Biosafety in 

Microbiological 

and Biomedical 

Laboratories

5th Edition

U.S. Department of Health and Human Services

Public Health Service

Centers for Disease Control and Prevention

National Institutes of Health

HHS Publication No. (CDC) 21-1112

Revised December 2009

iii

Foreword

Biosafety in Microbiological and Biomedical Laboratories (BMBL) quickly became 

the cornerstone of biosafety practice and policy in the United States upon first 

publication in 1984. Historically, the information in this publication 

has been advisory is nature even though legislation and regulation, in some 

circumstances, have overtaken it and made compliance with the guidance 

provided mandatory. We wish to emphasize that the 5th edition of the BMBL 

remains an advisory document recommending best practices for the safe 

conduct of work in biomedical and clinical laboratories from a biosafety 

perspective, and is not intended as a regulatory document though we 

recognize that it will be used that way by some.

This edition of the BMBL includes additional sections, expanded sections 

on the principles and practices of biosafety and risk assessment; and revised 

agent summary statements and appendices. We worked to harmonize the 

recommendations included in this edition with guidance issued and regulations 

promulgated by other federal agencies. Wherever possible, we clarified both the 

language and intent of the information provided. The events of September 11, 

2001, and the anthrax attacks in October of that year re-shaped and changed, 

forever, the way we manage and conduct work in biological and clinical 

laboratories and drew into focus the need for inclusion of additional information 

in the BMBL. To better serve the needs of our community in this new era, this 

edition includes information on the following topics:

■ Occupational medicine and immunization

■ Decontamination and sterilization

■ Laboratory biosecurity and risk assessment

■ Biosafety level 3 (Ag) laboratories

■ Agent summary statements for some agricultural pathogens

■ Biological toxins

At last count, over two hundred of our scientific and professional colleagues 

have assisted in the preparation of the 5th edition through participation in technical 

working groups, serving as reviewers and guest editors, and as subject matter 

experts. We wish to thank them all for their dedication and hard work for without 

them the 5th edition of the BMBL would not be possible. We also recognize the 

hard work and contributions made by all who participated in preparation of the 

previous editions of the BMBL; we have built on their solid work and commitment. 

It is impossible to publish this revision without recognizing the visionary leadership 

of the previous BMBL editors, Drs. John Richardson and W. Emmett Barkley, and 

Drs. Jonathan Richmond and Robert W. McKinney, without whom the BMBL 

would not be the widely and well-regarded resource it is today. The Executive 

Steering Committee did a stellar job in shepherding this massive revision effort 
iv Biosafety in Microbiological and Biomedical Laboratories

and not without many bumps and bruises along the way. It is through their absolute 

commitment to quality, technical accuracy, and dedication to the professional 

practice of biosafety that the 5th edition is born. We are truly grateful to Ms. 

Kerstin Traum, Council Rock Consulting for her expertise, keen eye for detail and 

seemingly tireless efforts in performing the duties of technical writer-editor. We 

also gratefully acknowledge Ms. Cheryl Warfield of Proven Practices, LLC for her 

copy-editing and formatting skills that significantly enhanced this edition’s 

readability and ease of use.

Finally, without the superb project management abilities and leadership of 

Dr. Joseph McDade and the technical/scientific editing expertise of Dr. Karl Johnson, 

especially in virology, the 5th edition of the BMBL would not be possible.

We hope you find this 5th edition of Biosafety in Microbiological and Biomedical 

Laboratories complete, timely and most of all, easy to use. Thank you for your 

patience and understanding during the long and comprehensive revision process. 

We believe you will find it was well worth the wait.

Deborah E. Wilson, DrPH, CBSP L. Casey Chosewood, M.D.

Director Director

Division of Occupational Office of Health and Safety

Health and Safety Centers for Disease Control

National Institutes of Health and Prevention

Bethesda, Maryland Atlanta, Georgia

September 2009
Guest Editors v

Editors

L. Casey Chosewood, MD 

Director, Office of Health and Safety 

Centers for Disease Control and Prevention 

Atlanta, GA 30333

Deborah E. Wilson, DrPH, CBSP 

Director, Division of Occupational Health and Safety 

Office of the Director 

National Institutes of Health 

Bethesda, MD 20892 

Steering Committee

Shanna Nesby-O’Dell, DVM, MPH 

Chief, External Activities Program and 

WHO Collaborating Center for Biosafety and Training 

Office of Health and Safety 

Centers for Disease Control and Prevention 

Robbin S. Weyant, PhD 

Chief, Laboratory Safety Branch 

Office of Health and Safety 

Centers for Disease Control and Prevention 

Martin L. Sanders, PhD, CBSP, RBP 

Deputy Director 

Office of Health and Safety 

Centers for Disease Control and Prevention 

Deborah E. Wilson, DrPH, CBSP 

Director, Division of Occupational Health and Safety 

Office of the Director 

National Institutes of Health 

Guest Editors 

Matthew J. Arduino, MS, DrPH 

Chief, Environmental and Applied Microbiology Section 

Division of Healthcare Quality Promotion 

Centers for Disease Control and Prevention 

W. Emmett Barkley, PhD 

President

Proven Practices, LLC
vi Biosafety in Microbiological and Biomedical Laboratories

Mark Q. Benedict 

Division of Parasitic Diseases 

Centers for Disease Control and Prevention 

Louisa Chapman, MD, MSPH 

Assistant to the Director for Immunization Policy 

National Immunization Program 

Centers for Disease Control and Prevention 

Dennis M. Dixon, PhD 

Chief, Bacteriology and Mycology Branch 

Division of Microbiology and Infectious Diseases 

National Institutes of Health 

Mark L. Eberhard, PhD 

Director, Division of Parasitic Diseases 

Centers for Disease Control and Prevention 

Martin S. Favero, PhD 

Director, Scientific and Clinical Affairs 

Advanced Sterilization Products 

Johnson and Johnson, Inc. 

Susan Gorsky 

Regulations Officer, Office of Hazardous Materials Standards 

Pipeline and Hazardous Materials Safety Administration 

United States Department of Transportation

Mary E. Groesch, PhD 

Senior Advisor for Science Policy 

Office of Science Policy, Office of the Director 

National Institutes of Health 

Ted Hackstadt, PhD 

Laboratory of Intracellular Parasites 

National Institute of Allergy and Infectious Diseases 

Rocky Mountain Laboratory 

Robert A. Heckert, DVM, PhD 

National Program Leader, Animal Health 

USDA, Agriculture Research Service 

Beltsville, MD 

Mark L. Hemphill, MS 

Chief of Policy 

Select Agent Program 

Centers for Disease Control and Prevention 
Guest Editors vii

Barbara L. Herwaldt, MD, MPH 

Medical Officer 

Parasitic Diseases Branch 

Division of Parasitic Diseases 

Centers for Disease Control and Prevention 

Julia K. Hilliard, PhD 

Viral Immunology Center, 

Department of Biology 

Georgia State University 

William C. Howard, MS 

Industrial Hygiene/Safety Manager 

Office of Health and Safety 

Centers for Disease Control and Prevention 

Herbert Jacobi 

Deputy Director

Division of Occupational Health and Safety 

National Institutes of Health 

Rachel E. Levinson, MA 

Director 

Government and Industry Liaison Office 

The Biodesign Institute 

Arizona State University 

Brian W. J. Mahy, MA, PhD, ScD, DSc 

Senior Scientific Advisor 

National Center for Infectious Diseases 

Centers for Disease Control and Prevention 

Charles B. Millard, PhD 

Lieutenant Colonel, U.S. Army 

Director, Division of Biochemistry 

Walter Reed Army Institute of Research 

Shanna Nesby-O’Dell, DVM, MPH 

Chief, External Activities Program and 

WHO Collaborating Center for Biosafety and Training 

Office of Health and Safety 

Centers for Disease Control and Prevention 
viii Biosafety in Microbiological and Biomedical Laboratories

Amy P. Patterson, MD 

Director 

Office of Biotechnology Activities/Office of Science Policy 

Office of the Director 

National Institutes of Health 

Jonathan Y. Richmond, PhD 

Biosafety Consultant 

Jonathan Richmond and Associates 

Southport, North Carolina 

Martin Sanders, PhD, CBSP, RBP 

Deputy Director 

Office of Health and Safety 

Centers for Disease Control and Prevention 

James Schmitt, MD, MS 

Medical Director 

Occupational Medical Service 

Division of Occupational Health and Safety 

National Institutes of Health 

Frank Simione, MS 

American Type Culture Collection 

Manassas, VA 

David G. Stuart, PhD 

Microbiologist 

The Baker Company 

Lee Ann Thomas, DVM 

Director 

Select Agent, Organisms and Vectors, and Animals 

Animal and Plant Health Inspection Service 

U.S. Department of Agriculture 

Larry G. Thompson 

Veterinary Diagnostic Laboratory 

Tifton, GA 

Robbin S. Weyant, PhD 

Chief, Laboratory Safety Branch 

Office of Health and Safety 

Centers for Disease Control and Prevention 
Contributors ix

Jeffrey I. Cohen 

Murray Cohen 

L. Eugene Cole II 

Chester Cooper 

David Cox 

Nancy Cox 

Rebecca Cox 

Jon Crane 

Jack Crawford 

Inger Damon 

Charles L. Divan 

Walter Dowdle 

Dennis Eagleson 

Eileen Edmondson 

Carol L. Eisenhauer 

Ana Espinel-Ingroff 

Joseph Esposito 

Michael T. Fallon 

Heinz Feldmann 

Barry Fields 

Howard Fields 

Michael J. Firko 

Collette Fitzgerald 

Diane O. Fleming 

Thomas Folks 

Ken Gage 

John Galgiani 

G. Gale Galland 

Leslie Garry Adams 

Mahmoud Ghannoum 

Mark Gibson 

Contributors

BMBL is by its nature a continuously revised manual, and each revision refines 

and extends the contributions to previous editions. Since 1984, when the first 

edition of BMBL was published, many scientists and biosafety specialists have 

contributed to this important reference work. The 5th edition is no exception, 

as specialists in multiple disciplines generously provided their considerable 

expertise to this revision. The Editors and Steering Committee gratefully 

acknowledge the contributions of all of these many contributors over the life of 

the BMBL, especially contributors to the current edition, who are listed below. 

Contributors to BMBL 5th Edition

David Abraham 

L. Garry Adams 

Michael Adler 

Lee Alderman 

Christopher E. Ansell 

Amy Barringer 

Ronald A. Barry 

Raymond L. Beets 

Ermias Belay 

Kathryn Bernard 

Carolyn Black 

Walter Bond 

Cheryl Bopp 

Sandra Bragg 

David Bressler 

Charles Brokopp 

Elizabeth J. Browder 

Bobby Gene Brown 

Corrie Brown 

Douglas R. Brown 

Michael Buchmeier 

Robert Bull 

Karen B. Byers 

Jane Caputo 

Arturo Casadevall 

Christina Cassetti 

Byron Caughey 

Vishnu Chaturvedi 

Louisa Chapman 

Bruce Chesebro 

May Chu 
x Biosafety in Microbiological and Biomedical Laboratories

Chester Gipson 

Irene Glowinski 

Richard Green 

William Grizzle 

Mary E. Groesch 

Ted Hackstadt 

Ted Hadfield 

Susan B. Harper 

Lynn Harding 

Kathryn L. Harris 

Robert J. Hawley 

Mark L. Hemphill 

David K. Henderson 

Richard Henkel 

Sherry Henry 

Robert H. Hill 

Julia Hilliard 

Alex Hoffmaster 

James D. Holt 

William C. Howard

Melony Ihrig 

Eddie L. Jackson 

Peter Jahrling 

Robert C. Jambou 

J. Michael Janda 

Jackie Katz 

Carole Kauffman 

Molly Kellum 

Fred Khoshbin 

Maxim Kiefer 

Michael P. Kiley 

Susan K. Kingston 

Bruce Klein 

Phillip H. Klesius 

Joseph P. Kozlovac 

Randy Kray 

Katrina Kretsinger 

Steve Kridel 

Thomas G. Ksiazek 

Robert Lamb 

Linda Lambert 

Ross D. LeClaire 

Geoffrey J. Letchworth 

Paul N. Levett 

Randall Levings 

Stuart Levitz 

Douglas Luster 

Keither Mansfield 

Dale Martin 

Al Mateczun 

Henry Mathews 

Michael McGinnis 

John J. McGlone 

Farhad Memarzadeh 

Claudia A. Mickelson

Barry Miller 

John G. Miller 

Thomas L. Miller 

Douglas M. Moore 

Mario Morales 

Ronald L. Morgan 

Rand Mortimer 

Bernard Moss 

Waleid I. Muhmed 

Brian Murphy 

Irving Nachamkin 

Janet K. A. Nicholson 

Alison D. O’Brien 

Marina O’Reilly 

Peter Palese 

Ross Pastel 

Bill Peters 

CJ Peters 

Jeannine Petersen 

Janet S. Peterson 

Louise M. Pitt 

Mark A. Poli 

Tanja Popovic 

Nathaniel Powell 

Ann M. Powers 

Suzette Priola 

Robert Purcell 

Greg Raymond

Yvonne M. Reid

Eric Resultan 

Judith Rhodes 

Robert L. Rice 

Jonathan Richmond 
Contributors xi

Betty Robertson 

Luis Rodriguez 

Pierre Rollin 

Nancy Rosenstein 

Eugene Rosenthal 

Michael D. Ruff 

Charles Rupprecht 

Scott Rusk 

Janice M. Rusnak 

Elliot Ryser 

Reynolds M. Salerno 

Jim Samuel 

Gary Sanden 

Thomas Sawicki 

Michelle Saylor 

Linda R. K. Schlater 

Connie Schmaljohn 

James J. Schmidt 

Beverly Schmitt 

James M. Schmitt 

Gregg N. Schuiling 

Lynne Sehulster 

Dennis Senne 

Tom Shih 

Allan Shipp 

Lance Simpson 

Theresa J. Smith 

Christine Spiropolou 

Gregory J. Stewart 

Yvonne J. Stifel 

Rudy Stotz 

Janet E. Stout 

Nancy Strockbine 

Kanta Subbarao 

Bala Swaminathan 

David Swayne 

James R. Swearengen 

Bill Switzer 

Mallory K. Tate 

James F. Taylor 

Louise D. Teel 

Robert B. Tesh 

Larry Thompson 

Alfonso Torres 

Kerstin E. Traum 

Theodore J. Traum 

David Trees 

Charles Trimarchi 

Robert G. Ulrich 

Cristina Vargas 

Don Vesley 

Paul E. Vinson 

David Waag 

Robert W. Wannemacher 

Cheryl Warfield 

David Warnock 

William Watson 

Mike Weathers 

Robert Webster 

Elizabeth Weirich 

Louis S. Welker 

Diane L. Whipple 

Denise Whitby 

Richard Whitley 

Catherine L. Wilhelmsen 

Axel Wolff 

David Woods 

Scott D. Wright 

Jack Wunder 

Robert Yarchoan 

Uri Yokel 

Lisa Young 

Gary L. Zackowitz 

Contents xiii

Contents

Section I—Introduction.......................................................................... 1

The Occurrence of Laboratory-Associated Infections .................................... 1

Evolution of National Biosafety Guidelines .................................................... 3

Risk Criteria for Establishing Ascending Levels of Containment.................... 4

Agent Summary Statements ......................................................................... 5

Biosecurity...................................................................................................... 6

Using BMBL ................................................................................................... 6

Looking Ahead ............................................................................................... 6

References..................................................................................................... 7

Section II—Biological Risk Assessment.............................................. 9

Hazardous Characteristics of an Agent........................................................ 10

Hazardous Characteristics of Laboratory Procedures.................................. 14

Potential Hazards Associated with Work Practices, Safety 

Equipment and Facility Safeguards ...................................................... 15

An Approach to Assess Risks and Select Appropriate Safeguards.............. 16

Conclusion.................................................................................................... 19

References................................................................................................... 19

Section III—Principles of Biosafety .................................................... 22

Laboratory Practices and Technique............................................................ 22

Safety Equipment (Primary Barriers and Personal Protective Equipment) ... 22

Facility Design and Construction (Secondary Barriers)................................ 23

Biosafety Levels ........................................................................................... 24

Animal Facilities ........................................................................................... 27

Clinical Laboratories..................................................................................... 27

Importation and Interstate Shipment of Certain Biomedical Materials ......... 28

Select Agents ............................................................................................... 28

References................................................................................................... 28

Section IV—Laboratory Biosafety Level Criteria............................... 30

Biosafety Level 1.......................................................................................... 30

A. Standard Microbiological Practices................................................ 30

B. Special Practices ........................................................................... 32

C. Safety Equipment (Primary Barriers and Personal

Protective Equipment).................................................................... 32

D. Laboratory Facilities (Secondary Barriers)..................................... 33
xiv Biosafety in Microbiological and Biomedical Laboratories

Biosafety Level 2.......................................................................................... 33

A. Standard Microbiological Practices................................................ 33

B. Special Practices ........................................................................... 35

C. Safety Equipment (Primary Barriers and Personal 

Protective Equipment).................................................................... 36

D. Laboratory Facilities (Secondary Barriers)..................................... 37

Biosafety Level 3.......................................................................................... 38

A. Standard Microbiological Practices................................................ 38

B. Special Practices ........................................................................... 40

C. Safety Equipment (Primary Barriers and Personal 

Protective Equipment).................................................................... 41

D. Laboratory Facilities (Secondary Barriers)..................................... 42

Biosafety Level 4.......................................................................................... 45

A. Standard Microbiological Practices................................................ 45

B. Special Practices ........................................................................... 47

C. Safety Equipment (Primary Barriers and Personal 

Protective Equipment).................................................................... 49

D. Laboratory Facilities (Secondary Barriers)..................................... 51

Section V—Vertebrate Animal Biosafety Level Criteria for 

Vivarium Research Facilities............................................................... 60

Animal Biosafety Level 1.............................................................................. 61

A. Standard Microbiological Practices................................................ 61

B. Special Practices ........................................................................... 64

C. Safety Equipment (Primary Barriers and Personal 

Protective Equipment).................................................................... 64

D. Laboratory Facilities (Secondary Barriers)..................................... 65

Animal Biosafety Level 2.............................................................................. 67

A. Standard Microbiological Practices................................................ 67

B. Special Practices ........................................................................... 70

C. Safety Equipment (Primary Barriers and Personal 

Protective Equipment).................................................................... 72

D. Laboratory Facilities (Secondary Barriers)..................................... 73
Contents xv

Animal Biosafety Level 3.............................................................................. 75

A. Standard Microbiological Practices................................................ 76

B. Special Practices ........................................................................... 79

C. Safety Equipment (Primary Barriers and Personal 

Protective Equipment).................................................................... 80

D. Laboratory Facilities (Secondary Barriers)..................................... 82

Animal Biosafety Level 4.............................................................................. 85

A. Standard Microbiological Practices................................................ 86

B. Special Practices ........................................................................... 89

C. Safety Equipment (Primary Barriers and Personal 

Protective Equipment).................................................................... 92

D. Laboratory Facilities (Secondary Barriers)..................................... 94

References................................................................................................. 102

Section VI—Principles of Laboratory Biosecurity........................... 104

Biosafety and Biosecurity........................................................................... 105

Risk Management Methodology................................................................. 106

Developing a Biosecurity Program............................................................. 107

Example Guidance: A Biosecurity Risk Assessment and 

Management Process ......................................................................... 107

Elements of a Biosecurity Program............................................................ 109

References................................................................................................. 113

Section VII—Occupational Health and Immunoprophylaxis .......... 114

Occupational Health Support Service Elements ........................................ 115

Occupational Health in the BSL-4 Setting.................................................. 120

References................................................................................................. 120

Section VIII—Agent Summary Statements....................................... 123

Section VIII-A: Bacterial Agents ................................................................. 123

Bacillus anthracis ................................................................................ 123

Bordetella pertussis............................................................................. 125

Brucella species.................................................................................. 126

Burkholderia mallei.............................................................................. 127

Burkholderia pseudomallei.................................................................. 129

Campylobacter (C. jejuni subsp. jejuni, C. coli, C. fetus subsp. 

fetus, C. upsaliensis).................................................................... 130
xvi Biosafety in Microbiological and Biomedical Laboratories

Chlamydia psittaci (Chlamydophila psittaci), C. trachomatis, 

C. pneumoniae (Chlamydophila pneumoniae)............................. 131

Neurotoxin-producing Clostridia species............................................. 134

Clostridium tetani and Tetanus toxin ................................................... 136

Corynebacterium diphtheriae.............................................................. 137

Francisella tularensis .......................................................................... 138

Helicobacter species........................................................................... 139

Legionella pneumophila and Other Legionella-like Agents ................. 140

Leptospira ........................................................................................... 142

Listeria monocytogenes ...................................................................... 143

Mycobacterium leprae......................................................................... 144

Mycobacterium tuberculosis complex ................................................. 145

Mycobacterium spp. other than M. tuberculosis complex 

and M. leprae............................................................................... 147

Neisseria gonorrhoeae........................................................................ 148

Neisseria meningitidis ......................................................................... 149

Salmonella serotypes, other than S. Typhi.......................................... 151

Salmonella Typhi................................................................................. 152

Shiga toxin (Verocytotoxin)-producing Escherichia coli ...................... 154

Shigella ............................................................................................... 155

Treponema pallidum............................................................................ 157

Vibrio enteritis species (V. cholerae, V. parahaemolyticus)................. 158

Yersinia pestis..................................................................................... 159

References................................................................................................. 161

Section VIII-B: Fungal Agents .................................................................... 170

Blastomyces dermatitidis .................................................................... 170

Coccidioides immitis and Coccidioides posadasii............................... 171

Cryptococcus Neoformans.................................................................. 172

Histoplasma capsulatum..................................................................... 173

Sporothrix schenckii............................................................................ 175

Dermatophytes (Epidermophyton, Microsporum, and Trichophyton).. 176

Miscellaneous Molds........................................................................... 177

References................................................................................................. 178
Contents xvii

Section VIII-C: Parasitic Agents ................................................................. 182

Blood and Tissue Protozoal Parasites ................................................ 182

Intestinal Protozoal Parasites.............................................................. 186

Trematode Parasites........................................................................... 188

Cestode Parasites............................................................................... 190

Nematode Parasites............................................................................ 192

References................................................................................................. 194

Section VIII-D: Rickettsial Agents............................................................... 195

Coxiella burnetii................................................................................... 195

Rickettsia prowazekii; Rickettsia typhi (R. mooseri); Orientia 

(Rickettsia) tsutsugamushi and Spotted Fever Group agents 

of human disease; Rickettsia rickettsii, Rickettsia conorii, 

Rickettsia akari, Rickettsia australis, Rickettsia siberica, 

and Rickettsia japonicum............................................................. 196

References................................................................................................. 199

Section VIII-E: Viral Agents ........................................................................ 200

Hantaviruses ....................................................................................... 200

Hendra Virus (formerly known as Equine Morbillivirus) 

and Nipah Virus ........................................................................... 201

Hepatitis A Virus, Hepatitis E Virus ..................................................... 202

Hepatitis B Virus, Hepatitis C Virus (formerly known as 

nonA nonB Virus), Hepatitis D Virus ............................................ 204

Herpesvirus Simiae (Cerocopithecine Herpesvirus I, Herpes B Virus).. 205

Human Herpes Virus........................................................................... 208

Influenza.............................................................................................. 211

Lymphocytic Choriomeningitis Virus ................................................... 215

Poliovirus............................................................................................. 216

Poxviruses........................................................................................... 218

Rabies Virus (and related lyssaviruses).............................................. 220

Retroviruses, including Human and Simian 

Immunodeficiency Viruses (HIV and SIV).................................... 221

Severe Acute Respiratory Syndrome (SARS) Coronavirus ................ 224

References................................................................................................. 227
xviii Biosafety in Microbiological and Biomedical Laboratories

Section VIII-F: Arboviruses and Related Zoonotic Viruses......................... 233

Viruses with BSL-2 Containment Recommended............................... 235

Viruses with BSL-3 Containment Recommended............................... 236

Viruses with BSL-4 Containment Recommended............................... 238

Dealing with Unknown Arboviruses..................................................... 238

Chimeric Viruses ................................................................................. 239

West Nile Virus (WNV)........................................................................ 240

Eastern Equine Encephalitis (EEE) Virus, Venezuelan 

Equine Encephalitis (VEE) Virus, and Western Equine 

Encephalitis (WEE) Virus............................................................. 242

Rift Valley Fever Virus (RVFV)............................................................ 244

References................................................................................................. 265

Section VIII-G: Toxin Agents....................................................................... 268

Botulinum Neurotoxin.......................................................................... 268

Staphylococcal Enterotoxins (SE)....................................................... 270

Ricin Toxin........................................................................................... 272

Selected Low Molecular Weight (LMW) Toxins................................... 274

References................................................................................................. 278

Section VIII-H: Prion Diseases................................................................... 282

References................................................................................................. 289

Appendix A – Primary Containment for Biohazards: 

Selection, Installation and Use of Biological Safety Cabinets....... 290

Section I—Introduction............................................................................... 290

Section II—The High Efficiency Particulate Air (HEPA) Filter 

and the Development of Biological Containment Devices .................. 291

Section III—Biological Safety Cabinets...................................................... 292

Section IV—Other Laboratory Hazards and Risk Assessment .................. 298

Section V — BSC Use by the Investigator: 

Work Practices and Procedures.......................................................... 300

Section VI—Facility and Engineering Requirements.................................. 305

Section VII — Certification of BSCs ........................................................... 307

Referrences................................................................................................ 323

Appendix B—Decontamination and Disinfection............................ 326

Environmentally Mediated Infection Transmission ..................................... 326

Principles of Sterilization and Disinfection.................................................. 326
Contents xix

Decontamination in the Microbiology Laboratory ....................................... 329

Special Infectious Agent Issues.................................................................. 334

References................................................................................................. 334

Appendix C—Transportation of Infectious Substances ................. 336

Transportation Regulations ........................................................................ 336

General DOT Packaging Requirements for Transport of 

Infectious Substances by Aircraft........................................................ 340

Appendix D—Agriculture Pathogen Biosafety ................................ 343

I. Introduction ......................................................................................... 343

II. BSL-3-Ag for Work with Loose-housed Animals ................................. 343

III. BSL-3 and ABSL-3 Plus Potential Facility Enhancements 

for Agriculture Agent Permitting........................................................... 347

IV. Pathogens of Veterinary Significance ................................................. 350

V. Summaries of Selected Agriculture Agents......................................... 352

African Swine Fever Virus (ASFV)............................................... 352

African Horse Sickness Virus (AHSV) ......................................... 353

Akabane Virus (AKAV)................................................................. 354

Bluetongue Virus (BTV) ............................................................... 355

Classical Swine Fever Virus (Hog Cholera)................................. 357

Contagious Bovine Pleuropneumonia Agent (CBPP).................. 358

Contagious Caprine Pleuropneumonia Agent (CCPP) ................ 359

Foot and Mouth Disease Virus (FMD) ......................................... 360

Heartwater Disease Agent (HD)................................................... 362

Infectious Salmon Anemia (ISA) Virus ......................................... 363

Lumpy Skin Disease (LSD) Virus................................................. 364

Malignant Catarrhal Fever Virus (MCFV) (Exotic Strains) ........... 365

Menangle Virus (MenV) ............................................................... 366

Newcastle Disease (ND) Virus..................................................... 367

Peste Des Petits Ruminants Virus (PPRV).................................. 368

Rinderpest Virus (RPV)................................................................ 369

Sheep and Goat Pox Virus (SGPV)............................................. 370

Spring Viremia of Carp Virus (SVCV) .......................................... 371

Swine Vesicular Disease Virus (SVDV) ....................................... 372

References................................................................................................. 373

VI. Additional Information ......................................................................... 376
xx Biosafety in Microbiological and Biomedical Laboratories

Appendix E—Arthropod Containment Guidelines (ACG)............... 377

References................................................................................................. 378

Appendix F—Select Agents and Toxins........................................... 379

Appendix G—Integrated Pest Management (IPM)........................... 380

References................................................................................................. 382

Appendix H—Working with Human, NHP and Other Mammalian 

Cells and Tissues ............................................................................... 383

References................................................................................................. 384

Appendix I—Guidelines for Work with Toxins of 

Biological Origin................................................................................. 385

References................................................................................................. 392

Appendix J—NIH Oversight of Research Involving 

Recombinant Biosafety Issues ......................................................... 394

Appendix K—Resources.................................................................... 396

Appendix L—Acronyms..................................................................... 401

Index .................................................................................................... 405
Tables and Figures xxi

Tables

Sections II through VII

Table Page

1 Classification of Infectious Microorganisms by Risk Group 10 

2 Summary of Recommended Biosafety Levels for Infectious Agents 59 

3 Summary of Recommended Animal Biosafety Levels for 103

Activities in which Experimentally or Naturally Infected 

Vertebrate Animals are Used

4 Explanation of Symbols Used in Table 6 to Define Basis 234

for Assignment of Viruses to Biosafety Levels

5 Vaccine Strains of BSL-3 and BSL-4 Viruses that 235

May Be Handled as BSL-2 

6 Alphabetic Listing of 597 Arboviruses and Hemorrhagic 246

Fever Viruses

7 The Human Prion Diseases 283

8 The Animal Prion Diseases 284 

9 Tissue Preparation for Human CJD and Related Diseases 287

10 Prion Inactivation Methods for Reusable Instruments and Surfaces 288

Appendix A: Primary Containment for Biohazards: Selection, 

Installation and Use of Biological Safety Cabinets

Table Page

1 Selection of a Safety Cabinet through Risk Assessment 311 

2 Comparison of Biosafety Cabinet Characteristics 311

3 Field Performance Tests Applied to the Three Classes 312

of Biological Safety Cabinets

4 Reference for Applicable Containment Test 313 

Appendix B: Decontamination and Disinfection

Table Page

1 Descending Order of Resistance to Germicidal Chemicals 330 

2 Activity Levels of Selected Liquid Germicides 333

Appendix I: Guidelines for Work with Toxins of Biological Origin

Table Page

1 Physical Inactivation of Selected Toxins 390 

2 Chemical Inactivation of Selected Toxins 391
xxii Biosafety in Microbiological and Biomedical Laboratories

Figures

Appendix A: Primary Containment for Biohazards: Selection, 

Installation and Use of Biological Safety Cabinets

Figure Page

1 HEPA filters 314 

2 The Class I BSC 315

3 The Class II, Type A1 BSC 315

4 Canopy (thimble) 316 

5A The Class II, Type B1 BSC (Class Design) 316

5B The Class II, Type B1 BSC (Bench Top Design) 317

6 The Class II, Type B2 BSC 318 

7 The tabletop model of a Class II, Type A2 BSC 318 

8 The Class III BSC 319 

9A The horizontal laminar flow “clean bench” 319 

9B The vertical laminar flow “clean bench” 320 

10 A modified containment cabinet or Class I BSC 321 

11 A typical layout for working “clean to dirty” 322 

12 One method to protect a house vacuum system 322 

13 A bag-in-bag-out filter enclosure 323 

Appendix C: Transportation of Infectious Substances

Figure Page

1 A Category A UN Standard Triple Packaging 341 

2 A Category B Non-specification Triple Packaging 342
Introduction 1

Section I—Introduction

Over the past two decades, Biosafety in Microbiological and Biomedical 

Laboratories (BMBL) has become the code of practice for biosafety—the discipline 

addressing the safe handling and containment of infectious microorganisms and 

hazardous biological materials. The principles of biosafety introduced in 1984 in 

the first edition of BMBL1

 and carried through in this fifth edition remain steadfast. 

