the most 

common clinical signs, with 87% of the patients becoming 

stuporous or comatose during the first 3 days of hospitalization.184 Radiographic lesions were noted in the basal 

ganglia, thalami, and cerebral cortex. This study found 

an important association between a short prodrome 

(ie, the time between initial nonspecific symptoms and 

the first major neurologic symptom) and an increased 

risk for death or for severe disease. The eight patients 

who had a poor outcome all had a prodrome of 2 days 

or less, and all four deaths occurred in this group.184

Clinical laboratory findings in patients with EEE 

often demonstrate an early leukopenia followed by a 

leukocytosis. Elevated opening pressure is commonly 

noted on lumbar puncture and, especially in children, 

the CSF lymphocytic pleocytosis may reach a cell 

count of thousands of mononuclear cells per microliter. However, in a recent report, neutrophilic pleocytosis with elevated levels of protein were the most 

consistent findings when CSF was evaluated within 

the first week of symptom onset.184 Specific diagnosis 

of EEE depends on virus isolation or serologic testing 

in which rising titers of HI, complement-fixing, or 

neutralizing antibodies are observed. IgM antibodies are usually detectable in acute-phase sera.167 As 

with other alphaviruses, neutralization tests are the 

most specific. Immunohistochemistry can also be 

performed postmortem on fixed brain samples.145 In 

NHPs exposed by aerosol to EEEV, the period from 

fever onset until the animal is moribund is less than 

48 hours regardless of dose.145

Western Equine Encephalitis

Like VEEV, naturally acquired WEEV (by mosquito 

bite) is less virulent for adult humans than for equids 

and children, with lower rates of fatalities and neurological sequelae.185 As with EEEV, infants and elderly 

people are especially susceptible to severe clinical illness 

and neurological sequelae, with case fatality rates of 

about 10%. Highlands J virus, an antigenically related 

member of the WEE complex that is isolated frequently 

in the eastern United States, rarely infects humans.

The incubation period is 5 to 10 days for natural 

WEEV infection. In NHPs infected by aerosol, the 

incubation period is 4 to 5 days.149 A large percentage of patients with vectorborne infections are either 

asymptomatic or present with a nonspecific febrile 

illness or aseptic meningitis. The ratio of encephalitis 

cases per infection has been estimated to vary from 1 

per 1,150 in adults, to 1 per 58 in children, to 1 per 1 

in infants.74 However, the severity of the syndrome 

and the incidence of inapparent infection almost 

certainly depend on the strain and dose of the virus, 

and the route of infection. Some unusual isolates 

show high virulence in laboratory animals138,186,187

and in one study of laboratory-acquired infections 

in adults, two of five patients died.35 Symptoms 

usually begin with malaise, headache, and fever, 

followed by nausea and vomiting.188 Telemetry data 

from NHPs exposed to WEEV by aerosol revealed, in 

addition to fever, increases in heart rate and changes 

in electrocardiograph readings, indicative of sinus 

tachycardia.189 A transient leukopenia followed by a 

pronounced leukocytosis composed almost entirely 

of segmented neutrophils correlated with a poor 

prognosis. Fever severity also correlated with a poor 

prognosis. Over the next few days the symptoms intensified, and in some cases, somnolence or delirium 

progressed into coma. The severity of neurological 

involvement is inversely related to age, with more 

than 90% of children younger than 1 year exhibiting 

focal or generalized seizures.190 Physical examination typically reveals nuchal rigidity, impaired 

sensorium, and upper motor neuron deficits with 

pathologically abnormal reflexes.

Patients with the most severe disease usually die 

within the first week of clinical illness, with case 

fatalities averaging 10%. Other patients begin a gradual convalescence after the first week of encephalitic 

symptoms. Most adults recover completely, but it may 

take months to years to recuperate from fatigability, 

recurrent headaches, emotional lability, and impaired 

concentration.191 Some patients have permanent 

residua of motor weakness, cognitive deficits, or a 

seizure disorder. Children carry a higher incidence of 

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Alphavirus Encephalitides

neurological sequelae, ranging from less than 1% 

in those older than 1 year, to 10% in infants 2 to 3 

months old, to more than 50% in newborns. Congenital 

infection in the last trimester of pregnancy has been 

described, with resultant encephalitis in the infants.192

Laboratory accidents involving aerosol exposure to 

WEEV have been documented and the mortality of 

those limited cases was 40%.35 In NHPs, aerosol exposure to a dose equivalent to 10 times the median 

infective dose produced fever, and 50% of the animals 

developed clinical signs indicative of encephalitis. 

Twenty-five percent of those animals died from the 

infection by day 9 postexposure.189

Viremia is rarely detectable by the time patients 

present with encephalitic symptoms, but IgM, HI, 

and neutralizing antibodies are generally detected 

by the end of the first week of illness, and they increase in titer during the following week.167,193,194 In 

NHPs exposed to aerosolized WEEV, the virus was 

not detectable in the serum or nasopharynx postexposure.189 However, low levels of virus were detected 

in CSF. Antibody responses were not detectable by 

an enzyme-linked immunosorbent assay or plaquereduction neutralization test until day 9 postexposure in survivors, which was after control animals 

had died from the infection. Complement-fixing 

serologic responses generally appear in the second 

week and rise thereafter. Isolation of virus with up 

to 4-fold increase in titer is diagnostic, but because 

of serologic cross-reactions with other alphaviruses, 

neutralization tests are preferred. Examination of the 

CSF reveals a lymphocytic pleocytosis ranging from 

10 to 400 mononuclear cells per microliter. WEEV may 

occasionally be isolated from the CSF taken within 

the first 2 days of fever, and it is frequently recovered 

from brain tissue on postmortem examination.195

Survival from natural infection presumably confers 

long-term immunity; however, it may not protect 

against aerosol exposure.196

Differential Diagnosis of Alphavirus Encephalitis

Most acute infections with VEEV and WEEV produce a moderately severe but nonspecific clinical 

illness, consisting of fever, headache, and myalgias. 

Therefore, in a potential biological warfare scenario, 

alphaviruses should be considered in the differential 

diagnosis whenever epidemic febrile illness occurs, 

especially if several patients progress to neurological 

disease. Sick or dying equids near an epidemic febrile 

illness among troops should immediately suggest the 

possibility of large-scale alphavirus exposure. Other 

potential biowarfare agents that may infrequently 

produce or imitate a meningoencephalitic syndrome 

include Rift Valley fever virus, Brucella species, Yersinia 

pestis, Salmonella typhi, Coxiella burnetii, and botulinum 

toxin. As with any meningoencephalitis diagnosis, it 

is imperative to rule out any potential cause that may 

be specifically treatable.

For encephalitis cases that are more sporadic in their 

occurrence, other important viral etiologies that might 

not be readily discriminated from the alphaviruses by 

TABLE 20-3

SOME IMPORTANT VIRAL CAUSES* OF 

ENDEMIC ENCEPHALOMYELITIS

Virus Family Genus Species

Togaviridae Alphavirus Eastern equine 

encephalitis virus

Western equine 

encephalitis virus

Venezuelan equine 

encephalitis virus

Flaviviridae St Louis encephalitis 

virus

 Murray Valley 

encephalitis virus

 West Nile virus

 Japanese encephalitis 

virus

 Dengue virus

 Tickborne complex 

viruses

Bunyaviridae LaCrosse virus

 Rift Valley fever virus

 Toscana virus

Paramyxoviridae Paramyxovirus Mumps virus

Morbillivirus Measles virus

Henipavirus Hendra virus

Nipah virus

Arenaviridae Arenavirus Lymphocytic choriomeningitis virus

Machupo virus

Junin virus

Guanarito virus

Picornaviridae Enterovirus Poliovirus

Coxsackievirus

Echovirus

Reoviridae Colorado tick fever 

virus

Rhabdoviridae Lyssavirus Australian bat lyssavirus

Rabies virus

Herpesviridae Herpesvirus Herpes simplex virus 

types 1 and 2

Epstein-Barr virus

Cytomegalovirus

Adenoviridae Adenovirus

*Not all-inclusive.

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clinical features are listed in Table 20-3. This list is not 

all-inclusive but suggests other viral encephalitides 

that should be considered if a patient presents, a priori, 

with an encephalitic syndrome. 

Epidemiological, historical, and laboratory information remains critical to differential diagnosis. Immediate and careful consideration must be given to 

treatable infections that may mimic viral encephalitis 

(Exhibit 20-1) because prompt and appropriate intervention can be lifesaving. In addition, vascular, 

autoimmune, and neoplastic diseases may imitate 

infectious meningoencephalitis.

For endemic meningoencephalitic disease that occurs outside biowarfare theaters, the geographical locale and the patient’s travel history are of preeminent 

importance in diagnosing an arboviral encephalitis. 

Risk for disease is increased relative to the patient’s 

amount of arthropod contact near swampy or forested areas during the summer in temperate climates 

or year-round in the tropics. Encephalitic illness of 

equids in the surrounding locale is an important 

indication of ongoing transmission of encephalitic 

alphaviruses. Animal studies have indicated that the 

virus may not be detectable in the serum during the 

febrile period, and antibody responses may be weak 

or nonexistent, making diagnosis difficult, which 

is particularly true for WEEV. Examination of the 

CSF, including viral cultures or reverse transcription 

polymerase chain reaction, is critical in differentiating 

bacterial from viral infections, and infectious from 

noninfectious etiologies. Serum and CSF tests based 

on antibody or genetic detection hold great promise 

in more rapid diagnosis of infectious encephalitis. 

In some instances it will be necessary to (a) institute 

therapy for possible, treatable, infecting organisms 

and (b) await definitive laboratory diagnostic tests. 

Medical Management and Prevention

No licensed vaccines or therapeutics currently exist 

for the alphaviral encephalitides; therefore, treatment 

is aimed at management of specific symptoms (eg, 

anticonvulsant medication and airway protection). 

The high fever occasionally produced by WEEV 

infection in humans is a special problem among the 

arboviral encephalitides that may require aggressive 

antihyperthermia measures.194,197 The US Army has 

extensive experience with IND live-attenuated and 

formalin-inactivated vaccines in humans (which are 

discussed later in this chapter). 

Use of an effective vaccine in horses would also 

prevent outbreaks of epizootic VEE, as equines are 

the major amplifying species for VEEV. However, 

vaccination of horses is not a useful public health 

tool for EEEV, WEEV, or enzootic VEEV, because 

horses are not important as amplifying hosts for these 

viruses. Integrated mosquito control measures can 

also have significant impact on ameliorating epidemic 

transmission.

EXHIBIT 20-1

NONVIRAL CAUSES OF 

ENCEPHALOMYELITIS

Treatable infectious conditions that can mimic viral 

encephalitis:

Partially treated bacterial meningitis

Brain abscess

Subdural empyema

Embolic encephalitis associated with bacterial 

endocarditis

Lyme disease

Tuberculous meningitis

Fungal meningitis

Rocky Mountain spotted fever

Cat scratch disease

Cerebral malaria

Trypanosomiasis

Toxoplasmosis

Vascular, autoimmune, and neoplastic diseases that 

can mimic infectious meningoencephalitis:

Lupus cerebritis

Cerebral and granulomatous arteritis

Lymphomatous cerebritis

Whipple’s disease

Behçet syndrome

Carcinomatosis meningitis

IMMUNOPROPHYLAXIS

Relevant Immune Effector Mechanisms

The equine encephalitis viruses constitute both an 

endemic disease threat as well as a biological warfare 

threat; therefore, adequate immunoprophylaxis of 

military and civilian personnel will require protection against both vectorborne and aerosol-acquired 

infections. The requirements for protection against 

parenteral infection are well described, but the requirements for protection against infectious aerosols are 

more stringent and remain largely unidentified. Within 

a few days of infection with an alphavirus, specific 

antibodies can be detected in the serum of animals 

or humans. Within 7 to 14 days, a virus-neutralizing 

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antibody response develops, as measured by the ability 

of serum antibodies to block virus infectivity in vitro or 

in vivo. Protection from mosquito-vectored alphavirus 

disease is believed to be primarily mediated by this 

virus-specific neutralizing antibody response, which 

is largely directed against epitopes on the E2 glycoprotein.198,199 Protection mediated by nonneutralizing 

antibodies to alphaviruses, directed largely at epitopes 

on the E1 glycoprotein, has also been described.196,200

Protection from aerosol exposure correlated with serum neutralization or antibody titers in some studies 

in mice, hamsters, and NHPs,130,131,135,198,201 but this is 

not consistently the case.

There have also been reports of virus-specific cytotoxic T cell responses induced against alphaviruses.202–204

Although cytotoxic T cell activity was not detected in 

early studies with a VEEV vaccine in mice,205 more 

recent studies have demonstrated a role for certain 

subsets of T cells in protection against VEEV.206–208

Nonspecific immune responses that occur following alphavirus infection include the induction of 

secretion of interferon (IFN)209–213 and the activation 

of cytotoxic macrophages.214 Several studies have 

demonstrated the importance of the innate immune 

response, specifically IFN-α, in resistance to alphavirus infection. Studies with Semliki Forest virus and 

VEEV have shown that IFNα/β receptor knockout 

mice are more susceptible to infection.215–217 Pre- and 

postexposure administration of IFN or inducers of 

interferon in vivo may be effective for protection 

against alphaviruses.218,219 IFN-β was beneficial in 

protection against the Semliki Forest virus peripheral challenge when administered up to 6 days postexposure. Mice were resistant to subcutaneous challenge with VEEV TrD and partially protected from 

inhalation challenge when administered pegylated 

IFN-α on days -2 and +5 relative to exposure.220

Pretreating mice with polyinosinic:polycytidylic 

acid (poly I:C) afforded partial protection against 

peripheral challenge with EEEV,215 and poly I:C with 

added carboxymethylcellulose and poly-L-lysine 

similarly induces protection against respiratory 

challenge with WEEV.219 Although these studies 

clearly indicate the importance of interferons in host 

resistance to alphavirus infections, further study 

is necessary to determine the efficacy of IFN-α for 

prophylactic or therapeutic use in humans.

Passive Immunization

Passive transfer of neutralizing antisera or 

monoclonal antibodies to naive recipients protects 

animals from subsequent parenteral challenge with 

homologous VEEV strains.201,212,221 Passive transfer 

of nonneutralizing, anti-E1 monoclonal antibodies directed against appropriate epitopes is also 

protective against SINV,200 WEEV,198 and VEEV.201

Monoclonal antibodies specific for the E3 protein 

of VEEV IAB do not neutralize VEEV IAB TrD in 

vitro; however, they inhibit VEEV IAB TrD production in infected cells and protect against intraperitoneal challenge with VEEV IAB TrD after passive 

transfer in mice.106 In contrast, for the respiratory 

route of infection, uniform protection was not observed after passive transfer of hyperimmune serum 

to hamsters198 neutralizing monoclonal antibodies 

to mice,222 suggesting that either additional immune 

mechanisms or the presence of protective antibodies 

along the respiratory tract may be needed. The time 

between the administration of immune serum and 

virus exposure may also be relevant. Protection of 

mice from intracerebral inoculation with WEEV was 

observed if immune serum was given no more than 3 

days before virus exposure.223,224 Similarly, monkeys 

passively immunized with horse antiserum to EEE or 

WEE resisted intranasal challenge from homologous 

virus 24 hours later, but they were unable to resist a 

second challenge with the same virus 7 weeks later.225

However, as the immune serum given in both studies 

was xenogeneic, the loss of protective capacity was 

presumably related in part to active clearance of the 

immune serum by the recipients.

The effect of administering immune serum to 

animals after the establishment of intracerebral infections has also been evaluated. Several studies using 

different alphaviruses demonstrated at least partial 

protection if the immune serum was administered 

within 24 hours of infection.223–228 Other researchers 

have suggested that postinfection serum transfer may 

also cause a more severe pathology, or may merely 

delay the onset of disease symptoms.41 Aggressive 

serotherapy following infections of two laboratory 

workers who developed acute WEE encephalitis resulted in the survival of one patient,39 but was ineffective in the second patient.225

In an EEE outbreak in New Jersey in 1959, 22 of 32 

diagnosed patients died. Most patients had demonstrable antibody during the onset or progression of 

encephalitis, and neutralizing antibody titers in sera 

from patients who died were generally similar to those 

observed in patients who recovered.229 This finding, 

coupled with animal studies indicating that transfer 

of virus-neutralizing anti-sera was unable to prevent 

progression of disease if infection of the brain was 

firmly established as described above, suggests that 

serotherapy would be an ineffective means of treatment for these virus infections unless initiated early 

in the course of disease.

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Medical Aspects of Biological Warfare 

Active Immunization

Investigational New Drug Vaccines

Although no vaccines exist against the encephalitic 

alphaviruses that are licensed for use in humans, the 

US Army has developed vaccines that are currently 

used under IND status to protect at-risk personnel 

including the live-attenuated VEEV vaccine TC-83 

and inactivated vaccines for VEEV (C-84), EEEV, and 

WEEV. The characteristics of these vaccines and the 

responses induced in human vaccinees are summarized in Table 20-4. 

Live Vaccines

The TC-83 VEEV vaccine, which was developed in 

1961 by serial passage of the virulent TrD strain in 

fetal guinea pig heart cells,230 is administered subcutaneously at 1 x 104

 to 2 x 104 plaque-forming units 

per 0.5 mL dose. The vaccine was used initially in 

laboratory and field personnel at risk for exposure 

to VEEV,231 and more than 6,000 people received the 

vaccine between 1964 and 1972.231 For reasons that 

remain unclear, approximately 20% of the people 

who receive TC-83 fail to develop a detectable neutralizing antibody response and presumably would 

not be protected if exposed to the virus. Another 20% 

to 25% of vaccine recipients experience clinical reactions ranging from mild transient symptoms to fever, 

chills, sore throat, and malaise in some cases sufficient 

to require bedrest.230,232 However, for recipients who 

respond with postvaccination titers of at least 1 per 

20, long-term follow-up studies have shown that titers 

persist for several years.233 In humans, documented 

vaccine-breakthrough infections have been attributed 

largely to exposure to heterologous, enzootic strains 

of VEEV.37,61,62 Although pregnant mares were not 

adversely affected by TC-83,234 pregnant women are 

advised not to receive the TC-83 vaccine because 

wild-type VEEV may have been associated with 

spontaneous abortions or stillbirths during epidemics 

in Venezuela in 1962 and 1995.27,121 

In animals, TC-83 vaccination protects hamsters 

from a lethal VEEV subcutaneous or aerosol challenge,135 although up to 20% of hamsters may die 

from reactions to the vaccine.127,235 Subcutaneous immunization of monkeys with the vaccine produces 

neutralizing antibody responses and protection from 

virulent VEEV delivered by peripheral or intranasal 

challenge.134 However, TC-83 provides only partial 

protection against aerosol challenge in outbred mice.139

TC-83 has been extensively administered to horses, 

burros, and mules, in part because large numbers of 

equids were vaccinated during the 1969–1970 and 1995 

epizootics.236,237 TC-83 immunization produces febrile 

TABLE 20-4

VACCINES AVAILABLE FOR VEE, EEE, AND WEE VIRUSES

Dose (mL)/

Route of Responding

Vaccine Form/Strain Administration Schedule Booster Dose/% Duration* Route

VEE (TC-83) TrD 0.5 mL/sc Day 0 82% 92% C-84/sc

Attenuated

VEE (C-84)† Inactivated TC-83 0.5 mL/sc After TC-83 76% NR‡ 60% 0.5 mL/sc

100% WT§ 100%

EEE Inactivated PE-6¥ 0.5 mL/sc Days 0, 28 58% 75% 0.1 mL/id

WEE Inactivated CM-4884¥ 0.5 mL/sc Days 0, 7, 28 50% 20% 0.5 mL/sc

*% of responders whose virus-neutralizing titers persist for at least 1 year

†current IND protocols specify use of C-84 only as a booster vaccine

‡TC-83 nonresponders

§

TC-83 responders given C-84 to boost waning titers

¥

laboratory designation

EEE: Eastern equine encephalitis

id: intradermal

IND: investigational new drug

sc: subcutaneous

TC: cell culture

TrD: Trinidad donkey

VEE: Venezuelan equine encephalitis

WEE: Western equine encephalitis

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responses and leukopenia in some equids,238,239 but 

neutralizing antibody responses to homologous (serotype IAB) virus eventually develop in 90% of these 

animals.238,240 Although it was difficult to accurately 

assess vaccine efficacy under the conditions of an 

ongoing epizootic, herds of animals known to have 

been immunized at least 2 weeks before any disease occurrence in the area did not sustain any VEEV-related 

deaths, whereas unimmunized herds experienced up 

to 60% mortality rates.232

The phenomenon of vaccine interference, in which 

prior immunity to heterologous alphaviruses inhibits 

vaccine virus replication and subsequent immune 

responses, is an unresolved problem with the use of 

TC-83 and presumably with other live-attenuated alphavirus vaccines. This occurrence has been observed 

in horses,241,242 in which preexisting antibodies to EEE 

and WEE may have interfered with TC-83 vaccination. 

Interference has also been observed in humans, in 

which preexisting immunity to a live alphavirus vaccine inhibited effective subsequent immunization with 

a second, different alphavirus vaccine.243 However, a 

recent study found no evidence for interference when 

vaccines for VEE, WEE, and EEE were administered 

simultaneously to NHPs. Interference may greatly 

depend on the nature of the vaccine and the virus 

strain(s) selected for both the vaccine and in vitro 

neutralization studies.244

Inactivated Vaccines

Early attempts to develop an inactivated VEEV vaccine resulted in preparations that contained residual live 

virus and caused disease in 4% of those who received 

it.226,245 Development of a formalin inactivated TC-83 

VEEV vaccine (C-84) was initiated because of the problems associated with incomplete inactivation.246 Initial 

clinical trials with the C-84 inactivated vaccine were 

begun in 1976 in 14 volunteers previously immunized 

with TC-83, and subsequently in 14 naive volunteers.247

The vaccine was found to be safe and elicited only mild 

tenderness at the injection site. Although C-84 was 

immunogenic, three doses were required to maintain 

detectable neutralizing antibody titers in recipients. A 

subsequent study has shown that most of the TC-83 

nonresponders and all of the individuals with waning 

titers responded to a booster dose of C-84 with a high 

probability of maintaining a titer for 3 years.231 However, 

the observation that hamsters given C-84 vaccine were 

protected from subcutaneous challenge but not from 

an aerosol exposure to VEE virus135 raised concerns 

that C-84 vaccination may not protect at-risk laboratory workers from aerosol exposure. Therefore, C-84 is 

currently administered only as a booster immunogen.

The PE-6 strain of EEEV was passed in primary 

chick-embryo cell cultures, and then it was formalin 

treated and lyophilized to produce an inactivated 

vaccine for EEEV.248 This vaccine is administered as a 

0.5-mL dose subcutaneously on days 0 and 28, with 

0.1-mL intradermal booster doses given as needed to 

maintain detectable neutralizing antibody titers. In 

initial clinical trials, only mild reactions to the vaccine 

were observed, and immunogenicity was demonstrated.249 The vaccine was given to 896 at-risk laboratory 

workers between 1976 and 1991 with no significant 

clinical reactions observed. A long-term follow-up 

study of 573 recipients indicated a 58% response rate 

after the primary series, and a 25% chance of failing to 

maintain adequate titers for 1 year. Response rates and 

persistence of titers increased with the administration 

of additional booster doses.231

A formalin inactivated WEEV vaccine was similarly prepared using the B-11 or CM-4884 virus 

strain, and it caused only mild clinical reactions when 

administered to WEEV-naive individuals, according 

to Phillip Pittman, when he was the former chief of 

the special immunization program at the US Army 

Medical Research Institute of Infectious Diseases in 

1996. Between 1976 and 1990, 359 laboratory workers were immunized with this vaccine. Long-term 

follow-up studies have indicated that administration 

of three 0.5 mL doses subcutaneously on days 0, 7, 

and 28 results in a 50% response rate (neutralization titer >1:40) after the primary series. Only 20% 

of the recipients maintain a titer for 1 year, although 

this level can be increased to 60% to 70% with additional booster immunizations, according to Pittman. 

As with the live-attenuated alphavirus vaccines, 

immune interference has also been observed after 

vaccinations with the inactivated alphavirus vaccines. 

Volunteers who received the inactivated EEEV and 

WEEV vaccines before receiving the live-attenuated 

VEEV vaccine had significantly lower rates of neutralizing antibody response than those receiving the VEEV 

vaccine before the EEEV and WEEV vaccines.250 

Next Generation Alphavirus Vaccines

Significant limitations are associated with the liveattenuated VEEV and formalin-inactivated VEEV, 

EEEV, and WEEV IND vaccines used to protect 

at-risk personnel. These limitations include the reactogenicity of the live-attenuated vaccine, the poor 

immunogenicity of the formalin-inactivated vaccines, 

and the demonstrated immune interference issues 

associated with these vaccines. As a result, efforts 

are underway using many different platforms to 

develop next-generation vaccines that can safely and 

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effectively protect against VEEV, EEEV, and WEEV; 

next-generation subunit, live-attenuated, inactivated, 

DNA, virus replicon particle, and SINV-based chimeric vaccines are all at various stages of development. Subunit vaccines consisting of glycoproteins 

produced in Escherichia coli or baculovirus expression 

systems have provided limited success in mouse models.251–254 Inactivated virus vaccines provided efficacy 

against aerosol challenge in mice.255 However, further 

study is required to determine the efficacy of subunit 

and inactivated virus vaccines in NHPs. SINV-based 

chimeric virus vaccines are immunogenic and protect 

mice against VEEV, EEEV, and WEEV.256–260 A recent 

study demonstrated a chimeric SINV–EEEV vaccine 

candidate protected most NHPs (82%) from lethal 

EEE disease following aerosol infection.261 The US 

Army has extensive experience in the development 

of next-generation live-attenuated, DNA, and viruslike replicon particle vaccines to protect against the 

encephalitic alphaviruses. 

V3526

The next-generation live-attenuated VEEV vaccine, 

V3526, was created by mutation of the furin cleavage 

site of PE2 in wild-type VEEV IAB combined with a 

second-site suppressor mutation in the E1 protein.262

These mutations significantly reduced the neurovirulence of V3526 as compared to the parent clone and 

TC-83 in mice, NHPs, and horses, and they stabilized 

the attenuated phenotype.130,150,151,263,264 V3526 has 

been shown to effectively elicit protective immune 

responses in rodents, NHPs, and horses against lethal subcutaneous or aerosol challenges with VEEV 

IAB TrD as well as other VEEV subtypes and related 

viruses (IC, IE, and Mucambo virus).135,130,150,151,263

Mice were protected from aerosol challenge with 

VEEV IAB TrD for up to 1 year following vaccination 

with V3526.151 Furthermore, immune interference 

was not observed when V3526 was administered to 

mice previously vaccinated with other alphavirus 

vaccines. Based on the success of V3526 in nonclinical studies, a phase 1 clinical trial was conducted to 

evaluate the safety, tolerability, and immunogenicity of this vaccine candidate in humans. V3526 was 

immunogenic in virtually all recipients, with robust 

immune responses elicited after administration of a 

single dose of the vaccine down to doses as low as 25 

plaque-forming units. However, a significant number 

of the vaccinated subjects experienced adverse events 

consistent with a viral syndrome to include headache, 

fever, malaise, myalgia, and sore throat.265 Based 

on these findings, clinical development of V3526 

was discontinued. Gamma irradiated and formalininactivated V3256 vaccines have subsequently been 

tested in mice, but these have not progressed beyond 

animal studies.266–268

DNA Vaccines

DNA vaccination with plasmids that express 

protein antigens within cells has numerous inherent 

advantages as a platform for the development of nextgeneration vaccines. Some of these benefits include 

that DNA vaccines:

• can be rapidly produced using well-established Good Manufacturing Practices and 

without the need to propagate a pathogen or 

inactivate an infectious organism; 

• avoid problems of preexisting immunity resulting from a lack of a host immune response 

to the vector backbone; and 

• have been demonstrated to be safe in numerous human clinical trials.269

Although a DNA vaccine expressing the structural 

proteins (C-E3-E2-6K-E1) of VEEV IAB TrD from the 

wild-type genes delivered by particle-mediated epidermal delivery or “gene gun” elicited strong overall 

antibody responses in multiple animal species, the 

neutralizing antibody responses were low and only 

partial protection against VEEV IAB TrD aerosol 

challenge was observed in mice and NHPs.270–272 A 

codon-optimized DNA vaccine construct expressing the structural proteins of VEEV IAB TrD minus 

the capsid protein delivered by intramuscular electroporation elicited improved antibody responses, 

including high levels of neutralizing antibodies in 

multiple animal species, and it provided protective 

immunity against VEEV IAB TrD aerosol challenge 

in mice and NHPs.273 Based on these results, a phase 

1 clinical trial to evaluate the safety, tolerability, and 

immunogenicity of this vaccine candidate in humans 

has been initiated. A trivalent formulation of VEEV, 

EEEV, and WEEV DNA vaccine constructs has also 

been extensively evaluated in animals to reach the 

goal of developing a vaccine capable of simultaneously eliciting protective immunity against VEEV, 

EEEV, and WEEV. Important to this goal, the immunogenicity of the combined VEEV, EEEV, and 

WEEV DNA vaccines was not significantly reduced 

as compared to the individual DNA vaccines, and 

protection against VEEV, EEEV, and WEEV aerosol 

challenge has been observed in mice and NHPs. As 

a result, nonclinical studies required to advance this 

trivalent DNA vaccine formulation into phase 1 clinical testing in humans are being conducted.

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Virus Replicon Particle Vaccines

Alphavirus-based replicon systems, derived by 

deletion of the genes encoding the viral structural proteins from full-length genomic complementary DNA 

clones and replacing these with heterologous genes of 

interest, represent a promising method for the development of next-generation vaccines.274 Virus-like replicon particles (VRPs) are produced in vitro following 

cotransfection of cells with the replicon RNA, which 

express the nonstructural proteins in cis and helper 

RNAs, which supply the structural proteins in trans.275

The immunogenicity and protective efficacy of VRPs 

expressing VEEV, EEEV, or WEEV envelope glycoprotein genes containing the furin cleavage site mutation 

in PE2 have been extensively evaluated in mice and 

NHPs.244 The VEEV, EEEV, and WEEV VRP vaccines 

elicited strong neutralizing antibody responses when 

administered individually and in combination to mice. 

In addition, mice receiving the individual or combined 

VRP vaccines were protected from respective VEEV, 

EEEV, or WEEV aerosol challenge up to 12 months 

after vaccination. NHPs receiving the individual VEEV 

or EEEV or the combined VRP vaccines developed 

strong neutralizing antibody responses and were 

protected against VEEV and EEEV aerosol challenge, 

respectively. However, the individual WEEV and 

combined VRP vaccines elicited low or no neutralizing 

antibodies against WEEV in NHPs, and incomplete 

protection against WEEV aerosol challenge was observed. The VEEV, EEEV, and WEEV VRP vaccines 

have not yet progressed beyond nonclinical studies.

THERAPEUTICS 

No licensed therapeutics are available for the specific treatment of alphavirus infections in humans. 

However, several studies have reported the identification of compounds with in vitro efficacy against alphaviruses.276–280 In three of these studies, the identified 

compounds targeted proteins of the viral polymerase 

complex.276–278 Two other studies identified compounds 

targeting host protein kinases.279,280 The efficacy of several of these compounds was demonstrated in rodent 

models of VEEV or WEEV infection276–279; however, no 

studies have reported efficacy of any compounds in 

NHP models of alphavirus infection.

SUMMARY

The equine encephalitis viruses consist of three 

antigenically related viruses within the Alphavirus

genus of the family Togaviridae: VEEV, WEEV, and 

EEEV. These viruses are vectored in nature by various 

species of mosquitoes and cause periodic epizootics 

among equines. Infection of equines with virulent 

strains of any these viruses produces a similar clinical course of severe encephalitis with high mortality. 

However, the clinical course following infection of 

humans differs. EEE is the most severe of the arbovirus encephalitides, with case fatality rates of 50% to 

70%. WEEV is generally less virulent for adults, but 

the infection commonly produces severe encephalitis 

in children, with case fatality rates approaching 10%. 

In contrast, encephalitis is rare following VEEV infection, but essentially all infected individuals develop a 

prostrating syndrome of high fever, headache, malaise, 

and prolonged convalescence.

Although natural infections are acquired by mosquito bite, these viruses are also highly infectious in 

low doses as aerosols. These viruses can be produced 

in large quantities using inexpensive and unsophisticated systems, are relatively stable, and are readily 

amenable to genetic manipulation. For these reasons, 

the equine encephalomyelitis viruses are considered 

credible biological warfare threats.

No specific therapy exists for infections caused 

by these viruses. A live-attenuated vaccine for VEEV 

(TC-83) and inactivated vaccines for VEEV, EEEV, 

and WEEV have been developed and are used under IND status. Although these vaccines are useful 

in protecting at-risk individuals, they have certain 

disadvantages, and improved vaccines are being 

developed.

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232. Burke DS, Ramsburg HH, Edelman R. Persistence in humans of antibody to subtypes of Venezuelan equine encephalomyelitis (VEE) virus after immunization with attenuated (TC-83) VEE virus vaccine. J Infect Dis. 1977;136:354–359.

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233. Eddy GA, Martin DH, Reeves WC, Johnson KM. Field studies of an attenuated Venezuelan equine encephalomyelitis 

vaccine (strain TC-83). Infect Immun. 1972;5:160–163.

234. Wenger F. Venezuelan equine encephalitis. Teratology. 1977;16:359–362.

235. Spertzel RO, Kahn DE. Safety and efficacy of an attenuated Venezuelan equine encephalomyelitis vaccine for use in 

Equidae. J Am Vet Med Assoc. 1971;159:731–738.

236. Calisher CH, Maness KS. Laboratory studies of Venezuelan equine encephalitis virus in equines, Texas, 1971. J Clin 

Microbiol. 1975;2:198–205.

237. Zehmer RB, Dean PB, Sudia WD, Calisher CH, Sather GE, Parker RL. Venezuelan equine encephalitis epidemic in 

Texas, 1971. Health Serv Rep. 1974;89:278–282.

238. Monlux WS, Luedke AJ, Bowne J. Central nervous system response of horses to Venezuelan equine encephalomyelitis 

vaccine (TC-83). J Am Vet Med Assoc. 1972;161:265–269.

239. Walton TE, Alvarez O Jr, Buckwalter RM, Johnson KM. Experimental infection of horses with an attenuated Venezuelan 

equine encephalomyelitis vaccine (strain TC-83). Infect Immun. 1972;5:750–756.

240. Jochim MM, Barber TL, Luedke AJ. Venezuelan equine encephalomyelitis: antibody response in vaccinated horses 

and resistance to infection with virulent virus. J Am Vet Med Assoc. 1973;162:280–283.

241. Calisher CH, Sasso DR, Sather GE. Possible evidence for interference with Venezuelan equine encephalitis virus vaccination of equines by pre-existing antibody to eastern or western equine encephalitis virus, or both. Appl Microbiol. 

1973;26:485–488.

242. McClain DJ, Pittman PR, Ramsburg HH, et al. Immunologic interference from sequential administration of live attenuated alphavirus vaccines. J Infect Dis. 1998;177:634–641.

243. Sutton LS, Brooke CC. Venezuelan equine encephalomyelitis due to vaccination in man. J Am Med Assoc. 1954;155:1473–

1476.

244. Reed DS, Glass PJ, Bakken RR, et al. Combined alphavirus replicon particle vaccine induces durable and cross-protective 

immune responses against equine encephalitis viruses. J Virol. 2014;88:12077–12086.

245. Cole FE Jr, May SW, Eddy GA. Inactivated Venezuelan equine encephalomyelitis vaccine prepared from attenuated 

(TC-83 strain) virus. Appl Microbiol. 1974;27:150–153.

246. Edelman R, Ascher MS, Oster CN, Ramsburg HH, Cole FE, Eddy GA. Evaluation in humans of a new, inactivated 

vaccine for Venezuelan equine encephalitis virus (C-84). J Infect Dis. 1979;140:708–715.

247. Maire LF III, McKinney RW, Cole FE Jr. An inactivated eastern equine encephalomyelitis vaccine propagated in chickembryo cell culture. I. Production and testing. Am J Trop Med Hyg. 1970;19:119–122.

248. Bartelloni PJ, McKinney RW, Duffy TP, Cole FE Jr. An inactivated eastern equine encephalomyelitis vaccine propagated 

in chick-embryo cell culture. II. Clinical and serologic responses in man. Am J Trop Med Hyg. 1970;19:123–126.

249. Bartelloni PJ, McKinney RW, Calia FM, Ramsburg HH, Cole FE Jr. Inactivated western equine encephalomyelitis 

vaccine propagated in chick embryo cell culture: clinical and serological evaluation in man. Am J Trop Med Hyg. 

1971;20:146–149.

250. Pittman PR, Liu CT, Cannon TL, Mangiafico JA, Gibbs PH. Immune interference after sequential alphavirus vaccine 

vaccinations. Vaccine. 2009;27:4879–4882.

251. Das D, Gares SL, Nagata LP, Suresh MR. Evaluation of a western equine encephalitis recombinant E1 protein for 

protective immunity and diagnostics. Antiviral Res. 2004;64:85–92.

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252. Hodgson LA, Ludwig GV, Smith JF. Expression, processing, and immunogenicity of the structural proteins of Venezuelan equine encephalitis virus from recombinant baculovirus vectors. Vaccine. 1999;17:1151–1160.

253. Das D, Nagata LP, Suresh MR. Immunological evaluation of Escherichia coli expressed E2 protein of Western equine 

encephalitis virus. Virus Res. 2007;128:26–33.

254. Toth AM, Geisler C, Aumiller JJ, Jarvis DL. Factors affecting recombinant Western equine encephalitis virus glycoprotein production in the baculovirus system. Protein Expr Purif. 2011;80:274–282.

255. Honnold SP, Bakken RR, Fisher D, et al. Second generation inactivated eastern equine encephalitis virus vaccine 

candidates protect mice against a lethal aerosol challenge. PLoS One. 2014;9:e104708.

256. Atasheva S, Wang E, Adams AP, et al. Chimeric alphavirus vaccine candidates protect mice from intranasal challenge 

with western equine encephalitis virus. Vaccine. 2009;27:4309–4319.

257. Wang E, Petrakova O, Adams AP, et al. Chimeric Sindbis/eastern equine encephalitis vaccine candidates are highly 

attenuated and immunogenic in mice. Vaccine. 2007;25:7573–7581.

258. Paessler S, Weaver SC. Vaccines for Venezuelan equine encephalitis. Vaccine. 2009;27(Suppl 4):D80–D85.

259. Paessler S, Fayzulin RZ, Anishchenko M, Greene IP, Weaver SC, Frolov I. Recombinant sindbis/Venezuelan equine 

encephalitis virus is highly attenuated and immunogenic. J Virol. 2003;77:9278–9286.

260. Paessler S, Ni H, Petrakova O, et al. Replication and clearance of Venezuelan equine encephalitis virus from the brains 

of animals vaccinated with chimeric SIN/VEE viruses. J Virol. 2006;80:2784–2796.

261. Roy CJ, Adams AP, Wang E, et al. A chimeric Sindbis-based vaccine protects cynomolgus macaques against a lethal 

aerosol challenge of eastern equine encephalitis virus. Vaccine. 2013;31:1464–1470.

262. Davis NL, Brown KW, Greenwald GF, et al. Attenuated mutants of Venezuelan equine encephalitis virus containing lethal mutations in the PE2 cleavage signal combined with a second-site suppressor mutation in E1. Virology. 

1995;212:102–110.

263. Fine DL, Roberts BA, Teehee ML, et al. Venezuelan equine encephalitis virus vaccine candidate (V3526) safety, immunogenicity and efficacy in horses. Vaccine. 2007;25:1868–1876.

264. Kenney JL, Volk SM, Pandya J, Wang E, Liang X, Weaver SC. Stability of RNA virus attenuation approaches. Vaccine. 

2011;29:2230–2234.

265. Martin SS, Bakken RR, Lind CM, et al. Telemetric analysis to detect febrile responses in mice following vaccination 

with a live-attenuated virus vaccine. Vaccine. 2009;27:6814–6823.

266. Martin SS, Bakken RR, Lind CM, et al. Evaluation of formalin inactivated V3526 virus with adjuvant as a next generation vaccine candidate for Venezuelan equine encephalitis virus. Vaccine. 2010;28:3143–3151.

267. Martin SS, Bakker RR, Lind CM, et al. Comparison of the immunological responses and efficacy of gamma-irradiated 

V3526 vaccine formulations against subcutaneous and aerosol challenge with Venezuelan equine encephalitis virus 

subtype IAB. Vaccine. 2010;28:1031–1040.

268. Fine DL, Jenkins E, Martin SS, Glass P, Parker MD, Grimm B. A multisystem approach for development and evaluation of inactivated vaccines for Venezuelan equine encephalitis virus (VEEV). J Virol Methods. 2010;163:424–432.

269. Dupuy LC, Schmaljohn CS. DNA vaccines for biodefense. Expert Rev Vaccines. 2009;8:1739–1754.

270. Riemenschneider J, Garrison A, Geisbert J, et al. Comparison of individual and combination DNA vaccines for B. 

anthracis, Ebola virus, Marburg virus and Venezuelan equine encephalitis virus. Vaccine. 2003;21:4071–4080.

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271. Dupuy LC, Locher CP, Paidhungat M, et al. Directed molecular evolution improves the immunogenicity and protective efficacy of a Venezuelan equine encephalitis virus DNA vaccine. Vaccine. 2009;27:4152–4160.

272. Dupuy LC, Richards MJ, Reed DS, Schmaljohn CS. Immunogenicity and protective efficacy of a DNA vaccine against 

Venezuelan equine encephalitis virus aerosol challenge in nonhuman primates. Vaccine. 2010;28:7345–7350.

273. Dupuy LC, Richards MJ, Ellefsen B, et al. A DNA vaccine for Venezuelan equine encephalitis virus delivered by 

intramulscular electroporation elicits high levels of neutralizing antibodies in multiple animal models and provides 

protective immunity to mice and nonhuman primates. Clin Vaccine Immunol. 2011;18:707–716.

274. Pushko P, Parker M, Ludwig GV, Davis NL, Johnston RE, Smith JF. Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous 

pathogens in vivo. Virology. 1997;239:389–401.

275. Kamrud KI, Custer M, Dudek JM, et al. Alphavirus replicon approach to promoterless analysis of IRES elements. 

Virology. 2007;360:376–387.

276. Julander JG, Smee DF, Morrey JD, Furuta Y. Effect of T-705 treatment on western equine encephalitis in a mouse 

model. Antiviral Res. 2009;82:169–171.

277. Chung DH, Jonsson CB, Tower NA, et al. Discovery of a novel compound with anti-Venezuelan equine encephalitis 

virus activity that targets the nonstructural protein 2. PLoS Pathog. 2014;10:e1004213.

278. Schroeder CE, Yao T, Sotsky J, et al. Development of (E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-Nphenylbenzamide, ML336: novel 2-amidinophenylbenzamides as potent inhibitors of Venezuelan equine encephalitis 

virus. J Med Chem. 2014;57:8608–8621.

279. Kehn-Hall K, Narayanan A, Lundberg L, et al. Modulation of GSK-3[insert Beta] activity in Venezuelan equine encephalitis virus infection. PLoS One. 2012;7:e34761.

280. Voss K, Amaya M, Mueller C, et al. Inhibition of host extracellular signal-regulated kinase (ERK) activation decreases 

new world alphavirus multiplication in infected cells. Virology. 2014;468–470:490–503.

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Chapter 21

HEMORRHAGIC FEVER- CAUSING 

MAMMARENAVIRUSES

SHELI R. RADOSHITZKY, PhD*; JENS H. KUHN, MD, PhD, PhD, MS†; PETER B. JAHRLING, PhD‡; and SINA BAVARI, PhD§

INTRODUCTION

HISTORY AND EPIDEMIOLOGY

Old World Mammarenaviruses

New World Mammarenaviruses

RESERVOIRS OF HEMORRHAGIC FEVER MAMMARENAVIRUSES

CLINICAL PRESENTATION

Old World Mammarenaviral Hemorrhagic Fevers

New World Mammarenaviral Hemorrhagic Fevers 

TAXONOMY AND PHYLOGENETIC RELATIONSHIPS

MOLECULAR CHARACTERISTICS 

PATHOLOGY AND PATHOGENESIS

Old World Mammarenaviral Hemorrhagic Fevers 

New World Mammarenaviral Hemorrhagic Fevers

COAGULOPATHIES

IMMUNE RESPONSE

Old World Mammarenaviral Hemorrhagic Fevers 

New World Mammarenaviral Hemorrhagic Fevers 

DIAGNOSIS

Detection of Virus-Specific Antibodies and Viral Antigens 

Detection of Viral Nucleic Acids

Virus Isolation

TREATMENT AND VACCINES

Passive Antibody Therapy

Vaccines

Antiviral Agents

SUMMARY

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*Principal Investigator, Molecular and Translational Sciences Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, 

Room 902-U, Fort Detrick, Maryland 21702; formerly, Postdoctoral Fellow, Toxicology Division, US Army Medical Research Institute of Infectious 

Diseases, 1425 Porter Street, Fort Detrick, Maryland

†

Virology Lead, National Institutes of Health/National Institute of Allergy and Infectious Diseases/Division of Clinical Research/Integrated Research 

Facility, B-8200 Research Plaza, Room 1A-132, Fort Detrick, Maryland 21702; formerly, Research Scholar, Harvard Medical School, Microbiology and 

Molecular Genetics Department, New England Primate Research Center, 1 Pine Hill Drive, Southborough, Massachusetts 

‡

Captain (Retired), Medical Service Corps, US Army; Director, National Institutes of Health/Division of Clinical Research/National Institute of Allergy and Infectious Diseases/Integrated Research Facility, 8200 Research Plaza, Room 1A-111A, Fort Detrick, Maryland 21702; formerly, Principal 

Scientific Advisor (Senior Research Scientist), US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

§

Science Director, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Room 900, Fort Detrick, Maryland 21702; formerly, 

Division Chief, Toxicology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Room 900, Fort Detrick, Maryland

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INTRODUCTION 

chupo virus (MACV), and Sabiá virus (SABV). The 

clinical course and pathology of the viral hemorrhagic fevers caused by these viruses can differ, and 

therefore various diagnosis and treatment options 

are available. This chapter summarizes similarities 

and disparities between the viruses and the diseases 

they cause. 

The family Arenaviridae includes the two genera 

Mammarenavirus and Reptarenavirus, which are established to accommodate mammalian and reptilian 

arenaviruses, respectively. Seven mammarenaviruses 

cause viral hemorrhagic fever in humans: Lassa virus 

(LASV), Lujo virus (LUJV), Chapare virus (CHAPV), 

Guanarito virus (GTOV), Junín virus (JUNV), MaHISTORY AND EPIDEMIOLOGY

Old World Mammarenaviruses

Lassa fever is a severe disease common in areas of 

western sub-Saharan Africa (Nigeria, Liberia, Guinea, 

Sierra Leone; Figure 21-1). Lassa fever was first described in Jos, Nigeria, in 1969 in a hospitalized patient 

and a caretaker, both of whom became severely ill and 

subsequently died.1 The etiologic agent of Lassa fever 

is LASV. The case fatality rate of Lassa fever is about 

Figure 21-1. Global distribution of viral hemorrhagic fever-causing mammarenaviruses. Mammarenaviruses are phylogenetically and serologically differentiated into Old World (orange) and New World (yellow) groups. Old World mammarenaviruses: Lujo virus (Zambia) and Lassa virus (other highlighted countries). New World mammarenaviruses: Guanarito virus 

(Venezuela), Sabiá virus (Brazil), Chapare and Machupo viruses (Bolivia), and Junín virus (Argentina).

1% to 2% in the endemic areas, with an estimated 

300,000 to 500,000 infections annually. The disease is 

especially severe late in pregnancy.2 Infections tend 

to be more common in February to April compared 

to the rest of the year. 

In September and October of 2008, LUJV was 

discovered during a nosocomial viral hemorrhagic 

fever outbreak. The index case became infected 

in Lusaka, Zambia (Figure 21-1), but the origin of 

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infection remains unknown. The patient was transferred to Johannesburg, South Africa, for medical 

management. Three secondary infections and one 

tertiary infection were reported, from which only 

one person survived.3

New World Mammarenaviruses

Argentinian hemorrhagic fever (AHF) cases were 

first described in the humid Pampas of Argentina in 

1955 (Figures 21-1 and 21-2).4 However, AHF epidemics may have occurred as early as 1943. The etiologic 

agent of AHF—JUNV—was later isolated from humans.5,6 Since the 1950s, JUNV is estimated to have 

caused about 30,000 AHF cases. Without treatment, 

the case fatality rate is approximately 20%. The AHFendemic region has expanded progressively into 

north-central Argentina to the extent that currently 5 

million people are considered to be at risk of infection.7

Bolivian hemorrhagic fever (BHF) was recognized 

in 1959 on the island of Orobayaya in the Beni Department in northeastern Bolivia (Figures 21-1 and 21-3). 

However, it was not until 1964, after initial outbreaks 

of this emerging hemorrhagic fever (1959–1962) caused 

470 cases, that BHF was first described by Mackenzie 

and coworkers.8 MACV, the etiological agent of BHF, is 

named after a river close to the outbreak area. MACV 

was isolated in 1963 from the spleen of a fatal human 

case in San Joaquín.9 Between 1962 and 1964, another 

series of localized BHF outbreaks occurred, which 

involved more than 1,000 patients, of whom 180 died. 

After 20 years of no reported cases, mainly as a result 

of rodent control measures,9 an outbreak of 19 cases 

occurred in 1994. Eight additional BHF cases were 

recognized in 1999, and 18 cases occurred in 2000. A 

larger outbreak, with 200 suspected cases, occurred in 

2008.10 The case fatality rate of BHF is approximately 

5% to 30%.

Figure 21-2. Geographic distribution of Junín virus in Argentina. Hyperendemic areas are shown in pink.

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Hemorrhagic Fever-Causing Mammarenaviruses

GTOV emerged in 1989 as the cause of a yet officially unnamed disease that is often referred to 

as “Venezuelan hemorrhagic fever (VHF).” This 

severe hemorrhagic illness was recognized when 

settlers moved into cleared forest areas in the municipalities of Guanarito and Guanare in the state 

of Portuguesa in central Venezuela (Figures 21-1 

and 21-4). The outbreak was initially misdiagnosed 

as severe dengue. Between 1990 and 1991, a total 

of 104 cases was reported with an approximately 

25% case fatality rate.11 The virus was isolated 

from the spleen of a 20-year-old male farm worker 

during autopsy.12 After a seemingly spontaneous 

drop in human cases between 1989 and 1992, a new 

outbreak occurred in 2002 with 18 reported cases.13

By 2006, approximately 600 cases of “VHF” have 

been reported.14,15

SABV, the cause of “Brazilian hemorrhagic fever,” 

was isolated in 1990 from a single patient with a fatal 

infection in São Paulo, Brazil (Figure 21-1).16 Subsequently, two laboratory infections were reported, one 

of which was successfully treated with ribavirin.17

In 2003 to 2004, CHAPV was recovered from a single 

fatal case of viral hemorrhagic fever in the Chapare 

River region in rural Bolivia (Figures 21-1 and 21-3). 

Additional cases were reported from this outbreak; 

however, details and laboratory confirmation are 

lacking.18

Figure 21-3. Geographic distribution of Machupo virus and Chapare virus in Bolivia (yellow). Hyperendemic areas are shown 

in pink. The only known location of Chapare virus infection is shown as a red dot.

RESERVOIRS OF HEMORRHAGIC FEVER MAMMARENAVIRUSES

Rodents of the superfamily Muroidea are the 

natural hosts of most mammarenaviruses. Old World 

mammarenaviruses are found in rodents of the family 

Muridae, subfamily Murinae. New World mammarenaviruses are found in rodents of the family Cricetidae, 

subfamily Sigmodontinae. Bats may transmit Tacaribe 

virus, and reservoirs for Chapare, Lujo, and Sabiá 

viruses have not yet been identified. 

The range of the corresponding rodent/bat host(s) 

determines the geographical distribution of each 

mammarenavirus. Field studies strongly support 

the concept of a single major reservoir host for each 

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virus.19 Principal hosts for LASV, MACV, JUNV, and 

GTOV are the natal mastomys (Mastomys natalensis), 

the big laucha (Calomys callosus), the drylands laucha 

(Calomys musculinus), and the short-tailed zygodont 

(Zygodontomys brevicauda), respectively.2,20–23

Current evidence suggests a long-term “diffuse 

co-evolution” between mammarenaviruses and their 

rodent hosts. According to this model, a parallel phylogeny between the viruses and their corresponding 

rodent host(s) allows for host switches between closely 

related rodents.19,24 Mammarenaviruses establish 

chronic infections in their respective reservoirs accompanied by chronic viremia or viruria without clinical 

signs of disease.25–28

Humans become infected with mammarenaviruses 

through contact with infected rodents or inhalation of 

aerosolized virus from contaminated rodent blood, 

excreta or secreta, or body parts caught in mechanical 

harvesters.13 In Western Africa, peridomestic rodents 

are also part of the diet of inhabitants of LASV-endemic 

areas, and therefore contaminated meat may be another route of virus transmission.29 New World mammarenavirus infections peak during harvest season 

when rodent populations are active. Infected cases are 

predominantly male agricultural workers who come in 

contact with infected rodents. Person-to-person transmission of LASV or MACV is not frequent, but it is 

possible by direct contact with body fluids or excreta of 

infected patients. Such transmission is probably not the 

principal mode of disease dissemination. Only small 

quantities of MACV can be isolated from human blood 

or from throat or oral swabs of infected patients.30

Figure 21-4. Geographic distribution of Guanarito virus in Venezuela. Clusters of infections areas are shown in pink.

CLINICAL PRESENTATION

Old World Mammarenaviral Hemorrhagic Fevers

The signs and symptoms of Lassa fever vary depending on the disease’s severity.31,32 The disease is 

mild or asymptomatic in about 80% of infected people, 

but 20% develop acute Lassa fever. The incubation 

period can range from 1 to 24 days with an average of 

7 to 18 days. Disease onset is insidious with low-grade 

fever, weakness, and general malaise. Within 2 to 4 

days, many patients experience symptoms including 

myalgia; arthralgia; lower back, abdominal, and/or 

retrosternal pain; headache; dizziness; or sore throat. 

Hypotension, productive cough, vomiting, and diarrhea are also common. Pharyngitis or conjunctivitis 

can occur as the disease progresses, and mucosal 

bleeding (gums, nose, and other sites), pleural or 

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pericardial effusions, or facial or neck edema occur in 

more severe cases.33 In the second week after onset, 

acute respiratory distress syndrome, moderate-tosevere diffuse encephalopathy, or shock develops in 

severe cases. Confusion, followed rapidly by tremors, 

convulsions, abnormal posturing, or coma, sometimes occur just before death.34 Another neurological 

manifestation is unilateral or bilateral sensorineural 

deafness, which occurs in about 30% of convalescent 

patients.33,35

Lassa fever presents with symptoms and signs 

indistinguishable from those of other febrile illnesses, 

such as malaria or other viral hemorrhagic fevers. 

Therefore, Lassa fever is difficult to diagnose clinically, but it should be suspected in patients with fever 

(≥38°C) not responding adequately to antimalarial and 

antibiotic drugs. Fever, pharyngitis, retrosternal pain, 

and proteinuria are the most useful clinical predictors 

for a Lassa fever diagnosis. Fever, sore throat, and 

vomiting are the best predictors for negative outcome.33

Disease outcome is also related to the degree of viremia and not to antibody response. The probability of 

fatal disease increases with high viremia, and survival 

rate is lowest in patients with both high viremia and 

high concentrations of aspartate aminotransferase. In 

patients recovering from Lassa fever, virus is cleared 

from blood circulation about 3 weeks after onset of 

illness.36 Survivors of LASV infection often recover 

without sequelae. However, severe sensorineural 

hearing deficits, which may develop during disease, 

may persistent permanently in approximately 13% to 

30% of survivors.37–39

Patients infected with LUJV initially present with 

symptoms and signs of nonspecific febrile illness such 

as severe headache, malaise, vomiting, fever, retrosternal pain, or myalgia. Disease manifestations increase 

in severity over 7 days with the development of diarrhea or pharyngitis. In some patients, morbilliform 

rash or facial edema is evident. Terminal features are 

acute respiratory distress syndrome, cerebral edema, 

neurologic signs, deteriorating renal function, or circulatory collapse. No overt hemorrhage is observed 

besides gingival bleeding, petechial rash, or oozing 

from injection sites in some of the patients.3 However, 

the clinical description of disease caused by LUJV infection is currently based on the observation of only 

five patients. 

New World Mammarenaviral Hemorrhagic Fevers 

New World mammarenaviral hemorrhagic fevers 

caused by CHAPV, GTOV, JUNV, MACV, or SABV 

are clinically similar.4,15,18,40–43 Disease begins insidiously after an incubation period of 1 to 2 weeks. Initial 

symptoms/signs often include fever and malaise, 

headache, myalgia, epigastric pain, or anorexia. After 

3 to 4 days, signs become increasingly severe with 

multisystem involvement: prostration; abdominal 

pain; nausea and vomiting; constipation; or mild diarrhea. In some cases, dizziness, photophobia, retroorbital pain, or disorientation may also appear, as 

well as the earliest signs of vascular damage, such as 

conjunctival injection, skin petechiae, mild (postural) 

hypotension, or flushing over the head and upper 

torso. About 30% of patients develop more severe 

hemorrhagic or prominent neurologic manifestations 

(convulsions, tremor of the hands or tongue, coma) 

or secondary bacterial infections during the second 

week of illness. Hemorrhagic manifestations, such as 

bleeding from mucous membranes (gums, nose, vagina/uterus, gastrointestinal tract) and ecchymoses at 

needle puncture sites, are common in these patients. 

However, blood loss is minor overall. Capillary leakage is a hallmark of disease, and elevated hematocrit 

occurs during the peak of capillary leak syndrome.13

Death usually occurs 7 to 12 days after disease onset 

from organ failure and shock. 

Patients who survive begin to improve during the 

second week of disease onset. Convalescence often 

lasts several weeks with fatigue, hair loss, dizziness, 

or Beau’s lines in digital nails. “VHF” convalescent 

symptoms also include sore throat or pharyngitis.15,44,45

TAXONOMY AND PHYLOGENETIC RELATIONSHIPS

The family Arenaviridae includes two genera, Mammarenavirus (mammalian arenaviruses) and Reptarenavirus (reptilian arenaviruses). The genus Mammarenavirus includes 31 species, and the majority of these 

species have only a single virus member each.46,47 Based 

on antigenic properties (serological cross-reactivity), 

sequence phylogeny, and geographical distribution, 

mammarenaviruses have been divided into two 

distinct groups. The Old World mammarenaviruses 

(also named the Lassa–lymphocytic choriomeningitis 

serocomplex) include viruses indigenous to Africa, 

such as LASV and LUJV, as well as the ubiquitous 

lymphocytic choriomeningitis virus. The New World 

mammarenaviruses (also named the Tacaribe serocomplex) include viruses indigenous to the Americas,48–51

such as CHAPV, GTOV, JUNV, MACV, and SABV.

The basis for mammarenaviruses phylogenetic 

analysis typically relies on the sequence of the nucleoprotein (N) gene. This analysis supports the previously defined antigenic grouping, further defines 

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virus relationships, and is largely consistent with 

analyses based on sequence data derived from other 

regions of mammarenaviruses genomes. According to 

N-based phylogenetic analysis, the member viruses 

of the 31 species represent four distinct phylogenetic 

groups: an Old World mammarenaviruses group and 

three New World mammarenavirus lineages (A, B, 

and C).52–55 New World mammarenaviruses Group A 

includes Allpahuayo virus, Flexal virus, Paraná virus, 

Pichindé virus, and Pirital virus from South America, 

together with Bear Canyon virus, Tamiami virus, and 

Whitewater Arroyo virus from North America. Group 

B contains the human pathogenic viruses CHAPV, 

GTOV, JUNV, MACV, and SABV, and the nonpathogenic Amaparí virus, Cupixi virus, and Tacaribe virus. 

Group C includes Latino virus and Oliveros virus.

Figure 21-5. Replication cycle of an idealized mammarenavirus. 

GPC: glycoprotein precursor 

IGR: intergenic region

NP: nucleoprotein 

RNA: ribonucleic acid

MOLECULAR CHARACTERISTICS 

Arenaviruses produce enveloped and spherical 

to pleomorphic virions, ranging from 50 to 300 nm 

in diameter (Figure 21-5).13,56–58 The particles’ sandy 

appearance in electron microscopy sections earned 

these viruses their name (Latin arena = sand). The 

arenavirus genome consists of two single-stranded 

ribonucleic acid (RNA) molecules, designated L (large) 

and S (small). Each of these genomic segments encodes 

two different proteins in two nonoverlapping reading 

frames of opposite orientation (ambisense coding arrangement; Figure 21-5). The L segment encodes the 

viral RNA-dependent RNA polymerase (L) and a zincbinding matrix protein (Z). The S segment encodes 

a nucleoprotein (NP) and an envelope glycoprotein 

precursor (GPC).59–61 Extracted virion RNA is not infectious, and, therefore, arenaviruses are considered 

by some as negative-sense RNA viruses despite the 

presence of the ambisense coding strategy.

Arenavirus cell entry and fusion with the host membrane is mediated by the arenavirion spike complex 

(Figure 21-5). In the case of mammarenaviruses, the 

spike is composed of the two envelope glycoprotein 

subunits, GP1 and GP2, derived from posttranslational 

cleavage of GPC and a stable signal peptide, cleaved 

off during GPC synthesis.61–65 Reptarenavirus spikes 

are fundamentally different from mammarenaviral 

spike proteins and are closely related structurally to 

the glycoproteins of filoviruses (GP1,2). Therefore, a 

stable signal peptide is absent.66 To enter cells, arenaviral GP1 binds to cell-surface receptors, and virions 

are internalized by endocytosis into intracellular endosomal compartments.67–71 Following pH-dependent 

membrane fusion mediated by GP2 and uncoating, 

viral ribonucleoprotein (RNP) complexes are released 

into the cytoplasm (Figure 21-5).58

Interestingly, genes required for the proper functioning of α-dystroglycan (αDG), the receptor for Lassa 

virus (LASV), are preferential targets of LASV-driven 

selective pressure (or natural selection) in populations of Western Africa where LASV is endemic. A 

genome-wide screen for recent selective sweeps in 

humans has identified positive selection of two genes 

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(LARGE and dystrophin) in a Nigerian population.72–74

Dystrophin is a cytosolic adaptor protein that is necessary for αDG to function properly. The cellular likeacetylglucosaminyltransferase (LARGE) is required 

for the posttranslational modification of αDG and 

consequently for LASV GP binding to αDG.75 In the 

identified population, more than 21% of individuals showed evidence of exposure to LASV infection, 

which may be responsible for the increase in the allelic 

variants of LARGE and dystrophin. Positive selection in LARGE was confined to introns and putative 

regulatory regions, suggesting that natural selection 

may have targeted variants giving rise to alternative 

splicing or differential gene expression of LARGE. 

These polymorphisms in LARGE and dystrophin may 

alter the ability of LASV GP to bind αDG and mediate 

entry, providing an advantage to the immune system, 

and thereby protecting these individuals from severe 

LASV infection.72–74

RNPs consist of NP, L, and viral genomic RNA. NP 

is the arenavirus major structural protein. It forms a 

bead-like polymer that associates with viral RNA. 

L, the arenaviral RNA-dependent RNA polymerase, 

mediates RNA replication and transcription (Figure 

21-5).76–79 L initiates transcription from the genome 

promoter at the genome 3’ end, and NP and L genomic 

complementary mRNA are synthesized and translated 

to proteins from the S and L segments, respectively.80–82

L together with NP also generate a full-length copy 

of antigenome RNA from the L and S segments. Antigenome S and L RNA segments then serve as a template for the synthesis of GP and Z mRNAs, which 

are translated into the respective proteins. Newly 

synthesized full-length arenavirus antigenomic and 

genomic RNAs are encapsidated by NP to generate 

the RNP complexes for further mRNA transcription 

and for production of virus progeny (Figure 21-5).83

The negative regulatory matrix protein Z inhibits 

arenaviral RNA synthesis in a dose-dependent manner.82,84–88 Z contains a zinc-binding RING motif89,90

that is essential for interaction with L and resultant 

inhibitory activity.86

Together with NP, Z mediates arenavirion assembly91 and budding.80,91,92 Virion budding occurs from 

the plasma membrane, where the virus RNP core associates with host-derived membrane that is highly 

enriched with the viral GP spike complex to form the 

virion envelope (Figure 21-5).80,91–93

In addition to the roles of NP and Z in viral replication, these proteins interfere with antiviral signaling. 

NP encoded by Old World and New World mammarenaviruses is involved in virus-induced inhibition of 

type I interferon.94,95 Z protein encoded by New World 

mammarenaviruses also interferes with this pathway.96

PATHOLOGY AND PATHOGENESIS

The mechanism of pathogenesis of mammarenaviral hemorrhagic fevers is not well understood. In 

both Old World and New World mammarenavirus 

infections, the pathological findings do not provide 

the basis to explain the relatively high case fatality 

rate and severity of disease.11,97–101 According to the 

current pathogenesis model, mammarenaviruses enter 

humans by inhalation and deposit in the lung terminal 

respiratory bronchioles. The viruses then gain entry to 

the lymphoid system and spread systematically in the 

absence of a detectable pneumonic focus.102 Mesothelial 

surfaces are infected next, perhaps a source of some of 

the observed effusions of parenchymal cells of several 

organs, particularly lymphoid tissues. Macrophages 

are early and prominent targets of mammarenavirus 

infection.17,103,104

Old World Mammarenaviral Hemorrhagic Fevers 

In the case of patients infected with LASV, failure 

to develop cellular and humoral immune responses, 

indicated by high levels of serum virus titers and virus 

replication in tissues, leads to the development of fatal 

Lassa fever.105 Nonhuman primate models of Lassa 

fever indicate that dendritic cells are prominent targets of LASV in the initial stages of infection, whereas 

Kupffer cells, hepatocytes, adrenal cortical cells, and 

endothelial cells are more frequently infected with 

LASV in the terminal stages of infection.106

Macroscopic abnormalities in Lassa fever patients 

include pleural effusions, pulmonary edema, ascites, 

and hemorrhagic manifestations in the gastrointestinal 

mucosa.1,31 Microscopic findings include multifocal 

hepatocellular necrosis and apoptosis or regeneration 

(mitosis), splenic necrosis in the marginal zone of the 

splenic periarteriolar lymphocytic sheath, adrenocortical necrosis, mild mononuclear interstitial myocarditis 

without myocardial fiber necrosis, alveolar edema with 

capillary congestion and mild interstitial pneumonitis, lymph nodal sinus histiocytosis, gastrointestinal 

mucosal petechiae, renal tubular injury, or interstitial 

nephritis.1,31,107,108

The most severe pathological hallmark of Lassa fever in humans is found in the liver.1,31,107,109 In addition 

to hepatocellular necrosis, mononuclear phagocytosis 

of necrotic hepatocytes and focal hepatocellular cytoplasmic degeneration are typical. However, the degree 

of hepatic tissue damage is insufficient to cause hepatic 

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failure, and only minimal recruitment of inflammatory 

cells into this organ is detected. Furthermore, no correlation has been observed between the degree of hepatic 

necrosis and chemical indicators of liver damage, such 

as elevated concentrations of aspartate aminotransferase, alanine transaminase, and lactate dehydrogenase 

in serum.107,108 Finally, the degree of liver damage can 

vary dramatically among patients that die from Lassa 

fever. Therefore, liver involvement is necessary—but 

not sufficient—in the chain of pathological events that 

lead to fatal Lassa fever.

Based on the degree of hepatic damage, three 

general phases have been proposed for the categorization of patients with fatal Lassa fever.66 The first 

phase, active hepatocellular injury, is defined by the 

presence of focal cytoplasmic degeneration with less 

than 20% of hepatocytes undergoing necrosis. This 

phase may represent the late stage of viremic spread 

and early cellular injury. This phase is most likely 

caused by direct viral action rather than mediated 

by a cellular immune response, since lymphocytic 

infiltration is not detected. The second phase, the 

peak of Lassa hepatitis, is characterized by 20% 

to 50% necrosis of hepatocytes, widespread focal 

cytoplasmic degeneration, and limited phagocytic 

infiltration. Progressive hepatocellular damage occurs during this phase, but early liver recovery is 

evident through the phagocytic removal of necrotic 

hepatocytes and regeneration of new cells. The third 

phase, hepatic recovery, is defined by less than 10% 

of hepatocellular necrosis, absence of focal cytoplasmic degeneration, and clear evidence of mitoses, 

which indicates liver regeneration.108

In addition to hepatic necrosis, splenic and adrenocortical cellular necrosis is observed in patients with 

Lassa fever. The predominant distribution of splenic 

necrosis is in the marginal zone of the periarteriolar 

lymphocytic sheath. Close examination of thin tissue 

sections reveals the presence of fibrin in addition to 

the debris of necrotic cells. The splenic venous subendothelium appears to be infiltrated by lymphocytes 

and other mononuclear cells. Additionally, multifocal 

adrenocortical cellular necrosis is most prominent in 

the zona fasciculata and is often associated with focal inflammatory reaction. However, in all examined 

cases, adrenal necrosis was mild, and greater than or 

equal to 90% of the cells of adrenal cortex appeared 

viable.107 Microscopic examination of adrenal glands 

shows prominent spherical, hyaline, and acidophilic 

cytoplasmic inclusions in cells near the junction of 

zona reticularis and medulla. In most cases, these 

cells appear to be adrenocortical cells of the zona 

reticularis; however, some cells are of adrenal medulla origin. 

LASV-induced impairment of vascular function 

is most likely central for the pathology observed in 

infected patients. LASV, which is a nonlytic virus, 

does not cause cytopathic effects or cellular damage 

in infected monocytes, macrophages, and endothelial 

cells.110 Nevertheless, infection of these cells is crucial 

for the pathology caused by the virus. In both experimentally infected animals and Lassa fever patients, 

the disruption of vascular endothelium function is 

closely followed by shock and death.107,111 Edema and 

pleural and pericardial effusions that are associated 

with fatal cases most likely result from increased vascular permeability. Only minimal vascular lesions are 

detected in fatal human Lassa fever cases and infected 

nonhuman primates, which can be explained by the 

nonlytic nature of LASV infection of the endothelium. 

The mechanism of LASV-induced increase in vascular 

permeability is not yet understood. Virus infection 

of the endothelium is commonly thought to cause 

changes in cellular function leading to increased fluid 

flow and subsequently to edema.

New World Mammarenaviral Hemorrhagic Fevers

The most common macroscopic abnormality in 

severe cases of New World mammarenaviral hemorrhagic fevers is widespread hemorrhage, particularly 

in the skin and mucous membranes (gastrointestinal 

tract), intracranium (Virchow-Robin space), kidneys, 

pericardium, spleen, adrenal glands, and lungs. 

Microscopic lesions include acidophilic bodies and 

focal necroses in the liver (in the case of BHF, hepatic 

petechiae are common, and the number and size of 

the Kupffer cell are also increased), acute tubular and 

papillary necrosis in the kidneys, reticular hyperplasia 

of the spleen and lymph nodes, or secondary bacterial 

lung infections in the case of AHF (acute bronchitis and 

bronchopneumonia, myocardial and lung abscesses) 

or interstitial pneumonia in the case of BHF97,98 or 

“VHF.”11,112 In AHF, the sites of cellular necrosis (hepatocytes, renal tubular epithelium, macrophages, 

dendritic reticular cells of the spleen and lymph nodes) 

correspond to sites of viral antigen accumulation, and 

both JUNV and MACV could be isolated from the 

blood, spleen, and lymph nodes of patients.20,30,113,114

Patterns of clinical AHF illness are JUNV strainspecific and can be hemorrhagic (Espindola strain), 

neurologic (Ledesma strain), mixed (P-3551 strain), 

and common (Romero strain).115–117 In animal models 

of AHF, each isolate induces a disease that faithfully 

replicates the clinical variant of the disease in the human from whom the viral strain was obtained. Animals 

infected with JUNV Espindola strain (hemorrhagic) 

demonstrate a pronounced bleeding tendency with 

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disseminated cutaneous and mucous membrane 

hemorrhage. In contrast, animals infected with JUNV 

Ledesma strain (neurologic) show little or no hemorrhagic manifestations, but develop overt and generally 

progressive signs of neurologic dysfunction: limb 

paresis, ataxia, tremulousness, or hyperactive startle 

reflexes. In guinea pigs, the Espindola strain replicates 

predominantly in the spleen, lymph nodes, and bone 

marrow, the major sites of necrosis, whereas lower virus loads are present in blood and brain. The Ledesma 

strain, however, is found predominantly in the brain, 

where moderate polioencephalitis is observed, and 

only low amounts of virus are recovered from the 

spleen and lymph nodes.

COAGULOPATHIES

Although the mammarenaviruses discussed in this 

chapter cause viral hemorrhagic fevers in humans, 

blood loss does not typically account for the diseases’ 

fatal outcome.107 Furthermore, pathogenic mammarenaviruses differ in their capacity to cause hemorrhages 

or coagulopathies in infected individuals, which is 

particularly true in Lassa fever patients in whom bleeding is uncommon and limited primarily to mucosal 

surfaces.118 In general, coagulation dysfunction is not 

considered to be associated with Lassa fever as neither 

disseminated intravascular coagulation nor a decrease 

in coagulation factors has been observed.119 However, 

in the few severe cases characterized by bleeding, it is 

typically associated with moderate thrombocytopenia 

and platelet dysfunction.119,120 The platelet malfunction 

appears to be mediated by a plasma inhibitor of platelet 

aggregation, which has yet to be characterized.120

Hemorrhages are more common in patients infected with New World mammarenaviruses than with 

Old World mammarenaviruses. However, similar to 

Lassa fever patients, relatively few vascular lesions 

are seen in patients with AHF.121 Endothelial cells, 

which highly express the New World mammarenavirus receptor transferrin receptor 1, are permissive 

to infection in culture, but minimal cytopathic effects 

are observed.70,122,123 Therefore, indirect effects may 

be responsible for the increased permeability seen in 

patients,104 and profuse bleeding is presumably a consequence of vascular damage caused by both cytokines 

and virus replication. Thrombocytopenia, which is 

commonly found in patients with AHF and in animals 

experimentally infected with JUNV or MACV, and 

elevated concentrations of factor VIII-related antigen 

(von Willebrand factor, vWF), which is synthesized 

and released from endothelial cells, could contribute to 

the observed endothelial dysfunction.124–128 However, 

vWF concentrations are low in JUNV-infected human 

umbilical vein endothelial cells, suggesting that vWF 

might originate from another source rather than from 

endothelial cells. Human umbilical vein endothelial 

cells infected with virulent JUNV strains increase production of the vasoactive mediators nitric oxide and 

prostaglandin PGI2

 compared to that observed with 

avirulent strains, providing a possible link between 

viral infection and the increased permeability observed 

in patients with AHF.123

In animal models of AHF and BHF, progressive 

thrombocytopenia is observed within 7 days following infection onset, with platelet counts reaching a 

nadir at or near the time of death. Coincident with the 

dropping platelet count, progressive necrosis of bone 

marrow occurs, suggesting that the decrease in the 

number of platelets may be related to impaired production.101,117,127,128 Furthermore, intracytoplasmic viral 

particles have been demonstrated in megakaryocytes 

by electron microscopy.129 The coagulative activity 

of blood in infected patients with AHF is also low.130

Similar to Lassa fever patients, plasma from patients 

with AHF contains an inhibitor of platelet function.35

Platelet inhibition appears to be reversible in vitro,131,132

and its effects are not neutralized by immune plasma 

containing a high titer of (neutralizing) antibodies 

to JUNV. Thus, the available evidence suggests that 

abnormal platelet function in patients does not result 

from an intrinsic platelet defect, but rather from inhibition by an extrinsic factor in plasma. 

Overall, specific coagulation abnormalities do 

not correlate with the severity of New World mammarenaviral infections, suggesting minimal involvement of coagulopathy in pathogenesis. Furthermore, 

limited evidence (four human cases)98,133 suggests 

that disseminated intravascular coagulation is not an 

important pathogenic phenomenon in mammarenaviral disease. However, several modest abnormalities 

of clotting factors and activation of fibrinolysis are 

observed in AHF patients and animal models. These 

abnormalities can vary depending on which virus 

variant is involved.124–126,128,134–136 The concentration of 

factor V is uniformly elevated (starting from day 8 of 

onset of AHF), and fibrinogen concentration is normal 

in mild cases and elevated in severe cases in the later 

stages of infection (after day 10). The activated partial 

thromboplastin time is prolonged during the acute 

phase of illness. A lower concentration ratio of factor 

VIII:C to vWF has been noted during the illness, but 

returns to normal during the convalescence period. 

In the guinea pig model, and to a lesser extent in 

humans, factor IX and XI concentrations are slightly 

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reduced.124,126–128,130,134 Levels of prothrombin fragment 

1+2 and thrombin-antithrombin III complexes are 

increased. However, antithrombin III activity levels 

in patients with AHF are within the normal range.130

In another study in patients with AHF, antigenic and 

functional levels of antithrombin III were below normal in the early stages of disease.137 In most cases of 

AHF, no significant changes in factor II, VII, prekallikrein, and kallikrein inhibitor, protein C, protein S, and 

C4b binding protein are observed.124,130 Plasminogen 

activity is below normal in AHF patients in the earlier 

stages of the disease (days 6–11),138 although normal or 

slightly elevated concentrations of α2-antiplasmin can 

be detected.124,130 Tissue plasminogen activator and ddimer concentrations are high in the early stages of the 

disease, whereas the plasminogen activator inhibitor-I 

concentration is increased only in severe cases during 

the second week of illness.130

IMMUNE RESPONSE

Old World Mammarenaviral Hemorrhagic Fevers 

Antibodies do not seem to play a significant role in 

LASV infection because production of LASV-specific 

antibodies is not correlated with Lassa fever survival. 

Such antibodies are detected in all patients, regardless 

of outcome.36 Low neutralizing antibodies titers occur 

only months after an acute LASV infection is resolved, 

long after virus has been cleared.139 Instead, resolution of LASV infection seems to depend primarily on 

cellular immunity, in particular the antiviral T-cell 

response.140,141 Data from experimental nonhuman 

primate studies show a correlation between surviving 

animals and high concentrations of activated T lymphocytes and control of viral replication. In contrast, 

animals that died had delayed, low T-cell activation 

and uncontrolled viral replication.142 In addition, seropositive individuals residing in Lassa fever-endemic 

areas have very strong memory CD4+ T-cell responses, 

and the antigenic epitopes have been mapped to NP 

and an N-terminal conserved region within GP2.141,143

However, results of a recent study using mice expressing humanized major histocompatibility complex 

class I and a single, exotic variant of LASV (Ba366) 

suggest that in the presence of persistent viremia, 

T cell responses may also contribute to deleterious 

innate inflammatory reactions and Lassa fever pathogenesis.105 Whether these results can be generalized to 

other LASV variants remains to be determined.

In contrast to other viral hemorrhagic fevers, such as 

Ebola virus disease,144 LASV infection does not result 

in a “cytokine storm” that interferes with the integrity 

of the vascular endothelium.145 Virus-induced immunosuppression may be involved in the pathogenesis 

of severe Lassa fever. Antigen-presenting cells, such 

as dendritic cells and macrophages, and endothelial 

cells are early targets of LASV infection, with dendritic cells producing more virus than macrophages. 

However, these cell types are not activated to produce 

proinflammatory cytokines, including tumor necrosis 

factor-α (TNF-α) and interleukin-8 (IL-8).110,146,147 These 

results are consistent with clinical data from Lassa 

fever patients showing correlation of fatal outcome 

and low concentrations or absence of proinflammatory 

cytokines such as IL-8 and interferon (IFN)-inducible 

protein 10.145 Although increased vascular permeability does not seem to be caused by inflammatory 

mediators, LASV infection may affect endothelial cell 

integrity via another mechanism. 

New World Mammarenaviral Hemorrhagic Fevers 

Similar to Lassa fever, the acute phase of New World 

mammarenaviral hemorrhagic fevers is associated 

with significant depression of host immunity. The 

frequency of pyogenic secondary bacterial infections 

in humans and animal models97,98,115 suggests that polymorphonuclear leukocyte function is compromised. 

Leukocyte dysfunction may be a result of bone marrow 

necrosis, maturation arrest, and direct interactions of 

JUNV with polymorphonuclear cells148 that lead to 

leukopenia.149,150 AHF is associated with a profound 

decrease in the recall of delayed hypersensitivity, 

diminished responsiveness of lymphocytes to nonspecific mitogens, decreased levels of circulating B- and 

T-cells, lymphoid necrosis, and inversion of CD4+/CD8+

lymphocyte ratios.114,151–154 Abnormalities reported 

in animal models include necrosis of macrophages, 

T- and B-lymphocyte depletion, decreased primary 

and secondary antibody responses, and blunted Arthus reaction and anergy after established tuberculin 

sensitivity.20,129,149,155–157 Defective macrophage function 

and high concentrations of IFN are highly plausible 

causes for these observed abnormalities. JUNV infects 

macrophages and monocytes extensively in vivo,114

and circulating monocytes contribute to viral spread 

in the acute phase of AHF.158 Virulent JUNV strains 

replicate in both spleen-derived dendritic cells and 

macrophages from guinea pigs, whereas attenuated 

strains, which are not immunosuppressive, replicate 

only in dendritic cells.159

Unlike LASV infection, in which generalized immune suppression is observed, AHF patients have 

elevated concentrations of proinflammatory as well 

as antiinflammatory cytokines that correlate with the 

disease’s severity. Both patients and animal models 

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have high serum concentrations of IFN-α. The extraordinarily high serum concentrations of IFN-α in JUNVinfected patients are indicative of a negative disease 

outcome.160,161 In patients who survive, high concentrations of IFN-α only occur during the first week after 

disease onset and fall to low concentrations during the 

second and following weeks. However, concentrations 

of interleukin-1β (IL-1β) remain normal.102,160–162

High serum concentrations of TNF-α, IL-6, IL-8, IL10, and elastase-α1-antitrypsin complex are found in 

patients with AHF. Significant correlations are found 

between concentrations of both IL-8 and IL-10 with 

TNF-α concentrations, and between IL-8 and elastaseα1-antitrypsin complex. Thus, IL-8 is suggested to 

play an essential role in neutrophil activation in AHF 

patients.163 Elevated TNF-α concentrations may be 

the trigger for some of the observed hemostatic and 

endothelial abnormalities observed in AHF patients. 

Results of several studies characterize the procoagulant 

activity164–166 and changes in vascular permeability167–169

of endothelial cells following exposure to TNF-α. 

JUNV–infected macrophages show no increase in cytokine production indicating that perhaps dendritic cells 

may increase cytokine concentrations.170 At present, 

it is unclear if the increased cytokine concentrations 

reflect high viral replication and widespread infection 

or if they play a role in immune-mediated pathology 

similar to other viral hemorrhagic fevers like Ebola 

virus disease.

In contrast to Lassa fever, in which the antibody 

response seems to be ineffective in controlling LASV 

infection, patients who recover from AHF develop 

antibodies in the second week of disease and clear the 

virus.171 In convalescent plasma from AHF survivors, 

robust titers of neutralizing antibodies, mainly immunoglobulin G (IgG), can be detected. After transfusion 

with immune plasma, viremia in patients is reduced.172

The case fatality rate of AHF patients treated early 

during the course of infection with immune plasma 

from surviving cases is reduced to 1%.173

DIAGNOSIS

Detection of Virus-Specific Antibodies and Viral 

Antigens

Mammarenavirus antibodies can be detected by 

enzyme-linked immunosorbent assays (ELISAs), virus 

neutralization tests, and indirect immunofluorescence assays (IFAs). ELISAs using recombinant proteins, infected 

cells, or blood as antigen have been developed for detection of pathogenic mammarenavirus antibodies.174–177

An immunoglobulin M (IgM) or IgG-specific ELISA is 

suitable for determining exposure to mammarenaviruses, 

but the relevance of IgM or IgG testing for acute infection 

depends on the virus and duration of illness.176 An early 

immunosuppression resulting from Lassa fever seems 

to result in depressed production of IgM at the early 

phase of the infection and, as a result, some patients fail 

to elaborate IgM at the time of presentation. However, an 

increase in IgG titers is observed during convalescence. 

Thus, neither IgM nor IgG titers alone should be used 

as a screening tool for early detection of Lassa fever.

The virus neutralization assay is accepted as a standard serodiagnostic assay to quantify the antibody 

response to infection of a wide variety of viruses.176

However, this test can be used for diagnosis of mammarenavirus infections only if a biosafety level 3 or 4 

laboratory is available. Virus neutralization tests are 

highly specific, but neutralizing antibodies may appear 

too late in the course of mammarenaviral disease to be 

useful for prompt diagnosis. For example, patients with 

Lassa fever do not usually develop neutralizing antibodies until weeks after they became ill,178 and patients with 

fatal Lassa fever may not develop, antibodies at all.179

IFA tests also detect antibodies in serum that bind to 

a fixed monolayer of virus-infected cells.33,180 However, 

the interpretation of IFA is complicated by positive 

staining results in both the acute- and convalescentphase of infection, as well as the subjective nature of 

the assay.174 ELISAs are thought to be more sensitive 

and specific than IFA.174,175,181,182 Cross-reactions can 

occur between different arenaviruses in these tests. 

Compared to antibody detection, antigen-capture 

ELISA using polyclonal or monoclonal antibodies 

for detection of viral antigens is valuable for rapid 

diagnosis of acute phase viral hemorrhagic fevers, 

such as Lassa fever, AHF, BHF, “VHF,” and “Brazilian 

hemorrhagic fever.”174,183 The sensitivity of sandwich 

antigen-capture ELISA is comparable to that of reverse 

transcription polymerase chain reaction (RT-PCR) 

for detection of Lassa fever.184 In a comparison of the 

diagnostic markers in a large cohort of potential Lassa 

fever patients, LASV antigens detected in blood using 

antigen-capture ELISAs or lateral flow immunoassays 

are more indicative of an acute LASV infection than 

positive antibody titers.177

Detection of Viral Nucleic Acids

RT-PCR, real-time PCR, and real-time RT-PCR 

tests are valuable tools for rapid and early diagnosis 

of mammarenavirus infections.177,179,185–192 However, 

the use of these assays in a clinical or environmental setting for the early detection of human cases 

has been limited by the expense of equipment and 

by expertise. RT-PCR has been used routinely for 

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confirmation of Lassa fever in Africa during collaborative missions following antigen detection by 

ELISA and lateral flow immunoassay.177,193 Some 

PCR tests detect a wide range of mammarenaviruses 

by targeting the highly conserved termini of the S 

RNA segment, but an RT-PCR assay detecting Old 

World mammarenaviruses targeting the L gene has 

been also developed.194 Other PCR tests are more 

virus-specific. Serum, plasma, cerebrospinal fluid, 

throat washings, and urine can be used for sample 

preparation. Real-time PCR may be advantageous 

because the risk of contamination is greatly reduced 

by using closed tubes and because the test quantifies 

viral RNA in serum. However, specimens containing a high concentration of viral RNA may produce 

false-negative results resulting from inhibition of 

the enzymatic reaction.178 Given the high degree of 

genetic variability of mammarenaviruses, selection 

of primers that can detect all strains of the viruses 

can be difficult,176 and PCR techniques may fail to 

amplify sequences of mammarenavirus strains even 

with limited sequence deviations.

Virus Isolation

Virus isolation is the gold standard for diagnosis 

of mammarenavirus infections.174,177,193 Mammarenaviruses can easily be recovered in cell cultures, 

particularly from Vero cells. Initial passaging of a 

virus isolate in laboratory rodents, such as suckling 

laboratory mice, guinea pigs, or newborn hamsters, 

may be even more sensitive. The presence of virus 

can then be confirmed by PCR or by detection of 

virus antigen in cells using immunohistochemical or 

IFA assays. However, considering the time required 

for virus isolation (days to weeks) and the need for 

special facilities (biosafety level 3 or 4 laboratories), 

which are unavailable in many mammarenavirusendemic areas, this method is less suitable for rapid 

diagnosis of mammarenaviral disease than PCR or 

antigen-capture ELISA.174,177,195 Recent discoveries of 

novel mammarenaviruses relied on the use of IFA, 

PCR, and pyrosequencing technology.3,18,194,196,197 Nextgeneration sequencing technology may be used in the 

near future for diagnostic purposes.

TREATMENT AND VACCINES

Few prophylactic and therapeutic treatments are 

approved for use against mammarenaviral hemorrhagic fevers. Treatment, therefore, consists primarily 

of supportive care and passive antibody therapy.

Passive Antibody Therapy

Transfusion of immune convalescent plasma with 

defined doses of JUNV-neutralizing antibodies is the 

present therapeutic intervention and treatment method 

against AHF. Immune serum treatment providing an 

adequate dose of neutralizing antibodies is effective 

in attenuating disease and reducing lethality to less 

than 1% if administered within the first 8 days of disease.173,198–200 However, about 10% of treated patients 

develop a transient cerebellar-cranial nerve syndrome 

3 to 6 weeks later.173,198–200

Studies with animal models suggest that passive 

antibody therapy may be useful for the treatment of 

BHF,201 but such therapy has not been thoroughly 

evaluated in a clinical setting. An in vitro study with 

Vero E6 cells shows that convalescent sera from 6 of 

7 putative “VHF” cases neutralized the infectivity of 

GTOV, and the neutralizing titers in the positive sera 

range from 160 to 640.14 However, even if a similar 

plasma therapy could be developed for BHF and 

“VHF,” maintaining adequate plasma stocks would 

be a challenge because of the limited number of cases 

and the absence of a program for convalescent serum 

collection. The additional risk of transfusion-borne 

diseases emphasizes that alternative treatments ought 

to be developed.202

In contrast, treatment of Lassa fever patients with 

convalescent serum of survivors did not confer protection when treated within 24 hours after hospital 

admission.203 Treatment of nonhuman primates and 

guinea pigs with plasma from convalescent animals 

containing high titers of neutralizing antibodies protects the animals from developing disease. However, 

protection is observed only if administration of plasma 

is performed directly after infection with LASV.204,205

Vaccines

Despite the bioterrorism and public health risks associated with pathogenic mammarenavirus infection, 

FDA-licensed vaccines are currently not available. Vaccines for the prevention of human mammarenavirus 

diseases are limited to a single, safe, efficacious, and 

live attenuated vaccine designated Candid 1 (Candidate no. 1), for the prevention of JUNV infection.200,206–208

Candid 1, which is classified as an investigational new 

drug in the United States, was derived from the wild 

type JUNV strain XJ13 through serial passage both in 

vivo and in vitro.209 A recent study suggests that the 

major determinant of attenuation in mice is located 

in the transmembrane domain of the G2 glycoprotein 

(F427I mutation).210 Candid 1 has been evaluated in 

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large-scale controlled trials among at-risk populations 

of agricultural workers in Argentina, where it showed 

a protective efficacy greater or equal to 84%. Vaccination of more than 150,000 high-risk individuals in the 

endemic areas has led to a consistent reduction in AHF 

cases with an excellent safety profile.208,211,212 The vaccine also cross-protects experimental animals against 

MACV infection,213 which suggests that Candid 1 

could be used during a BHF outbreak as an emergency 

containment measure. A summary of the historical 

development and biological properties of the vaccine 

can be found in a recent review.214

Another approach for vaccine development 

against AHF involves using a nonpathogenic mammarenavirus relative, Tacaribe virus, as a live vaccine 

in guinea pigs or the marmoset model of AHF.215–218

Animals develop measurable immune responses 

as early as 3 weeks following exposure to Tacaribe 

virus, and no clinical signs of AHF or histopathological changes are observed following exposure to 

a lethal dose of JUNV.

Several promising studies have focused on the development and preclinical testing of LASV vaccines. 

Nevertheless, no vaccine candidate has advanced 

toward human clinical trials. Early strategies involved 

the usage of an apathogenic mammalian Old World 

mammarenavirus, Mopeia virus, as a live vaccine. 

Rhesus monkeys that were inoculated with Mopeia 

virus and subsequently exposed to LASV developed 

no sign of LASV disease.219,220 However, since little is 

known about human infections with Mopeia virus, and 

some of the infected primates developed pathological 

alterations of the livers and kidneys,221 the safety of 

Mopeia virus should be proved before any efficacy 

studies are performed in humans.

Another live attenuated vaccine candidate against 

LASV infection is the chimeric ML29 virus. This recombinant virus carries the LASV S segment and the 

Mopeia virus L segment and is efficacious in nonhuman primates. Immunity is conferred via cellular 

responses, and no transient elevation of liver enzymes 

in the plasma is noted.222,223 However, as in the case 

with candidate vaccines based on Mopeia virus only, 

caution must be exerted, as the safety of LASV-Mopeia 

chimeric vaccines in humans is unclear.

Recombinant viruses expressing mammarenaviral 

antigens have also been tested as potential vaccines. 

Different viral platforms, such as vaccinia virus, vesicular stomatitis Indiana virus, attenuated yellow fever 

strain 17D virus, and Venezuelan equine encephalitis 

virus replicon particles expressing mammarenaviral 

NP, GP, GP1, or GP2, have been evaluated in various 

animal models.140,224–230 The most promising results 

were obtained using the whole GP of LASV.

Other approaches based on inactivated mammarenaviruses231,232 or mammarenavirus-like particles233

have not been successful or have yet to be fully evaluated, respectively.

Antiviral Agents

Current antimammarenaviral therapy is limited 

to an off-label use of the nonimmunosuppressive 

guanosine analogue, ribavirin (1-β-d-ribofuranosyl1-H-1,2,4-triazole-3-carboxamide), an IMP dehydrogenase inhibitor. Recent studies suggest that the 

antiviral activity of ribavirin on mammarenaviruses 

is not mediated by depletion of the intracellular GTP 

pool, but may be exerted—at least partially—by lethal mutagenesis.234–236 Unfortunately, ribavirin has 

only partial efficacy against some mammarenavirus 

infections and is associated with significant toxicity 

in humans.17,200,203,212,237–244 Ribavirin can lead to adverse 

side effects such as thrombocytosis, severe anemia, and 

birth defects.241,245

Promising antivirals have been identified by smallmolecule high-throughput screens. These antivirals 

can be divided into six chemically distinct classes of 

small-molecule compounds that specifically inhibit 

GP-mediated membrane fusion with different selectivities against New World and/or Old World 

mammarenaviruses.236,246–248 One highly active and 

specific small-molecule inhibitor, ST-294, inhibits 

MACV, JUNV, GTOV, and SABV at concentrations 

in the nanomolar range. This molecule also demonstrates favorable pharmacodynamic properties 

(metabolically stable, orally bioavailable) and in vivo 

anti-mammarenaviral activity in a newborn mouse 

model.246 Mechanism-of-action studies suggest that 

this compound is a viral entry inhibitor targeting 

GP2.246 Another compound, ST-193, a benzimidazole 

derivative, inhibits cell entry of MACV and GTOV 

in vitro.236 Finally, two lead compounds, 16G8 and 

17C8, are highly active against MACV and GTOV, 

as well as LASV. These compounds act at the level of 

GP-mediated membrane fusion (IC50 ≈200–350 nM).247

Despite chemical differences, evidence suggests that 

these diverse inhibitors act through the pH-sensitive 

interface of the signal peptide and GP2 subunits in the 

GP spike complex. The inhibitors prevent virus entry 

by stabilizing the prefusion spike complex against pHinduced activation in the endosome.236,246,248

Other types of inhibitors that target viral RNA 

synthesis have also been reported. T-705 (favipiravir), 

a pyrazine derivative with broad antiviral activity 

against RNA viruses249–251 and several nonpathogenic 

mammarenaviruses,252,253 is also active in vitro against 

MACV, JUNV, and GTOV. T-705 most likely acts as 

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a purine nucleoside analog specifically targeting the 

viral RNA-dependent RNA polymerase.254 Results of 

studies using the Pichindé virus hamster model of 

acute mammarenaviral disease or a guinea pig model 

with an adapted Pichindé virus demonstrate that T-705 

could effectively protect against mammarenaviral 

disease after onset of clinical signs or in the late stage 

of illness.252,255

SUMMARY

Arenaviruses represent a large and taxonomically 

diverse group of animal viruses that are maintained 

by small rodents, bats, and snakes in nature. The majority of arenaviruses is not known to cause disease in 

humans. Seven mammarenaviruses, however, are the 

etiological agents of severe viral hemorrhagic fevers 

associated with high case fatality rates. LUJV, CHAPV, 

GTOV, and SABV, which are geographically restricted, 

have been associated with only a few to a few dozen 

cases. They are, therefore, relatively unimportant to 

clinicians or the warfighter compared to many other 

viruses that are usually coendemic. However, MACV, 

JUNV, and especially LASV have caused large outbreaks (LASV has caused hundreds of thousands of 

infections per year). Visitors to countries in which these 

viruses are endemic, or warfighters that are deployed 

to these countries, need to be aware of how to prevent 

and suspect a mammarenavirus infection. Distance 

from or safe handling of rodents and their bodily fluids 

or tissues and general rodent control around human 

settlements or camps should be the first priority to 

prevent mammarenavirus infections. 

Acknowledgments

We are grateful for the technical writing services offered by Laura Bollinger (IRF-Frederick) and the support by IRF-Frederick’s medical illustrator, Jiro Wada. The content of this publication does not necessarily reflect the views or policies of the US Department of Defense, US Department of Health and Human 

Services, the Department of the Army, or the institutions and companies affiliated with the authors. This 

work was funded in part through Battelle Memorial Institute’s prime contract with the US National Institute 

of Allergy and Infectious Diseases under Contract No. HHSN272200700016I. A subcontractor to Battelle 

Memorial Institute who performed this work is JHK, an employee of Tunnell Government Services, Inc.

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251. Morrey JD, Taro BS, Siddharthan V, et al. Efficacy of orally administered T-705 pyrazine analog on lethal West Nile 

virus infection in rodents. Antiviral Res. 2008;80:377–379.

252. Gowen BB, Smee DF, Wong MH, et al. Treatment of late stage disease in a model of arenaviral hemorrhagic fever: 

T-705 efficacy and reduced toxicity suggests an alternative to ribavirin. PLoS One. 2008;3:e3725.

253. Gowen BB, Wong MH, Jung KH, et al. In vitro and in vivo activities of T-705 against arenavirus and bunyavirus infections. Antimicrob Agents Chemother. 2007;51:3168–3176.

254. Mendenhall M, Russell A, Juelich T, et al. T-705 (favipiravir) inhibition of arenavirus replication in cell culture. Antimicrob Agents Chemother. 2011;55:782–787.

255. Mendenhall M, Russell A, Smee DF, et al. Effective oral favipiravir (T-705) therapy initiated after the onset of clinical 

disease in a model of arenavirus hemorrhagic fever. PLoS Negl Trop Dis. 2011;5:e1342.

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Chapter 22

HENIPAVIRUSES 

ARNOLD PARK, PhD*; SHELI R. RADOSHITZKY, PhD†; JENS H. KUHN, MD, PhD, PhD, MS‡; and BENHUR LEE, MD§

INTRODUCTION

HISTORY AND EMERGENCE OF HENIPAVIRUSES

Hendra Virus

Nipah Virus

Newly Identified Henipaviruses

EPIZOOTIOLOGY AND EPIDEMIOLOGY OF HENIPAVIRUSES

Hendra Virus

Nipah Virus

CLINICAL PRESENTATION OF HENIPAVIRUS INFECTIONS

Clinical Signs and Symptoms

Pathology

MOLECULAR BIOLOGY OF HENIPAVIRUSES

Virus Structure

Virus Genome

Virus Proteins

PATHOGENESIS

HENIPAVIRUSES AND BIOLOGICAL WEAPONS

Availability

Ease of Production and Dissemination

Potential for High Morbidity and Lethality

DIAGNOSIS

Reverse Transcription-Polymerase Chain Reaction

Detection of Henipavirus Antigens

Detection of Anti-henipavirus Immune Responses

Virus Isolation

Next-generation Sequencing

MEDICAL MANAGEMENT

Passive Immunotherapy

Ribavirin

Innate Immunotherapy

“Off-the-shelf” Therapies Evaluated In Vitro

SUMMARY

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*Postdoctoral Fellow, Microbiology Department, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Annenberg Building, Room 

17-24, New York, New York 10029

 †Principal Investigator, Molecular and Translational Sciences Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, 

Fort Detrick, Maryland 21702; formerly, Postdoctoral Fellow, Toxicology Division, US Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland

‡

Virology Lead, National Institutes of Health/National Institute of Allergy and Infectious Diseases/Division of Clinical Research/Integrated Research 

Facility, B-8200 Research Plaza, Room 1A-132, Fort Detrick, Maryland 21702; formerly, Research Scholar, Harvard Medical School, Department of 

Microbiology and Molecular Genetics, New England Primate Research Center, 1 Pine Hill Drive, Southborough, Massachusetts

§

Professor, Microbiology Department, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Annenberg Building, Room 17-24, New 

York, New York 10029

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INTRODUCTION

horses. The recent discovery of new henipaviruses of 

unknown virulence and spillover potential in bats1–3

emphasizes the need to understand this emergent 

group of viruses. 

As disease agents posing a severe threat to both agricultural livestock and human health, HeV and NiV are 

considered “overlap” select agents regulated by both 

the US Animal and Plant Health Inspection Service and 

the Centers for Disease Control and Prevention. As is 

the case for most select agents, approved therapeutics 

for human henipavirus infections do not exist. Promisingly, however, there have been rapid advances in 

developing new therapies and in repurposing existing 

Food and Drug Administration (FDA)-approved drugs 

for use in henipavirus infection treatment.

Henipaviruses were not discovered until 1994, 

when a novel virus, later called Hendra virus (HeV), 

was identified as the etiological agent of a fatal viral 

encephalitis in Hendra, Australia. Nipah virus (NiV), 

another virus closely related to HeV, emerged in Malaysia in 1998. Subsequently, the genus Henipavirus

was established within the family Paramyxoviridae as 

a taxonomic home for both HeV and NiV. HeV and 

NiV are high-consequence pathogens that are thought 

to be potential starting materials for biological weapons construction because they cause diseases with 

high case fatality rates, have the potential for aerosol 

transmission, are easily grown in cell culture to high 

titers, and have an unusually wide host range that 

encompasses humans and livestock, such as pigs and 

HISTORY AND EMERGENCE OF HENIPAVIRUSES

Hendra Virus 

Thoroughbred horse racing has been one of the great 

pastimes of Australia. The public imagination was 

struck in September 1994 when a prominent racehorse 

trainer, Vic Rail, 13 of his horses, and another horse 

from a neighboring property died of a sudden illness 

in Hendra, Australia. Rail’s stablehand also became ill, 

but recovered after an extended convalescence. The 

illness in both horses and humans was characterized 

by respiratory distress with neurological signs, and it 

culminated in blood-tinged, frothy nasal discharge in 

the horses.4,5

A major outbreak investigation ensued resulting 

in the identification of a new “equine morbillivirus” 

from infected horse and human samples. Experimental inoculation of this virus into naïve horses 

reproduced the disease.6 Preliminary phylogenetic 

analysis resulted in the placement of this new agent 

in the genus Morbillivirus (of which measles virus is 

the prototype member) in the family Paramyxoviridae. 

As this “equine morbillivirus” had unique molecular 

and pathogenic features that distinguished it from 

other morbilliviruses, the agent was renamed Hendra 

virus and finally reclassified in a novel paramyxoviral 

genus, Henipavirus.

7

A year after the Hendra outbreak, a horse stud 

owner died of relapsed encephalitis and was retrospectively diagnosed with HeV infection, originating 

from an encephalitic illness that predated the Hendra 

outbreak by several weeks. This single infection, which 

had no known epidemiological link to the Hendra 

outbreak, occurred 970 km north of Hendra, near 

Mackay. As in the Hendra outbreak, transmission to 

the stud owner involved horses, two of which died.8

Since the 1994 outbreaks, HeV infection emerged only 

periodically, and briefly, in 1999 and 2004. Then HeV 

infection emerged yearly from 2006 and proceeded in 

a truly accelerated fashion from 2011, all in Australia 

and ranging up and down the east coast (Figure 22-1).9

All disease outbreaks involving horses had an 84% case 

fatality rate,10 and of the seven human cases, including 

the 1994 outbreaks, four died. Considering that retrospective inspection of laboratory records and historical 

samples did not reveal signs of pre-1994 spillover of 

HeV to horses,11 HeV infection may have emerged 

from its natural reservoir in or around 1994. The lack 

of an epidemiological link between the outbreaks11

suggested potential spillover from another animal 

reservoir to the horses. However, extensive sampling 

of domestic animals and wildlife initially revealed no 

sign of HeV.11–14 A more targeted investigation based 

on outbreak characteristics (host should be present in 

both outbreak locales, be able to move or interchange 

between the locales, and plausibly have contact with 

horses) revealed that fruit bats of the genus Pteropus

(Figure 22-2), otherwise known as “flying foxes,” had 

anti-HeV antibodies15 and sometimes were infected 

with HeV itself.16

Nipah Virus

The emergence of NiV has several parallels to that 

of HeV, but with more dramatic consequences. NiV is 

named after Sungai Nipah, Malaysia, the home village 

of the patient from whom NiV was first isolated. NiV, 

like HeV, emerged from pteropid fruit bats, but in this 

case spilled over to domestic pigs (Sus scrofa domesti244-949 DLA DS.indb 549 6/4/18 
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Medical Aspects of Biological Warfare 

Figure 22-1. The bats that harbor henipaviruses, including Hendra virus and Nipah virus, have widespread distribution. 

However, outbreaks of Hendra and Nipah disease have been limited to Australia and Malaysia/Bangladesh, respectively. 

Each outbreak shown on the timeline may represent more than one spillover event from the bat reservoir.

Data sources: (1) Eaton BT, Broder CC, Middleton D, Wang LF. Hendra and Nipah viruses: different and dangerous. Nat 

Rev Microbiol. 2006;4:23–35. (2) Drexler JF, Corman VM, Müller MA, et al. Bats host major mammalian paramyxoviruses. 

Nat Commun. 2012;3:796. (3) Breed AC, Meers J, Sendow I, et al. The distribution of henipaviruses in Southeast Asia and 

Australasia: is Wallace’s line a barrier to Nipah virus? PLoS One. 2013;8:e61316. (4) Australian Veterinary Association. Hendra 

virus. http://www.ava.com.au/hendra-virus. Accessed February 13, 2014. (5) World Health Organization Regional Office for 

South-East Asia. Nipah virus outbreaks in the WHO South-East Asia Region. http://www.searo.who.int/entity/emerging_diseases/links/nipah_virus_outbreaks_sear/en/. Accessed February 13, 2014. (6) World Health Organization Regional Office 

for South-East Asia. Surveillance and outbreak alert: Nipah virus. http://www.searo.who.int/entity/emerging_diseases/links/

nipah_virus/en/. Accessed February 13, 2014.

cus), which served as highly effective amplifying hosts. 

The first recorded potential NiV spillover occurred in 

1997, with unusual pig deaths at pig farms near Ipoh, 

Malaysia, attributed to classical swine fever at the time. 

However, retrospective analysis of serum samples 

from several patients presenting with encephalitis in 

1997 detected anti-NiV antibodies.17

Beginning in September 1998, also near Ipoh, pigs 

and pig farmers became ill in a major outbreak of 

febrile encephalitis with respiratory symptoms. The 

mosquitoborne Japanese encephalitis virus (JEV), endemic to Southeast Asia, was immediately suspected, 

especially since domestic pigs are known to serve as 

an amplifying host for JEV,18 and also because 4 of 

28 initial human cases tested positive for anti-JEV 

antibodies.17 Despite extensive anti-JEV measures, 

however, including mosquito control and a JEV vaccination campaign,19 the outbreak continued unabated, 

spreading into neighboring districts and afflicting increasing numbers of pigs and humans. The outbreak 

also spread to Singapore via exported infected pigs.20

Furthermore, the outbreak was widespread with 

disease mainly afflicting adults,21 whereas Japanese 

encephalitis is asymptomatic in the vast majority of 

cases and more likely to cause disease in children.18

Also, cerebrospinal fluid from an infected patient 

gave rise to syncytia (multinucleated cells formed 

by cell-cell fusion) in Vero cell culture, indicative of a 

pH-independent fusion mechanism of the infectious 

agent.22 Such formation is uncharacteristic of JEV, 

which has a pH-dependent fusion mechanism. Finally, the Centers for Disease Control and Prevention 

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Henipaviruses

determined by cross-reactive immunofluorescence, 

serology, and sequence analysis that the novel agent 

was closely related to HeV.23

At the end of the 1998–1999 outbreak, at least 294 

human infections resulting in clinical encephalitis 

were recorded in Malaysia and Singapore, of which 

106 infections were fatal.17,20 The inclusion of milder 

nonencephalitic cases (as determined by exposure to 

infected pigs and seroconversion, a number of which 

later developed late onset encephalitis) increased the 

total number of cases to more than 360.24,25 More than 

a million pigs were culled in an effort to contain the 

outbreak, resulting in severe economic damage and 

widespread changes to the pig farming industry.26,27

Subsequent outbreaks of NiV infection have occurred 

in Bangladesh and adjacent areas of India on an almost 

yearly basis (Figure 22-1). In contrast to the Malaysian 

outbreak, these subsequent spillovers from pteropid 

bats have mainly occurred without involvement of 

a domestic animal intermediate. Generally these 

spillovers had higher case fatality rates, ranging from 

40% to 100%.28 These differences may be the result of 

differences among disparate NiV strains, the standard of care in Bangladesh/India versus Malaysia, or 

Figure 22-2. One of the reservoirs of Hendra virus: a spectacled flying fox (Pteropus conspicillatus) near Cairns, Australia. 

Photograph: Courtesy of Pål A Olsvik, Bergen, Norway.

Figure 22-3. (a) In Bangladesh, fresh date palm sap is collected by shaving the date palm tree, placing a tap, and collecting 

the sap in a clay pot overnight. Fruit bats are known to lick the sap stream and even urinate or defecate into the clay pots, 

thus potentially contaminating the raw sap with infectious Nipah virus. (b) Covering the sap stream with a bamboo skirt is 

highly effective at preventing contamination. 

Photographs: Courtesy of Nazmun Nahar, International Centre for Diarrhoeal Disease Research, Bangladesh.

a b

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sociocultural factors unique to Bangladesh/India.29,30

For example, a major route of spillover during the 

Bangladesh outbreaks is the consumption of fresh 

date palm sap, a seasonal delicacy that can become 

contaminated with fruit bat saliva or excreta during 

collection (Figure 22-3).31

Newly Identified Henipaviruses

After nearly two decades of intensive investigation of HeV and NiV, evidence of novel henipaviruses in diverse wildlife species has created 

increased public health concern.1–3,32–36 For example, 

the recent discovery of divergent new clades of 

henipaviruses in indigenous bat populations across 

Africa and even Central/South America,1,34 at least 

another henipavirus endemic to Australia but distinct from HeV (Cedar virus),32 and a henipavirus 

in southeastern China (Mòjiāng virus) from a putative rodent reservoir that may be linked to cases of 

fatal pneumonia in humans,35 all raise urgent questions about the possible global emergence of these 

zoonotic viruses. These new findings extended the 

potential geographic distribution of henipaviruses 

from Southeast Asia/Australia to Africa and Central/

South America (Figure 22-1). At least one of these 

novel henipaviruses, Cedar virus, could be isolated 

in culture, but was nonpathogenic in small animal 

models.32 The discovery of a potential henipavirus 

spillover to humans in Cameroon, however, emphasizes the need to both increase vigilance and knowledge of this latent pool of henipaviruses.3

 Human 

sera from Cameroon capable of neutralizing NiV 

were significantly associated with bat butchering 

in areas of intensive deforestation.3 This association 

highlights the role of environmental changes and 

specific human behaviors in determining the risk of 

zoonotic transmission. Although the pathogenicity 

and virulence of newly discovered henipaviruses 

remain to be determined, the repeated misdiagnosis 

of NiV as Japanese encephalitis in Southeast Asia remains a cautionary tale. Henipavirus-derived illness 

may often be ascribed to other encephalitic diseases 

known to occur in the affected area.

EPIZOOTIOLOGY AND EPIDEMIOLOGY OF HENIPAVIRUSES

Pteropid (fruit) bats appear to be the major reservoir 

host for HeV and NiV. No appreciable signs of HeV 

and NiV infections have been found in other wildlife 

or domestic animals.11,37 Anti-HeV or anti-NiV antibodies are highly prevalent in pteropid bat populations 

throughout Southeast Asia and Australia37,38; HeV 

and NiV have been isolated from Pteropus fruit bats 

throughout their geographic range37; and all known 

outbreaks of HeV and NiV infection are linked to exposure of domestic animals or humans to fruit bats or 

their excretions.30 Furthermore, experimental infection 

of Pteropus bats belonging to different species with 

HeV or NiV did not result in clinical signs of infection, despite inconsistent signs of seroconversion, viral 

replication in tissues, and virion excretion in urine.39–42

These data support the hypothesis that HeV and NiV 

are not pathogenic in their natural Pteropus hosts, thus 

persisting subclinically in these bat populations.

Despite their apparent long coevolutionary history with bats and likely endemic nature,37 HeV and 

NiV emerged to cause human disease suddenly and 

nearly simultaneously, likely signaling common factors driving their emergence. A retrospective study 

of environmental factors and man-driven changes in 

Malaysia identified slash-and-burn agriculture with 

concomitant loss of forest habitat, a resulting impenetrable smoke haze that led to crop failures, and severe 

drought as potential precipitating factors.43 Fruit bats 

were driven to populate cultivated fruit orchards, which 

were often located adjacent to pig farms, thus providing a means of transmission. In one plausible scenario, 

half-eaten fruits contaminated with NiV-infected bat 

saliva are dropped into a pig farm and then eaten by 

the pigs. Such half-eaten fruits were found in pig farms 

near the epicenter of the 1998–1999 NiV encephalitis 

outbreak.43 Similar environmental and manmade 

pressures facing fruit bat populations across Southeast 

Asia and Australia are likely behind the persistent 

upsurge in HeV and NiV spillovers in recent years.4

Hendra Virus

All seven known human cases of HeV infection 

resulted from intimate contact with sick horses. HeV 

is highly virulent in horses, and infection often culminates in copious production of infectious respiratory 

secretions.38 Human infections were traced back to 

efforts to save these horses without the use of personal 

protective equipment. A horse trainer, for example, 

attempted to force-feed a sick mare with abrased 

bare hands.5 However, such cases of horse-to-human 

transmission remain the exception. Many other people 

who were also highly exposed to contaminated horse 

bodily fluids, even to fluids from horses that were 

implicated in HeV transmission to other humans, did 

not develop signs of HeV infection.30 With increased 

public awareness of the risk of HeV transmission and 

the corresponding increase in proper use of personal 

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Henipaviruses

protective equipment, the risk of spillover to humans 

has been mitigated.4 The introduction of an HeV vaccine for horses in 2012, Equivac HeV (Pfizer Animal 

Health, Brisbane, Australia), will hopefully further 

minimize human transmission risk.

How horses initially become infected remains to 

be elucidated. Equine cases of infection may result 

from horses grazing on pastures contaminated with 

bat excreta or remains of half-eaten fruit.11,37 Infected 

horses inefficiently transmit the virus, even to other 

horses, but some horse-to-horse transmission may 

occur through licking infectious nasal discharge.11

Respiratory transmission of HeV has never been demonstrated experimentally or during natural infection.37

The inefficiency of HeV transmission is buttressed by 

findings that despite a continuous low prevalence of 

HeV in Pteropus bats in Queensland and New South 

Wales,44 people having extensive contact with Pteropus

bats in these areas, with bat bites and exposures to bat 

blood in many cases, had no sign of HeV exposure.45

Direct comparisons of NiV and HeV infection in some 

rodent models suggest that HeV may be less efficient 

in infecting animals through the intranasal route,46,47

although this lower efficiency remains to be verified 

in a more relevant nonhuman primate model.

Nipah Virus

Malaysia Outbreak, 1998–1999

During the large Malaysia outbreak, pigs served 

as a highly effective amplifying reservoir, contracting 

infection and transmitting NiV so efficiently that the 

infection rate among pigs at affected farms approached 

100%.27 The risk to humans posed by this spread was 

exacerbated by the mild illness NiV caused in the 

pigs, with a lethality of less than 1% to 5% and often 

presenting asymptomatically.27 Unsuspecting farmers 

thus moved asymptomatic pigs to other farms and 

slaughterhouses, quickly spreading the virus. A subset 

of infected pigs developed febrile illness, respiratory 

signs such as labored breathing and harsh nonproductive cough, and neurological signs such as myocloni 

and uncoordinated gait.27

Nearly all human cases during the Malaysia NiV 

encephalitis outbreak can be attributed to direct contact with or proximity to infected pigs, and most cases 

were pig farmers.30,48,49 One person who denied any 

recent proximity to pig farms repaired pig cages, suggesting that the virion-containing secretions remained 

infectious on surfaces for extended periods.50 A few 

human infections may have resulted from secondary 

transmission through dogs, which were commonly 

infected during the outbreak.11,49,51 Evidence of humanto-human transmission is limited. A large cohort study 

of 393 healthcare workers intimately involved in caring 

for NiV-infected patients identified only three nurses 

who seroconverted and had potential illness, despite 

many reported high-risk exposures.52 Risk may have 

been minimized by precautions taken by the healthcare workers, because patients clearly shed infectious 

NiV in respiratory secretions and urine, especially 

during the early phase of illness.53 Human-to-human 

transmission possibly may have played a role in the 

NiV outbreak, although the epidemiological record 

does not provide unambiguous evidence.30 Finally, 

retrospective investigations appear to show that NiV 

was causing disease in pigs as early as 1996, but that 

the mild symptoms and rough similarity to other diseases (eg, classical swine fever) did not raise suspicion 

of anything unusual.11,54 The advent of increasingly 

inexpensive, high-throughput sequencing may make 

disease surveillance and agent identification more 

likely to catch emerging pathogens such as NiV in 

the future.

Bangladesh and India, 2001 to Present

In comparison to the Malaysia NiV encephalitis 

outbreak, subsequent outbreaks in Bangladesh and 

India are of greater concern because of evidence of 

clear chains of human-to-human transmission.30 As 

in Malaysia, infected patients shed NiV in their bodily 

fluids including saliva.55,56 The intimate care for sick 

family members, involving being in physical contact, 

sharing utensils and food, and sleeping in the same 

bed to provide them comfort, greatly increased the risk 

of transmission.57 The longest documented transmission chain involved five generations, with the third 

generation involving a religious leader who infected 

22 family members and followers.57,58 A major 2001 

outbreak in Siliguri, India, was also characterized by 

hospital-associated transmission: one admitted patient 

became the source of more than 40 subsequent infections within a hospital and nursing homes. 59 As with 

the outbreak in Malaysia, Japanese encephalitis was 

initially suspected, and the causative agent was not 

identified as NiV until samples were retrospectively 

analyzed several years later.59

The outbreaks in Bangladesh and India are also 

notable for the apparent lack of a domestic animal 

intermediate between the Pteropus bat reservoir and 

humans. Although a few incidents appear to involve 

domestic animals such as cows, pigs, or goats,60–62 the 

major route of spillover has been the consumption of 

contaminated fresh date palm sap.31 Date palm trees 

are tapped for their sweet sap in the winter, and bats 

often lick the sap stream. Defecation and urination 

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into or near the collection pots, or even drowned dead 

bats, have been observed.31,63 Fortunately, the use of 

a simple bamboo skirt to cover and protect the sap 

stream, a method local to northwest Bangladesh that 

has not been consistently or widely used, appears to be 

highly effective in preventing contamination (Figure 

22-3).64 More widespread use of this method and other 

interventions to minimize risk would help the affected 

regions of the Indian subcontinent break out of its cycle 

of yearly NiV encephalitis outbreaks.

CLINICAL PRESENTATION OF HENIPAVIRUS INFECTIONS

The incubation period of HeV or NiV infection and 

illness typically ranges from a few days to 14 days.65

Although information on HeV infection is limited 

because of the few human cases, the clinical signs and 

pathology of HeV and NiV infections are similar.66 The 

hallmarks of henipavirus pathogenesis are extensive 

vasculopathy, respiratory distress, and encephalitic 

disease with corresponding neurological symptoms. 

Respiratory and encephalitic symptoms may appear to 

varying degrees. During the first Malaysia NiV disease 

outbreak, clinical signs were mainly encephalitic in 

nature with minor pulmonary involvement, whereas 

in subsequent outbreaks in Bangladesh, encephalitis 

was more commonly joined by severe respiratory 

distress.29,62 Researchers are actively investigating 

whether these differences result from genetic differences among viral variants, the route of transmission, 

or other factors.

Clinical Signs and Symptoms

Neurological signs and symptoms may include 

fever, headache, confusion, myocloni, seizures, meningism, and motor deficits including areflexia and 

hypotonia.5,29,48,59,67,68 Brain stem involvement, a poor 

prognostic factor, may be evidenced by a reduced 

level of consciousness, vomiting, abnormal pupillary 

and doll’s eye reflex, hypertension, and tachycardia.48

Figure 22-4. (a) Multinucleated endothelial syncytium (arrow) in brain with perivascular hemorrhage, hematoxylin 

and eosin stain. (b) Necrotic plaque in cerebral parenchyma 

with adjacent thrombosis (arrow), hematoxylin and eosin 

stain. (c) Typical magnetic resonance imaging for acute 

henipavirus encephalitis, with discrete, hyperintense lesions. 

Photographs: Courtesy of KT Wong, University of Malaya, 

Kuala Lumpur, Malaysia, with permission from Elsevier (a

and b) and Neurology Asia (c).

a b

c

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Henipaviruses

Cerebrospinal fluid is characterized by elevated white 

blood cell counts and/or protein concentration in a 

substantial proportion of cases.48,69

A minority of survivors (<10%) of HeV and NiV 

infection may experience relapsing encephalitis after 

apparent recovery or even initial asymptomatic or 

apparently nonencephalitic infection. Relapse may 

occur soon after apparent recovery or long after, with 

an average of 8 months and up to 11 years documented.65 Even without relapsing or progressive disease, 

a substantial proportion of survivors may experience 

long-term neurological deficits.70,71

Although the Malaysia outbreak of NiV disease 

was mainly characterized by encephalitic signs, a 

substantial proportion of patients still developed 

pulmonary signs such as cough and abnormal chest 

radiographs.20,48,68 More severe pulmonary symptoms, 

seen in subsequent NiV disease outbreaks and also 

some cases of HeV infection (eg, a horse trainer who 

developed progressive respiratory failure5), may also 

include atypical pneumonia, breathing difficulty, and 

acute respiratory distress syndrome.29,65

Pathology

Infection of microvascular endothelial cells leads 

to systemic vasculitis, thrombosis, and resultant 

microinfarction, especially in major organs such 

as kidneys, heart, lungs, and brain.65,72 Focal perivascular necrosis or hemorrhage is seen in highly 

vascular organs such as the spleen.73 Syncytia (giant 

multinucleated cell) formation is occasionally seen 

in the endothelium (Figure 22-4a) and among parenchymal cells of major organs. In the brain, discrete

plaque-like lesions with varying degrees of necrosis, 

edema, and inflammation (Figure 22-4b) likely correspond to the small hyperintense lesions in both 

grey and white matter seen by magnetic resonance 

(Figure 22-4c).65 The dual pathology of vasculitis with 

associated microinfarction and direct infection of parenchyma of major organs is a distinguishing feature 

of henipavirus pathogenesis.65 Tissue damage in the 

central nervous system from both microinfarction 

and direct infection of neuronal cells distinguishes 

henipavirus infections from other viral encephalitic 

diseases.74 Relapse encephalitis appears to result from 

recrudescent infection, with extensive parenchymal 

necrosis, edema, and inflammation corresponding to 

confluent lesions seen in magnetic resonance scans (as 

opposed to the more discrete foci usually seen during 

acute encephalitis during early illness). Pathology 

associated with relapsing encephalitis is only found 

in the central nervous system, and no vasculopathy 

is present, even in the brain.25,75

MOLECULAR BIOLOGY OF HENIPAVIRUSES

Virus Structure

Henipaviruses are negative-sense ribonucleic 

acid (RNA) viruses that produce enveloped virions. 

Henipaviruses are currently classified as members of 

the genus Henipavirus, family Paramyxoviridae, order 

Mononegavirales. 

Like other paramyxovirus particles, Hendra and 

Nipah virions have pleomorphic shapes, usually more 

spherical although sometimes filamentous as well (Figure 22-5a), and range in size from less than 200 nm to 

more than 1,000 nm in diameter.76,77 The virions contain 

helical ribonucleocapsids with the herringbone appearance characteristic of, and unique to, paramyxoviruses 

(Figure 22-5b). A unique feature of Hendra virions is 

the frequent presence of a double fringe surrounding 

the particle,77 which may result from differing lengths 

or conformations of the envelope proteins; in contrast, 

Nipah virions predominantly display a single fringe.

Virus Genome 

Like all mononegaviruses, henipaviruses have a 

linear, monopartite, single-stranded RNA genome of 

negative polarity. The overall structure of henipavirus 

genomes is similar to those of other paramyxoviruses, 

with 3’ leader and 5’ trailer sequences at the termini of 

the genomes that act as virus-specific promoters, 5’ and 

3’ untranslated regions flanking each gene, and a conserved intergenic signal between each gene.78 With a 

few recently described exceptions, henipaviruses have 

the longest known paramyxoviral genomes (≈18 kb). 

The especially long 3’ untranslated regions are unique 

features of henipavirus genomes and account for much 

of the extra length compared to other paramyxoviruses 

(≈15 kb).79 The functional relevance of these long untranslated regions remains to be determined. 

Viral Proteins 

Henipavirus genomes contain six genes, which 

encode a nucleocapsid protein (N), phosphoprotein 

(P), matrix protein (M), fusion protein (F), attachment glycoprotein (G), and large RNA-dependent

RNA polymerase (L). N encapsulates the genome, 

thereby forming the ribonucleoprotein complex. 

Like the genomes of other paramyxoviruses, 

henipavirus genomes have nucleotide lengths that 

are evenly divisible by six, a feature likely resulting 

from the periodicity of N protein encapsidation of 

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the genome. P and L form the polymerase complex, 

which replicates the viral genome and transcribes 

viral mRNAs. 

M organizes the assembly and budding of virions 

and underlies the viral envelope. M also transits 

through the nucleus, although the ultimate significance 

of this transit for henipavirus replication and pathogenesis remains unclear. G binds to the cellular receptor(s), 

and F catalyzes the membrane-membrane fusion 

responsible for viral entry. G and F can also catalyze 

cell-cell fusion, leading to syncytium (giant multinucleated cell) formation, which is a hallmark of henipavirus 

infection (Figure 22-5c). The henipavirus attachment 

protein is unique among paramyxoviruses in that it 

does not possess any hemagglutinin activity (although 

morbilliviruses such as measles virus will only agglutinate red blood cells from primates of certain species).80

The tissue tropism of HeV and NiV is determined 

by entry receptor use. The receptor tyrosine kinase 

ephrin-B2 serves as an entry receptor for all known 

henipaviruses,32,81,82 but at least HeV and NiV can 

also use the related ephrin-B3 as an alternative receptor.83,84 As cellular factors that are critical during 

embryogenesis, ephrin-B2 and -B3 are highly evolutionarily conserved. This high level of conservation 

contributes to the unusually wide potential host range 

of HeV and NiV, which have been shown to infect 

mammals spanning six orders.85 Ephrin-B2 and -B3 

from a wide range of mammals allow viral entry,86

chicken embryos are susceptible to NiV infection,87

and even zebrafish ephrin-B2 can serve as an entry 

receptor.72 The tropism of henipaviruses is also not 

restricted by the cellular protease required for F protein cleavage, a maturation step required to render 

it fusion-competent. Unlike some paramyxoviral 

F proteins that require a protease of limited tissue 

distribution, the henipavirus F protein uses the ubiquitously expressed endosomal protease cathepsin 

L,88,89 thereby further extending tissue and possibly 

host tropism. 

Figure 22-5. (a) Nipah virions produced from infected Vero E6 cells. (b) Nipah virus nucleocapsid, with the herringbone 

appearance characteristic of paramyxoviruses. (c) Multinucleated Nipah virus-induced syncytium in human umbilical vein 

endothelial cells. Blue represents nuclei; red represents actin filaments and illustrates cell boundaries; and green represents 

Nipah virus matrix protein. 

Photographs: (a and b) Courtesy of Cynthia Goldsmith, Centers for Disease Control and Prevention, Atlanta, Georgia. (c) 

Courtesy of Arnold Park, Icahn School of Medicine at Mount Sinai, New York, New York. 

a c

b

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PATHOGENESIS

macrophages. It is also possible that henipaviruses 

access the central nervous system more directly via 

infection of the olfactory epithelium and spread along 

the olfactory neurons.94,95

Clear differences exist between HeV and NiV infections regarding localization of initial infection in 

animal models despite their use of the same entry 

receptors,46,47 which has yet to be understood. The 

mechanism of foodborne NiV transmission also is unclear since digestive tract epithelium does not express 

ephrin-B2 or -B3.72 Virus infection most likely occurs 

via accessible and susceptible cells in the oropharyngeal mucosa, such as those of tonsillar tissues and 

salivary glands.

Henipaviruses partly owe their virulence to effective antagonism of host immune responses. Interferons 

(messengers of innate immunity that trigger extensive 

antiviral responses) bind extracellularly to interferon 

receptors, which usually trigger an intracellular signal 

cascade that includes the critical immune signaling 

factor signal transducer and activator of transcription 1 (STAT1). Like other paramyxoviruses, HeV 

and NiV encode several nonstructural proteins from 

their P genes, that is, the C, V, and W proteins, which 

antagonize innate immune signaling. The P, V, and W 

proteins have identical N-termini that bind and inhibit 

STAT1, partly by sequestering STAT1 and thus inhibiting transcription of interferon-inducible genes.96–99 The 

P gene products also antagonize signaling from other 

intracellular sensors of infection, such as TLR3 and 

Mda-5,100–102 which sense double-stranded RNA (an 

intermediate of viral replication) in endosomes and 

the cytoplasm, respectively.

In contrast to the host species-specific inhibition of 

interferon signaling induced by some paramyxoviruses,103 NiV can inhibit interferon signaling in cells 

from a number of tested mammals,104 consistent with 

the ability to cause disease in a wide range of hosts. 

The high virulence of henipaviruses in the “wrong” 

host may therefore be a function of their broad tissue 

tropism in vivo, their ability to gain systemic access, 

and their inhibitory effect on immune responses.

The pathogenesis of henipavirus encephalitis has 

been examined predominantly using animal models. 

The oronasal route of infection is used most often during such experiments as it reflects at least one type of 

natural transmission and might also simulate exposure 

during a biological attack.90 Similar to results obtained 

in vitro, in vivo tropism is predominantly determined 

by the absence or presence of henipavirus receptors on 

potential target cells. 

Ephrin-B2 is highly expressed on the endothelial 

and smooth muscle cells that line arterial vessels, 

lungs, and brain. The human airway epithelium 

expresses both ephrin-B2 and -B3,91 and upon infection, the virus likely crosses the epithelium via 

limited basolateral virion release, while disruption 

of the epithelium via cell-cell fusion may also facilitate systemic entry. In many animal models of 

Nipah encephalitis, the alveolar epithelium tests 

immunopositive for NiV antigens, and the alveolar 

wall frequently undergoes fibrinoid necrosis.92 Interestingly, in pigs, an increased number of alveolar 

macrophages is a consistent histological feature. In 

human lung tissue, viral antigen is found in multinucleated giant cells located in the alveolar space.73

Multinucleated giant cell formation is a product 

not only of alveolar macrophage fusion, which is 

generally a prominent feature of chronic inflammatory conditions, but also of NiV-induced syncytia 

formation. Transmigration of infected alveolar macrophages may serve as a “Trojan horse” for the virus 

to gain systemic access via the microvasculature. 

Cell-free and cell-associated viremia then result in 

systemic infection.93

Ephrin-B3 expression is mostly restricted to the 

central nervous system (eg, brainstem), which correlates with the observation of antemortal brainstem 

dysfunction noted during henipavirus encephalitis.48,83 Henipaviruses likely gain access to the brain 

via basolateral release from brain microvascular 

endothelial cells, disruption of the blood-brain 

barrier as a result of cell–cell fusion and vasculitis, 

and transmigration of “Trojan horse” inflammatory 

HENIPAVIRUSES AND BIOLOGICAL WEAPONS

HeV and NiV are classified as Centers for Disease 

Control and Prevention Category C Bioterrorism 

agents because of the following:

• their availability,

• ease of production and dissemination, and 

• potential for high morbidity and lethality.

Availability

HeV and NiV are readily available from tissues or 

fluids from patients during frequent, recurring disease 

outbreaks as well as by isolation from their natural bat 

reservoirs. Furthermore, reverse genetics systems are 

available in biosafety level 4 laboratories for the rescue 

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of henipaviruses directly from plasmids.105,106 De novo 

access to, and modification of, henipaviruses can be 

accomplished by a determined hostile group with access to resources and the relevant technical expertise. 

Ease of Production and Dissemination

Henipaviruses can be grown to very high titers, 

up to 108 TCID50/mL or PFU/mL,107 in a wide range 

of cell lines.108 Although henipaviruses are highly 

sensitive to temperature variation and dessication, 

henipaviruses can persist for days under certain 

conditions.109 Therefore, under optimal conditions 

henipaviruses could possibly be maintained at high 

titer for extended periods of time. Henipaviruses 

infect a wide range of hosts, including domestic 

animals such as dogs and livestock, that could serve 

as amplifiers of infection. Although NiV and HeV 

clearly have the potential to infect through aerosol, 

this route has not been conclusively shown, either 

experimentally or from the natural history90,110; 

however, limited epidemiological evidence suggests that some human cases of NiV infection in 

Bangladesh may have resulted from exposure to 

coughing.58,111 Also restricting the biological weapons potential of henipaviruses is the lack of sustained human-to-human transmission: the longest 

documented chain of transmission was five generations.57,58 A naturally occurring or intentionally 

mutated strain with higher transmission efficiency 

would be required to sustain an epidemic; an expanding pandemic, however, may not be a required 

or desired goal of a bioterror attack.

Potential for High Morbidity and Lethality

HeV and NiV cause disease with very high case 

fatality rates, ranging from 40% to 100% in recent 

outbreaks.28 Survivors of disease or asymptomatically 

infected people may present years later with relapsing or late onset encephalitis, indicating occasional 

persistence of viral infection.65

DIAGNOSIS

In a bioweapons attack or other mass casualty 

scenario, rapid diagnostic methods must be used 

to identify the causative agent(s). These methods 

may be differentiated by whether or not the specific agent must be suspected before testing and 

by how quickly the results can be obtained. Under 

normal circumstances, henipavirus etiology would 

not be suspected without exposure to risk factors 

(contact with bats, ill persons, or domestic animals, 

or consumption of raw date palm sap) in the currently affected areas of Southeast Asia and Australia. Henipavirus etiology may also be considered 

throughout the known geographic range of bats 

known to harbor henipaviruses (Figure 22-1) if 

the responsible agent is unknown, with the initial

misdiagnosis of NiV as Japanese encephalitis virus 

remaining a cautionary tale. These factors do not 

apply in a bioweapons or bioterrorism event, however. The clinical presentation of henipaviruses with 

encephalitic and/or respiratory symptoms cannot 

be readily distinguished from other viral and nonviral causes of encephalitis, and diagnosis requires 

epidemiological and laboratory investigation. Given 

the broad host range of henipaviruses, the involvement of sick domestic or local animals can suggest 

potential henipavirus etiology.

Detailed recent reviews of henipavirus diagnosis 

may be found elsewhere107,112,113; the available methods 

are discussed below.

Reverse Transcription-Polymerase Chain Reaction 

If henipaviruses are suspected, henipavirus-specific 

polymerase chain reaction or real-time polymerase chain 

reaction should be performed on RNA extracted from 

patient samples. Prospective samples include serum, 

whole blood (detectable viremia may be cell-associated93), urine, nasopharyngeal aspirates, throat swabs, 

cerebrospinal fluid, or tissue samples from highly affected tissues such as the brain, lungs, kidneys, or spleen.

Detection of Henipavirus Antigens 

Characterized anti-henipavirus antibodies can be 

used to detect viral antigens in formalin-fixed tissues. 

Similarly, immunofluorescence with anti-henipavirus 

antibodies can be performed on infected cell cultures. 

Detection of Anti-henipavirus Immune Responses

A number of methods exist for detection of antihenipavirus antibodies in sera of infected patients. Specific immunoglobulin M responses develop in virtually 

all patients within the first week postexposure, and the 

slower immunoglobulin G response encompasses virtually all patients after 2 weeks of infection.29,114 Detection 

of serum antibodies is useful for diagnostics because 

antibodies are more stable than viral RNA over time 

and under different conditions. Furthermore, viremia 

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Henipaviruses

can be difficult to detect when patients are symptomatic. 

Serum neutralization of replicating virus is considered 

the gold standard serological test, although this test 

requires biological safety level 4 containment.107 Surrogate neutralization tests, which can be performed 

at biological safety level 2 conditions, offer a combination of high sensitivity and high specificity. Such 

tests include the use of vesicular stomatitis Indiana 

virus pseudotyped with the henipavirus envelope proteins115–117 or a Luminex (Luminex Corporation, Austin, 

TX) platform-based assay assessing the ability of sera 

to inhibit the binding of the henipavirus receptor to microbeads coated with henipavirus attachment protein.118

Enzyme-linked immunosorbent assay variations on this 

test using infected cell lysate or recombinant henipavirus proteins have been commonly used as frontline 

assays because of their simplicity and affordability, but 

typically have a relatively high false-positive rate.107

Virus Isolation 

Viral etiology may be suspected if cells incubated 

with filtered patient samples develop cytopathic effects. Henipaviruses grow efficiently in a wide range of 

cell lines, including Vero E6 cells.119 Syncytia formation 

in cell culture would implicate enveloped viruses with 

a pH-independent fusion mechanism, which include 

henipaviruses (Figure 22-5c). Electron microscopy on 

viral preparations and infected cells could implicate 

a henipavirus as the potential agent. New or modified henipaviruses may have different characteristics, 

however, so any final diagnosis requires multiple 

routes of confirmation. In contrast to sequencing, virus isolation is a days-long process (2–5 days usually 

pass until cytopathic effects become visible), and two 

rounds of 5 days each are recommended before virus 

recovery is judged unsuccessful.107,112 Furthermore, for 

an outbreak suspected to be caused by a highly virulent 

agent, virus isolation attempt should be performed 

under high-level biosafety containment. Nevertheless, 

isolation and characterization of the agent remain the 

most conclusive demonstration of etiology.

Next-generation Sequencing 

Next-generation sequencing (NGS) is slowly becoming more widely available and will enable the 

identification of known and unknown henipaviruses. 

As the cost and speed of NGS continue to decrease, a 

rapid and routine measure using NGS in a suspected 

outbreak is increasingly possible following RNA extraction and reverse transcription polymerase chain 

reaction.107 Since henipaviruses only rarely infect humans, detection of henipavirus sequences would not 

normally be expected, and an appreciable presence 

of henipavirus sequence in multiple samples would 

suggest potential henipavirus etiology. Henipavirusspecific NGS is already becoming routinely used 

during postoutbreak investigations to determine the 

characteristics of new henipavirus isolates.107,120

MEDICAL MANAGEMENT

Licensed therapeutics to treat henipavirus infection 

in humans are not available. Thus, medical management 

of henipavirus infections is supportive. Mechanical 

ventilation is required if the patient becomes comatose 

or develops acute respiratory distress syndrome. Appropriate measures should be taken as for any potentially 

highly contagious pathogen, including quarantine and 

use of personal protective equipment and engineering 

controls such as negative air flow, if available; careful 

handling of clinical specimens; and rapid epidemiological investigation (with particular attention to potential 

spread via domestic animals) and identification of 

high-risk contacts. Despite the lack of specifically recommended therapeutics, several potential treatments with 

varying levels of supporting evidence should be considered in the event of a bioweapons or mass casualty 

event. Some of the proposed therapeutic interventions 

discussed below may also be appropriate in the case 

of accidental exposure or as prophylaxis for frontline 

responders to a potential outbreak.

Passive Immunotherapy

Active vaccination is highly effective in animal 

models83 and was the basis for the recently approved 

Equivac HeV vaccine for horses. This vaccine contains 

a soluble version of the HeV attachment envelope protein, which stimulates the production of neutralizing 

anti-HeV antibodies and provides protection against 

HeV infection.121 However, an active vaccination approach for henipaviruses is unlikely to be a practical 

strategy on a population-wide basis in humans for 

several reasons. Compared to veterinary vaccines, 

human vaccines have higher regulatory hurdles. In 

addition, such vaccines would be truly useful only for 

a few people. Few cases have occurred during natural 

outbreaks, and the likelihood that populations outside 

of affected locations will be exposed to a pathogenic 

henipavirus is low. However, the risk–benefit calculations may be different for frontline responders to 

suspected outbreaks.

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Passive immunotherapy may be highly efficacious 

as postexposure treatment. In recent postexposure 

prophylaxis studies, ferrets and grivets received 1 to 

2 doses of a human monoclonal antibody with neutralizing activity against HeV and NiV attachment 

proteins 10 to 72 hours after virus exposure, which 

protected the animals from disease.122,123 The antibody 

has been offered to individuals with high risk of HeV 

exposure, and human clinical trials started in May 2015 

in Australia.124,125 Although further development of this 

strategy may not be economically viable if left to the 

market, such monoclonal antibodies warrant serious 

consideration as a stockpiled resource that can be used 

in a limited outbreak or bioweapons attack.

Ribavirin

Ribavirin, a guanosine analog first synthesized in 

1970, has been known to have broad-spectrum activity against many RNA and DNA viruses. Ribavirin 

is mainly used against human respiratory syncytial 

virus infections and to treat persistent hepatitis C 

virus infections.126,127 Ribavirin has several potential 

antiviral properties, which may differ in importance 

for different viruses.128

Because of its broad-spectrum effect, ribavirin was 

used in an off-label, nonrandomized, unblinded trial 

during the first Malaysia outbreak of NiV encephalitis, 

despite the known adverse effects (primarily hemolytic 

anemia at high dose).129 The lethality in the treated group 

was reduced by 40%, without affecting the rate of anemia. 

Follow-up studies revealed that ribavirin inhibits HeV 

and NiV replication in vitro.67,130–133 Results from in vivo 

studies examining the efficacy of ribavirin in the hamster130,131 and grivet134 animal models consistently found 

that ribavirin extends time to death. These animal models 

may represent particularly susceptible models for HeV 

and NiV pathogenesis because of the reproducibility of 

human disease.90 One concern has been that ribavirin 

only inefficiently crosses the blood–brain barrier, which 

is particularly consequential for an encephalitic disease. 

Modifications to the drug administration method, however, have the potential to overcome this hurdle.135–138

Current treatment of chronic hepatitis C involves 

the combination of type I interferon (discussed further 

in the next section) and ribavirin, which act synergistically.139 Ribavirin may therefore be evaluated in 

combination with other promising therapeutics in the 

case of henipavirus infection.

Innate Immunotherapy

Henipavirus inhibition of cellular production of 

interferon and of cellular responses to exogenous interferon is incomplete.140,141 Therefore, a clear opportunity 

is available to inhibit HeV and NiV pathogenesis by 

augmenting the innate immune response in vivo. 

The investigational double-stranded RNA compound 

poly(I)-poly(C12U), which stimulates type I interferon 

production, is highly effective against NiV infection in 

the hamster model when administered immediately 

after exposure.131 However, follow-up studies need to 

address the postexposure therapeutic window. Furthermore, despite undergoing phase III clinical trials 

for treatment of chronic fatigue syndrome, poly(I)-

poly(C12U) (Rintatolimod, Hemispherx Biopharma, 

Philadelphia, PA) has not been FDA approved.

Recombinant and modified type I interferons (eg, 

PEGylated interferon-α), however, have been approved by the FDA for multiple uses, including the 

treatment of chronic hepatitis B and C. Exogenous 

interferon inhibits henipavirus replication in vitro141

and would be a more direct approach to treat henipavirus infections than stimulating interferon production 

with compounds like poly(I)-poly(C12U). However, 

these compounds have yet to be evaluated against 

henipavirus infections.

 “Off-the-Shelf” Therapies Evaluated In Vitro

Numerous potential therapies to treat henipavirus infections are in varying stages of development 

(reviewed elsewhere).142,143 Promising therapies that 

are already available for off-label use against henipaviruses, but have not been evaluated for efficacy in 

vivo, are briefly described below. 

As NiV M protein requires ubiquitinylation as 

part of its intracellular trafficking pathway, proteasome inhibitors, which deplete the intracellular 

pool of free ubiquitin, are potent inhibitors of NiV 

replication in vitro.144 Next-generation proteasome 

inhibitors with improved pharmacokinetics, such as 

the FDA-approved carfilzomib or orally bioavailable 

analogs such as oprozomib (currently in phase I/II 

oncology trials), have even greater efficacy against 

henipaviruses in vitro.145 If these effects on henipaviruses can be translated into in vivo potency, the 

possibility of using potential FDA-approved proteasome inhibitors for off-label use will be a significant 

and realistic option for exposed or infected frontline 

responders. 

Many clinically available drugs inhibit henipavirus replication in vitro in the low micromolar range, 

including the alpha blocker phenoxybenzamine as 

well as the beta blocker propranolol, the antifungal 

clotrimazole, and the estrogen receptor antagonist 

tamoxifen.146 However, it is unclear whether any of 

these therapies can achieve viral inhibitory concentrations in vivo.

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SUMMARY

all aspects of henipavirus ecology, molecular biology, 

and pathogenesis. Although HeV and NiV do not appear efficiently transmissible at this time, vigilance 

for variants (or other henipaviruses) with enhanced 

transmissibility should be maintained. Significant 

progress in the development and identification of effective therapeutics for henipaviruses will mitigate the 

risks involved in managing future outbreaks.

The recent emergence, high virulence, and pandemic potential of HeV and NiV have fueled public 

concern and even lead to repeated public calls in Australia for culling of the reservoir hosts, the flying foxes. 

The worldwide discovery of numerous henipaviruses 

of unknown pathogenicity, including evidence of a 

potential spillover event to humans in Africa, further 

emphasizes the need for continued investigations into 

Acknowledgments

We are grateful for the technical writing services offered by Laura Bollinger (IRF-Frederick). The content 

of this publication does not necessarily reflect the views or policies of the US Department of the Army, the 

US Department of Defense, the US Department of Health and Human Services, or of the institutions and 

companies affiliated with the authors. JHK performed this work as an employee of Tunnell Government 

Services, Inc, a subcontractor to Battelle Memorial Institute under its prime contract with NIAID, under 

Contract No. HHSN272200700016I.

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54. Aziz J, Olson J, Lee OB, et al. Nipah Virus Infection of Animals in Malaysia. Paper presented at XIth International Congress 

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104. Hagmaier K, Stock N, Goodbourn S, Wang LF, Randall R. A single amino acid substitution in the V protein of Nipah 

virus alters its ability to block interferon signalling in cells from different species. J Gen Virol. 2006;87:3649–3653.

105. Conzelmann KK. Reverse genetics of Mononegavirales. Curr Top Microbiol Immunol. 2004;283:1–41.

106. Yoneda M, Guillaume V, Ikeda F, et al. Establishment of a Nipah virus rescue system. Proc Natl Acad Sci U S A. 

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107. Wang LF, Daniels P. Diagnosis of henipavirus infection: current capabilities and future directions. Curr Top Microbiol 

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108. Aljofan M, Saubern S, Meyer AG, Marsh G, Meers J, Mungall BA. Characteristics of Nipah virus and Hendra virus 

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109. Fogarty R, Halpin K, Hyatt AD, Daszak P, Mungall BA. Henipavirus susceptibility to environmental variables. Virus 

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Chapter 23

FILOVIRUSES

SHELI R. RADOSHITZKY, PhD*; SINA BAVARI, PhD†; PETER B. JAHRLING, PhD‡; and JENS H. KUHN, MD, PhD, PhD, MS§

INTRODUCTION

NOMENCLATURE

Filovirus Taxonomy

Filovirus Disease Nomenclature

Filovirus Categorization

MOLECULAR BIOLOGY

Filovirion Structure

Filovirus Genomes and Proteins

Filovirus Lifecycle

Geographic Distribution

Natural Reservoirs of Filoviruses

Epizootiology of Filoviruses

Epidemiology of Filovirus Infections

Serological Surveys

Environmental Niche Modeling

TRANSMISSION

THREAT TO THE WARFIGHTER

Filoviruses and the Soviet Biological Warfare Program

PREVENTION

Behavioral Modification

Filovirus Inactivation and Decontamination

Vaccines

DISEASE

Pathogenesis

Clinical Presentation

DIAGNOSIS

TREATMENT

SUMMARY

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*Principal Investigator, Molecular and Translational Sciences Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, 

Fort Detrick, Maryland 21702; formerly, Postdoctoral Fellow, Toxicology Division, US Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland

†

Science Director, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Room 900, Fort Detrick, Maryland 21702; formerly, 

Division Chief, Toxicology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Room 900, Fort Detrick, Maryland 

‡

Captain (Retired), Medical Service Corps, US Army; Director, National Institutes of Health/Division of Clinical Research/National Institute of Allergy and Infectious Diseases/Integrated Research Facility, 8200 Research Plaza, Room 1A-111A, Fort Detrick, Maryland 21702; formerly, Principal 

Scientific Advisor (Senior Research Scientist), US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

§

Virology Lead, National Institutes of Health/National Institute of Allergy and Infectious Diseases/Division of Clinical Research/Integrated Research 

Facility, B-8200 Research Plaza, Room 1A-132, Fort Detrick, Maryland 21702; formerly, Research Scholar, Harvard Medical School, Department of 

Microbiology and Molecular Genetics, New England Primate Research Center, 1 Pine Hill Drive, Southborough, Massachusetts 

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INTRODUCTION

arguably the most notorious as they are associated with 

the highest lethality and receive the widest attention 

in the media.2 Importantly, filoviruses were included 

in the Soviet biological weapons research program.3

The actual achievements of this program are still under 

debate, but through their inclusion, filoviruses gained 

military significance. This chapter provides an overview 

of the diversity, epidemiology, and molecular biology 

of filoviruses; summarizes the clinical presentation 

and pathology of the human diseases they cause; and 

reviews current developments in prophylactics and 

antivirals for the prevention and treatment of infections. 

Human viral hemorrhagic fevers are typically caused 

by members of the four families: (1) Arenaviridae (several 

mammarenaviruses), (2) Bunyaviridae (several hantaviruses, nairoviruses, phleboviruses), (3) Filoviridae

(certain ebolaviruses, marburgviruses), and (4) Flaviviridae (several flaviviruses sensu stricto).1

 The viruses 

of these four families are distinct in their molecular 

biology, reservoir host spectrum, and transmission 

route. However, the human diseases these viruses cause 

are clinically and pathologically similar, and all of the 

diseases are associated with significant lethalities (case 

fatality rates).1 Among these viruses, filoviruses are 

NOMENCLATURE

Filovirus Taxonomy

According to the International Committee on 

Taxonomy of Viruses, the family Filoviridae is one 

of seven families included in the order Mononegavirales.

4,5 The eight members of the family Filoviridae, 

referred to as filoviruses, are assigned to the three 

genera—Cuevavirus, Ebolavirus, and Marburgvirus—

based on the evolutionary/phylogenetic relationship 

of their coding-complete genome sequences and 

differences in biological properties of their virions 

(Figure 23-1).4–6 The members of the three genera, 

referred to as cuevaviruses, ebolaviruses, and marburgviruses, respectively, also differ in their geographic distribution, virion antigenicity, and overall 

genome organization. The International Committee 

on Taxonomy of Viruses Filoviridae Study Group 

recognizes one cuevavirus, Lloviu virus (LLOV); 

five ebolaviruses, Bundibugyo virus (BDBV), Ebola 

virus (EBOV), Reston virus (RESTV), Sudan virus 

(SUDV), and Taï Forest virus (TAFV); and two marburgviruses, Marburg virus (MARV) and Ravn virus 

(RAVV) (Table 23-1, Figure 23-1).6,7 The different 

isolates of each filovirus are grouped into variants, 

which typically correspond to viruses circulating in 

particular human disease outbreaks.8

 For instance, 

several EBOV isolates were obtained during a disease outbreak in 1976 in Zaire (Democratic Republic 

of Congo).9 These isolates are more closely related 

to each other than to several EBOV isolates obtained 

during a disease outbreak in Guinea in 2014. These 

two groups of viruses are therefore assigned different variant names (in this case, Yambuku and Makona, respectively).10,11 The term “strain” is reserved 

for nonnatural, laboratory-animal-adapted or certain 

cDNA-derived filoviruses.12,13

Filovirus Disease Nomenclature

With the exception of LLOV and RESTV, all other 

filoviruses are associated with severe human illness. 

In the International Statistical Classification of Diseases 

and Related Health Problems by the World Health Organization, human disease names are standardized 

internationally. In its most current version, International Statistical Classification of Diseases and Related 

Health Problems, Tenth Revision (ICD-10), two filovirus 

diseases are distinguished: (1) Marburg virus disease 

(MVD; colloquially often referred to as Marburg 

hemorrhagic fever), which is caused by MARV or 

RAVV; and (2) Ebola virus disease (EVD; colloquially often referred to as Ebola hemorrhagic fever), 

which is caused by BDBV, EBOV, SUDV, or TAFV 

(Table 23-2).14

Filovirus Categorization

All filoviruses are considered World Health Organization Risk Group 4 infective microorganisms. 

Therefore, any research involving replicative forms of 

the viruses must be performed in maximum containment facilities.15 In the United States, such facilities are 

designated as (animal) biosafety level 4 laboratories. 

Given the associated high lethality with infections 

and the absence of licensed medical countermeasures 

(MCMs), filoviruses are considered high-consequence 

pathogens and are therefore categorized as Centers 

for Disease Control and Prevention (CDC) Bioterrorism Category A Agents16 and National Institute of 

Allergy and Infectious Diseases Category A Pathogens.17 Categorized as Tier 1 Select Agents, access to 

replicative forms of filoviruses is highly restricted 

by law,16 and their export is tightly controlled.18

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Figure 23-1. Phylogenetic relationships of members of the family Filoviridae. Bayesian coalescent analysis of representative 

cuevaviruses, marburgviruses, and ebolaviruses. Shown is the maximum clade credibility tree with the most recent common 

ancestor number at each node. Posterior probability values are shown beneath the most recent common ancestor estimates 

in years. The scale is in substitutions/site (based on data published by Serena Carroll/CDC). Appended to the virus abbreviation via a “/” is the variant abbreviation (eg, KiC, MtE, Ast, Yam, But, Pau, Gul, Phi) connected by a hyphen to the isolate 

designation (not all variant names are yet standardized, see data sources 3 and 4). MRCA: most recent common ancestor

Colors assigned to viruses in this table will be used in follow-up tables and figures: RAVV: purple; MARV: blue; LLOV: yellow; 

EBOV: red; BDBV: orange; TAFV: brown; SUDV: green; and RESTV: gray.

Data sources: (1) Carroll SA, Towner JS, Sealy TK, et al. Molecular evolution of viruses of the family Filoviridae based on 

97 whole-genome sequences. J Virol. 2013;87:2608–2616. (2) Peterson AT, Bauer JT, Mills JN. Ecologic and geographic distribution of filovirus disease. Emerg Infect Dis. 2004;10:40–47. (3) Kuhn JH, Bao Y, Bavari S, et al. Virus nomenclature below 

the species level: a standardized nomenclature for natural variants of viruses assigned to the family Filoviridae. Arch Virol. 

2013;158:301–311. (4) Kuhn JH, Andersen KG, Bào Y, et al. Filovirus RefSeq entries: evaluation and selection of filovirus type 

variants, type sequences, and names. Viruses. 2014;6:3663–3682.

Other countries differ from the United States and 

each other in the extent of implemented regulations 

or laws in regard to filovirus access and distribution. 

However, a worldwide general consensus exists on 

the overall threat associated with filoviruses and the 

need for proper containment.

MOLECULAR BIOLOGY

Filovirion Structure

Filoviruses are viruses that produce virions with 

filamentous morphology. Filovirions, which are enveloped particles that are greater than 800 nm long 

and approximately 90 nm in diameter, are covered 

with spike protrusions of approximately 10 nm long. 

The particles are flexible and appear pleomorphic, 

assuming shapes that are reminiscent of spaghetti 

(Figure 23-2), but they can also be branched or circularized.19–25

Filovirus Genomes and Proteins

Complete filovirions contain one or more genome 

copies.23 Each genome is a monopartite, approximately 

19 kb long, linear, uncapped, and polyadenylated 

single-stranded RNA of negative polarity that has 3’ 

and 5’ complementary termini. All filoviruses contain 

genomes with the same linear arrangement of six 

(LLOV) to seven genes (all other filoviruses) in the 

order 3’-NP-VP35-VP40-GP-VP30-VP24-L-5’.26,27 However, the various filovirus genomes differ from each 

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TABLE 23-1

FILOVIRUS CLASSIFICATION AND NOMENCLATURE

2010 to Present Outdated Virus Names and Abbreviations

Order Mononegavirales

 Family Filoviridae

 Genus Marburgvirus

 Species Marburg marburgvirus

 Virus 1: Marburg virus (MARV) Marburg virus (MBGV), Lake Victoria marburgvirus

 Virus 2: Ravn virus (RAVV) Marburg virus (MBGV), Lake Victoria marburgvirus

 Genus Ebolavirus

 Species Bundibugyo ebolavirus

 Virus: Bundibugyo virus (BDBV) Bundibugyo virus (BEBOV) 

 Species Reston ebolavirus

 Virus: Reston virus (RESTV) Reston ebolavirus (REBOV)

 Species Sudan ebolavirus

 Virus: Sudan virus (SUDV) Sudan ebolavirus (SEBOV)

 Species Taï Forest ebolavirus

 Virus: Taï Forest virus (TAFV) Côte d’Ivoire ebolavirus (CIEBOV), Ivory Coast ebolavirus (ICEBOV)

 Species Zaire ebolavirus

 Virus: Ebola virus (EBOV) Zaire ebolavirus (ZEBOV)

 Genus Cuevavirus

 Species Lloviu cuevavirus

 Virus: Lloviu virus (LLOV)

In taxonomy, taxa (orders, families, genera, and species; recognizable by italicization) are considered concepts of the mind 

that do not have properties. Taxa are represented by physical members, the viruses (names in color). Only virus names are 

to be abbreviated in technical writing. See Figure 23-1 for color explanations. 

Data sources: (1) Kuhn JH, Becker S, Ebihara H, et al. Family Filoviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz 

EJ, eds. Virus Taxonomy—Ninth Report of the International Committee on Taxonomy of Viruses. London, United Kingdom: Elsevier/Academic Press; 2011:665–671. (2) Kuhn JH, Becker S, Ebihara H, et al. Proposal for a revised taxonomy of the family 

Filoviridae: classification, names of taxa and viruses, and virus abbreviations. Arch Virol. 2010;155:2083–2103. (3) Bukreyev AA, 

Chandran K, Dolnik O, et al. Discussions and decisions of the 2012–2014 International Committee on Taxonomy of Viruses 

(ICTV) Filoviridae Study Group, January 2012–June 2013. Arch Virol. 2014;159:821–830.

other in sequence and in the number of gene overlaps, 

intergenic regions, and the proteins expressed from the 

GP gene (Figure 23-3).26–29 The seven filovirus genes, 

NP, VP35, VP40, GP, VP30, VP24, and L, encode at least 

seven structural proteins, respectively: nucleoprotein 

(NP), polymerase cofactor (VP35), matrix protein 

(VP40), glycoprotein (GP1,2), transcriptional activator 

(VP30), secondary matrix protein (VP24), and RNAdependent RNA polymerase (L).23,26,27,30,31 In the case 

of cuevaviruses and ebolaviruses, three nonstructural 

proteins are encoded: secreted glycoprotein (sGP), 

secondary secreted glycoprotein (ssGP), and Δ-peptide 

(Table 23-3).32–34 GP1,2 is also converted into a nonstructural secreted product (GP1,2Δ) by tumor necrosis factor 

α-converting enzyme.35 Under certain circumstances, 

sGP may become a structural component of ebolavirions.36 Five of the main structural proteins—NP, VP35, 

VP40, GP1,2, and L—are clearly functional analogs of 

the standard set of mononegaviral core proteins (N, 

P, M, G, and L, respectively).37

Filovirus Lifecycle

Filovirions bind to attachment factors on the host cell 

surface38 via GP1,2, a type 1 transmembrane and class I 

fusion protein,39,40 which determines filovirus host and 

cell tropism.41 After cell surface binding, filovirions enter 

the cell through endocytosis.42–44 In the endolysosome, 

after a proteolytic cleavage that reveals the receptorbinding site, GP1,2 engages Niemann-Pick C1 protein,45,46

which triggers a complex GP1,2 refolding process ensuing in fusion of the endolysosomal membrane and 

the virion envelope.47 The result of this fusion is the 

release of the filovirus ribonucleoprotein (RNP) complex into the cytosol, where filovirus replication occurs.

At the core of the RNP complex is a helical polymer 

of NPs that serves as a scaffold for the filovirus genome 

and VP40 and VP24, which wrap around the helix.21–23

The functional polymerase complex, which is part of 

the RNP, consists of filoviral L, VP35, and filovirusunique VP30, and is bound to the filoviral genome.48,49

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Upon release into the cytosol, these complexes move 

along the filoviral genome in the infected cell and 

transcribe the six/seven filoviral genes into polyadenylated typically monocistronic mRNAs that are then 

translated, or replicate the entire NP-encapsidated 

genome into encapsidated antigenomes and back into 

encapsidated progeny genomes.50,51 VP40 and VP24, 

which regulate these two processes,52 also regulate 

virion morphogenesis in the cell by recruiting newly 

formed RNPs and play major parts in the filovirion 

budding process from host cell membranes. Filovirions bud through endosomal multivesicular bodies 

followed by exocytic release or via direct budding 

through the plasma membrane at membrane/lipid 

rafts.53–57 GP1,2 is expressed and proteolytically cleaved 

into its two subunits (GP1 and GP2) during transport 

through the secretory pathway of the infected cell, 

and trimers of GP1-GP2 heterodimers are transported 

to and inserted into host cell membranes.58,59 Budding filovirions, which acquire their envelopes from 

the host cell membrane during egress, therefore also 

acquire the inserted GP1,2, which are the spikes seen on 

the filovirion surface in electron microscopy sections. 

Geographic Distribution

The still undefined geographic distribution of filoviruses in nature is deduced from natural host reservoir studies, epizootiology, epidemiology, serological 

surveys, and ecological niche modeling.2

Natural Reservoirs of Filoviruses

Although numerous studies were performed,2

the natural host(s) for BDBV, EBOV, LLOV, RESTV, 

SUDV, and TAFV remain elusive. MARV and RAVV 

are the only filoviruses for which at least one natural 

TABLE 23-2 

FILOVIRUS DISEASE CLASSIFICATION AND 

NOMENCLATURE

ICD-10 (1990–Present) Informal Designations

A98.3: Marburg virus disease Marburg hemorrhagic

(MVD) fever (MHF)

Caused by:

Marburg virus (MARV)

Ravn virus (RAVV)

A98.4: Ebola virus disease Ebola hemorrhagic fever 

(EVD) (EHF)

Caused by:

Bundibugyo virus (BDBV)

Ebola virus (EBOV)

Sudan virus (SUDV)

Taï Forest virus (TAFV)

See Figure 23-1 for color explanations. 