These principles are containment and risk assessment. The fundamentals of 

containment include the microbiological practices, safety equipment, and facility 

safeguards that protect laboratory workers, the environment, and the public 

from exposure to infectious microorganisms that are handled and stored in the 

laboratory. Risk assessment is the process that enables the appropriate selection 

of microbiological practices, safety equipment, and facility safeguards that can 

prevent laboratory-associated infections (LAI). The purpose of periodic updates 

of BMBL is to refine guidance based on new knowledge and experiences and 

to address contemporary issues that present new risks that confront laboratory 

workers and the public health. In this way the code of practice will continue to 

serve the microbiological and biomedical community as a relevant and valuable 

authoritative reference. 

We are living in an era of uncertainty and change. New infectious agents 

and diseases have emerged. Work with infectious agents in public and private 

research, public health, clinical and diagnostic laboratories, and in animal care 

facilities has expanded. Recent world events have demonstrated new threats 

of bioterrorism. For these reasons, organizations and laboratory directors are 

compelled to evaluate and ensure the effectiveness of their biosafety programs, 

the proficiency of their workers, as well as the capability of equipment, facilities, 

and management practices to provide containment and security of microbiological 

agents. Similarly, individual workers who handle pathogenic microorganisms must 

understand the containment conditions under which infectious agents can be 

safely manipulated and secured. Application of this knowledge and the use of 

appropriate techniques and equipment will enable the microbiological and 

biomedical community to prevent personal, laboratory and environmental 

exposure to potentially infectious agents or biohazards. 

The Occurrence of Laboratory-Associated Infections

Published reports of LAIs first appeared around the start of the twentieth century. 

By 1978, four studies by Pike and Sulkin collectively identified 4,079 LAIs resulting 

in 168 deaths occurring between 1930 and 1978.2-5 These studies found that 

the ten most common causative agents of overt infections among workers 

were Brucella spp., Coxiella burnetii, hepatitis B virus (HBV), Salmonella typhi, 

Francisella tularensis, Mycobacterium tuberculosis, Blastomyces dermatitidis, 

Venezuelan equine encephalitis virus, Chlamydia psittaci, and Coccidioides 

immitis. The authors acknowledged that the 4,079 cases did not represent all 

LAIs that occurred during this period since many laboratories chose not to 
2 Biosafety in Microbiological and Biomedical Laboratories

report overt cases or conduct surveillance programs to identify sub-clinical or 

asymptomatic infections. 

In addition, reports of LAIs seldom provided data sufficient to determine 

incidence rates, complicating quantitative assessments of risk. Similarly, there 

were no distinguishable accidents or exposure events identified in more than 

80% of the LAIs reported before 1978. Studies did show that in many cases 

the infected person worked with a microbiological agent or was in the vicinity 

of another person handling an agent.2-6

During the 20 years following the Pike and Sulkin publications, a worldwide 

literature search by Harding and Byers revealed 1,267 overt infections with 22 

deaths.7

 Five deaths were of fetuses aborted as the consequence of a maternal 

LAI. Mycobacterium tuberculosis, Coxiella burnetii, hantavirus, arboviruses, HBV, 

Brucella spp., Salmonella spp., Shigella spp., hepatitis C virus, and Cryptosporidium 

spp. accounted for 1,074 of the 1,267 infections. The authors also identified an 

additional 663 cases that presented as sub-clinical infections. Like Pike and 

Sulkin, Harding and Byers reported that only a small number of the LAI involved 

a specific incident. The non-specific associations reported most often by these 

authors were working with a microbiological agent, being in or around the 

laboratory, or being around infected animals. 

The findings of Harding and Byers indicated that clinical (diagnostic) and 

research laboratories accounted for 45% and 51%, respectively, of the total LAIs 

reported. This is a marked difference from the LAIs reported by Pike and Sulkin 

prior to 1979, which indicated that clinical and research laboratories accounted 

for 17% and 59%, respectively. The relative increase of LAIs in clinical laboratories 

may be due in part to improved employee health surveillance programs that are 

able to detect sub-clinical infections, or to the use of inadequate containment 

procedures during the early stages of culture identification. 

Comparison of the more recent LAIs reported by Harding and Byers with 

those reported by Pike and Sulkin suggests that the number is decreasing. Harding 

and Byers note that improvements in containment equipment, engineering controls, 

and greater emphasis on safety training may be contributing factors to the apparent 

reduction in LAIs over two decades. However, due to the lack of information on 

the actual numbers of infections and the population at risk, it is difficult to 

determine the true incidence of LAIs with any degree of certainty. 

Publication of the occurrence of LAIs provides an invaluable resource 

for the microbiological and biomedical community. For example, one report 

of occupational exposures associated with Brucella melitensis, an organism 

capable of transmission by the aerosol route, described how a staff member in 

a clinical microbiology laboratory accidentally sub-cultured B. melitensis on the 

open bench.8

 This error and a breech in containment practices resulted in eight 

LAIs with B. melitensis among 26 laboratory members, an attack rate of 31%. 
Introduction 3

Reports of LAIs can serve as lessons in the importance of maintaining safe 

conditions in biological research. 

Evolution of National Biosafety Guidelines 

National biosafety guidelines evolved from the efforts of the microbiological and 

biomedical community to promote the use of safe microbiological practices, 

safety equipment and facility safeguards that will reduce LAIs and protect the 

public health and environment. The historical accounts of LAIs raised awareness 

about the hazards of infectious microorganisms and the health risks to laboratory 

workers who handle them. Many published accounts suggested practices and 

methods that might prevent LAIs.9

 Arnold G. Wedum was the Director of Industrial 

Health and Safety at the United States Army Biological Research Laboratories, 

Fort Detrick from 1944 to 1969. His pioneering work in biosafety provided the 

foundation for evaluating the risks of handling infectious microorganisms and for 

recognizing biological hazards and developing practices, equipment, and facility 

safeguards for their control. Fort Detrick also advanced the field by aiding the 

development of biosafety programs at the United States Department of Agriculture 

(USDA), National Animal Research Center and the United States Department of 

Health and Human Services (DHHS), Centers for Disease Control and Prevention 

(CDC) and National Institutes of Health (NIH). These governmental organizations 

subsequently developed several national biosafety guidelines that preceded the 

first edition of BMBL. 

In 1974, the CDC published Classification of Etiologic Agents on the Basis 

of Hazard.10 This report introduced the concept for establishing ascending levels 

of containment that correspond to risks associated with handling infectious 

microorganisms that present similar hazardous characteristics. Human pathogens 

were grouped into four classes according to mode of transmission and the 

severity of disease they caused. A fifth class included non-indigenous animal 

pathogens whose entry into the United States was restricted by USDA policy. 

The NIH published National Cancer Institute Safety Standards for Research 

Involving Oncogenic Viruses in 1974.11 These guidelines established three levels of 

containment based on an assessment of the hypothetical risk of cancer in humans 

from exposure to animal oncogenic viruses or a suspected human oncogenic 

virus isolate from man.12,13 In 1976 NIH first published the NIH Guidelines for 

Research Involving Recombinant DNA Molecules (NIH Guidelines).14 The NIH 

Guidelines described in detail the microbiological practices, equipment, and 

facility safeguards that correspond to four ascending levels of physical containment 

and established criteria for assigning experiments to a containment level based 

on an assessment of potential hazards of this emerging technology. The 

evolution of these guidelines set the foundation for developing a code of practice 

for biosafety in microbiological and biomedical laboratories. Led by the CDC and 

NIH, a broad collaborative initiative involving scientists, laboratory directors, 
4 Biosafety in Microbiological and Biomedical Laboratories

occupational physicians, epidemiologists, public health officials and health and 

safety professionals developed the first edition of BMBL in 1984. The BMBL 

provided the technical content not previously available in biosafety guidelines 

by adding summary statements conveying guidance pertinent to infectious 

microorganisms that had caused LAIs. The fifth edition of BMBL is also the 

product of a broad collaborative initiative committed to perpetuate the value 

of this national biosafety code of practice. 

Risk Criteria for Establishing Ascending Levels of Containment

The primary risk criteria used to define the four ascending levels of containment, 

referred to as biosafety levels 1 through 4, are infectivity, severity of disease, 

transmissibility, and the nature of the work being conducted. Another important 

risk factor for agents that cause moderate to severe disease is the origin of the 

agent, whether indigenous or exotic. Each level of containment describes the 

microbiological practices, safety equipment and facility safeguards for the 

corresponding level of risk associated with handling a particular agent. The 

basic practices and equipment are appropriate for protocols common to most 

research and clinical laboratories. The facility safeguards help protect nonlaboratory occupants of the building and the public health and environment. 

Biosafety level 1 (BSL-1) is the basic level of protection and is appropriate 

for agents that are not known to cause disease in normal, healthy humans. 

Biosafety level 2 (BSL-2) is appropriate for handling moderate-risk agents that 

cause human disease of varying severity by ingestion or through percutaneous 

or mucous membrane exposure. Biosafety level 3 (BSL-3) is appropriate for 

agents with a known potential for aerosol transmission, for agents that may 

cause serious and potentially lethal infections and that are indigenous or exotic 

in origin. Exotic agents that pose a high individual risk of life-threatening disease 

by infectious aerosols and for which no treatment is available are restricted to 

high containment laboratories that meet biosafety level 4 (BSL-4) standards. 

It is important to emphasize that the causative incident for most LAIs 

is unknown.7,8 Less obvious exposures such as the inhalation of infectious 

aerosols or direct contact of the broken skin or mucous membranes with droplets 

containing an infectious microorganism or surfaces contaminated by droplets 

may possibly explain the incident responsible for a number of LAIs. Most 

manipulations of liquid suspensions of microorganisms produce aerosols and 

droplets. Small-particle aerosols have respirable size particles that may contain 

one or several microorganisms. These small particles stay airborne and easily 

disperse throughout the laboratory. When inhaled, the human lung will retain 

those particles. Larger particle droplets rapidly fall out of the air, contaminating 

gloves, the immediate work area, and the mucous membranes of unprotected 

workers. A procedure’s potential to release microorganisms into the air as 

aerosols and droplets is the most important operational risk factor that supports 

the need for containment equipment and facility safeguards. 
Introduction 5

Agent Summary Statements 

The fifth edition, as in all previous editions, includes agent summary statements 

that describe the hazards, recommended precautions, and levels of containment 

appropriate for handling specific human and zoonotic pathogens in the laboratory 

and in facilities that house laboratory vertebrate animals. Agent summary statements 

are included for agents that meet one or more of the following three criteria: 

1. the agent is a proven hazard to laboratory personnel working with 

infectious materials; 

2. the agent has a high potential for causing LAIs even though no 

documented cases exist; and 

3. the agent causes grave disease or presents a significant public 

health hazard. 

Scientists, clinicians, and biosafety professionals prepared the statements 

by assessing the risks of handling the agents using standard protocols followed 

in many laboratories. No one should conclude that the absence of an agent 

summary statement for a human pathogen means that the agent is safe to 

handle at BSL-1, or without a risk assessment to determine the appropriate 

level of containment. Laboratory directors should also conduct independent 

risk assessments before beginning work with an agent or procedure new to the 

laboratory, even though an agent summary statement is available. There may be 

situations where a laboratory director should consider modifying the precautionary 

measures or recommended practices, equipment, and facility safeguards 

described in an agent summary statement. In addition, laboratory directors 

should seek guidance when conducting risk assessments. Knowledgeable 

colleagues; institutional biosafety committees; biosafety officers; and public 

health, biosafety, and scientific associations are excellent resources. 

The agent summary statements in the fourth edition BMBL were reviewed 

in the course of preparing the fifth edition of BMBL. There are new and updated 

agent summary statements including those for agents now classified as Select 

Agents. For example, there is an updated section on arboviruses and related 

zoonotic viruses including new agent summary statements. There are also 

substantive revisions to the Influenza Agent Summary Statement that address 

non-contemporary human influenza strains and recommend safeguards for 

research involving reverse genetics of the 1918 influenza strain. 

The fifth edition also includes a revised section on risk assessment that 

gives more emphasis on the importance of this process in selecting the appropriate 

practices and level of containment. That section intentionally follows this introduction 

because risk assessment represents the foundation—a code of practice for safe 

handling of infectious agents in microbiological and biomedical laboratories. 
6 Biosafety in Microbiological and Biomedical Laboratories

Biosecurity

Today, the nation is facing a new challenge in safeguarding the public health 

from potential domestic or international terrorism involving the use of dangerous 

biological agents or toxins. Existing standards and practices may require adaptation to 

ensure protection from such hostile actions. In addition, recent federal regulations 

mandate increased security within the microbiological and biomedical community 

in order to protect biological pathogens and toxins from theft, loss, or misuse. 

The fifth edition of BMBL includes an important new section on biosecurity—the 

discipline addressing the security of microbiological agents and toxins and the 

threats posed to human and animal health, the environment, and the economy 

by deliberate misuse or release. A careful review of the biosecurity concepts and 

guidelines introduced in this new section is essential for all laboratory workers. 

Using BMBL

BMBL is both a code of practice and an authoritative reference. Knowledge 

sufficient to work safely with hazardous microorganisms requires a careful review 

of the entire BMBL. This will offer the reader an understanding of the biosafety 

principles that serve as the basis for the concepts and recommendations included 

in this reference. Reading only selected sections will not adequately prepare even 

an experienced laboratory worker to handle potentially infectious agents safely. 

The recommended practices, safety equipment, and facility safeguards 

described in the first edition of BMBL and expanded in the fifth edition are 

advisory in most circumstances. The intent was and is to establish a voluntary 

code of practice, one that all members of a laboratory community will together 

embrace to safeguard themselves and their colleagues, and to protect the public 

health and environment. 

Looking Ahead 

Laboratory-associated infections from exposure to biological agents known to 

cause disease are infrequent. It is critical that the microbiological and biomedical 

community continue its resolve to remain vigilant and not to become complacent. 

The LAIs reported in the last 25 years demonstrate that accidents and unrecognized 

exposures continue to occur. The absence of clear evidence of the means of 

transmission in most documented LAI should motivate persons at risk to be alert 

to all potential routes of exposure. The accidental release of microbial aerosols is 

a probable cause of many LAI15, which demonstrates the importance of worker 

training and the ability to recognize potential hazards and correct unsafe habits. 

Attention to and proficient use of work practices, safety equipment and 

engineering controls are also essential. 
Introduction 7

The nation’s response to recent world events brings with it a heightened 

concern for a potential increase in LAIs. In 2003, the United States federal 

government awarded significant funding for the construction of National 

Biocontainment Laboratories (NBL) and Regional Biocontainment Laboratories 

(RBL). The NBLs will house BSL-2, 3, and 4 laboratories; the RBLs will house 

BSL-2 and 3 laboratories. In addition, construction of new containment facilities 

by private and public institutions is underway nationwide. The expansion of 

biocontainment laboratories nationwide dramatically increases the need for 

training in microbiological practices and biosafety principles. 

Understanding the principles of biosafety and adherence to the microbiological 

practices, containment and facility safeguards described in BMBL will contribute 

to a safer and healthier working environment for laboratory staff and adjacent 

personnel, and the community. 

References

1. Richardson JH, Barkley WE, editors. Biosafety in microbiological and 

biomedical laboratories. 1st ed. Washington, DC. 1984. 

2. Sulkin SE, Pike RM. Survey of laboratory-acquired infections. Am J Pub Hlth. 

1951;41:769-81. 

3. Pike RM, Sulkin SE, Schulze ML. Continuing importance of laboratoryacquired infections. Am J Pub Hlth. 1965;55:190-99. 

4. Pike RM. Laboratory-associated infections: summary and analysis of 3921 

cases. Health Lab Sci. 1976;13:105-14. 

5. Pike RM. Past and present hazards of working with infectious agents. 

Arch Pathol Lab Med. 1978;102:333-36. 

6. Pike RM. Laboratory-associated infections: incidence, fatalities, causes, 

and prevention. Annu Rev Microbiol. 1979;33:41-66. 

7. Harding AL, Byers KB. Epidemiology of laboratory-associated infections. 

In: Fleming DO, Hunt DL, editors. Biological safety: principles and practices. 

3rd ed. Washington, DC: ASM Press; 2000:35-54. 

8. Staskiewicz J, Lewis CM, Colville J, et al. Outbreak of Brucella melitensis

among microbiology laboratory workers in a community hospital. J Clin 

Microbiol. 1991;29:287-90. 

9. Wedum AG. Laboratory safety in research with infectious diseases. Public 

Health Rep. 1964;79:619-33. 

10. Classification of etiological agents on the basis of hazard. 4th ed. Atlanta, 

Centers for Disease Control (US); 1974. 

11. National Cancer Institute safety standards for research involving oncogenic 

viruses. Bethesda: The National Institutes of Health (US), National Cancer 

Institute, Office of Research Safety; 1974, DHEW Publication No. (NIH) 75-790.

12. Wedum AG. History and epidemiology of laboratory-acquired infections 

(in relation to the cancer research program). JABSA. 1997;2:12-29. 
8 Biosafety in Microbiological and Biomedical Laboratories

13. West DL, Twardzik DR, McKinney RW, et al. Identification, analysis, and 

control of biohazards in viral cancer research. In: Fuscaldo AA, Erlick BJ, 

Hindman B, editors. Laboratory safety theory and practice. New York: 

Academic Press; 1980. p. 167-223. 

14. NIH guidelines for research involving recombinant DNA molecules. 

Bethesda: The National Institutes of Health (US), Office of Biotechnology 

Activities; 2002, April. 

15. Rusnak JM, Kortepeter MG, Hawley RJ, et al. Risk of occupationally 

acquired illnesses from biological threat agents in unvaccinated laboratory 

workers. Biosecur Bioterror. 2004;2:281-93. 
Biological Risk Assessment 9

Section II—Biological Risk Assessment

Risk assessment is an important responsibility for directors and principal 

investigators of microbiological and biomedical laboratories. Institutional biosafety 

committees (IBC), animal care and use committees, biological safety professionals, 

and laboratory animal veterinarians share in this responsibility. Risk assessment 

is a process used to identify the hazardous characteristics of a known infectious 

or potentially infectious agent or material, the activities that can result in a person’s 

exposure to an agent, the likelihood that such exposure will cause a LAI, and the 

probable consequences of such an infection. The information identified by risk 

assessment will provide a guide for the selection of appropriate biosafety levels 

and microbiological practices, safety equipment, and facility safeguards that can 

prevent LAIs.

Laboratory directors and principal investigators should use risk assessment 

to alert their staffs to the hazards of working with infectious agents and to the need 

for developing proficiency in the use of selected safe practices and containment 

equipment. Successful control of hazards in the laboratory also protects persons 

not directly associated with the laboratory, such as other occupants of the same 

building, and the public.

Risk assessment requires careful judgment. Adverse consequences are more 

likely to occur if the risks are underestimated. By contrast, imposition of safeguards 

more rigorous than actually needed may result in additional expense and burden 

for the laboratory, with little safety enhancement. Unnecessary burden may result 

in circumvention of required safeguards. However, where there is insufficient 

information to make a clear determination of risk, it is prudent to consider the 

need for additional safeguards until more data are available.

The primary factors to consider in risk assessment and selection of 

precautions fall into two broad categories: agent hazards and laboratory procedure 

hazards. In addition, the capability of the laboratory staff to control hazards must 

be considered. This capability will depend on the training, technical proficiency, 

and good habits of all members of the laboratory, and the operational integrity 

of containment equipment and facility safeguards.

The agent summary statements contained in BMBL identify the primary agent 

and procedure hazards for specific pathogens and recommend precautions for their 

control. The guest editors and contributors of this and previous editions of BMBL 

based their recommendations on an assessment of the risks associated with the 

handling of pathogens using generally routine generic laboratory procedures. A 

review of the summary statement for a specific pathogen is a helpful starting 

point for assessment of the risks of working with that agent and those for a 

similar agent.
10 Biosafety in Microbiological and Biomedical Laboratories

Hazardous Characteristics of an Agent

The principal hazardous characteristics of an agent are: its capability to infect and 

cause disease in a susceptible human or animal host, its virulence as measured 

by the severity of disease, and the availability of preventive measures and effective 

treatments for the disease. The World Health Organization (WHO) has recommended 

an agent risk group classification for laboratory use that describes four general 

risk groups based on these principal characteristics and the route of transmission 

of the natural disease.1

The four groups address the risk to both the laboratory 

worker and the community. The NIH Guidelines established a comparable 

classification and assigned human etiological agents into four risk groups on the 

basis of hazard.2

The descriptions of the WHO and NIH risk group classifications 

are presented in Table 1. They correlate with but do not equate to biosafety 

levels. A risk assessment will determine the degree of correlation between an 

agent’s risk group classification and biosafety level. See Section 3 for a further 

discussion of the differences and relatedness of risk groups and biosafety levels.

Table 1: Classification of Infectious Microorganisms by Risk Group

Risk Group 

Classification

NIH Guidelines for 

Research involving 

Recombinant DNA 

Molecules 20022

World Health Organization 

Laboratory Biosafety Manual 3rd

Edition 20041

Risk Group 1 Agents not associated with 

disease in healthy adult humans.

(No or low individual and community risk) 

A microorganism unlikely to cause human 

or animal disease.

Risk Group 2 Agents associated with human 

disease that is rarely serious 

and for which preventive or 

therapeutic interventions are 

often available.

(Moderate individual risk; low community 

risk) A pathogen that can cause human 

or animal disease but is unlikely to be a 

serious hazard 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 spread of infection is limited. 

Risk Group 3 Agents associated with serious 

or lethal human disease for 

which preventive or therapeutic 

interventions may be available 

(high individual risk but low 

community risk).

(High individual risk; low community risk) 

A pathogen that usually causes serious 

human or animal disease but does not 

ordinarily spread from one infected 

individual to another. Effective treatment 

and preventive measures are available. 

Risk Group 4 Agents likely to cause serious 

or lethal human disease for 

which preventive or therapeutic 

interventions are not usually 

available (high individual risk and 

high community risk).

(High individual and community risk)

A pathogen that usually causes serious 

human or animal disease and can be 

readily transmitted from one individual 

to another, directly or indirectly. Effective 

treatment and preventive measures are 

not usually available.3
Biological Risk Assessment 11

Other hazardous characteristics of an agent include probable routes of 

transmission of laboratory infection, infective dose, stability in the environment, 

host range, and its endemic nature. In addition, reports of LAIs are a clear 

indicator of hazard and often are sources of information helpful for identifying 

agent and procedural hazards, and the precautions for their control. The absence 

of a report does not indicate minimal risk. Reports seldom provide incidence 

data, making comparative judgments on risks among agents difficult. The number 

of infections reported for a single agent may be an indication of the frequency 

of use as well as risk. Nevertheless, reporting of LAIs by laboratory directors in 

the scientific and medical literature is encouraged. Reviews of such reports and 

analyses of LAIs identified through extensive surveys are a valuable resource 

for risk assessment and reinforcement of the biosafety principles. The summary 

statements in BMBL include specific references to reports on LAIs.

The predominant probable routes of transmission in the laboratory are: 

1) direct skin, eye or mucosal membrane exposure to an agent; 2) parenteral 

inoculation by a syringe needle or other contaminated sharp, or by bites from 

infected animals and arthropod vectors; 3) ingestion of liquid suspension of an 

infectious agent, or by contaminated hand to mouth exposure; and 4) inhalation 

of infectious aerosols. An awareness of the routes of transmission for the natural 

human disease is helpful in identifying probable routes of transmission in the 

laboratory and the potential for any risk to the public health. For example, 

transmission of infectious agents can occur by direct contact with discharges 

from respiratory mucous membranes of infected persons, which would be a clear 

indication that a laboratory worker is at risk of infection from mucosal membrane 

exposure to droplets generated while handling that agent. The American Public 

Health Association publication Control of Communicable Diseases Manual is an 

excellent reference for identifying both natural and often noted laboratory modes 

of transmission.3

 However, it is important to remember that the nature and 

severity of disease caused by a laboratory infection and the probable laboratory 

route of transmission of the infectious agent may differ from the route of 

transmission and severity associated with the naturally-acquired disease.4

An agent capable of transmitting disease through respiratory exposure to 

infectious aerosols is a serious laboratory hazard, both for the person handling 

the agent and for other laboratory occupants. This hazard requires special caution 

because infectious aerosols may not be a recognized route of transmission for 

the natural disease. Infective dose and agent stability are particularly important in 

establishing the risk of airborne transmission of disease. For example, the reports 

of multiple infections in laboratories associated with the use of Coxiella burnetii 

are explained by its low inhalation infective dose, which is estimated to be ten 

inhaled infectious particles, and its resistance to environmental stresses that 

enables the agent to survive outside of a living host or culture media long 

enough to become an aerosol hazard.5
12 Biosafety in Microbiological and Biomedical Laboratories

When work involves the use of laboratory animals, the hazardous 

characteristics of zoonotic agents require careful consideration in risk assessment. 

Evidence that experimental animals can shed zoonotic agents and other infectious 

agents under study in saliva, urine, or feces is an important indicator of hazard. 

The death of a primate center laboratory worker from Cercopithecine herpes 

virus 1 (CHV-1, also known as Monkey B virus) infection following an ocular 

splash exposure to biologic material from a rhesus macaque emphasizes the 

seriousness of this hazard.6 Lack of awareness for this potential hazard can 

make laboratory staff vulnerable to an unexpected outbreak involving multiple 

infections.7 Experiments that demonstrate transmission of disease from an 

infected animal to a normal animal housed in the same cage are reliable 

indicators of hazard. Experiments that do not demonstrate transmission, 

however, do not rule out hazard. For example, experimental animals infected 

with Francisella tularensis, Coxiella burnetii, Coccidioides immitis, or Chlamydia 

psittaci—agents that have caused many LAIs—rarely infect cagemates.8

The origin of the agent is also important in risk assessment. Non-indigenous 

agents are of special concern because of their potential to introduce risk of 

transmission, or spread of human and animal or infectious diseases from foreign 

countries into the United States. Importation of etiological agents of human disease 

requires a permit from the CDC. Importation of many etiological agents of livestock, 

poultry and other animal diseases requires a permit from the USDA’s Animal and 

Plant Health Inspection Service (APHIS). For additional details, see Appendix C.

Genetically modified agent hazards. The identification and assessment 

of hazardous characteristics of genetically modified agents involve consideration 

of the same factors used in risk assessment of the wild-type organism. It is 

particularly important to address the possibility that the genetic modification could 

increase an agent’s pathogenicity or affect its susceptibility to antibiotics or other 

effective treatments. The risk assessment can be difficult or incomplete, because 

important information may not be available for a newly engineered agent. Several 

investigators have reported that they observed unanticipated enhanced virulence 

in recent studies with engineered agents.9-12 These observations give reason to 

remain alert to the possibility that experimental alteration of virulence genes may 

lead to increased risk. It also suggests that risk assessment is a continuing 

process that requires updating as research progresses.

The NIH Guidelines are the key reference in assessing risk and establishing 

an appropriate biosafety level for work involving recombinant DNA molecules.2

The purpose of the NIH Guidelines is to promote the safe conduct of research 

involving recombinant DNA. The guidelines specify appropriate practices and 

procedures for research involving constructing and handling both recombinant 

DNA molecules and organisms and viruses that contain recombinant DNA. They 

define recombinant DNA as a molecule constructed outside of a living cell with 

the capability to replicate in a living cell. The NIH Guidelines explicitly address 

experiments that involve introduction of recombinant DNA into Risk Groups 2, 3, 
Biological Risk Assessment 13

and 4 agents, and experiments in which the DNA from Risk Groups 2, 3, and 4 

agents is cloned into nonpathogenic prokaryotic or lower eukaryotic host-vector 

systems. Compliance with the NIH Guidelines is mandatory for investigators 

conducting recombinant DNA research funded by the NIH or performed at, or 

sponsored by, any public or private entity that receives any NIH funding for 

recombinant DNA research. Many other institutions have adopted these 

guidelines as the best current practice.