ICD-10: International Statistical Classification of Diseases and 

Related Health Problems, Tenth Revision

Data source: World Health Organization. International Statistical Classification of Diseases and Related Health Problems, Tenth 

Revision (ICD-10). http://apps.who.int/classifications/icd10/

browse/2015/en. Accessed September 22, 2015.

Figure 23-2. Filovirion structure. (a) Colorized scanning electron micrograph of a single filamentous Ebola virion (original 

magnification × 100,000). (b) Colorized scanning electron micrograph of filamentous Ebola virions (red) budding from a 

chronically infected grivet (Vero E6) cell (blue) (original magnification × 35,000). 

Photographs: Courtesy of John Bernbaum and Jiro Wada, Integrated Research Facility at Fort Detrick, Maryland.

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Filoviruses

host has been unambiguously identified. Both viruses 

could be isolated repeatedly from several wild and 

seemingly healthy Egyptian rousettes (cavernicolous 

and frugivorous pteropodid bats of the species Rousettus aegyptiacus) inhabiting Kitaka Cave and Python 

Cave in Uganda.60,61 A few human MVD cases were 

recorded among visitors of these caves.60,61 Studies 

suggest that low-level transmission of both viruses 

among these bats occurs throughout the year with 

peaks of infection in older juveniles.60 Experimental 

infections of Egyptian rousettes demonstrated their 

capacity for oral shedding of MARV,62 suggesting that 

half-eaten and thereby contaminated fruit could be 

part of a bat–human transmission route. However, 

Egyptian rousettes are widely distributed across 

sub-Saharan and Northern Africa and Western and 

Southern Asia in colonies reaching up to 50,000 bats. 

Consequently, it is puzzling why MVD outbreaks 

among humans are rare events that seem to be confined to a few geographic zones of Africa.63

Only a loose association with bats is indicated in the 

cases of EBOV and RESTV. For instance, anti-EBOV 

immunoglobulin G antibodies or extremely short (≈300 

nt) EBOV genomic fragments were detected in individual bats of several pteropodid species collected in 

Gabon, Ghana, and Republic of the Congo, but never 

both at the same time.64–67 Anti-RESTV immunoglobulin G was detected in pteropodid bats sampled in the 

Philippines.68 However, neither replicating isolates nor 

coding-complete genomes have yet been recovered 

from any bat, which is puzzling given that filoviruses 

generally replicate to high titers in standard cell cultures.63 Potentially, these bats have only been exposed 

to, rather than infected with, EBOV/RESTV by a yet 

unidentified host. Finally, any connection to healthy 

bats is lacking for BDBV, LLOV, SUDV, and TAFV.63

Figure 23-3. Filovirus genome organization. All filovirus genomes have the same overall sequence of genes (rectangles) and 

open reading frames (horizontal arrows), but differ from each other in the number and position of gene overlaps (triangles) and 

intergenic regions. Cuevaviruses and ebolaviruses differ from marburgviruses in that their GP genes contain three—rather 

than one—open reading frames that are accessible through transcriptional editing. Cuevaviruses differ from ebolaviruses 

and marburgviruses in that VP24 and L open reading frames are transcribed from a single bicistronic transcript. Genomes 

are drawn to scale; waved lines indicate incomplete sequencing of 3’ and 5’ leader and trailer sequences. See Figure 23-1 for 

color explanations. ORF: open reading frame

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TABLE 23-3 

FUNCTION OF FILOVIRAL PROTEINS

Encoding

Protein Gene Protein Characteristics Protein Function

Nucleoprotein (NP)1–8 NP Second-most abundant protein in Major RNP component; nucleocapsid and

infected cells and in virions; consists of cellular inclusion body formation; 

two distinct functional modules; encapsidation of filovirus genome and

homooligomerizes to form helical antigenome; genome replication and

polymers; binds to genomic and transcription

antigenomic RNA, VP35, VP40, VP30, 

and VP24; phosphorylated; depending 

on filovirus, O-glycosylated and/or 

 sialylated

Polymerase cofactor = VP35 Homooligomer; phosphorylated; binds RNP component; Replicase-transcriptase

viral protein 35 to double-stranded RNA, NP, and L cofactor; inhibits innate immune response

(VP35)2,9–23 by interfering with MDA-5 and RIG-1 

pathways, IRF-3 and IRF-7, and the RNAi 

 pathway

Matrix protein = viral VP40 Most abundant protein in infected cells Matrix component; regulation of genome

protein 40 and in virions; consists of two distinct transcription and replication; regulation

(VP40)11,12,24–26,27–42 functional modules; homooligomerizes of virion morphogenesis and egress; 

to form dimers and circular hexamers sequence determines filovirus

and octamers; binds single-stranded pathogenicity in rodents.

RNA, α-tubulin, VP35; hydrophobic; Marburgviruses only: inhibits innate

membrane-associated; contains one immune response by JAK1 signaling

(marburgviruses) or three 

(cuevaviruses and ebolaviruses) latebudding motifs; binds NEDD4 and 

Tsg101; ubiquitinylated

Cuevaviruses and GP Mostly nonstructural; secreted as a Unknown. Hypothesized to be an

ebolaviruses only: parallel homodimer in high amounts antibody-decoy and antiinflammatory

secreted from infected cells; N-glycosylated, agent

glycoprotein C-mannosylated, sialylated

(sGP)43–46 

Glycoprotein GP Type 1 transmembrane and class I fusion Virion adsorption to filovirus-susceptible

(GP1,2)

47–60 protein; cleaved to GP1 and GP2 cells via cellular attachment factors;

subunits that heterodimerize; mature determines filovirus cell and tissue

protein is a trimer of GP1,2 tropism; induction of virus-cell

heterodimers; inserts into membranes; membrane fusion subsequent to

 heavily N- and O-glycosylated, endolysosomal binding to NPC1; inhibits

acylated, phosphorylated. Tumor innate immune response by interfering

necrosis factor α-converting enzyme with tetherin. Function of GP1,2Δ is

(TACE) converts GP1,2 into a soluble unknown

form (GP1,2Δ) 

Cuevaviruses and GP Nonstructural; secreted as a glycosylated Unknown

ebolaviruses only: monomer

secondary secreted 

glycoprotein (ssGP)61 

Cuevaviruses and GP Nonstructural; secreted; largely Unknown. Hypothesized to act as a

ebolaviruses only: unstructured; O-glycosylated and suppressor of filoviral superinfection

Δ-peptide62–64 sialylated and/or as a viroporin

Transcriptional VP30 Hexameric zinc finger protein; binds RNP component

activator = viral single-stranded RNA, NP, and L; Cuevaviruses and ebolaviruses only:

protein 30 (VP30)65–75 phosphorylated transcription initiation, reinitiation, and

 antitermination

(Table 23-3 continues)

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Secondary matrix VP24 Homotetramerizes; hydrophobic and Matrix component; regulation of genome

protein = viral membrane-associated transcription and replication; regulation

protein 24 of virion morphogenesis and egress;

(VP24)66,68,76–86 sequence determines filovirus

pathogenicity in rodents

Cuevaviruses and ebolaviruses only: 

Blocks phosphorylation of MAPK and

prevents karyopherin shuttling from 

cytoplasm into the nucleus; inhibits 

host-cell signaling downstream of 

IFN-α/β/γ 

RNA-dependent L Homodimerizes; binds to genomic and RNP component; genome replication and

RNA polymerase = antigenomic RNA, VP35, and VP30; transcription; transcriptional editing

large protein contains ATP-binding sites and a

(L)2,23,67,87–91 cap-1 MTAse domain

ATP: adenosine triphosphate 

IFN: interferon 

IRF: interferon regulatory factor 

JAK1: Janus kinase 1 

MAPK: mitogen-activated protein kinase 

MDA-5: melanoma differentiation-associated protein-5 

MTAse: methyltransferase 

NEDD4: neural precursor cell-expressed, developmentally down-regulated protein 4 

NPC1: Niemann-Pick C1 protein 

RIG-1: retinoic acid-inducible gene-1 

RNA: ribonucleic acid

RNAi: RNA interference 

RNP: ribonucleoprotein complex 

Tsg101: tumor susceptibility gene 101 protein

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its targeting for the inhibition of viral transcription. J Biol Chem. 2014;289:22723–22738. (76) Watt A, Moukambi F, Banadyga 

L, et al. A novel life cycle modeling system for Ebola virus shows a genome length-dependent role of VP24 in virus infectivity. J Virol. 2014;88:10511–10524. (77) Reid SP, Leung LW, Hartman AL, et al. Ebola virus VP24 binds karyopherin alpha1 and 

blocks STAT1 nuclear accumulation. J Virol. 2006;80:5156–5167. (78) Reid SP, Valmas C, Martinez O, Sanchez FM, Basler CF. 

Ebola virus VP24 proteins inhibit the interaction of NPI-1 subfamily karyopherin alpha proteins with activated STAT1. J 

Virol. 2007;81:13469–13477. (79) Mateo M, Reid SP, Leung LW, Basler CF, Volchkov VE. Ebolavirus VP24 binding to karyopherins is required for inhibition of interferon signaling. J Virol. 2010;84:1169–1175. (80) Warren TK, Whitehouse CA, Wells 

J, et al. A single phosphorodiamidate morpholino oligomer targeting VP24 protects rhesus monkeys against lethal Ebola 

virus infection. MBio. 2015;6:e02344–02314. (81) Han Z, Boshra H, Sunyer JO, Zwiers SH, Paragas J, Harty RN. Biochemical 

and functional characterization of the Ebola virus VP24 protein: implications for a role in virus assembly and budding. J 

Virol. 2003;77:1793–1800. (82) Lee MS, Lebeda FJ, Olson MA. Fold prediction of VP24 protein of Ebola and Marburg viruses 

using de novo fragment assembly. J Struct Biol. 2009;167:136–144. (83) Mateo M, Carbonnelle C, Reynard O, et al. VP24 is a 

molecular determinant of Ebola virus virulence in guinea pigs. J Infect Dis. 2011;204(Suppl 3):S1011–S1020. (84) Xu W, Edwards MR, Borek DM, et al. Ebola virus VP24 targets a unique NLS binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated STAT1. Cell Host Microbe. 2014;16:187–200. (85) Zhang AP, Bornholdt ZA, 

Abelson DM, Saphire EO. Crystal structure of Marburg virus VP24. J Virol. 2014;88:5859–5863. (86) Zhang AP, Bornholdt ZA, 

Liu T, et al. The Ebola virus interferon antagonist VP24 directly binds STAT1 and has a novel, pyramidal fold. PLoS Pathog. 

2012;8:e1002550. (87) Sanchez A, Trappier SG, Mahy BW, Peters CJ, Nichol ST. The virion glycoproteins of Ebola viruses are 

encoded in two reading frames and are expressed through transcriptional editing. Proc Natl Acad Sci U S A. 1996;93:3602–3607. 

(88) Volchkov VE, Becker S, Volchkova VA, et al. GP mRNA of Ebola virus is edited by the Ebola virus polymerase and by 

T7 and vaccinia virus polymerases. Virology. 1995;214:421–430. (89) Shabman RS, Hoenen T, Groseth A, et al. An upstream 

open reading frame modulates Ebola virus polymerase translation and virus replication. PLoS Pathog. 2013;9:e1003147. (90) 

Mühlberger E, Sanchez A, Randolf A, et al. The nucleotide sequence of the L gene of Marburg virus, a filovirus: homologies 

with paramyxoviruses and rhabdoviruses. Virology. 1992;187:534–547. (91) Volchkov VE, Volchkova VA, Chepurnov AA, et 

al. Characterization of the L gene and 5’ trailer region of Ebola virus. J Gen Virol. 1999;80(Pt 2):355–362.

Table 23-3 continued

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Epizootiology of Filoviruses

Filoviruses are highly virulent pathogens for 

humans. Experimentally, most of them also cause 

frequently fatal infections in all thus-far studied 

nonhuman primates (NHPs): common marmosets 

(Callithrix jacchus), crab-eating macaques (Macaca 

fascicularis), grivets (Chlorocebus aethiops), hamadryas 

baboons (Papio hamadryas), rhesus monkeys (Macaca 

mulatta), and common squirrel monkeys (Saimiri sciureus). After serial laboratory adaptation of filoviruses, 

rodents such as laboratory mice, guinea pigs (Cavia 

porcellus), and Syrian golden hamsters (Mesocricetus 

auratus) infected with adapted filoviruses can develop 

fatal infections.2,69,70 However, whether filoviruses are 

also natural pathogens for animals other than humans 

remains under discussion. 

Five filoviruses have been loosely associated with 

epizootics. MARV was discovered in 1967 in West 

Germany among sick and dying laboratory workers 

who had used captive grivets imported from Uganda 

for poliomyelitis vaccine development.71 However, it 

was never clarified at what point these monkeys became infected in captivity (Uganda or en route to West 

Germany) or before capture.72 No evidence indicates 

that grivets are infected with filoviruses in the wild. 

RESTV was discovered in 1989 when crab-eating 

macaques coinfected with a simian arterivirus (Arteriviridae: Arterivirus) were imported from the Philippines 

into the United States, fell sick, and died.73 Similar 

epizootics among captive crab-eating macaques imported from the same Philippine facility occurred in the 

United States in 1990 and 1996, and in Italy in 1992.74

In 2008, RESTV was identified in the Philippines in 

captive domestic pigs (Sus scrofa) coinfected with another arterivirus, porcine reproductive and respiratory 

disease syndrome virus. These pigs suffered and died 

from a respiratory and abortion disease.75 It remains 

unclear how RESTV was introduced into these animal 

populations and why only four such introductions 

occurred. Although domestic pigs can be infected 

experimentally with EBOV76,77 and experimentally infected piglets can transmit EBOV directly to cohoused 

crab-eating macaques,78 evidence of natural infection 

of wild suids with filoviruses is lacking. 

Indirect evidence for natural nonhuman animal filovirus infections exists for EBOV and LLOV. In the case of 

EBOV, catastrophic declines of central chimpanzee (Pan 

troglodytes troglodytes) and western lowland gorilla (Gorilla gorilla gorilla) populations correlated with EBOVcaused EVD epidemics in Gabon and in the Republic 

of the Congo.79–81 In addition, duiker (Cephalophus spp) 

populations seem to have been affected at the same time. 

However, supporting evidence of EBOV involvement 

in these epizootics is limited to polymerase chain reaction (PCR)-based detection of short genomic fragments 

and detection of antigen in three central chimpanzees, 

10 western lowland gorillas, and one duiker.81 Replicating EBOV isolates have not yet been obtained, and 

complete or coding-complete EBOV genomes have yet 

to be detected to directly prove animal infection and 

possibly a link between human and animal disease. 

LLOV, however, was discovered in wild animals. 

A coding-complete LLOV genome was assembled 

from tissues taken from insectivorous Schreibers’ 

long-fingered bats (Miniopterus schreibersii). These bats 

were among hundreds that died of an unknown cause 

in 2002 in Cueva del Lloviu in Spain.82 However, in the 

absence of a replicating LLOV isolate, determining 

whether LLOV caused the bat fatalities or whether 

the bats were infected subclinically with the virus and 

died of different causes is impossible.

The only direct evidence for filovirus infection of 

animals in the wild exists for TAFV. In 1994, a viral 

hemorrhagic fever-like epizootic killed most members 

of a wild western chimpanzee (Pan troglodytes verus) 

community in Taï National Park in Côte d’Ivoire

(Western Africa).83 A female ethologist accidentally 

infected herself with the viral-hemorrhagic-fevercausing pathogen while performing necropsies on the 

deceased animals. TAFV was isolated from clinical 

material, and serological testing demonstrated that 

western chimpanzees were infected with the same 

agent.84 It is unclear, however, how the chimpanzees 

became infected and whether such infections are common or unusual events. 

Epidemiology of Filovirus Infections 

Filoviruses were discovered in 1967 in West 

Germany.71 Since then, 37 human EVD and MVD 

outbreaks have been recorded (Figure 23-4).85 The 

incidence of MVD and EVD apparently has continued to increase over the years, but this increase 

may simply result from improved surveillance and 

reporting. Statistical support that any ebolavirus is 

more virulent than another is scant, although based 

on current case numbers, MVD appears to be more 

lethal compared to EVD (Figure 23-4). Close to all 

of the 37 filovirus disease outbreaks occurred in

Middle/Eastern Africa. Interestingly, “hot spots” 

for filovirus disease outbreaks seem to exist. For 

instance, EBOV reappears continuously in Gabon, 

Republic of the Congo, and western Democratic 

Republic of the Congo; whereas BDBV, SUDV, and 

MARV caused repeated outbreaks in the northeastern Democratic Republic of the Congo, southern 

South Sudan, and Uganda (Figure 23-5). 

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Figure 23-4. Ebola and Marburg virus disease outbreaks. Ebola virus disease and Marburg virus disease outbreaks are listed 

chronologically by virus (colored vertically on the left). International case exportations are pointed out by arrows; proven laboratory 

infections are highlighted in gray and italics. Total case numbers and total number of fatalities are itemized for each outbreak in 

the utmost right columns (updated from data sources 1 and 2). The lethality/case fatality rate (dots) for each outbreak is plotted 

in the middle column on a 0% to 100% scale along with 99% confidence intervals (gray horizontal bars). The average lethality of a 

particular virus or virus group is shown by vertical lines (99% confidence intervals are emphasized by dashed lines). The vertical 

line showing the average lethality of all Ebola virus disease outbreaks overlaps with the vertical line showing the average lethality 

of all filovirus disease outbreaks and the vertical line showing the average lethality of all disease outbreaks caused only by Ebola 

virus (red). At the time of this writing, the 2013–2015 Ebola virus-caused Ebola virus disease outbreak in Western Africa has not 

been brought under control. Consequently, the case and fatality numbers are still subject to change and lethality should rather be 

regarded as a proportion of fatal cases than lethality until final numbers become available. See Figure 23-1 for color explanations.

COD: Democratic Republic of the Congo; COG: Republic of the Congo; UK: United Kingdom; USSR: Union of Soviet Socialist 

Republics. 

Data sources: (1) Kuhn JH. Filoviruses: A Compendium of 40 Years of Epidemiological, Clinical, and Laboratory Studies. In: Calisher CH, 

ed. Archives of Virology Supplementa Series, Vol 20. Vienna, Austria: Springer-Verlag Wien; 2008. (2) Kuhn JH, Dodd LE, Wahl-Jensen 

V, Radoshitzky SR, Bavari S, Jahrling PB. Evaluation of perceived threat differences posed by filovirus variants. Biosecur Bioterror. 

2011;9:361–371. (2) Kuhn JH. Ebolavirus and Marburgvirus infections. In: Kasper DL, Fauci AS, Hauser SL, Longo DL, Jameson JL, 

Loscalzo J, eds. Harrison’s Principles of Internal Medicine. Vol 2. 19th ed. Columbus, OH: McGraw-Hill Education; 2015:1323–1329.

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Figure 23-5. Ebola and Marburg virus disease outbreaks. Middle/equatorial African countries affected by Ebola virus disease 

and/or Marburg virus disease outbreaks are shown in light brown with outbreak locations marked as dots colored according 

to the etiological filovirus (updated from data sources 1 and 2). Arrows mark international case exportation. Former country 

names are listed in parentheses under the present name. 

COD: Democratic Republic of the Congo; COG: Republic of the Congo. 

Data sources: (1) Kuhn JH. Filoviruses: A Compendium of 40 Years of Epidemiological, Clinical, and Laboratory Studies. In: Calisher 

CH, ed. Archives of Virology Supplementa Series, Vol 20. Vienna, Austria: Springer-Verlag Wien; 2008. (2) Kuhn JH, Dodd LE, 

Wahl-Jensen V, Radoshitzky SR, Bavari S, Jahrling PB. Evaluation of perceived threat differences posed by filovirus variants. Biosecur Bioterror. 2011;9:361–371. (3) Kuhn JH. Ebolavirus and Marburgvirus infections. In: Kasper DL, Fauci AS, Hauser 

SL, Longo DL, Jameson JL, Loscalzo J, eds. Harrison’s Principles of Internal Medicine. Vol 2. 19th ed. Columbus, OH: McGraw-Hill 

Education; 2015:1323–1329.

Almost all filovirus disease outbreaks began with 

a single introduction of a filovirus into an index case 

who subsequently transmitted the infection to other 

humans. Thus, initial human filovirus infections are 

extremely rare events and occurred probably less than 

50 times since 1967.2

 In general, past filovirus disease 

outbreaks occurred in rural and often secluded areas 

and affected only several dozens to a few hundred 

people.2

 However, a few outbreaks occurred in populated areas, such as the 1995 EVD outbreak caused 

by EBOV in Kikwit (Zaire) and the 1998–2000 MVD 

outbreaks from MARV and RAVV around Durba and 

Watsa, Democratic Republic of the Congo (the former 

Zaire). This pattern shifted dramatically in December 

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2013 when the largest EVD outbreak began in Western 

Africa from a single introduction of EBOV.86,87 As of 

October 11, 2015, this outbreak has thus far caused 

28,490 cases and 11,312 deaths in Guinea, Liberia, 

Mali, Nigeria, Senegal, and Sierra Leone (Figure 23-4). 

Serological Surveys

Numerous serological surveys for antibodies 

against filoviral antigens have been performed in 

human and animal populations to further define 

the geographic spread of filoviruses and to better 

estimate risk of infection.2 However, results of most 

of these surveys are puzzling. In some surveys, the 

seroprevalence of anti-EBOV antibodies is extremely 

high (>5%–20%) in humans indicating frequent 

exposure to EBOV or related agents in the absence of 

disease. In other surveys, the seroprevalence of antifilovirus antibodies is moderate among humans living 

in areas that never had filovirus disease outbreaks (eg, 

certain African countries, Belarus, Germany, Ukraine). 

Many of these studies used MARV, EBOV, or SUDV 

antigens in indirect immunofluorescence assays (IFAs), 

which are subjective and thus difficult to interpret. IFA 

serosurveys are therefore regarded as presumptive 

by most experts. Modern serosurveys rely on the use 

of enzyme-linked immunosorbent assays (ELISAs) in 

conjunction with confirmatory western blot for the 

detection of antifilovirus antibodies. Few such studies 

were published, and results of these studies most often 

did not confirm IFA results.2

Overall, three disparate possibilities arise from the 

performed serosurveys (IFA and/or ELISA). First, all 

obtained results may be artifacts based on common 

nonfilovirus antibodies in human sera that are crossreactive with the used filoviral antigens, thereby 

leading to false-positive results. Second, filoviruses 

could subclinically infect humans or cause only mild 

disease, thereby leading to high seropositivity rates. 

Current data on the possibility of such infections are 

scarce88,89 and hotly debated, but the currently ongoing EVD outbreak in Western Africa may reveal subclinical infections resulting from the sheer number of 

recorded infections. Third, the discovery of LLOV in 

Spain82 indicates the possibility that filovirus diversity 

and geographic distribution is broader than currently 

appreciated. Perhaps contact with possibly nonpathogenic filoviruses (eg, LLOV- or RESTV-related viruses) 

induces antibodies that are cross-reactive with closely 

related filoviral antigens. Without convincing data for 

any of these possibilities, serosurvey data should not 

be ignored, but they should be used with caution for 

prediction of filovirus distribution or infection risk 

assessments.

Environmental Niche Modeling

Environmental niche modeling (ie, the use of 

algorithms to predict the geographic distribution 

of organisms on the basis of their environmental 

distribution using meteorological and other data) 

indicates succinct distributions for filoviruses in the 

Afrotropic ecozone.90–95 According to these models, 

ebolaviruses are endemic in humid rain forests in 

Western and Middle Africa and South-Eastern Asia, 

whereas marburgviruses circulate in caves located 

in arid woodlands in Middle, Eastern, and Southern 

Africa.90,91 Filovirus emergence in human populations 

appears to be associated with the appearance of climate 

anomalies or drastic climate changes.92 For instance, 

ebolavirus activity is suggested to be correlated with 

unusually heavy rainfalls subsequent to extended dry 

periods.90,94,95 If these models prove correct, then filovirus disease outbreaks should be expected in numerous 

African countries that have thus far not experienced 

(or noticed) any outbreaks.93

TRANSMISSION

As the natural reservoir hosts for most filoviruses 

are unknown, how filoviruses are introduced into the 

human population is unclear. Researchers are tempted 

to speculate that initial infections occur after direct contact with tissues, secretions, or excretions of an animal 

or after a bite or sting.96,97 Even in the case of human 

infections in Ugandan caves that harbored MARV- and 

RAVV-infested Egyptian rousettes,60,61 it remains to 

be explained how these few people became infected, 

and why many others who visited these caves did not.

Human-to-human spread of filovirus infections is 

better understood. Epidemiological studies clearly 

demonstrate that filovirus transmission almost exclusively occurs through direct person-to-person contact 

or through direct contact with filovirus-contaminated 

material.2,98,99 Airborne spread has not been demonstrated for any filovirus during a natural outbreak, 

although healthcare workers risk infection during 

artificial aerosol creation performed as part of medical procedures such as centrifugation of samples, intubation of patients, or suction used during surgical 

procedures.100,101

Filoviruses replicate in humans to high titers (>106

plaque-forming units/mL) and at least in the case of 

EBOV, vast quantities of antigen deposit in the skin 

and around skin appendages.102 In animal models, the 

LD50 of EBOV has been estimated to be as low as 1 

plaque-forming unit.103 As a consequence, filoviruses 

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Medical Aspects of Biological Warfare 

are highly infectious and readily contagious through 

close contact with skin or mucous membranes, especially in the presence of small lesions.104–107 Filoviruses 

or filovirus RNA may be present in genital, nasal, and 

other bodily secretions. Transmission appears to be 

a rare event during the early, asymptomatic phase 

of disease.100,101 However, in the absence of personal 

protective equipment (PPE; disposable gowns, gloves, 

shoe covers, face-shields, or goggles), transmission 

occurs readily. Transmission is typical between sick 

people and their family members, friends, or healthcare workers who care for them; between deceased 

people and people who prepare bodies for funerals; 

and between medical personnel who handle medical samples, contaminated medical equipment, or 

decontamination. A second important transmission 

pathway is nosocomial spread through contaminated 

and reused disposable needles and syringes that, 

unfortunately, is still common in many chronically 

underfunded and therefore underequipped African 

hospitals.100,101,104,108–111 Implementation of quarantine 

measures and use of proper PPE usually suffice to 

interrupt human-to-human transmission and to terminate outbreaks.100,101,105,109,110,112

At this time, the question remains whether filoviruses truly adapt to the human host during prolonged 

interhuman transmission, and whether such adaptation could result in the natural selection of variants 

that is either more or less transmissible or more or less 

virulent. A recent study performed during the 2013–

2015 EVD outbreak in Africa indicates that mutations 

accumulate and particular subpopulations of EBOV 

arise during transmission,87 but these subpopulations 

have not been associated with particular phenotypes. 

THREAT TO THE WARFIGHTER

The warfighter could potentially be at risk of filovirus infection during humanitarian deployment, military campaigns, or war. Exposure to filoviruses could 

occur coincidentally through contact with unknown 

filovirus reservoir(s) and accidentally through exposure to infected people, deceased patients, or materials 

contaminated with human secretions or excretions. In 

addition, the warfighter may be exposed deliberately 

during an attack with biological weapons deployed 

by terrorists, hostile groups, or nation states.113 Coincidental and accidental risks can be dramatically 

reduced for the warfighter if common sense practices 

for tourists and standard operating procedures for 

healthcare workers are implemented. 

Filoviruses and the Soviet Biological Warfare Program

The Soviet Union maintained a highly clandestine 

biological weapons program from at least 1918 until 

at least 1991.114,115 In 1999, a published account from 

a high-ranking defector of the civilian “Biopreparat” 

arm of this program revealed that two filoviruses, 

EBOV and MARV, were included in the program.115

Additional revelations about the biological weapons 

program are scarce. Consequently, knowledge of the 

scope, goals, and achievements of especially the second generation of the program (1972–1991) is deduced 

from accounts from several additional defectors and 

a few researchers who were previously involved and 

legally left the Soviet Union/Russia, as well as from a 

few leaked classified reports or memos.114

Classified filovirus research probably began in the 

Soviet Union shortly after the discovery of MARV in 

1967 in Marburg and Frankfurt, West Germany, and 

Belgrade, Yugoslavia. West German and Yugoslavian 

scientists provided several isolates of the novel virus 

(most notably MARV Popp) to numerous international 

institutes for characterization studies to counter allegations that West Germany had developed a biological 

weapon. Among these institutes was the Union of Soviet Socialist Republics Academy of Medical Sciences 

Scientific Research Institute of Poliomyelitis and Viral 

Encephalitides (now named the M.P. Chumakov Institute of Poliomyelitis and Viral Encephalitides of the 

Russian Academy of Medical Sciences) in Moscow.114,116

Unclassified, nonmilitary-related research began at the 

institute immediately and resulted in a few published 

reports in Russian from 1968 to 1972.2 Current thinking 

is that filovirus research became classified thereafter 

and soon was abandoned at that institute after MARV 

cultures were transferred to the main military virology 

institute, the Scientific Research Institute of Sanitation 

of the Union of Soviet Socialist Republics Ministry of 

Defense in Zagorsk (now named the Virology Center 

under the Scientific-Research Institute of Microbiology 

in the renamed city of Sergiev Posad) close to Moscow.114

The Institute of Tropical Medicine in Antwerp, 

Belgium, which had received MARV during the 1967 

MVD outbreak and EBOV during the 1976 EVD outbreaks, provided MARV isolate Voege and an EBOV 

Yambuku isolate in the mid-1980s within a standard 

collaboration for diagnostics development to the Belorussian Scientific-Research Institute for Epidemiology 

and Microbiology of the Belorussian SSR Ministry 

of Health (now named the Republican Research and 

Practical Center for Epidemiology and Microbiology) 

in Minsk. Although the institute in Minsk continued 

international collaboration and published manuscripts 

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Filoviruses

using MARV and EBOV, both viruses were most 

likely also transported to the institute in Zagorsk. 

Probably from Zagorsk, MARV Popp and the EBOV 

Yambuku isolates were transferred to the highly secretive Scientific-Production Association “Vector” 

(now named the State Scientific Center for Virology 

and Biotechnology “Vector”) in the closed settlement 

of Kolcovo close to Novosibirsk. MARV Voege and 

the same EBOV isolate were also transferred to the 

Scientific-Research Anti-Plague Institute for Siberia and 

the Far East in Irkutsk (Figure 23-6).114 Judging from 

Russian publications released in the mid-1990s, the 

three institutes in Irkutsk, Kolcovo, and Zagorsk made 

significant progress in basic research in terms of EBOV 

Figure 23-6. Locations of clandestine filovirus research in the Soviet Union. Marburg virus was provided to the Union of 

Soviet Socialist Republics Academy of Medical Sciences Scientific Research Institute of Poliomyelitis and Viral Encephalitides in Moscow by West German and Yugoslavian scientists and then transported to the Scientific-Research Institute of 

Sanitation of the Union of Soviet Socialist Republics Ministry of Defense in Zagorsk. Belgian scientists provided Marburg 

virus and Ebola virus to the Belorussian Scientific-Research Institute for Epidemiology and Microbiology of the Belorussian 

SSR Ministry of Health, which also forwarded cultures to Zagorsk. From there, cultures were transferred to the ScientificProduction Association “Vector” in Kolcovo and the Scientific-Research Anti-Plague Institute for Siberia and the Far East in 

Irkutsk. Major offensive research and development most likely occurred in Zagorsk and Kolcovo at least until 1991. 

Data sources: (1) Zilinskas RA, Leitenberg M, with Kuhn J. The Soviet Biological Weapons Program: A History. Cambridge, MA: 

Harvard University Press; 2012. (2) Alibek K, Handelman S. Biohazard—The Chilling True Story of the Largest Covert Biological 

Weapons Program in the World—Told from Inside by the Man Who Ran It. New York, NY: Random House; 1999. (3) Zilinskas 

RA. The anti-plague system and the Soviet biological warfare program. Crit Rev Microbiol. 2006;32:47–64.

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and MARV genomic sequencing, established ELISAand PCR-based diagnostics, and developed parenteral 

and aerosol rodent and NHP MVD/EVD models.114

Little is known about bona fide offensive research 

and development efforts. Based on current data, 

EBOV research apparently did not progress beyond 

the research stage because of production problems.114

MARV, however, was specified in the Soviet 11th 

five-year plan (1981–1985) to be weaponized.114

Weaponization efforts probably began in 1983 at the 

Scientific-Production Association or “Vector,” focusing 

on characterization of the pathogen, high-titer production in rodents and later in tissue bioreactors, and 

production of dried and milled formulations. Finally, 

these formulations were tested in aerosol experiments 

using animals around 1991. However, most likely few 

or none of the formulations reached the validation 

stage, and type-classified weapons (ie, MARV-loaded 

weapons that had been tested and succeeded in openair testing) were not developed.114

The extensive effort within the Soviet biological 

weapons program and the ultimate failure to produce a reliable weapon indicate that the risk of attack with a biological weapon constructed to spread 

filoviruses is relatively low, but not negligible. 

Although filoviruses are not naturally airborne and 

transmission from person-to-person is negligible 

without direct contact, the Soviet program suggests 

that these hurdles are thought by some not to be 

unsurmountable. However, a large-scale attack on 

civilians or armed forces with filoviruses seems unlikely and possible only by nation states rather than 

by small adversary groups. Such groups could—in 

theory—attempt to introduce filoviruses into human populations by means other than weaponry 

(eg, direct injection with needles; self-infection) 

to induce panic and thereby affect the economy of 

target populations.113,117 Therefore, educating the 

general public about filoviruses is vital to reduce 

the psychological impact of such an attack.

PREVENTION

Behavioral Modification

Prevention of initial introduction of filoviruses 

into human or other animal populations is difficult 

to impossible as long as the ecology of the viruses 

is not understood and their natural host reservoirs 

remain unknown. However, general “good infection 

control behaviors” should be encouraged to minimize 

the risk of initial infection. Such control behaviors 

include the avoidance of direct contact with wild 

animals; consumption of uncooked or undercooked 

wildlife; and unprotected exposure to animal excretions, secretions, fluids, or tissues. Control behaviors 

further include consuming water that has been boiled, 

reducing contact with arthropods (eg, application of 

insect repellents, using mosquito nets, screening for 

ticks), avoiding contact with obviously sick people, 

and avoiding unprotected sex. During a filovirus 

disease outbreak, locals should be educated about 

the nature of filoviruses. Certain cultural practices, 

such as handshaking, or particular funeral rituals, 

such as ritual hand washing or embalming of bodies, 

should either be strongly discouraged or modified to 

decrease filovirus transmission risk.118–121 Quarantine 

of infected people and avoidance of direct person-toperson contact generally suffices to prevent further 

spread. Healthcare and other staff should don proper 

PPE before handling patients or suspected cases of 

filovirus infection, clinical samples, or potentially 

contaminated material. Strict implementation of barrier nursing techniques in patient care is also vital. 

N-95/N-100 and positive air pressure respirators, if 

available, should be used especially during clinical 

procedures that may generate aerosols. However, 

users should be aware that positive air pressure 

respirators may induce fear, especially among local 

populations. As fomites are an important route of 

filovirus transmission, reuse of medical equipment 

should be avoided whenever possible. At all times, 

disposables should be used only once and promptly 

discarded.100,112–127

Filovirus Inactivation and Decontamination

Filoviruses produce enveloped virions that contain 

single-stranded RNA genomes.26,27 These virions, 

which are relatively labile, are rapidly inactivated by 

heat, pressure, radiation, or contact with detergents. 

Cheap and commonly available detergents (diethyl 

ether, phenolic compounds, sodium deoxycholate) and 

oxidizing agents, such as bleach or bleaching powder, 

should be used to disinfect surfaces or patient excreta 

or secreta.128–132 However, despite their overall lability, filovirions are stable for several days in liquids 

such as drying blood and on surfaces typically found 

in treatment units for more than 5 days.133 Corpses, 

which may contain infectious filoviruses for extended 

periods of time,134 should be buried quickly. Ideally, 

potentially contaminated disposables are autoclaved, 

irradiated, or burned.

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Vaccines

Despite numerous and diverse efforts,2,135–138 no 

FDA-approved vaccine exists to prevent filovirus 

infections. Candidate vaccines include inactivated 

and attenuated filoviruses, subunit vaccines (adenovirus,139–145 alphavirus,146–150 lyssavirus,151,152 orthopoxvirus,153 paramyxovirus,154–156 and vesiculovirus157–162

vectors expressing filovirus NP, VP35, VP40, GP1,2, 

VP24, VP30, and/or VP24), naked DNA vaccines encoding filovirus proteins (alone or in combination with 

adenovirus-based vectors),140,163–166 and filovirus-like 

particles consisting only of VP40, NP, and GP1,2.

167–170

These candidate vaccines were variably efficacious in 

different animal models. All of these vaccines have 

advantages and disadvantages in regard to safety 

profiles, induction of long-term immune responses, 

or ease of production.2,135–138 In recent years, consensus 

has been reached that only platforms that are highly 

protective in NHP models of filovirus disease should 

be considered for further development.69 Among these 

platforms, the most promising candidate vaccines are 

those that have been built using adenoviral or vesiculoviral backbones or filovirus-like particles (Table 23-4).

DISEASE

EVD and MVD are largely characterized through 

clinical observation of patients in underequipped hospitals,171–178 individual observations of patients who were 

transported to developed countries or suffered from accidental infections,179–183 limited examination of tissues obtained during human outbreaks via biopsies,102,112,184 and 

a very low number of often incomplete autopsies.185–192

Most of the examinations, including biopsies and autopsies, were performed before techniques for characterization of molecular pathogenesis events were available. 

Consequently, a paucity of EVD and MVD biomarkers 

exists.193,194 Given the rarity of EVD and MVD outbreaks, 

disease characterization has therefore depended on 

the use of filovirus-susceptible animals (rodents and 

NHPs). Although frequently referred to as “models” 

of EVD and MVD, the human disease remains largely 

uncharacterized, and animal infections do not necessarily mimic EVD and MVD completely.195 For instance, 

hemorrhagic manifestations, disseminated intravascular 

coagulopathy, bystander apoptosis, and lethality differ 

among the types of animals used as well as humans. 

Wild type filoviruses do not cause disease in rodents 

and therefore require serial adaptation to produce 

disease in these rodents.103,196 Such adaptation has been 

challenging, especially with laboratory mice. Consequently, true mouse models are only available for three 

filoviruses (ie, EBOV, MARV, RAVV).103,197–199 Laboratory mice are frequently used for initial MCM evaluation efforts for many reasons including the following: 

• Ethical concerns are limited about such 

experiments. 

• Mice are easily maintained. 

• Experiments are possible involving large 

numbers of animals. 

• The clonal background of laboratory mice 

simplifies statistical analysis of observed 

MCM effects on infection.200

Golden hamsters are becoming increasingly 

popular as models for EBOV-induced EVD because 

they—in contrast to laboratory mice—develop pronounced coagulatory defects mimicking those seen in 

humans.201 Guinea pigs are typically used as a bridge 

between small rodent (laboratory mice, hamsters) 

and NHP models for infections caused by EBOV and 

MARV.196,202–204 In contrast to laboratory mice, MCM 

evaluation results recorded during guinea pig experiments often translate to similar observations in NHPs. 

In addition, these experiments are less expensive and 

not as logistically challenging as NHP experiments. 

Nevertheless, NHPs are considered the gold standard for MCM evaluation, which is largely a result 

of requirements specified in the US Food and Drug 

Administration’s “Animal Rule” for licensure of candidate vaccines and therapeutics that cannot be tested in 

human clinical trials.69 EBOV and MARV rapidly infect 

crab-eating macaques, grivets, hamadryas baboons, 

rhesus monkeys, and common marmosets and induce 

a usually uniformly lethal disease.2,69,205–210 SUDV and 

RAVV infections can also be studied in crab-eating macaques and rhesus monkeys,207 whereas experiments 

using other NHPs have not been reported. Truly useful 

NHP models for BDBV and TAFV infections have yet 

to be described.

Pathogenesis

GP1,2 embedded in the envelope of filovirions determines cell and therefore tissue tropism of filoviruses 

based on its interaction with cell surface attachment 

factors and the intracellular receptor, NPC1.40 Filoviruses have a broad tropism, that is, the cognate binding 

partners of GP1,2 are expressed on a wide variety of cell 

types41 (in vivo, notable exceptions are lymphocytes, 

myocytes, and neurons20). In addition, cuevaviruses 

and ebolaviruses, but not marburgviruses, produce 

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TABLE 23-4 

SELECTED PROMISING CANDIDATE VACCINES FOR FILOVIRUS INFECTIONS 

Efficacy in

Candidate Vaccine Antigen Nonhuman Primates Additional Information

Filovirus-like virions EBOV-like virion consisting of 100% survival of crab- Nonreplicating protein-based

(VLPs)1 EBOV NP, VP40, and GP1,2 eating macaques vaccine; low safety risks; 

exposed to EBOV clinical-grade materials will 

be required for further 

 development

HPIV-3 vector2 EBOV GP1,2 100% survival of rhesus Replicating, therefore safety

monkeys exposed to concerns; possibly

 EBOV background immunity to 

 vector

Naked DNA + recombinant EBOV GP1,2 + SUDV GP1,2 (in Cross protection; 100% Nonreplicating; possibly

adenovirus 5 (rAD5) individual vectors) survival of crab-eating background immunity to

vector3 macaques exposed to vector; high dose necessary

 BDBV

Naked DNA + recombinant EBOV GP1,2 100% survival of crab- Nonreplicating; possibly

adenovirus 5 (rAD5) eating macaques background immunity to

vector4 exposed to EBOV vector; high dose necessary

RABV vector5 EBOV GP1,2 100% survival of rhesus Replicating, therefore safety

monkeys exposed to may be of concern

 EBOV

RABV vector5 EBOV GP1,2 50% survival of rhesus Nonreplicating

monkeys exposed to 

 EBOV

Recombinant chimpanzee EBOV GP1,2 100% survival of crab- Nonreplicating; high dose

adenovirus 3 vector eating macaques necessary. Phase 1 clinical

(cAD3) 6 exposed to EBOV trials finished

Recombinant human EBOV NP+GP1,2 + MARV NP+ 100% survival of crab- Nonreplicating; possibly

adenovirus 5 vector GP1,2 + RAVV GP1,2 + SUDV eating macaques background immunity to

(CAdVax) 7 GP1,2 (in individual vectors) exposed to EBOV or vector; high dose necessary.

 MARV EBOV-exposed survivors 

also survived later SUDV

 exposure

Recombinant human EBOV GP1,2 100% survival of crab- Nonreplicating; possibly

adenovirus 5 vector eating macaques background immunity to

(rAD5) 8,9 exposed to EBOV vector; high dose necessary. 

Phase 1 clinical trials finished

VEEV vector10 EBOV GP1,2 + SUDV GP1,2 (in 100% survival of crab- Nonreplicating; possibly

individual vectors) eating macaques background immunity to

exposed to EBOV or vector; clinical-grade

 SUDV materials will be required for 

further development.

VEEV vector10 SUDV GP1,2 100% survival of crab- Nonreplicating; possibly

eating macaques background immunity to

exposed to SUDV vector; high dose necessary

Vesicular stomatitis Indiana EBOV GP1,2 100% survival of crab- Replicating, therefore safety

virus (VSIVΔG) vector11,12 eating macaques may be of concern

exposed to EBOV

Vesicular stomatitis Indiana MARV GP1,2 100% survival of crab- Replicating, therefore safety

virus (VSIVΔG) vector11–14 eating macaques may be of concern

exposed to MARV

(Table 23-4 continues)

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Vesicular stomatitis Indiana BDBV GP1,2 100% survival of crab- Replicating, therefore safety

virus (VSIVΔG) vector15 eating macaques may be of concern

exposed to BDBV

Vesicular stomatitis Indiana EBOV GP1,2 75% survival of crab- Replicating, therefore safety

virus (VSIVΔG) vector16 eating macaques may be of concern

exposed to BDBV

Vesicular stomatitis Indiana MARV GP1,2 Cross protection; 100% Replicating, therefore safety

virus (VSIVΔG) vector13 survival of crab-eating may be of concern

macaques exposed to 

 RAVV

Vesicular stomatitis Indiana EBOV GP1,2 + MARV GP1,2 + 100% survival of crab- Replicating, therefore safety

virus (VSIVΔG) vector17 SUDV GP1,2 (in individual eating macaques may be of concern

vectors) exposed to EBOV, 

 MARV, SUDV, or TAFV

Vesicular stomatitis Indiana EBOV GP1,2 + MARV GP1,2 + 100% survival of rhesus Replicating, therefore safety

virus (VSIVΔG) -vector17 SUDV GP1,2 (in individual monkeys exposed to may be of concern

vectors) SUDV

BDBV: Bundibugyo virus

EBOV: Ebola virus

HPIV3: human parainfluenza virus 3

MARV: Marburg virus

RABV: rabies virus

RAVV: Ravn virus

SUDV: Sudan virus

TAFV: Taï Forest virus

VEEV: Venezuelan equine encephalitis virus

Note: Information on the status of all ongoing filovirus-relevant clinical trials can be found at https://ClinicalTrials.gov with 

the search terms “Ebola” or “Marburg.” See Figure 23-1 for color explanations. 

Data sources: (1) Warfield KL, Swenson DL, Olinger GG, Kalina WV, Aman MJ, Bavari S. Ebola virus-like particle-based 

vaccine protects nonhuman primates against lethal Ebola virus challenge. J Infect Dis. 2007;196(Suppl 2):S430–S437. (2) 

Bukreyev A, Rollin PE, Tate MK, et al. Successful topical respiratory tract immunization of primates against Ebola virus. J 

Virol. 2007;81:6379–6388. (3) Hensley LE, Mulangu S, Asiedu C, et al. Demonstration of cross-protective vaccine immunity 

against an emerging pathogenic ebolavirus Species. PLoS Pathog. 2010;6:e1000904. (4) Sullivan NJ, Sanchez A, Rollin PE, 

Yang ZY, Nabel GJ. Development of a preventive vaccine for Ebola virus infection in primates. Nature. 2000;408:605–609. 

(5) Blaney JE, Marzi A, Willet M, et al. Antibody quality and protection from lethal Ebola virus challenge in nonhuman 

primates immunized with rabies virus based bivalent vaccine. PLoS Pathog. 2013;9:e1003389. (6) Stanley DA, Honko AN, 

Asiedu C, et al. Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus 

challenge. Nat Med. 2014;20:1126–1129. (7) Swenson DL, Wang D, Luo M, et al. Vaccine to confer to nonhuman primates 

complete protection against multistrain Ebola and Marburg virus infections. Clin Vaccine Immunol. 2008;15:460–467. (8) 

Sullivan NJ, Geisbert TW, Geisbert JB, et al. Accelerated vaccination for Ebola virus haemorrhagic fever in non-human primates. Nature. 2003;424:681–684. (9) Sullivan NJ, Geisbert TW, Geisbert JB, et al. Immune protection of nonhuman primates 

against Ebola virus with single low-dose adenovirus vectors encoding modified GPs. PLoS Med. 2006;3:e177. (10) Herbert 

AS, Kuehne AI, Barth JF, et al. Venezuelan equine encephalitis virus replicon particle vaccine protects nonhuman primates 

from intramuscular and aerosol challenge with ebolavirus. J Virol. 2013;87:4952–4964. (11) Geisbert TW, Daddario-Dicaprio 

KM, Geisbert JB, et al. Vesicular stomatitis virus-based vaccines protect nonhuman primates against aerosol challenge 

with Ebola and Marburg viruses. Vaccine. 2008;26:6894–6900. (12) Jones SM, Feldmann H, Ströher U, et al. Live attenuated recombinant vaccine protects nonhuman primates against Ebola and Marburg viruses. Nat Med. 2005;11:786–790. 

(13) Daddario-DiCaprio KM, Geisbert TW, Geisbert JB, et al. Cross-protection against Marburg virus strains by using a 

live, attenuated recombinant vaccine. J Virol. 2006;80:9659–9666. (14) Mire CE, Geisbert JB, Agans KN, et al. Durability of 

a vesicular stomatitis virus-based Marburg virus vaccine in nonhuman primates. PLoS ONE. 2014;9:e94355. (15) Mire CE, 

Geisbert JB, Marzi A, Agans KN, Feldmann H, Geisbert TW. Vesicular stomatitis virus-based vaccines protect nonhuman 

primates against Bundibugyo ebolavirus. PLoS Negl Trop Dis. 2013;7:e2600. (16) Falzarano D, Feldmann F, Grolla A, et al. 

Single immunization with a monovalent vesicular stomatitis virus-based vaccine protects nonhuman primates against 

heterologous challenge with Bundibugyo ebolavirus. J Infect Dis. 2011;204(suppl 3):S1082–S1089. (17) Geisbert TW, Geisbert 

JB, Leung A, et al. Single-injection vaccine protects nonhuman primates against infection with Marburg virus and three 

species of Ebola virus. J Virol. 2009;83:7296–7304.

Table 23-4 continued

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the secreted proteins GP1,2Δ, 34 sGP,211 ssGP,33 and 

Δ-peptide.212,213 Although the function of these molecules is unclear, large concentrations of at least sGP 

in the serum of infected animals suggest that they 

might interfere with the host immune response by, for 

instance, serving as decoys for anti-GP1,2 antibodies.211

Once inside the cell, several filovirus proteins actively 

suppress the innate cellular immune response.214 VP35 

protects double-stranded viral RNA intermediates 

produced during genome replication to prevent recognition by the host pattern recognition receptors 

melanoma-differentiation-associated protein-5 and 

retinoic-acid-inducible protein-1. Filovirus VP35 is 

also a powerful host cell RNA silencing suppressor,215

and GP1,2 antagonizes the cellular viral restriction factor tetherin.216 In addition, VP35 inhibits interferon 

response factor 3 and 7 phosphorylation and inhibits 

interferon (IFN) α/β production.217–219 Ebolavirus VP24 

prevents karyopherin shuttling from the cytoplasm 

into the nucleus and thus inhibits host cell signaling 

downstream of IFN-α/β/γ.220–222 In the case of marburgviruses, VP40—but not VP24—interferes with the IFN 

pathway. MARV VP40 inhibits signal transducer and 

activator transcription phosphorylation in response to 

type I and II IFN and interleukin-6 (IL-6).223

The sequence of events during MVD and EVD 

pathogenesis is likely determined by the accessibility 

of susceptible cell types for filovirions, the route of 

infection, and the responses of these cells to infection. 

Sessile and mobile cells of the mononuclear phagocytic 

system (alveolar, peritoneal, pleural macrophages; 

Kupffer cells; microglia) and dendritic cells are initially 

infected.20,102,187,224–229 Filoviruses then spread via the 

lymphatics to regional lymph nodes and via blood to 

the liver, spleen, and other organs.224,228,230

In fundamental ways, macrophages and dendritic 

cells react differently to filovirus infections. Macrophages are activated upon infection and react with the 

release of proinflammatory cytokines, such as IL-1β, 

IL-6, and IL-8, and tumor necrosis factor-α (TNF-α), 

while releasing progeny virions.208,227,231–236 This release 

results in a recruitment of additional macrophages to 

the infection site, resulting in a vicious cycle of infection of additional macrophages. Increasing amounts 

of virions are released and spread through the blood, 

and they are measurable as increased viremia.224 Dendritic cells, however, react with aberrant responses to 

infection. Major histocompatibility complex class II 

is partially suppressed, and expression of tissue factor and the TNF-related apoptosis-inducing ligand is 

increased.237,238

Together, these responses may be the cause of observed death of bystander lymphocytes and general 

lymphoid hypoplasia in lymph nodes, spleen, and 

thymus. Lymphoid hypoplasia with the inhibition 

of IFN pathways at least partially explains the pronounced immunosuppression observed in people with 

fatal infections.239,240

The combined effects of the initial events in filovirus 

infection probably lead to broad organ system dysregulation, exposure of previously shielded filovirussusceptible cells, or the transformation of resistant 

to susceptible cells. Endothelial cells are activated 

through filovirus infection-induced cytokines.241 This 

activation is marked by increased expression and/or 

release of intravascular adhesion molecule-1, vascular 

cell adhesion molecule-1, and E- and P-selectin. Consequently, breakdown of the endothelial barrier function 

results from induced changes in the cadherin/catenin 

composition of adherens junctions in vascular endothelial cells.230,234,242–244 The result of this dysregulation is 

probably massive fluid redistribution (third spacing), 

which is evidenced clinically by widespread edema 

and possibly hypovolemic shock. Later in infection, 

endothelial cells, adrenal cortical cells, epithelial cells, 

reticular fibroblasts, hepatocytes, and reproductive 

cells among others also become directly infected with 

filoviruses, leading to cytolytic infection.20,187,208,224,245–248

The increasing concentrations of proinflammatory 

cytokines (IL-1, IL-6, IL-8, TNF-α, MCP-1, MIP-1α) 

and other mediators, such as tissue factor, probably 

form the basis for the induction of disseminated intravascular coagulation (DIC).249–253 The destruction 

of adrenal cortical cells results in decreased steroid 

synthesis, leading to hypotension and therefore further stasis in blood vessels, which may fuel DIC.208

Consequently, numerous microthrombi form in the 

vascular system and occlude smaller blood vessels 

especially. Hypoxemic infarcts develop in downstream 

tissue, manifested as multiple focal necroses in gonads, 

kidneys, liver, spleen, and other organs. The continuous destruction of the liver, clinically measurable as 

an increase in liver enzyme concentrations, leads to 

a dramatic decrease in albumin and clotting factors. 

Decreased albumin leads to further fluid redistribution 

(edema). DIC ceases once all circulating clotting factors 

have been consumed and leads to petechial rashes, 

ecchymoses, and general uncontrolled (but rarely lifethreatening) hemorrhages.187,246,254,255 Multiple organ 

dysfunction syndrome frequently occurs as a result 

of this series of events.

Clinical Presentation

MVD and EVD cannot be distinguished on grounds 

of clinical observation alone. Based on the few larger cohort studies published, few to no statistically significant 

differences were noted in the onset, duration, frequency, 

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or type of clinical signs and symptoms of MVD and 

EVD caused by the six filoviruses. Clinical presentation is overall dynamic, with most “typical” signs occurring in many—but not all—cases of infection.171–177

The current understanding of human filovirus 

disease is based largely on observations made during 

the MVD and EVD outbreaks resulting from MARV 

infection in Frankfurt/Main and Marburg in West 

Germany in 196771,192,256–259 and in Durba and Watsa in 

the Democratic Republic of the Congo in 1998–2000174; 

EVD outbreaks resulting from EBOV infection around 

Yambuku, Zaire, 1976,260,261 Kikwit, Zaire, 1995,171

Boene (Democratic Republic of the Congo),178 and in 

Western Africa, 2013–2015172,173,262; an EVD outbreak 

resulting from SUDV infection around Yambio, Sudan, 1976263; an EVD outbreak resulting from BDBV 

infection in Bundibugyo, Uganda, 2007121,175 (Table 

23-5); and single-case exportations or laboratory accidents. The incubation period of filovirus disease is 

highly variable (3–25 days, probably dependent on the 

route of infection and the amount of virus transmitted). Phase 1, the prodromic phase of the disease that 

coincides with virema, lasts 5 to 7 days and generally 

resembles influenza. This phase is characterized by a 

sudden onset of fever (>38.6°C) and chills, abdominal 

pain, arthralgia, cough, chest pain and shortness of 

breath, severe headaches, myalgia, pharyngitis, and 

the appearance of a morbilliform/maculopapular rash 

(which, however, may be difficult to see on black skin).

Phase 2 begins after 1 to 2 days of relative remission with a more dramatic clinical presentation including almost all organ systems. Severe abdominal 

pain, vomiting, and watery diarrhea mark the gastrointestinal effects of infection. Confusion, tremors, 

psychosis, and coma demonstrate the involvement of 

the central nervous system. Edema and orthostatic 

hypotension are both primary and secondary effects 

of filovirus replication in a variety of cells in the liver, 

adrenal glands, and vascular system. Induced DIC 

followed by a total lack of coagulative responses 

lead to hemorrhagic manifestations. Hemorrhage, 

which appears in only about 50% of the cases, includes bleeding from mucosal surfaces (gums, nose, 

rectum, vagina) and venipuncture sites, resulting in 

detectable blood in sputum, feces, urine, and vomit. 

Other hemorrhagic manifestations are subconjunctival hemorrhage, petechiae, purpura, and ecchymoses.171,191,233,238,251,256,257,264–267 On palpation, hepatomegaly 

is usually prominent, but jaundice is typically absent. 

Given the filovirus-induced immunosuppression, secondary bacterial and/or fungal infections may develop.

Death is usually the result of multiple organ dysfunction syndrome. Although multiple organ dysfunction syndrome may be caused by fluid redistribution 

(third spacing) and the multiple necroses in organs, 

death resulting from blood loss is extremely rare and 

occurs most frequently among women in labor.268–270

Survival of EVD, which is inversely correlated with 

viremia, is associated with particular immunoglobulin M and immunoglobulin G responses and a strong 

proinflammatory response early in the course of 

disease,249,250,252,271–273 and it is probably influenced 

by host genetics.274 Survivors of MVD and EVD may 

experience a wide variety of long-term sequelae that 

include amnesia, anxiety, joint pain, skin peeling and 

hair loss, fatigue, hepatitis, myalgia, myelitis, ocular 

manifestations (choroiditis, iridocyclitis, iritis, uveitis), 

hearing loss, orchitis, and/or psychosis. At least three 

filoviruses, MARV, EBOV, and SUDV, may induce 

persistent infections in the liver, eye, or gonads beyond reconvalescence, and may later reactivate or be 

transmitted sexually.120,275–280

Typical clinical laboratory parameters of MVD 

and EVD are progressing leukopenia (as low as 

1,000/μl) caused by the loss of lymphocytes with a 

left shift followed by leukocytosis resulting from an 

increase in granulocytes, and mild thrombocytopenia (50,000–100,000/μl). Liver, kidney, and pancreas 

dysfunction are evident in the form of increased concentrations of aspartate aminotransferase, alanine 

aminotransferase, γ-glutamyltransferase, amylase, 

creatinine, and urea, as well as hypokalemia. The 

effect of filovirus infection on the coagulation cascade becomes evident via prolonged prothrombin 

time and partial thromboplastin time and increased 

D-dimer concentrations.84,171–173,189,191,257,261,281–283

DIAGNOSIS

MVD or EVD should be considered in any acutely 

febrile patient who resides or has travelled through a 

filovirus-endemic area. A history of rural travel, expeditions into the rain forest or natural or artificial caves, and 

contact with sick or deceased animals, including humans, 

should raise suspicion. However, as the recent 2013–2015 

EVD outbreak in Western Africa demonstrated, filoviruses may be more broadly distributed than previously 

thought. Thus, the possibility of filovirus infection in a 

febrile patient from an African country without recorded 

filovirus infection should not be discounted. Unfortunately, MVD and EVD patients present with rather unspecific, influenza-like clinical signs caused by numerous 

pathogens that are more commonly encountered. Even 

later stages of MVD and EVD are easily confused with 

the clinical presentation of other diseases (Table 23-6).

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TABLE 23-5

CLINICAL PRESENTATION OF MARBURG VIRUS DISEASE OR EBOLA VIRUS DISEASE 

(ADAPTED AND AVERAGED FROM DATA SOURCES) 

Survivors of Fatal MARV Survivors of Fatal BDBV Survivors of Fatal EBOV

Clinical Signs and MARV Infections BDBV Infections EBOV Infections 

Symptoms in Humans Infection (%) (%) Infection (%) (%) Infection (%) (%)

Abdominal pain 59 57 73 88 27 26

Anorexia/appetite loss 77 72 68 77 

Anuria 13 18 

Arthralgia or myalgia 55 55 74 80 25 51

Asthenia 73 82 13 61

Bleeding from the gums 23 36 9

Bleeding from any site 59 71 29 54 22 47

Bleeding from GI tract 6 19

Chest pain 18 4 13 45 

Confusion/disorientation 27 36 19

Conjunctival injection/ 14 42 47 55 13

conjunctivitis

Cough 9 5 7 36 13 17

Diarrhea 59 56 83 92 27 44

Difficulty breathing/ distress 36 58 18 88 8 23

Dizziness 13 56

Epistaxis 18 34 7 9 

Facial/neck edema 92 82 24

Fever 10 29 78 81 71 74

Headaches 73 79 82 85 57 46

Hematemesis 68 76 18 

Hemoptysis 9 4 9 

Hiccups 18 44 18 40 2 13

Lumbar pain 5 8 5 36 

Maculopapular rash/ rash 25 27

Malaise or fatigue 86 83 56 100

Melena 41 58 27 

Nausea and vomiting 77 76 76 88 59 62

Petechiae 9 7 3

Sore throat, odynophagia, or 43 43 45 50 13 31

dysphagia

See Figure 23-1 for color explanations.

BDBV: Bundibugyo virus

EBOV: Ebola virus

GI: gastrointestinal

MARV: Marburg virus 

Data sources: (1) Bwaka MA, Bonnet MJ, Calain P, et al. Ebola hemorrhagic fever in Kikwit, Democratic Republic of the Congo: 

clinical observations in 103 patients. J Infect Dis. 1999;179(Suppl 1):S1–S7. (2) Bah EI, Lamah MC, Fletcher T, et al. Clinical 

presentation of patients with Ebola virus disease in Conakry, Guinea. N Engl J Med. 2015;372:40–47. (3) Schieffelin JS, Shaffer 

JG, Goba A, et al. Clinical illness and outcomes in patients with Ebola in Sierra Leone. N Engl J Med. 2014;371:2092–2100. (4) 

Bausch DG, Nichol ST, Muyembe-Tamfum JJ, et al. Marburg hemorrhagic fever associated with multiple genetic lineages of 

virus. N Engl J Med. 2006;355:909–919. (5) MacNeil A, Farnon EC, Wamala J, et al. Proportion of deaths and clinical features 

in Bundibugyo Ebola virus infection, Uganda. Emerg Infect Dis. 2010;16:1969–1972. (6) Roddy P, Howard N, Van Kerkhove 

MD, et al. Clinical manifestations and case management of Ebola haemorrhagic fever caused by a newly identified virus 

strain, Bundibugyo, Uganda, 2007–2008. PLoS One. 2012;7:e52986. (7) Barry M, Traore FA, Sako FB, et al. Ebola outbreak in 

Conakry, Guinea: epidemiological, clinical, and outcome features. Med Mal Infect. 2014;44:491–494.

Once filovirus infection is suspected, it is imperative to contact the proper public health authorities and 

infectious disease specialists and to perform all patient 

contact and sample handling with utmost caution.284

Filovirus infection can be confirmed safely and relatively easily in mobile field laboratories, local hospitals, 

and/or reference laboratories as long as the necessary 

technology and trained staff are available. Reverse 

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TABLE 23-6 

MARBURG VIRUS DISEASE AND EBOLA VIRUS DISEASE DIFFERENTIAL DIAGNOSIS 

(ADAPTED FROM DATA SOURCES) 

Viral Bacterial Fungal Parasite Noninfectious

Infections Infections Infections Infections Diseases

Major: fulminant viral Major: Major: Major: falciparum Major: acute promyelocytic

hepatitides; measles; Enterohemorrhagic histoplasmosis malaria leukemia; factor VII, IX,

rubella; VHFs caused Escherichia coli and X deficiencies;

by Lassa virus or enteritis; gram- hemolytic uremic

yellow fever virus negative bacterial syndrome; hereditary

septicemia; hemorrhagic

leptospirosis; murine telangiectasia; Kawasaki

typhus; rickettsial disease; platelet and

diseases; shigellosis; vascular disorders; 

typhoid fever; typhus snake envenomation; 

 thrombotic 

thrombocytopenic 

purpura; warfarin 

intoxication

Minor: chikungunya, Minor: anthrax; Minor: candidiasis Minor: Minor: drug rashes

hepatitis A, B, bartonellosis; trypanosomiasis,

non-A/B; herpes campylobacteriosis; visceral

simplex; influenza; meningococcal leishmaniasis

mononucleosis; septicemia; plague;

Sindbis disease; Pseudomonas

West Nile virus infections; psittacosis

fever; VHFs caused with endocarditis; 

by other viruses Q fever; relapsing 

fever; staphylococcal 

septicemia; 

streptococcal 

septicemia/rheumatic 

fever

VHF: viral hemorrhagic fever

Data sources: (1) Kuhn JH. Filoviruses. A Compendium of 40 Years of Epidemiological, Clinical, and Laboratory Studies. In: Carlisher CH, ed. Archives of Virology Supplementa Series, Vol 20. Vienna, Austria: Springer-Verlag Wien; 2008. (2) Grolla A, 

Lucht A, Dick D, Strong JE, Feldmann H. Laboratory diagnosis of Ebola and Marburg hemorrhagic fever. Bull Soc Pathol 

Exot. 2005;98:205–209. (3) Boisen ML, Schieffelin JS, Goba A, et al. Multiple circulating infections can mimic the early stages 

of viral hemorrhagic fevers and possible human exposure to filoviruses in Sierra Leone prior to the 2014 outbreak. Viral 

Immunol. 2015;28:19–31.

transcriptase-PCR is the method of choice for detection of filovirus genomes (detection limit: ≈1,000–2000 

genome copies/ml of serum) in, for instance, guanidinium isothiocyanate-inactivated samples.285–287 The less 

sensitive antigen-capture ELISA and antibody-capture 

ELISA are alternative or complementary assays for the 

detection of filovirus proteins and anti-filovirus antibodies in 60Co-irradiated samples.288,289 Samples from 

skin biopsies can be inactivated by formalin fixation 

and then used to diagnose filovirus infection using 

immunohistochemistry or in situ hybridization.102,290

Noninactivated samples, such as acute-phase serum 

or blood which typically contain high filovirus titers and 

antifilovirus antibodies,291 must not be handled outside 

of a maximum-containment (biosafety level 4) laboratory. Such samples should be collected with utmost caution using proper PPE and then sent to the appropriate 

World Health Organization reference laboratories using 

suitable transport media. Filoviruses typically grow 

quickly and to high titers in standard cell cultures such 

as grivet Vero E6, rhesus monkey MA-104, or human 

adrenal carcinoma SW-133 cells.292,293 From these infected cells, additional studies can be performed, such as 

variant isolation and typing, sample virus quantification 

by plaque assays, standard (consensus) Sanger genome 

sequencing, and easy visualization of typical shapes of 

filovirions using electron microscopy.294,295 An overview 

of current diagnostic options is provided in Table 23-7.

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TABLE 23-7

LABORATORY DIAGNOSIS OF FILOVIRUS INFECTION (ADAPTED FROM DATA SOURCES) 

PRIMARY ASSAYS

Diagnostic Test Target Clinical Material Advantage Disadvantage

RT-PCR Filovirus Blood, serum, Rapid; ultra-sensitive; specific; Requires PCR machine;

subgenomic, tissue can be performed on laboratory cross-contamination

genomic, or inactivated samples can lead to false-positive results;

antigenomic release of RT-PCR inhibitors from

nucleic acids tissue can lead to false negative 

 results

Antigen-capture Filovirus Blood, serum, Rapid; sensitive; specific; can Requires ELISA reader

ELISA antigen/ tissue be performed on inactivated

proteins samples; high-throughput 

 possible

IgG-capture Antifilovirus Serum Rapid; sensitive; specific; can Requires ELISA reader and large

ELISA antibodies be performed on inactivated amounts of purified native or

(late in samples recombinant filoviral antigen;

infection; some patients do not seroconvert

survivors) 

IgM-capture Antifilovirus Serum Rapid; sensitive; specific; can Requires ELISA reader and large

ELISA antibodies be performed on inactivated amounts of purified native or

(early in samples recombinant filoviral antigen;

infection) some patients do not seroconvert

SECONDARY/CONFIRMATORY ASSAYS

Diagnostic Test Target Source Advantage Disadvantage

Virus isolation Filoviruses Blood, tissue Specific Requires maximum-containment 

 laboratory and time; filovirus 

 isolation may fail or filovirus 

 replication may not cause CPE in 

 cell cultures during initial passages

Electron Complete or Blood, serum, Specific Insensitive; requires electron

microscopy fragmented tissue microscope

filovirions or 

characteristic 

cellular 

inclusion 

bodies 

Indirect Antifilovirus Serum Rapid; simple; safe Insensitive; possible cross 

immuno- antibodies reactions leading to false positive

fluorescent results; subjective interpretation; 

assay some patients do not seroconvert

Fluorescent Filovirus Tissue culture/ Rapid; simple; safe Insensitive; requires infectious

assay antigen/ isolated virus material and specific antibodies;

proteins subjective interpretation

Next-generation Filovirus Blood, serum, Very specific; ultra-sensitive; New and expensive technology;

sequencing subgenomic, tissue can determine coding- not yet widespread; requires

genomic, or complete filovirus genomes highly trained personnel and

antigenomic in absence of virus culture; bioinformatics support

nucleic acids allows molecular epidemiology

Immunohisto- Filoviral Tissue (skin, liver) Tissue can be fixed Requires time and specific

chemistry antigen antibodies

(Table 23-7 continues)

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In situ Filoviral Tissue Tissue can be fixed Requires special equipment and

hybridization nucleic acids specific probes

Western blot Antifilovirus Serum Specific Difficult interpretation; requires

antibodies specific antibodies

CPE: cytopathic effect

ELISA: enzyme-linked immunosorbent assay

IgG: immunoglobulin G

IgM: immunoglobulin M

RT-PCR: reverse transcriptase polymerase chain reaction

Data sources: (1) Kuhn JH. Filoviruses: A Compendium of 40 Years of Epidemiological, Clinical, and Laboratory Studies. In: Calisher 

CH, ed. Archives of Virology Supplementa Series, Vol 20. Vienna, Austria: Springer-Verlag Wien; 2008. (2) Gire SK, Goba A, 

Andersen KG, et al. Genomic surveillance elucidates Ebola virus origin and transmission during the 2014 outbreak. Science. 

2014;345:1369–1372. (3) Grolla A, Lucht A, Dick D, Strong JE, Feldmann H. Laboratory diagnosis of Ebola and Marburg hemorrhagic fever. Bull Soc Pathol Exot. 2005;98:205–209. (4) Wang YP, Zhang XE, Wei HP. Laboratory detection and diagnosis 

of filoviruses. Virol Sin. 2011;26:73–80.

Table 23-7 continued

TREATMENT

Treatment of MVD and EVD patients is challenging 

because of the healthcare workers’ risk of infection. 

Patients should be isolated, infection control precautions/strict barrier-nursing techniques need to be 

implemented, and healthcare personnel must wear 

proper PPE. In addition, standard operating procedures should be in place for safe clinical sample management; decontamination of possibly contaminated 

tools, materials or surface equipment and personnel; 

point-of-care laboratory testing; and infectious waste 

management. 

No FDA-approved specific therapy is available to 

treat human infections. Although statistical reports 

are still lacking, chances for survival from MVD and 

EVD are now thought to be dramatically increased 

through aggressive supportive therapy.262,296,297

Treatment should follow the guidelines for severe 

sepsis management and therefore aim to reestablish 

fluid and electrolyte balance and reversal of DIC, 

hemorrhage, hypotension/hypoperfusion, acute 

kidney injury, and shock. As filoviruses potently 

suppress the immune system, empirical and possibly prophylactic treatment of secondary bacterial 

and/or fungal infections with broad-spectrum antibiotics (eg, vancomycin, piperacillin/tazobactam) 

and antimycotics is advised. Pain management and 

administration of antiemetics should always be 

considered.180,181,262

Numerous drugs have been evaluated in vitro and 

in various animal models over the years to identify candidate MCMs to treat MVD and EVD.298–302 Although 

none of them has reached medical licensure, several 

have been sufficiently promising for emergency use in 

humans. An overview of the most commonly discussed 

MCMs for filovirus disease treatment is provided in 

Table 23-8.

SUMMARY

Due to extremely low human case numbers, filovirus infections were long considered exotic infectious 

diseases of no larger consequence to global public 

health. The still ongoing EVD outbreak in Western 

Africa, which by now includes close to 28,500 cases 

and 11,300 deaths, brought awareness to the fact that 

single filovirus introductions into the human population may lead to devastating and large epidemics 

that can spread quickly across international borders. 

Unfortunately, despite considerable scientific progress, 

many key questions regarding filoviruses remain to 

be answered. First, and foremost, the natural filovirus 

host reservoirs have to be identified so that preventive 

measures against initial zoonotic spillover into humans 

can be established. Second, almost all filovirus research 

currently focuses only on EBOV, SUDV, and MARV, 

while next to nothing is known about the molecular or 

pathogenic features of other filoviruses. Yet, medical 

countermeasures should be created that can be used 

against any human (and possibly) animal infection 

because it is currently unpredictable which filovirus 

will strike next.

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TABLE 23-8 

SELECTED PROMISING CANDIDATE THERAPEUTICS FOR FILOVIRUS INFECTIONS 

Candidate MCM Mechanism of Action Efficacy Additional Information

ZMapp (Mapp Cocktail of three monoclonal 100% survival of rhesus Produced in genetically

Biopharmaceutical, antibodies (c13C6, c2G4, monkeys up to 5 days after modified tobacco plants; may

Inc/LeafBio, Inc)1 c4G7) targeting EBOV GP1,2 EBOV exposure not be scalable; requires frozen

 shipping. Currently in phase 

 1/2 clinical trials 

MB-003 (USAMRIID/ Cocktail of three monoclonal 100% survival of rhesus Produced in genetically

Mapp antibodies (c13C6, h-13F6, monkeys 1 h after EBOV modified tobacco plants;

Biopharmaceutical) 2,3 c6D8) targeting EBOV GP1,2 exposure; 67% and 43% scalable; requires frozen

 survival at 2 days and 4–5 shipping

 days after exposure, 

 respectively

ZMAb (PHAC/Defyrus Cocktail of three monoclonal 100% survival of crab-eating Requires frozen shipping

Inc)4–7 antibodies (1H3, 2G4, 4G7) macaques 1 day after EBOV

targeting EBOV GP1,2 exposure; 50% survival at 2 

 days after exposure. 100% 

 survival of survivors after 

 reexposure to EBOV 10 

 weeks after challenge, 67% 

 survival after 13 weeks

Immucillin-A/BCX4430 Nucleoside analog that inhibits 100% survival of crab-eating In phase 1 clinical trial

(BioCryst the MARV RNA-dependent macaques 48 h after MARV

Pharmaceuticals) 8 RNA polymerase and causes exposure

lethal mutagenesis

Favipiravir/T-705 Nucleotide analog that inhibits 100% survival of IFNAR-/- Used as a licensed antiinfluenza

(Fujifilm/Toyama the filovirus RNA-dependent laboratory mice 6 days drug in Japan. Contraindicated

Chemical Co, Ltd) 9,10 RNA polymerase and causes after parenteral mouse- in pregnancy because of

lethal mutagenesis adapted EBOV exposure; possibility of teratogenicity

 17% survival of rhesus and embryotoxicity. In phase 

 macaques 3 clinical trials (FLUAV). 

 Currently is being evaluated

 on EVD patients in Guinea in 

 a single-arm phase 2 clinical

 trial

JK-05 (Sihuan Nucleotide analog that inhibits Efficacy in laboratory mice Considered for use in emergency

Pharmaceutical the filovirus RNA-dependent situations

Holdings Group, Ltd RNA polymerase and causes 

and Academy of Military lethal mutagenesis

Medical Sciences) 11 

TKM-Ebola/Tekmira- Lipid nanoparticle cocktail of 100% survival of rhesus Phase 1 clinical trial aborted

100802 (Tekmira siRNAs targeting EBOV VP35, monkeys 30–60 min after

Pharmaceuticals VP24, and L EBOV exposure; 83%, 50%, 

Corp) 12,13 and 67% survival at 1, 2, 

 and 3 days after exposure, 

 respectively

“TKM-Marburg” Lipid nanoparticle cocktail of 100% survival of rhesus

(Tekmira siRNAs targeting MARV NP monkeys 30–45 min, 1 day,

Pharmaceuticals Corp) 14 2 days, and 3 days after 

 MARV exposure

AVI-7537 (Sarepta Phosphorodiamidate 63% survival of rhesus Phase 1 clinical trial

Therapeutics) 15 morpholino oligomer monkeys 1 h after EBOV

targeting EBOV VP24 exposure

(Table 23-8 continues)

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AVI-6002 (Sarepta Phosphorodiamidate >60% survival of rhesus Phase 1 clinical trial completed

Therapeutics) 16 morpholino oligomer monkeys 30–60 min after

targeting EBOV VP35 and EBOV exposure

VP24

rVS[I]VΔG-ZEBOV-GP/ Postexposure vaccine 50% survival of rhesus Easy to produce; requires

BPSC1001 (Newlink consisting of a recombinant monkeys 20–30 min after frozen shipping; concerns

Genetics/PHAC) 17 replicating vesicular EBOV exposure; 100% and about immunocompromised

stomatitis Indiana virus 50% survival of laboratory patients. Currently in phase 1

expressing EBOV GP1,2 to mice and guinea pigs, clinical trials

stimulate anti-GP1,2 immune respectively, 24 h after

responses EBOV exposure

Other rVS[I]V Postexposure vaccine MARV: 100% survival of Easy to produce; requires

formulations18–20 consisting of a recombinant rhesus monkeys 20–30 min frozen shipping; concerns

replicating vesicular after MARV exposure; 83% about immunocompromised

stomatitis Indiana virus and 33% survival at 1 day patients

expressing filovirus GP1,2 to and 2 days after exposure,

stimulate anti-GP1,2 immune respectively

responses

SUDV: 100% survival of 

rhesus monkeys 20–30 min 

 after SUDV exposure

Recombinant Nematode Inhibits factor VIIa/tissue 33% survival of rhesus In phase 2 clinical trial for

Anticoagulant Protein c2 factor complex and blood monkeys 10 min and 1 day second-line treatment of

(rNAPc2) 21 clot formation after EBOV exposure metastatic colorectal carcinoma 

in combination with 

contemporary 5-FU-based 

 chemotherapy

Activated drotrecogin Recombinant human 20% survival of rhesus Withdrawn from market

alfa/Xigris (Eli Lilly and activated protein C; inhibits monkeys 1 h after EBOV

Company) 22 coagulation factors Va and exposure

VIIIa (antithrombotic) 

Hyperimmune equine23,24 Filovirions 50%–100% survival of Licensed in Russia for treatment

immunoglobulin G23–25 hamadryas baboons 5–15 of occupational accidents.

min after EBOV exposure; Highly immunogenic in

80%, 20%–100%, and 29% humans. Evaluation in rhesus

survival at 30 min, 1 h, and monkeys (using different dose

2 h after exposure, and virus variant) not

 respectively successful

Passive transfer of Filovirions Passive transfer of Uncontrolled experiment during

convalescent or concentrated polyclonal the 1995 EVD/EBOV outbreak

postimmunization IgG from immune rhesus in Kikwit, Zaire, suggested

plasma26–28 monkeys resulted in 100% passive transfer of whole blood

survival of rhesus monkeys to be protective for 7 of 8

at 15–30 min and 48 h after patients

 MARV exposure

See Figure 23-1 for color explanations.

ADE: adverse effects 

ADV: adenovirus

CMV: cytomegalovirus

EBOV: Ebola virus

EVD: Ebola virus disease

FLUAV: influenza A virus

5-FU: fluorouracil

GP: glycoprotein

IFN: interferon

(Table 23-8 continues)

Table 23-8 continued

IFNAR: interferon-α/β receptor 

IgG: immunoglobulin G

MARV: Marburg virus

MCM: medical countermeasures 

PHAC: Public Health Agency of Canada

siRNA: short interfering RNA 

SUDV: Sudan virus

USAMRIID: US Army Medical Research Institute of Infectious 

Diseases

VP: viral protein

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Note: Information on the status of all ongoing filovirus-relevant clinical trials can be found at https://ClinicalTrials.gov with the search terms 

“Ebola” or “Marburg.”

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2014;514:47–53. (2) Shurtleff AC, Biggins JE, Keeney AE, et al. Standardization of the filovirus plaque assay for use in preclinical studies. 

Viruses. 2012;4:3511–3530. (3) Pettitt J, Zeitlin L, Kim do H, et al. Therapeutic intervention of Ebola virus infection in rhesus macaques with 

the MB-003 monoclonal antibody cocktail. Sci Transl Med. 2013;5:199ra113. (4) Qiu X, Audet J, Wong G, et al. Successful treatment of Ebola 

virus-infected cynomolgus macaques with monoclonal antibodies. Sci Transl Med. 2012;4:138ra181. (5) Audet J, Wong G, Wang H, et al. 

Molecular characterization of the monoclonal antibodies composing ZMAb: a protective cocktail against Ebola virus. Sci Rep. 2014;4:6881.(6) 

Qiu X, Audet J, Wong G, et al. Sustained protection against Ebola virus infection following treatment of infected nonhuman primates with 

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Table 23-8 continued

Acknowledgments

We are grateful for the technical writing services offered by Laura Bollinger (IRF-Frederick) and the support 

by IRF-Frederick’s medical illustrator, Jiro Wada. The content of this publication does not necessarily reflect 

the views or policies of the US Department of Defense, US Department of Health and Human Services, the 

US Department of the Army, or of the institutions and companies affiliated with the authors. This work 

was funded in part through Battelle Memorial Institute’s prime contract with the US National Institute 

of Allergy and Infectious Diseases under Contract No. HHSN272200700016I. A subcontractor to Battelle 

Memorial Institute who performed this work is: JHK, an employee of Tunnell Government Services, Inc.

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Chapter 24

SMALLPOX AND RELATED 

ORTHOPOXVIRUSES

ARTHUR J. GOFF, PhD*; SARA C. JOHNSTON, PhD†; JASON KINDRACHUK, PhD‡; KENNY L. LIN, MS§; PETER B. 

JAHRLING, PhD¥; JOHN W. HUGGINS, PhD¶; M. SOFI IBRAHIM, MSC, PhD**; JAMES V. LAWLER, MPH, MD††; and 

JAMES W. MARTIN, MD, FACP‡‡

INTRODUCTION

AGENT CHARACTERISTICS 

Classification

Morphology

Entry

Replication

Morphogenesis and Egress

Phylogenetic Relationships

Pathogenesis

ORTHOPOXVIRUSES AS BIOLOGICAL WARFARE AND BIOTERRORISM 

THREATS

CLINICAL ASPECTS OF ORTHOPOXVIRUS INFECTIONS 

Smallpox

Monkeypox

Other Orthopoxviruses Infecting Humans

DIAGNOSIS

Clinical Diagnosis

Specimen Collection and Handling

Phenotypic Diagnosis

Immunodiagnosis

Nucleic Acid Diagnosis

MEDICAL MANAGEMENT 

Prophylaxis

Treatment

SUMMARY

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*Research Microbiologist, Department of Virology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

†

Research Microbiologist, Department of Virology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702 

‡

Staff Scientist, Critical Care Medicine Department, Clinical Center, National Institutes of Health, 10 Center Drive, Bethesda, Maryland 20814

§

GLP Study Coordinator, Nonclinical Development Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702; formerly, Regulatory Scientist, Department of Virology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, 

Fort Detrick, Maryland 

¥

Captain (Retired), Medical Service Corps, US Army; Director National Institutes of Health/Division of Clinical Research/National Institute of Allergy and 

Infectious Diseases/Integrated Research Facility, 8200 Research Plaza, Room 1A-111A, Fort Detrick, Maryland 21702; formerly, Principal Scientific Advisor 

(Senior Research Scientist), US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

¶

Retired; formerly, Chief, Viral Therapeutics Branch, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702 

**Lieutenant Colonel, Medical Service Corps, US Army Reserve; Research Microbiologist, Edgewood Chemical Biological Center, Ricketts Point Road, Gunpowder, Maryland 21010; formerly, Microbiologist, Virology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort 

Detrick, Maryland 

††Commander, Medical Corps, US Navy Reserve; ACESO Director and Chief, Clinical Research Department, Biodefense Research Directorate, Naval Medical 

Research Center, Fort Detrick, Maryland 21702; formerly, Chief Medical Officer, Integrated Research Facility, Fort Detrick, Maryland

‡‡Colonel (Retired), Medical Corps, US Army; Chief of Internal Medicine, US Army Healthcare Clinic, Vicenza, APO AE 09630-0040; formerly, Chief, Operational Medicine Department, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

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INTRODUCTION

Revelations in the 1990s that the former Soviet 

Union produced ton quantities of variola virus as a 

strategic weapon and conducted open air testing of 

aerosolized variola on Vozrozhdeniya Island in the 

Aral Sea have increased the plausibility of variola 

being used as a bioterrorism agent.3,7 Considerable 

investment is being made in biopreparedness measures 

against smallpox and related orthopoxviruses, including emergency response plans for mass immunization 

and quarantine, as well as development of improved 

countermeasures such as new vaccines and antiviral 

drugs.8,9 These countermeasures are also needed to 

respond to the public health threat of the closely related 

monkeypox virus, which occurs naturally in western 

and central Africa and produces a disease in humans 

that closely resembles smallpox. Alibek claimed that 

the monkeypox virus was weaponized by the former 

Soviet Union.10 The monkeypox virus was first isolated 

from cynomolgus macaques in Denmark in 1958; 

however, scientific interest did not increase until the 

1970s when it was demonstrated that monkeypox virus 

can cause lethal infection in humans.11,12 A dramatic 

increase in monkeypox virus incidence has occurred 

in the Democratic Republic of the Congo (DRC) over 

the past 30 years following the cessation of routine 

smallpox vaccination, and current estimates state that 

approximately 50% of the DRC general population is 

not protected against either the monkeypox virus or 

variola virus.13 Monkeypox virus was imported inadvertently into the United States in 2003 via a shipment 

of rodents originating in Ghana, where, in contrast to 

the significant morbidity and mortality seen in the 

DRC, little morbidity was associated with infection.14,15

Consequently, more than 50 human infections were 

documented in the United States, thus demonstrating the public health importance of this agent and its 

potential bioterrorist threat.14,15

Variola virus, the causative agent of smallpox, is 

one of the most significant bioterrorist threat agents. 

During the 20th century, smallpox was estimated to 

have caused more than 500 million human deaths.1

The disease and the naturally circulating virus itself 

were eradicated by the World Health Organization’s 

(WHO) global eradication campaign, which was 

declared a success in 1980.2 This program, which 

involved vaccinating all humans in a ring surrounding every suspected case of variola infection, was 

successful in part because smallpox is solely a human disease with no animal reservoirs to reintroduce 

the virus into the human population. The impact 

of a potential smallpox virus attack in the human 

population would be more catastrophic now than in 

previous outbreaks. The pace of viral spread would 

be accelerated since most vaccination programs were 

abandoned worldwide in the 1970s, the prevalence 

of immune-suppressed individuals with human 

immunodeficiency virus (HIV) infections and those 

undergoing chemotherapy for various cancers have 

grown, and human mobility including intercontinental air travel has increased. 

Variola virus is stable, highly infectious via the 

aerosol route, highly transmissible from infected to 

susceptible persons, and has a relatively long asymptomatic incubation period making contact tracing difficult.3

 Mathematical models of the reintroduction of 

variola into contemporary human populations indicate 

dire consequences.4 Public health experts have argued 

that a significant portion of the population should be 

prevaccinated to blunt the impact of a potential attack.5

However, the current licensed vaccine is associated 

with significant adverse events,6 which are more serious in persons who are immunocompromised, and 

prerelease vaccination is contraindicated for a significant portion of the population.

AGENT CHARACTERISTICS

Classification 

Poxviruses infect most vertebrates and invertebrates, causing various diseases of veterinary and medical importance. The poxvirus family is divided into 

two main subfamilies: (1) the Chordopoxvirinae, which 

infects vertebrates; and (2) the Entomopoxvirinae, which 

infects insects. Subfamily Chordopoxvirinae is divided 

into eight genera, one of which is Orthopoxvirus, which 

consists of numerous genetically similar pathogens 

capable of causing disease in humans including variola 

virus (Figure 24-1), monkeypox virus, cowpox virus, 

and vaccinia virus.16 Members of the Orthopoxvirus genus are mostly zoonotic pathogens (Table 24-1) that are 

antigenically similar.17 Antigenic similarity was vital 

to the smallpox eradication in 1980 and remains a key 

component for the protection of military personnel, 

healthcare workers, and researchers who are likely to 

come into contact with orthopoxviruses.9,18

Morphology

Orthopoxviruses are oval, brick-shaped particles 

with a geometrically corrugated outer surface (Figure 

24-2). Their size ranges from 220 nm to 450 nm long and 

140 nm to 260 nm wide.18 The outer envelope consists 

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Medical Aspects of Biological Warfare 

of a lipoprotein layer embedded with surface tubules 

enclosing a core described as biconcave. The core 

contains the viral DNA and core fibrils, and it is surrounded by the core envelope and a tightly arranged 

layer of rod-shaped structures known as the palisade 

layer.19 Between the palisade layer and the outer envelope are two oval masses known as the lateral bodies. 

Two forms of orthopoxviruses result from the replication cycle: intracellular mature virion (IMV) and 

enveloped virion (EV). EV can be further categorized 

as intracellular enveloped virion (IEV), cell-associated 

enveloped virion (CEV), and extracellular enveloped 

virion (EEV). IMV, CEV, and EEV are fully infectious, 

whereas the ability of IEV to promote productive infection in a naïve cell has not been demonstrated.18,19

These virus forms will be discussed in greater detail 

in a later section.

Entry

Entry of orthopoxviruses into permissive cells has 

been extensively studied and, depending on the methodology used, these studies have produced conflicting 

results. Electron microscopy has suggested that the 

attachment of enveloped virions (EEV and CEV) to the 

cell surface results in the disruption of the envelope 

and exposes the IMV particle that subsequently binds 

to the cell surface. In this model, the outer membrane 

of the IMV particle fuses with the plasma membrane 

of the cell, releasing the viral core into the cell.20

Endocytosis has been suggested as an alternative 

model of orthopoxvirus entry using video microscopy of fluorescently labeled virus particles. IMV 

and enveloped virions (CEV and EEV) bind to the 

cell surface and, following a complex series of signaling events, are internalized by endocytosis. For 

enveloped particles, it is predicted that low pH and/

or exposure to glycosaminoglycans results in the 

disruption of the envelope and exposure of the IMV 

particle. The outer membrane of the IMV then fuses 

with the endosomal membrane, releasing the viral 

core into the cell cytoplasm.21

It is likely that both suggested mechanisms of entry 

are used by orthopoxviruses and that the method used 

is dependent on the virus strain and the target cell type. 

Regardless of how the virus enters a given cell, the final 

step in entry is the initiation of early gene transcription 

that is followed by core uncoating and replication.

Figure 24-1. A transmission electron micrograph of a tissue 

section containing variola viruses. 

Photograph: Courtesy of FA Murphy, University of Texas 

Medical Branch, Galveston, Texas. 

TABLE 24-1

POXVIRUSES THAT CAUSE HUMAN DISEASE

Genus Species Animal Reservoir

Orthopoxvirus Variola virus None

Vaccinia virus Unknown (none?)

Cowpox virus Rodents

Monkeypox virus Rodents 

Parapoxvirus Bovine popular stomatitis virus Cattle

Orf virus Sheep

Pseudocowpox virus Cattle

Seal parapoxvirus Seals

Parapoxvirus Tanapox Rodents (?)

Yabapox virus Monkeys (?)

Molluscipoxvirus Molluscum contagiosum virus None

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The late transcripts are translated into structural and 

nonstructural proteins of the virions.19

Morphogenesis and Egress

Late (structural and nonstructural) proteins, along 

with DNA concatemers that are formed during the 

early phase of replication, are assembled into genomic 

DNA and packaged into immature virions, which then 

evolve into the brick-shaped IMV. IMVs, which represent the majority of virus particles produced during 

infection, are fully infectious; however, their release 

from the cell is dependent on cell lysis. A small subset 

of IMV particles gains two additional membranes at 

either the trans-Golgi network or early endosomes 

to become IEVs. IEVs migrate to the cell surface via 

microtubules where their outermost membrane fuses 

with the cell membrane to form CEVs. CEVs induce 

the polymerization of actin to form filaments that 

promote the direct transfer of CEVs to adjacent cells. 

If CEVs become dissociated from the cell membranes, 

they become EEVs. Although IMVs are produced in 

greatest abundance in cell culture and are the most 

stable to environmental degradation, CEVs and EEVs 

probably play a more critical role in cell-to-cell spread 

in the intact animal.22

Many of the Orthopoxvirus gene products, known 

as virokines and viroceptors, interact with and 

modulate essential functions of host cells and immune 

processses.18,23 Furthermore, the genomes of many 

poxvirus species also encode intracellular immune 

modulators, thus providing a broad and complex 

mechanism through which these viruses can subvert 

host immune responses that would be deleterious 

for infection. Interestingly, cowpox virus, which predominantly causes localized skin lesions in healthy 

humans, possesses the largest genome (~220 kbp) of 

the orthopoxvirus species and thus may contain the 

greatest number of immunomodulatory components 

to escape host immunity. In light of this, it is unsurprising that cowpox virus is also believed to have 

the broadest host range species of the orthopoxviruses.24 The limited host range of variola may relate 

to the unique association of viral gene products with 

various host signaling pathways. Therefore, strategies 

that block such key pathways in the replication and 

maturation of poxviruses provide potential targets 

for therapeutic intervention.19

Significant efforts have been made to dissect the 

molecular mechanisms that orthopoxviruses use to 

modulate host cell signaling networks.25–27 Investigations such as these provide important information 

regarding viral pathogenesis and may provide the 

identities of therapeutic targets for the development 

of novel orthopoxvirus specific antivirals.

Figure 24-2. Thin section of smallpox virus growing in the 

cytoplasm of an infected chick embryo cell. Intracellular mature virions (brick-shaped) and immature virions (spherical) 

are visible. Magnification is approximately × 25,000.

Photograph: Courtesy of FA Murphy, University of Texas 

Medical Branch, Galveston, Texas. 

Replication

Orthopoxvirus genomes are linear, double-stranded 

DNA approximately 200 kb long. The genomes encode 

between 176 and 266 proteins, including enzymes 

and factors that are necessary for self-replication and 

maturation. The central region of the genome contains 

highly conserved genes that are essential for viral 

replication, while the terminal regions contain less 

conserved genes that are important for virus–host 

interactions. The virus contains a number of virusencoded enzymes, in particular, a DNA-dependent 

RNA polymerase that transcribes the viral genome. 

Early transcripts encode proteins involved in the 

modulation of the host immune response as well as 

the DNA polymerase and other enzymes that promote 

the replication of the viral genome. Uncoating of the 

core exposes viral DNA and replication occurs in cytoplasmic factories referred to as B-type inclusions, in 

which virions at various stages of assembly are seen. 

Whether host cell nuclear factors are involved in viral 

replication or maturation is unclear. Cells infected 

with some poxviruses (eg, cowpox, avian poxviruses) 

also contain electron-dense A-type inclusions, usually 

containing mature virions; A-type inclusions are easily 

seen by light microscopy (Figure 24-3).18,19,21,22

Nascent viral genomes are used as the templates for 

the production of intermediate transcripts that encode 

factors that promote the production of late transcripts. 

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Phylogenetic Relationships

The evolutionary relationships within the Orthopoxvirus genus have been facilitated by the recent availability of complete DNA sequences for more than 30 

species. Phylogenetic analysis (Figure 24-4) reveals that 

at the sequence level, any two virus species contain at 

least 96% homogeny, variola is more closely related to 

camelpox and taterapox (isolated from rodents) viruses 

than any other members of the genus, and vaccinia is 

most closely related to cowpox virus strain GRI-90.28–31

Cowpox virus strain GRI-90 appears to be less closely 

related to cowpox virus strain Brighton, indicating that 

at least two separate species are included under the 

name cowpox virus. Monkeypox virus does not group 

closely with any other orthopoxvirus, which indicates 

that it diverged from the rest of the genus members 

long ago. Virulence or attenuation may hinge on a 

few genetic determinants. For example, variola major 

(associated with a 30% fatality rate) and variola minor 

(<1% fatality rate) are greater than 98% identical over 

the length of the 185,000-kilobase (kb) genome.

When a phylogeny is developed based on gene 

content instead of genetic sequence, the clustering is 

slightly different. In the gene content phylogeny, all 

cowpox virus strains belonging to the same clade are 

distinctly divergent from vaccinia virus, and camelpox virus is in the same clade as monkeypox virus 

instead of variola virus.30 Variola virus, monkeypox 

virus, and camelpox virus can all be traced back to a 

common branching point that is divergent from the 

less pathogenic orthopoxviruses vaccinia virus and 

a b

Figure 24-3. Cytoplasmic inclusion bodies in cells infected 

with orthopoxviruses. (a) B-type (pale red, irregular) inclusion, or Guarnieri, bodies, and A-type (large eosinophilic, 

with halo) inclusion bodies in ectodermal cells of the chorioallantoic membrane, in a pock produced by cowpox virus. A 

number of nucleated erythrocytes are in the ectoderm and 

free in the mesoderm, and the surface of the pock is ulcerated. Hematoxylin-eosin stain. (b) This section of the skin of a patient 

with hemorrhagic-type smallpox shows Guarnieri bodies and free erythrocytes below an early vesicle. Hematoxylin-eosin 

stain. Reproduced with permission from Fenner F, Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. 

Geneva, Switzerland: World Health Organization; 1988: 85.

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cowpox virus. The discrepancies that exist between the 

sequence-based and gene content-based phylogenies 

can predominantly be traced to the variable terminal 

ends of the genome.30 Most of the proteins encoded 

at the terminal ends are known to be involved in 

virus:host interaction (immunomodulation, virulence, 

host range restriction).19 Variola virus, which is strictly 

a human pathogen, has the fewest genes at its terminal ends whereas cowpox virus, which is capable of 

causing disease in numerous mammalian species, has 

the greatest number of genes at its terminal ends. It 

has been postulated that, through genetic reduction, 

variola virus has maintained only the genes essential 

to productive infection in humans. By minimizing 

the number of genes with potential overlapping or 

antagonistic functions, it is thought that variola virus 

became a highly adapted and efficient pathogen. In 

contrast, cowpox virus is capable of infecting a wide 

variety of mammalian species (including humans) but 

is not highly pathogenic in any of those species. The 

large number of genes at its terminal ends improves the 

host range of cowpox virus but decreases its overall efficiency. Regardless of phylogeny, all orthopoxviruses 

are antigenically similar allowing for cross protection.30

Viral neutralizing epitopes are associated with 

structural proteins encoded by genes located in the 

central conserved region of the viral genome, and 

they are present on the surface of IMVs as well as on 

the envelope of EEVs and CEVs.32–34 Given the conserved nature of these proteins, epitopes are relatively 

uniform for all members of the genus Orthopoxvirus. 

Cross protection allowed for the development of the 

vaccine that eradicated smallpox, and it continues to 

be a fundamental key to the development of vaccinia 

based countermeasures against orthopoxviruses.35–37

Pathogenesis

Most knowledge about smallpox pathogenesis 

is inferred from animal studies of vaccinia in mice, 

mousepox, rabbitpox, monkeypox, and from vaccinia 

in humans.16,38–40 Studies using nonhuman primates 

infected with variola corroborate these findings and 

lend further insight into human smallpox and monkeypox infections; however, certain deficiencies exist with 

the model.41 The production of a clinical syndrome in 

cynomolgus macaques that resembles human smallpox 

requires intravenous infection (an unnatural route) 

Figure 24-4. Orthopoxvirus gene sequence phylogenetic tree. Species names are noted on the branch line that separates different clades. Specific virus strains are indicated at the terminal point of the line. The numerical values represent the confidence 

predication using Bayesian inference for each clade. Reproduced (in accordance with the creative commons attribution license 

associated with open access articles [http://creativecommons.org/licenses/by/3.0]) from Hendrickson RC, Wang C, Hatcher 

EL, Lefkowitz EJ. Orthopoxvirus genome evolution: the role of gene loss. Viruses. 2010;2:1933–1967. 

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

0.96

0.95

HSPV

VACV-WR

VACV-MVA

VACV-COP Vaccinia virus

Cowpox virus

Cowpox virus

Monkeypox virus

RPXV

CPXV-GRI

MPXV-WR

MPXV-ZAI

Variola virus

Taterapox virus

Camelpox virus

Ectromelia virus

CPXV-GER

CPXV-BR

CMLV

ECTV

TATV

VARV-BRZ

VARV-SLN

VARV-KUW

VARV-SAF

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Medical Aspects of Biological Warfare 

with an extremely high dose of inoculum; and even 

then, mortality in this model is inconsistent.41 In both 

natural and experimental aerosol infections, the virus 

is introduced via the respiratory tract, where it first 

seeds the mucous membranes. Although it is unclear 

whether alveolar macrophages are responsible for the 

transport of virus, the first round of replication occurs in the lung draining lymph nodes, followed by a 

transient viremia, which seeds tissues, especially those 

of the reticuloendothelial system, including regional 

lymphatics, spleen, and tonsils.42,43 A second, brief 

viremia transports the virus to the skin and visceral 

tissues immediately before the prodromal phase. In 

humans, the prodrome is characterized by an abrupt 

onset of headache, backache, fever, and a sore throat 

as a result from viral replication in the oral mucosa. 

Characteristic skin lesions develop with a centrifugal 

distribution and synchronous progression following 

viral invasion of the capillary epithelium of the dermal layer. The virus may also be present in urine and 

conjunctival secretions.43 At death, most visceral tissues 

contain massive virus concentrations.

In a review of all pathology reports published in 

English over the past 200 years,44 Martin suggested that 

generally healthy patients who died of smallpox usually died of renal failure, shock secondary to volume 

depletion, and difficulty with oxygenation and ventilation as a result of viral pneumonia and airway compromise, respectively. Degeneration of hepatocytes 

may have caused a degree of compromise, but liver 

failure was not usually the proximate cause of death.

Much of the human pathogenesis of smallpox 

remains a mystery because of the limited tools that 

were available when it was an endemic disease. Comparisons between the limited clinical and pathological 

data from human smallpox victims with the pathophysiology of disease from monkeypox and variola 

nonhuman primate infection models suggest a role for 

the dysregulation of immune responses responsible 

for the production of proinflammatory cytokines, 

lymphocyte apoptosis, and the development of coagulation abnormalities. High viral burdens, which 

were identified in numerous target tissues in the 

animal models, were likely associated with organ 

dysfunction and multisystem failure. Immunohistochemistry staining confirmed the distribution of viral 

antigen, while electron microscopy demonstrated 

evidence of replicating virus, which correlated with 

pathology observed in the lymphoid tissues, skin, oral 

mucosa, gastrointestinal tract, reproductive system, 

and liver. Apoptosis, particularly within the T-cell 

population, was a prominent observation in lymphoid tissues, although the cause of this widespread 

apoptosis remains unknown. However, the strong 

production of proinflammatory cytokines is due at 

least in part to the upregulation of various proapoptotic genes. The strong upregulation of cytokines 

may have also contributed to the development of a 

hemorrhagic diathesis. The detection of D-dimers and 

other changes in hematologic parameters in monkeys 

that developed classical or hemorrhagic smallpox 

suggests that activation of the coagulation cascade is 

a component of both disease syndromes. However, in 

human populations, the occurrence of hemorrhagic 

smallpox was approximately 1% to 3% of the total 

cases observed.2,23,39,44,45

From these recent studies of variola and monkeypox 

virus infection in nonhuman primates, the “toxemia” 

described by clinicians for human smallpox may be 

fundamentally related to the processes underlying septic shock.2,46 Common denominators include lymphocyte apoptosis; proinflammatory cytokines (exuberant 

production of type I interferon [IFN], interleukin-6, 

tumor necrosis factor-α, and IFN-γ measurable in 

plasma); and disseminated intravascular coagulation. 

Aberrant activation of these pathways, which contributes to toxic shock, is a hallmark of pathological 

activation of the innate immune system.

To facilitate viral replication, orthopoxviruses generally modulate their host’s immune response to the 

pathogen’s advantage. Poxviruses encode proteins that 

target or interrupt the natural inflammatory response 

and interfere with apoptosis, synthesis of steroids, and 

initiation of the complement system. In general, these 

proteins block either extracellular immune signals (by 

mimicking or interfering with cytokine/chemokine 

proteins and/or receptors), or they work intracellularly 

by interfering with apoptosis, targeting by the immune 

system, or intracellular immune cell signaling. A combination of these mechanisms may allow the virus to 

overcome immunological surveillance and establish 

clinical disease in the host.47

ORTHOPOXVIRUSES AS BIOLOGICAL WARFARE AND BIOTERRORISM THREATS

Using variola virus in warfare is an old concept. 

British colonial commanders used blankets from smallpox victims as a biological weapon, distributing them 

among Native Americans.48–50 During the American 

Civil War, allegations were made about the use of 

variola as a biological weapon, although no definite 

evidence existed.51,52 In the years leading up to and 

during World War II, the Japanese military explored 

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weaponization of variola during the operations of 

Unit 731 in Mongolia and China. More recently, the 

former Soviet Union developed smallpox as a strategic 

weapon and produced ton quantities of liquid variola 

virus on a continuing basis well into the 1980s.10,53 The 

former Soviet Union also conducted open air testing of 

weaponized variola and demonstrated that infectious 

virus could infect humans 15 km downwind.7

Although declared stocks of variola virus exist only 

at the two WHO repositories (the Centers for Disease 

Control and Prevention [CDC] in Atlanta, Georgia, 

USA, and at the State Research Center of Virology and 

Biotechnology Vector in Koltsovo, Novosibirsk Oblast, 

Russia), it is of concern that undeclared stocks may 

exist in military sites within the former Soviet Union, 

or that they were transferred from the Soviet program 

to programs in Iraq, Iran, North Korea, or elsewhere.53

The probability that such stocks exists is impossible to 

assess, but the catastrophic consequences of smallpox 

release in a biological attack cannot be discounted.4

Variola is a significant threat for use as a biological 

weapon because of its stability, infectivity in aerosol 

form, small infectious dose, severe disease manifestations, and interhuman transmissibility. Furthermore, 

the anticipated morbidity and mortality for the 

general population may be higher than historical 

averages resulting from waning immunity following 

vaccinations in the distant past and immunosuppression as a result of HIV, cancer, organ transplants, and 

old age.3

 Other members of the Orthopoxvirus genus 

share many of variola’s properties and are potential 

agents of a deliberate bioterrorist attack. Of the poxviruses other than variola, monkeypox virus presents 

the greatest threat for biological warfare or terrorism 

use. Monkeypox can naturally produce severe disease 

in humans that closely resembles smallpox, with mortality exceeding 15% in some outbreaks.54 The disease 

can be transmitted from person to person, is highly 

transmissible by aerosol and, in at least some nonhuman primate models, has an infectious dose as low as 

one tissue culture infecting dose (TCID50).39,55–57 Monkeypox virus, like variola, is relatively stable and can 

resist desiccation in both heat and cold.58 Monkeypox 

virus also grows to high titers in cell culture systems, 

including the chick chorioallantoic membrane of embryonated eggs, a simple methodology described in 

older microbiology texts using equipment and supplies available at agricultural supply stores. A large 

dose of monkeypox delivered by aerosol can produce 

a rapidly progressive and overwhelming pneumonia 

in nonhuman primate models.40 Furthermore, monkeypox virus may have already been weaponized by 

the former Soviet military.10

Cowpox and buffalopox produce limited cutaneous disease in humans in natural infection.17 Buffalopox, like cattlepox, may be essentially identical 

to vaccinia.59 The effect of altering route of delivery, 

dose of virus, or the actual viral agent itself on human disease manifestation is unclear. Several studies 

demonstrate that orthopoxviruses produce different 

clinical syndromes and immunological responses 

in animal models depending on the route of infection.40,60–64 Aerosol infection has the potential to 

produce more pronounced pulmonary disease.40,56,65

In addition, all orthopoxviruses share a significant 

amount of homology with variola and monkeypox.29

If the critical virulence factors for systemic human 

disease were determined, then cowpox, buffalopox, 

or other orthopoxviruses could potentially be genetically modified to express these critical factors. When 

designed as a weapon and delivered by aerosol, these 

viruses could have a significant impact in humans, 

even without genetic modification.

Camelpox rarely, if ever, causes disease in humans. 

However, because of Iraqi admissions of research with 

camelpox as part of the country’s biological warfare 

program, some concern exists over its potential use 

as a biological weapon.66 Camelpox virus is a close 

relative of variola virus; the major difference between 

camelpox virus and variola major virus strain Bangladesh-1975 genomes is four additional insertions, 

elongated inverted terminal repeats, and a small area 

of gene rearrangement present in camelpox virus.28

As with other orthopoxviruses, slight modifications in 

the camelpox virus genome may dramatically change 

its pathogenicity in humans. Although prohibited by 

US law, genetic modification of camelpox would be 

a likely starting point by any group that wanted to 

construct variola based on published sequences. In 

addition, it is now technically feasible to create infectious variola using an oligonucleotide synthesizer, 

analogous to the recent demonstration for creation of 

the much simpler polio virus.31,67,68

The possibility of genetically engineered orthopoxviruses remains unknown in biodefense research. 

Studies have shown increased mousepox and vaccinia 

virus virulence in mouse models by the incorporation 

of cloned host cytokine genes into the virus genome.69,70

Whether these results represent findings unique to 

the virus–host model used or reflect a more general 

premise of enhanced virulence is unclear.71,72 The possibility of similar genetic engineering only increases 

the threat of orthopoxviruses that are not significant 

natural threats for human disease. Further research is 

warranted to ensure that present and future countermeasures are effective with modified viruses.

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CLINICAL ASPECTS OF ORTHOPOXVIRUS INFECTIONS

transmission. Following subsequent eruptions on 

the lower extremities, the rash spreads centrally to 

the trunk. Lesions quickly progressed from macules 

to papules and eventually to pustular (umbilicated) 

vesicles, and were more abundant on the extremities 

and face (Figure 24-5). This centrifugal distribution 

of lesions is an important diagnostic feature, typical 

of smallpox disease. In contrast to the lesions seen in 

varicella (chickenpox), smallpox lesions on various 

segments of the body remain generally synchronous 

in their stage of development. From 8 to 14 days after 

onset, the pustules form scabs, which leave depressed 

depigmented scars upon healing. Although variola 

titers in the throat, conjunctiva, and urine diminish 

with time, virus can readily be recovered from scabs 

throughout convalescence.2,82 Therefore, patients 

should be isolated and considered infectious until 

all scabs separate.

Two distinct forms of smallpox were recognized in 

the last century of smallpox occurrence: variola major 

and variola minor. Variola major, the highly virulent, 

prototypical, and historically significant form of the 

disease, remained prevalent in Asia and parts of Africa during the 20th century. Analysis conducted on 

human viral isolates indicated a distinct evolution of 

three virus clades (A, B, C) associated with geographical distribution and case fatality rates.31

Although isolates from clade C, found predominantly in Asia, were associated with an overall higher 

case fatality rate compared to A and B, virus evolution 

was present, as evidenced by the attenuated strains 

detected in Africa. Variola minor was distinguished by 

milder systemic toxicity and more diminutive pox lesions.2

 However, Dixon reported many cases that were 

indistinguishable from variola major in his extensive 

comparison of lesion types.83 Korte first described variola minor, found in Africa, in 1904.2

 Chapin found a 

similar mild form known as alastrim that occurred in 

North America as early as 1896 and subsequently was 

exported to South America, Europe, and Australia.84

Two distinct viral strains of reduced virulence caused 

variola minor and alastrim, and both typically caused 

1% mortality in unvaccinated victims.2

The Rao classification specified five clinical presentations of variola.85 Three-quarters of variola major 

cases were designated classic or ordinary type (Figure 24-5). After prodromal fever and constitutional 

symptoms appeared, patients developed the typical 

variola rash, centrifugal in distribution, with synchronous progression from macules to papules, to vesicles 

to pustules, and then to scabs. The fatality rate was 

3% in vaccinated and 30% in unvaccinated patients. 

Smallpox

Variola virus is stable and retains its infectivity for 

long periods outside the host.73 Variola virus is infectious by aerosol, but natural airborne spread other than 

among close contacts is unusual.3,74,75 Approximately 

30% of susceptible contacts became infected during the 

era of endemic smallpox,76 and the WHO eradication 

campaign was predicated upon the requirement of 

close person-to-person proximity for reliable transmission to occur. Nevertheless, two hospital outbreaks 

demonstrated that variola virus can be spread through 

airborne dissemination in conditions of low relative 

humidity.77 The patients in these outbreaks were infectious from the onset of their eruptive exanthem, most 

commonly from days 3 through 6 after fever onset. If 

the patient had a cough, then chances of virus transmission were greatly increased. Indirect transmission 

via contaminated bedding or other fomites was infrequent.78 Some people in close contact with patients 

harbored virus in their throats without developing 

disease and may have been a means of secondary 

transmission.43,79

After exposure to aerosolized virus, variola travels from the upper or the lower respiratory tract to 

regional lymph nodes, where it replicates and gives 

rise to a primary viremia.2 The incubation period of 

smallpox averages 12 days (range 9–14 days). Those 

in contact with infected patients were quarantined 

for a minimum of 16 to 17 days following exposure 

to ensure an adequate window of time had passed 

without the appearance of symptoms.2 During normal smallpox disease, variola virus was sporadically 

recovered from the blood, but not nearly at the levels 

detected in patients with hemorrhagic smallpox.80

After replication in regional lymph nodes virus disseminated systemically to other lymphoid tissues, 

spleen, liver, bone marrow, and the lungs, and created a secondary viremia. Clinical manifestations 

began acutely with malaise, fever, rigors, vomiting, 

headache, and backache; 15% of patients developed 

delirium. Approximately 10% of light-skinned patients exhibited an observable erythematous rash 

during this phase. After 2 to 3 more days, an exanthem appeared concomitantly with a discrete rash 

about the face, hands, and forearms. Given the lack 

of a keratin layer on mucous membranes, lesions 

shed infected epithelial cells and give rise to infectious oropharyngeal secretions in the first few days 

of the eruptive illness, and occasionally 24 hours 

before eruption.81 These respiratory secretions were 

the most significant but not the sole means of virus 

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Figure 24-5. This series of photographs illustrates the evolution of skin lesions in an unvaccinated infant with the classic 

form of variola major. (a) The third day of rash shows synchronous eruption of skin lesions; some are becoming vesiculated. 

(b) On the fifth day of rash, almost all papules are vesicular or pustular. (c) On the seventh day of rash, many lesions are 

umbilicated, and all lesions are in the same general stage of development. Reproduced with permission from Fenner F, 

Henderson DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. Geneva, Switzerland: World Health Organization; 

1988: 10–14. Photographs by I Arita.

a b c

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Other clinical presentations of smallpox occurred 

less frequently, probably because of the difference 

in host immune response. Flat-type smallpox, noted 

in 2% to 5% of smallpox patients, was characterized 

by both severe systemic toxicity and the slow evolution of flat, soft, and focal skin lesions that did not 

resemble the classical variola exanthem (Figure 24-6). 

This syndrome caused 66% mortality in vaccinated 

patients and 95% mortality in unvaccinated patients.85

Fewer than 3% of smallpox patients developed 

hemorrhagic-type smallpox, which was accompanied 

by extensive petechiae (Figure 24-7), mucosal hemorrhage, and intense toxemia; death usually occurred 

before typical pox lesions developed.86 However, on 

occasion hemorrhagic smallpox also occurred as a 

late stage complication of classical smallpox disease. 

Both hemorrhagic-type and flat-type smallpox may 

have indicated underlying immunodeficiency; hemorrhagic forms occurred more commonly in pregnant 

women and young children.87 The modified type, 

which occurred typically (but not exclusively) in previously vaccinated individuals, was characterized by 

moderation of constitutional symptoms and typically 

reduced numbers of and a rapid evolution of lesions, 

with scabs formed by the 9th day of the illness. The 

variola sine eruptione was characterized by prodromal 

fever and constitutional symptoms. These patients, 

most of whom had been vaccinated, never developed 

a rash.85 In actuality, the manifestations of variola 

infection fell along a spectrum, and classification was 

primarily for the purpose of prognosis.

Bacterial superinfection of pox lesions was relatively common in the preantibiotic era, especially in 

the absence of proper hygiene and medical care in 

tropical environments.2 Arthritis and osteomyelitis 

developed late in the disease in about 1% to 2% of 

patients, occurred more frequently in children, and 

often manifested as bilateral joint involvement, particularly of the elbows.88 Viral inclusion bodies could 

be demonstrated in the joint effusion and bone marrow 

of the involved extremity. Cough and bronchitis were 

occasionally reported as prominent manifestations of 

smallpox, with implications for spread of contagion; 

however, pneumonia was unusual.2 Pulmonary edema 

occurred frequently in hemorrhagic-type and flat-type 

smallpox. Orchitis was noted in approximately 0.1% 

of patients. Encephalitis developed in 1 in 500 cases of 

variola major, compared with 1 in 2,000 cases of variola 

minor. Keratitis and corneal ulcers were important 

complications of smallpox, progressing to blindness in slightly less than 1% of cases. Disease during 

pregnancy precipitated high perinatal mortality, and 

congenital infection was also recognized.2

Partial immunity through vaccination resulted in 

modified-type smallpox, in which sparse skin lesions 

evolved variably, often without pustules, and quickly, 

with crusting occurring as early as the 7th day of illness. When exposed to smallpox, some fully immune 

individuals developed fever, sore throat, and conjunctivitis (called contact fever), which lasted several 

days but did not give rise to the toxicity or minor skin 

lesions that signify variola sine eruptione. Persons who 

recovered from smallpox possessed long-lasting immunity, although a second attack may have occurred 

in 1 in 1,000 persons after an intervening period of 15 

to 20 years.89 Both humoral and cellular responses are 

important components of recovery from infection. 

Neutralizing antibodies peak 2 to 3 weeks following 

onset and last longer than 5 years and up to several 

decades in some individuals.34,45,90

Figure 24-6. Flat-type smallpox in an unvaccinated woman on the sixth day of rash. Extensive flat lesions (a and b) and systemic toxicity with fatal outcome were typical. Reproduced with permission from Fenner F, Henderson DA, Arita I, Jezek Z, 

Ladnyi ID. Smallpox and Its Eradication. Geneva, Switzerland: World Health Organization; 1988: 33. Photographs by F Dekking.

a b

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Monkeypox

The clinical features of human monkeypox are 

classically described as being similar to those of 

smallpox.91 Disease begins with a 2- to 4-day disruptive phase with high fever and prostration. The 

rash develops and progresses synchronously over 

2 to 4 weeks, evolving from macules to papules, to 

vesicles and pustules, to scabs. Lesions are usually 

umbilicated, have a centrifugal distribution, and 

involve the palms and soles. Sore throat and frank 

tonsillitis frequently occur during the eruptive 

phase of human monkeypox.91,92 Lymphadenopathy 

is a common finding that differentiates monkeypox 

from smallpox. This has been documented in up to 

83% of unvaccinated persons with monkeypox and 

arises most frequently early in the course of infection, 

involving the submandibular and cervical nodes and 

less frequently the axillary and inguinal nodes.

Clinical manifestations of human monkeypox are 

likely more diverse, and not as stereotypical as those 

of smallpox. Mild infections were frequent in the first 

recognized African cases, with 14% of patients having 

fewer than 25 lesions and no incapacity.91 In a series of 

282 patients (of which 250 were not vaccinated), the exanthema first appeared somewhere other than the face 

in 18% of the unvaccinated patients; 31% of vaccinated 

patients had pleomorphic or “cropping” appearance 

of rash lesions, and 9.4% had centripetal distribution.93

All of these features were inconsistent with a mimic 

of smallpox. Patients in the 2003 US outbreak tended 

to have fewer mild lesions than most African patients. 

Patients were hospitalized in only 19 of 78 suspected 

cases in the United States, and only 2 had significant illness requiring some form of medical intervention.94,95 A 

surveillance study conducted from 2005 to 2007 in the 

DRC laboratory confirmed 760 cases, which suggests a 

20-fold increase in human monkeypox incidence since 

the 1980s in the same health zone.13

Monkeypox virus can be subdivided into two 

distinct clades that are genetically, clinically, and 

geographically distinct. The Congo Basin monkeypox virus clade has associated case fatality rates of 

approximately 10% in nonvaccinated individuals,96

as opposed to the reduced pathogenicity and transmissibility of the West African clade of monkeypox 

(which caused the US outbreak).57,97 Comparative 

infection models in nonhuman primates, mice, prairie 

dogs, and ground squirrels have all demonstrated 

greater lethality or morbidity associated with Congo 

Basin monkeypox virus infection as compared to 

West African monkeypox virus.98–101 A sine eruptione

form of monkeypox has not been described, but the 

number of serologically diagnosed infections without 

consistent rash illness suggests it is a possibility.102 A 

hemorrhagic form of human monkeypox has not been 

documented.103,104

Complications of monkeypox are more common in 

unvaccinated persons and children.96 During intensive surveillance in the DRC between 1980 and 1986, 

secondary bacterial superinfection of the skin was the 

most common complication (19.2% of unvaccinated 

patients), followed by pulmonary distress/pneumonia 

(11.6% of unvaccinated patients), vomiting/diarrhea/

dehydration (6.8% of unvaccinated patients), and keratitis (4.4% of unvaccinated patients). With the exception 

of keratitis, the incidence of these complications in 

vaccinated persons was at least 3-fold less. Alopecia 

has been noted in some cases.105 Encephalitis was 

detected in at least one monkeypox case in the DRC 

and in one of the cases in the US outbreak of 2003.93,95

As in smallpox, permanent pitted scars are often left 

after scabs separate.

Severity of disease and death is related to age 

and vaccination status, with younger unvaccinated 

children faring worse.91,105–107 The case fatality rate in 

Africa varied in different outbreaks and periods of 

increased surveillance. The fatality rate was 17% from 

1970 through 1979, 10% from 1981 through 1986, and 

1.5% from 1996 through 1997.54 The low fatality rates 

in certain outbreaks were influenced by the lack of 

proper laboratory confirmation to exclude cases of 

varicella virus from monkeypox virus infections. It is 

believed no fatalities occurred among the 78 suspected 

Figure 24-7. Early hemorrhagic-type smallpox with cutaneous signs of hemorrhagic diathesis. Death usually occurred 

before the complete evolution of pox lesions. Reproduced 

with permission from Herrlich A, Munz E, Rodenwaldt E. 

Die pocken; Erreger, Epidemiologie und klinisches Bild. 2nd ed. 

Stuttgart, Germany: Thieme; 1967. In: Fenner F, Henderson 

DA, Arita I, Jezek Z, Ladnyi ID. Smallpox and Its Eradication. 

Geneva, Switzerland: World Health Organization; 1988: 35.

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Medical Aspects of Biological Warfare 

cases in the 2003 US monkeypox outbreak, at least in 

part because the less virulent West African strain was 

responsible.94 The presence of comorbid illnesses, such 

as measles, malaria, or diarrheal disease, may have a 

significant impact on mortality in children.96 Cause of 

death in monkeypox is not universally clear, although 

19 of 33 fatalities in one series of patients involved 

pulmonary distress or bronchopneumonia, suggesting 

superimposed bacterial pneumonia.

Other Orthopoxviruses Infecting Humans

Cowpox is primarily a localized, cutaneous disease.17 Baxby, Bennett, and Getty reviewed 54 cases 

of cowpox infection with a detailed discussion of 

clinical manifestations.108 Disease usually consists of 

single pock-like lesions on the hands or face, although 

multiple lesions are seen in roughly one-quarter of 

cases. Typical lesions progress from macule to papule 

to vesicle to pustule to dark eschar, with a hemorrhagic base being common in the late vesicular stage. 

Progression from macule to eschar is slow, often evolving over 2 to 3 weeks. Local edema, induration, and 

inflammation are common and can be pronounced. 

Lesions are painful and are accompanied by regional 

lymphadenopathy. Complete healing and scab separation usually occur within 6 to 8 weeks of onset, but 

may take 12 weeks or longer. A majority of patients 

experience some constitutional symptoms before the 

eschar stage.

The majority of human cowpox infections are selflimited and without complication. Ocular involvement, including the cornea, can occur, but it usually 

resolves without permanent damage. A few severe 

generalized cowpox infections have been reported including one fatality.108,109 Three of these four described 

cases included a history of atopic dermatitis, indicating a risk of increased severity of disease analogous 

to vaccinia.

Buffalopox and cattlepox infections in humans 

have not been extensively described but have been 

observed in areas of Brazil. Limited data suggest human infection usually occurs on the hands and consists 

of inflamed and painful pustular lesions progressing 

through a Jennerian evolution.110–112 Regional lymphadenopathy and fever can accompany local disease.112

DIAGNOSIS

Clinical Diagnosis

The clinical presentation of smallpox is similar to 

many vesicular and pustular rash illnesses, including 

varicella, herpes simplex, drug reactions, and erythema 

multiforme. Although the index of suspicion for an 

eradicated disease may be low, the failure to recognize a case of smallpox could result in the exposure 

of hospital contacts and the seeding of an outbreak. 

The smallpox diagnosis and evaluation page on the 

CDC website (http://www.bt.cdc.gov/agent/smallpox/

diagnosis/) is an essential resource to assist a clinician 

in evaluating a febrile patient presenting with a rash. 

This site contains an algorithm to quickly determine 

the likelihood of clinical smallpox and a standardized 

worksheet to classify the risk of smallpox using the 

CDC criteria. 

Specimen Collection and Handling

Collection of appropriate specimens is paramount 

for accurate laboratory diagnosis of orthopoxvirus 

infection. Ideally, cutaneous tissue (from skin lesions) 

and blood are sent for diagnostic testing, with other 

samples being sent at the request of public health officials or experts in the field.104 Detailed instructions for 

specimen collection can be found in the Department 

of Defense Smallpox Response Plan (http://www.

bt.cdc.gov/agent/smallpox/responseplan/index.asp) 

or on the CDC website (http://www.cdc.gov/ncidod/

monkeypox/index.htm). Briefly, vesicles or pustules 

should be unroofed, the detached vesicle skin sent in 

a dry tube, and the base of the lesion scraped to make 

a touch-prep on a glass slide. Biopsy specimens should 

be split (if possible) and sent in formalin and in a dry 

tube. If scabs are collected, two scabs should be sent in 

a dry tube. Dacron or polyester swabs should be used 

for oropharyngeal swabs and transported in dry tubes. 

Blood should be collected in a serum separator tube 

(which is then centrifuged to separate serum) and in 

an anticoagulant tube for whole blood. Clinical specimens potentially containing orthopoxviruses other 

than variola virus, including monkeypox virus, may 

be handled in a biosafety level 2 using biosafety level 

3 practices (specimens potentially containing variola 

virus must be handled in biosafety level 4).113 

Many phenotypic and genotypic methods involving 

virological, immunological, and molecular approaches 

have been used to identify orthopoxviruses.

Phenotypic Diagnosis

In the past, a presumptive diagnosis of orthopoxviruses required a laboratory with capabilities and 

expertise in viral diagnostics. Microscopists with 

experience in poxvirus infections can often recognize 

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the characteristic inclusion bodies (Guarnieri bodies, 

corresponding to B-type poxvirus inclusions [see 

Figure 24-3]) in tissue samples under light microscopy. These cytoplasmic inclusions are hematoxylinophilic, stain reddish purple with Giemsa stain, 

and contain Feulgen-positive material.114 Electron 

microscopy reveals the unmistakable brick-like 

morphology of orthopoxviruses in thin sections of 

infected materials. 

Microscopy alone cannot differentiate members of 

the genus Orthopoxvirus, yet the epidemiological setting 

can suggest which species is involved. The orthopoxviruses with pathogenicity in humans (with the exception of molluscum contagiosum) can be grown on the 

chorioallantoic membranes of 12-day-old embryonated 

chicken eggs, where they form characteristic pocks. 

These viruses also grow readily in easily obtained 

cell cultures, including VERO cells and additional 

monkey kidney cell lines, A549 cells, and others. Variola could characteristically be differentiated from 

other viruses by a strict temperature cut-off at 39°C. 

Methods for isolation and identification of individual 

virus species have been reviewed.115–117 For example, 

immunogold staining prior to electron microscopy 

permits a more precise identification to the species level.

Immunodiagnosis

Serologic testing for anti-Orthopoxvirus antibodies 

is an old technique, and various assays were used extensively in the study of smallpox.2

 Current common 

approaches include enzyme-linked immunosorbent 

assay (ELISA), plaque reduction neutralization test 

(PRNT), and immunofluorescence microscopy. Although these assays are proficient at demonstrating 

the presence of an orthopoxvirus infection, antigenic 

similarity that results in serologic cross-reactivity 

makes species differentiation extremely difficult. 

ELISAs and PRNTs can be used to detect orthopoxvirus antibodies in a serum sample; however, data 

interpretation is different. ELISA assays measure the 

total amount of antibody present in a given serum 

sample, and they can measure both immunoglobulin 

M (IgM) and IgG antibodies, allowing for the identification of markers for both active and prior infection. 

Since IgM antibodies disappear within 6 months, 

IgM ELISAs can be used to detect recent infections 

when virus detection is not possible after lesions have 

healed and scabs have separated. In the investigation of the 2003 US monkeypox outbreak, the CDC 

relied on anti-Orthopoxvirus IgG and IgM ELISAs for 

serologic diagnosis.95 More recently, a combination 

of T-cell measurements and a novel IgG ELISA were 

used to enhance epidemiological follow-up studies 

to this outbreak.118,119 It has also been suggested that, 

by using linear peptides as antigens, species-specific 

orthopoxvirus ELISA assays can be developed. 

Although ELISAs can be sensitive, quick, and easy 

to perform, they do not provide information as to 

antibody functionality and their ability to neutralize 

orthopoxviruses in vitro, or any inferred protective 

immunity. To determine the neutralizing (ie, inferred 

protective immunity) antibody titer, a PRNT must 

be performed. The theoretical protective immunity 

value for serum antibody levels against variola virus is greater than or equal to 1:40 as determined by 

PRNT.120 PRNT cannot differentiate between IgM and 

IgG antibodies, and orthopoxvirus species-specific 

PRNT assays have not been developed. 

Similar to ELISAs, immunofluorescence microscopy 

has been used to detect IgM in acute infection directed 

against cowpox.109 The technique used is similar to 

ELISA except that a fluorescent tag attached to the 

detection antibody allows visual, colorimetric observation of orthopoxvirus antibodies. Immunofluorescence 

microscopy is not a quantitative assay and only allows 

determinations of presence versus absence. Quantification can be performed using a fluorescence plate 

reader. Similar to ELISA, this assay will not provide 

information on protective antibody levels.

Nucleic Acid Diagnosis

The molecular diagnostic approaches, including 

DNA sequencing, polymerase chain reaction (PCR), 

restriction fragment-length polymorphism (RFLP), 

real-time PCR, and microarrays, are more sensitive 

and specific than the conventional virological and 

immunological approaches. Of these techniques, sequencing provides the highest level of specificity for 

species or strain identification, but current sequencing 

techniques are not yet as practical as rapid diagnostic 

tools in most laboratories. RFLP analysis and microarray genotyping also provide high levels of specificity 

and when combined with PCR, these approaches can 

offer high levels of sensitivity.121–123

Successful performance of PCR-based diagnostics 

requires extraction of DNA from body fluid and tissue 

samples, careful design of oligonucleotide primers and 

probes, and optimization of amplification and detection conditions. Numerous commercial nucleic acid 

purification methods are available for various sample 

types, which involve cell lysis and protein denaturation followed by DNA precipitation or fractionation 

by reversible binding to an affinity matrix. Selection 

of appropriate primers and probes, and optimization 

of assay conditions require knowledge of genome 

sequences and molecular biology techniques.

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One of the basic techniques used in PCR-based diagnostics uses PCR-amplified regions of the genome 

separated on agarose gels by electrophoresis, where 

the amplicon sizes are used to identify the sample. 

Several PCR gel-analysis assays have been used to 

identify cowpox, monkeypox, vaccinia, and variola 

viruses from clinical specimens.117,124–126

Large fragment (LPCR-RFLP) analysis requires amplifying large DNA fragments with high fidelity DNA 

polymerase enzymes. The amplified LPCR products 

are purified on agarose gels and digested with a restriction enzyme. The digested DNA fragments are then 

electrophoresed on polyacrylamide gels for a constant 

period at constant voltage and stained with ethidium 

bromide. The restriction pattern is then visualized and 

photographed with a digital camera. The positions 

for all DNA fragments in each restriction pattern are 

determined and digitized by appropriate fingerprinting software. From this pattern, a similarity coefficient 

is calculated for every pair of restriction patterns and 

used as an index for species differentiation.

Real-time PCR methods provide exquisite levels of 

sensitivity and specificity.127 Real-time PCR is the measurement, by fluorescence detection, of the amount of 

nucleic acids produced during every cycle of the PCR. 

Several detection chemistries, such as the intercalating dyes (SYBR Green, Applied Biosystems, Foster 

City, CA), hydrolysis probes (5’ nuclease or Taqman, 

Minor Groove Binding Proteins [MGBP]), hybridization probes (Fluorescence Resonance Energy Transfer 

[FRET]) and molecular beacons, are used. There are 

several commercially available instruments for realtime PCR, such as the ABI—7900 (Applied Biosystems, 

Foster City, CA), Smart Cycler (Cepheid, Synntvale, 

CA), LightCycler (Roche Diagnostics Corporation, 

Indianapolis, IN), MJ Opticon (Bio-Rad, Hercules, CA), 

RotorGene (Corbett Life Science, Sydney, Australia), 

RAPID (Idaho Technology, Salt Lake City, UT), and 

others. When combined with portable analytical platforms such as the Smart Cycler or LightCycler, realtime PCR systems can be readily deployed to field sites 

for rapid testing. Real-time PCR assays, which can be 

performed in a few hours, can test clinical specimens 

for all orthopoxviruses or for specific species such as 

vaccinia, variola, or monkeypox.127–130

Real-time PCR was one of the diagnostic techniques 

used in the investigation of the 2003 US monkeypox 

outbreak.95 It has also been used during monkeypox 

active disease surveillance studies in the DRC, identifying 760 new human cases of monkeypox between 

November 2005 and November 2007.13 Because of its 

sensitivity, rapidity, and ease of use, real-time PCR 

will likely become the primary method of preliminary 

diagnosis of Orthopoxvirus infection, with isolation 

and growth in a high-level containment laboratory 

reserved for confirmation.

MEDICAL MANAGEMENT

Prophylaxis

Vaccination

History. Attempts to use infected material to induce 

immunity to smallpox date to the first millennium; 

the Chinese used scabs or pus collected from mild 

smallpox cases to infect recipients usually via insertion of bamboo splinters into the nasal mucosa. This 

procedure produced disease in a controlled situation 

that was typically milder than naturally occurring 

disease and allowed for isolation or controlled exposure of nonimmune individuals. The practice spread 

to India and from there to Istanbul, where Europeans 

encountered it in the early 18th century. In Europe, 

the inoculation of the skin with infected pock material 

was later referred to as variolation to distinguish the 

procedure from vaccination. Inducing immunity using 

variola-contaminated materials had been known to the 

British Royal Medical Society through Joseph Lister’s 

reports from China as early as 1700, but the procedure 

was not practiced until Lady Mary Wortley Montagu, 

wife of the British ambassador to Turkey, introduced it 

to British society. Lady Montagu, who had been badly 

disfigured from smallpox, had her son inoculated in 

Constantinople in 1717 and subsequently arranged for 

surgeon Charles Maitland to inoculate her daughter in 

1722. In the British American colonies, Cotton Mather 

of Boston persuaded Dr Zabdiel Boylston to conduct 

variolation on 224 people in 1721 after reading about 

inoculation in a Royal Medical Society publication.83

During a smallpox outbreak in Boston in 1752, more 

than 2,000 persons underwent variolation, resulting 

in a 90% reduction in mortality among the population immunized. During the Revolutionary War, the 

Canadian Campaign failed largely because the American reinforcements contracted smallpox. Continued 

problems with recurring smallpox epidemics among 

recruits to the Continental Army resulted in a directive in 1779 for variolation of all new recruits. General 

Washington, who had undergone variolation himself 

as a young man, was the first military commander to 

order immunization of his forces.131

The practice of variolation, which was never widely 

accepted, was outlawed at times because many of those 

inoculated developed grave clinical illness. Variolation 

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often caused a 1% to 2% mortality rate, and the individuals who died had the potential to transmit natural 

smallpox. Edward Jenner overcame problems of inoculation with variola by capitalizing on the long-held 

observation that milkmaids had clear complexions 

(without smallpox scars), presumably because they 

had had cowpox, which caused milder disease in 

humans. Folklore maintained that human infection 

with cowpox conferred lifelong immunity to smallpox. In 1796 Jenner scientifically demonstrated that 

inoculation with material obtained from a milkmaid’s 

cowpox lesions would result in immunity and protection from infection with smallpox when introduced by 

inoculation.83 Jenner published his findings in 1798, 

and in 1801 he reported that 100,000 persons had been 

vaccinated in England. By the 1820s vaccination had 

become widespread throughout Britain and much of 

Europe. Although derivation of current vaccinia strains 

is uncertain, it is not a form of cowpox, and because 

Jenner lost his original material used for vaccination, 

the specific source of current vaccinia strains remains 

unknown.83 The United States began regulating production of the vaccine in 1925. Since then, the New 

York City Board of Health strain of vaccinia has been 

used as the primary US vaccine strain. The WHO 

global vaccination program eventually led to smallpox 

eradication, with the last serially transmitted smallpox 

case reported in 1977. Routine vaccination of children 

in the United States ceased in 1971, and vaccination 

of hospital workers ceased in 1976. Vaccination of 

military personnel was continued because of Cold War 

concerns about its intentional use but eventually halted 

in 1989. The risk of bioterrorism prompted smallpox 

vaccination in at-risk military personnel and civilian 

healthcare workers to be resumed in 2003.132,133

During the WHO global eradication program, most 

of the human population received vaccinia virus by 

scarification. Although there were multiple manufacturers worldwide, and vaccine lots varied with respect 

to potency and purity, almost all vaccinia administered 

was derived from one of two lineages, the New York 

Board of Health and Lister strains.2 Live vaccinia virus 

suspension was placed as a drop on the skin or drawn 

up by capillary action between the tines of a bifurcated 

needle; the nominal dose of live vaccinia was roughly 

105

 virions. Usually, primary vaccination is uneventful; following introduction into the skin, the virus 

replicates in basal layer keratinocytes, spreads cell-tocell, and leads to discrete vesicle formation. Within a 

week, the vesicle evolves into a pustule surrounded 

by inflammatory tissue. This lesion scabs over within 

10 to 14 days; eventually, the scab is shed. Vaccinees 

in the global campaign often experienced tender 

axillary lymph nodes, fever, and malaise for brief 

periods. Occasionally, however, complications arose 

with varying degrees of severity. Accidental transfer 

of vaccinia from the inoculation site was common, 

but of little consequence unless transferred to the eye. 

Generalized vaccinia, which involved systemic spread 

of the virus and eruption of multiple pocks at distant 

sites, was more serious. In individuals with eczema or 

atopic dermatitis, however, it sometimes led to extensive inflammation and secondary bacterial infection. 

More serious, life-threatening complications arose in 

vaccinees with defects in cell-mediated immunity; the 

vaccination site frequently enlarged to form an ulcer, 

secondary ulcers appeared, and the infection cleared 

slowly or not at all. The most serious event was postvaccinial encephalitis. Although rare, this condition 

was frequently fatal. Death occurred in approximately 

one in one million primary vaccinations.134,135 Adverse 

events may be more frequent and severe if mass immunization were to be resumed in an unscreened general 

population that now includes transplant recipients on 

immunosuppressive drugs, HIV-infected individuals, 

and geriatric patients.

Recent Vaccination Campaigns. The requirement 

that any alternative vaccine must not be inferior to live 

vaccinia sets a high standard. The successful immunization or “take rate” has been greater than 95%, both 

historically and in a more recent series of more than 

450,000 military vaccinees.132 In this series, one case 

of encephalitis and 37 cases of myopericarditis were 

documented in a prescreened, healthy, young adult 

population. Although the incidence of myopericarditis 

was below the historical average and the cases were 

mild, this adverse event contributed to the general reluctance of the civilian healthcare population to accept 

vaccination.133 Live (replicating) vaccinia immunization has also been used as postexposure prophylaxis 

and is believed effective if administered within 4 days 

of exposure. As a potential replacement for vaccine 

strategies used during the eradication campaign, a 

new vaccine was prepared in massive quantities (>300 

million doses) by selection of plaque-purified progeny virus from the New York Board of Health strain 

(Dryvax, Wyeth Laboratories, Marietta, PA), which 

was amplified in VERO cell cultures. This vaccine is 

of greater purity and free of adventitious agents in 

comparison with its predecessor, which was prepared 

on calf skin. Phase I safety and immunogenicity trials 

for ACAM2000 indicate greater than 95% take rates and 

adverse events comparable to those of the predecessor 

vaccine.136 ACAM2000 was approved for use in 2007 by 

the Food and Drug Administration (FDA), effectively 

replacing Dryvax as the vaccine of choice for licensed 

use. Additionally, this vaccine is currently being 

maintained as part of the Strategic National Stockpile. 

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ACAM2000 is no less reactogenic than Dryvax; therefore, vaccination remains limited to groups at the 

greatest risk of contracting orthopoxviruses. 

Vaccination is performed with a bifurcated needle 

onto which the reconstituted vaccinia preparation has 

been drawn, using 15 jabs with enough strength to produce a visible trace of bleeding. The resulting vaccination lesion is then kept covered with a nonadherent and 

nonimpervious dressing. Care must be taken to prevent 

inadvertent inoculation of the vaccinee or others. In primary vaccinees, a papule forms within 5 days, developing into a vesicle on the 5th or 6th day postvaccination, 

which signifies a major reaction, or take. The vesicle 

subsequently becomes pustular, swelling subsides, and 

a crust forms, which comes off in 14 to 21 days. At the 

height of the primary reaction, known as the Jennerian 

response, regional lymphadenopathy usually occurs, 

which may be accompanied by systemic manifestations of fever and malaise. Primary vaccination with 

vaccine at a potency of 100 million pock-forming units 

per milliliter elicits a 97% response rate both by major 

reaction and neutralizing antibody response. Allergic 

sensitization to viral proteins can persist so that the 

appearance of a papule and redness may occur within 

24 hours of revaccination, with vesicles occasionally 

developing within 24 to 48 hours. This allergic response 

peaks within 3 days and does not constitute a “major 

reaction or take.” The immunological response occurring after 3 days is an accelerated but otherwise similar 

appearance of papule, vesicle, and/or pustule to that 

seen in the primary vaccination response. Revaccination 

is considered successful if a vesicular or pustular lesion 

or an area of definite palpable induration or congestion 

surrounding a central lesion (scar or ulcer) is present on 

examination at 6 to 8 days after revaccination.

The immunization of military and civilian individuals has provided an opportunity to study the nature 

of adverse events using modern tools of immunology. 

A strong association was established between adverse 

events and increased systemic cytokines, in particular, 

IFN-γ, tumor necrosis factor-α, interleukin-5, and 

interleukin-10.137 Some researchers have speculated 

that cardiac events, although rare, may be related to 

dramatic alterations in cytokine profiles.

Protective immunity elicited by live vaccinia is 

thought to depend on a combination of humoral and 

cellular immune responses. Using a monkey model 

in which animals were immunized with vaccinia and 

challenged with monkeypox virus, Edghill-Smith et 

al showed that vaccinia specific B-cells are critical for 

protection.138 Antibody depletion of B-cells (but not 

CD4+ or CD8+ T-cells) abrogated vaccinia-induced 

protection. Edghill-Smith et al have also shown 

that simian immunodeficiency virus compromised 

monkeys could withstand monkeypox infection if it 

was preceded by a dose of nonreplicating Modified 

Vaccinia Ankara (MVA) strain vaccinia, but they were 

not protected against monkeypox challenge when their 

CD4+ T-cell counts were < 300 mm.138

MVA is an alternative vaccine that has promise as a 

nonreplicating immunogen. MVA, which was used in 

Germany in the later stages of global eradication, was 

shown to be safe and immunogenic, but its protective 

efficacy has not been established in humans. MVA 

was generated by more than 500 serial passages in 

chick embryo fibroblasts, which resulted in multiple 

deletions and mutations and an inability to replicate 

efficiently in human and most other mammalian 

cells.139 Ultrastructural examination of purified MVA 

reveals that most of the particles are enveloped; the 

host restriction occurs at a late stage of maturation. 

The presence of enveloped particles is believed to be 

important to the elicitation of protective immunity. Experimentally, MVA was demonstrated to protect monkeys against a monkeypox virus challenge after one 

or two doses of MVA or MVA followed by Dryvax.140

Surprisingly, a single dose of MVA also protected when 

a challenge followed immunization by as little as 10 

days, although protection was not absolute; a modest 

number of pocks and a low-level viremia occurred in 

the MVA recipients following challenge. 

Aside from live and attenuated virus vaccines, a 

number of other approaches are being investigated. 

Most of these vaccine strategies involve the use of viral 

DNA or viral protein(s). Prime-boost strategies (ie, an 

initial vaccination with a DNA-based vaccine followed 

by a protein-based vaccine) have also been used. All 

of these strategies have shown some or significant 

promise in animal models; however, none of these 

have been approved by the FDA and are not currently 

being used as investigational new drugs.141

Outcome. Successful smallpox vaccination provides 

high-level immunity for the majority of recipients for 3 

to 5 years followed by decreasing immunity. In Mack’s 

review of importation cases in Europe from 1950 

through 1972, he provided epidemiological evidence 

of some relative protection from death, if not from disease severity, in individuals who had been immunized 

more than 20 years before exposure. However, for the 

older population in particular, vaccination within 10 

years of exposure did not prevent all cases but did 

prevent some smallpox deaths.142 Multiple vaccinations 

are thought to produce more long-lasting immunity. 

Vaccination has been effective in preventing disease 

in 95% of vaccinees.143 Vaccination was also shown to 

prevent or substantially reduce the severity of infection 

when given as a secondary prophylaxis within a few 

days of exposure.2

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Contraindications. Smallpox vaccination is contraindicated in the preoutbreak setting for individuals 

with the following conditions or those having close 

contact with individuals with the following conditions:

• a history of atopic dermatitis (eczema);

• acute, chronic, or exfoliative skin conditions 

that disrupt the epidermis;

• pregnancy or the possibility of becoming 

pregnant; or

• a compromised immune system as a consequence of HIV infection, acquired immunodeficiency syndrome, autoimmune disorders, 

cancer, radiation treatment, immunosuppressive therapy, or other immunodeficiencies.

Additional relative contraindications for potential vaccinees, but not close contacts, are smallpox 

vaccine-component allergies, moderate or severe acute 

intercurrent infections, topical ophthalmologic steroid 

medications, age younger than 18, and maternal breastfeeding. A history of Darier’s disease and household 

contact with active disease also are contraindications 

for vaccination.6

Adverse Events. Vaccinia can be transmitted from a 

vaccinee’s unhealed vaccination site to other persons 

by close contact and the same adverse events as with 

intentional vaccination can result. To avoid inadvertent transmission, vaccinees should wash their hands 

with soap and water or use antiseptic hand rubs immediately after touching the vaccination site and after 

dressing changes. Vaccinia contaminated dressings 

should be placed in sealed plastic bags and disposed 

in household trash.144

Adverse reactions to smallpox vaccination are diagnosed by a clinical examination. Most reactions can be 

managed with observation and supportive measures. 

Self-limited reactions include fever, headache, fatigue, 

myalgia, chills, local skin reactions, nonspecific rashes, 

erythema multiforme, lymphadenopathy, and pain 

at the vaccination site. Adverse reactions that require 

further evaluation and possible therapeutic intervention include inadvertent inoculation involving the eye, 

generalized vaccinia, eczema vaccinatum, progressive 

vaccinia, postvaccinial central nervous system disease, 

and fetal vaccinia.6,145

Inadvertent inoculation generally results in a condition that is self-limited unless it involves the eye or eyelid, which requires an ophthalmologist’s evaluation. 

Topical treatment with trifluridine (Viroptic, Glaxo/

Smith/Kline, Brentford, Middlesex, United Kingdom) 

or vidarabine (ViraA, King Pharmaceuticals, Bristol, 

TN) is often recommended, although the FDA does not 

specifically approve the treatment of ocular vaccinia 

for either of these drugs. Most published experience 

is with use of vidarabine, but this drug is no longer 

manufactured.146

Generalized vaccinia is characterized by a disseminated maculopapular or vesicular rash, frequently on 

an erythematous base, and typically occurring 6 to 9 

days after primary vaccination. Treatment with vaccinia immune globulin (VIG) is restricted to those who 

are systemically ill or have an immunocompromising 

condition or recurrent disease that can last up to a year. 

Contact precautions should be used to prevent further 

transmission and nosocomial infection.6

Eczema vaccinatum occurs in individuals with a history of atopic dermatitis, regardless of current disease 

activity, and can be a papular, vesicular, or pustular 

rash. This rash may be generalized, or localized with 

involvement anywhere on the body, with a predilection for areas of previous atopic dermatitis lesions. 

Mortality ranges from 17% to 30% and is reduced by 

use of VIG. Contact precautions should be used to prevent further transmission and nosocomial infection.6 

Progressive vaccinia is a rare, severe, and often fatal 

complication of vaccination that occurs in individuals 

with immunodeficiency conditions and is characterized by painless progressive necrosis at the vaccination 

site with or without metastases to distant sites.

This condition carries a high mortality rate; therefore, progressive vaccinia should be aggressively 

treated with VIG, intensive monitoring, and tertiary 

medical center level support. Persons with the following conditions are at the highest risk:

• congenital or acquired immunodeficiencies;

• HIV infection/acquired immunodeficiency 

syndrome;

• cancer;

• autoimmune disease; 

• immunosuppressive therapy; or

• organ transplant.

Anecdotal experience has shown that despite treatment with VIG, individuals with cell-mediated immunity defects have a poorer prognosis than those with 

humoral defects. Infection control measures should 

include contact and respiratory precautions to prevent 

transmission and nosocomial infection.6

Central nervous system disease, which includes 

postvaccinial encephalopathy and postvaccinial 

encephalomyelitis, occurs rarely after smallpox vaccination. Postvaccinial encephalopathy occurs more 

frequently, typically affects infants and children 

younger than age 2, and reflects vascular damage to the central nervous system. Symptoms that 

typically occur 6 to 10 days postvaccination include 

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Medical Aspects of Biological Warfare 

seizures, hemiplegia, aphasia, and transient amnesia. 

Histopathologic findings include cerebral edema, 

lymphocytic meningeal inflammation, ganglion degeneration, and perivascular hemorrhage. Patients with 

postvaccinial encephalopathy who survive can be left 

with cerebral impairment and hemiplegia. Postvaccinial encephalomyelitis, which affects individuals 

who are age 2 or older, is characterized by abrupt onset 

of fever, vomiting, malaise, and anorexia occurring 

approximately 11 to 15 days postvaccination. Symptoms progress to amnesia, confusion, disorientation, 

restlessness, delirium, drowsiness, and seizures. The 

cerebral spinal fluid has normal chemistries and cell 

count. Histopathology findings include demyelization 

and microglial proliferation in demyelinated areas, 

with lymphocytic infiltration but without significant 

edema. The cause for central nervous system disease 

is unknown, and no specific therapy exists. Therefore, 

intervention is limited to anticonvulsant therapy and 

intensive supportive care. Fetal vaccinia, which results 

from vaccinial transmission from mother to fetus, is a 

rare but serious complication of smallpox vaccination 

during or immediately before pregnancy.6

In the Department of Defense 2002–2003 vaccination 

program involving 540,824 vaccinees,66 symptomatic 

cases of myopericarditis were reported, for a rate of 

1.2 per 10,000. Mean time from vaccination to evaluation for myopericarditis was 10.4 days, with a range 

of 3 to 25 days. Reports of myocarditis in vaccinees in 

2003 raised concerns of carditis and cardiac deaths in 

individuals undergoing smallpox vaccination. That 

year, 21 cases of myo/pericarditis of 36,217 vaccinees 

were reported, with 19 (90%) occurring in revaccinees. 

The median age of those affected was 48, and they were 

predominantly women. Eleven of the individuals were 

hospitalized, but there were no fatalities. Of the 540,824 

total vaccinees over the 2 years, 449,198 were military 

personnel (the rest were civilians), and of these there 

were 37 cases, for an occurrence rate of 1 per 12,000 

vaccinees.131 Ischemic cardiac events including fatalities have also been reported as a consequence of the 

use of vaccinia vaccine (Dryvax) during the campaign. 

Although no clear association has been found, history 

of ischemic heart disease and significant cardiac risk 

pose relative contraindications for smallpox vaccination. Consequently, individuals with a history of myocarditis, pericarditis, or ischemic heart disease should 

refrain from vaccination.147,148

Smallpox Biothreat Policy. In a smallpox release 

from a bioterrorist event, individuals would be vaccinated according to the current national policy, which 

recommends initial vaccination of higher risk groups 

(individuals directly exposed to the release and those 

with close contact to smallpox patients) and medical 

and emergency transport personnel. Vaccination of the 

general population would then be extended in concentric rings around the initial cases to impede the spread. 

There are no absolute contraindications to vaccination 

for individuals with high-risk exposure to smallpox. 

Persons at the greatest risk of complications of vaccination are those for whom smallpox infection poses the 

greatest risk. If relative contraindications exist for an 

individual, the risks must be weighed against the risk 

of a potentially fatal smallpox infection.

Postexposure prophylaxis with vaccine offers 

protection against smallpox but is untried in human 

infections with other orthopoxviruses.2

 Despite a lack 

of hard evidence in humans, postexposure vaccination 

is likely efficacious against other orthopoxviruses, and 

during the 2003 US monkeypox outbreak the CDC 

recommended vaccination of potentially exposed 

persons.93

Treatment

Passive Immunization

VIG is available from the CDC as an investigational 

new drug in two formulations: intramuscular and 

intravenous. VIG may be beneficial in treating some 

of the adverse effects associated with vaccination. VIG 

has no proven benefit in smallpox treatment, and its 

efficacy in treatment of monkeypox infections is unknown. Monoclonal antibodies have been shown to be 

beneficial in animal models under certain conditions, 

but this concept has not yet been sufficiently developed 

for efficacy testing in humans. 

Antiviral Drugs

The introduction of monkeypox virus to the United 

States, the endemic nature of monkeypox virus in certain regions of Africa, and the continued threat of an act 

of bioterrorism with monkeypox or variola indicate the 

need for anti-Orthopoxvirus therapeutic drugs. In addition, a therapeutic would be useful for the treatment of 

adverse events associated with vaccination. The only 

FDA approved antiviral drug available for treating 

orthopoxviruses is cidofovir. However, it is approved 

for treatment of cytomegalovirus in HIV patients so 

it can only be offered for treatment of orthopoxvirus 

infections under emergency use protocols maintained 

by both the Department of Health and Human Services 

and the Department of Defense.149

The elaborate replication strategy of poxviruses offers a number of potential targets for therapeutic intervention.150 Initial studies to identify effective antiviral 

agents for orthopoxviruses tested drugs developed for 

other viruses that share similar molecular targets.149

The effort to discover effective drugs against DNA 

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viruses initially focused on treatment of herpesvirus 

infections. The discovery of acyclovir led to practical 

therapy and a better understanding of the importance 

of viral and cellular enzymes involved in phosphorylation of acyclovir to acyclovir triphosphate, the active 

chemical entity. Acyclovir failed to inhibit cytomegalovirus because unlike the thymidine kinase of herpes 

simplex, cytomegalovirus thymidine kinase lacked 

the appropriate specificity, which was overcome by 

synthesis of a series of phosphorylated analogues using a stable phosphonate bond. The most promising 

candidate using this approach was cidofovir, which is 

a dCMP analog.151 Cidofovir is licensed for treatment 

of cytomegalovirus-associated retinitis under the trade 

name Vistide (Gilead Sciences Inc, Foster City, CA), 

and inhibits the cytomegalovirus DNA polymerase, a 

target shared with the poxviruses. Cidofovir has been 

demonstrated to protect nonhuman primates against 

severe disease in both the monkeypox and variola 

nonhuman primate models, when administered within 

48 hours of intravenous or respiratory exposure to 

the virus.152–157 Although the drug formulation used 

in these studies has been criticized for requiring intravenous administration, patients with advanced 

disease would already be receiving intravenous fluids 

as part of their supportive care, and once weekly or 

every other day cidofovir administration would not 

significantly increase the healthcare burden. Cidofovir 

has been associated with nephrotoxicity; therefore, 

careful attention to fluid management is important and 

patient hydration and coadministration of probenecid 

is required.

Cidofovir requires bolus dosing to allow drug entry 

into cells by pinocytosis; however, bolus dosing results 

in transiently high concentrations in the kidney. For 

this reason, an oral formulation with lower toxicity 

is more desirable. CMX001 is a lipid conjugate of 

cidofovir with a 1-0-hexadecyl-oxypro-pyl (HDP) 

covalently linked to the nucleotide analogue. Attachment of the lipid moiety allows CMX001 to be taken 

up into cells through lysophosphatidylcholine (LPC) 

uptake pathways,158 which results in lower toxicity and 

increased bioavailability. This formulation dramatically reduced transient drug levels in the kidney and 

eliminated nephrotoxicity in toxicology studies using 

mice.159 CMX001 has also demonstrated protection in 

mouse and rabbit models of orthopoxvirus infection. 

Demonstrating efficacy of CMX001 in nonhuman primates is not possible because of the higher oxidative 

metabolism in monkeys. However, both cidofovir and 

CMX001 produce the same antiviral product in vivo 

(cidofovir diphosphate), allowing cidofovir to be used 

as a surrogate. Increased oxidative metabolism is not 

observed in humans. Although an oral formulation 

of cidofovir is not yet available for human use, it is in 

phase I/II clinical trials and is used under emergency 

authorization for the treatment of systemic adenovirus 

infections of pediatric hematopoietic stem cell transplant recipients.160

An alternative approach to identifying compounds 

with anti-Orthopoxvirus activity was the use of a 

high-throughput screen using vaccinia and cowpox 

virus. More than 300,000 compounds were evaluated 

and several potent lead structures were identified 

for optimization and evaluation against vaccinia, 

monkeypox, and variola viruses.161 From this effort 

ST-246 [4-trifluoromethyl-N-(3,3a,4,4a,5,5a,6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop[f]isoindol2(1H)-yl)-benzamide] was identified and is under 

advanced development by the Biomedical Advanced 

Research and Development Authority. ST-246 is both 

potent (EC50 < 0.010 μM), selective (CC50 > 40 mM), 

and active against multiple orthopoxviruses, including monkeypox, camelpox, cowpox, ectromelia 

(mousepox), vaccinia, and variola viruses in vitro; 

and against monkeypox, variola, cowpox, vaccinia, 

and ectromelia in vivo.161 The viral target of ST-246 

is the F13L (homologue) gene product p37. This 

viral phospholipase plays a critical role in egress of 

viral particles from the host cell and the inhibition of 

this process with ST-246 reduced extracellular virus 

by 10-fold.161 ST-246 has demonstrated efficacy in 

multiple animal models of orthopoxvirus infection 

both prophylactically and therapeutically, and more 

importantly, it has prevented morbidity and mortality against monkeypox virus and prevented mortality from variola virus in nonhuman primates.162

In addition, the compound was well tolerated by 

human subjects after daily oral administration for 

21 consecutive days.163 ST-246 has been placed in 

the Strategic National Stockpile, but FDA has not 

approved it. 

Cidofovir, ST-246, CMX001, and VIG have been 

used successfully in combination to treat the adverse 

effects of vaccination. In a recent case, vaccinia virus 

mutants resistant to ST-246 were isolated from a marine who developed progressive vaccinia following 

vaccination and was subsequently treated with ST246, CMX001, and VIG.164 The patient recovered, but 

it highlighted the importance of combination therapy; 

and as stated previously, VIG is not in development 

as an antiviral against monkeypox or variola virus. 

Myriad alternative approaches to orthopoxvirus 

treatment, such as interferon mimetics, interferon beta, 

RNAi, mixantrone, and terameprocol (to name a recent 

few), are under investigation. These compounds have 

shown varying degrees of success in a wide range 

of test systems; however, none of them has reached 

the stage of clinical development for use as an antiOrthopoxvirus treatment.157,165–170

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Medical Aspects of Biological Warfare 

SUMMARY

by modification of a closely related virus such as 

camelpox, or chemical synthesis using increasingly 

powerful automated equipment.

The potential threat from smallpox specifically 

and orthopoxvirus infections in general will expand 

as the technology to create these viruses becomes 

increasingly available in laboratories around the 

world. Furthermore, scientists have been successful 

in making orthopoxviruses more virulent through 

genetic manipulation. The biodefense community 

has made considerable progress in developing new 

drugs such as ST-246 and CMX001 for treatment of 

orthopoxvirus infections and safer vaccines. There is 

still no approved treatment for smallpox; however, 

FDA approved ACAM2000 as a smallpox vaccine in 

2007. MVA, although not FDA approved, is placed in 

the Strategic National Stockpile. 

Smallpox no longer causes human disease thanks 

to the dedicated efforts of public health officials 

who participated in the WHO smallpox eradication 

program. Although the former Soviet Union participated in the eradication program, it is believed 

that the Soviets continued developing smallpox 

for biowarfare into the 1980s. The Soviet Union is 

dissolved and its offensive program has been dismantled, but the institutions and technology that 

developed this and other offensive weapons systems 

remain. Because the submission and destruction of 

smallpox virus stores was a voluntary program, it 

cannot be ascertained with certainty that smallpox 

viruses do not exist outside US and Russian storage facilities. Since the sequence of several variola 

isolates is known to a high degree of certainty, it is 

technically possible to generate viable virus either 

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154. Stittelaar KJ, Neyts J, Naesens L, et al. Antiviral treatment is more effective than smallpox vaccination upon lethal 

monkeypox virus infection. Nature. 2006;439:745–748.

155. Huggins JW, Zwiers SH, Baker RO, et al. Cidofovir treatment of variola (smallpox) in the hemorrhagic smallpox 

primate model and the IV monkeypox primate model. Antivir Res. 2003;57:A78–A78.

156. Huggins JW, Raymond JL, Fisher R, Jahrling P, Hensley L. Sequential determination of virus in blood and tissues of 

the variola cynomolgus monkey model of classical smallpox reveals that IV cidofovir can effectively treat monkeys 

with extensive viral burden. Antivir Res. 2006;70:A36–A37.

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monkeypox primate models. Antivir Res. 2004;62:A57–A58.

158. Painter W, Robertson A, Trost LC, Godkin S, Lampert B, Painter G. First pharmacokinetic and safety study in humans 

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160. Paolino K, Sande J, Perez E, et al. Eradication of disseminated adenovirus infection in a pediatric hematopoietic stem 

cell transplantation recipient using the novel antiviral agent CMX001. J Clin Virol. 2011;50:167–170.

161. Yang G, Pevear DC, Davies MH, et al. An orally bioavailable antipoxvirus compound (ST-246) inhibits extracellular 

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162. Huggins J, Goff A, Hensley L, et al. Nonhuman primates are protected from smallpox virus or monkeypox virus challenges by the antiviral drug ST-246. Antimicrob Agents Chemother. 2009;53:2620–2625.

163. Jordan R, Chinsangaram J, Bolken TC, et al. Safety and pharmacokinetics of the antiorthopoxvirus compound ST-246 

following repeat oral dosing in healthy adult subjects. Antimicrob Agents Chemother. 2010;54:2560–2566.

164. Lederman ER, Davidson W, Groff HL, et al. Progressive vaccinia: case description and laboratory-guided therapy 

with vaccinia immune globulin, ST-246, and CMX001. J Infect Dis. 2012;206:1372–1385.

165. Ahmed CM, Burkhart MA, Subramaniam PS, Mujtaba MG, Johnson HM. Peptide mimetics of gamma interferon 

possess antiviral properties against vaccinia virus and other viruses in the presence of poxvirus B8R protein. J Virol. 

2005;79:5632–5639.

166. Alkhalil A, Strand S, Mucker E, Huggins JW, Jahrling PB, Ibrahim SM. Inhibition of monkeypox virus replication by 

RNA interference. Virol J. 2009;6:188.

167. Altmann SE, Smith AL, Dyall J, et al. Inhibition of cowpox virus and monkeypox virus infection by mitoxantrone. 

Antiviral Res. 2012;93:305–308.

168. Johnston SC, Lin KL, Connor JH, Ruthel G, Goff A, Hensley LE. In vitro inhibition of monkeypox virus production 

and spread by Interferon-beta. Virol J. 2012;9:5.

169. Kinch MS, Yunus AS, Lear C, et al. FGI-104: a broad-spectrum small molecule inhibitor of viral infection. Am J Transl 

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170. Pollara JJ, Laster SM, Petty IT. Inhibition of poxvirus growth by Terameprocol, a methylated derivative of nordihydroguaiaretic acid. Antiviral Res. 2010;88:287–295.

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Chapter 25

EMERGING INFECTIOUS DISEASES 

AND FUTURE THREATS

CHRIS A. WHITEHOUSE, PhD*; BRETT BEITZEL, PhD†; ZYGMUNT F. DEMBEK, PhD, MS, MPH, LHD‡; and

ALAN L. SCHMALJOHN, PhD§

INTRODUCTION

What Are Emerging Infectious Diseases?

Factors That Contribute to Emerging Infectious Diseases

EMERGING BACTERIAL DISEASES 

Waterborne Diseases

Foodborne Diseases

Tickborne Diseases

Emerging Antibiotic Resistance

Genomic Epidemiology—Use of Whole Genome Sequencing to Track Epidemics 

of Bacterial Pathogens 

EMERGING VIRAL DISEASES 

Avian Influenza and the Threat of Pandemics

Swine Influenza and the H1N1 Influenza Pandemic, 2009

Human Infections With Avian Influenza Viruses

Influenza Viruses in Bats

Diseases Caused by Emerging Coronaviruses

Diseases Caused by Emerging Paramyxoviruses

Emerging Mosquitoborne Diseases: Dengue, West Nile, and Chickungunya

Emerging Tickborne Phleboviruses

Ebola Epidemic in West Africa

Viral Pathogen Discovery by High-Throughput DNA Sequencing

FUTURE THREATS

Genetically Engineered Organisms

Synthetic Biology 

SUMMARY

*Principal Investigator, Molecular and Translational Sciences Department, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 

Maryland 21702; formerly, Chief, Disease Investigation Branch, US Geological Survey, National Wildlife Health Center, 6006 Schroeder Road, Madison, Wisconsin

†

Principal Investigator, Center for Genomic Sciences, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

‡

Colonel (Retired), Medical Service Corps, US Army Reserve; Associate Professor, Department of Military and Emergency Medicine, Uniformed Services University 

of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814; formerly, Chief Biodefense Epidemiology and Education & Training Programs, Division 

of Medicine, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

§

Professor, Department of Microbiology and Immunology, University of Maryland School of Medicine, 655 West Baltimore Street, HSF-1, 322B, Baltimore, 

Maryland 21201 

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INTRODUCTION

Although it is controversial whether HIV/AIDS should 

be considered a zoonotic disease,4 it is now clear that 

both HIV-1 and HIV-2 had zoonotic origins.5–7 In 

addition, as was observed with the 2003 outbreak of 

monkeypox in the United States, increasing trade in 

exotic animals for pets has led to increased opportunities for pathogens to “jump” from animal reservoirs 

to humans. The use of exotic animals (eg, Himalayan 

palm civets) for food in China and the close aggregation of numerous animal species in public markets may 

have led to the emergence of the 2002–2003 outbreak 

of severe acute respiratory syndrome (SARS).8

Many of the viruses or bacteria that cause concern 

as potential bioweapons are considered emerging 

pathogens, and most are also of zoonotic origin. In 

particular, some of these agents have appeared in new 

geographical locations where they were not previously seen (eg, the sudden occurrence of monkeypox 

in the midwest of the United States in 2003, and the 

largest recorded outbreak of Marburg hemorrhagic 

fever in Angola in 2005). In some cases, the specific 

use of a pathogen in an act of bioterrorism could 

classify that pathogen as an emerging or reemerging 

disease agent, as was the case for Bacillus anthracis

during the 2001 anthrax attacks in the United States. 

Through increasingly accessible molecular biology 

techniques, completely new organisms—or significant modification of existing ones—can now be made 

in the laboratory (ie, synthetic biology). The use of 

these methods is beneficial and necessary for modern 

biomedical research to proceed. However, the same 

methods and techniques can be used for nefarious 

purposes and, along with naturally occurring emerging infections, represent significant future threats to 

both military and civilian populations. 

More than 20 years after the Institute of Medicine 

Report, much progress on emerging infectious diseases 

has been made, including a greater awareness; use of 

next-generation sequencing for the characterization of 

pathogens, vectors, and their hosts and for enhanced 

diagnostics; and increased laboratory infrastructure 

including additional biocontainment laboratories (ie, 

biosafety level 3 laboratories and biosafety level 4 laboratories) to safely work with these pathogens.9

 Despite 

this progress, new diseases continue to emerge. This 

continual emergence of new infectious diseases poses a 

continuing challenge, requiring constant surveillance, 

the ability to promptly respond with new diagnostics 

and new vaccines and drugs, and ongoing research 

into the basic biology of novel pathogens. 

What Are Emerging Infectious Diseases?

Infectious diseases have caused the deadliest pandemics in recorded human history. Some of these have 

included the Black Death (bubonic plague resulting 

in 25–40 million deaths), the 1918–1919 influenza 

pandemic (“Spanish Flu” resulting in an estimated 50 

million deaths), and the ongoing human immunodeficiency virus/acquired immunodeficiency syndrome 

(HIV/AIDS) pandemic (resulting in 35 million deaths 

to date).1

 Emerging infectious diseases, as defined in 

the landmark report by the Institute of Medicine in 

1992, are those diseases whose incidence has increased 

within the past 20 years or whose incidence threatens 

to increase in the near future.2 Although some “emerging” diseases have been now recognized for more than 

20 years (eg, HIV/AIDS, Lyme disease or Legionnaires’ 

disease), their importance has not diminished, and 

the factors associated with their emergence are still 

relevant. Emerging infections include those that are 

recognized in their host (humans, animals, or plants) 

for the first time or caused by new or newly described 

agents. Reemerging diseases are diseases that historically have infected humans, but appear in new locations, or whose incidence had previously declined, 

but now are increasing. In addition, this definition 

includes organisms that are developing antimicrobial 

resistance and established chronic diseases with a 

recently discovered infectious origin. 

Factors That Contribute to Emerging Infectious 

Diseases

Many factors contribute to the emergence of new 

diseases. In the United States, in particular, these factors include increasing population density and urbanization; immunosuppression (resulting from aging, 

malnutrition, cancer, or infection with HIV); changes 

in land use (eg, deforestation, reforestation, and 

fragmentation), climate, and weather; international 

travel and commerce; microbial or vector adaptation 

and change (mutations resulting in drug/pesticide 

resistance or increased virulence).2

 Internationally, 

many of these factors also hold true; however, many 

developing countries also have to deal with war, 

political instability, inadequate healthcare, and basic 

sanitation needs. 

The numerous examples of novel infections originating from animal species (ie, zoonoses) suggest that 

animals are an important source of emerging diseases.3

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EMERGING BACTERIAL DISEASES

by bacteriologists studying the pathogen.15 This event 

underscores the potential for cholera to cause widespread illness where water is not disinfected with a 

modern bacteriocide such as chlorine.15

In 1991, after almost a century without cholera, 

outbreaks in Latin America resulted in about 400,000 

cases of cholera and more than 4,000 deaths.16 Off 

the Peruvian coast, a significant correlation existed 

between cholera incidence and elevated sea surface 

temperature from 1997 to 2000, which included the 

1997 to 1998 El Niño event.17 Some people believe 

that the eighth worldwide pandemic began in 1992.18

During 2011, 58 countries reported a total of 589,854 

cases including 7,816 deaths from cholera.13 Cholera 

cases in the United States have decreased to about 

10 cases per year from 1995 through 2009; however, 

42 cases were reported in 2011.13 Most of these cases 

were either travel-associated or associated with consumption of undercooked seafood harvested along 

the Gulf Coast. 

Cholera occurs through fecal-oral transmission 

brought about by deterioration of sanitary conditions. 

Epidemics are strongly linked to the consumption 

of unsafe water, poor hygiene, poor sanitation, and 

crowded living conditions (Figure 25-1). Water or food 

contaminated by human waste is the major vehicle for 

disease transmission. Cholera transmission is thought 

to require 103

 organisms to exert an effect in the gut, 

with 1011 organisms as a minimum infective dose 

needed to survive stomach acid.19

Before 1992, all previous cholera pandemics were 

caused by the V cholerae serogroup O1 (classical) or El 

Tor biotypes. Large outbreaks in 1992 resulted from 

transmission of a previously unknown serogroup, V 

cholerae O139, which has since spread from India and 

Bangladesh to countries throughout Asia, including 

Pakistan, Nepal, China, Thailand, Kazakhstan, Afghanistan, and Malaysia.20

Enterotoxin produced by V cholerae O1 and O139 

can cause severe fluid loss from the gut. In severe 

cases, profuse watery diarrhea, nausea, and vomiting 

can lead to rapid dehydration, acidosis, circulatory 

collapse, and renal failure. Successful treatment of 

cholera patients depends on rapid fluid and electrolyte 

replacement. Antimicrobial therapy can also be useful. 

Mixed success has been obtained with cholera vaccines. Historically, live attenuated vaccines have been 

more effective than killed whole-cell vaccines.21 No 

licensed cholera vaccines are available in the United 

States.

Waterborne Diseases

Emerging waterborne diseases constitute a major 

health hazard in both developing and developed 

countries. From 2007 to 2008, 48 disease outbreaks 

associated with contaminated drinking water were 

reported in the United States, resulting in 4,128 ill 

people and 3 deaths.10 During this same time, more 

than 13,966 cases of illness were associated with 134 

recreational water-associated outbreaks of disease.11

Although these numbers represent disease caused 

by a range of pathogenic organisms (ie, viruses, 

bacteria, parasites), the majority (58%) of drinking 

water-associated outbreaks were caused by bacterial 

pathogens. Bacterial pathogens associated with drinking water disease outbreaks included Legionella (12 

outbreaks), Campylobacter (4 outbreaks), Salmonella (3 

outbreaks), Escherichia coli O157:H7 and Providencia (1 

outbreak each).10 Bacterial pathogens were responsible 

for 21% of the outbreaks of disease associated with 

recreational water exposure. Diseases associated with 

exposure to recreational water are more diverse than 

those associated with drinking water and include acute 

gastrointestinal illness, acute respiratory illness, and 

dermatologic illness. Accordingly, the list of bacterial 

pathogens responsible is more diverse and includes 

E coli O157:H7, Shigella sonnei, Legionella, Plesiomonas 

shigelloides, and Vibrio vulnificus. Dermatitis outbreaks 

were most often attributed to Pseudomonas, primarily P aeruginosa.

12 Internationally, cholera (caused by 

Vibrio cholerae) is still a major killer, as demonstrated 

by recent large outbreaks in Sierra Leone, Zimbabwe, 

and Democratic Republic of Congo in Africa resulting in more than 120,000 cases combined and more 

than double that number of cases in Haiti following 

a massive earthquake in 2010 (see Cholera and Vibrio 

cholerae). 

Cholera and Vibrio cholerae

Cholera is one of the most rapidly fatal diseases 

known, capable of killing within 12 to 24 hours after 

onset of diarrhea. The World Health Organization 

(WHO) estimates 3 to 5 million cholera illnesses and 

up to 130,000 deaths occur globally each year.13 Cholera 

accounts date back to Hippocrates.14 Seven worldwide 

cholera pandemics have occurred. An 1892 cholera 

outbreak in Hamburg, Germany, affecting 17,000 

people and causing 8,605 deaths, was attributed to the 

inadvertent contamination of the city’s water supply 

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Medical Aspects of Biological Warfare 

On January 12, 2010, a magnitude 7.0 earthquake 

decimated the island nation of Haiti, leaving a quarter 

of a million people dead, 300,000 injured, and 2 million 

homeless. Nine months later, in October, a cholera 

outbreak was confirmed in Haiti,22 indicating the first 

occurrence of cholera in Haiti in at least 100 years. 

Some have suggested that cholera may never have been 

in Haiti before 2010.23 Based on epidemiological data, 

the cholera outbreak began in the upstream region of 

the Artibonite River (Figure 25-2).24 The presumed first 

case was a 28-year-old man with a history of severe 

untreated psychiatric disease.25 The patient had a history of wandering nude through town throughout the 

day and both bathing in and drinking the water from 

the Latem River, one of the tributaries of the Artibonite River. On October 12, 2010, he developed acute 

onset of profuse watery diarrhea. In less than 24 hours 

after the onset of symptoms, he died at home without 

seeking medical attention. The first hospitalized case 

of cholera in Haiti occurred at the Mirebalais Government Hospital on October 17, 2010.24

By mid-November, cholera had spread to every part 

of the country and to neighboring Dominican Republic, and by mid-December a total of 121,518 cases of 

cholera, resulting in 63,711 hospitalizations and 2,591 

deaths, had been reported from Haiti.22 The cholera 

outbreak in Haiti has continued since 2010 with more 

than 734,983 cases and 8,761 deaths as of April 3, 2015, 

according to the Pan American Health Organization.26

The outbreak strain was identified as V cholerae O1, 

serotype Ogawa, biotype El Tor.27 Whole genome DNA 

sequencing and epidemiological analysis confirmed 

that the outbreak strain was inadvertently introduced 

into Haiti by United Nations security forces from 

Nepal.28–30

A cholera outbreak was reported in Kathmandu 

(Nepal’s capital city) on September 23, 2010, shortly 

before troops left for Haiti.31 The first cholera cases 

in Haiti came from a village named Meye, located 

150 meters downstream from the Nepalese military 

camp.24,30 Taken together, evidence strongly supports 

the conclusion that the United Nations military camp, 

housing the Nepalese peacekeeping troops in Meye, 

was the source of the Haitian cholera epidemic. These 

findings led to considerable political unrest and have 

forever changed the global response to natural disasters. In late 2013, survivors and family members of the 

nearly 700,000 Haitians who contracted cholera sued the 

United Nations, accusing them of covering up its role 

in starting the cholera outbreak in Haiti. In early 2015, 

a US federal judge ruled that the Haitians could not sue 

the United Nations because the organization has legal 

Figure 25-1. Typical conditions that can lead to a cholera 

epidemic. This photograph was taken in 1974 during a 

cholera research and nutrition survey amidst floodwaters 

in Bangladesh. 

Photograph: Courtesy of Dr Jack Weissman, Centers for 

Disease Control and Prevention Public Health Image Library.

Figure 25-2. The Artibonite River is the longest and most 

important river in Haiti. It forms part of the international 

border between Haiti and the Dominican Republic and 

empties into the Gulf of Gonâve. It is believed that the 2010 

cholera outbreak began in the upstream region of this river. 

Photograph: Courtesy of Kendra Helmer, US Agency for 

International Development. 

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immunity against lawsuits. In August 2016, the Court 

upheld the United Nations’ immunity from claims 

(http://www.ijdh.org/cholera/cholera-litigation/).

Other Vibrioses

In recent years, some noncholera vibrios have 

acquired increasing importance because of their association with human disease. More than 70 members 

are in the family Vibrionaceae, 12 of which have been 

isolated from human clinical specimens and apparently are pathogenic for humans.32 Vibrio species are 

primarily aquatic and common in marine and estuarine 

environments and on the surface and in the intestinal 

tracts of marine animals. V parahemolyticus and V 

vulnificus are halophilic vibrios commonly associated 

with consumption of undercooked seafood. Diarrhea, 

cramping, nausea, vomiting, fever, and headache are 

commonly associated with V parahemolyticus infections. 

Cases of diarrhea related to seafood consumption 

increased worldwide with the emergence of pandemic 

strain O3:K6, which was originally observed in Southeast Asia.33 V vulnificus is the most common source 

of vibrio infections in the United States resulting in 

gastrointestinal symptoms similar to V parahemolyticus, 

but may also lead to ulcerative skin infections if open 

wounds are exposed to contaminated water. Septicemia can occur in those infected with V vulnificus who 

are immunosuppressed or have liver disease or chronic 

alcoholism, and septicemic patients can have a mortality rate of up to 50%. In most cases the disease begins 

several days after the patient has eaten raw oysters. 

Other human pathogenic species include V mimicus, V 

metschnikovii, V cincinnatiensis, V hollisae, V damsela, V 

fluvialis, V furnissii, V alginolyticus, and V harveyi; most 

of these have been associated with sporadic diarrhea, 

septicemia, and wound infections.32

Legionellosis

Legionnaires’ disease was first recognized in 1976 

after a large outbreak of severe pneumonia occurred 

among attendees at a convention of war veterans in 

Philadelphia. A total of 182 people, all members of 

the Pennsylvania American Legion, developed an 

acute respiratory illness, and 29 individuals died from 

the disease.34 The cause of the outbreak remained a 

mystery for 6 months until the discovery by Joseph 

McDade, a Centers for Disease Control and Prevention (CDC) microbiologist, of a few gram-negative 

bacilli, subsequently named Legionella pneumophila,

35

in a gram stain of tissue from a guinea pig inoculated 

with lung tissue from a patient who died from the 

disease.36 Using the indirect immunofluorescence 

assay, McDade showed that the sera of patients from 

the convention mounted an antibody response against 

the newly isolated bacterium,36 marking the discovery 

of a whole new family of pathogenic bacteria. Retrospective analysis, however, showed that outbreaks of 

acute respiratory disease from as far back as 1957 have 

now been attributed to L pneumophia.

37,38 The earliest 

recorded isolate of a Legionella species was recovered 

by Hugh Tatlock in 1943 during an outbreak of Fort 

Bragg fever.39,40

Legionnaires’ disease is normally acquired by 

inhalation or aspiration of L pneumophila or other 

closely related Legionella species. Water is the major 

reservoir for legionellae, and the bacteria are found 

in freshwater environments worldwide. Legionnaires’ 

disease has been associated with various water sources 

where bacterial growth is permitted, including cooling towers,41 whirlpool spas,42 and grocery store mist 

machines.42 The association between a portable shower 

and nosocomial legionellosis was demonstrated more 

than 30 years ago.43 The most common source of legionellosis in hospitals is from the hot water system,44

and sustained transmission of Legionnaires’ disease 

in the hospital environment can be difficult to control.42 Community-acquired legionellosis is thought 

to account for most infections.45 An Italian survey of 

household hot water systems in 2000 found bacterial 

contamination, with Legionella species in 23% of the 

homes and Pseudomonas species in 38%. One Legionella

species, L longbeachae, has been associated with disease 

transmission from potting soil.16

Legionnaires’ disease is an acute bacterial illness 

that initially presents with anorexia, malaise, myalgia, 

and headache, with a rapidly rising fever and chills. 

Temperatures commonly reach 102°F to 105°F and are 

associated with nonproductive cough, abdominal pain, 

and diarrhea. The disease may eventually progress to 

respiratory failure and has a case-fatality rate as high 

as 39% in hospitalized cases. Nonpneumonic legionellosis, or Pontiac fever, occurs after exposure to aerosols 

of water colonized with Legionella species.46–48 Attack 

rates after exposure to an aerosol-generating source, 

which often range from 50% to 80%, are exceptionally 

high. After a typical asymptomatic interval of 12 to 48 

hours after exposure, patients note the abrupt onset of 

fever, chills, headache, malaise, and myalgias. Pneumonia is absent and those who are affected recover 

in 2 to 7 days without receiving specific treatment.49

Legionella is now recognized around the world as an 

important cause of community-acquired and hospitalacquired pneumonia, occurring both sporadically and 

in outbreaks. Although 90% of Legionella infections 

in humans are caused by L pneumophila, there are 50 

named species of Legionella, with approximately 20 

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known to cause human infections.50 Some unusual 

strains of bacteria, which infect amoebae and have been 

termed Legionella-like amoebal pathogens (LLAPs), 

appear to be closely related to Legionella species on 

the basis of 16S ribosomal RNA gene sequencing.51,52

Three LLAP strains are now named Legionella species53; 

one of them, LLAP-3, which was first isolated from the 

sputum of a patient with pneumonia by coculture with 

amoebae, is considered a human pathogen.54

Foodborne Diseases

More than 200 diseases are transmitted through 

food, including illnesses resulting from viruses, bacteria, parasites, toxins, metals, and prions. In the United 

States, the burden of foodborne illness is estimated at 

approximately 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths each year.55 Among the bacterial 

pathogens estimated to cause the greatest number of 

US foodborne illnesses are Campylobacter, Salmonella, 

Shigella, Clostridium, and Staphylococcus.

55 Emerging 

bacterial illnesses include E coli O157:H7 and other 

enterhemorrhagic and enterotoxigenic E coli, as well 

as antibiotic resistant bacteria. Many of the pathogens 

of greatest concern today (eg, C jejuni, E coli O157:H7, 

Listeria monocytogenes, Cyclospora cayetanensis) were not 

recognized as causes of foodborne illness just 20 years 

ago. Other rare pathogens, such as E coli O104:H4, recently emerged as a cause of a foodborne outbreak of hemolytic uremic syndrome (HUS) in northern Germany 

resulting in more than 4,000 cases and 54 deaths (see 

section on Disease Caused by Escherichia coli O104:H4). 

The majority of gastrointestinal illnesses are caused 

by foodborne agents not yet identified. It is estimated 

that 62 million foodborne-related illnesses and 3,200 

deaths occur in the United States each year from unknown pathogens.55 Bacillus anthracis, although rarely 

seen as a gastrointestinal illness in the United States, 

has become a concern since cases occurred in 2000 

and 2009 (see next section). Even in areas of the world 

where gastrointestinal anthrax is more common, the 

oropharyngeal form is underreported because physicians are unfamiliar with it.56 Unreported foodborne 

disease, deaths from unknown food agents,57 and 

chronic sequelae58 may be a huge unrecognized burden of illness.

Gastrointestinal Anthrax

Bacillus anthracis is the causative agent of anthrax, 

a naturally occurring zoonotic disease. The greatest 

bioweapons threat from anthrax is through aerosol 

dispersion and subsequent inhalation of concentrated 

spores (for more details, see Chapter 6, Anthrax). Gastrointestinal anthrax, however, is contracted through 

the ingestion of B anthracis spores in contaminated 

food or water. This form of the disease occurs more 

commonly than inhalational anthrax in the developing 

world, but it is rare in the United States and other developed nations.56,59 In one large outbreak in Uganda, 

155 villagers ate the meat of a zebu (bovine) that had 

died of an unknown disease. Within 15 to 72 hours, 

143 (92%) persons developed presumed anthrax. Of 

these, 91% had gastrointestinal complaints and 9% 

oropharyngeal edema; nine of them—all children—

died within 48 hours of illness onset.59

Although rare in the United States, gastrointestinal anthrax does occur naturally, and anthraxcontaminated meat was found to be associated with 

gastrointestinal illness in Minnesota in 2000.16 Another 

case occurred in 2009 from exposure to animal-hide 

drums.22 Purposeful contamination of food or water 

is possible, but it would require a high infective dose. 

Misdiagnosis of gastrointestinal anthrax may lead 

to a higher mortality than other forms of anthrax; 

thus awareness of this disease remains important in 

anthrax-endemic areas and in the setting of possible 

bioterrorism. 

Disease Caused by Campylobacter jejuni

Campylobacter was first identified in 1909 (then 

called Vibrio fetus) from the placentas and aborted 

fetuses of cattle. The organism was not isolated from 

humans until nearly 40 years later when it was found in 

the blood of a pregnant woman who had an infectious 

abortion in 1947.60 Campylobacter jejuni (Figure 25-3), 

Figure 25-3. Scanning electron microscope image of Campylobacter jejuni illustrating its corkscrew appearance. Magnification ×11,734. 

Photograph: Courtesy of Janice Carr, Centers for Disease 

Control and Prevention Public Health Image Library.

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along with C coli, have been recognized as agents of 

gastrointestinal infection since the late 1970s. Today, C 

jejuni is considered the most commonly reported foodborne bacterial pathogen in the United States, affecting 

2.4 million persons annually.61 Campylobacteriosis is 

an enteric illness of variable severity including diarrhea (which may be bloody), abdominal pain, malaise, 

fever, nausea, and vomiting occurring 2 to 5 days after 

exposure. Many infections are asymptomatic; however, 

infection with this pathogen has also been associated 

with development of Guillain-Barré syndrome and 

arthritis.62,63 Infants are more susceptible to C jejuni

infections upon first exposure.64 Persons who recover 

from C jejuni infection develop immunity. Poultry 

colonized with Campylobacter species is a major source 

of infections for humans.65–67 The reported incidence of 

Campylobacter species on poultry carcasses has varied, 

but has been as high as 100%.66

Several virulence properties, including motility, 

adherence, invasion, and toxin production, have been 

recognized in C jejuni.

68 Along with several other 

enteric bacteria, C jejuni produces a toxin called cytolethal distending toxin that works by a completely 

novel mechanism; mammalian cells exposed to the 

toxin distend to almost 10 times their normal size from 

a molecular blockage in their cell cycle.69 Although 

cytolethal distending toxin is the best characterized 

Campylobacter toxin, its role in the pathogenesis of human campylobacteriosis is still unclear.70

Because illness from Campylobacter infection is generally self-limited, no treatment other than rehydration 

and electrolyte replacement is generally recommended. 

However, in more severe cases (ie, high fever, bloody 

diarrhea, or septicemia), antibiotic therapy can be used 

to shorten the duration of symptoms if it is given early 

in the illness. Because infection with C jejuni in pregnant 

women may have deleterious effects on the fetus, infected pregnant women receive antimicrobial treatment. 

Erythromycin, the drug of choice for C jejuni infections, 

is safe, lacks serious toxicity, and is easy to administer. 

However, most clinical trials performed in adults or 

children have not found that erythromycin significantly 

alters the clinical course of Campylobacter infections.71,72

Other antimicrobial agents, particularly the quinolones 

(eg, fluoroquinolones such as ciprofloxacin) and newer 

macrolides including azithromycin are also being used. 

Unfortunately, as the use of fluoroquinolones has expanded (especially in food animals), the rate of resistance 

of campylobacters to these agents has increased.73 For 

example, a 1994 study found that most clinical isolates 

of C jejuni from US troops in Thailand were resistant to 

ciprofloxacin. Additionally, nearly one-third of isolates 

from US troops located in Hat Yai were resistant to 

azithromycin.74 In another study conducted in 1997 in 

Minnesota, 13 (14%) of 91 chicken products purchased 

in grocery stores were contaminated with ciprofloxacin-resistant C jejuni,

75 illustrating the need for more 

prudent antimicrobial use in food–animal production.

Disease Caused by Clostridium botulinum

Clostridium botulinum produces botulinum toxin, 

which causes the clinical manifestations of botulism. 

Botulinum toxin, with a lethal dose of about 1 μg/kg, is 

the most potent of the natural toxins.76 There are seven 

antigenic types of toxin, designated A through G with 

most human disease caused by types A, B, and E. Botulinum toxins A and B are most often associated with 

home canning and home-prepared foods, whereas 

botulinum toxin E is exclusively associated with ingestion of aquatic animals. Interestingly, the incidence of 

botulism in Alaska is among the highest in the world, 

and all cases of foodborne botulism in Alaska have 

been associated with eating traditional Alaska native 

foods, mostly from marine mammals; most of these 

cases were caused by toxin type E.77 From 1990 to 2000, 

160 foodborne botulism events affected 263 persons in 

the United States. Of these, 67 required intubation, and 

11 deaths occurred.77 Food items commonly associated 

with botulinum intoxication included homemade salsa 

and home-bottled garlic in oil. 

Clinical illness is characterized by cranial nerve 

palsies, followed by symmetric descending flaccid 

muscle paralysis, which may involve the respiratory 

muscles. Full recovery may take weeks to months. 

Therapy includes intensive care support, mechanical 

ventilation as necessary, and timely administration 

of equine antitoxin.78 See Chapter 14 for an in-depth 

discussion of the botulinum toxin.

Disease Caused by Escherichia coli O157:H7

Diarrheagenic E coli strains are important causes of 

diarrhea in humans. These strains have been divided 

into different pathotypes, according to their virulence 

attributes and mechanisms involved in the disease 

process. The major groups of intestinal pathogenic 

E coli strains include enteropathogenic E coli (EPEC), 

enteroaggregative E coli (EAEC), enterotoxigenic E coli, 

enteroinvasive E coli (EIEC), and enterohemorrhagic E 

coli.

79 Sometimes enterohemorrhagic E coli are known 

as Shiga toxin-producing E coli (STEC) and can be 

spread by food or water. 

One STEC strain in particular, E coli O157:H7, has 

emerged as a cause of serious pediatric illness worldwide. Production of Shiga toxins depends on the presence of stx genes, located in the bacterial genome on 

lambdoid prophages, which are classified as mobile 

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genetic elements. It is these intrinsic Shiga toxins that 

can initiate a cascade of events that includes bloody 

diarrhea and HUS (exhibited by microangiopathic 

hemolytic anemia), acute renal failure, and thrombocytopenia.80 HUS occurs in about 4% of all reported cases, 

and those younger than 5 years of age are at greatest 

risk for HUS subsequent to E coli O157:H7 infection.55

The mortality rate for HUS is 3% to 5% and about 5% 

of the survivors have severe consequences, including end stage renal disease or permanent neurologic 

damage.81 Antibiotic treatment of E coli O157:H7 is 

not recommended.82 There is anecdotal evidence for 

an increase in the risk of HUS with the use of some 

antimicrobial agents. However, conclusive proof of 

this occurrence is lacking. Fluid replacement is the 

cornerstone of the treatment of diarrheal illness caused 

by enterohemorrhagic E coli.

The primary source of E coli O157:H7 is beef cattle. 

Current animal agricultural practices of grain (rather 

than hay) feeding of these animals decreases the pH 

in the colon, thereby promoting acid resistance in the 

bacteria and enhanced growth promotion for E coli

pathogens.83

Disease Caused by Escherichia coli O104:H4

In May through June of 2011, two separate outbreaks 

of bloody diarrhea and HUS occurred in Europe. One 

was centered in Germany and comprised 3,816 cases 

of bloody diarrhea, 845 cases of HUS and 54 deaths; 

whereas, the other occurred in France and comprised 

15 cases of bloody diarrhea, 9 of which progressed to 

HUS.84,85 These cases, however, were not caused by E 

coli O157:H7. These outbreaks were caused by a more 

virulent form of Shiga toxin-producing E coli called E coli

O104:H4 and represented the highest frequency of HUS 

and death recorded from an STEC strain. Epidemiological investigation determined that contaminated sprouts 

were the source of the outbreak and was a consequence 

of tainted fenugreek seeds from an exporter in Egypt 

that were obtained by a German seed distributor supplying a German sprout farm.84 Tainted water may have 

led to contamination of sprout seeds that were exported 

from Egypt and distributed to farms in Europe. A portion of the original seed shipment was also sent to an 

English seed distributor, which repackaged the seeds 

and supplied them to French garden stores, leading to 

the outbreak in France.86

Disease Caused by Salmonella Species

Salmonella species infect an estimated 1.4 million 

persons annually in the United States. Severe infections are not uncommon, although most infections 

are self-limiting with diarrhea, vomiting, abdominal 

cramps, and fever. Estimates suggest that approximately 15,000 people are hospitalized and more than 

500 deaths occur each year from Salmonella infections.55

Food animals are the primary reservoir for human 

nontyphoidal Salmonella infections. Thousands of 

Salmonella serotypes exist, and many naturally inhabit 

the avian, mammalian, and reptilian gastrointestinal 

tracts. Poultry is the main source of the salmonellae in 

the food supply, but other vehicles for disease transmission include raw salads, milk, water, and shellfish. 

Infection with many Salmonella serotypes cause gastroenteritis with associated diarrhea, vomiting, febrile 

illness, headache, and dehydration. Septicemia, enteric 

fever, and localized infections may also evolve from 

Salmonella infection. The most highly pathogenic of 

the salmonellae is S typhi, which causes typhoid fever, 

for which symptoms include septicemia, high fever, 

headache, and gastrointestinal illness. S typhimurium

was the pathogen used in 1984 by an Oregon cult with 

intent to make people ill by deliberate contamination of 

salad bars.87 More than 750 cases of illness resulted, but 

no deaths occurred, which may have not been the case 

had S typhi been chosen as the pathogenic biological 

weapon. A 1985 salmonellosis outbreak affecting more 

than 16,000 persons caused by cross-contamination of 

pasteurized with unpasteurized milk demonstrates the 

potential for large-scale illness caused by the salmonellae in the current food distribution system.88

Tickborne Diseases

Borreliosis

Lyme arthritis, as a distinct clinical entity, was 

recognized as early as 1972 in residents of three communities in eastern Connecticut.89 Lyme disease or 

Lyme borreliosis is now the most commonly reported 

arthropod-borne illness in North America and Europe. 

In 1981, Dr Willy Burgdorfer and colleagues at the 

Rocky Mountain Laboratories in Hamilton, Montana, 

first observed spirochetes in adult deer ticks (Ixodes 

scapularis; then called Ixodes dammini) collected from 

vegetation on Shelter Island, New York, a known 

endemic focus of Lyme disease.90 The bacteria were 

shown to react specifically with antibodies from 

Lyme disease patients,90,91 and later, spirochetes were 

isolated from the blood of two patients with Lyme 

disease,92 proving the infection’s spirochetal etiology.91

The spirochetes were later named, Borrelia burgdorferi

(Figure 25-4), after Dr Burgdorfer. I scapularis (Figure 

25-5) is now considered the primary vector of Lyme 

disease in the northeastern and north central United 

States. Other vectors are closely related ixodid ticks, 

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Emerging Infectious Diseases and Future Threats

including I pacificus in the western United States, I 

ricinus in Europe, and I persulcatus in Asia. Based 

on genotyping of bacterial isolates, B burgdorferi has 

now been subdivided into multiple Borrelia species or 

genospecies.93 In North America, all strains belong to 

the first group, B burgdorferi sensu stricto. This species, 

along with two others, B afzelii and B garinii, are found 

in Europe, although most of the disease there results 

from the latter two species. Also, interestingly, only B 

afzelii and B garinii seem to be associated with the illness in Asia.93,94 B japonica, which was isolated in Japan, 

is not known to cause human disease.95

Lyme disease evolves from a red macule or papule 

that expands annularly like a bulls-eye rash, known 

as erythema migrans, which may exhibit as a single 

lesion or as multiple lesions. However, the erythema 

migrans rash does not occur in all Lyme disease cases. 

Early systemic manifestations can include malaise, 

fatigue, fever, headache, stiff neck, myalgia, migratory 

arthralgias, and lymphadenopathy, which may last for 

several weeks if untreated. In weeks to months after 

erythema migrans onset, neurological abnormalities 

may develop, including facial palsy, chorea, cerebellar ataxia, motor or sensory radiculoneuritis, myelitis, 

and encephalitis; these symptoms fluctuate and may 

become chronic. Cardiac abnormalities and chronic 

arthritis also may result.82

Surveillance for Lyme disease in the United States 

began in 1982, and it was designated a nationally 

notifiable disease in 1991. Since then, the number of 

reported cases has increased steadily with 17,029 cases 

reported in 2001.96 In 2002, 23,763 cases were reported, 

an increase of 40% from the previous year.96 In 2015, approximately 300,000 people were diagnosed with Lyme 

disease in the United States. As with other tickborne 

diseases, this continued emergence of Lyme disease 

underscores the need for persons living in endemic 

areas to reduce their risk for infection through proper 

pest management, landscaping practices, repellent use, 

and prompt removal of ticks. 

A newly recognized tick-transmitted disease that 

produces a rash (erythema migrans) similar to, and 

often indistinguishable from, that seen in Lyme disease has been identified in the southeastern and south 

central United States.97–99 Unlike Lyme disease, however, symptoms develop following the bite of a lone 

star tick, Amblyomma americanum (Figure 25-6). The 

disease is named southern tick-associated rash illness,

Figure 25-4. Darkfield photomicrograph of the Lyme disease 

spirochete, Borrelia burgdorferi, magnified 400x. 

Photograph: Courtesy of Centers for Disease Control and 

Prevention Public Health Image Library.

Figure 25-5. Ixodes scapularis tick, also called the blacklegged tick, is found on a wide range of hosts and is considered the main vector of the Lyme disease spirochete, 

Borrelia burgdorferi. I scapularis is also a vector of Anaplasma 

phagocytophilum and Babesia microtii, the causative agents of 

human granulocytic ehrlichiosis and babesiosis, respectively. 

Photograph: Courtesy of James Gathany and provided by 

Michael L Levin, PhD, Centers for Disease Control and Prevention Public Health Image Library. Image 1669.

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Medical Aspects of Biological Warfare 

but it has also been referred to as Master’s disease, or 

southern Lyme disease. A americanum ticks are not 

known to be competent vectors of B burgdorferi, and 

serological testing for Lyme disease in southern tickassociated rash illness patients are typically negative, 

despite microscopic evidence of spirochetes in biopsy 

samples. Physicians and researchers speculated that 

a new tick-associated spirochete may be responsible. 

Subsequently, molecular evidence of a novel Borrelia

species was reported from A americanum ticks, from 

white-tailed deer, and from the skin of a patient with 

southern tick-associated rash illness.100–103 The organism, named Borrelia lonestari, was initially described 

only by polymerase chain reaction (PCR) amplification of the flagellin B gene (fla B) and 16S ribosomal 

DNA,104 but it has now been isolated in culture and 

more extensively studied.105

Another new tickborne Borrelia species has emerged 

to cause disease in humans. A novel Borrelia species 

was first isolated from ixodid ticks from Japan in 1995 

and named B miyamotoi.

106 Subsequently, the bacterium 

was detected in ixodid ticks from North America107,108

and Europe.109,110 In 2011, B miyamotoi infection was 

detected in 46 patients from Russia.111 All patients 

reported recent tick bite and were hospitalized with 

influenza-like illness with fever, headache, fatigue, 

myalgia, proteinuria, and elevated hepatic aminotransferase levels.111 Cases were first described in North 

America in 2013.112,113 All cases in North America have 

been in persons living in Lyme disease-endemic regions of the northeastern United States. Interestingly, 

B miyamotoi is genetically more similar to the tickborne 

relapsing-fever borreliae, which are transmitted by soft 

(argasid) ticks, not hard (ixodid) ticks. Some patients 

infected with B miyamotoi have even presented with 

clinical symptoms of relapsing fever.111

True relapsing fever borreliae have been known for 

many decades and are transmitted by ticks or lice. Relapsing fever caused by the spirochete B recurrentis can 

be transmitted by the body louse Pediculus humanus. B 

hermsii, which is the causative agent of tickborne relapsing fever, is transmitted by the soft tick Ornithodoros 

hermsi.

114 The disease results in fever lasting 2 to 9 days 

with 1 to 10 relapses. Although the total duration of 

louseborne disease usually averages 13 to 16 days, the 

tickborne disease is often longer. Gastrointestinal and 

respiratory involvement is common. Neuropsychiatric 

symptoms have also occurred.82 Relapsing fever was 

first reported in the United States in 1915115 and normally occurs in the higher elevations of the western 

United States and southern British Columbia (Canada). 

A tickborne relapsing fever outbreak occurred for the 

first time in Montana in 2002 among five persons visiting 

a cabin in the western part of the state.114 Spirochetes 

were isolated from two of the patients and were identified as B hermsii and O hermsi ticks were collected 

from the cabin where the patients slept. This was the 

first report of both B hermsii and O hermsi in Montana, 

suggesting the risk of infection may be expanding 

beyond the previously recognized geographic range.

Anaplasmosis/Ehrlichiosis

Human granulocytic anaplasmosis is caused by 

infection with Anaplasma phagocytophilum, whereas the 

agent of human monocytotropic ehrlichiosis is Ehrlichia 

chaffeensis. Monocytotropic ehrlichiosis occurs in rural 

and suburban areas south of New Jersey to Kansas 

and in California, while granulocytic anaplasmosis 

occurs in areas where Lyme disease is endemic.82 The A 

americanum tick (see Figure 25-6) transmits E chaffeensis, 

while I scapularis (see Figure 25-5), the Lyme disease 

vector, also transmits A phagocytophilum. A spectrum 

of mild-to-severe, life-threatening, or fatal disease occurs with anaplasmosis. About 20% of patients have 

meningoencephalitis. Infection with A phagocytophilum

Figure 25-6. A female lone star tick, Amblyomma americanum, 

found throughout the southeastern United States. These 

ticks are considered the main vectors of Ehrlichia chaffeensis

and Borrelia lonestari, the agents of human monocytotropic 

ehrlichiosis and southern tick-associated rash illness, respectively. 

Photograph: Courtesy of James Gathany and provided by 

Michael L Levin, PhD, Centers for Disease Control and Prevention Public Health Image Library. Image 4407.

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is characterized by acute and often self-limited fever, 

malaise, myalgia, thrombocytopenia, leucopenia, and 

increased hepatic transaminases.82 Illness ranges from 

mild to severe, with less than 1% case fatality.

As the I scapularis tick is the vector for transmission 

of B burgdorferi, B miyamotoi, A phagocytophilum, and B 

microti, coinfections of Lyme disease (and Lyme-like 

disease), anaplasmosis, and babesiosis (caused by the 

protozoan Babesia microti) can occur from the bite of 

this tick. In the United States, ticks of the Ixodes genus 

can transmit all of these diseases as well as the viral 

pathogens Powassan virus and the related deer–tick 

virus.82,116 Coinfections with babesiosis and Lyme 

disease are known at times to increase the severity of 

both diseases.82

Emerging Antibiotic Resistance 

Antimicrobial resistance is not a new phenomenon. 

Sulfonamide-resistant Streptococcus pyogenes emerged 

in military hospitals in the 1930s, and penicillinresistant Staphylococcus aureus appeared in London 

civilian hospitals soon after the introduction of penicillin in the 1940s.117 However, the number of resistant 

organisms, the geographic regions affected by drug 

resistance, and the number of bacterial species that 

are multidrug resistant (MDR) is increasing. Since the 

1980s, a reemergence of tuberculosis has occurred that 

is often caused by MDR Mycobacterium tuberculosis118

and requires the use of several—sometimes six to seven 

different—drugs to treat.119 After initial reports in 2006 

from South Africa of extensively drug-resistant tuberculosis (defined as tuberculosis caused by strains of M 

tuberculosis resistant to rifampicin, isoniazid, fluoroquinolones, and any of the second-line injectable drugs 

such as capreomycin, amikacin, and kanamycin), the 

number of countries reporting cases of extensively 

drug-resistant tuberculosis has increased to at least 

84.120 Additionally, cases of vaguely defined totally 

drug-resistant tuberculosis have been reported.121,122

Other notable examples of MDR strains worldwide 

include Enterococcus faecium, Enterobacter cloacae, Klebsiella pneumoniae, S aureus, Acinetobacter baumannii, and 

P aeruginosa.

117 In developing countries, MDR enteric 

bacteria such as Salmonella enteritidis, Shigella flexneri, 

and V cholerae are major threats to public health.

Salmonella antibiotic resistance has emerged to 

become a serious concern in agriculture as well as 

patient management.73,123,124 Antibiotic resistance 

in E coli O157:H7 has been shown to occur rapidly 

following exposure to various antibiotics, including 

triclosan, chloramphenicol, erythromycin, imipenem, 

tetracycline, and trimethoprim, as well as to a number 

of biocides.125 

Few antibiotics are more potent than vancomycin. 

The emergence of microbial vancomycin resistance 

continues to be of increasing concern to clinicians and 

public health professionals, and surveillance systems 

have been instituted to monitor these pathogens.126

S aureus is an important cause of illness and death 

and accounts for about one-fifth of bacteremia cases 

in the United States.127 The discovery of vancomycin 

resistance in S aureus clinical isolates could portend 

the end of the antibiotic era in medicine.42,75

Both hospital and home healthcare patients are 

significantly affected by the growing emergence of 

antibiotic resistance.127,128 Restrictive guidelines have 

therefore been developed for the use of vancomycin 

and other glycopeptide antimicrobials. These guidelines include a recommendation against the routine 

use of vancomycin as perioperative antibiotic prophylaxis for surgical site infections.129 Vancomycinintermediate resistance among S aureus has also 

been identified, and subsequent guidance has been 

developed for their identification and control of 

transmission.42

The carbapenem class of antimicrobials, which 

comprises imipenem, meropenem, ertapenem, and 

doripenem, is often the last resort for the safe and effective treatment of infections caused by MDR gramnegative bacteria, including the extended-spectrum 

β-lactamase (ESBL)-producing Enterobacteriaceae. 

Resistance to carbapenems occurs through several 

mechanisms, including the production of carbapenemases. The vast majority of acquired carbapenemases belong to one of three classes of β-lactamases, 

namely class B (metallo-β-lactamases) or classes A 

and D (serine carbapenemases). The class A group 

includes Klebsiella pneumoniae carbapenemase (KPC), 

which is currently the most common carbapenemase 

and was first detected in North Carolina in 1996 and 

has since spread worldwide.130 KPC made headlines 

when it caused an outbreak among 18 patients at 

the National Institutes of Health Clinical Center in 

2011.131 Six of the patients died from their infections. 

The use of genomic sequencing to determine the 

source of this outbreak illustrates the application 

of this technique in epidemiological investigations 

(see next section). 

The past few years have seen an emergence of 

a new type of carbapenemase, designated New 

Delhi metallo-β-lactamase-1 (NDM-1). It was first 

described in 2009 in Klebsiella pneumoniae (Figure 

25-7) isolated from a patient receiving treatment 

for a urinary tract infection in a Swedish hospital, 

but who was of Indian origin and had previously 

received medical care in New Delhi, India.132 Since 

this first reported case in 2009, NDM-1 producing 

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bacteria have rapidly spread to every continent 

except for Central and South America. In most of 

these cases, patients had been hospitalized in India, 

Pakistan, or Bangladesh, or had spent some time in 

that part of the world. It therefore suggests that the 

Indian subcontinent is currently the main reservoir 

of NDM-1 producers.133

A substantial number of patients have been part 

of the growing phenomenon of “medical tourism” 

resulting from delays for medical interventions 

such as hip and knee replacements, spinal surgery, 

and ophthalmologic procedures. It is estimated that 

in 2012 as many as 1.6 million Americans received 

healthcare outside of the United States.134 Many of 

these medical tourists undergo such procedures in 

India, which may put them at risk of contracting 

NDM-1 strains of bacteria. NDM-1 has been identified 

mostly in E coli and K pneumoniae, in many cases in 

strains that are already MDR, making these bacterial pathogens resistant to virtually every clinically 

available antibiotic.

This is even more alarming considering the decreasing number of potentially new antibiotics that have 

come through the pharmaceutical pipeline in recent 

decades. The reasons for this decline are many and 

diverse.135 Some of these reasons include the nature of 

antibiotic use, which is typically short term, compared 

to other drugs; the drug’s uncertain future because of 

the constantly evolving nature of antibiotic resistance; 

and governmental over-regulation. Thus, appropriate 

antibiotic use will continue to be an important issue 

for clinicians and epidemiologists for the foreseeable 

future.136

Genomic Epidemiology—Use of Whole Genome 

Sequencing to Track Epidemics of Bacterial 

Pathogens 

Historically, public health investigators have used 

techniques such as DNA–DNA hybridization, patterns of restriction endonuclease digestion of DNA 

on agarose gels, or pulsed-field gel electrophoresis, 

to determine the relatedness of bacterial pathogens 

isolated from different patients or different geographic 

regions. Although these methods can provide some 

information regarding strain relatedness, they vary 

greatly in their resolution. Bacterial genotyping techniques commonly used in outbreak investigations 

have limited power of resolution because they target 

only small parts of the genome. More recently, whole 

genome sequencing has emerged as a rapid and highresolution method to investigate bacterial disease outbreaks137–139; this application of the technique is often 

referred to as “genomic epidemiology.”140 Two recent 

high-profile examples of using this technology to track 

the origin and transmission of bacterial pathogens 

during outbreaks include the 2010 cholera epidemic in 

Haiti24 and the 2011 German E coli O104:H4 outbreak84

discussed previously. 

Cholera had not been previously reported from 

Haiti; thus, the main question was from where did the 

strain of V cholerae responsible for the outbreak come? 

The source of the cholera in Haiti has been controversial, with three main hypotheses being suggested. 

The first hypothesis was that the pathogen arrived to 

Haiti from the Gulf of Mexico because of tectonic shifts 

resulting from the earthquake. The second hypothesis 

was that the pathogen evolved into disease-causing 

strains from nonpathogenic strains naturally present 

in Haiti. The third hypothesis was that the pathogen 

was somehow inadvertently introduced into the Haitian environment, triggering the epidemic.24 A specific 

form of this hypothesis, that Nepalese soldiers from a 

United Nations military camp were the direct source 

of the cholera, was a commonly held belief in Haiti. 

To resolve this question, Matthew Waldor of Harvard 

Medical School collected several samples of the V 

cholerae strain circulating in Haiti and sent them to 

colleagues at Pacific Biosciences, a biotech company 

that manufactures third-generation single-molecule 

real-time DNA sequencers. Scientists at Pacific Biosciences sequenced DNA from two samples from 

the Haitian outbreak, one strain that caused cholera 

in Latin America in 1991, and two V cholerae clinical 

strains isolated from Bangladesh in 2002 and 2008 and 

compared them to reference genomes already in the 

database. They analyzed single nucleotide and copy 

Figure 25-7. Colonies of Klebsiella pneumoniae, the bacterium 

in which New Delhi metallo-β-lactamase-1 was first identified. Magnification ×10.

Photograph: Courtesy of Centers for Disease Control and 

Prevention Public Health Image Library.

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number variations to determine the likely phylogeny 

of the Haitian strain and found that the Haitian isolates 

were more closely related to the strains from Bangladesh (South Asia) and more distantly related to isolates 

circulating in South America. Their conclusions were 

that the Haitian epidemic was probably the result of 

the introduction of a V cholerae strain from a distant 

geographic source.28

Unfortunately, this determination was as specific as 

these investigators could get without having a more 

extensive collection of strains to compare. Using a 

different next-generation DNA sequencer (Genome 

AnalyzerIIx, Illumina, San Diego, CA), Frank M Aarestrup and his team at the Technical University of 

Denmark sequenced 24 V cholerae isolates collected 

from August to November 2010 from five different 

districts in Nepal. Phylogenetic analysis showed that 

all 24 V cholerae isolates from Nepal belonged to a single 

well-supported clade that also contained isolates from 

Bangladesh and Haiti. Furthermore, direct comparison between the three Haiti outbreak strains and the 

three most closely related strains from Nepal show a 

near perfect match.29 This finding, along with epidemiological data, strongly supports the hypothesis that 

the V cholerae strains responsible for the 2010 Haitian 

cholera epidemic were brought to Haiti from Nepal, 

most likely via Nepalese soldiers serving as United 

Nations peacekeepers.

A similar approach (ie, rapid whole genome sequencing) was taken to fully characterize the E coli

from the 2011 German outbreak in near real-time.141

This comprehensive analysis took place in the first 

days and weeks of the outbreak, rapidly enough to 

inform physicians treating infected patients and epidemiologists tracing the source of the pathogen. Only 

this kind of rapid whole genome sequencing allowed 

investigators to determine that the outbreak strain 

was an extremely rare form of bacterium that was a 

“hybrid” of enteroaggregative E coli and enterohemmorhagic E coli. Researchers also determined that this 

was distinct from other E coli O104:H4 strains because 

it contained a prophage encoding a Shiga toxin and a 

distinct set of other virulence and antibiotic-resistance 

factors.141

These are only two examples of the use of genomic 

epidemiology. However, it is clear that as the speed 

and accuracy of next-generation DNA sequencing increases (and the cost decreases), it is likely that in the 

near future, it will be as common a diagnostic tool as 

the PCR is today. 

EMERGING VIRAL DISEASES

Avian Influenza and the Threat of Pandemics

Influenza is a highly contagious, acute respiratory 

illness, with clear evidence of human infections dating back to the Middle Ages, and probably occurring 

as far back as ancient Greece and Rome. The influenza 

viruses are members of the Orthomyxoviridae family 

and contain a segmented negative-sense RNA genome.142 There are three genera of influenza viruses: 

influenza A, B, and C. Influenza A and B viruses are 

associated with seasonal epidemic illness in humans; 

whereas, influenza C infections in humans are sporadic. Because influenza A viruses are the only type 

of influenza viruses that have caused pandemics 

in the human population, this section will focus on 

this influenza virus type. The genome of influenza 

A viruses comprises eight gene segments, encoding 

10 to 12 proteins.142,143 The segmented nature of the 

genome allows for reassortment, or the exchange of 

segments (and genes) between two or more virus 

strains co-infecting the same cell. The major surface 

glycoproteins of influenza A viruses, hemagglutinin 

(HA) and neuraminidase (NA), are the major antigens 

of the virus. HA and NA are involved in the interactions between the virus and host cells, and they are 

the major targets of neutralizing antibodies. These 

proteins are seen as spikes in electron micrographs 

(Figure 25-8). The HA binds to sialic acid-containing 

moieties on the cell surface, mediating attachment 

and entry of the virus, and the NA is a receptordestroying enzyme that cleaves sialic acids from the 

glycan backbone, thus facilitating release and spread 

of the virus. Subtypes of influenza A viruses are designated by their particular HA and NA types (to date, 

distinct subtypes of influenza B and C viruses have 

not been observed). Sixteen HA and 9 NA subtypes 

have been identified in aquatic birds, which act as the 

major reservoir for influenza A viruses in nature. In 

addition, influenza A viruses can infect many mammalian species, including pigs, horses, dogs, cats, 

ferrets, mink, whales, and seals. Influenza A viruses 

of the most recently described subtypes, H17N10 and 

H18N11, have not been isolated; partial genome sequences of these highly divergent influenza A viruses 

were identified in bats from Guatemala and Peru 

(see section on Influenza Viruses in Bats).144,145 Thus 

far, only influenza A viruses carrying one of three 

HA subtypes (H1, H2, H3) have been able to achieve 

sustained transmission and establish themselves in 

the human population, causing subsequent seasonal 

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epidemics. For example, one circulating influenza 

virus strain is designated subtype H3N2 and has 

been the most commonly isolated strain during the 

last 4 decades. 

Antigenic diversity in influenza A viruses can result 

from changes in the HA and NA genes. One type of 

variation called “antigenic drift” occurs as a result of 

accumulation of point mutations in the genes encoding HA and NA proteins. These point mutations occur 

randomly as the virus is copied in infected cells and 

are largely responsible for the annual epidemics of 

influenza seen during the winter months, and for the 

frequent need to reformulate the seasonal influenza 

vaccine. 

Another type of change that can occur is called 

“antigenic shift,” which results from the reassortment 

of genes that occurs when two different influenza 

viruses infect the same host cell, causing a shift in the 

HA and/or NA type of the virus. This phenomenon 

results in the emergence of novel influenza A strains 

that have the potential to cause widespread infection 

and disease in a susceptible population. Since 1933, 

when the influenza A virus was first isolated (an H1N1 

subtype), major antigenic shifts (and pandemics) have 

occurred in 1957 (“Asian influenza,” an H2N2 subtype 

virus) and in 1968 (“Hong Kong influenza,” an H3N2 

subtype virus). In 1977, the H1N1 subtype virus reappeared after a more than 20-year hiatus; however, this 

time it did not cause severe disease, most likely because 

of the immunity of persons older than 20 years of age 

who had been infected with the virus when it circulated earlier in the century. It is highly unlikely that 

this virus was maintained in an animal host for more 

than 20 years without changes. One possible explanation is that the virus was maintained in a laboratory 

freezer until it somehow was reintroduced into the 

human population. 

In 2009, a swine-origin H1N1 influenza A virus 

caused the first influenza pandemic of the 21st century 

(see section on Swine Influenza and the H1N1 Influenza Pandemic, 2009). This H1N1 influenza A virus 

then replaced the circulating seasonal H1N1 influenza 

viruses, and continues to cause seasonal epidemic 

infections, with typically mild-to-moderate illness.

Of the three influenza pandemics that occurred in 

the 20th century, the pandemic of 1918 to 1919 was 

the most devastating, causing an estimated 20 to 40 

million deaths worldwide. Unusually, young healthy 

adults between 20 and 40 years of age accounted for 

almost half of the influenza deaths during this pandemic. The epidemic spread rapidly, moving around 

the globe in less than 6 months. It is estimated that 

the pandemic killed 675,000 Americans, including 

43,000 servicemen who were mobilized for World War 

I (Figures 25-9 and 25-10), and it may have played a 

significant role in ending the war.146 Its impact was 

so profound that the average life expectancy in the 

United States temporarily declined by more than 10 

years.147

Analysis of survivor antibody titers from the late 

1930s suggested that the 1918 strain was an H1N1 

subtype virus closely related to classical swine influenza viruses.148 Researchers at the Armed Forces 

Institute of Pathology in Washington, DC—who 

isolated influenza viral RNA from preserved lung 

tissue of US servicemen who died during the 1918 

pandemic, and also from a victim of the pandemic 

who was buried in a mass grave in Brevig Mission, 

Alaska—ultimately confirmed this theory. Over the 

next decade, all eight gene segments of the 1918 

influenza virus were reconstructed, sequenced, and 

characterized.149 Unfortunately, no obvious genetic 

changes were observed in any of these gene sequences that would account for the exceptional virulence 

of this pandemic virus.150 The reconstructed virus 

was highly virulent in animal models, including 

Figure 25-8. Negative-stained transmission electron micrograph showing the reconstructed 1918 influenza virons collected from the supernatants of virus-infected Madin-Darby 

Canine Kidney cell culture 18-hours postinfection. Surface 

spikes (hemagglutinin and neuraminidase) can be clearly 

seen extending from the surface of the virons. 

Photograph: Courtesy of Cynthia Goldsmith and provided 

by Dr Terrence Tumpey, Centers for Disease Control and 

Prevention Public Health Image Library. Image 8160.

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mice, ferrets, and nonhuman primates.151,152 It was 

determined that the HA and polymerase complex 

genes played important roles in virulence, although 

no single property of the virus has been identified 

to fully explain the devastating mortality seen in 

1918 and 1919.143 

Host factors undoubtedly played some role. It has 

been suggested that an uncontrolled cytokine response 

was elicited by the virus, leading to immunopathology. 

However, most of the mortality during the pandemic 

appeared to be attributable to secondary bacterial 

pneumonia.9 Viral and host factors could be involved 

in increased susceptibility to bacterial infections, 

and this remains an active area of investigation. In 

addition, the fact that the pandemic occurred in the 

preantibiotic era also likely contributed to the high 

mortality observed. 

Much has been learned from the remarkable 

achievement of the resurrection of the 1918 virus. Continued study of this pathogen will continue to provide 

valuable information for the development of vaccines 

and treatments for future pandemic influenza viruses. 

Countermeasures do exist for the treatment and 

prevention of influenza. Annual vaccines include 

two influenza A strains (H1N1 and H3N2) and two 

influenza B strains (one from each of the two influenza 

B lineages that circulate in humans). The component 

strains are selected based on surveillance of the strains 

that are circulating in humans about 6 months prior 

to when vaccine will be needed for immunizations 

before the start of the influenza season. In some cases, 

the vaccine does not match the circulating strain, and 

low vaccine effectiveness is then observed. The strains 

may need to be updated annually due to antigenic 

drift. Influenza vaccines against potentially pandemic 

influenza viruses (eg, H5 and H7 subtypes) have been 

manufactured and evaluated in clinical trials, but 

these vaccines have been found to be suboptimally 

immunogenic, requiring higher doses or adjuvants 

to achieve the antibody responses needed for protection.153 In recent years, there has been much interest 

in developing a universal vaccine that would provide 

broad cross-protection against multiple subtypes of 

influenza, including pandemic strains that may emerge 

in the future.154 The identification of a highly conserved 

region of the influenza HA (the stem or stalk region) 

against which broadly cross-neutralizing antibodies 

have been detected in humans has spurred a great effort to develop ways of using the conserved HA stem 

region as an immunogen.155–157 However, the availability of such a vaccine—if this strategy is successful—is 

still years in the future. 

Figure 25-9. Emergency hospital during the 1918 influenza 

pandemic, Camp Fuston, Kansas. NCP 1603. 

Photograph: Courtesy of the Otis Historical Archives, National Museum of Health and Medicine, Washington, DC.

Figure 25-10. Influenza wards, US Army camp hospitals at 

(a) Aix-Les-Bains, France (Reeve 14682), and (b) Hollerich, 

Luxembourg (Reeve 15183). 

Photographs: Courtesy of the Otis Historical Archives, National Museum of Health and Medicine, Washington, DC. 

a

b

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Two classes of drugs are available to treat influenza: 

(1) the NA inhibitors (oseltamivir, zanamivir) and (2) 

the M2 ion channel inhibitors (adamantanes). As with 

many antivirals, development of resistant strains is 

a problem that limits their use, and successful treatment with NA inhibitors must be initiated early after 

symptom onset to be effective. 

Swine Influenza and the H1N1 Influenza 

Pandemic, 2009

Influenza infections in pigs were first recognized clinically during the 1918 Spanish influenza 

pandemic, and the first isolation of an influenza 

virus was from pigs in 1930.158 Transmission of influenza virus to humans from swine has since been 

documented on several occasions.159,160 Before 2009, 

infections of humans with swine influenza viruses 

were sporadic and did not result in large outbreaks 

of illness. In all cases, illness was indistinguishable 

from typical influenza virus infection in humans. 

Between 1958 and 2005, 37 cases of swine influenza 

virus infections in humans were reported, 19 of 

which were in the United States (reviewed in Meyers 

et al159). In 22 (61%) of these cases, recent exposure 

to swine was reported, and 13 of these cases resulted 

from occupational exposure. In the United States in 

1976, there was an outbreak of swine influenza at 

Fort Dix in New Jersey.161 Infection with an H1N1 

swine influenza virus resulted in one soldier’s death 

and respiratory illness in 12 additional soldiers. No 

exposures to pigs were reported. It was subsequently 

found by serological analysis that as many as 230 

soldiers were infected.161

Influenza A viruses of the H1, H2, and H3 subtype 

are all present in swine. The first influenza pandemic 

of the 21st century occurred in 2009, and was caused 

by an H1N1 virus that originated in swine. The emergence of a pandemic virus from the swine reservoir 

was unanticipated, particularly with many influenza 

researchers focusing their efforts in recent years on 

avian influenza (AI) viruses, particularly H5N1 (see 

section on Human Infections with Highly Pathogenic 

H5N1 Avian Influenza Viruses), as possible agents for 

the next pandemic. 

Influenza-like illness was reported in two children 

in southern California in March 2009, and the number 

of pneumonia cases increased in Mexico City around 

the same time. A novel H1N1 influenza A virus was 

isolated from individuals in the United States in April 

2009. The virus was soon characterized as a quadruple 

reassortant virus of the H1N1 subtype, with gene 

segments from swine and avian influenza viruses.162

The novel virus spread rapidly throughout the world, 

and the WHO declared a pandemic on June 11, 2009. 

Although illness caused by the nascent pandemic virus 

was generally mild to moderate, severe illness was 

observed in individuals with underlying conditions 

such as obesity and diabetes, in pregnant women, 

and—surprisingly—in older children and young 

adults.163,164 This is in contrast to seasonal influenza 

epidemics, where the burden of disease is usually 

greatest in the very young and the elderly. 

Antigenic characterization of the 2009 pandemic 

H1N1 virus and serological studies revealed that the 

HA was related to that of H1N1 influenza viruses that 

circulated in the 1930s, 1940s, and earlier, including 

the 1918 H1N1 influenza virus.9,165 In addition, the 

2009 pandemic H1N1 virus was antigenically similar 

to the H1N1 virus that caused the swine influenza at 

Fort Dix that triggered a national vaccination campaign 

in 1977.161 Vaccination in 1977 likely afforded some 

protection against the pandemic virus. 

The 2009 pandemic H1N1 viruses were found to 

be sensitive to NA inhibitors and were resistant to 

ion channel inhibitors. Resistance to adamantanes 

was conferred by a mutation in the M2 gene of the 

2009 H1N1 viruses. An immediate response to the 

emergence of the 2009 H1N1 virus was the production 

of a vaccine. Vaccine manufacturers in the Northern 

Hemisphere had recently finished distribution of the 

trivalent vaccine for the 2008 to 2009 winter influenza 

season when the pandemic H1N1 virus emerged. 

Delays occurred in vaccine production, resulting from 

the difficulty in generating reassortant seed viruses for 

vaccine manufacture and the instability of the pH1N1 

HA protein used for potency testing for vaccine lot 

release. As a result of these delays, vaccine was not 

distributed until the peak of infections had passed. 

Human clinical testing of the monovalent inactivated 

H1N1 vaccine revealed a high titer antibody response 

to vaccination in most age groups tested, providing 

more evidence of preexisting immunity to the pandemic H1N1 virus.166,167

The WHO declared the end of the 2009 influenza 

pandemic in August 2010. The WHO reported 18,631 

laboratory-confirmed deaths caused by the H1N1 virus 

between April 2009 and August 2010. However, recent 

estimates of global mortality from this pandemic are 

reported to be approximately 10- to 15-fold higher.168,169

In 2010, the pandemic H1N1 virus replaced the 

circulating seasonal H1N1 influenza viruses, and it 

has continued to circulate concomitantly with H3N2 

human influenza viruses, causing mild to moderate 

disease. Since the winter of 2010–2011, the pandemic 

H1N1 virus has been a component of the seasonal 

influenza vaccine. The HA of the H1N1 virus does not 

appear to have undergone significant antigenic drift, 

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and so the original vaccine strain, A/California/7/2009, 

has not changed (http://www.who.int/influenza/

vaccines/virus/recommendations/en/).

The first influenza pandemic of the 21st century was 

the first influenza pandemic to occur in the molecular 

biology era. Much information about this virus will 

continue to be generated. Although delays occurred 

in vaccine production and deployment, the novel 

virus was rapidly identified and characterized. Key 

questions about this virus remain, including the precise point of origin of the virus and the reason for the 

severity of disease in pregnant women infected with 

this virus. These questions and other features of the 

virus are the subjects of intensive study. 

Fortunately, the morbidity and mortality from the 

2009 influenza pandemic were not on the scale of the 

1918, 1957 or 1968 pandemics. The 2009 H1N1 influenza pandemic underscored several important aspects 

of influenza biology: 

• the unpredictability of the emergence of novel 

influenza viruses from an animal reservoir; 

• the diversity of reassortant influenza viruses 

in nature derived from a variety of animal 

hosts; 

• the importance of preexisting immunity in the 

human population; 

• the rapidity with which a human-adapted 

virus can spread globally; and 

• the importance of surveillance of swine influenza viruses, as well as their avian counterparts. 

Swine influenza viruses continue to present a 

pandemic threat. In 2011, and particularly in the 

summer of 2012, a number of cases of human infection with quadruple reassortant swine H3N2 viruses 

were reported. These viruses are genetically identical 

to the pandemic H1N1 viruses, except that the HA 

and NA genes are derived from circulating swine 

H3N2 triple reassortant viruses.170 Importantly, 

the M gene segment is derived from the Eurasian 

swine lineage, perhaps increasing the likelihood of 

sustained transmission of these viruses in humans. 

Influenza viruses that circulate in swine are referred 

to as “variant” viruses when isolated in humans, so 

that the human cases are considered to be infections 

with the H3N2v virus.

From July to September 2012, 306 cases of human infection with H3N2v influenza viruses were 

reported.171 H3N2v has been associated with typical 

influenza illness, and 16 H3N2v-associated hospitalizations and one death occurred. Almost all 

cases have documented histories of swine exposure, 

and the majority of cases were associated with attendance at state fairs. However, some cases have 

suggested the presence of limited person-to-person 

transmission.

Influenza viruses in swine do not appear to be subjected to the same immunologic pressure that leads 

to antigenic drift in human influenza viruses. Once 

introduced into swine populations, influenza viruses 

therefore tend to be antigenically stable. The H3N2v viruses isolated from humans are phylogenetically most 

closely related to human influenza viruses from the 

mid 1990s.170,172 Clinical cases of H3N2v have occurred 

primarily in children 12 and younger, ie, individuals 

born after these viruses last circulated in humans.

Several studies have assessed the degree of baseline 

population immunity to H3N2v viruses by measuring 

antibody against these viruses using serum samples 

from different age groups. These studies have also suggested that children younger than 10 would be largely 

susceptible to infection based on lack of preexisting 

antibody.171,172 Current seasonal inactivated influenza 

vaccine does not induce an antibody that recognizes 

H3N2v in children, although some cross-reactive antibodies are observed in adults.

These observations suggest that H3N2v viruses pose 

a potential pandemic risk. The viruses are prevalent 

in domestic swine and have a demonstrated ability 

to infect humans. They possess genotypes that have 

features that potentially enable human transmission, 

and some cases of human-to-human transmission have 

been observed. Previous swine origin viruses have already caused pandemics, and influenza viruses of the 

H3 subtype are clearly capable of causing widespread 

human disease. Although the pattern of baseline antibody possibly suggests that the impact of an H3N2v 

pandemic would be focused on young children, the 

majority of adults would also be predicted to be susceptible. Thus, development of effective vaccines for 

H3N2v candidate viruses is a high priority.

Human Infections With Avian Influenza Viruses

Wild aquatic birds are the major reservoirs of all 

subtypes of influenza A virus that have been isolated, 

and the viruses do not cause symptomatic infections 

in these species. It was generally accepted—until recently—that for an influenza pandemic to occur, AI 

viruses would reassort with human influenza viruses 

in an intermediate host, and a novel strain capable of 

infecting humans (with no preexisting immunity to the 

new virus) would emerge. Rare transmission events 

directly from birds or transmission of AI from other 

animals to humans have been reported.173 Transmission of AI from birds to humans before 1997 occurred 

with AI viruses mainly of the H7 subtype.174 Human 

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infections with other AI subtypes have since been 

reported: for example, in 1999 and 2003 with H9N2 

viruses175,176; in 2003 with H7N7 viruses177; from 1997 

to present with H5N1 viruses178; in Egypt with H10N7 

viruses,175 and in 2013 in Taiwan with H6N1.179 These 

cases confirmed that AI viruses are capable of directly 

infecting humans without the requirement for reassortment in an intermediate mammalian host. 

Human infections with AI viruses have resulted in 

a wide spectrum of disease, ranging from mild febrile 

and respiratory illness in some H5 and H9N2 influenza 

infections, conjunctivitis in the case of H7 influenza 

infections, to severe disease and death, as seen with the 

highly pathogenic avian influenza (HPAI) H5N1 cases 

that have occurred between 1997 and the present.178

In contrast to the rarity of the isolation of AI viruses 

from humans, serosurveys of farmers in rural southern 

China suggest that many other subtypes of AI viruses 

have crossed the species barrier and infected humans. 

Specifically, seroprevalence levels of 2% to 7% for H5 

viruses alone were reported,180 and the seropositivity of human sera for H7, H10, and H11 viruses was 

estimated to be as high as 38%, 17%, and 15%, respectively. The dogma had been that because of receptor 

specificity, AI viruses were incapable of efficiently 

infecting humans. It has long been believed that this 

host restriction of AI viruses prevents the emergence 

of new pandemic strains via direct avian-to-human 

transmission. However, human cases of direct infection 

by AI viruses are becoming increasingly frequent; it is 

now known that the potential of an AI virus to infect 

humans is polygenic in nature, and it is widely accepted that this is not solely attributable to its HA receptor 

specificity. The most significant zoonotic transmissions 

of AI viruses in recent years have been caused by H5N1 

and H7N9 AI viruses. These outbreaks are described 

in more detail in the following sections. 

Human Infections With Highly Pathogenic H5N1 

Avian Influenza Viruses

The first reported cases of H5N1 influenza infections 

in humans occurred in 1997 in Hong Kong. The first 

case was a 3-year-old boy.181 The child died 12 days 

after the onset of symptoms with several complications, including respiratory failure, renal failure, and 

disseminated intravascular coagulopathy. An H5N1 AI 

virus was isolated from a tracheal aspirate specimen 

obtained on day 10 of illness. The nucleotide sequence 

of the isolate revealed a multibasic amino acid sequence 

at the HA cleavage site, a motif characteristic of HPAI 

viruses,182 which—until this point—had only been 

known to cause severe disease in poultry. Phylogenetic 

analysis of the H5N1 Hong Kong isolate revealed no 

evidence of genetic reassortment with recent human 

influenza A viruses. The isolate was highly pathogenic 

for chickens and the virus displayed an AI virus-like 

receptor specificity. No clear epidemiological link was 

established between the infected child and infected 

poultry. However, outbreaks of influenza occurred in 

poultry on farms in Hong Kong between late March 

and early May 1997, and two viruses from one of these 

outbreaks were identified as H5N1 influenza viruses. It 

was reported that sick chickens were at the preschool 

attended by the child, although no evidence indicates 

that these chickens were infected with AI or that the 

child was in close contact with them. 

Additional cases of H5N1 in humans in Hong Kong 

were confirmed in 1997.183 In total, 18 Hong Kong 

residents became infected with HPAI H5N1 influenza 

in 1997, of whom 6 died. The cases, which were not 

geographically related or confined to a specific age 

group, occurred in children and adults with ages 

ranging from 1 to 60 years. In 7 of the 18 cases, histories of possible exposure to poultry existed, where 

the patients had either bought chickens before they 

became ill or had worked in proximity to chicken 

stalls near their homes.184 Seven of the patients had 

severe complications, most prominently pneumonia, 

gastrointestinal manifestations, elevated liver enzyme 

levels, and renal failure. With one exception, patients 

younger than age 13 recovered from their illness 

whereas older patients had more severe disease that 

resulted in death in five cases. 

An epidemiological study of the human H5N1 cases 

in Hong Kong in 1997 suggested that the viruses were 

transmitted directly from birds to humans, and serological evidence of human-to-human transmission was 

limited.184 In most cases, infection was associated with 

recent exposure to live poultry. Sequence analysis of AI 

viruses circulating in China around that time resulted 

in the hypothesis that the H5N1 influenza viruses 

that infected humans in 1997 arose by reassortment 

between an H5N1 influenza A/goose/Guangdong/1/96-

like virus and an H9N2 or H6N1 virus similar to those 

circulating in the live bird markets of Hong Kong in 

1997. However, the actual sequence of reassortment 

events cannot be definitively determined from the 

small number of viruses available for analysis from 

preceding years.173

The human cases of HPAI H5N1 infection that occurred in 1997 coincided with further outbreaks of 

highly pathogenic H5N1 influenza in poultry on farms 

and in live bird markets in Hong Kong. Slaughter of 

the 1.5 million poultry in the Hong Kong Special Administrative Region was conducted between December 

29 and December 31, 1997. Many experts believe that 

because of this action, an influenza pandemic caused 

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by the H5N1 virus was averted, although it has since 

become apparent that the H5N1 AI viruses have not 

adapted for efficient transmission in humans. Reintroduction of poultry to the Hong Kong Special Administrative Region began in February 1998. At this time, 

new practices were introduced for the live bird markets 

in Hong Kong. Waterfowl (eg, ducks and geese) are 

now sold at separate markets from chickens; ducks and 

geese are now slaughtered at the markets; and markets 

have a monthly rest day when they close for thorough 

cleaning, the remaining birds are culled, and restocked 

with fresh imported poultry. Surveillance of birds in 

the markets has continued since the 1997 outbreaks. 

In 2003, again in Hong Kong, two cases of HPAI 

H5N1 infection were confirmed in a father and son of 

a family who had recently visited mainland China.8

HPAI H5N1 AI infections again appeared in the human 

population in 2004 in Vietnam and Thailand, and they 

were confirmed in Cambodia, Indonesia, and China in 

2005. HPAI viruses of the H5N1 subtype in Asia continued to evolve and spread in avian populations, and 

human cases were eventually reported in the Middle 

East, Europe, and Africa. Multiple genotypes and 

several clades of H5N1 influenza viruses have been 

identified.96,185 It is believed that the highly pathogenic 

H5N1 viruses in Asia originated from viruses in ducks 

in southern China. HPAI H5N1 viruses have been 

isolated from dead migratory birds in Hong Kong and 

parts of China,186 implicating wild birds in their spread 

across Asia and other parts of the world. 

Human cases and fatalities have since been reported 

in 15 countries. As of October 2013, 641 laboratoryconfirmed cases and 370 deaths have been reported to 

the WHO.178 The WHO’s website contains a comprehensive timeline that chronicles the panzootic spread 

of HPAI H5N1 viruses since 1997 and the associated 

incursions of the virus into the human population.178

The HPAI H5N1 viruses of the A/goose/Guangdong 

lineage continue to evolve by antigenic drift, resulting 

in efforts to continually update stockpiled vaccine 

strains for use should these viruses gain the ability to 

transmit efficiently and cause widespread infections. 

Of particular concern are the HPAI H5N1 viruses 

circulating in Egypt since 2009, because these isolates 

display increased affinity for human-type sialic acid 

receptors.187,188 Fortunately, HPAI H5N1 viruses have 

not acquired the ability to transmit efficiently from 

person to person, although several small family clusters of cases have been reported.189,190

Despite the global prevalence of HPAI H5N1 infections in birds, and the number of reported infections in 

humans, these viruses have not yet acquired the necessary genetic changes required for efficient, sustained 

transmission in the largely immunosusceptible human 

population. Recent controversial studies involving 

intentional introduction of mutations into HPAI H5N1 

viruses to confer efficient transmissibility in the ferret 

model—the preferred animal model for the study of 

influenza transmission—resulted in the identification 

of several changes in the viral genome that achieved 

this state.188,191 Changes in the HA gene and the PB2 

polymerase gene were found to be necessary—but 

not sufficient—to confer transmissibility in ferrets.192

Human Infections With H7N9 Avian Influenza 

Viruses, 2013

Human cases of H7 AI have typically been associated with large outbreaks of H7 AI infection in birds, 

caused by either highly pathogenic or low pathogenicity viruses. With the exception of a fatal infection 

by a HPAI H7N7 in the Netherlands in 2003,177 illness 

associated with H7 AI infections in humans has been 

relatively mild. 

Recent human cases of H7N9 AI infection in China 

have caused great concern regarding the potential 

emergence of an influenza pandemic. Human infections with H7N9 AI viruses were first reported in 

China on March 31, 2013.193 The first three cases reported were in two individuals from Shanghai and 

one individual from Anhui.194 All three patients died. 

Between April and the end of May 2013, 132 laboratory 

confirmed cases had been reported to the WHO, 37 of 

them fatal. Cases occurred in eight contiguous provinces of eastern China and in the two municipalities of 

Beijing and Shanghai, and a single case was reported 

in Taiwan.195 The infected individual from Taiwan had 

recently travelled to Jiangsu Province in China. 

In the majority of the laboratory-confirmed cases 

of H7N9 in China and in the case reported in Taiwan, 

illness was severe.194,195 In the initial three fatal cases 

reported in China, all three patients presented with 

fever, cough, and dyspnea. Radiologic findings were 

consistent with pneumonia, with diffuse opacities 

and consolidation.194 The patients progressed rapidly 

to acute respiratory distress syndrome (ARDS) and 

multiorgan failure. Gao et al194 reported the clinical 

features of an additional 111 laboratory confirmed 

cases in China. The most common early symptoms 

were fever and cough. Ninety-seven percent of these 

patients had findings consistent with pneumonia 

upon admission to the hospital, 77% were admitted to 

the intensive care unit, and 27% of patients died. The 

median age of patients was 61 years; 68% were male 

and 61% had at least one underlying medical condition—most commonly, coronary heart disease, hypertension, diabetes, or chronic obstructive pulmonary 

disease. Presence of an underlying medical condition 

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was identified in this study as the only independent 

risk factor for progression to ARDS. The most common 

complications of H7N9 AI infection in these patients 

were ARDS (71%), shock (26%), acute kidney injury 

(16%), and rhabdomyolysis (10%). Pneumonia and 

ARDS occurred in all fatal cases; progression to severe 

pneumonia, ARDS, and shock was rapid. The leading 

cause of death was refractory hypoxemia. In all cases, 

patients were treated with neuraminidase inhibitors.

It has been suggested that many mild cases may 

have occurred but were not reported.179 Nevertheless, 

it is clear that the avian-origin H7N9 virus is capable 

of causing severe disease and death in humans, and 

age and underlying medical conditions are risk factors 

for severe disease. 

The possible origin of the H7N9 AI viruses that 

emerged in China in 2013 has been extensively studied. 

Phylogenetic analyses determined that the viruses are 

the result of multiple reassortment events, and they 

have gene segments related to those from at least 

three different types of AI viruses (Figure 25-11).194,196

The HA genes are most closely related to those from 

low pathogenicity AI H7N3 viruses that were isolated 

in parts of Asia, including China, in 2011 (A/duck/

Zhejiang/12/2011-like). The NA genes are most closely 

related to those from low pathogenicity AI H7N9 viruses that circulated in South Korea in 2011 (A/wild 

bird/Korea/A14/2011-like); although notably, the human H7N9 viruses have a 15-amino acid deletion in 

the NA stalk region that had previously been reported 

to be associated with the adaptation of AI viruses to 

terrestrial poultry.194,196,197 The donors of the six internal 

protein genes were H9N2 viruses.194,198 It appears that 

ducks and chickens were probably intermediate hosts 

for the H7N9 reassortant viruses. 

The H7N9 AI viruses isolated from humans and from 

birds at the time of the outbreak lack the multibasic 

amino acid cleavage motif in the HA gene that is seen 

in HPAI viruses. In addition, infection of avian species 

with H7N9 isolates did not result in disease, although 

virus was shed and the birds developed antibodies. 

These observations underscore the challenge for surveillance of these viruses in avian populations, since they 

do not cause overt disease.193 The source of the H7N9 

AI viruses appears to be live bird markets.199,200 H7N9 

AI viruses isolated from live bird markets were almost 

identical in sequence to the human isolates. However, 

H7N9 virus was isolated from only a small percentage 

of samples taken from birds and the environment all 

across China. All samples taken from swine and from 

slaughterhouses were negative for H7N9 AI. 

In summary, AI viruses of the H7 subtype have been 

directly transmitted to humans on numerous occasions. The recent emergence of H7N9 AI infections in 

humans in China confirmed that H7 AI viruses have 

the potential to cause severe disease. Studies have 

demonstrated that the H7N9 AI viruses isolated from 

human cases bear some genetic markers that are associated with adaptation to mammals,196,198 and their ability to transmit efficiently in the ferret model by direct 

contact and by respiratory droplets has been demonstrated.201,202 Characterization of the crystal structure 

of the H7N9 AI HA glycoprotein showed preferential 

recognition of avian-like receptors by H7N9 AI viruses 

isolated from humans, suggesting that the virus may 

be poorly adapted for mammalian transmission at 

this time.179 Although sustained, efficient human-tohuman transmission has not been observed, the pandemic potential of these viruses is of great concern. It 

is expected that more human cases will occur in the 

winter months. In December 2013, two human cases 

of H7N9 AI infection in Hong Kong were reported. In 

both cases, the infections were thought to have been 

acquired in the neighboring city of Shenzhen (http://

www.chp.gov.hk/en/view_content/32486.html). As of 

January 31, 2015, 677 laboratory-confirmed cases of 

human infection with H7N9 AI had been reported to 

the WHO, with at least 275 deaths.203

Influenza Viruses in Bats

In recent years, an increasing interest has emerged 

in the role of bats as reservoirs of viral pathogens.204

The coronavirus that caused the SARS outbreak in 

2003 (SARS-CoV) is closely related to CoV genomic 

sequences found in bats in China (see section on 

Diseases Caused by Emerging Coronaviruses). The 

paramyxoviruses Hendra and Nipah were isolated 

from bats. Bats are thought to be possible reservoirs of 

filoviruses, including Ebola virus and Lloviu virus, a 

novel filovirus205,206 since viral nucleic acid sequences 

have been identified in a variety of bat species. Additionally, Marburg virus has been isolated from 

Egyptian fruit bats (Rousettus aegyptiacus).207–209

In 2012, Tong and colleagues144 reported the identification of novel influenza virus sequences in little 

yellow-shouldered bats in two locations in Guatemala. 

Virus was not isolated, but nucleic acid sequences were 

derived from rectal swab samples, and from liver, intestine, and kidney tissue samples. The sequences were 

identified as originating from a highly divergent influenza virus. The novel virus was designated as belonging 

to a new subtype of influenza A viruses, H17N10. The 

NA gene was the most divergent gene segment, and 

it was found to have an older ancestral relationship to 

known influenza A and B viruses. The solution of the 

N10 crystal structure determined that, although it shares 

general structural features with the other influenza A 

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Figure 25-11. Diagram showing the likely genetic evolution of the H7N9 virus that emerged in China in 2013. The eight genes 

of the H7N9 virus are closely related avian influenza viruses found in domestic ducks, wild birds, and domestic poultry in 

Asia. The virus likely emerged from “reassortment,” a process in which two or more influenza viruses coinfect a single host 

and exchange genes. This process can result in the creation of a new influenza virus, and it is likely that multiple reassortment events led to the creation of the H7N9 virus. These events may have occurred in habitats shared by wild and domestic 

birds and/or in live bird/poultry markets, where different species of birds are bought and sold for food. As the above diagram 

shows, the H7N9 virus likely obtained its HA (hemagglutinin) gene from domestic ducks, its NA (neuraminidase) gene from 

wild birds, and its six remaining genes from multiple-related H9N2 influenza viruses in domestic poultry. 

M: matrix; NP: nucleoprotein; NS: nonstructural; PA: polymerase subunit A; PB1: polymerase subunit B1; PB2: polymerase 

subunit B2

Diagram: Courtesy of Centers for Disease Control and Prevention. 

NAs whose structures have been determined, it does 

not have the conserved amino acids that are involved 

in sialic acid binding and cleavage210,211 and the protein 

does not display enzymatic neuraminidase activity 

necessary for its function in mediating spread of the 

virus from infected cells. The HA structure suggests 

that the H17N10 virus does not use sialic acid as a 

receptor.179 The polymerase complex encoded by the 

sequences found in the bats did function in human 

cells, but the sequences of the polymerase genes suggest that they may be incompatible with other influenza A subtypes.

In 2013, Tong et al145 reported the identification 

of RNA encoding another distinct influenza virus, 

designated H18N11, in flat-faced fruit bats in Peru. 

Again, the sequences were highly divergent from 

known influenza A viruses, and they indicated a 

long-standing virus–host relationship. The sequences 

were most closely related to the H17N10 influenza 

sequences previously reported by this group.144

Structural and functional studies of the HA and NA 

encoded by these sequences suggest that sialic acid is 

not a receptor for the virus and is not used for virus 

release from the infected cell. The H18N11 influenza 

virus was not isolated, but viral sequences were 

identified in rectal swabs and intestines of the bats. 

The overall H18 structure was found to be similar to 

that of the known influenza A trimers, but unlike the 

known HAs, the H18 structure infers no requirement 

of low pH for fusion. In addition, the receptor-binding 

domain of the H18 glycoprotein is dramatically different. Like the N10 NA, the general N11 structure is 

similar to the known influenza A NAs (ie, tetrameric), 

but the N11 active site is different, even from that of 

the N10 protein. The N11 protein does not display 

glycan binding or enzymatic neuraminidase activity. 

Seroprevalence studies found that approximately 38% 

of the Guatemalan bats tested had detectable antibodies to H17,144 and 50% of bats tested had antibodies 

to either the recombinant H18 or N11.145

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Some debate exists as to whether the viruses encoded by the viral sequences identified in the bats 

in Guatemala and Peru should even be classified as 

influenza A viruses. They may represent ancient ancestral viruses. The significance of these findings for the 

potential for the emergence of novel influenza viruses 

that may infect humans, or for reassortment with other 

influenza viruses in nature remains to be determined. 

Diseases Caused by Emerging Coronaviruses

SARS, which first emerged in Guangdong province of China in November 2002, is a classic example 

of a newly emerging viral disease. By January 2003, 

the disease had spread to Guangzhou, the capital of 

Guangdong province, and caused major outbreaks, 

primarily affecting healthcare workers. In February 

2003, a physician from Guangdong spent a single 

day in a hotel in Hong Kong, during which time he 

transmitted the infection to 16 other guests. These 

individuals quickly spread the disease to Hong Kong, 

Singapore, Vietnam, and Toronto.212 Within weeks, 

SARS had spread to affect thousands of people in 25 

countries across five continents and, by the end of the 

global outbreak (July 2003), more than 8,000 reported 

cases existed, with 744 fatalities.213 Within 4 months 

of the beginning of the outbreak, a novel coronavirus 

(SARS-CoV) was identified as the infectious agent 

of the syndrome.214–216 Cases of SARS have not been 

reported since 2003. 

Middle Eastern Respiratory Syndrome

In June 2012, a 60-year-old man was admitted with a 

history of fever, cough, expectoration, and shortness of 

breath to a hospital in Jeddah, Saudi Arabia.217 Despite 

treatment in an intensive care unit, the patient died 

11 days after admission from respiratory and renal 

failure. Clinical isolates were initially tested and found 

negative for influenza, parainfluenza, enterovirus, and 

adenovirus. A sputum sample that was obtained upon 

admission was inoculated in Vero and LLC-MK2 cells, 

resulting in visible cytopathic effect. A viral familywide PCR performed on nucleic acid extracted from 

infected cells gave a positive result for coronaviruses. 

Sequencing of the PCR amplicon resulted in a novel 

sequence that indicated the newly discovered virus 

was most closely related to bat coronaviruses. The first 

report of the novel coronavirus was made in ProMEDmail on September 20, 2012, by Dr Ali Mohamed Zaki 

of the Dr Soliman Fakeeh Hospital.218

Virus samples were sent for full genome deep sequencing to the Erasmus Medical Center in Rotterdam, 

Netherlands.219 Full-genome sequencing confirmed 

that the novel virus was similar to BtCoV-HKU4 and 

BtCoV-HKU5, members of the C lineage of the betacoronavirus, but it was sufficiently different enough to 

warrant classification as a new species that was named 

HCoV-EMC/2012.

Coronaviruses have relatively large, singlestranded, positive-sense RNA genomes. The HCoVEMC/2012 genome is approximately 30 kb in length 

and encodes both structural and nonstructural proteins. Before 2003, only two coronaviruses (HCoV229E and HCoV-OC43) were known to infect humans, 

and those caused only mild respiratory disease.220,221 As 

noted previously, SARS-CoV previously caused an epidemic in 32 countries, infecting more than 8,000 people. 

Since 2003, two additional human coronaviruses have 

been identified, HCoV-NL63177,222 and HCoV-HKU1,223

both of which can cause pneumonia.

On September 23, 2012, the United Kingdom 

Health Protection Agency reported on the case of a 

49-year-old man who had become sick while in Saudi 

Arabia in August 2012. That illness resolved, but he 

subsequently presented to a physician in Qatar with 

a cough, myalgia, and arthralgia on September 3, 

2012. Five days later he was admitted to the hospital 

with fever and hypoxia. His condition worsened and 

he was transferred to London by air ambulance. His 

condition deteriorated once in London, and he was 

placed on extracorporeal membrane oxygenation on 

September 20.144 Initially, the patient was screened for 

common viral and bacteriological causes of respiratory 

illness with no positive results. After the September 

20 ProMED report of a novel coronavirus identified 

in the Middle East, patient samples were screened by 

using a pan-coronavirus reverse transcriptase polymerase chain reaction (RT-PCR) assay. Sequencing of 

the PCR product showed it was nearly identical to the 

EMC/2012 virus.

Following the initial description of these two cases 

which came to be called Middle East Respiratory Syndrome (MERS), a retrospective investigation of an 

outbreak of acute respiratory disease was performed 

at the Zarqa hospital in Jordan.224 In April 2012, the 

Zarqa hospital had 13 patients who presented with 

high fever and acute lower respiratory symptoms. 

Laboratory tests performed at the time of the outbreak 

were inconclusive. The cluster consisted of two phases. 

In the first phase, four patients had onset of symptoms 

between March 21 and April 2. The patient with the 

earliest onset (a 25-year-old student) and a 40-year-old 

nurse who worked at the hospital died within 2 to 4 

weeks of symptom onset. MERS-CoV infection was 

confirmed in both of these cases by specific RT-PCR. 

A second wave of disease followed with onset of 

symptoms between April 11 and April 26. This second 

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wave consisted of seven healthcare workers from the 

hospital and two family members of patients from the 

first wave of disease. Three of the healthcare workers 

and the two family members had close contact with 

individuals in the first wave, raising the possibility of 

limited person-to-person transmission of the virus.

A published report in June 2013 confirmed personto-person transmission in a cluster of 23 confirmed 

and 11 probable MERS-CoV infections in hospitals in 

the Al-Hasa governorate of Saudi Arabia.200 The first 

patient was admitted to the hospital on April 5, 2013, 

with dizziness and diaphoresis. He was not tested for 

MERS-CoV, but infection was subsequently confirmed 

in his son. The first patient is thought to have transmitted the virus to a patient in an adjacent room (in addition to his son), who then transmitted the virus to an 

additional seven patients (six in the dialysis unit and 

one in the intensive care unit). Further transmission of 

the virus was documented to an additional 10 patients, 

two healthcare workers, and three family members.

In May 2015, Republic of Korea health officials reported a case of MERS-CoV infection in a 68-year-old 

man who had been traveling in the Middle East for 

several weeks. He was asymptomatic while traveling, 

Figure 25-12. History of travel from in or near the Arabian Peninsula within 14 days of illness onset for confirmed cases 

(N = 130) of Middle East respiratory syndrome coronavirus infection reported to the World Health Organization from 2012 

to 2013. All cases have been directly or indirectly linked through travel to or residence in Saudi Arabia, Qatar, Jordan, and 

the United Arab Emirates. Figure does not include recent cases in South Korea. 

Data source: Centers for Disease Control and Prevention. Updated information on the epidemiology of Middle East respiratory syndrome coronavirus (MERS-CoV) infection and guidance for the public, clinicians, and public health authorities, 

2012–2013. MMWR Morb Mortal Wkly Rep. 2013;62:793–796.

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but developed symptoms within a week of returning 

to Korea. He was seen at four separate hospitals and 

was admitted to the fourth one in Seoul on May 18, 

where it was confirmed that he was infected with 

MERS-CoV.225 During his medical visits, before being 

diagnosed, he infected around 30 other individuals 

who were present in the hospital at the same time. 

A secondary case from the second hospital went on 

to infect more than 80 additional people. In total, 186 

cases of MERS (185 in the Republic of Korea and 1 in 

China226) were confirmed during the Korean outbreak, 

more than half of which had been infected by one of 

the two “superspreaders.”

As of September 2, 2015, 1,493 confirmed cases of 

MERS-CoV infection resulted in 527 deaths (35% case 

fatality rate).226 The majority of these cases were from 

Saudi Arabia, but Korea, France, Italy, Jordan, Qatar, 

Tunisia, the United Kingdom, and the United Arab 

Emirates also have reported cases. During the recent 

outbreak in South Korea, secondary and tertiary transmission occurred from the index case who traveled to 

the Middle East. All other cases outside of the Middle 

East had recorded recent travel to the Middle East 

(Figure 25-12).227

A meta-analysis of 47 laboratory-confirmed cases 

of MERS-CoV infection from Saudi Arabia found that 

most patients presented with fever, cough, shortness of 

breath, and myalgia.200 Almost all of the identified patients had underlying comorbidities, including diabetes (68%), chronic kidney disease (49%), hypertension 

(34%), chronic heart disease (28%), and chronic lung 

disease (26%). Twenty-eight (60%) of the patients died, 

and the fatality rate increased with increasing age.

The receptor for MERS-CoV was identified within 

6 months of the original characterization of the virus. 

Dipeptidyl peptidase 4 (DPP4) from extracts of cells 

susceptible to virus infection co-purified with the 

receptor-binding domain of the EMC/2012 spike protein.228 Transient expression of DPP4 in nonsusceptible 

cells also rendered them susceptible to infection and 

preincubation of cells with anti-DPP4 polyclonal antibodies made them resistant to MERS-CoV infection. 

The rapid identification of the receptor opens several 

avenues for generating antiviral therapeutics. Manipulation of DPP4 levels or the development of small 

molecules or monoclonal antibodies that can block the 

interaction of MERS-CoV with DPP4 could potentially 

alter the course of disease in infected individuals.

Other therapeutic interventions may prove useful for treatment of MERS-CoV infection. The virus 

has been shown to be sensitive to type I interferon 

in vitro, with its replication limited by two to four 

orders of magnitude when cells are pretreated with 

interferon.229,230 In addition, in a rhesus macaque model 

of MERS-CoV infection,231 treatment with interferon 

α2b and ribavirin was shown to ameliorate some of 

the disease symptoms.231 Additionally, a monoclonal 

antibody isolated from an infected patient has shown 

prophylactic and postexposure protection against 

MERS-CoV infection in a mouse model of infection.232

Vaccination of mice, camels, and rhesus macaques 

with a DNA vaccine expressing the MERS-CoV spike 

protein was shown to elicit neutralizing antibody responses in all three species, and it could also protect 

macaques from challenge with MERS-CoV.233

Parallels can be drawn between the current MERSCoV epidemic and the SARS epidemic of 2002 to 2003. 

Although the reservoir for MERS-CoV has not been 

identified, both viruses are thought to circulate in bats. 

SARS-like coronaviruses were identified in three species of bats from the genus Rhinolophus.

186 The virus 

may have spilled over into palm civets (Paguma larvata) 

(Figure 25-13), which served as an amplifying host. 

The virus could then be transmitted to humans when 

they came into contact with infected civets in wild 

animal markets. The genome sequence of MERS-CoV 

indicates that it is closely related to two bat coronaviruses, BtCoV-HKU4 and BtCoV-HKU5. A bat survey 

conducted in Saudi Arabia identified a fecal sample 

from a Taphozous perforatus bat that yielded a PCR 

product with 100% identity to the sequence of MERSFigure 25-13. The masked palm civet was originally implicated as the possible animal source for the SARS coronavirus 

after SARS-like coronaviruses were isolated from animals 

found in a live animal market in Guangdong, China. These 

animals are trapped and butchered for food in southern 

China. This photograph was taken at a wet market in Guangzhou in May 2003. 

SARS: severe acute respiratory syndrome

Photograph: Courtesy of Dr Meirion Evans, Cardiff University, United Kingdom.

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CoV EMC/2012.234 In another survey, a fecal sample 

from a South African Neoromicia zuluensis bat yielded 

a PCR product whose nucleotide sequence indicated 

that it was closely related to the MERS-CoV.235

Although MERS-CoV-like viruses have been identified in bats, nothing indicates that MERS-CoV is jumping directly from bats into humans. One possibility 

is that MERS-CoV may move from bats through an 

intermediate host that has a closer association with 

humans, as was the case of the SARS-CoV. Serum 

surveys of livestock in Egypt, Oman, and Spain identified high levels of MERS-CoV reactive antibodies 

in dromedary camels.236,237 Subsequently, MERS-CoV 

RNA was detected in three camels that had close association with two human cases.218 In November and 

December 2013, a large, nationwide serosurvey of 

livestock in the Kingdom of Saudi Arabia found that 

74% of the sampled dromedary camels had antibodies 

reactive to MERS-CoV.238 Testing of archived serum 

samples found MERS-CoV reactivity dating back to 

1992, indicating that the virus has been circulating in 

the Kingdom of Saudi Arabia since at least that time. 

In addition to the serological data, two groups isolated 

replication-competent MERS-CoV from dromedary 

camels in late 2013 through early 2014.239,240

Public health measures were an important aspect of 

halting the spread of the SARS epidemic, and public 

health officials worldwide have been proactive with 

measures intended to reduce the possibility that MERS 

could become another pandemic. One of the main 

concerns has centered on the Hajj, the annual event 

in which millions of Muslim pilgrims from around 

the world travel to Mecca in Saudi Arabia. The Saudi 

Arabian Ministry of Health recommended that persons 

older than 65, pregnant women, children younger than 

12, or those with chronic diseases should postpone 

performing the Hajj in 2013. Many may have heeded 

those warnings, as participation in the 2013 Hajj was 

estimated at just less than 2 million pilgrims, down 

from 3.2 million pilgrims in 2012. 

As of this writing, cases of MERS are still occurring on the Arabian Peninsula. Unfortunately, unlike 

SARS-CoV, MERS-CoV shows no sign of abating and 

continued efforts to understand this virus will be important to control this emerging disease. 

Diseases Caused by Emerging Paramyxoviruses

Hendra Virus

In 1994, a new member of the paramyxoviruses 

emerged for the first time in Brisbane, Australia, killing 

14 race horses and a horse trainer.241 Another worker 

at the stable survived with an influenza-like illness. 

One year later, a farmer from Mackay (800 km north 

of Brisbane) died as a result of encephalitis caused by 

this novel virus.242 Two of his horses were subsequently 

shown to have died from the same virus 13 months 

earlier. Genetic analysis of the virus showed it was 

distantly related to the morbilliviruses, which contain 

other members such as rinderpest, measles, and canine 

distemper viruses, and so the virus was initially named 

equine morbillivirus,241 but was later renamed Hendra 

virus after the Brisbane suburb where the outbreak 

occurred. Serologic evidence243 and later evidence of 

infection was found in several species of Australian 

flying foxes (ie, fruit bats of the genus Pteropus) (Figure

25-14), supporting epidemiological evidence that fruit 

bats are the natural reservoir for Hendra virus. Field, 

experimental, and molecular investigations indicate 

that Hendra virus is an endemic fruit bat virus that 

has probably co-evolved with its pteropid hosts.53,244,245

Although additional occurrences of Hendra virus 

have been relatively rare and sporadic, as of June 2014, 

50 outbreaks of Hendra virus occurred in Australia, all 

involving infection of horses. Four of these outbreaks 

have spread to humans as a result of direct contact 

with infected horses. The case fatality rate in humans 

is 60% and in horses 75%.246

Nipah Virus

Nearly 5 years after the discovery of the Hendra virus, a massive outbreak of porcine respiratory disease 

occurred in Malaysia and subsequently caused the 

deaths of 105 pig farm or abattoir workers, the eventual 

culling of more than 1 million pigs, and the discovery 

of a new virus closely related to Hendra called Nipah 

virus.247 The predominant clinical syndrome in humans was encephalitic (not respiratory as was seen in 

the infected pigs) with clinical signs including fever, 

headache, myalgia, drowsiness, and disorientation 

sometimes leading to coma within 48 hours.248,249 The 

majority of human cases had a history of direct contact 

with infected pigs, most of whom were pig farmers. 

Preliminary research on the new virus revealed that it 

had ultrastructural, antigenic, serologic, and molecular 

characteristics similar to Hendra virus.247 Follow-up 

molecular studies showed the genome of Nipah virus 

to be highly homologous to that of Hendra virus, with 

specific genes sharing from 70% to 88% nucleotide 

homologies and amino acid homologies ranging from 

67% to 92%.247 Given the degree of similarity and other 

unique features of these viruses, they both were placed 

in a new genus, Henipavirus, within the family Paramyxoviridae.

250 With the knowledge of the similarities 

between Nipah and Hendra viruses, it was natural that 

attention focused on Malaysian bats as the source of the 

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infection in pigs.53 Initial surveillance efforts identified 

the presence of neutralizing antibodies to Nipah virus 

in the sera of 21 bats from five species (four species 

of fruit bat, including two flying fox species, and one 

insectivorous bat species).251 Although no virus was 

isolated or viral RNA amplified from these seropositive 

bats, later attempts proved successful and virus was 

isolated from pooled urine samples collected from a 

colony of seropositive flying foxes from Tioman Island 

off the coast of Malaysia.252

The virus reemerged in Bangladesh in 2001, each 

resulting in a cluster of febrile neurologic illnesses with 

nine reported deaths.253 Since 2001, outbreaks of Nipah 

have occurred nearly every year in Bangladesh. More 

than 70% of those infected have died and one-third of 

the survivors have permanent neurological deficits.254

Outbreak investigations in Bangladesh have identified 

consumption of raw date palm sap as the primary route 

of transmission of Nipah virus from Pteropus bats to 

people. Date palm sap is harvested in the winter in 

Bangladesh by shaving the bark from the sugar date 

palm tree and collecting the sap into open clay pots. 

Pteropus bats (see Figure 25-14) that shed Nipah virus 

in their saliva frequently visit the trees during sap collection and lick the sap as it is running into the pot, 

thereby contaminating the sap.255 However, similar to 

other viruses, such as Ebola virus, transmission can 

also occur by direct contact with infected individuals, particularly during patient care (ie, nosocomial 

transmission) or exposure to infected patients’ bodily 

secretions during traditional burial practices.256 Thus, 

patients from regions where Nipah virus is known to 

occur who present with meningoencephalitis should 

be placed in an isolation room or ward and healthcare 

workers caring for these patients should wear gloves 

and masks.

Emerging Mosquitoborne Viruses: Dengue, West 

Nile, and Chikungunya

Mosquitoborne viruses are members of the more 

general category of arthropodborne viruses or arboviruses. Human infection with arboviruses can be 

asymptomatic or can cause diseases ranging from 

a mild febrile illness to encephalitis or even severe 

hemorrhagic fever in some cases. Others cause rash 

and epidemic arthralgia. Most arboviruses require a 

reservoir host such as a bird or small mammal while 

using a vector—usually a mosquito or tick—for transmission to another host.257 From this complex life cycle, 

many arboviruses are restricted to specific geographical regions. For example, Ross River and Murray Valley encephalitis viruses are restricted to Australia and 

surrounding islands, whereas o’nyong-nyong virus 

Figure 25-14. Flying foxes (Pteropus spp) are the natural reservoir of the Nipah and Hendra viruses, and possibly other 

emerging paramyxoviruses. Other species of bats have been 

found to be reservoirs of SARS-like coronaviruses. Photos 

show the little red flying fox (Pteropus scapulatus) in flight 

(a) and roosting (b). 

Photographs: Courtesy of Raina Plowright, Department of Veterinary Medicine and Epidemiology, University of California, 

Davis, California.

a b

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occurs only in Africa. However, because of various 

ecological or environmental changes (whether natural 

or manmade) that lead to changes in the mosquito 

vector distribution or genetic changes in the viruses 

themselves, some arboviruses may not always remain 

restricted to their previously known geographical 

regions.

Dengue Virus

Dengue fever, which is caused by one of four viral 

subtypes (designated DENV-1 to DENV-4), is one of 

the most common mosquitoborne viral infections of 

humans, with up to 100 million cases reported annually and some 2.5 billion people living at risk of 

infection in tropical and subtropical regions of Africa, 

Asia, and the Americas.258 Infection with dengue virus 

(DENV) can present in several clinical manifestations. 

Classical dengue fever is an acute febrile illness that 

often occurs in children and is characterized by fever, 

severe headache and muscle aches, nausea, vomiting, 

and rash. This acute illness, which usually lasts for 8 to 

10 days, is rarely fatal. A more severe form of dengue 

infection is dengue hemorrhagic fever/dengue shock 

syndrome (DHF/DSS). DHF usually begins during the 

first week of the acute illness and can lead to hemorrhagic manifestations, including petechiae, ecchymoses, epistaxis, bleeding gums, and gastrointestinal tract 

bleeding.259 DSS occurs if the patient goes on to develop 

hypotension and shock due to plasma leakage and circulatory failure, which happens in about one-third of 

severe dengue cases (especially children) and is often 

associated with higher mortality. Convalescence for 

patients with DHF is usually short and uneventful, 

and if shock is overcome, patients usually recover 

within 2 to 3 days.259

The pathogenesis of DHF/DSS is complicated and 

not well understood. Two theories are frequently cited 

to explain the pathogenetic changes that occur in DHF/

DSS. The most commonly accepted theory is known 

as immune enhancement.260,261 This idea suggests that 

patients experiencing a second infection with a heterologous DENV serotype have a significantly higher 

risk of developing DHF and DSS. Preexisting heterologous dengue antibody recognizes the infecting virus 

and forms an antigen-antibody complex, which is then 

bound to and internalized by immunoglobulin Fc receptors on macrophages. Thus, it is hypothesized that 

prior infection, through a process known as antibodydependent enhancement, enhances the infection and 

replication of DENV in mononuclear cells.259 The other 

theory assumes that DENV changes genetically as a 

result of selective pressures as it replicates in humans 

and/or mosquitoes and that the phenotypic expression of these genetic changes may include increased 

virus replication and virulence. All the data suggest 

that a combination of one’s viral, immunopathogenic, 

age, and genetic background plays a role in disease 

severity.258

Although first identified in southeast Asia in the 

1940s and 1950s, evidence suggest that DENVs were 

derived from a primitive progenitor introduced to 

Asia from Africa about 1,000 years ago.262 Studies of 

DENV ecology in sylvatic habitats of west Africa and 

Malaysia have identified transmission cycles involving 

nonhuman primates as reservoir hosts and arboreal, 

tree-hole dwelling Aedes species mosquitoes as vectors.263,264 Efficient interhuman DENV transmission 

probably requires a human population of 10,000 to 

1 million people, a feature of urban civilization that 

did not exist until about 4,000 years ago, suggesting 

the sylvatic cycle is probably ancestral.265 Further support for this idea comes from studies suggesting that a 

zoonotic transfer of DENV from sylvatic to sustained 

human transmission occurred between 125 and 320 

years ago.262 In the past 300 years, these viruses have 

become established in the urban centers of the tropics. The principal urban vector, Aedes aegypti, is highly 

domesticated and is adapted to humans, preferring to 

feed on them and lay their eggs in artificial containers in and around houses. Ae. albopictus (the Asian 

tiger mosquito) (Figure 25-15) is a secondary vector 

of DENVs. 

In the past 25 years, a marked global emergence of 

epidemic dengue has occurred, with more frequent 

and larger epidemics associated with more severe 

disease.259,266,267 The reasons are not fully understood, 

but are thought to stem from major demographic and 

Figure 25-15. A female Aedes albopictus mosquito feeding on 

a human host. This mosquito, along with Aedes aegypti, are 

competent vectors of dengue virus.

Photograph: Courtesy of James Gathany, Centers for Disease Control and Prevention Public Health Image Library. 

Image 4490.

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societal changes that have occurred over the past 50 

years. In particular, unprecedented global population 

growth and associated unplanned and uncontrolled 

urbanization occurred, especially in the tropical developing countries.259 Other potential factors associated 

with the global emergence of dengue include the lack 

of effective mosquito control in many tropical areas 

where dengue is endemic, increased international air 

travel, and a general decay in public health infrastructure in most countries over the past 30 years.259 Dengue does occur—albeit rarely—in the United States, 

primarily in southern Texas and Florida. Because the 

mosquito vectors that transmit DENVs are distributed 

throughout much of the southeastern United States, 

there is likely a greater potential for emergence of 

dengue. This situation may be unfolding in southern 

Florida.267a Florida has a history of epidemic DENV 

transmission, but more recent cases were most likely 

imported by tourism or triggered by infected individuals traveling into the area. However, in the late summer 

of 2009, DENV-1 infection was confirmed in a person 

who acquired the virus while traveling to Key West in 

Monroe County, Florida. DENV-1 infections were subsequently confirmed in two Monroe County residents 

without history of recent travel.268 In 2010, additional 

dengue cases from Monroe County were reported, and 

DENV-1 was isolated from a mosquito pool269 and a 

blood donor from Key West.270 Phylogenetic analyses 

of these viral isolates indicated that endemic DENV-1 

was transmitted in Key West over at least a 2-year period.268 In 2013, Martin County in east-central Florida 

reported 29 cases of locally transmitted DENV-1, and 

occasional locally transmitted cases have continued to 

occur since then.267a

West Nile Virus

West Nile virus (WNV) was first isolated in 1937 

from the blood of a febrile patient in the West Nile 

district of northern Uganda. It is now one of the most 

widely distributed of all mosquitoborne arboviruses, 

and it is found in areas throughout Africa, Europe, 

Asia, and the Americas. Yet, until 1999, it was completely exotic to the Western Hemisphere. In the late 

summer of 1999, WNV emerged in the New York City 

area as the cause of an outbreak of meningoencephalitis resulting in 7 deaths among 62 confirmed cases.75 A 

concurrent outbreak occurred among the horse population on Long Island, resulting in 25 equine cases, 

including 9 fatalities.271 The principal mosquito vectors 

were Culex pipiens or other related Culex species; however, the virus has been isolated from other mosquito 

species and even from ticks in some cases.266,272 The 

virus has been shown to be capable of infecting more 

than 50 species of mosquitoes and ticks.272,273 Since the 

introduction of WNV into New York in 1999, the virus 

Figure 25-16. Yearly spread of West Nile virus activity across the United States, 1999 to 2004. Data represent counties reporting West Nile virus activity in humans (red) and nonhuman (eg, birds, mosquitoes, equines, and other mammals) (blue) in 

the United States. 

Data source: National Center for Infectious Diseases, Centers for Disease Control and Prevention.

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has spread across the United States (Figure 25-16), 

north into Canada, and south into Mexico, Central 

America, and the Caribbean Islands. 

Recent years have seen a high incidence of human infection with WNV through blood transfusion, 

mother-to-fetus transmission, and transmission in 

breast milk, and also by organ transplantation, causing even greater public health concerns.96,212,274,275 After 

several years of low WNV activity in the United States, 

a multistate outbreak was seen in 2012, with more than 

5,600 cases and 286 deaths recorded.276

Chikungunya

The first recorded outbreak of chikungunya (CHIK) 

occurred in the Newala District of Tanzania (formerly 

Tanganyika) in 1952 to 1953.277,278 The outbreak was 

initially thought to have been caused by DENV because 

it shared many clinical features with dengue infection and was thought to be transmitted by Ae aegypti

mosquitoes. The infection manifested with a sudden 

onset of incapacitating joint pain and high fever, leading locals to call it chikungunya, meaning “that which 

bends up” in the local Makonde language. The disease 

also often led to development of a maculopapular rash, 

anorexia, and constipation. Most symptoms usually resolved within 7 to 10 days, but the arthralgia could last 

for months following the infection. In some patients, 

the joint pain was so severe months after infection that 

they were unable to change position without help.

A viral agent was recovered from the serum of 

acutely ill patients by intracerebral inoculation into 

mice.279 Hyperimmune serum raised against the virus 

could cross neutralize Semliki Forest virus (an alphavirus) but not DENV, indicating that the virus was more 

closely related to the alphaviruses than flaviviruses.

The virus isolated from the outbreak in the Newala 

District, chikungunya virus (CHIKV), is an Old World 

Figure 25-17. Predicted dispersal pattern of chikungunya virus from Africa to the Indian Ocean and Europe during the past 

20 to 50 years. 

DR Congo: Democratic Republic of the Congo

Photograph: Courtesy of Creative Commons, licensed under CC BY 2.0. https://virologyj.biomedcentral.com/

articles/10.1186/1743-422X-5-33. 

Data source: de Lamballerie X, Leroy E, Charrel RN, Ttsetsarkin K, Higgs S, Gould EA. Chikungunya virus adapts to tiger 

mosquito via evolutionary convergence: a sign of things to come? Virol J. 2008;5:33. 

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alphavirus in the Semliki Forest antigenic complex 

that is found mainly in Africa and Southeast Asia. 

African transmission follows a sylvatic cycle between 

nonhuman primates, small mammals, and Aedes species mosquitoes, with occasional spillover into human 

populations when vector populations are high.263 Asian 

transmission follows an urban cycle, with the virus 

transmitted between humans via the urban dwelling 

Ae aegypti and Ae albopictus mosquito vectors.

Between 1960 and 1980 numerous documented outbreaks of CHIK occurred throughout Africa and Asia, 

followed by relative quiet between 1980 and 2000.280 In 

2000, the virus reemerged when an estimated 50,000 

people were infected in Kinshasa, Democratic Republic 

of the Congo, the first reappearance of the virus there 

in 39 years.281,282 From May until July 2004, an outbreak 

occurred on Lamu Island off the coast of Kenya. There 

were 1,300 reported cases of CHIKV infection of a total 

population of 18,000 on the island. A seroprevalence 

study conducted after the epidemic found that 75% 

of the population had detectable IgG and/or IgM 

antibodies to the virus, indicating that approximately 

13,500 people had been infected.283 The virus spread 

to Mombasa, Kenya, and then to the Comoros Islands, 

where an estimated 215,000 people contracted the 

disease on Grand Comore Island between February 

and May of 2005.284

Additional outbreaks occurred on the Indian 

Ocean islands of Mauritius, the Seychelles, Madagascar, and Mayotte, culminating in a large outbreak on 

Reunion Island between March 1, 2005 and April 30, 

2006. During the Reunion Island outbreak, an estimated 255,000 people were infected.285 The outbreak 

on Reunion Island was unusual because the main 

mosquito vector, Ae aegypti, was not abundant on the 

island. It appeared that the main vector responsible 

for transmission during the Reunion outbreak was 

Ae albopictus. Genetic characterization of the virus 

from Reunion Island identified a key single amino 

acid change (A226V, ie, the alanine at position 226 

was changed to valine) in the envelope glycoprotein 

that enabled the virus to infect Ae albopictus more 

efficiently.286,287

A large outbreak of CHIKV occurred in India in 

2006, marking a return of the virus that had been absent for 33 years (Figure 25-17; note: the yellow arrow

indicating the presence of CHIKV in India in 2000 

is derived from a virus isolated from mosquitoes in 

Yawat, Maharashtra, not a human case). It is estimated 

that 1.4 million people were infected. Genetic analysis 

of the virus showed that it was related to the East 

African and Indian Ocean strains from the previous 

couple of years, but it lacked the A226V mutation.288

During a second wave of infection in 2007 in Kerala, 

India, the virus had obtained the A226V mutation 

(likely independently from the Reunion strains of 

the virus), indicating that it had adapted to the high 

population densities of Ae albopictus in the area at 

the time.288,289

The rapid adaptation of CHIKV to Ae albopictus

mosquitoes may represent a threat to Europe and 

North America. Ae aegypti mosquitoes have only been 

detected in a small swath of the southern United States. 

Ae albopictus, however, has been detected as far north 

as Pennsylvania, New Jersey, and southern New York 

in the United States, and as far north as Germany and 

the Netherlands in Europe. An outbreak in Ravenna, 

Italy during the summer of 2007 may foreshadow 

potential future outbreaks in the United States and Europe. A visitor from the active outbreak area of Kerala, 

India became ill with CHIKV 2 days after arriving in 

Ravenna on June 21. Virus was transmitted locally by 

Ae albopictus mosquitoes, resulting in 205 autochthonous cases identified between July 4 and September 27, 

peaking during the third week of August.290 This was 

the first observation of sustained CHIKV transmission 

in a temperate climate.

In December 2013, the WHO reported confirmed 

cases of CHIKV infection on the Caribbean Island of 

Saint Martin including two confirmed cases, four probable cases, and another 20 suspected cases.291 None of 

the cases reported recent travel outside of Saint Martin, 

indicating that these were the first reported cases of local transmission of CHIKV in the Western Hemisphere.

After the initial detection in Saint Martin, the virus 

spread rapidly throughout the Caribbean, and South 

and Central America. The cumulative case number for 

2014 throughout the Americas reached nearly 25,000 

confirmed and more than 1.1 million suspected cases, 

with the highest incidence rate of 56% occurring on 

the island of Martinique.292

In the summer of 2014, the Florida Department of 

Health reported detection of the first autochthonous 

transmission of CHIKV in the United States.293 Eleven 

cases of transmission were detected in Miami-Dade, 

Palm Beach, Saint Lucie, and Broward counties.

No treatment is available for CHIKV infection. The 

sole remedy available consists of treating to alleviate 

the symptoms. An attenuated CHIKV vaccine developed by the US Army Medical Research Institute for 

Infectious Disease and the University of Maryland progressed through phase II clinical trials in 2000 before 

it was discontinued because of a change in funding 

priorities.294 The recent explosion in the size of CHIK 

outbreaks and the demonstration that the virus can 

cause outbreaks in Europe and potentially the United 

States may call for a reinvestment in the development 

of CHIKV vaccines.

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Emerging Tickborne Phleboviruses

Until recently, most bunyaviruses (family Bunyaviridae) within the genus Phlebovirus that were of 

concern to human health were transmitted by either 

mosquitoes or sandflies, including viruses such as Rift 

Valley fever virus or sandfly fever virus, respectively. 

Recently, new tickborne diseases caused by novel 

phleboviruses have emerged in China (and later seen 

in Japan and South Korea) and in the midwestern 

United States. 

Severe Fever With Thrombocytopenia Syndrome 

Virus

Severe fever with thrombocytopenia syndrome 

(SFTS) is an emerging tickborne disease first described 

from rural areas of central China.295,296 The major clinical symptoms include fever, thrombocytopenia (low 

platelet count), gastrointestinal symptoms, and leukocytopenia (low white blood cell count). Initial cases (79 

cases with 10 deaths, case fatality rate of 12.7%) were 

found in 2007 from the Henan Province.295 Interestingly, because of the similarity in clinical symptoms, 

investigators first suspected human granulocytic 

anaplasmosis, another tickborne disease caused by the 

bacterium Anaplasma phagocytophilum.

295,296 However, 

when no bacterial DNA or antibodies against this bacterium could be detected in the blood samples from the 

majority of the patients, a viral etiology was suspected. 

In 2009, a novel virus was isolated from the blood of a 

patient from Xinyang City in Henan Province.296 Independently, another group identified the virus from the 

same region of China using high-throughput sequencing of acute-phase sera from 10 patients who had fever, 

thrombocytopenia, leukopenia, and a history of tick 

bite.295 The group also isolated a virus that reacted with 

patients’ sera in immunoflorescence assays and had 

characteristic virion morphology consistent with that 

of a bunyavirus (Figure 25-18).295 These investigators 

named the disease thrombocytopenia and leukopenia 

syndrome and the new virus, Henan fever virus, after 

the location of the index patient.295 However, SFTS and 

SFTS virus (SFTSV) are generally accepted. 

As the name implies, prominent manifestations of 

the disease include thrombocytopenia and leukopenia. 

Other major symptoms include sudden onset of fever 

and gastrointestinal symptoms (vomiting, diarrhea, 

and upper abdominal pain).295,296 Multiorgan failure developed rapidly in most patients as shown by elevated 

levels of serum alanine aminotransferase, aspartate 

aminotransferase, creatine kinase, and lactate dehydroFigure 25-18. Thin-section electron microscopy of the novel 

bunyavirus (red arrows) associated with fever, thrombocytopenia, and leukopenia syndrome (now called severe 

fever with thrombocytopenia syndrome) in China. Original 

magnification ×50,000. 

Photograph: Courtesy of Creative Commons, licensed 

under CC BY 2.0. http://journals.plos.org/plospathogens/

article?id=10.1371/journal.ppat.1002369.

Data source: Xu B, Liu L, Huang X, et al. Metagenomic 

analysis of fever, thrombocytopenia and leukopenia syndrome (FTLS) in Henan Province, China: discovery of a new 

bunyavirus. PLoS Pathog. 2011;7:e1002369. 

Figure 25-19. Phylogenetic tree for the RNA-dependent RNA 

polymerase (RdRP) gene sequences of the large segment of 

an isolate obtained from a fatal case of severe fever with 

thrombocytopenia syndrome (SFTS) in South Korea (black 

dot) compared with representative SFTS virus strains from 

China and Japan. The tree was constructed on the basis of 

the nucleic acid sequences of the RdRP genes by using the 

neighbor-joining method. Location, year of isolation, and 

GenBank accession numbers are indicated. Branch length of 

the tree shows the evolutionary distance. Scale bar indicates 

2.0% sequence distance. 

Data source: Kim KH, Yi J, Kim G, et al. Severe fever with 

thrombocytopenia syndrome, South Korea, 2012. Emerg Infect 

Dis. 2013;19:1892–1894.

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genase. In one study of 285 patients from the Henan 

Province, investigators reported that a small number 

of patients experienced mental status alterations, ecchymosis, gastrointestinal hemorrhage, pulmonary 

hemorrhage, and disseminated intravascular coagulation.295 Interestingly, many of these symptoms are 

similar to those of hemorrhagic fevers caused by other 

bunyaviruses such as Crimean-Congo hemorrhagic 

fever, hemorrhagic fever with renal syndrome, and 

Rift Valley fever. Although no indications of person-toperson transmission exist in the initial clusters of cases, 

several recent reports demonstrate person-to-person 

transmission of SFTSV among healthcare workers, 

family members, and mortuary workers.211,297,298

Epidemiological investigations during the initial 

cluster of cases showed that the majority of patients 

were farmers living in wooded and hilly areas and 

were working in the fields before the onset of disease.296

Also, mosquitoes and ticks were commonly found 

in the patients’ home environment. Thus, the role of 

arthropod vectors was highly suspected. Although 

no viral RNA was found in any of 5,900 mosquitoes 

tested, more than 5% of Haemaphysalis longicornis ticks 

collected from animals in the areas were the patients 

lived contained SFTSV RNA.296 H longicornis is widely 

distributed in the Asia-Pacific region, including China, 

Korea, Japan, Australia, the Pacific Islands, and New 

Zealand.299 SFTSV-specific antibodies and viral RNA 

have also been found in several domesticated animals 

in China.300 In a sampling of more than 3,000 domesticated animals in Shandong Province, specific antibodies were detected in 69.5% of sheep, 60.5% of cattle, 

37.9% of dogs, 3.1% of pigs, and 47.4% of chickens. 

SFTSV RNA was detected in all these animal hosts as 

well, albeit at a somewhat lower prevalence, ranging 

from 1.7% to 5.3%.300 These findings demonstrate that 

natural infections of SFTSV occur in several domesticated animals in disease-endemic areas and that the 

virus has a wide host range. However, the role of domesticated animals in the circulation and transmission 

of SFTSV remains unclear.300

The disease has also been detected in Japan and 

South Korea, killing at least eight people in each 

country thus far.301 This occurrence is perhaps not 

too surprising given that the range of the tick vector 

includes these countries and SFTSV was detected 

in H longicornis ticks collected during 2011 to 2012 

in South Korea.301 The strains isolated from South 

Korea were closely related to those from China, but 

were somewhat more distantly related to those from 

Japan (Figure 25-19),301 which is consistent with the 

geographic distance between these countries and 

presumably reflects the greater evolutionary history 

between these viruses. 

Heartland Virus

At about the same time that SFTSV was discovered 

in rural China (ie, 2009), a similar but distinct virus 

infected two men in rural Missouri in the United States. 

The men, one in his late 50s and the other in his late 

60s who both lived on large farms in northwestern 

Missouri, independently presented to Heartland 

Regional Medical Center in Saint Joseph, Missouri, 

in early June 2009.302 Both individuals had elevated 

temperatures exceeding 39°C, thrombocytopenia, and 

leukopenia. Other symptoms included elevation of the 

liver enzymes alanine aminotransferase and aspartate 

aminotransferase, nonbloody diarrhea, fatigue, and 

anorexia. The two men were hospitalized for 10 and 12 

days, respectively, and both had short-term memory 

difficulty, which slowly improved over 4 to 6 weeks.302

These symptoms sound remarkably similar to those of 

the Chinese patients suffering from SFTS. 

Because all the specimens collected from these two 

individuals were negative for all the known pathogens, 

blood was sent to the CDC in Atlanta, Georgia, for 

further testing. Electron microscopy revealed viruses 

consistent with members of the family Bunyaviridae. 

Next-generation sequencing and phylogenetic analysis identified the viruses as novel members of the 

Phlebovirus genus. The authors named it the Heartland 

virus. The Heartland virus is most closely related to the 

SFTSV, but is clearly distinct because it shows amino 

acid differences in the viral polymerase and nucleoprotein of 27% and 38%, respectively.302 This novel virus 

was also distinct from an uncharacterized bunyavirus 

called Lone Star virus, which was isolated in 1967 from 

an A americanum tick found on a woodchuck in western 

Kentucky. Comparison of the polymerase amino acid 

sequence showed that the Lone Star virus shared only 

34% identity with the Heartland virus.302

Both patients infected with the Heartland virus in 

Missouri had reported being bitten by ticks 5 to 7 days 

before the onset of their illness. Given the similarity 

to SFTS, both in terms of the disease symptoms and 

the high percent identity of the virus to SFTSV, it was 

highly likely that Heartland virus was also tickborne. 

In 2012, investigators from the CDC collected and 

tested arthropods in areas of northwestern Missouri, 

including the farms of the two patients, to identify 

potential arthropod vectors for this new pathogen.303

These investigators collected 56,428 ticks at 12 sites 

including both patients’ farms. A americanum was the 

most frequently encountered tick and represented 

97.5% of the collected ticks.303 They grouped the ticks 

into pools by site, collection date, species, sex, and life 

stage. Ten pools composed of nymphs of A americanum

were RT-PCR positive for the Heartland virus, and 

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eight pools yielded viable virus in cell culture. None 

of the 758 specimens (representing 12 species) of mosquitoes collected was positive for the virus.303 Although 

more epidemiological and laboratory work is needed, 

these data strongly incriminate the A americanum tick 

as the vector of Heartland virus, at least in Missouri.

Ebola Epidemic in West Africa 

On December 26, 2013, an 18-month-old boy from 

the village of Meliandou in the Guéckédou District of 

Guinea had experienced an illness characterized by fever, vomiting, and black stools. He died 2 days later.304

Within a few weeks, his 3-year-old sister, mother, 

grandmother, and a nurse who had treated him all 

developed similar symptoms, and all died within a 

week.305 Others in the surrounding area continued to 

get sick and die for the next several months. By the 

end of March 2014, the disease that had circulated in 

the southeastern corner of Guinea was identified as 

Ebola (for more detailed information on Ebola virus, 

see Chapter 23, Filoviruses).306,307 By then, 111 clinically 

suspected cases and 79 deaths had occurred.

Full-genome sequencing of viral isolates from 

Guinea identified the virus as Zaire ebolavirus, but it 

represented a new genetic clade of virus, different from 

those circulating in the Democratic Republic of the 

Congo and Gabon. These data suggest that the virus 

may have been indigenous to Guinea and had not just 

“jumped” from another area where Ebola outbreaks 

have occurred. Ebola had not been seen in West Africa 

before, although serological evidence from patients 

with febrile illnesses had detected Ebola virus-specific 

antibodies in a subset of the population in Sierra Leone.308 An epidemiological investigation found that the 

likely source of the virus that had infected the young 

boy was a colony of insectivorous free-tailed bats (Mops 

condylurus) that lived in a hollow tree in the village. 

Villagers reported that children played regularly in 

the tree and that a colony of bats lived in the tree.309

Subsequent sequencing of a large number of Ebola 

virus isolates from the outbreak also indicated that the 

outbreak had likely started with a single transmission 

from the animal reservoir to human, followed by human-to-human transmission to sustain the outbreak.310

By March 30, 2014, cases had begun to be reported 

from the Foya district across the border in Liberia, and 

on May 24, Sierra Leone reported its first laboratory 

confirmed case.311 The West African outbreak quickly 

became the largest outbreak of Ebola in recorded history, surpassing the previous record of 425 cases from 

the 2000 to 2001 outbreak in Uganda.312 Previous outbreaks had occurred in relatively remote areas, limiting the ability of the virus to spread to large numbers 

of people. With the West African outbreak, however, 

cases were reported from the large capital cities of 

Monrovia, Liberia (~1 million inhabitants), Conakry, 

Guinea (~1.6 million inhabitants), and Freetown, Sierra Leone (~1.2 million inhabitants). Some models 

predicted that—if left unchecked—the total number of 

Ebola cases could exceed 1 million cases.313 Spurred in 

part by worst-case scenario predictions such as these, 

international partners tried to bring the outbreak under 

control. US assistance focused on Liberia, and included 

the deployment of 3,000 soldiers to Liberia under Operation United Assistance to support logistics, train 

health workers, and build 17 Ebola treatment units.314

British assistance efforts focused on Sierra Leone, and 

the French assisted the Guinean government.

The number of new cases per week peaked in late 

2014 (September in Liberia, November in Sierra Leone, 

and December in Guinea).315 During the height of 

the outbreak, Ebola virus-infected individuals were 

exported to Italy, Mali, Nigeria, Senegal, Spain, the 

United Kingdom, and the United States. Nigeria, Mali, 

and the United States also had limited local transmission of the virus originating from the imported cases. 

In the United States, a traveler from Liberia presented 

at a hospital in Dallas County, Texas, on September 25, 

2014.316 He presented with fever, abdominal pain, and 

headache, but was misdiagnosed with sinusitis and 

discharged. He returned to the hospital on September 

28 with worsening symptoms, was admitted, and tested 

positive for Ebola virus infection on September 30. The 

patient died on October 8, but two nurses became infected while caring for him. The nurses both survived.

As of April 13, 2016, there were a total of 28,616 

cases of Ebola (confirmed, probable, and suspected) 

with 11,310 deaths (40% case fatality rate).317 Sierra 

Leone was the last country to have active cases of Ebola 

virus disease and was declared Ebola-free on March 17, 

2016. An investigational vaccine for Ebola was tested 

in Guinea in 2015 and was shown to be 100% effective 

in preventing disease in contacts of confirmed Ebola 

cases.318 The trial used a “ring vaccination” design in 

which contacts and contacts of contacts of confirmed 

Ebola patients were either immediately vaccinated 

or vaccinated after a delay of 21 days. After interim 

analysis of the data from the trial showed the high effectiveness of immediate vaccination, the data safety 

and monitoring board recommended that the delayed 

vaccination arm be dropped and all participants be 

offered immediate vaccination. An additional Ebola 

vaccine trial, the Sierra Leone Trial to Introduce a 

Vaccine Against Ebola (STRIVE) was performed in 

Sierra Leone between April and August 2015. Nearly 

9,000 volunteers consisting of healthcare and frontline 

workers were vaccinated in a phase II/III trial. Due 

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to the decrease in incidence of Ebola virus disease 

during the duration of the STRIVE trial, efficacy data 

could not be obtained for the vaccine, but a subset of 

the participants was enrolled in safety and immunogenicity substudies. No adverse events were reported 

in the safety study, and the immunogenicity study is 

ongoing as of October 2016. 

Viral Pathogen Discovery by High-Throughput 

DNA Sequencing

Traditional diagnosis of viral infections has required some foreknowledge of the viral pathogen 

that is suspected to be causing disease. For example, 

enzyme-linked immunosorbent assays require either 

recombinant antigens or antigens isolated from whole 

organisms. Real-time PCR assays require sequence 

information from the genomes of the organisms that 

are suspected to be present. Any novel agent that has 

a genome that is sufficiently divergent enough to alter 

the structure of an antigen or change a binding site for 

a real-time PCR probe may not be detected in these assays. The advent of microarrays that contain millions of 

short nucleic acid probes increases the likelihood that 

a pathogen can be detected. These arrays can screen 

for hundreds to thousands of pathogens in a single 

sample,319,320 but still require the genome sequences of 

each pathogen to design probes. Novel or divergent 

pathogens may also escape identification by arrays. 

When high-throughput massively parallel sequencing became available in 2005,321 it was quickly realized 

that the hundreds of thousands to millions of sequencing reads obtained by this new form of sequencing could 

be used as a diagnostic tool. The first demonstration of 

this occurred in 2007 with the detection of Israeli acute 

paralysis virus in colonies of bees that suffered from 

colony collapse disorder (CCD).319 RNA was isolated 

from bees from CCD and non-CCD bee colonies, amplified by random RT-PCR, and then sequenced on a 454 

sequencer. Examination of the sequence reads identified 

large numbers of bacteria, fungi, and viruses, but only 

reads from Israeli acute paralysis virus seemed to correlate with the presence of CCD in a colony.

The first demonstrated use of massively parallel 

sequencing for the detection of a novel human pathogen occurred in 2008, when a novel arenavirus was 

detected in patients that had received visceral organ 

donations from a single donor.322 The donor had died 

of cerebral hemorrhage 10 days after returning from 

a 3-month trip to the former Yugoslavia. His liver 

and kidneys were transplanted into three recipients, 

whose initial recoveries were unremarkable. Within 

4 to 6 weeks of receiving the transplants, however, 

all three recipients died displaying various levels of 

encephalopathy. Tissues were collected from each 

recipient, and RNA was extracted for sequencing. 

After sequencing, 14 sequence reads out of more 

than 100,000 reads obtained showed amino-acid level 

similarity to lymphocytic choriomeningitis virus. The 

reads obtained by massively parallel sequencing were 

used to design PCR primers, and standard PCR and 

sequencing enabled the recovery of the full genome 

sequence of the novel virus. The virus was 72% to 

87% identical to lymphocytic choriomeningitis virus 

at the nucleotide level, and 79% to 97% identical at the 

amino acid level. Despite these relatively high levels of 

identity to lymphocytic choriomeningitis virus, initial 

tests with diagnostic microarrays had failed to identify 

any candidate viruses as a cause of the infection.

Another example of the use of massively parallel 

sequencing to identify a novel pathogen was published in 2009.323 In September 2008, a patient in critical 

condition with hemorrhagic fever was airlifted from 

Lusaka, Zambia to Standton, South Africa (a suburb of 

Johannesburg). Although the index patient was under 

care in the hospital, the disease spread to a paramedic 

who had been on the air ambulance flight from Lusaka, a nurse who attended to the index patient, and 

a hospital worker who had cleaned the index patient’s 

room. A tertiary case of disease also spread to a nurse 

who attended to the sick paramedic. The first four 

patients died, and the fifth patient, who was treated 

with ribavirin, survived. Liver and skin sections were 

submitted to the CDC, where immunohistochemical 

staining with a monoclonal antibody broadly crossreactive for Old World arenaviruses gave a positive result.323 Subsequent RT-PCR with conserved arenavirus 

primers yielded partial sequences of the glycoprotein 

and nucleoprotein genes, indicating the presence of 

a novel arenavirus. Serum and tissue samples from 

some of the cases were submitted for massively parallel 

sequencing, yielding 5.6 kb of sequence from the novel 

arenavirus. The partial genome was used to design 

PCR amplicons, and those were used to recover the 

full genome of the virus. Analysis of the full genome 

indicated that the novel virus was phylogenetically 

distinct from previously known arenaviruses, and it 

was named Lujo virus after Lusaka and Johannesburg.

Both of the cases described previously used relatively low numbers of sequencing reads from a 454 

sequencer (around 100,000 per sample) to identify 

genome fragments of the novel viruses. The fragments 

were then joined by PCR and conventional Sanger 

sequencing to obtain the full genome sequences. A 

third example demonstrated a different approach 

using the extremely high capacity of the Illumina 

HiSeq sequencer. In 2009, three people from a remote 

village in the Bas-Congo province of the Democratic 

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Republic of the Congo were stricken with a hemorrhagic fever of unknown origin.324 The first case was 

a 15-year-old boy who presented to the hospital with 

malaise, epistaxis, conjunctival injection, gingival 

bleeding, hematemesis, and bloody diarrhea. The 

hemorrhagic symptoms had only started the previous 

day, and the patient died 2 days later from sudden 

circulatory collapse. The second case was a 13-yearold girl who presented with similar symptoms and 

died 3 days after onset of her disease. The final case 

was a 32-year-old male nurse who worked in the 

clinic where the original two patients had been seen. 

He was transferred to a regional hospital 2 days after the onset of disease, where he was treated with 

fluid resuscitation, blood transfusion, and antibiotics. He recovered spontaneously a few days later.

A serum sample that was taken from the nurse before his recovery tested negative by PCR for all viruses 

known to cause acute hemorrhagic fever in Africa. 

RNA was extracted from the serum and sequenced by 

454, yielding a single read (of approximately 4,500) that 

had 41% identity to known rhabdoviruses. Attempts 

to recover more of the virus genome sequence by 

PCR were stymied by limited sample, so the sample 

was subjected to sequencing on the HiSeq. The HiSeq 

run yielded more than 140 million reads, with 30,000 

of them mapping to the novel rhabdovirus. The large 

number of reads obtained allowed reconstruction of 

98.2% of the genome and showed that the new virus, 

named Bas-Congo virus, was only distantly related to 

other rhabdoviruses.

Several other examples of the utility of next generation sequencing are applied to viral pathogen discovery, 

including Heartland virus and SFTSV mentioned previously in this chapter. However, several bottlenecks still 

remain to the widespread adoption of this technology 

in diagnostic settings. One of the most difficult aspects 

is simply analyzing the extremely large datasets that 

can be generated. In many cases, the overwhelming 

majority of sequence reads will be from the host (human) and trying to identify a pathogen is like looking 

for a needle in a haystack. Many groups are working 

on computer algorithms that can sort through the datasets to rapidly and correctly identify pathogen reads. 

No one algorithm has been successful, prompting the 

US Defense Threat Reduction Agency in 2013 to offer a $1 million prize through Innocentive.com to the 

team that can develop the most reliable and efficient 

algorithm. The prize ultimately went to a bioinformatics team at the University of Tübingen in Germany. 

The unprecedented ability to detect all of the nucleic 

acid present in a diagnostic sample is a powerful tool 

for pathogen discovery, but it does have some pitfalls. 

An example of this pitfall was published in September 

2013 when the genome of a highly divergent single 

stranded DNA virus was detected in samples from 

patients with chronic seronegative hepatitis and diarrhea of unknown etiology.325 Deep sequencing of 

serum samples from patients with chronic hepatitis 

identified a virus that was related to both circoviruses 

and parvoviruses, and it was provisionally named 

parvovirus-like hybrid virus (PHV-1). Deep sequencing of diarrheal stool samples in a separate laboratory 

independently identified a virus that had 99% identity 

to PHV-1, which were named PHV-2. Both PHV-1 and 

PHV-2 were 99% identical to a virus named National 

Institutes of Health-Chonqing virus that had been identified in samples from Chinese seronegative hepatitis 

patients.179 Suspicions about the frequency at which 

these viruses were being detected led to a reanalysis 

of the samples using different nucleic acid extraction 

reagents, and eventually led to the conclusion that all 

of the detections of these viruses were likely linked to 

commercial nucleic acid isolation spin-columns that 

had been used in all of the studies. PHV sequences 

were identified in metagenomic sequencing datasets 

from the coastal marine waters of North America, suggesting that PHV was linked to diatoms present in the 

marine waters that generate the silica matrix used in 

the commercial spin-columns. 

As next-generation sequencing continues to increase 

in throughput and decreases in price, its utility for 

identifying novel viral pathogens will continue to increase. One can imagine a scenario in the not so distant 

future in which a clinician will be able to test for every 

pathogen present in a patient’s sample without needing to pre-select tests for specific pathogens based on 

the patient’s symptoms.

FUTURE THREATS

Genetically Engineered Organisms

Without human intervention, the natural world has 

produced innumerable microbial agents that continue 

to emerge as new or newly observed causes of disease. 

Human activity has also played a huge role in the 

emergence of many diseases, but this effect has—for 

the most part—been inadvertent, rather than deliberate. 

The spread of HIV, for example, can be attributed almost 

entirely to human behavior, and the same was true of 

the spread of smallpox. Historically, both microbial 

agents and the affected populations have tended toward 

change during the disease outbreaks. Examples from the 

human experience include the way in which diseases 

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such as smallpox and measles favored the survival of 

several generations of Europeans who were most resistant to these diseases, followed later by unchecked 

contagion and decimation of new populations when the 

same diseases were introduced to isolated islands and 

the New World.326,327 A classic example of agent–host 

adaptation in animals was the intentional introduction 

of myxoma virus (an orthopoxvirus, reminiscent of 

smallpox in rabbits) into Australia to control or eliminate 

a scourge of rabbits. At first, mortalities were high in 

the Australian rabbits, but in time the rabbits acquired 

a degree of genetic resistance. In parallel, the circulating virus became diminished in its virulence, persisting 

and being shed over a longer period of time in infected 

rabbits.328 For both rabbit and virus, natural selection 

blindly favored survival of the species. This natural 

order has been intentionally perturbed by humans, 

from the lifesaving selection of relatively benign forms 

of disease to use as vaccines against the most virulent 

forms (eg, variolation, or the classical adaptation of 

measles, mumps, and rubella vaccines) to the intentional 

selection of the most virulent disease agents in biological 

weapons programs (the latter finally stigmatized and 

outlawed as such in the Biological Weapons Convention Treaty). Other microbial perturbations have been 

unintended, such as the treatment-based selection of antibiotic resistant bacteria now widespread in hospitals.329

More recently, humankind has acquired the technical capacity to create microbial threats far more deadly 

than natural evolution could create or sustain. Genetic 

engineering, the intentional molecular manipulation 

of genes, has proven to have capacity for both good 

and ill. A few examples from open scientific literature 

will follow to illustrate the seriousness of the threat of 

genetically engineered microorganisms. 

Antibiotic resistant strains of B anthracis, the causative agent of anthrax, have been derived not only by 

biological selection, but also more directly by genetic 

engineering.330–332 Scientifically, the capacity to do so 

with any bacterial threat is unsurprising, but the implications are ominous. Similarly, for anyone moderately 

skilled in microbiology, it is obvious that otherwise 

harmless bacteria may be engineered to synthesize 

toxins made by unrelated lethal strains of bacteria. 

Buffering the threat, unauthorized conduct of most 

such experimentation has become not only difficult 

but also illegal—subject to fines and incarceration—in 

many countries including the United States. In the 

United States, federally funded research that may 

result in knowledge that could be used for nefarious 

purposes, so called dual use research of concern, is 

subject to review before initiation of research and also 

at the stage when the findings from such research are 

ready for submission for publication. 

Viral genomes can now be easily manipulated in 

the laboratory and infectious viruses can be generated from plasmid DNA. The progression of this 

technology with human pathogens began some 20 

years ago with the simpler viruses (positive sense, 

single-strand, small genomes) such as poliovirus,333

alphaviruses,205 and flaviviruses.334 It has grown to include negative-strand viruses (eg, vesicular stomatitis 

virus, respiratory syncytial virus, Ebola virus) and 

segmented viruses (eg, influenza virus, CrimeanCongo hemorrhagic fever virus). Even the relatively 

large genome of vaccinia virus can be derived from 

DNA cloned into bacteria.335 In a parenthetical observation that was alarming to some in its simplicity, the 

capacity to derive a human pathogenic virus (poliovirus) by chemical synthesis was demonstrated.336 Even 

more controversial were the efforts to genetically 

resurrect the 1918 influenza virus that killed some 

20 million persons before disappearing152,337–339 and 

the proposals to genetically manipulate smallpox 

virus.340 Experiments designed to create or improve 

vaccines, to understand interactions between virus 

and host, or to unveil some arcane mysteries of the 

viruses themselves have simultaneously proven the 

ease with which bioactive and sometimes harmful 

molecules may be inserted into viruses. Symbolizing this, a large body of work with recombinant 

poxviruses was widely considered to be entirely 

benign until it was reported that a mouse poxvirus 

(ectromelia virus) was rendered more virulent by its 

modification to co-express a molecule of the immune 

system (ie, interleukin-4).341 This result was merely 

part of a progression of studies of similar design 

and outcome,342 but its timing (2001) crystallized the 

potential problem. 

Perhaps the most prominent example of dual use 

research of concern in recent years occurred in late 

2011, when two independent research groups prepared 

to publish research studies in which mutations were 

introduced into highly pathogenic influenza H5N1 

viruses that facilitated efficient transmission of the 

viruses in the ferret model.188,191 The ensuing debate 

resulted in a self-imposed moratorium on such research by influenza scientists in the United States and 

internationally,343 while a regulatory framework for the 

review of proposals for such gain-of-function studies 

was constructed.344 As a result, research proposals for 

this type of study submitted for US federal funding are 

subject to additional layers of review. It is expected that 

other countries will follow suit, if they do not already 

have such a framework. For more detailed information, the reader is directed to a special issue of Science

specifically devoted to the H5N1 gain-of-function 

research debate.345

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Ultimately, the capacity to create deadly pathogens 

through genetic engineering is restrained in large part 

by technical knowledge and opportunity, and in the final analysis, by intent. That is, what is straightforward 

for skilled scientists is impossibly difficult for the 

untrained and unequipped. However, a determined 

person with the appropriate set of knowledge and 

skills may succeed in creating genetically engineered 

microorganisms. Unfortunately, such organisms 

could also be created by well-intentioned scientists 

who underestimate the unexpected consequences of 

their work.

What countermeasures and solutions exist? Laws 

and regulations to emphatically restrict accidental or 

intentional creation of new deadly organisms, or possession of the deadly agents already existing in nature, 

have been implemented in the United States (eg, 7 

CFR Part 331, 9 CFR Part 121, and 42 CFR Part 73), 

but these bounds are difficult—if not impossible—to 

enforce internationally. Also helpful are the myriad 

coordination meetings and rehearsals for public 

health responses to pandemic natural threats such as 

smallpox or a pandemic influenza virus; in the case of 

the outbreak of a contagious genetically engineered 

microorganism, classical methods of epidemiology 

and quarantine would likely be exceedingly helpful. 

Also encouraging, the likely period of ignorance as to 

the nature and design of a newly emerged causative 

agent has been compressed as the newest technologies 

have been applied to both diagnostics and bioforensics. 

Less encouraging, development of specific medical 

countermeasures (vaccines, therapeutic drugs) for a 

previously unknown organism usually takes years. 

Some regard this as impetus to redirect greater funding toward discovery of generic methods of boosting 

innate immunity in persons to provide increased 

resistance to most or all infectious agents. A related 

approach is to target common cellular pathways used 

and shared by many unrelated agents, especially 

viruses. As with conventional agents, great localized 

harm could be done and widespread panic produced 

by genetically engineered microorganisms, even if 

medical countermeasures were nominally available.

Synthetic Biology

Genome synthesis is no longer limited to the realm of 

viral genomes. In 2008, Gibson et al published a paper 

describing the complete chemical synthesis of all 582,970 

bases of the Mycoplasma genitalium genome.346 The starting material for the synthesis was short oligonucleotides 

of the kind that can be purchased for $0.10 per base or 

less. These were gradually assembled into larger and 

larger pieces of DNA until the researchers cloned and 

maintained the complete genome in the form of a yeast 

artificial chromosome in Saccharomyces cerevisiae.

Following closely on the heels of this achievement, 

the same group published a second paper in 2010 

detailing the complete chemical synthesis of the 1.08 

megabase-pair genome of M mycoides.

347 This genome 

was synthesized in a manner similar to that described 

above, but the group went one step farther. The group 

transplanted the synthetic genome into the husk of a 

M capricolum cell from which the normal genome had 

been removed. The cellular materials left behind after 

removing the normal genome accepted the new, synthetic genome and kick-started replication of the novel 

bacterium called M mycoides JCVI-syn1.0 after the J. 

Craig Venter Institute where the work was performed. 

To prove that the new bacterium had the synthetic genome, the group had included watermarks encoded in 

the genome during synthesis. These watermarks used 

a cipher made of short nucleotide sequences to encode 

e-mail and web addresses, the names of the authors, 

and the following famous quotes:

• “To live, to err, to fall, to triumph, to recreate 

life out of life” (by James Joyce), 

• “See things not as they are, but as they might 

be” (by Robert Oppenheimer), and 

• “What I cannot create, I cannot understand” 

(by Richard Feynman).

SUMMARY

Emerging infectious diseases are among some of the 

most important future threats facing both military and 

civilian populations. These diseases are caused by a 

variety of infectious agents (ie, bacteria, viruses, fungi, 

and parasites), some of which are new to mankind, 

whereas others have been around for millennia, but 

are only newly recognized. Still others may be common commensals that have acquired virulence factors 

(eg, toxins) or antimicrobial resistance genes though 

natural or unnatural (ie, genetic engineering) means. 

Despite many successes in disease control and prevention, infectious diseases remain the leading cause 

of death worldwide and the third leading cause of 

death in the United States. HIV/AIDS, which was first 

recognized in 1981, is the most dramatic example of 

an infectious disease that has rapidly emerged during 

the last 35 years. Despite the significant advances in 

treatment of HIV/AIDS, the pandemic will continue 

to put large numbers of people at risk for new and 

reemerging opportunistic infections. The rapid spread 

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of the West Nile virus across the United States after 

its introduction in 1999 and the increasing problem 

of antimicrobial resistance are other examples of the 

ability of microbes to emerge, adapt, and spread.

Future threats are difficult to predict but will likely 

include many of the topics covered in this chapter, 

including the following: 

• increasingly complex challenges of foodborne 

and waterborne diseases, 

• the threat of another influenza pandemic, 

• emerging antibacterial and antiviral resistance, and 

• the increasing incidence of zoonotic diseases. 

Meeting these challenges will require a multidisciplinary approach using the expertise of physicians and 

veterinarians trained in public health, microbiologists, 

pathologists, ecologists, vector biologists, and military 

and civilian public health officials. 

Emerging infectious diseases have been defined 

as those diseases which have been newly recognized or whose incidence has increased within 

the past 20 years. What new diseases will be encountered in the next 20 years? What role will the 

increasingly advanced fields of molecular biology, 

genomics, and synthetic biology play? Will infectious agents from the past be resurrected, as has 

been done with the 1918 influenza virus? Or will 

increasingly advanced bioterrorists or rogue nations create weapons though genetic engineering or 

synthetic biology? Only through increased knowledge gained from continued research in infectious 

diseases will we be able to meet the challenges of 

these future threats. 

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314. Gostin LO, Friedman EA. A retrospective and prospective analysis of the west African Ebola virus disease epidemic: 

robust national health systems at the foundation and an empowered WHO at the apex. Lancet. 2015;385:1902–1909.

315. World Health Organization. Ebola Data and Statistics. http://apps.who.int/gho/data/node.ebola-sitrep. Accessed September 17, 2015. 

316. Chevalier MS, Chung W, Smith J, et al. Ebola virus disease cluster in the United States—Dallas County, Texas, 2014. 

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ebola-situation-report-9-september-2015. Accessed September 15, 2015. 

318. Henao-Restrepo AM, Longini IM, Egger M, et al. Efficacy and effectiveness of an rVSV-vectored vaccine expressing Ebola surface glycoprotein: interim results from the Guinea ring vaccination cluster-randomised trial. Lancet. 

2015;386:857–866.

319. Cox-Foster DL, Conlan S, Holmes EC, et al. A metagenomic survey of microbes in honey bee colony collapse disorder. 

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320. Wang D, Coscoy L, Zylberberg M, et al. Microarray-based detection and genotyping of viral pathogens. Proc Natl Acad 

Sci U S A. 2002;99:15687–15692.

321. Margulies M, Egholm M, Altman WE, et al. Genome sequencing in microfabricated high-density picolitre reactors. 

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322. Palacios G, Druce J, Du L, et al. A new arenavirus in a cluster of fatal transplant-associated diseases. N Engl J Med. 

2008;358:991–998.

323. Briese T, Paweska JT, McMullan LK, et al. Genetic detection and characterization of Lujo virus, a new hemorrhagic 

fever-associated arenavirus from southern Africa. PLoS Pathog. 2009;5:e1000455.

324. Grard G, Fair JN, Lee D, et al. A novel rhabdovirus associated with acute hemorrhagic fever in central Africa. PLoS 

Pathog. 2012;8:e1002924.

325. Naccache SN, Greninger AL, Lee D, et al. The perils of pathogen discovery: origin of a novel parvovirus-like hybrid 

genome traced to nucleic acid extraction spin columns. J Virol. 2013;87:11966–11977.

326. Diamond J. Guns, Germs, and Steal: The Fates of Human Societies. New York, NY: WW Norton; 1997.

327. McMichael AJ. Environmental and social influences on emerging infectious diseases: past, present and future. Philos 

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328. Fenner F. The Florey lecture, 1983. Biological control, as exemplified by smallpox eradication and myxomatosis. Proc 

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329. Barrett JF. MRSA: status and prospects for therapy? An evaluation of key papers on the topic of MRSA and antibiotic 

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330. Athamna A, Athamna M, Abu-Rashed N, Medlej B, Bast DJ, Rubinstein E. Selection of Bacillus anthracis isolates resistant 

to antibiotics. J Antimicrob Chemother. 2004;54:424–428.

331. Brook I, Elliott TB, Pryor HI 2nd, et al. In vitro resistance of Bacillus anthracis Sterne to doxycycline, macrolides and 

quinolones. Int J Antimicrob Agents. 2001;18:559–562.

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332. Gilligan PH. Therapeutic challenges posed by bacterial bioterrorism threats. Curr Opin Microbiol. 2002;5:489–495.

333. Racaniello VR. Studying poliovirus with infectious cloned cDNA. Rev Infect Dis. 1984;6(Suppl 2):S514–S515.

334. Rice CM, Grakoui A, Galler R, Chambers TJ. Transcription of infectious yellow fever RNA from full-length cDNA 

templates produced by in vitro ligation. New Biol. 1989;1:285–296.

335. Domi A, Moss B. Cloning the vaccinia virus genome as a bacterial artificial chromosome in Escherichia coli and recovery 

of infectious virus in mammalian cells. Proc Natl Acad Sci U S A. 2002;99:12415–12420.

336. Cello J, Paul AV, Wimmer E. Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of 

natural template. Science. 2002;297:1016–1018.

337. Palese P. Influenza: old and new threats. Nat Med. 2004;10:s82–s87.

338. Kaiser J. Biocontainment. 1918 flu experiments spark concerns about biosafety. Science. 2004;306:591.

339. Kobasa D, Takada A, Shinya K, et al. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 

pandemic virus. Nature. 2004;431:703–707.

340. Vogel G. Infectious diseases. WHO gives a cautious green light to smallpox experiments. Science. 2004;306:1270–1271.

341. Jackson RJ, Ramsay AJ, Christensen CD, Beaton S, Hall DF, Ramshaw IA. Expression of mouse interleukin-4 by a recombinant ectromelia virus suppresses cytolytic lymphocyte responses and overcomes genetic resistance to mousepox. 

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342. Ramshaw IA, Ramsay AJ, Karupiah G, Rolph MS, Mahalingam S, Ruby JC. Cytokines and immunity to viral infections. Immunol Rev. 1997;159:119–135.

343. Fouchier RA, Garcia-Sastre A, Kawaoka Y, et al. Pause on avian flu transmission research. Science. 2012;335:400–401.

344. Patterson AP, Tabak LA, Fauci AS, Collins FS, Howard S. Research funding: a framework for decisions about research 

with HPAI H5N1 viruses. Science. 2013;339:1036–1037.

345. Alberts B. H5N1. Introduction. Science. 2012;336:1521.

346. Gibson DG, Benders GA, Andrews-Pfannkoch C, et al. Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science. 2008;319:1215–1220.

347. Gibson DG, Glass JI, Lartigue C, et al. Creation of a bacterial cell controlled by a chemically synthesized genome. 

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Chapter 26 

LABORATORY IDENTIFICATION 

OF THREATS

DAVID A. NORWOOD, PhD*; TIMOTHY D. MINOGUE, PhD†; RANDAL J. SCHOEPP, PhD‡; and MARK J. WOLCOTT, PhD§

INTRODUCTION

THE LABORATORY RESPONSE 

Role of the Military Clinical and Field Laboratories

Military Field Laboratories

Defense Laboratory Network 

Identification Levels

Civilian

Military

Allies

IDENTIFICATION APPROACHES

Specimen Collection and Processing

Culture-Based Microbiological Methods 

Automated Identification Systems

Antibiotic and Antimicrobial Susceptibility Testing

Microbial Culture Versus Rapid Methods

Integration of In Vivo and In Vitro Diagnostic Tests

Immunodiagnostic Methods

Enzyme-Linked Immunosorbent Assay 

Electrochemiluminescence

Flow Cytometry

MAGPIX

Hand Held Assays 

Future Perspectives

Molecular Detection Methods

Real-Time Polymerase Chain Reaction

Next Generation Molecular Diagnostics

The Horizon–Agnostic Diagnostic Applications

BIOSURVEILLANCE AND EMERGING THREATS

FUTURE APPROACHES

Early Recognition of the Host Response

SUMMARY

*Chief, Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

†

Chief, Molecular Diagnostics Department, Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter 

Street, Fort Detrick, Maryland 21702

‡

Chief, Immunodiagnostics and Biologics Department, Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland 21702

§

Chief, Transitional and Operational Diagnostics Department, Diagnostic Systems Division, US Army Medical Research Institute of Infectious Diseases, 

1425 Porter Street, Fort Detrick, Maryland 21702

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INTRODUCTION

Amendments (CLIAs), which were passed in 1988 

(CLIA ‘88), regulate the quality of the clinical laboratory performing the testing, whereas section 210(h) of 

the Federal Food, Drug, and Cosmetic Act regulates in 

vitro diagnostic tests. As such, the FDA has oversight 

and regulatory authority to clear in vitro diagnostic 

tests (medical devices) for commercial sale and use. 

The combination of a CLIA-accredited laboratory 

performing an FDA-cleared diagnostic test results in 

a regulatory compliant diagnostic result that can be 

used for the patient’s treatment and prognosis. For the 

Department of Defense (DoD), maintaining regulatory 

compliance in performing in vitro diagnostic tests in a 

deployed environment poses a significant challenge.

CLIA sets the standards for any laboratory that 

performs testing on human samples for use in disease diagnosis and treatment. The goal of CLIA was 

to improve the quality of any testing conducted for 

medical purposes, and DoD facilities are not exempt 

from the requirements. However, the DoD was allowed 

to develop a separate plan for ensuring quality and 

standards in diagnostic testing, the Clinical Laboratory Improvement Program (CLIP; DoD Instruction 

6440.2).2

 CLIP is similar to CLIA with certain exceptions to meet military operational requirements. Both 

CLIA and CLIP govern the quality of the laboratory 

performing the diagnostic test and include standards 

for personnel, quality control, quality assurance, procedure manuals, proficiency testing, and inspections 

for adherence to the standards. CLIA and CLIP require 

laboratory registration to perform testing, and registrations are based on the level of test complexity that the 

laboratory is accredited to perform. 

Minimal complexity tests (waived) are simple tests 

that do not require significant quality oversight, such 

as tests cleared by the FDA for home use. Moderate and 

high complexity tests require increased knowledge, 

training and experience, quality control, and interpretation and judgment. Moderate tests are typically 

more automated while high complexity tests require 

significant technical manipulation by personnel. The 

current FDA-cleared diagnostic system, the Joint 

Biological Agent Identification and Diagnostic System 

(JBAIDS), is a high complexity test, which can make it 

difficult to maintain a high complexity CLIA registration in a deployed setting. 

Movement of laboratory diagnostic capabilities to 

forward locations is driving long-term goals for DoD 

medical diagnostic devices to be CLIA-waived devices. 