The NIH Guidelines were first published in 1976 and are revised on an 

ongoing basis in response to scientific and policy developments. The guidelines 

outline the roles and responsibilities of various entities affiliated with recombinant 

DNA research, including institutions, investigators, and the NIH. Recombinant 

DNA research subject to the NIH Guidelines may require: 1) approval by the NIH 

Director, review by the NIH Recombinant DNA Advisory Committee (RAC), and 

approval by the IBC; or 2) review by the NIH Office of Biotechnology Activities 

(OBA) and approval by the IBC; or 3) review by the RAC and approvals by the 

IBC and Institutional Review Board; or 4) approval by the IBC prior to initiation 

of the research; or 5) notification of the IBC simultaneous with initiation of the 

work. It is important to note that review by an IBC is required for all non-exempt 

experiments as defined by the NIH Guidelines.

The NIH Guidelines were the first documents to formulate the concept of an 

IBC as the responsible entity for biosafety issues stemming from recombinant 

DNA research. The NIH Guidelines outline the membership, procedures, and 

functions of an IBC. The institution is ultimately responsible for the effectiveness 

of the IBC, and may define additional roles and responsibilities for the IBC apart 

from those specified in the NIH Guidelines. See Appendix J for more information 

about the NIH Guidelines and OBA.

Cell cultures. Workers who handle or manipulate human or animal cells 

and tissues are at risk for possible exposure to potentially infectious latent and 

adventitious agents that may be present in those cells and tissues. This risk is well 

understood and illustrated by the reactivation of herpes viruses from latency,13,14

the inadvertent transmission of disease to organ recipients,15,16 and the persistence 

of human immunodeficiency virus (HIV), HBV, and hepatitis C virus (HCV) within 

infected individuals in the U.S. population.17 There also is evidence of accidental 

transplantation of human tumor cells to healthy recipients which indicates that 

these cells are potentially hazardous to laboratory workers who handle them.18

In addition, human and animal cell lines that are not well characterized or are 

obtained from secondary sources may introduce an infectious hazard to the 

laboratory. For example, the handling of nude mice inoculated with a tumor cell 

line unknowingly infected with lymphocytic choriomeningitis virus resulted in 

multiple LAIs.19 The potential for human cell lines to harbor a bloodborne 

pathogen led the Occupational Health and Safety Administration (OSHA) to 

interpret that the occupational exposure to bloodborne pathogens final rule 

would include primary human cell lines and explants.17
14 Biosafety in Microbiological and Biomedical Laboratories

Hazardous Characteristics of Laboratory Procedures

Investigations of LAIs have identified five principal routes of laboratory 

transmission. These are parenteral inoculations with syringe needles or other 

contaminated sharps, spills and splashes onto skin and mucous membranes, 

ingestion through mouth pipetting, animal bites and scratches, and inhalation 

exposures to infectious aerosols. The first four routes of laboratory transmission 

are easy to detect, but account for less than 20 percent of all reported LAIs.20

Most reports of such infections do not include information sufficient to identify the 

route of transmission of infection. Work has shown that the probable sources of 

infection—animal or ectoparasite, clinical specimen, agent, and aerosol—are 

apparent in approximately 50 percent of cases.21

Aerosols are a serious hazard because they are ubiquitous in laboratory 

procedures, are usually undetected, and are extremely pervasive, placing the 

laboratory worker carrying out the procedure and other persons in the laboratory 

at risk of infection. There is general agreement among biosafety professionals, 

laboratory directors and principal investigators who have investigated LAIs that 

an aerosol generated by procedures and operations is the probable source of 

many LAIs, particularly in cases involving workers whose only known risk factor 

was that they worked with an agent or in an area where that work was done.

Procedures that impart energy to a microbial suspension will produce 

aerosols. Procedures and equipment used routinely for handling infectious 

agents in laboratories, such as pipetting, blenders, non-self contained 

centrifuges, sonicators and vortex mixers are proven sources of aerosols. These 

procedures and equipment generate respirable-size particles that remain airborne 

for protracted periods. When inhaled, these particles are retained in the lungs 

creating an exposure hazard for the person performing the operation, coworkers 

in the laboratory, and a potential hazard for persons occupying adjacent spaces 

open to air flow from the laboratory. A number of investigators have determined the 

aerosol output of common laboratory procedures. In addition, investigators have 

proposed a model for estimating inhalation dosage from a laboratory aerosol source. 

Parameters that characterize aerosol hazards include an agent’s inhalation infective 

dose, its viability in an aerosol, aerosol concentration, and particle size.22,23,24

Procedures and equipment that generate respirable size particles also 

generate larger size droplets that can contain multiple copies of an infectious 

agent. The larger size droplets settle out of the air rapidly, contaminating the 

gloved hands and work surface and possibly the mucous membranes of the 

persons performing the procedure. An evaluation of the release of both respirable 

particles and droplets from laboratory operations determined that the respirable 

component is relatively small and does not vary widely; in contrast hand and 

surface contamination is substantial and varies widely.25 The potential risk from 

exposure to droplet contamination requires as much attention in a risk 

assessment as the respirable component of aerosols.
Biological Risk Assessment 15

Technique can significantly impact aerosol output and dose. The worker 

who is careful and proficient will minimize the generation of aerosols. A careless 

and hurried worker will substantially increase the aerosol hazard. For example, 

the hurried worker may operate a sonic homogenizer with maximum aeration 

whereas the careful worker will consistently operate the device to assure minimal 

aeration. Experiments show that the aerosol burden with maximal aeration is 

approximately 200 times greater than aerosol burden with minimal aeration.22

Similar results were shown for pipetting with bubbles and with minimal bubbles. 

Containment and good laboratory practices also reduce this risk.

Potential Hazards Associated with Work Practices, Safety Equipment 

and Facility Safeguards

Workers are the first line of defense for protecting themselves, others in the 

laboratory, and the public from exposure to hazardous agents. Protection depends 

on the conscientious and proficient use of good microbiological practices and the 

correct use of safety equipment. A risk assessment should identify any potential 

deficiencies in the practices of the laboratory workers. Carelessness is the most 

serious concern, because it can compromise any safeguards of the laboratory 

and increase the risk for coworkers. Training, experience, knowledge of the agent 

and procedure hazards, good habits, caution, attentiveness, and concern for the 

health of coworkers are prerequisites for a laboratory staff in order to reduce the 

inherent risks that attend work with hazardous agents. Not all workers who join a 

laboratory staff will have these prerequisite traits even though they may possess 

excellent scientific credentials. Laboratory directors or principal investigators 

should train and retrain new staff to the point where aseptic techniques and 

safety precautions become second nature.26

There may be hazards that require specialized personal protective 

equipment in addition to safety glasses, laboratory gowns, and gloves. For 

example, a procedure that presents a splash hazard may require the use of a 

mask and a face shield to provide adequate protection. Inadequate training in 

the proper use of personal protective equipment may reduce its effectiveness, 

provide a false sense of security, and could increase the risk to the laboratory 

worker. For example, a respirator may impart a risk to the wearer independent 

of the agents being manipulated.

Safety equipment such as biological safety cabinets (BSC), centrifuge safety 

cups, and sealed rotors are used to provide a high degree of protection for the 

laboratory worker from exposure to microbial aerosols and droplets. Safety 

equipment that is not working properly is hazardous, especially when the user is 

unaware of the malfunction. Poor location, room air currents, decreased airflow, 

leaking filters, raised sashes, crowded work surfaces, and poor user technique 

compromise the containment capability of a BSC. The safety characteristics 

of modern centrifuges are only effective if the equipment is operated properly. 

Training in the correct use of equipment, proper procedure, routine inspections 
16 Biosafety in Microbiological and Biomedical Laboratories

and potential malfunctions, and periodic re-certification of equipment, as needed, 

is essential.

Facility safeguards help prevent the accidental release of an agent from the 

laboratory. Their use is particularly important at BSL-3 and BSL-4 because the 

agents assigned to those levels can transmit disease by the inhalation route or can 

cause life-threatening disease. For example, one facility safeguard is directional 

airflow. This safeguard helps to prevent aerosol transmission from a laboratory 

into other areas of the building. Directional airflow is dependent on the operational 

integrity of the laboratory’s heating, ventilation, and air conditioning (HVAC) 

system. HVAC systems require careful monitoring and periodic maintenance 

to sustain operational integrity. Loss of directional airflow compromises safe 

laboratory operation. BSL-4 containment facilities provide more complex 

safeguards that require significant expertise to design and operate.

Consideration of facility safeguards is an integral part of the risk assessments. 

A biological safety professional, building and facilities staff, and the IBC should 

help assess the facility’s capability to provide appropriate protection for the 

planned work, and recommend changes as necessary. Risk assessment may 

support the need to include additional facility safeguards in the construction of 

new or renovation of old BSL-3 facilities.

An Approach to Assess Risks and Select Appropriate Safeguards

Biological risk assessment is a subjective process requiring consideration of 

many hazardous characteristics of agents and procedures, with judgments 

based often on incomplete information. There is no standard approach for 

conducting a biological risk assessment, but some structure can be helpful 

in guiding the process. This section describes a five-step approach that gives 

structure to the risk assessment process.

First, identify agent hazards and perform an initial assessment of risk. 

Consider the principal hazardous characteristics of the agent, which include its 

capability to infect and cause disease in a susceptible human host, severity of 

disease, and the availability of preventive measures and effective treatments.

Several excellent resources provide information and guidance for making an 

initial risk assessment. The BMBL provides agent summary statements for some 

agents that are associated with LAIs or are of increased public concern. Agent 

summary statements also identify known and suspected routes of transmission 

of laboratory infection and, when available, information on infective dose, host 

range, agent stability in the environment, protective immunizations, and 

attenuated strains of the agent.

A thorough examination of the agent hazards is necessary when the 

intended use of an agent does not correspond with the general conditions 

described in the summary statement or when an agent summary statement is 
Biological Risk Assessment 17

not available. Although a summary statement for one agent may provide helpful 

information for assessing the risk of a similar agent, it should not serve as the 

primary resource for making the risk determination for that agent. Refer to other 

resources for guidance in identifying the agent hazards.

The Control of Communicable Diseases Manual provides information on 

communicable diseases including concise summaries on severity, mode of 

transmission, and the susceptibility and resistance of humans to disease.3

 In 

addition, it is always helpful to seek guidance from colleagues with experience 

in handling the agent and from biological safety professionals.

Often there is not sufficient information to make an appropriate assessment 

of risk. For example, the hazard of an unknown agent that may be present in a 

diagnostic specimen will be unknown until after completing agent identification 

and typing procedures. It would be prudent in this case to assume the specimen 

contains an agent presenting the hazardous classification that correlates with 

BSL-2, unless additional information suggests the presence of an agent of higher 

risk. Identification of agent hazards associated with newly emergent pathogens 

also requires judgments based on incomplete information. Consult interim 

biosafety guidelines prepared by the CDC and the WHO for risk assessment 

guidance. When assessing the hazards of a newly attenuated pathogen, 

experimental data should support a judgment that the attenuated pathogen 

is less hazardous than the wild-type parent pathogen before making any 

reduction in the containment recommended for that pathogen.

Make a preliminary determination of the biosafety level that best correlates 

with the initial risk assessment based on the identification and evaluation of the 

agent hazards. Remember that aerosol and droplet routes of agent transmission 

also are important considerations in specification of safety equipment and facility 

design that result in a given BSL level.

Second, identify laboratory procedure hazards. The principal laboratory 

procedure hazards are agent concentration, suspension volume, equipment and 

procedures that generate small particle aerosols and larger airborne particles 

(droplets), and use of sharps. Procedures involving animals can present a 

number of hazards such as bites and scratches, exposure to zoonotic agents, 

and the handling of experimentally generated infectious aerosols.

The complexity of a laboratory procedure can also present a hazard. 

The agent summary statement provides information on the primary laboratory 

hazards associated with typically routine procedures used in handling an agent. 

In proposed laboratory procedures where the procedure hazards differ from the 

general conditions of the agent summary statement or where an agent summary 

statement is not available, the risk assessment should identify specific hazards 

associated with the procedures.
18 Biosafety in Microbiological and Biomedical Laboratories

Third, make a determination of the appropriate biosafety level and 

select additional precautions indicated by the risk assessment. The 

selection of the appropriate biosafety level and the selection of any additional 

laboratory precautions require a comprehensive understanding of the practices, 

safety equipment, and facility safeguards described in Sections III, IV, and V of 

this publication.

There will be situations where the intended use of an agent requires greater 

precautions than those described in the agent’s Summary Statement. These 

situations will require the careful selection of additional precautions. An obvious 

example would be a procedure for exposing animals to experimentally generated 

infectious aerosols.

It is unlikely that a risk assessment would indicate a need to alter the 

recommended facility safeguards specified for the selected biosafety level. If this 

does occur, however, it is important that a biological safety professional validate 

this judgment independently before augmenting any facility secondary barrier.

It is also important to recognize that individuals in the laboratory may differ in 

their susceptibility to disease. Pre-existing diseases, medications, compromised 

immunity, and pregnancy or breast-feeding that may increase exposure to infants 

to certain agents, are some of the conditions that may increase the risk of an 

individual for acquiring a LAI. Consultation with an occupational physician 

knowledgeable in infectious diseases is advisable in these circumstances.

Fourth, evaluate the proficiencies of staff regarding safe practices and 

the integrity of safety equipment. The protection of laboratory workers, other 

persons associated with the laboratory, and the public will depend ultimately on the 

laboratory workers themselves. In conducting a risk assessment, the laboratory 

director or principal investigator should ensure that laboratory workers have 

acquired the technical proficiency in the use of microbiological practices and 

safety equipment required for the safe handling of the agent, and have developed 

good habits that sustain excellence in the performance of those practices. An 

evaluation of a person’s training, experience in handling infectious agents, 

proficiency in the use of sterile techniques and BSCs, ability to respond to 

emergencies, and willingness to accept responsibility for protecting one’s 

self and others is important insurance that a laboratory worker is capable 

of working safely.

The laboratory director or principal investigator should also ensure that the 

necessary safety equipment is available and operating properly. For example, a 

BSC that is not certified represents a potentially serious hazard to the laboratory 

worker using it and to others in the laboratory. The director should have all 

equipment deficiencies corrected before starting work with an agent.
Biological Risk Assessment 19

Fifth, review the risk assessment with a biosafety professional, 

subject matter expert, and the IBC. A review of the risk assessment and 

selected safeguards by knowledgeable individuals is always beneficial and 

sometimes required by regulatory or funding agencies, as is the case with 

the NIH Guidelines.

2

 Review of potentially high risk protocols by the local IBC 

should become standard practice. Adopting this step voluntarily will promote 

the use of safe practices in work with hazardous agents in microbiological 

and biomedical laboratories.

Conclusion

Risk assessment is the basis for the safeguards developed by the CDC, the NIH, 

and the microbiological and biomedical community to protect the health of 

laboratory workers and the public from the risks associated with the use of 

hazardous biological agents in laboratories. Experience shows that these 

established safe practices, equipment, and facility safeguards work.

New knowledge and experience may justify altering these safeguards. Risk 

assessment, however, must be the basis for recommended change. Assessments 

conducted by laboratory directors and principal investigators for the use of 

emergent agents and the conduct of novel experiments will contribute to our 

understanding of the risks these endeavors may present and the means for 

their control. Those risk assessments will likely mirror progress in science and 

technology and serve as the basis for future revisions of BMBL.

References

1. World Health Organization. Laboratory biosafety manual. 3rd ed. Geneva; 2004.

2. The National Institutes of Health (US), Office of Biotechnology Activities. NIH 

guidelines for research involving recombinant DNA molecules. Bethesda; 

2002, April.

3. American Public Health Association. Control of communicable diseases 

manual. 18th ed. DL Heymann, editor. Washington, DC; 2005.

4. Lennette EH, Koprowski H. Human infection with Venezuelan equine 

encephalomyelitis virus. JAMA. 1943;123:1088-95.

5. Tigertt WD, Benenson AS, Gochenour WS. Airborne Q fever. Bacteriol Rev. 

1961;25:285-93.

6. Centers for Disease Control and Prevention. Fatal Cercopithecine 

herpesvirus 1 (B virus) infection following a mucocutaneous exposure and 

interim recommendations for worker protection. MMWR Morb Mortal Wkly 

Rep. 1998;47:1073-6,1083.

7. Centers for Disease Control and Prevention. Laboratory management of 

agents associated with hantavirus pulmonary syndrome: interim biosafety 

guidelines. MMWR Recomm Rep. 1994;43:1-7.

8. Wedum AG, Barkley WE, Hellman A. Handling of infectious agents. 

J Am Vet Med Assoc. 1972;161:1557-67.
20 Biosafety in Microbiological and Biomedical Laboratories

9. Jackson RJ, Ramsay AJ, Christensen CD, et al. Expression of mouse 

interleukin-4 by a recombinant ectromelia virus suppresses cytolytic 

lymphocyte responses and overcomes genetic resistance to mousepox. 

J Virol. 2001;75:1205-10.

10. Shimono N, Morici L, Casali N, et al. Hypervirulent mutant of Mycobacterium 

tuberculosis resulting from disruption of the mce1 operon. Proc Natl Acad 

Sci U S A. 2003;100:15918-23.

11. Cunningham ML, Titus RG, Turco SJ, et al. Regulation of differentiation to 

the infective stage of the protozoan parasite Leishmania major by 

tetrahydrobiopterin. Science. 2001;292:285-7.

12. Kobasa D, Takada A, Shinya K, et al. Enhanced virulence of influenza 

A viruses with the haemagglutinin of the 1918 pandemic virus. Nature. 

2004;431:703-7.

13. Efstathiou S, Preston CM. Towards an understanding of the molecular 

basis of herpes simplex virus latency. Virus Res. 2005;111:108-19.

14. Oxman MN, Levin MJ, Johnson GR, et al. A vaccine to prevent herpes zoster 

and postherpetic neuralgia in older adults. N Engl J Med. 2005;352:2271-84.

15. Centers for Disease Control and Prevention. Update: Investigation of rabies 

infections in organ donor and transplant recipients—Alabama, Arkansas, 

Oklahoma, and Texas. MMWR Morb Mortal Wkly Rep. 2004;53:615-16.

16. Centers for Disease Control and Prevention. Lymphocytic choriomeningitis 

virus infection in organ transplant recipients—Massachusetts, Rhode Island, 

2005. MMWR Morb Mortal Wkly Rep. 2005;54:537-39.

17. US Department of Labor. Occupational exposure to bloodborne pathogens. 

Final Rule. Standard interpretations: applicability of 1910.1030 to established 

human cell lines. June 24, 1994.

18. Gartner HV, Seidl C, Luckenbach C, et al. Genetic analysis of a sarcoma 

accidentally transplanted from a patient to a surgeon. N Engl J Med. 

1996;335:1494-7.

19. Dykewicz CA, Dato VM, Fisher-Hoch SP, et al. Lymphocytic choriomeningitis 

outbreak associated with nude mice in a research institute. JAMA. 

1992;267:1349-53.

20. Pike RM. Laboratory-associated infections: incidence, fatalities, causes, 

and prevention. Annu Rev Microbiol. 1979;33:41-66.

21. Harding AL, Byers KB. Epidemiology of laboratory-associated infections. 

In: Fleming DO, Hunt DL, editors. Biological safety: principles and practices. 

3rd ed. Washington, DC: ASM Press; 2000:35-54.

22. Dimmick RL, Fogl WF, Chatigny MA. Potential for accidental microbial 

aerosol transmission in the biology laboratory. In: Hellman A, Oxman MN, 

Pollack R, editors. Biohazards in biological research. Proceedings of a 

conference held at the Asilomar conference center; 1973 Jan 22-24; Pacific 

Grove, CA. New York: Cold Spring Harbor Laboratory; 1973. p. 246-66.
Biological Risk Assessment 21

23. Kenny MT, Sable FL. Particle size distribution of Serratia marcescens 

aerosols created during common laboratory procedures and simulated 

laboratory accidents. Appl Microbiol. 1968;16:1146-50.

24. Chatigny MA, Barkley WE, Vogl WF. Aerosol biohazard in microbiological 

laboratories and how it is affected by air conditioning systems. ASHRAE 

Transactions. 1974;80(Pt 1):463-469.

25. Chatigny MA, Hatch MT, Wolochow H, et al. Studies on release and survival 

of biological substances used in recombinant DNA laboratory procedures. 

National Institutes of Health Recombinant DNA Technical Bulletin. 1979.

26. Lennette EH. Panel V common sense in the laboratory: recommendations 

and priorities. Biohazards in biological research. Proceedings of a 

conference held at the Asilomar conference center; 1973 Jan 22-24; Pacific 

Grove, CA. New York: Cold Spring Harbor Laboratory; 1973. p. 353.
22 Biosafety in Microbiological and Biomedical Laboratories

Section III—Principles of Biosafety

A fundamental objective of any biosafety program is the containment of potentially 

harmful biological agents. The term “containment” is used in describing safe 

methods, facilities and equipment for managing infectious materials in the 

laboratory environment where they are being handled or maintained. The 

purpose of containment is to reduce or eliminate exposure of laboratory workers, 

other persons, and the outside environment to potentially hazardous agents. 

The use of vaccines may provide an increased level of personal protection. 

The risk assessment of the work to be done with a specific agent will determine 

the appropriate combination of these elements.

Laboratory Practices and Technique

The most important element of containment is strict adherence to standard 

microbiological practices and techniques. Persons working with infectious agents 

or potentially infected materials must be aware of potential hazards, and must be 

trained and proficient in the practices and techniques required for handling such 

material safely. The director or person in charge of the laboratory is responsible 

for providing or arranging the appropriate training of personnel.

Each laboratory should develop or adopt a biosafety or operations manual 

that identifies the hazards that will or may be encountered, and that specifies 

practices and procedures designed to minimize or eliminate exposures to these 

hazards. Personnel should be advised of special hazards and should be required 

to read and follow the required practices and procedures. A scientist, trained and 

knowledgeable in appropriate laboratory techniques, safety procedures, and 

hazards associated with handling infectious agents must be responsible for the 

conduct of work with any infectious agents or materials. This individual should 

consult with biosafety or other health and safety professionals with regard to 

risk assessment.

When standard laboratory practices are not sufficient to control the hazards 

associated with a particular agent or laboratory procedure, additional measures 

may be needed. The laboratory director is responsible for selecting additional 

safety practices, which must be in keeping with the hazards associated with the 

agent or procedure.

Appropriate facility design and engineering features, safety equipment, and 

management practices must supplement laboratory personnel, safety practices, 

and techniques.

Safety Equipment (Primary Barriers and Personal Protective Equipment)

Safety equipment includes BSCs, enclosed containers, and other engineering 

controls designed to remove or minimize exposures to hazardous biological 

materials. The BSC is the principal device used to provide containment of 
Principles of Biosafety 23

infectious droplets or aerosols generated by many microbiological procedures. 

Three types of BSCs (Class I, II, III) used in microbiological laboratories are 

described and illustrated in Appendix A. Open-fronted Class I and Class II BSCs 

are primary barriers that offer significant levels of protection to laboratory personnel 

and to the environment when used with good microbiological techniques. The Class 

II biological safety cabinet also provides protection from external contamination 

of the materials (e.g., cell cultures, microbiological stocks) being manipulated 

inside the cabinet. The gas-tight Class III biological safety cabinet provides the 

highest attainable level of protection to personnel and the environment.

An example of another primary barrier is the safety centrifuge cup, an 

enclosed container designed to prevent aerosols from being released during 

centrifugation. To minimize aerosol hazards, containment controls such as 

BSCs or centrifuge cups must be used when handling infectious agents.

Safety equipment also may include items for personal protection, such 

as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety 

glasses, or goggles. Personal protective equipment is often used in combination 

with BSCs and other devices that contain the agents, animals, or materials being 

handled. In some situations in which it is impractical to work in BSCs, personal 

protective equipment may form the primary barrier between personnel and the 

infectious materials. Examples include certain animal studies, animal necropsy, 

agent production activities, and activities relating to maintenance, service, or 

support of the laboratory facility.

Facility Design and Construction (Secondary Barriers)

The design and construction of the facility contributes to the laboratory workers’

protection, provides a barrier to protect persons outside the laboratory, and 

protects persons or animals in the community from infectious agents that may be 

accidentally released from the laboratory. Laboratory directors are responsible 

for providing facilities commensurate with the laboratory’s function and the 

recommended biosafety level for the agents being manipulated.

The recommended secondary barrier(s) will depend on the risk of transmission 

of specific agents. For example, the exposure risks for most laboratory work in 

BSL-1 and BSL-2 facilities will be direct contact with the agents, or inadvertent 

contact exposures through contaminated work environments. Secondary barriers 

in these laboratories may include separation of the laboratory work area from 

public access, availability of a decontamination facility (e.g., autoclave), and 

hand washing facilities.

When the risk of infection by exposure to an infectious aerosol is present, 

higher levels of primary containment and multiple secondary barriers may 

become necessary to prevent infectious agents from escaping into the 

environment. Such design features include specialized ventilation systems to 
24 Biosafety in Microbiological and Biomedical Laboratories

ensure directional airflow, air treatment systems to decontaminate or remove 

agents from exhaust air, controlled access zones, airlocks at laboratory entrances, 

or separate buildings or modules to isolate the laboratory. Design engineers for 

laboratories may refer to specific ventilation recommendations as found in the 

ASHRAE Laboratory Design Guide published by the American Society of 

Heating, Refrigerating, and Air-Conditioning Engineers (ASHRAE).1

Biosafety Levels

Four BSLs are described in Section 4, which consist of combinations of 

laboratory practices and techniques, safety equipment, and laboratory facilities. 

Each combination is specifically appropriate for the operations performed, the 

documented or suspected routes of transmission of the infectious agents, and 

the laboratory function or activity. The BSLs described in this manual should be 

differentiated from Risk Groups, as described in the NIH Guidelines and the 

World Health Organization Laboratory Biosafety Manual. Risk groups are the 

result of a classification of microbiological agents based on their association with, 

and resulting severity of, disease in humans. The risk group of an agent should 

be one factor considered in association with mode of transmission, procedural 

protocols, experience of staff, and other factors in determining the BSL in which 

the work will be conducted.

The recommended biosafety level(s) for the organisms in Section VIII 

(Agent Summary Statements) represent those conditions under which the agent 

ordinarily can be safely handled. Of course, not all of the organisms capable of 

causing disease are included in Section VIII and an institution must be prepared 

to perform risk assessments for these agents using the best available information. 

Detailed information regarding the conduct of biological risk assessments can be 

found in Section II. The laboratory director is specifically and primarily responsible 

for assessing the risks and applying the appropriate biosafety levels. The 

institution’s Biological Safety Officer (BSO) and IBC can be of great assistance 

in performing and reviewing the required risk assessment. At one point, under 

the NIH Guidelines, BSOs were required only when large-scale research or 

production of organisms containing recombinant DNA molecules was performed 

or when work with recombinant DNA molecules was conducted at BSL-3 or 

above. IBCs were required only when an institution was performing non-exempt 

recombinant DNA experiments. Today, however, it is strongly suggested that an 

institution conducting research or otherwise working with pathogenic agents have 

a BSO and properly constituted and functioning IBC. The responsibilities of each 

now extend beyond those described in the NIH Guidelines and depend on the 

size and complexity of the program.

Generally, work with known agents should be conducted at the biosafety 

level recommended in Section VIII. When information is available to suggest that 

virulence, pathogenicity, antibiotic resistance patterns, vaccine and treatment 

availability, or other factors are significantly altered, more (or less) stringent 
Principles of Biosafety 25

practices may be specified. Often an increased volume or a high concentration 

of agent may require additional containment practices.

Biosafety Level 1 practices, safety equipment, and facility design and 

construction are appropriate for undergraduate and secondary educational 

training and teaching laboratories, and for other laboratories in which work is 

done with defined and characterized strains of viable microorganisms not known 

to consistently cause disease in healthy adult humans. Bacillus subtilis, Nigeria 

gruberi, infectious canine hepatitis virus, and exempt organisms under the NIH 

Guidelines are representative of microorganisms meeting these criteria. Many 

agents not ordinarily associated with disease processes in humans are, however, 

opportunistic pathogens and may cause infection in the young, the aged, and 

immunodeficient or immunosuppressed individuals. Vaccine strains that have 

undergone multiple in vivo passages should not be considered avirulent simply 

because they are vaccine strains.

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.

Biosafety Level 2 practices, equipment, and facility design and construction 

are applicable to clinical, diagnostic, teaching, and other laboratories in which 

work is done with the broad spectrum of indigenous moderate-risk agents that 

are present in the community and associated with human disease of varying 

severity. With good microbiological techniques, these agents can be used safely 

in activities conducted on the open bench, provided the potential for producing 

splashes or aerosols is low. Hepatitis B virus, HIV, the Salmonella, and 

Toxoplasma are representative of microorganisms assigned to this containment 

level. BSL-2 is appropriate when work is done with any human-derived blood, 

body fluids, tissues, or primary human cell lines where the presence of an 

infectious agent may be unknown. (Laboratory personnel working with humanderived materials should refer to the OSHA Bloodborne Pathogen Standard2

 for 

specific required precautions).

Primary hazards to personnel working with these agents relate to accidental 

percutaneous or mucous membrane exposures, or ingestion of infectious 

materials. Extreme caution should be taken with contaminated needles or sharp 

instruments. Even though organisms routinely manipulated at BSL-2 are not 

known to be transmissible by the aerosol route, procedures with aerosol or high 

splash potential that may increase the risk of such personnel exposure must be 

conducted in primary containment equipment, or in devices such as a BSC or 

safety centrifuge cups. Personal protective equipment should be used as 

appropriate, such as splash shields, face protection, gowns, and gloves. 