The Next Generation Diagnostic System (NGDS), the 

Biofire Defense FilmArray, will likely be a moderate 

complexity device, an incremental improvement over 

Medical diagnosis is the process by which clinicians 

attempt to deduce the cause of a particular disease or 

disorder in a sick individual. The goal of diagnosis is 

to assist in making correct medical decisions about the 

patient’s treatment and prognosis. For infectious diseases, a variety of medical information is used to make 

a diagnosis including a physical examination, interview 

with the patient, medical history of the patient, and 

clinical findings as reported by laboratory tests. The 

focus of this chapter will center on laboratory tests used 

to diagnose biological threat agents. These tests represent a piece of the diagnostic puzzle and should not be 

used solely for diagnosis and treatment. Physical and 

clinical findings as well as medical history are critical 

to an accurate diagnosis, and integrating all available 

medical information as well as all available laboratory information reduces the chance for misdiagnosis. 

Diagnosis requires the synthesis of multiple pieces of 

information into a medical judgment that will be used 

to affect patient care; therefore, getting the right answer 

must always take priority over getting a quick answer. 

The content of this review will focus on the current 

and future state of in vitro diagnostics, as defined by 

the Food and Drug Administration (FDA): 

Those reagents, instruments, and systems intended 

for use in diagnosis of disease or other conditions, 

including a determination of the state of health, in 

order to cure, mitigate, treat, or prevent disease or 

its sequelae. Such products are intended for use in 

the collection, preparation, and examination of specimens taken from the human body.1

 

For the purposes of this chapter, a biological threat 

is any infectious disease entity or biological toxin 

encountered, either through natural distribution or 

intentionally delivered by an opposing force to deter, 

delay, or defeat US or allied military forces. The majority of biological threats of military and public health 

relevance are contained in the Department of Health 

and Human Services (DHHS) select agents and toxins 

list of regulated biological select agents and toxins (Table 26-1). Many of these biological threats were part of 

offensive biological weapons programs at one time. As 

with other infectious disease assays, biological threat 

laboratory assays are subject to the same requirements 

and regulations to be regulatory compliant, meaning 

that the test can be used for patient care. 

For laboratory assays, two critical elements must 

meet minimal standards to be considered regulatory 

compliant: (1) the laboratory performing the test must 

be qualified, and (2) the test being performed must 

be validated. The Clinical Laboratory Improvement 

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HHS SELECT AGENTS AND TOXINS

• Abrin

• Botulinum neurotoxins*

• Botulinum neurotoxin-producing Clostridium*

• Conotoxins

• Coxiella burnetii

• Crimean-Congo hemorrhagic fever virus

• Diacetoxyscirpenol

• Eastern equine encephalitis virus

• Ebola virus*

• Francisella tularensis*

• Lassa fever virus

• Lujo virus

• Marburg virus*

• Monkeypox virus

• 1918 pandemic influenza virus

• Ricin

• Rickettsia prowazekii

• SARS-associated coronavirus (SARS-CoV)

• Saxitoxin

• South American hemorrhagic fever viruses:

 Chapare

 Guanarito

 Junin

 Machupo

 Sabia

• Staphylococcal enterotoxins A,B,C,D,&E subtypes

• T-2 toxin

• Tetrodotoxin

• Tick-borne encephalitis complex (flavi) viruses:

 Far Eastern subtype

 Siberian subtype

• Kyasanur Forest disease virus

• Omsk hemorrhagic fever virus

• Variola major virus (Smallpox virus)*

• Variola minor virus (Alastrim)*

• Yersinia pestis* 

OVERLAP SELECT AGENTS AND TOXINS

• Bacillus anthracis*

• Bacillus anthracis Pasteur strain

• Brucella abortus

• Brucella melitensis

• Brucella suis

• Burkholderia mallei*

• Burkholderia pseudomallei*

• Hendra virus

• Nipah virus

• Rift Valley fever virus

• Venezuelan equine encephalitis virus

USDA SELECT AGENTS AND TOXINS

• African horse sickness virus

• African swine fever virus

• Avian influenza virus

• Classical swine fever virus

• Foot-and-mouth disease virus*

• Goat pox virus

• Lumpy skin disease virus

• Mycoplasma capricolum

• Mycoplasma mycoides

• Newcastle disease virus

• Peste des petits ruminants virus

• Rinderpest virus*

• Sheep pox virus

• Swine vesicular disease virus

USDA PLANT PROTECTION AND QUARANTINE 

SELECT AGENTS AND TOXINS

• Peronosclerospora philippinensis

• Peronosclerospora sacchari

• Phoma glycinicola

• Ralstonia solanacearum

• Rathayibacter toxicus

• Sclerophthora rayssiae

• Synchytrium endobioticum

• Xanthomonas oryzae

TABLE 26-1 

REGULATED BIOLOGICAL SELECT AGENTS AND TOXINS

HHS and USDA Select Agents and Toxins (7 CFR Part 331, 9 CFR Part 121, and 42 CFR Part 73)

*Denotes Tier 1 agent.

CFR: Code of Federal Regulations; HHS: Health and Human Services; SARS: severe acute respiratory syndrome; USDA: US Department 

of Agriculture.

Data source: http://www.selectagents.gov/SelectAgentsandToxinsList.html (valid June 2016).

the currently deployed system. The system is already 

FDA cleared for several infectious disease diagnostics 

and received an Emergency Use Authorization (EUA) 

during the 2014 Ebola outbreak in West Africa. FDAcleared assays for biological threats on the FilmArray 

will likely be available in 2017 and will replace the 

JBAIDS. 

Although CLIA/CLIP requirements are based on 

test complexity, FDA requirements for clearance are 

based on the risk associated with the test, and risk is 

dependent on the potential harm associated with obtaining the wrong diagnostic result. The FDA classifies 

in vitro diagnostic tests as either class I (lowest risk), 

class II (moderate to high risk), or class III (highest 

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risk) medical devices. The currently fielded JBAIDS 

system is an FDA regulated class II device and currently resides in combat support hospitals within the 

US Army and within other medical treatment facilities 

for the Air Force and Navy. For the DoD, the challenge 

remains maintaining regulatory compliance in far 

forward operational settings. 

The availability of FDA-cleared assays for biological 

threats remains somewhat limited. In vitro diagnostic 

tests for biological threats are orphan products, that is, 

there is not a large enough market to incentivize private 

industry to develop the tests because they are performed 

on an infrequent basis and, therefore, sales are limited. 

Consequently, most of the cleared diagnostic tests for 

biological threats have resulted from DoD acquisition 

programs. Currently cleared biological threat assays 

on the JBAIDS system include tests for Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Coxiella 

burnetii. The absence of useful and cleared infectious 

disease assays on JBAIDS hampers the utility of the 

system for clinicians. Future diagnostic devices for DoD 

would benefit from expanded capabilities for common 

infectious diseases of military relevance, not just those 

that are most likely to be used in a biological attack. 

Although biological science technology continues to 

advance, it must be emphasized that the DoD currently 

fielded and regulatory compliant in vitro diagnostic 

tests for biological threat agents are based on nucleic 

acid amplification chemistry that is 30 years old and 

a rapid cycling polymerase chain reaction (PCR) platform that is more than 10 years old. With the NGDS 

acquisition program underway, the platform is likely 

to be only an incremental improvement over the currently fielded system. The two most likely improvements will be onboard integrated sample processing 

and a sample in/answer out analysis flow. In essence, 

the system is likely to be an automated nucleic acid 

amplification in vitro diagnostic platform. During 

this time, microarrays, mass spectrometry, and DNA 

sequencing have advanced significantly for the identification of infectious agents.3

 Yet none of these approaches has matured to the point of receiving FDA 

clearance for medical diagnostic use or offer the hope 

of a simplified test that can be performed in a deployed 

setting for biological threat agents. 

Unlike technology, the ability of military laboratories to identify and confirm the presence of biological 

threats using regulatory compliant diagnostics matures 

at a much slower rate. This is not to discount the use of 

newer technologies by the DoD for environmental testing, vector surveillance, and population surveillance. 

These results can be used to make operational decisions, but they cannot be used for individual patient 

treatment, a concept often lost within the research and 

development community. The combination of using 

multiple diagnostic devices, multiple diagnostic markers, medical intelligence, medical acumen clinical signs 

and symptoms, and classical microbiology (Figure 26-1) 

still provides the most reliable approach for medical 

diagnosis of diseases to affect medical treatment or 

response after a biological threat attack. 

To enhance readiness and ensure the availability 

of laboratory testing capabilities, military and civilian 

clinical laboratories are linked into a series of laboratory response networks. The Centers for Disease Control and Prevention (CDC) sponsors the preeminent 

Laboratory Response Network (LRN) for bioterrorism. 

More recently, the DoD also has established the Defense Laboratory Network to further enhance military 

readiness. Together, these efforts have improved the 

national preparedness for biological threat identification, but continuing research and development are 

needed to improve the speed, reliability, robustness, 

and user friendliness of the new diagnostic technologies. This chapter will review currently available and 

future capabilities for agent identification and diagnostic technologies to protect and sustain the health 

of military personnel.

Figure 26-1. Orthogonal diagnostic testing uses an integrated 

testing strategy where more than one technology, technique, 

or biomarker is used to produce diagnostic results, which 

are then interpreted collectively. Although orthogonal 

diagnostic testing is a statistically independent approach, 

the combination of independent sensitivity and specificity 

values becomes highly valuable when combined. Orthogonal 

diagnostic testing improves the probability of reaching a 

“correct” result when the assays are less than 100% specific 

independently.

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THE LABORATORY RESPONSE

tative capability for identifying biological warfare 

agents. At the time of publication, the capability 

for biological threat agent detection is a mission 

primarily accomplished using JBAIDS. Specimens 

requiring more comprehensive analysis still require 

forwarding to the nearest reference or confirmatory 

laboratory, including the currently deployable assets 

for each service. 

Army

The Area Medical Laboratory (AML) is a modular, 

task-organized, and corps-level asset providing comprehensive laboratory support to theater commanders.5,7 The AML has transitioned from the original 

mission of testing primarily clinical specimens, with 

a capability for environmental samples (supporting 

force health protection) to being strictly an environmental sample testing lab. The AML can test for a 

broad range of biological, chemical, and radiological 

hazards. For biological agents, the laboratory uses a 

variety of rapid analytical methods, including molecular methods (such as real-time PCR), immunoassays (such as electrochemiluminescence [ECL] and 

enzyme-linked immunosorbent assay [ELISA]), and 

more advanced analyses involving bacterial culture, 

fatty acid profiling, and immunohistochemistry. The 

AML, which is the largest of the service deployable 

laboratories, can typically staff missions with a mix 

of microbiologists, biochemists, veterinary pathologists, and physicians. The AML maintains a degree 

of redundant equipment for long-term or split-base 

operations. 

The 20th CBRNE Command (CBRNE—Chemical, 

Biological, Radiological, Nuclear, and Explosives), 

previously called the 20th Support Command 

CBRNE, fields a multitude of assets under a single 

operational headquarters. Its mission is to detect, 

identify, assess, render safe, dismantle, transfer, 

and dispose of unexploded ordnance, improvised 

explosive devices, and other CBRNE hazards, including biological warfare agents (see reference 

14). The CBRNE Analytical & Remediation Activity 

Mobile Expeditionary Laboratory (CARA MEL), 

a unit within the 20th, provides high-throughput 

chemical, explosives, and biological sample analysis. 

It also has three mobile lab packages (a light mobile 

expeditionary lab, a heavy mobile expeditionary 

lab, and a chemical air monitoring system platform) 

that deploy to support weapons of mass destruction elimination and remediation efforts in forward 

deployed areas. 

Role of the Military Clinical and Field Laboratories

Military clinical and field laboratories play a critical role in the early recognition of biological threats. 

Intentionally delivered biological agents can also 

be used in bioterrorism scenarios to create terror or 

panic in civilian and military populations to achieve 

political, religious, or strategic goals. Although the 

principal function of military clinical laboratories is 

to provide data to support a clinical diagnosis, laboratory staff also provides subject matter expertise in 

theaters of operation on the handling and identification of hazardous microorganisms and biological 

toxins. In addition, these laboratories have a global 

view of disease in the theater and they play an important sentinel role by recognizing unique patterns 

of disease. Military field laboratory personnel may 

also evaluate environmental samples and veterinary 

medicine specimens as part of force health protection 

or a preventive medicine surveillance system in a 

theater of operations.4

 Military biological laboratory 

capabilities also exist to provide chemical, biological, radiological, and nuclear (CBRN) response, and 

elimination and remediation activities.5

Military Field Laboratories

Military field laboratories, which have many different configurations, are often incorporated into most of 

the services’ basic deployable treatment facilities. If a 

complete medical treatment facility (MTF) is part of 

a deployment, its intrinsic medical laboratory assets 

can be used. However, a medical laboratory may not 

be available for short duration operations. In this case, 

medical laboratory support would be provided by a 

facility outside the area of operations.6 A typical Army 

MTF in a theater of operations will have a limited 

initial microbiology capability even with the intrinsic 

laboratory component. 

Following the removal of the microbiology capability from most Army medical treatment facilities 

under the 1994 Medical Reengineering Initiative, 

the capability has been restored with adding a microbiology augmentation set (Medical Materiel Set, 

laboratory [microbiology] augmentation UA N403 

NSN 6545-01-505-2714 LIN M48987) and JBAIDS 

(UA 9409 NSN 6545-01-537-1100 LIN J00447). The 

N403 set contains necessary equipment and reagents 

to identify commonly encountered pathogenic bacteria. Susceptibility testing is not included. Although 

this medical set supports diagnostics of common 

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Navy

The Navy’s forward deployable preventive medicine units are medium-sized mobile laboratories that 

use multiple rapid techniques (to include PCR and 

ELISA) for identifying biological warfare agents on 

the battlefield. The forward deployable preventive 

medicine units are modular and can analyze samples 

containing chemical and radiological hazards. These 

laboratories specialize in providing high confidence 

identification of biological threat agents in concentrated 

environmental samples, and they can identify endemic 

infectious disease in clinically relevant specimens. 

Air Force

Air Force biological augmentation teams (unit 

type code FFBAT) and home station medical response 

laboratory biodetection teams use rapid analytical 

methods (such as real-time PCR) and immunological 

methods to screen environmental and clinical samples 

for threat agents.8,9 The biological augmentation 

teams are small (two members), easily deployed, 

and typically housed in a separate facility designed 

to be collocated with preexisting or planned medical 

facilities. The units are capable of providing early 

warning to commanders about the potential presence 

of biological threat agents, typically in support of 

installation protection programs. The theater commander, in conjunction with the theater surgeon 

and nuclear, biological, and chemical officer, must 

decide which and how many of these laboratories 

are needed, based on factors such as the threat of a 

biological attack, the size of the theater, the number 

of detectors and sensitive sites in the theater, and the 

confidence level of results needed. 

Defense Laboratory Network 

The response to future CBRN threats will require 

an integrated military laboratory network that can 

respond with agility and competence. The logistical 

and technical burden of preparing for all possible 

health threats will be too great for the military clinical or field laboratories, which have limited space and 

weight restrictions. The most important role of these 

laboratories is to provide rapid and accurate laboratory 

support for medical diagnosis, rule out the most common threats, and alert the command about suspicious 

disease occurrences. The military Defense Laboratory 

Network consists of the front-line MTF clinical laboratories or deployed military laboratories backed by 

regional MTFs or military reference laboratories with 

access to more sophisticated diagnostic capabilities. 

The clinical laboratories in the regional medical centers 

or large medical activities are the gateways into the 

civilian LRN sponsored by the CDC. 

At the top of the military response capability are 

research laboratories, such as the US Army Medical 

Research Institute of Infectious Diseases (USAMRIID; 

Fort Detrick, MD) and the Naval Medical Research 

Center (Silver Spring, MD). Other laboratories, such 

as the US Air Force Institute for Operational Health 

(Wright Patterson Air Force Base, OH) and the Naval 

Health Research Center (San Diego, CA), also provide 

reference laboratory services for a myriad of endemic 

infectious diseases. Military research laboratories have 

traditionally solved some of the most complex and difficult diagnostic problems, but they are not routinely 

organized to perform high-throughput clinical sample 

processing and evaluation. Sentinel laboratories are 

generally supported by the network’s designated 

confirmatory laboratories, but they may communicate 

directly with national laboratories if necessary. 

The network of military laboratories with connections to federal and state civilian response systems 

provides unparalleled depth and resources to the 

biological threat response (Figure 26-2). The Defense 

Laboratory Network is a standing member of the federal Integrated Consortium of Laboratory Networks 

(ICLN). The ICLN was established in 2005 under a 

memorandum of agreement signed by senior officials 

of federal agencies including the Departments of Agriculture, Defense, Commerce, Energy, Health and 

Figure 26-2. The network of military laboratories with 

connections to federal and state civilian response systems 

provides unparalleled depth and resources to the biological 

threat response. National Laboratory Response Network for 

Bioterrorism. CDC: Centers for Disease Control and Prevention; NMRC: Naval Medical Research Center; USAMRIID: 

US Army Medical Research Institute of Infectious Diseases.

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Laboratory Identification of Threats

Human Services, Homeland Security, Interior, Justice, 

and State, and the Environmental Protection Agency 

(https://www.icln.org/ valid February 2014). The ICLN 

was charged with promoting enhanced commonality 

and integration of network functions. Although the 

ICLN does not direct resources or operations, it does 

provide an environment for integrating network operations and strategies. The Department of Homeland 

Security (DHS) is charged with overall leadership and 

coordination.

Identification Levels

Rapid infectious disease diagnostics are not 

quantitative, not linked to traceable standards, and, 

overall, are not as well developed as other laboratory 

technologies. The inherent biological variability that 

exists between any two organisms (mammalian and 

microbial) complicates the ability to discern with absolute certainty the perpetrator of an infectious disease 

event. Laboratory tests for many infectious agents are 

not highly automated and still rely on decades-old 

technologies and techniques. Culture remains the 

gold standard for identifying organisms, but not all 

infectious disease agents can be grown in culture, or 

are difficult to culture in routine microbiology laboratories, making alternative methods necessary. These 

constraints significantly affect the confidence at which 

results on diagnostic or detection assays for infectious 

agents can be reported. It often goes unstated that the 

best that can be done in biology is that, with high confidence, what is incriminated as the infectious disease 

agent has high probability of being correct.

When microbiology culture capability is difficult or 

not available (eg, virus cultures in field laboratories), 

serological diagnosis (use of the antibody response) to 

the organism is still a useful method and sometimes 

the only way to discern some infections. The problem 

with both traditional culture and serodiagnosis is 

the time required to obtain results. Culture may take 

several days and serodiagnosis is constrained by the 

time required to mount an antibody response, which 

can exceed a week or more (Figure 26-3). 

Within the past few decades molecular and immunodiagnostic technologies have been developed to 

improve the specificity and time to obtain diagnostic 

and detection information on infectious agents. Immunodiagnostic technologies are based on the use 

of antibodies as diagnostic reagents. Diagnostic and 

detection assays have been developed that can decrease detection times down to the range of minutes. 

Molecular diagnostics are based on the detection of 

specific nucleic acids characteristic of the infectious 

disease agent. Often the molecular diagnostic assay 

has to rely on the amplification of specific DNA sequences from extracted nucleic acids, DNA or RNA. 

Amplification techniques take tiny amounts of nucleic 

acid material and replicate them many times through 

enzymatic reactions, some that occur through cycles of 

heating and cooling. These techniques may bring more 

ambiguity on interpreting the results of the assays. 

Unlike cultured microbial agents, which can provide 

definitive results, immunodiagnostic and molecular 

diagnostic assays have various levels of false-positive 

and false-negative results. Discerning false-positive 

and false-negative results from true results becomes 

a risk management effort, aided by different levels of 

identification to express the degree of confidence associated with various testing methodologies. 

Civilian

The CDC LRN uses two levels of identification: (1) 

presumptive and (2) confirmed.10 In 1998,11 following 

a demonstration that Iraq sponsored state activities 

involving production and use of biological weapons, 

President Clinton issued Presidential Decision Directive 62, Combating Terrorism, and assigned specific 

missions to federal departments and agencies. The 

Figure 26-3. The typical infection and response time course 

begins with the initial pathogen encounter and leads to the 

formation and maintenance of active immunological memory 

(IgM and IgG) where serological detection is useful. Clinical 

disease, however, typically occurs around days 3 to 5 where 

detection of the infectious agent is possible. Often, by the time 

clinical disease is manifest, especially for the biological threat 

agents, clinical intervention to ensure survivability is not as 

effective as desired. To provide the most effective medical 

intervention on infectious agents, the closer to time 0 laboratory data is available, the more successful the outcome. IgG: 

immunoglobulin G; IgM: immunoglobulin M.

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Medical Aspects of Biological Warfare 

directive included a request to Congress to provide 

funding to the DHHS to support a renewed program 

of public health preparedness. In 1999, the LRN was 

established by the DHHS, the CDC in collaboration 

with founding partners, the Federal Bureau of Investigation, DoD, and the Association of Public Health 

Laboratories. The mission statement for the LRN is 

as follows:

The LRN is a critical national security infrastructure 

asset that, with its partners, will develop, maintain, 

and strengthen an integrated domestic and international network of laboratories to respond rapidly to 

biological, chemical, and radiological threats and other high priority public health emergencies through 

training, rapid testing, timely notification and secure 

messaging of laboratory results.11

The LRN includes a biological network (LRN-B) 

and a chemical network. Identification of biological 

threat agents within LRN-B is—in part—based on 

the level of testing, which is based on the level of the 

laboratories where testing is performed. The different 

levels of laboratories within the LRN are sentinel, reference, and national laboratories (Figure 26-2). Sentinel 

laboratories represent the thousands of community 

based hospital laboratories that have direct contact 

with patients and may be the first to spot atypical infectious disease presentations. Sentinel laboratories do 

not actually confirm the presence of biological agents 

but rather are trained to recognize and appropriately 

handle biological agents that could potentially be extremely dangerous pathogens. Sentinel laboratories 

then refer these presumptive cultures to their closest 

LRN reference laboratory for more definitive testing. 

These reference laboratories perform standardized 

tests to detect, and typically confirm, the presence of 

biological agents that may represent a biological threat. 

Reference laboratories, which are normally located 

within the respective state public health laboratories, 

perform reference-level tasks in biological safety level 

3 (BSL-3) facilities.12 Some LRN reference laboratories 

are located at county public health laboratories, animal health/veterinarian laboratories, military medical 

treatment facilities, and food safety laboratories. Public 

health directors can use LRN reference laboratory results to determine when a broad range of public health 

responses can be implemented. The CDC LRN protocols are currently limited to several bacterial agents, 

orthopoxviruses, and a couple of biologic toxins, and 

not all of the protocols have full confirmation methods 

for reference laboratory use (Table 26-2). 

A recent programmatic change to the LRN system 

will subdivide the network configuration for the reference laboratories. The LRN reference laboratories will 

be broken down into limited (RL3), standard (RL2), 

and advanced levels (RL1). The standards for each level 

will be based in part on the minimum operational BSL, 

the core instrumentation and equipment available (including advanced platforms), and testing capabilities 

(number of agents and technologies). Limited laboratories (RL3) will typically do limited, specialized testing not meeting RL2 standards. Standard laboratories 

(RL2) will be typical state public health laboratories 

capable of the full agent testing capability on clinical 

and high risk environmental samples. Advanced laboratories (RL1), typically state public health laboratories 

in regional locations that cover a risk-based, priority 

population center (under the DHS Urban Area Security 

Initiative13), will be capable of additional testing capabilities using advanced detection and characterization 

methods such as mass spectrometry. The three national 

laboratories have unique resources to handle highly 

infectious agents (typically at BSL-3 and BSL-414) and 

the ability to identify and characterize more agents, 

including BSL-4 viruses. 

The CDC LRN, as a network, includes laboratories, 

secure communications, training, protocols, reagents, and 

proficiency testing. LRN member laboratories encompass 

federal laboratories (including laboratories at CDC, 

the US Department of Agriculture [USDA], the FDA, 

and other facilities run by federal agencies), state and 

local public health, military (DoD laboratories located 

both within the United States and abroad), food testing 

(FDA and USDA laboratories), environmental (water 

and other environmental samples), veterinary (USDA 

and state), and international laboratories (Canada, the 

United Kingdom, Australia, Mexico, and South Korea). 

As the LRN-B continues programmatic maturation, it 

will not only continue to address biological terrorism 

preparedness and response (national security and public health emergency preparedness), but also address 

emerging infectious disease preparedness and response 

(eg, newly emerging viruses such as Middle East respiratory syndrome coronavirus) and biosurveillance. 

Military

Military identification levels differ from the civilian 

system in two specific aspects: 

1. Current military doctrine includes four 

levels of identification (presumptive, field 

confirmation, theater level validation, and 

definitive) based, in part, on what level or 

what unit does the testing; and

2. Testing algorithms are based on the concept 

of testing for biological markers (biomarker) 

rather than culturing the specific agents. 

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TABLE 26-2 

PRESUMPTIVE AND CONFIRMATION METHODS

Bacillus 

anthracis 

Anthrax Nucleic acid 

amplification 

(PCR) 

Immunoassay 

(spore vs 

cell) - HHA or 

plate based

Culture and gamma 

phage with capsule 

or PCR of a culture 

with three assays

Culture; PCR; Not needed

immunoassay

B anthracis is one 

of more than 260 

different Bacillus

spp. but is readily 

distinguishable from 

the others by the 

production of betahemolysin that is 

readily apparent on 

blood agar plates. 

B anthracis exists as 

both a vegetative 

cell and as an 

environmentally 

stable spore. B 

anthracis contains 

2 plasmids, pXO1 

and pXO2 that 

impart virulence 

characteristics and 

serve as diagnostic 

markers for both 

immunoassay and 

nucleic acid assays. 

Immunoassays 

will differ when 

testing for the 

vegetative cell or 

the spore. Whereas 

immunoassay 

and nucleic acid 

analysis can be 

used for diagnostic 

confirmation, 

culture is required 

for confirmation.

Gram-positive rod; 

spore-forming; 

aerobic; nonmotile 

catalase positive; 

large, gray-white to 

white; nonhemolytic 

colonies on sheep 

blood agar plates. 

Organism Disease* Presumptive† Confirmatory (LRN)‡ Key Identity Markers BSL-2 BSL-3

(Table 26-2 continues)

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Medical Aspects of Biological Warfare 

Brucellosis

Brucellosis

Brucellosis

Brucella 

abortus

Brucella 

melitensis

Brucella 

suis 

Nucleic acid 

amplification 

(PCR) (not 

species 

specific)

Immunoassay 

(not species 

specific) - 

HHA or plate 

based

Culture with 

biochemical testing

Depending on the taxonomy being used, 

brucellae contain 10 

recognized species that include B 

abortus, B melitensis, 

and B suis, the most 

common and important human pathogens. Differentiating 

the human pathogenic species from 

the other brucellae, 

however, is not 

easy and requires 

several growth and 

biochemical determinations. Immunoassay and nucleic 

acid assays are 

currently not helpful 

in distinguishing 

the pathogens from 

the nonpathogens. Culture and 

biochemical testing 

are required for 

confirmation.

Gram-negative coccobacilli or short rods; 

white, nonmotile, 

nonencapsulated, 

nonspore forming, 

slow-growing, nonhemolytic colonies 

on sheep blood agar 

plates; some species 

require enhanced 

CO2 for growth.

Initial culture; 

PCR; 

immunoassay

Culture 

confirmation

Organism Disease* Presumptive† Confirmatory (LRN)‡ Key Identity Markers BSL-2 BSL-3

(Table 26-2 continues)

Table 26-2 continued

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Laboratory Identification of Threats

Burkholderia mallei

Burkholderia pseudomallei

Glanders

Melioidosis

Nucleic acid 

amplification 

(PCR)

Culture with biochemical testingB mallei and B 

pseudomallei are two 

of the 60 currently 

recognized species 

that include other 

human pathogens. 

As part of their 

environmental 

saprophytic 

lifestyle, the 

Burkholderia are 

complex organisms 

that are readily 

culturable, but 

often display 

colony morphology 

variations that 

confound routine 

microbiological 

analysis. 

Biochemical 

differentiation, 

including 

gentamicin 

and polymyxin 

susceptibility, 

determination 

of arginine 

dihydrolase 

and lysine 

decarboxylase, 

and arabinose 

fermentation 

are required for 

differentiation and 

confirmation.

Gram-negative rod; 

oxidase-positive 

to variable, 

small, nonmotile, 

nonsporulating, 

nonencapsulated; 

primary isolation 

requires 48–72 h at 

37°C; nonhemolytic, 

typically about 

1 mm in width, 

white (turning 

yellow with age), B 

pseudomallei grows 

well on MacConkey 

agar, but B mallei

does not.

Initial culture; 

PCR

Culture 

confirmation

Organism Disease* Presumptive† Confirmatory (LRN)‡ Key Identity Markers BSL-2 BSL-3

Table 26-2 continued

(Table 26-2 continues)

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Medical Aspects of Biological Warfare 

Botulism 

A–E

Epsilon 

toxin

Q fever

Clostridium botulinum 

Clostridium 

perfringens 

Coxiella 

burnetii 

Nucleic acid 

amplification 

(PCR) 

Immunoassay - 

HHA or plate 

based

Nucleic acid 

amplification 

(PCR) 

Nucleic acid 

amplification 

(PCR) 

Immunoassay - 

HHA or plate 

based

Mouse testing

Not in LRN

Send to CDC

Gram-positive rod; 

spore-forming; 

obligate anaerobe 

catalase negative; 

lipase production 

on egg yolk agar; 

150,000 Da protein 

toxin (types A–G); 2 

subunits.

Gram-positive rod; 

spore-forming; 

obligate anaerobe 

catalase negative; 

5 types (A-E), but 

only types B and D 

produce the epsilon 

toxin; on a blood 

agar plate produces 

double zone beta 

hemolysis.

C burnetii is an 

obligate intracellular parasite that 

makes routine 

culture difficult. 

Culture in eggs or 

cells has previously 

been required so 

routine laboratory 

diagnostics are not 

common. Although 

highly infectious, C 

burnetii is typically 

not fatal and often 

serology is used for 

diagnosis. Direct 

fluorescent antibody 

and nucleic acid assays are often used 

for presumptive 

and confirmatory 

diagnostics.

Initial culture; 

PCR; immunoassay; 

toxin-antitoxin neutralization test

Initial culture; 

PCR; immunoassay

PCR; immunoassay

Not 

needed

Not 

needed

Culture 

confirmation

Table 26-2 continued

Organism Disease* Presumptive† Confirmatory (LRN)‡ Key Identity Markers BSL-2 BSL-3

(Table 26-2 continues)

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Laboratory Identification of Threats

Francisella Tularemia

tularensis 

Nucleic acid 

amplification 

(PCR) 

Immunoassay - 

HHA or plate 

based

Culture with direct 

fluorescent antibody 

stain

F tularensis subspecies 

tularensis (type A) 

and F tularensis subspecies holarctica 

(type B) are the two 

most virulent strains 

of this expanding 

group of organisms. Until recently, 

F tularensis type A 

or B was restricted 

to the Northern 

Hemisphere where 

F tularensis type A 

or B is common in 

North America, but 

only F tularensis

type B is typically 

found in Europe 

and Asia. F tularensis

is relatively easy to 

grow and growth is 

required for confirmation, typically by 

the direct fluorescent antibody assay.

Extremely small, 

pleomorphic, 

gram-negative coccobacilli; nonspore 

forming; facultative intracellular 

parasite; nonmotile; 

catalase positive 

opalescent smooth 

colonies on cysteine 

heart agar.

Initial culture; 

PCR; immunoassay

Culture 

confirmation

Louseborne 

typhus, 

Typhus 

exanthematicus

Spotted 

fever

Rickettsia 

prowazekii 

Rickettsia 

rickettsii

JBAIDS nucleic 

acid amplification (PCR) 

Nucleic acid 

amplification 

(PCR) 

Immunoassay - 

HHA or plate 

based

Not in LRN Gram-negative, 

obligate intracellular 

parasitic, aerobic 

bacteria.

PCR Culture 

confirmation

(Table 26-2 continues)

Organism Disease* Presumptive† Confirmatory (LRN)‡ Key Identity Markers BSL-2 BSL-3

Table 26-2 continued

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Medical Aspects of Biological Warfare 

Enterotoxins 

A and B 

(SEA & 

SEB)

Plague

Staphylococcus 

aureus

Yersinia 

pestis 

Immunoassay - 

HHA or plate 

based

Nucleic acid 

amplification 

(PCR) 

Immunoassay - 

HHA or plate 

based

Not in LRN

Culture with phage 

testing

Gram-positive, cocci; 

facultative anaerobic, large round 

white to yellow, 

beta-hemolytic colonies on sheep blood 

agar; characteristic 

“grape-cluster” on 

Gram stain; catalase 

and coagulase-positive; multiple toxins 

depend on strain.

Y pestis belongs to a 

smaller group of 

organisms, but is 

much more difficult 

to correctly identify. 

Y pestis has several 

plasmids that confer 

various virulence 

traits and are useful 

diagnostic assay 

targets, but the plasmids are promiscuous and can be found 

in non-Y pestis causing the potential for 

false-positive assays. 

Capsule (F1) is a 

good marker for the 

diagnosis of Y pestis,

but does not get produced at the optimal 

growth temperature 

for Y pestis (28°C). Instead, it is produced 

at 35°–37°C, making 

this marker less reliable for environmental Y pestis detection. 

Immunoassay and 

nucleic acid assays 

are available for diagnostics, but confirmation of Y pestis is 

done using phage on 

cultural growth.

Gram-negative coccobacilli often pleomorphic; nonspore 

forming; facultative 

anaerobe; nonmotile 

beaten copper colonies (MacConkey 

agar).

Initial culture; 

immunoassay

Initial culture; 

PCR; immunoassay

Not 

needed

Culture 

confirmation

(Table 26-2 continues)

Organism Disease* Presumptive† Confirmatory (LRN)‡ Key Identity Markers BSL-2 BSL-3

Table 26-2 continued

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Laboratory Identification of Threats

Viral hemorrhagic 

fevers

Viral hemorrhagic 

fevers

Viral hemorrhagic 

fevers

Smallpox

Viral encephalitic 

disease

Viral encephalitic 

disease

CrimeanCongo 

hemorrhagic 

fever 

virus/

bunyaviruses

Ebola, 

Marburg 

virus/filoviridae 

viruses

Lassa/

arenaviruses

Variola 

major

Venezuelan 

equine 

encephalitis 

virus/

alpha 

viruses

Yellow 

fever 

virus/flaviruses 

Nucleic acid 

amplification 

(PCR) 

Nucleic acid 

amplification 

(PCR) 

Nucleic acid 

amplification 

(PCR) 

Nucleic acid 

amplification 

(PCR) 

Immunoassay - 

HHA or plate 

based

Nucleic acid 

amplification 

(PCR) 

Immunoassay - 

HHA or plate 

based

Nucleic acid 

amplification 

(PCR) 

Not in LRN

Not in LRN

Not in LRN

Send to CDC

Not in LRN

Not in LRN

Single negativestranded, tripartite 

genomes (large 

[RNA-polymerase], 

medium [glycoproteins], small [nucleocapsid protein]) 

exist in a helical/

pseudo-circular 

structure; enveloped 

RNA viruses.

Linear, negative-sense 

single-stranded 

RNA virus; enveloped; filamentous or 

pleomorphic, with 

extensive branching, or U-shaped, 

6-shaped, or circular 

forms; limited cytopathic effect in Vero 

cells.

Two single-stranded 

RNA segments ambisense RNA virus; 

beaded nucleocapsid, spherical with 

glycoprotein spikes.

Large double-stranded DNA virus; 

enveloped, brickshaped morphology; 

Guarnieri bodies 

(virus inclusions) 

under light microscopy.

Linear positive-sense 

single-stranded 

RNA virus; enveloped, spherical 

virions with distinct 

glycoprotein spikes; 

cytopathic effect in 

Vero cells.

Linear positive-sense 

single-stranded 

RNA virus; enveloped, icosahedral 

nucleocapsid; cytopathic effect in Vero 

cells.

PCR

PCR

PCR

PCR; immunoassay

PCR

PCR

Culture 

confirmation/

BSL-4

BSL-4

BSL-4

BSL-4 

(CDC 

ONLY)

Culture 

confirmation

Culture 

confirmation

(Table 26-2 continues)

Organism Disease* Presumptive† Confirmatory (LRN)‡ Key Identity Markers BSL-2 BSL-3

Table 26-2 continued

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Medical Aspects of Biological Warfare 

The military concept of testing for biomarkers follows the logic that if the biomarker is present, then the 

agent of interest is also present. Some complications 

exist with using biomarkers. One problem is defining a biomarker. Multiservice Tactics, Techniques, and 

Procedures (MTTP) for Biological Surveillance Editor15

provided a definition, but that document was replaced 

by Army Techniques Publication 3-11.377 in 2013 and 

the definition was lost. The current doctrinal revision 

to Army Techniques Publication ATP 4-02.7/MCRP 

4-11.1F/NTTP 4-02.7/AFTTP 3-42.3 15 March 2016, 

MTTP for Health Services Support in a CBRN Environment, reestablishes the definition as: 

A biomarker refers to a detectable/measurable substance that is correlated with the presence of a BW 

[biological warfare] agent (bacteria, virus, or toxin). 

Biomarkers should be unique to the biological agent, 

often associated with virulence, and can be independent of the biological agent’s viability/infectivity/

functionality. 

The types of biomarkers listed included nucleic acid 

sequences, antigens or toxins for immunological methods, growth properties (as demonstrated on biochemical 

tests or selective media), and microscopic characteristics. The revised doctrinal definition will help guide 

correct application in the absence of specific details. 

Another scientific concern with the use of biomarkers 

is that some biomarkers are present due to nonthreat 

infectious agents inducing similar biomarker profiles 

to threat agents. Although these results are considered 

false-positives for biothreats, induction of disease specific 

profiles still indicates infection and therefore can remain 

useful in the overall determination of the etiologic agent. 

Biomarkers also do not necessarily reflect viability 

of the infectious agent. Although the simple presence 

or absence of an agent can be important, determination 

of viability may be a significant component, especially 

in nonclinical samples where the biomarker could be 

simply background flora. When laboratories rely on 

biomarkers in lieu of culture, the ability to determine 

other critical information is often lost, such as antimicrobial resistance, epidemiological strain typing, 

or legal evidence for forensic science and attribution 

purposes. Concentrating on biomarkers may lead to 

a myopic result that limits the full understanding of 

medical implications for an incident or outbreak.

The military identification levels are well defined 

in doctrine as follows5,7 (Figure 26-4). Presumptive 

identification of a biological threat agent is achieved 

by the detection of a biological marker using a single 

test methodology (eg, hand held assay [HHA]). Presumptive identification uses technologies with limited 

specificity and sensitivity by general purpose forces 

in a field environment to determine the presence of a 

biological hazard with a low level of confidence but 

with a degree of certainty necessary to support immediate tactical decisions. Since identification at this 

level is based on specific technologies, it is limited to 

the assays deployed and cannot detect or identify new 

or emerging infectious disease agents for which the 

technologies assays are not available.

Organism Disease* Presumptive† Confirmatory (LRN)‡ Key Identity Markers BSL-2 BSL-3

Table 26-2 continued

Ricin intoxicationRicin toxin Nucleic acid 

amplification 

(PCR) 

Immunoassay - 

HHA or plate 

based

Send to CDC 60,000–65,000 Da 

protein toxin; 2 

subunits castor bean 

origin.

PCR; immunoassayNot 

needed

*Disease refers to the disease state induced by the agent or the disease-causing entity of the agent.

†

Presumptive refers to typical diagnostic assay techniques used for reporting the presumptive evidence of a disease-causing agent.

‡

Confirmatory (LRN) refers to the diagnostic assay techniques used for reporting the confirmed evidence of a disease-causing agent being 

present.

BSL: biological safety level

CDC: Centers for Disease Control and Prevention

HHA: hand held immunoassay

JBAIDS: Joint Biological Agent Identification and Diagnostic System

LRN: Laboratory Response Network

PCR: polymerase chain reaction

RNA: ribonucleic acid

SEA: Staphylococcal enterotoxin A 

SEB: Staphylococcal enterotoxin B

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Laboratory Identification of Threats

Field confirmatory identification is achieved when 

two or more independent technologies confirm the 

identification of a biological agent. This may be an 

immunoassay (eg, HHA, ECL, ELISA, nucleic acid amplification result, and/or culture growth/microscopy). 

According to doctrine, a single result from JBAIDS 

can be used as a field confirmatory identification. A 

genomic biomarker must be included. Field confirmatory identification uses technologies with increased 

specificity and sensitivity, by technical forces in a field 

environment, to identify the presence of biological 

agents with a moderate level of confidence and a degree of certainty necessary to support follow-on tactical 

decisions. Depending on the technologies deployed 

(eg, culture), some limited ability exists to detect or 

identify infectious disease agents beyond the limits 

of deployed assays.

Theater validation is achieved using devices, materials, or technologies that detect biomarkers using two or 

more independent biomarker results (ie, one biomarker 

is detected by two or more independent methodologies 

or more than one biomarker is detected by a single methodology). Examples are: (1) hand held immunological 

assay plus nucleic acid amplification or (2) nucleic acid 

amplification using two different biomarkers (eg, gene 

targets). Theater validation identification uses multiple 

independent, established protocols and technologies 

by scientific experts in a controlled environment of a 

fixed or mobile/transportable laboratory to characterize 

biological materials with a high level of confidence and 

the degree of certainty necessary to support operational 

level decisions. After a preventive medicine detachment, a combat support hospital or CBRN reconnaissance assets identify a biological/clinical specimen as a 

biological threat agent, the specimen is sent by courier 

to those specialized laboratories/teams with advanced 

microbiological capabilities and highly skilled medical 

personnel. These could include laboratories/teams such 

as an AML, 20th CBRNE CARA MEL, the US Air Force 

biological augmentation team, or the US Navy forward 

Figure 26-4. Military identification levels correspond with both the technology used and the facility doing the laboratory 

analysis. Although there is some correspondence to the civilian Centers for Disease Control and Prevention’s Laboratory 

Response Network system, the military system has significantly unique aspects. 

AML: area medical laboratory; BAT: biological augmentation team; CARA: CBRNE analytical remediation activity; CBRN: 

chemical, biological, radiological, and nuclear; CBRNE: chemical, biological, radiological, nuclear, and explosives; CONUS: 

continental United States; CVN: aircraft carrier, nuclear; FDPMU: forward deployed preventive medicine unit; Hosp: hospital; LHA: amphibious assault ship (general purpose); LHD: amphibious assault ship (multipurpose); LRN: Laboratory 

Response Network; NEPMU: Navy environmental and preventive medicine unit; OCONUS: outside the continental United 

States; PVNTMED: preventive medicine; T-AH: hospital ship; VET: veterinary

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Medical Aspects of Biological Warfare 

deployable preventive medicine unit when available in 

the operational area. Although the units listed here have 

the potential to produce theater validation level results, 

they may not inherently have that capability deployed 

in all circumstances. 

The theater validation laboratories must implement 

a quality assurance program, preferably with independent audits, proficiency testing, scientist level data 

review, document control, demonstration of procedure 

traceability, some level of electronic sample management, documentation of personnel training, and accreditation (if available). These laboratories would 

typically conduct initial field confirmatory analysis 

(quick report) followed by theater validation (more 

testing and time). If these specialized laboratories/

teams are unavailable, biological specimens that are 

presumptively positive for a biological threat agent 

will have to be forwarded to the nearest reference 

laboratory, even if this is in the continental United 

States (CONUS). 

Definitive identification is the correlation of a biological agent to a known substance, or in the case where 

the substance is previously unknown, the substance is 

type classified and analyzed. Definitive identification 

is the use of multiple state-of-the-art, independent, 

established protocols and technologies by scientific 

experts in a nationally recognized laboratory to determine the unambiguous identity of a biological agent 

with the highest level of confidence and degree of certainty necessary to support strategic level decisions. It 

also supports the initiation of attribution to implicate 

or point to the source of the identified material. In all 

cases a definitive identification occurs at a US-based and 

sanctioned reference laboratory specifically equipped to 

perform detailed analysis on the type of suspect material to be identified. Definitive identification typically 

includes the ability to propagate the biological agent 

so that there is sufficient material available for analysis 

by the multiple methods and protocols, and the ability 

to look at strains by epidemiological methods, but also 

so material is available to initiate attribution analysis. 

Definitive identification is performed using the highest level quality assurance measures in a controlled 

laboratory. Definitive identification or “confirmation” 

testing is performed at sanctioned reference laboratories, including reference laboratories of the CDC LRN 

as appropriate. Specific LRN protocols and reagents 

are proprietary, but any definitive identification or confirmation typically follows a well-established scheme, 

including the use of well characterized reagents by 

well-practiced personnel. 

Like biomarkers, there are also inherent problems 

with the application and details involved in the identification levels that need to be understood to correctly 

apply the inherent concepts contained within the 

definitions and an appropriate application of the term 

confirmed. In one definition of confirmation, it states 

“the occurrence of two or more indicators corresponding with one another and thereby corroborating the 

predicted outcome.” Confirmation of an identification of a biological agent, however, often needs to be 

grounded in more information, especially given the 

consequences of an incorrect identification to both 

the military member as well as the military operation 

being conducted. In addition, identification of a biological agent based on nonmetabolic methods, in the 

absence of morbidity or mortality, always presents the 

possibility that the identification is detecting inactive 

materials.16

Biological materials, microbes and toxins, are fragile 

compared to nuclear or chemical agents. They can be 

inactivated during the course of dispersal (especially 

dissemination from munitions), through natural biocidal activity (sunlight both desiccates as well as 

inactivates through ultraviolet irradiation), ineffective weaponization processes, or myriad physical or 

chemical activities. The confidence in an identification of a biological attack is also affected by how it 

has been detected.7,8 Doctrinally, low, medium, and 

high confidence are part of the identification levels, 

yet the level of confidence an assay provides is also 

governed by factors that include the scientific quality and accuracy of the test methods, the target or 

purpose of the assay(s), experience and knowledge of 

testing personnel, and the environment in which the 

lab is operating.8 Detection by one biological detector system has a lower confidence level than if two 

detectors have made the detection. Theater validation 

identification (including two biomarkers) endorses and 

bolsters those automated detections, but confirmation should still be viewed with a level of suspicion 

resulting from inherent biological diversity. Until a 

full characterization of the agent can be undertaken, 

the term confirmed should be used with some level of 

reservation; and military commanders, responsible for 

both the mission and the welfare of service members, 

should proceed with the realization of the ambiguous 

nature that biological threats present.

Allies

US allies, including members of the North Atlantic 

Treaty Organization, have different doctrinal identification levels. Before 1995, the North Atlantic Treaty 

Organization recognized the need for common approaches for sampling and identification of biological 

and chemical warfare agents. Within its doctrine, three 

levels of identification also exist16: 

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1. Provisional identification: A biological agent 

may be considered provisionally identified 

when one of three criteria is met (presence 

of a unique antigen, presence of a unique 

nucleic acid sequence, or positive culture or 

multi-metabolic assay); 

2. Confirmed identification: The identification 

of a biological agent is confirmed when any 

two of the three criteria for provisional identification have been met in the presence of 

authentic reference standards (positive and 

negative controls) under identical experimental conditions; and

3. Unambiguous identification: The unambiguous identification of a biological agent 

provides the highest level of certainty required for the development of strategic and 

political positions. Confirmed identification 

becomes unambiguous under four criteria: 

(1) positive response is obtained by a genetic 

identification method; (2) positive response 

is obtained by an immunological method; 

(3) positive match is obtained by in vitro 

culture or multimetabolic assay; and (4) the 

disease properties of the microbial agent are 

confirmed in an accepted animal model.

IDENTIFICATION APPROACHES

Specimen Collection and Processing 

Clinical specimens can be divided into three different categories based on the ability to affect the 

disease course: (1) early postexposure, (2) clinical, and 

(3) convalescent/terminal/postmortem.5,17 Common 

specimens for biological warfare agents are similar to 

those collected for diagnosis of any infectious disease 

and typically correspond to clinical manifestations 

(Table 26-3). Specimens often include swabs, induced 

respiratory secretions, blood cultures, serum, sputum, 

urine, stool, skin scrapings, lesion aspirates, and 

biopsy materials.5,18 Nasal and facial swab samples 

should not be used for making decisions about individual medical care; however, they could support the 

rapid identification of a biological threat (postattack) 

and help direct force health protection efforts.19,20

Baseline serum samples (presymptomatic) should be 

collected on all potentially exposed personnel after an 

overt attack. These samples will help to both define 

the forces exposed but could also provide diagnostic 

information in the event that nontraditional agents 

are being used. 

In cases of sudden or suspicious deaths, autopsy 

samples should be taken. Specimens and cultures 

containing possible highly infectious agents should 

be handled in accordance with established biosafety 

precautions. Specimens should be sent rapidly (within 

24 hours) on wet ice (2°C–8°C) to an analytical laboratory capable of handling them. Blood cultures should 

be collected before the administration of antibiotics. If 

necessary, the blood cultures should be shipped to the 

laboratory within 24 hours at room temperature (21°C–

23°C). Overseas laboratories should not attempt to ship 

clinical specimens to CONUS reference laboratories 

using only wet ice unless the provisions for reicing the 

samples are made with the carrier. Shipments requiring more than 24 hours should be frozen on dry ice or 

liquid nitrogen if possible. Specific shipping guidance 

should be obtained from the supporting laboratory 

before shipment. Specimens should not be treated with 

permanent fixatives (ie, formalin or formaldehyde) 

unless that is the only way to ensure sample stability. 

Storage and shipping of samples at –20°C to 25°C is 

contraindicated. 

Environmental samples, while not patient specific, 

are often highly useful to medical decision making. 

These samples include several different categories of 

materials such as buffers and filters from air sampling 

devices, powders, soil and vegetation, animals (including rodents and insects as potential vectors), food 

samples from both fresh and packaged materials if ingestion is suspected, and nearly everything else that is 

not a clinical sample. These samples, when taken before 

any overt disease onset, can help identify a causative 

agent and potentially lead to prophylactic treatment. 

Nonclinical samples represent the biggest challenge in 

the detection of biological agents because of the vast 

repertoire of sample types and microorganisms in the 

environment that cause false-positive and false-negative 

detection reactions in many laboratory assays. 

A substantial amount of guidance exists—both 

military specific5,16 and general21—so details of taking 

and processing of environmental samples is beyond 

the scope of this chapter. Environmental samples will 

contain myriad physical and chemical agents that can 

potentially interfere with detection technologies and 

cause false negative results. Environmental samples 

include samples that are both highly stable as well as 

samples that will degrade with time similar to clinical 

samples. Guidelines for the submission of environmental samples are not as well detailed as those for clinical 

samples. In general, environmental samples should be 

maintained at nearly the same state as when they were 

collected. Dry samples should be kept dry, moist or 

wet samples should be preserved from desiccation, and 

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TABLE 26-3 SPECIMEN COLLECTION FOR SELECT BIOLOGICAL WARFARE AGENTS Postexposure† Clinical ConOrganism Incubation Period* Time Samples Time Samples Time Bacillus anthracis 1–6 d; 3 d 0–72 h Nasal and throat swabs, 48–72 h Serum for toxin assays; 72 h–

and induced respiratory whole blood (blood secretions cultures) and tissue 

smears for direct fluorescent antibody§

Brucella 5–60 d; 5 d 0–36 h Nasal and throat swabs, 72–168 h Whole blood (blood 7–28 

and induced respiratory; cultures); note: notify 

note: notify laboratory laboratory for extended 

for extended culture blood culture incubation protocol incubation protocol 

Burkholderia pseudomallei/ 1–21 d; 3 d 0–48 h Nasal and throat swabs, 24–96 h Serum for capsular 7–28 

mallei and induced respiratory polysaccharide assays; 

secretions whole blood (blood 

cultures) 

Clostridium botulinum/ 0–24 h Nasal and throat swabs, 24–72 h Blood or serum for toxin 7–28 

botulinum toxins A/B/E and induced respiratory detection 

secretions for toxin detection

Coxiella burnetii 7–41 d 0–72 h Nasal and throat swabs, 3–14 d Whole blood (blood 14–60 

and induced respiratory cultures) and directsecretions (egg, tissue molecular detection¥culture, or axenic media)

Encephalitic viruses/ 2–6 d 0–24 h Nasal and throat swabs, 24–72 h Throat swabs up to 5 6–21 

alpha viruses/VEE/etc and induced respiratory days, then cerebrosecretions spinal fluid and serum

Francisella tularensis 1–21 d; 3 d 0–24 h Nasal and throat swabs, 24–72 h Whole blood (blood 6–21 

and induced respiratory cultures); directsecretions fluorescent antibody§

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Hemorrhagic fever 4–21 d 0–24 h Nasal and throat swabs, 2–5 d Serum 6–21 

viruses/Ebola/Marburg/ and induced respiratoryDengue/etc secretions

Ricin 18–24 h 0–24 h Nasal and throat swabs, 24–48 h Serum/plasma for toxin 6–21 

and induced respiratory assays; urine forsecretions ricinine is questionable

Staphylococcal 3–12 h 0–4 h Nasal and throat swabs, 2–6 h Blood or serum 

enterotoxins A/B/C and induced respiratory 

secretions for toxin detection

Vesicular and pustular 7–17 d 0–72 h Nasal and throat swabs, 2–5 d Serum and lesions/ 6–21 

rash illnesses/Orthopox and induced respiratory scrapings for (Variola) secretions microscopy and viral 

culture 

Yersinia pestis 1–7 d; 2 d 0–72 h Nasal and throat swabs, 24–72 h Whole blood (blood 7–10 

and induced respiratory cultures); direct secretions fluorescent antibody§ 

*Typical period; initial presentation of a high-dose exposure; dependent on dose; aerosol route.†Rapid molecular and immunoassays can be done, but none are FDA cleared for patient treatment.‡Serology and other tests may not be FDA cleared for patient treatment, but convalescent/terminal/postmortem testing is rarely used to influen§Direct fluorescent antibody tests are not FDA approved, but accepted if done as a laboratory-developed test and validation data available. ¥FDA approved for direct patient treatment.IgA: immunoglobulin A; IgG: immunoglobulin G; IgM: immunoglobulin M; VEE:Venezuelan equine encephalitisTable 26-3 continued

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Medical Aspects of Biological Warfare 

cold samples should be kept cool. One especially critical 

requirement for any environmental sample is the 

initiation and maintenance of chain of custody documentation,7,16,22 from the sample collection through to 

the analysis laboratory. Again, like shipping clinical 

samples, guidance should be obtained from the supporting laboratory before shipment.

A multitude of international, domestic, and commercial regulations mandate the proper packing 

and documentation (including labeling) of biological 

materials (Table 26-4). Biological samples, infectious 

agents, and biological select agents and toxins all 

represent some level of dangerous goods that need 

special handling to protect the public, airline workers, 

couriers, and other persons who work for commercial 

shippers and who handle the dangerous goods within 

the shipping process. In addition, proper packing and 

shipping of dangerous goods reduces the exposure of 

the shipper to the risks of criminal and civil liabilities 

associated with shipping dangerous goods, particularly infectious substances. Each of the regulations 

deals with specific shipping requirements, but in 

general, all define an infectious substance as a material 

known or reasonably expected to contain a pathogen 

(a microorganism that can cause disease in humans 

or animals). Universal examples of pathogens include 

bacteria, viruses, fungi, and other infectious agents. An 

infectious substance is assigned to one of the following 

three potential categories: 

1. Category A: An infectious substance transported in a form capable of causing permanent disability or life-threatening or fatal disease in otherwise healthy humans or animals 

when exposure occurs. Category A infectious 

substances are assigned the identification 

number UN 2814 or UN 2900, based on the 

known medical history or symptoms of the 

source patient or animal, endemic local conditions, or professional judgment concerning 

the individual circumstances of the source 

human or animal. 

2. Category B: An infectious substance that does 

not meet the criteria for inclusion in Category 

A. Category B infectious substances bear the 

shipping term “Biological substance, Category B” and are assigned the identification 

number UN 3373. 

3. Toxins from plant, animal, or bacterial sources that do not contain an infectious substance 

and are not contained in an infectious substance may be considered for classification 

as toxic substances; and they are assigned 

the identification number UN 3172. 

In addition, other requirements may exist, including 

requirements for dry ice (dry ice is classified by the 

Department of Transportation and the International 

Air Transport Association as a “miscellaneous” hazard, 

class 9). The International Air Transport Association 

manual, Dangerous Goods Regulations, is the leading 

guide to shipping dangerous goods, including infectious agents by air, which generally includes most 

shipments from CONUS and outside of the continental 

United States (OCONUS). Dangerous Goods Regulations

provided requirements for packaging a shipment to 

classify, mark, pack, label, and document dangerous 

goods to meet international requirements. Key issues 

in shipping biological materials include—at a minimum—the following: 

• maintaining the sample integrity (especially 

metabolic viability); 

• some identification of the sample if possible 

(determining appropriate Category A, Category B, or toxin); 

• packaging requirements (packaging corresponding to category such as Category A must 

consist of three components: [1] a primary 

receptacle[s]; [2] a secondary packaging; and 

[3] a rigid outer packaging); and 

• documentation (International Air Transport 

Association Shipper’s Declaration for Dangerous Goods, DD Form 2890, DoD Multimodal 

Dangerous Goods Declaration, APHIS/CDC 

Form 2, Request to Transfer Select Agents 

and Toxins, and any import or export permits 

required). 

Other considerations for shipping biological 

samples may exist5,23,24 and typically require personnel who have been trained and are certified to package hazardous materials for shipment (including but 

not limited to Transport of Biomedical Materials at 

https://phc.amedd.army.mil/Pages/CourseDetails.

aspx?CourseID=89 [valid September 2016]). Specific 

specimen collection and handling guidelines for the 

bioterrorism agents are available from CDC and 

the American Society for Microbiology (see http://

emergency.cdc.gov/bioterrorism/ or http://www.asm.

org/index.php/guidelines/sentinel-guidelines; both 

valid September 2016). 

Culture-Based Microbiological Methods

Microbes that cause infectious disease are an example of a classic host–parasite relationship. Suspecting, 

or even having some evidence of a microbe’s ability 

to produce disease, is still inferential science. Having 

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Laboratory Identification of Threats

unequivocal proof of a specific etiological agent as 

the cause of an infectious disease requires the application of conventional microbial culture to validate 

Koch’s postulates (the four standards of a logical 

chain of experimental evidence designed to establish 

a causal relationship between a causative microbe and 

a disease). Microorganisms can cause tissue damage 

(disease) by releasing a variety of toxins or destructive 

enzymes into the host. Although a number of ways exist to obtain indirect evidence of a microbe’s effect on 

the host, propagating the causative microbial agent is 

still considered the gold standard for linking a specific 

microbial agent to the disease status.

Specific guidelines for identifying bioterrorism 

agents can be obtained from the CDC (http:\www.

bt.cdc.gov) or the American Society for Microbiology 

(http://www.asm.org/index.php/guidelines/sentinelguidelines). Guidelines for identification of additional 

agents that cause other infectious diseases can be found 

in diagnostic microbiology textbooks. Although the 

ability to propagate infectious disease microbes in routine culture has been available for more than a century, 

TABLE 26-4 

INTERNATIONAL AND DOMESTIC STANDARDS FOR SHIPPING

International Domestic

United Nations Economic Commission for Europe “Recommendations on the 49 CFR transportation (highway

Transport of Dangerous Goods,” also called the “Orange Book” 2009 transportation regulations)

(http://www.unece.org/trans/danger/publi/unrec/rev16/16files_e.html; 

valid February 2014)

European Agreement concerning the International Carriage of Dangerous 42, 7, & 9 CFR for select agents 

Goods by Road (ADR) (http://www.unece.org/trans/danger/publi/adr/

adr_e.html; valid February 2014)

Final Governing Standards (each country; DoD Instruction 4715.5, 9 and 21 CFR for biological products

Management of Environmental Compliance at Overseas Installations) 

(https://www.fedcenter.gov/Login/index.cfm?pge_id=3739&

NotAuthorized=1&returnto=%2Fprograms%2Fcompliance%2Ffgs%2F

index%2Ecfm%3F; valid February 2014)

International Air Transport Association Dangerous Goods Regulations (2014; US Postal Service 

55th edition) (http://www.iata.org/publications/dgr/Pages/manuals.aspx; Domestic Mail Manual (10.17 Infectious

valid February 2014) Substances, Hazard Class 6, Division 

6.2) and International Mail Manual 

(135.1 Infectious Substances)

International Civil Aviation Organization Regulations (http://www.icao.int DoD 4500.9-R, Defense Transportation 

/safety/DangerousGoods/Pages/default.aspx; valid February 2014) Regulation; Part II, Chapter 204

International Maritime Dangerous Goods Code (http://www.imo.org/blast Air Force Manual 24-204/(Interservice) 

/mainframe.asp?topic_id=158; valid February 2014) (for maritime shipments) TM 38-250 NAVSUP PUB/505 MCO 

P4030.19J DLAI 4145.3 (2012), 

Transportation; Preparing Hazardous 

Materials for Military Air Shipments

World Health Organization Guidelines Army Regulation 50-1, Nuclear and

Guidance on Regulations for the Transport of Infectious Substances 2007– Chemical Weapons and Materiel

2008 (http://www.who.int/csr/resources/publications/biosafety/WHO_CDS Biological Surety (2008)

_EPR_2007_2cc.pdf valid February 2014)

Laboratory Biosafety Manual - Third Edition

(http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_CSR

_LYO_2004_11/en/; valid February 2014)

Globally Harmonized System of Classification and Labeling of Chemicals Army Regulation 190-17, Biological 

(https://www.osha.gov/dsg/hazcom/global.html; valid July 2016) Select Agents and Toxins Security 

Program (2009)

CFR: Code of Federal Regulations; DLAI: Defense Logistics Agency Information; DoD: Department of Defense; MCO: Marine Corps Publications; NAVSUP PUB: Navy Supplement Publication; TM: Technical Manual

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Medical Aspects of Biological Warfare 

many bioterrorism and infectious disease agents—

especially the viruses—are not always easily cultured. 

In addition, culturing a specific microbial agent from 

a clinical sample is often routine; culturing the same 

microbial agent from an environmental sample is 

manyfold more difficult. In either case, knowing which 

microbial agent(s) is needed will greatly help to create 

the right conditions for propagation. A physician’s 

clinical observations or medical intelligence should 

help guide the analytical plan (see Table 26-3).18,25

The bioterrorism and infectious disease agents are 

separated into aerobic and anaerobic bacterial agents 

and viruses. Fungal and parasitic microbial agents are 

not often encountered as bioterrorism and infectious 

disease agents targeted against humans. Most aerobic 

bacterial threat agents can be isolated by using three 

common clinical bacteriological media: (1) 5% sheep 

blood agar (SBA); (2) MacConkey agar; and (3) chocolate 

agar (CHOC). Cystine heart agar supplemented with 

5% sheep blood has been suggested as a preferred medium for F tularensis, but CHOC agar usually suffices in 

clinical samples. Although Brucella agar was developed 

as a preferred medium for Brucella, improvements in 

SBA and CHOC agars support the growth of fastidious 

microorganisms such as Brucella. Nonselective SBA supports the growth of most bacterial agents, including B 

anthracis, Brucella, Burkholderia, and Y pestis. MacConkey 

agar, which is the preferred selective medium for gramnegative Enterobacteriaceae, supports Burkholderia and Y 

pestis. Liquid medium, such as trypticase soy broth, can 

also be used followed by subculturing to SBA or CHOC 

when solid medium initially fails to produce growth. 

Anaerobic organisms (those organisms that do not 

require oxygen for growth; some of which may react 

negatively or even die if oxygen is present), such as 

Clostridium species, require the use of anaerobic media 

and methods. Anaerobic methods reduce the exposure 

of microorganisms to molecular oxygen through the 

use of anaerobic jars or anaerobic chambers, and use 

culture media that are especially designed to dissolve or deplete oxygen, allowing the anaerobes to 

propagate. The liquid medium thioglycollate readily 

supports anaerobic microorganisms and should be 

considered a routine medium if Clostridium species 

could be encountered. 

The use of multiple bacteriological media is recommended both for redundancy as well as an aid to initial 

notification. Propagation of viruses is more complex 

and usually takes longer than those for bacteria. Since 

viruses are obligate intracellular parasites, propagation in various host systems is required. Most readily 

viruses are typically propagated in cultures of various 

cell lines, but laboratory animals and embryonated 

eggs are also used. Although no single cell culture is 

sensitive to all the viruses encountered, Vero (African 

green monkey kidney) cells are commonly used for 

many of the viruses (Table 26-5). 

Cells used for propagating viruses require growth at

an appropriate temperature and gas mixture (typically, 

37°C, 5% CO2 for mammalian cells) in an incubator. 

In addition, cell cultures also require special growth 

media that have stringent requirements for pH, glucose, antibiotics, growth factors, and other nutrients. 

Growth factors used to supplement media are often 

derived from the serum of animal blood, such as fetal 

bovine serum. Cell plating density (number of cells 

per volume of culture medium) and inoculation density of the virus are critical factors. Viruses manifest 

their presence in cell culture by different mechanisms 

including cellular degeneration (cytopathic effect), 

plaque formation, and metabolic inhibition testing. 

Some viruses require other means to demonstrate their 

presence in cell culture including fluorescent antibody 

testing or nucleic acid amplification methods.

Automated Identification Systems

Many automated identification systems are commercially available that have some capability to 

identify the major bacterial biological threat agents (B 

anthracis, Brucella spp, Burkholderia mallei, Burkholderia 

pseudomallei, F tularensis, and Y pestis). These systems 

include the BioMérieux (Durham, NC) VITEK 2, 

Siemens (Tarrytown, NY) MicroScan, MIDI Sherlock 

Microbial Identification System (Newark, DE), Trek 

(Cleveland, OH) ARIS 2X, Biolog (Hayward, CA), and 

the Bruker (Billerica, MA) Biotyper matrix-assisted 

laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). The Becton Dickinson 

(Franklin Lakes, NJ) Phoenix Automated Microbiology 

System does not appear to be capable of identification 

of the major bacterial biological threat agents listed. 

An advantage to the automated identification systems 

is that if a laboratory is routinely using one of these 

commercial systems, personnel are already trained and 

reagents are typically on-hand. The primary disadvantage is that often false-positives or false-negatives occur, including misidentifications as another organism 

(Table 26-6). Although some identifications on some 

systems are problematic, identification of some agents

by the automated systems are very accurate and often 

highly discriminatory. The identification of B anthracis and F tularensis by the MIDI Sherlock Microbial 

Identification System is very specific and an accepted 

method.26–28 Blind acceptance of results from one of 

the automated commercial systems, however, needs 

to be avoided, and results need to be substantiated, or 

refuted, by other assay information.

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TABLE 26-5 

VIRAL HEMORRHAGIC FEVER CULTURE INFORMATION

Viral Hemorrhagic Fever 

Virus Endemic Area Mortality Cells and Incubation Time Growth Characteristics

Arenaviruses Lassa virus West Africa 1%–2% Vero E6–Vero: 3–5 d No CPE; requires 2nd

assay; plaques

Junin Argentinian 30% Vero: 3–5 d No CPE; requires 2nd 

pampas assay; plaques are 

difficult, but possible

Machupo Bolivia 25%–35% Vero E6: 3–5 d No CPE; plaques fine

Bunyaviruses Crimean-Congo Africa, SE Europe, 30% <0.5% SM 3–14 d; possible to Plaque assays just

hemorrhagic Central passage in E6, SW13, or as difficult

fever virus CER cells after initial

isolation, but may require 

>1 blind passages

Rift Valley fever Asia, India, Africa Vero: 2–4 d CPE/plaques

Hanta virus Europe, Asia, 5% for Vero E6: 10–14 d No CPE; requires 2nd

(Hantaan, South America HFRS assay such as IFA or

Dobrava, Seoul, (rare) PCR; often requires

Puumala, Sin blind serial passages to

Nombre Andes) isolate; hard to plaque

Filoviruses Ebola virus Africa, Philippines 50%–90% Vero E6: 6–12 d CPE/plaques

(Ebola Reston) for 

 Sudan/

Zaire

Marburg virus Africa 23%–70% Vero E6: 6–12 d CPE/plaques

Flavivirus Yellow fever Africa, South Overall 3% MK2 cells (also BHK21): Little to no CPE; 

virus America to 12%, 3–6 d requires 2nd assay

20% to such as PCR or IFA to

50% if confirm; plaques fine

severe in Vero cells

 second 

phase 

develops

Kyasanur Forest Southern India 3%–5% Vero/Vero E6 SM: 3–6 d CPE/plaques

disease virus

Omsk Siberia 0.2%–3% Vero/Vero E6 SM: 3–6 d CPE/plaques

hemorrhagic 

fever virus

CER: chicken embryo related; CPE cytopathic effect; HFRS: hemorrhagic fever with renal syndrome; IFA: immunofluorescence assay; PCR: 

polymerase chain reaction; SE: southeast; SM: suckling pig

Although not an automated identification system, 

identification of bacteria with sequence data of rRNA 

genes (16S or 23S) needs to be mentioned. Carl Woese 

pioneered this use of 16S rRNA in the late 1970s for use 

in phylogenetic studies.29 16S rRNA gene sequencing 

has become a standard reference method for identification of many microbes. Bacterial 16S rRNA gene 

sequences are available on public databases such as the 

National Center for Biotechnology Information and the 

Michigan State University Ribosomal Database Project. 

Commercially, Applied Biosystems (Foster City, CA) 

sells 16S rDNA bacterial identification kits under the 

MicroSeq name that provide standardized reagents 

and protocols, but they are not yet FDA approved 

for direct patient care. Although implementation in 

a routine clinical microbiology laboratory has several 

drawbacks for microbial identification (time and cost 

predominately), the accuracy and practicality for many 

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TABLE 26-6 AUTOMATED IDENTIFICATION SYSTEMS FOR BIOLOGICAL THREAT AGENTS MIDI Sherlock Microbial Siemens MicroScan Identification System Trek ARIS 2X Biolog 

Rapid Neg Neg ID Biodefense Library 

bioMérieux/ ID/Type Type 3.0/BTR3 and RBTR3 Dangerous PathVITEK 2 3 Plate 2 Plate Instant FAME GNID Plate Identification D

Bacillus anthracis Yes – BCL card — — Yes‡ — Yes – GP plaBrucella spp Yes – GN card§ Yes Yes Yes Yes – GN plaBurkholderia mallei/ Yes – GN card¥ — — Yes — Yes – GN pla

pseudomallei

Francisella tularensis Yes – GN card§, ¥ — — Yes Yes Yes – GN plaYersinia pestis Yes – GN card Yes Yes¥ Yes Yes Yes – GN pla*Another system, bioMérieux VITEK MS is similar. †GENIII plate has been evaluated for all biological threat agents, but database is not commercially available.‡AOAC INTERNATIONAL cleared for Bacillus anthracis ID.§Noted as a species that may be nonreactive.¥Known false results for this organism on this system.¶Differentiation of Burkholderia mallei and Burkholderia pseudomallei may not be possible.

AOAC: Association of Official Analytical ChemistsBCL: Bacillus identification cardGN: Gram negativeGNID: Gram-negative IDGP: Gram positiveID: identificationNeg: negativeFAME: fatty acid methyl esterification

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Laboratory Identification of Threats

of the biological threat agents is useful. But like all 

systems, there are limitations to full implementation, 

predominately in that B anthracis, Brucella species, 

and Y pestis are often unable to be differentiated from 

near neighbors with sufficient resolution to make the 

system practical.

Antibiotic and Antimicrobial Susceptibility Testing

A principal reason for propagation of bioterrorism 

or infectious disease agents in culture is to screen 

the agent for antibiotic or antimicrobial agent resistance or susceptibility. Although most of the bacterial biological threat agents have well-characterized 

susceptibility to antibiotics (Table 26-7), it will be 

critical to distinguish those organisms that acquire 

natural or laboratory modifications to normal or 

traditional antimicrobial susceptibility.30 Strains of 

B anthracis,

31–33 Brucella abortus, Burkholderia spp,30

F tularensis,

34,35 and Y pestis36,37 have been reported 

to have natural antimicrobial drug resistance, including multiple drug resistances.38–43 The Clinical 

and Laboratory Standards Institute (www.clsi.org) 

has published standard protocols that include the 

biological threat agents to ensure accuracy and 

reproducibility of results. For the biological threat 

agents, classical minimum inhibitory concentration 

determinations are the preferred method.44 Although 

commercial antibiotic susceptibility testing devices 

are available,45,46 they have not been standardized to 

ensure correspondence to the reference method. The 

CDC LRN does include the use of the Epsilometer 

test (E-test) for antimicrobial susceptibility testing of 

selected microorganisms. The E-test is a direct quantification agar dilution method47 that has been adopted 

by many laboratories because of its ease of use and 

quantification capabilities. Molecular methods that 

screen for unique genetic markers of resistance have 

been developed37,48–54; however, molecular analysis 

approaches can be cumbersome when multiple loci 

are involved50,51 and do not always correlate with 

therapeutic effectiveness nor laboratory data.30 DNA 

microarrays offer the potential for simultaneous testing for specific antibiotic resistance genes, loci, and 

markers,49,50,55 but are not sufficiently developed for 

routine use.

Microbial Culture Versus Rapid Methods

With the introduction of newer rapid methods for 

biological threat agent detection and the codification 

of the term biomarkers in the military doctrine, there 

has been avoidance on the discussion of classical 

microbiological culture in the detection of biological 

threat agents. Classical microbiology culture, whether 

for bacteria or viruses, has been stigmatized as archaic 

and overly time consuming. The concept of obtaining 

a result in less than an hour—and being able to do 

something with that result—has taken center stage. Although the newer rapid methods for biological threat 

agent detection have matured over the past decade, 

there are still problematic areas in the sole reliance on 

these newer methods.

Current concepts of operations for theater validation 

laboratories are for multiple technologies that do not 

necessarily include culture of the organism. Most often, 

the use of nucleic acid amplification (through PCR) 

and immunoassays are the predominant methods for 

rapid identification. Operation of a theater validation 

laboratory with PCR and immunoassay technologies 

does not require the containment of a BSL-3 facility. 

To cause disease, microbial agents must be living 

or toxin agents must be biologically (metabolically) 

active. Unless an identification of a biological agent 

is based on some metabolic method, in the absence 

of morbidity or mortality, there is a possibility that 

the implicated agent has been inactivated.16 Inactivation of biological materials, especially in nonclinical 

TABLE 26-7 

STANDARD ANTIBIOTIC SUSCEPTIBILITY TESTING FOR BIOLOGICAL THREAT AGENTS

Bacillus anthracis Brucella spp Burkholderia mallei/pseudomallei Francisella tularensis Yersinia pestis

Penicillin Gentamicin Doxycycline Gentamicin Gentamicin

Doxycycline Streptomycin Tetracycline Streptomycin Streptomycin

Tetracycline Doxycycline Imipenem Doxycycline Doxycycline

Ciprofloxacin Tetracycline Amoxicillin-clavulanate Tetracycline Tetracycline

Trimethoprim/ Trimethoprim/ Ciprofloxacin Ciprofloxacin

sulfamethoxazole sulfamethoxazole Levofloxacin Chloramphenicol

Chloramphenicol Trimethoprim/

 sulfamethoxazole

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Medical Aspects of Biological Warfare 

samples, readily occurs and culture (or multimetabolic assays for toxins) is the only way to ensure that 

an implicated biological agent is actually capable of 

causing disease.

CDC LRN reference laboratories typically include 

the use of BSL-3 facilities because the CDC LRN identification requires identification based on culture of 

the organism(s). Current DoD doctrine, at the theater 

validation level, does not include culture as a requirement. The CDC LRN, however, does not include 

viral diagnostics/detection capabilities other than the 

inclusion of smallpox and other orthopoxviruses. In 

some areas of operations, consideration for viral threat 

agents is just as high, if not higher, than for the more 

traditional bacterial agents. Although deployed assets 

for the diagnostic/detection of viruses are not robust 

for practical reasons, consideration for those agents 

must be included in operations planning. Bacterial 

culturing can be done in BSL-2 facilities for the majority of the biological threat agents (Department of 

the Army Pamphlet [DA PAM] 385-69)56; however, it 

invokes enhanced requirements on facilities engaged 

in culturing any organisms, even those less than BSL-3. 

Any laboratory doing culture work will have to comply 

with all the provisions of that reference. Laboratories 

not doing culture work do not invoke the requirements 

of DA PAM 385-69.

Another consideration for inclusion of microbial 

culturing technologies includes the ability to provide 

sufficient samples for forensic science analysis and 

attribution. Without the propagation of the causative 

agents, the ability to conclusively confirm the agent as 

well as the ability to share samples among attribution 

laboratories will be greatly hindered. 

Integration of In Vivo and In Vitro Diagnostic Tests 

Integrated diagnostics, or orthogonal testing, is 

a recommended testing strategy for both clinical as 

well as environmental samples. Orthogonal diagnostic 

testing is the key to improving the reliability of rapid 

diagnostic technologies. Orthogonal testing refers to 

tests that are statistically independent or nonoverlapping but—in combination—provide a higher degree 

of certainty of the final result. Although orthogonal 

testing is not a standard perspective in the clinical 

diagnostic industry, the concept and its application 

are paramount when investigating some infectious 

agents. Any single detection technology has a set of 

limits with regard to sensitivity and, most importantly, 

specificity. Orthogonal testing seeks to overcome the 

inherent limitations of individual test results with the 

strength of data combinations.18 The application of 

orthogonal diagnostic testing uses an integrated testing 

strategy where more than one technology, technique, 

or biomarker is used to produce diagnostic results, 

which are then interpreted collectively (Figure 26-1).

Immunodiagnostic Methods

An integrated approach to agent detection and 

identification, using both immunological and nucleic 

acid-detection, will provide the most reliable laboratory data and is essential for a complete and accurate 

disease diagnosis.18 Understanding the strengths and 

weaknesses of each assay is paramount in the interpretation of results. Nucleic acid-detection assays are 

exquisitely sensitive and specific; this is the strength 

of the assay, but it can also be a weakness in particular 

situations. Immunodiagnostic assays are comparatively less sensitive, but have broader specificity; this 

is a weakness of the assay, but it can also be a strength 

in certain situations. 

In an orthogonal system, the advantages of the 

nucleic acid and immunological assays will offset the 

disadvantages. Detection of an endemic pathogen will 

rely on the high sensitivity of the nucleic acid-detection 

assay; however, for a newly emerging genetic variant 

the specificity of the nucleic acid-detection assay may 

result in a false negative. A detection system that incorporates immunodiagnostic assays will detect the 

variant with the broader specificity of antibodies. This 

can be illustrated with the detection of the newest ebolavirus, Bundibugyo. Initially, PCR-based assays failed 

to detect the virus because of the genetic variation. 

Only when the less sensitive but more broadly reactive antigen detection and capture immunoglobulin M 

ELISAs were used was the virus detected and identified as an ebolavirus.57 Clearly, both immunodiagnostic and nucleic acid-detection assays are vital when 

detecting pathogens that exhibit genetic variation 

whether natural or intentionally engineered.

Immunodiagnostic techniques diagnose disease by 

detection of agent-specific antigens and/or antibodies present in clinical samples. The most significant 

problem associated with development of an integrated 

diagnostic system is the inability of immunodiagnostic 

technologies to detect agents with sensitivities approaching those of more sensitive nucleic acid-detection technologies. These differences in assay sensitivity 

increase the probability of obtaining disparate results, 

and they could therefore actually complicate medical 

decisions. However, continued advances in immunodiagnostic technologies provide the basis for developing 

antigen- and antibody-detection platforms capable of 

meeting requirements for sensitivity, specificity, assay 

speed, robustness, and simplicity. Detection of specific 

proteins or other antigens or host-produced antibodies 

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Laboratory Identification of Threats

Figure 26-5. Representation of common enzyme-linked immunosorbent assay formats. The assay can be configured to detect 

antigen or antibodies. The target of interest (direct and indirect assays) or a capture antibody (sandwich assay) is immobilized 

by direct adsorption to a solid support such as a 96-well plate or magnetic bead. Detection of the target is accomplished using 

an enzyme-conjugated primary antibody (direct assay) or a matched set of unlabeled primary and conjugated secondary 

antibodies (indirect and sandwich assays). 

E: enzyme; 1°: first degree; 2°: second degree

directed against such antigens constitutes one of the 

most widely used and successful methods for identifying biological agents and for diagnosing the diseases 

they cause. Nearly all methods for detecting antigens 

and antibodies rely on production of complexes made 

of one or more receptor molecules and the entity being 

detected (Figure 26-5). 

Diagnosing disease using immunodiagnostic technologies is a multistep process involving formation 

of complexes bound to a solid substrate. This process 

is like making a sandwich in which detecting the 

biological agent or antibody depends on incorporation of all of the sandwich components. The assays 

are relatively simple and robust, but elimination 

of any one part of the sandwich results in a failure 

and a negative response. Primary ligands used in 

most immunoassays are polyclonal or monoclonal 

antibodies or antibody fragments. Generally, the first 

step in an immunodiagnostic assay is binding one or 

more antibodies for the target of interest onto a solid 

support. Immunoassays are either heterogeneous or 

homogeneous depending on the nature of the solid 

substrate. A heterogeneous assay requires physical 

separation of bound from unbound reactants by using 

techniques such as washing or centrifugation. These 

types of assays can remove interfering substances and 

are, therefore, usually more specific. 

Heterogeneous assays require more steps and increased manipulation that cumulatively affect assay 

precision. A homogeneous assay requires no physical separation but may require pretreatment steps to 

remove interfering substances. Homogeneous assays 

are usually faster and more conducive to automation because of their simplicity. However, the cost of 

these assays is usually greater because of the types of 

reagents and equipment required.

Once the test sample is reacted with the capture element, the final step in any immunoassay is detection of 

a signal generated by one or more assay components. 

This detection step is typically accomplished by using antibodies bound to (or labeled with) inorganic 

or organic molecules that produce a detectable signal 

under specific chemical or environmental conditions. 

The earliest labels used were molecules containing 

radioactive isotopes. However, radioisotope labels 

have generally been replaced with less cumbersome 

labels such as enzymes. Enzymes are effective labels 

because they catalyze chemical reactions, which can 

produce a signal. Depending on the nature of the signal, reactants may be detected visually, electronically, 

chemically, or physically. A single enzyme molecule 

can catalyze many chemical reactions without being 

consumed in the reaction; therefore, these labels are 

effective at amplifying assay signals. Most common 

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Medical Aspects of Biological Warfare 

enzyme-substrate reactions used in immunodiagnostics produce a visual signal that can be detected with 

the naked eye or by a spectrophotometer.

Fluorescent dyes and other organic and inorganic 

molecules capable of generating luminescent signals 

are also commonly used labels in immunoassays. 

Assays using these molecules are often more sensitive than enzyme immunoassays, but require specialized instrumentation and often suffer from high 

background contamination resulting from intrinsic 

fluorescent and luminescent qualities of some proteins 

and light-scattering effects. Signals in assays using 

these types of labels are amplified by integrating light 

signals over time and cyclic generation of photons. 

Other commonly used labels include gold, latex, and 

magnetic or paramagnetic particles. Each can be visualized by the naked eye or by instruments and are stable 

under a variety of environmental conditions. However, 

these labels are essentially inert and therefore do not 

produce an amplified signal. Signal amplification is 

useful and desirable because it results in increased 

assay sensitivity. 

Advances in the fields of biomedical engineering, 

chemistry, physics, and biology have led to an explosion of new diagnostic platforms and assay systems 

that offer great promise for improving diagnostic 

capabilities. An overview of technologies currently 

being used for identification of biological agents and 

either being used for diagnosing or being developed 

for use in diagnosing the diseases they cause will be 

presented.

Enzyme-Linked Immunosorbent Assay 

Since the 1970s, ELISA has remained a core technology for diagnosing disease caused by a wide 

variety of infectious and noninfectious agents. As a 

result, ELISA is perhaps the most widely used and 

best understood immunoassay technology. Assays, 

which have been developed in many formats, can be 

designed to detect either antigens associated with the 

agents themselves or antibodies produced in response 

to infection. ELISAs that detect biological agents or 

agent-specific antibodies are heterogeneous assays 

that capture agent-specific antigen or host-derived 

antibody onto a plastic multi-well plate by an antibody or antigen previously bound to the plate surface 

(capture element). Complexed antigen or antibody 

is then detected using a secondary antibody (detector antibody). The detector antibody can be directly 

labeled with a signal-generating molecule such as 

in a direct ELISA, or it can be detected with another 

antibody that is labeled with an enzyme such as in an 

indirect or capture (sandwich) ELISA formats. These 

enzymes catalyze a chemical reaction with substrate 

that results in a colorimetric change. Intensity of this 

color can be measured by a modified spectrophotometer that determines the optical density of the reaction 

using a specific wavelength of light. In many cases, 

the assay can be interpreted without instrumentation by simply viewing the color that appears in the 

reaction vessel. 

The major advantages of ELISAs are their ability to 

be configured for a variety of uses and applications. 

ELISAs can be used in field laboratory settings, but 

they require power for temperature-controlled incubators and refrigerators and other ancillary equipment 

needs. In addition, ELISAs:

• are commonly used and understood by clinical laboratories and physicians; 

• are amenable to high-throughput laboratory 

use and automation; 

• do not require highly purified antibodies; and 

• are relatively inexpensive to perform.

The major disadvantages are that they are labor 

intensive, temperature dependent, have a narrow 

antigen concentration dynamic range that makes 

quantitation difficult, and are relatively slow.

At the US Army Medical Research Institute of 

Infectious Diseases, antigen-detection ELISAs have 

been developed for nearly 40 different biological 

agents, and antibody-detection ELISAs have been 

developed for nearly 90 different agents. All of these 

assays were developed to use the same solid phase, 

buffers and other reagents, with similar incubation 

periods, incubation temperatures, and general procedures (Table 26-8). Although significant variation 

exists in assay limits of detection, ELISAs typically

are capable of detecting as little as 1 ng of antigen 

per ml of sample.

Electrochemiluminescence 

Immunodiagnostic technologies based on ECL detection are of continued military interest. ECL technology, 

commercially developed by BioVeris (Gaithersburg, 

MD), was incorporated into a field ready immunodiagnostic system, the M1M. The assay formats are similar 

to those of ELISA; however, magnetic beads serve as the 

solid support and magnets are used to concentrate target 

agents. The detection of target uses a chemiluminescent 

label (ruthenium, Ru). The small size of Ru (1,057 kDa) 

makes it easily conjugated to any protein ligand (antigen 

or antibody) using standard chemistries without affecting immunoreactivity or solubility of the protein. The 

heart of the M1M ECL analyzer is an electrochemical 

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Laboratory Identification of Threats

flow cell with a photo-detector placed just above the 

electrode. A magnet positioned just below the electrode 

captures the magnetic bead-Ru-tagged immune complex and holds it against the electrode. Application of 

an electric field results in a rapid electron transfer reaction between the substrate (tripropylamine) and the Ru. 

Excitation with as little as 1.5 V results in light emission, 

which in turn is detected by a charge-coupled device 

camera. The system’s strengths come from its speed, 

sensitivity, accuracy, and precision over a wide dynamic 

range. Magnetic beads provide a greater surface area 

than conventional surface-binding assays like ELISA. 

The reaction does not suffer surface steric and diffusion 

limitations encountered in solid-phase immunoassays; 

instead it occurs in a turbulent bead suspension, thus 

allowing for rapid reaction kinetics and short incubation 

time. Detection limits as low as 200 fmol/L are possible 

with a linear dynamic range that can span six orders of 

magnitude.58,59

Assay configurations can be identical to ELISA, 

direct, indirect, or sandwich assays. For antigen 

detection assays, the beads are coated with capture 

antibody, whereas for antibody detection assays the 

beads are coated with antigen or capture antibody. 

The coated paramagnetic beads, in the presence of 

biological agent (target), form immune complexes 

that are detected by the Ru-conjugated detector 

antibody. After a short 15-minute incubation period the analyzer draws the sample into the flow 

cell, captures and washes the magnetic beads, and 

measures the electrochemiluminescent signal (up 

to 1 minute per sample cleaning and reading time). 

Conveniently, the reagents can be lyophilized. The 

system uses 96-well plates that allow high sample 

throughput.

The ECL system effectively can detect staphylococcal enterotoxin B, ricin toxin, botulinum toxin, F 

tularensis, Y pestis F1 antigen, B anthracis protective 

TABLE 26-8 

COMPARISON OF IMMUNODIAGNOSTIC METHODS

ELISA ECL Luminex HHA

Antibody Requirements 

Purity None Required Required Required

Labeling None Biotin/ruthenium Biotin/beads Beads

Assay Parameter 

Coating time 12 h 0 0 0

Incubation time 3.5 h 15 m 30 m 15 m

Read time 1 sec/well 1 m/tube 20–120 sec/well 30 sec

No. of steps 5 1 1 1

No. of buffers required 3 1 1 1

Specialized reagents Conjugate Assay buffer Sheath fluid Sample buffer

Substrate Cell cleaner 

Solid phase used Microtiter well Magnetic bead Colored latex bead Nitrocellulose

Reaction Bound In solution In solution Bound

Detector label used HRP Ru PE Gold

Detection method Colorimetric Chemiluminescence Fluorescence Visual

Amount of sample per test 100 ml 50 ml 50 ml 200 ml

Prozone No Yes Yes No

Sample matrix effects No Yes Yes Yes

Multiplexing No No Yes Potential

Intraassay variation (%) 15%–20% 2%–12% 10%–25% Undetermined

Potential for PCR analysis Yes Yes Yes Yes

Limit of Detection (per ml) Single Multiplexed

Y pestis F1 (CFU) 250,000 500 62,500 125,000 ND

Staphylococcal enterotoxin B (ng) 0.63 0.05 3.13 6.25 ND

Venezuelan equine encephalitis 1.25 x 107 1.0 x 107 3.13 x 108 6.25 x 108 1 x 108

virus (PFU)

CFU: colony-forming unit; ECL: enhanced chemiluminescence; ELISA: enzyme-linked immunosorbent assay; HHA: hand held assay; HRP: 

horseradish peroxidase; ND: not detected; PCR: polymerase chain reaction; PE: phycoerythrin; PFU: plaque-forming unit; Ru; ruthenium

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Medical Aspects of Biological Warfare 

antigen (PA) and capsule, and Venezuelan equine 

encephalitis virus.18,60–63 The system, which had been 

demonstrated in field settings, was used as one part of 

an integrated diagnostic system in several deployable 

and/or deployed laboratories. In 2007, Roche (Basel, 

Switzerland) acquired BioVeris to expand its ECLbased Elecsys Systems, which ultimately led to the 

demise of the M1M platform and its use by the DoD. 

The platform remains in use, but Roche is no longer 

producing reagents and the system will be forced into 

obsolescence when supplies are no longer available. 

Critical assay performance characteristics and detection limits from three typical ECL agent-detection 

assays are shown in Table 26-8.

Meso Scale Diagnostics (Rockville, MD) has developed a line of immunodiagnostic instruments based on 

the ECL technology. Unlike the M1M that was single 

plex, analyzing a single sample for a single target, 

the MSD instrument is capable of multiplex analysis, 

analyzing for multiple targets on a single sample. The 

Meso Scale Diagnostics MULTI-ARRAY technology 

uses ECL to detect binding events on patterned arrays. In multiwell microplates, capture antibodies are 

bound to carbon electrodes integrated into the bottom 

of the plate. The plates can have up to 10 electrodes 

per well, with each electrode coated with a different 

capture antibody. Similar to the sandwich ELISA, 

the target of interest is captured on the electrode and 

detected by the target-specific Ru-conjugated detector antibody. As in the M1M system, electrochemical 

stimulation results in the Ru label emitting light at the 

surface of the electrodes, from which the concentration 

of target associated with the particular electrode can 

be determined. 

Evaluation of the technology at USAMRIID found 

sample testing in simple matrices, like the high volume 

air handler buffer, worked well, but the assays suffered from increased backgrounds in more complex 

matrices, like blood or serum. The ECL analyzer PR2 

is available in a manual configuration, Model 1800, 

and a fully automated configuration, Model 1900, each 

of which is capable of high-throughput analysis. For 

environmental testing, the Model 1500 is designed for 

automated aerosol sample testing. 

This multiplexed immunoassay platform has more 

than 400 assays commercially available for use in 

clinical, environmental, and research applications, 

with kits that are specifically designed for biodefense. 

MSD assays can be customized; however, antibody 

printing onto the electrodes must be done by the company, rendering laboratory derived tests less flexible, 

more complicated, and most likely more expensive. 

The NGDS acquisition program has identified the 

MSD PR2 instruments for possible inclusion as the 

immunodiagnostic component in its portable human 

diagnostic system. Dependence on any single company 

for both instrument and assays increases the risk to 

the DoD diagnostic and detection programs, which is 

reminiscent of the BioVeris experience.60 

Flow Cytometry

Flow cytometry, the measurement of physical and 

chemical characteristics of small particles, has many 

current applications in research and healthcare and is 

commonplace in most large clinical laboratories. Applications include cytokine detection, cell differentiation, chromosome analysis, cell sorting and typing, 

bacterial counting, hematology, DNA content, and 

drug discovery. The technique works by placing biological samples (ie, cells or other particles) into a liquid 

suspension. A fluorescent dye, the choice of which is 

based on its ability to bind to the particles of interest, 

is added to the solution. The suspension is made to 

flow in a stream past a laser beam. Light is scattered, 

and the distribution and intensity of scattered light 

is characteristic of the sample passing through. The 

wavelength of light is selected such that it causes the 

dye—bound to the particle of interest—to fluoresce. 

A computer counts and/or analyzes the fluorescent 

sample as it passes through the laser beam. Using the 

same excitation source, fluorescence may be split into 

different color components so that several different 

fluorophores can be measured simultaneously and signals interpreted by specialized software. Multiplexed 

flow cytometry assays have been demonstrated for a 

variety of cytokine targets.64 Particles can also be sorted 

from the stream and diverted into separate containers 

by applying a charge to the particles of interest.

The Luminex xMAP technology (Austin, TX) has 

resulted in significant improvements in multiplex flow 

cytometry-based diagnostics. The xMAP technology is 

based on polystyrene bead sets encoded with different 

intensities of red and infrared dyes (unique address to 

a bead set) and coated with a specific-capture antibody 

against one of the analytes of interest. Interrogation of 

the beads by two lasers identifies the spectral property 

of the bead (address) and hence the associated analyte, 

in addition to the phycoerythrin labeled secondary 

antibody against the specific analyte.

The Luminex 100/200 (Austin, TX) and the FLEXMAP 3D systems are flow cytometry-based instruments that can rapidly perform up to 100 tests simultaneously on a single sample. They incorporate three 

familiar technologies: (1) bioassays, (2) microspheres, 

and (3) fluorescence. Assays occur in solution; thus, 

reaction kinetics are rapid and incubation times are 

shorter. Capture antibodies or ligands are bound to 

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Laboratory Identification of Threats

microspheres labeled with two spectrally distinct 

fluorochromes. By adjusting the ratio of each fluorochrome, microspheres can be distinguished based on 

their spectral address. Bioassays are conducted on the 

surfaces of these microspheres. Detector antibodies 

are labeled with any of a number of different green 

fluorescent dyes. This detector-bound fluorochrome 

measures the extent of interaction that occurs at the 

microsphere surface; that is, it detects antigen in a typical antigen-detection assay. The instruments use two 

lasers: one for detection of the microsphere itself, and 

the other for the detector. Microspheres are analyzed 

individually as they pass by two separate laser beams, 

are classified based on their spectral address, and are 

measured in real time. Thousands (20,000) of microspheres are processed per second resulting in an assay 

system theoretically capable of analyzing up to 100 

different reactions on a single sample in just seconds. 

The manufacturer reports assay sensitivities in the 

femtomole level, dynamic range of 3 to 4 orders of magnitude, and claims results are highly consistent and 

reproducible.65 Because the intensity of the fluorescent 

label is read only at the surface of each microsphere, 

any unbound reporter molecules remaining in solution 

do not affect the assay, making homogeneous assay 

formats possible. The system, which can use tubes as 

well as 96- and 384-well plates, can be automated. In 

addition to the Luminex instrument, a plate shaker 

and liquid handling devices are required to complete 

assays. As with most technologies, many different 

formats can be used. Many multiplexed assay kits are 

commercially available from different manufacturers 

for various cytokines, phosphoproteins, and hormones.

The FLEXMAP 3D instrument is capable of high 

throughput and can be automated, which makes it 

better suited for a large clinical laboratory. No fieldready versions of the Luminex 100/200 are available, 

which limits the practical use of this instrument in 

deployment situations. No commercial or DoD sources 

for biological threat agent assays are available for this 

platform. 

MAGPIX

Flow cytometry-based systems can be accommodated in large diagnostic laboratories where environmental conditions are controlled and qualified technicians 

perform preventative maintenance to ensure the flow 

cells and lasers are clean, aligned, and functioning 

properly. Recently, the MAGPIX instrument based 

on the Luminex xMAP technology was introduced. 

The instrument, which eliminates some of the shortcomings of the flow cytometry-based instruments, 

has tremendous potential for forward laboratory 

applications in such resource-limited environments. 

MAGPIX uses magnetic color-coded microspheres to 

perform multiplexed assays. Fifty different individually addressable bead sets can be used on an instrument. Instead of interrogating individual microspheres 

sequentially through flow cytometry, MAGPIX uses 

magnetic force to move the microspheres to a stage and 

then images all the magnetic microspheres from that 

sample at once using a charge-coupled device camera. 

Three images, each taken with a different filter, are 

used to discriminate bead sets and determine assay 

signals. Two images are used to identify the unique 

bead address and the third image measures the presence of tracer fluorophore, indicating the presence of 

target analyte. The MAGPIX carries sufficient drive 

fluid onboard (650 mL) to analyze eight full microtiter plates (768 samples) and has a throughput rate of 

approximately 96 samples per hour, or 1.6 samples 

per minute. The system is fully compatible with all 

magnetic bead-based assays currently performed on 

the Luminex flow cytometers; all assay, sample, and 

reagent preparation protocols for both systems are 

analogous. The sensitivity of the MAGPIX system is 

similar or identical to the Luminex 100/200 instrument, 

which can detect ricin in the pg/mL range.

Sensitivities of bead-based assays are typically in the 

same range as—or in some cases superior to—those obtained in ELISAs.66,67 Previous limitations in fieldability 

for the Luminex flow cytometric instruments (large 

size, susceptibility of the laser alignment to shock or 

vibration) have also been largely overcome in the new 

MAGPIX instrument; this latter platform is smaller 

and more rugged. Per instrument cost has also been 

significantly decreased, which may also make it more 

affordable for widespread deployment in forward 

facilities. Featuring a flexible, open-architecture design, xMAP technology can be configured to perform 

a wide variety of bioassays quickly, cost effectively, 

and accurately. Six assays are commercially available 

for biodefense toxin targets: botulinum toxins A, B, E, 

F, staphylococcal enterotoxin B (SEB), and ricin.

Hand Held Assays 

HHAs are immunodiagnostic assays that are ideally 

suited for field-based diagnostics. Commonly found 

on the commercial market, they are simple enough to 

use and interpret that some types are even approved 

for over-the-counter use by the FDA; the best known 

one is the home pregnancy test. HHAs are typically 

designed on natural or synthetic membranes contained 

within a plastic or cardboard housing. A capture antibody (for antigen detection) or antigen (for antibody 

detection) is bound to the membrane and a second 

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Medical Aspects of Biological Warfare 

antibody labeled with some visible marker element is 

placed on a sample application pad. As sample flows 

across the membrane, antigen or antibody present in 

the sample binds to labeled antibody and is captured 

as the complex passes the bound antibody or antigen 

(Figure 26-6). Colloidal gold, carbon, paramagnetic, or 

colored latex beads are commonly used particles that 

create a visible line in the capture zone of the assay 

membrane.

HHAs are advantageous because they are relatively 

inexpensive, simple, and require little training to use, 

and results can be obtained in only 5 to 15 minutes. 

One of the greatest advantages of HHAs is the lack of 

reliance on instrumentation and logistical needs associated with those instruments. However, this lack 

of instrumentation decreases the utility of the tests 

because results cannot be quantified. To respond to this 

deficiency, several technologies are available to make 

these assays more quantitative and have the added 

benefit of increasing their sensitivity. One technology, produced by Response Biomedical Corporation 

(Vancouver, British Columbia, Canada), allows for 

quantitative interpretation of the HHA.68–71 The Rapid 

Analyte Measurement Platform (RAMP) cartridges 

for biodefense can detect B anthracis, ricin, botulinum 

toxin, and smallpox virus. Another method for quantitative detection of antibody/antigen complex formation in HHAs is use of up-converting phosphors.72,73

Paramagnetic particles have similarly been used in 

assays: instruments capable of detecting changes in 

magnetic flux within the capture zone (Quantum Design, San Diego, CA) have proven useful by improving 

sensitivity by as much as several orders of magnitude 

over more traditional HHAs.

DoD commonly uses HHAs to detect biological 

threat agents. The DoD Medical Countermeasure Systems, Critical Reagent Program, a repository for DoD 

diagnostic reagents, offers lateral flow assays for this 

purpose. In addition, several commercial companies 

have begun to market a variety of threat agent tests for 

use by first responders. However, independent evaluation of these assays has not typically been performed, 

so data acquired from the use of these assays must be 

interpreted carefully. Another common disadvantage 

of HHAs is their inability to incorporate the capability to run a full spectrum of control assays on a single 

strip assay. Recently, FDA approved two lateral flow 

assays for the detection of B anthracis for use in clinical 

settings.74 As with any diagnostic test, understanding 

its strengths and weaknesses will aid in proper interpretation of the results. HHAs are useful in initial 

screening of samples for biological threat agents, but 

results should be followed with confirmatory testing 

using an orthogonal system.

Future Perspectives

Traditionally, assays for detecting proteins and 

other nonnucleic acid targets, including antigens, 

antibodies, carbohydrates, and other organic molecules were conducted using antibodies produced 

in appropriate host animals. As a result, these assays 

were generically referred to as immunodiagnostic or 

immunodetection methods. In reality, numerous nonantibody molecules, including aptamers, peptides, and 

engineered antibody fragments, are now being used 

in affinity-based detection technologies.75–83 

Since an immunodiagnostic assay is directly related 

to the characteristics of the antibody components used, 

improved antibodies or antibody-like elements have 

the potential to significantly improve the sensitivity, 

specificity, and robustness of the assays. Naturally 

occurring single domain antibodies (sdAbs) derived 

from camelids and sharks possess unique properties 

that could improve present day immunodiagnostics. 

Through convergent evolutionary processes, both 

camelid and shark immune systems naturally possess nonconventional antibody subsets composed 

only of heavy chain homodimers and a single variable domain.84,85 The variable (V) domains of these 

antibodies represent the smallest naturally occurring 

antigen binding domains known. These extremely 

small (12–15 kDa) sdAbs can target enzyme clefts and 

Figure 26-6. Illustration of a typical hand held immunoassay. 

Photograph: Courtesy of US National Aeronautics and Space 

Administration.

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Laboratory Identification of Threats

cryptic antigens that conventional antibodies cannot. 

Unique structural characteristics provide them a high 

temperature (>60–90°C), proteolytic and pH stability,86–93 high solubility,94 and efficient and economical 

expression in a variety of microorganisms (including 

Escherichia coli).95 The unique features of these naturally 

occurring molecules could vastly improve the utility 

of any immunodiagnostic assay. 

Antibody-based biosensors provide the most reliable detection capability across the broadest range of 

biowarfare agents. They are, therefore, the preferred 

platform for DoD biosensor applications. However, 

the fragility of the antibody molecule together with the 

short shelf life (typically 2 weeks or less) of antibodybased biosensors severely complicates their use outside 

of a clinical laboratory environment. In addition, the 

variability in affinity across various antibody systems 

has precluded the development of multiplexing antibody arrays for biosensor applications. The Defense 

Advanced Research Projects Agency sponsored 

the Antibody Technology Program to develop and 

demonstrate approaches for achieving revolutionary 

improvements in the stability of antibodies while 

simultaneously demonstrating the ability to control 

antibody affinity for use in immunological detection.96–98 Each performer was supplied with the same 

starting material, single chain fragments (scFvs), and 

was asked to improve the antibodies by engineering 

them for improved stability and affinity. The desired 

metrics for improvements were decreasing the affinity 

of the antibody by at least 100-fold and increasing the 

stability of the supplied antibody such that it maintained its activity at 70°C for 1 hour. 

Initially, the performers achieved these requirements in separate proteins before attempting to 

meet both requirements in one protein. Each group 

Figure 26-7. Generic overview of PLA reactants and assay. (a) In addition to PCR reagents, PLA consists of antibodies 

to two different epitopes, each labeled with a unique oligonucleotide (proximity probe) and a connector oligonucleotide 

complementary to the free ends of each proximity probe. Unique to our design is the inclusion of magnetic beads coated 

with antigen-specific antibodies. (b) After formation of a bead/antigen/proximity probe complex, the free 5’ and 3’ ends of 

the antibody-bound oligonucleotides that are in close proximity to each other hybridize onto the connector oligonucleotide 

and are covalently joined by DNA ligase. Once joined, these provide a template for PCR amplification. Ab: antibody; Comp: 

complementary; Hyb: hybridization; PCR: polymerase chain reaction; PLA: proximity ligation assay

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Medical Aspects of Biological Warfare 

approached the solution differently, but produced 

antibodies with greater binding to the target ligand 

and improved thermostability. The Antibody Technology Program increased antibody affinity by a factor of 

400. Temperature stability of antibody molecules was 

improved by a factor of 36, which translated into an 

increased shelf life at room temperature from about 

1 month to 3 years. Similarly, antibody survival at 

70°C increased from 5 to 10 minutes to 48 hours. By 

creating these stable antibodies, it was postulated 

that different variable regions could be grafted onto 

the developed backbone to increase the stability of 

antibodies in general, without altering the affinity. 

These improvements would translate into improved 

immunodiagnostic assays that would function well in 

more austere environments, as well as decreasing the 

cold chain needs for these reagents.

Often the Achilles heel of immunodiagnostic assays is the lower sensitivity when compared to PCRbased assays. Advances in antibody development or 

engineering can improve antibody characteristics and 

therefore the resulting assays, but other advancements 

combine antibody detection with PCR to achieve 

sensitivity levels equivalent to PCR. Immuno-PCR 

assays are similar to ELISAs, but substitute the detector antibodies conjugated to enzymes with antibodies 

that are labeled with DNA.99 Using label-specific PCR 

primers, the DNA label is amplified and can result 

in increased sensitivity of 105-fold. These assays that 

relied on a single DNA-labeled antibody exhibited 

high background signals that frequently resulted in 

false-positive results.100,101 The proximity ligation assay 

eliminated the background limitations of immunePCR by requiring the binding of antibodies to at least 

two different epitopes on the target antigen.102 Each 

antibody is labeled with a specific oligonucleotide 

containing a PCR primer site and having either a free 

5’ or 3’ end (Figure 26-7). 

When the antibodies bind the target, the DNA labels 

are brought into proximity and the two complementary ends hybridize to a connector oligonucleotide with 

compatible ends. The hybridized strands are joined by 

DNA ligase and serve as a template for amplification 

and fluorescent probe detection. The amplified DNA 

is a surrogate marker for the target protein of interest. 

The 5’ or 3’ oligonucleotide ends that fail to hybridize 

completely with connectors cannot be amplified and 

reduce the background and the possibility of false positives. Proximity ligation assay detection of viruses and 

bacterium has proven to be more sensitive than ELISA 

and as sensitive as real-time PCR.103 In addition, the 

assays work in a wide variety of biological matrices, 

serum, plasma, cerebrospinal fluid, cell culture media, 

and lysates of cells and tissues.102,104 Improvements in 

technology and the components of immunodiagnostic 

assays continue to close the gap in sensitivity between 

protein detection and nucleic-acid detection making 

an orthogonal system ever more powerful. 

Molecular Detection Methods

PCR is the predominant methodology for detection 

of molecular signatures. Originally conceived in 1983 

by Kary Mullis,105 the first published application of 

PCR was by Saiki et al amplifying beta-globin genomic 

sequences and thus hallmarking the advent of the 

molecular biology field.106 In its simplest form, PCR 

consists of target genomic material, two oligonucleotide primers that flank the target sequence, a heatstable DNA polymerase, a defined solution of salts, 

and an equimolar mixture of deoxyribonucleotide 

triphosphates. This mixture is subjected to repeated 

cycles of defined temperature changes that facilitate 

denaturation of the template, annealing of the primers to the target, and extension of the primers so that 

the target sequence is amplifying. With each cycle, a 

theoretical doubling of the target sequence occurs. The 

whole procedure is carried out in a programmable 

thermal cycler that precisely controls the temperature 

at which the steps occur, the length of time the reaction 

is held at the different temperatures, and the number 

of cycles. Under ideal conditions, a single copy of a 

nucleic acid target can be amplified over a billionfold after 30 cycles, thus allowing amplification from 

targeted genomic signature with potential detection of 

etiologic agents down to a single copy.107–109 Genomic 

material, DNA or RNA (in the form of cDNA), can be 

targeted by this method of amplification. Rapid detection methods typically rely on real-time PCR where 

targeted genomic signatures are amplified via primers 

and detection accomplished through oligonucleotide 

probe hybridization. To this end, numerous PCR-based 

technologies are currently implemented in the clinical 

setting for diagnosis of infectious agents. 

Real-Time Polymerase Chain Reaction 

The most important development in rapid identification of biological agents is real-time PCR methods. 

Although traditional PCR is a powerful analytical tool 

that launched a revolution in molecular biology, it is 

difficult to use in clinical and field laboratories. As 

originally conceived, gene amplification assays can 

require 5 to 6 hours to complete, not including the sample processing required to remove PCR inhibitors.110

The improvement of assay time-to-answer came with 

the development of assay chemistries that allowed the 

PCR reaction to be monitored during the exponential 

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Laboratory Identification of Threats

amplification phase, that is, real-time (Figure 26-8). 

In this context, Lee et al and Livak et al developed 

real-time assays for detection and quantification of 

fluorescent reporters where fluorescence increase was 

directly proportional to the amount of PCR product 

generated in the reaction.111,112 In this scenario, higher 

starting copy numbers of the nucleic acid target resulted in earlier amplification where significant increase 

in fluorescence is observed. 

Three main probe-based fluorescence-monitoring 

systems exist for DNA amplification: (1) hydrolysis 

probes; (2) hybridization probes; and (3) DNAbinding agents. Hydrolysis probes, most exemplified 

by TaqMan (Applied Biosystems, Foster City, CA) 

chemistries, have been the most successful for rapidly 

identifying biological threats.108 Numerous assays have 

been developed against biological threat and infectious 

agents using these approaches by the DoD, the CDC, 

and the US Department of Energy.108,109

The JBAIDS is the current DoD fielded platform 

for molecular diagnostic/real-time PCR detection in 

reference laboratory, combat support hospital, and 

forward operating settings. This system supports assays primarily in the identification of several biological 

threat agents for clinical diagnostic application while 

also supporting assays for biosurveillance screening of 

biological threats as well as some infectious diseases. 

FDA-cleared assays for clinical diagnostics include B 

anthracis, F tularensis, Y pestis, C burnetii, and several 

forms of influenza (H5N1, A, B and A subtyping). 

Other assays for biosurveillance purposes cover additional biological threat targets, toxins, and foodborne 

pathogens. These assays can be run in approximately 

30 minutes with up to 32 samples per run. With this 

Figure 26-8. Overview of real-time PCR reactants and reaction conditions, generic. (a) Real-time PCR reactions (TaqMan probes 

depicted) consist of the canonical PCR reactants, such as forward and reverse primers as well as a DNA template. In addition 

to these reactants, real-time PCR contains either a fluorescently labeled probe or intercalating dye that is used to monitor 

amplicon quantities. In the depicted scenario, a sequence of DNA complementary to target sequence separates a fluorophore 

(F) and a quencher (Q). Fluorescence from the fluorophore in proximity to the quencher is greatly diminished compared to 

absence or distal fluorescence. (b) Similar to conventional PCR, real-time PCR reactions begin with a denaturing of the DNA 

template. Reducing the temperature allows amplicon-specific primers to anneal to the target sequence and amplification to 

begin. In some type of real-time reactions, amplified double-stranded DNA is directly quantified through measurement of 

DNA intercalating dyes such as SYBR green, which only fluoresces when intercalated. In the instance depicted, the probe 

anneals to the DNA template in similar fashion to the primers. When DNA polymerase encounters the probe, the enzyme’s 

exonuclease function cleaves the probe liberating the fluorophore. No longer in proximity to the quencher, fluorophore 

fluorescence can be monitored and then correlated to target sequence concentration. Subsequent cycling and amplification 

yield progressively more DNA template and, consequently, more fluorophore fluorescence. PCR: polymerase chain reaction

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Medical Aspects of Biological Warfare 

system, a presumptive identification of most biological 

agents can be completed in 3 hours or less. Although 

it is an excellent system for detecting biological threat 

agents, this system suffers from lack of use in the field 

setting because of the lack of assays for more commonly acquired pathogens that are more routinely 

seen in the clinical setting. To mitigate this issue, future 

generations of molecular detection instruments should 

have regulatory cleared assays for common infectious 

diseases to make use and maintenance worthwhile.

Next Generation Molecular Diagnostics

The JBAIDS device is currently fielded in DoD medical laboratories, and several of the aforementioned 

problems exist with this system including the lack of 

routine usage resulting from limited assay availability 

and the limited capability to run independent or replicate samples (32 samples per run). To address some 

of these issues, the Joint Program Executive Office, the 

office that fielded the JBAIDS, acquired the Biofire FilmArray platform for the NGDS. While the FilmArray 

(Biofire Diagnostics, Salt Lake City, UT) was chosen 

as the NGDS device, several other viable diagnostics 

were considered within source selection, including the 

Liat Analyzer (IQuum, Marlborough, MA) and the 3M 

Focus Integrated Cycler (Focus Diagnostics, Cypress, 

CA). Overall, the FilmArray was chosen based on ease 

of use, sensitivity, and available FDA-cleared assays 

for respiratory or other commonly acquired infectious 

diseases. 

FilmArray is an integrated sample prep and multiplex PCR diagnostic platform capable of detecting 

bacteria and viruses in a single reaction. This system 

can run FDA-cleared assays for common respiratory 

organisms or assays for biological threat detection in 

a pouch-based array, thus providing a routine application for the instrument in a clinical setting. In addition to the respiratory pouch, several other pouches 

have been evaluated, to include the blood culture and 

biological threat pouches verifying performance characteristics.113–116 Up to 48 independent reactions can be 

run in a single run; however, only a single sample can 

be run per pouch thereby limiting the throughput of 

this device. Overall, the system is a simple use instrument using syringe and closed pouch-based system to 

bead-beat and extracts nucleic acid with downstream 

application to an array-based set of real-time PCR reactions. Given the low complexity of operation, FDA is 

evaluating it for a CLIA-waver; however, currently, it 

is considered a moderate complexity device. 

Overall, this system provides an incremental step 

forward in technology compared to the JBAIDS that 

it will replace. Assay versatility will be sacrificed for 

integrated sample processing and clinically applicable 

assays upon deployment. While these additions to the 

DoD portfolio will augment current biosurveillance 

and biological threat detection capabilities, further 

development is required to truly advance the frontline military diagnostic applications. The current 

forerunner for filling this capability is next-generation 

sequencing (NGS) applications.

The Horizon–Agnostic Diagnostic Applications

The current endpoint and desired capability for diagnostics in the DoD is an agnostic molecular platform. 

All the aforementioned technologies require some a 

priori knowledge of the organism; for instance, realtime PCR requires sequence information regarding 

the target of interest to design primers and probe. In 

addition, in the application of real-time PCR, guidance 

from medical intelligence, symptomology, or endemic 

diseases is required because there are limitations to 

the number of discrete targets and samples that can 

be queried in a single run. These limitations could be 

overcome by application of agnostic diagnostic approaches such as NGS pathogen detection strategies.

NGS has many potential benefits over current molecular diagnostic approaches. In terms of agnostic 

detection, NGS has the capability to sequence an entire 

genome of an organism, thus obviating the need for 

specific a priori knowledge of the pathogen. For example, the detection of novel filovirus variants such 

as Lujo virus was accomplished via NGS discovery.117

While numerous methodologies have come and gone 

throughout technology development, current field 

leaders are Illumina’s sequence-by-synthesis (Illumina 

Inc, San Diego, CA) and PacBio’s (Pacific Biosciences, 

Menlo Park, CA) single molecule real-time sequencing. Each system has advantages and disadvantages. 

Illumina is the current leader with shorter sequence 

reads (72–250 bp), but generating significantly more 

sequence data (>10 GBp). PacBio, however, generates 

much longer reads (1–10 kb), but has significantly 

higher error rates. As the field progresses, newer 

nanopore technologies, such as the MinION (Oxford 

Nanopore, Oxford, UK), may supplant these current 

leaders in the near future.

Combinatory approaches between these two technologies have been applied to mitigate independent 

disadvantages while retaining platform-specific 

advantages.118 Numerous lab-derived tests and even 

510(k) submissions have cleared the FDA for use in 

detecting cancer. However, several steps and obstacles require mitigation before these technologies 

can be applied to regulatory compliant detection 

of a pathogenic organism. Principal in these issues 

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Laboratory Identification of Threats

include mitigation of high amounts of background 

host-derived nucleic acid, lack of specificity resulting 

from agnostic nature, and sensitivity issues. Current 

efforts within academia and DoD show promise toward mitigating these issues and bringing NGS into 

the diagnostic toolbox.

BIOSURVEILLANCE AND EMERGING THREATS

The emergence of new biological threats is a particular challenge for the military clinical or field laboratory. In the past, the biological defense research 

program for diagnostics has focused on agentspecific identification using collections of biological 

threats in the biological weapons programs of the 

United States (ended in 1969) and the former Soviet 

Union.119,120 However, several critical events have 

broadened the scope of the biological threat over the 

past 3 decades. The maturation and proliferation of 

biotechnology have resulted in several laboratory 

demonstrations of genetically engineered threats 

with new, potentially lethal characteristics.121–125

Jackson et al demonstrated that the virulence of 

orthopoxviruses was enhanced by the insertion of 

immunoregulatory genes, such as interleukin-4.124 In 

other work, Athamna et al demonstrated the intentional selection of antibiotic-resistant B anthracis.

121

Borzenkov, Pomerantsev, and Ashmarin modified 

Francisella, Brucella, and Yersinia species by inserting 

beta-endorphin genes.122,123

As a result of the proliferation of these biotechniques, public health officials can no longer depend 

on an adversary choosing any of the 15 to 20 biological threats of past generations, but now must prepare 

for a future of an infinite number of threats, some 

of which may have been genetically engineered to 

enhance virulence or avoid detection. Secondly, the 

emergence of more virulent and/or infectious strains 

of naturally occurring infectious diseases has posed 

significant public health challenges to civilian and 

military populations. The emergence of the H5N1 

and H1N1 variants of influenza is a recent example 

of the challenge that naturally occurring infectious 

diseases can present, the latter resulting in a pandemic 

from 2009 to 2010. These new threats will require the 

development of identification and diagnostic systems 

that can be used flexibly to allow early recognition 

of a unique biological threat, representing one of the 

next major research and development challenges for 

the DoD, DHHS, and DHS. The ability to identify and 

characterize genetically engineered threats or naturally 

emerging infectious diseases before they negatively 

affect military and public health is the focus of new 

initiatives in biosurveillance. 

A national effort on biosurveillance was formally 

initiated on October 18, 2007 in Homeland Security 

Presidential Directive-21,126 which defines biosurveillance as the process of active data gathering with 

appropriate analysis and interpretation of biosphere 

data that might relate to disease activity and threats 

to human or animal health—whether infectious, toxic, 

metabolic, or otherwise, and regardless of intentional 

or natural origin—to achieve early warning of health 

threats, early detection of health events, and overall 

situational awareness of disease activity. The DoD 

community has accepted biosurveillance as defined 

above as a working definition, and as synonymous 

with health surveillance as defined in DoD Directive 

6490.02E, Comprehensive Health Surveillance, which 

establishes policies and assigns responsibility for routine, comprehensive health surveillance of all military 

service members.127 The DoD has an extensive health 

surveillance program for all military personnel, and 

the Armed Forces Health Surveillance Center executes 

this effort.128 However, in addition to human health 

surveillance, biosurveillance encompasses active data 

gathering and interpretation of data from the entire 

biosphere, including animal health surveillance, vector 

surveillance, and environmental surveillance.129

The challenge 7 years removed from the Homeland 

Security Presidential Directive-21 is accessing, collecting, and interpreting all of the surveillance data that are 

available in a way that provides actionable information 

to affect public health. Specific challenges that must 

be addressed include information sharing, information technology tools to assimilate and analyze data, 

and algorithms to interpret and report the subset of 

data that affects public health. Within the confines of 

biosurveillance, diagnostic testing results are a very 

small percentage of the health surveillance data, and 

an even smaller percentage of the biosurveillance data. 

Therefore, care must be exercised to ensure that diagnostic testing data feed into biosurveillance without 

allowing the biosurveillance mission to become the 

critical requirements for diagnostic assay and platform 

development. Diagnostics must continue to focus on 

assisting clinicians in making correct medical decisions about the treatment and prognosis of individual 

patients. The ultimate goal and the significant challenge for the biosurveillance enterprise is translating 

the identification of a potential public health threat 

through biosurveillance to a medical countermeasure, 

such as an in vitro diagnostic test. Doing so in a timely 

manner will be critical to maintain military readiness 

and minimize public health impacts.

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Medical Aspects of Biological Warfare 

Ultimately, the success of biosurveillance depends 

on the tools and technologies available to survey the 

biological space that affects human health. These tools 

must move away from agent specific identification, 

which is currently the foundation of most FDA-cleared 

in vitro diagnostic tests, to a more agnostic approach. 

Unlike diagnostic tests, which are typically chosen 

based on clinical suspicion of a particular disease, 

biosurveillance platforms must attempt to identify all 

agent(s) in a particular sample. This identification can 

be approached through the use of multiple complementary identification technologies or agent agnostic 

platforms. The service lab component of the NGDS 

acquisition program will deliver several complementary platforms to OCONUS research laboratories to 

enhance the DoD’s biosurveillance capability. The instruments will include the Applied Biosystems (Foster 

City, CA) 7500 Fast Dx Real-Time PCR Instrument, 

the Luminex (Austin, TX) MAGPIX, and the Illumina 

(San Diego, CA) MiSeq instrument. The 7500 Fast Dx 

is an FDA-cleared molecular diagnostic device for the 

detection of nucleic acids by real-time PCR, whereas 

the MAGPIX is a highly multiplexed combined immunoassay/molecular assay platform for the detection of 

proteins or nucleic acids. Combined, these instruments 

could potentially cover the nucleic acid and protein 

biological space to include identification of viruses, 

bacteria, and toxins. The critical challenge for these 

two instruments will be the availability of assays that 

are capable of extensively surveying the infectious 

disease space. 

In addition, the Illumina MiSeq instrument is a 

nucleic acid sequencing instrument that may potentially be used as an agnostic approach to agent 

identification.130,131 Metagenomic sequencing has 

become a favored approach to identify all biological 

components in clinical and environmental samples, 

and significant investments have been made to stand 

up genomic sequencing centers within the DoD. The 

roll out of MiSeq instruments in overseas laboratories 

is the DoD’s attempt to take this capability beyond 

reference laboratories. Although sequencing has advanced significantly in the past decade, it has proven 

most useful in samples where the amount of organisms 

is not limiting, which is often not the case in clinical 

samples where the concentration of organism is extremely low in relation to the host nucleic acid in the 

sample. Teasing out the sequences that are significant 

for biosurveillance and public health purposes is the 

critical biochemical and bioinformatic challenge for 

metagenomic sequencing approaches.132

Although sequencing provides a wealth of information, sequence data alone does not substitute for the 

need to propagate and maintain the viable organisms 

necessary for medical countermeasure development 

efforts. This capability is critical, especially for unknown or emerging threats, as all vaccine, therapeutic, and diagnostic development will require enough 

purified agent material to perform the necessary 

investigations. Overall, a rapid response capability 

from agent identification to therapeutic delivery to 

the warfighter requires integration across program 

areas with logical transition from one capability area to 

another within DoD. A comprehensive biosurveillance 

plan will include sample acquisition, identification, 

and characterization capability that allows for rapid 

development of medical countermeasures. Transition 

of the deliverables from biosurveillance should bridge 

pathogen discovery with diagnostics, animal model 

development, and vaccine and therapeutic evaluation, 

thereby shortening the timeline between agent identification and fielding of medical countermeasures. 

Ultimately, data from biosurveillance efforts must 

lead to actionable information to respond rapidly with 

medical countermeasures such as vaccines, therapeutics, and diagnostics. 

Ultimately, the information provided by biosurveillance needs to translate into products that can be used 

in an emergency situation to enhance military readiness and public health. The nation’s ability to react to 

a biological event to minimize casualties and impacts, 

or biopreparedness, is critical during an emerging 

outbreak or intentional release of a biological threat 

agent. The emergence of H1N1 and H5N1 strains of 

influenza was a valuable lesson for the US government 

to provide medical countermeasures in a response that 

included the availability of in vitro diagnostic tests. In 

2004, the Project BioShield Act amended the Federal 

Food and Drug Cosmetic Act (21 USC 360bbb-3; sec 

564) to include a process by which non-FDA approved 

products or off-label uses of approved products could 

be rapidly fielded in declared emergency situations. 

Only the Secretary of Defense, Secretary of Homeland 

Security, or Secretary of Health and Human Services 

can determine whether an emergency situation meets 

the criteria established in the act. Once this occurs, the 

US Secretary of Health and Human Services issues a 

declaration allowing EUA submissions to the FDA for 

consideration and potential use. Declared emergencies are not limited to ongoing emergencies, but also 

include situations that may present a heightened risk 

for potential attacks or events. 

Any potential situation that would pose a significant 

risk to the public or to US military forces, or has the 

potential to adversely affect national security could 

be declared an emergency situation. This process was 

activated, refined, and used for in vitro diagnostics 

during the H1N1 pandemic in 2009–2010.133 The typical 

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Laboratory Identification of Threats

process to use an in vitro diagnostic test during an 

emergency involves the declaration of emergency, 

the submission of performance data to the FDA, FDA 

review, and FDA authorization to use the test under 

the EUA. One outcome of the H1N1 EUA process for 

diagnostics was the development of a pre-EUA process to streamline this process. Based on the FDA’s 

H1N1 guidance document,133 the DoD and FDA 

worked together to define a process for prepositioning performance data for in vitro diagnostic tests that 

were not yet FDA cleared but could be invaluable 

during a declared emergency. By allowing pre-EUA 

submissions for diagnostic tests, the FDA can review 

data, request additional data, and make preliminary 

decisions on utility before an emergency is declared, 

greatly reducing the time between the declaration of 

an emergency and the authorization to use the test. 

Pre-EUA approval does not grant permission to use 

or market the product under nonemergency conditions, but greatly enhances biopreparedness should 

a biological threat event occur. The DoD submitted 

73 assays for pre-EUA consideration to the FDA in 

July 2010, and eight assays have been accepted after 

providing additional performance data on the JBAIDS 

and Applied Biosystems 7500 FAST DX real-time PCR 

platforms. The pre-EUA process continues to expand 

the immediate availability of in vitro diagnostics 

during a declared emergency, and it adds previously 

unavailable biopreparedness capability for the DoD 

and the nation.

Success in responding to emerging or genetically engineered biological threats is dependent on identifying, 

characterizing, and reducing the health impacts of the 

threat, which requires a continuum from identification 

of the threat at the point of presentation (clinically or 

environmentally) through rapid medical countermeasure deployment. Doing so quickly requires the 

assimilation of all available biological data, determination of which data are meaningful, and identification 

of actionable information signifying a threat to public 

health. These are the underlying goals of biosurveillance. However, the collection of samples, characterization of the threat agent, development or identification 

of the appropriate countermeasures, and deployment 

of those countermeasures to be used under regulatory 

compliance are necessary to achieve the desired end 

state, thus minimizing the public health and military 

readiness impacts of emerging and engineered threats. 

FUTURE APPROACHES

Early Recognition of the Host Response

Early recognition is critical for the diagnosis and 

treatment of biological threat agents because of their 

disease progression, persistence, and lethality (Table 

26-9). The host responds to microbial invasion immunologically and also responds to pathological factors 

expressed by the foreign organism or toxin. Identifying 

early changes in the host gene response may provide 

an immediate indication of exposure to an agent and 

subsequently lead to early identification of the specific 

agent before the onset of disease. Several biological 

agents and toxins directly affect components important 

for innate immunity, such as macrophage or dendritic 

cell functions or immunomodulator expression. 

Host gene responses to biological threat insults 

can manifest in multiple ways. Studies suggest that 

the anthrax lethal factor may induce apoptosis in peripheral blood mononuclear cells, inhibit production 

of proinflammatory cytokines in peripheral blood 

mononuclear cells, and impair dendritic cells.134,135

Poxviruses may possess several mechanisms to inhibit 

innate immunity.136 Gibb, Norwood, Woollen, and 

Henchal reported that alveolar macrophages infected 

with Ebola virus demonstrated transient increases 

in cytokine and chemokine mRNA levels that were 

markedly reduced after 2 hours postexposure.137

Others have shown that Ebola virus infections are 

characterized by dysregulation of normal host immune responses.138 However, directly detecting these 

effects, especially inhibition of cytokine expression, is 

technically difficult to measure in potentially exposed 

populations.

New approaches that evaluate the regulation 

of host genes in microarrays may allow for early 

disease recognition.139,140 A complicated picture is 

emerging that goes beyond dysregulation of genes 

related to innate immunity. Relman suggested that 

there are genome-wide responses to pathogenic 

agents.141 Mendis identified cDNA fragments that 

were differentially expressed after 16 hours of in vitro 

exposure of human peripheral blood mononuclear 

cells to staphylococcal enterotoxin B.142 By using 

custom cDNA microarrays and real-time analysis, 

these investigators found a unique set of genes associated with staphylococcal enterotoxin B exposure. 

By 16 hours, there was a convergence of some gene 

expression responses: many of those genes code for 

proteins such as proteinases, transcription factors, 

vascular tone regulators, and respiratory distress. 

Additional studies are needed to characterize normal 

baseline parameters from a diverse group of individuals undergoing common physiological responses to 

the environment, as well as responses to the highest 

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TABLE 26-9 BIOLOGICAL WARFARE AGENT DISEASE CHARACTERISTICS Human-to-Human Infective Dose Incubation Duration of Disease Transmission (Aerosol) Period Illness LAnthrax No 8,000–1,000 spores 1–6 d 3–5 d (fatal if untreated) HBrucellosis No 10–100 cells 5–60 d; usually 30–60 d Weeks to months LGlanders Low 5,000–10,000 cells (NHP) 10–14 d 7–10 d (fatal if untreated) MMelioidosis Low 50–80 cells (NHP) 1–21 d; up to years 2–3 d (fatal if untreated) MPlague Moderate 500–15,000 cells 1–7 d; usually 2–3 d 1–6 d (fatal if untreated) HTularemia No 10–50 cells 1–21 d; usually 3–6 d Fatal if untreated MQ fever Rare 1–10 cells 7–41 d 2–14 d or longer if not treated LSmallpox High 10–100 organisms 7–17 d; average 12 d 4 weeks HVEE Low 10–100 organisms 2–6 d Days to weeks LViral hemorrhagic Moderate 1–10 organisms 4–12 d Death between 7–16 d M

fevers

Botulism No 0.003 μg/kg for type A 12 h–5 d Death in 24–72 h; lasts for H

months if not lethal

SEB No 0.0004 μg/kg 3–12 h Hours LRicin No 3–5 μg/kg (mouse LD50) 18–24 h Days HLD: lethal doseNHP: nonhuman primate SEB: staphylococcal enterotoxin BVEE: Venezuelan equine encephalitis

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Laboratory Identification of Threats

priority biological agents and toxins in appropriate 

animal models. Approaches that integrate detection 

of early host responses with the sensitive detection 

of biological agent markers can decrease morbidity 

and mortality by encouraging optimal therapeutic 

intervention.

SUMMARY

Military clinical and field laboratories play a critical role in the early recognition of biological threats, 

serving as unique sentinels in CONUS and OCONUS 

areas for biological threats and emerging infectious diseases. While performing regulatory compliant patient 

diagnostics for biological threats is difficult in a theater 

of operation, the fielding of the JBAIDS real-time PCR 

platform has had some success. The NGDS acquisition 

program will incrementally improve this capability by 

providing a highly multiplexed “sample in/answer 

out” capability for molecular biological threat identification. Although these fielded platforms provide a 

diagnostic capability in theater, they are not definitive 

means of identification and are based on targets that 

are currently well understood. Definitive identification 

requires orthogonal testing to improve the reliability 

of rapid diagnostic technologies and reduce risk. 

The integration of culture as well as nucleic acid 

and immunological biomarkers for the identification of 

biological threat agents is critical to elevate the level of 

confidence in identifying these high consequence infectious diseases. The network of laboratories available 

for confirmatory and definitive testing is strong and 

has improved significantly within the past 5 years. Future technologies will further increase the orthogonal 

capabilities of diagnostic platforms and strive toward 

agent agnostic agent identification. The integration of 

molecular and immunological identification on a single 

platform using common analytical chemistries may 

be realized within the next 5 to 10 years, and whole 

genome metagenomic sequencing holds the promise 

of identifying all infectious agents in a given sample. 

These approaches will be critical to accommodate 

the identification of emerging as well as genetically 

engineered agents. 

Although indications show that these future approaches are making progress, regulatory challenges 

will occur for diagnostic use of highly multiplexed 

and sequencing technologies. Fortunately, the FDA 

has been forward thinking and is currently engaged 

in identifying the key standards required for both 

highly multiplexed and whole sample sequencing 

based approaches for clearance of diagnostics. Biosurveillance initiatives may provide a means to evaluate 

and improve future platforms that could ultimately 

transition to diagnostic devices if costs permit. In the 

meantime, medical diagnostics for biological threat 

agents will rely on proven technologies that incorporate incremental improvements to simplify and 

improve the reliability and robustness of diagnostic 

devices for use throughout the military clinical and 

field laboratories. 

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Chapter 27

MEDICAL COUNTERMEASURES

PHILLIP R. PITTMAN, MD, MPH*; ELIZABETH S. BROWN, PhD†; and MATTHEW S. CHAMBERS, MD, MPH‡

INTRODUCTION 

BACTERIAL AND RICKETTSIAL DISEASES 

Anthrax

Tularemia 

Plague

Glanders and Melioidosis

Brucellosis

Q Fever

VIRAL DISEASES 

Encephalitic New World Alphaviruses 

Smallpox

Viral Hemorrhagic Fevers

TOXINS

Botulinum Neurotoxin

Staphylococcal Enterotoxin B

Ricin

SUMMARY

*Colonel (Retired), US Army; Chief, Department of Clinical Research, Division of Medicine, US Army Medical Research Institute of Infectious Diseases, 

1425 Porter Street, Fort Detrick, Maryland 21702

†

Medical–Technical Writer and Editor, Department of Clinical Research, Division of Medicine, US Army Medical Research Institute of Infectious Diseases, 

1425 Porter Street, Fort Detrick, Maryland 21702

‡

Lieutenant Colonel, Medical Corps, US Army; Chief, Preventive Medicine, Fort Wainwright, 4077 Neely Road, Fort Wainwright, Alaska 99703; formerly, 

Chief, Field Studies, Division of Medicine, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

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INTRODUCTION

or death (Table 27-2). Any effective plan for countering bioterrorism should address the logistics of 

maintaining adequate supplies of drugs and vaccines 

as well as personnel coordinating and dispensing 

needed supplies to the affected site.

FDA-approved vaccines against anthrax and 

smallpox are available; however, for many potential 

bioterrorism agents, only investigational vaccines 

that were developed and manufactured more than 

30 years ago are available. These vaccines have 

demonstrated efficacy in animal models and safety 

in at-risk laboratory workers; however, they did 

not qualify for FDA approval because studies to 

demonstrate their efficacy in humans were deemed 

unsafe and unethical. Although they can be obtained 

under investigational new drug (IND) protocols at 

limited sites in the United States, these vaccines are 

in extremely limited supply and some are declining 

in immunogenicity with age. 

Under the FDA Animal Rule instituted in 2002, approval of vaccines, antimicrobials, and other drugs can 

now be based on demonstration of efficacy in animal 

models alone if efficacy studies in humans would be 

unsafe or unethical. This rule provides an opportunity 

to develop many new and improved vaccines and other 

medications, with the ultimate goal of FDA licensure. 

However, drug development generally is a long process. In vaccine development, for example, generally 

3 to 5 years is required to identify a potential vaccine 

candidate and conduct animal studies to test for vaccine immunogenicity and efficacy, followed by 5 years 

of clinical trials for FDA approval and licensure. FDA 

vaccine approval then takes from 7 to 10 years, and 

under the FDA Animal Rule, additional time must be 

devoted to animal studies to identify correlates of protection. Thus, vaccine replacements are not expected 

to be available in the near future.

Countermeasures against bioterrorism, intended 

to minimize morbidity and mortality and to prevent or limit the number of secondary infections 

or intoxications, include (a) early identification of 

the bioterrorism event and persons exposed, (b) 

appropriate decontamination, (c) infection control, 

and (d) medical countermeasures. The first three 

countermeasures are nonmedical and are discussed 

in other chapters. This chapter covers medical countermeasures, which include interventions such as 

active immunoprophylaxis (ie, vaccines), passive 

immunoprophylaxis (immunoglobulins and antitoxins), and chemoprophylaxis (antimicrobial medications). Medical countermeasures may be initiated 

before an exposure (if individuals can be identified 

as being at high risk for exposure), after a confirmed 

exposure event, or after the onset of symptoms in 

infected individuals.

Because medical countermeasures may be associated with adverse events, the recommendation 

for their use must be weighed against the risk of 

exposure and disease. Vaccines, including both 

investigational vaccines and those approved by 

the US Food and Drug Administration (FDA), are 

available for some bioterrorism agents (Table 27-1). 

In the event of a bioterrorism incident, preexposure 

vaccination—if safe and available—may modify or 

eliminate the need for postexposure chemoprophylaxis. However, preexposure vaccination may not 

be possible or practical in the absence of a known 

or expected release of a specific bioterrorism agent, 

particularly with vaccinations that require multiple 

primary (or priming) doses to achieve immunity 

or repeated booster doses to maintain it. In these 

cases, chemoprophylaxis—and, in some cases, immunoprophylaxis—after identifying an exposure 

or infection may be effective in preventing disease 

BACTERIAL AND RICKETTSIAL DISEASES

Anthrax

Anthrax is caused by Bacillus anthracis, a sporeforming, gram-positive bacillus that can be found in 

many soil environments worldwide. It occurs in a 

vegetative state and in a spore state; the spore state, 

which can remain viable for decades, is the infectious form.1–3 Ruminants acquire spores by ingesting contaminated soil while grazing. Humans can 

become infected through skin contact, ingestion, or 

inhalation of B anthracis spores from infected animals 

or animal products.3

 Anthrax is not transmissible 

from person to person. Cutaneous anthrax is the 

most common naturally occurring form of anthrax, 

and gastrointestinal anthrax is the least common 

form. Inhalational anthrax, which occurs as a result 

of exposure to aerosolized spores, is considered the 

form of disease most likely to result from an act of 

bioterrorism. Meningitis can occur, as secondary 

seeding from bacteremia, with any form of anthrax.4

Because of its virulence, ease of preparation, the 

potential to aerosolize spores, and the stability and 

prolonged survival of the spore stage, B anthracis is 

an ideal agent for bioterrorism.4,5

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TABLE 27-1

VACCINES, VACCINE DOSAGE SCHEDULES, AND CORRELATES OF POSTVACCINATION 

PROTECTION 

Vaccine 

Disease (Dose and Route) Type of Vaccine Primary Series Booster Doses Immunogenicity Criteria

BACTERIA

Anthrax AVA (BioThrax) Sterile, acellular filtrate Months 0, 1, Months 12 & 18, 3 weeks after 3rd dose 

(0.5 mL IM) & 6 annually

Tularemia NDBR 101* Live attenuated Day 0 None Take reaction† by day 7

(15 punctures, 1 after vaccination; day 28

drop [0.06 mL] microagglutination titer

PC)	 ≥4-fold rise from

prevaccination baseline

Q fever NDBR 105*,‡,§ Inactivated Day 0 None 3–5 weeks after vaccination

(0.5 mL SC)

VIRUSES

VEE TC-83 NDBR 102* Live attenuated Day 0 None; boost with PRNT80 titer ≥1:20

(0.5 mL SC) C-84 per titer

C-84 TSI-GSD 205* Inactivated None¥ Initial responders PRNT80 titer ≥1:20

(0.5 mL SC) to TC-83 and 

past recipients 

of C-84: single

boost of C-84

Initial nonresponders to TC-83:

boost with C-84

WEE TSI-GSD 210*,§	 Inactivated	 Days 0, 7, & 28	 Mandatory boost: 	 PRNT80 titer ≥1:40

(0.5 mL SC) month 6; then as 

needed per titer

EEE TSI-GSD 104*	 Inactivated	 Days 0 & 28	 Mandatory boost: 	 PRNT80 titer ≥1:40

(0.5 mL SC) month 6; before 

month 6 and 

after: as needed

per titer (0.1 mL 

 ID)

Smallpox ACAM2000 Cell culture–based live Day 0 Every 1, 3, or 10 Take reaction** after primary

(15 punctures vaccinia virus years¶ vaccination

PC)

Yellow YF-Vax Live attenuated Day 0 Every 10 years 1 month after vaccination

fever (0.5 mL SC)

RVF TSI-GSD 200*,†† Inactivated Days 0, 7, & 28 Initial respon- PRNT80 titer ≥1:40

(1 mL SC) ders‡‡: mandatory

boost at month 

6; then as needed 

per titer

Initial nonrespon-

 ders‡‡: boost

within 90 days 

of low titer

(Table 27-1 continues)

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Medical Aspects of Biological Warfare 

Vaccination

History of the Anthrax Vaccine. In 1947, a factor 

isolated from the edema fluid of cutaneous B anthracis

lesions was found to successfully vaccinate animals.6

This factor, identified as the protective antigen (PA), 

was subsequently recovered by incubating B anthracis

in special culture medium.7,8 This work led to the development in 1954 of the first anthrax vaccine, which 

was derived from an alum-precipitated cell-free filtrate 

of an aerobic culture of B anthracis.

9

This early version of the anthrax vaccine was found 

to protect small laboratory animals and nonhuman 

primates (NHPs) from inhalational anthrax.9 The vaccine also demonstrated protection against cutaneous 

anthrax infections in employees working in textile 

mills processing raw imported goat hair. In particular, 

only three cases of cutaneous anthrax occurred in 379 

vaccinated employees, whereas 18 cases of cutaneous anthrax and all five cases of inhalational anthrax 

occurred in the 754 unvaccinated employees. Based 

on these results, the vaccine efficacy for anthrax was 

estimated to be 92.5%. The vaccine failures were found 

in one person who had received only two doses of vaccine, a second person who had received the initial three 

doses of vaccine but failed to receive follow-up doses at 

6 and 12 months (and was infected at 13 months), and 

a third person who was within a week of the fourth 

(month 6) vaccine dose, when titers are known to be 

lower.10 Vaccine breakthroughs were uncommon; the 

few documented cases of cutaneous anthrax occurred 

in individuals who had not completed the primary 

series or who were within days of a scheduled primary 

or booster dose.10,11

Anthrax Vaccine Adsorbed (BioThrax). The current 

FDA-approved anthrax vaccine adsorbed (AVA; see 

Table 27-1) was derived through improvements of the 

early alum-precipitated anthrax vaccine, specifically:

• using a B anthracis strain that produced a 

higher fraction of PA; 

• growing the culture under microaerophilic 

instead of aerobic conditions; and 

• substituting an aluminum hydroxide adjuvant in place of the aluminum potassium salt 

adjuvant.12,13

Notes: Vaccines listed are those available in the United States and used (under current or recent protocols) in the Special Immunizations

Program at the US Army Medical Research Institute of Infectious Diseases. Vaccines are available elsewhere for Argentine hemorrhagic 

fever (Candid #1, available in Argentina, which may have cross-protection for Bolivian hemorrhagic fever); Omsk hemorrhagic fever (for 

which cross-protection is provided from the tickborne encephalitis vaccine FSME-IMMUN); Kyasanur Forest disease (for which a vaccine 

is available in India); Crimean–Congo hemorrhagic fever (for which a vaccine is available in Bulgaria); and hemorrhagic fever with renal 

syndrome (Hantavax, which is available in South Korea). 

*

Investigational product. 

†

The take reaction is an erythematous papule, vesicle, and/or eschar, with or without induration, at the vaccination site. Compared with 

smallpox vaccination (using ACAM2000), tularemia vaccination (using NDBR 101) results in a smaller take reaction with less induration. 

‡

Prevaccination skin test is required before administration of NDBR 105.

§

Q fever and WEE vaccines are not currently administered in the SIP.

¥

Used only as booster (if needed per titer) after vaccination with TC-83.

¶

Booster doses of the smallpox vaccine are recommended every 3–10 years, depending on risk; for example, laboratory researchers working 

with variola virus (only at CDC) may receive yearly boosters.

**The take reaction after smallpox vaccination (using ACAM2000) is a clear vesicle or pustule, approximately 1 cm in diameter.

††A live attenuated RVF MP12 vaccine has recently undergone phase 1 and 2 clinical trials.

‡‡Titer is assessed 28 days after 3rd primary dose of RVF vaccine. Initial responders (PRNT80 titer ≥1:40 after 3rd dose) receive a boost at 6

months with titer assessed 28 days later. For responders to 6-month booster, titer is assessed annually with subsequent booster doses as needed. 

For initial nonresponders (PRNT80 titer <1:40), boosteris given within 90 days and titeris assessed 28 days later. For a nonresponse, boost/titer

may be repeated up to 4 times before declaring individual a nonresponder. (The month 6 booster does not apply to initial nonresponders.)

AVA: anthrax vaccine adsorbed; EEE: eastern equine encephalitis; ID: intradermal; IM: intramuscular; PC: percutaneous (scarification);

PRNT80: 80% plaque reduction neutralization titer; RVF: Rift Valley fever; SC: subcutaneous; TSI-GSD: The Salk Institute-Government Services Division; VEE: Venezuelan equine encephalitis; WEE: western equine encephalitis; YF-Vax: yellow fever vaccine

Data sources: (1) Dembek Z, ed. USAMRIID’s Medical Management of Biological Casualties Handbook. 7th ed. Fort Detrick, MD: US Army

Medical Research Institute of Infectious Diseases; 2011. (2) Emergent BioSolutions. BioThrax (Anthrax Vaccine Adsorbed) [package insert]. 

Lansing, MI: Emergent BioDefense Operations Lansing LLC; 2015. (3) Rusnak J, ed. Occupational Health Manual for Laboratory Exposures to 

Select (BSL-3 & BSL-4) and Other Biological Agents. 3rd ed. Fort Detrick, MD: US Army Medical Research Institute of Infectious Diseases; 2011.

(4) Rusnak JM, Kortepeter MG, Aldis J, Boudreau E. Experience in the medical management of potential laboratory exposures to agents of

bioterrorism on the basis of risk assessment at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). J Occ 

Env Med. 2004;46:801–811. (5) Sanofi Pasteur Biologics. ACAM2000. Cambridge, MA: Sanofi Pasteur Biologics; 2009. (6) Rotz LD, Dotson

DA, Damon IK, BecherJA;Advisory Committee on Immunization Practices. Vaccinia (smallpox) vaccine:recommendations of theAdvisory

Committee on Immunization Practices (ACIP), 2001. MMWR Recomm Rep. 2001;50(RR-10):1–25. (7) Monath TP, Cetron MS. Prevention of

yellow fever in persons traveling to the tropics. Clin Infect Dis. 2002;34:1369–1378. (8) Pittman PR, Liu CT, Cannon TL, et al. Immunogenicity

of an inactivated Rift Valley fever vaccine in humans: a 12-year experience. Vaccine. 1999;18:181–189.

Table 27-1 continued

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Medical Countermeasures

TABLE 27-2

TREATMENT AND POSTEXPOSURE PROPHYLAXIS FOR BACTERIAL DISEASES

Disease Preferred/Recommended Antimicrobials* Vaccine Passive Immunotherapy or Antitoxin

Anthrax 

(inhalational)

Treatment

Meningitis not ruled out:

Initial IV treatment for ≥14 days (or until

clinically stable) of ≥3 antibiotics: ciprofloxacin (400 mg IV every 8 h) AND 

meropenem (2 g IV every 8 h) AND 

linezolid (600 mg IV every 12 h) 

Followed by oral treatment as described 

for PEP, for a total of ≥60 days of antibiotic treatment

Meningitis ruled out:

Initial IV treatment for ≥14 days (or until

clinically stable) of ≥2 antibiotics: ciprofloxacin (400 mg IV every 8 h) AND 

clindamycin (900 mg IV every 8 h) OR 

linezolid (600 mg IV every 12 h)

Followed by oral treatment as described 

for PEP, for a total of ≥60 days of antibiotic treatment

Treatment

Not recommended

Treatment

Raxibacumab (40 mg/kg IV) infused 

over 2 h, 15 min, within 1 h after 

premedication with diphenhydramine, in combination with antibiotics

OR 

AIGIV (7 vials [420 units] IV), infused 

at 0.5 mL/min (first 30 min), 1 mL/

min incremental infusion rate if tolerated (every 30 min), max infusion 

rate of 2 mL/min 

PEP†

60 days of ciprofloxacin (500 mg PO every 

12 h) OR doxycycline (100 mg PO every 

12 h)

PEP

AVA‡ (up to 3 doses 

SC)

PEP§

Raxibacumab (40 mg/kg IV), infused 

over 2 h, 15 min, within 1 h after 

premedication with 

diphenhydramine

Tularemia Treatment

10 days of streptomycin (1 g IM every 12 h) 

OR gentamicin (5 mg/kg IM or IV daily)

Treatment

Not recommended

No passive immunotherapy available

PEP†

14 days of doxycycline (100 mg PO every 12 

h) OR ciprofloxacin (500 mg PO every 12 h)

PEP

Not recommended

Plague Treatment

≥10 days of streptomycin (1 g IM every 12

h) OR gentamicin (5 mg/kg IM or IV daily)

No vaccine availableNo passive immunotherapy available

PEP†

7 days of doxycycline (100 mg PO every 12 

h) OR ciprofloxacin (500 mg PO every 12 h)

Glanders or 

melioidosis

Treatment

Intensive therapy, no complications¥

:

10–14 days of ceftazidime (50 mg/kg [up 

to 2 g] IV every 8 h)

Intensive therapy, with complications¥

:

10–14 days of meropenem (25 mg/kg [up 

to 1 g] IV every 8 h) 

Eradication therapy¶

:

≥12 weeks of TMP-SMZ (PO) OR amoxicillin–clavulanic acid (PO)

No vaccine availableNo passive immunotherapy available

PEP**

21 days of TMP-SMZ (PO) OR amoxicillin–clavulanic acid (PO)

(Table 27-2 continues)

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Medical Aspects of Biological Warfare 

Brucellosis Treatment

Uncomplicated:

6 weeks of doxycycline (100 mg PO every 

12 h) AND 2–3 weeks of streptomycin 

(15 mg/kg IM daily) 

Complicated:

≥12 weeks of triple-antibiotic therapy (see

text and sources cited therein)

No vaccine availableNo passive immunotherapy available

PEP††

21 days of doxycycline (100 mg PO every 

12 h) AND rifampin (450–600 mg PO 

daily)

Q Fever Treatment

Acute:

14 days of doxycycline (100 mg PO every 

12 h)

Chronic:

Prolonged treatment with doxycycline 

(100 mg PO every 12 h) AND hydroxychloroquine (200 mg PO every 8 h) (see 

text and sources cited therein)

Treatment

Not recommended

No passive immunotherapy available

PEP

Not recommended; monitor for ≥21 days

(see text and sources cited therein)

PEP

Not recommended

*

Antimicrobials listed are the first-line therapies or those most often recommended. For alternative antimicrobials, see text and sources cited 

therein. Dosages are for adults (men and nonpregnant women); for children and for pregnant women, see text and references cited therein.

†

The antibiotic regimen described for PEP may also be used for treatment in a mass casualty situation where parenteral antibiotics are not 

available. In the case of plague, treatment in a mass casualty situation would be extended to 10 days.

‡

Individuals who were not vaccinated before exposure should receive three doses of AVA as PEP; those who previously received one or two 

doses of AVA should receive two doses of AVA as PEP, and those who received three or more doses of AVA preexposure should receive 

one dose of AVA as PEP.

§

Raxibacumab may be used for PEP in high-risk spore exposure cases.

¥

Initial IV antibiotic therapy for glanders or melioidosis should be continued for a minimum of 10 days and until the patient’s condition 

improves; the intensive IV therapy may need to be extended to ≥4 weeks. For severe neurologic, cutaneous, bone, joint, or prostate infections,

TMP-SMZ could be added to the regimen, administered as described for PEP, or see Lipsitz et al (see data source reference 9).

¶

In the eradication phase of therapy, the dosage of TMP-SMZ depends on weight: for adult >60 kg, give 160 mg/800 mg tablets (2 tablets

every 12 h); for adult 40–60 kg, give 80 mg/400 mg tablets (3 tablets every 12 hours); for adult <40 kg, give 160 mg/800 mg tablets (1 tablet 

every 12 hours) or 80 mg/400 mg tablets (2 tablets every 12 hours). The dosage of amoxicillin–clavulanic acid (which can be given to those 

who are intolerant of TMP-SMZ or those with strains that are resistant to TMP-SMZ) also depends on weight: for adult ≥60 kg, give 500

mg/125 mg tablets (3 tablets every 8 hours); for adult <60 kg, give 500 mg/125 mg tablets (2 tablets every 8 hours).

**This recommendation is based on animal studies and PEP used for possibly exposed laboratory workers; efficacy in preventing disease 

among exposed humans has not been evaluated. The dosage of TMP-SMZ and amoxicillin–clavulanic acid depends on weight, as described 

in the previous note.

††For high-risk exposures.

AIGIV: Anthrax Immune Globulin Intravenous; AVA: anthrax vaccine adsorbed; IM: intramuscular; IV: intravenous; PEP: postexposure

prophylaxis; PO: by mouth; SC: subcutaneous; TMP-SMZ: trimethoprim and sulfamethoxazole, or co-trimoxazole

Data sources: (1) Hendricks KA, Wright ME, Shadomy SV, et al. Centers for Disease Control and Prevention expert panel meetings on

prevention and treatment of anthrax in adults. Emerg Infect Dis. 2014;20(2). (2) Wright JG, Quinn CP, Shadomy S, Messonnier N; Centers for

Disease Control and Prevention. Use of anthrax vaccine in the United States:recommendations of theAdvisory Committee on Immunization

Practices (ACIP), 2009. MMWR Recomm Rep. 2010;59(RR-6):1–30. (3) Dembek Z, ed. USAMRIID’s Medical Management of Biological Casualties 

Handbook. 7th ed. Fort Detrick, MD: US Army Medical Research Institute of Infectious Diseases; 2011. (4) Emergent BioSolutions. BioThrax

(Anthrax Vaccine Adsorbed) [package insert]. Lansing, MI: Emergent BioDefense Operations Lansing LLC; 2015. (5) GlaxoSmithKline.

Raxibacumab. Research Triangle Park, NC: GlaxoSmithKline; 2014. (6) Rusnak J, ed. Occupational Health Manual for Laboratory Exposures to 

Select (BSL-3 & BSL-4) and Other Biological Agents. 3rd ed. Fort Detrick, MD: US Army Medical Research Institute of Infectious Diseases;

2011. (7) Dennis DT, Inglesby TV, Henderson DA, et al. Tularemia as a biological weapon: medical and public health management. JAMA. 

2001;285:2763–2773. (8) Inglesby TV, Dennis DT, Henderson DA, et al. Plague as a biological weapon: medical and public health management. JAMA. 2000;283:2281–2290. (9) Lipsitz R, Garges S, Aurigemma R, et al. Workshop on treatment of and postexposure prophylaxis for

Burkholderia pseudomallei and B mallei infection, 2010. Emerg Infect Dis. 2012;18:e2. (10) Ariza J, Bosilkovski M, Cascio A, et al. Perspectives for

Table 27-2 continued

(Table 27-2 continues)

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Originally produced by the Michigan Department 

of Public Health, AVA is manufactured by Emergent 

BioDefense Operations Lansing LLC (Lansing, MI) and 

marketed under the name BioThrax. It is licensed for 

preexposure use (and postexposure use, see below) in 

adults aged 18 to 65.14,15

AVA is derived from a sterile cell-free filtrate (with 

no dead or live bacteria) from cultures of an avirulent, 

nonencapsulated strain of B anthracis (toxinogenic, 

nonencapsulated v770-np1-R) that produces predominantly PA in the relative absence of other toxin 

components, such as lethal factor or edema factor.12,16

The filtrate used to produce AVA is adsorbed to 

aluminum hydroxide (Amphogel [Wyeth Laboratories, Madison, NJ]) as an adjuvant and contains PA,

formaldehyde, and benzethonium chloride, with 

trace lethal factor and edema factor components.16

Currently, AVA is given as an intramuscular injection (in the lower two-thirds of the deltoid muscle) 

of 0.5 mL at months 0, 1, and 6 (the primary series) 

with boosters at months 12 and 18 followed by yearly 

boosters as long as the individual remains at risk for 

anthrax infection.15

Both the earlier alum-precipitated vaccine and AVA 

have demonstrated efficacy against aerosol challenge 

in animal models.8,9,13,17–23 In their summary of several 

NHP studies, Friedlander et al24 noted that, of 55 monkeys given two doses of AVA, 52 (95%) survived lethal 

aerosol challenge without antibiotics. More recently, 

the Centers for Disease Control and Prevention (CDC) 

showed that three doses of AVA, administered intramuscularly at months 0, 1, and 6, protected rhesus macaques against inhalational anthrax for up to 4 years.23

Evidence suggests that both humoral and cellular 

immune responses against PA are critical to protection against disease after exposure.12,17,18 Vaccinating 

rhesus macaques with one dose of AVA elicited antiPA immunoglobulin (Ig) M titers peaking at 2 weeks 

after vaccination, IgG titers peaking at 4 to 5 weeks, 

and PA-specific lymphocyte proliferation present at 

5 weeks.19 In the CDC study, survival of macaques 

was correlated with cellular and humoral immune 

responses measured during and after administration 

of the first three doses of the primary series.23 After 

three doses of AVA, 83% to 100% of human vaccinees 

seroconvert.25–27 A missed dose of AVA does not necessitate restarting the primary series as recent evidence 

has demonstrated that the immune response among 

vaccinees whose month 6 dose is delayed by as much 

as 7 years is noninferior to that of individuals receiving this dose on schedule.28 However, the correlation 

between protection against anthrax infection and a 

specific antibody titer in humans is not yet clear.17

Vaccine Adverse Events. Adverse reactions to AVA 

are rarely severe when they occur. Adverse reactions 

in 6,985 persons who received a total of 16,435 doses 

of AVA, administered subcutaneously according to 

the original dosing schedule (at weeks 0, 2, and 4 and 

months 6, 12, and 18 followed by annual boosters), 

were primarily local reactions (edema or induration). 

These reactions were severe (>12 cm) in fewerthan 1%

of vaccinations, moderate (3–12 cm) in 3% of vaccinations, and mild (<3 cm) in 20% of vaccinations. Systemic 

reactions were uncommon, occurring in fewer than 

0.06% of vaccinees, and included fever, chills, body 

aches, or nausea.29 After the distribution of around 

2 million doses of vaccine, according to the original 

dosing schedule and route, to more than 500,000 

military personnel, data from the Vaccine Adverse 

Event Reporting System (VAERS) from 1998 to 2001 

showed approximately 1,841 reports describing 3,991 

adverse events following AVA vaccination. The most 

frequently reported events were injection site inflammation (752), “flu-like symptoms” (254), systemic 

rash (251), malaise/fatigue (236), arthralgia (229), and 

headache (196). Only 96 events (2%) were serious; of 

those, only 19 were deemed possibly, probably, or 

certainly related to the receipt of AVA, including anaphylaxis reported in two cases. Three additional cases 

of anaphylactic-like reactions were reported, but were 

not deemed serious.30

With intramuscular injections administered according to the current dosing schedule (which extends the 

interval between the first and second primary doses, 

such that doses are given at months 0, 1, 6, 12, and 18 

followed by annual boosters), the incidence of injection site (but not systemic) reactions has been reduced 

compared with the original AVA dosing schedule and 

route, with immune responses that are, by month 7, 

noninferior to those elicited using the original route 

and schedule.27,31,32

Women are more likely than men to experience 

adverse reactions, particularly certain local reactions, 

after AVA administration. In an anthrax vaccine study 

the treatment of brucellosis in the 21st century: the Ioannina recommendations. PLoS Med. 2007;4:e317. (11) Centers for Disease Control and

Prevention. Laboratory-acquired brucellosis—Indiana and Minnesota, 2006. MMWR Morb Mortal Wkly Rep. 2008;57:39–42. (12) Anderson

A, Bijlmer H, Fournier P-E, et al. Diagnosis and management of Q fever–United States, 2013: recommendations from CDC and the Q fever

working group. MMWR Recomm Rep. 2013;62(RR-03):1–30.

Table 27-2 continued

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conducted in laboratory workers and maintenance 

personnel at the US Army Medical Research Institute 

of Infectious Diseases (USAMRIID) over 25 years, 

female vaccinees were more likely than male vaccinees to have injection site reactions, including edema 

and lymphadenopathy, after subcutaneous injections 

of AVA.33 A recent phase 4 clinical trial comparing the 

subcutaneous and intramuscular routes of administration found that female vaccinees are more likely than 

male vaccinees to experience injection site reactions. 

Although this was true regardless of the route of administration, adverse reactions in vaccinees of both genders 

were reduced with intramuscular administration. For 

example, erythema occurred in 34% (intramuscular 

injection of AVA) vs 76% (subcutaneous injection of 

AVA) of female vaccinees and in 24% vs 48% of male 

vaccinees; induration occurred in 14% (intramuscular) 

vs 43% (subcutaneous) of female vaccinees and 10% 

vs 24% of male vaccinees; and edema occurred in 

19% vs 36% of female vaccinees and in 14% vs 27% of 

male vaccinees.27 Other factors also appear to predict 

reactogenicity; in particular, obese women are more 

likely than those of normal weight to experience local 

reactions, at least with subcutaneous administration.34,35

Race may also be associated with reactogenicity.35

A 2002 report by the Institute of Medicine’s Committee to Assess the Safety and Efficacy of the Anthrax 

Vaccine found that immediate, short-term adverse 

effects occur after AVA administration at rates similar 

to those associated with other licensed vaccines. Rare 

but serious problems have been reported, but this is 

true of other licensed vaccines as well. No evidence 

suggests that AVA causes long-term health problems; 

however, as with all vaccines, data regarding potential 

long-term effects are limited.35a

Protocols for managing adverse events associated 

with AVA administration have not yet been evaluated 

in randomized trials. However, individuals with local 

adverse events may be managed with ibuprofen or 

acetaminophen for pain, second-generation antihistamines if localized itching is a dominant feature, and ice 

packs for severe swelling extending below the elbow.36

In persons who have experienced an anaphylactic 

reaction to the vaccine or any of the vaccine components, subsequent anthrax vaccine doses are contraindicated. AVA is also contraindicated in persons with a 

history of anthrax infection because of previous observations of an increase in severe adverse events.37 The 

vaccine may be given in pregnancy only if the benefit 

outweighs the risk. 