Secondary barriers, such as hand washing sinks and waste decontamination 

facilities, must be available to reduce potential environmental contamination.
26 Biosafety in Microbiological and Biomedical Laboratories

Biosafety Level 3 practices, safety equipment, and facility design and 

construction are applicable to clinical, diagnostic, teaching, research, or 

production facilities in which work is done with indigenous or exotic agents 

with a potential for respiratory transmission, and which may cause serious and 

potentially lethal infection. Mycobacterium tuberculosis, St. Louis encephalitis 

virus, and Coxiella burnetii are representative of the microorganisms assigned 

to this level. Primary hazards to personnel working with these agents relate to 

autoinoculation, ingestion, and exposure to infectious aerosols. At BSL-3, more 

emphasis is placed on primary and secondary barriers to protect personnel in 

contiguous areas, the community, and the environment from exposure to 

potentially infectious aerosols. For example, all laboratory manipulations should 

be performed in a BSC or other enclosed equipment, such as a gas-tight aerosol 

generation chamber. Secondary barriers for this level include controlled access 

to the laboratory and ventilation requirements that minimize the release of 

infectious aerosols from the laboratory.

Biosafety Level 4 practices, safety equipment, and facility design and 

construction are applicable for work with dangerous and exotic agents that pose 

a high individual risk of life-threatening disease, which may be transmitted via the 

aerosol route and for which there is no available vaccine or therapy. Agents with 

a close or identical antigenic relationship to BSL-4 agents also should be handled 

at this level. When sufficient data are obtained, work with these agents may 

continue at this level or at a lower level. Viruses such as Marburg or CongoCrimean hemorrhagic fever are manipulated at BSL-4.

The primary hazards to personnel working with BSL-4 agents are respiratory 

exposure to infectious aerosols, mucous membrane or broken skin exposure to 

infectious droplets, and autoinoculation. All manipulations of potentially infectious 

diagnostic materials, isolates, and naturally or experimentally infected animals, 

pose a high risk of exposure and infection to laboratory personnel, the 

community, and the environment.

The laboratory worker’s complete isolation from aerosolized infectious 

materials is accomplished primarily by working in a Class III BSC or in a full-body, 

air-supplied positive-pressure personnel suit. The BSL-4 facility itself is generally 

a separate building or completely isolated zone with complex, specialized 

ventilation requirements and waste management systems to prevent release of 

viable agents to the environment.

The laboratory director is specifically and primarily responsible for the safe 

operation of the laboratory. His/her knowledge and judgment are critical in assessing 

risks and appropriately applying these recommendations. The recommended 

biosafety level represents those conditions under which the agent can ordinarily 

be safely handled. Special characteristics of the agents used, the training and 

experience of personnel, procedures being conducted and the nature or function of 

the laboratory may further influence the director in applying these recommendations.
Principles of Biosafety 27

Animal Facilities

Four standard biosafety levels are also described for activities involving infectious 

disease work with commonly used experimental animals. These four combinations 

of practices, safety equipment, and facilities are designated Animal Biosafety 

Levels 1, 2, 3, and 4, and provide increasing levels of protection to personnel 

and the environment.

One additional biosafety level, designated BSL-3-Agriculture (or BSL 3-Ag) 

addresses activities involving large or loose-housed animals and/or studies involving 

agents designated as High Consequence Pathogens by the USDA. BSL 3-Ag 

laboratories are designed so that the laboratory facility itself acts as a primary 

barrier to prevent release of infectious agents into the environment. More information 

on the design and operation of BSL 3-Ag facilities and USDA High Consequence 

Pathogens is provided in Appendix D.

Clinical Laboratories

Clinical laboratories, especially those in health care facilities, receive clinical 

specimens with requests for a variety of diagnostic and clinical support services. 

Typically, the infectious nature of clinical material is unknown, and specimens are 

often submitted with a broad request for microbiological examination for multiple 

agents (e.g., sputa submitted for “routine,” acid-fast, and fungal cultures). It is 

the responsibility of the laboratory director to establish standard procedures in 

the laboratory that realistically address the issue of the infective hazard of 

clinical specimens.

Except in extraordinary circumstances (e.g., suspected hemorrhagic fever), 

the initial processing of clinical specimens and serological identification of isolates 

can be done safely at BSL-2, the recommended level for work with bloodborne 

pathogens such as HBV and HIV. The containment elements described in BSL-2 

are consistent with the OSHA standard, “Occupational Exposure to Bloodborne 

Pathogens.2,3 This requires the use of specific precautions with all clinical 

specimens of blood or other potentially infectious material (Universal or Standard* 

Precautions).4,5 Additionally, other recommendations specific for clinical laboratories 

may be obtained from the Clinical Laboratory Standards Institute (formerly known 

as the National Committee for Clinical Laboratory Standards).6

BSL-2 recommendations and OSHA requirements focus on the prevention 

of percutaneous and mucous membrane exposures to clinical material. Primary 

barriers such as BSCs (Class I or II) should be used when performing procedures 

that might cause splashing, spraying, or splattering of droplets. Biological safety 

cabinets also should be used for the initial processing of clinical specimens when 

the nature of the test requested or other information suggests the likely presence 

of an agent readily transmissible by infectious aerosols (e.g., M. tuberculosis), or 

when the use of a BSC (Class II) is indicated to protect the integrity of the specimen.
28 Biosafety in Microbiological and Biomedical Laboratories

The segregation of clinical laboratory functions and limited or restricted 

access to such areas is the responsibility of the laboratory director. It is also the 

director’s responsibility to establish standard, written procedures that address 

the potential hazards and the required precautions to be implemented.

Importation and Interstate Shipment of Certain Biomedical Materials

The importation of etiologic agents and vectors of human diseases is subject to 

the requirements of the Public Health Service Foreign Quarantine regulations. 

Companion regulations of the Public Health Service and the Department of 

Transportation specify packaging, labeling, and shipping requirements for 

etiologic agents and diagnostic specimens shipped in interstate commerce. 

(See Appendix C.)

The USDA regulates the importation and interstate shipment of animal 

pathogens and prohibits the importation, possession, or use of certain exotic 

animal disease agents that pose a serious disease threat to domestic livestock 

and poultry. (See Appendix F.)

Select Agents

In recent years, with the passing of federal legislation regulating the possession, 

use, and transfer of agents with high adverse public health and/or agricultural 

consequences (DHHS and USDA Select Agents), much greater emphasis has 

been placed in the emerging field of biosecurity. Biosecurity and select agent 

issues are covered in detail in Section 6 and Appendix F of this document. In 

contrast with biosafety, a field dedicated to the protection of workers and the 

environment from exposures to infectious materials, the field of biosecurity 

prevents loss of valuable research materials and limits access to infectious 

materials by individuals who would use them for harmful purposes. Nevertheless, 

adequate containment of biological materials is a fundamental program 

component for both biosafety and biosecurity.

References

1. McIntosh IBD, Morgan CB, Dorgan CE. ASHRAE laboratory design guide. 

Atlanta: American Society of Heating, Refrigerating and Air-Conditioning 

Engineers, Inc; 2001.

2. U.S. Department of Labor. Occupational exposure to bloodborne pathogens. 

Final Rule. 29 CFR. 1910.1030 (1991).

3. Richmond JY. HIV biosafety: guidelines and regulations. In: Schochetman G, 

George JR, editors. AIDS testing. 2nd ed. New York: Springer-Verlag; 1994. 

p. 346-60.

4. Centers for Disease Control and Prevention. Update: universal precautions 

for prevention of transmission of human immunodeficiency virus, hepatitis 
Principles of Biosafety 29

B virus and other bloodborne pathogens in health-care settings. MMWR 

Morb Mortal Wkly Rep. 1988;37:377-82,387-8.

5. Garner JS. Guideline for isolation precautions in hospitals. Infect Control 

Hosp Epidemiol. 1996;17:53-80.

6. National Committee for Clinical Laboratory Standards. Protection of 

laboratory workers from occupationally-acquired infections; approved 

guideline, 3rd ed.

* In 1996 the United States Hospital Infection Control Practices Advisory Committee 

introduced a new set of guidelines, “Standard Precautions,” to synthesize the major 

features of Universal Precautions (blood and body fluid) with Body Substance Isolation 

Precautions (designed to reduce the risk of transmission of pathogens from moist body 

substances).6 Standard Precautions apply to 1) blood; 2) all body fluids, secretions, 

and excretions except sweat, regardless of whether or not they contain visible blood; 

3) non-intact skin; and 4) mucous membranes. For additional information on Standard 

Precautions, see reference 6 or the CDC Web site: www.cdc.gov. 
30 Biosafety in Microbiological and Biomedical Laboratories

Section IV—Laboratory Biosafety Level Criteria

The essential elements of the four biosafety levels for activities involving 

infectious microorganisms and laboratory animals are summarized in Table 2 of 

this section and discussed in Section 2. The levels are designated in ascending 

order, by degree of protection provided to personnel, the environment, and the 

community. Standard microbiological practices are common to all laboratories. 

Special microbiological practices enhance worker safety, environmental protection, 

and address the risk of handling agents requiring increasing levels of containment.

Biosafety Level 1

Biosafety Level 1 is suitable for work involving well-characterized agents not 

known to consistently cause disease in immunocompetent adult humans, and 

present minimal potential hazard to laboratory personnel and the environment. 

BSL-1 laboratories are not necessarily separated from the general traffic patterns 

in the building. Work is typically conducted on open bench tops using standard 

microbiological practices. Special containment equipment or facility design is 

not required, but may be used as determined by appropriate risk assessment. 

Laboratory personnel must have specific training in the procedures conducted in 

the laboratory and must be supervised by a scientist with training in microbiology 

or a related science.

The following standard practices, safety equipment, and facility requirements 

apply to BSL-1.

A. Standard Microbiological Practices

1. The laboratory supervisor must enforce the institutional policies that 

control access to the laboratory.

2. Persons must wash their hands after working with potentially hazardous 

materials and before leaving the laboratory.

3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, 

and storing food for human consumption must not be permitted in 

laboratory areas. Food must be stored outside the laboratory area in 

cabinets or refrigerators designated and used for this purpose.

4. Mouth pipetting is prohibited; mechanical pipetting devices must be used.

5. Policies for the safe handling of sharps, such as needles, scalpels, 

pipettes, and broken glassware must be developed and implemented. 

Whenever practical, laboratory supervisors should adopt improved 

engineering and work practice controls that reduce risk of sharps 

injuries.Precautions, including those listed below, must always be taken 

with sharp items. These include:
Laboratory Biosafety Level Criteria: BSL-1 31

a. Careful management of needles and other sharps are of primary 

importance. Needles must not be bent, sheared, broken, recapped, 

removed from disposable syringes, or otherwise manipulated by 

hand before disposal.

b. Used disposable needles and syringes must be carefully placed 

in conveniently located puncture-resistant containers used for 

sharps disposal.

c. Non-disposable sharps must be placed in a hard walled container 

for transport to a processing area for decontamination, preferably 

by autoclaving.

d. Broken glassware must not be handled directly. Instead, it must 

be removed using a brush and dustpan, tongs, or forceps. Plastic 

ware should be substituted for glassware whenever possible.

6. Perform all procedures to minimize the creation of splashes 

and/or aerosols.

7. Decontaminate work surfaces after completion of work and after 

any spill or splash of potentially infectious material with appropriate 

disinfectant.

8. Decontaminate all cultures, stocks, and other potentially infectious 

materials before disposal using an effective method. Depending on 

where the decontamination will be performed, the following methods 

should be used prior to transport.

a. Materials to be decontaminated outside of the immediate 

laboratory must be placed in a durable, leak proof container 

and secured for transport.

b. Materials to be removed from the facility for decontamination 

must be packed in accordance with applicable local, state, and 

federal regulations.

9. A sign incorporating the universal biohazard symbol must be posted 

at the entrance to the laboratory when infectious agents are present. 

The sign may include the name of the agent(s) in use, and the name 

and phone number of the laboratory supervisor or other responsible 

personnel. Agent information should be posted in accordance with 

the institutional policy.

10. An effective integrated pest management program is required. 

(See Appendix G.)
32 Biosafety in Microbiological and Biomedical Laboratories

11. The laboratory supervisor must ensure that laboratory personnel receive 

appropriate training regarding their duties, the necessary precautions 

to prevent exposures, and exposure evaluation procedures. Personnel 

must receive annual updates or additional training when procedural or 

policy changes occur. Personal health status may impact an individual’s 

susceptibility to infection, ability to receive immunizations or prophylactic 

interventions. Therefore, all laboratory personnel and particularly women 

of childbearing age should be provided with information regarding 

immune competence and conditions that may predispose them to 

infection. Individuals having these conditions should be encouraged 

to self-identify to the institution’s healthcare provider for appropriate 

counseling and guidance.

B. Special Practices

None required.

C. Safety Equipment (Primary Barriers and Personal Protective Equipment)

1. Special containment devices or equipment, such as BSCs, are not 

generally required.

2. Protective laboratory coats, gowns, or uniforms are recommended to 

prevent contamination of personal clothing.

3. Wear protective eyewear when conducting procedures that have the 

potential to create splashes of microorganisms or other hazardous 

materials. Persons who wear contact lenses in laboratories should 

also wear eye protection.

4. Gloves must be worn to protect hands from exposure to hazardous 

materials. Glove selection should be based on an appropriate risk 

assessment. Alternatives to latex gloves should be available. Wash 

hands prior to leaving the laboratory. In addition, BSL-1 workers should:

a. Change gloves when contaminated, glove integrity is compromised, 

or when otherwise necessary.

b. Remove gloves and wash hands when work with hazardous 

materials has been completed and before leaving the laboratory.

c. Do not wash or reuse disposable gloves. Dispose of used gloves 

with other contaminated laboratory waste. Hand washing protocols 

must be rigorously followed.
Laboratory Biosafety Level Criteria: BSL-2 33

D. Laboratory Facilities (Secondary Barriers)

1. Laboratories should have doors for access control.

2. Laboratories must have a sink for hand washing.

3. The laboratory should be designed so that it can be easily cleaned. 

Carpets and rugs in laboratories are not appropriate.

4. Laboratory furniture must be capable of supporting anticipated loads 

and uses. Spaces between benches, cabinets, and equipment should 

be accessible for cleaning.

a. Bench tops must be impervious to water and resistant to heat, 

organic solvents, acids, alkalis, and other chemicals.

b. Chairs used in laboratory work must be covered with a non-porous 

material that can be easily cleaned and decontaminated with 

appropriate disinfectant.

5. Laboratories windows that open to the exterior should be fitted with screens.

Biosafety Level 2

Biosafety Level 2 builds upon BSL-1. BSL-2 is suitable for work involving agents 

that pose moderate hazards to personnel and the environment. It differs from 

BSL-1 in that: 1) laboratory personnel have specific training in handling 

pathogenic agents and are supervised by scientists competent in handling 

infectious agents and associated procedures; 2) access to the laboratory is 

restricted when work is being conducted; and 3) all procedures in which 

infectious aerosols or splashes may be created are conducted in BSCs or other 

physical containment equipment.

The following standard and special practices, safety equipment, and facility 

requirements apply to BSL-2.

A. Standard Microbiological Practices

1. The laboratory supervisor must enforce the institutional policies that 

control access to the laboratory.

2. Persons must wash their hands after working with potentially hazardous 

materials and before leaving the laboratory.

3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, 

and storing food for human consumption must not be permitted in 

laboratory areas. Food must be stored outside the laboratory area in 

cabinets or refrigerators designated and used for this purpose.
34 Biosafety in Microbiological and Biomedical Laboratories

4. Mouth pipetting is prohibited; mechanical pipetting devices must be used.

5. Policies for the safe handling of sharps, such as needles, scalpels, 

pipettes, and broken glassware must be developed and implemented. 

Whenever practical, laboratory supervisors should adopt improved 

engineering and work practice controls that reduce risk of sharps 

injuries. Precautions, including those listed below, must always be 

taken with sharp items. These include:

a. Careful management of needles and other sharps are of primary 

importance. Needles must not be bent, sheared, broken, recapped, 

removed from disposable syringes, or otherwise manipulated by 

hand before disposal.

b. Used disposable needles and syringes must be carefully placed 

in conveniently located puncture-resistant containers used for 

sharps disposal.

c. Non-disposable sharps must be placed in a hard walled container 

for transport to a processing area for decontamination, preferably 

by autoclaving.

d. Broken glassware must not be handled directly. Instead, it must be 

removed using a brush and dustpan, tongs, or forceps. Plastic ware 

should be substituted for glassware whenever possible.

6. Perform all procedures to minimize the creation of splashes and/or aerosols.

7. Decontaminate work surfaces after completion of work and after any spill 

or splash of potentially infectious material with appropriate disinfectant.

8. Decontaminate all cultures, stocks, and other potentially infectious 

materials before disposal using an effective method. Depending on 

where the decontamination will be performed, the following methods 

should be used prior to transport:

a. Materials to be decontaminated outside of the immediate 

laboratory must be placed in a durable, leak proof container 

and secured for transport.

b. Materials to be removed from the facility for decontamination 

must be packed in accordance with applicable local, state, and 

federal regulations.

9. A sign incorporating the universal biohazard symbol must be posted 

at the entrance to the laboratory when infectious agents are present. 

Posted information must include: the laboratory’s biosafety level, the 
Laboratory Biosafety Level Criteria: BSL-2 35

supervisor’s name (or other responsible personnel), telephone number, 

and required procedures for entering and exiting the laboratory. Agent 

information should be posted in accordance with the institutional policy.

10. An effective integrated pest management program is required. 

(See Appendix G.)

11. The laboratory supervisor must ensure that laboratory personnel receive 

appropriate training regarding their duties, the necessary precautions to 

prevent exposures, and exposure evaluation procedures. Personnel must 

receive annual updates or additional training when procedural or policy 

changes occur. Personal health status may impact an individual’s 

susceptibility to infection, ability to receive immunizations or prophylactic 

interventions. Therefore, all laboratory personnel and particularly 

women of childbearing age should be provided with information 

regarding immune competence and conditions that may predispose 

them to infection. Individuals having these conditions should be 

encouraged to self-identify to the institution’s healthcare provider for 

appropriate counseling and guidance.

B. Special Practices

1. All persons entering the laboratory must be advised of the potential 

hazards and meet specific entry/exit requirements.

2. Laboratory personnel must be provided medical surveillance, as 

appropriate, and offered available immunizations for agents handled 

or potentially present in the laboratory.

3. Each institution should consider the need for collection and storage 

of serum samples from at-risk personnel.

4. A laboratory-specific biosafety manual must be prepared and adopted 

as policy. The biosafety manual must be available and accessible.

5. The laboratory supervisor must ensure that laboratory personnel 

demonstrate proficiency in standard and special microbiological 

practices before working with BSL-2 agents.

6. Potentially infectious materials must be placed in a durable, leak proof 

container during collection, handling, processing, storage, or transport 

within a facility.

7. Laboratory equipment should be routinely decontaminated, as well as, 

after spills, splashes, or other potential contamination.
36 Biosafety in Microbiological and Biomedical Laboratories

a. Spills involving infectious materials must be contained, 

decontaminated, and cleaned up by staff properly trained and 

equipped to work with infectious material.

b. Equipment must be decontaminated before repair, maintenance, 

or removal from the laboratory.

8. Incidents that may result in exposure to infectious materials must be 

immediately evaluated and treated according to procedures described 

in the laboratory biosafety manual. All such incidents must be reported 

to the laboratory supervisor. Medical evaluation, surveillance, and 

treatment should be provided and appropriate records maintained.

9. Animal and plants not associated with the work being performed must 

not be permitted in the laboratory.

10. All procedures involving the manipulation of infectious materials that 

may generate an aerosol should be conducted within a BSC or other 

physical containment devices.

C. Safety Equipment (Primary Barriers and Personal Protective Equipment)

1. Properly maintained BSCs, other appropriate personal protective 

equipment, or other physical containment devices must be used whenever:

a. Procedures with a potential for creating infectious aerosols or 

splashes are conducted. These may include pipetting, centrifuging, 

grinding, blending, shaking, mixing, sonicating, opening containers 

of infectious materials, inoculating animals intranasally, and 

harvesting infected tissues from animals or eggs.

b. High concentrations or large volumes of infectious agents are used. 

Such materials may be centrifuged in the open laboratory using 

sealed rotor heads or centrifuge safety cups.

2. Protective laboratory coats, gowns, smocks, or uniforms designated for 

laboratory use must be worn while working with hazardous materials. 

Remove protective clothing before leaving for non-laboratory areas, 

e.g., cafeteria, library, and administrative offices). Dispose of protective 

clothing appropriately, or deposit it for laundering by the institution. It is 

recommended that laboratory clothing not be taken home.

3. Eye and face protection (goggles, mask, face shield or other splatter 

guard) is used for anticipated splashes or sprays of infectious or other 

hazardous materials when the microorganisms must be handled 

outside the BSC or containment device. Eye and face protection 

must be disposed of with other contaminated laboratory waste or 
Laboratory Biosafety Level Criteria: BSL-2 37

decontaminated before reuse. Persons who wear contact lenses in 

laboratories should also wear eye protection.

4. Gloves must be worn to protect hands from exposure to hazardous 

materials. Glove selection should be based on an appropriate risk 

assessment. Alternatives to latex gloves should be available. Gloves 

must not be worn outside the laboratory. In addition, BSL-2 laboratory 

workers should:

a. Change gloves when contaminated, glove integrity is compromised, 

or when otherwise necessary.

b. Remove gloves and wash hands when work with hazardous 

materials has been completed and before leaving the laboratory.

c. Do not wash or reuse disposable gloves. Dispose of used gloves 

with other contaminated laboratory waste. Hand washing protocols 

must be rigorously followed.

5. Eye, face and respiratory protection should be used in rooms containing 

infected animals as determined by the risk assessment.

D. Laboratory Facilities (Secondary Barriers)

1. Laboratory doors should be self-closing and have locks in accordance 

with the institutional policies.

2. Laboratories must have a sink for hand washing. The sink may be 

manually, hands-free, or automatically operated. It should be located 

near the exit door.

3. The laboratory should be designed so that it can be easily cleaned and 

decontaminated. Carpets and rugs in laboratories are not permitted.

4. Laboratory furniture must be capable of supporting anticipated loads 

and uses. Spaces between benches, cabinets, and equipment should 

be accessible for cleaning.

a. Bench tops must be impervious to water and resistant to heat, 

organic solvents, acids, alkalis, and other chemicals.

b. Chairs used in laboratory work must be covered with a non-porous 

material that can be easily cleaned and decontaminated with 

appropriate disinfectant.

5. Laboratory windows that open to the exterior are not recommended. 

However, if a laboratory does have windows that open to the exterior, 

they must be fitted with screens.
38 Biosafety in Microbiological and Biomedical Laboratories

6. BSCs must be installed so that fluctuations of the room air supply 

and exhaust do not interfere with proper operations. BSCs should 

be located away from doors, windows that can be opened, heavily 

traveled laboratory areas, and other possible airflow disruptions.

7. Vacuum lines should be protected with liquid disinfectant traps.

8. An eyewash station must be readily available.

9. There are no specific requirements for ventilation systems. However, 

planning of new facilities should consider mechanical ventilation 

systems that provide an inward flow of air without recirculation to 

spaces outside of the laboratory.

10. HEPA filtered exhaust air from a Class II BSC can be safely recirculation 

back into the laboratory environment if the cabinet is tested and 

certified at least annually and operated according to manufacturer’s 

recommendations. BSCs can also be connected to the laboratory 

exhaust system by either a thimble (canopy) connection or directly 

exhausted to the outside through a hard connection. Provisions to 

assure proper safety cabinet performance and air system operation 

must be verified.

11. A method for decontaminating all laboratory wastes should be available 

in the facility (e.g., autoclave, chemical disinfection, incineration, or 

other validated decontamination method).

Biosafety Level 3

Biosafety Level 3 is applicable to clinical, diagnostic, teaching, research, or 

production facilities where work is performed with indigenous or exotic agents 

that may cause serious or potentially lethal disease through the inhalation route 

of exposure. Laboratory personnel must receive specific training in handling 

pathogenic and potentially lethal agents, and must be supervised by scientists 

competent in handling infectious agents and associated procedures.

All procedures involving the manipulation of infectious materials must be 

conducted within BSCs or other physical containment devices.

A BSL-3 laboratory has special engineering and design features.

The following standard and special safety practices, equipment, and facility 

requirements apply to BSL-3.

A. Standard Microbiological Practices

1. The laboratory supervisor must enforce the institutional policies that 

control access to the laboratory.
Laboratory Biosafety Level Criteria: BSL-3 39

2. Persons must wash their hands after working with potentially hazardous 

materials and before leaving the laboratory.

3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, 

and storing food for human consumption must not be permitted in 

laboratory areas. Food must be stored outside the laboratory area in 

cabinets or refrigerators designated and used for this purpose.

4. Mouth pipetting is prohibited; mechanical pipetting devices must be used.

5. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, 

and broken glassware must be developed and implemented. Whenever 

practical, laboratory supervisors should adopt improved engineering 

and work practice controls that reduce risk of sharps injuries.

Precautions, including those listed below, must always be taken with 

sharp items. These include:

a. Careful management of needles and other sharps are of primary 

importance. Needles must not be bent, sheared, broken, recapped, 

removed from disposable syringes, or otherwise manipulated by 

hand before disposal.

b. Used disposable needles and syringes must be carefully placed 

in conveniently located puncture-resistant containers used for 

sharps disposal.

c. Non-disposable sharps must be placed in a hard walled container 

for transport to a processing area for decontamination, preferably 

by autoclaving.

d. Broken glassware must not be handled directly. Instead, it must 

be removed using a brush and dustpan, tongs, or forceps. Plastic 

ware should be substituted for glassware whenever possible.

6. Perform all procedures to minimize the creation of splashes 

and/or aerosols.

7. Decontaminate work surfaces after completion of work and after any spill 

or splash of potentially infectious material with appropriate disinfectant.

8. Decontaminate all cultures, stocks, and other potentially infectious 

materials before disposal using an effective method. A method for 

decontaminating all laboratory wastes should be available in the facility, 

preferably within the laboratory (e.g., autoclave, chemical disinfection, 

incineration, or other validated decontamination method). Depending 

on where the decontamination will be performed, the following methods 

should be used prior to transport:
40 Biosafety in Microbiological and Biomedical Laboratories

a. Materials to be decontaminated outside of the immediate laboratory 

must be placed in a durable, leak proof container and secured 

for transport.

b. Materials to be removed from the facility for decontamination 

must be packed in accordance with applicable local, state, and 

federal regulations.

9. A sign incorporating the universal biohazard symbol must be posted 

at the entrance to the laboratory when infectious agents are present. 

Posted information must include the laboratory’s biosafety level, the 

supervisor’s name (or other responsible personnel), telephone number, 

and required procedures for entering and exiting the laboratory. Agent 

information should be posted in accordance with the institutional policy.

10. An effective integrated pest management program is required. 

(See Appendix G.)

11. The laboratory supervisor must ensure that laboratory personnel receive 

appropriate training regarding their duties, the necessary precautions 

to prevent exposures, and exposure evaluation procedures. Personnel 

must receive annual updates or additional training when procedural or 

policy changes occur. Personal health status may impact an individual’s 

susceptibility to infection, ability to receive immunizations or prophylactic 

interventions. Therefore, all laboratory personnel and particularly 

women of childbearing age should be provided with information 

regarding immune competence and conditions that may predispose 

them to infection. Individuals having these conditions should be 

encouraged to self-identify to the institution’s healthcare provider 

for appropriate counseling and guidance.

B. Special Practices

1. All persons entering the laboratory must be advised of the potential 

hazards and meet specific entry/exit requirements.

2. Laboratory personnel must be provided medical surveillance and 

offered appropriate immunizations for agents handled or potentially 

present in the laboratory.

3. Each institution should consider the need for collection and storage 

of serum samples from at-risk personnel.

4. A laboratory-specific biosafety manual must be prepared and adopted 

as policy. The biosafety manual must be available and accessible.
Laboratory Biosafety Level Criteria: BSL-3 41

5. The laboratory supervisor must ensure that laboratory personnel 

demonstrate proficiency in standard and special microbiological 

practices before working with BSL-3 agents.

6. Potentially infectious materials must be placed in a durable, leak proof 

container during collection, handling, processing, storage, or transport 

within a facility.

7. Laboratory equipment should be routinely decontaminated, as well as, 

after spills, splashes, or other potential contamination.

a. Spills involving infectious materials must be contained, 

decontaminated, and cleaned up by staff properly trained and 

equipped to work with infectious material.

b. Equipment must be decontaminated before repair, maintenance, or 

removal from the laboratory.

8. Incidents that may result in exposure to infectious materials must be 

immediately evaluated and treated according to procedures described 

in the laboratory biosafety manual. All such incidents must be reported 

to the laboratory supervisor. Medical evaluation, surveillance, and 

treatment should be provided and appropriate records maintained.

9. Animals and plants not associated with the work being performed must 

not be permitted in the laboratory.

10. All procedures involving the manipulation of infectious materials must 

be conducted within a BSC, or other physical containment devices. No 

work with open vessels is conducted on the bench. When a procedure 

cannot be performed within a BSC, a combination of personal protective 

equipment and other containment devices, such as a centrifuge safety 

cup or sealed rotor must be used.

C. Safety Equipment (Primary Barriers and Personal Protective Equipment)

1. All procedures involving the manipulation of infectious materials must 

be conducted within a BSC (preferably Class II or Class III), or other 

physical containment devices.

2. Workers in the laboratory where protective laboratory clothing with 

a solid-front, such as tie-back or wrap-around gowns, scrub suits, or 

coveralls. Protective clothing is not worn outside of the laboratory. 

Reusable clothing is decontaminated before being laundered. Clothing 

is changed when contaminated.
42 Biosafety in Microbiological and Biomedical Laboratories

3. Eye and face protection (goggles, mask, face shield or other splash 

guard) is used for anticipated splashes or sprays of infectious or other 

hazardous materials. Eye and face protection must be disposed of with 

other contaminated laboratory waste or decontaminated before reuse. 

Persons who wear contact lenses in laboratories must also wear 

eye protection.