Other Anthrax Vaccines. Another PA-based anthrax 

vaccine, anthrax vaccine precipitated (AVP), is made 

by alum precipitation of a cell-free culture filtrate of a 

derivative of the attenuated B anthracis Sterne strain. 

This vaccine, which is currently licensed in the United 

Kingdom, is administered as a primary series of four 

vaccinations at weeks 0, 3, 6, and 32 followed by annual boosters.21,38,39

A live attenuated anthrax vaccine (LAAV), which 

is produced in Russia, is licensed for use in humans 

in Georgia and Azerbaijan; it is unclear whether the 

vaccine is licensed elsewhere, such as other former 

Soviet Union republics or China. LAAV is reported 

to be protective in mass field trials, in which anthrax 

occurred less commonly in vaccinated persons (2.1 

cases per 100,000 persons), a risk reduction of cutaneous anthrax by a factor of 5.4 in the 18 months after 

vaccination.40–42

Vaccine Research. Although AVA and AVP are 

safe and effective, the lengthy dosing regimens for 

these vaccines are onerous, even with the recent dose 

reduction for AVA,43 and do not lend themselves 

to use for rapid prophylactic protection of military 

personnel deploying to high-risk regions. Ongoing 

research to improve the current vaccines includes efforts to enhance their efficacy by combining them with 

alternative adjuvants44 and by extending the intervals 

between some doses.28,39

The ability to prepare purified components of anthrax toxin by recombinant technology has presented 

the possibility of new anthrax vaccines. For example, a 

phase 1 clinical trial has found that an anthrax vaccine 

using recombinant Escherichia coli–derived B anthracis

PA was safe and well tolerated and elicited a robust 

humoral and cellular response after two doses.45

Other new PA-based vaccine candidates combine 

PA with other components of B anthracis, such as 

formaldehyde-inactivated spores, or use alternative 

delivery systems, such as intranasal or transdermal 

routes. DNA vaccines, in which immunogen-encoding 

genetic material is introduced into a host cell, may 

provide longer-lasting immunity. Such vaccines have 

only been explored in animal models.43,46

Passive Immunotherapy

The passive administration of polyclonal or monoclonal antibodies directed against PA or other B anthracis components is receiving attention as potential 

postexposure prophylaxis (PEP) or treatment. The 

recombinant, fully humanized monoclonal antibody 

raxibacumab and the polyclonal antiserum anthrax immune globulin intravenous (human) (AIGIV; marketed 

as Anthrasil by Emergent BioSolutions, Lansing, MI), 

which is derived from the plasma of AVA-vaccinated 

individuals, both have shown promise in animal studies of efficacy and appear to be safe and well tolerated 

in humans.3

 This approach to neutralizing anthrax 

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toxins may be especially effective when used in combination with antibiotic treatment or vaccination.46–48

Recently, raxibacumab obtained FDA approval for 

the treatment—combined with antibiotics—of adults 

and children with inhalational anthrax. Raxibacumab 

may also be used as PEP for possible aerosol exposure 

to B anthracis when other options are not available or 

appropriate.49,50 In March 2015, AIGIV received FDA 

approval for the treatment of inhalational anthrax, 

in combination with appropriate antibacterial drugs. 

MDX-1303 (marketed as Valortim by PharmAthene, 

Annapolis, MD), is another fully human monoclonal 

antibody being developed for therapeutic and PEP 

uses.

Antibiotic Agents

Antibiotics are effective against only the vegetative 

form of B anthracis, not the spore form. In the NHP 

model of inhalational anthrax, spores have survived in 

lung tissue for months (with 15%–20% spore survival 

at 42 days, 2% at 50 days, <1% at 75 days, and trace 

spores present at 100 days) in a dormant state.37,51,52 The 

1979 outbreak of inhalational anthrax in humans after 

an accidental release of spores from a Soviet biological 

weapons production facility (the Sverdlovsk outbreak) 

further supports the notion that lethal spores can persist in lung tissue after the initial exposure because 

cases of human anthrax developed as late as 43 days 

after the release.53 For this reason, a 60-day course of 

antibiotics is recommended both for the treatment 

of inhalational anthrax and as prophylaxis after inhalational exposure (but before symptom onset) in 

vaccinated and unvaccinated individuals. Prolonged 

spore survival has not been observed for other routes 

of exposure. 

Ciprofloxacin, doxycycline, levofloxacin, and 

penicillin G procaine have been FDA approved for 

treatment of inhalational anthrax and for PEP.16,37,51,54–56

Ciprofloxacin, doxycycline, and penicillin have 

reduced the incidence or progression of disease in 

NHPs after aerosol exposure to B anthracis.

37,51,55,57

In macaques exposed to 240,000 to 560,000 anthrax 

spores (8 median lethal doses), postexposure antibiotic 

prophylaxis with 30 days of penicillin, doxycyline, or 

ciprofloxacin resulted in survival of 7 of 10, 9 of 10, 

and 8 of 9 monkeys, respectively. All animals survived 

while on prophylaxis, but three monkeys treated with 

penicillin died between days 39 and 50 postexposure, 

one monkey treated with doxycycline died on day 58 

postexposure, and one monkey treated with ciprofloxacin died on day 36 postexposure.51 These deaths 

were attributed to the germination of spores that had 

persisted in lung tissue after inhalational exposure. 

Among human patients with inhalational anthrax 

between 1900 and 2005, Holty et al58 found that mortality was significantly lower for those who received 

(a) multidrug antibiotic regimens, (b) treatment (with 

antibiotics or anthrax antiserum) during the prodromal phase of the illness, or (c) pleural fluid drainage. 

Compared with historical cases, patients who were 

treated for inhalational anthrax during the fall 2001 

bioterrorism incident at the Brentwood Post Office 

and Senate office building in the United States were 

more likely to have had therapy initiated during the 

prodromal phase of the disease, to have received several antibiotics, or to have had pleural fluid drainage. 

These patients were also less likely to die (45% vs 92%). 

Adverse events associated with the prolonged, 

60-day, antibiotic prophylaxis regimen have had a 

significant impact on compliance. Overall compliance 

was reported to be around 44% among the 10,000 

persons at six eastern US sites in the 2001 incident for 

whom the regimen (using ciprofloxacin, doxycycline, 

or amoxicillin) was recommended.59 At least one adverse event was reported by 45% and 77% (at day 10 

and day 30, respectively) of the individuals receiving 

PEP most recently with ciprofloxacin. Among those 

receiving PEP most recently with doxycycline, 49% 

(day 10) and 71% (day 30) reported experiencing at 

least one adverse event. Adverse events at day 30 for 

ciprofloxacin and doxycycline were primarily gastrointestinal symptoms, including nausea, vomiting, 

diarrhea, abdominal pain, or heartburn (42% and 49% 

for ciprofloxacin and doxycycline, respectively); fainting, dizziness, light-headedness, or seizures (23% and 

18%); rash, hives, or itchy skin (14% and 14%); and joint 

problems (25% and 16%). Among the 2,631 individuals 

who took at least one dose of an antibiotic as PEP but 

stopped taking the drug before completing the full 

60-day course, reasons cited for early discontinuation 

included adverse events (43%), fear of long-term side 

effects from PEP (7%), and a perception of having a 

low risk for anthrax (25%).59 Other adverse events that 

can occur with quinolones but were not reported in 

this survey include headache, tremors, restlessness, 

confusion, and Achilles tendon rupture.

Because of the long-term persistence of spore forms 

of B anthracis in lung tissue after an inhalational exposure, antibiotic prophylaxis combined with vaccination would provide more prolonged protection than 

postexposure antibiotic prophylaxis alone.3,37,52 Several 

studies in rabbits and NHPs have demonstrated that 

PEP that combines antimicrobial treatment with two or 

three doses of AVA is protective.51,60 However, postexposure vaccination without concomitant antimicrobial 

treatment will not prevent disease from inhalational 

anthrax.

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Some strains of B anthracis have shown resistance to 

certain broad-spectrum antibiotics, such as penicillin, 

trimethoprim combined with sulfamethoxazole (TMPSMZ, also called co-trimoxazole), and cefuroxime. 

Because B anthracis strains could be engineered to be 

resistant to multiple antibiotics, including the current 

first-line treatments, more selective antibiotic drugs 

(eg, triclosan derivatives and oligochlorophens) and 

drug targets (eg, the bacterial cell division protein 

FtsZ) are being studied.61–63

Postevent Countermeasures: Current Options

Treatment. The recommended treatment for inhalational anthrax—and for other forms of anthrax with 

systemic involvement—varies somewhat depending 

on whether meningitis has been ruled out. According 

to the CDC’s guidelines,3 if meningitis has not been 

ruled out, patients with inhalational anthrax (adults 

including pregnant women and children)—whether 

vaccinated or not—should be treated initially with a 

combination of at least three antimicrobial drugs—all 

with good central nervous system (CNS) penetration—administered intravenously. This treatment 

should be continued for at least 2 to 3 weeks or until 

the patient is clinically stable, whichever is longer.3,64,65

The drug combination should include at least one bactericidal agent—although Bradley et al64 recommend 

two bactericidal agents for children—and at least 

one protein synthesis inhibitor (see Table 27-2). The 

preferred bactericidal agents for adults and children 

are ciprofloxacin (with levofloxacin or moxifloxacin 

as alternatives) and meropenem (with imipenem and 

doripenem as alternatives); for penicillin-susceptible 

strains, penicillin G or ampicillin can serve as the 

second bactericidal agent.3,64,65 For pregnant women, 

ciprofloxacin is the preferred bactericidal agent.65

The preferred protein synthesis inhibitor for adults 

(including pregnant women) and children is linezolid 

(with clindamycin, rifampin, or chloramphenicol as 

alternatives).3,64,65 For pregnant women, at least one 

antibiotic in the combination should be able to cross 

the placenta (eg, ciprofloxacin, levofloxacin, amoxicillin, or penicillin).65

If meningitis has been ruled out, the CDC’s guidelines indicate that the initial intravenous treatment for 

patients of all age groups (including pregnant women) 

should consist of a combination of at least two antimicrobial drugs, administered intravenously, for at 

least 2 weeks or until the patient is stable. In this case, 

CNS penetration is not crucial, but again, at least one 

agent should be bactericidal and at least one should 

be a protein synthesis inhibitor. Ciprofloxacin remains 

the first-choice bactericidal agent for adults (including 

pregnant women) and children, though penicillin G 

or ampicillin could be used if the strain is susceptible. 

Alternative bactericidal agents include meropenem, 

levofloxacin, imipenem, and vancomycin3,64 for adults. 

Hendricks et al3 and Meaney-Delman et al65 additionally include moxifloxacin and doripenem as alternative 

bactericidal agents. Clindamycin or linezolid are the 

first-choice protein synthesis inhibitors for patients of 

all ages in whom meningitis has been ruled out,3,64,65

though Bradley et al64 indicate that clindamycin is 

preferred over linezolid for children. Alternative 

protein synthesis inhibitors include doxycycline and 

rifampin.3,64,65 For pregnant women, at least one antibiotic in the combination should be able to cross the 

placenta.65

The CDC’s guidelines recommend adding AIGIV 

or raxibacumab, when available, to combination antibiotic therapy for adults (including pregnant women) 

and children with inhalational anthrax or other forms 

of anthrax with systemic involvement.3,64,65

For adults, whether or not meningitis has been 

ruled out, intravenous combination therapy should be 

followed by oral administration of a single antibiotic, 

as described below for PEP, such that antibiotic treatment continues for a total of at least 60 days.3,64,65 For 

children, Bradley et al64 recommend follow-up therapy 

that is essentially the same as that for adults, except 

that a combination of two antibiotics—one bactericidal 

agent and one protein synthesis inhibitor—should be 

used for children who are slower to recover or who, at 

the end of the initial intravenous treatment, continue 

to show signs of infection. Ciprofloxacin (with levofloxacin as an alternative) is the preferred bactericidal 

agent unless the strain is susceptible to penicillins, 

in which case, amoxicillin (or penicillin VK) would 

be preferred. Clindamycin is the preferred protein 

synthesis inhibitor, with doxycycline or linezolid as 

alternatives.64

Postexposure Prophylaxis. Any individual with 

known or suspected exposure (of greater than negligible risk) to aerosolized B anthracis, whether vaccinated or not, should receive antibiotic prophylaxis 

starting as soon as possible and continuing until B 

anthracis exposure has been excluded (see Table 27-2). 

If exposure is confirmed or cannot be excluded, PEP 

should continue for at least 60 days (to clear germinating spores). Prophylaxis should be initiated without 

delay for the greatest chance of success, but the specific drugs chosen should be subsequently modified 

if necessary based on the results of strain sensitivity 

testing.3,4,14,64–66

First-line drugs for PEP (prior to symptom onset) 

for adults (including pregnant women) and children are ciprofloxacin or doxycycline, administered 

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orally3,64,65; ciprofloxacin is preferred over doxycycline 

for pregnant women.65 Alternatives, if first-line drugs 

are contraindicated, not tolerated, or unavailable, 

include levofloxacin, moxifloxacin, and clindamycin. 

Amoxicillin and penicillin VK are also acceptable 

alternatives if the B anthracis strain is susceptible to 

penicillins.3,4,14,66 Although permanent dental staining 

has been associated with use of tetracyclines in young 

children, Bradley et al64 suggest that doxycycline may 

be less likely than older tetracyclines to have this effect and argue that such risks are outweighed by the 

benefits of its use in the event of possible exposure 

to anthrax. Similarly, Bradley et al64 suggest that the 

potential risk of cartilage toxicity from ciprofloxacin 

is outweighed by the benefits of its use as PEP in this 

context. If the strain of B anthracis involved is found 

to be susceptible to penicillins, amoxicillin would be 

the first choice for children.64

As of November 2015, AVA is licensed by FDA for 

PEP—when used in conjunction with recommended 

antibiotics—in adults aged 18 to 65 years who have 

been exposed to aerosolized spores.15 The recommended BioThrax PEP vaccination schedule for those 

not previously vaccinated is 0.5 mL subcutaneously at 

weeks 0 (diagnosis), 2, and 4. Individuals who received 

one or two doses of AVA before exposure should receive two doses of AVA (at weeks 0 and 2). Those who 

received three or more doses of AVA before exposure 

should receive a single booster as soon as possible after 

exposure.3,15 The CDC additionally recommends the 

use of AVA and antimicrobial therapy in individuals 

of all ages, including children and pregnant women, 

after an aerosol exposure,3,4,64,65 although Bradley et 

al64 recommend delaying administration of AVA for 

newborns until 6 weeks of age.

Tularemia

Francisella tularensis is a highly infectious, aerobic, 

non-spore-forming, gram-negative coccobacillus responsible for serious illness and occasionally death.67,68

Humans can acquire tularemia through (a) contact of 

skin or mucous membranes with the tissues or body 

secretions of infected animals, (b) bites of infected arthropods, (c) ingestion of contaminated food or water, 

or (d) inhalation of aerosolized agent from infected 

animal secretions.67 Person-to-person transmission of 

tularemia—although theoretically possible and reported at least once69—is considered rare and unlikely.67

Most patients with naturally occurring tularemia 

present with the ulceroglandular form of the disease 

(generally from intradermal exposure), and up to about 

one-quarter of patients have typhoidal tularemia (usually resulting from inhalation of infectious aerosols 

but occasionally from other exposure routes).14,66,70

Other presentations of tularemia include glandular, 

oculoglandular, oropharyngeal, and pneumonic (from 

inhalation or from hematogenous spread from other 

sites).66 Pneumonic tularemia and typhoidal tularemia 

with pulmonary symptoms are the most lethal forms 

of the disease, yet antibiotics have greatly reduced 

mortality from all forms of tularemia.66,70 Disease severity varies by subspecies (or biovar); in particular, 

two subspecies—F tularensis subspecies tularensis

(type A) and F tularensis subspecies holarctica (type 

B)—cause the majority of human disease.71 Outbreaks 

of tularemia—particularly inhalational tularemia—in 

nonendemic areas should alert officials to the possibility of a bioterrorism event.

Vaccination

Investigational Live Tularemia Vaccine. No licensed vaccine protecting against tularemia is available. Vaccination of at-risk laboratory personnel with 

an inactivated phenolized tularemia vaccine (Foshay 

vaccine) during the US offensive biological warfare 

program at Fort Detrick (before 1959) ameliorated 

disease, but did not prevent infection.72–74 A sample 

of the Soviet live F tularensis subspecies holaractica

vaccine (known as strain 15), which the Soviet Union 

used to vaccinate millions of persons during epidemics 

of type B tularemia beginning in the 1930s, was made 

available to Fort Detrick in 1956.71,73 Both a gray-variant 

and a blue-variant colony were cultivated from this 

vaccine (colonies appeared blue when illuminated 

with oblique light under a dissecting microscope). The 

blue-variant colony proved to be both more virulent 

and more immunogenic than the gray-variant colony. 

To improve protection against the virulent F tularensis

subspecies tularensis SCHU S4 strain, the blue-variant 

colony was passaged through white mice. These passages resulted in the derivative vaccine strain known 

as the live vaccine strain (LVS). The strain was used to 

prepare a lyophilized preparation known as the live 

tularemia vaccine, which was composed of 99% bluevariant and 1% gray-variant colonies. 

During the 27 years of the US offensive biological 

warfare program at Fort Detrick, tularemia was the 

most common laboratory-acquired infection. Most of 

the 161 cases were acquired from aerosol exposures.11

Beginning in 1959, the live attenuated tularemia vaccine—prepared from, and known as, LVS—was administered to the program’s at-risk laboratory personnel 

until the program closed in 1969 (Figure 27-1).72 After 

vaccination using LVS was instituted, the incidence of 

typhoidal/pneumonic tularemia decreased from 5.7 to 

0.27 cases per 1,000 at-risk employee-years. Although 

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no decrease in ulceroglandular tularemia was noted, 

the vaccine did ameliorate symptoms from ulceroglandular tularemia, and, unlike those who were unvaccinated before the start of the vaccination program, 

vaccinated persons did not require hospitalization.11

The occurrence of ulceroglandular tularemia in vaccinated persons was consistent with the observation 

that, although natural disease confers immunity to subsequent infections of typhoidal/pneumonic tularemia, 

it fails to protect against ulceroglandular tularemia. In 

1961, commercial production of LVS was initiated by 

the National Drug Company (Swiftwater, PA), under 

contract to the US Army Medical Research and Materiel Command. This vaccine, designated NDBR 101, 

continues to be given to at-risk laboratory workers at 

USAMRIID under an IND protocol (see Table 27-1).

The live attenuated NDBR 101 tularemia vaccine is 

supplied as a lyophilized preparation and reconstituted with sterile water before use, resulting in approximately 7 × 108

 viable organisms per mL. The vaccine 

is administered by scarification, with 15 to 30 pricks to 

the ulnar side of the forearm using a bifurcated needle 

and a droplet (approximately 0.1 mL) of the vaccine. 

The individual is examined after vaccination for a take 

reaction, similar to the examination done after smallpox vaccination. A take with tularemia vaccine is defined as the development of an erythematous papule, 

vesicle, and/or eschar with or without induration at 

the vaccination site; however, the postvaccination skin 

lesion is markedly smaller and has less induration than 

is generally seen in vaccinia vaccinations. Although a 

take is related to immunity, its exact correlation has 

not yet been determined (Figure 27-2).

Protective immunity against F tularensis is considered to be primarily cell mediated. Cell-mediated 

immunity has been correlated with a protective effect, 

and lack of cell-mediated immunity has been correlated with decreased protection.75,76 Cell-mediated 

immune responses occur within 1 to 4 weeks after 

naturally occurring infection or after LVS vaccination 

and reportedly last 1 to 3 decades.75,77–85 Absolute levels 

of agglutinating antibodies in persons vaccinated with 

aerosolized LVS could not be correlated with immunity, although the presence of agglutination antibodies 

in vaccinated persons suggested that they were more 

resistant to infection than those in the unvaccinated 

control group.86 A similar experience was observed in 

studies of the inactivated Foshay tularemia vaccine, Figure 27-1. Live attenuated NDBR 101 tularemia vaccine. 

Vaccination of at-risk laboratory workers, beginning in 1959, 

resulted in a decreased incidence of typhoidal tularemia 

from 5.7 to 0.27 cases per 1,000 at-risk employee-years, and 

ameliorated symptoms from ulceroglandular tularemia. The 

vaccine is administered by scarification with 15 to 30 pricks 

on the forearm, using a bifurcated needle.

Figure 27-2. “Take” from the live attenuated NDBR 101 

tularemia vaccine at day 7 postvaccination. 

Photograph: Courtesy of Special Immunizations Program,

US Army Medical Research Institute of Infectious Diseases, 

Fort Detrick, Maryland.

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in which antibodies were induced by the vaccine but 

were not protective against tularemia.72,74 Although 

nearly all vaccinees develop a humoral response, with 

microagglutination titers appearing between 2 and 4 

weeks postvaccination,75,82,87 a correlation could not be 

demonstrated between antibody titers and the magnitude of lymphocyte proliferative responses.76,84,88,89 An 

explanation for this discrepancy may be that the two 

types of immune responses are directed toward different antigenic determinants of the organism, with a 

protein determinant responsible for the cell-mediated 

immune response and a carbohydrate determinant 

causing the humoral response.88

NDBR 101 has not been licensed in the United States 

because of drawbacks, including the following:

• the vaccine’s uncertain history; 

• its unclear mechanism of attenuation (and 

therefore risk of reversion to virulence); and 

• the inconsistency across lots in the proportion 

of blue and gray colonies present.71,90,91

In experimental aerosol exposures of human volunteers, this vaccine protected only 71% to 83% of 

individuals from inhalational tularemia.92,93 Because of 

the short incubation period of tularemia, postexposure 

use of this vaccine is not recommended. NDBR 101 is 

recommended for laboratory personnel working with 

F tularensis.

67

The local skin lesion after vaccination (the take) is an 

expected occurrence and may result in the formation of 

a small scar. At the site of inoculation, a slightly raised 

erythematous lesion appears, which may become 

papular or vesicular and then forms a scab lasting 

approximately 2 to 3 weeks. Local axillary lymphadenopathy is reported in 20% to 36% of vaccinees. Systemic reactions are uncommon (<1%) and may include 

mild fever, malaise, headache, myalgias, arthralgias, 

and nausea. Mild elevation of liver function tests was 

noted in some vaccinees but was not determined to be 

vaccine related. The main contraindications of the vaccine are prior tularemia infection, immunodeficiency, 

liver disease, and pregnancy.

Vaccine Research 

Research is ongoing to develop a new LVS tularemia vaccine (using NDBR 101 as starting material) 

as well as subunit vaccines against tularemia.94 Improvements to the LVS vaccine have included efforts 

to produce LVS under current good manufacturing 

practice conditions. Subunit vaccines have shown 

some promise with newly developed adjuvants, such 

as immune stimulating complexes and CpGs.91 Live 

attenuated mutant strains of F tularensis derived from 

LVS and SCHU S4 are also being studied for their 

attenuation and protection against lethal F tularensis

challenge.71

Antibiotic Agents

The early initiation and adequate duration of therapy are key to the successful treatment of patients 

with tularemia.95 Streptomycin, the traditional treatment choice, has proven to be highly efficacious with 

a low risk of relapse, based on documented cases.96

However, this aminoglycoside carries the risk of side 

effects, such as vestibular toxicity and nephrotoxicity, and is often unavailable. Gentamicin, which 

has also proven efficacious in documented cases, 

appears to be an acceptable substitute. Fluoroquinolones, such as ciprofloxacin, have shown promise as 

therapy based on evidence in mice and from use in 

a human outbreak.70,95 Fluoroquinolones are highly 

active against F tularensis types A and B in vitro, and 

evidence from animal studies as well as human cases 

suggest that ciprofloxacin, levofloxacin, and moxifloxacin are likely to be effective.95,97 Ciprofloxacin, 

in particular, has generally had high efficacy with 

few side effects in adults and children.67,96 Treatment 

with 15 days of tetracycline (2 g daily, beginning 

within 48 hours of symptom onset) was effective in 

human volunteers exposed to an aerosol challenge 

of F tularensis. Reducing the treatment duration to 

10 days or reducing the dose to 1 g daily increased 

the incidence of relapse.98 However, tetracyclines 

may be associated with a greater risk of treatment 

failure or relapse.67

In humans challenged intradermally with an 

inoculation of F tularensis, 5 days of streptomycin, 

which is bactericidal at concentrations achieved 

in humans,95 successfully prevented tularemia.99

However, neither chloramphenicol nor tetracycline 

given in a 5-day course was effective as PEP.99 Tetracycline—given as a 1-g dose twice daily for 14 days, 

starting within 24 hours of exposure—prevented the 

development of tularemia in eight humans exposed 

to aerosols of 25,000 F tularensis SCHU S4 spores. 

However, decreasing the tetracycline dose to only 

1 g daily was not as effective in preventing tularemia, with 2 of 10 persons becoming ill. The failure 

of once-daily tetracycline to prevent tularemia may 

be caused by considerable fluctuations in tissue 

levels of the antibiotic, as demonstrated in monkeys 

given once daily tetracycline, in which the antibiotic 

ameliorated symptoms but did not prevent tularemia.98 F tularensis is an intracellular pathogen that is 

cleared slowly from host cells, even in the presence of 

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bacteriostatic antibiotics. Even in high concentrations, 

tetracyclines and chloramphenicol merely suppress 

multiplication of the organisms,94 which may explain 

the need for a somewhat prolonged 14-day course of 

these bacteriostatic antibiotics.

Ongoing research seeks to find:

• treatments that are safer, especially for children and pregnant women; 

• therapeutic agents more effective in preventing relapse; and 

• alternative treatments for antibiotic-resistant 

strains.97

Postevent Countermeasures: Current Options 

Treatment. According to the consensus-based 

recommendations developed by the Working Group 

on Civilian Biodefense, the first-line therapy in a contained casualty situation (in which a modest number 

of individuals require treatment) is 10 days of parenteral streptomycin, with gentamicin as an acceptable 

substitute, for nonpregnant adults and children. Alternatives include 15 to 21 days of doxycycline or chloramphenicol or 10 to 14 days of ciprofloxacin, although 

treatment with tetracyclines or chloramphenicol may 

be more likely to result in treatment failure or relapse. 

For pregnant women, gentamicin (for 10 days) is preferred over streptomycin; if aminoglycosides cannot be 

used, alternatives include doxycycline (14–21 days) or 

ciprofloxacin (10 days) if the risks of their use during 

pregnancy are outweighed by the benefits of treating 

tularemia.67

In a mass casualty setting, in which logistics and 

supply limitations may preclude the use of parenteral 

antibiotics, treatment recommendations are identical 

to those described below for PEP. Treatment should 

begin immediately after symptom onset and continue for at least 14 days. The choice of antimicrobial 

should be modified based on the results of strain susceptibility testing and clinical response. Antibiotics 

initially administered by the intravenous route may 

be administered orally once the patient’s condition 

improves.14,66,67,70,95

Postexposure Prophylaxis. PEP for asymptomatic 

individuals who have been exposed to F tularensis

is most effective when initiated within 24 hours of 

exposure and continued for at least 14 days. Firstline antibiotics for PEP, for adults (including pregnant women) and children, are oral doxycycline 

or ciprofloxacin (see Table 27-2).67,70,95 Doxycycline 

and ciprofloxacin both have the potential to cause 

adverse effects in the fetus and young child. For an 

asymptomatic potentially exposed pregnant woman 

or child, the risk of disease must be weighed against 

the potential toxicity of the antibiotics. A patient at 

low risk of exposure could be instructed to closely 

monitor body temperature for 14 days, with treatment 

initiated if symptoms appear.66,67,96

Plague 

Plague is an acute bacterial disease caused by 

Yersinia pestis, a nonmotile, gram-negative bacillus. 

Naturally occurring disease in humans is generally 

acquired when the bites of infected fleas result in lymphatic and blood infections (bubonic and septicemic 

plague, respectively). Pneumonic plague, the most 

deadly form of the disease, may be acquired by inhaling droplets emitted from an infected person, inhaling 

aerosols from infected animal tissues, inhaling Y pestis

as an aerosolized weapon, or as a result of secondary 

hematogenous seeding from bubonic or septicemic 

plague.100,101 Given the high mortality and person-toperson transmissibility associated with pneumonic 

plague, Y pestis is a candidate for use as a biological 

warfare or terrorism agent to cause pneumonic plague.

Vaccination

Formalin-Killed Plague Vaccine. The US-licensed 

formalin-killed whole bacillus vaccine (Greer Laboratories, Inc, Lenoir, NC) for preventing bubonic plague was 

discontinued in 1999. Although this vaccine and other 

formalin-killed plague vaccines demonstrated efficacy 

in the prevention or amelioration of bubonic plague 

based on retrospective, indirect evidence in vaccinated 

military troops, evidence did not support its efficacy 

in preventing pneumonic plague.101–109 The vaccine’s 

efficacy against aerosolized plague was demonstrated 

to be poor in animal models, and several individuals 

developed pneumonic plague despite vaccination.103–109

Furthermore, these vaccines caused significant adverse 

reactions and required frequent boosting.101

Other Vaccines. A live attenuated vaccine made 

from an avirulent strain of Y pestis (the EV76 strain) 

has been available since 1908. This vaccine offers 

protection against both bubonic and pneumonic 

plague in animal models, but it is not fully avirulent 

and has resulted in disease in mice.104 EV76 has been 

licensed for human use in the former Soviet Union 

and China for decades and has apparently caused no 

vaccine-related deaths, though adverse reactions are 

significant.110 For safety reasons, EV76 is not used for 

humans in most countries.

Vaccine Research. Because of safety issues with 

live vaccines, recent efforts have focused on the development of a subunit vaccine using virulence factors 

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from the surface of the plague bacterium to induce immunity.103,111 Two virulence factors—identified as the 

fraction 1 (F1) capsular antigen and the virulence (V) 

antigen—induced immunity and provided protection 

against plague in animal models. At USAMRIID, a new 

plague vaccine was developed by fusing the F1 capsular 

antigen with the V antigen to produce the recombinant 

F1-V vaccine. (A similar recombinant subunit vaccine 

formulation mixes the two antigens [F1+V].) In mice 

and rabbits, evidence indicates that the F1-V vaccine 

candidate is protective against both pneumonic and 

bubonic plague. In NHPs, it provided better protection than either the F1 antigen or the V antigen alone 

during aerosol challenge experiments.111–113 Ongoing 

approaches for improving F1-V–type vaccine candidates include genetic modification of antigens and the 

use of different adjuvants. Phase 1 and 2 clinical trials 

exploring subunit plague vaccines have been recently 

completed. Other researchers are exploring the use of 

bacterial, viral, and plant live carrier platforms.111

Antibiotic Agents. In general, studies are lacking 

on the relative effectiveness of various antibiotics in 

the treatment or PEP of pneumonic plague in humans. 

Streptomycin has traditionally been the preferred 

treatment for plague and has been effective when 

initiated promptly. Gentamicin, which is more widely 

available than streptomycin, has also been used successfully but is not currently FDA approved to treat 

plague.100 In particular, a randomized clinical trial 

conducted in Tanzania found that both gentamicin 

and doxycycline were highly effective in the treatment of all forms of plague.114 In murine models of 

pneumonic plague, doxycycline and tetracycline have 

not consistently performed as well as other antibiotics.100,115,116 However, the weight of experimental and 

anecdotal evidence for the effectiveness of doxycycline led the Working Group on Civilian Biodefense 

to recommend the use of the tetracycline class of 

antibiotics to treat plague when aminoglycosides 

cannot be used.100 Fluoroquinolones also have been 

used successfully to treat severe cases of plague.100

Recently, the FDA approved the fluoroquinolones 

levofloxacin and moxifloxacin for the treatment (and 

PEP) of plague based on studies demonstrating the 

efficacy of these antibiotics in NHPs.117,118

PEP with ciprofloxacin for 5 days was highly effective in mice when initiated within 24 hours after aerosol 

exposure to Y pestis, but not when initiated 48 hours after 

exposure.115 Doxycycline was relatively ineffective as 

prophylaxis in a mouse model, even if initiated within 

24 hours after aerosol exposure with mean inhibitory 

concentrations (MICs) ranging from 1 to 4 mg/L.115,116

The effectiveness of doxycycline, a bacteriostatic drug, 

generally requires antibiotic levels to be four times the 

MIC. Two lines of evidence suggest that the treatment 

failure may be related, in part, to increased metabolism 

of doxycycline in mice. First, tetracycline has been used 

successfully in humans to treat or prevent pneumonic 

plague.100,119,120 Second, doxycycline stabilized the bacterial loads in the spleens of mice infected with Y pestis

strains with lower MICs (≤1 mg/L).121 

Postevent Countermeasures: Current Options

Treatment. The prompt initiation of treatment 

(within 18–24 hours of symptom onset) is crucial, especially for primary pneumonic plague. According to 

consensus-based recommendations developed by the 

Working Group on Civilian Biodefense,100 the first-line 

antibiotic for treatment of plague in adult men, nonpregnant women, and children in a contained casualty 

situation is parenteral streptomycin or gentamicin 

(although gentamicin is not FDA approved for this 

use); alternatives include doxycycline, ciprofloxacin, 

levofloxacin, moxifloxacin (in adults), or chloramphenicol. For pregnant women, gentamicin is the preferred 

choice, with doxycycline and ciprofloxacin as alternatives. Treatment should be continued for at least 10 

days. Antibiotics initially administered intravenously 

can be administered orally pending improvement in 

the patient’s condition.14,66,100

In a mass casualty setting, in which parenteral 

administration of antibiotics may not be feasible, oral 

doxycycline or ciprofloxacin are the preferred choices, 

as described below for PEP, except that treatment 

duration should be 10 days.100

Postexposure Prophylaxis. Asymptomatic individuals exposed to aerosolized Y pestis—as well as 

persons who have had unprotected face-to-face contact (within 2 meters) with patients with pneumonic 

plague or those potentially exposed to aerosolized Y 

pestis—should receive PEP, beginning as soon as possible and continuing for 7 days after the last known or 

suspected Y pestis exposure or until exposure has been 

excluded. Individuals with cough or fever within an 

area in which cases of pneumonic plague are known 

or suspected to be occurring should also be given PEP. 

The first-line antibiotics for PEP in adults (including 

pregnant women) and children are doxycycline or 

ciprofloxacin; chloramphenicol is an alternative, but 

this drug carries the risk of causing aplastic anemia. 

Moxifloxacin (in adults) or levofloxacin may also be 

appropriate (see Table 27-2).14,66,100,105,115,116,122,123 Antibiotic sensitivity testing should be performed to assess 

for resistant strains. For an asymptomatic potentially 

exposed pregnant woman or child, the risk of disease 

must be weighed against the potential toxicity of the 

antibiotics.66,100

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Glanders and Melioidosis

Glanders and melioidosis are zoonotic diseases 

caused by the gram-negative bacteria, Burkholderia 

mallei and B pseudomallei, respectively.124–126 Equids 

serve as the natural reservoir for B mallei, which is 

generally restricted to parts of the Middle East, Asia, 

and South America.127 Glanders in humans is not common and has typically been associated with contact 

with equids or laboratory exposure. The mode of 

acquisition is believed to be primarily from inoculation with infectious secretions of the animal through 

broken skin or the nasal mucosa and less commonly 

from inhalation.11,14,66,125,128

B pseudomallei is a natural saprophyte that can be 

isolated from soil, stagnant waters, rice paddies, and 

market produce primarily in endemic areas, such as 

Southeast Asia and northern Australia. However, it 

has been found in many tropical and subtropical regions.124,129,130 Infection in humans is generally acquired 

through soil contamination of skin abrasions, but it 

may also be acquired by ingesting or inhaling the organism. Although symptoms of B pseudomallei infection 

are variable, the pulmonary form of melioidosis is the 

most common and may occur as a primary pneumonia 

or from secondary hematogenous seeding.14,66,124,129

Both B mallei and B pseudomallei have been studied 

in the past as potential biowarfare agents. The recent 

increase in biodefense concerns has renewed research 

interest in these organisms because of their potentially 

high mortality, availability, resistance to many antibiotics, and inhalational infectivity.130–132

Vaccination

No vaccines are available for preventing glanders or 

melioidosis in humans or animals. Efforts to develop 

vaccines are made more challenging by the propensity 

for both of these pathogens to develop into chronic or 

recurring disease.130,131 Among the more promising 

lines of research are vaccines using live attenuated 

bacteria (which are more immunogenic in animal 

models) and recombinant subunit vaccines (which are 

less immunogenic but appear to be safer).130,131 

Antibiotic Agents

No FDA-approved therapy or PEP exists for 

glanders or melioidosis. Treatment and PEP are complicated by the tendency for many strains of both B 

mallei and B pseudomallei to be resistant to a variety of 

antibiotics.133–135 For glanders, effective treatment and 

PEP strategies are especially uncertain because of the 

rarity of the disease in humans. 

Most strains of both B mallei and B pseudomallei have 

generally been susceptible to ceftazidime, meropenem, 

imipenem, ciprofloxacin, and tetracyclines. B mallei

is also generally sensitive to rifampin and aminoglycosides, to which most isolates of B pseudomallei are 

resistant.133–135 Resistance of B pseudomallei to TMPSMZ is rare in Australia135; in Thailand, however, the 

percentage of B pseudomallei isolates that are resistant 

to TMP-SMZ may be increasing.136

Because of the potential for latent or recurrent B 

pseudomallei infection, which can occur several decades 

after exposure, treatment of melioidosis is biphasic. The 

first phase consists of short-term, intensive, parenterally 

administered antibiotics; in the second phase, antibiotics are administered orally as long-term eradication 

therapy.134,137 In human cases of melioidosis, intravenous 

ceftazidime—with or without TMP-SMZ—has been effective during the initial intensive phase of treatment. 

For example, a randomized trial found a significant 

reduction in mortality among patients with severe 

melioidosis who were treated during the intensive 

phase with intravenous ceftazidime alone compared 

with those who received the conventional treatment 

of the time—a combination of chloramphenicol, 

doxycycline, and TMP-SMZ.138 Amoxicillin/clavulanic 

acid (or co-amoxiclav), imipenem, meropenem, and 

cefoperazone–sulbactam have also been effective.134

In particular, Cheng et al139 found that patients with 

severe melioidosis who were treated with meropenem 

during the intensive phase had outcomes similar to 

those treated with ceftazidime. However, imipenem 

has been associated with a higher frequency of CNS 

adverse effects and problems for patients with impaired renal function, and co-amoxiclav may be more 

likely than ceftazidime to result in treatment failure.140

In the eradication phase of melioidosis treatment, a 

combination of TMP-SMZ plus doxycycline has been 

used successfully, as has co-amoxiclav alone.134,141

However, a recent trial comparing combination antibiotic regimens during the eradication phase found 

that TMP-SMZ alone was noninferior to TMP-SMZ 

plus doxycycline, a combination that has been commonly recommended in Thailand. Excluding doxycycline may reduce adverse reactions and improve 

adherence by patients.141 An adequate duration of the 

eradication phase of treatment is crucial for preventing relapse. Limmathurotsakul et al142 have found that 

the most significant risk factors for relapse are choice 

and duration of oral antimicrobial therapy. Among 

patients treated during the eradication phase with an 

appropriate oral antibiotic regimen, such as TMP-SMZ 

plus doxycycline, a 12- to 16-week treatment duration 

reduced the risk of relapse by 90% compared with a 

treatment lasting no longer than 8 weeks.

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Treatment of patients with glanders is based largely 

on experience with treating melioidosis as well as the 

results of animal studies. In addition to antibiotic treatment, surgical drainage of abscesses may be required 

for some patients.128,134

Most evidence on the efficacy of antibiotics as PEP 

for melioidosis comes from laboratory exposures. For 

example, among 17 laboratory workers who manipulated cultures of B pseudomallei, 13 individuals experienced high-risk exposure to B pseudomallei from sniffing culture plates and/or performing routine laboratory procedures, such as subculturing and inoculation 

of the organism outside a biosafety cabinet (before the 

organism was identified). Beginning 0 to 4 days after 

the exposure, 16 of the exposed workers were treated 

with a 3-week course of TMP-SMZ, and 1 was treated 

with a 3-week course of doxycycline. None of the 17 

individuals developed symptoms consistent with 

melioidosis for 5 months after exposure.133 However, 

this response may reflect the low risk of laboratoryacquired illness from the organism as opposed to the 

effectiveness of antibiotic prophylaxis.143

Animal studies are also informative for the use of 

antibiotics as PEP for both B pseudomallei and B mallei. 

In mice, TMP-SMZ initiated 6 hours after exposure 

to aerosolized B pseudomallei or B mallei effectively 

prevented acute melioidosis and acute glanders. 

However, these mice nevertheless succumbed to 

melioidosis or glanders after relapse or immunosuppression, indicating that chronic infections had been 

established. In rats, PEP with 10 days of quinolones 

or TMP-SMZ, initiated within 3 hours of subcutaneous exposure to 105 organisms of B pseudomallei, was 

completely effective in preventing disease (verified 

by necropsy after animals were sacrificed at 2 months 

postexposure).144

Administration of either doxycycline or ciprofloxacin (twice daily for 5 or 10 days) protected mice from 

disease if started 48 hours before or immediately after 

intraperitoneal challenge with B pseudomallei, though 

relapses occurred in a few animals within 5 weeks of 

discontinuation of the antibiotics. However, when the 

initiation of antibiotic prophylaxis was delayed to 24 

hours after exposure, the treatment provided minimal 

protection, resulting in only a delay of infection, which 

occurred at least 5 weeks after discontinuation of the 

antibiotic.127

Doxycycline or ciprofloxacin (twice daily for 5 

days), initiated 48 hours before or immediately after 

intraperitoneal challenge with B mallei, had a protective effect in hamsters. But the effect was temporary in 

some animals, with disease occurring after discontinuation of the antibiotic. Relapses were associated with 

both ciprofloxacin and doxycycline beginning at day 18 

and day 28, respectively, after challenge. Necropsies of 

fatalities revealed splenomegaly with splenic abscesses 

from which B mallei could be isolated; necropsies of 

surviving animals revealed splenomegaly with an 

occasional abscess. Delay of ciprofloxacin or doxycycline prophylaxis initiation to 24 hours after exposure 

merely delayed disease, with relapses occurring in 

hamsters within 4 weeks of the challenge.127

The differences in results among animal models 

may be related—in part—to differential susceptibility 

among species to melioidosis and glanders. In particular, hamsters are highly susceptible to infection from 

B mallei; the protective effect of chemoprophylaxis in 

humans may be greater.

Postevent Countermeasures: Current Options 

Treatment. According to consensus recommendations developed at the 2010 US Department of Health 

and Human Services Burkholderia Workshop,140

patients with suspected or confirmed glanders or 

melioidosis should receive intensive therapy with intravenous antibiotics for 10 to 14 days, and until the 

patient’s condition improves, followed by prolonged 

oral eradication therapy for a minimum of 12 weeks 

to minimize the risk of relapse. The initial intravenous treatment should be extended to greater than 

or equal to 4 weeks for severe disease or lack of improvement. To reduce the likelihood of relapse, the 

duration of oral eradication therapy should depend 

on disease severity and the response to treatment.140

These consensus recommendations do not provide 

separate guidelines for children or pregnant women 

for the intensive phase of treatment. However, based 

on 24 years of pediatric melioidosis treatment at the 

Royal Darwin Hospital in the Northern Territory, 

Australia, McLeod et al145 similarly recommend 

a minimum of 14 days of intensive intravenous 

therapy followed by at least 12 weeks of eradication therapy for children aged 16 years or younger 

with disseminated disease. For pediatric patients 

with localized cutaneous melioidosis, McLeod et 

al indicate that 12 weeks of oral antibiotic therapy 

(without the initial intensive parenteral treatment 

phase) is generally sufficient.

For intensive intravenous therapy in adults and 

children, ceftazidime is adequate in most cases without 

complications. Meropenem is an acceptable alternative 

and may be preferable in cases with complications, 

such as neuromelioidosis or persistent bacteremia, 

or when the patient must be admitted to an intensive 

care unit. Patients whose condition worsens while 

taking ceftazidime should be switched to meropenem. 

TMP-SMZ (administered intravenously, orally, or via 

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nasogastric tube, using the dosing for eradication therapy) may be added to this regimen for patients with 

severe neurologic, cutaneous, bone, joint, or prostate 

infections.140,145 The optimal intravenous therapy for 

pregnant women is not clear; however, Wuthiekanun 

and Peacock146 indicate that intravenous co-amoxiclav 

is used to treat pregnant women (and children) in 

Thailand during the intensive phase of therapy.

For eradication therapy in nonpregnant adults 

and in children, TMP-SMZ is the first-line antibiotic. 

However, potential side effects include mild allergic 

reactions, Stevens–Johnson syndrome, bone marrow

suppression, renal failure, and liver damage. In addition, TMP-SMZ may result in adverse pregnancy 

outcomes. For pregnant women, patients who cannot 

tolerate TMP-SMZ, and cases in which the organism 

is resistant to TMP-SMZ, co-amoxiclav (at an amoxicillin to clavulanic acid ratio of 4:1) is an alternative

eradication-phase antibiotic, but it may be associated 

with a greater risk of relapse.140,145 McLeod et al145 indicate that co-amoxiclav is also used as an alternative 

to TMP-SMZ in pediatric patients with melioidosis in 

Thailand.

Postexposure Prophylaxis. Current recommendations for PEP after suspected B mallei or B pseudomallei

exposure are based largely on animal studies and in 

vitro work. Ideally, PEP should be initiated promptly 

after a known or suspected exposure and continued 

for a total duration of 21 days. The first-line agent 

for adults and children is TMP-SMZ. For cases in 

which the organism is resistant to TMP-SMZ or the 

patient cannot tolerate this antibiotic, co-amoxiclav 

is the second-line choice. However, although these 

recommendations are appropriate for small-scale 

(eg, laboratory) exposures, it is not clear whether 

the provision of PEP to all individuals potentially 

exposed in a large exposure event would be feasible 

or advisable.140

Because of the potential for delayed-onset disease 

and relapse, monitoring (including serologic testing) 

should continue for at least 6 months after cessation 

of antibiotic PEP in exposed individuals; infected individuals may require lifelong monitoring following 

treatment.11,14,66,147 Seroconversion may be indicative of 

relapse. If relapse is suspected, treatment (as described 

above) should be initiated. Antibiotic regimens should 

be adjusted based on results of sensitivity testing.14,66

Brucellosis

Brucellosis, a common zoonotic disease with a global 

distribution, is caused by infection with one of several 

Brucella spp, including B abortus, B melitensis, and B 

suis. These intracellular, nonspore-forming, gram-negative coccobacilli can cause severe disease in humans; 

mortality is low, but chronic, debilitating illness can 

result.54,148–150 Infection is transmitted to humans by 

direct contact with infected animals or their carcasses, 

ingestion of unpasteurized milk or milk products, and 

via laboratory exposure.148 Person-to-person transmission of brucellosis has been documented, but is rare.54,151

Brucella are highly infectious by aerosol and remain 

one of the most common causes of laboratory-acquired 

exposure,11,152 with an infective dose of only 10 to 100 

organisms.54 In untreated survivors, chronic illness can 

last for years. Infection with Brucella spp during pregnancy, if untreated, can cause spontaneous abortion or 

intrauterine fetal death.14,66 Brucella spp are potential 

agents of bioterrorism because of their widespread 

availability, the ease with which they can be aerosolized, 

their stability in the environment, and their ability to 

induce chronic disease.149

Vaccination

Live vaccines licensed for use in animals have 

eliminated brucellosis in most domestic animal herds 

in the United States, but no licensed human vaccine 

exists. Ongoing research is evaluating the following:

• live, attenuated vaccine candidates, in some 

cases encapsulated within microspheres for 

slow release; 

• subunit vaccines; 

• vaccines based on recombinant proteins; 

• vectored vaccines; and 

• DNA vaccines.150

Antibiotic Agents

No approved chemoprophylaxis exists for brucellosis, whether as treatment or as PEP. Although few studies have compared monotherapy with combination 

therapy in treating brucellosis, the existing evidence 

suggests that monotherapy is more likely to result in 

relapse and treatment failure.153–155 An adequate duration of therapy is also crucial to the effective treatment 

of brucellosis.

In a meta-analysis of randomized controlled trials 

comparing various treatment regimens for brucellosis, Skalsky et al155 found that treatment consisting of 

doxycycline combined with rifampin was more likely 

to fail (generally from relapse) than was a regimen 

of doxycycline plus streptomycin. Triple-antibiotic 

therapy with doxycycline, rifampin, and an aminoglycoside was even less likely to result in treatment 

failure, whereas use of a quinolone plus rifampin was 

among the least effective of the regimens compared. 

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These authors concluded that the preferred treatment 

should consist of two or three antibiotics, including an 

aminoglycoside. 

In a review, Franco, Mulder, and Smits156 noted 

that relapse rates with the doxycycline–rifampin 

regimen ranged from 16% to 40% (depending—in 

part—on duration of treatment), whereas the relapse 

rates for doxycycline–streptomycin and rifampin–

minocycline were 5.3% and 2%, respectively. Monotherapy has resulted in a combined treatment failure 

and relapse rate as high as 50%. In a more recent 

meta-analysis, Solís García del Pozo and Solera153

examined clinical trials using various antimicrobial 

combinations in the treatment of human brucellosis. With relapse, therapeutic failure, and adverse 

effect rates as the primary outcome variables, they 

found that the doxycycline–streptomycin combination outperformed a combination of doxycycline 

and rifampin. For example, across 15 studies and a 

total of 700 patients with brucellosis, 6 to 8 weeks 

of treatment with doxycycline–rifampin resulted 

in treatment failure or relapse in 15.2% to 16.6% of 

patients (in trials employing this combination for 

only 4 weeks, treatment failure or relapse occurred 

in 26.5% of 83 patients). In contrast, in 11 studies 

evaluating more than 700 patients, doxycycline (45 

days) plus streptomycin (15–21 days) resulted in 

treatment failure or relapse in 6.7%–7.6% of patients. 

Rates of serious side effects were similar (around 1%) 

for both of these combinations. The doxycycline–

gentamicin combination appeared to be equivalent 

to doxycycline–streptomycin.153

Evidence regarding effective PEP regimens comes 

largely from laboratory exposures. One study reported prophylaxis using the doxycycline–rifampin 

combination administered to nine asymptomatic laboratory workers who seroconverted after exposure 

to B abortus serotype 1 atypical strain (a strain with 

low virulence).157 These individuals subsequently 

developed symptoms of fever, headache, and chills 

that lasted a few days. In contrast, three persons 

who did not receive prophylaxis had symptoms of 

fever, headache, and chills for 2 to 3 weeks as well as 

anorexia, malaise, myalgia, or arthralgia lasting an 

additional 2 weeks. No relapses occurred in the nine 

persons who received antibiotic prophylaxis, which 

may be a result of either the low virulence of this particular strain in humans or the early administration 

of antibiotic prophylaxis. In another hospital laboratory incident, six laboratory workers were identified 

as having had a high-risk exposure to B melitensis

because they had sniffed and manipulated cultures 

outside a biosafety cabinet. Five of these individuals were given PEP for 3 weeks (four individuals 

received doxycycline twice daily plus rifampin once 

daily, and one pregnant laboratory worker received 

TMP-SMZ twice daily). One individual declined 

prophylaxis and subsequently developed brucellosis (confirmed by culture). The five individuals 

who received PEP remained healthy and did not 

seroconvert.158 In late 2007, the CDC became aware 

of 916 laboratory workers in 254 laboratories with 

potential exposure to RB51, an attenuated vaccine 

strain of B abortus used to vaccinate cattle, during a 

laboratory preparedness proficiency test. PEP was 

recommended for the 679 individuals characterized 

as having had high-risk exposures and was also offered to the 237 laboratory workers with low-risk 

exposures. No cases of brucellosis were reported, 

but the number of individuals who actually received 

PEP has not been documented.159

Postevent Countermeasures: Current Options

Treatment. For uncomplicated brucellosis in adults, 

a combination of oral doxycycline (for 6 weeks) and 

intramuscular streptomycin (for 2–3 weeks) is recommended as the “gold standard” treatment in the position 

paper that resulted from a 2006 consensus meeting (the 

Ioannina recommendations).154 Parenteral gentamicin 

(for 7 days) is an acceptable substitute for streptomycin. 

Six weeks of oral doxycycline plus oral rifampin is an 

alternative first-line regimen because the convenience 

of (and therefore, presumably, better adherence to) an 

entirely oral therapy is likely to overcome the drawbacks of this combination.154 The optimal treatment 

for pregnant women with brucellosis has not been 

sufficiently studied. TMP-SMZ and/or rifampin could 

be considered, with risks to the fetus of antimicrobial 

treatment balanced against the risk of spontaneous 

abortion resulting from the disease (and the risk of relapse in the case of monotherapy with rifampin).154,160,161

In adult patients with serious complications, such as 

neurobrucellosis or Brucella endocarditis, the optimal 

antibiotic combination and treatment duration are not 

clear. In general, however, the duration of treatment 

should extend to at least 3 months. The World Health 

Organization (WHO) recommends the addition of 

either TMP-SMZ or rifampin—both of which cross 

the blood–brain barrier—to the doxycycline–streptomycin combination for the treatment of neurobrucellosis. Because rifampin and TMP-SMZ penetrate cell 

membranes, the WHO also recommends the addition 

of one of these antibiotics to the combination therapy 

for Brucella endocarditis.160,162

The WHO160 recommends that children aged 8 

years and older receive the same antibiotics as adults 

for the same duration. For younger children, the 

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WHO indicates that satisfactory results have been 

achieved using a combination of TMP-SMZ for 6 

weeks and parenteral streptomycin (for 3 weeks) or 

gentamicin (for 7–10 days). Alternatives include TMPSMZ–rifampin for 6 weeks or rifampin with an aminoglycoside.160 More recent guidelines163 developed 

for the treatment of children in Saudi Arabia—where 

brucellosis is endemic—recommend 6 weeks of treatment with a combination of rifampin and TMP-SMZ 

or 6 weeks of rifampin and 7 days of gentamicin for 

children younger than 8 years old. For more severe 

disease in young children, these authors recommend 

rifampin, TMP-SMZ, and ciprofloxacin for 3 to 9 

months with gentamicin added for the first 14 days. 

They recommend avoidance of doxycycline in young 

children because of the potential for dental staining. 

For older children (≥8 years), they recommend doxycycline and rifampin for 6 weeks or doxycycline for 6 

weeks and either streptomycin (14 days) or gentamicin (7 days). For more severe disease in older children, 

they suggest using doxycycline–TMP-SMZ–rifampin 

for 3 to 9 months with gentamicin added during the 

first 14 days.163

Postexposure Prophylaxis. For asymptomatic individuals who have had a high-risk exposure (such as 

exposure to laboratory aerosols or biowarfare exposure) to Brucella isolates, the CDC164 recommends a 

combination of doxycycline and rifampin for 3 weeks 

(see Table 27-2). If that combination cannot be used, 

TMP-SMZ could be offered.14,66,160,164 For an asymptomatic pregnant woman with a high-risk exposure, PEP 

should be considered in consultation with an obstetrician, weighing the risk of disease against the potential 

toxicity of the antibiotics.164

Q Fever

Q fever is a zoonotic disease caused by a rickettsia, 

Coxiella burnetii, a gram-negative, obligately intracellular coccobaccillus with a global distribution.165 C 

burnetii is environmentally stable and remains viable 

in the soil and other substrates for weeks or potentially longer.166 Humans typically acquire C burnetii

infection by inhaling aerosols contaminated with the 

organisms (generally from the excreta of infected animals).167 Less common routes of transmission include 

the consumption of unpasteurized dairy products 

and transmission via tick bites.168,169 Person-to-person 

transmission has been reported only rarely.170,171 Cases 

of Q fever among US military personnel in Iraq have 

been linked to tick bites and helicopter-generated aerosols.172,173 Q fever manifests in an acute form—which 

may be asymptomatic—as well as a rare but potentially 

more serious chronic form (most often presenting as 

endocarditis) that can occur weeks, months, or years 

after the initial acute infection.14,66,165,167,174–177 Long-term 

sequelae—notably, chronic fatigue and cardiovascular 

disease—often occur after acute infection.175

Vaccination

C burnetii has two major antigens, known as phase 

I and phase II antigens. Strains in phase I have been 

propagated mainly in mammalian hosts, whereas 

strains in phase II have been adapted to yolk sacs or 

embryonated eggs. Although early vaccines were made 

from phase II egg-adapted strains, later vaccines were 

made from phase I strains and demonstrated protective 

potencies in guinea pigs 100 to 300 times greater than 

vaccines made from phase II strains.178 

No FDA-approved vaccine is available for vaccination against Q fever in the United States. However, one 

vaccine (Q-Vax) is approved in Australia and a similar 

IND vaccine (NDBR 105) has been used in at-risk researchers at Fort Detrick since 1965.

Q-Vax. Currently licensed in Australia, Q-Vax (CSL 

Ltd, Parkville, Victoria, Australia) has been demonstrated to be safe and effective for preventing Q fever. Q-Vax 

is a formalin-inactivated, highly purified C burnetii

whole-cell vaccine derived from the Henzerling strain, 

phase I antigenic state.179,180 More than 4,000 abattoir 

workers were vaccinated subcutaneously with 0.5 mL 

(30 μg) of the vaccine from 1981 to 1988. In an analysis 

of data through August 1989, only eight vaccinated 

persons developed Q fever, with all infections occurring within 13 days after vaccination (before vaccineinduced immunity) versus 97 cases in unvaccinated 

persons (among approximately 2,200 unvaccinated 

individuals, but the exact number is not known).179 In 

another study, among 2,555 vaccinated abattoir workers, only two cases of Q fever were diagnosed between 

1985 and 1990, with both cases occurring within a few 

days of vaccination (before immunity developed).181

Nearly 49,000 individuals (primarily abattoir workers 

and farmers) were vaccinated between 2001 and 2004 

during a national Q fever vaccination campaign in 

Australia. Compared with Q fever notification rates 

in 2001 and 2002, those in 2005 and 2006 declined by 

more than 50% to the lowest levels on record.182 A recent 

meta-analysis of four studies assessing the effectiveness of Q-Vax in a total of 4,956 subjects found that, 

after excluding patients who developed symptoms of 

Q fever within 15 days after vaccination, the vaccine’s 

effectiveness was 100% (with those cases included, the 

effectiveness was 98%).183

The main adverse event noted with this vaccine was 

the risk of severe necrosis (which resulted in sterile 

abscesses) at the vaccine site in vaccinees with prior 

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Medical Countermeasures

exposure to Q fever.179,184 Therefore, a skin test using 

0.02 mg of the vaccine is required before vaccination. 

Because of the risk of vaccine-site necrosis, vaccination 

against Q fever is contraindicated in persons with previous exposure to C burnetii as denoted by a positive 

skin test, which is defined as either (a) erythema of at 

least 30 mm or induration of at least 20 mm at day 1 or 

later after the skin test or (b) erythema and induration 

of at least 5 mm on day 7 after the test. Persons with 

a positive skin test are considered to be naturally immune and do not require vaccination. The exclusion 

from vaccination of individuals with a positive skin 

test has eliminated sterile abscesses (Figure 27-3).185,186

NDBR 105 Q Fever Vaccine. The NDBR 105 (IND 

610) Q fever vaccine is an inactivated, lyophilized 

vaccine whose preparation is similar to that of Q-Vax. 

The vaccine originates from chick fibroblast cultures 

derived from specific pathogen-free eggs infected with 

the phase I Henzerling strain. NDBR 105 has been 

effective in animal studies.185,187,188 The vaccine also 

prevented further cases of Q fever in at-risk laboratory 

workers in the final 4 years (1965–1969) of Fort Detrick’s 

offensive biological warfare program, compared to an 

average of three cases per year before the vaccine was 

available.11,189 In the 45 years of the biodefense research 

program at USAMRIID, only one case of Q fever (mild 

febrile illness with serologic confirmation)—attributed 

to a high-dose exposure from a breach in the filter of 

a biosafety cabinet—has occurred among vaccinated 

laboratory workers. However, the vaccine may have 

ameliorated disease symptoms in this case.190

As with Q-Vax, a skin test is required before vaccination to identify persons with prior exposure to C 

burnetii. For NDBR 105, skin testing is performed by 

injecting 0.1 mL of skin-test antigen (a 1:1500 dilution

of the vaccine with sterile water) intradermally into 

the forearm. 

The vaccine is given only once, both because it is 

presumed to result in lifelong immunity and because 

of the potential for serious local reactions in individuals with prior exposure via disease or vaccination. The vaccine is administered by injecting 0.5 mL 

subcutaneously in the upper outer aspect of the arm 

(see Table 27-1). Protection against Q fever is primarily cell-mediated immunity. Markers to determine 

vaccine immunity to NDBR 105 have been studied 

(ie, cell-mediated immunity studies, skin testing, and 

pre- and postimmunization antibody studies), but reliable markers have not yet been identified for NDBR 

105. After vaccination with the similar Q-Vax, skintest seroconversion occurred in only 31 of 52 persons 

(60%), but lymphoproliferative responses to C burnetii

antigens persisted for at least 5 years in 85% to 95% of 

vaccinated persons.179,186,191–193 Vaccine breakthroughs 

have been rare in vaccinated persons.

Adverse events from NDBR 105, which were reported by 72 (17%) of 420 skin-test–negative vaccinees, 

comprised mainly local reactions, including erythema, 

induration, or a sore arm. Most local reactions were 

classified as mild or moderate, but one person required 

prednisone secondary to erythema extending to the 

forearm. Some vaccinees experienced self-limited 

systemic adverse events, but these were uncommon 

and generally were characterized by headache, chills, 

malaise, fatigue, myalgia, and arthralgia.194 

NDBR 105 is available only at USAMRIID on an 

investigational basis, although it is on hold (as of November 2015) because lot release data for the skin test 

antigen are unavailable. 

Other Vaccines. Several studies are underway to 

explore new techniques for vaccine development, 

including research focusing on Th1 peptides from the 

major immunodominant proteins.195,196

Antibiotic Agents

Antibiotics are known to be effective for the treatment of Q fever, but the recommended treatment 

varies with the form (acute vs chronic) and severity 

of disease. Acute Q fever often resolves without 

treatment within 2 to 3 weeks.197,198 Doxycycline, 

which is considered the most effective antibiotic to 

treat Q fever, reduces elevated body temperature 

within 2 to 3 days from the start of treatment; in 

untreated patients, fever resolves in 12.5 days (on 

average). Other antibiotics, including macrolides, 

TMP-SMZ, quinolones, and rifampin, can also be 

helpful, yet typically less so than doxycycline. Some 

Figure 27-3. Positive Q fever skin test. Skin testing, performed by injecting 0.1 mL of skin test antigen intradermally 

in the forearm, is required before vaccination against Q fever 

to identify persons with prior exposure. Vaccination is contraindicated in individuals with a positive skin test because 

they are at risk for severe necrosis at the vaccine site. 

Photograph: Courtesy of Herbert Thompson, MD, MPH.

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doxycycline-resistant isolates of C burnetii have been 

reported, but such resistance does not appear to be 

common.198

Among 438 patients with Q fever during an outbreak in the Netherlands, doxycycline and moxifloxacin were the first and second most commonly 

prescribed initial antibiotics, respectively. However, 

several other antibiotics were also prescribed, including potentially effective alternatives (eg, low-dose 

doxycycline, TMP-SMZ, ciprofloxacin, clarithromycin, 

and cefuroxim) as well as beta-lactam antibiotics and 

azithromycin, which are considered ineffective against 

Q fever. Patients who were treated initially with 

beta-lactams or azithromycin were at greatest risk of 

hospitalization after at least 2 days of treatment. Those 

receiving doxycycline at the recommended dosage 

(200 mg/day) had the lowest risk of hospitalization.176

Doxycycline has also been the most effective antibiotic to treat chronic Q fever, particularly when 

combined with hydroxychloroquine, which increases 

the bactericidal activity of the treatment. Although 

treatment must be continued for 18 to 24 months, the 

use of doxycycline alone required treatment for up to 

5 years.198 Lifelong follow-up, and sometimes lifelong 

treatment, may be required.175 

Q fever infection during pregnancy, particularly 

during the first trimester, can result in obstetric 

complications as well as a greater risk of chronic 

Q fever for the mother, with spontaneous abortions of future pregnancies more likely.168,175,199

Carcopino et al199 compared maternal and fetal 

outcomes for 53 women who were diagnosed with 

Q fever during pregnancy, including 16 women 

who received long-term (≥5 weeks) treatment with

TMP-SMZ and 37 who did not. Among the women 

who did not receive long-term TMP-SMZ, 81% 

experienced obstetric complications, including 

spontaneous abortion, intrauterine growth retardation, intrauterine fetal death, and premature 

delivery. They found that long-term TMP-SMZ 

during pregnancy protected against chronic Q fever in the mother, placental infection by C burnetii, 

and obstetric complications.

Postevent Countermeasures: Current Options

Treatment. According to recommendations from 

the CDC and the Q Fever Working Group, nonpregnant adults and older children (≥8 years old) with

symptomatic acute Q fever should be treated with 

doxycycline. Ideally, treatment should be initiated 

within the first 3 days of symptom onset and continued for 14 days. Alternative antibiotics include 

moxifloxacin, clarithromycin, TMP-SMZ, or rifampin. 

Asymptomatic individuals and those whose symptoms 

have resolved without treatment generally should not 

receive antibiotic treatment, with the possible exception of individuals who are at high risk of developing 

chronic Q fever.197

For young children (<8 years old) with mild or 

uncomplicated illness, doxycycline should be administered for 5 days (which should not result in dental 

staining). If the patient remains febrile after this short 

course of doxycycline—or if the healthcare provider 

decides not to administer doxycycline at all—TMPSMZ should be administered for 14 days.197

For pregnant women with acute Q fever, a longer 

(≥5 weeks) course of TMP-SMZ may be effective in

reducing the risk of intrauterine fetal death, conversion to chronic Q fever in the mother, and adverse 

outcomes in future pregnancies. Treatment should 

not continue beyond 32 weeks’ gestation because of 

the risk of hyperbilirubinemia. Concomitant use of 

folic acid may prevent antifolate effects of TMP-SMZ. 

However, data on the safety of Q fever treatment 

during pregnancy are limited; consultation with an 

infectious disease expert is recommended.197

After an acute infection, healthy patients with 

no risk factors for the development of chronic Q 

fever should be regularly evaluated for clinical and 

serologic signs of illness for at least 6 months after 

diagnosis, as described by Anderson et al.197 Persons 

with risk factors for development of chronic disease 

should be serologically and clinically monitored more 

frequently and for a longer duration (at 3, 6, 12, 18, 

and 24 months after diagnosis of acute infection or, 

for pregnant women, after delivery). All patients who 

have recovered from an acute Q fever infection should 

be advised to seek immediate medical attention if 

symptoms of chronic Q fever reoccur at any time 

throughout their lives; this vigilance is particularly 

important for those with valvular defects or vascular 

abnormalities.197

Treatment of chronic Q fever typically involves 

a long course of doxycycline combined with hydroxychloroquine. A discussion of the appropriate 

duration of treatment, contraindications, and recommendations for the treatment of pregnant women and 

young children is beyond the scope of this chapter. 

However, these topics are discussed in depth by 

Anderson et al.197

Postexposure Prophylaxis. Limited data are available on the effectiveness of PEP for Q fever. The CDC 

and the Q Fever Working Group do not recommend 

PEP after potential exposure to C burnetii. Serologic 

and clinical (fever) monitoring is recommended for at 

least 3 weeks after exposure. At the first sign of fever, 

treatment should be initiated.197

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VIRAL DISEASES 

WEE are also available on an IND basis at USAMRIID. 

These vaccines, which have demonstrated efficacy in 

animal models, have been used in at-risk laboratory 

workers at the institute for more than 50 years in the 

case of TC-83. However, the live attenuated vaccine, 

TC-83, has high reactogenicity, and the inactivated 

vaccines have lower immunogenicity. Also, because 

of their investigational status and limited supply, use 

of these vaccines in a bioterrorism event would be 

extremely limited.

Venezuelan Equine Encephalitis TC-83 Vaccine. 

Live attenuated VEE TC-83 vaccine (IND 142, NDBR 

102) was manufactured at the National Drug Company (Swiftwater, PA) in 1965 using serial propagation 

of the Trinidad strain (subtype IAB) of VEEV in fetal 

guinea pig heart cells. The virus was plaqued once in 

chick embryo fibroblasts. Several VEE viral plaques 

were then picked and inoculated by the intracranial 

route into mice. The plaques that did not kill the mice 

were judged attenuated. One of the nonlethal plaques 

of VEEV was used as seed stock to propagate in the 

81st passage in fetal guinea pig heart cells.207 The TC83 designation refers to the number of passages in cell 

culture. The seed stock (81-2-4), which was provided by 

FortDetrick, wasdiluted1:100. Five lots were produced.

The bulk vaccine was stored at −80°C in 2- to 3-liter

quantities at the National Drug Company (Swiftwater, 

PA). In 1971, the bulk was diluted 1:400 with modified

Earle’s medium and 0.5% human serum albumin, and 

then lyophilized. The freeze-dried product was then 

distributed under vacuum into 6-mL vials to provide 

convenient 10-dose vials at 0.5 mL per dose. The components of the TC-83 vaccine include 0.5% human 

serum albumin and 50 μg/mL each of neomycin and 

streptomycin. The vaccine is administered as a single 0.5 

mL subcutaneous injection (approximately 104 plaqueforming units per dose) in the deltoid area of the arm. 

Lot release testing was performed in animals, 

including a guinea pig safety test, mouse safety test, 

and guinea pig protection (potency) tests. The initial 

safety test challenge in the animals was a 0.5 mL dose 

of the vaccine (containing approximately 106

 virions) 

administered intraperitoneally. All animals survived. 

Additional rabbit, suckling mouse, mouse virulence, 

and monkey neurovirulence testing were conducted. 

The vaccine was protective against both subcutaneous and aerosol challenge with VEEV in mice and 

hamsters. In a monkey model of aerosol exposure, 

protection was inconsistent. Periodic postrelease potency analyses have shown that infectivity for all lots 

has declined by one to two logs from the original data 

in the IND 142 submitted in 1965.208

Vaccination is the mainstay of medical countermeasures against viral agents of bioterrorism. FDAapproved vaccines (eg, smallpox and yellow fever 

vaccines) and investigational vaccines (eg, vaccines 

against Rift Valley fever virus [RVFV] and Venezuelan, Eastern, and Western equine encephalitis viruses 

[VEEV, EEEV, and WEEV]) are available in the United 

States. Although antiviral agents and immunotherapy 

may be given postexposure, many of these therapies 

are investigational drugs with associated toxicities, 

and they may be in limited supply.

Encephalitic New World Alphaviruses

VEEV, WEEV, and EEEV are lipid-enveloped RNA 

viruses of the genus Alphavirus (family Togaviridae). 

These viruses, found in regions of North, Central, and 

South America, can cause severe neurologic disease in 

humans and equids, which typically are infected via 

the bite of an infected mosquito.200,201 Infections may 

also be acquired via respiratory exposure to aerosolized virus, as may occur in a laboratory setting or a 

bioterrorism event. The VEEV complex consists of at 

least 13 subtypes and varieties, including epidemic/

epizootic viruses, which are pathogenic to humans 

and equids, and enzootic viruses that are generally 

avirulent in equids but, in some cases, pathogenic to 

humans.202–205 Humans with VEEV infections typically present with nonspecific febrile illness. However, 

in up to 14% of patients, VEEV causes neurologic 

disease.202,203 WEEV infections typically are either asymptomatic or cause mild, nonspecific symptoms; 

a minority of patients experience encephalitis or 

encephalomyelitis. Of the three New World encephalitic alphaviruses, EEEV is the most likely to cause 

severe disease or death.203 Among the survivors of 

encephalitis, up to 30% may experience neurologic 

sequelae.14,66,202,203 Young children tend to develop more 

severe illness, such as encephalitis, compared with 

adults and older children.200,202

Vaccination

Vaccines are licensed for use in equids, but the only 

vaccines available for humans against VEE, WEE, and 

EEE are investigational (see Table 27-1).201 Laboratoryacquired infections with VEEV in particular became 

problematic soon after discovery of the agent in 

1938206 and remain a concern.11 Both a live attenuated 

VEE vaccine (TC-83) and an inactivated VEE vaccine 

(C-84) are available under IND status at USAMRIID. 

Formalin-inactivated vaccines against both EEE and 

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At-risk laboratory workers at Fort Detrick have 

received the TC-83 vaccine since 1963. Administration 

of this vaccine to more than 6,000 individuals in initial 

evaluations demonstrated its excellent immunogenicity.209 In a study of 624 vaccinees, Pittman and colleagues210 found that 513 (82%) responded to one dose 

of TC-83 with an 80% plaque reduction neutralization 

titer (PRNT80) of at least 1:20. However, because the

vaccine is derived from epizootic strains of VEEV, it 

may not protect against enzootic strains and may not 

adequately protect against distantly related VEEV 

subtype IAB variants.190

The severity and frequency of adverse events 

from the VEE TC-83 vaccine vary with the vaccine 

lot. Among 624 vaccinees, for example, 134 (21.5%) 

reported self-limited reactions, primarily systemic 

reactions such as malaise (reported by 90 vaccinees), 

headache (68), fever (65), chills (50), and myalgia 

(43). In some vaccinees, these symptoms were severe 

enough to require bedrest, but in all cases symptoms 

resolved without permanent effects.210 No person-toperson transmission of VEE has been documented after 

vaccination with TC-83. Local reactions are rarely seen. 

Some evidence has hinted at an association between 

glucose metabolism or insulin release and either infection with VEE or inoculation with the VEE TC-83 vaccine. In most studies in humans and in animal models, 

results have been inconclusive or negative.211–214 However, out of an abundance of caution, the vaccine is not 

given to individuals with a family history of diabetes 

in first-degree relatives. 

The VEE TC-83 vaccine has never been evaluated 

in pregnant women. In 1975, one spontaneous abortion occurred as a probable complication of TC-83 

vaccination. In 1985, a severe fetal malformation 

in a stillborn infant occurred in a woman whose 

pregnancy was unidentified at the time of vaccination.215 This kind of event has been reproduced in 

many animal models. Rhesus monkey fetuses were 

inoculated with VEE vaccine virus by the direct 

intracerebral route at approximately gestational 

day 100. Congenital microcephaly, hydrocephalus, 

and cataracts were found in all animals and porencephaly in 67% of the cases. The virus replicated 

in the brain and other organs of the fetus.216 VEE 

vaccine virus, which is teratogenic for NHPs, must 

be considered a potential teratogen of humans. The 

wild type VEE virus is known to cause fetal malformations, abortions, and stillbirths.217

Venezuelan Equine Encephalitis C-84 Vaccine. The 

VEE C-84 formalin-inactivated vaccine (IND 914, TSIGSD 205) was developed in part because of the high 

rate of adverse reactions in humans vaccinated with 

TC-83. C-84, which is made from the TC-83 production seed, has undergone one more passage through 

chick embryo fibroblasts (the number 84 refers to the 

number of passages). The vaccine is then inactivated 

with formalin, and the resultant product is freeze 

dried. The VEE C-84 vaccine contains neomycin and 

streptomycin at a concentration of 50 μg/mL, sodium 

bisulfite, chicken eggs, and formalin.

In animal models, the VEE C-84 vaccine’s efficacy, 

particularly in protecting against aerosol challenge, 

has been inconsistent.218–222 However, it has successfully been used to boost human vaccinees who 

have previously received the VEE TC-83 vaccine.210

Therefore, although the C-84 vaccine is not used for 

primary vaccination against VEE, it has been used in 

at-risk laboratory workers at Fort Detrick as a booster 

for those individuals who have received the VEE TC83 vaccine and have either (a) an inadequate initial 

response with a PRNT80 of no more than 1:20, or (b) 

an adequate initial response to VEE TC-83 but PRNT80

levels that subsequently drop below 1:20.

Adverse events tend to be minor. Among 128 individuals who received C-84 as a booster, only minor 

local reactions occurred in 6.3% of vaccinees.210 From 

2002 to 2006 at USAMRIID, 8% to 33% of individuals 

receiving C-84 as a booster through the Special Immunizations Program (SIP) reported a discernible adverse 

event. Most reactions were mild and self-limiting local 

reactions of swelling, tenderness, and erythema at the 

vaccine site. Systemic reactions were uncommon and 

consisted of headache, arthralgia, fatigue, malaise, 

influenza-like symptoms, and myalgia. All symptoms 

resolved without sequelae.

The vaccine is administered as a 0.5 mL subcutaneous injection above the triceps area. The current protocol used in the SIP allows for a maximum of four doses 

per year if postvaccination titers are not adequate. 

Western Equine Encephalitis Vaccine. The inactivated WEE vaccine (IND 2013, TSI-GSD 210) is a 

lyophilized product originating from the supernatant 

harvested from primary chicken fibroblast cell cultures.223 The vaccine was prepared from pathogen-free 

eggs infected with the attenuated CM4884 strain of 

WEEV. The supernatant was harvested and filtered, 

and the virus was inactivated with formalin. The residual formalin was neutralized by sodium bisulfite. 

The medium contains 50 μg each of neomycin and 

streptomycin and 0.25% (weight/volume) of human 

serum albumin (US Pharmacopeia). The freeze-dried 

vaccine must be maintained at −25°C (±5°C) in a designated vaccine storage freezer. The National Drug 

Company originally manufactured the inactivated 

WEE vaccine. The current product, lot 3-1-92, was 

manufactured at the Salk Institute, Government Services Division (Swiftwater, PA) in 1992. Potency tests 

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have been conducted every 2 to 3 years since then, 

initially at the Salk Institute and then at Southern 

Research Institute (Frederick, MD).

Animal studies showed that the vaccine protected 

mice against intracerebral challenge with WEEV224 and 

protected hamsters against intraperitoneal challenge.225

The inactivated WEE vaccine protected 17 of 17 horses 

against intradermal challenge 12 months after vaccination, even in the absence of detectable WEE protective 

neutralizing antibodies.226 Human subjects who were 

administered the WEE vaccine subcutaneously (either 

0.5 mL at days 0 and 28 or 0.5 mL at day 0 and 0.25 

mL at day 28) showed similar serologic responses. 

Neutralizing antibody titers did not occur until day 14 

after the first dose of vaccine in each group. The mean 

log neutralization index was 1.7 and 1.8, respectively, 

at day 28 after the first dose. The antibody remained 

at acceptable levels through day 360 in 14 of 15 volunteers. Side effects from the vaccine were minimal, 

consisting primarily of headache, myalgias, malaise, 

and tenderness at the vaccination site.223

The inactivated WEE vaccine has been administered to at-risk personnel at Fort Detrick since the 

1970s. Pittman and colleagues evaluated the vaccine for its immunogenicity and safety in 363 at-risk 

workers enrolled in evaluation trials at USAMRIID 

between 1987 and 1997. All volunteers received subcutaneous injections with 0.5 mL of the inactivated 

WEE vaccine (lot 81-1) in an initial series of three 

doses, administered up to day 42 (the intended schedule was 0, 7, and 28 days). For individuals whose 

PRNT80 fell below 1:40, a booster dose (0.5 mL) was

administered subcutaneously. Serum samples for 

neutralizing antibody assays were collected before 

vaccination and approximately 28 days after the last 

dose of the initial series and each booster dose. Of 

these vaccinees, 151 subjects (41.6%) responded with 

a PRNT80 of greater than or equal to 1:40. Of 115 initial nonresponders, 76 (66%) converted to responder 

status after the first booster dose. A vaccination 

regimen of three initial doses and one booster dose 

provided protection lasting for 1.6 years in 50% of 

initial responders (unpublished data). 

Passive collection was used to record local and 

systemic adverse events from the inactivated WEE 

vaccine from 1987 to 1997. Of the 363 vaccinees who 

received three initial injections, only 5 reported local 

or systemic reactions. These reactions usually occurred 

between 24 and 48 hours after vaccine administration. 

Erythema, pruritus, and induration were reported after just one of the initial vaccinations. Two volunteers 

also reported influenza-like symptoms after the initial 

dose. All reactions were self-limited. No reactions were 

reported after 153 booster doses.

Recent active collection of adverse events from 2002 

through 2006 in the SIP revealed a reaction rate of 

15% to 20% following the primary series. The reaction 

rate was less for booster doses than for primary series 

doses. The majority of these symptoms were systemic 

and consisted of headache, sore throat, nausea, fatigue, 

myalgia, low-grade fever, and malaise. The duration 

of these adverse events was less than 72 hours. The 

vaccine has not been tested for teratogenicity or abortogenicity in any animal model, nor has it been tested 

in pregnant women; therefore, it is not advisable to 

vaccinate pregnant women.

According to the current SIP protocol, the primary 

series of the WEE vaccine is given subcutaneously 

at days 0, 7, and 28; a mandatory booster is given at 

month 6, with subsequent booster doses (up to four 

in a 12-month period) administered if and when the 

PRNT80 titer falls below 1:40.

Eastern Equine Encephalitis Vaccine. The Salk 

Institute manufactured the formalin-inactivated EEE 

vaccine (TSI-GSD 104) in 1989.227 The seed for the EEE 

vaccine was passaged twice in adult mice, twice in 

guinea pigs, and nine times in embryonated eggs.228

The final EEE vaccine was derived from supernatant 

fluids bearing virus accumulated from three successive 

passages in primary chick embryo fibroblast cell cultures prepared from pathogen-free eggs infected with 

the attenuated PI-6 strain of virus. The supernatant 

was harvested and filtered and the virus inactivated 

with formalin. The product was then lyophilized for 

storage at −20°C.

Animal studies have demonstrated that the EEE vaccine is 95% protective against intracerebral challenge 

with EEEV in guinea pigs, with survival correlating 

to serum neutralizing antibody titers.229 Vaccination 

of horses was also protective against intradermal 

challenge at 12 months postvaccination, even with an 

absence of detectable neutralizing antibody titers in 

16 of the 17 animals.226

The vaccine has been given to at-risk laboratory 

workers at Fort Detrick for more than 25 years. The 

response rate of 255 volunteers who received two 

primary vaccinations between 1992 and 1998 was 

77.3% (197 individuals), with a response defined 

as a PRNT80 of 1:40 or greater. Intradermal vaccination with the EEE vaccine resulted in an adequate 

titer in 66% of the initial nonresponders. Adverse 

events from the EEE vaccine, which occurred in 

approximately 20% of these individuals, consisted 

of headache, myalgias, and light-headedness. All 

symptoms subsided within several days. Mild and 

self-limiting local reactions of induration, erythema, 

pruritus, or pain at the vaccination site have also 

been reported (unpublished data). 

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The EEE vaccine contains 50 μg/mL of both neomycin and streptomycin and 0.25% (weight/volume) 

of human serum albumin. The initial vaccine dose 

is given as a 0.5 mL injection subcutaneously above 

the triceps area. A postvaccination PRNT80 of 1:40 or

greater is considered adequate. If the titer falls below 

1:40, a booster dose of 0.1 mL should be given intradermally on the volar surface of the forearm. Booster 

doses must be given at least 8 weeks apart.

Vaccine Research. The live attenuated VEE vaccine 

candidate V3526 was scheduled to replace the 50-yearold VEE TC-83 IND vaccine. This VEE vaccine candidate, a recombinant vaccine derived from the Trinidad 

donkey strain of VEEV, had improved activity against 

VEE enzootic strains. In phase 1 clinical trials, the vaccine elicited strong immune responses. However, because of high rates of severe neurologic adverse events 

in these trials, further development of this product was 

halted. These high rates were unexpected with V3526 

because it demonstrated less reactogenicity in NHP 

studies than the VEE TC-83 product. Recently, research 

in mice has suggested that a formalin-inactivated 

V3526 vaccine could replace C-84.230

Another line of research has explored the use of 

live chimeric Sindbis virus (an Old World alphavirus 

that is among the least pathogenic alphaviruses in 

humans) engineered to express structural proteins 

of VEEV, EEEV, or WEEV. Studies in animal models 

suggest that this approach has promise for all three 

New World alphaviruses. Other approaches include 

DNA vaccines expressing proteins of the TC-83 and 

Trinidad donkey strains of VEEV, viral-vectored vaccines, and nonreplicating virus-like particles.200,201 In 

a recent phase 1 clinical trial, a DNA vaccine against 

VEEV was well tolerated, with VEEV-neutralizing 

antibodies detected in 100% of subjects receiving the 

vaccine via intramuscular electroporation and in 63% 

to 88% of those receiving the vaccine via intradermal 

electroporation.230a

Many of the existing New World encephalitic alphavirus vaccines have been under IND status for more 

than 30 years. For several reasons, including funding 

shortfalls, these products have never been transitioned 

from development to licensure. 

Passive Immunotherapy

Hyperimmune serum has protected animals from 

lethal challenge with VEEV, WEEV, and EEEV. This 

line of work has progressed toward safer approaches 

using humanized murine monoclonal antibodies. 

Administration of humanized murine monoclonal 

antibodies against a VEEV envelope protein protected 

75% to 100% of mice challenged with lethal doses of 

VEEV if the antibodies were given within 24 hours 

after exposure; delaying administration to 48 hours 

postexposure greatly reduced the efficacy. Similar 

results have been found in animal models with the 

administration of human antibodies or human-like 

(macaque) antibody fragments.200

Antiviral Agents

Research on antiviral compounds effective against 

the encephalitic New World alphaviruses remains at 

an early stage. Although approaches using interferons (IFNs) and toll-like receptors have shown some 

promise in animal models, they must be administered 

before and after exposure.200 Some evidence suggests 

that carbodine (carbocyclic cytosine) may have potential as an antiviral agent to treat VEE postexposure.231

Recently, Chung et al232 reported their discovery and 

characterization of a novel anti-VEEV and anti-WEEV 

compound, the quinazolinone CID15997213. They 

found that this small molecule inhibited VEEV and 

WEEV by inhibiting viral RNA, protein, and progeny 

synthesis, specifically by targeting the nsP2 protein. In 

mice, administration of CID15997213 did not result in 

any signs of acute toxicity, and it provided complete 

protection from a lethal VEEV challenge at 50 mg/kg/

day.232

Postevent Countermeasures: Current Options

No treatment has been shown to alter the course 

of VEE, WEE, or EEE in humans once disease has 

been contracted. At this time, treatment is limited to 

supportive care. No PEP exists for the New World 

encephalitic alphaviruses. In the context of a laboratory exposure, previously vaccinated individuals 

who are exposed to EEEV, WEEV, or VEEV may be 

offered a booster dose of the appropriate vaccine if 

their antibody levels are inadequate66; however, in a 

mass casualty event, limited vaccine supplies would 

likely preclude large-scale vaccination. 

Smallpox

Smallpox is caused by variola virus, a DNA virus 

of the genus Orthopoxvirus. Once distributed globally, 

this disease was the greatest infectious cause of human 

mortality for centuries. In 1980, after an intensive vaccination program, the WHO declared that the disease 

was eradicated.233 Subsequently, all known stocks of 

variola virus were destroyed, with the exception of 

stocks at two WHO collaborating centers: (1) the CDC

and (2) the Russian State Research Center of Virology 

and Biotechnology. 

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Smallpox is readily transmitted from person to 

person via direct contact, droplets, aerosol, and contaminated fomites such as clothing and bedding.234,235

Smallpox has been designated a category A biothreat 

agent because of its high mortality, high transmissibility, the potential for aerosol dissemination and 

transmission, and history of massive weaponization 

by the former Soviet Union.

Vaccination

History of Smallpox Vaccination. Vaccination 

against smallpox was recorded in 1,000 bce in India and 

China, where individuals were inoculated with scabs 

or pus from smallpox victims (in either the skin or the 

nasal mucosa), producing disease that was milder than 

naturally occurring smallpox. In the 18th century in 

Europe, scratching and inoculation of the skin with 

material taken from smallpox lesions, known as variolation, was performed, resulting in a 90% reduction 

in mortality and long-lasting immunity. (Variolation 

was also performed using the pustules of a previously 

variolated individual.) In 1722, variolation of 242 individuals in Boston resulted in a smallpox death rate of 

2.5% (6 persons) compared to a death rate of 14% in unvaccinated persons (849 deaths among 5,889 cases).236

In 1770, Edward Jenner noticed that milkmaids

who had been exposed to cowpox virus (another orthopoxvirus) rarely had smallpox scars. Subsequently, 

Jenner discovered that inoculation of the skin with

cowpox virus taken from a milkmaid’s hand resulted 

in immunity. This early form of vaccination began in 

1796. Beginning in the mid-1840s, the smallpox vaccine was manufactured in calfskin. The virus used as 

the vaccine, though originally cowpox virus, changed 

over time and eventually was found to be a distinct 

virus whose precise origins were unknown; this virus 

became known as vaccinia virus.236 Production of the 

vaccine became regulated in 1925, with the New York 

City Board of Health strain of vaccinia as the primary 

US vaccine strain. Global vaccination efforts eventually led to eradication of the disease; the last known 

case of naturally occurring smallpox was reported in 

1977.233 Routine vaccination of US children ceased in 

1971, and vaccination of hospital workers ceased in 

1976. Finally, vaccination of military personnel was 

discontinued in 1989.234,236,237

Because of renewed concerns over the risk of bioterrorism, vaccination against smallpox in at-risk military 

personnel was resumed in 2003 using Dryvax (Wyeth 

Laboratories, Marietta, PA), a live-virus preparation 

of vaccinia virus (the New York City Board of Health 

strain) made from concentrated, lyophilized calf 

lymph. 

Dryvax and similar first-generation smallpox vaccines, which had been used in the global smallpox 

eradication campaign, were known to prevent smallpox. However, Dryvax was manufactured from the 

lymph collected from the skin of live animals scarified 

with vaccinia virus. Because of risks from adventitious 

viruses and subpopulations of virus with undesirable 

virulence properties, the manufacture of a cell culture–

derived (second-generation) vaccine was preferable to 

the animal-derived product.238 Dryvax was replaced by 

ACAM2000 in 2007.239

Current Smallpox Vaccine. The smallpox vaccine 

used in the United States today, ACAM2000 (Sanofi 

Pasteur Biologics, Cambridge, MA), is a cell culture–

based live vaccinia virus vaccine licensed by the FDA 

for prophylaxis against variola virus (see Table 27-1).238

ACAM2000 is a lyophilized preparation that is free 

of adventitious agents and contains trace amounts of 

neomycin and polymyxin B. The diluent for the vaccine contains 50% glycerin and 0.25% phenol in US 

Pharmacopeia sterile water.240

Protection against smallpox is from both humoral 

and cell-mediated immunity; the latter provides the 

main protection. Humoral responses of neutralizing 

and hemagglutination inhibition antibodies to the 

vaccine appear between days 10 and 14 after primary 

vaccination, and within 7 days after secondary vaccination. Clinical trials have shown that administration 

of ACAM2000 results in cutaneous, antibody, and T 

cell responses that are comparable to those elicited 

by Dryvax. The safety profile of the two vaccines also 

appears to be similar.241,242 

ACAM2000 is administered by scarification (percutaneously) to the upper arm over the deltoid muscle 

area with 15 jabs using a bifurcated needle.14,240 The 

individual is followed after vaccination to document a 

take reaction, a vesiculo-papular response that indicates 

immunity against smallpox. Six to 8 days after the primary vaccination, a primary major reaction to the vaccine 

develops—a clear vesicle or pustule with a diameter of 

approximately 1 cm. The site then scabs over by the end 

of the second week, with the scab drying and separating 

generally by day 14 to 21 (Figure 27-4). First-time vaccinees who do not exhibit either a primary major reaction or an immune response require revaccination. If no 

primary reaction is noted after revaccination (and after 

ensuring that proper technique in vaccine administration 

was used), these revaccinees are considered immune.240 

At some point in the future (which may be years), the 

immunity of vaccinated individuals may wane, and 

revaccination at that time may again result in a take.

The CDC recommends vaccination with confirmation of a take at least every 10 years for laboratory researchers working with nonhighly attenuated vaccinia 

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viruses, recombinant viruses developed from vaccinia 

viruses, and other nonvariola orthopoxviruses. For 

increased protection against more virulent nonvariola 

orthopoxviruses, such as monkeypox, revaccination 

every 3 years may be appropriate.190,243 Individuals 

working with variola virus in the laboratory (at CDC) 

are required to receive a smallpox vaccination every 3 

years (K S Meadows, Centers for Disease Control and 

Prevention, written communication, December 2015). 

In the event of a smallpox release from a bioterrorism attack, individuals would be vaccinated according to the national policy. The current national 

policy244 recommends vaccination initially of higher 

risk groups, including individuals directly exposed to 

the agent, household contacts or individuals with close 

contact to smallpox cases, and medical and emergency 

transport personnel. Ring vaccination—vaccination of 

contacts and contacts of the contacts in concentric rings 

around an identified active case—is the strategy that 

was used to control smallpox during the final years 

of the eradication campaign. In a postevent setting, 

there are no absolute contraindications to vaccination 

for an individual with high-risk exposure to smallpox. 

Persons at greatest risk of complications of vaccination 

are those for whom smallpox infection also poses the 

greatest risk. If relative contraindications exist for an 

exposed individual, then risks of adverse complications from vaccination must be weighed against the 

risk of a potentially fatal smallpox infection.

Secondary attack rates (ie, estimates of the risk 

of transmission from a primary case to secondary 

contacts of that case) from smallpox in unvaccinated 

persons have generally ranged from 36% to 88%, with 

an average rate of 58%. Household contacts in close 

proximity to the smallpox case for 4 hours or longer 

are at a higher risk for acquiring infection. In an outbreak recorded in the Shekhupura District of Pakistan 

during the smallpox era, the secondary attack rate was 

only 4% in persons vaccinated with a first-generation 

vaccinia virus vaccine within the previous 10 years 

(5/115) and 12% in persons vaccinated more than 10 

years before (8/65) compared with 96% in unvaccinated 

persons (26/27).245,246 Estimates of vaccine protection 

from imported cases of variola major between 1950 

and 1971 in Western countries, where immunity from 

smallpox would be expected to be mainly from vaccination, showed a case fatality rate (CFR) of only 1.4% 

in individuals who had received the smallpox vaccine 

within the previous 10 years, compared with a 52% 

mortality rate in individuals who had never received 

the vaccine, 7% mortality in individuals vaccinated 11 

to 20 years before, and 11% mortality in individuals 

vaccinated more than 20 years before. Postexposure 

vaccination resulted in 27% less mortality when compared (retrospectively) with smallpox patients who 

were never vaccinated.245

The effectiveness of postexposure vaccination appears to be greatest in the first 3 to 4 days after exposure 

to variola virus. In a recent review of historical data 

before the eradication of smallpox, Keckler and colleagues247 found that vaccination of contacts decreased 

mortality and/or reduced morbidity in 100%, 75%, 67%, 

58%, and 42% of reports when the (first-generation) 

smallpox vaccine was administered less than 1, 3, 5, 7, 

or 9 days postexposure, respectively. However, these 

historical data have a number of limitations, including the potential underestimation of prior immunity 

(from previous vaccination or exposure) in the patients 

described. Thus, Keckler et al also analyzed modern 

studies using animal models to assess the efficacy of 

postexposure vaccination (which, in these surrogate 

models, is also postinfection). In several of these 

studies using NHP and murine models, vaccination 

on postexposure day 0 or 1 resulted in 80% to 100% 

survival, and vaccination on postexposure day 2 or 

3 resulted in 15% to 100% survival. However, in two 

studies, survival was consistently nearly 0% regardless 

of the day of vaccination. Conclusions from the animal 

models are difficult because of the diversity among 

models and the variability across species in the course 

of the disease.247

Vaccine Contraindications. According to the Advisory Committee on Immunization Practices (ACIP) and 

the Healthcare Infection Control Practices Advisory 

Committee,248 smallpox vaccination is contraindicated 

in the pre-event setting for individuals who:

Figure 27-4. Take reaction in response to primary smallpox 

vaccination at (a) day 4, (b) day 7, (c) day 14, and (d) day 21. 

Reproduced from: Centers for Disease Control and Prevention website. http://www.bt.cdc.gov/agent/smallpox/

smallpox-images/vaxsit5a.htm. Accessed September 16, 2014. 

a b

c d

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TABLE 27-3 

CONTRAINDICATIONS TO AND PRECAUTIONS FOR PRE-EVENT SMALLPOX VACCINATION

Condition Contraindication or Precaution

Allergy Do not administer to those with allergies to vaccine components (eg, neomycin, polymyxin B). 

Where risk is great, vaccine should be administered with subsequent use of antihistamine or 

other appropriate medication.

Eczema (atopic Do not administer to those with a history of eczema or Darier disease, even if no rash is present.

dermatitis) or Darier Recent vaccinees should be counseled to avoid contact with individuals who have eczema or

disease (keratosis Darier disease.

follicularis) 

Other skin conditions	 Do not administer to those with disruptive or eruptive skin conditions, such as:

• Severe acne

• Burns

• Impetigo

• Contact dermatitis or psoriasis

• Chicken pox

The vaccine may be administered after the condition resolves or if the (noneczema/atopic) skin 

condition is sufficiently small and the patient is counseled to take great care to prevent transfer 

of vaccinia virus from vaccination site to affected skin. 

Vaccinees should be counseled to avoid contact with individuals who have a disruptive or 

eruptive skin condition.

Pregnancy or Do not administer to patients who are pregnant or breastfeeding. Advise vaccinees not to become 

breastfeeding 	 pregnant for ≥1 month after vaccination.

Recent vaccinees should be counseled to avoid contact with individuals who are pregnant or 

breastfeeding.

Infancy Do not administer to patients younger than 1 year old.

Recent vaccinees should be counseled to avoid contact with infants.

Immunodeficiency	 Do not administer to patients with diseases that have an immunodeficiency component, such as:

• Human immunodeficiency virus infection

• AIDS

• Many cancers

• Autoimmune diseases

Immunosuppressive	 Do not administer to patients who are currently taking immunosuppressive therapies, such as:

therapy • Cancer treatments

• Some treatments for autoimmune diseases

• Organ transplant maintenance

• Steroid therapy (equivalent to 2 mg/kg or greater of prednisone daily or 20 mg/day if given 

 for 14+ days), including medication for treatment of inflammatory eye disease

Immunosuppression from some medications may last for up to 3 months after discontinuation. 

Cardiovascular Do not administer vaccine to patients with a history of, or significant risk factors for, ischemic 

disease or risk heart disease, myocarditis, or pericarditis or those with significant cardiac risk factors (eg, 

factors hypertension, high cholesterol, or diabetes).

Simultaneous Do not administer these vaccines simultaneously because the resulting skin lesions are difficult to 

administration of distinguish.

varicella vaccine

Moderate or severe Do not administer vaccine to patients who are moderately or severely ill at the time of vaccination.

illness

(Table 27-3 continues)

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• have a history or presence of atopic dermatitis 

(eczema);

• have active acute, chronic, or exfoliative skin 

conditions disruptive of the epidermis or have 

Darier disease (keratosis follicularis);

• are pregnant or breastfeeding;

• have conditions associated with immunosuppression;

• have a serious allergy to any of the vaccine 

components;

• are younger than 1 year old; or

• have close physical contact with a person who 

(a) has a history or presence of atopic dermatitis or other acute, chronic, or exfoliative skin 

conditions; (b) has a condition associated with 

immunosuppression; or (c) is pregnant (Table 

27-3).248

The CDC has recently added underlying cardiac 

disease (eg, a history of ischemic heart disease, myocarditis, or pericarditis) or significant cardiac risk factors (eg, hypertension, high cholesterol, or diabetes) as 

relative contraindications to the vaccine in a pre-event 

setting; however, these exclusions may be temporary, 

pending the results of further research into the possible link between smallpox vaccination and cardiac 

disease.249

In addition to the contraindications listed above, 

the ACIP does not recommend vaccination of persons 

younger than 18 years old in the pre-event setting.248

Furthermore, although the presence of an infant in the 

household is not an absolute contraindication for vaccination of an adult, the ACIP recognizes that vaccination programs should defer vaccination of individuals 

whose households include infants younger than 1 year 

old because of data indicating a higher risk for adverse 

events among primary vaccinees in this age group. Because skin lesions resulting from the varicella vaccine 

may be confused with vaccinia lesions, simultaneous 

administration of the smallpox and varicella vaccines 

is not recommended.248

During an outbreak or after an intentional release of 

variola virus, there are no absolute contraindications 

to vaccination for any person who has been exposed 

to smallpox. However, if pregnant or eczematous 

persons are vaccinated under such circumstances, vaccinia immune globulin (VIG) could be administered 

concomitantly.234 

Complications of Vaccination. Vaccinia virus can 

be transmitted (shed) from a vaccinee’s unhealed 

vaccination site—or from lesions caused by autoinoculation, generalized vaccinia, eczema vaccinatum, 

or progressive vaccinia (see below)—to other persons 

by close contact. The virus can survive on fomites for 

at least several days.250 Contact transmission can lead 

to adverse events that are identical to those that could 

be caused by intentional vaccination. In addition, viral 

shedding from the vaccination site can cause autoinoculation, in which the vaccinee spreads infection 

from the vaccination site to other areas, such as the 

eye, where vaccinia virus infection is associated with 

significant morbidity (Figures 27-5 and 27-6). 

Although medical personnel are currently taught 

that vaccinia virus is shed from the vaccination site 

only until the scab (from the take reaction) separates,240,251 Pittman et al252 recently found that up to 

23% of vaccinees continued to shed vaccinia virus 

after scab separation and as late as postvaccination 

day 42. From December 2002 to March 2011, a period 

when approximately 2.1 million military personnel 

and 40,000 civilian emergency responders were vaccinated against smallpox with Dryvax or ACAM2000, 

the incidence of vaccinia transmission through contact 

was 5.4 per 100,000 vaccinees. Generally, the virus was 

transmitted to household members, intimate contacts, 

or sports contacts. Most cases were mild; only 1 of 115 

cases was life threatening.253 Between March 2008 and 

August 2010 (when only ACAM2000 was used) another 

group reported an incidence of contact transmission of 

4.4 per 100,000 vaccinations and an incidence of autoinoculation of up to 20.6 per 100,000 vaccinations (Table 

27-4).254 To avoid inadvertent transmission, vaccinees 

Active eye disease of Patients with inflammatory eye diseases may be at increased risk for autoinoculation of the eye by 

the conjunctiva or touching or rubbing the eye after touching the vaccination site. Such patients can be vaccinated 

cornea but should be counseled to take great care to prevent transfer of vaccinia virus to the eye. 

Notes: During an outbreak, after a biological terrorism event, orforindividuals with a high risk of exposure to variola virus, no contraindications are absolute. Persons at greatest risk of complications from vaccination are those for whom smallpox infection poses the greatest risk.

Data sources: (1) Dembek Z, ed. USAMRIID’s Medical Management of Biological Casualties Handbook. 7th ed. Fort Detrick, MD: US Army

Medical Research Institute of Infectious Diseases; 2011. (2) Wharton M, Strikas RA, Harpaz R, et al. Recommendations for using smallpox 

vaccine in a pre-event vaccination program: supplementalrecommendations of the Advisory Committee on Immunization Practices (ACIP)

and the Healthcare Infection Control Practices Advisory Committee (HICPAC). MMWR Recomm Rep. 2003;52(RR-7):1–16.

Table 27-3 continued

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should wash their hands with soap and water or use 

antiseptic hand rubs immediately after touching the 

vaccination site and after dressing changes. Vacciniacontaminated dressings should be placed in sealed 

plastic bags and disposed of in household trash. Two 

recent studies have demonstrated that the application 

of povidone iodine ointment to the vaccination site can 

reduce viral shedding.252,255

Smallpox vaccine adverse reactions are diagnosed 

by clinical exam. Most reactions can be managed with 

observation and supportive measures. Self-limited 

reactions include fever, headache, fatigue, myalgia, 

chills, local skin reactions, nonspecific rashes, erythema multiforme, lymphadenopathy, and pain at the 

vaccination site. Adverse reactions that require further 

evaluation and possible therapeutic intervention 

include inadvertent inoculation involving the eye, 

generalized vaccinia, eczema vaccinatum, progressive 

vaccinia, postvaccinial central nervous system disease, 

and fetal vaccinia (Tables 27-4 and 27-5).256,257

Inadvertent inoculation generally results in a condition that is self-limited unless the inoculation involves 

the eye or eyelid, which requires evaluation by an 

ophthalmologist (see Figure 27-6).258

Generalized vaccinia is characterized by a disseminated maculopapular or vesicular rash, frequently on 

an erythematous base and typically occurring 6 to 9 

days after primary vaccination (Figure 27-7). Generalized vaccinia must be distinguished from other 

postvaccination exanthems, such as erythema multiforme and roseola vaccinatum (Figure 27-8). Lane et al 

reported 242.5 cases per million primary vaccinations 

and 9.0 cases per million revaccinations in a 1968 tenstate survey of smallpox vaccination complications.259

The rash usually resolves without therapy. Contact 

precautions should be used to prevent further transmission and nosocomial infection.258

Eczema vaccinatum may occur in individuals with 

a history of atopic dermatitis, regardless of current 

disease activity, and it can be a papular, vesicular, or 

Figure 27-6. Ocular vaccinia. This 2-year-old child presented 

with a case of ocular vaccinia from autoinoculation. Ocular 

vaccinia is an eye infection that can be mild to severe and 

can lead to a loss of vision. It usually results from touching 

the eye when the vaccinia virus is on the hand. Image 5219.

Reproduced from: Centers for Disease Control and Prevention Public Health Image Library website, http://phil.CDC.

gov. Accessed September 16, 2014. Photograph: Courtesy

of Allen W Mathies, MD, and John Leedom, MD, California

Emergency Preparedness Office, Immunization Branch. 

Figure 27-5. Accidental autoinoculation. This 22-month-old 

child presented after having autoinoculated his lips and 

cheek 9 days postvaccination. Autoinoculation involves the 

spread of the vaccinia virus to another part of the vaccinee’s 

body, caused by touching the vaccination site and then touching another part of the body. Image 4655.

Reproduced from: Centers for Disease Control and Prevention Public Health Image Library website, http://phil.CDC.

gov. Accessed September 16, 2014. Photograph: Courtesy

of Allen W Mathies, MD, and John Leedom, MD, California

Emergency Preparedness Office, Immunization Branch.

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TABLE 27-4

RATES OF ADVERSE EVENTS AFTER SMALLPOX VACCINATION

Type of Event

Rate per 100,000 Vaccinations 

Mar 2008–Jun 2013*

(only serious AEs)

Mar 2008–

Aug 2010†

Dec 2002–

Mar 2011‡

Dec 2002–

May 2003§

Historic 

Estimates¥

Autoinoculation 0.6 6.4–20.6 — 10.7 60.6

Contact transmission 0.5 3.5–4.4 5.4 4.7 0.8–2.7

Myo/pericarditis 1.9 — — 8.2 10

Ischemic cardiac event 1.8 — — — —

Eczema vaccinatum 0.1 — — 0 0.2–3.5

Progressive vaccinia 0.1 — — 0 0.1–0.7

Meningitis 0.5 — — — —

Encephalitis — — — 0.2 0.3–0.9

Death 0.1 — — 0 0.1–0.2

Note: Dash indicates event not assessed. *

During this period, a total of approximately 834,465 doses of ACAM2000 were administered (approximately 832,035 to US military service members and 2,430 to US civilians). Events noted here are only those considered “serious” (those resulting in permanent disability, 

hospitalization or prolongation of hospitalization, life-threatening illness, or death). Reports of events were those submitted to the Vaccine 

Adverse Event Reporting System (VAERS) (see data source reference 4).

†

During this period, 451,518 doses of ACAM2000 were administered (450,284 to US military service members and 1,234 to US civilians). 

The first number in each range includes only “suspect” and “confirmed” cases; the second number also includes “possible” cases. Reports 

included those submitted to VAERS as well as other sources (see data source reference 2).

‡

During this period, approximately 2.1 million doses of smallpox vaccine (Dryvax until 2008, ACAM2000 thereafter) were administered to 

US military personnel and approximately 40,000 doses were administered to US civilians. Reports of contact transmission were assessed 

using the medical literature, VAERS, and the Defense Medical Surveillance System (see data source reference 1). §

During this period, approximately 450,293 doses of Dryvax were administered to military service members. Events noted here include 

those the authors considered “moderate or serious” (encephalitis, myopericarditis, eczema vaccinatum, progressive vaccinia, and death) as 

well as those they considered “mild or temporary” (inadvertent autoinoculation and contact transmission). Adverse events were collected 

in a variety of ways, including from VAERS (see data source reference 3). ¥

From smallpox vaccinations in US civilians (adults and adolescents) and (for myopericarditis) Finnish military personnel. See Grabenstein 

and Winkenwerder (data source reference 3) and sources cited therein.

AE: adverse event

Data sources: (1) Wertheimer ER, Olive DS, Brundage JF, Clark LL. Contact transmission of vaccinia virus from smallpox vaccinees in the

United States, 2003–2011. Vaccine. 2012;30:985–988. (2) Tack DM, Karem KL, Montgomery JR, et al. Unintentional transfer of vaccinia virus

associated with smallpox vaccines ACAM2000 compared with Dryvax. Human Vaccin Immunother. 2013;9:1489–1496. (3) Grabenstein JD,

Winkenwerder WJ Jr. US military smallpox vaccination program experience. JAMA. 2003;289:3278–3282. (4) McNeil MM, Cano M, Miller

ER, Petersen BW, Engler RJ, Bryant-Genevier MG. Ischemic cardiac events and other adverse events followingACAM2000 smallpox vaccine

in the Vaccine Adverse Event Reporting System. Vaccine. 2014;32:4758–4765.

pustular rash (Figure 27-9). Historically, eczema vaccinatum occurred at a rate of 14.1 and 3.0 per million 

primary and revaccinations, respectively259; however, 

in more recent military experience, no cases of eczema 

vaccinatum occurred in 450,293 smallpox vaccinations 

(of which 70.5% were primary vaccinations).257 The 

rash may be generalized or localized with involvement 

anywhere on the body, especially areas of previous 

atopic dermatitis lesions. 

Progressive vaccinia is a rare, severe, and often fatal 

complication of vaccination that occurs in individuals 

with immunodeficiency conditions. It is characterized 

by painless progressive necrosis at the vaccination site 

with or without metastases to distant sites (Figures 

27-10 and 27-11). Those at highest risk include persons

with congenital or acquired immunodeficiencies, HIV 

infection/AIDS, cancer, or autoimmune disease and 

those who have undergone organ transplantation 

or immunosuppressive therapy. Estimated rates of 

progressive vaccinia ranged from 1 to 3 per million 

vaccinees historically,259 no cases in 450,293 US military 

vaccinees,257 and no cases (that met case definition) in 

38,440 US civilian vaccinees in 2003.260

Although rare, central nervous system disease, which 

includes postvaccinial encephalopathy and postvaccinial encephalomyelitis, is the most frequent cause of 

death related to smallpox vaccination.259 Postvaccinial 

encephalopathy occurs more frequently than encephalomyelitis, typically affects infants and children younger 

than 2 years old, and reflects vascular damage to the central nervous system. Symptoms typically occur 6 to 10 

days after vaccination and include seizures, hemiplegia, 

aphasia, and transient amnesia. Histopathologic findings include cerebral edema, lymphocytic meningeal 

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TABLE 27-5

VACCINIA IMMUNE GLOBULIN ADMINISTRATION FOR COMPLICATIONS OF SMALLPOX 

(VACCINIA) VACCINATION

Indicated Not Recommended

• Inadvertent autoinoculation, extensive • Inadvertent autoinoculation, mild

 lesions or severe ocular vaccinia • Generalized vaccinia, mild (most cases)

 (without evidence of vaccinial keratitis) • Erythema multiforme

• Eczema vaccinatum • Vaccinial keratitis*

• Generalized vaccinia, severe or recurrent • Central nervous system complications 

• Progressive vaccinia

Note: Data are not available on the efficacy of prophylactic vaccinia immune globulin (VIG) for a pregnant woman to prevent fetal vaccinia

or on the efficacy of VIG as a treatment for an infant born with fetal vaccinia. 

*

VIG is contraindicated for persons with vaccinial keratitis as it may produce severe corneal opacities. An exception may be made in persons 

with additional, potentially fatal complications that may respond to VIG; in such cases, the choice may be one of life vs vision.

Data sources: (1) Rusnak J, ed. Occupational Health Manual for Laboratory Exposures to Select (BSL-3 & BSL-4) and Other Biological Agents. 3rd ed. 

Fort Detrick, MD: USArmy Medical Research Institute of Infectious Diseases; 2011. (2) Cono J, Casey CG, Bell DM; Centers for Disease Control

and Prevention. Smallpox vaccination and adverse reactions: guidance for clinicians. MMWR Recomm Rep. 2003;52(RR-4):1–28. (3) Lane JM,

Ruben FL, Neff JM, Millar JD. Complications of smallpox vaccination, 1968: results of ten statewide surveys. J Infect Dis. 1970;122:303–309.

(4) Lane JM, Ruben FL, Abrutyn E, Millar JD. Deaths attributable to smallpox vaccination, 1959 to 1966, and 1968. JAMA. 1970;212:441–444.

(5) Sejvar JJ, Labutta RJ, Chapman LE, Grabenstein JD, Iskander J, Lane JM. Neurologic adverse events associated with smallpox vaccination in the United States, 2002–2004. JAMA. 2005;294:2744–2750. (6) Ruben FL, Lane JM. Ocular vaccinia: an epidemiologic analysis of 348

cases. Arch Ophthalmol. 1970;84:45–48. (7) Fulginiti VA, Winograd LA, Jackson M, Ellis P. Therapy of experimental vaccinal keratitis: effect

of idoxuridine and VIG. Arch Ophthalmol. 1965;74:539–544. (8) Kempe CH. Studies on smallpox and complications of smallpox vaccination.

Pediatrics. 1960;26:176–189. (9) Military Vaccine Agency. Pregnancy Discovered after Smallpox Vaccination–IV-VIG. Silver Spring, MD: Vaccine

Healthcare Centers Network; 2013. Information Paper.

inflammation, ganglion degeneration, and perivascular 

hemorrhage. Patients with postvaccinial encephalopathy who survive can be left with cerebral impairment 

and hemiplegia. Postvaccinial encephalomyelitis, 

which generally affects individuals aged 2 years or 

older, is characterized by abrupt onset of fever, vomiting, malaise, and anorexia occurring approximately 11 

to 15 days after vaccination.258,261 Neff’s 1963 national 

survey262 detected 12 cases of postvaccinial encephalitis 

among 14,014 vaccinations. Symptoms progress to amnesia, confusion, disorientation, restlessness, delirium, 

drowsiness, and seizures. The cerebrospinal fluid has 

normal chemistries and cell counts. Histopathologic 

findings include demyelination and microglial proliferation in demyelinated areas with lymphocytic 

infiltration without significant edema. The cause for 

central nervous system disease is unknown, and no 

specific therapy exists. Intervention is limited to anticonvulsant therapy and intensive supportive care.258,263,264

Fetal vaccinia, which results from vaccinial transmission from mother to fetus, is a rare but serious 

complication of smallpox vaccination during or immediately before pregnancy (Figure 27-12). Fewer 

than 40 cases have been documented in the world’s 

literature.256

Myopericarditis, although previously reported as a 

rare complication of vaccination using vaccinia strains 

other than the New York City Board of Health strain, 

Figure 27-7. Generalized vaccinia. This 8-month-old infant 

developed a generalized vaccinia reaction after vaccination. 

Generalized vaccinia is a widespread rash involving sores 

on parts of the body away from the vaccination site, which 

results from vaccinia virus traveling through the bloodstream. Image 4644.

Reproduced from: Centers for Disease Control and Prevention Public Health Image Library website, http://phil.CDC.

gov. Accessed September 16, 2014. Photograph: Courtesy of

Allen W Mathies, MD, California Emergency Preparedness 

Office, Immunization Branch. 

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through June 2013. During this period, when more

than 830,000 vaccinations were administered to DoD 

personnel and an additional 2,430 were administered 

to civilians (such as research personnel), 169 reports 

of serious adverse events were submitted to VAERS, 

including 138 for which a diagnosis was verified. 

Of these reports, cardiac diagnoses were the most 

frequent, representing 54.4% of the reports; among 

cardiac events, myopericarditis (40.2% of cardiac reports), myocarditis (35.9%), and pericarditis (14.1%) 

Figure 27-8. After receiving a smallpox vaccination on the 

small of his back, this 14-month-old child manifested a nonspecific rash in the form of extensive, roseola-like erythematous macules and patches over his entire body. Eruptions 

such as this one are common after vaccination; although 

often dramatic in appearance, they are largely benign and 

usually self-limited. There is no evidence of systemic or cutaneous spread of the vaccinia virus, and live virions cannot 

be recovered from the involved sites. Image 3318.

Reproduced from: Centers for Disease Control and Prevention Public Health Image Library website, http://phil.CDC.

gov. Accessed September 16, 2014. Photograph: Courtesy of

Arthur E Kaye, Centers for Disease Control and Prevention.

was not well recognized until reported during active 

surveillance of the 2002–2003 US Department of Defense (DoD) vaccination program (Figure 27-13).265,266

In 2003, reports of myocarditis among vaccinees raised 

concerns about carditis and cardiac deaths in military 

personnel receiving the smallpox vaccine (which, 

at the time, was Dryvax). Among 450,293 vaccinees, 

37 individuals (all men receiving their first smallpox vaccination) experienced myopericarditis, for a 

rate of 82 per million vaccinees.257 Ischemic cardiac 

events, including (rarely) fatalities, have also been 

reported following vaccination with both Dryvax and 

ACAM2000. Most recently, McNeil and colleagues267

reviewed ACAM2000 reports in VAERS for March 2008 

Figure 27-9. Eczema vaccinatum. This 28-year-old woman 

with eczema vaccinatum contracted it from her vaccinated 

child. She had a history of atopic dermatitis, which was inactive when her child was vaccinated. As a therapy, she was 

given vaccinia immune globulin, idoxuridine eye drops, and 

methisazone, which resulted in healed lesions, no scarring, 

and no lasting ocular damage. Image 4621.

Reproduced from: Centers for Disease Control and Prevention Public Health Image Library website, http://phil.CDC.

gov. Accessed September 16, 2014. Photograph: Courtesy of

Allen W Mathies, MD, California Emergency Preparedness 

Office, Immunization Branch. 

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were the most common diagnoses.267 Although no clear 

association with vaccination has been found, a history 

of ischemic heart disease and the presence of significant cardiac risk pose relative contraindications for 

smallpox vaccination.265–268 Consequently, individuals 

with a history of myocarditis, pericarditis, or ischemic 

heart disease should not be vaccinated.

Vaccine research. Because of the contraindications 

and adverse events associated with first- and secondgeneration smallpox vaccines, including ACAM2000, 

development of third- and fourth-generation smallpox 

vaccines is ongoing. 

The highly attenuated modified vaccinia virus Ankara (MVA), a third-generation smallpox vaccine, was 

produced by 572 serial passages in chicken embryo 

fibroblasts, which rendered the virus unable to replicate in most mammalian cells. MVA, which was used 

toward the end of the worldwide smallpox eradication 

campaign, is immunogenic and safe for use even in immunocompromised individuals. MVA has been safely 

given to approximately 150,000 persons since it was first 

developed in the 1970s.242,269,270 The safety and immunogenicity of recently developed versions of MVA, such as 

Imvamune (Bavarian Nordic, Martinsried, Germany), 

which is stored in the Strategic National Stockpile 

(SNS), are currently being assessed in clinical trials.271,272

In completed clinical trials, Imvamune has been safe, 

well tolerated, and immunogenic, producing immune 

responses comparable to those elicited by Dryvax273,274; 

a trial comparing Imvamune to ACAM2000 is underway. Imvamune has received marketing authorization 

from the European Commission and Health Canada 

for immunization against smallpox in adults, including healthy individuals as well as those with immune 

deficiencies and skin disorders, such as atopic dermatitis 

and HIV infection.275 The CDC has submitted a preEUA request to the FDA for potential use of Imvamune 

during a public health emergency; if granted, it would 

allow Imvamune to be administered to HIV-infected 

individuals and those with atopic dermatitis.275

Aventis Pasteur smallpox vaccine (APSV, or WetVax) is a live, replication-competent liquid calf lymphderived vaccinia virus vaccine that results in strong 

humoral and cellular immune responses276; APSV is 

also stored in the SNS. The CDC also has submitted a 

pre-EUA for the use of diluted APSV during a public 

emergency to increase the supply of smallpox vaccine.275

Fourth-generation vaccine candidates include 

subunit and DNA vaccines composed of vaccinia 

virus membrane and/or virion proteins or variola 

homologs.239,270

Figure 27-11. Progressive vaccinia after debridement. Image 4594.

Reproduced from: Centers for Disease Control and Prevention Public Health Image Library website, http://phil.CDC.

gov. Accessed September 16, 2014. Photograph: Courtesy

of Centers for Disease Control and Prevention, California 

Department of Health Services. 

Figure 27-10. Progressive vaccinia. This patient presented 

with progressive vaccinia after receiving a smallpox vaccination. Progressive vaccinia, though rare, is one of the most 

severe complications of smallpox vaccination and is almost 

always life threatening. Image 4592. 

Reproduced from: Centers for Disease Control and Prevention Public Health Image Library website, http://phil.CDC.

gov. Accessed September 16, 2014. Photograph: Courtesy

of Centers for Disease Control and Prevention, California 

Department of Health Services. 

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Passive Immunotherapy

VIG, which is administered intravenously, is used 

primarily for complications from the smallpox vaccine 

(Table 27-5); it does not currently play a role in smallpox prevention.277 In particular, VIG may be recommended in severe cases of ocular vaccinia; however, it 

is contraindicated in individuals with vaccinial keratitis because of the risk of corneal clouding. Corneal 

clouding was observed in 4 of 22 persons with vaccinial 

keratitis who received VIG.278 A subsequent study in 

rabbits showed that treatment of vaccinial keratitis 

with VIG was associated with both corneal scarring 

and persistent and larger satellite lesions compared 

with control animals.279 VIG should not be withheld 

from a patient with keratitis if a comorbid condition 

exists that requires VIG administration and if the risk 

of the comorbid condition is greater than that of VIGassociated complications of keratitis.258

Treatment of generalized vaccinia with VIG is 

restricted to those who are systemically ill or have an 

immunocompromising condition.258

Individuals with eczema vaccinatum are generally 

systemically ill and require immediate therapy with 

VIG, which is the only currently approved treatment 

for this condition.258 The mortality rate of individuals with eczema vaccinatum was 7% (9/132), even 

with VIG therapy. A measurable antibody response 

developed in 55 of 56 survivors who had antibody 

titers obtained after VIG administration. No antibody 

response was detected in five persons with fatal eczema vaccinatum cases who had post-VIG antibody 

titers measured.280 

Progressive vaccinia carries a high mortality rate 

and should be aggressively treated with VIG, debridement, intensive monitoring, and tertiary medical center–level support.258 However, anecdotal experience 

has shown that, despite treatment with VIG, individuals with cell-mediated immunity defects have a poorer 

prognosis than those with humoral defects. 

Prophylactic VIG to prevent fetal vaccinia could be 

considered for a woman who discovers she is pregnant 

shortly after receiving the smallpox vaccine; however, 

data on the efficacy of this approach are not available.281 VIG could be considered for an infant born 

with lesions, but again, data regarding efficacy or the 

appropriate dosage are not available.258

Limited historical data are available on the effect 

of VIG in conjunction with the smallpox vaccine 

for postexposure prevention of smallpox in contact 

Figure 27-13. Histopathology of vaccine-related myocarditis 

showing a nonspecific lymphocytic infiltrate. Reproduced 

with permission of Department of Pathology, Brooke Army 

Medical Center, Texas.

Figure 27-12. Fetal vaccinia. This child contracted vaccinia 

virus while in utero. Image 3338.

Reproduced from: Centers for Disease Control and Prevention Public Health Image Library website, http://phil.CDC.

gov. Accessed September 16, 2014. Photograph: Courtesy of

Arthur E Kaye, Centers for Disease Control and Prevention. 

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cases.282–284 A 1961 study by Kempe et al282 demonstrated a statistically significant difference in 

smallpox cases among exposed contacts: smallpox

occurred in 5.5% of contacts (21/379) who received 

the smallpox vaccine alone compared with 1.5% of 

contacts (5/326) who received both the smallpox 

vaccine and VIG therapy. Research published a year 

later by Marennikova283 studied the effect of antivaccinia gamma globulin given to 13 of 42 persons who 

had been in close contact with smallpox patients. 

None of the 13 persons developed smallpox. Only 4 

of the 13 individuals had a history of prior smallpox 

vaccination, and all but 3 of the patients were not 

revaccinated until day 4 after the contact. Of the 29 

persons not given antivaccinia gamma globulin, 13 

developed smallpox.283 However, no clinical trials 

have provided evidence that giving VIG in conjunction with the smallpox vaccine as prophylaxis has a 

greater survival benefit than vaccination alone.285–287 

Monoclonal antibodies represent another approach 

to passive immunotherapy. Postexposure administration of human monoclonal antibodies has, for 

example, protected rabbits from a lethal dose of an 

orthopoxvirus.288

Antiviral Agents

Antiviral agents have been used successfully, sometimes in combination with VIG, in the treatment of 

complications of smallpox vaccination. Animal studies 

suggest that some of these antivirals would also be 

helpful in treating smallpox infection. 

Cidofovir has broad-spectrum activity against DNA 

viruses, including the herpes viruses, papillomavirus, 

adenovirus, and poxviruses.289–291 Cidofovir provides a 

pronounced, long-lasting inhibition of viral DNA synthesis, allowing for infrequent (weekly or bimonthly) 

dosing.292 Cidofovir has been approved by the FDA 

for treating cytomegalovirus retinitis in patients with 

AIDS. Treatment of vaccinia complications or smallpox 

with cidofovir would be an off-label use of the drug. 

However, both the DoD and the CDC currently have 

IND protocols for the use of cidofovir in these two 

conditions.

Studies of cidofovir have demonstrated improved 

or prolonged survival in BALB/c mice and in mice 

with severe combined immunodeficiency infected 

with vaccinia virus, as well as cowpox-infected mouse 

models, when treatment was initiated as long as 5 days 

before or up to 96 hours after infection.293 The greatest 

benefit of cidofovir prophylaxis was observed when it 

was administered within 24 hours before or after exposure.294–296 NHP studies have demonstrated improved 

survival in monkeypox and smallpox models.297

In humans, cidofovir has been effective in the 

treatment of the poxvirus infection molluscum contagiosum in patients with AIDS. Dose-related nephrotoxicity has been associated with cidofovir therapy 

in humans; however, this may be minimized by concomitant intravenous hydration with saline and oral 

probenecid (generally administered as a 2-g dose 3 

hours before the cidofovir infusion, and again at 2 and 

8 hours after infusion).298–300 An investigational drug, 

brincidofovir (or hexadecyloxypropyl-cidofovir, 

previously referred to as CMX-001; Chimerix Inc, 

Durham, NC), is an oral formulation of cidofovir that 

has shown no evidence of a link to nephrotoxicity.299

The US Government recently announced plans to add 

brincidofovir to the SNS for the treatment of patients 

with smallpox. 

Tecovirimat, previously known as ST-246, is a 

potent and specific inhibitor of orthopoxvirus replication under development by SIGA Technologies 

(Corvallis, OR). The drug is active against multiple 

species of orthopoxviruses, including variola virus 

and cidofovir-resistant cowpox variants. In animal 

models, this oral drug has been effective in preventing death from infection with variola virus and other 

orthopoxviruses; it also reduced shedding of vaccinia virus after smallpox vaccination.301,302 A recent 

study found that tecovirimat resulted in survival 

of 100% of cynomolgus macaques challenged with 

intravenous variola virus, whether the antiviral was 

administered beginning on the 2nd or 4th day after 

infection (only 50% of placebo-treated macaques 

survived). Disease in tecovirimat-treated macaques 

was milder, and oropharyngeal viral shedding was 

reduced, compared with placebo-treated survivors.303

Clinical trials have demonstrated that tecovirimat is 

safe and well tolerated.304 For treatment of orthopoxvirus infections, tecovirimat and brincidofovir may be 

more effective if used in combination. Furthermore, 

based on experience with the use of tecovirimat to 

treat progressive vaccinia,305 topical tecovirimat may 

need to be administered instead of—or in addition 

to—oral tecovirimat.304 The US Government recently 

added tecovirimat to the SNS for the treatment of 

patients with smallpox.

For the treatment of eczema vaccinatum, cidofovir 

can be used off-label or under an IND protocol, and 

the investigational antiviral drugs, tecovirimat and 

brincidofovir, are available under an emergency IND 

application.305,306

One animal study showed that both topical and 

intravenous cidofovir were effective in treating vaccinia necrosis in mice deficient in cell-mediated immunity. Topical cidofovir was more effective than 

intravenous cidofovir, and the administration of both 

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cidofovir preparations was superior to either preparation alone.307 Again, tecovirimat and brincidofovir are 

available under an emergency IND application.305,306

Because of the potential for renal toxicity with cidofovir, brincidofovir may be a better choice to treat 

progressive vaccinia, especially in immunocompromised individuals. It may be necessary to administer 

tecovirimat (particularly the topical preparation to 

improve absorption in individuals unable to eat) in 

addition to brincidofovir and repeated doses of VIG, 

as described by Lederman et al.305

The animal and human data suggest that cidofovir 

may be effective in therapy and in short-term prophylaxis of smallpox if initiated within 4 days after exposure. One dose of intravenous cidofovir may provide 

protection for 7 days.292

Topical treatment with trifluridine (viroptic; Catalytica Pharmaceuticals, Greenville, NC) is often recommended for ocular vaccinia resulting from inadvertent 

inoculation, although the FDA has not specifically 

approved this use of trifluridine.258,308

Postevent Countermeasures: Current Options

A suspected or confirmed case of human smallpox 

would be considered an international emergency and 

should be immediately reported to local and state 

public health authorities and the CDC. Individuals 

who have been exposed to smallpox patients or to 

animals infected with variola virus and laboratory 

workers with an aerosol or percutaneous exposure to 

variola virus must be quarantined and monitored (for 

fever, rash, or flu-like symptoms) for at least 17 days 

after the last contact with the index case or exposure, 

regardless of whether they have been vaccinated.14,66

Treatment. The FDA has not approved any antiviral 

agent to treat smallpox. However, tecovirimat and 

brincidofovir could be made available under an IND 

protocol to treat patients with smallpox. Tecovirimat 

is also available under a DoD IND and expanded access protocol for the treatment of smallpox, complications resulting from smallpox vaccination, or other 

orthopoxvirus infections. Intravenous cidofovir also 

is available to treat smallpox under an IND protocol. 

Clinical guidelines from the CDC for the use of antiviral medications in the event of a smallpox release or 

outbreak are under development. 

Postexposure Prophylaxis. The CDC recently 

provided detailed clinical guidance on postevent use 

of smallpox vaccines for individuals who have been 

exposed to smallpox virus and those who are at high 

risk for smallpox infection but have not had a known 

exposure.275 A brief summary of these recommendations follows.

For children and adults (including pregnant 

women) without severe immunodeficiency or relative 

contraindications who have been exposed to smallpox 

virus or are at high risk for infection, the CDC recommends the administration of ACAM2000. The CDC 

guidelines describe in detail the recommended procedure for vaccinating, in a postevent context, persons 

with a severe allergy to ACAM2000 or a component 

of that vaccine who have been exposed to the virus or 

are at high risk for infection. Briefly, such individuals should be vaccinated with ACAM2000, ASPV, or 

another vaccine, depending on the situation and the 

vaccines available; vaccination of such individuals may 

need to occur in a facility capable of treating an anaphylactic reaction. Individuals with atopic dermatitis 

who have been exposed to smallpox virus should be 

vaccinated with ACAM2000; those with atopic dermatitis who are at high risk of infection but without 

a known exposure should receive Imvamune instead 

unless they have previously received ACAM2000 

without complications. 

Some immunocompromised individuals—such 

as recipients of a solid organ transplant within the 

previous 3 months—are expected to benefit from 

vaccination with ACAM2000 (if exposed to smallpox 

virus) or Imvamune (if at high risk for smallpox 

infection without a known exposure). However, 

severely immunodeficient persons—such as those 

with HIV infection whose CD4 cell count is less 

than 50 cells/mm3 or those who recently received 

a bone marrow transplant—are, in general, not expected to benefit from vaccination with any of the 

smallpox vaccines currently available. Vaccination 

of severely immunocompromised individuals with 

Imvamune may be considered if antiviral agents are 

not available.275

Viral Hemorrhagic Fevers

Viral hemorrhagic fevers (VHFs)—severe illnesses 

characterized by fever, vascular dysregulation, and 

vascular damage—are caused by a subset of the lipidenveloped RNA viruses belonging to fourfamilies: (1)

Arenaviridae, (2) Bunyaviridae, (3) Filoviridae, and (4) 

Flaviviridae. Transmission to humans occurs in a variety 

of ways, such as via infected aerosols of rodent excreta, 

contact with infected blood or body fluids, or through 

the bites of infected arthropods. Not all patients infected with these viruses develop VHF.14,165,309,310 Because 

of their ability to cause widespread, severe illness and 

death and the potential for either aerosol dissemination 

and infectivity or person-to-person transmission, the 

viral agents of VHFs are considered potential agents 

of biological warfare.

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Vaccination

The only vaccine for any VHF that is licensed in 

the United States is the live attenuated yellow fever 

vaccine, YF-Vax (Sanofi Pasteur Biologics, Cambridge, 

MA), derived from the 17D yellow fever virus strain 

(see Table 27-1). This vaccine has substantially diminished the burden of yellow fever infection worldwide 

and is well tolerated, although it is contraindicated in 

infants and immunosuppressed patients and is used 

with caution in elderly people.311 Because of yellow 

fever’s short (3- to 6-day) incubation period, postexposure use of this vaccine is unlikely to be effective.309

A number of human vaccines developed and licensed in other countries may have efficacy against 

VHFs. In particular, a live attenuated Argentine hemorrhagic fever (AHF) vaccine, known as Candid #1, demonstrated efficacy against Junin virus in a field study

among 6,500 agricultural workers in Argentina who 

were randomly assigned to receive either placebo or 

Candid #1. Of the 23 patients who developed AHF, 22 

had received placebo compared to only 1 patient who 

had received the vaccine.312 Candid #1, which is the 

first vaccine used to control an arenaviral hemorrhagic 

fever, is the first live viral vaccine to be manufactured 

and registered in Argentina.313

Hantavax, a suckling mouse brain-derived hantavirus vaccine (Korea Green Cross Corporation, Yonginsi, Korea), has been licensed in South Korea since 

1990. Observational trials in North Korea and China 

and a randomized, placebo-controlled trial in Yugoslavia supported the vaccine’s efficacy314; however, 

the humoral immune response, when measured by 

PRNT80 antibodies, was protective in only 33.3% of 

vaccine recipients.315 Clinical trials and animal studies 

of other hantavirus vaccine candidates, such as DNA 

vaccines316,317 and vaccinia-vectored constructs,318 have 

suggested other potential vaccine options. 

In 1974, an inactivated Crimean–Congo hemorrhagic fever virus (CCHFV) vaccine developed by 

Soviet scientists was licensed in Bulgaria and is used 

in CCHFV-endemic areas of the country for military 

personnel and medical and agricultural workers. 

Although data are lacking on the total number of 

vaccinated civilians who have contracted CCHF, 

no vaccinated military personnel have contracted 

the disease since 1997, and none of the vaccinated 

laboratory personnel working with CCHFV have 

become infected, even after accidental exposure via 

needle prick.319 However, a recent study found that, 

although CCHFV-vaccinated individuals developed 

high CCHFV-specific antibodies after a single dose, 

neutralizing activity against CCHFV was low even 

after repeated doses.320

A formalin-inactivated RVFV vaccine (TSIGSD-200), currently under IND status, is used in the 

SIP at USAMRIID for laboratory workers who may be 

exposed to the virus (see Table 27-1).321,322 However, 

no human RVFV vaccines are commercially available. The primary focus of RVFV vaccine research is 

the vaccination of livestock to prevent abortions and 

deaths in these species and spillover into humans during epizootic outbreaks.323,324 A live attenuated vaccine 

for RVFV, the Smithburn strain, is the only vaccine 

approved for use in livestock. 

Phase 1 and 2 studies have been conducted at 

USAMRIID on a live attenuated RVF MP12 vaccine, 

which was found to be safe and immunogenic in human volunteers.325,326 Because inactivated vaccines are 

expensive (requiring multiple boosts) and live vaccines 

have side effects in animals (abortions and teratogenicity), other vaccine types are being explored for use 

in endemic areas. RVFV vaccines based on virus-like 

particles327 or recombinant viral vectors328 and DNA 

vaccines329 have recently demonstrated potential.

A formalin-inactivated Kyasanur Forest disease virus vaccine, licensed in India since 1990, was 62.4% effective (with a 95% confidence interval of 26.1%–80.8%) 

and 82.9% effective (with a 95% confidence interval of 

71.3%–89.8%) among those who received two doses 

and those who received an additional booster dose, respectively, compared with unvaccinated individuals.330

A neutralizing antibody study in humans and a 

viral challenge study in African green monkeys and 

crab-eating macaques demonstrated at least partial 

protection against two flaviviruses—Omsk hemorrhagic fever virus and tickborne encephalitis virus 

(TBEV)—using FSME-IMMUN, an inactivated TBEV 

vaccine licensed for use in Canada and Europe.331,332

Substantial research has focused on the development of an effective vaccine for protection against the 

five known antigenically distinct ebolaviruses:

1. Zaire ebolavirus (ZEBOV); 

2. Sudan ebolavirus (SEBOV); 

3. Taï Forest ebolavirus (also called Côte 

d’Ivoire ebolavirus); 

4. Reston ebolavirus; and 

5. Bundibugyo ebolavirus (BEBOV).333,334

Among the more promising approaches, several 

investigators have used vaccines based on viral vectors, 

such as recombinant vesicular stomatitis virus (VSV), 

that express the transmembrane glycoproteins of one or 

more ebolaviruses. For example, a single immunization 

with a replication-competent VSV vector expressing the 

ZEBOV glycoprotein, a vaccine candidate referred to as 

rVSV-ZEBOV, protected cynomolgus macaques from 

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lethal challenge with ZEBOV.335,336 A single injection of a 

recombinant VSV vaccine expressing glycoprotein from 

BEBOV provided 100% protection against BEBOV challenge in macaques, as did a short prime–boost regimen 

using recombinant VSV–based ZEBOV and SEBOV vaccines.337 A single immunization with a bivalent recombinant complex adenovirus vaccine (CAdVax), which 

expresses glycoproteins from both SEBOV and ZEBOV, 

protected macaques against ZEBOV challenge; with 

the addition of a boosting vaccination, CAdVax also 

provided protection against SEBOV challenge.338 Two 

injections of a recombinant human parainfluenza virus 

type 3 vaccine vector encoding ZEBOV glycoprotein 

protected rhesus macaques challenged with ZEBOV.339

Two intramuscular injections with a replicon vaccine 

based on SEBOV glycoprotein–expressing VEEV completely protected cynomolgus macaques challenged 

with aerosolized SEBOV.340 In another approach, 

macaques challenged with ZEBOV were protected by 

three immunizations with virus-like particles containing 

ebolavirus glycoprotein, VP40, and nucleoprotein.341

Since the 2014 Ebola virus disease (EVD) outbreak 

in West Africa, several candidate Ebola vaccines have 

moved rapidly forward in development. One candidate, a recombinant chimpanzee adenovirus type 

3–vectored ebolavirus Zaire vaccine (cAd3-EBOZ), 

recently underwent a phase 1 clinical trial in healthy 

adults. In this trial, a single intramuscular injection 

of cAd3-EBOZ was safe (2 of 10 subjects developed 

transient fever 1 day after vaccination) and resulted in 

antibody responses in the range reported to be protective in an NHP challenge model.342 A phase 1 trial has 

also found rVSV-ZEBOV to be safe and immunogenic 

in healthy adults.343 A phase 2/3 trial of cAd3-EBOZ 

and rVSV-ZEBOV is ongoing in Liberia. 

Vaccines may also prove useful as PEP against 

some VHF-causing pathogens. In one case report,344 a 

physician who experienced an accidental needlestick 

while working in an Ebola treatment facility in Sierra 

Leone during the 2014 Ebola outbreak was vaccinated 

43 hours postexposure with VSV-ZEBOV through 

an emergency IND. Strong innate and Ebola-specific 

adaptive immune responses were detected after vaccination, and the patient survived. 

Antiviral Agents

Antiviral medications prescribed to treat VHFs are 

important primarily after patients have developed 

symptoms because data are—in general—insufficient 

to support their use as PEP. 

Ribavirin. The antiviral medication with the most 

evidence of efficacy is ribavirin, a nonimmunosuppressive nucleoside analogue with activity against a 

number of viruses, including at least some arenaviruses and bunyaviruses, but not filoviruses or flaviviruses.309,345 Ribavirin inhibits the conversion of inosine 

5’-phosphate (IMP) to xanthosine 5’-phosphate, disrupting the synthesis of guanosine monophosphate, 

a vital nucleotide needed to form viral nucleic acid.346

However, because ribavirin does not efficiently cross 

the blood–brain barrier, it may not protect against 

neurologic effects of VHFs.309,347 Another caveat to 

the use of ribavirin is its association with serious side 

effects, including hemolytic anemia, hypocalcemia, 

hypomagnesemia,348 and genotoxicity.349 Ribavirin 

has demonstrated teratogenicity and embryotoxicity 

in animal studies; for this reason, it is generally contraindicated during pregnancy.309,350

Ribavirin appears to be effective in the treatment 

of Lassa fever if it is begun early in the course of the 

illness. Among patients with Lassa fever who were 

treated within the first 6 days after the onset of fever, 

intravenous ribavirin was more effective than passive immunotherapy in reducing mortality: the CFR

was reduced from 55% among patients treated with 

passive immunotherapy to 5% among those treated 

with ribavirin.351 Results from NHP studies support 

this finding.352,353 Ribavirin is less beneficial when 

administered starting after day 7 of illness.345 Data are 

extremely limited regarding the efficacy of ribavirin 

PEP for Lassa fever in humans.309,354

Ribavirin’s efficacy in treating AHF and other arenaviruses is less clear. In macaques inoculated with Junin

virus on day 0, ribavirin treatment begun on day 6 (after viremia and clinical signs of illness were detected) 

provided minimal protection. Of the four animals, 

one died early in the course of illness; although initial 

improvement was observed in the three remaining animals, all three subsequently developed a CNS infection 

that was fatal in two animals.347 However, Enria and 

colleagues found a survival benefit among humans 

with AHF who were treated with ribavirin.355,356 An 

anecdotal report described recovery from Bolivian 

hemorrhagic fever, which is caused by Machupo virus, 

in two patients treated with ribavirin.357

In macaques, ribavirin appears to provide a benefit 

when used as PEP against AHF. Among macaques 

inoculated with Junin virus on day 0, four animals

treated with ribavirin beginning on day 0 survived, 

whereas four that received placebo died during the 

4th week after infection.347

In a double-blind, placebo-controlled trial, ribavirin effectively reduced mortality and viremia in 

patients with hemorrhagic fever with renal syndrome 

(HFRS).358,359 However, a meta-analysis of the use of 

ribavirin in the treatment of HFRS and hantavirus 

pulmonary syndrome found mixed results.360

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Ribavirin also has demonstrated in vitro activity 

against CCHFV.348,361 The results of human studies 

assessing the efficacy of ribavirin in the treatment of 

CCHF are highly variable.362–365 However, as Ergonul 

and colleagues argue, this variability may be due—at 

least in part—to variability in the delay between symptom onset and the start of treatment.366,367 In one study, 

for example, patients admitted to a hospital and started 

on ribavirin within 2 days of symptom onset were less 

likely than others to become more severe cases.367

As PEP, evidence of ribavirin’s efficacy in humans 

is limited. In one case study,368 six healthcare workers 

who were exposed to CCHFV (via needlestick or contact with skin and mucosal surfaces) received ribavirin 

beginning within 1 hour of exposure; none of these 

individuals developed symptoms. One healthcare 

worker who was exposed to CCHFV (probably via 

aerosolization of contaminated blood or secretions) 

and did not receive ribavirin later developed CCHF 

(she recovered fully after treatment with ribavirin).368

Favipiravir. A viral RNA polymerase inhibitor, 

favipiravir (also known as T-705 and marketed as 

Avigan; Toyama Chemical Co, Shinjuku-ku, Tokyo) 

was initially developed as an antiinfluenza drug. It has 

been approved in Japan for specifically defined cases

of influenza. However, it may also be effective against 

several other virus families, including arenaviruses, 

bunyaviruses, and flaviviruses. 

During and after the 2014 EVD outbreak in West 

Africa, interest in favipiravir increased dramatically. A 

phase 2 clinical trial is underway in Guinea, aiming to 

assess the efficacy of favipiravir in reducing mortality 

in individuals with EVD. Preliminary results posted 

by the group Médecins sans Frontièrs suggest that 10 

days of favipiravir may be beneficial among patients 

(children older than the age of 1 year and nonpregnant 

adults) with high or moderate levels of viral replication 

who have not yet developed severe visceral lesions. 

However, the drug appears not to be efficacious among 

those with a very high level of viral replication along 

with serious visceral involvement.369 Based on the apparently greater benefit of favipiravir for patients with 

moderate to high viremia versus very high viremia, 

Van Herp et al370 argue for the use of favipiravir as 

PEP for contacts of patients with EVD.

In animal models, favipiravir has also shown promise to treat AHF and CCHF. Favipiravir resulted in 78% 

survival of guinea pigs infected with Junin virus when

administered intraperitoneally for 2 weeks beginning 

2 days after challenge; by comparison, only 11% of 

placebo-treated animals survived. Among ribavirintreated guinea pigs, survival ranged from 33% to 40%. 

Oral administration of favipiravir was less protective 

than was the intraperitoneal route, with 20% of orally 

treated guinea pigs surviving; however, animals that 

succumbed survived longer than placebo-treated animals.371 In a small animal model of CCHF, mice treated 

with favipiravir, initiated up to 2 days after infection 

with CCHFV, survived with no signs of disease and 

no virus detectable in blood or organs.372,373

BCX4430. The synthetic adenosine analogue 

BCX4430 has broad-spectrum antiviral activity against 

many viruses, including bunyaviruses, arenaviruses, 

flaviviruses, and filoviruses. In particular, Warren et 

al373 recently found that BCX4430, administered as 

late as 48 hours after infection, completely protected 

macaques from disease caused by Marburg virus. 

This product also conferred significant protection in 

guinea pigs challenged with Marburg virus and in mice 

challenged with Ebola virus. A phase 1 clinical trial to 

assess the safety, tolerability, and pharmacokinetics of 

BCX4430 in healthy adults was recently completed. 

Interferons. Stimulating the immune system is 

another potential therapeutic modality, but no human 

studies using this technique have been conducted for 

any of the VHF viruses. IFN combinations may be 

useful in such an approach, particularly with VHF 

infections in which the immune response is impaired. 

However, IFN compounds may be deleterious in some 

VHF infections, such as AHF, in which high IFN levels 

are associated with worse outcomes.310,374 IFNs have 

demonstrated a benefit in bunyavirus murine models.375 In NHPs inoculated with Ebola virus, early postexposure treatment with either IFN α-2b376 or IFN-β377

prolonged survival but did not prevent death. Similar 

findings were obtained in Marburg virus–inoculated 

macaques that received early postexposure treatment 

with IFN-β.

377 

Other Drugs. Although in vitro data suggest that 

the Mx family of proteins has antiviral activity against 

a wide variety of RNA viruses, further study is needed.378,379 Recently, FDA-approved IND applications 

and phase 1 clinical trials have been initiated for two 

small-molecule therapeutics: (1) anti-sense phosphorodiamidate morpholino oligomers (AVI-6002, AVI6003) and (2) lipid nanoparticle/small interfering RNA 

(TKM-Ebola). However, the need for multiple doses to 

achieve therapeutic efficacy makes these compounds 

less than ideal with regard to patient compliance and 

outbreak scenarios.380

Pathogenesis studies with Ebola virus have implicated tissue factor-induced disseminated intravascular 

coagulation as a critical component of fatal outcomes.381

In a rhesus macaque model of Ebola virus infection, 

treatment with a factor VIIa/tissue factor inhibitor 

(recombinant nematode anticoagulation protein c2 

or rNAPc2) led to a survival advantage382; however, 

rNAPc2 was not effective against Marburg virus.383

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This compound has not been tested in humans for 

treating EVD, and tissue factor inhibitors have not been 

effective in the treatment of septic shock.384

IMP dehydrogenase inhibitors (similar to ribavirin) 

have been tested in both in vitro and animal models 

against arenaviruses; however, because of their toxicity, such compounds have been used only experimentally for cancer patients in crisis.385,386 Other compounds 

that have demonstrated in vitro activity against 

arenaviruses include 3′-fluoro-3′-deoxyadenosine,387

phenothiazines,388 and myristic acid compounds.389,390

When challenged with Lassa virus, guinea pigs treated 

with ST-193, a small-molecule inhibitor of arenavirus 

entry into cells, had an overall survival rate of 62.5% 

compared with 0% in the ribavirin-treated and vehicle 

groups.391

Although using steroids to treat VHFs has not been 

recommended,309 evidence suggests that corticosteroids may be effective among severely ill patients with 

CCHF. In a recent study, among 16 severely ill patients 

with CCHF who received corticosteroid therapy in 

addition to ribavirin, 8 died (a CFR of 50%), whereas 

among 8 severely ill patients who did not receive additional corticosteroid therapy, all 8 died (a CFR of 

100%; P = 0.014). Among moderately ill patients, corticosteroid was not associated with a reduced CFR.392

Several antivirals have been tested in a bunyavirus 

(Punta Toro virus) murine model,375 suggesting possible compounds for further testing.

Passive Immunotherapy

Studies on the benefits of passive immunotherapy 

for treating VHFs have yielded mixed results.309 Serum collected from donors after infection with Junin

virus has been used successfully to treat AHF.355,393 In 

a cynomolgus macaque model of Lassa virus infection, treatment with serum from immune monkeys 

led to a survival advantage; the benefit was greater 

when this passive immunotherapy was combined 

with ribavirin.353 In humans, however, serum from 

convalescent patients used to treat Lassa fever did 

not reduce mortality in patients with a high risk of a 

fatal outcome.351 Human-derived antibodies against 

Bolivian hemorrhagic fever dosed in rhesus macaques 

to achieve neutralizing antibody titers of 1:4 to 1:8 protected monkeys against severe clinical manifestation 

of illness after Machupo virus challenge.394

Anecdotal evidence suggests that immunoglobulins 

and/or transfusions from convalescent patients may 

improve outcomes in human EVD.395,396 Postexposure 

treatment with concentrated polyclonal IgG antibodies 

collected from vaccinated macaques that survived an 

Ebola or Marburg challenge was completely protective in macaques challenged with Ebola or Marburg 

virus397; in contrast, an earlier study using equine IgG 

did not produce a mortality benefit in NHPs.376 ZMapp, 

a product composed of three humanized monoclonal 

antibodies produced in the plant Nicotiana benthamiana

completely protected macaques when treatment was 

initiated up to 5 days after Ebola virus challenge.398,399

ZMapp is now undergoing a clinical trial to assess its 

safety and efficacy in the treatment of EVD. 

Substantial supportive data are lacking for the use of 

immunoglobulin from survivors for treating CCHF,319

but 15 high-risk patients (viral load of at least 108 

copies/mL) treated with convalescent hyperimmunoglobulin had a survival rate of 86.6%,400 and an earlier 

small case series found 100% survival among treated 

patients.401 Monoclonal antibodies against HFRS 

viruses have been effective in murine models,402 and 

such treatment appears to be well tolerated in healthy 

human volunteers.403

Yellow fever virus immunotherapy data from human studies are lacking; however, specific monoclonal 

antibody therapy with MAb 2C9-cIgG resulted in 

substantial improvement in survival among hamsters 

infected with yellow fever virus.404

As with passive immunotherapy for treating other 

diseases, concerns about the transmission of bloodborne pathogens, such as hepatitis C,405 may limit treatment with donated serum or may—at a minimum—necessitate a rigorous screening process. In addition, the 

impracticality of obtaining large quantities of donated 

serum from previously infected individuals with no 

such population available (particularly in the United 

States) limits the utility of this treatment. Revolutionary advances in plant virus-based transient expression 

to manufacture large quantities of monoclonal antibodies may facilitate passive treatment with antibodies to 

counteract the effects of VHFs.405

Other Countermeasures

Good infection control practices, particularly 

the isolation of patients and barrier precautions, 

are a crucial countermeasure in efforts to limit the 

impact of VHF viruses used as biological weapons. 

The specific infection control needed for each virus 

is discussed elsewhere in this volume. Management 

measures also must overcome the fear and panic 

associated with use of a VHF virus whose potential 

lethality tends to be exaggerated in popular culture, 

such as Ebola.406 Modern intensive care unit support with careful fluid management will probably 

improve the outcome for patients infected with VHF 

viruses,407 but access to this care may be limited in 

a mass casualty scenario. 

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Postevent Countermeasures: Current Options

Treatment. Supportive care is the primary form 

of treatment of individuals with VHFs. For adults 

(including pregnant women) and children with a 

VHF of unknown etiology, the Working Group on 

Civilian Biodefense309 recommends treatment with 

ribavirin and supportive care, beginning as soon as 

possible after symptom onset. In the case of a VHF, 

the potential teratogenic and embryotoxic effects of 

ribavirin are thought to be outweighed by the benefits 

of treatment. If the VHF is found to be caused by an 

arenavirus or bunyavirus, then the ribavirin should be 

continued such that the patient is treated for 10 days. 

If the infection is caused by a filovirus or flavivirus, 

ribavirin should be discontinued. In a contained 

casualty situation, ribavirin should be administered 

intravenously (under an IND protocol); in a mass 

casualty situation, it should be administered orally 

(an off-label use).309 The DoD maintains expanded 

access protocols for the IND use of intravenous ribavirin to treat Lassa fever, CCHF, and HFRS caused 

by Hantaan, Seoul, Puumala, and Dobrava viruses. 

Patients with AHF or Bolivian hemorrhagic fever may 

benefit from convalescent plasma, which is used as 

an investigational therapy.14

Postexposure Prophylaxis. In the context of a bioterrorism event, the Working Group on Civilian Biodefense309 recommends careful observation of exposed 

patients for 21 days, with antiviral treatment begun 

only if fever or other signs and symptoms of infection 

appear. Persons with a high-risk exposure to a VHFcausing virus and close contacts of patients with a VHF 

(other than RVF or a flavivirus-caused VHF, which are 

not transmitted person to person) should be instructed 

to record their temperature twice daily and report any 

symptom of a VHF, including a temperature of 101°F

or higher. The appearance of symptoms should prompt 

the initiation of treatment as described previously.309

For asymptomatic laboratory workers or healthcare 

workers, a high-risk exposure (eg, via needlestick) to 

Lassa virus, CCHFV, or a hantavirus could warrant 

PEP with ribavirin408–410; although this recommendation is not a product of a consensus process.

TOXINS

Botulinum Neurotoxin

Clostridium botulinum is an anaerobic, gram-positive, 

spore-forming bacillus that produces a potent toxin, 

botulinum neurotoxin (BoNT). The most poisonous 

substance known, BoNT is found in soil and water 

worldwide and is commercially available for cosmetic and medical uses.165,411 By blocking the release 

of acetylcholine, a neurotransmitter that causes muscle 

contraction, BoNT may result in muscle weakness, 

flaccid paralysis, and subsequent respiratory impairment. Eight immunologically distinct toxin serotypes 

(A through H) are produced by discrete strains of the 

organism. Although botulism is generally acquired 

from ingestion of food contaminated with BoNT, it 

may also occur from toxin production by C botulinum

if present in the intestine or wounds. Botulism is not 

acquired naturally by aerosolization; this route of acquisition would suggest a possible bioterrorism event 

but may also occur from exposure to aerosolized toxin 

in a research laboratory.1

Vaccination

Pentavalent Botulinum Toxoid. No FDA-licensed 

vaccines are available for preexposure vaccination 

against botulism. An investigational product, pentavalent botulinum toxoid (PBT), was used from 1959 

through 2011 for persons at risk for exposure to BoNT 

serotypes A through E. The PBT was available as an 

IND through the CDC (IND-161, for at-risk laboratory workers) until it was discontinued based on data 

indicating a decline in immunogenicity of some of the 

toxin serotypes.412 The PBT has also been available 

through the US Army Office of the Surgeon General 

(IND-3723, for at-risk military personnel). Although 

IND-3723 remains active, the PBT is now effective 

only against toxin serotype A. Thus, the PBT could 

still be used to vaccinate military personnel against 

toxin serotype A.413 Derived from formalin-inactivated, 

partially purified toxin serotypes A, B, C, D, and E, the 

PBT was developed by the DoD and manufactured first 

by Parke Davis and later (beginning in the early 1970s) 

by the Michigan Department of Public Health. Each of 

the five toxin serotypes was propagated individually 

in bulk culture and then underwent acid precipitation, 

filtration, formaldehyde inactivation, and adsorption 

onto an aluminum phosphate adjuvant. The five individual toxin serotypes were then blended to produce 

the end product.413–415

Vaccine Research. Vaccine candidates include 

formalin-inactivated toxoids (A through F), which are 

made in nearly the same way as formalin-inactivated 

PBT, and recombinant BoNT vaccines.416,417 The production of formalin-inactivated toxoids is expensive 

and relatively time consuming because it (a) requires 

partially purified culture supernatants to be treated 

exhaustively with formaldehyde and (b) must be 

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Medical Aspects of Biological Warfare 

performed by a highly trained staff within a dedicated 

high-containment laboratory space. Furthermore, the 

resulting toxoid is relatively impure, containing only 

10% neurotoxoid (the remainder is irrelevant material). 

However, the use of pure and concentrated antigen 

in recombinant vaccines offers advantages—increased 

immunogenicity and decreased reactogenicity—over 

formalin-inactivated toxoids.414 Recombinant techniques use a fragment of the toxin that is immunogenic 

but is not capable of blocking cholinergic neurotransmission. Both Escherichia coli and yeast expression systems have been used in the production of recombinant 

fragments, mainly the carboxy-terminal fragment of 

the heavy chain (Hc) of the toxin.414,418 Phase 1 trials 

on the bivalent recombinant vaccine (for protection 

against toxin serotypes A and B) have been completed, 

with promising preliminary serologic results at 12 

months after two doses of vaccine (administered at 

0 and 6 weeks).419 DynPort Vaccine Company LLC 

(Frederick, MD) sponsored a phase 2 randomized, 

double-blind, placebo-controlled, multicenter study 

to evaluate the safety, dosing schedule, and antibody 

kinetics of recombinant botulinum vaccine A/B (rBV 

A/B-40) in healthy adults. It was completed in December 2010, but results have not been published. 

A phase 3 randomized study to assess the safety, lot 

consistency, and clinical benefit of rBV A/B is planned. 

Recombinant vaccines given by aerosol420,421 and by the 

mucosal route422 are also being investigated.

Because the BoNT Hc has been produced as a stable 

recombinant protein and is an excellent immunogen, it 

has been assessed in diverse viral delivery platforms.413

In particular, BoNT Hc has been virally vectored using attenuated human adenovirus,423,424 inactivated 

rabies virus virions,425 and Semliki Forest virus (SFV) 

DNA replicon426,427 or recombinant SFV viral replicon 

particles,428 conferring substantial protection against 

lethal challenge in murine models.

Passive Immunotherapy

In March 2013, the FDA approved BAT (Botulism 

Antitoxin Heptavalent [A, B, C, D, E, F, G] – Equine) 

to treat individuals with symptoms of botulism following a known or suspected exposure. BAT was developed at USAMRIID, as one of two equine-derived 

heptavalent BoNT antitoxins, and manufactured 

by Cangene Corporation (Winnipeg, MB, Canada), 

which is now Emergent BioSolutions (Rockville, MD). 

The first approval of a plasma derivative under the 

Animal Rule, BAT is a sterile solution of fragments 

of antibodies to seven of the eight BoNT serotypes 

known to cause botulism (A, B, C, D, E, F, and G, 

but not H). The antibody fragments are derived 

from the processing of whole antibodies obtained 

from horses previously immunized with a specific 

serotype. When administered to humans, the most 

commonly observed side effects include headache, 

fever, chills, rash, itching, and nausea. However, BAT 

has the potential to cause hypersensitivity reactions, 

including anaphylactic and anaphylactoid reactions, 

in individuals sensitive to equine proteins; delayed 

allergic reactions may occur 10 to 21 days after administration. Therefore, a skin test before administration 

of BAT and careful monitoring is advised. BAT is 

approved for use in adults and children, including 

infants with botulism caused by serotypes other than 

A or B. The safety of BAT in pregnant and lactating 

women is unknown; evidence regarding safety and 

efficacy in pediatric and geriatric populations is limited. BAT is maintained in the SNS and is available 

through the CDC’s Drug Service.429,430

In October 2003, the FDA approved the Botulism 

Immune Globulin Intravenous (Human) (BabyBIG), 

a human botulism immune globulin derived from 

pooled plasma of adults immunized with PBT, for 

the treatment of infants with botulism from toxin 

serotypes A and B. Because the product is derived 

from humans, BabyBIG does not carry the high 

risk of anaphylaxis observed with equine antitoxin 

products or the risk of lifelong hypersensitivity to 

equine antigens. BabyBIG may be obtained from the 

California Infant Botulism Treatment and Prevention Program through the California Department of 

Health Services.431

Although passive antibody prophylaxis has been 

effective in protecting laboratory animals from toxin 

exposure,432 the limited availability and short-lived 

protection of antitoxin preparations make preexposure or postexposure prophylaxis with these agents 

impractical for large numbers of people. Additionally, 

the administration of equine antitoxin in asymptomatic persons is not recommended because of the risk 

of anaphylaxis from the foreign proteins. However, if 

passive immunotherapy is given, it should be administered within 24 hours of a high-dose aerosol exposure 

to botulinum toxin.

Postevent Countermeasures: Current Options

Treatment. Immediately after clinical diagnosis 

of botulism, adults (including pregnant women) and 

children should receive a single intravenous infusion 

of antitoxin (BAT or, for infants with botulism from 

serotypes A or B, BabyBIG) to prevent further disease 

progression. The administration of antitoxin should 

not be delayed for laboratory testing to confirm the 

diagnosis.14,66,430,433–435 Skin testing should be conducted 

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before the administration of BAT to detect sensitivity 

to serum or antitoxin.430,435 Intensive supportive care 

(eg, artificial ventilation or feeding by enteral tube) 

should also be provided.434 Although antibiotics may 

be necessary for the treatment of wound botulism or 

secondary infections, aminoglycosides and clindamycin should be avoided because they may further impair 

neuromuscular transmission.434

Postexposure Prophylaxis. Asymptomatic individuals with suspected exposure to BoNT should 

be carefully monitored, preferably near critical care 

services, for evidence of botulism; the patient’s vital 

capacity and maximal expiratory force should be assessed frequently. Such individuals should be treated 

promptly with antitoxin at the first sign of illness.434 In 

rare instances, it may be appropriate to administer antitoxin as PEP to asymptomatic persons after a high-risk 

laboratory exposure. PEP may also be appropriate for 

asymptomatic persons who are thought to have been 

exposed concurrently with persons already diagnosed 

with botulism.14,66

Staphylococcal Enterotoxin B

Staphylococcal enterotoxin B (SEB) is one of more 

than 20 antigenically distinct enterotoxin proteins 

produced by the bacterium Staphylococcus aureus. 