4. Gloves must be worn to protect hands from exposure to hazardous 

materials. Glove selection should be based on an appropriate risk 

assessment. Alternatives to latex gloves should be available. Gloves must 

not be worn outside the laboratory. In addition, BSL-3 laboratory workers:

a. Changes gloves when contaminated, glove integrity is 

compromised, or when otherwise necessary. Wear two pairs 

of gloves when appropriate.

b. Remove gloves and wash hands when work with hazardous 

materials has been completed and before leaving the laboratory.

c. Do not wash or reuse disposable gloves. Dispose of used gloves 

with other contaminated laboratory waste. Hand washing protocols 

must be rigorously followed.

5. Eye, face, and respiratory protection must be used in rooms containing 

infected animals.

D. Laboratory Facilities (Secondary Barriers)

1. Laboratory doors must be self-closing and have locks in accordance 

with the institutional policies. The laboratory must be separated from 

areas that are open to unrestricted traffic flow within the building. 

Laboratory access is restricted. Access to the laboratory is through two 

self-closing doors. A clothing change room (anteroom) may be included 

in the passageway between the two self-closing doors.

2. Laboratories must have a sink for hand washing. The sink must be 

hands-free or automatically operated. It should be located near the exit 

door. If the laboratory is segregated into different laboratories, a sink 

must also be available for hand washing in each zone. Additional sinks 

may be required as determined by the risk assessment.

3. The laboratory must be designed so that it can be easily cleaned and 

decontaminated. Carpets and rugs are not permitted. Seams, floors, 

walls, and ceiling surfaces should be sealed. Spaces around doors 

and ventilation openings should be capable of being sealed to facilitate 

space decontamination.
Laboratory Biosafety Level Criteria: BSL-3 43

a. Floors must be slip resistant, impervious to liquids, and resistant 

to chemicals. Consideration should be given to the installation of 

seamless, sealed, resilient or poured floors, with integral cove bases.

b. Walls should be constructed to produce a sealed smooth finish 

that can be easily cleaned and decontaminated.

c. Ceilings should be constructed, sealed, and finished in the same 

general manner as walls.

Decontamination of the entire laboratory should be considered when 

there has been gross contamination of the space, significant changes 

in laboratory usage, for major renovations, or maintenance shut 

downs. Selection of the appropriate materials and methods used to 

decontaminate the laboratory must be based on the risk assessment.

4. Laboratory furniture must be capable of supporting anticipated loads 

and uses. Spaces between benches, cabinets, and equipment must 

be accessible for cleaning.

a. Bench tops must be impervious to water and resistant to heat, 

organic solvents, acids, alkalis, and other chemicals.

b. Chairs used in laboratory work must be covered with a non-porous 

material that can be easily cleaned and decontaminated with 

appropriate disinfectant.

5. All windows in the laboratory must be sealed.

6. BSCs must be installed so that fluctuations of the room air supply and 

exhaust do not interfere with proper operations. BSCs should be located 

away from doors, heavily traveled laboratory areas, and other possible 

airflow disruptions.

7. Vacuum lines must be protected with HEPA filters, or their equivalent. Filters 

must be replaced as needed. Liquid disinfectant traps may be required.

8. An eyewash station must be readily available in the laboratory.

9. A ducted air ventilation system is required. This system must provide 

sustained directional airflow by drawing air into the laboratory from “clean” 

areas toward “potentially contaminated” areas. The laboratory shall be 

designed such that under failure conditions the airflow will not be reversed.

a. Laboratory personnel must be able to verify directional airflow. A 

visual monitoring device, which confirms directional airflow, must 

be provided at the laboratory entry. Audible alarms should be 

considered to notify personnel of air flow disruption.
44 Biosafety in Microbiological and Biomedical Laboratories

b. The laboratory exhaust air must not re-circulate to any other area 

of the building.

c. The laboratory building exhaust air should be dispersed away from 

occupied areas and from building air intake locations or the exhaust 

air must be HEPA filtered.

HEPA filter housings should have gas-tight isolation dampers, 

decontamination ports, and/or bag-in/bag-out (with appropriate 

decontamination procedures) capability. The HEPA filter housing 

should allow for leak testing of each filter and assembly. The filters 

and the housing should be certified at least annually.

10. HEPA filtered exhaust air from a Class II BSC can be safely re-circulated 

into the laboratory environment if the cabinet is tested and certified at 

least annually and operated according to manufacturer’s recommendations. 

BSCs can also be connected to the laboratory exhaust system by either 

a thimble (canopy) connection or directly exhausted to the outside 

through a hard connection. Provisions to assure proper safety cabinet 

performance and air system operation must be verified. BSCs should be 

certified at least annually to assure correct performance. Class III BSCs 

must be directly (hard) connected up through the second exhaust HEPA 

filter of the cabinet. Supply air must be provided in such a manner that 

prevents positive pressurization of the cabinet.

11. A method for decontaminating all laboratory wastes should be available 

in the facility, preferably within the laboratory (e.g., autoclave, chemical 

disinfection, or other validated decontamination method).

12. Equipment that may produce infectious aerosols must be contained 

in primary barrier devices that exhaust air through HEPA filtration or 

other equivalent technology before being discharged into the laboratory. 

These HEPA filters should be tested and/or replaced at least annually.

13. Facility design consideration should be given to means of decontaminating 

large pieces of equipment before removal from the laboratory.

14. Enhanced environmental and personal protection may be required by 

the agent summary statement, risk assessment, or applicable local, 

state, or federal regulations. These laboratory enhancements may include, 

for example, one or more of the following: an anteroom for clean storage 

of equipment and supplies with dress-in, shower-out capabilities; gas 

tight dampers to facilitate laboratory isolation; final HEPA filtration of 

the laboratory exhaust air; laboratory effluent decontamination; and 

advanced access control devices, such as biometrics.
Laboratory Biosafety Level Criteria: BSL-4 45

15. The BSL-3 facility design, operational parameters, and procedures 

must be verified and documented prior to operation. Facilities must 

be re-verified and documented at least annually.

Biosafety Level 4

Biosafety Level 4 is required for work with dangerous and exotic agents that pose 

a high individual risk of aerosol-transmitted laboratory infections and life-threatening 

disease that is frequently fatal, for which there are no vaccines or treatments, or a 

related agent with unknown risk of transmission. Agents with a close or identical 

antigenic relationship to agents requiring BSL-4 containment must be handled at 

this level until sufficient data are obtained either to confirm continued work at this 

level, or re-designate the level. Laboratory staff must have specific and thorough 

training in handling extremely hazardous infectious agents. Laboratory staff must 

understand the primary and secondary containment functions of standard and 

special practices, containment equipment, and laboratory design characteristics. 

All laboratory staff and supervisors must be competent in handling agents and 

procedures requiring BSL-4 containment. The laboratory supervisor in 

accordance with institutional policies controls access to the laboratory.

There are two models for BSL-4 laboratories:

1. A Cabinet Laboratory—Manipulation of agents must be performed in a 

Class III BSC; and

2. A Suit Laboratory—Personnel must wear a positive pressure supplied 

air protective suit.

BSL-4 cabinet and suit laboratories have special engineering and design 

features to prevent microorganisms from being disseminated into the environment.

The following standard and special safety practices, equipment, and facilities 

apply to BSL-4.

A. Standard Microbiological Practices

1. The laboratory supervisor must enforce the institutional policies that 

control access to the laboratory.

2. Eating, drinking, smoking, handling contact lenses, applying cosmetics, 

and storing food for human consumption must not be permitted in 

laboratory areas. Food must be stored outside the laboratory area in 

cabinets or refrigerators designated and used for this purpose.

3. Mechanical pipetting devices must be used.

4. Policies for the safe handling of sharps, such as needles, scalpels, 

pipettes, and broken glassware must be developed and implemented. 
46 Biosafety in Microbiological and Biomedical Laboratories

Precautions, including those listed below, must be taken with any sharp 

items. These include:

a. Broken glassware must not be handled directly. Instead, it must 

be removed using a brush and dustpan, tongs, or forceps. Plastic 

ware should be substituted for glassware whenever possible.

b. Use of needles and syringes or other sharp instruments should be 

restricted in the laboratory, except when there is no practical alternative.

c. Used needles must not be bent, sheared, broken, recapped, 

removed from disposable syringes, or otherwise manipulated by 

hand before disposal or decontamination. Used disposable needles 

must be carefully placed in puncture-resistant containers used for 

sharps disposal, located as close to the point of use as possible.

d. Whenever practical, laboratory supervisors should adopt 

improved engineering and work practice controls that reduce 

risk of sharps injuries.

5. Perform all procedures to minimize the creation of splashes and/or aerosols.

6. Decontaminate work surfaces with appropriate disinfectant after 

completion of work and after any spill or splash of potentially infectious 

material.

7. Decontaminate all wastes before removal from the laboratory by an 

effective and validated method.

8. A sign incorporating the universal biohazard symbol must be posted 

at the entrance to the laboratory when infectious agents are present. 

Posted information must include the laboratory’s biosafety level, the 

supervisor’s name (or other responsible personnel), telephone number, 

and required procedures for entering and exiting the laboratory. Agent 

information should be posted in accordance with the institutional policy.

9. An effective integrated pest management program is required. 

(See Appendix G.)

10. The laboratory supervisor must ensure that laboratory personnel receive 

appropriate training regarding their duties, the necessary precautions 

to prevent exposures, and exposure evaluation procedures. Personnel 

must receive annual updates or additional training when procedural or 

policy changes occur. Personal health status may impact an individual’s 

susceptibility to infection, ability to receive immunizations or prophylactic 

interventions. Therefore, all laboratory personnel and particularly 

women of childbearing age should be provided with information 
Laboratory Biosafety Level Criteria: BSL-4 47

regarding immune competence and conditions that may predispose 

them to infection. Individuals having these conditions should be 

encouraged to self-identify to the institution’s healthcare provider for 

appropriate counseling and guidance.

B. Special Practices

1. All persons entering the laboratory must be advised of the potential 

hazards and meet specific entry requirements in accordance with 

institutional policies.

Only persons whose presence in the facility or individual laboratory rooms 

is required for scientific or support purposes are authorized to enter.

Entry into the facility must be limited by means of secure, locked doors. 

A logbook, or other means of documenting the date and time of all 

persons entering and leaving the laboratory must be maintained.

While the laboratory is operational, personnel must enter and exit 

the laboratory through the clothing change and shower rooms except 

during emergencies. All personal clothing must be removed in the outer 

clothing change room. All persons entering the laboratory must use 

laboratory clothing, including undergarments, pants, shirts, jumpsuits, 

shoes, and gloves (as appropriate). All persons leaving the laboratory 

must take a personal body shower. Used laboratory clothing must 

not be removed from the inner change room through the personal 

shower. These items must be treated as contaminated materials and 

decontaminated before laundering.

After the laboratory has been completely decontaminated and all 

infectious agents are secured, necessary staff may enter and exit 

without following the clothing change and shower requirements 

described above.

2. Laboratory personnel and support staff must be provided appropriate 

occupational medical services including medical surveillance and 

available immunizations for agents handled or potentially present in the 

laboratory. A system must be established for reporting and documenting 

laboratory accidents, exposures, employee absenteeism and for the 

medical surveillance of potential laboratory-associated illnesses. An 

essential adjunct to such an occupational medical services system is 

the availability of a facility for the isolation and medical care of personnel 

with potential or known laboratory-acquired infections.

3. Each institution should consider the need for collection and storage of 

serum samples from at-risk personnel.
48 Biosafety in Microbiological and Biomedical Laboratories

4. A laboratory-specific biosafety manual must be prepared. The 

biosafety manual must be available, accessible, and followed.

5. The laboratory supervisor is responsible for ensuring that 

laboratory personnel:

a. Demonstrate high proficiency in standard and special 

microbiological practices, and techniques for working with 

agents requiring BSL-4 containment.

b. Receive appropriate training in the practices and operations 

specific to the laboratory facility.

c. Receive annual updates and additional training when procedural 

or policy changes occur.

6. Removal of biological materials that are to remain in a viable or intact 

state from the laboratory must be transferred to a non-breakable, 

sealed primary container and then enclosed in a non-breakable, sealed 

secondary container. These materials must be transferred through a 

disinfectant dunk tank, fumigation chamber, or decontamination shower. 

Once removed, packaged viable material must not be opened outside 

BSL-4 containment unless inactivated by a validated method.

7. Laboratory equipment musts be routinely decontaminated, as well as 

after spills, splashes, or other potential contamination.

a. Spills involving infectious materials must be contained, decontaminated, 

and cleaned up by appropriate professional staff, or others properly 

trained and equipped to work with infectious material. A spill 

procedure must be developed and posted within the laboratory.

b. Equipment must be decontaminated using an effective and 

validated method before repair, maintenance, or removal from 

the laboratory. The interior of the Class III cabinet as well as all 

contaminated plenums, fans and filters must be decontaminated 

using a validated gaseous or vapor method.

c. Equipment or material that might be damaged by high temperatures 

or steam must be decontaminated using an effective and validated 

procedure such as a gaseous or vapor method in an airlock or 

chamber designed for this purpose.

8. Incidents that may result in exposure to infectious materials must be 

immediately evaluated and treated according to procedures described 

in the laboratory biosafety manual. All incidents must be reported to 

the laboratory supervisor, institutional management and appropriate 
Laboratory Biosafety Level Criteria: BSL-4 49

laboratory personnel as defined in the laboratory biosafety manual. 

Medical evaluation, surveillance, and treatment should be provided and 

appropriate records maintained.

9. Animals and plants not associated with the work being performed must 

not be permitted in the laboratory.

10. Supplies and materials that are not brought into the BSL-4 laboratory 

through the change room, must be brought in through a previously 

decontaminated double-door autoclave, fumigation chamber, or airlock. 

After securing the outer doors, personnel within the laboratory retrieve 

the materials by opening the interior doors of the autoclave, fumigation 

chamber, or airlock. These doors must be secured after materials 

are brought into the facility. The doors of the autoclave or fumigation 

chamber are interlocked in a manner that prevents opening of the outer 

door unless the autoclave or fumigation chamber has been operated 

through a decontamination cycle. 

Only necessary equipment and supplies should be stored inside the 

BSL-4 laboratory. All equipment and supplies taken inside the laboratory 

must be decontaminated before removal from the laboratory.

11. Daily inspections of essential containment and life support systems must be 

completed and documented before laboratory work is initiated to ensure 

that the laboratory is operating according to established parameters.

12. Practical and effective protocols for emergency situations must be 

established. These protocols must include plans for medical emergencies, 

facility malfunctions, fires, escape of animals within the laboratory, and 

other potential emergencies. Training in emergency response procedures 

must be provided to emergency response personnel and other 

responsible staff according to institutional policies.

C. Safety Equipment (Primary Barriers and Personal Protective Equipment)

Cabinet Laboratory

1. All manipulations of infectious materials within the laboratory must be 

conducted in the Class III biological safety cabinet.

Double-door, pass through autoclaves must be provided for 

decontaminating materials passing out of the Class III BSC(s). The 

autoclave doors must be interlocked so that only one can be opened 

at any time and be automatically controlled so that the outside door to 

the autoclave can only be opened after the decontamination cycle has 

been completed.
50 Biosafety in Microbiological and Biomedical Laboratories

The Class III cabinet must also have a pass-through dunk tank, 

fumigation chamber, or equivalent decontamination method so that 

materials and equipment that cannot be decontaminated in the 

autoclave can be safely removed from the cabinet. Containment 

must be maintained at all times.

The Class III cabinet must have a HEPA filter on the supply air intake 

and two HEPA filters in series on the exhaust outlet of the unit. There 

must be gas tight dampers on the supply and exhaust ducts of the 

cabinet to permit gas or vapor decontamination of the unit. Ports for 

injection of test medium must be present on all HEPA filter housings.

The interior of the Class III cabinet must be constructed with smooth 

finishes that can be easily cleaned and decontaminated. All sharp edges 

on cabinet finishes must be eliminated to reduce the potential for cuts 

and tears of gloves. Equipment to be placed in the Class III cabinet 

should also be free of sharp edges or other surfaces that may damage 

or puncture the cabinet gloves.

Class III cabinet gloves must be inspected for damage prior to use 

and changed if necessary. Gloves should be replaced annually during 

cabinet re-certification.

The cabinet should be designed to permit maintenance and repairs of 

cabinet mechanical systems (refrigeration, incubators, centrifuges, etc.) 

to be performed from the exterior of the cabinet whenever possible.

Manipulation of high concentrations or large volumes of infectious 

agents within the Class III cabinet should be performed using physical 

containment devices inside the cabinet whenever practical. Such 

materials should be centrifuged inside the cabinet using sealed rotor 

heads or centrifuge safety cups.

The Class III cabinet must be certified at least annually.

2. Workers in the laboratory must wear protective laboratory clothing 

with a solid-front, such as tie-back or wrap-around gowns, scrub 

suits, or coveralls. No personal clothing, jewelry, or other items 

except eyeglasses should be taken past the personal shower area. 

All protective clothing must be removed in the dirty side change room 

before showering. Reusable clothing must be autoclaved prior to 

removal from the laboratory for laundering.

3. Eye, face and respiratory protection should be used in rooms containing 

infected animals as determined by the risk assessment. Prescription 

eyeglasses must be decontaminated before removal through the 

personal body shower.
Laboratory Biosafety Level Criteria: BSL-4 51

4. Disposable gloves must be worn underneath cabinet gloves to protect 

the worker from exposure should a break or tear occur in a cabinet glove. 

Gloves must not be worn outside the laboratory. Alternatives to latex 

gloves should be available. Do not wash or reuse disposable gloves. 

Dispose of used gloves with other contaminated laboratory waste.

Suit Laboratory

1. All procedures must be conducted by personnel wearing a one-piece 

positive pressure supplied air suit.

All manipulations of infectious agents must be performed within a BSC 

or other primary barrier system.

Equipment that may produce aerosols must be contained in primary 

barrier devices that exhaust air through HEPA filtration before being 

discharged into the laboratory. These HEPA filters should be tested 

annually and replaced as needed.

HEPA filtered exhaust air from a Class II BSC can be safely re-circulated 

into the laboratory environment if the cabinet is tested and certified at 

least annually and operated according to manufacturer’s specifications.

2. Workers must wear laboratory clothing, such as scrub suits, before 

entering the room used for donning positive pressure suits. All 

laboratory clothing must be removed in the dirty side change room 

before entering the personal shower. 

3. Inner disposable gloves must be worn to protect against break or tears 

in the outer suit gloves. Disposable gloves must not be worn outside 

the change area. Alternatives to latex gloves should be available. Do 

not wash or reuse disposable gloves. Inner gloves must be removed 

and discarded in the inner change room prior to entering the personal 

shower. Dispose of used gloves with other contaminated waste.

4. Decontamination of outer suit gloves is performed during laboratory 

operations to remove gross contamination and minimize further 

contamination of the laboratory.

D. Laboratory Facilities (Secondary Barriers)

Cabinet Laboratory

1. The BSL-4 cabinet laboratory consists of either a separate building or 

a clearly demarcated and isolated zone within a building. Laboratory 

doors must have locks in accordance with the institutional policies.
52 Biosafety in Microbiological and Biomedical Laboratories

Rooms in the facility must be arranged to ensure sequential passage 

through an inner (dirty) changing area, a personal shower and an 

outer (clean) change room upon exiting the room(s) containing the 

Class III BSC(s).

An automatically activated emergency power source must be provided 

at a minimum for the laboratory exhaust system, life support systems, 

alarms, lighting, entry and exit controls, BSCs, and door gaskets. 

Monitoring and control systems for air supply, exhaust, life support, 

alarms, entry and exit controls, and security systems should be on an 

uninterrupted power supply (UPS).

A double-door autoclave, dunk tank, fumigation chamber, or ventilated 

airlock must be provided at the containment barrier for the passage of 

materials, supplies, or equipment.

2. A hands-free sink must be provided near the door of the cabinet room(s) 

and the inner change room. A sink must be provided in the outer change 

room. All sinks in the room(s) containing the Class III BSC must be 

connected to the wastewater decontamination system.

3. Walls, floors, and ceilings of the laboratory must be constructed to form 

a sealed internal shell to facilitate fumigation and prohibit animal and 

insect intrusion. The internal surfaces of this shell must be resistant to 

chemicals used for cleaning and decontamination of the area. Floors 

must be monolithic, sealed and coved.

All penetrations in the internal shell of the laboratory and inner change 

room must be sealed.

Openings around doors into the cabinet room and inner change room must 

be minimized and capable of being sealed to facilitate decontamination.

Drains in the laboratory floor (if present) must be connected directly to 

the liquid waste decontamination system.

Services and plumbing that penetrate the laboratory walls, floors, or ceiling 

must be installed to ensure that no backflow from the laboratory occurs. 

These penetrations must be fitted with two (in series) backflow prevention 

devices. Consideration should be given to locating these devices outside 

of containment. Atmospheric venting systems must be provided with two 

HEPA filters in series and be sealed up to the second filter.

Decontamination of the entire cabinet must be performed using a 

validated gaseous or vapor method when there have been significant 

changes in cabinet usage, before major renovations or maintenance 

shut downs, and in other situations, as determined by risk assessment. 
Laboratory Biosafety Level Criteria: BSL-4 53

Selection of the appropriate materials and methods used for 

decontamination must be based on the risk assessment.

4. Laboratory furniture must be of simple construction, capable of 

supporting anticipated loading and uses. Spaces between benches, 

cabinets, and equipment must be accessible for cleaning and 

decontamination. Chairs and other furniture must be covered with 

a non-porous material that can be easily decontaminated.

5. Windows must be break-resistant and sealed.

6. If Class II BSCs are needed in the cabinet laboratory, they must be 

installed so that fluctuations of the room air supply and exhaust do not 

interfere with proper operations. Class II cabinets should be located 

away from doors, heavily traveled laboratory areas, and other possible 

airflow disruptions.

7. Central vacuum systems are not recommended. If, however, there is a 

central vacuum system, it must not serve areas outside the cabinet room. 

Two in-line HEPA filters must be placed near each use point. Filters 

must be installed to permit in-place decontamination and replacement.

8. An eyewash station must be readily available in the laboratory.

9. A dedicated non-recirculating ventilation system is provided. Only 

laboratories with the same HVAC requirements (i.e., other BSL-4 labs, 

ABSL-4, BSL-3-Ag labs) may share ventilation systems if gas-tight 

dampers and HEPA filters isolate each individual laboratory system.

The supply and exhaust components of the ventilation system must be 

designed to maintain the laboratory at negative pressure to surrounding 

areas and provide differential pressure or directional airflow, as 

appropriate, between adjacent areas within the laboratory.

Redundant supply fans are recommended. Redundant exhaust fans 

are required. Supply and exhaust fans must be interlocked to prevent 

positive pressurization of the laboratory.

The ventilation system must be monitored and alarmed to indicate 

malfunction or deviation from design parameters. A visual monitoring 

device must be installed near the clean change room so proper differential 

pressures within the laboratory may be verified prior to entry.

Supply air to and exhaust air from the cabinet room, inner change room, 

and fumigation/decontamination chambers must pass through HEPA 

filter(s). The air exhaust discharge must be located away from occupied 

spaces and building air intakes.
54 Biosafety in Microbiological and Biomedical Laboratories

All HEPA filters should be located as near as practicable to the cabinet 

and laboratory in order to minimize the length of potentially contaminated 

ductwork. All HEPA filters must be tested and certified annually.

The HEPA filter housings should be designed to allow for in situ 

decontamination and validation of the filter prior to removal. The design 

of the HEPA filter housing must have gas-tight isolation dampers, 

decontamination ports, and ability to scan each filter assembly for leaks.

10. HEPA filtered exhaust air from a Class II BSC can be safely re-circulated 

into the laboratory environment if the cabinet is tested and certified at least 

annually and operated according to the manufacturer’s recommendations. 

If BSC exhaust is to be recirculated to the outside, BSCs can also be 

connected to the laboratory exhaust system by either a thimble (canopy) 

connection or a hard ducted, direct connection ensuring that cabinet 

exhaust air passes through two (2) HEPA filters—including the HEPA in 

the BSC—prior to release outside. Provisions to assure proper safety 

cabinet performance and air system operation must be verified.

Class III BSCs must be directly and independently exhausted through 

two HEPA filters in series. Supply air must be provided in such a manner 

that prevents positive pressurization of the cabinet.

11. Pass through dunk tanks, fumigation chambers, or equivalent 

decontamination methods must be provided so that materials and 

equipment that cannot be decontaminated in the autoclave can be 

safely removed from the cabinet room(s). Access to the exit side of 

the pass-through shall be limited to those individuals authorized to 

be in the BSL-4 laboratory.

12. Liquid effluents from cabinet room sinks, floor drains, autoclave chambers, 

and other sources within the cabinet room must be decontaminated by 

a proven method, preferably heat treatment, before being discharged 

to the sanitary sewer.

Decontamination of all liquid wastes must be documented. The 

decontamination process for liquid wastes must be validated physically 

and biologically. Biological validation must be performed annually or 

more often if required by institutional policy.

Effluents from showers and toilets may be discharged to the sanitary 

sewer without treatment.

13 A double-door, pass through autoclave(s) must be provided for 

decontaminating materials passing out of the cabinet laboratory. 

Autoclaves that open outside of the laboratory must be sealed to the 

interior wall. This bioseal must be durable and airtight and capable of 
Laboratory Biosafety Level Criteria: BSL-4 55

expansion and contraction. Positioning the bioseal so that the equipment 

can be accessed and maintained from outside the laboratory is strongly 

recommended. The autoclave doors must be interlocked so that only 

one can be opened at any time and be automatically controlled so 

that the outside door to the autoclave can only be opened after the 

decontamination cycle has been completed.

Gas and liquid discharge from the autoclave chamber must be 

decontaminated. When feasible, autoclave decontamination processes 

should be designed so that unfiltered air or steam exposed to infectious 

material cannot be released to the environment.

14. The BSL-4 facility design parameters and operational procedures must 

be documented. The facility must be tested to verify that the design and 

operational parameters have been met prior to operation. Facilities must 

also be re-verified annually. Verification criteria should be modified as 

necessary by operational experience.

15. Appropriate communication systems must be provided between the 

laboratory and the outside (e.g., voice, fax, and computer). Provisions 

for emergency communication and emergency access or egress must 

be developed and implemented.

Suit Laboratory

1. The BSL-4 suit laboratory consists of either a separate building or a 

clearly demarcated and isolated zone within a building. Laboratory 

doors must have locks in accordance with the institutional policies.

Rooms in the facility must be arranged to ensure exit by sequential 

passage through the chemical shower, inner (dirty) change room, 

personal shower, and outer (clean) changing area.

Entry into the BSL-4 laboratory must be through an airlock fitted with 

airtight doors. Personnel who enter this area must wear a positive pressure 

suit supplied with HEPA filtered breathing air. The breathing air systems 

must have redundant compressors, failure alarms and emergency backup.

A chemical shower must be provided to decontaminate the surface of 

the positive pressure suit before the worker leaves the laboratory. In the 

event of an emergency exit or failure of the chemical shower system, a 

method for decontaminating positive pressure suits, such as a gravity 

fed supply of chemical disinfectant, is needed.

An automatically activated emergency power source must be provided, 

at a minimum, for the laboratory exhaust system, life support systems, 

alarms, lighting, entry and exit controls, BSCs, and door gaskets. 
56 Biosafety in Microbiological and Biomedical Laboratories

Monitoring and control systems for air supply, exhaust, life support, alarms, 

entry and exit controls, and security systems should be on a UPS.

A double-door autoclave, dunk tank, or fumigation chamber must 

be provided at the containment barrier for the passage of materials, 

supplies, or equipment in or out of the laboratory.

2. Sinks inside the suit laboratory should be placed near procedure areas 

and be connected to the wastewater decontamination system.

3. Walls, floors, and ceilings of the laboratory must be constructed to form 

a sealed internal shell to facilitate fumigation and prohibit animal and 

insect intrusion. The internal surfaces of this shell must be resistant to 

chemicals used for cleaning and decontamination of the area. Floors 

must be monolithic, sealed and coved.

All penetrations in the internal shell of the laboratory, suit storage room 

and the inner change room must be sealed.

Drains, if present, in the laboratory floor must be connected directly 

to the liquid waste decontamination system. Sewer vents must have 

protection against insect and animal intrusion.

Services and plumbing that penetrate the laboratory walls, floors, or ceiling 

must be installed to ensure that no backflow from the laboratory occurs. 

These penetrations must be fitted with two (in series) backflow prevention 

devices. Consideration should be given to locating these devices 

outside of containment. Atmospheric venting systems must be provided 

with two HEPA filters in series and be sealed up to the second filter.

4. Laboratory furniture must be of simple construction, capable of supporting 

anticipated loading and uses. Sharp edges and corners should be avoided. 

Spaces between benches, cabinets, and equipment must be accessible 

for cleaning and decontamination. Chairs and other furniture must be 

covered with a non-porous material that can be easily decontaminated.

5. Windows must be break-resistant and sealed.

6. BSCs and other primary containment barrier systems must be installed 

so that fluctuations of the room air supply and exhaust do not interfere 

with proper operations. BSCs should be located away from doors, 

heavily traveled laboratory areas, and other possible airflow disruptions.

7. Central vacuum systems are not recommended. If, however, there is a 

central vacuum system, it must not serve areas outside the BSL-4 

laboratory. Two in-line HEPA filters must be placed near each use point. 

Filters must be installed to permit in-place decontamination and replacement.
Laboratory Biosafety Level Criteria: BSL-4 57

8. An eyewash station must be readily available in the laboratory area for 

use during maintenance and repair activities.

9. A dedicated, non-recirculating ventilation system is provided. Only 

laboratories with the same HVAC requirements (i.e., other BSL-4 labs, 

ABSL-4, BSL-3 Ag labs) may share ventilation systems if gas-tight 

dampers and HEPA filters isolate each individual laboratory system.

The supply and exhaust components of the ventilation system must be 

designed to maintain the laboratory at negative pressure to surrounding 

areas and provide differential pressure or directional airflow as 

appropriate between adjacent areas within the laboratory.

Redundant supply fans are recommended. Redundant exhaust fans 

are required. Supply and exhaust fans must be interlocked to prevent 

positive pressurization of the laboratory.

The ventilation system must be monitored and alarmed to indicate 

malfunction or deviation from design parameters. A visual monitoring 

device must be installed near the clean change room so proper 

differential pressures within the laboratory may be verified prior to entry.

Supply air to the laboratory, including the decontamination shower, 

must pass through a HEPA filter. All exhaust air from the suit laboratory, 

decontamination shower and fumigation or decontamination chambers 

must pass through two HEPA filters, in series, before discharge to the 

outside. The exhaust air discharge must be located away from occupied 

spaces and air intakes.

All HEPA filters must be located as near as practicable to the laboratory 

in order to minimize the length of potentially contaminated ductwork. All 

HEPA filters must be tested and certified annually.

The HEPA filter housings must be designed to allow for in situ 

decontamination and validation of the filter prior to removal. The design 

of the HEPA filter housing must have gas-tight isolation dampers, 

decontamination ports, and ability to scan each filter assembly for leaks.

10. HEPA filtered exhaust air from a Class II BSC can be safely re-circulated 

back into the laboratory environment if the cabinet is tested and 

certified at least annually and operated according to the manufacturer’s 

recommendations. Biological safety cabinets can also be connected to 

the laboratory exhaust system by either a thimble (canopy) connection 

or a direct (hard) connection. Provisions to assure proper safety cabinet 

performance and air system operation must be verified.
58 Biosafety in Microbiological and Biomedical Laboratories

11. Pass through dunk tanks, fumigation chambers, or equivalent 

decontamination methods must be provided so that materials and 

equipment that cannot be decontaminated in the autoclave can be 

safely removed from the BSL-4 laboratory. Access to the exit side 

of the pass-through shall be limited to those individuals authorized 

to be in the BSL-4 laboratory.

12. Liquid effluents from chemical showers, sinks, floor drains, autoclave 

chambers, and other sources within the laboratory must be decontaminated 

by a proven method, preferably heat treatment, before being discharged 

to the sanitary sewer.

Decontamination of all liquid wastes must be documented. The 

decontamination process for liquid wastes must be validated physically 

and biologically. Biological validation must be performed annually or 

more often if required by institutional policy.

Effluents from personal body showers and toilets may be discharged 

to the sanitary sewer without treatment.

13. A double-door, pass through autoclave(s) must be provided for 

decontaminating materials passing out of the cabinet laboratory. Autoclaves 

that open outside of the laboratory must be sealed to the interior wall. 

This bioseal must be durable, airtight, and capable of expansion and 

contraction. Positioning the bioseal so that the equipment can be accessed 

and maintained from outside the laboratory is strongly recommended. 

The autoclave doors must be interlocked so that only one can be opened 

at any time and be automatically controlled so that the outside door to 

the autoclave can only be opened after the decontamination cycle has 

been completed.

Gas and liquid discharge from the autoclave chamber must be 

decontaminated. When feasible, autoclave decontamination processes 

should be designed so that unfiltered air or steam exposed to infectious 

material cannot be released to the environment.

14. The BSL-4 facility design parameters and operational procedures must 

be documented. The facility must be tested to verify that the design and 

operational parameters have been met prior to operation. Facilities must 

also be re-verified annually. Verification criteria should be modified as 

necessary by operational experience.

15. Appropriate communication systems must be provided between the 

laboratory and the outside (e.g., voice, fax, and computer). Provisions 

for emergency communication and emergency access or egress must 

be developed and implemented.
Laboratory Biosafety Level Criteria 59

Table 2. Summary of Recommended Biosafety Levels for Infectious Agents

BSL Agents Practices Primary Barriers and

Safety Equipment

Facilities

(Secondary Barriers)

1 Not known to consistently cause 

diseases in healthy adults

Standard microbiological practices No ■ primary barriers required.

PPE: ■ laboratory coats and gloves; eye, face protection, as needed

Laboratory bench and sink required

2 Agents associated with human ■

diseaseRoutes of transmission include per- ■cutaneous injury, ingestion, mucous membrane exposure

BSL-1 practice plus:

Limited access ■

Biohazard ■ warning signs“ ■ Sharps” precautionsBiosafety manual defining any ■needed waste decontamination or medical surveillance policies

Primary barriers:

BSCs or other physical containment ■

devices used for all manipulations of agents that cause splashes or aerosols of infectious materialsPPE: ■ Laboratory coats, gloves, face and eye protection, as needed

BSL-1 plus:

Autoclave available ■

3 Indigenous or exotic agents that may 

cause serious or potentially lethal disease through the inhalation route of exposure

BSL-2 practice plus:

Controlled access ■

■ Decontamination of all waste■ Decontamination of laboratory 

clothing before laundering 

Primary barriers:

BSCs or other physical containment ■

devices used for all open manipulations of agentsPPE: Protective laboratory clothing, ■gloves, face, eye and respiratory protection, as needed

BSL-2 plus:

Physical separation from access ■

corridorsSelf-closing, double-door access ■Exhausted air not recirculated ■Negative airflow into laboratory ■Entry through airlock or anteroom ■Hand washing sink near laboratory ■exit

4 Dangerous/exotic agents which post ■

high individual risk of aerosol-transmitted laboratory infections that are frequently fatal, for which there are no vaccines or treatmentsAgents with a close or identical anti- ■genic relationship to an agent requiring BSL-4 until data are available to redesignate the levelRelated agents with unknown risk of ■transmission

BSL-3 practices plus:

Clothing change before entering ■

Shower on exit ■All material decontaminated on ■exit from facility

Primary barriers:

All procedures conducted in ■ Class III 

BSCs or Class I or II BSCs in combination with full-body, air-supplied, positive pressure suit

BSL-3 plus:

Separate building or isolated zone ■

Dedicated supply and exhaust, ■vacuum, and decontamination systemsOther requirements outlined in ■the text
60 Biosafety in Microbiological and Biomedical Laboratories

Section V—Vertebrate Animal Biosafety Level Criteria for 

Vivarium Research Facilities

This guidance is provided for the use of experimentally infected animals housed 

in indoor research facilities (e.g., vivaria), and is also useful in the maintenance 

of laboratory animals that may naturally harbor zoonotic infectious agents. In 

both instances, the institutional management must provide facilities, staff, and 

established practices that reasonably ensure appropriate levels of environmental 

quality, safety, security and care for the laboratory animal. Laboratory animal 

facilities are a special type of laboratory. As a general principle, the biosafety 

level (facilities, practices, and operational requirements) recommended for 

working with infectious agents in vivo and in vitro are comparable.

The animal room can present unique problems. In the animal room, the 

activities of the animals themselves can present unique hazards not found in 

standard microbiological laboratories. Animals may generate aerosols, they 

may bite and scratch, and they may be infected with a zoonotic agent. The 

co-application of Biosafety Levels and the Animal Biosafety Levels are 

determined by a protocol-driven risk assessment.

These recommendations presuppose that laboratory animal facilities, 

operational practices, and quality of animal care meet applicable standards 

and regulations (e.g., Guide for the Care and Use of Laboratory Animals1

 and 

Laboratory Animal Welfare Regulations2

) and that appropriate species have 

been selected for animal experiments. In addition, the organization must have 

an occupational health and safety program that addresses potential hazards 

associated with the conduct of laboratory animal research. The following 

publication by the Institute for Laboratory Animal Research (ILAR), Occupational 

Health and Safety in the Care and Use of Research Animals3

, is most helpful in 

this regard. Additional safety guidance on working with non-human primates is 

available in the ILAR publication, Occupational Health and Safety in the Care 

and Use of Nonhuman Primates.4

Facilities for laboratory animals used in studies of infectious or non-infectious 

disease should be physically separate from other activities such as animal 

production and quarantine, clinical laboratories, and especially from facilities 

providing patient care. Traffic flow that will minimize the risk of cross 

contamination should be incorporated into the facility design.

The recommendations detailed below describe four combinations of 

practices, safety equipment, and facilities for experiments with animals involved 

in infectious disease research and other studies that may require containment. 

These four combinations, designated Animal Biosafety Levels (ABSL) 1-4, 

provide increasing levels of protection to personnel and to the environment, 

and are recommended as minimal standards for activities involving infected 

laboratory animals. The four ABSLs describe animal facilities and practices 
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-1 61

applicable to work with animals infected with agents assigned to Biosafety Levels 

1-4, respectively. Investigators that are inexperienced in conducting these types 

of experiments should seek help in designing their experiments from individuals 

who are experienced in this special work.

In addition to the animal biosafety levels described in this section, the USDA 

has developed facility parameters and work practices for handling agents of 

agriculture significance. Appendix D includes a discussion on Animal Biosafety 

Level 3 Agriculture (BSL-3-Ag). USDA requirements are unique to agriculture 

because of the necessity to protect the environment from pathogens of economic 

or environmental impact. Appendix D also describes some of the enhancements 

beyond BSL/ABSL-3 that may be required by USDA-APHIS when working in the 

laboratory or vivarium with certain veterinary agents of concern.

Facility standards and practices for invertebrate vectors and hosts are not 

specifically addressed in this section. The reader is referred to Appendix E for 

more information on the Arthropod Containment Guidelines.

Animal Biosafety Level 1

Animal Biosafety Level 1 is suitable for work in animals involving well-characterized 

agents that are not known to cause disease in immunocompetent adult humans, 

and present minimal potential hazard to personnel and the environment.

ABSL-1 facilities should be separated from the general traffic patterns of the 

building and restricted as appropriate. Special containment equipment or facility 

design may be required as determined by appropriate risk assessment. (See 

Section 2, Biological Risk Assessment.)

Personnel must have specific training in animal facility procedures and must 

be supervised by an individual with adequate knowledge of potential hazards and 

experimental animal procedures.

The following standard practices, safety equipment, and facility requirements 

apply to ABSL-1.

A. Standard Microbiological Practices

1. The animal facility director establishes and enforces policies, 

procedures, and protocols for institutional policies and emergencies.

Each institute must assure that worker safety and health concerns 

are addressed as part of the animal protocol review.

Prior to beginning a study animal protocols must also be reviewed 

and approved by the Institutional Animal Care and Use Committee 

(IACUC)5

 and the Institutional Biosafety Committee.
62 Biosafety in Microbiological and Biomedical Laboratories

2. A safety manual specific to the animal facility is prepared or adopted 

in consultation with the animal facility director and appropriate safety 

professionals. The safety manual must be available and accessible. 

Personnel are advised of potential hazards and are required to read and 

follow instructions on practices and procedures.

3. The supervisor must ensure that animal care, laboratory and support 

personnel receive appropriate training regarding their duties, animal 

husbandry procedures, potential hazards, manipulations of infectious 

agents, necessary precautions to prevent exposures, and hazard/

exposure evaluation procedures (physical hazards, splashes, 

aerosolization, etc.). Personnel must receive annual updates and 

additional training when procedures or policies change. Records are 

maintained for all hazard evaluations, employee training sessions and 

staff attendance.

4. An appropriate medical surveillance program is in place, as determined 

by risk assessment. The need for an animal allergy prevention program 

should be considered.

Facility supervisors should ensure that medical staff is informed of 

potential occupational hazards within the animal facility, to include those 

associated with research, animal husbandry duties, animal care and 

manipulations.

Personal health status may impact an individual’s susceptibility to 

infection, ability to receive immunizations or prophylactic interventions. 

Therefore, all personnel and particularly women of childbearing age 

should be provided information regarding immune competence and 

conditions that may predispose them to infection. Individuals having 

these conditions should be encouraged to self-identify to the institution’s 

healthcare provider for appropriate counseling and guidance.

Personnel using respirators must be enrolled in an appropriately 

constituted respiratory protection program.

5. A sign incorporating safety information must be posted at the entrance 

to the areas where infectious materials and/or animals are housed or 

are manipulated. The sign must include the animal biosafety level, 

general occupational health requirements, personal protective equipment 

requirements, the supervisor’s name (or other responsible personnel), 

telephone number, and required procedures for entering and exiting the 

animal areas. Identification of specific infectious agents is recommended 

when more than one agent is being used within an animal room.

Security-sensitive agent information should be posted in accordance 

with the institutional policy.
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-1 63

Advance consideration should be given to emergency and disaster 

recovery plans, as a contingency for man-made or natural disasters.1,3,4

6. Access to the animal room is limited. Only those persons required for 

program or support purposes are authorized to enter the facility.

All persons including facility personnel, service workers, and visitors 

are advised of the potential hazards (natural or research pathogens, 

allergens, etc.) and are instructed on the appropriate safeguards.

7. Protective laboratory coats, gowns, or uniforms are recommended to 

prevent contamination of personal clothing.

Gloves are worn to prevent skin contact with contaminated, infectious 

and hazardous materials, and when handling animals.

Gloves and personal protective equipment should be removed in a manner 

that minimizes transfer of infectious materials outside of the areas where 

infectious materials and/or animals are housed or are manipulated.

Persons must wash their hands after removing gloves, and before 

leaving the areas where infectious materials and/or animals are 

housed or are manipulated.

Eye and face and respiratory protection should be used in rooms 

containing infected animals, as dictated by the risk assessment.

8. Eating, drinking, smoking, handling contact lenses, applying cosmetics, 

and storing food for human consumption must not be permitted in 

laboratory areas. Food must be stored outside of the laboratory in 

cabinets or refrigerators designed and used for this purpose.

9. All procedures are carefully performed to minimize the creation of 

aerosols or splatters of infectious materials and waste.

10. Mouth pipetting is prohibited. Mechanical pipetting devices must be used.

11. Policies for the safe handling of sharps, such as needles, scalpels, 

pipettes, and broken glassware must be developed and implemented.

When applicable, laboratory supervisors should adopt improved 

engineering and work practice controls that reduce the risk of sharps 

injuries. Precautions, including those listed below, must always be 

taken with sharp items. These include:

a. Use of needles and syringes or other sharp instruments in the 

animal facility is limited to situations where there is no alternative 
64 Biosafety in Microbiological and Biomedical Laboratories

for such procedures as parenteral injection, blood collection, or 

aspiration of fluids from laboratory animals and diaphragm bottles.

b. Disposable needles must not be bent, sheared, broken, recapped, 

removed from disposable syringes, or otherwise manipulated by hand 

before disposal. Used disposable needles must be carefully placed 

in puncture-resistant containers used for sharps disposal. Sharps 

containers should be located as close to the work site as possible.

c. Non-disposable sharps must be placed in a hard-walled container 

for transport to a processing area for decontamination, preferably 

by autoclaving.

d. Broken glassware must not be handled directly. Instead, it must be 

removed using a brush and dustpan, tongs, or forceps. Plastic ware 

should be substituted for glassware whenever possible.

e. Equipment containing sharp edges and corners should be avoided.

12. Equipment and work surfaces are routinely decontaminated with an 

appropriate disinfectant after work with an infectious agent, and after 

any spills, splashes, or other overt contamination.

13. Animals and plants not associated with the work being performed must 

not be permitted in the areas where infectious materials and/ or animals 

are housed or are manipulated.

14. An effective integrated pest management program is required. 

(See Appendix G.)

15. All wastes from the animal room (including animal tissues, carcasses, 

and bedding) are transported from the animal room in leak-proof, 

covered containers for appropriate disposal in compliance with 

applicable institutional, local and state requirements.

Decontaminate all potentially infectious materials before disposal 

using an effective method.

B. Special Practices

None required.

C. Safety Equipment (Primary Barriers and Personal Protective 

Equipment)

1. A risk assessment should determine the appropriate type of personal 

protective equipment to be utilized.
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-1 65

2. Special containment devices or equipment may not be required as 

determined by appropriate risk assessment.

3. Protective laboratory coats, gowns, or uniforms may be required to 

prevent contamination of personal clothing.

Protective outer clothing is not worn outside areas where infectious 

materials and/or animals are housed or manipulated. Gowns and 

uniforms are not worn outside the facility.

4. Protective eyewear is worn when conducting procedures that have 

the potential to create splashes of microorganisms or other hazardous 

materials. Persons who wear contact lenses should also wear eye 

protection when entering areas with potentially high concentrations or 

airborne particulates.

Persons having contact with NHPs must assess risk of mucous 

membrane exposure and wear protective equipment (e.g., masks, 

goggles, face shields, etc.) as appropriate for the task to be performed.

5. Gloves are worn to protect hands from exposure to hazardous materials.

A risk assessment should be performed to identify the appropriate glove 

for the task and alternatives to latex gloves should be available.

Change gloves when contaminated, glove integrity is compromised, 

or when otherwise necessary.

Gloves must not be worn outside the animal rooms.

Gloves and personal protective equipment should be removed in a 

manner that prevents transfer of infectious materials.

Do not wash or reuse disposable gloves. Dispose of used gloves with 

other contaminated waste.

6. Persons must wash their hands after handling animals and before leaving 

the areas where infectious materials and/or animals are housed or are 

manipulated. Hand washing should occur after the removal of gloves.

D. Laboratory Facilities (Secondary Barriers)

1. The animal facility is separated from areas that are open to unrestricted 

personnel traffic within the building. External facility doors are selfclosing and self-locking.

Access to the animal facility is restricted.
66 Biosafety in Microbiological and Biomedical Laboratories

Doors to areas where infectious materials and/or animals are housed, 

open inward, are self-closing, are kept closed when experimental animals 

are present, and should never be propped open. Doors to cubicles inside 

an animal room may open outward or slide horizontally or vertically.

2. The animal facility must have a sink for hand washing.

Sink traps are filled with water, and/or appropriate liquid to prevent the 

migration of vermin and gases.

3. The animal facility is designed, constructed, and maintained to facilitate 

cleaning and housekeeping. The interior surfaces (walls, floors and 

ceilings) are water resistant. Floors must be slip resistant, impervious 

to liquids, and resistant to chemicals.

It is recommended that penetrations in floors, walls and ceiling surfaces 

be sealed, including openings around ducts, doors and doorframes, to 

facilitate pest control and proper cleaning.

4. Cabinets and bench tops must be impervious to water and resistant to 

heat, organic solvents, acids, alkalis, and other chemicals. Spaces between 

benches, cabinets, and equipment should be accessible for cleaning.

Chairs used in animal area must be covered with a non-porous material 

that can be easily cleaned and decontaminated. Furniture must be 

capable of supporting anticipated loads and uses. Sharp edges and 

corners should be avoided.

5. External windows are not recommended; if present windows must be 

resistant to breakage. Where possible, windows should be sealed. If the 

animal facility has windows that open, they are fitted with fly screens. 

The presence of windows may impact facility security and therefore 

should be assessed by security personnel.

6. Ventilation should be provided in accordance with the Guide for Care and 

Use of Laboratory Animals.

1

 No recirculation of exhaust air may occur. 

It is recommended that animal rooms have inward directional airflow.

Ventilation system design should consider the heat and high moisture 

load produced during the cleaning of animal rooms and the cage 

wash process.

7. Internal facility appurtenances, such as light fixtures, air ducts, and utility 

pipes, are arranged to minimize horizontal surface areas to facilitate 

cleaning and minimize the accumulation of debris or fomites.

8. If floor drains are provided, the traps are filled with water, and/or 

appropriate disinfectant to prevent the migration of vermin and gases.
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-2 67

9. Cages are washed manually or preferably in a mechanical cage washer. 

The mechanical cage washer should have a final rinse temperature of at 

least 180°F. If manual cage washing is utilized, ensure that appropriate 

disinfectants are selected.

10. Illumination is adequate for all activities, avoiding reflections and glare 

that could impede vision.

11. Emergency eyewash and shower are readily available; location is 

determined by risk assessment.

Animal Biosafety Level 2

Animal Biosafety Level 2 builds upon the practices, procedures, containment 

equipment, and facility requirements of ABSL-1. ABSL-2 is suitable for work involving 

laboratory animals infected with agents associated with human disease and pose 

moderate hazards to personnel and the environment. It also addresses hazards 

from ingestion as well as from percutaneous and mucous membrane exposure.

ABSL-2 requires that: 1) access to the animal facility is restricted; 2) personnel 

must have specific training in animal facility procedures, the handling of infected 

animals and the manipulation of pathogenic agents; 3) personnel must be supervised 

by individuals with adequate knowledge of potential hazards, microbiological 

agents, animal manipulations and husbandry procedures; and 4) BSCs or other 

physical containment equipment is used when procedures involve the manipulation 

of infectious materials, or where aerosols or splashes may be created.

Appropriate personal protective equipment must be utilized to reduce 

exposure to infectious agents, animals, and contaminated equipment. Implementation 

of employee occupational health programs should be considered.

The following standard and special practices, safety equipment, and facility 

requirements apply to ABSL-2:

A. Standard Microbiological Practices

1. The animal facility director establishes and enforces policies, 

procedures, and protocols for institutional policies and emergencies.

Each organization must assure that worker safety and health concerns 

are addressed as part of the animal protocol review.

Prior to beginning a study, animal protocols must also be reviewed 

and approved by the IACUC5

 and the Institutional Biosafety Committee.

2. A safety manual specific to the animal facility is prepared or adopted 

in consultation with the animal facility director and appropriate safety 

professionals.
68 Biosafety in Microbiological and Biomedical Laboratories

The safety manual must be available and accessible. Personnel are 

advised of potential hazards, and are required to read and follow 

instructions on practices and procedures.

Consideration should be given to specific biohazards unique to the 

animal species and protocol in use.

3. The supervisor must ensure that animal care, laboratory, and support 

personnel receive appropriate training regarding their duties, animal 

husbandry procedure, potential hazards, manipulations of infectious 

agents, necessary precautions to prevent hazard or exposures, and 

hazard/exposure evaluation procedures (physical hazards, splashes, 

aerosolization, etc.). Personnel must receive annual updates or 

additional training when procedures or policies change. Records are 

maintained for all hazard evaluations, employee training sessions 

and staff attendance.

4. An appropriate medical surveillance program is in place, as determined 

by risk assessment. The need for an animal allergy prevention program 

should be considered.

Facility supervisors should ensure that medical staff is informed of 

potential occupational hazards within the animal facility, to include 

those associated with research, animal husbandry duties, animal 

care and manipulations.

Personal health status may impact an individual’s susceptibility to 

infection, ability to receive immunizations or prophylactic interventions. 

Therefore, all personnel and particularly women of childbearing age 

should be provided information regarding immune competence and 

conditions that may predispose them to infection. Individuals having 

these conditions should be encouraged to self-identify to the institution’s 

healthcare provider for appropriate counseling and guidance.

Personnel using respirators must be enrolled in an appropriately 

constituted respiratory protection program.

5. A sign incorporating the universal biohazard symbol must be posted 

at the entrance to areas where infectious materials and/ or animals 

are housed or are manipulated when infectious agents are present. 

The sign must include the animal biosafety level, general occupational 

health requirements, personal protective equipment requirements, the 

supervisor’s name (or names of other responsible personnel), telephone 

number, and required procedures for entering and exiting the animal 

areas. Identification of all infectious agents is necessary when more 

than one agent is being used within an animal room.
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-2 69

Security-sensitive agent information and occupational health requirements 

should be posted in accordance with the institutional policy.

Advance consideration should be given to emergency and disaster 

recovery plans, as a contingency for man-made or natural disasters.1,3,4

6. Access to the animal room is limited. Only those persons required for 

program or support purposes are authorized to enter the animal facility 

and the areas where infectious materials and/or animals are housed 

or manipulated.

All persons including facility personnel, service workers, and visitors are 

advised of the potential hazards (physical, naturally occurring, or research 

pathogens, allergens, etc.) and are instructed on the appropriate safeguards.

7. Protective laboratory coats, gowns, or uniforms are recommended to 

prevent contamination of personal clothing.

Gloves are worn to prevent skin contact with contaminated, infectious 

and hazardous materials and when handling animals.

Gloves and personal protective equipment should be removed in a manner 

that prevents transfer of infectious materials outside of the areas where 

infectious materials and/or animals are housed or are manipulated.

Persons must wash their hands after removing gloves, and before 

leaving the areas where infectious materials and/or animals are housed 

or are manipulated.

Eye, face and respiratory protection should be used in rooms containing 

infected animals, as dictated by the risk assessment.

8. Eating, drinking, smoking, handling contact lenses, applying cosmetics, 

and storing food for human consumption must not be permitted in 

laboratory areas. Food must be stored outside of the laboratory in 

cabinets or refrigerators designated and used for this purpose.

9. All procedures are carefully performed to minimize the creation of 

aerosols or splatters of infectious materials and waste.

10. Mouth pipetting is prohibited. Mechanical pipetting devices must be 

used.

11. Policies for the safe handling of sharps, such as needles, scalpels, pipettes, 

and broken glassware must be developed and implemented. When 

applicable, laboratory supervisors should adopt improved engineering 

and work practice controls that reduce the risk of sharps injuries. 

Precautions must always be taken with sharp items. These include:
70 Biosafety in Microbiological and Biomedical Laboratories

a. The use of needles and syringes or other sharp instruments in the 

animal facility is limited to situations where there is no alternative 

such as parenteral injection, blood collection, or aspiration of fluids 

from laboratory animals and diaphragm bottles.

b. Disposable needles must not be bent, sheared, broken, recapped, 

removed from disposable syringes, or otherwise manipulated by hand 

before disposal. Used, disposable needles must be carefully placed 

in puncture-resistant containers used for sharps disposal. Sharps 

containers should be located as close to the work site as possible.

c. Non-disposable sharps must be placed in a hard-walled container 

for transport to a processing area for decontamination, preferably 

by autoclaving.

d. Broken glassware must not be handled directly; it should be 

removed using a brush and dustpan, tongs, or forceps. Plastic 

ware should be substituted for glassware whenever possible.

e. Use of equipment with sharp edges and corners should be avoided.

12. Equipment and work surfaces are routinely decontaminated with an 

appropriate disinfectant after work with an infectious agent, and after 

any spills, splashes, or other overt contamination.

13. Animals and plants not associated with the work being performed must 

not be permitted in the areas where infectious materials and/ or animals 

are housed or manipulated.

14. An effective integrated pest management program is required. 

(See Appendix G.)

15. All wastes from the animal room (including animal tissues, carcasses, 

and bedding) are transported from the animal room in leak-proof 

containers for appropriate disposal in compliance with applicable 

institutional, local and state requirements.

Decontaminate all potentially infectious materials before disposal 

using an effective method.

B. Special Practices

1. Animal care staff, laboratory and routine support personnel must 

be provided a medical surveillance program as dictated by the risk 

assessment and administered appropriate immunizations for agents 

handled or potentially present, before entry into animal rooms.

When appropriate, a base line serum sample should be stored.
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-2 71

2. Procedures involving a high potential for generating aerosols should 

be conducted within a biosafety cabinet or other physical containment 

device. When a procedure cannot be performed within a biosafety 

cabinet, a combination of personal protective equipment and other 

containment devices must be used.

Restraint devices and practices that reduce the risk of exposure during 

animal manipulations (e.g., physical restraint devices, chemical restraint 

medications) should be used whenever possible.

3. Decontamination by an appropriate method (e.g. autoclave, chemical 

disinfection, or other approved decontamination methods) is necessary for 

all potentially infectious materials and animal waste before movement 

outside the areas where infectious materials and/or animals are housed 

or are manipulated. This includes potentially infectious animal tissues, 

carcasses, contaminated bedding, unused feed, sharps, and other refuse.

A method for decontaminating routine husbandry equipment, sensitive 

electronic and medical equipment should be identified and implemented.

Materials to be decontaminated outside of the immediate areas where 

infectious materials and/or animals are housed or are manipulated must 

be placed in a durable, leak proof, covered container and secured for 

transport. The outer surface of the container is disinfected prior to moving 

materials. The transport container must have a universal biohazard label.

Develop and implement an appropriate waste disposal program in 

compliance with applicable institutional, local and state requirements. 

Autoclaving of content prior to incineration is recommended.

4. Equipment, cages, and racks should be handled in a manner that 

minimizes contamination of other areas.

Equipment must be decontaminated before repair, maintenance, or 

removal from the areas where infectious materials and/or animals are 

housed or are manipulated.

5. Spills involving infectious materials must be contained, decontaminated, 

and cleaned up by staff properly trained and equipped to work with 

infectious material.

6. Incidents that may result in exposure to infectious materials must be 

immediately evaluated and treated according to procedures described 

in the safety manual. All such incidents must be reported to the animal 

facility supervisor or personnel designated by the institution. Medical 

evaluation, surveillance, and treatment should be provided as 

appropriate and records maintained.
72 Biosafety in Microbiological and Biomedical Laboratories

C. Safety Equipment (Primary Barriers and Personal Protective Equipment)

1. Properly maintained BSCs, personal protective equipment (e.g., 

gloves, lab coats, face shields, respirators, etc.) and/or other physical 

containment devices or equipment, are used whenever conducting 

procedures with a potential for creating aerosols, splashes, or other 

potential exposures to hazardous materials. These include necropsy of 

infected animals, harvesting of tissues or fluids from infected animals 

or eggs, and intranasal inoculation of animals.

When indicated by risk assessment, animals are housed in primary 

biosafety containment equipment appropriate for the animal species, such 

as solid wall and bottom cages covered with filter bonnets for rodents or 

other equivalent primary containment systems for larger animal cages.

2. A risk assessment should determine the appropriate type of personal 

protective equipment to be utilized.

Scrub suits and uniforms are removed before leaving the animal facility. 

Reusable clothing is appropriately contained and decontaminated 

before being laundered. Laboratory and protective clothing should 

never be taken home.