Ingestion of SEB is a common cause of food poisoning, with symptoms (including nausea, vomiting, and 

diarrhea) typically beginning within 1 to 6 hours of 

exposure. Ocular exposure can result in conjunctivitis 

and localized periocular swelling and sometimes gastrointestinal symptoms. Inhalation of SEB may cause 

fever, fatigue, respiratory symptoms, and sometimes 

gastrointestinal symptoms, generally within 2 to 12 

hours of exposure, which may progress to overt pulmonary edema, acute respiratory disease syndrome, 

septic shock, and death.66,436 Because it can be disseminated in a variety of ways and can cause lethal 

shock in humans, even at low doses (especially by 

the inhalational route), SEB is considered a potential 

bioterrorism agent.

Vaccination

No vaccine against SEB is available. However, 

several candidate vaccines have demonstrated protection against SEB challenge in animal models. These 

vaccines are based on a correlation between human 

antibody titers and the inhibition of T cell response to 

bacterial superantigens. A recombinantly attenuated 

SEB vaccine given by nasal or oral routes, using cholera 

toxin as a mucosal adjuvant, induced both systemic 

and mucosal antibodies and provided protection in 

mice against intraperitoneal and mucosal challenge 

with wild type SEB.437 Intramuscular vaccination with 

recombinantly attenuated SEB using an Alhydrogel 

(Accurate Chemical & Scientific Corporation, Westbury, NY) adjuvant was protective in rhesus monkeys 

challenged by aerosols of lethal doses of SEB. All 

monkeys developed antibody titers, and the release of 

inflammatory cytokines was not triggered.438 A phase 

1 clinical trial assessing the safety and immunogenicity of a recombinant SEB vaccine has recently been 

completed. 

A candidate SEB vaccine using a VEEV replicon 

as a vector has also been studied. The gene encoding 

mutagenized SEB was cloned into the VEEV replicon 

plasmid, and the product was then assembled into 

VEEV replicon particles. The vaccine elicited a strong 

antibody response in animal models and was protective against lethal doses of SEB.439

SEB toxoids (formalin-inactivated) incorporated 

into meningococcal proteosomes or microspheres 

have been found to be immunogenic and protective against aerosol SEB challenge in NHPs. The 

proteosome-toxoid, given intratracheally, elicited 

serum IgG and IgA antibody titers as well as a strong 

IgA response in bronchial secretions.440 Vaccination 

by an intratracheal route with formalinized SEB 

toxoid-containing microspheres resulted in higher 

antibody titers in the serum and respiratory tract, 

a higher survival rate, and a lower illness rate than 

booster doses given by intramuscular or oral routes. 

(Microspheres provide controlled release of the toxoid, which results in both a primary and an anamnestic secondary antitoxin response and thereby may 

require fewer doses.)441 However, enteric symptoms 

such as vomiting still occurred in many vaccinees 

with both vaccine candidates.440–442

Passive Immunotherapy, Postexposure Prophylaxis, 

and Treatment

No PEP is available for SEB. The only current treatment modality is intravenous human immunoglobulin. This form of passive immunotherapy can reduce 

mortality in animal models if given within 4 to 8 hours 

after inhalation.66

Ongoing work is assessing whether currently 

FDA-approved medications provide effective PEP or 

treatment for SEB. One of the most promising lines of 

research focuses on antiinflammatory and immunosuppressant agents as well as antioxidants. In particular, the immunosuppressant rapamycin (also known 

as sirolimus) has protected mice from intranasal and 

systemic exposure to SEB.443 A recent study in a murine 

model of SEB-induced lethal shock found that 75% of 

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Medical Aspects of Biological Warfare 

mice receiving a combination of the antiinflammatory 

drug dexamethasone (at 2 and 5 hours after SEB challenge) and the antioxidative drug N-acetyl cysteine 

(at 24, 30, 48, 54, 72, 78, and 96 hours after challenge) 

survived; by comparison, only 10% of untreated mice 

survived.444 

Postevent Countermeasures: Current Options

Treatment is limited to supportive care, which 

should focus on oxygenation and hydration; severe 

cases with pulmonary edema may require ventilation, vasopressors, and diuretics. At this time, no PEP 

is available; individuals potentially exposed to SEB 

should be closely monitored for symptoms of intoxication and treated accordingly.14,66

Ricin

Ricin is a protein toxin derived from castor beans 

(the seeds of the castor oil plant, Ricinus communis). 

Ricin, a cytotoxic lectin, consists of an A-chain, the 

toxic portion of the protein, bound to a B-chain, which 

serves to bind the toxin to surface receptors found on 

mammalian cells, enabling the A-chain to enter the 

cell. Once inside the cell, the A-chain inhibits protein 

synthesis, which ultimately results in cell death.445–449

Ricin can be delivered by aerosol, ingestion, or injection.450 Inhalation of ricin as a small-particle aerosol 

may produce pathological changes beginning within 

8 hours, manifested as severe respiratory symptoms 

associated with fever and followed by acute respiratory failure within 36 to 72 hours. Ingestion of 

ricin may result, beginning within 3 to 20 hours, in 

severe gastrointestinal symptoms (nausea, vomiting, cramps, and diarrhea) followed by vascular 

collapse and death. Injection can result, beginning 

within 6 hours, in general weakness and myalgias, 

followed by vomiting, fever, multiorgan failure, and 

death.14,66,448,449

Vaccination

No vaccine is available, but several vaccine candidates are being studied.451 Because passive prophylaxis 

with monoclonal antibodies in animals is protective 

against ricin challenge, the vaccine candidates are 

based on induction of a humoral response.452,453

The most promising development for a vaccine 

has been to genetically engineer the ricin toxin A 

chain (RTA) subunit to eliminate both its enzymatic 

activity and its ability to induce vascular leaking. The 

nontoxic RTA subunit has been demonstrated to induce antibodies in animal models and to protect mice 

against intraperitoneal challenge with large doses of 

ricin.451 A pilot clinical trial in humans demonstrated 

that a recombinant RTA vaccine (RiVax), given as 

three monthly intramuscular injections at doses of 

10, 33, or 100 μg (five volunteers at each dose), was 

safe and elicited ricin-neutralizing antibodies in one 

of five individuals in the low-dose group, four of 

five in the intermediate-dose group, and five of five 

in the high-dose group.454 However, the antibody 

response was of short duration. More recently, a 

phase 1B trial of Alhydrogel-absorbed RiVax found 

positive titers of anti-RiVax antibodies in four of five 

volunteers receiving three 10-μg doses and four of 

four individuals receiving three 100-μg doses. All 

of the eight individuals who seroconverted still had 

positive titers on day 252; five of these individuals 

continued to exhibit titers on day 364. The vaccine 

appeared to be safe and well tolerated.455 A recently 

developed heat-stable version of RiVax could extend the vaccine’s shelf life at high temperatures, 

potentially simplifying storage and distribution.456

A ricin vaccine candidate (RTA 1–33/44–198 or 

RVEc) developed at USAMRIID demonstrated high 

relative stability to thermal denaturation, no detectable cytotoxicity, and immunogenicity in animal 

studies. The vaccine demonstrated protective immunity against aerosol challenge with ricin in rodents, 

rabbits, and NHPs. Additionally, no toxicity was 

observed in two animal models.457–460 In a phase 1 

escalating, multiple-dose study, this vaccine was 

found to be safe, well tolerated, and immunogenic 

in healthy adults who received three doses of either 

20 μg (10 volunteers) or 50 μg (10 volunteers) of 

RVEc. Among 10 volunteers who received a single 

100-μg dose, 2 individuals developed elevated creatine phosphokinase levels, which resolved without 

sequelae; no further vaccinations were administered 

at this dosage. Four individuals in the 50-μg group 

received a single booster dose, which was safe and 

well tolerated; all booster recipients developed a 

robust anamnestic response.461 Further studies are 

planned to optimize dose, scheduling, and route of 

administration.

A ricin toxoid vaccine encapsulated in polylactide 

microspheres or poly(lactide-co-glycolide) microspheres and given intranasally was demonstrated to 

be protective against aerosolized ricin intoxication in 

mice. Both systemic and mucosal immune responses 

were observed, with high titers of antiricin IgG2a at 2 

weeks postvaccination and still present and protective 

in mice 1 year later.462 Oral vaccination of mice with 

the ricin toxoid vaccine encapsulated in poly(lactideco-glycolide) microspheres was also protective against 

lethal aerosol ricin challenge.463

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Treatment and Postexposure Prophylaxis

No therapeutic or PEP agent for ricin intoxication 

has been developed. Although passive immunoprophylaxis of mice can reduce mortality against 

intravenous or intraperitoneal ricin challenge if 

given within a few hours of exposure, passive immunoprophylaxis is not effective against aerosol 

intoxication.452,453 The development of prophylactic and therapeutic medical countermeasures for 

ricin intoxication is challenging in part because 

ricin is taken up into cells rapidly464 and has high 

enzymatic efficiency,465 leaving a narrow treatment 

window. 

Postevent Countermeasures: Current Options

Individuals who may have been exposed to ricin 

should be monitored closely. Diagnostic testing could 

include nasal swabs, sputum, and induced respiratory 

secretions for assay via polymerase chain reaction or 

antigen enzyme-linked immunosorbent assay (AgELISA) and serum for baseline toxin assays via AgELISA or polymerase chain reaction.66 Treatment consists primarily of supportive care, such as oxygenation, 

maintenance of electrolyte balance, and hydration for 

inhalational exposure and gastric lavage, administration of cathartics, and volume replacement of fluid loss 

for gastrointestinal intoxication.14,66,466

SUMMARY

Although medical countermeasures are effective in 

preventing disease, the greater challenge is to develop 

a balanced approach that may provide preexposure 

and postexposure medical countermeasures to protect 

both military and civilian populations. Generally, 

military personnel undergo prophylactic vaccination 

against a broad array of endemic diseases as deployments into areas not travelled by the masses could 

be required without significant advance notice. In 

addition, the military has recognized the benefit of 

vaccinating troops for protection against exposure to 

a biological weapons release in a battlefield setting. 

However, vaccination of civilians in advance may not 

be feasible because of the larger host of potential biological threat agents in a civilian population and the 

infrequent occurrence of bioterrorism events expected 

in a civilian population. 

Vaccine recommendations for the civilian and military populations must weigh the risks and benefits as 

well as the logistics of maintaining immunity with 

vaccine booster doses. More studies to assess the longterm medical effects of repeated vaccination with multiple vaccines are needed to assure civilian and military 

populations about the safety of the long-term use of 

vaccines. Protection of the public from bioterrorism 

will require the development, production, stockpile 

maintenance, and distribution of effective medical 

countermeasures for both prevention and treatment 

of illness, with careful forethought about the balance 

of preexposure and postexposure countermeasures. 

It is likely that the military will be involved with both 

the distribution of medical supplies and the management of bioterrorism events within the continental 

United States; therefore, military physicians must be 

properly trained and prepared for managing bioterrorism events.

Acknowledgments

The following individuals were the authors of the previous edition of this chapter and contributed to this edition:

Janice M Rusnak, Ellen F Boudreau, Matthew J Hepburn, James W Martin, and Sina Bavari.

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	457.	 Olson MA, Carra JH, Roxas-Duncan V, Wannemacher RW, Smith LA, Millard CB. Finding a new vaccine in the ricin

protein fold. Protein Eng Des Sel. 2004;17:391–397.

	458.	 Carra JH, Wannemacher RW, Tammariello RE, et al. Improved formulation of a recombinant ricin A-chain vaccine

increases its stability and effective antigenicity. Vaccine. 2007;25:4149–4158.

	459.	 McLain DE, Lewis BS, Chapman JL, Wannemacher RW, Lindsey CY, Smith LA. Protective effect of two recombinant

ricin subunit vaccines in the New Zealand white rabbit subjected to a lethal aerosolized ricin challenge: survival, immunological response, and histopathological findings. Toxicol Sci. 2012;126:72–83.

	460.	 McLain DE, Horn TL, Detrisac CJ, Lindsey CY, Smith LA. Progress in biological threat agent vaccine development: a

repeat-dose toxicity study of a recombinant ricin toxin A-chain (rRTA) 1-33/44-198 vaccine (RVEc) in male and female 

New Zealand white rabbits. Int J Toxicol. 2011;30:143–152.

461. Pittman PR, Reisler RB, Lindsey CY, et al. Safety and immunogenicity of ricin vaccine, RVEC (TM), in a phase 1 clinical 

trial. Vaccine. 2015;33:7299–7306.

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462. Yan CH, Rill WL, Malli R, et al. Intranasal stimulation of long-lasting immunity against aerosol ricin challenge with 

ricin toxoid vaccine encapsulated in polymeric microspheres. Vaccine. 1996;14:1031–1038.

	463.	 Kende M, Yan CH, Hewetson J, Frick MA, Rill WL, Tammariello R. Oral immunization of mice with ricin toxoid vaccine encapsulated in polymeric microspheres against aerosol challenge. Vaccine. 2002;20:1681–1691.

464. Fodstad O, Olsnes S, Pihl A. Toxicity, distribution and elimination of cancerostatic lectins abrin and ricin after parenteral injection into mice. Br J Cancer. 1976;34:418–425.

465. Endo Y, Tsurugi K. The RNA N-glycosidase activity of ricin A chain–the characteristics of the enzymatic activity of 

ricin A chain with ribosomes and ribosomal RNA. J Biol Chem. 1988;263:8735–8739.

	466.	 Ramasamy S, Liu CQ, Tran H, et al. Principles of antidote pharmacology: an update on prophylaxis, post-exposure

treatment recommendations and research initiatives for biological agents. Br J Pharmacol. 2010;161:721–748.

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Future Prospects of Vaccines and Antibodies in Biodefense

Chapter 28

FUTURE PROSPECTS OF VACCINES 

AND ANTIBODIES IN BIODEFENSE

JEFFREY FROUDE II, PhD*; CRYSTAL W. BURKE, PhD†; and JEAN-NICOLAS TOURNIER, MD, PhD‡

INTRODUCTION

TRENDS IN BIODEFENSE VACCINE DEVELOPMENT

Live Attenuated Vaccines

Recombinant Vaccine Vectors

Subunit Vaccines

Nucleic Acid Vaccines

Mucosal Vaccines

Adjuvants

THE FUTURE OF ANTIBODIES IN BIODEFENSE

Antibody Generations and the Development of New Therapeutic Formats

Antitoxin Monoclonal Antibodies

Antibacterial Monoclonal Antibodies

Antiviral Monoclonal Antibodies

SUMMARY

*Clinical Pharmacology Fellow, Experimental Therapeutics Division, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, Maryland 20910

†

Research Microbiologist, Virology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Room 6, Fort Detrick, Maryland 21702

‡

Colonel, French Armed Forces Health Service, Department of Infectious Diseases, Unité Biothérapies anti-infectieuses et immunité, Institut de recherche biomédicale 

des armées, 1 place du Général Valérie André, Brétigny-sur-Orge, 91220 France; École du Val-de-Grâce, Paris, France; Unité Génomique virale et vaccination, 

Institut Pasteur, Paris, France

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INTRODUCTION

transgenic mice that carry human antibody gene loci.1

The growing problem of increased bacterial resistance to antimicrobials, together with the need 

for alternative strategies to treat infectious diseases 

resistant to therapeutics, has stimulated a renewed 

interest in antibody therapy in the fields of infection 

and intoxication within biodefense. Although the risk 

of antimicrobial resistance is acute, an agent selected 

for deliberate dissemination may also be selected or 

engineered for antimicrobial resistance to existing 

treatments. Antibody therapy may be one way to mitigate that risk. Furthermore, utilization of antibodies in 

combination with existing therapeutics may provide 

synergistic benefits.

The pathway for monoclonal antibody (mAb) use 

in infectious disease follows proof-of-concept studies 

utilizing species matched fractionated serum, the use 

of human convalescent serum, and the early success 

of pAbs. Although initial results are promising, only 

a few mAbs are biodefense-related; namely raxibacumab, which obtained approval by the Food and 

Drug Administration (FDA) in 2012 for treatment of 

inhaled anthrax. However, several biodefense-specific 

mAb cocktails are showing success in early clinical 

phase trials.2

 The FDA has approved antibody therapy 

for emergency use when no other therapeutic options 

are available.3 Throughout this chapter, monoclonal 

antibodies will be abbreviated as mAbs. Some of these 

therapeutics are also abbreviated as rAbs, or recombinant antibodies, in other texts, defined by their selection or engineering, and expression utilizing a range 

of DNA-based molecular biology techniques. Here, 

novel antibody designs (ie, fusion proteins) will be 

identified by their construct.

Vaccine development has also greatly benefited 

from advanced biotechnology. New vaccines may 

reveal previously unrecognized or underappreciated 

pathways to providing protection against biological 

weapons. This chapter will first examine the possible 

future of vaccines in a biodefense perspective, then 

consider how antibodies may provide novel and efficient ways to protect soldiers.

Novel and next-generation vaccine approaches are 

being developed in parallel with new generations of 

antibody-based therapeutics. Each of these approaches 

has advantages specific to the disease, timeline, development status, and therapeutic or treatment window 

of its targeted pathogen. Usually, a specific or broadly 

neutralizing antibody response is required to provide 

either innate protection or vaccine-mediated protection, but this is not always the case. The increasing 

demand for antibody-drug conjugates, vaccine and 

small-molecule synergistic effects, and vaccine prophylactic use highlights the need to develop these two 

strategies individually and in combination to discover 

the optimal forms of protection. 

Edward Jenner’s 1794 discovery of the cross-protection afforded by a cowpox virus to variola virus led 

to the first vaccine, and ultimately to the successful 

eradication of variola on May 8, 1980, when the World 

Health Assembly certified the world free of naturally 

occurring smallpox. In the early 1880s, Louis Pasteur 

led the development of live attenuated vaccines, and 

in the late 1890s Emil von Behring and Kitasato Shibasaburo developed serum therapy against diphtheria 

and other microbes. Serum therapy was effective, 

but the administration of large amounts of animal 

proteins often led to undesirable side effects such as 

serum sickness. Within the same time period, vaccine 

developments provided efficient protective active immunity against rabies, tuberculosis, typhoid fever, and 

diphtheria and tetanus toxins. 

The use of serum therapy declined after the discovery of antibiotics in the 1940s. It was later recognized 

that the critical protection afforded by crude serum 

was linked to polyclonal antibodies (pAbs), the “magic 

bullets” imagined by Paul Ehrlich. The development of 

biotechnology during the 20th century allowed Georges Köhler and César Milstein to isolate the first murine 

monoclonal antibody by immortalizing B cells in 1975. 

Hybridoma technology revolutionized antibody therapeutics and was later enriched by immortalization of 

human peripheral B cells, direct cloning of variable 

genes into phage expression libraries, and creation of 

TRENDS IN BIODEFENSE VACCINE DEVELOPMENT

The FDA has fully licensed only two biodefense-related vaccines (Table 28-1). The development of vaccines 

is generally an expensive undertaking; a single vaccine 

usually takes 10 to 15 years to reach licensure, at a cost 

estimated in the hundreds of millions of dollars.4

 The 

pharmaceutical industry’s evaluation of the cost is not 

simple, but a recent rotavirus vaccine is estimated to have 

cost between $205 million and $878 million.5 For vaccine 

manufacturing companies, the biodefense market is limited, and as defense budgets globally tend to decrease, 

decisions to develop novel vaccines will be carefully 

examined. However, healthcare authorities are increasingly recognizing the benefit and cost-effectiveness of 

vaccines, so vaccine research in biodefense makes sense. 

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Future Prospects of Vaccines and Antibodies in Biodefense

TABLE 28-1

MAIN VACCINE STRATEGIES APPLIED TO BIODEFENSE AGENTS UNDER INVESTIGATION OR 

AUTHORIZED BY THE US FOOD AND DRUG ADMINISTRATION

Strategy Vaccine Agent Manufacturer Status

Live attenuated vaccine Vaccinia virus Smallpox Sanofi (Paris, France) Authorized by FDA

Francisella LVS strain Tularemia DynPort Vaccine Co LLC Under investigation

(Frederick, MD)

Stern strain Anthrax Colorado Serum Co Not authorized by FDA

(Denver, CO)

EV76 Plague Not currently Not authorized by FDA

 manufactured

Recombinant vaccine c-Ad3-EBO Ebola virus NIAID-GSK Under clinical

vectors (Brentford, UK) investigation

VSV-EBOV Ebola virus NewLink Genetics Under clinical 

(Ames, IA); Merck investigation

 (Summit, NJ)

Subunit vaccine AVA Anthrax protective Emergent BioSolutions, Authorized by FDA

 antigen (Rockville, MD)

LcrV-F1 Plague Dynport Vaccine Co LLC Under clinical 

 investigation

AVA: anthrax vaccine adsorbed

FDA: Food and Drug Administration

LVS: live vaccine strain 

NIAID-GSK: National Institute of Allergy and Infectious Diseases-GlaxoSmithKline

VSV-EBOV: vesicular stomatitis virus-Ebola virus vaccine

The future of vaccine development can be seen as 

responding to two different demands: On one hand, 

health authorities are pushing for safer, well-defined, 

purified vaccines, including vaccines with minimalist 

compositions that are able to protect against a single 

agent. Future biodefense vaccines developed in this 

context will need to follow these requirements. On the 

other hand, defense authorities want vaccines effective 

against dozens of different agents, protective against 

pulmonary contamination, triggering a rapid immune 

response, easy to administer, easy to produce on a 

large scale, and stable enough for long-term storage. In 

addition to these specifications, development and production costs should be kept as low as possible. These 

two global trends are clearly antagonistic, but emerging technologies may help in the development of safer 

vaccines with a larger range of efficacy. This section 

will examine how emerging technologies may improve 

old vaccines or give rise to new, multivalent vaccines. 

Live Attenuated Vaccines

Jenner’s serendipitous discovery of an attenuated 

vaccinia virus (VACV)6

 was later rationally extended 

and developed by Pasteur to produce vaccines against 

anthrax7 and later rabies.8 Live vaccines have since proven to be very efficient, as evidenced by the eradication of 

smallpox,9 the prevention and control of poliomyelitis10

and yellow fever,11 and, more generally, the control of 

infectious diseases throughout the 20th century. The 

only attenuation technique available before the 20th 

century was the time-consuming and rudimentary 

passage of virus in transformed culture cell lines or 

in atypical hosts. Ideally, live attenuated vaccines are 

composed of live virus or bacterium that establishes a 

mild infection at the site of replication, which the immune system then controls by mounting an immune 

response. Live attenuated vaccines use a weakened 

agent, allowing for a controlled infection, which is why 

these vaccines optimally activate immune effectors and 

are more effective than inactivated vaccines.12 Live attenuated vaccines must respect a delicate balance. They 

should be active enough to be immunogenic, but also 

sufficiently attenuated to be safe. The present regulatory environment imposes strong limitations on the 

development of new live attenuated vaccines to limit 

the risk of wild type virulence reversion, especially 

for virus-based vaccines. Discussed later are promising technologies that may aid in the development of 

live attenuated viruses as safer vaccine candidates. 

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Medical Aspects of Biological Warfare 

Replication-defective viruses represent the first approach to improving safety and limiting the risk of uncontrolled infection in an immune-compromised host. 

As mentioned previously, this attenuation technique 

has historically occurred through serial passage; for 

example, a VACV was passed more than 570 times in 

chicken embryo fibroblasts, leading to the production 

of an attenuated vaccine called modified vaccinia virus 

Ankara (MVA) in the 1970s.13 Through passage, MVA 

lost approximately 15% of its genome, was rendered 

replication-defective, and thus has proven to be safe 

in large clinical studies. Capitalizing on sequencing 

technology, researchers have synthetically developed 

an MVA homolog on a Lister strain vaccine virus 

background by deleting five regions similar to the regions lost by MVA.14 This demonstrates one way that 

genetic-based technology can help in the development 

of rational vaccines. These viruses, however, are less 

immunogenic than their parental strains, illustrating 

the delicate balance needed between immunogenicity 

and safety. Because they are defective in replication, 

these vaccines are too efficiently eliminated, leading 

to a poor immune response. 

Codon deoptimization is a novel strategy for developing live attenuated vaccines against RNA viruses, 

which represent a unique challenge due to their high 

genetic instability. Numerous studies have shown how 

the low-fidelity, RNA-dependent RNA polymerase can 

allow a virus population to rapidly drift or potentially 

revert any mutation.12 Mammalians, as well as many 

other organisms, have codon bias, using synonymous 

codons more frequently than others. This bias is probably correlated to the efficiency of translation, and 

observed in virus structural genes. Codon deoptimization in the genome capsid of poliovirus induced a 

profoundly attenuated strain that triggered a strong 

immune response in animals.15 As a general strategy 

for other vaccine development, codon deoptimization 

offers the major advantage that the genome is not mutated; therefore, it does not affect the antigenicity of the 

proteins. Codon deoptimization has been applied to 

different RNA viruses16–18; however, more studies are 

needed to evaluate whether this promising approach 

is safe and applicable for large-scale production. 

Another strategy for the production of live attenuated vaccines is using microRNAs (miRNAs), which 

are genetically encoded short RNAs that have tissuespecific or developmental expressions and that play 

a large role in gene regulation.19 Researchers have 

inserted miRNA sequences into the poliovirus genome 

to restrict its replication in the central nervous system 

(CNS).20 However, it cannot be ruled out that the virus, 

by replicating outside of the CNS, could insert mutations in the miRNA sequence and revert its virulence. 

Finally, zinc-finger (ZF) nuclease-controlled live 

attenuated virus is another experimental strategy 

that may be exploitable in the future as a vaccine.12

This strategy has used ZF domains coupled to other 

functional domains to produce novel transcription 

factors that increased or decreased gene transcription 

with promoter specificity.21–23 In other studies, ZF 

domains were fused to a restriction enzyme nuclease 

domain, resulting in the cleavage of specific sequences 

of double-stranded DNA.24–26 This strategy could be 

useful in vaccination efforts to prevent or eliminate 

persistent viral infections.

The first live attenuated bacteria-based vaccine 

for human use was developed against tuberculosis 

through 230 serial passages of Mycobacterium bovis over 

a period of 14 years on artificial medium by Calmette 

and Guérin.27 Live attenuated vaccines have historically been developed by the empirical technology of 

passage, and used in humans against anthrax (in the 

former USSR)28 and plague (in the former USSR and 

in developing countries where plague is endemic),29

but they are not authorized in Western countries. 

Francisella live attenuated vaccine live vaccine strain 

is licensed by the FDA, but could be improved. Advances in molecular biology and a better knowledge 

of host–pathogen interactions will help in the future 

development of rational strategies for novel live attenuated bacterial vaccines. 

Recombinant Vaccine Vectors

Viruses can be engineered as carriers of heterologous antigens. This was elegantly demonstrated 

more than 30 years ago using a VACV expressing the 

hepatitis B surface antigen (HBsAg), which induced 

a heterologous protective immunity against hepatitis 

B in chimpanzees.30,31 Although very promising, this 

strategy has yet to lead to a licensed vaccine for use in 

humans. Many viruses have been tested for their utility 

as a vaccine vector through their capacity to express 

heterologous proteins and induce an appropriate 

immune response. The myriad virus genera used to 

express recombinant protective antigen (PA) of anthrax 

can be seen as a paradigm of the extensive possibility 

of molecular biology in vaccinology. Anthrax PA has 

been successfully expressed by members from very 

diverse families of viruses, including poxvirus,32 rabies 

virus,33 hepatitis B virus,34 adenovirus,35 and influenza 

virus,36 as well as in baculovirus and vaccina virus 

recombinants.37 

Interest in the virus vector strategy was recently 

tested by the quick development of vector vaccines 

to help control the 2014 Ebola virus outbreak in West 

Africa. A replication-defective chimpanzee adenovirus 

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Future Prospects of Vaccines and Antibodies in Biodefense

type 3 vector (cAd3-EBO) vaccine has shown interesting preliminary results in phase I trials,38 while a 

vesicular stomatitis virus-based vector will soon enter 

the clinical phase.39 Only a limited number of viruses 

have successfully undergone large-scale manufacturing practices and clinical trials and may one day reach 

human licensure. These include poxviruses, adenoviruses, Alphaviruses, and Flavivirus.40–42

Poxviruses represent good vector candidates because they grow to high titers, are very stable when 

lyophilized, and can accept very large transgenes 

due to their large genome sizes.43 Moreover, the issue 

of preexisting immunity is limited because the adult 

population born after 1980 is naïve, and the protection 

afforded by the vector against variola provides dualpurpose use in a biodefense perspective. This last point 

has been exemplified by the development of a bivalent 

vaccine of a VACV vector expressing anthrax PA that 

provides immunity against anthrax and variola.32 MVA 

has been used as vector for multiple viruses, such as 

human immunodeficiency virus (HIV), influenza, and 

dengue, but it has been largely supplanted by avipoxviruses, especially canarypox, which have a better 

safety profile because they do not replicate in mammalian cells.44 Canarypoxvirus-vectored vaccines have 

been developed against rabies and measles and studied 

in HIV.44 Although they proved to induce significant 

immune responses, the immune response is reduced 

in hosts with preexisting cross-protective immunity 

against the vector. This relatively new field would 

benefit immensely from a greater understanding of 

precisely which viral genes are crucial to triggering 

an efficient immune response. 

Human adenoviruses (Ads) are also live vectors well 

suited for vaccine development, with a large genome 

of 38 kb of double-stranded DNA.41 Recombinant 

Ads vectors have the E1 segment deleted, allowing 

the insertion of 5 kb of heterologous antigens, and are 

replication-defective in human cells. Most Ads vectors 

have been developed using Ad5, Ad26, and Ad35.41,45,46

Ad5 vectors can be administered by nasal spray, allowing targeted vaccination of the mucosal surface. 

The main drawback of Ads vectors is the presence of 

preexisting immunity for Ad5 and the recombination 

hazard during the production process. 

Another viable vector platform is virus-like replicon 

particles (VRPs). VRPs have been generated using the 

members of the Alphavirus or Vesculovirus genera. 

Alphaviruses contain a single-stranded, positive RNA 

of 12 kb and are members of the family Togaviridae. 

Replicons derived from Sindbis virus, Semliki Forest virus, and Venezuelan equine encephalitis virus 

(VEEV) were engineered by replacing structural genes 

with heterologous genes.47 Vesicular stomatitis virusbased VRPs expressing Marburg virus glycoproteininduced immune cross-protection against various 

strains of Marburg virus.48

Yellow fever virus (YFV) was developed under the 

ChimeriVax technology by Sanofi (Paris, France) as a 

chimeric virus for other Flaviviruses, such as dengue, 

West Nile virus, and Japanese encephalitis virus.42 The 

platform consists of the live YFV 17D vaccine backbone 

virus deleted of its premembrane and envelope genes, 

which were replaced by heterologous genes from other 

Flaviviruses. This vector has been closely examined 

regarding its safety and the risk of recombination in 

the environment. Although safe and tested in large 

phase III clinical trials for dengue, this technology is 

limited to the Flaviviridae family.49

Bacteria can also be used as a vector for carrying 

vaccine antigens. Listeria monocytogenes and Salmonella

species have been used as vaccine vectors in numerous 

clinical trials, mainly as cancer vaccine candidates.50

Salmonella has shown some promise from a biodefense 

perspective as a vector for vaccines against pathogens 

such as anthrax51 and plague52; however, for a vaccine strategy in a general population, less pathogenic 

strains are desired. 

Among many models, lactobacilli are regarded as 

interesting bacteria because they can induce a mucosal 

immune response by expressing an antigen locally. 

It has been shown that PA from Bacillus anthracis expressed by a Lactobacillus gasseri induces significant 

protection against the infection in mice.53 Because 

lactobacilli are probiotic, they can be administered 

easily and carry multiple genes, providing potential 

immunity against diverse agents.

Additionally, Bacillus spores can be used as an antigen carrier and vaccine platform.54 This platform is 

promising because the spores are very stable and can 

be used as a mucosal vaccine. As an example, Bacillus 

subtilis-expressing PA has been shown to induce protective immunity against anthrax in a murine model.55

One of the main hurdles for vaccine vectors is the 

evaluation of efficacy in a human population that may 

have acquired various levels of preexisting immunity. 

Limited data has been published so far on this topic, 

but a recent review of the literature suggests that for 

Salmonella vectors preexisting immunity can enhance 

subsequent induction of immunity, while for adenovirus preexisting immunity is a hindrance.56

Subunit Vaccines

Since the inception of vaccinology, the hunt for 

ways to address attenuated vaccine safety concerns 

has resulted in the search for immunogenic subunits. This was first exemplified in the 1920s by the 

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Medical Aspects of Biological Warfare 

discovery that formaldehyde-inactivated exotoxins 

are immunogenic.57 However, beyond exotoxins, 

large-scale production of subunit antigens was not 

feasible at the time. During the “golden age” of vaccinology, in the 1960s and 1970s, most vaccines were 

inactivated viruses or bacteria. Renewed interest in 

the development of subunit vaccines occurred in 

the 1980s with the emergence of a hepatitis B virus 

subunit vaccine composed of the HBsAg protein, 

initially purified from patient sera and later produced 

by genetic engineering.57 Subunit vaccines are attractive for use in biodefense because they offer stimulation of targeted immune responses with minimal 

side effects. Indeed, the anthrax PA-based vaccine 

BioThrax (Emergent BioSolutions, Rockville, MD) 

and next-generation plague fusion protein LcrV-F1 

vaccine currently under clinical investigation are both 

antigen-targeted subunit vaccines produced through 

genetic engineering. 

First demonstrated 35 years ago, synthetic viruslike particles (VLPs) are molecular structures based 

on viral protein subunits that form artificial capsid 

units.58 VLPs mimic native virions by displaying 

repetitive subunits of the viral surface proteins that 

present the immune system with conformational 

viral epitopes that can elicit strong T cell and B cell 

immune responses; however, they can be distinguished from virus by the absence of nucleic material. The absence of replication renders them safe 

antigens for use as a vaccine. In 1986, at the dawn 

of recombinant technologies, HBsAg was produced 

with yeast to form the first synthetic type of VLP to 

come on the market (Recombivax B [Merck, Summit, 

NJ] and Engerix B [GlaxoSmithKline, Brentford, 

United Kingdom]). Some 20 years later, two human papilloma virus (HPV) vaccines (HPV 16 VLP 

vaccine and HPV 18 VLP vaccine)—based on the 

L1 protein produced in yeast or insect cells—were 

licensed (Cervarix [GlaxoSmithKline] and Gardasil 

[Merck]).59 More recently, a VLP-based hepatitis E 

virus vaccine was licensed for use in the Chinese 

market (Hecolin [Xiamen Innovax Biotech, Xiamen, 

China]).60

Numerous VLP-based vaccines are currently in 

preclinical development and clinical trials, which 

shows the potential of this strategy.61 Of particular 

interest for biodefense, VLPs have shown promising initial results in protection against the Ebola and 

Marburg viruses.62 VLPs consisting of up to three 

antigens—(1) the glycoprotein, (2) the matrix protein 

(VP40), and (3) the nucleoprotein—as well as “hybrid 

VLPs”—containing both Ebola and Marburg glycoprotein and nucleoprotein—can confer protection 

against homologous challenge with either virus in 

rodents and nonhuman primates.62 VLPs can be genetically engineered, incorporating peptides, proteins, 

or pathogen-associated molecular pattern ligands to 

create elaborate scaffolds.63

The plasticity and potential of VLPs are quite remarkable and limited only by the human imagination. 

Their plasticity is exemplified by insect Flock House 

virus VLP nanoparticles that display anthrax antigen. When the VLPs are coated with the PA-binding 

domain of anthrax toxin receptor 2, they serve as a 

decoy receptor for anthrax toxins in vitro and in vivo. 

Alternatively, when coated with PA protein, the VLPs 

can be used as an efficient anthrax vaccine.64 VLPs 

can be used as a molecular platform to help the immune system recognize heterologous antigen. These 

characteristics place synthetic VLPs at the boundary 

between subunit antigen and adjuvant (see the discussion under Adjuvants).

Initially launched in the 1980s with the first pneumococcal vaccine, recent technological advances in 

glycan synthesis, glycan structure analysis, and glycochemistry are paving the way for a new era in carbohydrate vaccine design.65 In this approach, the surface of 

bacteria is covered by a dense array of polysaccharide 

on the lipopolysaccharide and the capsule, forming a 

unique feature with strong antigenic properties. It is 

important to note that glycan synthesis is complex and 

its support of antigenic specificity is not genetically 

encoded. The conjugation of glycans to a carrier protein or to an adjuvant induces long-lasting protection 

against encapsulated bacteria, which could potentially 

be extended to viruses. This novel and booming field 

may help develop new vaccines against gram-negative 

or capsulated bacteria such as Yersinia pestis, Francisella 

tularensis, and Burkholderia pseudomallei.

Advances in genomics, including high-throughput 

DNA sequencing, have provided access to complete 

antigenic repertoires of pathogens. Capitalizing on 

this information, in a process coined “reverse vaccinology,” previously unknown vaccine antigens have 

been identified.66,67 The first success story of this strategy was the long-sought vaccine against meningococcus B.68 In contrast to Neisseria meningitidis serogroups 

A, C, Y and W135, carbohydrate vaccines could 

not be used for serogroup B due to the similarities 

between the capsular polysaccharide and a human 

neural antigen. The first step of the lengthy process 

of developing this vaccine was a computer analysis 

of the targeted genome sequence to identify all genes 

coding for predicted antigens. The 600 predicted 

antigens were screened for their expression by the 

pathogen and their immunogenicity (referred as the 

antigenome).69 Twenty-nine selected antigens were 

further tested for their immunogenicity in animal 

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Future Prospects of Vaccines and Antibodies in Biodefense

models. Lastly, the selected PAs were evaluated for 

their presence in worldwide representative pathogen 

collections. Five stable PAs were industrially produced for clinical trials. At each step of the process, 

the number of antigens dramatically decreased, but 

the power of the strategy comes from the complete 

genome analysis.68 Although this strategy is costly, it 

may be applied to diverse biothreat agents for which 

other methods of vaccine development have so far 

been ineffective.

“Systems vaccinology” is a generic term recently 

coined to describe the use of systems approaches to 

identify signatures that can be used to predict vaccine immunity in humans.70 The two major goals 

of systems vaccinology are to characterize the host 

response by identifying genes and pathways whose 

expressions are altered in those receiving vaccines, 

and then to identify predictors of vaccine efficacy. 

Systems vaccinology is still in its infancy, but it 

may help in the future design of efficient vaccines, 

especially for pathogens that constantly change their 

surface-expressed molecules, such as plasmodia and 

other eukaryotic parasites.71

Structural vaccinology, the use of three-dimensional 

structural information to design novel and improved 

vaccine antigens, is another trend in rational vaccine 

design. Advances in X-ray crystallography and nuclear 

magnetic resonance spectroscopy have enabled researchers to pinpoint new structures and antigenic 

epitopes at atomic resolution.72 Recent advances may 

have solved one of the hottest issues in vaccinology: 

respiratory syncytial virus (RSV). RSV has been a 

challenge for vaccine development after a disastrous 

clinical trial in the 1960s showed that a killed vaccine 

enhanced the disease, increasing hospitalization rates 

of children and causing two deaths.73 RSV fusion (F) 

glycoprotein, which helps the virus merge its membranes with the host cell, exists in two conformational 

states, the prefusion metastable state and the postfusion stable state.74 By engineering soluble variants of 

RSV F with a stably exposed antigenic site, researchers have identified one easy-to-produce variant that 

induces a strong neutralizing immune response in 

murine and nonhuman primate models.75 Complete 

understanding of antigenic structure may aid the 

identification of key sites to target in order to disable 

a protein’s function, stabilize select conformations of a 

protein, or determine antibody–antigen complexes, all 

of which would allow the development of more stable, 

homogeneous, efficiently produced vaccine antigens. 

This strategy might be applied with great success to 

viruses for which neutralization could be obtained 

through surface glycoproteins, such as hemorrhagic 

fever viruses. 

Nucleic Acid Vaccines

Proof-of-concept studies for nucleic acid vaccines 

occurred more than 2 decades ago, when it was shown 

that intramuscular injection of mRNA or DNA resulted 

in local production of a reporter gene76 and the induction of an immune response.77,78 For DNA vaccination 

with this approach, the gene of an antigen is encoded 

and expressed from a plasmid-based system. In theory 

this method provides a subunit vaccination; however, 

by expression of the antigen within the host, rather than 

exogenous supplementation of the protein, the antigen 

is expressed in its natural form. As a result, the antigen 

can be processed by the immune system to activate 

both humoral and cellular immune responses.79 DNA 

vaccines are particularly attractive in the biodefense 

field because they offer many advantages: (a) they can 

be engineered without the need to culture a pathogen; 

(b) manufacturing processes to produce plasmid DNA 

are well-established; (c) plasmid DNA manufacture 

is extremely rapid and can be designed for any engineered or emerging pathogen; and (d) the vaccines are 

safe and pose no risk of integration into the genome. 

Despite these obvious advantages and many clinical 

trials, no DNA vaccines have been authorized for human use by the FDA.80 Many hurdles need to be overcome if there is to be a future for this vaccine class. One 

such challenge is the manufacturing of plasmid DNA 

on a large scale with good manufacturing processes.81

In the biodefense field, DNA-based vaccines have been 

tested against anthrax with somewhat disappointing 

results, but they have shown efficiency against Filoviruses, poxviruses, and encephalitic Alphaviruses.82

The greatest challenge, though, is the suboptimal 

immunogenicity elicited by the vaccines in humans, 

which has been found to be significantly lower than 

that observed in rodents. Several strategies have been 

developed to improve the vaccines’ immunogenicity.79

As with live vaccines, codon optimization has been 

tested and helps with transgene expression in human 

cells. Another strategy has been to optimize the design of the transgene, including adding untranslated 

regions which may be critical regulators of vaccine 

gene expression located 5’ and 3’ to the encoded gene. 

One additional strategy has been to include unmethylated cytosine-phosphate-guanine (CpG) motifs in the 

sequence. CpG motifs are absent from eukaryotic DNA 

and are recognized as pathogen-associated molecular 

patterns by toll-like receptor (TLR) 9. The presence of 

CpG motifs may increase immunogenicity by stimulating a robust innate immune response.

In addition to making alterations in the plasmid 

DNA itself, changes in the delivery of DNA vaccines 

may enhance the quality and magnitude of the immune 

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Medical Aspects of Biological Warfare 

response elicited. Many technologies have been tested, 

and the debate over their efficiencies is as long-standing as DNA vaccine technology itself.83 So far, needle 

injection (subcutaneous or intramuscular), particle 

bombardment (“gene gun”), high-pressure liquid delivery, and electroporation have been evaluated, and 

each has specific advantages and disadvantages.83,84

RNA vaccines have been developed more recently, 

mainly because RNA molecules were known to be less 

stable. Despite this instability, RNA vaccines retain three 

major advantages over DNA vaccines.85,86 First, RNA 

must be delivered only into the cytoplasm of target 

cells (in contrast to DNA, which must be transported 

to the nucleus). Second, genomic integration of the 

RNA is not possible, circumventing this safety concern 

(as well as the risk of inducing anti-DNA antibodies). 

Thus, RNA vaccines are not classified by the FDA as 

“gene therapy.” Third, RNA interactions with the host 

are very versatile because the intracellular host innate 

immune response is focused on the detection and 

elimination of exogenous (mainly viral) RNAs. Due 

to the transient nature of RNA and the requirement of 

translation by host machinery, the load of antigen produced is more controlled, thereby minimizing the risk 

of tolerance induction by long-term antigen exposure. 

Proof-of-concept studies demonstrating the efficiency of RNA vaccines has been established for 

numerous antigens.85–87 However, RNA vaccines are 

still very experimental and require improvements 

to overcome the hurdles of clinical vaccine development. RNA stability and delivery methods need to 

be improved. Ribonucleases are present on the skin 

and in tissues, and mRNAs are negatively charged, 

which impairs their entry in the cytosol through the 

cell membrane. New methods for improving the RNA 

transfection efficiency may help. A recent study has 

shown that encapsulating a self-amplifying RNA into 

a lipid nanoparticle protected it from ribonuclease 

digestion and elicited a broad, potent, and protective 

immune response in rodents.88

High production costs and low RNA yield from in 

vitro production of mRNAs presents the second major 

challenge to the development of RNA vaccines.89,90

So far, the longest chemically synthetized RNA with 

biological activity is 117 nucleotides.90 Next-generation 

nucleic acid vaccines will also require an improved 

delivery technology, assessed in conjunction with the 

development of the vaccine.

Mucosal Vaccines

During an attack, biothreat agents would likely be 

presented in one of two primary routes of administration: aerosols or introduction into food or water supplies. Thus, an infectious agent would enter the host 

through the respiratory or intestinal mucosae. For this 

reason, vaccines targeting the mucosal surfaces seem 

logical for biodefense. However, a distinction between 

agents that elicits mucosal infections and those that 

simply exploit mucosal tissues as a mean to gain access to the body should be made.91 When developing a 

vaccine to protect the mucosal surfaces, two strategies 

exist: (1) to increase the mucosal response of existing 

systemic vaccines and (2) to design a vaccine targeted 

for mucosal delivery and immune response,: the latter will be examined in this section. Mucosal vaccine 

development should be focused on agents that provoke 

a mucosal-associated infection, but not on agents that 

are controlled by systemic immunity. Mucosal vaccines offer many advantages from a production and 

regulatory point of view92: (a) oral vaccines do not 

need purification steps because the gut microbiota is 

already complex; (b) mucosal vaccines do not require 

injection, so they are subject to a better compliance and 

are suited for mass vaccination as they do not require 

medical personnel for administration; and (c) mucosal 

vaccines do not carry risk of spreading blood-borne 

infection because no needles are involved. 

Mucosal immunology is an expanding field that has 

led to a better understanding of the mucosal immune 

system and response. Nevertheless, few mucosal vaccines have made it to licensure. Thus far, the only live 

attenuated virus vaccine administered intranasally 

are FluMist (MedImmune, Cambridge, United Kingdom), against influenza virus, and Vaxchora (PaxVax, 

Redwood City, CA), a suspension of Vibrio cholerae 

strain CVD 103-HgR) against cholera.93 Two other live 

attenuated vaccines administered orally against rotavirus94 and S typhi have also been efficiently launched.95

Notably, two inactivated oral vaccines against cholera have been marketed, providing an interesting 

proof-of-principle for future inactivated vaccines.96 

In the biodefense field, Y pestis has been a model for 

the development of live mucosal vaccines providing 

protection against bubonic and pulmonary plague.91

A major challenge for mucosal vaccine development 

is the production of candidates that strike a balance between immunogenicity and attenuation. Historically, 

most mucosal vaccines have been produced through 

passage of virus in host cell culture, as was done for 

the oral polio vaccine. It is clear that mucosal vaccines 

will benefit immensely from a more rational approach 

using genetic manipulation to increase their safety 

and stability, as exemplified by the typhoid Ty21A 

vaccine.97

Another area of mucosal vaccine development that 

needs attention is characterizing the optimal route of 

immunization. The biodefense community frequently 

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Future Prospects of Vaccines and Antibodies in Biodefense

focuses on intranasal, oral, and sublingual routes of 

immunization. The mucosal immune system is compartmentalized into nasopharynx-associated lymphoid 

tissues, bronchus-associated lymphoid tissues, and 

gut-associated lymphoid tissues.98,99 Although some 

functional connections between the respiratory and 

gut immune systems have recently been described,100

the presumably limited connectivity between the respiratory tract and the gut places a constraint on the 

definition of the optimal route of immunization. As a 

result, different routes of immunization tend to result 

in compartmentalized responses, with (a) intranasal 

immunization inducing a strong immune response 

(measured by the secretory immunoglobulin A [IgA] 

production) in the upper and lower respiratory tract 

and the gastric and genital tracts; (b) sublingual immunization inducing a response in the upper and lower 

respiratory tract and the gastrointestinal tract; and (c) 

oral immunization triggering an immune response in 

the salivary glands, gastrointestinal tract, and mammary glands.98,99

Development of a strong immune response to 

oral vaccination is especially challenging because 

of oral tolerance and the host’s need to maintain 

homeostasis to protect against immune responses to 

digested antigens. Even after preclinical success with 

oral vaccination, many clinical studies have failed to 

induce an effective immune response.97,99 Therefore, 

oral vaccine formulation studies are needed to gain 

a better understanding of the effects of stabilizing 

the antigens in a harsh environment and targeting 

them to the gut-associated lymphoid tissues. Intranasal administration remains attractive because of 

the large mucosal surface area the nose provides and 

the access to the nasopharynx-associated lymphoid 

tissues, which can activate immune responses in the 

respiratory and gastrointestinal tracts.99 The seemingly 

global mucosal stimulatory potential after intranasal 

immunization may represent an important path for 

novel vaccines. Moreover, intranasal administration 

lowers antigen and adjuvant doses compared with oral 

vaccinations, making them more cost effective. Aerosol 

spray, droplets, and powders have been optimized 

and represent an attractive field for the development 

of new devices.101,102

However, the primary role of the nasal mucosa 

is to protect the respiratory airways, not to convey 

antigens to the immune system.101 A challenge for 

intranasal immunization is that the nasal mucosa is 

intricately connected to the olfactory nerve and the 

CNS. As a result, intranasal immunizations using 

Escherichia coli heat labile toxin as an adjuvant in 

humans has been correlated with Bell’s palsy development.103 In contrast, sublingual immunization has 

gained interest because it stimulates a broad activation of the immune system while avoiding perturbation of the CNS.104

Adjusting mucosal vaccine formulations may be 

another way to improve their efficacy. Little is known 

about how to formulate a better mucosal vaccine, 

although there are currently two main approaches: 

using either (1) soluble or (2) particulate vaccines.92

Defining the most effective vaccine formulation is a 

universal challenge in vaccinology (see the discussion 

in Adjuvants). 

Finally, the development of new mucosal adjuvants could significantly improve the effectiveness of 

mucosal vaccines. Stimulating the appropriate type 

of immune response can dramatically affect the immediate and long-term immune response to a vaccine, 

ultimately determining protection from disease. The 

use of heat labile enterotoxin and cholera toxin has 

been abandoned due to neurologic effects and overt 

diarrhea, respectively.105 In the future, mucosal adjuvants may be improved by combining particles with 

TLR ligands.106 Alternative adjuvants are discussed in 

the following section. 

Adjuvants

The word “adjuvant” is derived from the Latin 

verb “adjuvare” meaning “to help.” Adjuvant properties were discovered by Gaston Ramon in 1920.107

Although adjuvants have proven to be crucial for 

most vaccines, the field has been relatively neglected 

until recently. The most widely used adjuvant to date, 

alum, has been empirically instilled in billions of 

vaccine doses since the 1920s, even though its mode 

of action was not discovered until 2008.108 The only 

other category of adjuvant authorized for human 

use is the squalene derivatives: MF59 by Novartis 

(Basel, Switzerland),109 AS03 by GlaxoSmithKline,110

and AF03 by Sanofi-Pasteur (Lyon, France).111 These 

adjuvants have been used in the pandemic influenza 

vaccine since 2009.112 Over the past decade, many 

preclinical studies have expanded the list of potential adjuvants.113,114 Historically, adjuvants have been 

recognized as a “perfect mix” of old ingredients, 

including water in oil (w/o) and oil in water (o/w).115

Their adjuvant properties are thought to be based 

on nonspecific inflammatory stimulation that brings 

all the cellular players to the site of inoculation. 

Instead of the typical adjuvant formulations of the 

past, some large pharmaceutical companies such as 

GlaxoSmithKline have developed new mixes adapted 

for specific vaccines that require strong and longlasting immune responses.116 The rediscovery of the 

central role the innate immune response plays in the 

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Medical Aspects of Biological Warfare 

development of overall immunity has encouraged the 

design of TLR and nucleotide-binding oligomerization domain receptor (or NOD-like receptor [NLR]) 

ligands as adjuvant components. For example, AS04, 

developed by GlaxoSmithKline, is composed of the 

TLR4 agonist 3-O-desacyl-4’-monophosphoryl lipid 

A (MPL), a component of lipopolysaccharide.116 Interestingly, this bacterial component has proven to be a 

good adjuvant for virus vaccines such as the human 

papilloma virus vaccine Cervarix, demonstrating that 

viral and bacterial motifs can cooperate to activate the 

immune system. More intricate mixes have also been 

tested by GlaxoSmithKline, including AS02, which 

contains MPL and QS-21, a saponin-based adjuvant 

derived from the bark of Quillaja saponaria.

116

In the same context, there is potential that the 

emerging nanotechnology field may help improve adjuvants in the future.63 Nanoparticles (1–1,000 nm size 

particles) retain adjuvant activity by improving antigen 

delivery and triggering innate immune responses. 

Numerous polymer motifs, such as poly(lactide-coglycolide) nanoparticles, co-polymers hydrogels, and 

cationic liposomes, have been tested as carriers of antigens. Some biodegradable nanoparticles are safe and 

already available for use in vaccine formulation.117,118

These particles can be decorated by molecular motifs 

either to target specific cells, such as dendritic cells or 

macrophages, expressing specific pattern-recognition 

receptors. Nanotechnology may supply an opportunity 

to improve vaccine adjuvants used in biodefense.

THE FUTURE OF ANTIBODIES IN BIODEFENSE

Just as with vaccine discovery, the development 

of antibody-based therapeutics can be an expensive 

and time-consuming effort, with new products taking 

years to reach licensure; however, most of the more 

recent products are of human or human-like origin, 

often requiring less time than older products to reach 

this milestone. Antibody-based therapeutics represent 

the fastest growing class of biological therapeutics: 43 

therapeutic mAbs had been approved as of December 

2013, and 36 of these are still active in the European 

Union or US market.119 However, few of these are 

specific to biodefense agents. Biodefense-specific 

infectious agents and toxins are generally not as well 

studied, limiting the targets and antigenic material 

required for antibody generation. The production of 

these therapeutics has predominantly been hindered 

by the historically high cost of antibody therapeutic 

development, primarily in the production of a final 

protein in the concentration and format necessary 

to elicit protection. Also, in contrast to the “one-bug, 

one-drug” approach often used for vaccines designed 

to combat multiple agents (utilization of a single drug 

for each agent), antibody development costs may be 

increased by the need for oligoclonals, or cocktails, of 

multiple antibodies that focus on a single agent, often 

acting via different mechanisms or protecting against 

escape mutants. Despite these constraints, antibody 

therapeutics are increasingly necessary to fill the gap 

when vaccine development has yet to produce an 

efficacious product, as in the 2014 West Africa Ebola 

virus outbreak.120

Antigen-specific protection afforded by antibodies 

can have advantages compared to the protection elicited by vaccines, especially for biodefense. Vaccines 

must elicit an effective, long-lasting immune response 

whose maintenance may require multiple booster 

injections. Antibodies, however, may be administered 

in quantities that achieve a titer of protection that 

exceeds that elicited by vaccines due to the decreased 

immunogenicity of antibodies when used in a human 

or humanized format. These mAbs may then provide 

a higher level of protection, a level necessary in biodefense, because bioweapon exposure is often intended to 

use elevated levels of the agent or toxin compared with 

natural exposures. Emerging diseases and toxins do 

not always represent a threat exclusively, and several 

agents have been evaluated for their therapeutic potential. For example, botulinum neurotoxins (BoNTs) 

have a therapeutic application, making vaccination 

clinically disputable. Antibodies can serve multiple 

roles: as a therapeutic alternative when no vaccine is 

available or as the sole treatment when a vaccine would 

not be efficacious. This section will discuss new and 

emerging technologies that have improved antibody 

discovery; events specific to antitoxin, antibacterial, 

and antiviral antibody development; and the future 

formats and production challenges of these molecules.

Antibody Generations and the Development of 

New Therapeutic Formats

The most representative and recognizable antibody 

format, immunoglobulin G (IgG), is often the simple 

fractionation or isolation of antibodies from human or 

animal sera to produce protective antibodies (Figure 

28-1). Additional antibody formats will be discussed 

later in this chapter. 

Sera, the first antibody product used, has recently 

seen a resurgence of use. Generally, sera is fractionated 

to produce pAbs that are used as a first line of defense 

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Future Prospects of Vaccines and Antibodies in Biodefense

against emerging pathogens; pAbs have been essential 

to providing critical treatments for infectious diseases. 

The use of these pAbs, as well as their role as future 

therapeutic countermeasures, will be discussed later 

within each subsequent section. 

Historically, mAbs have been associated with several advancing generations of molecules (Figure 28-2). 

The first generations of mAbs were strictly murine in 

origin, as described in the early 1980s by Köhler and 

Milstein, who used hybridomas from stable fusions of 

immortalized myeloma cells with B cells from immunized mice.1

 Several of these mAbs made it to clinical 

trials for the treatment of cancers and transplant rejection, but were ultimately withdrawn due to a variety 

of concerns, including the high immunogenicity of 

these foreign proteins in humans, first identified by 

responses from human anti-mouse antibodies.121

Due to the immunogenicity of these antibodies, 

murine mAbs had little utility and often caused adverse effects. Following the publication of Köhler and 

Milstein’s hybridoma article, human/mouse chimeric 

antibodies were developed as second generation therapeutic molecules with a human Fc region to decrease 

immunogenicity.1 Several biodefense mAbs have been 

produced in this format, primarily due to the ability 

to develop antibodies using mouse models for exotic 

infectious diseases that could be rapidly chimerized 

to a human Fc region for a single clinical application. 

Even with the addition of a human portion to the antibody, the administration of these chimeric antibodies continued to produce an immune response to the 

remaining murine domains. 

Third- and fourth-generation mAbs were developed 

by using selection and engineering advances in recombinant DNA-based molecular biology techniques 

to reduce immunogenicity. This was accomplished 

through humanizing the framework regions and 

transferring only the antigen-binding loops made up 

by the complementary-dependent regions. Further 

humanization can be achieved by making point mutations within the complementary dependent regions 

themselves. Humanization of each mAb generation 

requires special care because multiple residues of the 

framework regions are often required for stability of 

the antibody; therefore, it is essential to ensure that 

these modifications do not negatively impact the 

binding or activity of the mAbs when changing residues within the complementary dependent regions. 

Although the overall immune systems of mice and 

Figure 28-2. Representations of the progression of antibodies through the progressive generations of humanization from 

mouse (-omab), chimeric (-ximab), humanized (-zumab), and human (-umab) antibodies.

Figure 28-1. Representation of the immunoglobulin G antibody isotype. Specific regions are identified by color. The 

variable light (VL) and variable heavy (VH) regions, in pink

and orange, respectively, together make up the Fv region. 

Each Fv and CH1 domain (upper green and purple for each V 

domain) makes up a Fab fragment. The combination of the 

CH2 and CH3 (purple) domains makes up the Fc fragment.

Fab: fragment antigen-binding; Fc: fragment crystallizable; 

Fv: variable fragment

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Medical Aspects of Biological Warfare 

humans are similar, researchers should understand 

that the response in mice might not always correlate 

to the human response.122 The most recent techniques 

used to produce human or humanized mAbs include 

DNA-based selection libraries (eg, phage display123), 

isolation from peripheral blood B cells,124 and use of 

transgenic humanized animals. 

The future of antibody development may not be in 

the isolation of the antibody to obtain a fully human 

product, but rather in the production of new formats 

that are able to enhance a particular antibody function, 

or in the administration of the antibody to the target or 

host. New mAb therapies being explored use not only 

antibody–vaccine combinations, but also antibody–

drug conjugates125 and many new antibody-based 

formats, such as bispecific and multispecific antibodies,126 intrabodies,127 and transbodies128 (Figure 28-3). 

The IgG1 isotype, depicted in Figure 28-1, is the 

most abundant isotype of immunoglobulin produced 

against protein antigens. Other isotypes include IgA, 

IgD, IgE, IgM, and multiple subclasses of IgG. As 

therapeutics, IgGs have seen the greatest use because 

they are the most predominant and abundant antibody in the serum and have historically been easier 

to isolate and purify. IgMs are large-molecular-weight 

pentamers and are the first low-affinity antibodies 

expressed in response to an infection. These antibodies are secreted from the surface of B cells before their 

maturation into a plasmocyte prior to the IgG isotype 

switch. The lack of recombinant and isolated IgMs 

available as therapeutics may predominantly be due 

to their overall molecular weight and the subsequent 

difficulty of purifying them. 

IgAs consist of monomers in human serum and 

dimers after secretion at the mucosal surface through 

polymeric Ig receptors, are most prominent within 

the mucosal and gastrointestinal tract and serve as 

the first line of defense against natural infections.129

The lack of alternative antibody isotypes beyond 

IgGs available for use against therapeutic targets can 

partly be attributed to the lack of appropriate in vivo 

models and partly to the difficulty in purifying these 

products in their dimer form. New transgenic species 

and advanced antibody production methodologies 

will most certainly provide the necessary tools to test 

alternative classes of antibody therapeutics. 

IgG antibody isotypes, and to a lesser extent, fragment antigen-binding (Fab) fragments, have been the 

primary therapeutic format of antibodies that have 

been FDA approved for use. These antibodies have an 

extremely large size (around 150 and 55 kDa, respectively) and a half-life averaging 21 days for a human 

antibody, but they may not have optimal presentation 

to the epitope or tissue. Functional CH2 domains (called 

nanoantibodies),130 single-domain heavy chain (abbreviated VHH) camelid-derived nanobodies,131 and fusion 

antibody fragments are progressing through late stage 

development and into clinical trials. As in the case of 

single domain nanoantibodies and nanobodies, these 

smaller, independently folded domains with antigen 

Figure 28-3. Representative antibody fragments demonstrating therapeutic potential through particular function, access, or 

host interaction. Immunoglobulin G (IgG) shown on left for reference. 

Fab: fragment antigen-binding; Fc: fragment crystallizable; scFv: single-chain variable fragment; VH: variable heavy

Illustration: Adapted with permission from Frenzel A, Hust M, Schirrmann T. Expression of recombinant antibodies. Front Immunol. 2013;4:126. This work is licensed under a Creative Commons Attribution 3.0 Generic License (https://creativecommons.

org/licenses/by/3.0/legalcode).

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Future Prospects of Vaccines and Antibodies in Biodefense

binding capabilities may serve as highly specific and 

extremely stable therapeutics. Although they possess 

remarkably stable physiochemical properties, these 

antibodies are rapidly cleared from the bloodstream 

in a matter of hours, hindering their development as a 

potential therapeutic in their base format. Methods to 

increase the half-life of these antibodies include binding and linkage to Fc domains, albumin, and polyethylene glycol. For both full-length antibodies and new 

single-domain formats, a range of methods is being 

developed to increase the antibodies’ serum half-lives. 

Antitoxin Monoclonal Antibodies

Treatment for intoxication using mAbs has seen a resurgence of utility in the past several years. Early work 

with serum therapy and passive transfer of antibodies 

provided successful proof-of-concept studies against 

diphtheria and tetanus via toxin neutralization.132

Subsequent development of antitoxin antibodies, using high antibody affinity as a primary discriminator 

for selection, has shown advances against bacterial 

and viral targets.133–135 Selection for these affinities 

has spurred vast improvements in anti-ricin and 

anti-BoNT, often into the sub-nM and pM levels. 

However, the narrow specificity of the antibodies to 

the toxin targets often limits their efficacy when used 

against toxins with multiple serotypes, as with BoNT, 

which has seven known serotypes (A through G).136

Selection of these antibodies has been further driven 

by improvements in humanization. This section will 

primarily focus on toxins that have the capability for 

weaponization as biowarfare toxins in the absence of 

the producing pathogen. 

Ricinus communis, a plant extensively cultivated 

around the world produces the Ricin toxin. Ricin is a 

type 2 ribosome-inactivating protein consisting of two 

parts. The B-subunit (RTB) binds to sugars on the cell 

surface for cytosolic entry of an A-subunit (RTA), which 

inhibits protein synthesis. Both of these subunits of the 

toxin have epitopes that have been targeted for therapeutic and vaccine development. Although two vaccines 

are undergoing early phase clinical trials (NCT01317667 

and NCT00812071), there are currently no licensed 

measures for treatment, leaving options for supportive care only. (See Table 28-2137–145 for more details.) A 

proof-of-concept study using direct inhalation of ricin 

toxin followed by treatment with pAbs 20 minutes 

postchallenge demonstrated protection.146 Monoclonal 

antibody development has progressed through multiple 

antibody formats against both RTA and RTB.147,148 Chimeric mouse/human antibodies are currently the only 

format that has been successfully used to demonstrate 

protection postintoxication.141,149 Future use of antibodies against ricin intoxication may require a mixture of 

antibodies to both RTA and RTB to provide protection against the multiple mechanisms of intoxication. 

Humanizing these early protective antibodies will be 

essential in reducing the therapeutic agent’s immunogenicity and expanding its pharmacokinetic capabilities.

Clostridium botulinum is a gram-positive, sporeforming, anaerobic bacteria that secretes neurotoxins 

causing botulism.150 C botunlinum produce multiple 

serotypes of the neurotoxin BoNT, from A to G (denoted BoNT/A to BoNT/G), of which, types A, B, E, 

and F have been shown to cause human botulism.136 In 

2011, the Centers for Disease Control and Prevention 

discontinued the investigational new drug (IND) use of 

the pentavalent (ABCDE) botulinum vaccine, leaving a 

therapeutic gap in the protection of at-risk workers.151

Although most of the treatments for botulism are based 

on supportive measures (eg, intensive respiratory 

care), passive administration of anti-BoNT immune 

sera has been used for treatment. A civilian working 

group on biodefense recommended the utilization of 

antisera immediately following diagnosis,152 and treatment with equine antisera within 24 hours has demonstrated success.153 Two therapeutic products have been 

derived from equine pAbs for biodefense use.137 Wellsuited to protect against the various serotypes of BoNT, 

these formulations were prepared, although not used, 

during Operation Desert Storm and Operation Desert 

Shield in the 1990s. The new heptavalent botulinum 

antitoxin (HBAT, Cangene Corp, Winnipeg, Canada) 

is composed primarily of Fab and F(ab’)2 fragments 

and is available from the Centers for Disease Control 

and Prevention under an IND protocol for naturallyacquired botulism.137 New formulations of oligoclonal 

mixtures are being developed to recapitulate broad serotypic capacity, providing a more efficacious product 

and extended half-lives in humanized formats. Two 

parallel efforts, currently under development by the 

United States and European Union, are to develop a 

panel of antibodies against BoNT/A/B and /E (XOMA 

3AB and AntiBotABE, respectively).139,147,154

Even given the rapid clearance and success of polyclonal and novel monoclonal antibody mixtures, the 

antibodies are effective only against the circulating 

toxin, and often against just a single serotype or subtype each. Once the toxin is attached to the receptor, 

it is internalized by receptor-mediated endocytosis, 

ultimately blocking neuromuscular communication. 

However, the administration of antitoxin may still be 

necessary because, even after the onset of symptoms, 

BoNT may remain in the bloodstream throughout infection, and clearance is essential for subsequent recovery. The oligoclonal approach to developing a cocktail 

against multiple serotypes is necessary for any future 

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TABLE 28-2 EMERGING ANTIBODY-BASED BIODEFENSE THERAPEUTICS International Nonproprietary Name, Targeted Product Name, or Code Targeted Agent Antigen Category* Antibody Type (Isolation) HBAT (Cangene Corp, Clostridium botulinum BoNT (A–G) A Polyclonal Fab and F(ab’)2 fragments 

Winnipeg, Canada) toxin (botulism) XOMA 3AB (Xoma Corp, C botulinum toxin (botulism) BoNT (A) A Recombinant IgG1 mAbs (human 

Berkeley, CA) and murine origin)

AntiBotABE (EU Consortium, C botulinum toxin (botulism) BoNT (A) A Recombinant IgG mAb (phage library 

7th Framework Programme, derived)Brussels, Belgium)

RAC18 Ricin toxin from Ricin A chain B Murine mAb 

Ricinus communis

c4C13 Ricin toxin from Ricin A chain B Chimeric mouse/human mAb 

R communis

43RCA Ricin toxin from R communis Ricin A chain B scFv antibody fragment HuMAb-154 SEB SEB B Human IgG1 FL9, FL10 SEB SEB B Human IgG mAbs (phage library 

derived)

Shigamabs cαStx1 and Food- and water-borne cαStx1 and B Chimeric mouse/human mAb 

cαStx2v (Thallion pathogens cαStx2 ShigaPharmaceuticals, Inc, toxinsMontreal, Canada)

*National Institute of Allergy and Infectious Diseases category A, B, and C priority pathogens. (See https://www.niaid.nih.gov/research/emer

breakdown of the classification of these biothreat categories.)HBAT: heptavalent botulinum antitoxin; IgG: immunoglobulin G; IND: investigational new drug; mAb: monoclonal antibody; R&D: research enterotoxin BData sources (as per chapter reference list): (137) Centers for Disease Control and Prevention. Investigational heptavalent botulinum antitoxiantitoxin AB and investigational botulinum antitoxin E. MMWR Morb Mortal Wkly Rep. 2010;59:299. (138) Avril A, Miethe S, Popoff MR, et al. Isoprimate origin, cross-neutralizing botulinum neurotoxins A1 and A2 by targeting their heavy chain. BMC Biotechnol. 2015;15:86. (139) Miethe S, R

ment of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library. Mabs. 2014;6:446–459. (140) Rasetti-Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B. Mabs. 2015;7:1161–1177. (141) Pratt TS, Pincus SH, HaKM. Oropharyngeal aspiration of ricin as a lung challenge model for evaluation of the therapeutic index of antibodies against ricin A-chain fo2007;33:459–481. (142) Guo JW, Shen BF, Feng JN, Sun YX, Yu M, Hu MR. A novel neutralizing monoclonal antibody against cell-binding po

2005;24:263–266. (143) Pelat T, Hust M, Hale M, Lefranc MP, Dübel S, Thullier P. Isolation of a human-like antibody fragment (scFv) that neutralize

2009;9:60. (144) Drozdowski B, Zhou Y, Kline B, et al. Generation and characterization of high affinity human monoclonal antibodies that neutraliBased Ther Vaccines. 2010;8:9. (145) Larkin EA, Stiles BG, Ulrich RG. Inhibition of toxic shock by human monoclonal antibodies against staphylococc

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product because rapid diagnostic determination of the 

toxin may not be available at the time of intoxication.

Staphylococcal enterotoxin B (SEB), one of multiple virulence factors of the gram-positive bacterium 

Staphylococcus aureus, is an extremely antigenic toxin in 

a family of many staphylococcal enterotoxins. Contact 

with naturally occurring S aureus can occur with these 

bacteria as they grow on the skin, mucosal surface, and 

can be found in food items. Previous proof-of-concept 

studies have demonstrated protection against toxic 

shock syndrome associated with staphylococcal enterotoxins utilizing human-derived IgG.155 Currently, 

only a few mAbs targeting SEB toxins have been tested, 

and these used mouse models of protection. Derived 

from a human phage-display library, the first two 

mAbs, FL9 and FL10, demonstrated protection when 

premixed with the toxin.145 HuMAb-154, a human 

antibody derived from hybridoma technology, demonstrated partial protection and delayed time to death 

when administered following increasing lethal doses 

of intraperitoneally administered SEB.144 The human 

origin of these three antibodies supplies the advantage 

of reduced immunogenicity when intended for human 

use. However, these antibodies have yet to be used in 

a primate model or against aerosol exposure, experiments necessary to establish efficacy of the therapeutic 

potential against SEB for biodefense. 

One of the main issues concerning toxin therapy using mAbs is treatment beyond 24 hours postexposure. 

Antitoxin antibodies are most effective when given 

early after exposure, and these timelines can be restrictive given the time it takes to detect and identify the 

toxin of interest. Although anti-ricin antibodies, when 

administered effectively, clear all toxins, antitoxin antibodies inhibit the activity of the toxin only, and not the 

pathogen itself (ie, neutralizing BoNT versus treating 

C botulinum). Thus, in the case of an infection, a treatment plan should be developed to eliminate both the 

toxin and the pathogen responsible for its production. 

Antibacterial Monoclonal Antibodies 

Many bacterial agents produce toxins. In addition 

to using mAbs against these bacterial toxins, mAb 

prophylaxis and therapy has targeted components 

on the surface of the bacterium, as with anthrax.156

As early as 1890, antibacterial serum therapy proved 

successful, with the application of immune serum 

against diphtheria and tetanus developed by Emil 

von Behring, a German military doctor, who received 

a Nobel Prize for the initial development of pAbs.132

With the germ theory growing more widely accepted 

throughout the 1900s, the first antibiotics were concurrently developed and often overshadowed the use of 

serum and antibody therapy. However, an increase 

in bacterial resistance has been observed across all 

existing antibiotic classes highlighting the need for the 

identification of new therapeutic options.157 The early 

success of antibody-based therapies starting with polyclonal formulations and now more recent mAbs may 

constitute a therapeutic class capable to fill therapeutic 

gaps or even augment existing current therapies. Given 

the success of mAbs against cancer inflammatory and 

autoimmune diseases, it is surprising that more antiinfective mAb treatments have failed to make it to 

market. The primary factor inhibiting the progress of 

antibacterial mAbs is their lack of efficacy in animal 

studies and clinical testing, despite promising preclinical data.156 (See Table 28-3157–163 for more details.) 

Anthrax, from B anthracis, is an agent that has 

been previously weaponized by state-sponsors and 

more recently used in an intentional dissemination of 

spores in the United States in 2001.164 Early development of human antibody-based therapeutics against 

anthrax began after earlier antibodies of animal origin increased the therapeutic window of the disease 

and also shortened the duration of antibiotic treatment. Anthrax-specific antibody-based therapeutics 

act by a variety of mechanisms, either by targeting 

the capsule or by neutralizing the toxins that treat 

or augment therapy.156 These antibodies represent 

the most advanced biodefense-related therapeutics, 

with the only two biodefense class specific mAbs, 

approved, and several others advancing through 

preclinical and clinical trials. These represent some of 

the most widely studied antibodies in the biodefense 

arena, with multiple mechanisms of action identified. 

Raxibacumab and Anthim (Elusys Therapeutics, Pine 

Brook, NJ) act by inhibiting PA-receptor interactions. 

Murine-derived antibodies 7.5G and 48.3 inhibit 

PA cleavage by furin, but thus far they have only 

been tested in murine models.165,166 Thravixa (previously AVP-21D9; Emergent BioSolutions), currently 

in phase I clinical trial, inhibits PA heptamerization.

Although no mAbs that directly inhibit the PA-LF/

EF complex interaction are currently undergoing 

clinical evaluation, the potential for therapies has 

been demonstrated by human mAbs IZNLF, SS87, 

Fab A8, and 2LF, as well as the cross-reactive mAb 

H10. Other mechanisms have been shown by the chimeric chimpanzee/human mAbs LF10E and LF11H, 

which act by inhibiting endocytosis and translocation. 

However, LF10E and LF11H have only been tested in 

rodent models. Within all of these second generation 

therapeutics to anthrax, the specific mechanisms are 

expected to increase the therapeutic window, decrease length of treatment and assist in overcoming 

antibiotic resistance. 

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TABLE 28-3

ADVANCEMENTS OF TREATMENTS FOR BIODEFENSE BACTERIAL AGENTS UTILIZING 

ANTIBODY-BASED THERAPEUTICS

International Approval, US Patent

Nonproprietary Clinical or

Name, Product Targeted Antibody Type Phase, Reference

Name, or Code Targeted Agent Antigen Category* (Isolation) or Stage No.

Raxibacumab/ABthrax Bacillus anthracis Anti-PA A Recombinant Approved 601351

(GlaxoSmithKline, (anthrax) IgG1 mAb (2012)

Brentford, UK) (naïve library)

Valortim (Bristol- B anthracis Anti-PA A Human mAb Phase 1 7456264

Myers Squibb, New (anthrax) (transgenic

York, NY)/MAb-1303 mice)

Anthim Elusys B anthracis Anti-PA A Chimeric Phase 1 7446182

Therapeutics (Elusys (anthrax) deimmunized

Therapeutics, Pine mAb (murine 

Brook, NJ)/ETI-204 origin)

Thravixa (Emergent B anthracis Anti-PA A Human mAb Phase 1 7438909, 

BioSolutions, Rockville, (anthrax) (hybridoma) 7442373

MD)/AVP-21D9

Anthriving (Emergent B anthracis Anti-PA A Polyclonal Phase 1/2 N/A

BioSolutions)/AIG (anthrax) antibody from 

AVA human 

 plasma

mAb 7.3 Yersinia pestis LcrV A Murine mAb R&D (157)

(plague) (hybridoma)

m252/m252/m254 Y pestis F1/LcrV A Recombinant R&D (158)

(plague) human mAb 

(naïve library)

mAb 3 Francisella tularensis LPS A Murine IgG2a R&D (159)

(tularemia)

6B3 Brucella species B melitensis LPS B Murine IgG3 R&D (160)

(brucellosis)

2C8 Brucella species B abortus LPS B Murine IgG3 R&D (160)

(brucellosis)

Ps6F6 Burkholderia Exopolysaccharide B Murine IgG3 R&D (161)

pseudomallei

(melioidosis)

1G2-1D3 B mallei (glanders) LPS B Murine IgG2a R&D (162, 163)

*

National Institute of Allergy and Infectious Diseases category A, B, and C priority pathogens.

AIG: anthrax immune globulin; AVA: Anthrax Vaccine Absorbed; IgG: immunoglobulin G; LPS: lipopolysaccharide; mAb: monoclonal 

antibody; N/A: not applicable; PA: protective antigen; R&D: research and development; IgG: immunoglobulin G

Data sources (as per chapter reference list): (157) Hughes JM. Preserving the lifesaving power of antimicrobial agents. JAMA. 2011;305:1027–

1028. (158) Hill J, Copse C, Leary S, Stagg AJ, Williamson ED, Titball RW. Synergistic protection of mice against plague with monoclonal 

antibodies specific for the F1 and V antigens of Yersinia pestis. Infect Immun. 2003;71:2234–2238. (159) Xiao X, Zhu Z, Dankmeyer JL, et al. 

Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a bubonic plague model. PLoS One. 2010;5:e13047. (160) 

Lu Z, Roche MI, Hui JH, et al. Generation and characterization of hybridoma antibodies for immunotherapy of tularemia. Immunol Lett. 

2007;112:92–103. (161) Laurent TC, Mertens P, Dierick JF, Letesson JJ, Lambert C, De Bolle X. Functional, molecular and structural characterisation of five anti-Brucella LPS mAb. Mol Immunol. 2004;40:1237–1247. (162) Bottex C, Gauthier YP, Hagen RM, et al. Attempted passive 

prophylaxis with a monoclonal anti-Burkholderia pseudomallei exopolysaccharide antibody in a murine model of melioidosis. Immunopharmacol 

Immunotoxicol. 2005;27:565–583. (163) Trevino SR, Permenter AR, England MJ, et al. Monoclonal antibodies passively protect BALB/c mice 

against Burkholderia mallei aerosol challenge. Infect Immun. 2006;74:1958–1961.

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Bubonic, septicemic, and pneumonic plague are 

all caused by the gram-negative bacterium Y pestis.

Y pestis contains two dominant targets for antibodybased therapeutics: (1) the surface polymer F1 and (2) 

the surface protein LcrV (as previously described in 

Plague, Chapter 10 in this volume). Early proof-ofconcept studies using the murine anti-LcrV mAb 7.3 

demonstrated complete protection against an aerosolized Y pestis challenge.158 Other murine antibodies 

demonstrated similar protection, with mAb 7.3 and 

F1-04-A-G1 providing protection when administered 

as an aerosol cocktail prior to aerosolized challenge.167

Naïve libraries have been used to produce the recombinant mAbs m252, m253, and m254. When used in 

a cocktail, these antibodies demonstrate synergistic 

protection, but they have been tested in murine models only and in the absence of antibiotics.159 Because 

antibiotics are generally the first line of treatment, an 

understanding of the combined efficacy of antibody 

therapies and antibiotics is currently lacking in the 

antibody-based treatment of plague.

F tularensis is extremely virulent and difficult to 

identify with serological tests. Although F tularensis is susceptible to current antibiotics, an effective 

antibody-based therapeutic may be necessary because 

the various strains use different virulence mechanisms. 

Although these differences may explain the difficulty 

in obtaining an efficacious therapeutic, they also highlight the unique nature of each strain, which could 

be treated individually by using specific antibodies. 

The potential for effective antibody therapy against F 

tularensis in an aerosolized challenge model was first 

demonstrated by serum transfer of F tularensis LVS, 

which increased the mean time to death against the 

SchuS4 challenge strain.168 The current antibody-based 

therapeutic options for F tularensis are of murine origin 

and have only been tested in rodent models.160,169 Additional research and development will be needed in 

these areas to advance an effective biologic for clinical 

application. 

The most successful utilization of antibacterial 

mAbs to date targeted the toxins of these organisms. 

These mAbs often possess extremely high affinity to 

their toxin targets, binding to and clearing the damaging toxins while allowing time for the host’s immune 

response to clear the underlying bacterial infection. In 

contrast to toxin-exclusive neutralization, clearance 

mechanisms play a critical role in the therapeutic 

efficacy of these antibodies. In addition to the Fcmediated clearance, it has been shown that antibodies 

can paradoxically increase the cytotoxic effects of these 

agents, as was seen in the enhancement of anthrax 

lethal toxin.170 This complex interaction between the 

requirement for antibody-mediated clearance and the 

potential for negative antibody-dependent enhancement has also been recently reported for virus-specific 

antibody therapeutics.171

Antiviral Monoclonal Antibodies

Historically, vaccination has been the primary 

means of providing any medical countermeasure 

against viruses with bioterrorism agent potential. 

Following the eradication of smallpox, outbreaks 

of biodefense-related viruses in populations, with 

the exception of yellow fever, have been relatively 

infrequent. Several of these agents remain poorly understood due to their sporadic occurrence or orphan 

nature and limitations in resources or facilities capable 

of researching these viral diseases. The high mutation 

rate and poor understanding of many of these viruses 

account for the therapeutic gaps in protection, while 

the high infectivity and mortality make these an optimal bioweapon. For many viruses considered to have 

potential for use as biological weapons, including viral 

hemorrhagic fevers (VHFs) (eg, Ebola virus, Sudan 

virus, and Marburg virus) and Togaviridae encephalitides (eg, VEEV, eastern (EEEV), and western (WEEV) 

equine encephalitis viruses), only supportive therapy 

exists following exposure. Ribavirin, a nonimmunosuppressive nucleoside-analogue with broadly protective antiviral properties, has demonstrated protection 

only against the VHF Lassa virus and is available only 

for compassionate use under an IND application.172

Additional studies have indicated that the use of ribavirin would be effective against other arenaviruses as 

well as Bunyaviruses (see Alphaviruses, Chapter 20 

in this volume). Beyond ribavirin, few other drug options exist, leaving only supportive care in many cases. 

The successful application of therapeutic antibodies has been demonstrated previously. The National

Institute of Allergy and Infectious Diseases (NIAID) 

categorizes biodefense agents based on several factors 

including, but not limited to, the agent’s pathogenesis, 

dissemination, available treatments, as well as the 

agent’s mortality. Category A agents are considered the 

most dangerous, based on ease of dissemination, high 

mortality, public health impact, and absence of available 

therapeutics, with Categories B and C following to a 

lesser extent in one or all of these areas. This chapter has 

focused primarily on Categories A and B agents; however, two category C agents, Hendra virus and Nipah 

virus, have well-characterized therapeutic mAbs for 

postexposure treatment. (See Table 28-4173–183 for more 

details.) The human mAb m102.4 demonstrated protection against Nipah in the ferret model184 and Hendra in 

a nonhuman primate (NHP) model183 as a postexposure 

therapeutic as late as 7 to 8 days after infection. 

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TABLE 28-4

ADVANCEMENTS OF TREATMENTS APPLIED ON BIODEFENSE VIRAL AGENTS UTILIZING 

ANTIBODY-BASED THERAPEUTICS

International Approval, Patent, Trial

Nonproprietary Clinical Number, or

Name, Product Targeted Targeted Antibody Type Phase, Reference

Name, or Code Agent Antigen Category* (Isolation) or Stage No.

ZMapp (13C6, 2G4, Filovirus Ebola Zaire A Cocktail of three Phase 1/2, NCT02389192,

4G7) (Ebola) GP recombinant human utilized NCT02363322

 mAbs EUA (173)

ZMab (1H3, 2G4, 4G7) Filovirus Ebola Zaire A Cocktail of three Utilized (174)

(Ebola) GP recombinant human EUA

 mAbs

MB-003 (13C6, 13F6, Filovirus Ebola Zaire A Cocktail of three Utilized (175)

6D8) (Ebola) GP recombinant human EUA

 mAbs

8AH8AL Variola major B5 A Chimeric chimpanzee/ R&D (176)

(smallpox) human mAb

6C Variola major A33 A Chimeric chimpanzee/ R&D (177)

(smallpox) human mAb

hV26 / h101 Variola major H3/B5 A Human mAbs R&D (178)

(smallpox) (transgenic mice)

hB5RmAb Variola major B5 A Human mAb R&D USP 7811568

(smallpox) (derived from rat)

Vaccinia immune Variola major Whole A 16.5% IgG from Approved (179)

globulin (smallpox) antigen vaccinia virus for 

 individuals vaccine

 SE

Hu1A3B-7 VEE E2 GP B Humanized mAb IgG1 R&D (180)

 (derived from mouse)

Hu1A4A-IgG1-2A VEE E2 GP B Humanized mAb IgG1 R&D (181)

 (derived from mouse)

ToR67-3B4 VEE E1 GP B scFv-Fc fusion protein R&D (182)

 (NHP phage display)

m102.4 Nipah / GP G C Recombinant IgG1 Preclinical (183)

Hendra mAb development

*

National Institute of Allergy and Infectious Diseases category A, B, and C priority pathogens.

EUA: Food and Drug Administration Emergency Use Authorization; GP: glycoprotein; mAb: monoclonal antibody; NHP: nonhuman primate; R&D: research and development; SE: side effects; USP: US Pharmacopeia; VEE: Venezuelan equine encephalitis

Data sources (as per chapter reference list): (173) Qiu X, Wong G2, Audet J, et al. Reversion of advanced Ebola virus disease in nonhuman 

primates with ZMapp. Nature. 2014;514:47–53. (174) Qiu X, Audet J, Wong G, et al. Sustained protection against Ebola virus infection following treatment of infected nonhuman primates with ZMAb. Sci Rep. 2013;3:3365. (175) Pettitt J, Zeitlin L, Kim do H, et al. Therapeutic 

intervention of Ebola virus infection in rhesus macaques with the MB-003 monoclonal antibody cocktail. Sci Transl Med. 2013;5:199ra113. 

(176) Chen Z, Earl P, Americo J, et al. Chimpanzee/human mAbs to vaccinia virus B5 protein neutralize vaccinia and smallpox viruses and 

protect mice against vaccinia virus. Proc Natl Acad Sci U S A. 2006;103:1882–1887. (177) Chen Z, Earl P, Americo J, et al. Characterization of 

chimpanzee/human monoclonal antibodies to vaccinia virus A33 glycoprotein and its variola virus homolog in vitro and in a vaccinia virus 

mouse protection model. J Virol. 2007;81:8989–8995. (178) McCausland MM, Benhnia MR, Crickard L, et al. Combination therapy of vaccinia 

virus infection with human anti-H3 and anti-B5 monoclonal antibodies in a small animal model. Antivir Ther. 2010;15:661–675. (179) Hopkins 

RJ, Lane JM. Clinical efficacy of intramuscular vaccinia immune globulin: a literature review. Clin Infect Dis. 2004;39:819–826. (180) Goodchild 

SA, O’Brien LM, Steven J, et al. A humanised murine monoclonal antibody with broad serogroup specificity protects mice from challenge 

with Venezuelan equine encephalitis virus. Antiviral Res. 2011;90:1–8. (181) Hu WG, Phelps AL, Jager S, et al. A recombinant humanized 

monoclonal antibody completely protects mice against lethal challenge with Venezuelan equine encephalitis virus. Vaccine. 2010;28:5558–5564. 

(182) Rulker T, Voss L, Thullier P, et al. Isolation and characterisation of a human-like antibody fragment (scFv) that inactivates VEEV in 

vitro and in vivo. PLoS One. 2012;7:e37242. (183) Geisbert TW, Mire CE2, Geisbert JB, et al. Therapeutic treatment of Nipah virus infection 

in nonhuman primates with a neutralizing human monoclonal antibody. Sci Transl Med. 2014;6:242ra82.

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Before the development of mAb-based therapeutics, 

passive antibody therapy had been the only option 

for treating VHFs, but these efforts yielded mixed results.185 Despite previous successful results, concerns 

about the transmissibility of blood-borne pathogens 

from donor sera to recipients remain. In many cases, 

the diagnostic screening capabilities needed to confirm 

that a sample is pathogen-free before administration within the therapeutic window are not present. 

However, passive immunotherapy has provided the 

initial necessary evidence that therapeutic antibodies can be an effective preexposure or postexposure 

therapeutic.185

Despite the promise shown by passive immunotherapy, early negative experimental evidence 

limited interest in the application of antibody-based 

therapeutics against VHFs, specifically the Filoviruses. Previous passive transfer studies against Lassa 

virus demonstrated effectiveness if given early in the 

course of infection.186 During the 1995 Kikwit Ebola 

outbreak, crude blood transfusions were used as an 

immunotherapy, resulting in a death rate of one in 

eight, in comparison to an 80% mortality rate for those 

untreated.187 Studies using hyperimmunized equine 

serum against Ebola virus administered to macaques 

demonstrated delayed time to death but no change 

in the survival rate.188 The first attempt at protecting 

against Ebola virus used KZ52, a human antiglycoprotein mAb.189 KZ52 effectively neutralized Ebola virus 

in plaque assays and, when passively administered, 

protected guinea pigs,190 but it failed to protect or affect 

disease progression when given to NHPs.191

One explanation for the failures of passive immunization of pAbs or single mAbs to protect 

against Filoviruses is that this therapy controls the 

viral burden initially, but once depleted, the virus 

overwhelms the system. However, in 2012 passively 

transferred species-matched pAbs were found to 

provide complete protection in an NHP model, 

demonstrating the capability of antibody-based 

therapies against Filoviruses.192 Recent oligoclonal, or 

cocktail, mixtures of antibodies that target multiple 

epitopes of the virus—such as MB-003,175 ZMAb,174 

and ZMapp173—have demonstrated protection in infected NHPs. Furthermore, ZMapp was administered 

under emergency use authorization to two healthcare 

providers infected with Ebola during the 2014 West 

Africa outbreak who subsequently survived the 

infection.3 Similar to vaccines that provide humoral 

immunity, such as VEEV replicon particles,193 ZMAb 

provided complete sustained protection 10 weeks 

posttreatment upon re-challenge in NHPs.174 These 

studies, taken together with published data specific 

to the correlates of Filovirus protection,194 provide 

evidence of the ability of antibody therapy to provide 

protection until the host’s humoral immune system 

initiates a response. 

Historically, smallpox is one of the most concerning of all biothreat agents. Even with a successful 

vaccination program, smallpox remains a potential 

biological weapon because of the large nonvaccinated population. Other than vaccination, the only 

approved therapeutic is vaccinia immune globulin, 

a pAb extracted from vaccinated humans. Despite its 

limited potency against the disease,179,195 this product 

is approved to reduce the potential side effects of 

the vaccine and has been shown to reduce morbidity 

and mortality associated with smallpox.179 Several 

products have been developed using animal-based 

isolation techniques, although none are in clinical trials. Given the restricted access to the smallpox virus, 

new antibody-based therapeutics have been compared 

with vaccinia immune globulin using the vaccine 

strain for challenge. Several products have emerged 

in recent years from phage-display technology using 

chimpanzees,176,177 transgenic mAbs,178 and humanized 

rat mAbs196; mAbs developed against smallpox have 

been isolated from either vaccinated or infected animals. The humanized mAb hB5RmAb, whose parental 

antibody was isolated from a rat, and 8AH8AL, a chimeric chimpanzee/human recombinant antibody (rAb) 

derived from phage display, are both directed against 

the B5 surface protein of the extracellular enveloped 

virions.176,196 Additionally, two human mAbs, hV26 and 

h101, were isolated from transgenic mice and bind to 

the H3 protein found at the surface of mature virus and 

to the B5 protein, respectively.178 All of these antibodies 

elicited protection in various mouse models; however, 

none have been tested in NHP models of disease, as 

recommended by the World Health Organization. 

Several viruses of the Alphavirus genus cause 

encephalitis, and of these, VEEV and EEEV are classified as category B select agents. Early animal studies 

using passive transfer of neutralizing antisera and 

mAbs demonstrated this therapy’s protection against 

Alphaviruses.197–199 Early proof-of-concept studies have 

shown human constructs and recombinant mAbs to be 

successful in providing protection in mice.181,200,201 The 

humanized mAb Hu1A3B-7 (IgG1 isotype) binds the E2 

glycoprotein and is broadly specific to VEEV subtypes, 

neutralizing type IAB (Trinidad donkey or TrD), type 

II (Fe37c), and type IIIA (Mucambo BeAn8) in vitro. 

Hu1A3B-7 administered intraperitoneally 24 hours 

postchallenge provided complete protection against 

subcutaneous challenge of 100 times the median lethal 

dose (LD50) of VEEV TrD, as well as 90% protection 

when challenged against 100 LD50 by aerosol.200 Hy4 

is a humanized antibody that binds to the VEEV E2 

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Medical Aspects of Biological Warfare 

protein. When administered intraperitoneally 1 or 24 

hours postchallenge with VEEV TrD, just 10 μg elicited 

90% and 75% protection. Administration of 500 μg of 

Hy4 24 hours prechallenge provided 80% protection 

against intranasal challenge of 1,350 plaque-forming 

units.201 The mAb Hu1A4AIgG1-2A, humanized 

from murine 1A4A, binds to the E2 glycoprotein of 

VEEV with high affinity. The Hu1A4AIgGIgG1-2A 

provided both prophylactic and therapeutic protection 

against subcutaneous administration of VEEV TrD.181

The recently developed ToR67-3B4, an NHP phagedisplay derived scFv-Fc fusion antibody directed to 

the E1 protein, represents one example of the next 

generation of constructs providing protection against 

Alphaviruses.182 This antibody provided 83% protection against an aerosol challenge of VEEV TrD, with 

limited protection at later times in the mouse model.182

The antibodies that protected against VEEV were only 

tested in mice. None of the above antibodies have been 

tested in larger models; any potential therapeutic for 

biodefense would need to be used in models currently 

under development.202

Unlike other Department of Defense biothreat agents, 

Alphaviruses have a neuroinvasive component, which 

limits the effectiveness of antibody-based therapeutics 

due to their inability to cross the blood–brain barrier 

(BBB). Although active infection can inhibit the BBB’s 

ability to filter, allowing some immune therapeutics 

to pass through late during infection, new therapeutic 

designs with the ability to cross the BBB would facilitate treatments. Developers of the next generation of 

Alphavirus-based antibodies should endeavor to design 

antibodies with a capability to cross the BBB. One possible format for these therapeutics would be bispecific 

antibodies (see Figure 28-3). Several antibodies have been 

developed for diseases and conditions unrelated to biodefense that could serve as a model for the development of 

BBB-crossing therapeutic mAbs against Alphaviruses.203

Other antibody formats developed outside of biodefense 

include complete trifunctional chimeric IgGs and the 

scFv-scFv constructs.204 While a broadly reactive mAb is 

ideal, the high mutation frequency of Alphaviruses and 

other RNA viruses raises concern about the potential 

emergence of resistant strains.147 A monoclonal therapeutic will most likely require an oligoclonal product, 

or cocktail of several antibodies, each broadly reactive 

and capable individually of neutralizing the virus. 

Any successful viral infection requires viral particles to be released into extracellular space; however, 

with some biodefense-related viruses, such as variola 

major, that require just one inhaled particle-forming 

unit to initiate an infection, the utility of circulating 

antibodies becomes limited.205 Viral latency has been 

one of the major challenges in developing effective 

antibody-based therapeutics against HIV.206 Furthermore, as demonstrated by the brief protection windows described above, many biodefense-related viral 

pathogens require rapid identification and antibody 

administration within the first 48 to 72 hours to be effective. This is typically the case for Alphaviruses that 

cross the BBB and Filoviruses that rapidly overwhelm 

the immune system with high viral load. 

In addition to rapid diagnosis, having a greater understanding of biodefense agents and their pathogenesis in host model systems will greatly aid in the ability 

to more quickly identify and develop therapeutics. 

For example, the identification of Alphavirus glycoprotein glycosylation sites and successful production 

of recombinant glycoproteins would allow for rapid 

screening of target antibodies. Similarly, the development of an animal model for Crimean-Congo hemorrhagic fever would supply an in vivo model system to 

test therapeutic efficacy. These are just two examples 

of basic tools and knowledge that could significantly 

enhance the productivity and efficacy of vaccine and 

therapeutic development efforts. To achieve the greatest success, future work should therefore focus on 

the development of appropriate reagent material and 

model development in parallel with the programmatic 

development of therapeutic and vaccine candidates. 

SUMMARY

Vaccines and antibody-based therapeutics are 

some of the greatest achievements in global health 

improvement in previous centuries. Despite these 

advances, biodefense vaccines and treatments remain 

scarce, and there is a great need to define future 

requirements specific to biodefense vaccines and 

antibodies. Enormous advances have been made in 

the fields of vaccines and antibody-based medical 

countermeasures, and many creative strategies have 

been developed that may address the current needs; 

however, the barriers between an idea or concept and 

a product are vast, and costs to develop one product 

can surmount $100 million. 

The first challenges in the future development of 

vaccine or immunotherapy medical countermeasures 

will be how to prioritize the funding for an ever-growing pipeline of products and whether to develop vaccines or antibodies (or a combination of both). Within 

the limited funding environment of infectious diseases 

and toxins, focus should be agent-specific for the development of specific vaccines and antibody-based 

therapeutics. One option would be to focus vaccine 

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Future Prospects of Vaccines and Antibodies in Biodefense

development primarily on communicable diseases, for 

which the threat of epidemic outbreaks is a primary 

concern. Then, noncommunicable diseases and toxins, 

or those diseases that are poorly transmitted, could be 

addressed by development of antibody-based therapeutics. Alternative approaches could then be used to 

augment the initial round of medical countermeasures. 

New vaccine and therapeutic development should 

not only be aligned with the relative ease in obtaining 

many of the more historical biological agents (eg, ricin 

or anthrax) as a determining factor, but also should be 

aligned to the categorization of the agents and availability or absence of availability of effective vaccines 

and/or therapeutics for the higher category agents. 

The Amerithrax anthrax attacks highlight the panic 

and fear that can quickly disrupt public, commercial, 

and governmental activities with localized instances 

of infection. This public fear perception is the principal 

reason why Filovirus infections, specifically Ebola 

virus, attract so much attention in contrast to other 

infectious diseases that kill far more people annually 

from ongoing epidemic outbreaks (eg, influenza virus). 

In the United States, a new strategy using a “wholegovernment” approach has been implemented by the 

National Interagency Confederation for Biological 

Research to coordinate efforts for the development of 

medical countermeasures.207 Internationally, NATO 

(North Atlantic Treaty Organization) panels, cooperative agreements, and basic science partnerships are 

also being used to reduce the overall cost and impact 

of incorporating more novel means for developing 

countermeasures on tighter budgets. Involvement in 

these cooperative strategies should be leveraged at 

the interagency and international level as a means of 

cost reduction, as well as a diversification of expertise 

as the community searches for the next generation of 

medical countermeasures.

Biodefense vaccines may also face several more 

general issues of approval and licensure, much like 

common vaccines, but with the added requirements 

of approval under the Animal Rule because many of 

these diseases do not exist for an adequate Phase II/

III study. Vaccine approval, even in the military, can 

be challenging, as illustrated by the anthrax vaccine 

campaign that was interrupted by the Department 

of Defense in 1999.208 Vaccine safety standards have 

become more stringent over the last few decades, 

and biodefense vaccines must be held to these standards. Whereas confidence in vaccine efficacy has 

also improved, the task of ensuring vaccine safety 

can be daunting, as exemplified in spring 2010 by the 

increased risk of narcolepsy and catalepsy observed 

in patients in several countries after H1N1 vaccination.209 Another concept that may need to be integrated 

into biodefense vaccines is individualized medicine. 

Next-generation vaccines may be targeted to specific 

subpopulations according to their HLA genotypes or 

the capacity of their immune system to mount an appropriate immune response.

The next generation of antibody development 

should focus on the mechanism of how antibodies 

enter into or influence a cellular environment. For 

example, “transbodies” are cell-permeable antibodies 

made through conjugation of an antibody to a ligand 

to facilitate entry of the antibody into the cell or to 

inhibit a specific function, as with immunotoxins. 

Conversely, “intrabodies” are antibodies developed to 

achieve intracellular expression using the application 

of recombinant DNA technology.210

Even with these novel means of antibody delivery 

and action, target identification remains one of the 

largest challenges in developing the next effective 

antibody therapeutics. Secondarily, antigen-binding 

specificity and access are other points to consider 

because antibodies are highly specific to the target, 

and corresponding antigens from different species, 

such as NHPs or rodents, are dependent on the antigen alignment between species. In many cases, the 

critical epitopes targeted by vaccines and therapeutics 

are conserved and additional mitigation has been 

achieved by the use of oligoclonals, or cocktails of 

antibodies. These critical epitopes are of particular 

concern for both vaccines and therapeutic antibodies 

because biodefense-related treatments often require 

special use of the FDA Animal Rule for advancement 

through clinical trials. 

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treatment modalities. Disaster Med Public Health Prep. 2007;1:122–134.

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152. Arnon SS, Schechter R, Inglesby TV, et al. Botulinum toxin as a biological weapon: medical and public health management. JAMA. 2001;285:1059–1070.

153. Chang GY, Ganguly G. Early antitoxin treatment in wound botulism results in better outcome. Eur Neurol. 2003;49:151–

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154. Chahboun S, Hust M, Liu Y, et al. Isolation of a nanomolar scFv inhibiting the endopeptidase activity of botulinum toxin 

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155. Yanagisawa C, Hanaki H, Natae T, Sunakawa K. Neutralization of staphylococcal exotoxins in vitro by human-origin 

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157. Hughes JM. Preserving the lifesaving power of antimicrobial agents. JAMA. 2011;305:1027–1028.

158. Hill J, Copse C, Leary S, Stagg AJ, Williamson ED, Titball RW. Synergistic protection of mice against plague with 

monoclonal antibodies specific for the F1 and V antigens of Yersinia pestis. Infect Immun. 2003;71:2234–2238.

159. Xiao X, Zhu Z, Dankmeyer JL, et al. Human anti-plague monoclonal antibodies protect mice from Yersinia pestis in a 

bubonic plague model. PLoS One. 2010;5:e13047.

160. Lu Z, Roche MI, Hui JH, et al. Generation and characterization of hybridoma antibodies for immunotherapy of tularemia. Immunol Lett. 2007;112:92–103.

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162. Bottex C, Gauthier YP, Hagen RM, et al. Attempted passive prophylaxis with a monoclonal anti-Burkholderia pseudomallei exopolysaccharide antibody in a murine model of melioidosis. Immunopharmacol Immunotoxicol. 2005;27:565–583.

163. Trevino SR, Permenter AR, England MJ, et al. Monoclonal antibodies passively protect BALB/c mice against Burkholderia 

mallei aerosol challenge. Infect Immun. 2006;74:1958–1961.

164. Inglesby TV, O’Toole T, Henderson DA, et al. Anthrax as a biological weapon, 2002: updated recommendations for 

management. JAMA. 2002;287:2236–2252.

165. Brossier F, Lévy M, Landier A, Lafaye P, Mock M. Functional analysis of Bacillus anthracis protective antigen by using 

neutralizing monoclonal antibodies. Infect Immun. 2004;72:6313–6317.

166. Rivera J, Nakouzi A, Abboud N, et al. A monoclonal antibody to Bacillus anthracis protective antigen defines a neutralizing epitope in domain 1. Infect Immun. 2006;74:4149–4156.

167. Hill J, Eyles JE, Elvin SJ, Healey GD, Lukaszewski RA, Titball RW. Administration of antibody to the lung protects 

mice against pneumonic plague. Infect Immun. 2006;74:3068–3070.

168. Kirimanjeswara GS, Golden JM, Bakshi CS, Metzger DW. Prophylactic and therapeutic use of antibodies for protection 

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169. Savitt AG, Mena-Taboada P, Monsalve G, Benach JL. Francisella tularensis infection-derived monoclonal antibodies 

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171. Tirado SM, Yoon KJ. Antibody-dependent enhancement of virus infection and disease. Viral Immunol. 2003;16:69–86.

172. McCormick JB, King IJ, Webb PA, et al. Lassa fever. Effective therapy with ribavirin. N Engl J Med. 1986;314:20–26.

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175. Pettitt J, Zeitlin L, Kim do H, et al. Therapeutic intervention of Ebola virus infection in rhesus macaques with the MB003 monoclonal antibody cocktail. Sci Transl Med. 2013;5:199ra113.

176. Chen Z, Earl P, Americo J, et al. Chimpanzee/human mAbs to vaccinia virus B5 protein neutralize vaccinia and smallpox viruses and protect mice against vaccinia virus. Proc Natl Acad Sci U S A. 2006;103:1882–1887.

177. Chen Z, Earl P, Americo J, et al. Characterization of chimpanzee/human monoclonal antibodies to vaccinia virus 

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179. Hopkins RJ, Lane JM. Clinical efficacy of intramuscular vaccinia immune globulin: a literature review. Clin Infect Dis. 

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180. Goodchild SA, O’Brien LM, Steven J, et al. A humanised murine monoclonal antibody with broad serogroup specificity protects mice from challenge with Venezuelan equine encephalitis virus. Antiviral Res. 2011;90:1–8.

181. Hu WG, Phelps AL, Jager S, et al. A recombinant humanized monoclonal antibody completely protects mice against 

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184. Bossart KN, Zhu Z, Middleton D, et al. A neutralizing human monoclonal antibody protects against lethal disease in 

a new ferret model of acute nipah virus infection. PLoS Pathog. 2009;5:e1000642.

185. Borio L, Inglesby T, Peters CJ, et al. Hemorrhagic fever viruses as biological weapons: medical and public health 

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186. Frame JD, Verbrugge GP, Gill RG, Pinneo L. The use of Lassa fever convalescent plasma in Nigeria. Trans R Soc Trop 

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190. Parren PW, Geisbert TW, Maruyama T, Jahrling PB, Burton DR. Pre- and postexposure prophylaxis of Ebola virus 

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monkeys. PLoS Pathog. 2007;3:e9.

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disease. Proc Natl Acad Sci U S A. 2012;109:5034–5039.

193. Herbert AS, Kuehne AI, Barth JF, et al. Venezuelan equine encephalitis virus replicon particle vaccine protects nonhuman primates from intramuscular and aerosol challenge with ebolavirus. J Virol. 2013;87:4952–4964.

194. Wong G, Richardson JS, Pillet S, et al. Immune parameters correlate with protection against Ebola virus infection in 

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197. Jahrling PB, Stephenson EH. Protective efficacies of live attenuated and formaldehyde-inactivated Venezuelan equine 

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200. Goodchild SA, O’Brien LM, Steven J, et al. A humanised murine monoclonal antibody with broad serogroup specificity protects mice from challenge with Venezuelan equine encephalitis virus. Antiviral Res. 2011;90:1–8.

201. Hunt AR, Frederickson S, Hinkel C, Bowdish KS, Roehrig JT. A humanized murine monoclonal antibody protects mice 

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202. Reed DS, Glass PJ2, Bakken RR, et al. Combined alphavirus replicon particle vaccine induces durable and crossprotective immune responses against equine encephalitis viruses. J Virol. 2014;88:12077–12086.

203. Yu YJ, Zhang Y, Kenrick M, et al. Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target. Sci Transl Med. 2011;3:84ra44.

204. Herrmann I, Baeuerle PA, Friedrich M, et al. Highly efficient elimination of colorectal tumor-initiating cells by an 

EpCAM/CD3-bispecific antibody engaging human T cells. PLoS One. 2010;5:e13474.

205. Nicas M, Hubbard AE, Jones RM, Reingold AL. The infectious dose of variola (smallpox) virus. Appl Biosafety. 

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207. Gilman JK, Wright M, Clifford Lane H, Schoomaker EB. A model of federal interagency cooperation: the National 

Interagency Confederation for Biological Research. Biosecur Bioterror. 2014;12:144–150.

208. Cybulski RJ Jr, Sanz P, O’Brien AD. Anthrax vaccination strategies. Mol Aspects Med. 2009;30:490–502.

209. Dauvilliers Y, Arnulf I, Lecendreux M, et al. Increased risk of narcolepsy in children and adults after pandemic H1N1 

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210. Messer A, Joshi SN. Intrabodies as neuroprotective therapeutics. Neurotherapeutics. 2013;10:447–458.

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Aerobiology: History, Development, and Programs

Chapter 29

AEROBIOLOGY: HISTORY, 

DEVELOPMENT, AND PROGRAMS

DOUGLAS S. REED, PhD*; AYSEGUL NALCA, MD, PhD†; and CHAD J. ROY, PhD‡

INTRODUCTION

CONCEPTUAL BASIS OF AEROBIOLOGY IN INFECTIOUS DISEASE

OFFENSIVE BIOLOGICAL DEVELOPMENT AND CLINICAL APPLICATIONS IN 

THE UNITED STATES

Camp Detrick, Black Maria, and the US Army Medical Unit (1954–1970)

Clinical Exposure Trials: Operation Whitecoat (1954–1973)

BIOLOGICAL AEROSOL EXPOSURE SYSTEMS

Exposure Systems

Sampling

Monitoring and Control

Dosimetry

Parameters Impacting Aerosol Dosimetry

SUMMARY

*Associate Professor, Aerobiological Manager, RBI, Department of Immunology, University of Pittsburgh, 3501 Fifth Avenue, Pittsburgh, Pennsylvania 

15261; formerly, Microbiologist, Center for Aerobiological Sciences, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, 

Fort Detrick, Maryland

†

Chief, Department of Animal Studies, Center for Aerobiological Sciences, US Army Medical Research Institute of Infectious Diseases, 1425 Porter 

Street, Fort Detrick, Maryland 21702

‡

Associate Professor, Department of Microbiology and Immunology, Tulane School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 70112; 

and Director, Infectious Disease Aerobiology, Division of Microbiology, Tulane National Primate Research Center, 18703 Three Rivers Road, Covington, 

Louisiana 70433

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INTRODUCTION

The concept of using inhaled infectious particles as 

biological weapons is not new. The significance of this 

route was first appreciated and truly understood in 

the early 20th century, although the concept of infection by inhalation has been intermittently influenced 

by the study of infectious disease epidemiology. The 

question of whether diseases are “air-caused” has 

had, in past centuries, ardent believers and equally 

passionate cynics. Historically, for example, the prevailing theory was that all infections originated from 

“miasma,” or contaminated air. The cyclic nature of 

disease transmitted by aerosol among people living in 

groups is described in basic terms in ancient preserved 

artifacts. The Smith papyrus, dating from 1600 bce

and held in the Field Museum of Natural History in 

Chicago, describes prayers recited to gods of disease 

to purify the “winds” of the “pestilence of the time.”1

Epidemics were thought to be transmitted by aerosol 

even in the early days of medical science. Around 

400 bce, Hippocrates dictated that “airs, waters, and 

places” directly influenced the health of people, and 

he used the knowledge of seasonal change to guide 

diagnosis of differing ailments. In the Middle Ages, 

few pathogens impacted understanding of epidemic 

spread of disease as Yersinia pestis, the causative agent 

of the “black death.” At-risk populations eventually 

learned that the only defense against infection and 

death was to avoid contact with victims dying or 

dead from the bacterial disease. Pneumonic plague, 

the corollary form of infection from an infected host, 

is now recognized to transmit from expectoration of 

respiratory droplets. People may have unwittingly 

avoided respiratory exposure to aerosolized Y pestis

by avoiding contact with infected hosts and thereby 

not contracting the most feared (and deadly) form of 

the bacterial disease. 

Advancements in the field of chemistry in the 19th 

century gave rise to the concept of miasmic theory 

of disease. Sir Edwin Chadwick (1850) in Britain advanced the public health practices associated with 

the avoidance of the malodorous vapors to preserve 

the overall health of at-risk populations. The concept 

of spontaneous generation of disease-causing agents 

in vapors, however, was countered and ultimately 

refuted by Louis Pasteur (1860) during the same era. 

Pasteur demonstrated the presence of living organisms 

that was the root cause of fermentation and decomposition. His work in this area was instrumental in 

the understanding that infection could only appear 

miasmic if airborne microorganisms were present. 

By the end of the 19th century, most communicable 

bacterial pathogens had been identified, and there 

were only a few effective airborne agents. At roughly 

the same time, seminal work in vector-borne disease, 

including demonstration of parasitic disease cycles for 

malaria and filariasis, further improved public health 

measures and reduced disease burden. The concept 

that the majority of disease agents affecting large 

numbers of people were food- or water-borne greatly 

minimized aerosol transmission as an important pathway of infection. 

In the early 20th century, it was also shown that 

respiratory droplets from diseased individuals, never 

traveling more than an arm’s length from the infected 

person, could readily transmit disease. The theory of 

large-droplet infection, coupled with the recognition 

that arthropods were vectors for disease, nearly negated the respiratory route of infection from consideration in natural endemic spread of disease.2

 It was not 

until the great influenza pandemic of 1917–1918 that 

airborne disease transmission was again considered a 

medically important infection route. The notion that 

near-instant dehydration takes place in the environment once numerous particles are expelled from an 

infected host, causing submicron infectious biological 

aerosols to “float” for hours, solidified the concept of 

ecological transport from an infected host to an otherwise naïve host and ultimately successful disease transmission. A more modern understanding of airborne 

contagion also dictated that the probability and rate 

of disease transmission through air differs from, for 

example, a contaminated well.3 Although the number 

and tempo of infections from a contaminated well 

are horizontal, arithmetic, and limited to the number 

of interactions with a single source, airborne disease 

transmission is truly a vertical and geometric process 

and is not limited to interactions with one infected 

source, but rather the general vicinity of one or many 

infection sources.4 The indoor environment that now 

comprises most of the modern world amplifies probability of vertical transmission from airborne pathogens. 

There is a sharp distinction between naturally 

communicable airborne disease and those that are 

artificially induced through human-made biological 

aerosols. Modern military and ancillary industrial 

development activities, primarily associated with offensive biological weapon development in the 20th 

century, exploited the characteristics of aerosols that 

would promote maximum potential impact upon enemies.5

 This was primarily achieved by modern and 

sophisticated manipulation of the particulars, such as 

particle size distribution and environmental dehydration, to assure successful delivery to the respiratory 

system of the target host population. An early scientific 

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Aerobiology: History, Development, and Programs

concept in the process of designing and producing biological aerosols as modern weapons was the research 

and understanding of naturally occurring airborne 

disease. A basic, empirically derived understanding 

of natural epidemics from human source generators 

(respiratory expectoration) and indirect sources (eg, 

fomites on bed sheets) was essential to better appreciate important environmental and physiochemical 

factors when designing biological aerosols. It was soon 

recognized that airborne infection, when left up to the 

natural transmission process, was an overwhelmingly 

variable process influenced by a number of intrinsic 

and extrinsic factors, many of which cannot be readily 

controlled. The process of natural spread of disease 

by the aerosol route was described in detail in studies 

predating World War II; comprehensive descriptions 

were first published in the eminent text, Airborne 

Contagion and Air Hygiene, by WF Wells.1 Many of the 

early tenets of infection from droplet nuclei are presented in this work, with descriptions of experiments 

that demonstrate the most basic mechanisms dictating 

infection from an airborne microbial source. 

CONCEPTUAL BASIS OF AEROBIOLOGY IN INFECTIOUS DISEASE

The basic mechanism for transmitting airborne 

disease is by droplet nuclei. Droplet nuclei have been 

described as small, air-suspended residues arising 

from the evaporation of droplets emanating from the 

mouth and nose. These nuclei-containing infectious 

microbes (bacteria or viruses) or toxic components 

collectively comprise biological aerosols that are medically important. Such aerosols are readily produced 

artificially by spraying or atomizing wet or dried 

preparations of microorganisms or toxins. 

There are many experimental uses of aerosols, but 

those used for respiratory disease studies are especially important.3

 The study of disease pathogenesis 

in animal models can be more meaningful if subjects 

are infected by the same route that occurs naturally in 

humans. In contrast to intratracheal or intranasal instillation, infectious challenge with aerosol particulates 

greatly increases natural dispersion in the respiratory 

system and is consistent with “natural” aerosol infection. Dosage, aerosol particle size, age, environmental 

temperature, and humidity can all be measured, controlled, and analyzed to some extent.6

 Moreover, the 

interplay of these features can be studied in the context 

of microbial viability and resulting virulence. 

There are disadvantages, however, that are inherent 

in aerobiological experimentation. The significance of 

aerosol age on airborne organism virulence is not fully 

known. Finally, respiratory doses are difficult to reliably calculate because the degree of lung retention of inhaled aerosol particles, while predictable, usually is not 

measured.7 Experiments involving aerosol challenge 

of animals include determining the host species’ susceptibilities, estimating or establishing dose-response 

curves, evaluating the effect of therapy or stress, and 

testing the efficacy of experimental vaccines. 

These early studies made clear that measuring and 

controlling as many of the variables as possible associated with stability, viability, and corresponding infectivity of virulent biological aerosols was required for 

the first biological weapons produced using modern 

technological methods. Rapid industrialization of the 

microbiological and evaluation aspects of developing 

biological weapons was pursued by the militaries of 

world powers at the time, which ushered in an era 

of aerobiological research that was performed on a 

grand scale. 

Military programs throughout the mid-20th century 

engaged in researching and developing biological 

weapons selected aerosol as the predominant modality and route of battlefield delivery to the enemy. A 

historic brain trust, comprised of the personnel and 

physical resources capable in this scientific area, was 

developed among the superpowers to support this 

effort. The extensive network that developed was 

uniquely qualified to harness and perfect the biological, physiochemical, and logistical characteristics preferential to aerosol stability and survival for industrial 

production and eventual delivery in munitions. 

OFFENSIVE BIOLOGICAL DEVELOPMENT AND CLINICAL APPLICATIONS 

IN THE UNITED STATES

Camp Detrick, Black Maria, and the US Army 

Medical Unit (1954–1970)

In 1941, the Secretary of War asked the National 

Academy of Sciences (NAS) to review the risk of 

biological warfare if the United States were to become engaged in World War II. The War Bureau of 

Consultants from the NAS advised the Department 

of Defense to prepare for biological warfare and to 

provide the resources for both defensive and offensive capabilities. In the spring of 1942, the Army 

determined that the first US Army biological warfare 

laboratories would be located at Camp Detrick (Army 

Air National Guard [ANG] Airfield) in Frederick, 

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Medical Aspects of Biological Warfare 

Maryland.8 Before the offensive and defensive efforts 

were pursued at Camp Detrick, the Safety Division 

made great strides in developing capabilities for 

biocontainment, decontamination, and sterilization 

of hazardous disease agents. Biological weapon production and testing facilities were initially built at 

Camp Detrick for the purpose of producing anthrax 

and botulinum neurotoxin for weapons. The first research facility was located in the ANG hangar, which 

was modified to include laboratories. A seven-story 

pilot plant facility was built in 1943 to test fermenters 

to find the most optimal configuration for culturing 

large amounts of organisms such as B anthracis. A 

free-standing building was constructed to house this 

operation; it was covered with the most impervious 

material available at the time (tar paper), which gave 

the structure the appearance of an ominous black box 

and invoked the moniker “Black Maria.” This facility 

was later dismantled to make way for larger, more 

modern buildings. The US expanded its offensive 

biological warfare efforts to include production and 

storage facilities at the Pine Bluff Arsenal in Arkansas, 

and in Terre Haute, Indiana, during the Cold War in 

the wake of World War II. The capability at Fort Detrick and ancillary production facilities throughout the 

United States provided the source material for initial 

efforts in preparing and packaging biological agents 

capable of being dispersed as aerosols via munitions. 

The microbiological expertise and industrial-sized 

production capabilities during these early efforts were 

essential for biological stability, which was required 

for continual production of microbial product that 

could survive the rigors of the aerosol environment. 

Maintaining strain virulence, toxin production, and 

corresponding lot comparability were critical to successful aerosol delivery.

In 1942, President Roosevelt dedicated an initial 

126,720 acres of Utah desert land for use by the War 

Department. Another biological weapons laboratory 

was opened 6 days later at Dugway Proving Ground 

in Utah as a testing and evaluation facility. The remoteness and massive land area of this base was 

ideal for evaluating how aerosolized biological agents 

performed in the natural environment. A series of experiments were commenced to evaluate the utility of 

aerosol dispersal as a means of executing a biological 

weapon attack, including open-air experiments with 

active biological agents. Aerosolized organisms were 

detected as far as 30 or more miles away in large-scale 

aerosol tests. Clandestine dispersals of surrogate organisms, such as Serratia marcescens and Bacillus globigii

(now B atrophaeus) were also conducted in a number 

of urban locations, including New York and San Francisco. Years later it was realized that these experiments 

actually resulted in a number of illnesses and possibly 

at least one death, despite the “harmlessness” of the 

bacteria used. These tests, while highly unethical, 

demonstrated the potential for an aerosol attack with 

a biological weapon.9–11

Clinical Exposure Trials: Operation Whitecoat 

(1954–1973)

Concurrent and subsequent to the massive operational efforts that were underway to produce and 

evaluate biological agents, limited human clinical 

studies began with a program called Camp Detrick-22 

(CD-22) in 1954. Initially, this program of testing 

biological agents on human volunteers was to assess 

incapacitating agents’ delivery and effects on soldiers, 

and was very similar to human evaluation of lachrymatory chemical agents (tear gas). The program was 

eventually expanded to test the efficacy of medical 

interventions and vaccines, and became known as 

Project or Operation Whitecoat. Program volunteers 

were primarily chosen from US enlisted soldiers who, 

based on their stated religious preference, were affiliated with the Seventh-day Adventist (SDA) church. 

These soldiers were promised to serve in the military 

only in noncombat positions if they were enrolled into 

Operation Whitecoat as volunteers for testing. In addition to SDA-affiliated soldiers, Ohio State Penitentiary 

prisoners also attended as volunteers of the program. 

Both soldiers and the prisoners signed the consent 

forms before they enrolled to the program. They were 

free to withdraw from the program any time and they 

were informed about the possible effects of each study. 

Overall, more than 2,300 volunteers were tested in 

137 protocols to develop and test for safety, vaccines, 

and therapeutics against tularemia, Q fever, viral encephalitis, Rift Valley fever, sandfly fever, and plague 

between 1954 and 1973 during Operation Whitecoat.9 

Because this kind of testing is now recognized as unethical, Operation Whitecoat constituted one of the few 

times in history when aerosolized agent delivery was 

directly tested in the targeted host, the human being, 

rather than a surrogate animal species.12 Although 

unethically obtained by modern standards, data from 

these early clinical studies remain highly relevant as 

true indicators of delivery of biological agents by the 

aerosol route in humans. 

Of the list of potential biological agents tested in 

this manner, only studies involving Q fever (Coxiella 

burnetti) and tularemia (Francisella tularensis) were 

considered safe enough for use in aerosol challenge 

in humans. Both agents produced infections that were 

not rapidly progressive, and antibiotic treatment (ie, 

chloramphenicol, streptomycin) was readily available 

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and proven to be effective. Consequently, aerosol studies in humans were performed with these agents, in 

which a 1-million-liter cloud chamber (Figure 29-1) 

was employed for the initial aerosol dispersion. This 

unique structure, with 1-1/4-inch-thick steel walls, was 

truly remarkable in that it was one of the only configuration facilities where small munitions loaded with 

prepared biological agent could be detonated and aerosol dispersion could be studied over an appreciable 

amount of time. The black rubber bladders integrated 

in the otherwise gray exterior of the chamber, which 

absorbed the percussion from the detonation of the 

munitions used at the time, gave the enormous sphere 

its nickname, “Eight Ball.” In addition to the study of 

agent survival estimates, aerosols from the interior 

could also be used for exposure studies with volunteers from Operation Whitecoat. These controlled 

clinical exposures were a critical aspect of the ongoing characterization of biological agents because they 

represented the only opportunity to study the interaction of aerosols originating from detonated munitions 

with the human respiratory system. These studies 

provided information on the physical size distribution, biological stability, and corresponding viability 

of the microbial payloads prepared for delivery on the 

battlefield. The Eight Ball was used for exposures first 

with C burnetii and then F tularensis aerosol trials, and 

decontaminated in 1970 during the decommissioning 

of the US offensive biological program. No longer in 

use, the testing chamber remains at Fort Detrick and 

was listed in 1977 on the US National Historic Register 

as a landmark site (National Park Service landmark 

77000696). In addition to these aerosol trials, outdoor 

aerosolized C burnetti studies that emulated biological 

warfare scenarios at Fort Detrick were performed in 

Dugway Proving Ground, as well.

Studies with aerosolized F tularensis indicated that 

when the aerosol residence time increased, infectivity 

of airborne bacteria decreased. This information, critical to understanding the environmental susceptibility 

of an organism, opened the door to the development 

of an attenuated vaccine for F tularensis. Early killed 

and live attenuated tularemia vaccine testing studies 

with volunteers from the inmates of Ohio State Penitentiary used intracutaneous and respiratory challenge 

of F tularensis. Of the unvaccinated volunteers, 16 of 

20 (80%) showed signs of disease following low-dose 

aerosol challenge ranging from 10 to 52 organisms.10

Aerosol challenge of vaccinated volunteers resulted in 

signs of tularemia systemic infection in 8 of 14 (57%) 

killed vaccine vaccinated volunteers, while only 3 of 

18 (16%) live attenuated vaccine vaccinated volunteers 

had any systemic signs of infection. 10,13 

The potency of an attenuated tularemia vaccine 

delivered as an aerosol against aerosolized F tularensis

was tested during follow-up studies.11 Minor systemic 

signs and symptoms, such as sore throat and cough, 

were seen in 30% of aerosolized live vaccine strain 

(LVS) vaccinated volunteers; pea-sized cervical lymphadenopathy was observed in all vaccinated subjects. 

Control group and aerosol-vaccinated volunteers 

were then exposed to approximately 2.5E+04 colonyforming units (CFUs) of aerosolized F tularensis; this 

challenge dose was estimated to be over 2,500-fold 

more than the minimum dose required to cause disease 

in humans. Almost all (94%) control group subjects had 

fever greater than 100°F after a 3- to 5-day incubation 

period. Following the clinical signs of sudden onset of 

fever (103°F/104°F), some patients had headache, chills, 

Figure 29-1. Design of the 1-million-liter sphere ball known 

as “Eight Ball,” which was used to expose the Operation 

Whitecoat volunteers to Francisella tularensis and C burnetii

at Fort Detrick, Maryland. 

Photograph reproduced from US Army Medical Department, Medical Research and Materiel Command, Office of 

Public Affairs, Fort Detrick, MD. R3086, no. 1.

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Medical Aspects of Biological Warfare 

and sore throat accompanied by malaise, noticeable 

myalgia and backache, nausea, and anorexia. Nearly 

all (89%) of the control group required treatment 

with antibiotics, while 70% of the vaccinated group 

had fever and only 23% required treatment. Other 

delivery routes (oral, cutaneous, and intradermal) for 

the tularemia vaccine were also evaluated against different challenge routes (intracutaneous, intradermal, 

aerosol).13,14

A similar study was performed to test prophylactic 

efficacy of tetracycline against aerosolized F tularensis.

15 Preceding a 2- to 6-day incubation period, all 

control group subjects (100%) experienced fever and 

the other hallmark clinical signs of the disease. The 

group receiving antibiotic 24 hours postexposure and 

continuing for 15 days showed no signs of disease. 

Interestingly, following the cessation of the treatment, 

2 of the 10 (20%) volunteers developed acute tularemia. 

The group that received treatment 28 days initiating 

24 hours after exposure did not experience any signs 

of the disease during or after antibiotic treatment. 10

A portion of the ongoing clinical efficacy trials with 

Operation Whitecoat personnel involving Q fever 

(C burnetti) were performed with prisoners from the 

Maryland State House of Correction.9 The efficacy of 

Phase I strain Henzerling and Phase II strain Nine 

Mile vaccine was tested against aerosolized C burnetti

in these subjects. These studies indicated that a vaccine of adequate potency was effective in protecting 

humans against Q fever disease; the protection afforded by these vaccines lasted nearly 1 year after 

vaccination. Collectively, clinical studies using aerosol 

infection to develop offensive biological capabilities (at 

the beginning) and defensive biological capabilities 

(later) developed and improved medically important 

countermeasures (vaccines and therapeutic). These 

studies also contributed to a clear and scientifically 

realistic understanding of clinical disease progression, 

signs, symptoms, and diagnostic parameters of many 

of the priority biological pathogens of interest, namely 

C burnetti, F tularensis, sandfly fever, the alphaviral 

encephalitides, Rift Valley fever, and staphylococcal 

enterotoxins.9 This line of investigation also provided 

clinical insight into the comparative pathophysiology 

of a disease experimentally induced through a nonnatural route of exposure (aerosol), which was crucial 

for the viral disease agents that are naturally vectorborne (eg, alphaviruses). 

BIOLOGICAL AEROSOL EXPOSURE SYSTEMS

One of the cornerstones in the development of aerobiology capabilities during the former US offensive 

program and in the present-day defensive biological program is the operational capability to conduct 

animal studies that incorporate aerosol exposure as a 

modality for delivering biological agents. In contrast 

to the clinical studies that took place during the offensive biological program, animal studies presently 

serve as the only source for data on pathogenesis and 

performance of medical countermeasures to priority 

pathogens, such as Tier 1 select agents (those for which 

there is the most concern regarding their potential for 

use and the resulting consequences). Appreciation of 

the componentry in studies involving aerosol challenge is an essential part of the collective required for 

successful integration into animal experimentation, 

and remains a core competency of any infectious disease aerobiology program. 

Significant efforts to place engineering controls to 

protect and contain biological aerosols were integrated 

early and remain the approach in modern facilitates 

engaged in this type of experimentation. In the modern 

era in the United States, experimenting with aerosol 

exposures with select agents requires approval of the 

Centers for Disease Control and Prevention’s (CDC’s) 

Division of Select Agent and Toxins (DSAT) and must 

follow the recommendations in the Biosafety in Microbiological and Biomedical Laboratories (BMBL) manual. 

Aerosol exposures of animals to infectious agents or 

toxins, particularly those that are potential biological 

threat agents, are performed in laboratory environments that are negatively pressurized and rigidly 

controlled, typically at biosafety level-3 (BSL-3) or 

higher. Most aerosol exposures are performed inside 

class III biological safety cabinets (BSCs), which are 

expensive, completely contained environments with 

HEPA-filtered supply and exhaust. However, this is 

not always the case. Some exposures are performed 

under standard class II BSC or in self-contained equipment, such as the Glas-Col (Terra Haute, IN) inhalation 

exposure chamber (which is typically used for tuberculosis studies and not select agents and toxins). In 

some institutions, aerosol exposures are performed in 

the same room where the exposed animals will be subsequently housed, while in others aerosol exposures 

take place in separate suites and animals are transported from the holding room to the exposure suite 

using negatively pressurized transport devices, such 

as a negatively pressurized and filtered mobile transfer cart. The use of a class III BSC in a separate suite 

provides the greatest flexibility for decontamination 

and reuse of the aerosol equipment between multiple 

pathogens or animal species. The different options also 

alter the need for personal protective equipment (PPE). 

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Using aerosols in a class III BSC in a dedicated suite 

separate from animal holding requires only minimal 

PPE, while other options, depending on the pathogen, 

typically require the use of N-95 or powered-air purifying respirators (PAPRs). Beyond the engineering 

controls and PPE described here, a number of other 

issues must be considered, including decontamination 

of the space and security and administrative controls 

(eg, standard operating procedures, training, and 

oversight). The laboratory space needed to prepare 

for aerosol exposures (both the pathogenic agent and 

the aerosol equipment) and the dose required should 

be considered and determined.

Exposure Systems

Henderson Apparatus 

In 1952, David Henderson described an aerosol 

exposure system designed for ease of operation that 

could ensure reproducibility between experiments 

exposing animals to “clouds” containing infectious 

organisms.16 This system also incorporated engineering controls to ensure the safety of those using it, to 

prevent exposure of laboratory personnel. It consisted 

of a spraying apparatus (an aerosol generator), an 

exposure tube (analogous to the exposure chambers 

used today), and an impinger (an aerosol sampling 

device), as well as a number of points for monitoring 

and controlling airflow, vacuum, and pressure. The 

system was dynamic, with air continuously pumped 

into and exhausted from the exposure apparatus 

throughout the exposure to eliminate effects resulting 

from aerosol decay of the organism, as would occur 

in a static system. As originally described, the system 

recirculated waste air that was filtered and reused as 

dilution air in the exposure. 

Modern Exposure Systems

Most aerosol exposure systems used in present-day 

laboratories that perform bioaerosol studies with select 

agents are generally some derivation of the original 

Henderson apparatus.17 Most are also dynamic and 

incorporate some measure of safety for laboratory personnel in addition to performing the aerosol in a BSC 

and with the primary engineering controls described 

above. These systems are designed with greater flexibility for incorporation of other aerosol generators, 

exposure chambers, or sampling devices, as well 

as improved monitoring and control of the aerosol. 

Exhaust air is filtered but, unlike the Henderson apparatus, waste air is not subsequently recirculated into 

the exposure loop.

Generators

Although a wide range of aerosol generators can 

be and are employed, the Collison nebulizer is by far 

the most commonly used aerosol generator for exposures using select agents (viruses or bacteria) and 

toxins. This generator has become a standard for three 

primary reasons: (1) Collison nebulizers are relatively 

inexpensive and easy to maintain; (2) the Collison 

generates a relatively uniform, nearly monodisperse 

particle distribution; and (3) aerosol particles in the 

size range generated by a Collison (approximately 1 to 

2 μm in diameter) will reach the deep lung (alveolar 

regions) of most mammalian species with minimal 

deposition in the upper respiratory tract. However, 

other generators have been used, including in recent 

years the spinning top aerosol generator (STAG)18,19

and flow-focusing aerosol generator (FFAG).20–23

These generators allow for customization to larger 

particle size distributions, thereby allowing study 

of differential effects based on deposition in distinct 

compartments of the respiratory tract. Where it has 

been examined, in most instances particle deposition 

in the upper respiratory tract (as compared to deposition in the lower) increases the dose required to cause 

morbidity and mortality and alters the pathogenesis 

of the disease, and countermeasures are often more 

efficacious.18,21–24 It has been postulated that deposition of encephalitic viruses in the upper respiratory 

tract might more readily lead to infection of the brain 

as a result of infection in the olfactory region, but the 

data accumulated to date is contradictory and needs 

further examination.19,25–27

All aerosol generators described in modern exposure systems utilize “wet,” liquid aerosols rather 

than the dry powder aerosol systems that were used 

in the past during offensive development. The use 

of dry powder systems to aerosolize biologically active microbial aerosols raises concerns regarding the 

potential for “dual-use” research and harkens back 

to the type of technical expertise common during 

the now-decommissioned offensive biological development program. Dual-use research is defined by 

federal policy as, “life sciences research that, based 

on current understanding, can be reasonably anticipated to provide knowledge, information, products, 

or technologies that could be directly misapplied to 

pose a significant threat with broad potential consequences to public health and safety, agricultural 

crops and other plants, animals, the environment, 

materiel, or national security.”28 In particular, it is 

noted among the scope that experiments of concern 

include those that, “increase the stability, transmissibility, or the ability to disseminate the agent or toxin.”28

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Generation of dry-powder forms of virulent agents has 

been interpreted as falling into that category; however, 

that has not precluded the use of dry-powder research 

in biodefense, and in the pharmaceutical industry there 

is considerable interest and research into dry-powder 

delivery of vaccines and therapeutics.29–37

Exposure Chamber

The choice of exposure chamber is greatly dictated 

by the animal species being exposed.17 Rabbits and 

nonhuman primates are typically exposed one or 

two at a time using nose-only or head-only exposure 

chambers because of the animals’ size and laboratory 

space limitations. Rodents (mice and rats) and ferrets 

are exposed using either nose-only or whole-body 

chambers. Nose-only exposure chambers deliver the 

aerosol to the respiratory tract without contaminating 

the surface of the animal with the pathogenic organism, alleviating concerns regarding infection via swallowing and/or fomites as opposed to true inhalation. 

However, the current designs of nose-only systems 

place far greater stress upon rodents as evidenced by 

increased corticosterone in the blood that could alter 

the outcome of infectious disease studies.38 In addition, 

recent studies evaluating deposition and retention of 

select agents and toxins have demonstrated that the 

majority of what is initially inhaled is removed from 

the respiratory tract and ends up in the gastrointestinal 

system.21–23 The choice of nose-only or whole-body exposure chambers should be carefully considered prior 

to initiating studies.

Sampling

Traditionally, aerosol sampling of infectious 

organisms and toxins has been done using liquid 

impingement as a means to collect a representative 

sample to quantify both concentration and viability 

of the pathogenic agent (or activity, if a toxin) in the 

aerosol at the time of exposure. Impingers come in a 

variety of shapes and sizes but invariably reply upon 

impaction of an aerosol into a liquid interface. Impingers allow assessment of viable bacteria or viruses in 

the aerosol but do not provide a means for assessing 

particle size or the number of bacteria or viruses per 

particle. Filters and cyclones are also routinely used 

for sampling bioaerosols to determine concentration 

of viable microorganisms.39–41

When selecting sampling devices, sampling efficiency should be evaluated, as well as the effects of 

sampling processes (eg, flow rate, collection media) 

on the viability of the organism being measured. Particle aerodynamic size can be measured during these 

types of exposures using either viable impactor-type 

devices, such as an Andersen cascade collector, or 

analytical devices that employ dual time-of-flight laser technology. Other optical technologies for particle 

sizing are also employed, such as laser-scattering type 

instruments (eg, Grimm Technologies, Incorporated 

[Douglasville, GA] or TSI Incorporated [Shoreview, 

MN] particle spectrometers). Size characterization of 

liquid bioaerosols, whose malleability depends on 

prevailing environmental conditions in the exposure 

chamber, and the density of the formulation used are 

usually expressed as the mass median aerodynamic 

diameter (MMAD). The MMAD provides a median 

size of the particle distribution based on the behavior 

of the particles through the air and the corresponding 

velocity, rather than an actual physical measurement 

of size. This type of characterization is appropriate 

for liquid-based aerosols whose size can be dynamic 

within the exposure systems. Great care should also 

be taken to sample in the “breathing zone” where 

the animal is likely to inhale particles, as particle 

size could be different outside of that zone. Larger 

particles can break into smaller particles or shrink 

through evaporation, which can greatly influence 

where particles will deposit in the respiratory tract. 

This can also influence the viability of the microorganism in the aerosol, as has been seen with F tularensis, 

in which increased sodium chloride concentration 

resulting from particle evaporation resulted in a loss 

of viability, thereby requiring higher concentrations 

to achieve a lethal dose.42 Raising the relative humidity in the chamber improved bacterial viability in the 

aerosol, but it does not alter infectivity.42,43 Particle 

density is also an important consideration because it 

will influence particle size based on the percentage of 

solids and volatile components that will be lost due 

to evaporation.44 

Monitoring and Control

Older biological aerosol exposure systems allow for 

monitoring and control of environmental parameters, 

although typically in a crude fashion via a manually 

controlled instrumentation panel that must be continuously manipulated by a laboratory aerosol technician 

operator. Modern biological aerosol exposure systems, 

in contrast, are operated using fully integrated, process 

flow-control computer systems in addition to constantly monitoring and recording changes in environmental 

parameters (relative humidity, temperature, pressure) 

and flow rates (nebulizer, secondary air, exhaust, sampling).45 This improves accuracy in exposure timing 

and control as well as improving dosimetry precision 

in nonhuman primates. With the increased monitoring 

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Aerobiology: History, Development, and Programs

comes the improved ability to control and alter these 

parameters during the exposure to evaluate the impact 

on aerosol concentration.

Dosimetry

The most critical aspect of biological aerosol exposure systems is the ability to determine the dose delivered to the exposed animal based on the operational 

characteristics and performance of the system to the 

users’ requirements. Aerosol “dose” is reported in one 

of three ways: (1) inhaled (also called presented dose, 

the total number of infectious organisms or mass of 

toxin inhaled), (2) deposited (the amount that deposits within the respiratory tract), and (3) retained (the 

amount that remains in the respiratory tract after a 

specific time).17,46 The delineation between deposited 

and retained dose is time-dependent and is not fixed 

except within a given system. For example, in 1962 

Harper and Morton defined retention of Bacillus globigii

spores as the number of spores remaining in the lungs 

of guinea pigs 1 day after an aerosol exposure.47 A 

considerable amount of what is inhaled is exhaled, removed, or destroyed by the host’s innate mechanisms 

for clearing the respiratory tract (eg, the mucociliary escalator, mucin, defensins, and surfactants). Deposited 

and retained dose are difficult to measure for infectious 

organisms, which begin to replicate or escape from the 

respiratory tract into the circulation almost as soon as 

they deposit in the respiratory tract. Further, measuring deposited and retained dose requires sacrificing the 

animal and harvesting tissues in the respiratory tract. 

Understanding deposition and retention is useful for 

understanding the aerosol biology and pathology of 

infectious organisms, but the impact on the efficacy 

of medical countermeasures is less clear.21,22,24,48,49 This 

must be evaluated on a pathogen- and host-specific 

basis, and the results from one system should not be 

generally applied to other systems (including other 

animal species infected with the same pathogen). Further research is desperately needed.

Parameters Impacting Aerosol Dosimetry

Aerosol Performance

System performance between aerosols is compared 

using the ratio between the aerosol concentration and 

the nebulizer concentration, also known as the spray 

factor. Spray factor can be used to determine the nebulizer concentration required to achieve a desired inhaled 

dose in future studies with that pathogen. Spray factors 

can only be compared within a given aerosol system 

for a particular agent, and different systems are not 

comparable using spray factor performance. Spray 

factor is essential to building a microbial database of 

relative aerosol viability within a particular aerosol 

system, and ultimately dictates the capability (and 

limitations) of dosing animal species within an aerosol 

system with that pathogen. Aerosols performed prior 

to animal exposures determine the spray factor as well 

as assess the impact of environmental parameters on 

aerosol performance. Relative humidity in particular has 

been shown to impact particle size distribution as well 

as viability of a number of bacteria and viruses.43,50–57

Aerosol efficiency (the ratio of viable agent in the aerosol 

sample to the quantity of agent nebulized) and relative 

recovery (aerosol sampling of the challenge agent relative to a known standard included in the aerosol) have 

been used as alternatives to spray factor and serve an 

equivalent function.50,58,59 These effects are pathogenand system-specific and require careful evaluation in developing new systems or working with new pathogens.

Anatomy and Physiology

Numerous studies have highlighted differences in 

the respiratory anatomy between mammalian species. 

In particular, the length and degree of branching in the 

bronchus and bronchioles vary greatly between species, getting smaller in length and with less branching 

as species get smaller.60 Differences in the amount of 

branching have been noted between strains of inbred 

mice. There is also considerable difference in the thickness of bronchial epithelium and the production of 

mucus,61 all factors that can impact particle deposition 

and retention in the respiratory tract.

Respiratory Function

To determine the inhaled dose requires measuring 

the respiratory minute volume (Vm) of the experimental animal.62 For experiments with smaller animals 

like rodents and ferrets, where multiple animals are 

exposed at a time, minute volume is determined using 

a simplistic formula based on the animal’s weight and 

corresponding surface area, and was developed by 

Arthur Guyton over six decades ago.63 Other methods 

to determine respiratory function were developed by 

Bide and Alexander and are also used.64,65 Respiratory 

function is typically measured using plethysmography 

if larger animals, such as rabbits and nonhuman primates, are employed in experimentation. Plethysmography is typically performed either immediately before 

or during the aerosol exposure. Plethysmography 

(and the aerosol exposure) of nonhuman primates is 

performed while animals are anesthetized, which can 

dramatically suppress respiratory function. Rabbits, 

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although not anesthetized, are typically restrained, 

which can increase respiration and minute volume 

between rabbits of similar size, age, and gender, and 

can vary dramatically. Because of space limitations 

in the class III BSC, plethysmography of rabbits and 

nonhuman primates can typically be done more easily 

before the aerosol exposure. If plethysmography is performed prior to exposure, minute volume is presumed 

not to change during exposure (or changes only minimally), although data have shown tremendous variation in minute volume in larger animals irrespective 

of weight and other factors such as age, gender, and 

level of anesthesia. If not accounted for, this can lead 

to tremendous variation in presented dose delivered 

to animals during exposure. However, most biological 

aerosol exposure systems do not account for individual 

variation in minute volume between animals, instead 

relying upon a fixed exposure time. This is an area 

that needs further development to ensure similar and 

consistent dosing between treated and control groups.

SUMMARY

The interface of aerobiology, infectious disease, 

and the transmissibility of disease are ever present. 

Harnessing, controlling, and delivering pathogenic 

agents by aerosol remains the primary and most 

predicted route of exposure for both military and 

state-sponsored terrorist acts using biological weapons. The threat of a biological agent being optimized 

for aerosol delivery holds the potential to reach a 

target population more efficiently and more completely than any other possible exposure modality 

available. Much of what is known in the scientific 

lexicon of aerobiology, as in many fields of study, 

is derived primarily from observation of nature and 

natural processes; namely the transmission of disease 

either through indirect sources, such as contaminated 

sewage aerosolized at a particle size distribution that 

approximates respirability, or direct sources, such as 

proximal contact with an infected host while coughing or sneezing.4

 The early challenge was to overcome 

the identified environmental and physiochemical factors that would most rapidly degrade or kill microbial 

preparations when in an aerosol form. Accordingly, 

modern development of aerobiological techniques 

was synthetically modeled after natural processes 

most efficient at disease transmission. Maintaining 

the physical characteristics and viability of a pathogenic organism for delivery into the environment 

by virtue of munitions or secondary direct aerosol 

generator was no small task, and by all accounts 

in the history of the offensive biological programs, 

overcoming these barriers required sophisticated approaches. Early biological weapons programs in the 

United States and Soviet Union focused initially on 

transferring laboratory bench-based microbiological 

propagation into industrial-class operational capability, first producing massive quantities of pathogenic 

agent. Preparation of live microbiological agents for 

airborne delivery relied heavily upon techniques for 

preservation and packaging that maintain viability 

and protect against environmental degradation once 

released. Concurrent to developing and perfecting 

industrial-class microbial propagation, preparation, 

preservation, and delivery techniques, significant efforts were made to determine pathophysiology and 

pathogenesis in animal models and even in limited 

human studies. Complex systems for testing and 

evaluating optimized microbial preparations using 

select animal species emerged at this time to better 

support this effort. Sophisticated testing systems 

that integrated aerosol delivery to a varied array of 

animal species developed during this time. The small 

modular aerosol exposure systems in use in many 

modern laboratories, which are mere shadows of the 

industrial versions of the past, continue to function 

under the same basic design and performance criteria. Conversely, the clinical studies that incorporated 

aerosol exposure with agents easily treated with 

available chemotherapeutic agents at the time were 

an advent that will forever remain in the annals of the 

offensive biological program. The massive dedication 

of scientific resources and infrastructure to respond 

to this effort was specifically focused on aerosol as 

the primary means of delivery to the enemy. This 

is an important consideration because a number of 

the biological agents selected for development were 

not naturally communicable through the airborne 

route; therefore no clinical experience with infection 

existed at the time. Predominant disease models and 

pathogens that catered to aerosol delivery emerged as 

cornerstones of the state-sponsored biological weapon programs. These very programs, at their zenith, 

optimized the industrial production, packaging, and 

prospective aerosol delivery of biological agents in a 

manner that history had never witnessed. The aerobiology resources and capabilities adjunctive to the 

biological weapon programs ultimately experienced 

a dramatic reduction, and a complete shutdown in 

many cases, that coincided with the signing of the 

biological weapons convention in 1969. Some countries, however, continued covert operations, including 

the aerosol research components, well into the 21st 

century. 

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Collectively, in the aftermath of the decommissioning of the offensive biological programs, much 

of the infrastructure needed to effectively perform 

research for medical countermeasures was effectively rebuilt, albeit on a much smaller scale with 

significant technical and engineering limitations in 

mind. Present-day research organizations that incorporate aerobiology resources and expertise, such as 

the program at the US Army Medical Research Institute of Infectious Diseases at Fort Detrick, Maryland, 

embody a small-scale, sophisticated support structure similar to many programs at other federally 

supported, contracting, and academic laboratories 

throughout the nation. 

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influenza virus. Vaccine. 2003;21(21-22):2805–2812.

33. Wong YL, Sampson S, Germishuizen WA, et al. Drying a tuberculosis vaccine without freezing. Proc Natl Acad Sci U 

S A. 2007;104(8):2591–2595.

34. Ohtake S, Martin RA, Saxena A, et al. Formulation and stabilization of Francisella tularensis live vaccine strain. J Pharm 

Sci. 2011;100(8):3076–3087.

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35. Ohtake S, Martin R, Saxena A, et al. Room temperature stabilization of oral, live attenuated Salmonella enterica serovar 

Typhi-vectored vaccines. Vaccine. 2011;29(15):2761–2771.

36. Matinkhoo S, Lynch KH, Dennis JJ, Finlay WH, Vehring R. Spray-dried respirable powders containing bacteriophages 

for the treatment of pulmonary infections. J Pharm Sci. 2011;100(12):5197–5205.

37. Jin TH, Tsao E, Goudsmit J, Dheenadhayalan V, Sadoff J. Stabilizing formulations for inhalable powders of an adenovirus 35-vectored tuberculosis (TB) vaccine (AERAS-402). Vaccine. 2010;28(27):4369–4375.

38. Corbanie EA, Remon JP, Van Reeth K, Landman WJM, van Eck JHH, Vervaet C. Spray drying of an attenuated live 

Newcastle disease vaccine virus intended for respiratory mass vaccination of poultry. Vaccine. 2007;25(49):8306–8317.

39. Gater ST, Peters KN, Kocsis AG, Dhariwala MO, Anderson DM, Anderson PE. Host stress and immune responses 

during aerosol challenge of Brown Norway rats with Yersinia pestis. Front Cell Infect Microbiol. 2012;2:147.

40. Dybwad M, Skogan G, Blatny JM. Comparative testing and evaluation of nine different air samplers: end-to-end sampling efficiencies as specific performance measurements for bioaerosol applications. Aerosol Sci Tech. 2014;48(3):282–295.

41. Fabian P, McDevitt JJ, Houseman EA, Milton DK. Airborne influenza virus detection with four aerosol samplers using 

molecular and infectivity assays: considerations for a new infectious virus aerosol sampler. Indoor Air. 2009;19(5):433–

441.

42. Dabisch P, Yeager J, Kline J, Klinedinst K, Welsch A, Pitt ML. Comparison of the efficiency of sampling devices for 

aerosolized Burkholderia pseudomallei. Inhal Toxicol. 2012;24(5):247–254.

43. Hood AM. Infectivity of Pasteurella tularensis clouds. J Hyg (Lond). 1961;59:497–504.

44. Faith SA, Smith LP, Swatland AS, Reed DS. Growth conditions and environmental factors impact aerosolization but 

not virulence of Francisella tularensis infection in mice. Front Cell Infect Microbiol. 2012;2:126.

45. Hinds WC. Production of Test Aerosols. Aerosol Technology—Properties, Behavior, and Measurement of Airborne Particles. 

New York, NY: Wiley & Sons; 1999.

46. Hartings JM, Roy CJ. The automated bioaerosol exposure system: preclinical platform development and a respiratory 

dosimetry application with nonhuman primates. J Pharmacol Toxicol Methods. 2004;49(1):39–55.

47. Lippmann M, Yeates DB, Albert RE. Deposition, retention, and clearance of inhaled particles. Br J Ind Med. 1980;37(4):337–

362.

48. Harper GJ, Morton JD. A method for measuring the retained dose in experiments on airborne infection. J Hyg (Lond). 

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49. White JD, Rooney JR, Prickett PA, Derrenbacher EB, Beard CW, Griffith WR. Pathogenesis of experimental respiratory 

tularemia in monkeys. J Infect Dis. 1964;114:277–283.

50. Thomas RJ, Davies C, Nunez A, et al. Particle-size dependent effects in the Balb/c murine model of inhalational melioidosis. Front Cell Infect Microbiol. 2012;2:101.

51. Kim SW, Ramakrishnan MA, Raynor PC, Goyal SM. Effects of humidity and other factors on the generation and 

sampling of a coronavirus aerosol. Aerobiologia. 2007;23(4):239–248.

52. Walter MV, Marthi B, Fieland VP, Ganio LM. Effect of aerosolization on subsequent bacterial survival. Appl Environ 

Microbiol. 1990;56(11):3468–3472.

53. Marthi B, Fieland VP, Walter M, Seidler RJ. Survival of bacteria during aerosolization. Appl Environ Microbiol. 

1990;56(11):3463–3467.

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54. Stephenson EH, Larson EW, Dominik JW. Effect of environmental factors on aerosol-induced Lassa virus infection. J 

Med Virol. 1984;14(4):295–303.

55. Cox CS. Inactivation kinetics of some microorganisms subjected to a variety of stresses. Appl Environ Microbiol. 

1976;31(6):836–846.

56. Schaffer FL, Soergel ME, Straube DC. Survival of airborne influenza virus: effects of propagating host, relative humidity, and composition of spray fluids. Arch Virol. 1976;51(4):263–273.

57. Cox CS, Goldberg LJ. Aerosol survival of Pasteurella tularensis and the influence of relative humidity. Appl Microbiol. 

1972;23(1):1–3.

58. Cox CS. Aerosol survival of Pasteurella tularensis disseminated from the wet and dry states. Appl Microbiol. 

1971;21(3):482–486.

59. Appert J, Raynor PC, Abin M, et al. Influence of suspending liquid, impactor type, and substrate on size-selective 

sampling of MS2 and Adenovirus aerosols. Aerosol Sci Tech. 2012;46(3):249–257.

60. Stewart SL, Grinshpun SA, Willeke K, Terzieva S, Ulevicius V, Donnelly J. Effect of impact stress on microbial recovery 

on an agar surface. Appl Environ Microb. 1995;61(4):1232–1239.

61. Hyde DM, Miller LA, Schelegle ES, et al. Asthma: a comparison of animal models using stereological methods. Eur 

Respir Rev. 2006;15(101):122–135.

62. Patton JS, Byron PR. Inhaling medicines: delivering drugs to the body through the lungs. Nat Rev Drug Discov. 

2007;6(1):67–74.

63. Guyton A. Measurement of the respiratory volumes of laboratory animals. Am J Physiol. 1947;150:70–77.

64. Bide RW, Armour SJ, Yee E. Allometric respiration/body mass data for animals to be used for estimates of inhalation 

toxicity to young adult humans. J Appl Toxicol. 2000;20(4):273–290.

65. Alexander DJ, Collins CJ, Coombs DW, et al. Association of Inhalation Toxicologists (AIT) working party recommendation for standard delivered dose calculation and expression in non-clinical aerosol inhalation toxicology studies 

with pharmaceuticals. Inhal Toxicol. 2008;20(13):1179–1189. 

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Chapter 30

BIOSAFETY

DAVID E. HARBOURT, PhD, SM(NRCM), RBP*; CATHERINE L. WILHELMSEN, DVM, PhD, CBSP†; 

KRISTIE M. YEAKLE, RBP‡; and ROBERT J. HAWLEY, PhD, RBP, CBSP§

INTRODUCTION

Biosafety

Evolution of Biosafety

RISK GROUPS AND BIOSAFETY LEVELS

Risk Groups

How Agents Are Placed in Risk Groups

Biosafety Levels

BIOSAFETY PROGRAM ELEMENTS REQUIRED FOR CONTAINMENT AND 

MAXIMUM CONTAINMENT LABORATORIES

Measures Taken in Research to Protect Laboratory Workers

Documenting Safety Procedures

Assessing Individual Risk

Physical Barriers

Personal Protective Equipment

Medical Surveillance

Protecting the Community and the Environment

Solid and Liquid Waste Inactivation and Disposal

Standard and Special Microbiological Practices

ROLE OF MANAGEMENT IN A BIOSAFETY PROGRAM

Laboratory Safety Audits

Biological Defense Research Program Laboratories

Laboratory Animal Care and Use Program

THE BIOSAFETY PROFESSION

SUMMARY

*Biosafety Officer, Office of Safety, Radiation, and Environment, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort 

Detrick, Maryland 21702; formerly, Fellow, National Biosafety and Biocontainment Training Program, National Institutes of Health, 13 South Drive, 

Bethesda, Maryland

†

Lieutenant Colonel (Retired), Veterinary Corps, US Army; Veterinary Medical Officer, Division of Pathology; formerly, Biosafety Officer, Office of Safety, 

Radiation Protection, and Environmental Health, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland; formerly, Chief, Division of Toxicology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

‡

Alternate Biosafety Officer, Office of Safety, Radiation, and Environment, US Army Medical Research Institute of Infectious Diseases, 1425 Porter 

Street, Fort Detrick, Maryland 21702

§

Consultant, Biological Safety and Security, Alliance Biosciences, 6810 Deerpath Road, Suite 315, Elkridge, Maryland 21075; formerly Senior Advisor, 

Science, Midwest Research Institute, 110 Thomas Johnson Drive, Suite 170, Frederick, Maryland; formerly, Chief, Safety and Radiation Protection, US 

Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

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INTRODUCTION

Biosafety

Biological safety, or “biosafety,” is the application 

of concepts pertaining to risk assessment, engineering technology, personal protective equipment (PPE), 

policies, training, and preventive medicine to promote 

safe laboratory practices, procedures, and the proper 

use of containment equipment and facilities. In biomedicine, laboratory workers apply these tenets to 

prevent laboratory-acquired infections and the release 

of pathogenic organisms into the environment. A biohazard is defined as any microorganism (including, but 

not limited to, bacteria, viruses, fungi, rickettsiae, or 

protozoa); parasite; vector; biological toxin; infectious 

substance; or any naturally occurring, bioengineered, 

or synthesized component of any such microorganism 

or infectious substance that is capable of causing the 

following:

• death, disease, or other biological malfunction 

in humans, animals, plants, or other living 

organisms;

• deleterious alteration of the environment; or

• an adverse impact on commerce or trade 

agreements.

These hazardous agents may be handled safely 

through careful integration of accepted microbiological 

practices and primary and secondary containments of 

potential biohazards.

Primary containment involves placing a barrier at 

the level of the hazard, confining the material to protect laboratory personnel and the immediate laboratory environment by adhering to prudent laboratory 

practices and appropriate use of engineering controls. 

Examples of primary containment include biological safety cabinets (BSCs), ventilated animal cages, 

and associated equipment. Secondary containment 

involves protecting the environment external to the 

laboratory from exposure to infectious or biohazardous materials through facility design and operational 

practices.

Combinations of laboratory practices, containment equipment, and special laboratory design are 

used to achieve different levels of physical containment. The current terminology in the United States 

is “biosafety level” (BSL), though historically, the 

designation “P” was used to indicate the level of 

physical containment (such as P-1 through P-4).1

 BSL 

is used in Biosafety in Microbiological and Biomedical 

Laboratories,

1

 which focuses on protecting laboratory 

employees. Biosafety level may also be abbreviated 

“BL,” which is used in Appendix G of the National 

Institutes of Health (NIH) publication Guidelines for 

Research Involving Recombinant DNA Molecules 

(also known as the NIH Guidelines).2

 However, Appendix G of the NIH Guidelines focuses primarily 

on physical containment involving work with recombinant deoxyribonucleic acid (DNA) molecules 

and organisms and viruses containing recombinant 

DNA molecules. 

There are four levels of biosafety (designated 1 

through 4) that define the parameters of containment 

necessary to protect personnel and the environment.1

BSL-1 is the least restrictive, whereas BSL-4 requires 

a special containment or maximum containment 

laboratory facility. Positive-pressure encapsulating 

suits (PPES), primarily manufactured by ILC Dover 

(ILC Dover LP, Frederica, DE) or Honeywell Safety 

Products (Smithfield, RI), or gas-tight Class III BSC 

systems are used in a maximum containment (BSL4) laboratory environment. Biosafety is not possible 

without proper and extensive training. The principal 

investigator or laboratory supervisor is responsible 

for providing or arranging for appropriate training 

of all personnel within the laboratory to maintain and 

sustain a safe working environment.

Evolution of Biosafety

Steps to limit the spread of infection have been 

practiced in the field of biomedicine since human illness was associated with infectious microorganisms 

and biologically derived toxins. However, Fort Detrick (in Frederick, MD) is considered the birthplace 

(beginning in the 1940s) of modern biosafety as a 

discrete discipline. During the early years of biosafety, 

safer working practices, principles, and engineering 

controls were developed,3,4 as individuals conducting biomedical research commonly became infected 

with the organism being studied. As the hazard of 

working with organisms increased, so did the need to 

protect laboratory personnel conducting the research. 

Contributions to the field of biosafety were a direct 

result of the innovations and extensive experiences 

of Fort Detrick personnel who worked with a variety 

of infectious microorganisms and biological toxins. 

Dr Arnold Wedum, director of industrial health and 

safety at Fort Detrick—and regarded by many as the 

father of the US biosafety profession—promoted the 

attitude that biosafety should be an integral part of 

biomedical research.5

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Biosafety

To enhance worker safety and environmental protection, Wedum4

 promoted use of the following:

• class III gas-tight BSC;

• noninfectious microorganisms in recombinant 

DNA research;

• P-4 (today’s BSL-4) principles, practices, and 

PPES facilities when working with potentially 

aerosol-transmitted zoonotic microorganisms 

(eg, those causing tularemia and Q fever if a 

class III cabinet system was not available); and

• vaccination or immunization of laboratory 

workers.

Another safety enhancement was demonstrating 

and publicizing the importance of prohibiting mouth 

pipetting for fluid transfers involving hazardous material.6,7 Dr Emmett Barkley8

 reiterated the hazard of 

oral pipetting, which should not be practiced in the 

laboratory. Barkley was chief of the Safety Division 

of the National Cancer Institute (Bethesda, MD) and 

subsequently director of research safety at NIH when 

the NIH Guidelines were developed and adopted. He 

was instrumental in developing physical containment 

parameters for recombinant DNA research.9

Critical to the advancement of modern biosafety was 

the development of air filtration technology. During 

the early 1940s, the US Army Chemical Warfare Service 

Laboratories (Edgewood, MD) studied the composition of filter paper captured from German gas mask 

canisters in search of better smoke filters. These early 

studies resulted in the design of collective protection 

filter units for use at the particulate-removal stage by a 

combined chemical, biological, and radiological purification unit of the US armed services. In the late 1940s, 

the Atomic Energy Commission (precursor of the 

Nuclear Regulatory Commission) adopted this type 

of filter to confine airborne radioactive particles in the 

exhaust ventilation systems of experimental reactors 

and in other areas of nuclear research. Subsequently, 

Arthur D Little Company, Incorporated (Boston, MA) 

and the US Naval Research Laboratory (Washington, 

DC) developed a prototype glass-fiber filter paper. 

Eventually, thin, corrugated, aluminum-alloy separators replaced the original asbestos, thermoplastics, and 

resin-treated papers. Throughout this development 

period, military specifications were developed and 

implemented to ensure the safe operating and optimal conditions of filters,10 ultimately leading to the 

production of high-efficiency particulate air (HEPA) 

filters, which are used today in a variety of engineering 

controls, as well as in laboratory heating, ventilation, 

and air conditioning systems.

HEPA filters are constructed of paper-thin sheets 

of borosilicate medium that are pleated to increase 

their surface area. The borosilicate sheets are tightly 

pleated over aluminum separators for added stability and affixed to a frame.10 A BSC first developed 

in 1964 for a pharmaceutical company used HEPA 

filter technology to provide clean air in the work area 

and containment as the primary barrier placed at the 

source of hazardous powders. Subsequent research 

led to the development of a class II, type A BSC that 

was delivered to the National Cancer Institute by 

the Baker Company (Sanford, ME).11 The National 

Cancer Institute also developed a specification for the 

first class II, type B console BSC. HEPA filters have 

been proven to be effective, economical, and reliable 

devices for removing radioactive and nonradioactive particulate aerosols at a high rate of collection 

frequency.10

Operation and retention efficiency of HEPA filters 

has been documented. Three mechanisms account 

for the collection (retention) of particles within HEPA 

filters:

1. Small particles ranging from 0.01 to 0.2 μm 

in diameter are collected in a HEPA filter by 

diffusion and are retained at an efficiency 

approaching 100%.

2. Particles in the respirable range (those of 

a size that may be inhaled and retained in 

the lungs, 0.5 to 5.0 μm in diameter) are retained in a HEPA filter by a combination of 

impaction and interception at an efficiency 

approaching 100%.

3. Particles with an intermediate size range 

(between 0.2 and 0.5 μm in diameter) are 

retained by a combination of diffusion and 

impaction.

The HEPA filter is least efficient at retaining particles with a diameter of 0.3 μm, with a minimum 

collection efficiency of 99.97%. Hence, a standard test 

of HEPA filter efficiency uses a generated aerosol of 

particles that are 0.3 μm in diameter; to pass the test, 

the HEPA filter must retain 99.97% of the particles.12

All the air exhausted from BSCs, within which 

infectious materials must be manipulated, is directed 

through a HEPA filter before recirculation to a laboratory room or discharge to the outside environment 

through the building exhaust system. Therefore, in addition to adherence to rigorous work practice controls, 

HEPA filtration of laboratory exhaust air provides an 

extra margin of safety for workers, the laboratory areas, 

and the outside environment.

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Medical Aspects of Biological Warfare 

RISK GROUPS AND BIOSAFETY LEVELS

risk, low community risk) includes pathogens that 

usually cause serious human or animal disease, but 

do not ordinarily spread from one infected individual 

to another efficiently. Effective treatment and preventive measures are likely available. An example is the 

causative agent of tularemia, Francisella tularensis, in 

humans and animals. Risk group 4 (high individual 

and community risk) pathogens usually cause serious human or animal disease and can be readily 

transmitted from one individual to another, either 

directly or indirectly. Effective treatment and preventive measures are not normally available. Examples 

include Variola virus, Ebola virus, Lassa fever virus, 

and Marburg fever virus. 

Assigning Agents to Risk Groups

It is important to understand how microorganisms 

are placed in risk groups and how that knowledge is 

used to develop procedures and physical infrastructure to contain these agents. The following criteria 

must be considered to assess risk while working in 

a laboratory or animal environment with a specific 

microorganism.

• Number of past laboratory infections. The 

most frequent laboratory-associated infections in humans are caused by the Brucella 

species. Extra caution must be taken when 

working with this agent because of its low 

TABLE 30-1

RELATIONSHIPS OF RISK GROUPS, BIOSAFETY LEVELS, PRACTICES, AND EQUIPMENT

Risk 

Group

Biosafety 

Level

Laboratory Type Laboratory Practices Safety Equipment

1 Basic: BSL-1 Basic teaching; researchGood microbiological techniquesNone; open bench work

2 Basic: BSL-2 Primary health 

services; diagnostic 

services; research

Universal precautions plus protective clothing and biohazard 

sign

Open bench plus BSC for potential 

aerosols

3 Containment: 

BSL-3

Special diagnostic 

services; research

As level 2 plus controlled access, 

double door entry, special clothing, and directional airflow 

BSC and/or other primary devices for 

all activities

4 Maximum 

containment: 

BSL-4

Dangerous pathogens; 

research

As level 3 plus airlock entry, 

shower exit and special waste 

disposal

Class III BSC, or positive-pressure protective suits in conjunction with class II 

BSCs, double-door autoclave (through 

the wall), and filtered air

BSC: biological safety cabinet

BSL: biosafety level

Risk Groups

Agents infectious to humans, including those used 

in research, are placed into risk groups based on the 

danger they pose to human health. Risk group assignment helps researchers determine the containment 

condition (or BSL) appropriate for handling a particular agent (Table 30-1). Multiple schemes for assigning 

risk groups have been developed. The NIH Guidelines, 

Health Canada (Ottawa, Ontario, Canada),13 other nations, and the World Health Organization (Geneva, 

Switzerland)14 all have risk group paradigms. The 

World Health Organization has categorized infectious 

agents and biological toxins into four risk groups. 

These risk groups relate to, but do not equate to, the 

BSLs of laboratories designed to work with organisms 

in each risk group.14 Risk group 1 (no or low individual 

and community risk) comprises microorganisms including Escherichia coli K12 and Candida albicans that are 

unlikely to cause human or animal disease in healthy 

adult individuals. Risk group 2 (moderate individual 

risk, low community risk) includes pathogens that can 

cause human or animal disease, but are unlikely to be 

serious hazards to laboratory workers, the community, 

livestock, or the environment. Laboratory exposures 

may cause serious infection, but effective treatment 

and preventive measures are available, and the risk 

of infection spreading is limited. An example is a 

causative agent of viral hepatitis, Hepatitis B virus, in 

humans and animals. Risk group 3 (high individual 

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Biosafety

infectious dose for humans. About 10 to 100 

organisms can cause an infection in a susceptible human host.15

• Natural mortality rate. The natural mortality 

or case-fatality rate of diseases varies widely 

(Table 30-2).15

• Human infectious dose. Working with an 

organism having a low infectious dose for 

humans will place the laboratory worker at a 

greater risk than working with an organism 

having a higher infectious dose. The infectious dose of organisms for humans varies 

and is also dependent on the immunological 

competency of the host (Table 30-3). Although 

the literature contains information about the 

potential infectious dose for humans as extrapolated from animal data (see Table 30-3), 

an attempt to provide quantitative human 

infectious doses is not possible.16

• Efficacy of vaccination and treatment (if 

available). Vaccines are available for some 

of the agents studied within the laboratory. 

Receiving a vaccination must be based on a 

risk assessment. Only those individuals who 

are considered at risk should be offered the 

vaccination. However, the potential risk of 

the adverse effects from the vaccination might 

outweigh the risk of acquiring an infection. 

In addition, a vaccination might not provide 

100% protection; an overwhelming infectious 

dose can overcome the protective capacity of 

a vaccination. Therefore, a vaccination should 

be considered only as an adjunct to safety, 

not as a substitute for safety and prudent 

practices. Treatment (chemoprophylaxis) in 

the form of antibiotic therapy may also be 

available to treat illnesses caused by many 

of the microorganisms being manipulated in 

the laboratory, specifically by the bacterial 

and rickettsial agents. It is necessary to determine the antibiotic sensitivity and resistance 

pattern (antibiogram) of the agent under 

investigation. The rationale is that treatment 

will be known in advance if an inadvertent 

laboratory exposure occurs. Treatment for 

exposure to a virus might be problematic 

because only symptomatic treatment may be 

available. There are few available antiviral 

agents that may be effective for postexposure 

prophylaxis. Specific antiviral agents include 

the following:

° Rabies: rabies immune globulin for passive 

therapy, followed by the human diploid cell 

rabies vaccine or rabies vaccine, adsorbed 

for active vaccination.

° Macacine herpesvirus 1 (formerly Cercopithecine herpesvirus; B virus): valacyclovir 

hydrochloride (VALTREX; GlaxoSmithKline, Research Triangle Park, NC). 

° Arenaviridae and bunyaviridae (including 

the viruses that cause Lassa fever, Argentine hemorrhagic fever, and Crimean-Congo hemorrhagic fever): ribavirin. This material can be used under an investigational 

new drug protocol (in the United States) 

TABLE 30-2

CASE-FATALITY RATE BY DISEASE

Disease (Untreated) Organism Case-Fatality 

Rate

Plague, bubonic Yersinia pestis 50%–60%

Cholera Vibrio cholerae 50% or more

Tularemia, pulmonaryFrancisella tularensis 30%–60%

Anthrax, cutaneous Bacillus anthracis 5%–20%

Tularemia, typhoidal Francisella tularensis 5%–15%

Brucellosis Brucella species 2% or less

Q fever Coxiella burnetii 1%–2.4%

TABLE 30-3

HUMAN INFECTIOUS DOSE BY ORGANISM

Organism Infectious Dose

Route of 

Exposure

Vibrio cholerae 108 Ingestion1

Yersinia pestis 100–20,000 Inhalation2

Bacillus anthracis ~ 1,300 Inhalation3

Brucella species 10–500 Inhalation2 

Francisella tularensis 10 Inhalation4

Coxiella burnetii 1 Inhalation5 

Data sources: (1) Sack DA, Sack RB, Nair GB, Siddique AK. Cholera. 

Lancet. 2004;363:223–233. (2) Franz DR, Jahrling PB, Friedlander AM, 

et al. Clinical recognition and management of patients exposed to 

biological warfare agents. JAMA. 1997;278:399–411. (3) Dull PM, 

Wilson KE, Kournikakis B, et al. Bacillus anthracis aerosolization associated with a contaminated mail sorting machine. Emerg Infect Dis. 

2002;8:1044–1047. (4) Jones RM, Nicas M, Hubbard A, Sylvester MD, 

Reingold A. The infectious dose of Francisella tularensis (tularemia). 

Appl Biosafety. 2005;10:227–239. (5) Jones RM, Nicas N, Hubbard A, 

Reingold A. The infectious dose of Coxiella burnetti (Q-fever). Appl 

Biosafety. 2006;11:32–41.

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Medical Aspects of Biological Warfare 

only for empirical treatment of hemorrhagic fever virus patients while awaiting 

identification of the etiological agent.

° Poxviridae (including the viruses that cause 

smallpox and monkeypox): in addition to 

the Dryvax (Wyeth Inc, Philadelphia, PA) 

vaccine variant derived from Vero cells, 

ACAM2000 (Acambis, Canton, MA), two 

small molecule poxvirus inhibitors are currently in clinical trials: the Cidofovir lipid 

conjugate CMX001 (Chimerix Pharmaceuticals, Durham, NC) and ST-246 (Tecovirimat; 

SIGA Technologies, New York, NY).17–19

° Retroviridae (including human immunodeficiency virus): the latest highly active 

antiretroviral therapy recommendations 

for postexposure prophylaxis are available 

through the US Public Health Service.20 

° There are currently small-molecule therapeutics under development for treating potential filoviradae (including Marburg and 

Ebola viruses) infection, but no products 

have yet been approved by the US Food 

and Drug Administration for clinical use.21

° Additional vaccines and antiviral treatments for flaviviradae and togaviradae 

infections are in varying stages of development or clinical trials. A US Food and 

Drug Administration licensed vaccine is 

available as a preventative treatment or 

prophylaxis against such agents as Japanese encephalitis virus and Yellow Fever 

virus, while other vaccines remain under 

investigational new drug status for Venezuelan equine encephalitis, Dengue fever, 

and tick-borne encephalitis virus.22–25

• Extent to which infected animals transmit 

the disease. This discussion involves the zoonotic diseases or diseases that can be transmitted from animals to humans. These diseases 

include, but are not limited to, the following:

° those transmitted directly from animals to 

humans (eg, rabies);

° those that can be acquired indirectly by 

humans through ingestion, inhalation, or 

contact with infected animal products, soil, 

water, or other environmental surfaces that 

have been contaminated with animal waste 

or a dead animal (eg, anthrax); and

° a disease that has an animal reservoir, but 

requires a mosquito or other arthropod to 

transmit the disease to humans (eg, St Louis 

encephalitis virus and Rocky Mountain 

spotted fever).

 Exposure risks in laboratories that use animals 

may differ from the exposure risks encountered in microbiology laboratories. Within 

microbiology laboratories, hazardous conditions may arise from human activities or from 

equipment within the laboratory. In animal 

facilities, the animals themselves may create 

hazards for the laboratory workers via:

° generation of infectious aerosols;

° animal bites or scratches to the person 

handling the animal; and

° shedding of infectious known or unknown 

zoonotic agents in animal secretions and 

excretions, contaminating the animal holding room, cage, bedding, equipment, or 

other fomites. For example, in addition to 

usual activities in the laboratory, handling 

materials contaminated with hantaviruses 

is a concern because viruses are spread 

as aerosols or dusts from rodent urine, 

droppings, or by direct contact with saliva 

through cuts or mucous membranes.

• Stability of the agent. An agent’s (microorganism’s) stability to environmental conditions and susceptibility or resistance to disinfectants results from its internal and external 

chemical composition. For instance, spores 

of the genus Bacillus are resistant to adverse 

environmental conditions and disinfectants in 

part because of the presence of dipicolinic acid 

(pyridine-2,6-dicarboxylic acid) in their spore 

coat. Dipicolinic acid plays a significant role in 

the survival of Bacillus spores exposed to wet 

heat and ultraviolet radiation.26 Many viruses 

and bacteria are sensitive to environmental 

conditions and disinfectants because of the 

high lipid content in their outermost layer.

Biosafety Levels

BSLs are guidelines that have evolved to protect 

laboratory workers from biological hazards. They 

do not take into account additional hazards found 

within the laboratory, including chemical, physical, 

or radiological hazards. These guidelines are based 

on data from laboratory-acquired infections and 

on an understanding of the risks associated with 

various manipulations of many agents transmissible by different routes. These guidelines operate 

on the premise that safe work sites result from a 

combination of engineering controls, management 

policies, work practices and procedures, and, occasionally, medical interventions. The different 

BSLs developed for microbiological and biomedical 

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laboratories provide increasing levels of personnel 

and environmental protection.1 BSL descriptions 

comprise a combination of facilities, equipment, and 

procedures used to handle infectious agents to protect the laboratory worker, the environment, and the 

community. This combination is proportional to the 

potential hazard level (risk group) of a given infectious agent. Equipment serving as primary barriers 

includes but is not limited to BSCs, centrifuge safety 

cups, and containment animal caging. Facilities also 

consist of secondary barriers, such as self-closing/

locking doors, hand-washing sinks, and unidirectional airflow from the least hazardous areas to the 

potentially most hazardous areas. Procedures consist 

of standard and special microbiological practices. 

Finally, PPE includes dedicated laboratory clothing 

and respiratory protection.

There are four BSLs described in Biosafety in Microbiological and Biomedical Laboratories.

1

 These levels range 

from a basic level (BSL-1) through maximum containment (BSL-4). BSL-1 consists of facilities, equipment, 

and procedures suitable for work, with infectious 

agents of no known or of minimal potential hazard 

to healthy laboratory personnel. BSL-1 represents 

a basic level of containment that relies on standard 

microbiological practices, with no special primary or 

secondary barriers recommended, other than a sink 

for hand washing.

BSL-2 consists of facilities, equipment, and procedures applicable to clinical, diagnostic, or teaching 

laboratories; suitable for work involving indigenous 

moderate-risk infectious agents present in the community; and associated with human disease of varying 

severity for which vaccines or therapeutics are usually 

available.1

Primary hazards to personnel working with these 

agents are accidental percutaneous or mucous membrane exposures and ingestion of infectious materials. 

(Inhalation exposure to agents at the BSL-2 level is 

uncommon; the main risk with aerosol generation 

is potential contamination of the laboratory with 

infectious agents that can result in exposure through 

breaks in the skin, ingestion, or injury. Therefore, all 

aerosol-generating procedures should be performed 

in a BSC or other primary containment equipment, 

but respiratory protection to mitigate aerosol exposure is rarely recommended unless there are other 

circumstances involved.) BSL-2 differs from BSL-1 

in five ways:

1. Laboratory personnel receive specific training in handling pathogenic agents.

2. Scientists experienced in handling specific 

agents direct the laboratory.

3. Access to the laboratory is limited when work 

is in progress.

4. A laboratory-specific biosafety manual is 

prepared or adopted.

5. Procedures capable of generating potentially infectious aerosols are conducted within 

class I or class II BSCs or other primary 

containment equipment. Personnel receive 

specific training in the proper use of primary containment equipment and adhere 

strictly to recommended microbiological 

practices.

BSL-3 includes facilities, equipment, and procedures applicable to clinical, diagnostic, research, 

or production facilities in which work is done with 

indigenous or exotic agents that may cause serious 

or potentially lethal disease, especially after inhalation exposure, and vaccines or therapeutics may be 

available.1 Hazards to personnel working with these 

agents include autoinoculation, ingestion, and exposure to infectious aerosols. BSL-3 differs from BSL-2 

in four ways:

1. At BSL-3, laboratory personnel receive more 

extensive training in handling potentially 

lethal pathogenic agents than the degree of 

training received at BSL-2.

2. All manipulations of infectious or toxin-containing materials are conducted within class 

II or class III BSCs or other primary containment equipment. Personnel are trained to use 

this safety equipment properly.

3. The laboratory has special engineering and 

design features that include access zones with 

two self-closing and locking doors, sealed 

penetrations or penetrations capable of being 

sealed, and directional airflow (from areas of 

low-hazard potential to areas of high-hazard 

potential). Laboratory personnel are trained 

to understand these special design features.

4. Only the laboratory director can approve a 

modification of these BSL-3 recommendations.

BSL-4 comprises facilities, equipment, and procedures required for work with dangerous and exotic 

agents that pose a high individual risk of life-threatening disease transmitted by the inhalation route 

and for which a vaccine or therapy are not usually 

available.1 Hazards to personnel working with these 

agents include autoinoculation, mucous membrane 

or broken skin exposure to infectious droplets, and 

exposure to infectious aerosols. BSL-4 differs from 

BSL-3 in six ways:

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Medical Aspects of Biological Warfare 

1. Laboratory personnel receive specific and 

thorough training to handle extremely hazardous infectious agents. Their supervisors 

are competent scientists who are trained and 

experienced in working with these agents.

2. Laboratory personnel understand the function of primary and secondary barriers and 

laboratory design features. They are trained 

in standard and special microbiological practices and the proper use of primary containment equipment.

3. The laboratory director strictly controls access to the laboratory.

4. The laboratory is in a controlled area within 

a building, completely isolated from all other 

areas of the building, or is in a separate building.

5. All activities involving agent manipulation 

within the work areas of the laboratory are 

conducted within a class III BSC, or within a 

class I or class II BSC used in conjunction with 

a one-piece, positive-pressure protective suit 

that is ventilated by a life-support system.

6. The BSL-4 laboratory, or maximum containment laboratory, has special engineering and 

design features to prevent dissemination of 

microorganisms to the environment.

BIOSAFETY PROGRAM ELEMENTS REQUIRED FOR CONTAINMENT AND MAXIMUM 

CONTAINMENT LABORATORIES

Measures Taken in Research to Protect Laboratory 

Workers

Although BSL-3 practices, safety equipment, and 

facility design and construction are applicable to clinical, 

diagnostic, teaching, research, and production (largescale) facilities, where work is done with indigenous or 

exotic agents with the potential for respiratory transmission and lethal infection, this section will emphasize 

BSL-3 research laboratories. BSL-4 practices, safety 

equipment, and facility design and construction apply 

to work in a reference diagnostic or research setting with 

dangerous and exotic agents that pose a high individual 

risk of life threatening disease. These agents may be 

transmitted by aerosol, and there may be no available 

vaccine or therapy. BSL-4 research facilities, both class 

III BSC laboratories and protective-suit laboratories, 

will be covered in this section. Due to the Biological 

and Toxin Weapons Convention of 1972, legitimate 

production (large-scale or greater than 10 L of culture) 

BSL-4 facilities do not currently exist (most BSL-4 operations are small scale only because of the working 

conditions inherent to a BSL-4 suit or cabinet laboratory; 

large-scale facilities would be used only in very special 

circumstances and do not exist in the United States).

Documenting Safety Procedures

A laboratory’s biological safety program manual 

is a laboratory-specific guide that should include 

safety standards and standing operating procedures 

(SOPs), guidelines, and documents for the containment laboratory (see the Lawrence Berkeley National 

Laboratory’s Biological Safety Program Manual27 as 

an example). These safety SOPs identify the special 

hazards of the laboratory and the procedures to abate 

or mitigate the associated risk. SOPs or documents 

specify the following:

• laboratory entry and exit in detail;

• proper use of laboratory-specific safety equipment (eg, BSCs, sterilizers, pass boxes, and 

dunk tanks);

• decontamination procedures for the specific 

laboratory;

• maintenance of laboratory safety and maintenance-related records (access logs, drain flush 

logs, emergency deluge shower, and eyewash 

periodic test logs);

• floor plan with hand-wash sinks and all other 

safety features annotated;

• emergency and routine communication procedures for the specific laboratory; and

• laboratory and agent-specific training for all 

laboratory personnel.

A compilation of existing SOPs, specifying how 

a laboratory worker would access the SOPs (online, 

paper copy in a binder, or both) is suggested. To meet 

the specific training and proficiency requirement, 

trainers should provide documentation for standard 

safety and laboratory essential training, with specific 

additions for the laboratory that cover orientation for 

workers new to the laboratory and laboratory-unique 

procedures and operations. Trainers should consider 

including in the manual material safety data sheets 

for the chemicals used in the laboratory. Material 

safety data sheets for chemicals can be obtained from 

vendors’ websites or from the institutional chemical 

hygiene officer.

Assessing Individual Risk

For each person working in a BSL-3 and BSL-4 

research laboratory, a supervisor conducts a detailed, 

thorough, individually tailored job hazard analysis or 

workplace hazard analysis (risk assessment). During 

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Biosafety

this analysis, each task the individual intends to perform within containment is evaluated in terms of its inherent risk (as described in Risk Groups and Biosafety 

Levels, above). Each task is considered in terms of a 

potential laboratory exposure to the infectious agent 

(and its associated toxins for toxin-producing [toxigenic] agents). Considerations include use of sharp 

instruments and animals that could potentially result 

in puncture injuries, operations that may generate 

infectious aerosols, and direct handling of infectious 

agents versus observing (auditing) others working 

with biological materials. The hazards, once identified, 

are mitigated, preferably by isolating operations that 

pose a risk within primary and secondary containment 

devices (barriers), by substituting unbreakable plastic 

laboratory vessels for glassware and blunt instruments 

for sharp instruments, and by chemically or physically 

immobilizing animals to prevent or reduce the risk 

of sudden or unpredictable behavior leading to bites 

and scratches. Once the risk assessment is written, this 

document is approved by the second-line supervisor 

and reviewed by both the biological safety officer and 

the occupational health physician for accuracy and 

completeness.

The preferred means to mitigate risk is by using 

engineering controls (eg, BSCs, chemical fume hoods, 

sealed centrifuge rotors, and safety cups) and partial 

containment caging for animals (eg, micro isolator 

cages; ventilated cage racks; and ventilated, negativepressure, HEPA-filtered rigid cubicles or flexible 

isolators). Where the hazard cannot be eliminated by 

physical means, it can be managed by administrative 

controls that provide specific training on procedures. 

Examples of such procedures include disposing used 

injection needles without recapping them or using an 

approved, one-handed practice to recap needles, either 

the one-handed scoop technique or a one-handed 

technique using a recapping device (an engineering 

control that holds the cap in place). Specific training 

is provided to encourage workers to use safe methods 

and operations to prevent aerosol generation, skin and 

mucosal contact with infectious agents, and handling 

of sharps where they cannot be eliminated.

If the hazard cannot be eliminated by engineering or 

administrative controls, it may be mitigated by using 

PPE to protect against contact, as well as mucosal and 

respiratory exposure. Vaccinations, when available and 

where medically indicated, may serve as an adjunct to 

PPE, but never as a substitution for PPE. Once all the 

tasks an individual will perform have been assessed 

and all the infectious and toxic agents the individual 

will work with have been identified, the tasks and 

agents are recorded in a document that the worker 

and the supervisor prepare together. The mitigating 

controls are then chosen with input from safety professionals and occupational health and medical staff 

to form a collection of primary barriers, approved 

practices, PPE, and vaccinations. Based on an individual worker’s current educational and experience 

levels and state of health, certain controls may not be 

feasible. High-risk tasks may have to be avoided, on 

a spectrum that may range from observing high-risk 

tasks (in-vivo work, such as manipulations of exposed 

animals) and performing low-risk tasks (in-vitro work 

with infected cell cultures in a BSC), to the extreme 

that the individual may not be granted access to the 

containment laboratory.

Physical Barriers

Primary barriers include class II and class III BSCs, 

PPESs, and containment animal housing. Class II BSCs 

are open-fronted cabinets with HEPA filtered laminar 

airflow. Class II type A1 and type A2 cabinets may 

exhaust HEPA-filtered air back into the laboratory 

or may exhaust the air to the environment through 

an exhaust canopy. Class II type B1 cabinets have 

HEPA-filtered down-flow air composed of uncontaminated, recirculated in-flow air (30%) and exhaust most 

(70%) of the contaminated air through a dedicated 

duct with a HEPA filter to the atmosphere. Class II 

type B2 (total exhaust) cabinets exhaust all in-flow 

and down-flow air to the atmosphere after passing 

through a HEPA filter located in a dedicated exhaust 

duct. To verify proper operation, all class II BSCs must 

be field certified in accordance with National Sanitation Foundation International Standard/American 

National Standard for Biosafety Cabinetry Class II 

(Laminar Flow) Biosafety Cabinetry Standard 4928 on 

initial installation, at least annually thereafter, or after 

every major repair or relocation of the cabinet.1,29,30 It 

is recommended that accredited certifiers be engaged 

for provision of class II BSC certification and repair 

service. Class II cabinets may be used in BSL-3 laboratories, when supplemented by use of PPE (gloves, 

gowns, and respiratory protection when warranted by 

a risk assessment), and may be used in BSL-4 laboratories in conjunction with wearing a one-piece, positivepressure, ventilated suit with a life-support system, an 

in-line HEPA or high-purity filter, and supplied with 

grade D breathing air.31

When working within a class II BSC, the equipment 

and materials are arranged in a clean-to-dirty layout, 

with clean materials in the center of the workspace, 

potentially contaminated materials at one end of 

the workspace within the cabinet, and potentially 

contaminated waste materials at the other end of the 

workspace.32 Class III cabinets are totally enclosed, 

ventilated, gas-tight cabinets. They provide the highest level of product, personal, and environmental 

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Medical Aspects of Biological Warfare 

protection against respiratory exposure to infectious or 

toxic aerosols and are most suitable for work in BSL3 and BSL-4 laboratories. Operations are conducted 

using shoulder-length gloves or half-suits connected 

to the cabinets. Air is supplied to the class III cabinet 

through a HEPA filter, and air exhausted from the 

cabinet to the atmosphere passes through two HEPA 

filters in series (or one HEPA filter and an exhaust air 

incinerator). Materials are removed from the cabinet 

by passing them through an interlocked, double-door 

sterilizer or through a chemical dunk tank filled with 

an appropriate disinfectant for the infectious agents 

or toxins in use at BSL-4, but some class III cabinets 

interlock with a class II BSC for removal at BSL-3. 

Several class III cabinets, housing a refrigerator, cell 

culture incubator, centrifuge, or aerosol-generating 

equipment, may be connected in a cabinet line as an 

integrated system for use in a BSL-3 laboratory or 

in a BSL-4 cabinet laboratory. A complete change of 

clothing is required for workers, including a dedicated laboratory scrub suit, jumpsuit or gown, shoes, 

and examination gloves for hand protection in case 

of a puncture or if a pinhole develops in the cabinet 

shoulder-length gloves, or half-suits.

Primary barriers for animal housing include the 

following: (a) micro isolator cages with filter tops for 

rodents; (b) ventilated rodent cage racks; (c) ventilated, 

negative-pressure, HEPA-filtered cubicles; (d) ventilated, negative-pressure, HEPA-filtered flexible film 

isolators; and (e) rigid, ventilated, negative-pressure, 

HEPA-filtered isolation cages.32 Rigid, ventilated, 

negative-pressure, HEPA-filtered, mobile animal 

transport carts have been developed at US Army 

Medical Research Institute of Infectious Diseases to 

isolate animals during transfer between containment 

animal facilities.33 Other primary containment devices 

include ventilated, filtered enclosures for continuous 

flow centrifuges and use of sealed rotors and centrifuge safety cups in conventional centrifuges. Primary 

containment devices used in necropsy rooms include 

downdraft necropsy tables, specially designed class II 

cabinets for conducting necropsies, and HEPA-filtered 

vacuum shrouds for oscillating bone saws.

Personal Protective Equipment

In BSL-3 containment, laboratory workers wear 

protective clothing, such as solid-front or wraparound 

gowns, scrub suits, or coveralls. This protective clothing is not to be worn outside the laboratory. To aid 

in enforcement of this rule, laboratory clothing may 

be color-coded, so that it can be readily identified if 

worn outside the laboratory. Scrub suits are typically 

two-piece ensembles composed of trousers and tunics. Tunics with long sleeves that terminate in knit 

wrist cuffs aid in donning protective gloves. Gloves 

are drawn over the cuffs and may be secured in place 

using tape. Long-sleeved tunics are favored over shortsleeved tunics because long sleeves with gloves taped 

to the sleeves can provide a physical barrier to protect 

the skin of the wrists and arms from potential exposure to infectious agents, including bacterial spores.33

Disposable clothing should not be reused. Reusable 

clothing is decontaminated, usually by autoclaving, 

before being laundered to prevent an exposure hazard to laundry workers.30,34 Clothing is changed when 

overtly contaminated or after every work session, depending on facility policy. The wearing of dedicated 

laboratory shoes or safety shoes may be required in 

BSL-3 facilities. Otherwise, disposable shoe covers 

should be worn. Wearing dedicated laboratory socks 

provides comfort to the feet and extra skin protection 

to exposed ankles if trousers are not long enough to 

cover the legs fully. Not all biocontainment facilities 

in the United States require workers to have a change 

of clothes. If a clothing change is required, dedicated 

socks and shoes are indicated. In the absence of a 

clothing change requirement, the dedicated shoes and 

socks may not be used in lieu of shoe covers, coveralls, 

or no additional PPE, depending on a risk assessment. 

Protective gloves must be worn when handling 

infectious materials, animals, and contaminated material. Gloves are selected to meet the needs of the risk 

assessment. Nitrile or latex gloves may be appropriate if they provide the worker with protection from 

the infectious agent being handled. However, gloves 

manufactured from other materials (eg, neoprene 

[DuPont Performance Elastomers LLC, Wilmington, 

DE], butyl rubber, and Hypalon [DuPont Performance 

Elastomers LLC]) may be indicated to protect against 

exposure to other contaminated materials, such as 

toxins, organic solvents, and caustics, or to serve as an 

alternative to personnel who may have allergic reactions or sensitivities to latex or nitrile. Gloves should 

be changed frequently, followed by thorough hand 

washing. Disposable gloves should never be reused. To 

ensure protection when working with highly hazardous materials, double gloving (wearing two pairs of 

gloves) should be practiced, with the inner glove taped 

to the wrist cuff to minimize potential contamination. 

If the outer glove is punctured or torn, the protective 

skin barrier should still be maintained by the inner 

glove if it was not breached (provision of redundant 

protection). If working with contaminated sharps (eg, 

needles, scalpels, glass slides, capillary tubes, pipettes) 

or with infected animals that may bite or scratch, laboratory workers should consider wearing cut-resistant 

over-gloves (eg, Kevlar [EI Du Pont de Nemours and 

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Biosafety

Company, Wilmington, DE]; armored, stainless-steel 

mesh; or leather gloves) for additional protection.35 If 

working with materials where there is a splash hazard, 

the use of safety goggles or face shields and head covers (bonnets, caps, hood) may be indicated, unless the 

individuals are using a full-face respirator, such as a 

powered air-purifying respirator (PAPR).

When entering rooms housing infected animals, 

additional PPE (wraparound gowns or Tyvek [DuPont Tyvek, Richmond, VA] coveralls, foot covers or 

boots, head covers, eye and respiratory protection, 

etc) is required. These PPE requirements will be 

indicated on the warning sign posted on the door 

of the animal’s cage. Respiratory protection is provided by using properly fitted respirators approved 

by the National Institute of Occupational Safety 

and Health (NIOSH).36 Surgical masks or nuisance 

dust masks do not meet the NIOSH definition of a 

respirator. NIOSH-approved respiratory protection 

systems are commonly used in BSL-3 laboratories 

and animal rooms when the respiratory hazard cannot be completely engineered out through the use 

of primary containment devices. Respirators used 

to filter particulates are classified into three series, 

corresponding to resistance to oil mist particles: 

(1) N, or least resistant, (2) P, or partially resistant, 

and (3) R, or resistant. They are further differentiated based on their efficiency at removal of 0.3 

μm aerosol particles, similar to HEPA filters (95%, 

99%, and 99.97% or –100).37 Useful and comfortable 

negative-pressure respirators include disposable 

N-100 filtering face pieces with integral exhalation 

valves and tight-fitting, half-face, negative-pressure 

respirators fitted with N-100 particulate filters. These 

respirators have an assigned protection factor of 10, 

meaning there are 10-fold fewer particulates at the 

breathing zone inside the respirator than outside 

the respirator, providing the respirator is properly 

fitted and worn. A properly fitted and worn full-face 

piece, negative-pressure respirator has an assigned 

protection factor of 50 to 100 and also provides eye 

protection. All users of respirators must be enrolled in 

a respiratory protection program in accordance with 

the Occupational Safety and Health Administration 

(OSHA) Respiratory Protection Standard.31 Users of 

tight-fitting respirators must be fit tested annually or 

when significant physical changes occur (weight gain 

or loss) using an approved qualitative or quantitative 

fit test. Wearers of tight-fitting respirators must not 

have facial hair that could interfere with the fit of the 

respirator, nor should eyeglasses interfere with the 

tight seal. Users of full-face, tight-fitting respirators 

who wear eyeglasses will need special optical inserts 

that may be worn inside the respirator face piece. 

Individuals fit tested for respirators must ensure that 

they only use respirators that they have been trained 

and certified to use during annual fit testing. 

When working in a BSL-3 environment, such as 

a room housing infected animals in open cages or a 

necropsy room equipped with a downdraft table and 

an oscillating bone saw, greater respiratory protection 

might be needed. A NIOSH-approved PAPR with a 

loose-fitting hood or a tight-fitting full-face piece is 

often used and provides an assigned protection factor of 1,000. Benefits of wearing a loose-fitting hood 

include comfort, no requirement for fit testing, and 

amenability to use by individuals with facial hair. 

Reusable turbo blowers for PAPRs are powered by 

rechargeable batteries. The blowers may be equipped 

with N-100 particulate filters or with combination 

cartridges that incorporate a particulate filter with 

activated charcoal or other chemical absorbent for use 

in atmospheres of between 19.5% and 23.5% oxygen 

that have contaminated particulates and low levels 

of organic or other specified chemical vapors. The 

airflow in cubic feet per minute, with cartridges installed, must be checked with a flow gauge before each 

work session. Because there are no OSHA standards 

or end-of-service life indicators for particulate filters 

when used with infectious agents, institutes have to 

develop local criteria for determining when to replace 

particulate filters. As a complete protective ensemble, 

PAPRs with loose-fitting hoods may be worn in conjunction with Tyvek suits or long-sleeved scrub suits, 

gloves, laboratory socks, and shoes with shoe covers 

or over-boots. All NIOSH-approved respirators are 

approved as a complete system, so components cannot be switched between different manufacturers’ 

products without negating the approval. For example, 

a NIOSH-approved PAPR system consists of the turbo 

blower unit, battery, belt, hose, filters or cartridges, 

and loose-fitting hood or tight-fitting face piece, all 

assembled and marketed by the manufacturer as a 

complete system. Only approved, compatible replacement components from the same manufacturer may 

be used with a given respiratory protection system.

To be approved to use a respirator, a user must be 

medically cleared based on a health history questionnaire and a pulmonary function test or other relevant 

medical examinations on a case-by-case basis; be 

enrolled in an employer-provided OSHA-compliant 

respiratory protection program31; receive initial and 

annual training on the use of the assigned respirator 

or additional training when a different type of respirator is assigned; and undergo annual fit testing for 

negative-pressure, tight-fitting respirators. Proper fit 

testing procedures are available in Appendix A of the 

OHSA Respiratory Protection Standard.31

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Medical Aspects of Biological Warfare 

In a class III BSC operation (BSL-4 cabinet laboratory), personnel must remove all personal clothing and 

undergarments and shoes. Complete laboratory clothing, including undergarments, pants, shirts, shoes, and 

gloves, is provided and worn by laboratory workers.1

Workers wear nitrile or latex examination gloves for 

extra protection when working in class III BSCs, just in 

case the shoulder-length box gloves develop pinholes, 

punctures, or tears.

In BSL-4 protective suit laboratories and BSL-4 

animal facilities, personnel must remove all personal 

clothing, including undergarments, socks, shoes, 

and jewelry. Personnel at USAMRIID may ask for an 

exemption for wedding bands, but only eyeglasses in 

addition to exempted wedding bands may be worn 

in the BSL-4 suites. Complete laboratory clothing, 

including undergarments, pants, shirts, jumpsuits, 

socks, and gloves, is provided for, and used by, laboratory workers. Workers don a fully encapsulating 

positive-pressure protective suit supported by an 

umbilical-supplied air system. It is common practice 

in BSL-4 laboratories for individuals to periodically 

verify PPES integrity prior to donning by taping the 

exhaust valves of the suit and inflating it to a set pressure point. This test is performed at USAMRIID at a 

minimum when the individuals change their gloves 

on a weekly basis, but practices vary at other BSL-4 

facilities. In addition, annual pressure decay testing 

is conducted at USAMRIID on all PPES used in the 

BSL-4 laboratory. The suit can be fitted with integral 

protective over-boots or with legs terminating in soft 

booties. If a suit of the latter design is used, the worker 

dons protective over-boots inside the BSL-4 suit facility, after passing through an airlock equipped with a 

decontaminating chemical suit shower. When not in 

use, protective over-boots are stored inside the BSL4 facility. As of this writing, PPES for use in a BSL-4 

environment are not federally regulated by OSHA as 

level A chemical suits or as respirators, and such suits 

are not currently NIOSH approved. However, the 

compressor and filter system must provide minimum 

grade D breathing air to the PPES.31

Medical Surveillance

Medical surveillance, if indicated, may comprise 

baseline and periodic (usually annual) studies, including the following:

• complete medical history,

• urinalysis,

• hematology (complete blood count),

• serum chemistry panel,

• serum protective antibody titers for specific 

disease agents,

• physical examination, and

• ancillary studies.

Ancillary studies can include the following:

• periodic chest radiograph,

• periodic electrocardiogram,

• annual audiogram,

• annual visual acuity testing,

• annual evaluation of respiratory capacity, and

• mental fitness, neurological examinations, 

and random testing for illicit substance use 

(as needed).

An effective occupational health program benefits 

both the employee and the employer and may reduce 

time lost to injuries. This occupational health program 

will comply with OSHA and other applicable federal 

and state laws and regulations. Medical surveillance is 

a critical part of a comprehensive occupational health 

and safety program. An occupational health and safety 

program has the following objectives38:

• protect workers against health and safety 

hazards in the work environment;

• properly place workers according to their 

physical, mental, and emotional abilities;

• maintain a pleasant, healthy work environment;

• establish preplacement examinations;

• establish regular, periodic health examinations (medical surveillance);

• diagnose and treat occupational injuries, exposures, and diseases;

• consult with the worker’s personal physician, 

with the worker’s consent, regarding other 

related health problems;

• provide health education and counseling for 

workers;

• provide safety education for workers;

• identify hazardous situations or find the 

means to prevent or mitigate hazardous situations; and

• establish surveys and studies of the industrial 

environment to protect workers, their families, and the community.

Laboratory workers employed in a BSL-4 suit facility are enrolled in a medical surveillance program, and 

they should be medically evaluated for fitness to use a 

PPES. At USAMRIID, workers in the BSL-4 suit laboratories are enrolled in a hearing protection program. 

When the 8-hour, time-weighted average level is 85 

dB (decibels) or greater, workers must be enrolled in 

an employer-provided hearing protection program 

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to comply with OSHA regulations.39 The program 

requires employees to undergo initial baseline and 

annual surveillance audiometry, fitting, and training 

to use hearing protectors (ear plugs or muffs).

Personnel are required to receive initial familiarization training on how to wear the protective suit, 

as well as receive extensive, documented, tailored 

training provided by an assigned mentor before being considered proficient to work independently in 

BSL-4 containment. Currently, USAMRIID enrolls new 

personnel who plan on working in BSL-4 containment 

in a specialized 3-day training course teaching them 

the fundamentals of the BSL-4 environment, suit use, 

entry and exit procedures, movement and dexterity 

exercises, and emergency response. Once employees 

complete the basic training course, they are then mentored for a set period of time before they may apply 

for independent access to BSL-4. After demonstrating 

proficiency, laboratory workers can begin independent 

work in the BSL-4 containment suite. 

During normal operations in the BSL-4 containment suite, workers may disconnect briefly from the 

breathing air supply to move about and then couple 

to an airline in a new location within the suite. One 

manufacturer advises that up to a 5-minute residual 

air supply may remain in the suit if there is an unanticipated loss or interruption of the breathing air supply.40 In regular operations, it is prudent not to remain 

disconnected from the air supply for more than 2 or 3 

minutes, because the carbon dioxide concentration and 

humidity level will quickly rise within the suit space. It 

is recommended as a best practice to remain connected 

to the air supply as much as possible when completing work tasks in BSL-4. Generally, the visor fogs ups 

before the carbon dioxide concentration builds up to 

a hazardous level, thus prompting the user to connect 

to the air supply expeditiously. 

It is important that personnel are fit for the physical 

challenges of working in a BSL-4 PPES laboratory. An 

ongoing medical surveillance program ensures that, 

in the event of occupational exposure to an infectious 

agent or toxin, the medical needs of the worker will 

be met immediately. If a laboratory worker should 

become ill without obvious exposure to an agent, the 

individual will be assessed to determine whether the 

illness is related to an unknown laboratory exposure.

At USAMRIID, all potential biological exposures are 

assessed through the combined efforts of the Safety, 

Radiation, and Environment Division; personnel supervisors; and the Medical Division. All employees are 

instructed to notify the Safety, Radiation, and Environment Division of any mishaps occurring either inside 

or outside containment suites. For mishaps in the containment suites, all personnel involved in the incident 

are instructed to report to the Medical Division in the 

absence of a life-threatening emergency for an initial 

briefing. The initial briefing is conducted with the affected personnel, supervisors, a safety representative, 

and the competent medical authority. After the briefing, initial exposure and disease risk are determined 

and postexposure prophylaxis is administered (as 

determined by subject matter experts).41,42 In the event 

of a medical emergency or potential exposure in BSL-4 

containment, the Department of Defense currently has 

a memorandum of agreement with the Department 

of Health and Human Services for potential exposure 

monitoring, care, and treatment of inpatients enrolled 

as clinical research subjects. Local arrangements are 

made for laboratories outside the United States.

Protecting the Community and the Environment

Secondary barriers are the elements of laboratory facility design and construction that (a) contribute to protection of laboratory personnel, (b) provide a barrier to 

protect persons outside the laboratory, and (c) protect 

persons and animals in the community from infectious 

agents in the event of an accidental release within the 

laboratory.1

 Secondary barriers in BSL-3 containment 

facilities at USAMRIID include entry vestibules or personnel airlocks that feature two self-closing and lockable doors, clothes change rooms and shower facilities 

based on a risk assessment, and a hand-washing sink 

in each laboratory room. The sink is located near the 

room exit door and can be operated using foot pedals 

or knee or elbow paddles, or is automatically activated 

by an infrared sensor. Other secondary barriers include 

floor, wall, and ceiling finishes constructed for easy 

cleaning and decontamination; sealed penetrations 

in floors, walls, and ceilings; and sealable openings to 

facilitate decontamination. Laboratory furniture has 

waterproof and chemical-resistant bench tops, and 

chairs are covered with nonfabric material to permit 

easy decontamination. An autoclave is available in the 

facility. The facility is equipped with a ducted exhaust 

ventilation system that creates inward directional airflow from areas of lower potential hazard to areas of 

higher potential hazard (negative-pressure gradient) 

without recirculation of air or airflow reversals under 

failure scenarios.1

 To confirm inward airflow, a visual 

monitoring device (eg, a magnehelic differential pressure gauge [Dwyer Instruments, Michigan City, IN]; 

photohelic gauge [Dwyer Instruments, Michigan City, 

IN]; rodimeter; or “telltail”) should be available at the 

laboratory entry. 

In animal biosafety level 3 (ABSL-3) facilities, room 

fittings and ventilation should be in accordance with 

the Institute of Laboratory Animal Resources Commission on Life Sciences and National Research Council’s 

Guide for Care and Use of Laboratory Animals43 and 

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Medical Aspects of Biological Warfare 

Biosafety in Microbiological and Biomedical Laboratories.

1

If the ABSL-3 facility has floor drains, the drain traps 

are always filled with an appropriate disinfectant. 

Additional environmental protection design features 

(enhancements) in BSL-3 laboratories and animalholding spaces (including provision of personnel 

showers and effluent decontamination, HEPA filtration of exhaust air, and containment of piped services) 

may be indicated, depending on the nature of the 

infectious agents to be used (eg, arboviruses, highly 

pathogenic influenza viruses and high-consequence 

animal pathogens); the risk assessment or maximum 

credible event analysis of the site (eg, laboratory to 

be located in a highly populated urban center or in a 

remote region having a low-density population); and 

applicable federal, state, and local regulations. 

Secondary barriers required in BSL-4 laboratories 

and ABSL-4 animal-holding spaces are all those specified for BSL-3 laboratories and ABSL-3 animal holding 

spaces, with additional provisions. Other required secondary barriers include a dedicated, non-recirculating 

ventilation system with supply and exhaust components balanced to ensure directional airflow from areas 

of lower potential hazard to areas of higher potential 

hazard. HEPA filtration of supply air and double 

HEPA filtration of exhaust air, with redundancy 

(backup exhaust duct with fan and in-line double 

HEPA filters), are also required, as is alarm and daily 

monitoring to prevent positive pressurization of the 

laboratory or animal-holding space. In large, complex 

operations, a supervisory control and data acquisition 

system (also known as a building automation system) 

may be installed to monitor and control room pressures 

automatically. An automatically starting emergency 

power source (usually a diesel-powered generator) 

is required as a minimum for the redundant exhaust 

ventilation systems, redundant life-support (breathing 

air) systems, alarms, lighting, entry and exit controls, 

and BSCs. Laboratories using PPES are required to 

have primary and backup breathing air compressors 

along with a secondary breathing air system capable 

of supporting the egress of all personnel in the BSL4 suites in the event of a breathing air compressor 

failure. In practice, the freezers and other laboratory 

equipment (incubators and refrigerators) are generally 

also on circuits that can switch to emergency backup 

power. Other infrastructure elements that contribute 

to the secondary barrier include change rooms, personnel showers, effluent decontamination by a proven 

method (preferably heat treatment), and containment 

of piped services. Floor and sink drain traps must be 

kept filled with an appropriate disinfectant (one with 

proven efficacy for the microorganisms handled within 

the BSL-4 facility). An autoclave with two interlocked 

doors, with the outer door sealed to the outer wall (a 

so-called “bioseal”), is required at the containment 

barrier. The autoclave is automatically controlled so 

the outer door cannot be opened until a sterilization 

cycle has been completed. A dunk tank, fumigation 

chamber, or a ventilated equipment airlock is also 

provided so materials may pass into the containment 

area. Materials that cannot be steam sterilized may be 

safely decontaminated either through a fumigation 

cycle in a ventilated airlock or by passage through the 

chemical shower cycle or dunk tank and removed from 

the containment area. The walls, floors, and ceilings are 

constructed as a sealed internal shell (the containment 

envelope) capable of being decontaminated using a 

fumigant. Bench tops have seamless surfaces impervious to water, resistant to chemicals, and free of sharp 

edges. Appropriate electronic communications are 

provided between the BSL-4 containment area and the 

noncontainment area, which may include a telephone, 

facsimile, two-way radio, intercom, and a computer 

system on a local area network or wireless network. 

BSL-4 protective suit laboratories also have a dedicated 

area for storing suits and boots, and a double-door 

personnel airlock equipped with a chemical shower for 

surface decontamination of protective suits. Animalholding rooms need to meet the standards specified 

in the Guide for Care and Use of Laboratory Animals.

43

Containment operational parameters are inspected 

and verified daily before work is initiated in the BSL4 facility.

Solid and Liquid Waste Inactivation and Disposal

The US Environmental Protection Agency (EPA) 

defines antimicrobial pesticides as substances or 

mixtures of substances used to destroy or suppress 

the growth of harmful microorganisms (eg, bacteria, 

viruses, or fungi) on inanimate objects and surfaces. 

Public health antimicrobial products are intended 

to control microorganisms infectious to humans in 

any inanimate environment. These products include 

sterilizers (sporicides) and disinfectants.44 Sterilizers 

(sporicides) are used to destroy or eliminate all forms 

of microbial life, including fungi, viruses, and all forms 

of bacteria and their spores. Sterilization is widely used 

in hospitals for infection control. Types of sterilizers 

include steam under pressure (autoclaves), dry-heat 

ovens, low-temperature gas (ethylene oxide), and liquid chemical sterilants. All types of sterilizers are also 

applicable for use in microbiological and biomedical 

laboratories. In laboratories, autoclaving is used to 

prepare sterile instruments, equipment, and microbiological nutrient media and to render microbiologically 

contaminated liquid and solid waste sterile before it 

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Biosafety

enters the waste disposal stream. Laboratory glassware 

is dried, sterilized, and depyrogenated (rendered free 

of endogenous pyrogens) in dry-heat ovens. Ethylene 

oxide sterilization is used to sterilize materials such as 

delicate instruments and laboratory notebooks, which 

cannot withstand steam sterilization, but is seldom 

used to sterilize solid waste. Liquid sterilants, used 

to sterilize delicate instruments by immersion and to 

sterilize impervious surfaces by surface application, 

can be added to suspensions of infectious materials to 

chemically inactivate them. Disinfectants, according 

to the EPA, are used on hard inanimate surfaces and 

objects to destroy or irreversibly inactivate infectious 

fungi and bacteria, but not necessarily their spores. 

The EPA divides disinfectant products into two major 

types: hospital and general use. Hospital disinfectants 

are most critical to infection control in hospitals and 

are used on medical and dental instruments and on 

hospital environmental surfaces. General disinfectants 

are products used in households, swimming pools, 

and water purifiers.

The decision about the type of biological inactivation required depends on a number of factors; the 

type of biological agent requiring inactivation along 

with whether the agent is present in a large amount 

of organic material can initially narrow the choice. In 

some cases where either large spills or large amounts 

of organic material are present, a detergent solution 

may be used prior to disinfectant to enhance efficacy at 

the site of cleanup. Other variables, including but not 

limited to pH, temperature, type of materials requiring 

biological inactivation (eg, neoprene, metals, or plastics), age of disinfectant, humidity, concentration, and 

contact time requirements can also affect the choice of 

inactivation method.

An example of a liquid sterilant-disinfectant is Alcide EXSPORE (Alcide Corporation, Redmond, WA) 

4:1:1 base concentrate (1.52% sodium chlorite; EPA 

Registration No. 45631-3), which comes with a separate 

activator concentrate (9.5% lactic acid) as a set. This 

sterilant-disinfectant must be freshly prepared by 

diluting the base with water per the manufacturer’s instructions before adding activator to generate chlorine 

dioxide.45 The prepared sterilant-disinfectant should be 

used immediately and must be freshly prepared daily.

An example of a hospital disinfectant is MicroChem Plus (National Chemical Laboratories, Inc, 

Philadelphia, PA; EPA Registration No. 1839-95-2296), 

a proprietary mixture of two quaternary ammonium 

compounds and inert ingredients that is labeled to 

kill listed microorganisms (specified viruses, fungi, 

and nonspore-forming bacteria) when mixed at the 

rate of 2 ounces of the concentrated product per gallon of water.46

An example of a general disinfectant used at 

USAMRIID is Clorox Ultra Germicidal Bleach (The 

Clorox Company, Oakland, CA; 6.15% sodium hypochlorite [5.84% available chlorine]; EPA Registration 

No. 67619-8). When mixed at the rate of 12 ounces 

per gallon of water (5,000 ppm), it is labeled to kill 

listed microorganisms (specified viruses, fungi, and 

nonspore-forming bacteria).47 Bleach is not registered 

by the EPA as a sterilant. During the subsequent cleaning and decontamination of spore-contaminated postal 

facilities after the 2001 anthrax-by-mail incidents, the 

EPA issued crisis exemptions on a case-by-case basis to 

use bleach for emergency decontamination subject to 

adherence with specified conditions of application (see 

http://www.epa.gov/pesticides/factsheets/chemicals/

bleachfactsheet.htm). Bleach solutions (1:10 dilution) 

are now sold premixed, as standard 1:10 dilutions must 

be made fresh daily. Clorox disinfecting wipes (EPA 

Registration No. 5813-79) also come ready-made with 

an extended shelf life compared to fresh 1:10 solutions. 

Wipes may be used in the laboratory or in the field 

with the same efficacy as fresh daily 1:10 solutions.

In BSL-4 laboratories and in BSL-3 and ABSL-3 

facilities, if indicated by the risk assessment, liquid 

effluent (laboratory sewage) must be inactivated by 

a proven process, generally heat treatment under 

pressure.1

 Effluent decontamination systems are 

available as six different types. Systems may be 

batch-based chemical systems using peracetic acid, 

sodium hypochlorite, quaternary ammonium compounds, or chlorine dioxide. Heat treatment effluent 

decontamination systems may be either continuous 

flow or batch process and can run at high temperature 

(>121°C) or sub-boiling temperatures. Batch process 

effluent decontamination systems models can also 

be designed to run as a heat treatment system augmented by chemicals depending on the demands 

of the laboratory. Solids suspended in the liquid 

waste are comminuted (finely ground). The effluent 

is heated to specified temperature and held at that 

temperature for a certain period of time. Then it is 

cooled, sampled for sterility testing, and released to 

a municipal or nonpublic sewer system. The time–

temperature relationship for the selected process 

depends on the inactivation profile of the infectious 

microorganisms that could be present in the liquid 

waste. The current process at Fort Detrick holds the 

heated effluent at 132°C (270°F) for a minimum of 12 

minutes, sufficient to inactivate fungal and bacterial 

spores. The standard liquid biowaste process used at 

the Canadian Science Centre for Human and Animal 

Health (Winnipeg, Manitoba, Canada) heats the effluent to 121°C (250°F) for a 30-minute holding time, but 

has the capability of achieving a temperature as high 

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Medical Aspects of Biological Warfare 

as 141°C (286°F).48 The standard process is sufficient 

to inactivate fungal and bacterial spores. The higher 

temperature is available, if needed, to inactivate prions (heat-resistant infectious proteins).49

Animal carcasses exposed to biological agents inside BSL-3 or BSL-4 laboratories require decontamination prior to removal from the containment suites. Per 

SOP, carcasses are bagged and decontaminated with 

the appropriate disinfectant prior to autoclaving. The 

autoclaved carcasses can be incinerated subsequently 

or disposed of in accordance with applicable local, 

state, and federal regulations. Animal carcasses can 

also be inactivated with an alkaline hydrolysis-based 

tissue digester. Digesters can be run either at boiling or 

sub-boiling temperatures using potassium hydroxide 

or sodium hydroxide alkali for a set cycle time. Cycle 

time depends on weight, composition, and surface area 

of animal carcasses, percent of alkali, temperature, and 

amount of water.50–52

After infectious materials have been inactivated by 

an appropriate method of sterilization or disinfection, 

they may be removed from the laboratory and disposed of in accordance with applicable federal, state, 

and local regulations. In the United States, disposal 

of several categories of solid waste (regulated medical waste, perceived medical waste, and pathological 

waste) is regulated at the state level. Many states have 

strict regulations that require that such waste be sterilized and rendered unrecognizable (by processes such 

as incineration, shredding, or grinding with steam 

sterilizing or irradiating) before final disposal in a 

sanitary landfill.

Standard and Special Microbiological Practices

Standard and special microbiological practices 

universal to all BSLs are as follows:

• The laboratory director limits or restricts access to the laboratory when experiments are 

in progress.

• A biohazard sign may be posted at the entrance of the BSL-1 laboratory if infectious 

agents are present or stored in the laboratory. 

A biohazard sign is posted at the entrance of 

BSL-2, BSL-3, and BSL-4 laboratories and animal rooms when infectious agents are present.

• Policies for the safe handling of sharps are 

instituted.

• All procedures are performed carefully to 

minimize the creation of aerosols.

• Work surfaces are decontaminated at least 

once daily and after any spill of viable 

material.

• All infectious waste is decontaminated by 

an approved process (eg, autoclaving before 

disposal).

• A pest (insect and rodent) control program 

must be in effect.

• Personnel wash their hands after handling 

viable materials, after removing gloves, and 

before leaving the laboratory.

• Eating, drinking, smoking, handling contact 

lenses, taking medication, and storing food 

for human consumption in the laboratory or 

animal-holding facility are not permitted. If 

contact lenses are worn in the laboratory or 

animal-holding area, goggles or a face shield 

should also be worn. Personnel should refrain 

from applying cosmetics or lip balm, chewing 

gum, and taking oral medications while in the 

laboratory or animal-holding facility.

• Mouth pipetting is prohibited. Only mechanical pipetting devices are to be used.

There are no special practices for the BSL-1 laboratory. The following special practices apply to BSL-2, 

BSL-3, and BSL-4 laboratories, as well as to ABSL-2, 

ABSL-3, and ABSL-4 animal-holding areas:

• Secure all laboratories registered for select 

agents and toxins.53 Keep BSL-2 and BSL-3 laboratory room doors closed when working with 

infectious agents. Keep doors in BSL-4 laboratories and in ABSL-2, ABSL-3, and ABSL-4 

animal-holding areas closed and locked.

• Only individuals advised of the potential 

hazards who meet specific entry requirements 

may enter the laboratory or animal-holding 

room.

• In ABSL-2, ABSL-3, and ABSL-4 animalholding facilities, the Institutional Animal 

Care and Use Committee and the Institutional 

Biosafety Committee approve special policies 

and procedures.

• Along with the biohazard sign, post the following information at the entrance to the laboratory or animal-holding room: the agents in 

use, the BSL, required vaccinations, any PPE 

required, the name and phone number of the 

principal investigator, and any procedures 

required to exit the laboratory or animalholding room.

• At-risk individuals entering the laboratory or 

animal-holding room may receive appropriate 

vaccinations if available for the agents being 

handled or agents potentially present in the 

room.

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Biosafety

• A tuberculosis skin test or other tuberculosis 

surveillance procedures are indicated on an 

annual basis if personnel are working with 

or around nonhuman primates. 

• Describe biosafety procedures for BSL-2 and 

ABSL-2 facilities in SOPs. Describe biosafety 

procedures for BSL-3 and BSL-4 laboratories 

and ABSL-3 and ABSL-4 animal-holding facilities in a biological safety manual specific to the 

laboratory or animal-holding facility. Advise 

personnel of the specific hazards, require them 

to read and ensure they understand the manual, 

and make certain that they comply with it.

• The laboratory director must ensure that 

laboratory and support personnel receive 

appropriate initial training and annual training, and additional training on potential 

hazards in the laboratory or animal facility; 

precautions to take to prevent exposures; and 

procedures on evaluating potential exposures. 

The laboratory director is also responsible for 

ensuring that the previously described training is appropriately documented.

• Use caution with needles and syringes. In 

BSL-3 and BSL-4 laboratories and in ABSL3 and ABSL-4 animal-holding facilities, use 

only needle-locking syringes or disposable 

syringe–needle systems in which the needle is 

integral to the syringe. Also consider tools that 

allow for one-handed recapping of syringe 

needles or systems without needles. Dispose of used sharps in conveniently located 

puncture-resistant containers.

• Place all potentially infectious materials in 

covered, leak-proof containers during collection, manipulation, storage, transport, or 

shipping. Place viable material to be removed 

from a class III BSC or a BSL-4 facility in an 

unbreakable, sealed primary container that is 

enclosed in an unbreakable, sealed secondary 

container. Pass this enclosed material through 

a chemical disinfectant dunk tank, fumigation chamber, or airlock with a chemical suit 

shower (in the case of a BSL-4 suit facility).

• Decontaminate work surfaces and laboratory 

equipment with an effective disinfectant routinely, after work with infectious materials is 

completed, and after any spills. Contaminated 

equipment must be appropriately decontaminated before repair or maintenance or packaging for transport.

• Immediately report to the laboratory director 

(supervisor) any spill or accident that results 

in exposure to infectious materials. Institute 

medical evaluation, surveillance, and treatment 

as appropriate and document this medical care 

in writing. In BSL-3 and BSL-4 containment 

facilities, develop and post spill procedures and 

conduct drills on an annual basis. Professional 

staff or other appropriately trained personnel must decontaminate, contain, and clean 

up any spill of infectious material. In BSL-4 

containment, establish practical and effective 

protocols for emergency situations, including 

the evacuation of incapacitated staff.

• Animals and plants unrelated to the work 

conducted are not permitted in the laboratory.

• In BSL-3 and BSL-4 containment facilities, 

the laboratory director must ensure that all 

personnel are proficient in standard microbiological practices, laboratory-specific practices, 

and operations before they begin work with 

microorganisms.

• In BSL-3 and BSL-4 containment facilities, conduct open manipulations of infectious agents 

in BSCs or other primary containment devices. 

Conducting work in open vessels on the open 

bench is prohibited. Vessels with tight-fitting 

covers (gasketed caps, O-ring seals) should 

be used to hold viable cultures within water 

baths and shaking incubators. Use sealed rotors or centrifuge safety containers fitted with 

O-ring seals to contain centrifuge tubes. Use 

plastic-backed paper towels on nonperforated 

surfaces to facilitate cleanup. Use plastic vessels in place of glass vessels.

• In BSL-3 and BSL-4 containment facilities, 

autoclave or decontaminate all materials other 

than materials to be retained in a viable state 

before removing them.

• At BSL-4, maintain a physical or electronic 

log of all personnel, with the time of each 

person’s laboratory entry and exit recorded. 

This requirement also applies to all personnel who have access to areas in which select 

agents and toxins are used or stored.53

• In BSL-4 containment (and in BSL-3 containment, if indicated by risk assessment, sitespecific conditions, or applicable regulations), 

enter and exit the laboratory only through the 

clothing change and shower rooms. Remove 

and leave personal clothing in the outer change 

room. Change completely into laboratory 

clothing. On exiting the laboratory, remove 

and leave all laboratory clothing in the inner 

change room. Take a decontaminating (soap 

and water) personal wet shower for a minimum of 3 minutes on exit from the laboratory. 

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Medical Aspects of Biological Warfare 

Autoclave soiled laboratory clothing before 

laundering. Use the equipment airlock to enter 

or exit the laboratory only in an emergency.

• Bring supplies and materials into the BSL-4 

facility through the double-door autoclave, 

fumigation chamber, or equipment airlock, 

which is decontaminated before and after each 

use. Secure the airlock outer door before the 

inner door is opened. Secure the airlock inner 

door after materials are brought into the facility.

• In BSL-4 containment, institutes are required 

to establish a system to report laboratory accidents and exposures, employee absenteeism, and medical surveillance of a potential 

laboratory-acquired illness.1

• Make available a facility for quarantine, isolation, and medical care of personnel who 

work in BSL-4 containment and who are affected with a potential or known laboratoryacquired illness.

ROLE OF MANAGEMENT IN A BIOSAFETY PROGRAM

Management must consider safety a top priority 

and, on a daily basis, work closely with and support 

safety personnel. Although management must provide 

a biosafety program as well as engineering features and 

equipment designed to reduce the risks associated with 

the research conducted at the institute, safety is also an 

individual responsibility. To illustrate this point (Figure

30-1), consider the mission or purpose of an institute as 

the hub of a wheel. All personnel, regardless of education, experience, or job description, are the spokes of the 

wheel and must be reminded regularly of the importance 

of their contributions to an institute. If one (or more) of 

the spokes is not functioning as designed, the wheel 

does not operate smoothly. Consequently, it takes longer to meet not only personal goals and objectives, but 

also institute goals and objectives. All personnel (each 

spoke of the wheel) in an institute must be considered 

important, regardless of their perceived contributions. 

The goals of a biosafety program include the 

following: (a) prevention of injury, infection, and 

death of employees and the public; (b) prevention 

MISSION

Security staff MISSION Custodial staff;

housekeeping

Safety staff

Facility 

engineers*

Laboratory 

technicians

Animal 

caretakers

Administration

Principle

investigators

Figure 30-1. Institute personnel are depicted as the spokes of 

a wheel that work together to accomplish a common mission.

*maintenance staff

of environmental contamination; (c) conformance to 

prudent biosafety practices; and (d) compliance with 

federal, state, and local regulations and guidelines. 

The ultimate objective of these goals is to keep everyone healthy while supporting productive research. 

Appropriate personnel training is paramount. Both 

initial and refresher personnel training must address 

the institutional biological safety program and the 

elements of biosafety. Training can be conducted as 

a discussion rather than as a formal lecture to promote audience participation. This technique allows 

individuals to have ownership over policies through 

an integrated program of safety engineering, vaccination, health surveillance, and medical management of 

illness. Risk encompasses awareness, assessment (or 

evaluation), mitigation, and management of the risk. 

Communication is a fundamental part of risk assessment and training. The US government has developed 

a 5-step risk management process (Figure 30-2).54 The 

five sequential steps of the risk management process 

include the following:

1. Identify hazards. What is the hazard?

2. Assess hazards. What is the danger of this 

hazard?

3. Develop controls and make risk decision. 

What controls can be used to remove this 

hazard, or make a decision to accept some 

risk?

4. Implement controls. Controls developed for 

the risk are implemented (or put into operation or practice).

5. Supervise and evaluate. After a period of 

evaluation as new data becomes available, 

the controls implemented are reviewed to 

determine whether they were adequate, or 

if additional controls must be added.

The philosophy of a biosafety program is based on 

an early estimation of risk, followed by application 

of appropriate containment and protective measures. 

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Biosafety

It is very important to investigate and review safety 

incidents at the institute because presentation of this 

data will heighten the awareness of individuals that 

accidents do happen despite safeguards. 

Laboratory Safety Audits

An audit is a methodical examination and review. In 

the present context, it is a systematic, critical review of 

laboratory safety features and procedures. The terms 

“survey” (comprehensive view) and “inspection” (a 

critical appraisal, description of some obvious hazards 

and how safety personnel try to minimize the risk of 

these hazards, an official examination, or checking or 

testing against established standards) are often used 

interchangeably with the term “audit.” Safety personnel must emphasize that their role is to try to identify 

hazards, conduct risk assessments, develop risk management strategies, and evaluate the effectiveness of 

those strategies over time while minimizing impact on 

the research. Safety personnel must actively engage 

with and seek the help of all administrative and laboratory personnel in hazard identification. It is important 

for safety personnel to remain actively engaged with 

laboratory personnel outside of laboratory audits to 

minimize potential negative associations that may be 

encountered with inspections. It must be understood 

that a safety department cannot provide absolute 

safety, but strives to provide reasonable safety. Safety 

personnel advise, guide, provide limited training, and 

implement institute and regulatory policies (in conjunction with the institutional biosafety committee). 

The safety department, with continued support from 

management and all facility personnel, can minimize 

the risk of hazards by implementing institute and 

regulatory guidelines. During the laboratory safety 

audit, safety practices and equipment are evaluated. 

General safety, life safety, biological safety, chemical 

hygiene, and radiation safety are topics covered in 

a typical laboratory safety audit. Laboratory audits 

should be scheduled on a regular basis and may be 

announced or unannounced.

Self-audits of required safety practices provide a 

measure for achieving compliance with safety rules 

and regulations.55 Designated safety specialists can 

conduct regular safety audits at quarterly intervals, 

accompanied by the laboratory supervisor and a facilities management representative. Deficiencies can be 

pointed out during the audit. Later, a written report 

with suggestions for corrective action may be sent 

to the laboratory supervisor. The supervisor reports 

progress on remediation to the safety specialist within 

a mutually agreed on, fixed-time period. Safety personnel should follow up on any deficiencies noted during 

a laboratory audit periodically to ensure laboratory 

personnel have taken the appropriate corrective actions. Support from higher management is essential 

for an audit to have the desired effect of improving 

employee safety, as well as instituting compliance with 

applicable regulations.56

Use of a checklist ensures a systematic, standardized audit, thus reducing the chance of missing critical 

items. Citing the pertinent requirement or applicable 

regulation on the checklist provides a ready reference 

and justification for each item listed on the checklist. 

Within the overall laboratory safety audit, the following list of biosafety elements should be covered57:

• autoclave repair and operational records 

where applicable,

• proper use of PPE,

• appropriate laboratory clothing,

• no food or drink in the laboratory,

• proper use of sharps and sharps disposal 

containers,

• decontamination of infectious materials before disposal,

• proper disposal of laboratory waste,

• proper signage (laboratory, equipment, 

materiel),

• current certification of BSCs and fume hoods, 

and

• use of in-line HEPA filters on laboratory 

vacuum outlets where applicable.

Additional biosafety elements audited at 

USAMRIID include: (a) weekly flushing floor and sink 

drains and recording the action in a drain flush log; 

(b) flushing the eyewash weekly and recording the 

action in an eyewash flush log; (c) testing (flushing 

and measuring the flow rate) the emergency deluge 

shower at least weekly and recording the action in an 

Figure 30-2. Five steps of the risk management process. 

Adapted from: US Army Safety Center, Fort Detrick, MD.

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Medical Aspects of Biological Warfare 

emergency shower test log; (d) recording during the 

audit differential pressures for laboratory rooms as 

displayed on the magnehelic and photohelic gauges; 

(e) checking documentation that emergency communication devices have been tested at least monthly; 

(f) testing and recording during the audit operating 

status of alarms, emergency lights, and emergency 

exit lights; and (g) spot checking laboratory SOPs, 

laboratory biosafety manuals, and laboratory personnel training records. 

Four events that warrant conducting a formal, 

unscheduled audit of a laboratory include the 

following57:

1. accident or injury in the workplace,

2. follow-up to implementation of new biosafety regulations or procedures,

3. a new funding source requesting documentation of workplace safety, and

4. new infectious agents proposed for use in the 

laboratory.

An important time for evaluation of biosafety SOPs 

may be before a major outside organization or agency 

conducts a site visit.57 Two examples of organizations 

conducting site visits are the Joint Commission on 

Accreditation of Healthcare Organizations and the 

Association for Assessment and Accreditation of 

Laboratory Animal Care (AAALAC) International. 

Examples of agencies that conduct inspections of 

laboratories registered for select agents are the US 

Centers for Disease Control and Prevention and US 

Department of Agriculture Animal and Plant Health 

Inspection Service Select Agent Program Laboratory 

Inspection Programs. Laboratories that do not work 

on select agents may be subject to a US Department of 

Agriculture inspection for specific biological agents or 

an NIH Office of Biotechnology Activities audit if they 

have a functioning Institutional Biosafety Committee.58

For subordinate laboratories of the US Army Medical 

Research and Materiel Command, safety office personnel conduct periodic safety site assistance visits.30 For 

Department of Defense (DoD) research, development, 

test, and evaluation (RDTE) laboratories, the director 

of Army safety conducts biological defense safety 

evaluation site visits.30

In DoD RDTE facilities, health and safety professionals must conduct internal inspections (audits) of 

BSL-1 and BSL-2 laboratories at least quarterly and 

must conduct internal inspections of BSL-3 and BSL4 laboratories at least monthly.34 Inspections must be 

documented, deviations from safe practices recorded, 

and recommended corrective actions taken. If deviations are life threatening, access to the laboratory area 

is restricted until corrective actions have been taken. 

New RDTE efforts involving biological agents must be 

evaluated and inspected before startup. Any Department of the Army headquarters agency can recommend special studies or reviews when (a) conditions or 

practices that may affect safety have changed, (b) major 

system modifications to facility design and physical 

configuration are made, and (c) safety, health, and 

environmental protection standards and requirements 

have changed significantly.30 Safety officials maintain 

safety inspection records for 3 years, and they review 

records annually to note trends that require corrective 

actions.30 Laboratory supervisors review their work 

areas at least weekly and take any needed corrective 

actions promptly.

At USAMRIID, safety professionals assigned to the 

Office of Safety, Radiation and Environment conduct 

semiannual comprehensive inspections of BSL-1 and 

BSL-2 and quarterly inspections of BSL-3 and BSL-4 

laboratories to identify potential problems. These 

quarterly inspections augment the monthly inspections 

conducted by laboratory suite supervisors or their 

designees. Inspections, which may be announced or 

unannounced, include coverage of general safety practices and safety practices specific to a particular BSL.57

Biological Defense Research Program Laboratories

All laboratories involved in DoD RDTE operations must comply with the Department of the Army 

Pamphlet, Safety Standards for Microbiological and Biomedical Laboratories.

34 These regulations specify safety 

policy, responsibilities, and procedures for military 

and contract laboratories conducting operations at 

BSL-2, BSL-3, and BSL-4 in support of the US military 

biological defense program. The DoD Biological Surety 

(Biosurety) Program is a new program implemented 

in DoD biological defense RDTE laboratories that 

use DoD-provided biological agents.59 This biosurety 

program is patterned after existing nuclear and chemical surety programs, and its purpose is to ensure the 

safe and secure use of biological agents. The program 

encompasses physical security, biological safety, biological agent accountability, and personal reliability 

as measures to prevent unauthorized access to agents 

of bioterrorism (select agents).59,60

Laboratory Animal Care and Use Program

Federal animal welfare regulations61–63 from the US 

Department of Agriculture Animal and Plant Health 

Inspection Service, state and local laws, and the Public 

Health Service Policy on Humane Care and Use of Animals64 regulate the care and use of laboratory animals 

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Biosafety

used in research. Many of the applicable regulations and 

policies are summarized in the Guide for the Care and Use 

of Laboratory Animals.

43 The responsible administrative 

official at each institution using laboratory research animals must appoint an Institutional Animal Care and Use 

Committee representative to oversee and evaluate the 

institution’s animal program, procedures, and facilities 

on a semiannual basis to ensure they are consistent with 

the animal welfare regulations, Public Health Service 

policy (for those institutions that receive NIH funding), 

and recommendations specified in the guide.43 The 

guide covers many aspects of an institutional animal 

care and use program, including the following:

• policies and responsibilities,

• monitoring care and use of animals,

• veterinary care,

• qualifications and training of personnel who 

work with animals, and

• occupational health and safety of personnel 

working with animals, physical facilities, and 

animal husbandry.

Under the heading of occupational health and 

safety, critical topics in an effective animal care and 

use program include the following:

• hazard identification and risk assessment;

• personnel training, hygiene, safe facilities, and 

procedures;

• health monitoring;

• animal experimentation involving biological 

and other hazardous agents;

• use of PPE;

• medical evaluation; and

• preventive medicine for personnel working 

with animals.

A voluntary program exists for the assessment and 

accreditation of institutional animal care and use programs. At the request of a given institution, the Association for Assessment and Accreditation of Laboratory 

Animal Care (AAALAC) will send laboratory animal 

technical experts to the institution to conduct a site visit 

and evaluate all aspects of an institution’s animal care 

and use program. If all aspects of the program meet 

the high standards of AAALAC, the institution may be 

granted the coveted designation “AAALAC accredited,” 

which is effective for 3 years. Triennial renewals require 

a complete, comprehensive reassessment of an institution’s animal care and use program. Accreditation by 

AAALAC is mandatory for DoD organizations and facilities maintaining animals for use in DoD programs.64

THE BIOSAFETY PROFESSION

Many biological safety professionals begin their careers as bench scientists in the biological sciences, particularly microbiology, or as professionals in medicine 

or the allied health sciences, and subsequently transfer 

into the biological safety field to work as biological 

safety officers, occupational health and safety managers or specialists, or in closely related positions. With 

the quickening tempo of biological defense research 

and the establishment of new, high (BSL-3 or BSL-4) 

biocontainment laboratories, the demand for competent biological safety professionals is increasing. 

Academic institutions and government agencies are 

beginning to recognize the need to establish didactic 

and practical training opportunities in biological 

safety. For example, the Division of Occupational 

Health and Safety and the National Institute of Allergy and Infectious Diseases of the NIH have jointly 

established a National Biosafety and Biocontainment 

Training Program offering 2-year postbaccalaureate 

and postdoctoral fellowships at the NIH campus in 

Bethesda, Maryland. This program specifically trains 

fellows to support BSL-3 and BSL-4 research environments by acquiring the necessary knowledge and 

skills to meet scientific, regulatory, biocontainment, 

biosafety, engineering, communications, management, and public-relations challenges associated with 

conducting research in such facilities.65 Education is 

carried out through extensive mentorship and training 

in pertinent safety and regulatory guidelines within 

the 27 institutes and centers at the NIH-Bethesda 

campus. Second-year fellows then apply their knowledge through a series of developmental assignments 

at external facilities outside the NIH system to better 

develop a well-rounded understanding of prudent 

safety practices that they may apply after departing 

the fellowship. Examples of academic fellowship 

programs include the biosafety fellowship program 

at Washington University School of Medicine in St 

Louis, Missouri, or the 1-year internship program at 

the Great Lakes Regional Center for Excellence at the 

University of Chicago.

Credentialing biological safety professionals is not 

currently mandated or regulated. A formal, voluntary credentialing process exists to enable biological 

safety professionals to meet minimum set standards 

of expertise and proficiency. The American Biological 

Safety Association (ABSA), the national organization 

of biological safety professionals, has established two 

levels of credentialing: (1) the Registered Biosafety 

Professional (RBP) and (2) the Certified Biological 

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Safety Professional (CBSP). The RBP is an individual 

with a documented university education or specialized 

training in relevant biological safety disciplines who 

has submitted an application and has been found to be 

eligible for registration by the ABSA RBP Evaluation 

Review Panel.66 The RBP has sufficient understanding 

of cell biology, pathogenic microbiology, molecular 

genetics, host immune responses, and concepts of 

infectious agent transmission to enable the RBP to 

apply safeguards when working with biohazardous 

materials. 

The CBSP is an individual who has a combination 

of documented university education, specialized training, and experience in relevant biological safety disciplines, and has further demonstrated knowledge and 

proficiency by passing the Specialist Microbiologist in 

Biological Safety Microbiology examination administered by the National Registry of Certified Microbiologists of the American Society for Microbiology. Every 

5 years, qualification as a specialist microbiologist may 

be renewed by submitting to the National Registry 

of Microbiologists evidence of acceptable continuing 

education credits or by retaking and passing the examination. The CBSP also participates in a certification 

maintenance program administered by ABSA in which 

the individual submits a certain number of acceptable certification maintenance points every 5 years to 

maintain certification.

SUMMARY

A successful biosafety program is based on an 

early estimation of risk and application of appropriate 

containment and protective measures. It is important 

to review safety incidents that occur in the institute, 

because these data will heighten individual awareness that accidents do happen despite implementing 

safeguards. The goals of a biosafety program are to:

• facilitate safe, productive research,

• prevent environmental contamination,

• conform to prudent biosafety practices, and

• comply with federal, state, local, and institutional regulations and guidelines.

To achieve the goals of the biosafety program, information pertaining to the program must be conveyed to 

the workforce, along with how it benefits the workforce. 

Presentation of concepts must be expressed in understandable terms. Initial and refresher training of personnel 

must address elements of biosafety and the institute’s 

biological safety program. To promote audience attentiveness, participation, and retention of information, training is best conducted in an informal discussion format. 

Training success is gauged by how well the workforce 

collectively internalizes the biosafety program, as evaluated within the overall context of a positive safety culture 

that permeates all work attitudes and operations. Elements of a positive safety culture include the following34:

• applying (regularly) safety practices and using 

safety terms in the workplace;

• including safety practices in the employee’s 

job description and performance appraisals;

• specifying and monitoring safe behaviors in 

the workplace;

• providing tangible rewards for promoting 

safety;

• articulating safety concerns in interactions 

with management, peers, and subordinates;

• emphasizing safety procedures when starting 

new tasks;

• briefing employees on safety procedures and 

the consequences of ignoring safety practices 

or engaging in unsafe behaviors;

• observing, reporting, and correcting hazards 

promptly; 

• keeping staff up to date on regulatory and 

institutional changes; and

• using PPE appropriately (always).

Management must consider safety a top priority and 

work closely on a daily basis with safety professionals, 

who need their support on policies to be implemented. 

Management must provide a safety program, engineering features, and equipment designed to reduce 

research-associated risks in the institute. Biosafety 

professionals strive to provide reasonable assurance of 

biological safety, but cannot guarantee absolute safety. 

In the end, the success of the safety program depends 

on the employees themselves. Safety is as much an 

individual responsibility as any other assigned performance objective.

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Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the US Army and Alliance Biosciences. Research was conducted in compliance with 

the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments 

involving animals, and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals.

43

The facility where this research was conducted is fully accredited by AAALAC International.

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49. Taylor DM, Woodgate SL, Atkinson MJ. Inactivation of the bovine spongiform encephalopathy agent by rendering 

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52. Kaye GI, Weber PB, Evans A and Venzia RA. Efficacy of alkaline hydrolysis as an alternative method for treatment 

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53. 42 CFR, Parts 72, 73, and 1003 (Possession, Use, and Transfer of Select Agents and Toxins; Final Rule), 2012.

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58. 7 CFR, Part 331 and 9 CFR, Part 121 (Agricultural Bioterrorism Protection Act of 2002: Possession, Use, and Transfer 

of Biological Agents and Toxins), 2005.

59. US Department of Defense. Safeguarding Biological Select Agents and Toxins. Washington, DC: DoD; 2004. DoD 

Directive 5210.88, USD(I).

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research laboratory. Biosecur Bioterror. 2004;2:7–16.

61. 7 USC § 54 (Transportation, Sale, and Handling of Certain Animals), 2004.

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Chapter 31

BIOLOGICAL SURETY

SAMUEL S. EDWIN, PhD*; VIRGINIA I. ROXAS-DUNCAN, PhD†; AMERICA M. CERALDE, MBA, MT(AMT)‡; 

SHELLEY C. JORGENSEN, PhD, MT (ASCP)§; and NEAL E. WOOLLEN, DVM, MSS, PhD¥

INTRODUCTION

BIOLOGICAL SURETY 

Control of Biological Select Agents and Toxins

Registration for Possession, Use, and Transfer of Biological Select Agents and 

Toxins

Security Risk Assessment

Biological Select Agents and Toxins Inventory and Accountability

Centralized Management of Long-Term Biological Select Agents and Toxins

Biological Select Agents and Toxins Inventory Audits

Biological Select Agent and Toxin Transfers

Reporting Theft, Loss, or Release of Biological Select Agents and Toxins

Identifying Select Agents and Toxins

Restricted Experiments

Biosafety

Personnel Reliability

Biosecurity

Incident Response and Emergency Management

SUMMARY

*Responsible Official and Surety Officer; Chief, Biosurety Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort 

Detrick, Maryland 21702

†

Alternate Responsible Official and Chief, Select Agent Management Branch, Biosurety Division, US Army Medical Research Institute of Infectious 

Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

‡

Alternate Surety Officer and Chief, Personnel Reliability Program Branch, Biosurety Division, US Army Medical Research Institute of Infectious 

Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

§

Lieutenant Colonel, Medical Services, US Army; Chief, Surety Branch, US Army Office of The Surgeon General/US Army Medical Command, 

G-34 Protection Division, DHHQ, 7700 Arlington Boulevard, Falls Church, Virginia 

¥

Colonel, Veterinary Corps, US Army; Director, Department of Defense Biological Select Agent and Toxin Biosafety Program Office, 1546 Porter 

Street, Fort Detrick, Maryland 21702

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INTRODUCTION

Evidence of human engagement in chemical and 

biological warfare to terrorize individuals or opposing 

armies and concurrent efforts to reduce these threats 

date back to the dawn of civilization. Some of the more 

prominent reports of possible biological warfare from 

the past millennium include the poisoning of enemy 

water wells with rye ergot fungus, a hallucinating 

agent, by the Assyrians; the use of hellebore roots to 

poison the drinking water of Kirrha by Solon of Athens 

(600 bce); the use of poison arrows dipped in gangreneand tetanus-causing agents by the Scythian archers of 

the Trojan war (400 bce); tossing of venomous snakes 

onto the opponent ships of Pergamus by Hannibal at 

Eurymedon (190 bce); hurling decomposing human 

bodies into enemy water wells by Emperor Barbarossa 

at the battle of Tortona (1155); catapulting the cadavers of plague victims over the city walls of Caffa (now 

Feodosia, Ukraine) by the Tartars (1346); distributing 

blankets and handkerchiefs from smallpox-infected 

patients to Native Americans by the British troops 

(1763); and sale of clothing from yellow fever and 

smallpox-infected patients by Confederate soldiers 

to unsuspecting Union troops during the American 

Civil War.1-5 Causative agents were linked to infectious 

diseases by 19th-century scientists Louis Pasteur and 

Robert Koch. Advances in the field of microbiology 

soon led to the isolation of microbial agents from 

diseased humans and animals. Moreover, the development of in vitro methods to grow these pathogens in 

large scale gave those interested in biological weapons 

a new perspective in selecting an agent based on its 

ability to cause fear, disease, and mass casualties. 

Recognizing the destructive powers of war, especially the devastation caused by chemical and biological weapons, developed nations of the world have attempted to establish international rules of engagement 

by drafting treaties and declarations that primarily 

focused on disarmament, laws of war, and war crimes 

(Table 31-1). The 1st International Peace Conference in 

1899 at Hague, The Netherlands, produced the Prohibition of the Use of Projectiles with the Sole Object to 

Spread Asphyxiating Poisonous Gases.6

 Ratified by all 

major powers except the United States of America, this 

declaration states that in any war between signatory 

powers, the parties will abstain from using projectiles, 

“the sole object of which is the diffusion of asphyxiating or deleterious gases.” The 2nd International Peace 

Conference held in 1907 prohibited the use of poisons 

and weapons with poisons.7 The major accomplishment of these peace conferences was the establishment 

of an international court for mandatory arbitration and 

dispute settlement between nations. 

Despite the declaration prohibiting projectiles that 

spread poisonous gases, biological weapons were 

not unequivocally prohibited. The advent of World 

War I (WWI) led to rapid progression of chemical 

and biological weapons, particularly those that were 

developed and used by the German Army. Various 

chemical weapons were used extensively during WWI 

primarily to demoralize, injure, and kill entrenched 

enemies indiscriminately. These ranged from disabling tear gas to deadly phosgene and chlorine gases. 

Due to the widespread use of chemical weapons and 

rapid development of high-explosive agents during 

this war, WWI is often referred to as “The Chemists’ 

War.” With advances in the understanding of bacterial agents during the 19th century, the German Army 

launched a massive biological weapons campaign 

against the Allied Forces during WWI. However, 

instead of targeting humans, they concentrated on 

infecting livestock (horses and mules) with Bacillus 

anthracis and Burkholderia mallei. Several animals died 

from these infections, but these biological tactics 

failed to match the success of the chemical warfare 

efforts.8

After the end of WWI and with no lasting peace in 

sight, the Biological Weapons Convention developed 

the “Protocol for the Prohibition of the Use in War of 

Asphyxiating, Poison or Other Gases and the BacteTABLE 31-1

TIMELINE OF INTERNATIONAL RULES AND 

TREATIES TO LIMIT OR BAN CHEMICAL AND 

BIOLOGICAL WEAPONS USE

Year Significant Event

1899

1st International Peace Conference

Prohibition of the use of projectiles to spread asphyxiating poisonous gases

1907

2nd International Peace Conference

Prohibition of the use of poisons and weapons with 

poisons

1925

The Geneva Protocol

Prohibition of germ (biological) and chemical 

warfare

1972

Biological Weapons Convention 

Prohibition of development, production, and stockpiling of biological weapons 

1986 The Second Review Conference

Establishment of confidence building measures 

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riological Methods of Warfare,” signed in 1925 at Geneva, Switzerland, as an extension of the international 

peace conferences of 1899 and 1907. Also known as the 

“Geneva Protocol,” this treaty permanently bans the 

use of all forms of chemical and biological warfare. 

However, it did not prohibit the use of biological or 

chemical agents for research and development, storage, and transfer. Many countries that signed on to the 

Geneva Protocol retained the right to retaliate against 

biological or chemical weapon attacks with their own 

arsenals. Treaties, declarations, and protocols produced by the international community continued to 

lack robust verification methods, leading to distrust 

among nations and reinvigoration of chemical and 

biological weapons programs prior to World War II 

(WWII). Several countries initiated biological warfare 

programs between the World Wars. The first scientifically informed use of biological agents as weapons 

began when the Japanese military conducted human 

experimentation with several infectious agents during 

combat, targeting military personnel and civilians in 

Manchuria and China.1,2,9 During WWII, many countries, including the United States, Canada, United 

Kingdom, Germany, Japan, and the Soviet Union 

had active bioweapons programs with stockpiles of 

military significance. The Japanese military used biological weapons, killing tens of thousands of civilians 

and military.1,2,9–12 

In 1972, US President Richard M Nixon made the 

decision to abandon biological weapons research and 

signed the Biological Weapons Convention, the first 

multilateral disarmament treaty banning development, production, and stockpiling of biological weapons. The US destroyed all biological weapon stockpiles 

and made the facilities that produced these weapons 

inoperable. Participant nations in the 2nd Review 

Conference in 1986 agreed to implement a number of 

confidence-building measures to prevent ambiguities, 

doubts, and suspicions and to improve international 

collaboration toward peaceful biological research.13

In 1995, an extremist microbiologist was arrested 

for obtaining Yersinia pestis by mail order in the United 

States. Concern about the ease with which diseasecausing agents could be obtained led the US Congress 

to pass the Antiterrorism and Effective Death Penalty 

Act of 1996.

14 This act directed the US Department of 

Health and Human Services (HHS) to establish: (a) a 

list of biological agents and toxins (“select agents”) that 

pose significant threat to public health and safety; (b) 

procedures for regulating the transfer of these agents; 

and (c) training requirements for entities working 

with these agents. HHS delegated this authority to the 

Centers for Disease Control and Prevention (CDC) to 

establish the Laboratory Registration and Select Agent 

Transfer Program in 1996. Congress significantly 

increased the oversight of biological select agents 

and toxins (BSAT) following the anthrax attacks of 

2001 by passing the USA PATRIOT Act (Uniting and 

Strengthening America by Providing Appropriate 

Tools Required to Intercept and Obstruct Terrorism 

Act of 2001),15 which restricted access to BSAT, and the 

Bioterrorism Act (Public Health Security and Bioterrorism Preparedness and Response Act of 2002),16 which 

included increased safeguards, security measures, 

and oversight of the possession and use of BSAT. The 

Bioterrorism Act also granted similar regulatory authority to the US Department of Agriculture (USDA) 

over select agents that pose severe threat to animal and 

plant health or products.17 This led to the establishment 

of the Federal Select Agent Program (FSAP).

The FSAP consists of the CDC Division of Select 

Agents and Toxins (CDC-DSAT) and the Animal and 

Plant Health Inspection Services (APHIS) Agricultural 

Select Agent Program that oversee the possession, 

use, transfer, and destruction of BSAT that has the 

potential to pose severe threat to public, animal, or 

plant health or to animal or plant products within the 

United States. This chapter details the key concepts of 

the FSAP and US Department of the Army’s (DA’s) 

Biological Surety Program (BSP) and highlights how 

implementation protects the worker, the community, 

and the environment. 

BIOLOGICAL SURETY

Biological surety, or “biosurety,” is a Department 

of Defense (DoD) program for commanders and directors to implement and monitor judicious application 

of core principles pertaining to control of BSAT, biosafety and occupational health, personnel reliability, 

biosecurity, and emergency response in all military 

laboratories involved in developing medical countermeasures to BSAT for service members and the public. 

The principles of safety, security, agent accountability, 

personnel reliability, and incident response plans 

formulated by chemical and nuclear surety programs 

were instrumental during the development of the 

DA’s biological surety regulations.18 Certain infectious agents and toxins, designated as BSAT, have 

the potential to pose a severe threat to public health 

and safety, animal or plant health, or animal or plant 

products, and their possession, use, and transfer are 

regulated by the HHS and the USDA under the Select 

Agent Regulations. In addition, research involving 

recombinant or synthetic nucleic acid molecules, 

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EXHIBIT 31-1

LIST OF TIER 1 BIOLOGICAL SELECT 

AGENTS AND TOXINS

Botulinum neurotoxins

Botulinum neurotoxin producing species of Clostridium

Ebola virus

Francisella tularensis

Marburg virus

Variola major virus (smallpox virus)

Variola minor virus (alastrim)

Yersinia pestis

Bacillus anthracis

Burkholderia mallei

Burkholderia pseudomallei

Foot-and-mouth disease virus

Rinderpest virus

Reproduced from: Centers for Disease Control and Prevention; Animal and Plant Health Inspection Services. List of 

Select Agents and Toxins. 12 September 2013. http://www.

selectagents.gov/Select%20Agents%20and%20Toxins%20

List.html. Accessed June 25, 2014.

including the creation and use of organisms and viruses containing recombinant or synthetic nucleic acid 

molecules, is regulated by National Institutes of Health 

(NIH) Office of Biotechnology Activities. The intent 

of the DoD BSP is to properly safeguard BSAT that is 

in the possession or custody of DoD facilities against 

theft, loss, diversion, or unauthorized access or use, 

and to ensure that operations involving such agents 

are conducted in a safe, secure, and reliable manner 

per regulatory requirements. 

The CDC-DSAT and APHIS Agriculture Select 

Agent Services monitor compliance of registered entities to HHS- and USDA-published final rules, outlined 

in 42 CFR Part 73,19 7 CFR Part 331,20 and 9 CFR Part 

121.21 One of the key components of the BSP that was 

unique to the DoD is the Biological Personnel Reliability Program (BPRP), which ensures that individuals 

with access to BSAT meet high standards of reliability 

and suitability. Recent updates to FSAP regulations 

require individuals with access to Tier 1 BSAT (Exhibit 

31-1) be enrolled in a “suitability” program similar to 

the DA’s BPRP program. With this change, the FSAP 

and the BSP correspondingly enhance the safety of 

individuals working with BSAT, protect and safeguard 

communities with biocontainment laboratories, and 

monitor the security of BSAT in entities registered 

and authorized to work with these agents and toxins 

(Figure 31-1).

Figure 31-1. Key elements of the Federal Select Agent Program and Biological Surety Program.

Control of Biological Select Agents and Toxins 

In accordance with 42 CFR Part 73,19 the CDC-DSAT 

regulates agents and toxins that pose a severe threat 

to public health and safety. The APHIS Agriculture 

Select Agent Services regulates biological agents that 

pose a significant threat to plant and plant products 

in accordance with 7 CFR Part 331.20 Agents that 

cause severe threat to humans, animals, and animal 

products are known as the “overlap agents” and are 

regulated by the CDC-DSAT and APHIS Agriculture 

Select Agent Services in accordance with 9 CFR Part 

121.21 In 2010, US President Barack Obama, through 

Executive Order 1354622 directed HHS and USDA to: 

(a) designate a subset of BSAT (Tier 1,23 see Exhibit 31-1) 

that presents the greatest risk of deliberate misuse with 

the most significant potential to cause mass casualties 

or devastating effects to the economy, critical infrastructure, or public confidence; (b) explore options for 

graded protection of Tier 1 BSAT to permit tailored risk 

management practices based on relevant contextual 

factors; and (c) consider reducing the overall number 

of agents and toxins on the select agents list. Federal 

BSAT regulations (42 CFR Part 73, 7 CFR Part 331, and 

9 CFR Part 121) have been revised in accordance with 

Executive Order 13546.20–22

The FSAP mandates the appointment of a responsible official (RO) and an alternate responsible official (ARO) within each registered entity to monitor 

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compliance with the regulations governing select 

agents and toxins (SATs). Entities are authorized to 

appoint multiple AROs. The RO is granted authority 

and control to ensure compliance with FSAP regulations. In the absence of the RO, the ARO monitors 

entity compliance to FSAP regulations. In the DoD, a 

unit commander with a mission to conduct BSAT work 

(eg, development of diagnostics, medical countermeasures, etc) appoints an RO to monitor compliance 

of the entity to DoD, Army, federal, state, and local 

regulations governing BSAT. Regulatory oversight on 

entities that have a need to possess, use, and transfer 

BSAT is initiated by submission of various CDC APHIS 

forms that are specific for each regulatory component 

(Exhibit 31-2).24

Registration for Possession, Use, and Transfer of 

Biological Select Agents and Toxins

The FSAP requires all individuals, laboratories, and 

entities to register for possession, use, and transfer of 

BSAT. The first step in this process involves providing 

information through the completion of APHIS/CDC 

Form 1, Registration for Possession, Use, and Transfer 

of Select Agents and Toxins;25 as described in 7 CFR 

331,20 9 CFR 121,21 and 42 CFR 73.19 This form consists 

of several sections targeted to provide the regulatory 

agency with critical information on the biocontainment facility, safety, security, personnel, training, and 

research plans using SAT. 

The entity is physically inspected by the FSAP following submission of the completed APHIS/CDC Form 

1. The primary focus of this inspection is compliance 

with applicable federal regulations governing BSAT (7 

EXHIBIT 31-2

ANIMAL AND PLANT HEALTH INSPECTION SERVICE/CENTERS FOR DISEASE CONTROL 

AND PREVENTION FORMS

• APHIS/CDC Form 1: Application for Registration for Possession, Use, and Transfer of Select Agents and 

Toxins 

• APHIS/CDC Form 2: Request to Transfer Select Agents and Toxins 

• APHIS/CDC Form 3: Report of Theft, Loss, or Release of Select Agents or Toxins

• APHIS/CDC Form 4: Report of the Identification of a Select Agent or Toxin

• APHIS/CDC Form 5: Request for Exemption of Select Agents and Toxins for an Investigational Product

APHIS: Animal and Plant Health Inspection Services

CDC: Centers for Disease Control and Prevention

Reproduced from: Centers for Disease Control and Prevention; Animal and Plant Health Inspection Services. Forms. 13 August 2013. 

http://www.selectagents.gov/Forms.html. Accessed June 25, 2014.

CFR 331,20 9 CFR 121,21 and 42 CFR 7319). During this 

visit, the inspectors verify the information provided in 

the submitted APHIS/CDC Form 1; evaluate personnel training, including mentorship programs; conduct 

interviews of personnel to identify issues related to 

biosafety, biosecurity, and training programs; check 

the engineering controls supporting the containment 

suites; and corroborate the commissioning or service 

records of all supporting machinery, including airhandling units, breathing-air systems, validation 

data for autoclaves, and all inactivation procedures 

to ensure that proper parameters are met and the 

methods used are determined to be efficacious with 

respect to producing nonviable waste. Ideally, entity 

registration is granted for 3 years after all inspection 

observations are satisfactorily resolved. However, 

a “conditional” registration may be granted under 

special circumstances (eg, during the interim when 

the entity needs to be operational to generate the data 

to satisfy a requirement). The FSAP inspectors ensure 

that the workers, communities, and the environment 

are not harmed by the operation of a containment or 

high containment laboratory. 

APHIS/CDC Form 1 is also used to request changes 

to an approved registration. The entity must submit 

a letter to the FSAP requesting amendment to its 

registration and furnish the revised sections of the 

APHIS/CDC Form 1 related to the modifications. Most 

common amendments to registration involve addition 

and removal of personnel, name changes, addition or 

removal of agents or toxins, and changes in statement 

of work, including changes in project design, agent 

strains, animal models, modes of agent administration, 

and new laboratory projects.

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Security Risk Assessment

Security risk assessment (SRA) is the method used 

to approve an individual for access to select agents or 

toxins in accordance with the USA PATRIOT Act of 

2001 and the Public Health Security and Bioterrorism 

Preparedness and Response Act of 2002. The Federal 

Bureau of Investigation Criminal Justice Information 

Services division determines if an individual who has 

been identified by a registered entity as having a legitimate need to access a select agent or toxin meets one 

of the statutory restrictors that would restrict access. 

A “restricted person” under 18 USC 175b (USA 

PATRIOT Act) is an individual26 who: 

• is under indictment for a crime punishable 

by imprisonment for a term exceeding 1 year 

or who has been convicted in any court of a 

crime punishable by imprisonment for a term 

exceeding 1 year;

• is a fugitive from justice;

• is an unlawful user of any controlled substance; 

• is an alien illegally or unlawfully in the United 

States;

• has been adjudicated as a mental defective or 

has been committed to any mental institution;

• is an alien (other than an alien lawfully admitted for permanent residence) who is a national 

of a country as to which the secretary of state 

has made a determination (that remains in 

effect) that such country has repeatedly provided support for acts of international terrorism; or

• has been discharged from the armed services 

of the United States under dishonorable conditions.

All individuals, including the RO, AROs, laboratory research staff, and animal-care workers requesting unescorted access to CDC- or APHIS-registered 

spaces containing BSAT require an approved SRA. 

Escorted individuals, such as inspectors and visitors 

with no access to BSAT, do not require an approved 

SRA. FSAP works closely with the Federal Bureau of 

Investigation Criminal Justice Information Services 

division to identify individuals who are prohibited 

to access BSAT based on the restrictions identified in 

the USA PATRIOT Act of 2001.15 This process involves 

submitting an amendment to the lead agency (CDC or 

APHIS) and adding the individual to the entity registration to obtain a unique Department of Justice number, which is recorded on a Bioterrorism Security Risk 

Assessment Form (FD-961). The completed FD-961 is 

reviewed, certified by the RO, and submitted to CJIS 

with two sets of fingerprints.26 The FSAP authorizes 

individual access to BSAT based on the results of the 

SRA. The SRA is renewed every 3 years. All individuals 

with approved SRA undergo a general initial training, 

which provides site-specific information on biosafety, 

security, incident response, and insider threat awareness. Refresher training is provided annually to all 

SRA-approved individuals.

Biological Select Agents and Toxins Inventory and 

Accountability

FSAP regulations require complete, current, and 

accurate inventory of all long-term (LT) BSAT. Materials that contain or have been exposed to infectious 

select agents, including (but not limited to) laboratory 

cultures, animals, animal tissues, confirmed clinical 

specimens, plants, and plant tissues, are subject to 

FSAP regulations. Select toxins and recombinant or 

synthetic nucleic acids encoding functional forms of 

select toxins are also regulated. Animals inoculated 

with select toxins and their tissues are exempt from 

FSAP regulations. Inventory records are not required 

for BSAT that the FSAP has excluded from the provisions of the Select Agent Regulations, nor for inactivated BSAT materials as long as an approved method 

for inactivation is used. 

CDC-DSAT defines LT storage as placement in a 

system designed to ensure viability for future use. As 

a rule, LT BSAT materials are not part of an ongoing 

experiment and have not been accessed for a significant period of time (eg, 30 calendar days).27 SAT 

are considered working stock (WS) if the materials 

are: (a) a part of an ongoing experiment, (b) accessed 

frequently, or (c) not stored for an extended period 

of time. FSAP regulations do not require inventory 

records for BSAT classified as WS; however, all WS 

must be kept and used in secure locations by approved 

individuals (ie, those with current SRAs enrolled in a 

suitability program, if accessing Tier 1 agents). The 

DA’s interim guidance on BSAT inventory management allows BSAT to remain in WS status for up to 180 

days; however, the DA guidance document requires 

individuals to maintain detailed records of all BSAT 

WS materials at all times. 

Significant amounts of BSAT WS can be generated 

in a containment laboratory on any given day. Accounting for these materials can be challenging, as 

they are continuously used or consumed in various 

experiments. Entities with large BSAT inventories must 

establish procedures to retain only “valuable” BSAT. 

Establishing peer-reviewed and accepted criteria for 

retention and destruction of LT BSAT materials can be 

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beneficial to the investigators and the host entity. An 

example of criteria developed for retention and destruction of LT BSAT materials is shown in Exhibit 31-3. 

Specimen boxes containing LT BSAT materials can 

be wrapped with tamper-evident materials after verification by two BPRP-certified individuals. Follow-up 

tube-by-tube inventory is not needed as long as the 

tamper-evident seals remain intact. Reducing access 

and repeated contact with LT BSAT materials will 

preserve specimen integrity and will also allow for 

accurate real-time inventory of these materials. Additionally, uniform labeling of LT BSAT specimens 

should remain a priority for research staff in order to 

have well-labeled research materials for all current and 

future investigations. Advances in labeling technologies permit for human-readable information, barcodes, 

and radiofrequency identification tags to be incorporated on any size of specimen labels. Specimen tags 

that adhere to frozen tubes are also available, making 

it possible to label archival materials. 

Centralized Management of Long-Term Biological 

Select Agents and Toxins 

Maintaining accurate and current inventory of LT 

BSAT materials at all times can be burdensome to 

principal investigators (PIs) and research staff who 

are focused on meeting timelines for deliverables 

and project goals. BSAT inventory discrepancies 

identified during internal audits or announced and 

unannounced inspections by regulatory agencies can 

result in serious consequences to the registered entity 

with respect to continuation of research and loss of 

public trust. One proposed solution to this dilemma 

is to establish centralized management of LT BSAT 

materials under the care of the RO and the AROs to 

alleviate considerable inventory and accountability 

burden from the PIs and research staff (Exhibit 31-4). 

Under this model, LT BSAT materials that have been 

verified by a third party would be labeled with PIspecific information, wrapped with tamper-evident 

materials, and centrally stored within the registered 

laboratory space in dedicated storage containers with 

restricted access. 

Centralized LT BSAT inventory management 

would enhance readiness for unannounced regulatory compliance inspections that include BSAT inventory verification, and would simplify the transition 

of BSAT inventory when a PI retires or leaves the 

institution. Verification of LT BSAT inventory by the 

PI or researcher and a third party would also allow 

for identification of archival specimens requiring new 

uniform labels. The PI or researcher will identify BSAT 

specimens no longer needed for current and future 

investigations, including potentially contaminated 

specimens, specimens with reduced or no bioactivity, 

and excess specimens.

Biological Select Agents and Toxins Inventory 

Audits

Registered entities are required to conduct complete 

inventory audits of a PI’s BSAT holdings in LT storage 

during physical relocation of a collection or inventory upon the departure of a registered PI with BSAT 

holdings, or in the event of a theft or loss of BSAT. In 

addition to the FSAP requirements, Army Regulation 

(AR) 50-1 requires annual 100% physical inventory of 

all BSAT holdings by each PI.28 If the LT BSAT materials are verified and wrapped, the inventory burden is 

dramatically reduced, as long as the tamper-evident 

seals are intact. Army regulation also requires BSAT 

inventory audits of each registered PI at least once 

annually by the biological surety program staff. These 

EXHIBIT 31-3

SAMPLE CRITERIA FOR RETAINING OR DESTROYING BIOLOGICAL SELECT AGENTS 

OR TOXINS

Retention Criteria

1. Unique materials (serotypes, strains, etc)

2. Support ongoing research activities and all existing 

agreements

3. High scientific value for future scientific investigations

4. Deemed evidence material by law enforcement

5. Materials retained from published studies

Destruction Criteria

1. Potentially contaminated and/or degraded materials (eg, samples that have been subjected to 

multiple freeze/thaw cycles)

2. Excess quantities from a specific microbe or toxin

3. No anticipated future scientific value with the understanding that projected future mission requirements can be difficult

4. Materials that lack expected bioactivity

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inventory audits include inspection of laboratory records of BSAT usage, physical inventory verification 

of both LT storage and WS BSAT, verification of the 

SAP registration of the PIs, BSAT transfer documentation, and BSAT destruction records. The annual 

BSAT inventory audits provide a great opportunity to 

interact with the registered PI and his or her technical 

staff and to identify areas where additional training 

may be warranted. 

Biological Select Agent and Toxin Transfers 

The Select Agent Regulations require entities to develop provisions and policies for shipping, receiving, 

and storing SAT, including documented procedures 

for receiving, monitoring, and shipping all SAT. There 

are primarily two types of BSAT transfers: intraentity 

and interentity. BSAT material must be packaged by 

individuals approved by the HHS secretary or APHIS 

administrator for access to SAT. If the transfer involves 

Tier 1 BSAT, the approved individuals must be certified in the entity’s suitability program or personnel 

reliability program. 

Intraentity transfers of SAT are performed between 

two registered PIs with a complete chain of custody 

document. The sender and receiver must be registered 

with the SAP for the BSAT being transferred. These 

transfers are physically performed by approved individuals in accordance with entity-specific standard 

operating procedures. An approved APHIS/CDC 

Form 2 is not required for intraentity transfer of BSAT 

materials. 

Interentity transfers of SAT require an approved 

APHIS/CDC Form 2 prior to physical transfer of 

these materials. Once issued, an approved APHIS/

CDC Form 2 is valid for 30 days. These transfers are 

governed by the US Department of Transportation 

Hazardous Material Regulations found in 49 CFR, 

parts 100 to 185.29 The approved individual packaging SAT must ensure compliance with all applicable 

laws concerning packaging and shipping. DA uses approved BSP personnel trained and certified in shipping 

EXHIBIT 31-4

CENTRALIZED BIOLOGICAL SELECT AGENTS AND TOXINS INVENTORY MANAGEMENT 

CONSIDERATIONS

Reduce inventory burden on PI/researcher

• Transfer long-term BSAT accountability responsibility to RO/ARO and dedicated biological surety staff (select 

agent managers)

• Limit principal investigator/researcher responsibility to working stock BSAT materials

Enhance accountability and security

• Manage long-term BSAT materials with dedicated staff

° 100% long-term BSAT inventory verification and tamper-evident wrapping

° Long-term BSAT consolidated within registered space 

° Eliminate variability in record keeping from multiple PIs/researchers

• Enhance security of BSAT materials

° Long-term BSAT in dedicated and locked freezers within registered spaces

° Limit physical access to long-term BSAT materials

Manageable process with economy of space and personnel

• Enhance real-time inventory awareness for long-term BSAT

• Consolidate long-term BSAT within containment spaces

Maintain mission capability with enhanced flexibility

• Retain all unique and critical BSAT materials 

• Capture all essential characterization and experimental data (eg, DoD BSAT database)

• Prepared to receive or send BSAT to other DoD entities at all times 

Inventory reduction

• Assist PIs in identifying and destroying BSAT with no current or future scientific value

• Encourage sharing of BSAT among PIs within the institute 

ARO: alternate responsible individual; BSAT: biological select agents and toxins; DoD: Department of Defense; PI: principal investigator; RO: responsible official

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procedures to verify the contents of the SAT shipments 

inside the containment laboratories prior to packaging. A completed chain of custody form is retained 

with copies of shipping documents for at least 5 years 

(DoD standard). The individual who witnesses packaging inside the containment laboratory also verifies 

the approved APHIS/CDC Form 2 and the shipping 

documents. The FSAP has amended the select agent 

regulations to accept and promote the recommendation of the report of the Defense Science Board Task 

Force, DoD Biological Safety and Security Program,30

regarding the “lost in crowd” approach for all SAT 

shipments. However, registered DoD laboratories are 

currently required to use a carrier that maintains positive control, ensures chain of custody, is certified to 

handle HAZMAT (hazardous materials) standards 6.1 

(poisons) and 6.2 (infectious substances), and requires 

two qualified drivers possessing current secret clearance, with at least one driver in the truck or within 25 

feet of the truck at all times. Harmonization of DoD 

regulations with the FSAP is being discussed to standardize the select agent and toxin shipping practices. 

Exempt quantity (permissible amount) transfers 

of select toxins (Table 31-2) are not regulated by the 

FSAP.31 The “toxin due diligence” provision was developed by FSAP to address concern that someone might 

stockpile toxins by receiving multiple orders below 

the excluded amount. It requires a person transferring 

toxins in amounts which would otherwise be excluded 

from the provisions to: (a) use due diligence to ensure 

that the recipient has a legitimate need to handle or 

use such toxins; and (b) report to FSAP if they detect a 

known or suspected violation of federal law or become 

aware of suspicious activity related to the toxin.32

Most “exempt” toxin transfers are to a nonregistered 

PI or a collaborator who demonstrates a legitimate 

need to handle or use the toxin being transferred. Due 

diligence must precede the transfer to ensure that the 

recipient does not exceed the exempt quantity limit 

established by the FSAP with any existing remnant 

quantities in their laboratories from previous investigations. The person initiating the transfer can require 

the recipient to complete documentation stating the 

intended use of the toxins and a statement indicating 

that receiving the requested amount of the toxin will 

not put them over the limits established for the select 

toxins by the FSAP. Tracking “exempt” select toxin 

transfers (sending and receiving) and monitoring their 

use must be an integral part of a due diligence effort 

at the entity level to avoid investigators accumulating quantities of select toxins above the permissible 

amounts at any time.

Reporting Theft, Loss, or Release of Biological 

Select Agents and Toxins

FSAP requires an entity to immediately notify 

CDC or APHIS and appropriate federal, state, or local law enforcement agencies (by e-mail, facsimile, or 

telephone) of incidents involving theft, loss, or release 

(occupational exposure or release of an agent or toxin 

outside of the primary barriers of the bio-containment 

area) of SAT.33 Thefts or losses also must be reported 

even if the SAT is subsequently recovered or the responsible parties are identified. A completed APHIS/

CDC Form 3 must be submitted within 7 calendar 

days. 

A BSAT inventory deficiency investigation may 

involve: (a) immediate notification to the physical 

security office; (b) 100% physical inventory of all of 

the registered PI’s BSAT holdings by the RO or ARO; 

(c) complete inspection of the PI’s BSAT usage records 

(laboratory notes); and (d) a complete database records 

check of the BSAT inventory holdings of the PI. If theft 

of BSAT is suspected, appropriate law enforcement 

agencies must be informed. 

Release of BSAT from “primary containment” or 

release resulting in “potential exposure” to individuals requires immediate notification to the FSAP. Spills 

of SAT in biological safety level (BSL)-4 laboratories 

(sealed laboratories with personnel wearing positive 

pressure encapsulated suits) can be safely cleaned up 

without potential human exposure; no FSAP reporting is necessary because the entire BSL-4 laboratory 

TABLE 31-2

PERMISSIBLE TOXIN AMOUNTS 

Health and Human Services Toxins Amount

Abrin 100 mg

Botulinum neurotoxins 0.5 mg

Short, paralytic alpha conotoxins 100 mg

Diacetoxyscirpenol 1,000 mg

Ricin 100 mg

Saxitoxin 100 mg

Staphylococcal enterotoxins (subtypes A, 

B, C, D, and E)

5 mg

T-2 toxin 1,000 mg

Tetrodotoxin 100 mg

Centers for Disease Control and Prevention; Animal and Plant 

Health Inspection Services. Permissible Toxin Amounts. 5 October 

2012. http://www.selectagents.gov/Permissible%20Toxin%20

Amounts.html. Accessed June 25, 2014.

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is considered “primary containment.” However, if an 

individual experiences a breach in his or her positive 

pressure encapsulating suit at the same time as a spill 

or work done with animals outside of primary containment, initial notification to FSAP reporting is required, 

followed by the completion of APHIS/CDC Form 3. In 

contrast, SAT spills in BSL-2 and BSL-3 laboratories 

(unsealed directional airflow laboratories with personnel not wearing positive pressure encapsulated suits) 

outside of a functioning biological safety cabinet are 

reportable to FSAP, as these laboratory spaces are considered “secondary containment.” The data collected 

and analyzed by the CDC on theft, loss, or release 

reporting from 2004 to 2010 indicate that the risk of 

exposure from BSAT managed by US laboratories to 

the general population is low.34

Identifying Select Agents and Toxins

Identifying BSAT as a result of diagnosis, verification, and proficiency testing, and final disposition of 

the identified agent or toxin must be reported to FSAP 

within 7 calendar days by completing APHIS/CDC 

Form 4. Identifying Tier 1 BSAT (see Exhibit 31-2) from 

diagnostic samples requires immediate (ie, within 24 

hours) reporting to FSAP via e-mail, facsimile, or telephone. BSAT identified from proficiency testing specimens must be reported within 90 days of receipt of the 

sample. Any amount of select toxin identified must be 

reported to FSAP. Entities not registered with the FSAP 

are also required to report BSAT that have been identified from diagnostic specimens. Unregistered entities 

have 7 calendar days to transfer to a registered entity 

or destroy the identified SAT to remain in compliance 

with current federal regulations. 

Restricted Experiments

An individual or an entity approved by the FSAP 

may not conduct restricted experiments without prior 

approval by the HHS secretary or APHIS administrator. 

Restricted experiments are: (a) experiments that involve 

the deliberate transfer of, or selection for, a drug resistance trait to select agents that are not known to acquire 

the trait naturally, if such acquisition could compromise 

the control of disease agents in humans, veterinary 

medicine, or agriculture; and (b) experiments involving 

the deliberate formation of synthetic or recombinant 

nucleic acids containing genes for the biosynthesis of 

select toxin lethal for vertebrates at an LD50 (the amount 

necessary to kill 50% of the subject population) that is 

less than 100 ng/kg body weight.19 Additional guidance 

on restricted experiments involving recombinant or synthetic nucleic acids is outlined in the NIH’s Guidelines for 

Research Involving Recombinant or Synthetic Nucleic Acid 

Molecules.

35 This guidance is mandated for research that 

is conducted at or sponsored by an entity that receives 

any support for recombinant or synthetic nucleic acid 

research from the NIH, including research performed 

directly by the NIH. 

Most registered entities designate the responsibility 

of identifying, reviewing, and approving restricted 

experiments to their Institutional Biosafety Committee 

(IBC). The biosafety officer and the RO are members 

of the IBC. Entity-specific IBC-approved research proposals with restricted experiments are forwarded to 

FSAP for review and approval. Restricted experiments 

containing HHS and overlap select agents will be further reviewed by the Intragovernmental Select Agents 

and Toxins Technical Advisory Committee. Restricted 

experiments involving USDA select agents will be further reviewed by subject matter experts from APHIS. 

A typical request to FSAP to review a restricted experiment includes, but is not limited to, description of:

• the proposed experiment, including intended 

objectives,

• nucleic acid insert and the intended biological characteristics of the recombinant gene 

product, 

• cloning/expression vector,

• host organism used for molecular cloning,

• selection methods (recombinant or passive),

• antimicrobial markers use, 

• BSL considerations, 

• estimated amount of toxin (recombinant or 

synthetic) to be produced (if applicable), and 

• any planned animal or plant experiments.36

Restricted experiments using recombinant and 

passive selection methods and all select agent products resulting from these experiments are also subject 

to FSAP regulations. Transfer of any products of 

restricted experiments must be coordinated through 

the FSAP. The DA and the DoD require all of their 

research laboratories to remain in full compliance with 

all federal regulations governing BSAT.

Biosafety

Biosafety in microbiological and biomedical laboratories is based on two key principles: “containment” 

and “risk assessment.” Core concepts of containment 

include microbiological practices, safety equipment, 

and facility safeguards that protect laboratory workers, the environment, and the public from exposure to 

infectious organisms. Risk assessment is a process that 

enables the appropriate selection of microbiological 

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practices, safety equipment, and facility safeguards 

that can prevent laboratory-associated infections. 

Modern biosafety practices described in the 5th edition of Biosafety in Microbiological and Biomedical Laboratories37 are accepted as standards of practice by all 

CDC-registered entities to conduct work with SAT. 

The DA mandates the use of the current version of 

the manual and DA pamphlet 385-69, Safety Standards 

for Microbiological and Biomedical Laboratories,

38 in all 

US Army activities and facilities in which infectious 

agents or toxins are used, produced, stored, handled, 

transported, transferred, or disposed of, including the 

Army National Guard, the US Army Reserve, and contractors and consultants conducting microbiological 

and biomedical activities for the Army. The detailed 

principles and practices of biosafety are covered in a 

separate chapter of this textbook. 

It is critically important to thoroughly train individuals in biosafety practices prior to providing access 

to the containment laboratories to handle, manipulate, 

and store BSAT. Training must include:

• microbiological laboratory techniques;

• use of personal protective equipment (PPE), 

safety equipment, and containment laboratory equipment;

• information on bloodborne pathogens;

• an entity-specific chemical hygiene plan;

• BSAT-specific information;

• emergency exit operations;

• immediate first aid; and 

• reporting requirements for potential exposures to infectious agents and toxins. 

Some of this initial training can be structured into 

mentorship programs in which individuals approved 

as mentors ensure new laboratory workers are able to 

work safely within the containment laboratories before 

they are granted independent access. In addition to 

project-specific training, the worker requesting access 

to the containment laboratories with BSAT is trained 

to recognize biohazards, understand potential health 

risks associated with exposures, provide appropriate 

first aid, and carry out follow-up reporting procedures. 

FSAP regulations require individuals with access to 

Tier 1 BSAT to be enrolled in an occupational health 

program.19–21 AR 50-128 also requires commanders 

and directors of the entities with a biological surety 

mission to establish and implement an occupational 

health program. Core elements of an occupational 

health program include: (a) risk assessment, (b) medical 

surveillance, (c) access to clinical occupational health 

services and management, and (d) hazard communication. Select agent risk assessments should consider: 

• route of exposure,

• infectious dose,

• agent virulence,

• incubation period,

• environmental stability,

• communicability,

• genetic modification,

• available resources for pre- and post-exposure 

prophylaxis,

• available vaccine options,

• PPE use, and 

• biocontainment requirements.39

Occupational health plans are required to comply 

with US Department of Labor and Occupational Safety 

and Health Administration regulations, as well as 

patient confidentiality laws. Promoting a safe and 

healthy work environment requires limiting exposures 

to infectious agents and toxins, promptly detecting 

and treating exposures, and using information gained 

from incidents to further improve safety measures and 

worker training. Occupational health and safety is a 

shared responsibility among the individual workers, 

their supervisors, PIs, biosafety specialists, healthcare 

providers, and the employer. 

Personnel Reliability

Personnel reliability programs existed for decades in 

the US military. The BPRP, modeled after the military’s 

nuclear and chemical personnel reliability programs, 

ensures that individuals with access to BSAT meet the 

highest standards of reliability.18,40–42 The concept of 

personnel reliability was implemented over a decade 

ago in DoD laboratories working with BSAT.43 AR 50-1, 

Biological Surety, outlines the BPRP described herein.28

Individuals with access to BSAT in DA and DoD 

laboratories are required to be enrolled in a BPRP. 

The FSAP added the “suitability” requirement for individuals with access to Tier 1 BSAT in October 2012. 

The FSAP’s suitability assessment of personnel is based 

in part on the DoD’s personnel reliability programs. 

The BPRP and the suitability assessment of personnel 

are primarily designed to reduce the risk of SAT misuse by an individual who has access to these agents 

(insider threat). The intent of the US Army’s BPRP18

and the FSAP’s suitability assessment of personnel44

is the same; however, there are significant differences 

between the two programs. 

The commander or director is the head of the organization’s BPRP and can serve as the reviewing official. The reviewing official appoints certifying officials 

(COs) who determine the reliability and suitability of 

individuals requiring access to SAT and ensure they 

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Medical Aspects of Biological Warfare 

are appropriately qualified and trained to perform their 

duties. Commanders and directors may appoint BPRP 

monitors to assist COs in administering day-to-day 

activities; however, COs are responsible for continuous 

monitoring of individuals enrolled in the personnel reliability program. The reviewing official monitors the CO’s 

decisions to disqualify individuals and may overturn 

them when procedures are unfair, inconsistent, or incorrectly applied. AR 50-1 requires the reviewing official to 

review all individual disqualification actions submitted 

by the CO.28 The FSAP recommends suitability decisions 

on individuals requesting access to Tier 1 BSAT be a 

combined decision of the CO, RO, and the entity leadership (eg, commander, director, or reviewing official).44

To begin enrollment in the BPRP, supervisors of 

individuals who need to access BSAT in the CDCregistered containment spaces contact the designated 

CO. The CO is the gatekeeper for access to BSAT, 

ensuring that persons requesting access have met all 

the qualifying conditions. FSAP ensures that restricted 

persons do not have access to BSAT through the SRA 

process (see section above); the Army’s BPRP further 

ensures that persons with access to BSAT are:

• trustworthy,

• mentally and emotionally stable,

• physically competent,

• free of unstable medical conditions,

• able to exercise sound judgment,

• willing to accept responsibility,

• able to adapt to changing work environments,

• free from drug and alcohol abuse, 

• willing to participate in random drug testing, 

and

• willing to comply with all training requirements.

Enrollment in the Army’s BPRP involves: 

• initial interview,

• personnel records review,

• personnel security investigation, 

• medical evaluation,

• drug testing, and 

• CO’s final evaluation and briefing.

The order of steps in the process is discretionary; 

nevertheless, each step must take place and be fully 

documented.

Initial Interview

The CO is required to conduct a personal interview of a potential enrollee in the BPRP to assess 

suitability and reliability. The CO must inform 

the candidate of the Privacy Act of 197445 and the 

Health Insurance Portability and Accountability 

Act46 to obtain consent to proceed with the screening process. Although not required by the regulations, the initial interview may also include a written questionnaire. The candidate is asked questions 

that will allow the CO to determine whether he or 

she has engaged in any activities that would be 

either mandatory or potentially disqualifying factors. Mandatory disqualifying factors are those that 

are beyond the discretion of the CO for deciding 

reliability and suitability. If extenuating circumstances exist, the reviewing official may request an 

exception for the individual’s enrollment through 

command channels. The following are mandatory 

disqualifying factors: 

• current substance or alcohol dependence;

• drug or substance abuse within 5 years prior 

to the initial interview;

• trafficking, cultivating, processing, or manufacturing illegal or controlled drugs within 

the past 15 years;

• drug or substance abuse while enrolled in 

BPRP;

• inability to meet safety requirements; or

• meeting the criteria of a restricted person as 

defined by 18 USC § 175b.47

Other potentially disqualifying factors include:

• alcohol-related incidents or alcohol abuse;

• drug or substance abuse more than 5 years 

prior to initial interview; or

• mental or physical medical condition, medication usage, or medical treatment that may 

result in:

° altered state of consciousness,

° impaired judgment or concentration,

° increased risk of impairment if exposed to 

BSAT,

° impaired ability to wear PPE,

° inability to meet physical requirements, or

° inappropriate attitude, conduct, or behavior.

The CO must inform the candidate that he or she 

will be subject to random, unannounced drug testing 

as part of continuous monitoring; an initial negative 

test is required prior to certification in the BPRP. 

The CO must also explain to the candidate about: (a) 

continuous monitoring, (b) the requirement for selfreporting, and (c) use of prescription drugs. The initial 

interview is a good opportunity for the CO to get to 

know the candidate and to begin a relationship based 

on mutual trust and respect. 

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Biological Surety

Personnel Records Screening 

Once the CO has completed the initial interview and 

found the candidate to be suitable for enrollment, the 

applicant’s personnel records are screened by a supporting personnel officer. The screening official will 

determine the individual’s citizenship and identify 

it to the CO. Any potentially disqualifying information (PDI) discovered during the screening process 

is immediately communicated to the CO. Individuals with extended federal government service may 

have information in their personnel records from the 

inception of their employment. In contrast, information in contract employees’ personnel records may be 

limited to the length of their employment with that 

company. Anything that may indicate unsatisfactory 

employment history or dereliction of duty, such as job 

applications, enlistment contracts, and any other available pertinent record should be reviewed for PDI. The 

CO acts on any PDI discovered during the personnel 

records screening; however, the CO does not retain 

any records of this information. 

Personnel Security Records Screening

The current minimum personnel security investigation (PSI) requirement for unescorted access to 

BSAT within DoD is a favorably adjudicated singlescope background investigation. This level of PSI is 

conducted to confer top secret clearance. However, 

a security clearance is not required for BPRP enrollment. The personnel security manager will request 

a copy of the PSI from the Office of Personnel Management on behalf of the CO. The personnel security 

officer will expeditiously provide any adverse information to the CO, ensuring Privacy Act requirements 

are not violated. Personnel scheduled for initial assignment to BPRP positions must have the appropriate and favorably adjudicated PSI completed within 

the 5 years preceding certification to the BPRP. PSI 

files contain sensitive information and should only 

be retained for the time necessary to determine suitability and reliability. The CO will review the results 

of the personnel security investigation to determine 

if the individual meets the suitability and reliability 

requirements of the BPRP.48 The FSAP is not prescriptive, with respect to PSI, above what is required to 

obtain an SRA for suitability assessment of individuals with access to Tier 1 BSAT. 

Medical Evaluation

The competent medical authority (CMA) medically 

evaluates the candidate to ensure that the individual 

seeking enrollment in the BPRP is physically, mentally, 

and emotionally stable; alert; competent; dependable; 

and free of unstable medical conditions that may 

impact BPRP duties.18 The CMA meets with the candidate and reviews the individual’s medical records 

to identify any PDI. Medical PDI includes any medical condition, medication use, or medical treatment 

that may result in an altered level of consciousness, 

impaired judgment or concentration, impaired ability 

to safely wear required PPE, or impaired ability to perform the physical requirements of the BPRP position, 

as substantiated by the medical authority to the CO. 

The candidate may also provide the CMA copies of 

medical records from a personal healthcare provider. 

If medical records are incomplete or inadequate, the 

CMA will conduct the appropriate medical evaluation. This may include a mental health evaluation if 

the CMA determines such an evaluation is prudent or 

upon request by the CO.18 Medical PDI is reported to 

the CO with recommendations regarding the person’s 

fitness for assignment to these duties or limitations in 

duties or reasonable accommodations that might allow 

the individual to perform his or her duties without 

compromising worker safety. 

Drug Testing

All candidates for BPRP must complete drug testing within 6 months prior to initial certification. All 

drug test results will be provided to the CO before 

the individual is certified in the BPRP. Positive drug 

test results indicating illegal drug use will result in 

disqualification. 

Certifying Official’s Final Evaluation and Briefing

After the candidate has completed all phases of the 

screening, the CO conducts a final evaluation of all 

the information received during the screening process and conducts a final interview. During the final 

interview, the candidate will have an opportunity to 

review and discuss any BPRP-relevant issues, including PDI discovered during the screening process and 

the circumstances surrounding such an event, and 

before the CO’s decision on the candidate’s suitability and reliability for the program. During this 

time the CO: 

• reviews the duties and responsibilities of 

the individual’s BPRP position, including 

required PPE use; 

• discusses the expectations for continuous 

monitoring; 

• reviews disqualifying factors, including any 

incidents or medical issues that may have occurred since the initial interview; 

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Medical Aspects of Biological Warfare 

• reminds the individual that prescription drug 

use must be under the supervision of a healthcare provider; and 

• reviews the self-reporting requirements of the 

BPRP. 

At the end of the interview, the CO should inform 

the candidate whether he or she is suitable for the 

program, and the individual signs DA Form 3180 indicating his or her understanding of the program and 

willingness to comply with the requirements. If the 

candidate is determined to be eligible, the CO ensures 

that the candidate has completed all the core safety, 

security, and emergency training. The CO will notify 

the RO immediately after the individual is certified 

in the BPRP.18

Individuals certified in the BPRP are subject to continuous monitoring. Continuous evaluation includes, 

but is not limited to: 

• self-reporting, 

• peer and supervisor observation and reporting,

• periodic unannounced drug testing,

• periodic personnel security investigations, 

• periodic medical evaluations by the CMA, and 

• CO observation and evaluation. 

The FSAP recommends the RO’s involvement in the 

development, implementation, and administration of 

the Tier 1 BSAT suitability assessment program. The 

RO must ensure that access to Tier 1 BSAT is limited to 

individuals in the suitability program with the entity’s 

ongoing suitability monitoring, and have current FSAP 

approval to access SAT. Ongoing efforts to harmonize 

the DoD regulations governing BSAT with the FSAP 

are expected to clarify the role of the RO in the BPRP 

or suitability assessment program. 

Biosecurity

Safeguarding BSAT is a high priority for the DoD41,43

and the FSAP.49 According to the Office of Science and 

Technology Policy, the term “biosecurity” refers to 

the protection, control of, and accountability for highconsequence biological agents and toxins and critically 

relevant biological materials and information within 

laboratories to prevent unauthorized possession, 

loss, theft, misuse, diversion, or intentional release.50

AR 190-17, Biological Select Agents and Toxins Security 

Program51 prescribes the policy, responsibilities, procedures, and minimum standards for safeguarding 

BSAT. Biosecurity plans are based on risk assessments, 

are entity specific, and constitute sensitive information. A site-specific security plan based on risk assessments must be developed by all CDC-registered 

entities with BSAT. An effective site-specific security 

plan will have initial and continuous input from and 

interactions with: 

• security personnel,

• commanders or directors,

• subject matter experts,

• local law enforcement officers,

• ROs and AROs,

• biosafety officers,

• occupational health CMAs,

• facilities management personnel, and

• information security management personnel. 

An effective biosecurity plan is based on operational 

processes, accounts for all BSAT from creation or acquisition to destruction, does not violate any laws, weighs 

both primary and secondary affects, and is reviewed 

and updated at least annually. 

The biosecurity program for CDC-registered entities 

with BSAT can be broadly divided into at least five 

major components: (1) BSAT security, (2) physical security, (3) personnel security, (4) operational security, 

and (5) information security. 

Biological Select Agents and Toxins Security

There are a number of factors that contribute to the 

challenge of effective BSAT inventory and accountability within containment laboratories. Temperaturesensitive microbes, confined spaces, sharing of limited 

freezer space by multiple investigators, co-existence 

of both LT and WS BSAT, multiple users, and illegible 

specimen labels can all contribute to ineffective BSAT 

inventory and accountability. Uniform labels with 

human-readable information and barcodes, inventory 

verification and wrapping of all LT BSAT with tamperevident materials, centralized storage of wrapped LT 

BSAT within the containment laboratory, and controlled access to LT BSAT materials can preserve the 

integrity of the stored specimens and provide an accurate real-time inventory of these materials. These LT 

BSAT management strategies can be instituted without 

affecting ongoing research. Entities must establish 

standard operating procedures for incoming, outgoing, and intraentity BSAT transfer. All transfers must 

be conducted with chain-of-custody documentation, 

which is retained and verified with BSAT inventory 

databases. BSAT destruction documents should be 

confirmed with the BSAT databases. BSAT inventory 

audit should include review of laboratory notes and 

verification of BSAT WS materials. All BSAT materials 

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must be maintained in CDC-registered laboratory 

spaces with restricted access to prevent theft, loss, or 

release of these materials. All personnel with access to 

BSAT must be trained in FSAP regulations, including 

reporting requirements. Entities must also conduct a 

complete inventory audit of a PI: (a) when the PI with 

BSAT holdings leaves the entity; (b) in the event of a 

theft or loss of BSAT; and (c) upon physical relocation 

of a collection of BSAT materials. These practices will 

also prepare the entity for any unannounced inspections. Effective BSAT inventory and accountability 

practices will preserve the integrity of the specimens 

and increase research efficiency within the containment laboratories. 

Physical Security

A physical security plan developed using site-specific risk assessment can detect, deter, or delay threat 

and provide sufficient time to respond to the threat. 

Security barriers such as perimeter fences, armed 

guards, walls, locked doors, secured laboratories, and 

locked freezers can deter intrusion and deny access to 

BSAT. FSAP regulations require: 

• controls limiting access to CDC-registered 

spaces to approved individuals with access 

to BSAT,

• provisions to safeguard animals and plants 

infected with select agents, 

• review and update of access logs to CDCregistered spaces, 

• prevention of access credentials sharing,

• procedures for reporting loss of access credentials,

• procedures for personnel changes, 

• three barriers (physical structures that are 

designed to prevent access to unauthorized 

individuals) to access Tier 1 BSAT, 

• intrusion detection systems where Tier 1 BSAT 

is manipulated or stored, 

• response time not exceeding 15 minutes for a 

force capable of interrupting a threat to Tier 

1 BSAT manipulation and storage spaces, and 

• procedures for access control in power 

failures.19,49

Personnel Security

The FSAP and DoD consider personnel security 

integral to detecting insider threat. The personnel 

security office at the entity level works with the RO 

to facilitate SRA documentation and fingerprinting 

for individuals requesting access to CDC-registered 

spaces. Personnel security also includes: verification 

of background information, security investigations, 

personnel dossier reviews, identifying violators of 

security and safety procedures, and identifying individuals who threaten or support those who threaten 

to do harm to others. The biosecurity plan should 

include personnel security measures based on a 

site-specific risk assessment. A robust “insider threat 

awareness” training program developed and continuously updated based on site-specific risk assessments 

is administered to individuals with access to Tier 1 

BSAT. Insider threat awareness training is an annual 

requirement.19 

Operational Security

Effective operational security posture builds on 

existing operational procedures and mitigates threats 

based on site-specific risk assessments.49 Operational 

security measures for an entity with BSAT should 

include: 

• training personnel on securing BSAT;

• monitoring individual access to areas containing SAT;

• monitoring BSAT activities inside containment suites through security closed-circuit 

television or by using an escort;

• control of after-hour and weekend access to 

containment laboratories with BSAT;

• screening visitors, packages, and delivery 

trucks at the entry point;

• procedures in place for immediate notification 

to the RO, commander or director, security 

forces, and law enforcement if theft or loss of 

SAT is suspected; 

• training personnel to identify and report suspicious activities; 

• prominently displayed identification badges 

on individuals within the entity;

• constant building security surveillance; 

• intrusion detection systems; 

• surveillance of backup power generators; 

and 

• peer reporting procedures for any sudden 

changes in behavior among approved individuals with access to SAT.49 

Information Security

FSAP regulations require registered entities to 

develop and implement procedures for information 

control and information security.52 Information security procedures and protocols must: 

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Medical Aspects of Biological Warfare 

• ensure all external connections to systems 

that manage security for the registered 

space are isolated or have controls that 

permit and monitor authorized and authenticated users; 

• ensure authorized and authenticated users 

only access information necessary to fulfill 

their roles and responsibilities; 

• prevent malicious code from compromising 

the confidentiality, integrity, or availability 

of information systems that control safety and 

emergency equipment, engineering controls 

for the containment laboratories, and access 

to registered space; 

• include regular patching and updates to 

operating systems as well as to individual 

applications; 

• protect network operating systems with security firewalls; 

• protect hardware assets; 

• include data encryption; 

• ensure remote access capability; 

• establish robust information backup systems 

in the event of primary system failure; 

• establish procedures for purging electronic 

storage media prior to disposal; and 

• establish procedures for shredding paper 

documents and computer disks.52 

Incident Response and Emergency Management

A robust incident response plan and a knowledgeable and competent emergency management team 

are critical to an entity involved in developing medical countermeasures against dangerous pathogens 

and toxins. An incident is an occurrence, natural or 

human-made, that requires a response to prevent the 

theft, loss, or release of an SAT or to protect human 

life and animal and plant health.53 FSAP, DA, and 

DoD regulations require entities with SAT to develop, 

exercise, and routinely update a comprehensive, sitespecific incident response plan to ensure the security 

and safeguarding of BSAT in the event of human-made 

threats and natural disasters. A site-specific incident 

response plan protects human life before property, is 

focused on laboratories and not just the entire facility, is developed as a result of collaboration between 

research staff and leadership, includes responder 

participation and training, and addresses primary 

and secondary effects and the impact on workers at 

the facility.53 Developing an incident response plan at 

the entity level should be a team effort involving (but 

not limited to) the RO, AROs, biosafety officer, facility 

engineers, PI or researcher, security manager, occupational health physician or CMA, and entity leadership, 

with input from local first responders (fire department, 

emergency medical and law enforcement). 

Laboratory leadership, supervisors, biosafety specialists and subject matter experts within a registered 

entity with SAT should develop incident response information specific to the agents, toxins, and procedures 

conducted in that laboratory. Individuals working in 

the laboratory must be trained on how to respond 

to an incident with the materials they handle in the 

laboratory, emergency exit procedures, and the use of 

communication devices within the laboratory. Laboratory incident response information must also include 

decontamination protocols, first-aid, and reporting 

requirements.19 Laboratory and facility incident response plans should be practiced via exercises with 

entity staff and external first responders (fire department, emergency medical and law enforcement); this 

practice is critical and will save lives and property in 

the event of a real incident. 

The incident response plan should consider and 

mitigate vulnerability assessments specific to the 

laboratory and the facility. The incident response plan 

must include provisions for theft, loss, or release of 

SAT, inventory discrepancies, and security breaches.19

Theft, Loss or Release 

Response to suspected theft or loss of SAT should 

include immediate notification to the entity RO and 

commander or director for an immediate investigation and verification of pertinent SAT inventory. An 

investigation should include physical inventory and 

reconciliation of all LT SAT with database records, 

review of laboratory usage records, transfer records, 

destruction records, and WS records. Once theft or 

loss has occurred, the investigation and recovery 

of SAT is a law enforcement function. Law enforcement, state, and federal agencies, including FSAP, 

must be notified of theft or loss of SAT; in terms of 

FSAP, initial notification is followed by a completed 

APHIS/CDC Form 3 within 7 days. The entity should 

be prepared to support law enforcement with all its 

recovery efforts. 

Release of SAT from primary containment could 

occur during movement (breakage of specimen 

tubes), due to loss of engineering controls (eg, 

equipment malfunction, power outage), or as a result of an unforeseen event inside the containment 

laboratory. SAT release can pose a significant additional risk of exposure to workers if they are not 

adequately protected with PPE and if the release is 

not captured and neutralized. Workers potentially 

exposed to SAT should be immediately evaluated by 

occupational medicine staff, and appropriate followup care must be provided to the affected workers. 

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Local and state public health agencies and FSAP 

must be notified of SAT release, including potential 

exposures to workers. Theft, loss, or release of SAT 

is also reported to the chain of command in the DA 

and DoD laboratories. 

Inventory Discrepancies

SAT inventory discrepancies (overage or shortage) 

should be immediately reported to the entity RO and 

AROs. The PI and research staff must conduct an 

investigation to resolve or confirm the inventory discrepancy. The memorandum of inventory discrepancy 

investigation should include: 

• identity of the SAT,

• amount of discrepancy,

• date of last inventory and by whom,

• current or last known storage location,

• names of individuals who discovered the 

discrepancy,

• names of individuals who are notified of the 

discrepancy, and 

• explanation or resolution, if available. 

Theft and loss of BSAT must be reported to FSAP.19

Security Breaches

A security breach can occur due to a disruption in 

an established security network or failure to follow 

established security procedures and policies, or during active and deliberate intrusion from unauthorized 

sources (eg, intruders, enemy forces). The RO and the 

commander or director must be notified of all security 

breaches to restricted areas containing SAT. Security 

breaches may include: 

• access to SAT by individuals not approved by 

the FSAP; 

• individuals “piggy backing” into restricted 

areas; 

• tampering of access controls, locks, and seals 

securing SAT; 

• unauthorized access to SAT inventory databases; 

• tampering of security badges, passcodes, or 

other entry credentials to restricted areas 

containing SAT; 

• unauthorized removal of SAT from restricted 

areas; 

• sharing of access credentials by workers; 

• damage to building infrastructure resulting 

in easy access to SAT; and 

• compromises due to hacking or deliberate 

manipulations in computer programing controlling containment access. 

Lessons learned should be incorporated to enhance 

security systems and decrease security breaches.56 The 

FSAP requires the RO to ensure that individuals with 

access to SAT are trained annually on entity incident 

response plans. 

SUMMARY

The intent of the FSAP and the DoD’s BSP is the 

same: to allow peaceful research to continue while 

restricting BSAT access to individuals and parties 

who intend to misuse them and do harm. Overall, 

current regulatory requirements promote laboratory 

safety and security of BSAT by requiring laboratory 

registration; prescreening of individuals requesting 

access to BSAT; personnel reliability or suitability 

assessments for individuals seeking access to Tier 

1 BSAT; BSAT inventory management; preapproval 

and monitoring of BSAT transfers; reporting requirements for theft, loss, release, or identification of 

BSAT; preapproval for certain genetic alterations to 

BSAT (restricted experiments); and periodic onsite inspections by regulatory agencies. Regulatory burden 

on entities with BSAT can be significant; however, 

it is critical for the public to have confidence that 

work involving BSAT is conducted in a manner that 

prioritizes laboratory and public safety and protection of the environment. 

Biological surety and security requirements to 

access BSAT in DoD laboratories currently meet or 

exceed that of the FSAP. DoD also imposes additional 

biological surety and security measures beyond those 

required by the FSAP, on contractors using DoDowned BSAT. Having different eligibility standards 

to access and work with BSAT can have significant 

impact on collaborative research; harmonization of 

administrative policies and practices of facilities registered with FSAP is expected to promote increased 

collaboration among scientists. Currently, DoD is 

synchronizing its biological surety regulations with 

the FSAP regulations in accordance with Executive 

Order 13546.22

Scientific advances in synthetic biology are likely 

to challenge the current regulations governing BSAT; 

however, current US regulations governing BSAT are 

consistent with the broad international framework 

of agreements intended to prevent development and 

proliferation of chemical and biological weapons. 

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10. Roffey R, Tegnell A, Elgh F. Biological warfare in a historical perspective. Clin Microbiol Infect. 2002; 8(8):450–454. 

11. Harris S. Japanese biological warfare research on humans: a case study on microbiology and ethics. Ann N Y Acad Sci. 

1992;666:21–52.

12. Harris S. The Japanese biological warfare programme: an overview, In: Biological and Toxin Weapons: Research, Development 

and Use from the Middle Ages to 1945. Edited by Geissler, E and Moon, J.E.v.C, 127-152. 1999. Stockholm International 

Peace Research Institute, Oxford University Press, Oxford, UK.

13. United Nations Office for Disarmament Affairs. Convention on the Prohibition of the Development, Production and 

Stockpiling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction. http://www.un.org/disarmament/WMD/Bio. Accessed June 25, 2014.

14. Pub L 104-132. Antiterrorism and Effective Death Penalty Act of 1996. 24 April 1996. http://www.gpo.gov/fdsys/pkg/

PLAW-104publ132/content-detail.html. Accessed June 25, 2014.

15. Pub L 107-56. Uniting and Strengthening America by Providing Appropriate Tools Required to Intercept and Obstruct 

Terrorism Act of 2001. 26 October 2001. http://www.gpo.gov/fdsys/pkg/PLAW-107publ56/content-detail.html. Accessed 

June 25, 2014.

16. Pub L 107-188. Public Health Security and Bioterrorism Preparedness and Response Act of 2002. 12 June 2002. http://

www.gpo.gov/fdsys/pkg/PLAW-107publ188/pdf/PLAW-107publ188.pdf. Accessed June 25, 2014.

17. US Department of Agriculture Animal and Plant Health Inspection Service. Agricultural Select Agent Program. http://

www.aphis.usda.gov/programs/ag_selectagent/. Accessed August 15, 2014. 

18. US Department of the Army. Biological Surety. Washington, DC: DA; 2008. Army Regulation 50-1. 

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Biological Surety

19. Select Agents and Toxins, 42 CFR Part 73 (2012).

20. Possession, Use, and Transfer of Select Agents and Toxins, 7 CFR Part 331 (2012).

21. Possession, Use, and Transfer of Select Agents and Toxins, 9 CFR Part 121 (2012).

22. Optimizing the Security of Biological Select Agents and Toxins in the United States. Fed Regist. 2010;75(130): 39439–39442. 

Executive Order 13546. http://www.gpo.gov/fdsys/pkg/FR-2010-07-08/pdf/2010-16864.pdf. Accessed June 25, 2014.

23. Centers for Disease Control and Prevention; Animal and Plant Health Inspection Services. List of Select Agents and 

Toxins. 12 September 2013. http://www.selectagents.gov/Select%20Agents%20and%20Toxins%20List.html. Accessed 

June 25, 2014.

24. Centers for Disease Control and Prevention; Animal and Plant Health Inspection Services. Forms. 13 August 2013. 

http://www.selectagents.gov/Forms.html. Accessed June 25, 2014.

25. Centers for Disease Control and Prevention; Animal and Plant Health Inspection Services. Registration for Possession, 

Use, and Transfer of Select Agents and Toxins. 22 October 2013. http://www.selectagents.gov/RegistrationForm.html. 

Accessed June 25, 2014.

26. Centers for Disease Control and Prevention; Animal and Plant Health Inspection Services. Security Risk Assessments. 

25 March 2013. http://www.selectagents.gov/sra.html. Accessed June 25, 2014.

27. US Department of Health and Human Services (HHS), Centers for Disease Control and Prevention (CDC)/Division 

of Select Agents and Toxins and US Department of Agriculture (USDA), Animal and Plant Health Inspection Service 

(APHIS)/Agriculture Select Agent Program (ASAP). Guidance on the Inventory of Select Agents and Toxins. 20 June 

2014. http://www.selectagents.gov/LongTermStorage.html. Accessed August 15, 2014. 

28. US Department of the Army. Nuclear and Chemical Weapons and Materiel: Biological Surety. Washington, DC: DA; 2008. 

Army Regulation 50-1. 

29. Transportation, 49 CFR, Parts 100–185 (1999).

30. US Department of Defense, Office of the Under Secretary of Defense for Acquisition, Technology, and Logistics. Report 

of the Defense Science Board Taskforce on DoD Biological Safety and Security Program. Washington, DC: Defense Science 

Board; 2008.

31. Centers for Disease Control and Prevention; Animal and Plant Health Inspection Services. Permissible Toxin Amounts. 

5 October 2012. http://www.selectagents.gov/Permissible%20Toxin%20Amounts.html. Accessed June 25, 2014.

32. Centers for Disease Control and Prevention; Animal and Plant Health Inspection Services. Federal Select Agent Program 

Policy Statement on Toxin Due Diligence Provision. 3 May 2013. http://www.selectagents.gov/Toxin_Due_Diligence_Provision.html. Accessed June 25, 2014.

33. US Department of Health and Human Services Centers for Disease Control and Prevention and US Department of 

Agriculture Animal and Plant Health Inspection Service Agriculture Select Agent Services. Select Agents and Toxins: 

Theft, Loss or Release Information Document. Atlanta, GA: CDC; June 8, 2010. 

34. Henkel RD, Miller T, Weyant RS. Monitoring select agent theft, loss and release reports in the United States, 2004–2010. 

Applied Biosafety. 2012;17(4):171–180.

35. US Department of Health and Human Services, National Institutes of Health, Office of Biotechnology Activities. NIH 

Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. Bethesda, MD: HHS/NIH; 2013.

36. Federal Select Agent Program, US Department of Agriculture/Animal and Plant Health Inspection Service; Department 

of Health and Human Services/Centers for Disease Control and Prevention. Restricted Experiment Guidance Document. 

Riverdale, MD, Atlanta, GA: USDA/APHIS, HHS/CDC; 2013. 

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37. US Department of Health and Human Services, Centers for Disease Control and Prevention, and National Institutes 

of Health. Chosewood LC, Wilson DE, eds. Biosafety in Microbiological and Biomedical Laboratories. 5th ed. Washington, 

DC: US Government Printing Office; 2009.

38. US Department of the Army. Safety Standards for Microbiological and Biomedical Laboratories. Washington, DC: DA; 2013. 

DA PAM 385-69, Rapid Action Revision.

39. US Department of Health and Human Services, Centers for Disease Control and Prevention/Division of Select Agents 

and Toxins; US Department of Agriculture/Animal and Plant Health Inspection Service Agriculture Select Agent Program. Occupational Health Program Guidance Document for Working With Tier-1 Select Agents and Toxins. Washington, 

DC: HHS; 5 July 2013.

40. US Department of Defense. Nuclear Weapon Personnel Reliability Program (PRP). Washington, DC: DoD; 2012. DoD 

Instruction 5210.42.

41. US Department of Defense. Minimum Security Standards for Safeguarding Chemical Agents. Washington, DC: DoD; 2007. 

DoD Instruction 5210.65. 

42. US Department of Defense. Minimum Security Standards for Safeguarding Biological Select Agents and Toxins. Washington, 

DC: DoD; 2006. DoD Instruction 5210.89.

43. Carr K, Henchal EA, Wilhelmsen C, Carr B. Implementation of biosurety systems in a Department of Defense medical 

research laboratory. Biosecur Bioterror. 2004;2(1):7–16.

44. US Department of Health and Human Services, Centers for Disease Control and Prevention/Division of Select Agents 

and Toxins; US Department of Agriculture/Animal and Plant Health Inspection Service. Guidance for Suitability Assessments. Washington, DC: HHS; 2013.

45. Privacy Act, 5 USC § 552a (1974).

46. Health Insurance Portability and Accountability Act, Pub L 104–191, 110 Stat 1936.

47. Crimes and Criminal Procedure, 18 USC § 175B (2011).

48. US Department of Defense. Personnel Reliability Program—Personnel Security Investigations and Adjudications. Policy and 

Implementation Procedures for Personnel Security Investigations and Adjudications. Washington, DC: DoD; 2012. Army 

Directive 2012-17.

49. US Department of Health and Human Services, Centers for Disease Control and Prevention/Division of Select Agents 

and Toxins; US Department of Agriculture/Animal and Plant Health Inspection Service/Agriculture Select Agent 

Program. Security Guidance for Select Agent or Toxin Facilities. Washington, DC: HHS; 2013.

50. The White House Office of Science and Technology Policy. Biosecurity website. http://www.whitehouse.gov/

administration/eop/ostp/nstc/biosecurity. Accessed July 3, 2014.

51. US Department of the Army. Biological Select Agents and Toxins Security Program. Washington, DC: DA; 2009. Army 

Regulation 190-17.

52. US Department of Health and Human Services, Centers for Disease Control and Prevention/Division of Select Agents 

and Toxins; US Department of Agriculture/Animal and Plant Health Inspection Service/Agriculture Select Agent 

Program. Information Systems Security Control Guidance Document. Washington, DC: HHS; 2012.

53. US Department of Health and Human Services, Centers for Disease Control and Prevention/Division of Select Agents 

and Toxins; US Department of Agriculture/Animal and Plant Health Inspection Service/Agriculture Select Agent 

Program. Incident Response Plan Guidance Document. Washington, DC: HHS; 2013.

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Ethical Issues in the Development of Drugs and Vaccines for Biodefense

Chapter 32

ETHICAL ISSUES IN THE 

DEVELOPMENT OF DRUGS AND 

VACCINES FOR BIODEFENSE 

ARTHUR O. ANDERSON, MD*; JEFFREY E. STEPHENSON, PhD†; and BRET K. PURCELL, PhD, MD‡

INTRODUCTION

OVERVIEW OF THE HISTORY OF BIODEFENSE DEVELOPMENT AND 

MEDICAL ETHICS

Biodefense and Ethics in the 18th and 19th Centuries

Biodefense, Ethics, and Research in the 20th Century

IMPACT OF REGULATING AGENCIES ON STRATEGIC RESEARCH 

CONFLICT BETWEEN REGULATIONS AND ETHICAL RESPONSIBILITIES

Department of Defense/Food and Drug Administration Memorandum of 

Understanding (1987)

Investigational New Drug Status of Therapeutics and Vaccines

Summary Points

OPTIONS FOR FULFILLING MISSION AND ETHICAL RESPONSIBILITIES TO 

MILITARY PERSONNEL

Option 1: Continue to Use Investigational New Drug Products Without

Full Compliance

Option 2: Negotiate for Accelerated Licensure

Option 3: Institute Waiver of Informed Consent

CURRENT MOVEMENTS IN THE REGULATORY ENVIRONMENT

The Public Health Security and Bioterrorism Preparedness and Response

Act of 2002

21 Code of Federal Regulations 314 Subpart I: The Animal Efficacy Rule

BioShield Act of 2004

The Turner Bill

Biodefense and Pandemic Vaccine and Drug Development Act Renewal of 2011

DILEMMAS FOR BIODEFENSE RESEARCH

SUMMARY

*Colonel (Retired), Medical Corps, US Army; Director, Office of Human Use and Ethics, US Army Medical Research Institute of Infectious Diseases, 1425 Porter 

Street, Fort Detrick, Maryland 21702 

†

Regulatory Compliance Specialist, US Army Medical Research and Materiel Command, Telemedicine and Advanced Technology Research Center, 1054 Patchel 

Street, Fort Detrick, Maryland 21702; formerly, Institutional Review Board Administrator, US Army Medical Research Institute of Infectious Diseases, 1425 

Porter Street, Fort Detrick, Maryland

‡

Colonel, Medical Corps, US Army; Deputy Chief, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort 

Detrick, Maryland 21702

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INTRODUCTION

Over the past 60 years the conditions that must be 

met to use many of these drugs and vaccine products 

have become more restrictive. Until the approval of 

an animal efficacy rule and passage of the Project 

BioShield Act of 2004, FDA regulations originating in 

the 1938 Food, Drug, and Cosmetic Act made emergent 

medical responses to bioterrorist attacks extremely 

complex by prohibiting use of investigational products until there was substantial evidence of human 

clinical efficacy. Gathering evidence in a scientifically valid clinical trial requires the participation of 

large numbers of subjects who have or are at risk of 

acquiring the disease, and accumulating these clinical 

observations requires a lot of time and considerable 

expense. Although some disease agents cause sporadic 

epidemics, others infect individuals randomly when 

a reservoir of contagion is present. Biowarfare attacks 

involving these highly infectious agents would likely 

affect many people suddenly, permitting neither the 

opportunity to enroll enough subjects in a study nor 

the time to establish the needed medical resources for 

detailed clinical observations. 

Although FDA restrictions are meant to protect the 

public from possible harm, delaying use of potentially 

beneficial products until clinical efficacy trial outcomes 

are known can be detrimental to a prompt and effective response in the event of a widespread biowarfare 

attack. Throughout most of the 20th century and into 

the 21st century, successful animal studies followed 

by substantial evidence of efficacy from human clinical trials have been required before a drug could be 

approved for market. In an emergency, however, it 

may be beneficial to allow animal study evidence if 

the circumstances cannot permit controlled human 

drug efficacy trials. 

Current regulations governing research related 

to biodefense development cover a wide swath of 

legal and ethical ground. However, the relationship 

between the military and the FDA is a complex one, 

partly because of the institutions’ different missions. The FDA regulates the manufacture, testing, 

promotion, and commerce of medical products, 

and it makes a legal distinction between products 

that are approved and not approved for marketing. 

Products with FDA approval for testing but not approved for marketing are classified as investigational 

new drugs (INDs). FDA regulations specify what is 

necessary to move from an investigational product 

to an approved drug. Under directive Department of 

Defense (DoD) Instruction 6200.02, dated February 27, 

2008, the DoD shall “make preferential use of products approved by the FDA for general commercial 

The anthrax postal mailings of October 2001 made 

the nation acutely aware of not only the possibility of 

biological weapons attacks on US soil, but also brought 

to the forefront concerns over the proper measures to 

be implemented to prepare for and prevent such biological warfare scenarios. It is evident that drugs and 

vaccines may be needed immediately to respond appropriately to emergency or battle situations; however, 

definitively identifying a realistic threat list and implementing a coherent, organized strategy of biodefense 

remain elusive. Government research funding and 

regulatory agencies, intelligence gathering agencies, 

private and government-sponsored pharmaceutical 

industries, and the armed services must work together 

more effectively to accurately identify the immediate 

and future threats so that countermeasure therapeutics—and in a limited fashion vaccines—are prioritized 

for research, development, and production. Many 

potential drugs that are not yet approved for marketing but have preclinical evidence of efficacy may be 

considered and used in the event of bioterrorist attacks 

or in times of war.

The pharmaceutical industry is not prepared or 

organized to respond to such situations; it is in the 

business of developing drugs to treat natural diseases 

afflicting patients of the civilian healthcare industry. 

Profit considerations and sustained business growth 

are the primary objectives of pharmaceutical companies and their shareholders, so drugs are more likely 

to be developed for common and chronic diseases 

rather than rare diseases. For such naturally occurring, 

often relatively common diseases, many potential test 

subjects are available to participate in drug safety and 

efficacy trials because of the possibility that the new 

drug might cure their diseases or help future patients.

This is not the case for products required as countermeasures against biological warfare agents. These 

infectious disease agents and toxins are usually found 

in areas of the world where they occur in sporadic, 

small epidemics that kill everyone affected and fail to 

spread. In any case, there are rarely sufficient numbers 

of “naturally” occurring disease outbreaks of this kind 

to conduct clinical trials yielding substantial evidence 

of human clinical efficacy. To fulfill this critical need 

to generate efficacy data on the product of interest, 

the US Food and Drug Administration (FDA) established the animal efficacy rule under 21 CFR [Code of 

Federal Regulations] 314.6. This regulation allowed 

the FDA to approve the use of an investigational drug 

in humans based on evidence of effectiveness from 

studies conducted using well-controlled, validated 

animal models.1,2

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Ethical Issues in the Development of Drugs and Vaccines for Biodefense

marketing, when available, to provide the needed 

medical countermeasure;” however “if at the time of 

the need under a force health protection program for 

a medical countermeasure against a particular threat, 

no satisfactory FDA-approved medical product is 

available, request approval by the ASD(HA) [Assistant Secretary of Defense for Health Affairs] to use an 

unapproved product under an EUA [emergency use 

authorization] or, if an EUA is not feasible, under an 

IND [investigational new drug] application.”3

Because members of the armed services are at the 

greatest risk for biowarfare attack, it is prudent for the 

military to research and develop effective biological 

defenses that may also be used for treatment in the 

civilian population in an emergency. FDA regulations 

pose three significant legal hurdles to the military’s 

ethical responsibility to protect service personnel. 

First, because organisms that are potential biological 

warfare agents, such as those causing inhalational 

anthrax, plague, or Ebola, are rare in nature (generally producing small, sporadic clusters of disease 

with case numbers insufficient for field trial efficacy 

studies) and can be life threatening, it is immoral to 

conduct clinical trials to determine clinical efficacy 

because of the inherent risk to participants. Second, 

outside of clinical trials, the systematic use of IND 

products (as opposed to single use instances) in 

emergency life-threatening situations is illegal. Third, 

it is illegal for the DoD to systematically use licensed 

drugs in large numbers of persons for uses other 

than those granted by the FDA product licensure 

agreement (off-label use) without the notice required 

under 10 USC [United States Code] 1107(1) or under 

the legitimate practice of medicine. Ultimately, however, researchers must find ways to overcome these 

limitations so that the FDA and DoD can fulfill their 

respective executive branch responsibilities while 

minimizing conflicts. 

Federal regulations serve as practical and praiseworthy legal and ethical safeguards for the conduct 

of human subjects’ research. However, as detailed 

above, regulations governing the conduct of human 

subjects research can also have the unintended consequence of slowing the development and advancement of biodefense-related medical products, which 

can result in the following ethical dilemma: on one 

hand, the military has the duty to adhere to regulations and obey the country’s laws; on the other hand, 

the military has the duty to use all available means to 

protect its personnel and civilians and accomplish the 

mission. Mechanisms to bridge the two horns of this 

dilemma are needed; in particular, there must be a legal 

way to make protective drugs and vaccines available 

when the normally required clinical trials cannot be 

conducted. Consequently, several avenues have been 

established to address these issues including the use 

of notice under 10 USC 1107(a), the use of the EUA 

provisions of section 564 of the Federal Food, Drug, 

and Cosmetic Act, enacted in 2004 by the Secretary of 

Health and Human Services, or the establishment of 

an FDA approved IND protocol.3,4

This chapter will demonstrate ways to protect military personnel and possibly even the civilian population. The history of the development of biodefense in 

military medicine and the ethics of biomedical research 

will be covered. In addition, a summary of the evolution of regulations that influence or inform human 

subjects research, including research intended and designed in part to meet the needs of the military personnel, will be presented. Then an analysis and discussion 

of the conflict between regulatory requirements and 

adherence to ethical principles in the military setting 

will demonstrate three options the DoD may pursue in 

relation to the issues outlined. Some of the legislated 

solutions recently proposed or implemented will also 

be included.