Gowns, uniforms, laboratory coats and personal protective equipment 

are worn while in the areas where infectious materials and/or animals 

are housed or manipulated and removed prior to exiting. Disposable 

personal protective equipment and other contaminated waste are 

appropriately contained and decontaminated prior to disposal.

3. Eye and face protection (mask, goggles, face shield or other splatter 

guard) are used for manipulations or activities that may result in 

splashes or sprays from infectious or other hazardous materials and 

when the animal or microorganisms must be handled outside the BSC 

or containment device. Eye and face protection must be disposed of 

with other contaminated laboratory waste or decontaminated before 

reuse. Persons who wear contact lenses should also wear eye 

protection when entering areas with potentially high concentrations or 

airborne particulates.

Persons having contact with NHPs should assess risk of mucous 

membrane exposure and wear protective equipment (e.g., masks, 

goggles, face shields) appropriate for the task to be performed. 

Respiratory protection is worn based upon risk assessment.

4. Gloves are worn to protect hands from exposure to hazardous materials. 

A risk assessment should be performed to identify the appropriate glove 

for the task and alternatives to latex gloves should be available.
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-2 73

Gloves are changed when contaminated, glove integrity is compromised, 

or when otherwise necessary.

Gloves must not be worn outside the animal rooms.

Gloves and personal protective equipment should be removed in a 

manner that prevents transfer of infectious materials.

Do not wash or reuse disposable gloves. Dispose of used gloves with 

other contaminated waste.

Persons must wash their hands after handling animals and before leaving 

the areas where infectious materials and/or animals are housed or are 

manipulated. Hand washing should occur after the removal of gloves.

D. Laboratory Facilities (Secondary Barriers)

1. The animal facility is separated from areas that are open to unrestricted 

personnel traffic within the building. External facility doors are self-closing 

and self-locking.

Doors to areas where infectious materials and/or animals are housed, open 

inward, are self-closing, are kept closed when experimental animals are 

present, and should never be propped open. Doors to cubicles inside an 

animal room may open outward or slide horizontally or vertically.

2. A hand-washing sink is located at the exit of the areas where infectious 

materials and/or animals are housed or are manipulated. Additional 

sinks for hand washing should be located in other appropriate locations 

within the facility.

If the animal facility has segregated areas where infectious materials 

and/or animals are housed or manipulated, a sink must also be 

available for hand washing at the exit from each segregated area.

Sink traps are filled with water, and/or appropriate disinfectant to 

prevent the migration of vermin and gases.

3. The animal facility is designed, constructed, and maintained to facilitate 

cleaning and housekeeping. The interior surfaces (walls, floors and 

ceilings) are water resistant.

Penetrations in floors, walls and ceiling surfaces are sealed, including 

openings around ducts, doors and doorframes, to facilitate pest control 

and proper cleaning.

Floors must be slip-resistant, impervious to liquids, and resistant 

to chemicals.
74 Biosafety in Microbiological and Biomedical Laboratories

4. Cabinets and bench tops must be impervious to water and resistant to 

heat, organic solvents, acids, alkalis, and other chemicals. Spaces between 

benches, cabinets, and equipment should be accessible for cleaning.

Furniture should be minimized. Chairs used in animal area must be 

covered with a non-porous material that can be easily cleaned and 

decontaminated. Furniture must be capable of supporting anticipated 

loads and uses. Sharp edges and corners should be avoided.

5. External windows are not recommended; if present, windows must be 

sealed and resistant to breakage. The presence of windows may impact 

facility security and therefore should be assessed by security personnel.

6. Ventilation should be provided in accordance with the Guide for Care 

and Use of Laboratory Animals.

1

 The direction of airflow into the animal 

facility is inward; animal rooms maintain inward directional airflow 

compared to adjoining hallways. A ducted exhaust air ventilation 

system is provided. Exhaust air is discharged to the outside without 

being recirculated to other rooms.

Ventilation system design should consider the heat and high moisture 

load produced during the cleaning of animal rooms and the cage 

wash process.

7. Internal facility appurtenances, such as light fixtures, air ducts, and 

utility pipes, are arranged to minimize horizontal surface areas, to 

facilitate cleaning and minimize the accumulation of debris or fomites.

8. Floor drains must be maintained and filled with water, and/or appropriate 

disinfectant to prevent the migration of vermin and gases.

9. Cages should be autoclaved or otherwise decontaminated prior to 

washing. Mechanical cage washer should have a final rinse temperature 

of at least 180°F. The cage wash area should be designed to 

accommodate the use of high-pressure spray systems, humidity, 

strong chemical disinfectants and 180°F water temperatures during 

the cage/equipment cleaning process.

10. Illumination is adequate for all activities, avoiding reflections and glare 

that could impede vision.

11. If BSCs are present, they must be installed so that fluctuations of the 

room air supply and exhaust do not interfere with proper operations. 

BSCs should be located away from doors, heavily traveled laboratory 

areas, and other possible airflow disruptions.
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-3 75

HEPA filtered exhaust air from a Class II BSC can be safely re-circulated 

back into the laboratory environment if the cabinet is tested and certified at 

least annually and operated according to manufacturer’s recommendations. 

BSCs can also be connected to the laboratory exhaust system by 

either a thimble (canopy) connection or directly to the outside through 

an independent, hard connection. Provisions to assure proper safety 

cabinet performance and air system operation must be verified. BSCs 

should be recertified at least once a year to ensure correct performance.

All BSCs should be used according to manufacturer’s specifications to 

protect the worker and avoid creating a hazardous environment from 

volatile chemicals and gases.

12. If vacuum service (i.e., central or local) is provided, each service 

connection should be fitted with liquid disinfectant traps and an in-line 

HEPA filter placed as near as practicable to each use point or service cock. 

Filters are installed to permit in-place decontamination and replacement.

13. An autoclave should be present in the animal facility to facilitate 

decontamination of infectious materials and waste.

14. Emergency eyewash and shower are readily available; location is 

determined by risk assessment.

Animal Biosafety Level 3

Animal Biosafety Level 3 involves practices suitable for work with laboratory 

animals infected with indigenous or exotic agents, agents that present a potential 

for aerosol transmission, and agents causing serious or potentially lethal disease. 

ABSL-3 builds upon the standard practices, procedures, containment equipment, 

and facility requirements of ABSL-2.

The ABSL-3 laboratory has special engineering and design features. 

ABSL-3 requires that: 1) access to the animal facility is restricted; 2) personnel 

must have specific training in animal facility procedures, the handling of infected 

animals, and the manipulation of potentially lethal agents; 3) personnel must 

be supervised by individuals with adequate knowledge of potential hazards, 

microbiological agents, animal manipulations, and husbandry procedures; and 

4) procedures involving the manipulation of infectious materials, or where 

aerosols or splashes may be created, must be conducted in BSCs or by use 

of other physical containment equipment.

Appropriate personal protective equipment must be utilized to reduce 

exposure to infectious agents, animals, and contaminated equipment. 

Employee occupational health programs must be implemented.
76 Biosafety in Microbiological and Biomedical Laboratories

The following standard and special safety practices, safety equipment, and 

facility requirements apply to ABSL-3.

A. Standard Microbiological Practices

1. The animal facility director establishes and enforces policies, 

procedures, and protocols for institutional policies and emergencies.

Each institute must assure that worker safety and health concerns are 

addressed as part of the animal protocol review.

Prior to beginning a study, animal protocols must be reviewed and 

approved by the IACUC5 and the Institutional Biosafety Committee.

2. A safety manual specific to the animal facility is prepared or adopted 

in consultation with the animal facility director and appropriate 

safety professionals.

The safety manual must be available and accessible. Personnel are 

advised of potential and special hazards, and are required to read 

and follow instructions on practices and procedures.

Consideration must be given to specific biohazards unique to the 

animal species and protocol in use.

3. The supervisor must ensure that animal care, laboratory and support 

personnel receive appropriate training regarding their duties, animal 

husbandry procedures, potential hazards, manipulations of infectious 

agents, necessary precautions to prevent hazard or exposures, and 

hazard/exposure evaluation procedures (physical hazards, splashes, 

aerosolization, etc.). Personnel must receive annual updates or additional 

training when procedures or policies change. Records are maintained for 

all hazard evaluations, employee training sessions and staff attendance.

4. An appropriate medical surveillance program is in place, as determined 

by risk assessment. The need for an animal allergy prevention program 

should be considered.

Facility supervisors should ensure that medical staff is informed of potential 

occupational hazards within the animal facility, to include those associated 

with the research, animal husbandry duties, animal care, and manipulations.

Personal health status may impact an individual’s susceptibility to 

infection, ability to receive immunizations or prophylactic interventions. 

Therefore, all personnel and particularly women of childbearing age 

should be provided information regarding immune competence and 

conditions that may predispose them to infection. Individuals having 
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-3 77

these conditions should be encouraged to self-identify to the institution’s 

healthcare provider for appropriate counseling and guidance.

Personnel using respirators must be enrolled in an appropriately 

constituted respiratory protection program.

5. A sign incorporating the universal biohazard symbol must be posted 

at the entrance to areas where infectious materials and/or animals are 

housed or are manipulated. The sign must include the animal biosafety 

level, general occupational health requirements, personal protective 

equipment requirements, the supervisor’s name (or other responsible 

personnel), telephone number, and required procedures for entering and 

exiting the animal areas. Identification of specific infectious agents is 

recommended when more than one agent is used within an animal room.

Security-sensitive agent information and occupational health requirements 

should be posted in accordance with the institutional policy.

Advance consideration should be given to emergency and disaster 

recovery plans, as a contingency for man-made or natural disasters.1,3,4

6. Access to the animal room is limited to the fewest number of individuals 

possible. Only those persons required for program or support purposes 

are authorized to enter the animal facility and the areas where infectious 

materials and/or animals are housed or are manipulated.

All persons, including facility personnel, service workers, and visitors, 

are advised of the potential hazards (natural or research pathogens, 

allergens, etc.) and are instructed on the appropriate safeguards.

7. Protective laboratory coats, gowns, or uniforms are recommended to 

prevent contamination of personal clothing.

Gloves are worn to prevent skin contact with contaminated, infectious/

hazardous materials and when handling animals. Double-glove 

practices should be used when dictated by risk assessment.

Gloves and personal protective equipment should be removed in a 

manner that prevents transfer of infectious materials outside of the areas 

where infectious materials and/or animals are housed or are manipulated.

Persons must wash their hands after removing gloves and before 

leaving the areas where infectious materials and/or animals are 

housed or are manipulated.

Eye, face and respiratory protection should be used in rooms containing 

infected animals, as dictated by the risk assessment.
78 Biosafety in Microbiological and Biomedical Laboratories

8. Eating, drinking, smoking, handling contact lenses, applying cosmetics, 

and storing food for human consumption must not be permitted in 

laboratory areas. Food must be stored outside the laboratory area in 

cabinets or refrigerators designated and used for this purpose.

9. All procedures are carefully performed to minimize the creation of 

aerosols or splatters of infectious materials and waste.

10. Mouth pipetting is prohibited. Mechanical pipetting devices must be used.

11. Policies for the safe handling of sharps, such as needles, scalpels, 

pipettes, and broken glassware must be developed and implemented.

When applicable, laboratory supervisors should adopt improved 

engineering and work practice controls that reduce the risk of sharps 

injuries. Precautions must always be taken with sharp items. These include:

a. Use of needles and syringes or other sharp instruments in the 

animal facility is limited to situations where there is no alternative 

such as parenteral injection, blood collection, or aspiration of fluids 

from laboratory animals and diaphragm bottles.

b. Disposable needles must not be bent, sheared, broken, recapped, 

removed from disposable syringes, or otherwise manipulated by hand 

before disposal. Used, disposable needles must be carefully placed 

in puncture-resistant containers used for sharps disposal. Sharps 

containers should be located as close to the work site as possible.

c. Non-disposable sharps must be placed in a hard-walled container 

for transport to a processing area for decontamination, preferably 

by autoclaving.

d. Broken glassware must not be handled directly; it should be 

removed using a brush and dustpan, tongs, or forceps. Plastic 

ware should be substituted for glassware whenever possible.

e. Use of equipment with sharp edges and corners should be avoided.

12. Equipment and work surfaces are routinely decontaminated with an 

appropriate disinfectant after work with an infectious agent, and after 

any spills, splashes, or other overt contamination.

13. Animals and plants not associated with the work being performed must 

not be permitted in the areas where infectious materials and/ or animals 

are housed or are manipulated.

14. An effective integrated pest management program is required. 

(See Appendix G.)
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-3 79

15. All wastes from the animal room (including animal tissues, carcasses, 

and bedding) are transported from the animal room in leak-proof 

containers for appropriate disposal in compliance with applicable 

institutional, local and state requirements.

Decontaminate all potentially infectious materials before disposal using 

an effective method.

B. Special Practices

1. Animal care staff, laboratory and routine support personnel must 

be provided a medical surveillance program as dictated by the risk 

assessment and administered appropriate immunizations for agents 

handled or potentially present, before entry into animal rooms.

When appropriate, a base line serum sample should be stored.

2. All procedures involving the manipulation of infectious materials, handling 

of infected animals or the generation of aerosols must be conducted 

within BSCs or other physical containment devices when practical.

When a procedure cannot be performed within a biosafety cabinet, a 

combination of personal protective equipment and other containment 

devices must be used.

Restraint devices and practices are used to reduce the risk of exposure 

during animal manipulations (e.g., physical restraint devices, chemical 

restraint medications).

3. The risk of infectious aerosols from infected animals or their bedding 

also can be reduced if animals are housed in containment caging 

systems, such as solid wall and bottom cages covered with filter 

bonnets, open cages placed in inward flow ventilated enclosures, 

HEPA-filter isolators and caging systems, or other equivalent primary 

containment systems.

4. Actively ventilated caging systems must be designed to prevent the 

escape of microorganisms from the cage. Exhaust plenums for these 

systems should be sealed to prevent escape of microorganisms if the 

ventilation system becomes static, and the exhaust must be HEPA 

filtered. Safety mechanisms should be in place that prevent the cages 

and exhaust plenums from becoming positive to the surrounding area 

should the exhaust fan fail. The system should also be alarmed to 

indicate operational malfunctions.

5. A method for decontaminating all infectious materials must be available 

within the facility, preferably within the areas where infectious materials 
80 Biosafety in Microbiological and Biomedical Laboratories

and/or animals are housed or are manipulated (e.g., autoclave, 

chemical disinfection, or other approved decontamination methods).

Consideration must be given to means for decontaminating routine 

husbandry equipment, sensitive electronic and medical equipment.

Decontaminate all potential infectious materials (including animal 

tissues, carcasses, contaminated bedding, unused feed, sharps, and 

other refuse) by an appropriate method before removal from the areas 

where infectious materials and/or animals are housed or manipulated.

It is recommended that animal bedding and waste be decontaminated 

prior to manipulation and before removal from the areas where 

infectious materials and/or animals are housed or are manipulated, 

preferably within the caging system.

Develop and implement an appropriate waste disposal program in 

compliance with applicable institutional, local and state requirements.

6. Equipment, cages, and racks should be handled in a manner that 

minimizes contamination of other areas.

Equipment must be decontaminated before repair, maintenance, or 

removal from the areas where infectious materials and/or animals are 

housed or are manipulated.

Spills involving infectious materials must be contained, decontaminated, 

and cleaned up by staff properly trained and equipped to work with 

infectious material.

7. Incidents that may result in exposure to infectious materials must be 

immediately evaluated and treated according to procedures described 

in the safety manual. All such incidents must be reported to the animal 

facility supervisor or personnel designated by the institution. Medical 

evaluation, surveillance, and treatment should be provided as 

appropriate and records maintained.

C. Safety Equipment (Primary Barriers and Personal Protective Equipment)

1. Properly maintained BSCs and other physical containment devices or 

equipment should be used for all manipulations for infectious materials 

and when possible, animals. These manipulations include necropsy, 

harvesting of tissues or fluids from infected animals or eggs, and 

intranasal inoculation of animals.

The risk of infectious aerosols from infected animals or bedding can be 

reduced by primary barrier systems. These systems may include solid 
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-3 81

wall and bottom cages covered with filter bonnets, ventilated cage rack 

systems, or for larger cages placed in inward flow ventilated enclosures 

or other equivalent systems or devices.

2. A risk assessment should determine the appropriate type of personal 

protective equipment to be utilized.

Personnel within the animal facility where protective clothing, such as 

uniforms or scrub suits. Reusable clothing is appropriately contained 

and decontaminated before being laundered. Laboratory and protective 

clothing should never be taken home. Disposable personal protective 

equipment such as non-woven olefin cover-all suits, wrap-around or 

solid-front gowns should be worn over this clothing, before entering 

the areas where infectious materials and/or animals are housed or 

manipulated. Front-button laboratory coats are unsuitable.

Disposable personal protective equipment must be removed when 

leaving the areas where infectious materials and/or animals are housed 

or are manipulated. Scrub suits and uniforms are removed before 

leaving the animal facility.

Disposable personal protective equipment and other contaminated 

waste are appropriately contained and decontaminated prior to disposal.

3. All personnel entering areas where infectious materials and/or animals 

are housed or manipulated wear appropriate eye, face and respiratory 

protection. To prevent cross contamination, boots, shoe covers, or other 

protective footwear, are used where indicated.

Eye and face protection must be disposed of with other contaminated 

laboratory waste or decontaminated before reuse. Persons who wear 

contact lenses should also wear eye protection when entering areas 

with potentially high concentrations or airborne particulates.

4. Gloves are worn to protect hands from exposure to hazardous materials.

A risk assessment should be performed to identify the appropriate 

glove for the task and alternatives to latex gloves should be available.

Procedures may require the use of wearing two pairs of gloves 

(double-glove).

Gloves are changed when contaminated, glove integrity is compromised, 

or when otherwise necessary.

Gloves must not be worn outside the animal rooms.
82 Biosafety in Microbiological and Biomedical Laboratories

Gloves and personal protective equipment should be removed in a 

manner that prevents transfer of infectious materials.

Do not wash or reuse disposable gloves. Dispose of used gloves with 

other contaminated waste.

Persons must wash their hands after handling animals and before leaving 

the areas where infectious materials and/or animals are housed or are 

manipulated. Hand washing should occur after the removal of gloves.

D. Laboratory Facilities (Secondary Barriers)

1. The animal facility is separated from areas that are open to unrestricted 

personnel traffic within the building. External facility doors are selfclosing and self-locking.

Access to the animal facility is restricted.

Doors to areas where infectious materials and/or animals are housed, 

open inward, are self-closing, are kept closed when experimental 

animals are present, and should never be propped open.

Entry into the containment area is via a double-door entry, which 

constitutes an anteroom/airlock and a change room. Showers may be 

considered based on risk assessment. An additional double-door access 

anteroom or double-doored autoclave may be provided for movement of 

supplies and wastes into and out of the facility.

2. A hand-washing sink is located at the exit of the areas where infectious 

materials and/or animals are housed or are manipulated. Additional 

sinks for hand washing should be located in other appropriate locations 

within the facility. The sink should be hands-free or automatically operated.

If the animal facility has multiple segregated areas where infectious 

materials and/or animals are housed or are manipulated, a sink must also 

be available for hand washing at the exit from each segregated area.

Sink traps are filled with water, and/or appropriate liquid to prevent the 

migration of vermin and gases.

3. The animal facility is designed, constructed, and maintained to facilitate 

cleaning, decontamination and housekeeping. The interior surfaces 

(walls, floors and ceilings) are water resistant.

Penetrations in floors, walls and ceiling surfaces are sealed, including 

openings around ducts and doorframes, to facilitate pest control, proper 

cleaning and decontamination. Walls, floors and ceilings should form a 

sealed and sanitizable surface.
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-3 83

Floors must be slip resistant, impervious to liquids, and resistant to 

chemicals. Flooring is seamless, sealed resilient or poured floors, with 

integral cove bases.

Decontamination of an entire animal room should be considered when 

there has been gross contamination of the space, significant changes 

in usage, for major renovations, or maintenance shut downs. Selection 

of the appropriate materials and methods used to decontaminate the 

animal room must be based on the risk assessment.

4. Cabinets and bench tops must be impervious to water and resistant 

to heat, organic solvents, acids, alkalis, and other chemicals. Spaces 

between benches, cabinets, and equipment should be accessible 

for cleaning.

Furniture should be minimized. Chairs used in animal areas must be 

covered with a non-porous material that can be easily cleaned and 

decontaminated. Furniture must be capable of supporting anticipated 

loads and uses. Equipment and furnishings with sharp edges and 

corners should be avoided.

5. External windows are not recommended; if present, all windows must 

be sealed and must be resistant to breakage. The presence of windows 

may impact facility security and therefore should be assessed by 

security personnel.

6. Ventilation of the facility should be provided in accordance with the 

Guide for Care and Use of Laboratory Animals.

1

 The direction of 

airflow into the animal facility is inward; animal rooms maintain inward 

directional airflow compared to adjoining hallways. A ducted exhaust 

air ventilation system is provided. Exhaust air is discharged to the 

outside without being recirculated to other rooms. This system creates 

directional airflow, which draws air into the animal room from “clean” 

areas and toward “contaminated” areas.

Ventilation system design should consider the heat and high moisture 

load produced during the cleaning of animal rooms and the cage wash 

process. HEPA filtration and other treatments of the exhaust air may 

not be required, but should be considered based on site requirements, 

specific agent manipulations and use conditions. The exhaust must be 

dispersed away from occupied areas and air intakes.

Personnel must verify that the direction of the airflow (into the animal 

areas) is proper. It is recommended that a visual monitoring device that 

indicates directional inward airflow be provided at the animal room entry. 

The ABSL-3 animal facility shall be designed such that under failure 
84 Biosafety in Microbiological and Biomedical Laboratories

conditions the airflow will not be reversed. Alarms should be considered 

to notify personnel of ventilation and HVAC system failure.

7. Internal facility appurtenances, such as light fixtures, air ducts, and utility 

pipes, are arranged to minimize horizontal surface areas, to facilitate 

cleaning and minimize the accumulation of debris or fomites.

8. Floor drains must be maintained and filled with water, and/or appropriate 

disinfectant to prevent the migration of vermin and gases.

9. Cages are washed in a mechanical cage washer. The mechanical cage 

washer has a final rinse temperature of at least 180°F. Cages should be 

autoclaved or otherwise decontaminated prior to removal from ABSL-3 

space. The cage wash facility should be designed and constructed to 

accommodate high-pressure spray systems, humidity, strong chemical 

disinfectants and 180°F water temperatures during the cage cleaning 

process.

10. Illumination is adequate for all activities, avoiding reflections and glare 

that could impede vision.

11. BSCs (Class II, Class III) must be installed so that fluctuations of the 

room air supply and exhaust do not interfere with proper operations. 

Class II BSCs should be located away from doors, heavily traveled 

laboratory areas, and other possible airflow disruptions.

HEPA filtered exhaust air from a Class II BSC can be safely re-circulated 

into the laboratory environment if the cabinet is tested and certified at 

least annually and operated according to manufacturer’s recommendations. 

BSCs can also be connected to the laboratory exhaust system by either 

a thimble (canopy) connection or exhausted directly to the outside 

through a direct (hard) connection. Provisions to assure proper safety 

cabinet performance and air system operation must be verified. BSCs 

should be certified at least annually to assure correct performance.

Class III BSCs must supply air in such a manner that prevents positive 

pressurization of the cabinet or the laboratory room.

All BSCs should be used according to manufacturers’ specifications.

When applicable, equipment that may produce infectious aerosols must 

be contained in devices that exhaust air through HEPA filtration or other 

equivalent technology before being discharged into the animal facility. 

These HEPA filters should be tested and/or replaced at least annually.

12. An autoclave is available which is convenient to the animal rooms where 

the biohazard is contained. The autoclave is utilized to decontaminate 
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-4 85

infectious materials and waste before moving it to the other areas of 

the facility. If not convenient to areas where infectious materials and/

or animals are housed or are manipulated, special practices should be 

developed for transport of infectious materials to designated alternate 

location/s within the facility.

13. Emergency eyewash and shower are readily available; location is 

determined by risk assessment.

14. The ABSL-3 facility design and operational procedures must be 

documented. The facility must be tested to verify that the design and 

operational parameters have been met prior to use. Facilities should be 

re-verified at least annually against these procedures as modified by 

operational experience.

15. Additional environmental protection (e.g., personnel showers, HEPA 

filtration of exhaust air, containment of other piped services, and the 

provision of effluent decontamination) should be considered if recommended 

by the agent summary statement, as determined by risk assessment of 

the site conditions, or other applicable federal, state or local regulations.

Animal Biosafety Level 4

Animal Biosafety Level 4 is required for work with animals infected with dangerous 

and exotic agents that pose a high individual risk of aerosol-transmitted laboratory 

infections and life-threatening disease that is frequently fatal, for which there are 

no vaccines or treatments; or a related agent with unknown risk of transmission. 

Agents with a close or identical antigenic relationship to agents requiring ABSL-4 

containment must be handled at this level until sufficient data are obtained either 

to confirm continued work at this level, or to re-designate the level. Animal care 

staff must have specific and thorough training in handling extremely hazardous, 

infectious agents and infected animals. Animal care staff must understand the 

primary and secondary containment functions of standard and special practices, 

containment equipment, and laboratory design characteristics. All animal care 

staff and supervisors must be competent in handling animals, agents and 

procedures requiring ABSL-4 containment. The animal facility director and/or 

laboratory supervisor control access to the animal facility within the ABSL-4 

laboratory in accordance with institutional policies.

There are two models for ABSL-4 laboratories:

1. A Cabinet Laboratory—All handling of agents, infected animals and 

housing of infected animals must be performed in Class III BSCs 

(see Appendix A); and

2. A Suit Laboratory—Personnel must wear a positive pressure protective 

suit (see Appendix A); infected animals must be housed in ventilated 

enclosures with inward directional airflow and HEPA filtered exhaust; 
86 Biosafety in Microbiological and Biomedical Laboratories

and infected animals should be handled within a primary barrier system, 

such as a Class II BSC or other equivalent containment system.

ABSL-4 builds upon the standard practices, procedures, containment 

equipment, and facility requirements of ABSL-3. However, ABSL-4 cabinet 

and suit laboratories have special engineering and design features to prevent 

microorganisms from being disseminated into the environment and personnel. 

The ABSL-4 cabinet laboratory is distinctly different from an ABSL-3 laboratory 

containing a Class III BSC. The following standard and special safety practices, 

equipment, and facilities apply to ABSL-4.

A. Standard Microbiological Practices

1. The animal facility directors must establish and enforce policies, 

procedures, and protocols for biosafety, biosecurity and emergencies 

within the ABSL-4 laboratory.

The animal facility director and/or designated institutional officials are 

responsible for enforcing the policies that control access to the ABSL-4 

facility. Laboratory personnel and support staff must be provided 

appropriate occupational medical service including medical surveillance 

and available immunizations for agents handled or potentially present in 

the laboratory.3

 A system must be established for reporting and documenting 

laboratory accidents, exposures, employee absenteeism and for the 

medical surveillance of potential laboratory-associated illnesses. An 

essential adjunct to such an occupational medical services system is 

the availability of a facility for the isolation and medical care of personnel 

with potential or known laboratory-acquired infections. Facility supervisors 

should ensure that medical staff are informed of potential occupational 

hazards within the animal facility including those associated with the 

research, animal husbandry duties, animal care, and manipulations.

An ABSL-4 laboratory specific, biosafety manual must be prepared in 

consultation with the animal facility director, the laboratory supervisor, 

and the biosafety advisor. The biosafety manual must be available and 

accessible. Personnel are advised of special hazards, and are required 

to read and follow instructions on practices and procedures.

Prior to beginning a study, appropriate policies and procedures for animal 

welfare during the conduct of research, must be developed and approved 

by the IACUC. The biosafety official, the IBC and/or other applicable 

committees, are responsible for review of protocols and polices to prevent 

hazardous exposures to personnel who manipulate and care for animals.

2. A complete clothing change is required in the ABSL-4 operation. 

Personnel within the animal facility where protective clothing, such as 

uniforms or scrub suits. 
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-4 87

All persons leaving the BSL-4/ABSL-4 laboratory are required to take a 

personal body shower.

3. Eating, drinking, smoking, handling contact lenses, applying cosmetics, 

and storing food for human consumption must not be permitted in 

laboratory areas. Food must be stored outside the laboratory area in 

cabinets or refrigerators designated and used for this purpose.

4. Mechanical pipetting devices must be used.

5. Policies for the safe handling of sharps, such as needles, scalpels, 

pipettes, and broken glassware must be developed and implemented.

When applicable, laboratory supervisors should adopt improved 

engineering and work practice controls that reduce the risk of sharps 

injuries. Precautions, including those listed below, must always be taken 

with sharp items. These include:

a. Use of needles and syringes or other sharp instruments are limited 

for use in the animal facility is limited to situations where there 

is no alternative such as parenteral injection, blood collection, or 

aspiration of fluids from laboratory animals and diaphragm bottles.

b. Disposable needles must not be bent, sheared, broken, recapped, 

removed from disposable syringes, or otherwise manipulated by 

hand before disposal. Used disposable needles must be carefully 

placed in puncture-resistant containers used for sharps disposal 

and placed as close to the work site as possible.

c. Non-disposable sharps must be placed in a hard walled container 

for transport to a processing area for decontamination, preferably 

by autoclaving.

d. Broken glassware must not be handled directly. Instead, it must be 

removed using a brush and dustpan, tongs, or forceps. Plastic ware 

should be substituted for glassware whenever possible.

e. Equipment containing sharp edges and corners should be avoided.

6. Perform all procedures to minimize the creation of splashes 

and/or aerosols.

Procedures involving the manipulation of infectious materials must 

be conducted within biological safety cabinets, or other physical 

containment devices. When procedures cannot be performed in a 

BSC, alternate containment equipment should be used.
88 Biosafety in Microbiological and Biomedical Laboratories

7. Decontaminate work surfaces after completion of work and after any 

spill or splash of potentially infectious material with appropriate disinfectant.