OVERVIEW OF THE HISTORY OF BIODEFENSE DEVELOPMENT AND MEDICAL ETHICS

Advances in biomedical research have led to 

considerable breakthroughs in the treatment of 

diseases that military personnel face. Although the 

focus of this chapter is on biodefense, the history of 

research to protect military personnel from disease 

has frequently targeted naturally occurring diseases 

unfamiliar to US troops. The need for development of 

medical treatment in military settings has frequently 

been the impetus for conceptual breakthroughs in the 

ethics of human participation in research. Biomedical research involving human subjects in military 

research facilities must be conducted with oversight 

from an institutional review board (IRB), per 32 CFR 

219.109,5 45 CFR Part 46,6 21 CFR 56,7 and 21 CFR 50. 

Acknowledgment of ethical dimensions in biodefense research requires the cooperation of all military personnel. However, the ethical principles that 

serve as the foundations of current ethical practices 

in military medical research did not come about de 

novo, and neither did the biodefense and protection 

methods. Military medical ethics standards evolved 

over centuries, often in tandem with or in reaction to 

biodefense needs, or in response to ethical lapses or 

controversies. At times the military has assumed the 

lead in establishing human subjects’ research ethics 

precedence.

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Medical Aspects of Biological Warfare 

Biodefense and Ethics in the 18th and 19th Centuries

In 1766, while still a general for England, George 

Washington and his soldiers were unable to take 

Quebec in the French and Indian War. In part this 

failure was due to smallpox outbreaks that affected 

his troops.8 Later when Washington led Continental 

Army troops against the British, a smallpox epidemic 

reduced his healthy troop strength to half while the 

British troops, who had been variolated, were already 

immune to the spreading contagion. Troops were often 

gathered together from remote parts of the fledgling 

nation and placed into crowded camps, mingling with 

local civilian populations, which expanded variola 

transmission even further into vulnerable populations.9

 Washington proclaimed smallpox to be his 

“most dangerous enemy,” and by 1777 he had all his 

soldiers variolated before beginning new military operations. In doing so, Washington fulfilled the ethical 

responsibility of ensuring the health of his military 

personnel, which in turn served to fulfill his professional responsibility as commander of a military force 

to preserve the nation. However, a public unfamiliar 

with the stakes or conditions weighing on this choice 

criticized Washington’s actions (Figure 32-1).

Advances in military medicine and hygiene developed through experiences gained in battlefield 

medicine during the American Civil War were adapted 

as standards of medical care during the latter part of 

the 19th century. New medical schools such as Johns 

Hopkins sought advice about the most advanced 

patient care facilities, medical practices, and medical 

treatment lessons learned on the battlefield. The most 

direct evidence of the influence of military medicine 

on standard medical care practice is provided by John 

Shaw Billings.10 While serving in the office of the Army 

surgeon general, he designed the Johns Hopkins Hospital building, applying concepts he learned about the 

importance of hygiene, light, and ventilation while 

evaluating medical care in Civil War field hospitals. 

Billings also created an indexing system for medical publications that was used for the Army surgeon 

general’s library and became the nidus of the National 

Library of Medicine. The Welch Medical Library at the 

Johns Hopkins University School of Medicine adopted 

this same system. Additionally, the Army ambulance 

system was developed during the Civil War because 

removing injured soldiers to field hospitals had a better 

outcome than treating soldiers in the field. Furthermore, soldiers suffering war wounds frequently died 

from infection. This lesson was not lost on military 

physicians. As the end of the war neared, the fledgling 

science of bacteriology and epidemiology became hot 

topics of battlefield military medical research. Surgical techniques and use of anesthesia and antiseptics 

became commonplace during the Civil War.11–13

The Civil War was also a testing ground for medical education. One lesson learned from the war was 

that many who served as military physicians did not 

have the skills needed to save lives in the battlefield. 

So the Army created its own medical school at what 

later became the old Walter Reed Army Institute of 

Research building. Those who created this school liked 

the training being done at Johns Hopkins, where some 

later became faculty. Later, civilian hospitals adopted 

the same surgical techniques and treatment methods. 

Johns Hopkins Medical School created new academic 

standards not found at “proprietary” medical schools. 

Thus, with the help and influence of military medical 

experience, Johns Hopkins set the stage for medical 

treatment in the modern era.

Surgeon General George Sternberg, who had been 

trained as a bacteriologist at Johns Hopkins Medical 

School, appointed Major Walter Reed, another Johns 

Hopkins medical trainee, to the Yellow Fever Commission in 1900. Reed used “informed consent” statements 

when he recruited volunteer subjects from among 

soldiers and civilians during the occupation of Cuba at 

the end of the Spanish-American War, and those statements could be considered “personal service contracts” 

(Figure 32-2). These documents clearly communicated 

the risks and benefits of participation, described the 

purpose of the study, provided a general timeline 

for participation, and stated that compensation and 

Figure 32-1. George Cruikshank, Vaccination against Small 

Pox or Mercenary and Merciless spreaders of Death and 

Devastation driven out of Society! London, England: SW 

Fores, 1808. General George Washington was strongly criticized in the press because of the risks and his decision to go 

ahead with forced variolation despite concerns. A political 

cartoon, published in the 1800s, shows how critically forced 

variolation was seen by the public despite the Army’s intent 

to benefit its soldiers.

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Ethical Issues in the Development of Drugs and Vaccines for Biodefense

Figure 32-2. (a) English translation of the yellow fever informed consent document. (b) Spanish version of the yellow fever 

informed consent documents. Major Walter Reed, who was appointed to the Yellow Fever Commission in 1900, used “informed consent” statements when he recruited volunteer subjects from among soldiers and civilians during the occupation 

of Cuba at the end of the Spanish-American War, which could be considered “personal service contracts.” However, these 

(Figure 32-2 continues)

a

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Medical Aspects of Biological Warfare 

documents clearly communicated the risks and benefits of participation, described the purpose of the study, provided a 

general timeline for participation, and stated that compensation and medical care would be provided. All of these are standard elements required in informed consent forms provided to research participants today.

Documents: Courtesy of Historical Collections and Services, Claude Moore Health Sciences Library, University of Virginia, 

Charlottesville, Virginia.

Figure 32-2 continued

b

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Ethical Issues in the Development of Drugs and Vaccines for Biodefense

medical care would be provided. All of these are standard elements required in informed consent forms 

provided to research participants today. Even if the 

yellow fever statements did not directly influence the 

creation of other military or civilian informed consent documents, it is at least plausible to claim that 

documentation of informed consent from research 

participants in the military predates the practice in 

civilian medicine.

Biodefense, Ethics, and Research in the 20th Century

Ethical issues surrounding informed consent continued into the 20th century. At the same time, the 

importance of strategic research was emphasized, 

which influenced the growth of epidemiological and 

infectious disease research. A 1925 Army regulation 

(AR) promoting infectious disease research noted 

that “volunteers” should be used in “experimental” 

research.14 In 1932, the Secretary of the Navy granted 

permission for experiments with divers, provided they 

were “informed volunteers.”15

The importance of strategic medical research was 

not unwarranted. In 1939, Japanese scientists attempted to obtain virulent strains of yellow fever virus from 

Rockefeller University. Vigilant scientists thwarted 

the attempt, but it did not take long before the threat 

of biological weaponry reached the War Department. 

In 1941, Secretary of War Henry L Stimson wrote to 

Frank B Jewett, president of the National Academy 

of Sciences, and asked him to appoint a committee to 

recommend actions. He wrote, “Because of the dangers that might confront this country from potential 

enemies employing what may be broadly described as 

biological warfare, it seems advisable that investigations be initiated to survey the present situation and 

the future possibilities.”16 In the summer of 1942, the 

War Research Service was established, under George 

W Merck, Jr, in the civilian Federal Security Agency 

to begin development of the US biological warfare 

program with offensive and defensive objectives. On 

October 9, 1942, the full committee of the War Research 

Service endorsed the chairman’s statement on the use 

of humans in research:

The use of human experimentation is not only desirable, but necessary in the study of many of the problems of war medicine which confront us. When any 

risks are involved, only volunteers should be utilized 

as subjects, and these only after the risks have been 

fully explained and after signed consent statements 

have been obtained which shall prove that the volunteer offered his services with full knowledge and 

that claims for damage will be waived. An accurate 

record should be kept of the terms in which the risks 

involved were described.17

Despite the War Research Service’s ethical commitment to adequately inform subjects of the risks 

involved in research, the statement includes an assertion of waiver of rights that is now considered 

unethical to include in military informed consent 

documents. The War Research Service also supported 

other experiments performed by civilian scientists that 

involved subjects whose capacity to give valid consent 

to participate was doubtful, including institutionalized 

people with cognitive disabilities.

Meanwhile, military involvement in the development of infectious diseases research was advancing. 

One of the military’s clear successes was the progress 

it made against acute respiratory disease. Because of 

crowded living conditions and other physical stresses, 

acute respiratory disease had consistently been a cause 

of morbidity and mortality among soldiers and an 

increasing economic liability for the military. In the 

early 1950s, military researchers under Maurice Hilleman at the Walter Reed Army Institute of Research 

identified seven distinct types of adenoviruses and 

created vaccines against them, a classic example of 

a quick, successful development by the military of 

medical countermeasures.

As the medical research community began preparing for biological threat and committing resources and 

time to attendant research, the undercurrent of doubts 

among human subjects research continued. It was not 

until Nazi and Japanese war crimes became public that 

human subjects research issues came to the forefront 

of the dialogue on the role and value of science in 

society. Dr Andrew Ivy compiled 10 conditions that 

must be met for research involving human subjects 

for the Nuremberg Tribunal in December 1946. This 

document, now famously referred to as the Nuremberg 

Code, was part of the Tribunal outcomes. In 1947, the 

Nuremberg Code was published in response to widespread knowledge of Nazi atrocities, including the 

unethical and traumatizing practices of Nazi doctors. 

The Nuremberg Code provided a clear statement of 

the ethical conditions to be met for humans as medical 

research subjects (Exhibit 32-1).

The DoD adopted all of the elements of the Nuremberg Code verbatim and added a prisoner-of-war 

provision.18 The Army included the code in directive 

Cs-385, which required that informed consent must be 

in writing, excluded prisoners of war from participation, and included a method for DoD compensation 

for research-related injuries sustained by participants. 

In 1962, Cs-385 became AR 70-25, Use of Volunteers as 

Subjects of Research,19 which regulated Army research 

until 1983.

In 1952, the Armed Forces Medical Policy Council 

noted that nonpathogenic biological warfare simulations conducted at Fort Detrick (formerly known as 

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Medical Aspects of Biological Warfare 

EXHIBIT 32-1

THE NUREMBERG CODE (1947)

1. The voluntary consent of the human subjects is absolutely essential.

This means that the person involved should have legal capacity to give consent; should be so situated as to be able 

to exercise free power of choice, without the intervention of any element of force, fraud, deceit, duress, overreaching, or other ulterior form of constraint or coercion; and should have sufficient knowledge and comprehension of the 

elements of the subject matter involved as to enable him to make an understanding and enlightened decision. This 

latter element requires that before the acceptance of an affirmative decision by the experimental subject there should 

be made known to him the nature, duration, and purpose of the experiment; the method and means by which it is to 

be conducted; all inconveniences and hazards reasonably to be expected; and the effects upon his health or person 

which may possibly come from his participation in the experiment. The duty and responsibility for ascertaining the 

quality of the consent rests upon each individual who initiates, directs or engages in the experiment. It is a personal 

duty and responsibility which may not be delegated to another with impunity.

2. The experiment should be such as to yield fruitful results for the good of society, unprocurable by other methods 

or means of study, and not random and unnecessary in nature.

3. The experiment should be so designed and based on the results of animal experimentation and a knowledge of 

the natural history of the disease or other problem under study that the anticipated results will justify the performance of the experiment.

4. The experiment should be so conducted as to avoid all unnecessary physical and mental suffering and injury.

5. No experiments should be conducted where there is an a priori reason to believe that death or disabling injury 

will occur; except, perhaps, in those experiments where the experimental physicians also serve as subjects.*

6. The degree of risk to be taken should never exceed that determined by the humanitarian importance of the 

problem to be solved by the experiment. 

7. Proper preparations should be made and adequate facilities provided to protect the experimental subject against 

even remote possibilities of injury, disability, or death. 

8. The experiments should be conducted only by scientifically qualified persons. The highest degree of skill and 

care should be required through all stages of the experiment of those who conduct or engage in the experiment. 

9. During the course of the experiment the human subject should be at liberty to bring the experiment to an end if 

he has reached the physical or mental state where continuation of the experiment seems to him to be impossible.

10. During the course of the experiment the scientist in charge must be prepared to terminate the experiment at 

any stage, if he has probable cause to believe, in the exercise of the good faith, superior skill, and careful judgment required of him, that a continuation of the experiment is likely to result in injury, disability, or death to the 

experimental subject.

*The self-experimentation clause of item 5 was omitted from the Wilson Memorandum and subsequent directives and regulations 

such as Cs-385 and AR 70-25 because it would be irresponsible for the person whose knowledge was essential for the safety and 

welfare of subjects to render himself incapacitated by taking the test agent along with his subjects. 

Note: The Nuremberg military tribunal’s decision in the case of the United States v Karl Brandt et al includes what is now called the 

Nuremberg Code, a 10-point statement delimiting permissible medical experimentation on human subjects. According to this statement, human experimentation is justified only if the results benefit society, and only if carried out in accord with basic principles 

that “satisfy moral, ethical and legal concepts.”

Data source: Permissible medical experiments. In: Trials of War Criminals before the Nuremberg Military Tribunals under Control Council 

Law No. 10. Vol 2. Washington, DC: US Government Printing Office; 1946–1949.

Camp Detrick before 1956) and at various locations 

across the United States showed that the population 

was vulnerable to biological attack. Additionally, 

experiments with virulent disease agents in animal 

models attested to the incapacitating and lethal effects 

of these agents when delivered as weapons. However, 

there was doubt among the council members that extrapolation of animal data to humans was valid, and 

human studies appeared necessary. Ad hoc meetings 

of scientists, Armed Forces Epidemiology Board advisors, and military leaders occurred at Fort Detrick during the spring of 1953.20,21 Thorough consideration of 

244-949 DLA DS.indb 922 6/4/18 
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Ethical Issues in the Development of Drugs and Vaccines for Biodefense

the ethical and legal basis for human subjects research 

resulted in the design of several prototype research 

protocols and creation of the US Army Medical Unit 

(Figures 32-3 and 32-4). This unit heavily invested in 

animal experimentation but aimed at modeling human infectious diseases to study pathogenesis and 

response to vaccines and therapeutics. Later, the US 

Army Medical Unit for offensive biological warfare 

was discontinued, and the US Army Medical Research 

Institute of Infectious Diseases (USAMRIID) was established in 1969 to conduct only defensive research 

and development of countermeasures to select agent 

infections.

In 1954, military research studies using human 

participants began in a program called CD-22 (Camp 

Detrick–22) that included soldier participants in a project called Operation Whitecoat. The participants were 

mainly conscientious objectors who were Seventh-day 

Adventists trained as Army medics. The program was 

designed to determine the extent to which humans are 

susceptible to infection with biological warfare agents. 

The soldier participants were exposed to actual disease 

agents such as those causing Q fever and tularemia to 

understand how these illnesses affected the body and 

to determine indices of human vulnerability that may 

be used to design clinical efficacy studies. In keeping 

with the charge in the Nuremberg Code to protect 

study participants, the US Army Medical Unit, under 

the direction of the Army surgeon general, carefully 

managed the project. Throughout the program’s history from 1954 to 1973, no fatalities or long-term injuries occurred among Operation Whitecoat volunteers.

Operation Whitecoat serves as a morally praiseworthy model for the conduct of biodefense research 

involving human subjects. The process of informed 

consent was successfully implemented from the inception of Operation Whitecoat. Each medical investigator 

prepared a protocol that was extensively reviewed 

and modified to comply with each of the elements of 

the Nuremberg Code. After a committee determined 

whether ethical requirements and scientific validity 

were met, Army officials approved the protocol. Then 

potential volunteers were briefed as a group regarding 

the approved protocol, and they attended a project 

interview with the medical investigator in which the 

potential volunteers could ask questions about the 

study. Informed consent documents (Figure 32-5) were 

signed after an obligatory waiting period that ranged 

from 24 hours to 4 weeks, depending on the risk involved in the study. Volunteers were encouraged to 

discuss the study with family members, clergy, and 

personal physicians before making a final decision. By 

allowing volunteers sufficient time and opportunity to 

ask questions about risks, potential benefits, and the 

conduct of the study, this multistage informed consent 

process ensured that participation was voluntary. Soldiers were told that their participation in the research 

was not compulsory. Approximately 20% of those 

soldiers approached for participation in Operation 

Whitecoat declined. Review of Operation Whitecoat 

records of interviews with many of the volunteers and 

investigators revealed that the researchers informed 

Figure 32-3. Aerial photograph of Fort Detrick, 1958. The 

US Army Medical Unit was assembled from existing Fort 

Detrick components concerned with occupational health 

and safety, the dispensary, and a small hospital referred to 

as Ward 200 of Walter Reed Army Medical Center. These 

components originated under separate Army commands, 

yet they formed an integrated, functional unit. 

Photograph: Courtesy of the Department of the Army.

Figure 32-4. The US Army Medical Unit at Fort Detrick, 

under Colonel William Tigertt (center) was staffed with 

personnel drawn from the US Army, Navy, Air Force, and 

Public Health Service, whose assignment was given the 

highest national priority because of their unique expertise in 

infectious disease medical care, research, and epidemiology, 

and because of their determination to provide the Operation 

Whitecoat volunteers the best care and support for their 

safety during the trials. Photograph taken in 1957.

Photograph: Courtesy of the Department of the Army.

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Medical Aspects of Biological Warfare 

Figure 32-5. Early (1955) informed consent used for one of the Camp Detrick-22 Operation Whitecoat experiments. 

Document: Courtesy of Medical Records Archives, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, 

Maryland.

participants that the research was scientifically valid 

and potentially dangerous, and that any harm to the 

participants would be minimized.

Approximately 153 studies related to the diagnosis, 

prevention, and treatment of various diseases were 

completed during Operation Whitecoat, including 

research on Q fever and tularemia infections and staphylococcal enterotoxins. Vaccines to be used against 

Venezuelan equine encephalitis, plague, tularemia, 

Rocky Mountain spotted fever, and Rift Valley fever 

were tested for evidence of safety in humans. However, 

scientists conducted animal studies before human 

244-949 DLA DS.indb 924 6/4/18 
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Ethical Issues in the Development of Drugs and Vaccines for Biodefense

including the principles of respect for persons, beneficence, and justice, were compiled from a review of 

codes of conduct and standard medical and research 

ethics practices. Respect for persons refers to those 

practices whereby the right of individuals to make 

fully informed decisions is respected, and the need 

for protection of persons who are less able to exercise 

autonomy is recognized. Beneficence refers to the deliberate intention to do good and the assurance that 

participation in the research is more likely to result in 

good than in harm. Justice demands that the potential 

benefit and harm of the research be distributed fairly 

in society, which has typically been understood to 

mean that the research cannot solely assist or exploit 

any certain demographic.

In practice, these three principles yield the research 

requirements respectively for informed consent, risk/

benefit analysis, and fair inclusion/exclusion criteria 

for participants. Much has been written about these 

principles, their flexibility and adequacy as guides, and 

their connection to philosophical foundations,24–26 and 

they remain appreciated as a practical approach to considering actions in biomedical contexts. The principles 

are secular but not incompatible with religious views, 

and they recognize the value of human individuals and 

the importance of collective benefits. The principles 

were incorporated into all federal institutions that fund 

research, including the DoD, as part of this common 

rule. Hence, “common rule” became the catch phrase 

used to refer to the institution-wide incorporation of 

explicit ethical requirements as identified in the Belmont Report. In 2011, the DoD Directive 3216.02, more 

recently updated as a DoD Instruction (DoDI) 3216.02, 

was implemented “to establish policy and assign responsibilities for the protection of human subjects in 

DoD-supported programs to implement part 219 of 

title 32, Code of Federal Regulations (CFR) (also known 

and hereinafter referred to as “the Common Rule”).27

Success in incorporating ethical principles into 

human subjects research in the military in the mid20th century was complemented by numerous early 

achievements of military researchers of high moral 

character who ethically developed vaccines for a 

variety of infections, including yellow fever (1900), 

typhoid fever (1909), pneumonia (1945), hepatitis A 

(1945), influenza (1957), rubella (1961), adenovirus 

(1952–1969), and meningococcal disease (1966) without 

written regulations.9

subjects research. For instance, researchers exposed 

Operation Whitecoat volunteers to aerosolized Q fever 

organisms only after completion of animal safety and 

efficacy studies. The first exposure occurred on January 25, 1955, with the use of a 1-million-liter stainless 

steel sphere at Fort Detrick known as the “Eight Ball.” 

This research device was designed to allow exposure of 

animals and humans to carefully controlled numbers 

of organisms by an aerosol route.

Research conducted during Operation Whitecoat 

also contributed to the development of equipment 

and procedures that established the standard for 

laboratory biosafety throughout the world. The ethical commitment to the safety of laboratory workers 

engaged with dangerous toxins, viruses, and diseases 

was manifested by the development of biological safety 

cabinets with laminar flow hoods, “hot suites” with 

differential air pressure to contain pathogens within 

the suites, decontamination procedures, prototype fermentors, incubators, refrigerated centrifuges, particle 

sizers, and various other types of specially fabricated 

laboratory equipment. Many of the techniques and 

systems developed at Fort Detrick to ensure worker 

safety while handling hazardous materials are now 

used in hospitals, pharmacies, and various manufacturing industries worldwide.

Operation Whitecoat was not the only example of 

US military involvement in human subjects research, 

and not all involvement in human subjects research 

reflects favorably on the US military. For example, 

the US military conducted unethical research involving LSD on uninformed human subjects from 1958 to 

1964.22 Congress enacted the National Research Act of 

1974 because US Public Health Service personnel and 

civilian collaborators at the Tuskegee Institute violated 

human subjects’ rights, most famously in the Tuskegee 

syphilis experiments.23 This act immediately imposed 

rules for the protection of human subjects involved in 

research, requiring informed consent from subjects and 

review of research by IRBs. The act created the National 

Commission for the Protection of Human Subjects of 

Biomedical and Behavioral Research, which published 

the Belmont Report, a compilation of the principles 

implicit in ethical medical practices, in 1979. The commission also provided a schema for the formal review 

of research by standing committees. Belmont Report 

findings were incorporated into AR 70-25 in 1988.19

The ethical principles identified in the report, 

IMPACT OF REGULATING AGENCIES ON STRATEGIC RESEARCH

The Public Readiness and Emergency Preparedness Act provides compensation to individuals for 

serious physical injuries or deaths from pandemic, 

epidemic, or security countermeasures identified 

in a declaration issued by the secretary pursuant to 

section 319F-3(b) of the Public Health Service Act 

(42 USC 247d-6d).28 The emergency use authorization (EUA) program was established in 2004, when 

244-949 DLA DS.indb 925 6/4/18 
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Medical Aspects of Biological Warfare 

The requirement for proof of efficacy of all medical 

countermeasures, premised on the principle of protecting the lives and other interests of human subjects, is 

a responsible action. But the Kefauver-Harris Drug 

Amendments also categorized the only available 

medical countermeasures against biological weapons as 

unapproved new drugs requiring approvals, which created an ethical dilemma for the DoD. Compliance with 

the FDA regulations meant that the DoD either had to 

risk the deaths of human subjects in a valid clinical trial, 

or withhold potentially life-saving drugs or vaccines 

because they lacked substantial evidence of human clinical efficacy. Currently, the FDA allows for “expanded 

access” IND applications for unapproved new drugs 

that have demonstrated sufficient safety and efficacy 

to warrant their limited use. (The drugs and vaccines in 

question would all require evidence of animal efficacy, 

unless no animal model of human disease could be 

found.) Additionally, AR 70-25 [1962, 1974, and 1988, 

but not 1990]32 contained clauses [3c] that exempted 

biodefense research and testing if there was intent to 

benefit the research subject. To resolve this issue, the 

DoD sought exceptions to these new regulations by 

negotiating memoranda of understanding (MOU) with 

the FDA in 1964, 1974, and 1987. An MOU provided the 

FDA an assurance that the DoD would conduct clinical 

testing of biologics, categorized as unapproved new 

drugs, under FDA regulations, including requirements 

for human subject informed consent, IRB review, and 

controlled clinical trials in medical research (see 21 CFR 

50 and 56).33 The MOU states that the DoD will meet 

these requirements without jeopardizing responsibilities related to its mission of protecting national interests 

and safety. Additional MOUs between the DoD and the 

FDA have been established, and include the following:

• MOU 224-75-30033,34 which “establishes the 

procedures to be followed regarding the 

investigational use of drugs, including antibiotics and biologics, and medical devices by 

DoD;” and 

• MOU 225-07-800335 for “sharing of information and expertise between the Federal partners” as well as regulation AR 40-7, which 

governs the Army’s use of investigational 

products.

the Project BioShield Act, among other measures, 

amended Section 564 of the Federal Food, Drug, and 

Cosmetic Act to include this provision.29 EUA permits 

the FDA commissioner to authorize the use of an 

unapproved medical product or an unapproved use 

of an approved medical product during a declared 

emergency involving a heightened risk of attack on the 

public or US military forces, or a significant potential 

to affect national security.30

The evolution of regulatory bodies overseeing 

human subjects’ research paralleled the evolution of 

military medical research ethics. These regulatory 

bodies influenced military research in positive and 

negative ways. In 1901 in Missouri, 13 children died 

of tetanus after receiving horse serum contaminated 

by Clostridium tetani for treatment of diphtheria. In 

1902, Congress enacted the Biologics Control Act (the 

Virus-Toxin Law), which gave the federal government 

authority to require standards for the production of 

biological products, including vaccines. The act contained provisions for establishing a board (including 

the surgeons general of the Navy, Army, and Marine 

Hospital Service) with the power to create regulations 

for licensing vaccines and antitoxins. Thereafter, only 

annually licensed, inspected facilities were permitted 

to produce biologics. This act marked the commencement of America’s federal public health policy for 

biologics.

The 1938 Food, Drug, and Cosmetic Act regulated 

biologics through mid-century. For the first time, drug 

production had to meet standards for safety before 

receiving approval for marketing. The 1944 Public 

Health Service Act reinforced or expanded public 

health policy standards in two ways: (1) it became the 

mechanism containing explicit regulation of biologics, 

and (2) it created the FDA. Under its new authority, 

the FDA approved the influenza vaccine, chiefly on 

the strength of data provided by the Army.31

In 1962, Congress passed the Kefauver-Harris 

Drug Amendments to the Food, Drug, and Cosmetic 

Act, which effectively launched the modern US drug 

regulatory system. These amendments stipulated an 

intense premarketing approval system, giving FDA the 

power to deny approval for products with safety concerns. The amendments also required proof of human 

efficacy for all drugs and biologics, including vaccines.

CONFLICT BETWEEN REGULATIONS AND ETHICAL RESPONSIBILITIES

The military situation is unique. In the tension 

between the good of the individual and the good for 

the social organization, the latter justifiably holds 

greater weight in decision-making procedures in the 

military context. Members of the military have unique 

responsibilities, which include being fit for duty. The 

military organization also has responsibilities to its 

service members, including providing healthcare 

specific to the dangers encountered in deployment 

locations.

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Ethical Issues in the Development of Drugs and Vaccines for Biodefense

Department of Defense/Food and Drug Administration Memorandum of Understanding (1987)

The 1991 Persian Gulf War brought into focus the 

inadequacy of the 1987 MOU and the conflicts between 

the duties of the two agencies. The DoD’s mission is 

to protect the interests of the United States. The DoD 

also recognizes its ethical responsibility to protect the 

health of military personnel. Thus, the DoD is doubly 

obligated to the mission and to service members. It is 

the responsibility of service members to keep themselves fit throughout the current mission and for future 

missions. When troops are threatened by biowarfare, 

in the absence of an approved biodefense product, one 

supported by preclinical data may be the only available 

option for troop protection. With a credible threat, the 

situation is similar to that of patients with an incurable 

disease who wish to try a potential remedy in advance 

of large clinical trials if it offers plausible expectation 

of some benefit. Such a product administered but 

proven ineffective would be analogous to sending 

troops to battle with faulty equipment. Such a product 

later proven unsafe would be analogous to friendly 

fire—perhaps an even more damaging situation for 

morale. Thus, the military requires a fine balance 

between necessity and caution. Proper biodefensive 

posture requires effective therapeutic countermeasure 

prophylaxis or treatment and, when appropriate, vaccination against credible threats. 

Currently, vaccinations include licensed anthrax 

and smallpox vaccines and unlicensed vaccines for 

tularemia, botulism toxin poisoning, and a variety 

of encephalitides, including Venezuelan equine 

encephalitis, western equine encephalitis, and eastern equine encephalitis. Data for these unlicensed 

vaccines support human safety and efficacy,36 even 

though efficacy has been demonstrated only in animals. Medical experts favor the use of these vaccines 

in protecting human beings when threat dictates. Because the vaccines are not licensed and will not—for 

ethical reasons—undergo the clinical efficacy trials 

required by FDA, they can only be used in an IND 

status unless testing and efficacy is demonstrated 

under FDA-approved Good Laboratory Practices 

efficacy studies animal rule.

Investigational New Drug Status of Therapeutics 

and Vaccines

FDA considers any administration of an investigational product to a human to constitute a clinical 

investigation and authorizes the administration of 

an investigational product only in the context of an 

IND, which permits clinical research trials to move 

forward37 or expanded access treatment to be administered under an emergency treatment IND application 

in which the investigational product use would not 

considered research. Because the therapeutic benefit 

of the investigational product is unknown, FDA also 

requires informed consent. Administration of an investigational product requires specific and detailed 

recordkeeping measures. However, the recordkeeping requirements relate specifically to research, not 

to emergency or preventive measures connected to 

imminent risk of biological attacks on the battlefield. 

It would take exponentially longer to collect data from 

and perform recordkeeping for 100,000 soldiers than to 

merely administer an unlicensed therapeutic or vaccine 

for treatment or prevention purposes. The consenting 

process alone for 100,000 individuals receiving an investigational product would take so long that strategic 

combat moves, such as immediate mobilization and 

deployment of a unit, would be impossible. Storing 

informed consent documents for 100,000 soldiers, and 

the accompanying logistical challenge of reconsenting soldiers if new risk information emerged during 

deployment, would also be daunting. It has been 

estimated that implementing only one protocol for 

an investigational product may generate up to 94,000 

lbs of paper records in a theater of operations, according to physicians discussing rewriting IND protocols 

in meetings held in 2002 and 2003 at Fort Detrick, 

Maryland. Furthermore, continuous data collection, 

as required by the FDA’s Good Clinical Practices 

(GCPs), is unfeasible and would effectively result in 

noncompliance problems, such as what occurred during the Persian Gulf War. FDA regulations governing 

storage and distribution of INDs (21 CFR 312.57 and 

59)33 are specific and limiting, which would render 

some therapeutics and any immunization schedule 

impossible in the field.

The FDA’s commitment to protecting the citizenry 

from the unknown effects of medical treatments has 

thus resulted in two legal quandaries. First, the FDA 

permits the use of unapproved products, including the 

vaccines in question, for research purposes under an 

IND.38 However, the situation in war is not a research 

situation but it still requires the use of an unapproved 

product under an IND protocol unless the notice requirement is fulfilled under 10 USC 1107(a); the product is administered during the practice of medicine; 

or it is provided under an IND protocol approved by 

the FDA. Giving these products to military personnel 

before engagement in war for purposes of preventing 

disease caused by a biowarfare agent constitutes a 

treatment application of the product and is not considered research. No benefit is believed to accrue to 

an individual receiving an investigational product. 

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Medical Aspects of Biological Warfare 

All IND protocols are clinical investigations, but not 

all clinical investigations are considered research. For 

example, off-label use of an approved product with no 

intention of reporting the safety and efficacy data to 

the FDA is not considered research under the Common 

Rule. However, both research and clinical investigations must receive approval by a formal IRB. Administration of investigational therapeutic or vaccination 

to military personnel in wartime does not constitute 

research, even though it is the only classification FDA 

permits for these unlicensed and untried vaccines. 

Continuing to categorize such vaccines and drugs as 

“investigational” also fails to inspire confidence in soldiers asked to receive the therapeutic or vaccine, even 

if limited evidence shows that the product is not only 

safe but likely efficacious based on extrapolation from 

animal data. The label “investigational” does not communicate the strength of the data from animal studies 

that supports the safety and efficacy of the product. 

It creates the perception that soldiers at risk of losing 

their lives in combat are also being used as subjects of 

research—or guinea pigs—despite the intent to use 

these products solely for the soldiers’ protection.

The FDA requires informed consent from subjects 

receiving investigational products. Consequently, subjects have the right to decide whether they will receive 

the investigational drug, and soldiers must understand 

that they cannot be required to take investigational 

drug products. The requirement for informed consent is based on the Nuremberg Trial findings related 

to research in which benefits did not directly accrue 

to research participants. In the context of preventive 

treatment in a military conflict, the requirement for 

informed consent is a misapplication of a principle of 

clinical research ethics. Enlisted and commissioned 

soldiers surrender much of their autonomy in matters 

of choice and accept the relinquishment of autonomy 

as a standard of military discipline and law. Specifically, one of the rights that military personnel forsake 

is the discretionary authority over their medical 

treatment under the rules and regulations governing 

force health protection (FHP). FHP can be defined 

as all services performed, provided, or arranged by 

the armed services to promote, improve, conserve, or 

restore the mental or physical well-being of personnel. The requirement for informed consent threatens 

to put a divisive wedge between commander and 

subordinates, and such discord is counterproductive 

to military recruitment, retention, and mission accomplishment. One solution to this problem may be 

to move IND products to licensure either by obtaining 

FDA approval through the use of the animal efficacy 

rule or by BioShield EUA, with all of the attendant 

medical subject matter expert board review and input 

afforded to products going before the FDA.

In the first Persian Gulf War, the DoD was acutely 

concerned with protecting military personnel from 

harm related to biological weapons. Intelligence indicated that Iraq had not only used chemical weapons 

against humans in the past, but it had also manufactured and stockpiled biological weapons that were 

believed to be ready for use. In documents sent to the 

FDA regarding implementing proper biodefense in 

military personnel against botulism, the DoD argued 

that waiver of informed consent was justified because 

a botulism vaccine (also referred to as the pentavalent 

botulinum toxoid vaccine) was to be administered as 

protection of and not as research on military personnel. 

The FDA accepted this DoD argument and exempted 

the DoD from the data gathering and recordkeeping 

requirements typically required during the administration of investigational products.39–44

This decision had historic consequences. Some 

commentators characterized the FDA’s accommodation of the DoD’s wishes as unethical. This accusation 

resulted in changes in the relationship between the 

FDA and DoD after veterans claimed “Gulf War syndrome” injuries. Gulf War syndrome is a phrase used 

to capture the constellation of injury claims stemming 

from symptoms experienced by Gulf War veterans 

after the conflict, some of which have been attributed 

to anthrax and/or botulism vaccination.45,46 Despite 

repeated high visibility detailed studies conducted by 

the Institute of Medicine of the National Academies of 

Science, no causal relation has been shown between 

these symptoms and receipt of either vaccine.47–51 Most 

soldiers who received inoculations from the same lots 

of vaccine as those who claim illness did not experience any of the associated symptoms. Furthermore, 

the majority of claims of illness were associated with 

receipt of the anthrax vaccine, which was already an 

FDA-licensed product for inhalational and cutaneous 

anthrax at the time of deployment for the first Persian Gulf War, rather than the botulism vaccination, 

which few soldiers received. Articles that summarize 

long-term outcomes after receipt of multiple vaccines, 

including those used during the Persian Gulf War, 

address the safety of these vaccines.38,52,53 But even if 

the existence of a causal relationship between receipt 

of the vaccine and the manifestations of the Gulf War 

syndrome is accepted, the DoD’s use of the vaccines to 

protect the force was an ethically supportable decision. 

It was an ethically supportable decision first and foremost because military intelligence indicated botulism 

was Iraq’s biological weapon of choice, which meant 

there was a likelihood of its use during military operations. Any use of botulism by the Iraqi forces would 

place American soldiers directly in harm’s way, but 

to an extent greater than would be faced during most 

traditional 20th century warfare. 

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Ethical Issues in the Development of Drugs and Vaccines for Biodefense

consent for receipt of an investigational product can 

undermine public trust and military morale. The FDA 

requirement for informed consent for receipt of an 

investigational product is mandated as necessary for 

research protocol approval and is premised on the 

idea that administration of an investigational product 

is for research purposes, and the safety and efficacy 

of the drug are unknown. The caveat to this requirement is the use of the EUAs and expanded access 

IND applications in support of deployments, both of 

which are not considered research. If countermeasures 

without medically significant contraindications were 

licensed for therapeutic purposes, this would lower the 

threshold for requiring informed consent. Licensure 

“for military use” would remove the stigma attached 

to use of an agent categorized as “investigational” for 

research purposes and mitigate the need for the use 

of investigational products along with the potential 

of failure in meeting the stringent regulatory requirements established for human subjects protection.

Realities of Deployment Conflict With Food and 

Drug Administration Regulations and Guidance

GCP data requirements support new product license applications, but GCP data collection does not 

serve the purposes of DoD military use of selected 

(unlicensed) medical products. The FDA enforces 

clinical data collection on IND products as a function of stringent protection of research integrity. 

Shortfalls in data management, such as missing 

data, missing vials, or missing forms, are inevitable 

during expediencies of real-time deployment and 

the exigencies of warfare, making it difficult for the 

DoD to meet FDA requirements. Unanticipated or 

unavoidable protocol violations and deviations inevitably occur, even under ideal investigational circumstances, and even when researchers fully intend 

to strictly follow GCP requirements. Unforeseen 

circumstances encountered in war are unavoidable. 

Scientific misconduct, then, may be suspected when 

the realities of deployment work against traditional 

scripted research strategies. Ultimately, force protection—not research—is the primary purpose of the 

military use of these countermeasures.

This scenario was one of the instigating factors that 

led Congress to enact 10 USC 1107(a) (notice for off-label 

uses required) and 10 USC 1107(f) (EUA provisions for 

DoD).54 The DoD had an obligation to meet this extra 

threat for its soldiers’ health and for the benefit of the 

military mission. To meet this threat as ethically as possible, subject matter experts weighed in on the risks and 

benefits of using the vaccine, and discussions between 

the DoD and FDA were held. The possibility of ill effects 

from the vaccine is an unintended consequence of the 

situation, but they could not have been known beforehand and do not alter the ethically supportable dimensions of the decision-making process, the intentions, 

or even the execution of the plan to vaccinate soldiers.

Summary Points

Historically Human Subjects Protections Regulations 

Had Been Incompatible With Department of Defense 

Deployments

The immediacy of war preparations and the chaos 

generated during military operations work against 

requirements of human subjects protection, including the requirement to solicit and obtain informed 

consent from subjects. Receipt of an IND drug must be 

voluntary. However, by definition, true FHP measures 

cannot be voluntary. The voluntary nature of FDAregulated research can undercut the effectiveness of 

FHP measures, which rely on universal compliance 

for their efficacy. FHP measures, which are necessary 

for successful operations in war, are imposed to safeguard the soldiers’ health. If left to the choice of individual soldiers, the health benefit to the soldier may 

be compromised and military success jeopardized. To 

mitigate this dilemma, under 10 USC 1107 a waiver 

may now be granted where informed consent would 

not be necessary, thus allowing the incorporation of 

unlicensed new drugs into the FHP program. Military 

personnel, who have ceded part of their autonomy to 

the government as a condition of service, are obligated 

to accept command-directed protective measures in 

the United States (immunizations are voluntary in 

the United Kingdom and in most European militaries). However, waiving the requirement for informed 

OPTIONS FOR FULFILLING MISSION AND ETHICAL RESPONSIBILITIES 

TO MILITARY PERSONNEL

Option 1: Continue to Use Investigational New 

Drug Products Without Full Compliance

Regardless of a presidential waver of informed 

consent, the DoD cannot use investigational products 

without many instances of noncompliance with GCP 

unless the needed product is approved under an 

EUA. However, the EUA was not available to DoD 

during the deployments to Desert Storm in 1991 and 

Bosnia in 1992, and the return deployment to Iraq in 

2003. Therefore, it was predictable that serious and 

continuing noncompliance would result from use of 

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IND products in a dynamic battlefield environment. 

GCP conflict with the requirements of countermeasure 

use during wartime, as seen during the first Persian 

Gulf War. The ethical responsibility of the DoD to 

protect soldier health and welfare does not commit 

the DoD to creating marketable products. However, 

if the data are gathered on these investigational products during wartime with the intention of increasing 

product knowledge, then GCP restrictions may need 

to be adaptable and flexible for wartime military use 

because of the inherent limitations imposed by military 

operations. These changes would permit the DoD to 

contribute to research by adding to the data gathered 

before bringing these investigational products to market. DoD can choose to move forward with a particular 

investigational product while doing its best to use the 

product according to FDA requirements, including 

adhering to GCP when practical.

Problems

Any relaxation of FDA standards could facilitate an 

impression of abuse of power by the DoD. Accusations 

of product approvals without sufficient consideration 

of safety issues could result in legal and economic 

fallout for the federal government. Most importantly, 

relaxing these standards, which the FDA has put in 

place to protect citizens, could result in a patient’s 

injury or death.

Option 2: Negotiate for Accelerated Licensure

The DoD can negotiate with the FDA for assistance 

in hastening licensure of products required in contingencies or for FHP. If the DoD negotiates directly 

with the FDA, then drugs and vaccines could be given 

without the burden of research format and documentation. Epidemiological follow-up—not case report 

forms—would determine benefit, and decisions to 

retain or withdraw approval could be based on epidemiological analyses. The DoD could ask the FDA to 

waive investigational drug requirements that cannot 

be practicably met in specific cases. Finally, there are 

now several mechanisms by which investigational 

products may be used by the DoD for FHP to include 

10 USC 1107(a) notice provision, presidential wavers 

to advanced informed consent, EUA, fast-tract product 

approval, accelerated approval, breakthrough therapy 

designation, and orphan drug status.55

Problems

The potential for DoD abuse of such power, or even 

the perception of abuse of such powers, will always 

be present. In addition, applications for FDA licensure 

must originate from the patent holder, not the DoD.

Option 3: Institute Waiver of Informed Consent

Although considered a necessary condition for research to be ethical, the requirements for obtaining informed consent (21 CFR 50.20-.27, 32 CFR 219.116-.117, 

45 CFR 46.116-.117)33,56,57 are not absolute. If informed 

consent is unfeasible or contrary to the best interests 

of recipients (21 CFR 50),33 such as in emergency 

situations or where the subject cannot give informed 

consent because of a medical condition and no representative for the subject can be found, the requirement 

can be waived. Executive Order 13139 and the Strom 

Thurmond National Defense Authorization Act of 1999 

give the president of the United States the power to 

waive the requirement for informed consent for the 

administration of an unlicensed product to military 

personnel in connection with their participation in a 

particular operation.58 The requirements are a formal 

request from the secretary of defense for such a waiver, 

based on evidence of safety and efficacy weighed 

against medical risks, and the requirement that a duly 

constituted IRB must approve the waiver, recordkeeping capabilities, and the information to be distributed 

to soldiers before receipt of the drug or vaccine.

One might argue that there is no need for a waiver of 

informed consent. If a soldier refuses receipt of a particular unlicensed product, he or she can be replaced by 

another soldier who is willing. But one does not have 

to search far for a scenario where waiver of informed 

consent might be warranted. The present day worries 

over recruitment and retention reflect this situation.

Problems

Some existing regulations conflict with the president’s power to waive informed consent requirements for military personnel, including conflicts and 

limitations posed by Title 10 USC Section 980 (10 USC 

980),59,60 AR 70-25.19 Title 10 USC 980 reads as follows:

Funds appropriated to the Department of Defense 

may not be used for research involving a human being as an experimental subject unless (1) the informed 

consent of the subject is obtained in advance; or (2) in 

the case of research intended to be beneficial to the 

subject, the informed consent of the subject or a legal 

representative of the subject is obtained in advance.35

10 USC 980 contains no provision for waiver of the 

requirement for informed consent, not even for the 

president, and neither of its two conditions for waiving the requirement would be met by a presidential 

waiver. However, DoDI 3126.02 allows for a waiver of 

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Ethical Issues in the Development of Drugs and Vaccines for Biodefense

the advance informed consent provision of 10 USC 980 

for research conducted under 21 CFR 50.24, Exception 

from Informed Consent for Emergency Research.

Chapter 3, section 1, paragraph (f) of AR 70-25 

states that “voluntary consent of the human subject 

is essential. Military personnel are not subject to punishment under the Uniform Code of Military Justice 

for choosing not to take part as human subjects. No 

administrative sanctions will be taken against military 

or civilian personnel for choosing not to participate as 

human subjects.”17 Therefore, when the DoD and USC 

regulations are compared and interpreted including 

DoDI 6200.02, Section 5.2.4 medical products for FHP, 

DoDI 3216.02, subject research definitions, 10 USC 980 

(informed consent for human subject research), and 10 

USC 1107 (f) (presidential consent waiver in specific 

military operations for FHP), these regulations are in 

agreement. Thus, the DoD and USC regulations are not 

in conflict because 10 USC 980 (informed consent for 

human subject research) and 10 USC 1107 (f) (presidential waiver) are not in conflict because the latter 

applies to a waiver for a specific military operation, 

which is FHP and not human subject research. 

An additional problem with presidential waiver of 

informed consent is the requirement that such a waiver 

be posted for public review in the Federal Register. This 

requirement makes operational secrecy impossible, especially given the length of time some vaccines require 

to elicit adequate titers in recipients.

Also, public perception is a looming issue. If the 

requirement for informed consent is waived—even 

by the president—public backlash is not likely to be 

quiet or short lived. Public awareness of research 

subject abuse has grown, and the public is aware that 

informed consent is essential for the ethical use of 

products for which the FDA cannot claim knowledge 

of safety and efficacy. Public outrage directed at the 

military, and the subsequent erosion of trust between 

the government and the governed, is a risk that also 

must be considered.

CURRENT MOVEMENTS IN THE REGULATORY ENVIRONMENT

Further restricting the ability of the DoD to properly protect military personnel with vaccines with 

preclinical evidence of efficacy would not be the best 

solution to this legal and ethical dilemma. If the DoD 

were to eschew unlicensed products and the IND issue 

entirely, an argument could be made that military personnel would be at greater risk from infectious agents. 

However, several options are available to address this 

issue,34 some of which have seen dialogue or attention 

in the form of legislation.

The Public Health Security and Bioterrorism 

Preparedness and Response Act of 2002

The Public Health Security and Bioterrorism 

Preparedness and Response Act of 2002, also called 

the Bioterrorism Act and the Guidance for Industry: Expedited Programs for Serious Conditions – 

Drugs and Biologics,60 contains several provisions 

to facilitate approval of vaccines and other priority 

countermeasures eligible for accelerated approval, 

clearance, or licensing. Title II of the act also contains 

the kernel of what is known as biosurety, which is 

a combination of biosafety, security, and personal 

reliability needed to safeguard select biological 

agents and toxins that could potentially be used in 

bioterrorism. Finally, this act approved the animal 

efficacy rule.61 The Guidance for Industry document 

outlines expedited programs to include fast track, 

breakthrough therapy, accelerated approval, and 

priority review designations.

21 Code of Federal Regulations 314 Subpart I: The 

Animal Efficacy Rule 

Another regulatory response that reflects a positive 

move toward reducing conflicts in responsibilities 

between the FDA and DoD was the creation of an 

animal efficacy rule. A draft animal efficacy rule was 

prepared by the FDA commissioner’s office and had 

been published for public comment 2 years before the 

terrorist attacks in fall 2001. The FDA recognized the 

acute need for an animal efficacy rule that would help 

make certain essential new pharmaceutical products 

available much sooner. These products, such as current 

IND vaccines, cannot be safely or ethically tested for 

effectiveness in humans because of the nature of the 

illnesses they are designed to treat.

The FDA amended its new drug and biological 

product regulations so that certain human drugs 

and biologics intended to relieve or prevent serious 

or life-threatening conditions may be approved for 

marketing based on evidence of effectiveness from appropriate animal studies when human efficacy studies 

are not ethical or feasible. The FDA took this action 

because it recognized the need for adequate medical 

responses to protect or treat individuals exposed to 

lethal or permanently disabling toxic substances or 

organisms. This new rule, part of FDA’s effort to help 

improve the nation’s ability to respond to emergencies, 

including terrorist events, applies when adequate and 

well-controlled clinical studies in humans cannot be 

ethically conducted because the studies would involve 

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Medical Aspects of Biological Warfare 

administering a potentially lethal or permanently disabling toxic substance or organism to healthy human 

volunteers.

Under the new rule, certain new drug and biological products used to reduce or prevent the toxicity of 

chemical, biological, radiological, or nuclear substances may be approved for use in humans based on 

evidence of effectiveness derived only from appropriate animal studies and any additional supporting data. 

Products evaluated for effectiveness under the rule will 

be evaluated for safety under preexisting requirements 

for establishing the safety of new drug and biological 

products. The FDA proposed this new regulation on 

October 5, 1999, and the rule took effect on June 30, 

2002. The advent of the animal efficacy rule shows 

the importance of animals in finding safe and effective countermeasures to the various toxic biological, 

chemical, radiological, and nuclear threats.

Using animal surrogates to prove clinical efficacy 

is not a perfect solution, even though it is the only 

ethical and moral solution in the case of drugs and 

vaccines aimed at mitigating biowarfare or bioterrorism threats. Additionally, use of animals in infectious 

disease research presents its own ethical and moral 

dilemma. Intentional infection of animal research 

subjects with deadly diseases requires strong consideration of the research harm versus benefit analysis 

by the institutional animal care and use committee as 

well as development of species and disease-specific 

humane early endpoints.62 Use of analgesics and humane early endpoints in animal studies is not always 

standardized among infectious disease researchers, 

potentially making comparison of research data difficult. Other challenges include conduct of research 

under animal biosafety level 3-4 and good laboratory 

practices conditions, development of adequate animal 

models, and gaining clear FDA guidance on research 

design and animal manipulation methods. 

To improve the validity of animal efficacy studies as 

models of human clinical efficacy, it is important to be 

rigorous in searches for the most optimal model that 

accurately mimics human disease. It is also necessary to 

draw precise comparisons between immune responses 

and drug kinetics in the animal surrogate and analogous 

responses in patients who participate in product safety 

but not clinical efficacy studies. Furthermore, because 

drugs approved by the animal efficacy rule may still 

not be “proven” efficacious in humans, postmarketing epidemiological studies are necessary to monitor 

outcomes. Finally, some diseases, such as dengue and 

smallpox, only affect human beings and do not affect 

animals. If animal efficacy data cannot be produced for 

a disease, the implication is that no vaccine could be 

created or used in human beings, which hardly seems 

a fitting solution. Testing of countermeasures against 

disease surrogates (closely related diseases) that do have 

animal models or the use of in vitro tissue culture assays 

systems may be the only alternatives to evaluating some 

diseases that lack a suitable animal model. 

BioShield Act of 2004

Project BioShield was designed to speed the development and availability of medical countermeasures 

in response to bioweapons threats by accelerating and 

streamlining government research on countermeasures, creating incentives for private companies to 

develop countermeasures for inclusion in a national 

stockpile, and giving the government the ability to 

make these products quickly and widely available in 

a public health emergency to protect citizens from an 

attack using an unmodified select agent.

The BioShield Act of 2004 created permanent funding for the procurement of medical countermeasures 

and gave the federal government the power to purchase available vaccines. The FDA and Department of 

Health and Human Services are tasked not only with 

determining that new vaccines and treatment measures 

are safe and efficacious, but also with the responsibility 

of making promising vaccines and treatment measures 

expeditiously available for emergency situations. The 

FDA Emergency Use Authorization for Promising 

Medical Countermeasures provides one of the best 

ways of getting such products to those who might 

need them most, including military personnel. The 

legislation also requires the secretary of the Department of Health and Human Services to approve such 

emergency use measures, with the added requirement 

of FDA expert opinion that the benefits of the vaccine 

or treatment outweigh the risks involved in its application. Just such an emergency use of anthrax vaccine 

adsorbed (Biothrax, BioPort Corporation, Lansing, 

MI) was approved by Health and Human Services 

Secretary Tommy G Thompson on January 14, 2005, 

authorizing its emergency use.

Under DoDI 1107a (emergency use products), a 

waiver by the president would permit the use of a 

product in the times of an emergency:

Waiver by the President. (1) In the case of the administration of a product authorized for emergency 

use under section 564 of the Federal Food, Drug, 

and Cosmetic Act to members of the armed forces, 

the condition described in section 564(e)(1)(ii)(III) of 

such act and required under paragraph (1)(A) or (2)

(A) of such section 564(e), designated to ensure that 

individuals are informed of an option to accept or refuse administration of a product may be waived only 

by the President only if the President determines in 

writing that complying with such requirement is not 

in the interest of national security. (2) The waiver 

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Ethical Issues in the Development of Drugs and Vaccines for Biodefense

authority provided in paragraph (1) shall not be construed to apply to any case other than a case in which 

an individual is required to be informed of an option to accept or refuse administration of a particular 

product by reason of a determination by the Secretary of Health and Human Services that emergency 

use of such product is authorized under section 564 

of the Federal Food, Drug and Cosmetic Act.

Provision of Information. If the President, under 

subsection (a) waives the condition described in section 564(e)(1)(A)(ii)(III) of the Federal Food, Drug 

and Cosmetic Act, and if the Secretary of Defense, 

in consultation with the Secretary of Health and Human Services, makes a determination that it is not 

feasible based on time limitations for the information 

described in section 564(e)(1)(A)(ii)(I) or (II) of such 

Act and required under paragraph (1)(A) or (2)(A) of 

such section 564(e), to be provided to a member of 

the armed forces (or next-of-kin in case of the death 

of a member) to whom the product was administered 

as soon as possible, but not later than 30 days after 

such administration. The authority provided for in 

this subsection may not be delegated. Information 

concerning the administration of the product shall 

be recorded in the medical record of the member. 

Applicability of Other Provisions. In the case of an 

authorization by the Secretary of Health and Human 

Services under section 564(a)(1) of the Federal Food, 

Drug, and Cosmetic Act based on a determination 

of the Secretary of Defense under section 564(b)(1)

(B) if such Act, subsections (a) through (f) of section 

1107 shall not apply to the use of a product that is 

the subject of such authorization within the scope of 

such authorization and while such authorization is 

effective.63,64

The Turner Bill

Another bill (HR 4258, Rapid Pathogen Identification to Delivery of Cures Act), introduced by Congressman Jim Turner et alia on May 4, 2004, allows 

research and development of medical countermeasures and diagnostics to move at a quicker pace so 

that new products can rapidly be made available for 

emergencies. In addition, the Turner Bill provides 

for research and development of drugs and vaccines 

against genetically modified pathogens not accounted 

for in the Project BioShield legislation, which covered 

only countermeasures related to existing unmodified 

threat agents.

Project BioShield and the Turner Bill together establish an FDA EUA for critical biomedical countermeasures. The FDA may approve solely for emergency use 

a product not approved for full commercial marketing. 

For products that are near final approval but may 

not have met all the criteria, the FDA has created a 

streamlined IND process, with the animal efficacy rule 

playing a central role, for products designed to protect 

against or treat conditions caused by nuclear, chemical, or biological terrorism. Such a process was used 

to obtain FDA approval for pyridostigmine, which 

is licensed for use in treating myasthenia gravis and 

was approved on February 5, 2003, for use to increase 

survival after exposure to soman nerve gas poisoning 

by the military in combat.65

Biodefense and Pandemic Vaccine and Drug Development Act Renewal of 2011

In October 2005, Senator Richard Burr of North 

Carolina introduced the Biodefense and Pandemic 

Vaccine and Drug Development Act of 2005 (S 1873). 

This bill establishes the Biomedical Advanced Research 

and Development Agency as the lead federal agency 

for the development of countermeasures against 

bioterrorism. The new agency would report directly 

to the secretary of Health and Human Services. The 

bill provides incentives for domestic manufacturing 

of vaccines and countermeasures, and it gives broad 

liability protections to companies that develop vaccines for biological weapons. This bill may appear to 

settle the residual concerns left unresolved by Project 

BioShield, but it has raised additional controversy 

because of public perceptions that it is too favorable 

to the pharmaceutical industry and issues related to 

secrecy provisions.

In March 2011, the Pandemic and All-Hazards Preparedness Act Reauthorization Act of 2011 received US 

Senate approval.66 The act renewed measures from the 

2006 legislation to promote the development and procurement of medical countermeasures against weapons of mass destruction agents. It reauthorized Project 

Bioshield Special Reserve Fund for purchasing vaccines 

and other treatments over a 10-year period. Funding 

for biodefense has now evolved to include nonbiodefense and emerging pathogens research, development, 

and product acquisition with continued government 

financial increases since the original act in 2005.67

DILEMMAS FOR BIODEFENSE RESEARCH

The potential devastating consequences of a bioterrorist event was revealed to the public in 2001, and this 

fear was enhanced by its proximity in time to the tragic 

events of 9/11. The immediate reaction of the government and the public was to support new legislation 

intended to protect the homeland (Patriot Act) and 

expand the law enforcement, military, intelligence, 

and defense industries. Funding was dramatically 

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Medical Aspects of Biological Warfare 

increased to agencies funding biodefense research and 

development. Tens of billions of dollars were spent 

on building infrastructure, establishing personal reliability/biosurety/biosecurity oversight, expanding the 

number of biocontainment laboratories, hiring containment specialists, training first responders, stockpiling 

personal protective equipment, establishing laboratory response networks and biodetection capabilities, 

establishing stockpiles of medical countermeasures, 

and greatly expanding biodefense research and 

development.68 

Dual use research consists of scientific studies 

that possess the potential of generating information 

or material that—if used nefariously—could harm 

public health.69–75 This dichotomy often results in the 

placement of potentially excessive restrictions and 

limitations upon the types of research permitted, 

and thus has the potential of preventing or severely 

restricting the ability of scientists to find solutions or 

answer key questions needed to mitigate biothreats. 

For example, performing genetic modifications to 

produce antibiotic resistant bacterial select agents or 

creating novel recombinant strains of influenza are 

prevented under the Biological Weapons and Toxins Convention and the United States Government 

Policy for Oversight of the Life Sciences Dual Use 

Research. Without access to these modified organisms, 

determining the efficacy of potential new therapies 

or vaccines using in vitro assays and in vivo animal 

models is hampered, creating fundamental gaps in our 

biodefensive posture and national security. A critical 

need exists for a review of ethics in biodefense76 and 

the researchers responsible to safely and securely 

find solutions to the biodefense related problems.77

SUMMARY

This chapter has provided a view of the history 

of ethically conducted human subject research in

the military and has presented some of the problems that still exist among the distinct regulatory 

bodies that impact this research. The DoD has an 

ethical responsibility to protect military personnel, 

yet there is disagreement over how to best protect 

them against biological warfare attacks, in light of 

equal commitments to respecting agency autonomy 

and limiting government power over individual 

decisions regarding what constitutes one’s own 

best interests. These issues and problems are not 

a mystery to those who confront them on a daily 

basis, and many thoughtful individuals are focusing their attention on resolving these dilemmas. 

Some progress is being made, at least in terms of 

productive dialogue and substantive attention to 

legislation that might impact research.

Acknowledgments

The authors would like to acknowledge the efforts of the following individuals in preparing this chapter. 

Lee L Zwanziger, PhD, while Director of Research on the Staff of the President’s Council on Bioethics, 

provided valuable advice and criticism during the early phases of writing. Jonathan D Moreno, PhD; Emily Davie; Joseph S Kornfeld, Professor of Biomedical Ethics at the University of Virginia and Director, 

Center for Biomedical Ethics; and Griffin Trotter, PhD, MD, Associate Professor in the Center for Health 

Care Ethics at Saint Louis University provided critical reviewers’ comments. 

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November 12, 2015.

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Index

ABBREVIATIONS and ACRONYMS

A

AAALAC: Association for Assessment and Accreditation of Laboratory Animal Care 

ABC: ATP-binding cassette

ABSA: American Biological Safety Association 

ABSL: animal biosafety level

ACB: Acinetobacter calcoaceticus baumannii

ACEI: angiotensin-converting enzyme inhibitor

ACIP: Advisory Committee on Immunization Practices

ADAMs: A Disintegrin-Like And Metalloproteinase-containing 

protein

Ads: adenoviruses

Ag-ELISA: antigen enzyme-linked immunosorbent assay

agr: accessory gene regulator 

AHF: Argentinian hemorrhagic fever

AI: avian influenza

AIDS: acquired immune deficiency syndrome; acquired immunodeficiency syndrome

AIGIV: anthrax immune globulin intravenous (human)

ALO: anthrolysin O

α-DG: α-dystroglycan

AML: Area Medical Laboratory

AMR: antimicrobial resistance monitoring 

ANG: Air National Guard

Ank: ankyrin repeat domain 

AnkG: ankyrin repeat-containing protein G

AP-1: activator protein 1 

APC: antigen-presenting cell 

APHIS: Animal and Plant Health Inspection Services 

APSV: Aventis Pasteur smallpox vaccine

AR: Army Regulation

ARDS: acute respiratory distress syndrome 

ARM: antimicrobial resistance monitoring

ARMoR: Antimicrobial Resistance Monitoring and Research 

(Program)

ARO: alternate responsible official

ASD(HA): Assistant Secretary of Defense for Health Affairs 

ASP: amnesic shellfish poisoning

AT: autotransporter

ATLS: Advanced Trauma Life Support

ATP: adenosine 5′-triphosphate 

AVA: anthrax vaccine adsorbed 

AVP: anthrax vaccine precipitated

B

BAMC: Brooke Army Medical Center 

BAT: botulism antitoxin heptavalent

BBB: blood–brain barrier

BDBV: Bundibugyo virus

BEBOV: Bundibugyo ebolavirus

BHF: Bolivian hemorrhagic fever

BIG: Botulism Immune Globulin Intravenous

BMBL: Biosafety in Microbiological and Biomedical Laboratories

BoNT: botulinum neurotoxin

BPRP: Biological Personnel Reliability Program

BSAT: biological select agents and toxins 

BSC: biological safety cabinet

BSL: biosafety level

BSP: Biological Surety Program

BT: bioterrorism

BTWC: Biological and Toxin Weapons Convention

BuHA: Burkholderia Hep_Hag autotransporter

BW: biological warfare

BWC: Biological Weapons Convention

C

C: capsid

cAd3-EBOZ: chimpanzee adenovirus type 3–vectored ebolavirus 

Zaire vaccine

CAdVax: complex adenovirus vaccine

CAFO: concentrated animal feeding operation

CAP: College of American Pathologists

CARA MEL: CBRNE Analytical & Remediation Activity Mobile 

Expeditionary Laboratory

CBDP: Chemical Biological Defense Program

CBIRF: Chemical-Biological Incident Response Force

CBRN: chemical, biological, radiological, and nuclear

CBRNE: chemical, biological, radiological, nuclear, and explosives

CBSP: Certified Biological Safety Professional 

CC50: modeling of cytotoxicity data

CCD: colony collapse disorder

CCHFV: Crimean–Congo hemorrhagic fever virus

CCP: critical control point

CD: Cluster of Differentiation

CD-22: Camp Detrick-22

CDC: Centers for Disease Control and Prevention

CDC-DSAT: Centers for Disease Control and Prevention-Division 

of Select Agents and Toxins

CEV: cell-associated enveloped virion

CFR: case fatality rate; Code of Federal Regulations

CFT: cell-free translation; complement fixation test

CFU/cfu: colony-forming unit

CHAPV: Chapare virus

CHIK: chikungunya

CHIKV: chikungunya virus

CHOC: chocolate agar

CLDC: cationic liposome DNA complex

CLIA: Clinical Laboratory Improvement Amendments

CLIP: Clinical Laboratory Improvement Program

CLSI: Clinical Laboratory Standards Institute

CMA: competent medical authority

CMI: cell-mediated immune

CNS: central nervous system

CO: certifying official 

CONUS: continental United States

CoV: Coronavirus

CP: capsid protein

CpG: cytosine-phosphate-guanine

CPS: capsular polysaccharide

CSF: cerebrospinal fluid

CSH: Combat Support Hospital

CST: Civil Support Teams

CT: computed tomography

CWC: Chemical Weapons Convention

D

DA: US Department of the Army

DA PAM: Department of the Army Pamphlet

dB: decibel

DEET: N,N-diethylmetatoluamide

DENV: dengue virus

DHF: dengue hemorrhagic fever

DHHS: Department of Health and Human Services

DHS: Department of Homeland Security

DIC: disseminated intravascular coagulation

DNA: deoxyribonucleic acid

DoD: Department of Defense

DoD-GEIS: Department of Defense-Global Emerging Infections 

Surveillance and Response System 

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DoDI: Department of Defense Instruction

DON: deoxynivalenol

dot/icm: defective in organelle trafficking/intracellular multiplication (lc)

DPP4: dipeptidyl peptidase 4

DRC: Democratic Republic of the Congo

DSAT: Division of Select Agent and Toxins

DSS: dengue shock syndrome

DURC: dual use research of concern

E

EAEC: enteroaggregative Escherichia coli

EBOV: Ebola virus

EC50: concentration of a drug that gives half-maximal response

ECL: electrochemiluminescence

EDTA: ethylenediaminetetraacetic acid

EEE: eastern equine encephalitis

EEEV: Eastern equine encephalitis virus

EEV: extracellular enveloped virion

EIEC: enteroinvasive Escherichia coli

EHEC: enterohemorrhagic Escherichia coli

ELISA: enzyme-linked immunosorbent assay

EMDG: Expeditionary Medical Dental Group

EPA: US Environmental Protection Agency

EPEC: enteropathogenic Escherichia coli

EPICON: epidemiological consultation

EPS: exopolysaccharide 

ER: endoplasmic reticulum

Erks: extracellular signal-regulated kinases

ESBL: extended-spectrum b-lactamase 

ESF: emergency support function

ESKAPE: Enterococcus faecium, Staphylococcus aureus, Klebsiella 

pneumoniae, Acinetobacter calcoaceticus baumannii, Pseudomonas 

aeruginosa, and Enterobacter

ET: edema toxin

EUA: Emergency Use Authorization

EV: enveloped virion

EVD: Ebola virus disease

F

F: fusion protein

F1: fraction 1

Fab: fragment antigen-binding

FBI: Federal Bureau of Investigation

FDA: Food and Drug Administration

FEMA: Federal Emergency Management Agency

FFAG: flow-focusing aerosol generator

FFBAT: Air Force Biological Augmentation Team

FHP: force health protection

fla B: flagellin B gene

FMD: foot and mouth disease

FPI: Francisella pathogenicity island

FSAP: Federal Select Agent Program

G

G: attachment glycoprotein

Gal/GalNac: N-acetyl galactosamine

GCP: Good Clinical Practices

G-CSF: granulocyte colony-stimulating factor 

GEIS: Global Emerging Infections Surveillance and Response 

System

GMP: good manufacturing practices

GPC: glycoprotein precursor

GTOV: Guanarito virus

GTX: gonyautoxin

H

HA: hemagglutinin

HACCP: hazard analysis critical control point

HAI: healthcare-associated infection/ hospital-associated infection 

HAVCR1: hepatitis A virus cellular receptor 1

HAZMAT: hazardous materials

HBAT: heptavalent botulinum antitoxin

HBsAg: hepatitis B surface antigen

Hc: heavy chain

HCoV: human Coronavirus

HDP: 1-0-hexadecyl-oxypro-pyl

HEPA: high-efficiency particulate air 

HeV: Hendra virus

HFRS: hemorrhagic fever with renal syndrome

HHA: hand held assay

HHS: US Department of Health and Human Services

HI: hemagglutination inhibition

HIV: human immunodeficiency virus

HLA: Human Leukocyte Antigen

HPAI: highly pathogenic avian influenza

HPV: human papilloma virus

HTS: high throughput screening

HUS: hemolytic uremic syndrome

I

IBC: Institutional Biosafety Committee

IC50: half-maximal inhibitory concentration

ICAM-1: intercellular adhesion molecule-1

ICC: International Clonal Complex

ICD-10: International Statistical Classification of Diseases and Related 

Health Problems, Tenth Revision

ICLN: Integrated Consortium of Laboratory Networks

ICS: Incident Command System

IEV: intracellular enveloped virion

IFA: indirect immunofluorescence assay

IFN: interferon

IFNγ: interferon gamma

IgG: immunoglobulin G

IgM: immunoglobulin M

IHA: direct hemagglutination assay

IHR: International Health Regulations

IL: interleukin

IM: intramuscular/intramuscularly

IMP: inosine 5’-phosphate

IMV: intracellular mature virion

IN: intranasal

IND: investigational new drug

IP: intraperitoneal

IQ: ilimaquinone

IRB: institutional review board

IT: intratracheal

IQ: ilimaquinone

IU: international unit

IV: intravenous 

J

JBAIDS: Joint Biological Agent Identification and Diagnostic 

System

JEV: Japanese encephalitis virus

JUNV: Junín virus

K

KPC: Klebsiella pneumoniae carbapenemase

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Index

L

L: large; large RNA-dependent RNA polymerase

LAAV: live attenuated anthrax vaccine

LAMP: lysosomal-associated membrane protein

LASV: Lassa virus

LC/MS: liquid chromatography/mass spectrometry

LCt50: acute toxicity

LD50: lethal dose (amount necessary to kill 50% of the subject 

population) 

LFA-1: lymphocyte function-associated antigen

LLAP: Legionella-like amoebal pathogen

LLOV: Lloviu virus

LOD: limit of detection

LPC: lysophosphatidylcholine

LPCR: large-fragment polymerase chain reaction

LPS: lipopolysaccharide

LRMC: Landstuhl Regional Medical Center 

LRN: Laboratory Response Network

LRN-B: Laboratory Response Network-Biological

LSD: lysergic acid diethylamide

LT: lethal toxin; long term

LUJV: Lujo virus

LVS: live vaccine strain

M

M: matrix protein

mAb/mAbs: monoclonal antibody/monoclonal antibodies 

MACV: Machupo virus

MADV: Madariaga virus

MALDI-TOF MS: matrix-assisted laser desorption/ionization-time 

of flight mass spectrometry

MAP: mitogen-activated protein

MAPK: mitogen-activated protein kinase

MARV: Marburg virus

MASCAL: mass casualty event or exercise

Mb: megabase pair 

MCM: medical countermeasure

MCP-1: monocyte chemoattractant protein-1

MDCK: Madin-Darby Canine Kidney cells

MDR: multidrug resistant 

MDRO: multidrug-resistant organism

MEDCOM: US Army Medical Command

MEDEVAC: medical evacuation

MERS: Middle East Respiratory Syndrome

MH: Madagascar hissing

MHC: major histocompatibility complex

MHS: Military Health System

MIC: mean inhibitory concentration

miRNA: microRNA

MLST: multilocus sequence typing

MLVA: multiple-locus variable number tandem repeat analysis

MMAD: mass median aerodynamic diameter

MNGC: multinucleated giant cell

MOU: memorandum of understanding

MPL: 3-O-desacyl-4′-monophosphoryl lipid A

MRSN: Multidrug-Resistant Organism Repository and Surveillance Network

MS: mass spectroscopy

MSD: Meso Scale Discovery

MTF: medical treatment facility

mTOR: mammalian target of rapamycin

MVA: Modified Vaccinia Ankara/modified vaccinia virus Ankara

MVD: Marburg virus disease

MW: molecular weight

N

N: nucleocapsid protein

NA: neuraminidase; North American

NAS: National Academy of Sciences 

NATO: North Atlantic Treaty Organization

NDM: New Delhi metallo-β-lactamase-1

NDMS: National Disaster Medical System

NDRF: National Disaster Recovery Framework

NF-AT: nuclear factor of activated T cells

NF-kB: nuclear factor kappa-light-chain-enhancer of activated B 

cells

NGDS: Next Generation Diagnostic System

NGS: next-generation sequencing

NHP: nonhuman primate 

NIAID: National Institute of Allergy and Infectious Diseases

NIH: National Institutes of Health

NIOSH: National Institute of Occupational Safety and Health

NiV: Nipah virus

NIV: nivalenol

NLR: NOD-like receptor

NMDA: N-methyl-d-aspartate

NNMC: National Naval Medical Center 

NOD: nucleotide oligomerization domain

NP: nucleoprotein

NPG: National Preparedness Goal

NRF: National Response Framework

NSABB: National Science Advisory Board for Biodefense

NSP: neurotoxic shellfish poisoning

O

OCONUS: outside of the continental United States

OEF: Operation Enduring Freedom

OFPBL: Oxidative-Fermentative-Polymyxin B-Bacitracin-Lactose 

(agar)

OIF: Operation Iraqi Freedom

OPS: O-polysaccharide

OR: operating room

OSHA: Occupational Safety and Health Administration

o/w: oil in water

P

P: phosphoprotein

PA: protective antigen

pAbs: polyclonal antibodies

PAPR: powered air-purifying respirator

PBT: pentavalent botulinum toxoid

PCR: polymerase chain reaction

PDI: potentially disqualifying information

PEG: polyethylene glycol

PEP: postexposure prophylaxis 

PFGE: pulsed field gel electrophoresis

PHV: parvovirus-like hybrid virus

PI: principal investigator

PI3K: phosphoinositide 3 kinase

PKC: protein kinase C

PLA2: phospholipase A2

Poly I:C: polyinosinic:polycytidylic acid

PPE: personal protective equipment

PPES: positive-pressure encapsulating suit

PRNT: plaque reduction neutralization test

PRNT80: 80% plaque reduction neutralization titer 

PS: polysaccharide

PSI: personnel security investigation

PSP: paralytic shellfish poisoning; phage shock protein

PTA: pteroic acid

PTK: protein tyrosine kinase 

PV: parasitophorous vacuoles 

Abbreviations and Acronyms

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Q

QS: quorum sensing

Q-Vax: Q fever vaccine

R

rAb: recombinant antibody

RAMP: Rapid Analyte Measurement Platform

RANTES: regulated upon activation normal T-cell expressed, and 

secreted

RAVV: Ravn virus

rBimA: recombinant Burkholderia intracellular motility A

RBP: Registered Biosafety Professional 

rDNA: ribosomal deoxyribonucleic acid

RDTE: research, development, test, and evaluation

RESTV: Reston virus

RFLP: restriction fragment-length polymorphism

RIFLE: risk, injury, failure, loss, and end-stage renal disease

RIP: ribosome inactivating protein

RNA: ribonucleic acid

RNP: ribonucleoprotein 

RO: responsible official

RR: relative risk 

RSV: respiratory syncytial virus

RTA: ricin A chain

RTB: lectin B-chain

RT-PCR: reverse transcription/transcriptase polymerase chain 

reaction

RVFV: Rift Valley fever virus

S

S: small

SA: South American

SABV: Sabiá virus

SARS: severe acute respiratory syndrome

SARS-CoV: severe acute respiratory syndrome-Coronavirus

SAT: select agents and toxins

SBA: sheep blood agar

SD: standard deviation

SDA: Seventh-day Adventist

SdAbs: single domain antibodies

SE: staphylococcal enterotoxin

SEA: staphylococcal enterotoxin A

SEB: staphylococcal enterotoxin B

SEBOV: Sudan ebolavirus

SFTS: severe fever with thrombocytopenia syndrome

SFTSV: severe fever with thrombocytopenia syndrome virus

SFV: Semliki Forest virus

sGP: secreted glycoprotein

SINV: Sindbis virus

SIP: Special Immunizations Program

SMX: sulfamethoxazole

SNARE: soluble N-ethylmaleimide-sensitive factor activating 

protein receptor

SNS: Strategic National Stockpile

SOP: standard operating procedure

SPE: streptococcal pyrogenic exotoxin

SRA: security risk assessment 

ssGP: secondary secreted glycoprotein

ST: sequence type 

STAG: spinning top aerosol generator

STEC: Shiga toxin-producing Escherichia coli

STRIVE: Sierra Leone Trial to Introduce a Vaccine Against Ebola

STX: saxitoxin

SUDV: Sudan virus

T

T3SS: type three secretion system

T6SS: type VI secretion system

TAFV: Taï Forest virus

Tat: twin arginine transport

TBEV: tickborne encephalitis virus

TCID50: median tissue culture infective/infecting dose

TCR: T-cell receptor 

3D: three-dimensional

TIR: toll/IL-1 receptor 

TLR: toll-like receptor 

TMP: trimethoprim

TMP-SMZ: trimethoprim combined with sulfamethoxazole

TNF-a/TNFa: tumor necrosis factor-alpha

TrD: Trinidad donkey

TSS: toxic shock syndrome

TSST-1: toxic shock syndrome toxin-1 

TTSS: type III secretion system

U

UK: United Kingdom

UN: United Nations

UNSCOM: UN Special Commission

USAMRIID: US Army Medical Research Institute of Infectious 

Diseases

USAMU: US Army Medical Unit

USA PATRIOT Act: Uniting and Strengthening America by Providing Appropriate Tools Required to Intercept and Obstruct 

Terrorism Act of 2001 

USC: United States Code

USDA: US Department of Agriculture

USNS: United States Naval Ship

USSR: Union of Soviet Socialist Republics (or the Soviet Union, 

now Russia) 

V

V: variable

VACV: vaccinia virus

VAERS: Vaccine Adverse Event Reporting System

VEE: Venezuelan equine encephalitis

VEEV: Venezuelan equine encephalitis virus

VHF: Venezuelan hemorrhagic fever; viral hemorrhagic fever

VIG: vaccinia immune globulin

VLP: virus-like particle 

VLS: vascular leak syndrome

Vm: minute volume

VRP: virus-like replicon particle

VS: virtual screening

VSV: vesicular stomatitis virus

vWF: von Willebrand factor 

W

WCV: whole cell vaccine

WEE: Western equine encephalitis

WEEV: Western equine encephalitis virus

WHO: World Health Organization

WMD: weapons of mass destruction

WNV: West Nile virus

w/o: water in oil

WRAIR: Walter Reed Army Institute of Research

WRAMC: Walter Reed Army Medical Center

WS: working stock

WWI: World War I 

WWII: World War II 

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Index

Y

Yaps: Yersinia autotransporter proteins

YFV: Yellow fever virus

Z

Z: zinc-binding matrix protein

ZEBOV: Zaire ebolavirus

ZF: zinc-finger

Abbreviations and Acronyms

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Medical Aspects of Biological Warfare 

A

Abbreviations, xxvii–xxxi

“ABCDE” algorithm, 116

Abortions, septic

Brucella and, 162–163

ACAM2000 vaccine, 115, 631–632

ACB. See Acinetobacter baumannii

Acellular vaccines, 235

Acinetobacter baumannii

antibiotic resistance, 328

characterization and identification of source, 327–328

epidemiological consultation, 324–327

genetic analysis, 327–328

historical perspectives, 322–324

inclusion in ESKAPE pathogens, 322, 328–332

molecular analysis, 327–328

strain collection and sampling, 327

U.S. Military Health System response, 328–332

Acronyms, xxvii–xxxi

Active immunization

alphavirus encephalitides, 498–501

Acute respiratory distress syndrome, 663–664

Adjuvants, vaccines, 831–832

Advanced Trauma Life Support, 110, 116, 117

Advisory Committee on Immunization Practices, 141

Aerobiology

biological aerosol exposure systems, 860–864

clinical applications in the U.S., 857–860

conceptual basis of, 857

dosimetry, 863

exposure chambers, 862

exposure systems, 860–862

generators, 861–862

Henderson apparatus, 861

monitoring and control, 862–863

offensive biological development in the U.S., 857–860

overview, 856–857

parameters impacting aerosol dosimetry, 863–864

sampling, 862

Aerosol dosimetry, 863–864

Afghanistan

Acinetobacter infections, 322, 323, 324–327

Al Qaeda biological warfare program, 15

Q fever outbreak, 307

ricin as biological weapon research, 376

Aflatoxins, 443, 445

Africa

Chikungunya outbreak, 674

Ebola virus epidemic, 677–678

African milk bush, 79

Agency for Healthcare Research and Quality, 329

Agent Orange, 79

Agent X, 338

Agglutination tests, 165

Agricultural terrorism, 78–80

Air Force field laboratories, 706

Al Qaeda

biological warfare program, 15, 16–17

Alcide EXSPORE, 883

Alibek, Dr. Kenneth, 254, 339

Alibekov, Colonel Kanatjan, 254, 339

Alphavirus encephalitides

active immunization, 498–501

antigenicity, 483–486

clinical disease, 492–496

diagnosis of, 492–496

differential diagnosis of, 495–496

eastern equine encephalitis, 484–485, 493–494

epidemiology and ecology, 486–488

genetic relationships, 483–486

historical perspectives, 481–483

immune effector mechanisms, 496–497

immunoprophylaxis, 496–501

medical countermeasures, 773–776

medical management, 496

passive immunization, 497–498

pathogenesis, 490–492

prevention of, 496

replication of, 488––490

significance of, 482–483

therapeutics, 501

Venezuelan equine encephalitis, 483–484, 485, 492–493, 773–774

virion structure, 488––490

western equine encephalitis, 484, 485–486, 494–495

American Biological Safety Association, 889–890

Aminoglycoside toxicity

differentiating from botulism, 343

Aminoglycosides

brucellosis treatment, 166–167

AML. See Area Medical Laboratory

Amnesic shellfish poisoning

description of toxin, 467–468

diagnosis of, 468–469

inhalation-acquired disease, 468

mechanism of action, 468

medical management, 469

oral ingestion, 468

signs and symptoms, 468

Amoxicillin

anthrax treatment, 140

glanders treatment, 204

melioidosis prophylaxis, 236

“AMPLE” history, 117

Anaplasmosis, 654–655

Angel’s trumpet, 449

Angiotensin-converting enzyme inhibitors, 417

Animal and Plant Health Inspection Service, 80, 897–899, 902–904

Animal care, 888–889

Animal disease models, 186–189, 382–384, 406, 587, 888–889

Animal diseases

African milk bush attack, 79

alphavirus encephalitides, 481–501

anthrax attacks by Germany in 1915-1916, 43–44, 78

Clostridium perfringens diseases, 362–367

coxiellosis, 310

deaths as indicator of biological agent attack, 40

filoviruses, 580

foot and mouth disease, 78–79

glanders, 186–189, 191–193

Hendra virus, 549, 552–553, 669

Nipah virus, 549–552, 553–554, 669–670

occurring simultaneously with human disease, 41

Animal Efficacy Rule, 272, 931–932

Animal poisons, 422

Anthrax

accidental release of, 49–50, 130

active immunization, 140–142

attacks by Germany in 1915-1916, 43–44, 78

attacks in US, 2001, 47–48, 130

INDEX 

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Aum Shinrikyo cult attacks, 13, 46–47

bioterrorism attacks, 13–15

characteristics of organism, 131–132

clinical disease, 135–138

cutaneous, 135–136

diagnosis of, 138–139

epidemiology of, 132

foodborne, 72

gastrointestinal, 138, 650

historical perspectives, 130–131

incidence in the US, 132

inhalational, 136–137

medical countermeasures, 752, 754, 757–761

meningitis, 137

oropharyngeal, 138

pathogenesis, 132–135

prophylactic treatment after exposure, 140, 760–761

Russian epidemic, 9

treatment of, 139–140

vaccine side effects, 142

vaccines, 10, 113, 114, 753–758

WHO surveillance and control guidelines, 140

Anthrax vaccine adsorbed, 754, 757–758

Anthrolysin O, 135

Antibacterial monoclonal antibodies, 837–839

Antibiotic resistance diseases, 655–656

Antibiotic susceptibility testing, 727

Antibiotics

Animal Efficacy Rule, 272

anthrax treatment, 139–140

bacterial disease treatment, 759–760

brucellosis treatment, 166–167, 768–769

Clostridium perfringens treatment, 363

glanders treatment, 202–204, 766–767

melioidosis treatment, 233–234, 766

multidrug-resistant bacterial infections, 328

plague treatment, 272, 765

Q fever, 771–772

tularemia treatment, 292–294, 763–764

Antibodies

antibacterial monoclonal antibodies, 837–839

antibody generations, 832–835

antitoxin monoclonal antibodies

antiviral monoclonal antibodies, 839–842

biodefense development, 832–842

development of new therapeutic formats, 832–835

ricin treatment, 388

Anticholinergic plants, 450

Anticrop agents

produced by the US military, 5

Anticrop terrorism, 79

Antigen-presenting cells, 404–406

Antigenic shift, 658

Antihypertensives, 417

Antimicrobial Resistance Monitoring and Research Program, 

331–332

Antimicrobial susceptibility testing, 727

Antitoxin monoclonal antibodies, 835–837

Antitoxins

C. botulinum pre- and postexposure prophylaxis, 347

for C. botulinum, 345–346

for Clostridium perfringens, 363

Antivenoms, 432

Antiviral agents

hemorrhagic fever-causing mammarenaviruses, 531–532

smallpox, 787–788

viral hemorrhagic fevers, 790–791

Antiviral monoclonal antibodies, 839–842

Aquariums

palytoxin exposure, 471–472

Arachnids, 419–420

ARDS. See Acute respiratory distress syndrome

Area Medical Laboratory, 117, 705

Argentinian hemorrhagic fever, 520, 526, 528, 530

Armed Forces Health Surveillance Center, 329

Armed Forces Medical Policy Council, 921

ARMoR. See Antimicrobial Resistance Monitoring and Research 

Program

Army field laboratories, 705

Army Public Health Command, 324

Army Techniques Publication, 716

Army Techniques Publication No. 5-19, 95

Arthritis

brucellosis and, 164

Asahara, Shoko, 46, 339

Asia

Chikungunya outbreak, 674

melioidosis incidence, 227

plague pandemic, 251

Aspergillus, 76

Association for Assessment and Accreditation of Laboratory 

Animal Care, 889

Association of Professionals in Infection Control and Epidemiology, 329

ATLS. See Advanced Trauma Life Support

ATP-binding cassette proteins, 190–191

Aum Shinrikyo cult

bioterrorism attacks, 13, 46–47

C. botulinum research program, 72, 339

Aureomycin

glanders treatment, 204

Australia

melioidosis studies, 233–234

paramyxovirus infections, 669

Q fever outbreak, 306

Automated identification systems, 724–727

Autotransporter proteins

glanders virulence mechanism, 190–191

plaque virulence factor, 263

Autumn crocus, 447

Avian influenza A virus, 19–22, 657–660, 661–662

Avian influenza H5N1 virus, 662–663

Avian influenza H7N9 virus, 663–664

Azithromycin

glanders treatment, 204

B

Bacillus aerogenes capsulatus, 362

Bacillus anthracis

accidental release of, 49–50, 130

active immunization, 140–142

attacks by Germany in 1915-1916, 43–44, 78

attacks in US, 2001, 47–48, 130

Aum Shinrikyo cult attacks, 13, 46–47

bioterrorism attacks, 13–15

characteristics of, 131–132

confirmatory tests for, 131–132

cutaneous anthrax, 135–136

diagnosis of, 138–139

epidemiology of, 132

foodborne, 72

gastrointestinal anthrax, 138, 650

historical perspectives, 130–131

inhalational anthrax, 136–137

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medical countermeasures, 752, 754, 757–761

meningitis, 137

oropharyngeal anthrax, 138

pathogenesis, 132–135

prophylactic treatment after exposure, 140, 760–761

Russian anthrax epidemic and, 9

treatment of, 139–140

vaccine for US military, 10

vaccines, 10, 113, 114, 753–758

Bacillus cereus, 132, 134

Bacillus welchii, 362

Bacterial culture techniques

glanders, 198–199

tularemia, 293

Bacteriophages, 202

Baghdad, Iraq

Acinetobacter infections, 325–326, 328

BALB/c mice, 186, 188, 205

Balkengrippe. See Q fever

Bangladesh

Nipah virus outbreak, 553–554, 670

BAT, 345

Bats

influenza viruses in, 664–666

BCA agar, 199

BCX4430, 791

Bedbugs

as plague vector, 256–257

Bee venom, 420, 421

Belladonna, 449

Belladonna toxicity

differentiating from botulism, 343

Bergendorff, Roger, 16

Beta-bungarotoxin, 367

Biochemical identification, 199

Biocrimes, 12

Biodefense and Pandemic Vaccine and Drug Development Act 

Renewal of 2011, 933

Biofire Defense FilmArray, 702

Biological aerosol exposure systems, 860–864

Biological agents. See also specific agents by name

accidental release of, 49–50

biocrimes, 12

biological surety, 896–911

biosafety levels, 122

characteristics of, 742

consequences of, 96–98

critical agents for health preparedness, 113

delivery systems developed by Iraq, 11

disarmament, 17–18

dual use research of concern, 19–22

early use of, 2–3

hospital infection control precautions, 121

the Iranian program, 12

the Iraq program, 10–11

the Libyan program, 12

the North Korean program, 12

pan-hazard preparedness, 17–24

produced by the US military, 5

prophylaxis against diseases caused by Category A agents, 119

regulated biological select agents and toxins, 703

risk assessment systems, 95

the South African program, 11–12

the Soviet program, 8–10

study of natural outbreaks for potential bioweapon use, 50–54

the Syrian program, 12

therapy of diseases caused by Category A agents, 119

the US program, 4–8

use during the World Wars, 3–4

Biological and Toxin Weapons Convention, 375

Biological Defense Research Program Laboratories, 888

Biological Incident Annex, 94, 98

Biological Integrated Detection System, 112

Biological Personnel Reliability Program, 898, 901, 905–908

Biological safety. See Biosafety

Biological safety cabinets, 860

Biological select agents and toxins

biosafety, 904–905

biosecurity, 908–910

centralized management of, 901

control of, 898–899

identifying, 904

incident response and emergency management, 910–911

inventory and accountability, 900–901

inventory audits, 901–902

inventory discrepancies, 911

list of Tier 1 agents and toxins, 898

permissible toxin amounts, 903

personnel reliability, 905–908

registration for possession, use, and transfer of, 899

reporting theft, loss, or release of, 903–904

restricted experiments, 904

sample criteria for retaining or destroying, 901

security breaches, 911

security risk assessment, 900

theft, loss or release of, 910

transfers, 902–903

Biological surety

biological select agent and toxin transfers, 902–903

biological select agents and toxins inventory and accountability, 900–901

biological select agents and toxins inventory audits, 901–902

biosafety, 904–905

biosecurity, 908–910

centralized management of long-term biological select agents 

and toxins, 901

control of biological select agents and toxins, 898–899

identifying select agents and toxins, 904

incident response and emergency management, 910–911

information security, 909–910

international rules and treaties to limit or ban biological 

weapons use, 896

inventory discrepancies, 911

operational security, 909

overview, 897–898

personnel reliability, 905–908

personnel security, 909

physical security, 909

registration for possession, use, and transfer of biological 

select agents and toxins, 899

reporting theft, loss, or release of biological select agents and 

toxins, 903–904

restricted experiments, 904

security breaches, 911

security risk assessment, 900

theft, loss or release, 910

Biological Surety Program, 888

Biological terrorism. See Bioterrorism

Biological warfare

impact of advanced molecular techniques on epidemiology of, 

58–59

Biological Weapons Convention, 8, 11, 17–18, 179, 338, 375, 897

Biologics Control Act, 926

Biomarkers, 716–718

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Biopreparat, 9, 10

Biosafety

assessing individual risk, 876–877

assigning agents in risk groups, 872–874

Biological Defense Research Program Laboratories, 888

biosafety levels, 874–876

case-fatality rate by disease, 873

definition of, 870

documenting safety procedures, 876

evolution of, 870–871

human infectious dose by organism, 873

laboratory animal care and use program, 888–889

laboratory safety audits, 887–888

laboratory worker protection, 876

medical surveillance, 880–881

microbiological practices, 884–886

personal protective equipment, 878–880

physical barriers, 877–878

precautions employed, 122

the profession, 889–890

program elements required for containment and maximum 

containment laboratories, 876–886

program management role, 886–889

protecting the community and environment, 881–882

risk groups, 872

solid and liquid waste inactivation and disposal, 882–884

BioSense 2.0 program, 57, 58

BioShield Act of 2004, 932–933

Biosurety Program, 888. See also Biological surety

Biosurveillance, 739–741

Bioterrorism. See also Medical countermeasures; specific agents by 

name

agricultural, 78–80

case studies, 43–49

casualty management, 110–124

consequence management, 94–96

consequences of, 96–98

historical perspectives, 13–17

hospital infection control precautions, 121

impact of advanced molecular techniques on epidemiology of, 

58–59

improving recognition and surveillance of, 56–58

infectious diseases resulting from, 121

local and national response, 98–102

recovery, 103–105

risk assessment systems, 95

samples to obtain from victims, 118

signs of C. botulinum attack, 346–347

Bioterrorism Act, 897

BioThrax, 140–141, 754, 757

BioThreat Alert Lateral Flow Assay, 386

BioWatch program, 56, 101

Bites, venomous, 418

Bivalent botulinum toxoid, 347

Bivalent recombinant botulinum vaccine, 348

Black Death plague, 2–3, 250–251

“Black Maria,” 858

Black rats

plague transmission role, 257

Black tar heroin, 342

Bloomberg, Michael

ricin attacks in 2004, 48, 377

Bolivian hemorrhagic fever, 520, 526, 530

Borreliosis, 652–654

BotDB resource, 348

Botulinum immune globulin, 347

Botulinum toxin

antitoxins, 345–346

bioterrorist attack signs, 346–347

botulinum neurotoxin production, 340–341

clinical disease, 341–342

description of agent, 72–73, 340–341

diagnosis of, 343–344

diagnostic assays, 343–344

foodborne botulism, 72–73, 342, 344

historical perspectives, 338–340

inhalation-acquired botulism, 342, 344

pathogenesis, 341

prophylactic treatment, 347, 794–795

symptoms of, 342

therapeutic drug research, 348–349

treatment of, 345–349

vaccine research, 348, 793–794

BPRP. See Biological Personnel Reliability Program

Brazilian hemorrhagic fever, 521

Brevetoxins, 463, 465–467

Brooke Army Medical Center

Acinetobacter infections, 324

Brucella

host specificity, 161

lipopolysaccharide epitopes, 161–162

nomenspecies, 160–161

virulence factors, 162

Brucella abortus, 163

Brucella canis, 162

Brucella melitensis, 164

Brucella suis

development as biological weapon, 160

Brucellosis

clinical manifestations, 164–165

diagnosis of, 165–166

epidemiology of, 162

historical perspectives, 160

infectious agent characteristics, 160–162

medical countermeasures, 768–770

minimum inhibitory concentration breakpoint ranges, 167

pathogenesis, 163

prophylactic treatment, 167, 769–770

symptoms and signs of, 164

treatment of, 166–167

vaccines, 768

BSAT. See Biological select agents and toxins

Bubonic plague, 265, 267

Buffalopox, 623, 628

Bundibugyo virus, 571–575, 589, 592

BurkDiff, 200

Burkholderia mallei

animal disease models, 186–189

attacks by Germany in 1915-1916, 43–44, 78

biochemical identification, 199

characteristics of, 181–182

clinical disease in animals, 191–193

clinical disease in humans, 193–198

clinical features of laboratory-acquired infections, 196–197

control of, 208–109

decontamination, 208–109

diagnosis in equines, 202

differentiation from B. pseudomallei, 181, 182

epidemiology of, 182–183

growth characteristics, 198

historical perspectives, 179–181

host immunity, 205–206

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identification of, 199–201

immunological detection, 201

immunotherapies, 208

intracellular characteristics, 186

isolation of organism, 198–199

laboratory diagnosis, 198–202

medical countermeasures, 766–768

military relevance, 178–179

morphology, 198

nucleic acid-based identification, 199–201

pathogenesis, 184–191

prophylactic treatment, 205–209, 767–768

septicemia, 195

serologic diagnosis, 201–202

transmission of, 183–184

treatment of, 202–204

vaccines, 206, 207–208, 766

virulence mechanisms, 189–191

Burkholderia pseudomallei

candidate virulence factors, 228–230

characteristics of, 224–225

clinical disease, 227, 230–232

diagnosis of, 232–233

differentiation from B. mallei, 181, 182

epidemiology of, 226

medical countermeasures, 766–768

military relevance, 225–226

pathogenesis, 226–227

prevention of, 234–236

prevention of disease, 234–236

treatment of, 233–234

Burnet, Dr. Frank Macfarlane, 306

Bush, George H., 10

Bush, George W., 22, 114

BWC. See Biological Weapons Convention

Byetta, 417

C

Caffa

plague pandemic, 250, 252

CAFOs. See Concentrated animal feeding operations

California

plague pandemic, 251

Camelpox, 623

Camp Detrick, Maryland

biological warfare agents research, 857–858, 923–925

Burkholderia mallei infections, 178, 183, 194–195, 196–197

Campylobacter jejuni, 73, 77, 650–651

Canada

alphavirus encephalitides outbreaks, 482

Capsular polysaccharides, 235

Captopril, 417

Cardiotoxic plants, 448–449

Cardiotoxins, 440–441, 448

Cardiovascular system

Staphylococcal enterotoxin B effects, 407–408

Caribbean Islands

Chikungunya outbreak, 674

Castor beans, 375, 377, 382, 446

Casualty management

alerting authorities, 118–123

chemical protection, 113

diagnosis of casualties, 118, 120

disinfecting or decontaminating, 116–117

epidemiological investigation, 123–124

establishing diagnosis, 117–118

immunologic protection, 113–116

infection control, 118, 121

maintaining level of proficiency, 124

maintaining level of suspicion, 110–12

managing psychological aftermath, 123–124

physical protection, 112–113

primary assessment, 116

prophylaxis against diseases caused by Category A agents, 119

protecting self, 112–116

providing therapy, 118

psychological effects of biological attacks, 123–124

saving patient’s life, 116

secondary assessment, 116–118

therapy of diseases caused by Category A agents, 119

Catalytic A-chain, 377–378, 380, 390

Cationic liposome DNA complexes, 208

Cats

plague transmission role, 257

Cavanaugh, Lieutenant Colonel Dan C., 252

C57Bl/6 mice, 186, 188

CBRNE Command, 705

CDC. See Centers for Disease Control and Prevention

Ceftazidime

glanders treatment, 204

melioidosis treatment, 233

Cell-free translation assay, 386, 387

Cell-mediated immune responses, 205

Cellular stress response inhibitors, 391

Centers for Disease Control and Prevention

BioSense 2.0 program, 57

BSAT forms, 899, 902–904

case definition for brucellosis, 165

Division of Select Agent and Toxins, 860, 897–898

Emergency Operations Center, 345

epidemiological investigative assistance, 123

Laboratory Registration and Select Agent Transfer Program, 897

Laboratory Response Network, 14, 22, 101, 122, 386, 704, 

727–728

Morbidity and Mortality Weekly Report, 259

plague diagnosis procedures, 270

plague treatment procedures, 272

procedures for isolation and presumptive identification of 

Y. pestis, 269

public health preparedness capabilities, 102

Select Agents and Toxins Program, 375

smallpox specimen collection, 628

Centipedes, 443

Central America

alphavirus encephalitides outbreaks, 482, 487–488

Chikungunya outbreak, 674

Central Intelligence Agency, 8, 377

CFT. See Cell-free translation assay; Complement fixation test

Chapare virus, 521, 523–524

Chemical Battalions, 117

Chemical-Biological Incident Response Force, 117, 123

Chemical Corps, 116

Chemical nerve agent poisoning

differentiating from botulism, 343

Chemical protection, 113

Chemical Warfare Service, 5

Chemical Weapons Convention, 375

CHEMPACKs, 102

Chikungunya, 673–674

China

accusations of US biowarfare attacks, 6–7

Avian influenza H7N9 virus infections, 663–664

plague pandemic, 251

severe fever with thrombocytopenia syndrome virus, 675–676

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Chloramphenicol

glanders treatment, 204

melioidosis treatment, 234

plague treatment, 272

Chloroform:methanol residue vaccine, 312

Chloroquine

Q fever treatment, 311

Cholera, 647–649

Cidofovir, 635

Ciguatoxic fish, 422, 462

Cilastatin

melioidosis treatment, 233

Ciprofloxacin

anthrax exposure prophylaxis, 10, 140

anthrax treatment, 139

plague treatment, 272

Civil Support Teams, 123

Clavulanate

glanders treatment, 204

melioidosis prophylaxis, 236

CLIA. See Clinical Laboratory Improvement Amendments

Clindamycin

anthrax treatment, 140

Clinical Laboratory Improvement Amendments, 702–703

Clinical Laboratory Improvement Program, 702–703

Clinical Laboratory Standards Institute, 166

CLIP. See Clinical Laboratory Improvement Program

Clorox Ultra Germicidal Bleach, 883

Clostridium argentinense, 340

Clostridium baratii, 340

Clostridium botulinum

antitoxins, 345–346

bioterrorist attack signs, 346–347

botulinum neurotoxin production, 340–341

clinical disease, 341–342

description of agent, 72–73

diagnosis of, 343–344

diagnostic assays, 343–344

foodborne botulism, 72–73, 342, 344, 651

historical perspectives, 338–340

inhalation-acquired botulism, 342, 344

pathogenesis, 341

prophylactic treatment, 347

symptoms of, 342

therapeutic drug research, 348–349

treatment of, 345–349

vaccine research, 348

Clostridium butyricum, 340

Clostridium perfringens alpha toxin, 362–363

Clostridium perfringens epsilon toxin

chemical properties, 364

description of, 363–365

diseases, 363

historical perspectives, 362–363

mechanism of action, 364–365

medical management, 366–367

natural occurrence of, 363–364

physical properties, 364

signs and symptoms of, 365–366

toxin types, 362

Clostridium perfringens type D, 365–366

Clostridium welchii, 362

Cnidarians, 420–422

Co-trimoxazole, 203

Colony collapse disorder, 678

Colorado potato beetle, 79

Common source outbreaks, 40

Community Emergency Response Team Program, 100

Competent medical authority, 907

Complement fixation test, 201, 202

Comprehensive Health Surveillance, 739

Comprehensive Preparedness Guide 201, 95

Concentrated animal feeding operations, 79

Confirmation identification methods, 709–716

Congo

incidence of human plague cases, 259

Congressional Office Building

ricin bioterrorism attack, 16

Consequence management

Comprehensive Preparedness Guide 201, 95

consequences of biological incidents, 96–98

definition of, 94

local and national response, 98–102

National Disaster Recovery Framework, 94

recovery, 103–105

risk assessment systems, 95

Convention on the Prohibition of the Development, Production 

and Stockpiling of Bacteriological and Toxin Weapons and on 

their Destruction, 8, 11, 17–18, 179, 338, 375

Cooperative Biological Engagement Program, 10

Corals

palytoxin exposure, 469, 471–472

Coronaviruses, 666–669

Countermeasures. See Medical countermeasures

Cowpox, 623, 628

Coxiella burnetti

characteristics of, 307–309

diagnosis of, 311

disease in animals, 310

disease in humans, 310

epidemiology of, 309–310

historical perspectives, 306

medical countermeasures, 770–772

military relevance, 306–307

pathogenesis, 309

postexposure prophylaxis, 772

treatment of, 311

vaccines, 312, 770–771

Coxiellosis, 310

Critical control points, 81

Crocus, 447

Crooker, Michael, 16

Crop diseases, 79

Cruikshank, George, 918

Cryptosporidiosis, 75

Cryptosporidium parvum, 75, 77, 78

Culture-based methods, 722–724, 727–728

Cutaneous anthrax, 135–136

Cutaneous plague, 268–269

Cyanogenic plants, 448

Cysticercosis, 77

Cytotoxic plants, 446–447

Czechoslovakia

use of biological agents during World War II, 4

D

Dalles, Oregon

Salmonella typhimurium attacks, 1984, 44–46

Darwin Prospective Melioidosis Study, 227, 230, 234

Daschle, Tom

anthrax attack, 2001, 47–48

Decontamination

glanders, 208–109

Decontamination procedures, 104, 116–117

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Defence Science and Technology Laboratory, 388

Defense Laboratory Network, 706–707

Delaware

alphavirus encephalitides outbreaks, 482

Dengue fever, 671–672

Department of Defense Global Emerging Infections Surveillance 

and Response System, 324, 329, 331

Dermal exposure

palytoxin, 471

Derrick, Dr. Edward Holbrook, 306

Despeciated equine heptavalent antitoxin, 345

DHHS. See U.S. Department of Health and Human Services

DHS. See U.S. Department of Homeland Security

Diagnostic tests, in vivo/in vitro, 728

Digital imaging, 202

Dilger, Anton, 44, 78, 178

Diphtheria

differentiating from botulism, 343

Disinfection procedures, 116–117

Division of Select Agent and Toxins, 860, 897–898

DNA assays, 199–201

DNA vaccines, 500

DoD-Geis. See Department of Defense Global Emerging Infections 

Surveillance and Response System

Dominant-negative inhibitors, 366

Dominican Republic

cholera outbreak, 648

Domoic acid, 463, 467–469

Dosimetry, 863

Doxycycline

anthrax exposure prophylaxis, 140

anthrax treatment, 139

brucellosis treatment, 166–167

glanders treatment, 203–204

melioidosis treatment, 234

plague postexposure prophylaxis, 273

plague treatment, 272

Q fever treatment, 311

Drug-resistant bacteria. See Multidrug-resistant organisms

Dryvax, 631–632

Dual use research of concern, 19–20

DURC. See Dual use research of concern

Dutschke, Everett, 16, 377

Dysentery, 363

E

Eastern equine encephalitis, 484–485, 493–494

Ebola virus, 118, 571, 573–575, 580–592, 596–598, 677–678

ECL. See Electrochemiluminescence

Eczema vaccinatum, 633

Edema toxin, 132–135

Ehrlich, Paul, 375

Ehrlichiosis, 654–655

“Eight Ball,” 859, 925

Electrochemiluminescence, 385, 386, 387, 730–732

Electronic Surveillance System for the Early Notification of 

Community-based Epidemics, 57

ELISA. See Enzyme-linked immunosorbent assays

Emergency management, 910–911

Emergency Operations Center, 345

Emergency support functions, 98, 101, 123

Emergency Use Authorization, 703, 740–741, 925–926, 929

Emerging infectious diseases

antibiotic resistance, 655–656

avian influenza, 657–660

bacterial diseases, 647–657

chikungunya, 673–674

coronaviruses, 666–669

definition of, 646

Dengue fever, 671–672

Ebola epidemic in West Africa, 677–678

factors contributing to, 646

foodborne diseases, 650–652

genetically engineered organisms, 679–681

genomic epidemiology, 656–657

high-throughput DNA sequencing, 678–679

H1N1 influenza pandemic, 660–661

human infection with avian influenza viruses, 661–664

influenza viruses in bats, 664–666

mosquitoborne viruses, 670–674

paramyxoviruses, 669–670

swine influenza, 660–661

synthetic biology, 681

tickborne diseases, 652–655

tickborne phleboviruses, 675–677

viral diseases, 657–679

waterborne diseases, 647–650

West Nile virus, 672–673

Encephalitis. See Alphavirus encephalitides

Encephalomyelitis

nonviral causes of, 496

viral causes of, 495

Endocarditis, Q fever, 310

Endopep-MS, 344

England

plague pandemic, 250

Enhanced Response Force Package Teams, 123

Enrofloxacin

glanders treatment, 204

Enterohemorrhagic E. Coli, 74

Enterotoxemia, 363, 365

Envenomation, 417, 418

Environmental niche modeling, 583

Environmental Protection Agency, 101

Enzootic

definition of, 249

Enzyme-linked immunosorbent assays

botulism diagnosis, 344

brucellosis diagnosis, 165, 166

filoviruses diagnosis, 593–595

glanders diagnosis, 201, 202

hemorrhagic fever-causing mammarenaviruses diagnosis, 

529–530

orthopoxviruses diagnosis, 629

overview, 728–730

plague diagnosis, 270

Q fever diagnosis, 311

ricin diagnosis, 385, 387

Staphylococcal enterotoxin B diagnosis, 408

EpiCenter application, 57

EPICON. See Epidemiological consultations

Epidemics

clues to an unnatural event, 39–41

definition of, 38

downwind plume patterns, 41

Ebola virus in West Africa, 38

epidemic curves, 42

multiple epidemics, 40

outbreak investigation, 41–43

recognition of, 38–39

Epidemiological consultations, 324–327

Epidemiology

accidental release of biological agents, 49–50

assessment tool, 54–56

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Index

bioterrorism events, 43–49

clues to an unnatural event, 39–41, 111

common source outbreaks, 40

of epidemics, 38–43

the epidemiological triangle, 38

impact of advanced molecular techniques, 58–59

improving recognition and surveillance of bioterrorism, 56–58

investigations after biological attacks, 123–124

point source outbreaks, 40

study of natural outbreaks for potential bioweapon use, 50–54

Epizootic

definition of, 249

Epsilon toxin vaccine, 367

Equine encephalitis. See Alphavirus encephalitides

Equines

C botulinum antitoxin, 347

glanders diagnosis, 202

Escherichia coli, 74, 77, 78, 377, 651–652

Escherichia coli O104:H4, 652

Escherichia coli O157:H7, 651–652

ESKAPE pathogens, 322, 328–332

Ethical issues

Animal Efficacy Rule, 931–932

biodefense, ethics, and research in the 20th Century, 921–925

biodefense and ethics in the 18th and 19th Centuries, 918–921

Biodefense and Pandemic Vaccine and Drug Development Act 

Renewal of 2011, 933

BioShield Act of 2004, 932–933

conflict between regulations and ethical responsibilities, 926–929

current movements in the regulatory environment, 931–933

dilemmas for biodefense research, 933–934

DoD/FDA Memorandum of Understanding, 927

impact of regulating agencies on strategic research, 925–926

investigational new drug status, 927–929

options for fulfilling mission and ethical responsibilities to 

military personnel, 929–931

overview of, 916–925

Public Health Security and Bioterrorism Preparedness and 

Response Act of 2002, 931

summary points, 929

The Turner Bill, 933

EUA. See Emergency Use Authorization

Europe

plague pandemic, 250–251

Excitatory neurotoxins, 430–432

Exendin-4, 417

Expeditionary Medical Dental Group, 326

Exposure chambers, 862

Exposure systems, 860–862

Eyes

Staphylococcal enterotoxin B effects, 408

F

F1 antigen, 262

F1-V vaccine, 273–274

Fairchild Air Force Base

ricin attacks in 2004, 377

Farcy. See Glanders

Favipiravir, 791

FBI. See Federal Bureau of Investigation

Federal Bureau of Investigation, 14, 45, 101

Federal Emergency Management Agency, 94, 100

Federal Experts Security Advisory Panel, 23

Federal Response Plan, 22

Federal Select Agent Program, 20, 897–900, 904–906

FEMA. See Federal Emergency Management Agency

Field laboratories, military, 705–706

FilmArray, 738

Filoviruses

behavioral modification for prevention, 586

categorization of viruses, 571–572

clinical presentation of disease, 590–591

decontamination, 586

diagnosis of, 591–595

disease nomenclature, 571, 574

environmental niche modeling, 583

epidemiology of, 580–583

epizootiology of, 580

filovirion structure, 572

function of, 576–579

genomes, 572–573, 575

geographic distribution, 574

inactivation of, 586

lifecyle, 573–574

molecular biology, 572–583

natural reservoirs of, 574–575

nomenclature, 571–572

pathogenesis, 587, 590

prevention of, 586–587

proteins, 572–573

serological surveys, 583

Soviet biological warfare program, 584–586

taxonomy, 571

transmission of, 583–584

treatment of, 595, 596–598

vaccines, 587, 588–589

Fimbrae, 262–263

Fish, venomous, 420–422

Fleas

as plague vector, 256–257

plague virulence and transmission factors, 264

Florida

plague pandemic, 251

Flow cytometry, 732–733

Flu Near You, 57

Fluorescence in situ hybridization assay, 202

Fluorescence Resonance Energy Transfer, 630

Fluorescence spectroscopy, 202

Fluorescent antibody staining, 166, 270–271

Food, Drug, and Cosmetic Act, 926

Food and Drug Administration, 82, 112, 272, 587, 702–704, 916, 

926–933

Food Emergency Response Network, 101

Food Safety and Modernization Act, 82

Food supply security, 80–82

Foodborne diseases

anthrax, 72

botulinum toxins, 72–73, 342, 344

Campylobacter jejuni, 73, 77, 650–651

Clostridium botulinum, 72–73, 342, 344, 651

Clostridium perfringens diseases, 363

cryptosporidiosis, 75

emerging diseases, 650–652

Escherichia coli, 74, 651–652

gastrointestinal anthrax, 650

hepatitis A, 75

listeriosis, 73–74

mycotoxicosis, 75–76

parasites, 76–77

salmonellosis, 44–46, 73, 652

shigellosis, 47, 74–75

threat potential, 76, 77

Foot and mouth disease, 78–79

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Medical Aspects of Biological Warfare 

Force health protection, 928–929

Fort Detrick, Maryland

Burkholderia mallei infections, 178, 183, 194–195, 196–197

Fox squirrels

plague transmission role, 257

Foxglove, 447

Francisella pathogenicity island, 289

Francisella tularensis

acquired, 289

aerosol transmission, 288

arthropod vectors, 288

bacterial culture techniques, 293

characteristics of, 286–287

clinical manifestations, 290–292

diagnosis of, 292–294

direct contact, 287

epidemiology of, 287–289

food and water ingestion, 288

laboratory-acquired disease, 289

laboratory worker exposure, 296

mammalian bites, 288

medical countermeasures, 761–764

pathogenesis, 289–290

postexposure prophylaxis, 295, 764

prophylactic treatment, 295–295

rapid diagnostic methods, 293–294

serology, 293

treatment of, 294–295

in unusual settings, 288–289

use as biological weapon, 296

vaccination with live vaccine strain, 289, 290, 295–296, 761–763

Freeman, Dr. Mavis, 306

Frist, Bill

ricin attacks in 2003-2004, 48–49

Fugu puffer fish, 422

Fungal toxins, 443, 445

G

Gas gangrene, 362–363

Gas masks, 112

Gastroenteritis, 78

Gastrointestinal diseases

anthrax, 138, 650

plague, 268

plant irritants, 451

tularemia, 291

Genetic analysis

Acinetobacter baumannii, 327–328

Genetically engineered organisms, 679–681

Geneva Convention, 8, 11

Geneva Protocol, 3, 6, 896–897

Genomic epidemiology, 656–657

Gentamicin

plague treatment, 272

Germany

cholera outbreak, 647

Shiga toxin deaths, 377

use of biological agents during World War I and II, 3–4, 43–44, 

78, 178

Giardia lamblia, 78

Gila monster, 417

Glanders

animal disease models, 186–189

attacks by Germany in 1915-1916, 43–44, 78, 178

biochemical identification, 199

clinical disease in animals, 191–193

clinical disease in humans, 193–198

clinical features of laboratory-acquired infections, 196–197

control of, 208–109

decontamination, 208–109

diagnosis in equines, 202

epidemiology of, 182–183

historical perspectives, 179–181

host immunity, 205–206

identification of, 199–201

immunological detection, 201

immunotherapies, 208

infectious agent characteristics, 181–182

isolation of organism, 198–199

laboratory diagnosis, 198–202

medical countermeasures, 766–768

military relevance, 178–179

morphology, 198

nucleic acid-based identification, 199–201

organism growth characteristics, 198

pathogenesis, 184–191

prophylactic treatment, 205–209, 767–768

septicemic, 195

serologic diagnosis, 201–202

transmission of, 183–184

treatment of, 202–204

vaccines, 206, 207–208, 766

virulence mechanisms, 189–191

Global Emerging Infections Surveillance and Response System, 

324, 329

Gonyautoxins, 463

Good Clinical Practices, 927, 929

Government Accountability Office, 24

Granulocyte colony-stimulating factor, 234

Great Britain

eradication of glanders, 180

use of biological agents during World War II, 4

Ground squirrels

plague transmission role, 257, 258

Guanarito virus, 521, 522, 523–524, 530–531

Guillain-Barré syndrome

differentiating from botulism, 343

Guinea pigs

as animal disease models, 186

Gulf War

Q fever outbreak, 307

H

HACCPs. See Hazard analysis critical control points

Haffkine, Waldemar M.W., 273

Hague Convention, 3

HAIs. See Healthcare-assisted infections

Haiti

cholera outbreak, 648–649

Hamsters

as animal disease models, 186

Handheld assay devices

overview, 733–734

ricin detection, 385, 387

Harris, Larry Wayne, 13–14

Hatfill, Dr. Steven J., 14

HAVCR1. See Hepatitis A virus cellular receptor 1

Hazard analysis critical control points, 81

Hazardous Material Regulations, 902

Hazardous Materials Response Unit, 117

HBAT, 345

Healthcare-assisted infections, 329

Heartland virus, 676–677

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Index

Hematology

Staphylococcal enterotoxin B, 408

Hemolytic toxins, 437–440

Hemorrhagic fever-causing mammarenaviruses

antiviral agents, 531–532

clinical presentation, 522–523

coagulopathies, 527–528

diagnosis of, 529–530

epidemiology of, 519–521

historical perspectives, 519–521

immune response, 528–529

molecular characteristics, 524–525

New World mammarenaviruses, 520–521, 523, 526–527, 

528–529

Old World mammarenaviruses, 519–520, 522–523, 525–526, 

528

passive antibody therapy, 530

pathogenesis, 525–527

pathology, 525–527

phylogenetic relationships, 523–524

reservoirs of, 521–522

taxonomy, 523–524

treatment of, 530–532

vaccines, 530–532

Hemorrhagic toxins, 437–440

Hemostasis-active toxins, 435–437

Hemostasis system, 438–439

Henderson apparatus, 861

Hendra virus, 549, 552–553, 669

Henipaviruses

anti-henipavirus immune response detection, 558–559

antigen detection, 558

availability of, 557–558

as biological weapons, 557–558

clinical presentation, 554–555

diagnosis of, 558–559

dissemination of, 558

emergence of, 549–552

epidemiology of, 552–554

epizootiology of, 552–554

Hendra virus, 549, 552–553

historical perspectives, 549–552

innate immunotherapy, 560

isolation of virus, 559

medical management, 559–560

molecular biology of, 555–556

newly identified viruses, 552

next-generation sequencing, 559

Nipah virus, 549–552, 553–554

passive immunotherapy, 559–560

pathogenesis, 557

pathology, 555

potential for high morbidity and lethality, 558

production of, 558

reverse transcription-polymerase chain reaction, 558

ribavirin, 560

signs and symptoms of, 554–555

therapies evaluated in vitro, 560

virus genome, 555

virus proteins, 555–556

virus structure, 555

Hepatitis A, 75, 77

Hepatitis A virus cellular receptor 1, 367

Heptavalent despeciated antitoxin, 346

Herbal medicine

ricin poisoning, 381

Hfab-BAT, 346

HHA. See Handheld assay devices

High-efficiency particulate air filters, 871, 877, 882

High throughput DNA sequencing, 678–679

High throughput screening, 390, 391

Highly pathogenic avian influenza A virus, 19–22, 662–663

H1N1 influenza A virus, 19, 22, 660–661

H5N1 virus, 19–22

Homeland Security Council, 22

Homoheptamers, 365

Hong Kong, China

Avian influenza H5N1 virus infections, 662–663

Horses

glanders diagnosis, 202

Host immunity, 205–206

Host response, 741–743

HTS. See High throughput screening

Human fleas

as plague vector, 256

Hussein, Saddam, 10–11

Hybridization probes, 630

Hydroxychloroquine

Q fever treatment, 311

Hyperimmune sera, 367

I

Iatrogenic botulism, 342, 344

IHA. See Indirect hemagglutination assay

Ilimaquinone, 391

Illumina MiSeq instrument, 740

Imipenem

glanders treatment, 204

melioidosis treatment, 233

Immunization. See also Vaccines

alphavirus encephalitides

anthrax, 140–142

Immunodiagnostic methods, 728–730

Immunologic protection, 113–116

Immunological detection, 201

Immunosorbent assays. See Enzyme-linked immunosorbent assays

Immunotherapies

glanders, 208

IMP dehydrogenase inhibitors, 792

In vivo/in vitro diagnostic tests, 728

Inactivated vaccines, 499

Incapacitating agents

produced by the US military, 5

Incident Command System, 121

Incident response and emergency management, 910–911

IND. See Investigational new drugs

India

Chikungunya outbreak, 674

Nipah virus outbreak, 553–554

plague pandemic, 251

Indian Ocean islands

Chikungunya outbreak, 674

Indian Plague Commission, 256

Indirect hemagglutination assay, 201

Indirect immunofluorescence assay, 311

Infants

botulism, 342, 346

Infection control, 118, 121

Infectious Diseases Society of America, 322

Influenza

Avian influenza pandemic threat, 657–660

H1N1 influenza pandemic, 660–661

swine influenza, 660–661

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Medical Aspects of Biological Warfare 

Inhalation-acquired disease

amnesic shellfish poisoning, 468

anthrax, 136–137

botulism, 342, 344

neurotoxic shellfish poisoning, 466

palytoxin, 471

paralytic shellfish poisoning, 464

ricin, 382–384

Injections

ricin administration, 382

Innate immunotherapy

henipaviruses, 560

Insect stings, 420, 421

Institutional Biosafety Committee, 904

Integrated Consortium of Laboratory Networks, 706–707

Intercellular adhesion molecule-1, 406

Interferon gamma, 163, 205–206, 406

Interferons, 791

International Clonal Complex, 323

International Committee of the Red Cross, 6

International Health Regulations, 259

International Society on Toxinology, 416

Invasive species, 80

Inventory audits, 901–902

Invertebrates

as disease models, 188–189

Investigational new drugs, 114–116, 345, 346, 347, 498, 927–929

Iran

biological warfare agents, 12

ricin as biological weapon research, 376

Iraq

Acinetobacter infections, 322, 323, 324–327

biological warfare agents, 10–11

C. botulinum research program, 339

Q fever outbreak, 2005, 53–54, 307

ricin as biological weapon research, 376

Iraq Survey Group, 11

Iron sequestration, 263

Irritants, local

plants causing, 451

Ishii, General Shiro, 3–4, 5, 338

Ivins, Dr. Bruce E., 15

J

Japan

Aum Shinrikyo cult bioterrorism research and attacks, 13, 

46–47, 72, 339

biocrimes, 12

biological agent research during World War I, 3–4

bioterrorism attacks, 13

C. botulinum research program, 338

development of Burkholderia mallei as potential biowarfare 

agent, 179

plague warfare research, 252–254

severe fever with thrombocytopenia syndrome virus, 676

Japanese encephalitis virus, 550

JBAIDS. See Joint Biological Agent Identification and Diagnostic 

Systems

Jellyfish stings, 420, 430, 432, 441

Jewett, Frank B., 921

Joint Bacterial Repository, 329

Joint Biological Agent Identification and Diagnostic Systems, 112, 

702–704, 738

Joint Biological Integrated Detection System, 112

Joint Task Force for Civil Support, 123

Junín virus, 519–520, 522, 523, 526–528, 530531

Justinian plague, 250

K

Kathmandu, Nepal

cholera outbreak, 648

Kefauver-Harris Drug Amendments, 926

Kennedy, John F., 7

Khrushchev, Nikita, 9

Killed whole cell vaccines, 206–207

Kosovo

tularemia outbreak, 1999-2000, 52–53

Kostov, Vladimir, 9, 376

Krishnaswami, Dr. C.S., 224

L

Laboratories

allies’ identification levels, 718–719

antibiotic and antimicrobial susceptibility testing, 727

assessing individual risk, 876–877

automated identification systems, 724–727

Biological Defense Research Program Laboratories, 888

biosafety program element requirements, 876–886

biosurveillance, 739–741

civilian identification levels, 707–708

Clinical Laboratory Improvement Amendments, 702–703

Clinical Laboratory Improvement Program, 702–703

containment and maximum containment laboratories, 876–886

culture-based microbiological methods, 722–724

Defense Laboratory Network, 706–707

documenting safety procedures, 876

early recognition of host response, 741–743

electrochemiluminescence, 730–732

emerging threat surveillance, 739–741

enzyme-linked immunosorbent assays, 730

flow cytometry, 732–733

future perspectives, 734–736

hand held assays, 733–734

horizon-agnostic diagnostic applications, 738–739

identification approaches, 719–739

identification levels, 707

immunodiagnostic methods, 728–730

integration of in vivo and in vitro diagnostic tests, 728

Joint Biological Agent Identification and Diagnostic Systems, 

112, 702, 704

laboratory animal care and use program, 888–889

laboratory worker protection, 876

MAGPIX, 733

medical surveillance, 880–881

microbiological culture methods, 727–728

microbiological practices, 884–886

military field laboratories, 705–706

military identification levels, 708, 716–718

molecular detection methods, 736

Next Generation Diagnostic System, 702

next generation molecular diagnostics, 738

personal protective equipment, 878–880

physical barriers, 877–878

presumptive and confirmation methods, 709–716

protecting the community and environment, 881–882

rapid methods, 727–728

real-time polymerase chain reaction, 736–738

regulated biological select agents and toxins, 703

research, development, test, and evaluation laboratories, 888

role of military clinical and field laboratories, 705

safety audits, 887–888

solid and liquid waste inactivation and disposal, 882–884

specimen collection and processing, 719–722

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Index

Laboratory-acquired diseases

botulinum intoxication, 342

tularemia, 289, 296

Laboratory of HealthCare Associated Infections, 327

Laboratory Registration and Select Agent Transfer Program, 897

Laboratory Response Network, 14, 22, 101, 122, 386, 704, 727–728

Lahore Polo Club, 204

Lambert-Eaton syndrome

differentiating from botulism, 343

Landstuhl Regional Medical Center

Acinetobacter infections, 323, 324–327

Lassa virus, 519, 522, 523–525, 528–531

Latin America

cholera outbreak, 647

LcrV, 261–262

Lectin B-chain, 377–378, 389–390

Legionella, 308–309

Legionellosis, 649–650

Lethal toxin, 132–135

Levofloxacin

plague postexposure prophylaxis, 273

plague treatment, 272

Libya

biological warfare agents, 12

Lice

as plague vector, 256–257

Linezolid

anthrax treatment, 140

Lipid rafts, 364–365

Lipopolysaccharide antibodies, 235, 308

Liquid chromatography, 386, 387

Listeria monocytogenes, 73–74

Listeriosis, 73–74

Liston, W.G., 256

Live attenuated vaccines, 207, 273, 758, 825–826

Live vaccines

alphavirus encephalitides, 498–499

tularemia, 289, 290, 295–296, 761–763

Livestock. See Animal diseases

Lloviu virus, 571–575, 580

Local response, 98–102

London, England

ricin bioterrorism attack, 16

Louisiana

plague pandemic, 251

LRN. See Laboratory Response Network

Lujo virus, 519–520, 523

LVS tularemia vaccine, 295–296

Lyme disease, 652–654

Lymphocyte function-associated antigen, 406

Lysosomal-associated membrane proteins, 308

M

M-44 vaccine, 312

Machupo virus, 520, 522, 523–524, 526–527, 531

MAGPIX, 733

Major histocompatibility complex, 404–405

Malaysia

Nipah virus outbreak, 553

paramyxovirus infections, 669–670

Malleomyces mallei, 178

Mammarenaviruses. See Hemorrhagic fever-causing mammarenaviruses

Manganese sequestration, 263

MAP. See Mitogen-activated protein kinase

Marburg virus, 571–575, 580–592, 596–598

Marine algal toxins

amnesic shellfish poisoning, 467–469

neurotoxic shellfish poisoning, 465–467

palytoxin, 469–472

paralytic shellfish poisoning, 463–465

Marine envenoming, 420–422

Markov, Georgi, 9, 375, 376, 382

Marshall, Lieutenant Colonel John D., 252

Marshall Plan, 7

Martha’s Vineyard, Massachusetts

tularemia outbreak, 2000, 51–52, 292

Maryland

alphavirus encephalitides outbreaks, 482

MASCAL. See Medical mass casualty event

Masks, 112–113

Mass median aerodynamic diameter, 862

Mass spectrometry, 202, 328, 344, 386, 387

Massachusetts

alphavirus encephalitides outbreaks, 482

tularemia outbreak, 2000, 51–52, 292

Mau-Mau attack, 79

MDCK cells, 366

MDR Organism Repository and Surveillance Network, 329–332

MDROs. See Multidrug-resistant organisms

Meadow saffron, 447

MEDCOM. See U.S. Army Medical Command

Medical countermeasures

anthrax, 752, 754, 757–761

bacterial diseases, 752–772

botulinum neurotoxin, 793–795

brucellosis, 768–770

encephalitic New World alphaviruses, 773–776

glanders, 766–768

melioidosis, 766–768

plague, 764–765

Q fever, 770–772

ricin, 796–797

rickettsial diseases, 752–772

smallpox, 776–788

Staphylococcal enterotoxin B, 795–796

toxins, 793–797

tularemia, 761–764

viral diseases, 773–793

viral hemorrhagic fevers, 788–793

Medical ethics. See Ethical issues

Medical mass casualty event

casualty management, 110–124

Medical Reengineering Initiative, 705

Medical treatment facilities, 705

Melioidosis

candidate virulence factors, 228–230

clinical disease, 227, 230–232

diagnosis of, 232–233

epidemiology of, 226

infectious agent characteristics, 224–225

medical countermeasures, 766–768

military relevance, 225–226

pathogenesis, 226–227

postexposure prophylaxis, 235–236, 767–768

prevention of, 234–236

treatment of, 233–234

vaccines, 766

Meningitis

anthrax meningitis, 137

plague meningitis, 268

Merck, George W., 5

Merck, George W., Jr., 921

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Medical Aspects of Biological Warfare 

Meropenem

glanders treatment, 204

melioidosis treatment, 233

MERS. See Middle Eastern respiratory syndrome

Meselson, Matthew, 9

Metropolitan Medical Response System, 121

Mexico

alphavirus encephalitides outbreaks, 487

Meyer, Karl F., 273

MHS. See Military Health System

Mice

as animal disease models, 186–188

Microbiological methods

culture-based, 722–724, 727–728

Microbiological practices

biosafety, 884–886

MicroChem Plus, 883

Middle Eastern respiratory syndrome, 666–669

MIDI Sherlock Microbial Identification System, 199

Military field laboratories, 705–706

Military Health System

response to Acinetobacter infections, 328–332

Military installations. See also U.S. military

plague risks, 252, 253

Military personnel

options for fulfilling mission and ethical responsibilities, 929–931

Minnesota Patriots Council, 13–14

Minor Groove Binding Proteins, 630

Missoula, Montana

Q fever outbreak, 306

Missouri

Heartland virus, 676–677

Mitogen-activated protein kinase, 134, 378

MNGCs. See Multinucleated giant cells

Modified Vaccinia Ankara, 632

Modified vaccinia virus, 826

Mold toxins, 443

Molecular detection methods

Acinetobacter baumannii, 327–328

next generation diagnostics, 738

overview, 736

Molecular sequencing technology, 58–59

Monkeypox, 623, 627–628

Monoclonal antibodies

antibacterial, 837–839

antitoxin, 835–837

antiviral, 839–842

Monocyte chemoattractant protein-1, 205, 406

Montana

Q fever outbreak, 306

Morbidity and Mortality Weekly Report, 259

Mosquitoborne viruses, 670–674

Mousepox virus, 19

MRSN. See MDR Organism Repository and Surveillance Network

Mucosal vaccines, 830–831

Multidrug-resistant organisms

antibiotic resistance, 328, 655–656

characterization and identification of source, 327–328

epidemiological consultation, 324–327

ESKAPE pathogens, 322, 328–332

genetic analysis, 327–328

historical perspectives, 322–324

molecular analysis, 327–328

strain collection and sampling, 327

U.S. Military Health System response, 328–332

Multilocus sequence typing, 181, 182

Multinucleated giant cells, 186, 190

Multiple-locus variable number tandem repeat analysis, 182–183

Mushrooms, 422, 442, 444–445, 452

Mussolini, Benito, 375

Myasthenia gravis

differentiating from botulism, 343

Mycotoxins, 75–76

Myonecrosis, 362, 363

Myotoxins, 432–435

N

National Academy of Sciences, 73, 857

National Biosurveillance Integration System, 101

National Committee for Clinical Laboratory Standards, 166

National Disaster Medical System, 123

National Disaster Recovery Framework, 94, 96, 104

National Guard, 100, 123

National Incident Management System, 96–97, 121

National Institute of Occupational Safety and Health, 879

National Institutes of Health

human genome project, 58

Office of Biotechnology Activities, 898

Office of Science Policy, 20

National laboratories, 122

National Mitigation Framework, 96

National Naval Medical Center, 328

National Preparedness Goal, 94, 98

National Prevention Framework, 96

National response, 98–102

National Response Framework, 94, 96, 98–99, 100

National Response Plan, 22

National Safety Council, 6

National Science Advisory Board for Biodefense, 20–21

National Security Council, 22

National Strategy for Countering Biological Threats, 23

Navy field laboratories, 706

NBC News

anthrax attack, 2001, 47–48

NDRF. See National Disaster Recovery Framework

Necrotizing enteritis, 363

Nectrotoxins, 442–443

Nepal

cholera outbreak, 648

Nephrotixic plants, 450

Nephrotoxins, 442

Nerve agent poisoning

differentiating from botulism, 343

Neuraminidases, 365, 367

Neurotoxic plants, 450

Neurotoxic shellfish poisoning

description of toxin, 465–466

diagnosis of, 467

inhalation-acquired disease, 466

mechanism of action, 466

medical management, 467

oral ingestion, 466

signs and symptoms, 466

Neurotoxins, 340–341, 424–432

New Orleans, Louisiana

plague pandemic, 251

New World mammarenaviruses, 520–521, 523, 526–527, 528–529

New York, New York

plague pandemic, 251

West Nile virus outbreak, 1999, 50–51

Next Generation Diagnostic System, 702, 704

Next generation molecular diagnostics, 738

Next-generation sequencing, 559, 738–739

NGDS. See Next Generation Diagnostic System

Nightshade, 449

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Index

NIH. See National Institutes of Health

Nine Mile variant, 307–308

Nipah virus, 549–552, 553–554, 669–670

Nixon, Richard M., 7, 338, 897

Nomenspecies, 160–161

North America

alphavirus encephalitides outbreaks, 482, 487

North Atlantic Treaty Organization, 718–719

North Carolina

alphavirus encephalitides outbreaks, 482

North Carolina Disease Event Tracking and Epidemiologic Collection Tool, 57

North Korea

accusations of US biowarfare attacks, 6

biological warfare agents, 12

Nosocomial infections, 326–327

NSABB. See National Science Advisory Board for Biodefense

Nucleic acid-based identification

glanders, 199–201

Nucleic acid vaccines, 829–830

Nunn-Lugar Biological Threat Reduction Program, 10

Nuremberg Code, 921, 922

Nuttall, George, 362

O

Obama, Barack, 48, 377, 898

Occupational Safety and Health Administration

Respiratory Protection Standard, 879

Oculoglandular tularemia, 291

Office International des Epizooties, 180–181

Office of Biotechnology Activities, 898

Office of Emergency Preparedness, 22

Office of Public Health Emergency Preparedness, 22

Office of Science and Technology Programs, 21

Office of Science Policy, 20

Ofloxacin

glanders, 203

Q fever treatment, 311

OFPBL agar, 199

Old World mammarenaviruses, 519–520, 522–523, 525–526, 528

Oleander, 440

Operation Whitecoat, 6, 858, 923

Operations Desert Shield/Storm

biological agents exposure, 10

immunologic protection, 113

pyridostigmine bromide prophylaxis, 113

Operations Enduring Freedom/Iraqi Freedom

Acinetobacter infections, 322, 323, 324–327

Q fever outbreak, 53–54, 307

Oral ingestion

amnesic shellfish poisoning, 468

neurotoxic shellfish poisoning, 466

palytoxin, 470–471

paralytic shellfish poisoning, 464

ricin, 380–382

Oropharyngeal anthrax, 138

Orthopoxviruses

as biological warfare agents, 622–623

characteristics of, 617–622

classification, 617

clinical aspects of infections, 624–628

diagnosis of, 628–630

entry into cells, 618

immunodiagnosis, 629

medical management, 630–635

monkeypox, 627–628

morphogenesis and egress, 619

morphology, 617–618

nucleic acid diagnosis, 629–630

pathogenesis, 621–622

phenotypic diagnosis, 628–629

phylogenetic relationships, 620–621

prophylaxis, 630–634

replication, 619

specimen collection and handling, 628

treatment of, 624–635

Outbreak investigations, 41–43

P

p38 mitogen activated protein kinase, 391

Pacific Yew tree, 449

Paclitaxel, 416–417

Pakistan

glanders outbreak, 204

Palytoxin

dermal exposure, 471

description of toxin, 469–470

diagnosis of, 472

exposure through home aquaria, 471–472

inhalation-acquired disease, 471

mechanism of action, 470

medical management, 472

oral ingestion, 470–471

signs and symptoms, 470–472

Pan-hazard preparedness, 17–24

Pandemics

Avian influenza threat, 657–660

Paralytic neurotoxins, 424–430

Paralytic shellfish poisoning

cause of death, 464

description of toxin, 463

diagnosis of, 465

differentiating from botulism, 343

inhalation-acquired disease, 464

mechanism of action, 463–464

medical management, 465

oral ingestion, 464

signs and symptoms, 464

Paramyxoviruses, 669–670

Parasites, 76–77

Passive antibody therapy

hemorrhagic fever-causing mammarenaviruses, 530

Passive hemagglutination antibody detection, 270

Passive immunotherapy

alphavirus encephalitides, 497–498

bacterial diseases, 758–759

botulinum toxins, 794

henipaviruses, 559–560

Staphylococcal enterotoxin B, 795–796

viral hemorrhagic fevers, 792

PBT. See Pentavalent botulinum toxoid

pCD, 261

PCR. See Polymerase chain reaction assays

Peake, Lieutenant General James B., 324

Penicillin

anthrax exposure prophylaxis, 140

anthrax treatment, 139

Pennsylvania

Legionnaires’ disease outbreak, 649

Pensacola, Florida

plague pandemic, 251

Pentavalent botulinum toxoid, 347, 793

Personal protective equipment, 860–861, 877–880

Personnel reliability programs, 898, 901, 905–908

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PFGE. See Pulsed field gel electrophoresis

Phage shock protein response, 263

Pharyngeal plague, 268

Phleboviruses, 675–677

Phospholipases, 135

Physical protection, 112–113

PLA2 toxins, 432–434

Plague

antibiotic treatment, 272

autotransporter proteins, 263

biochemistry of, 255–256

as biological warfare agent, 252–254

Black Death, 250–251

bubonic plague, 265, 267

clinical manifestations, 265–269

cutaneous, 268–269

diagnosis of, 269–271

endemic disease, 251–252

epidemiology of, 256–259

F1 antigen, 262

fimbrae, 262–263

first pandemic, 250

gastrointestinal, 268

growth characteristics, 254–255

historical perspectives, 249–251

incidence of, 259–261

infectious agent characteristics, 254–256

iron sequestration, 263

isolation of patients, 271–272

Justinian plague, 250

laboratory confirmation, 269–271

mammals known to harbor plague in the US, 257

manganese sequestration, 263

medical countermeasures, 764–765

meningitis, 268

morphology, 254

pathogenesis, 264–265

phage shock protein response, 263

pharyngeal, 268

plasminogen activator, 262

pneumonic, 259, 267, 268

postexposure prophylaxis, 273, 765

prevention of, 272–273

second pandemic, 250–251

septicemic, 265–267

signs and symptoms, 269

small RNAs, 263

surface structures, 263

taxonomy, 254

third pandemic, 251

transmission of, 2–3

treatment of, 271–274

twin arginine transport, 263

Type III secretion system, 261–262

vaccines, 115, 273–274, 764–765

virulence and transmission factors in the flea, 264

virulence determinants, 261–264

virulence factors in mammalian hosts, 262–263

warfare and, 251–254

Plague Manual, 269

Plague Vaccine USP, 273

Plant poisons, 422, 440, 446–452

Plaque reduction neutralization test, 629

Plasminogen activator, 262

pMT, 261

Pneumonic plague, 259, 267, 268

Pneumonic tularemia, 291–292

Point source outbreaks, 40

Poisons

animals, 422

as biological warfare agents, 423

definitions, 417

epidemiology of, 418–423

mushrooms, 422, 444–445

plants, 422, 446–452

research directions, 452–453

Poland

use of biological agents during World War II, 4

Poliovirus type 1, 19

Polymerase chain reaction assays

Acinetobacter infection diagnosis, 328

botulism diagnosis, 344

brucellosis diagnosis, 161, 165

filoviruses diagnosis, 593–595

glanders diagnosis, 200, 202

hemorrhagic fever-causing mammarenaviruses diagnosis, 

529–530

orthopoxviruses diagnosis, 629–630

overview, 728–730

real-time polymerase chain reaction, 678, 736–738

ricin diagnosis, 387

Staphylococcal enterotoxin B diagnosis, 408

tularemia diagnosis, 293

viral pathogen diagnosis, 678–679

Polysaccharide-based subunit vaccines, 207–208

Portal Shield, 112

Powered air-purifying respirators, 879

Poxviruses, 617

pPCP, 261

Prairie dogs

plague transmission role, 257, 258

President’s Disaster Relief Fund, 100

Presumptive identification methods, 709–716

Primary atypical pneumonia, 307

Primates, nonhuman

as animal disease models, 188

Project 112, 7

Project BioShield Act, 22–23, 740, 926

Pronase, 367

Protective clothing, 112–113

Protein kinases, 391, 406

Protein subunit vaccines, 207

Protein tyrosine kinases, 406

Proteolysis, 364

Proteus OX-19, 4

Protoxin, 367

PTA. See Pteroic acid

Pteroic acid, 390

Public health preparedness, 102

Public Health Security and Bioterrorism Preparedness and Response Act of 2002, 931

Public Health Service Act, 925, 926

Public Readiness and Emergency Preparedness Act, 925

Puffer fish, 422

Pulpy kidney disease, 365

Pulsed field gel electrophoresis, 325, 327, 328

Purple foxglove, 447

Putin, Vladimir, 10

Pyridostigmine bromide, 113

Q

Q fever

diagnosis of, 311

disease in animals, 310

disease in humans, 310

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epidemiology of, 309–310

Gulf War, 307

historical perspectives, 306

infectious agent, 307–309

Iraq outbreak, 2005, 53–54, 307

medical countermeasures, 770–772

military relevance, 306–307

outbreaks during World War II, 8, 306–307

pathogenesis, 309

postexposure prophylaxis, 772

treatment of, 311

vaccines, 115, 312, 770–771

Q-Vax, 312

Queensland, Australia

Q fever outbreak, 306

Quorum sensing systems, 191

R

Rajneesh, Bhagwan Shree, 13, 44–46

Rajneeshee cult

bioterrorism attacks, 13, 44–46

Raman spectroscopy, 202

RANTES protein, 206

Rat fleas

as plague vector, 256

Rats

plague transmission role, 257, 258

Ravn virus, 571–575

Raxibacumab

anthrax treatment, 140

Reagan, Ronald, 9

Real-time Outbreak and Disease Surveillance Laboratory, 57

Real-time polymerase chain reaction, 678, 736–738

Recombinant granulocyte colony-stimulating factor, 234

Recombinant vaccine vectors, 826–827

Recovery operations, 103–105

Red tides, 466

Reference laboratories, 122

Registered Biosafety Professional, 889–890

Research, development, test, and evaluation laboratories, 888

Research ethics. See Ethical issues

Respiratory Protection Standard, 879

Respiratory system

Staphylococcal enterotoxin B effects, 407

Respiratory tularemia, 291

Reston virus, 571–575, 580, 587

Restriction fragment-length polymorphism, 629–630

RevCons, 17

Reverse transcription-polymerase chain reaction, 558, 678

Review conferences, 17

Rhodesia

biological warfare agents, 11–12

Ribavirin

henipavirus treatment, 560

viral hemorrhagic fever treatment, 790–791

Ribosome inactivating proteins, 377

Richardson, Shannon Guess, 16, 48, 377

Ricin

activity assay, 386

antibody treatment, 388

attacks in US, 2003-2004, 48–49

biochemical identification, 377–378

as biological warfare agent, 376

bioterrorism attacks, 13, 15–16, 376–377

cause of death, 384–385

cellular stress response inhibitors, 391

description of agent, 377–379

detection of, 385–386

diagnosis of, 386–387

enzyme-linked immunosorbent assays, 385, 387

handheld assay detection devices, 385, 387

historical perspectives, 375–376

inhalation of, 382–384

injection of, 382

liquid chromatography/mass spectrometry, 386, 387

medical countermeasures, 796–797

medical management, 387–391

oral intoxication, 380–382

pathogenesis, 378–379

pathology, 379–385

ricin A chain inhibitors, 390–391

sample verification platforms, 385–386

signs and symptoms of, 379–385

small molecule inhibitors, 389–390

Soviet Union attacks, 9

supportive and specific therapy, 389

transport inhibitors, 391

vaccines, 387–388, 796

Rifampin

brucellosis treatment, 167

Riluzole, 366

Ring vaccination, 115

RIPs. See Ribosome inactivating proteins

Risk assessment systems, 95

Risk communication, 115, 124

RiVax, 388

Rock squirrels

plague transmission role, 257, 258

Roosevelt, Franklin D., 5, 858

RTA. See Catalytic A-chain

RTB. See Lectin B-chain

Russell, Findlay E., 416

RVEc, 388

S

Sabiá virus, 521, 523–524, 531

Sacroilitis

brucellosis and, 164

Safety audits, 887–888

Saint Joseph, Missouri

Heartland virus, 676–677

Salmonella

bioterrorism attacks, 13, 44–46

food- and waterborne, 44–46, 73, 652

Salmonella typhimurium

attacks in Dalles, Oregon, 1984, 44–46

food and waterborne, 73

Salmonellosis

attacks in Dalles, Oregon, 1984, 44–46

food and waterborne, 73

Sample verification platforms, 385–386

San Francisco, California

plague pandemic, 251

Sarin

bioterrorism attacks, 13

SARS, 666

Saudi Arabia

Middle Eastern respiratory syndrome, 666–669

Saxitoxins, 463–465

Scolopendrid centipede, 443

Scorpion stings, 419–420, 430–432, 442–443

Sea snakes, 420–422

Secreted proteins, 189–190

Secretory systems, 189–190

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Security risk assessment, 900

Select Agent List, 22

Select Agent Program, 14, 23

Select Agent Regulations, 900, 902

Select Agents and Toxins Program, 375

Senate Office Building

anthrax attack, 2001, 47–48

ricin attacks in 2004, 48–49, 377

Sentinel laboratories, 122

Septic abortions

Brucella and, 162–163

Septicemic disease

plague, 265–267

septicemic glanders, 195

tularemia, 292

Sequence types, 328

Sequencing technology, 58–59

Serologic diagnoses

filoviruses, 583

glanders, 201–202

plague, 270

tularemia, 293

Severe acute respiratory syndrome, 666

Severe fever with thrombocytopenia syndrome virus, 675–676

Shiga toxins, 74, 377, 651–652

Shigella dysenteriae

attacks in Dallas, Texas, 1996, 47

food and waterborne, 74–75

Shigella sonnei, 78

Shigellosis

attacks in Dallas, Texas, 1996, 47

food and waterborne, 74–75

Shipping standards, 723

Sialidases, 365

Simonson, Stewart, 340

Singapore Armed Forces

melioidosis cases, 225

Skin diseases

cutaneous anthrax, 135–136

cutaneous plague, 268–269

Skin irritation

plants causing, 451

Small molecule inhibitors, 389–390

Small RNAs, 263

Smallpox

as biological warfare agents, 622–623

biothreat policy, 634

characteristics of virus, 617–622

classification, 617

clinical aspects of infections, 624–626

diagnosis of, 628–630

entry into cells, 618

immunodiagnosis, 629

impact on Native Americans, 3

medical countermeasures, 776–788

medical management, 630–635

morphogenesis and egress, 619

morphology, 617–618

nucleic acid diagnosis, 629–630

outbreak in Yugoslavia, 39

pathogenesis, 621–622

phenotypic diagnosis, 628–629

phylogenetic relationships, 620–621

preparedness, 18–19

prophylaxis, 630–634

replication, 619

Soviet Union production of virus, 9

specimen collection and handling, 628

treatment of, 624–635

vaccine, 114–115, 777–787

WHO eradication program, 9

Smallpox Response Plan, 628

Smuggling risks, 80

Snakes, venomous, 418–419, 424–426, 428–430, 432–442

Soman

pyridostigmine bromide prophylaxis, 113

South Africa

biological warfare agents, 11–12

South America

alphavirus encephalitides outbreaks, 482

Chikungunya outbreak, 674

South Korea

severe fever with thrombocytopenia syndrome virus, 676

Soviet Union

accidental anthrax release, 49–50, 130

accusations of US biowarfare attacks, 6–7

biological warfare agents, 8–10

C. botulinum research program, 338–339

filoviruses biological warfare program, 584–586

plague warfare research, 254

ricin as biological weapon research, 376

Specimen collection, 719–722

Spider bites, 419–420, 421, 430, 432, 442–443

Spokane, Washington

ricin attacks in 2004, 377

Squirrels

plague transmission role, 257–258

Stafford Act, 98, 100

Stalin, Joseph, 9

Standing operating procedures, 876

Staphylococcal enterotoxin B

animal models, 405–406

cardiovascular symptoms, 407–408

characterization of, 404–405

clinical disease, 406–408

detection of, 408

development of therapeutics, 409

diagnosis of, 408

fever onset and duration, 407

headache onset and duration, 407

hematology, 408

host response, 405–406

medical countermeasures, 795–796

medical management, 408–409

nausea and vomiting, 407

ocular effects, 408

respiratory symptoms, 407

signs and symptoms, 406–408

vaccines, 409, 795

Staphylococcus aureus

animal models, 405–406

cardiovascular symptoms, 407–408

characterization of toxins, 404–405

clinical disease, 406–408

detection of, 408

development of therapeutics, 409

diagnosis of, 408

fever onset and duration, 409

headache onset and duration, 407

hematology, 408

host response, 405–406

medical management, 408–409

nausea and vomiting, 407

ocular effects, 408

respiratory symptoms, 407

signs and symptoms, 406–408

vaccines, 409

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State response planning, 102

Sternberg, Surgeon General George, 918

Stevens, Robert, 14

Stillmark, Peter Hermann, 375

Stimson, Henry L., 921

Stingrays, 421–422

Stings, venomous, 418

Strategic National Stockpile, 22–23, 102, 123, 631–632

Streptococcus pyogenes

animal models, 405–406

cardiovascular symptoms, 407–408

characterization of toxins, 404–405

clinical disease, 406–408

detection of, 408

development of therapeutics, 409

diagnosis of, 408

fever onset and duration, 409

headache onset and duration, 407

hematology, 408

host response, 405–406

medical management, 408–409

nausea and vomiting, 407

ocular effects, 408

respiratory symptoms, 407

signs and symptoms, 406–408

vaccines, 409

Streptomycin

brucellosis treatment, 166–167

glanders treatment, 204

Stroke

differentiating from botulism, 343

Structural vaccinology, 829

Subunit vaccines, 827–829

Sudan virus, 571–575, 587–589, 597

Sulfadiazine

glanders treatment, 204

Superantigens, 404

Supportive therapy

ricin, 389

Staphylococcal enterotoxin B, 408

Surface polysaccharides, 189

Surface structures, 263

Suzuki, Dr. Mitsuru, 12

Sverdlovsk, Soviet Union

accidental anthrax release, 49–50, 130

Swine influenza, 660–661

Syndromic surveillance, 56–57

Synthetic biology, 681

Syria

biological warfare agents, 12

ricin as biological weapon research, 376

Systems vaccinology, 829

T

T-cell receptors, 404–405

T-cells

response to Brucella, 163

Tactical Combat Casualty Care, 140

Taenia solium, 76–77

Taï virus, 571–575, 580, 587, 589

Tanzania

Chikungunya outbreak, 673–674

Tapeworms, 76–77

Technical Escort Units, 117

Terrorism, biological. See Bioterrorism

Tetrodotoxin, 422, 426, 430

Texas

plague pandemic, 251

Thailand

botulinum toxin foodborne outbreak, 339–340

melioidosis incidence, 227

Thompson, Diane, 12

Thrombocytopenia syndrome, 675–676

Tickborne disease

anaplasmosis, 654–655

borreliosis, 652–654

differentiating from botulism, 343

Ehrlichiosis, 654–655

Heartland virus, 676–677

paralysis caused by Ixodidae family, 419–420

phleboviruses, 675–677

severe fever with thrombocytopenia syndrome virus, 675–676

TMP-SMX

melioidosis treatment and prophylaxis, 234, 236

Tokyo, Japan

Aum Shinrikyo cult anthrax attacks, 13, 46–47

Toll-like receptors, 404

Torovirus, 78

Toxic shock syndrome, 404–405, 408

Toxins

arachnids, 419–420

biological surety, 896–911

as biological warfare agents, 423

bites, 418

botulinum toxins, 72–73, 340–349, 793–795

cardiotoxins, 440–441

classes, 424–452

cnidarians, 420–422

definitions, 417

epidemiology of, 418–423

excitatory neurotoxins, 430–432

fish, 420–422

hemolytic toxins, 437–440

hemorrhagic toxins, 437–440

hemostasis-active toxins, 435–437

insects, 420, 421

marine algal, 462–473

medical countermeasures, 793–797

myotoxins, 432–435

nectrotoxins, 442–443

nephrotoxins, 442

origin of word, 2

paralytic neurotoxins, 424–430

research directions, 452–453

scorpions, 419–420

sea snakes, 420–422

snakes, 418–419

spiders, 419–420, 421

staphylococcal enterotoxin B, 404–409, 795–796

stings, 418

Transport inhibitors, 391

Tree squirrels

plague transmission role, 257

Trimethoprim

glanders treatment, 204

Tularemia

aerosol transmission, 288

arthropod vectors, 288

bacterial culture techniques, 293

clinical manifestations, 290–292

diagnosis of, 292–294

direct contact, 287

epidemiology of, 287–289

food and water ingestion, 288

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infectious agent, 286–287

Kosovo outbreak, 1999-2000, 52–53

laboratory-acquired, 289

laboratory worker exposure, 296

mammalian bites, 288

Martha’s Vineyard outbreak, 2000, 51–52, 292

medical countermeasures, 761–764

pathogenesis, 289–290

postexposure prophylaxis, 295, 764

prophylactic treatment, 295–295

rapid diagnostic methods, 293–294

serology, 293

treatment of, 294–295

in unusual settings, 288–289

use as biological weapon, 296

vaccination with live vaccine strain, 289, 290, 295–296, 761–763

The Turner Bill, 933

Twin arginine transport, 263

Type III secretion system, 261–262

Typhoidal tularemia, 291, 292

U

U.K. Laboratory of HealthCare Associated Infections, 327

Ukraine

plague pandemic, 250, 252

Ulceroglandular tularemia, 291, 292

UN Special Commission, 10, 11

United States Naval Ship Comfort, 323, 325

University of Pittsburgh, 57

UNSCOM. See UN Special Commission

Uruguay Round Agreement on the Application of Sanitary and 

Phytosanitary Measures, 80

U.S. Army Center for Health Promotion and Preventive Medicine, 324

U.S. Army Medical Command, 329

U.S. Army Medical Research and Materiel Command, 329

U.S. Army Medical Research Institute of Infectious Diseases

anthrax sample testing, 14–15

biosafety, 880–881

casualty management, 110–124

creation of, 8, 923

ricin vaccine research, 388

tularemia vaccine, 295–296

U.S. Congress

anthrax attacks in US, 2001, 47–48, 130

ricin bioterrorism attack, 16, 377

U.S. Department of Agriculture, 5, 23, 44, 80, 101–102, 703, 897

U.S. Department of Defense, 57, 112, 324, 329, 331–332, 628, 704, 

888, 927–931

U.S. Department of Energy, 58

U.S. Department of Health and Human Services, 20, 21–22, 

100–102, 703, 897

U.S. Department of Homeland Security, 23, 56, 94, 101–102

U.S. Department of State, 102

U.S. Department of Transportation, 902

U.S. Environmental Protection Agency, 882–883

U.S. Food and Drug Administration, 82, 112, 272, 587, 702–704, 

916, 926–933

U.S. military. See also specific wars by name

biological warfare agents, 4–8

Health System response to Acinetobacter infections

laboratories, 705–708, 716–718

options for fulfilling mission and ethical responsibilities to 

military personnel, 929–931

plague risks at military installations, 252, 253

U.S. National Science Advisory Board for Biosecurity, 59

U.S. Postal Service

anthrax attacks in 2001, 47–48, 130

ricin attacks in 2003-2004, 48–49

ricin bioterrorism attack, 16

USA PATRIOT Act, 897, 900

USAMRIID. See U.S. Army Medical Research Institute of Infectious Diseases

USDA. See U.S. Department of Agriculture

V

V3526 vaccine, 500

Vaccine Adverse Event Reporting System, 757

Vaccines

ACAM2000 vaccine, 115

adjuvants, 831–832

adverse events, 757–758

alphavirus encephalitides, 498–501, 773–776, 777–787, 789–790

anthrax, 10, 113, 114, 140–141, 753–758

bacterial diseases, 753–756

botulinum toxin, 348, 793–794

brucellosis, 768

Clostridium perfringens, 363, 366–367

filoviruses, 587, 588–589

glanders, 206, 207–208, 766

inactivated, 499

live attenuated vaccines, 207, 273, 758, 825–826

live vaccine strains, 289, 290, 295–296, 498–499

melioidosis, 234–236, 766

mucosal vaccines, 830–831

nucleic acid vaccines, 829–830

orthopoxviruses, 630–634

plague, 115, 273–274, 764–765

Q fever, 115, 312, 770–771

recombinant vaccine vectors, 826–827

ricin, 387–388, 796

rickettsial diseases, 753–754

ring vaccination, 115

smallpox, 114–115, 777–787

Staphylococcal enterotoxin B, 409, 795

Staphylococcus aureus, 409

Streptococcus pyogenes, 409

subunit vaccines, 827–829

trends in biodefense development, 824–825

tularemia, 289, 290, 295–296, 761–763

viral hemorrhagic fevers, 788–790

whole cell vaccines, 312

yellow fever, 115

Vaccinia immune globulin, 633, 634

Vaccinia virus, 825–826

Variola virus, 617, 622–623–626

Vascular leak syndrome, 376

Venezuelan equine encephalitis, 483–484, 485, 492–493, 773–774

Venezuelan hemorrhagic fever, 521, 530

Venoms

arachnids, 419–420

as biological warfare agents, 423

bites, 418

cnidarians, 420–422

definitions, 417

epidemiology of, 418–423

fish, 420–422

insects, 420, 421

research directions, 452–453

scorpions, 419–420

sea snakes, 420–422

snakes, 418–419

spiders, 419–420, 421

stings, 418

Verotoxin, 377

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VHF. See Viral hemorrhagic fevers

Vibrio cholerae, 77, 647–649

Vibrioses, 649

Vietnam War

melioidosis cases, 225

plague incidence, 252

plague prevention, 249

Viral hemorrhagic fevers, 725, 788–793

Virginia

alphavirus encephalitides outbreaks, 482

Virtual screening, 390, 391

Virulence factors

glanders, 191

melioidosis, 228–230

Virus-like particles, 828

Virus replicon particle vaccines, 501, 827

Virus-Toxin Law, 926

W

W bombs, 376

Walter Reed Army Institute of Research, 324, 327, 328–330

Walter Reed Army Medical Center

Acinetobacter infections, 323, 324–327

War Bureau of Consultants, 857

War Research Service, 921

War Reserve Service, 5

Washington

plague pandemic, 251

ricin attacks in 2004, 377

Washington, George, 918

Waste disposal, 882–884

Water Sentinel program, 78

Water supply

bioterrorism concerns, 77–78

security of, 80–82

Waterborne diseases

cholera, 647–649

cryptosporidiosis, 75

emerging diseases, 647–650

Escherichia coli, 74

hepatitis A, 75

Legionellosis, 649–650

listeriosis, 73–74

parasites, 76–77

salmonellosis, 73

shigellosis, 74–75

threat potential, 76, 77

vibrioses, 649

Weapons of Mass Destruction-Civil Support Teams, 123

Weekly Epidemiological Record, 259

Welch, William, 362

West Africa

Ebola virus epidemic, 677–678

West Nile virus

New York City outbreak, 1999, 50–51

yearly spread of activity across the U.S., 1999-2004, 672–673

Western blot assay, 202

Western equine encephalitis, 484, 485–486, 494–495, 774–776

Whitmore, Captain Alfred, 224

WHO. See World Health Organization

Whole cell vaccines, 312

Whole genome sequencing, 656–657

Wicker, Roger

ricin attacks in 2004, 48–49, 377

Wood rats

plague transmission role, 258

Working Group on Civilian Biodefense, 272, 273

World Health Assembly, 18

World Health Organization

anthrax surveillance and control guidelines, 140

attempted investigation of biologic agent use during World 

War II, 6

Plague Manual, 269

smallpox eradication program, 9

Weekly Epidemiological Record, 259

World Trade Organization, 80

World War I and II

gas gangrene, 363

plague attacks, 252–254

plague outbreak, 252

Q fever outbreaks, 8, 306–307

ricin as biological weapon research, 376

use of biological agents, 3–4, 43–44, 78

Wound botulism, 342, 344

WRAIR. See Walter Reed Army Institute of Research

X

Xoma 3AB, 346

Y

Yellow fever, 115

Yellow Fever Commission, 918

Yellow fever virus, 827

Yellow oleander, 440

Yeltsin, Boris, 10, 338–339

Yersinia pestis

antibiotic treatment, 272

autotransporter proteins, 263

biochemistry of, 255–256

as biological warfare agent, 252–254

Black Death, 250–251

bubonic plague, 265, 267

characteristics of, 254–256

clinical manifestations, 265–269

cutaneous plague, 268–269

diagnosis of, 269–271

differentiation from Y. pseudotuberculosis, 254

endemic disease, 251–252

epidemiology of, 256–259

F1 antigen, 262

fimbrae, 262–263

first pandemic, 250

gastrointestinal plague, 268

growth characteristics, 254–255

historical perspectives, 249–251

incidence of, 259–261

iron sequestration, 263

isolation of patients, 271–272

Justinian plague, 250

laboratory confirmation, 269–271

manganese sequestration, 263

medical countermeasures, 764–765

morphology, 254

pathogenesis, 264–265

phage shock protein response, 263

pharyngeal plague, 268

plague meningitis, 268

plasminogen activator, 262

pneumonic plague, 267, 268

postexposure prophylaxis, 273, 765

prevention of, 272–273

second pandemic, 250–251

septicemic plague, 265–267

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l

Medical Aspects of Biological Warfare 

signs and symptoms, 269

small RNAs, 263

surface structures, 263

taxonomy, 254

third pandemic, 251

treatment of, 271–274

twin arginine transport, 263

Type III secretion system, 261–262

vaccines, 115, 273–274, 764–765

virulence and transmission factors in the flea, 264

virulence determinants, 261–264

virulence factors in mammalian hosts, 262–263

warfare and, 251–254

Yersinia pseudotuberculosis, 254, 255

YopK, 262

Yugoslavia

smallpox outbreak, 39

Z

Zmapp, 792

Zoonotic illnesses. See Animal diseases

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