Incidents that may result in exposure to infectious materials must be 

immediately evaluated and treated according to procedures described 

in the laboratory biosafety manual. All incidents must be reported to the 

animal facility director, laboratory supervisor, institutional management 

and appropriate facility safety personnel. Medical evaluation, surveillance, 

and treatment must be provided and appropriate records maintained.

8. Decontaminate all wastes (including animal tissues, carcasses, and 

contaminated bedding) and other materials before removal from the 

ABSL-4 laboratory by an effective and validated method. Laboratory 

clothing should be decontaminated before laundering.

Supplies and materials needed in the facility must be brought in through 

a double-door autoclave, fumigation chamber, or airlock. Supplies and 

materials that are not brought into the ABSL-4 laboratory through the 

change room must be brought in through a previously decontaminated 

double-door autoclave, fumigation chamber, or airlock. Containment 

should be maintained at all times. After securing the outer doors, 

personnel within the areas where infectious materials and/or animals 

are housed or are manipulated retrieve the materials by opening the 

interior doors of the autoclave, fumigation chamber, or airlock. These 

doors must be secured after materials are brought into the facility.

Only necessary equipment and supplies should be taken inside 

the ABSL-4 laboratory. All equipment and supplies taken inside the 

laboratory must be decontaminated before removal. Consideration 

should be given to means for decontaminating routine husbandry 

equipment and sensitive electronic and medical equipment.

The doors of the autoclave and fumigation chamber are interlocked in 

a manner that prevents opening of the outer door unless the autoclave 

has been operated through a decontamination cycle or the fumigation 

chamber has been decontaminated.

9. A sign incorporating the universal biohazard symbol must be posted at 

the entrance to the laboratory and the animal room/s when infectious 

agents are present. The sign must include the animal biosafety level, 

general occupational health requirements, personal protective equipment 

requirements, the supervisor’s name (or other responsible personnel), 

telephone number, and required procedures for entering and exiting the 

animal areas. Identification of specific infectious agents is recommended 

when more than one agent is being used within an animal room.
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-4 89

Security sensitive agent information and occupational health requirements 

should be posted in accordance with the institutional policy.

Advance consideration must be given to emergency and disaster 

recovery plans, as a contingency for man-made or natural disasters.1,3,4

10. An effective integrated pest management program is required. 

(See Appendix G.)

11. The laboratory supervisor must ensure that laboratory personnel receive 

appropriate training regarding their duties, the necessary precautions 

to prevent exposures, and exposure evaluation procedures. Personnel 

must receive annual updates or additional training when procedural 

or policy changes occur. Personal health status may impact an 

individual’s susceptibility to infection, ability to receive immunizations 

or prophylactic interventions. Therefore, all laboratory personnel 

and particularly women of childbearing age should be provided with 

information regarding immune competence and conditions that may 

predispose them to infection. Individuals having these conditions should 

be encouraged to self-identify to the institution’s healthcare provider for 

appropriate counseling and guidance.

12. Animals and plants not associated with the work being performed must 

not be permitted in the areas where infectious materials and/ or animals 

are housed or are manipulated.

B. Special Practices

1. All persons entering the ABSL-4 laboratory must be advised of the 

potential hazards and meet specific entry/exit requirements.

Only persons whose presence in the laboratory or individual animal rooms 

is required for scientific or support purposes are authorized to enter.

Entry into the facility must be limited by means of secure, locked doors. 

A logbook, or other means of documenting the date and time of all 

persons entering and leaving the ABSL-4 laboratory must be maintained.

While the laboratory is operational, personnel must enter and exit 

the laboratory through the clothing change and shower rooms except 

during emergencies. All personal clothing must be removed in the outer 

clothing change room. All personnel entering the laboratory must use 

laboratory clothing, including undergarments, pants, shirts, jumpsuits, 

shoes, and gloves.

All persons leaving the ABSL-4 laboratory are required to take a 

personal body shower. Used laboratory clothing must not be removed 
90 Biosafety in Microbiological and Biomedical Laboratories

from the inner change room through the personal shower. These items 

must be treated as contaminated materials and decontaminated before 

laundering or disposal.

After the laboratory has been completely decontaminated by validated 

method, necessary staff may enter and exit the laboratory without 

following the clothing change and shower requirements described above.

Personal health status may impact an individual’s susceptibility to infection, 

ability to receive immunizations or prophylactic interventions. Therefore, 

all laboratory personnel and particularly women of childbearing age 

should be provided with information regarding immune competence and 

conditions that may predispose them to infection. Individuals having 

these conditions should be encouraged to self-identify to the institution’s 

healthcare provider for appropriate counseling and guidance.

2. Animal facility personnel and support staff must be provided 

occupational medical services, including medical surveillance and 

available immunizations for agents handled or potentially present in the 

laboratory. A system must be established for reporting and documenting 

laboratory accidents, exposures, employee absenteeism and for the 

medical surveillance of potential laboratory-acquired illnesses. An 

essential adjunct to an occupational medical system is the availability 

of a facility for the isolation and medical care of personnel with 

potential or known laboratory-acquired illnesses.

3. Each institution must establish policies and procedures describing 

the collection and storage of serum samples from at-risk personnel.

4. The animal facility supervisor is responsible for ensuring that 

animal personnel:

a. Receive appropriate training in the practices and operations 

specific to the animal facility, such as animal husbandry procedures, 

potential hazards present, manipulations of infectious agents, 

and necessary precautions to prevent potential exposures.

b. Demonstrate high proficiency in standard and special microbiological 

practices, and techniques before entering the ABSL-4 facility or 

working with agents requiring ABSL-4 containment.

c. Receive annual updates and additional training when procedure 

or policy changes occur. Records are maintained for all hazard 

evaluations and employee training.

5. Removal of biological materials that are to remain in a viable or intact 

state from the ABSL-4 laboratory must be transferred to a non-breakable, 
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-4 91

sealed primary container and then enclosed in a non-breakable, sealed 

secondary container. These materials must be transferred through a 

disinfectant dunk tank, fumigation chamber, or decontamination shower. 

Once removed, packaged viable material must not be opened outside 

ABSL-4 containment unless inactivated by a validated method.

6. Laboratory equipment must be routinely decontaminated, as well as 

after spills, splashes, or other potential contamination. Equipment, 

cages, and racks should be handled in manner that minimizes 

contamination of other areas. Cages are autoclaved or thoroughly 

decontaminated before they are cleaned and washed.

a. All equipment and contaminated materials must be decontaminated 

before removal from the animal facility. Equipment must be 

decontaminated using an effective and validated method before 

repair, maintenance, or removal from the animal facility.

b. Equipment or material that might be damaged by high temperatures 

or steam must be decontaminated using an effective and validated 

procedure such as a gaseous or vapor method in an airlock or 

chamber designed for this purpose.

c. Spills involving infectious materials must be contained, 

decontaminated, and cleaned up by staff properly trained and 

equipped to work with infectious material. A spill procedure must 

be developed and posted within the laboratory. Spills and accidents 

of potentially infectious materials must be immediately reported 

to the animal facility and laboratory supervisors or personnel 

designated by the institution.

7. The doors of the autoclave and fumigation chamber are interlocked in a 

manner that prevents opening of the outer door unless the autoclave/

decontamination chamber has been operated through a decontamination 

cycle or the fumigation chamber has been decontaminated.

8. Daily inspections of essential containment and life support systems must 

be completed before laboratory work is initiated to ensure that the laboratory 

and animal facilities are operating according to established parameters.

9. Practical and effective protocols for emergencies must be established. 

These protocols must include plans for medical emergencies, facility 

malfunctions, fires, escape of animals within the ABSL-4 laboratory, 

and other potential emergencies. Training in emergency response 

procedures must be provided to emergency response personnel 

according to institutional policies.
92 Biosafety in Microbiological and Biomedical Laboratories

10. Based on site-specific risk assessment, personnel assigned to work with 

infected animals may be required to work in pairs. Procedures to reduce 

possible worker exposure must be instituted, such as use of squeeze cages, 

working only with anesthetized animals, or other appropriate practices.

C. Safety Equipment (Primary Barriers and Personal Protective Equipment)

Cabinet Laboratory

1. All manipulations of infectious animals and materials within the 

laboratory must be conducted in the Class III BSC. Double-door, pass 

through autoclaves must be provided for decontaminating materials 

passing out of the Class III BSC(s). The autoclave doors must be 

interlocked so that only one can be opened at any time and be 

automatically controlled so that the outside door to the autoclave can 

only be opened after the decontamination cycle has been completed.

The Class III cabinet must also have a pass-through dunk tank, 

fumigation chamber, or equivalent decontamination method so that 

materials and equipment that cannot be decontaminated in the 

autoclave can be safely removed from the cabinet. Containment 

must be maintained at all times.

The Class III cabinet must have a HEPA filter on the supply air intake 

and two HEPA filters in series on the exhaust outlet of the unit. There 

must be gas-tight dampers on the supply and exhaust ducts of the 

cabinet to permit gas or vapor decontamination of the unit. Ports for 

injection of test medium must be present on all HEPA filter housings.

The interior of the Class III cabinet must be constructed with smooth 

finishes that can be easily cleaned and decontaminated. All sharp edges 

on cabinet finishes must be eliminated to reduce the potential for cuts 

and tears of gloves. Equipment to be placed in the Class III cabinet 

should also be free of sharp edges or other surfaces that may damage 

or puncture the cabinet gloves.

Class III cabinet gloves must be inspected for leaks periodically and 

changed if necessary. Gloves should be replaced annually during 

cabinet re-certification.

The cabinet should be designed to permit maintenance and repairs of 

cabinet mechanical systems (refrigeration, incubators, centrifuges, etc.) 

to be performed from the exterior of the cabinet whenever possible.

Manipulation of high concentrations or large volumes of infectious 

agents within the Class III cabinet should be performed using physical 

containment devices inside the cabinet whenever practical. Such 
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-4 93

materials should be centrifuged inside the cabinet using sealed rotor 

heads or centrifuge safety cups.

The interior of the Class III cabinet as well as all contaminated plenums, 

fans and filters must be decontaminated using a validated gaseous or 

vapor method.

The Class III cabinet must be certified at least annually.

Restraint devices and practices that reduce the risk of exposure during 

animal manipulations must be used where practicable (e.g., physical 

restraint devices, chemical restraint medications, mesh or Kevlar 

gloves, etc.).

2. Workers must wear protective laboratory clothing such as solid-front or 

wrap-around gowns, scrub suits, or coveralls when in the laboratory. 

No personal clothing, jewelry, or other items except eyeglasses should 

be taken past the personal shower area. Upon exiting the laboratory, 

all protective clothing must be removed in the dirty side change room 

before showering. Reusable laboratory clothing must be autoclaved 

before being laundered.

3. Eye, face and respiratory protection should be used in rooms containing 

infected animals as determined by the risk assessment. Prescription eye 

glasses must be decontaminated before removal thought the personal 

body shower.

4. Gloves must be worn to protect against breaks or tears in the cabinet 

gloves. Gloves must not be worn outside the laboratory. Alternatives 

to latex gloves should be available. Do not wash or reuse disposable 

gloves. Dispose of used gloves with other contaminated waste.

Suit Laboratory

1. Infected animals should be housed in a primary containment system 

(such as open cages placed in ventilated enclosures, solid wall and 

bottom cages covered with filter bonnets and opened in laminar flow 

hoods, or other equivalent primary containment systems).

Personnel wearing a one-piece positive pressure suit ventilated with 

a life support system must conduct all procedures.

All manipulations of potentially infectious agents must be performed 

within a Class II BSC or other primary barrier system. Infected animals 

should be handled within a primary barrier system, such as a Class II 

BSC or other equivalent containment system.
94 Biosafety in Microbiological and Biomedical Laboratories

Equipment that may produce aerosols must be contained in devices 

that exhaust air through HEPA filtration before being discharged into 

the laboratory. These HEPA filters should be tested annually and 

replaced as need.

HEPA filtered exhaust air from a Class II BSC can be safely re-circulated 

into the laboratory environment if the cabinet is tested and certified at 

least annually and operated according to manufacturer’s recommendations.

2. Workers must wear protective laboratory clothing, such as scrub suits, 

before entering the room used for donning positive pressure suits. 

All protective clothing must be removed in the dirty side change room 

before entering the personal shower. Reusable laboratory clothing 

must be autoclaved before being laundered.

3. Inner gloves must be worn to protect against break or tears in the outer 

suit gloves. Disposable gloves must not be worn outside the change 

area. Alternatives to latex gloves should be available. Do not wash or 

reuse disposable gloves. Inner gloves must be removed and discarded 

in the inner change room prior to entering the personal shower. Dispose 

of used gloves with other contaminated waste.

4. Decontamination of outer suit gloves is performed during operations 

to remove gross contamination and minimize further contamination 

of the laboratory.

D. Laboratory Facilities (Secondary Barriers)

Cabinet Laboratory

1. The ABSL-4 cabinet laboratory consists of either a separate building or 

a clearly demarcated and isolated zone within a building. Laboratory 

doors must have locks in accordance with the institutional policies.

Rooms in the ABSL-4 facility must be arranged to ensure sequential 

passage through an inner (dirty) change area, personal shower and 

outer (clean) change room prior to exiting the room(s) containing the 

Class III BSC(s).

An automatically activated emergency power source must be provided 

at a minimum for the laboratory exhaust system, life support systems, 

alarms, lighting, entry and exit controls, BSCs, and door gaskets. 

Monitoring and control systems for air supply, exhaust, life support, 

alarms, entry and exit, and security systems should be on an 

uninterrupted power supply (UPS).
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-4 95

A double-door autoclave, dunk tank, fumigation chamber, or ventilated 

anteroom/airlock must be provided at the containment barrier for the 

passage of materials, supplies, or equipment.

2. A hands-free sink must be provided near the doors of the cabinet 

room(s) and the inner change rooms. A sink must be provided in the 

outer change room. All sinks in the room(s) containing the Class III 

BSC must be connected to the wastewater decontamination system.

3. Walls, floors, and ceilings of the laboratory must be constructed to form 

a sealed internal shell to facilitate fumigation and prohibit animal and 

insect intrusion. The internal surfaces of this shell must be resistant to 

liquids and chemicals used for cleaning and decontamination of the 

area. Floors must be monolithic, sealed and coved.

All penetrations in the internal shell of the laboratory and inner change 

room must be sealed.

Openings around doors into the cabinet room and inner change 

room must be minimized and capable of being sealed to facilitate 

decontamination.

All drains in ABSL-4 laboratory area floor must be connected directly 

to the liquid waste decontamination system.

Services and plumbing that penetrate the laboratory walls, floors or 

ceiling, must be installed to ensure that no backflow from the laboratory 

occurs. Services must be sealed and be provided with redundant backflow 

prevention. Consideration should be given to locating these devices 

outside of containment. Atmospheric venting systems must be provided 

with two HEPA filters in series and are sealed up to the second filter.

Decontamination of the entire cabinet must be performed using a validated 

gaseous or vapor method when there have been significant changes in 

cabinet usage, before major renovations or maintenance shut downs, 

and in other situations, as determined by risk assessment. Selection of 

the appropriate materials and methods used for decontamination must 

be based on the risk assessment of the biological agents in use.

4. Laboratory furniture must be of simple construction, capable of 

supporting anticipated loading and uses. Spaces between benches, 

cabinets, and equipment must be accessible for cleaning and 

decontamination. Chairs and other furniture should be covered 

with a non-porous material that can be easily decontaminated.
96 Biosafety in Microbiological and Biomedical Laboratories

5. Windows must be break-resistant and sealed.

6. If Class II BSCs are needed in the cabinet laboratory, they must be installed 

so that fluctuations of the room air supply and exhaust do not interfere 

with proper operations. Class II BSCs should be located away from doors, 

heavily traveled laboratory areas, and other possible airflow disruptions.

7. Central vacuum systems are not recommended. If, however, there is a 

central vacuum system, it must not serve areas outside the cabinet room. 

Two in-line HEPA filters must be placed near each use point. Filters 

must be installed to permit in-place decontamination and replacement.

8. An eyewash station must be readily available in the laboratory.

9. A dedicated non-recirculating ventilation system is provided. Only 

laboratories with the same HVAC requirements (i.e., other BSL-4 labs, 

ABSL-4, BSL-3-Ag labs) may share ventilation systems if gas-tight 

dampers and HEPA filters isolate each individual laboratory system. 

The supply and exhaust components of the ventilation system must 

be designed to maintain the ABSL-4 laboratory at negative pressure to 

surrounding areas and provide differential pressure/directional airflow 

between adjacent areas within the laboratory.

Redundant supply fans are recommended. Redundant exhaust fans 

are required. Supply and exhaust fans must be interlocked to prevent 

positive pressurization of the laboratory.

The ventilation system must be monitored and alarmed to indicate 

malfunction or deviation from design parameters. A visual monitoring 

device must be installed near the clean change room so proper 

differential pressures within the laboratory may be verified.

Supply air to and exhaust air from the cabinet room and 

fumigation/decontamination chambers must pass through HEPA filter(s). 

The air exhaust discharge must be located away from occupied spaces 

and building air intakes.

All HEPA filters should be located as near as practicable to the cabinet 

laboratory in order to minimize the length of potentially contaminated 

ductwork. All HEPA filters must be tested and certified annually.

The HEPA filter housings should be designed to allow for in situ 

decontamination and validation of the filter prior to removal. The design 

of the HEPA filter housing must have gas-tight isolation dampers; 

decontamination ports, and ability to scan each filter assembly for leaks.
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-4 97

10. HEPA filtered exhaust air from a Class II BSC can be safely recirculated into the laboratory environment if the cabinet is tested and 

certified at least annually and operated according to the manufacturer’s 

recommendations. Biological safety cabinets can also be connected to 

the laboratory exhaust system by either a thimble (canopy) connection 

or a direct (hard) connection. Provisions to assure proper safety cabinet 

performance and air system operation must be verified.

Class III BSCs must be directly and independently exhausted through 

two HEPA filters in series. Supply air must be provided in such a manner 

that prevents positive pressurization of the cabinet.

11. Pass through dunk tanks, fumigation chambers, or equivalent 

decontamination methods must be provided so that materials and 

equipment that cannot be decontaminated in the autoclave can be 

safely removed from the cabinet room(s). Access to the exit side of 

the pass though shall be limited to those individuals authorized to 

be in the ABSL-4 laboratory.

12. Liquid effluents from cabinet room sinks, floor drains, autoclave 

chambers, and other sources within the cabinet room must be 

decontaminated by a proven method, preferably heat treatment, 

before being discharged to the sanitary sewer.

Decontamination of all liquid wastes must be documented. The 

decontamination process for liquid wastes must be validated physically 

and biologically. Biological validation must be performed annually or 

more often as required by institutional policy.

Effluents from showers and toilets may be discharged to the sanitary 

sewer without treatment.

13. A double-door autoclave must be provided for decontaminating waste 

or other materials passing out of the cabinet room. Autoclaves that open 

outside of the laboratory must be sealed to the wall. This bioseal must 

be durable, airtight, and sealed to the wall. Positioning the bioseal so 

that the equipment can be accessed and maintained from outside the 

laboratory is recommended. The autoclave doors must be interlocked so 

that only one can be opened at any time and be automatically controlled 

so that the outside door can only be opened after the autoclave 

decontamination cycle has been completed.

Gas and liquid discharge from the autoclave chamber must be 

decontaminated. When feasible, autoclave decontamination processes 

should be designed so that over-pressurization cannot release unfiltered 

air or steam exposed to infectious material to the environment.
98 Biosafety in Microbiological and Biomedical Laboratories

14. The ABSL-4 facility design parameters and operational procedures must 

be documented. The facility must be tested to verify that the design and 

operational parameters have been met prior to operation. Facilities must 

also be re-verified annually. Verification criteria should be modified as 

necessary by operational experience.

15. Appropriate communication systems must be provided between the 

ABSL-4 laboratory and the outside (e.g., voice, fax, and computer). 

Provisions for emergency communication and access/egress must 

be considered.

Suit Laboratory

1. The ABSL-4 suit laboratory consists of either a separate building or 

a clearly demarcated and isolated zone within a building. Laboratory 

doors must have locks in accordance with the institutional policies.

Entry to this laboratory must be through an airlock fitted with airtight 

doors. Personnel who enter this laboratory must wear a positive 

pressure suit ventilated by a life support system with HEPA filtered 

breathing air. The breathing air system must have redundant 

compressors, failure alarms and an emergency backup system.

Rooms in the facility must be arranged to ensure sequential passage 

through the chemical shower, inner (dirty) change room, personal 

shower, and outer (clean) changing area upon exit.

A chemical shower must be provided to decontaminate the surface 

of the positive pressure suit before the worker leaves the ABSL-4 

laboratory. In the event of an emergency exit or failure of chemical 

shower, a method for decontaminating positive pressure suits, such 

as a gravity fed supply of chemical disinfectant, is needed.

An automatically activated emergency power source must be provided 

at a minimum for the laboratory exhaust system, life support systems, 

alarms, lighting, entry and exit controls, BSCs, and door gaskets. 

Monitoring and control systems for air supply, exhaust, life support, 

alarms, entry and exit, and security systems should be on a UPS.

A double-door autoclave, dunk tank, or fumigation chamber must 

be provided at the containment barrier for the passage of materials, 

supplies, or equipment.

2. Sinks inside the ABSL-4 laboratory must be placed near procedure 

areas, contain traps, and be connected to the wastewater 

decontamination system.
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-4 99

3. Walls, floors, and ceilings of the ABSL-4 laboratory must be constructed 

to form a sealed internal shell to facilitate fumigation and prohibit animal 

and insect intrusion. The internal surfaces of this shell must be resistant 

to liquids and chemicals used for cleaning and decontamination of the 

area. Floors must be monolithic, sealed and coved.

All penetrations in the internal shell of the laboratory, suit storage room 

and the inner change room must be sealed.

Drains, if present, in the laboratory floor must be connected directly 

to the liquid waste decontamination system. Sewer vents and other 

service lines must be protected by two HEPA filters in series and have 

protection against insect and animal intrusion.

Services and plumbing that penetrate the laboratory walls, floors, or 

ceiling must be installed to ensure that no backflow from the laboratory 

occurs. These penetrations must be fitted with two (in series) backflow 

prevention devices. Consideration should be given to locating these 

devices outside of containment. Atmospheric venting systems must 

be provided with two HEPA filters in series and be sealed up to the 

second filter.

Decontamination of the entire laboratory must be performed using a 

validated gaseous or vapor method when there have been significant 

changes in laboratory usage, before major renovations or maintenance 

shut downs, and in other situations, as determined by risk assessment.

4. Laboratory furniture must be of simple construction, capable of supporting 

anticipated loading and uses. Spaces between benches, cabinets, 

and equipment must be accessible for cleaning, decontamination and 

unencumbered movement of personnel. Chairs and other furniture 

should be covered with a non-porous material that can be easily 

decontaminated. Sharp edges and corners should be avoided.

5. Windows must be break-resistant and sealed.

6. BSCs and other primary containment barrier systems must be installed 

so that fluctuations of the room air supply and exhaust do not interfere 

with proper operations. BSCs should be located away from doors, 

heavily traveled laboratory areas, and other possible airflow disruptions.

7. Central vacuum systems are not recommended. If, however, there is 

a central vacuum system, it must not serve areas outside the ABSL-4 

laboratory. Two in-line HEPA filters must be placed near each use 

point. Filters must be installed to permit in-place decontamination 

and replacement.
100 Biosafety in Microbiological and Biomedical Laboratories

8. An eyewash station must be readily available in the laboratory area for 

use during maintenance and repair activities.

9. A dedicated non-recirculating ventilation system is provided. Only 

laboratories with the same HVAC requirements (i.e., other BSL-4 labs, 

ABSL-4, BSL-3-Ag labs) may share ventilation systems if gas tight 

dampers and HEPA filters isolate each individual laboratory system.

The supply and exhaust components of the ventilation system must be 

designed to maintain the BSL-4/ABSL-4 laboratory at negative pressure 

to surrounding areas and provide correct differential pressure between 

adjacent areas within the laboratory.

Redundant supply fans are recommended. Redundant exhaust fans 

are required. Supply and exhaust fans must be interlocked to prevent 

positive pressurization of the laboratory.

The ventilation system must be monitored and alarmed to indicate 

malfunction or deviation from design parameters. A visual monitoring 

device must be installed near the clean change room so proper 

differential pressures within the laboratory may be verified.

Supply air to the ABSL-4 laboratory, including the decontamination 

shower, must pass through a HEPA filter. All exhaust air from the BSL-4/

ABSL-4 suit laboratory, decontamination shower and fumigation or 

decontamination chambers must pass through two HEPA filters, in 

series before discharge to the outside. The exhaust air discharge must 

be located away from occupied spaces and air intakes.

All HEPA filters must be located as near as practicable to the 

areas where infectious materials and/or animals are housed or are 

manipulated in order to minimize the length of potentially contaminated 

ductwork. All HEPA filters must be tested and certified annually.

The HEPA filter housings are designed to allow for in situ 

decontamination and validation of the filter prior to removal. The design 

of the HEPA filter housing must have gas-tight isolation dampers; 

decontamination ports; and ability to scan each filter assembly for leaks.

10. HEPA filtered exhaust air from a Class II BSC can be safely recirculated back into the laboratory environment if the cabinet is 

tested and certified at least annually and operated according to the 

manufacturer’s recommendations. Biological safety cabinets can also 

be connected to the laboratory exhaust system by either a thimble 

(canopy) connection or directly to the outside through an independent, 

direct (hard) connection. Provisions to assure proper safety cabinet 

performance and air system operation must be verified.
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities: ABSL-4 101

11. Pass through dunk tanks, fumigation chambers, or equivalent 

decontamination methods must be provided so that materials and 

equipment that cannot be decontaminated in the autoclave can be 

safely removed from the ABSL-4 laboratory. Access to the exit side of 

the pass-through shall be limited to those individuals authorized to be in 

the ABSL-4 laboratory.

12. Liquid effluents from chemical showers, sinks, floor drains, autoclave 

chambers, and other sources within the laboratory must be 

decontaminated by a proven method, preferably heat treatment, before 

being discharged to the sanitary sewer.

Decontamination of all liquid wastes must be documented. The 

decontamination process for liquid wastes must be validated physically 

and biologically. Biological validation must be performed annually or 

more often as required by institutional policy.

Effluents from personal body showers and toilets may be discharged to 

the sanitary sewer without treatment.

13. A double-door, pass through autoclave(s) must be provided for 

decontaminating materials passing out of the cabinet laboratory. 

Autoclaves that open outside of the laboratory must be sealed to 

the wall through which the autoclave passes. This bioseal must be 

durable and airtight. Positioning the bioseal so that the equipment can 

be accessed and maintained from outside the laboratory is strongly 

recommended. The autoclave doors must be interlocked so that only 

one can be opened at any time and be automatically controlled so 

that the outside door to the autoclave can only be opened after the 

decontamination cycle has been completed.

The size of the autoclave should be sufficient to accommodate the 

intended usage, equipment size, and potential future increases in cage 

size. Autoclaves should facilitate isolation for routine servicing.

Gas and liquid discharge from the autoclave chamber must be 

decontaminated. When feasible, autoclave decontamination processes 

should be designed so that over-pressurization cannot release unfiltered 

air or steam exposed to infectious material to the environment.

14. The ABSL-4 facility design parameters and operational procedures must 

be documented. The facility must be tested to verify that the design and 

operational parameters have been met prior to operation. Facilities must 

also be re-verified. Verification criteria should be modified as necessary 

by operational experience.
102 Biosafety in Microbiological and Biomedical Laboratories

Consider placing ABSL-4 areas away from exterior walls of buildings to 

minimize the impact from the outside environmental and temperatures.

15. Appropriate communication systems must be provided between the 

laboratory and the outside (e.g., voice, fax, and computer). Provisions 

for emergency communication and access/egress should be considered.

References

1. Institute for Laboratory Animal Research. Guide for the care and use of 

laboratory animals. Washington, DC: National Academy Press; 1996.

2. Animal Welfare Act and Amendment, Title 9 CFR Subchapter A, Parts 1, 2, 3 

(1976).

3. National Research Council; Institute for Laboratory Animal Research. 

Occupational health and safety in the care and use of research animals. 

Washington, DC: National Academy Press; 1997.

4. National Research Council; Institute for Laboratory Animal Research. 

Occupational health and safety in the care and use of nonhuman primates. 

Washington, DC: National Academy Press; 2003.

5. National Institutes of Health, Office of Laboratory Animal Welfare. Public 

Health Service policy on humane care and use of laboratory animals, 

Bethesda (MD); The National Institutes of Health (US); 2000.
Vertebrate Animal Biosafety Level Criteria for Vivarium Research Facilities 103

Table 3. Summary of Recommended Animal Biosafety Levels for Activities in which Experimentally or Naturally Infected 

Vertebrate Animals are Used

BSL Agents Practices Primary Barriers and Safety Equipment Facilities

(Secondary Barriers)

1 Not known to consistently cause diseases 

in healthy adults

Standard animal care and management practices, including appropriate medical surveillance programs

As required for normal care of each species

PPE: ■ laboratory coats and gloves; 

eye, face protection, as needed

Standard animal facility:

No recirculation of exhaust air ■

Directional air flow recommended ■Hand washing sink is available ■

2 Agents associated with human disease ■

Hazard: percutaneous injury, ingestion, ■mucous membrane exposure

ABSL-1 practice plus:

Limited access ■

Biohazard ■ warning signs“ ■ Sharps” precautionsBiosafety manual ■

■ Decontamination of all infectious 

wastes and animal cages prior to washing

ABSL-1 equipment plus primary barriers:

Containment equipment appropriate ■

for animal specialPPE: ■ Laboratory coats, gloves, face, eye and respiratory protection, as needed

■

ABSL-1 plus:

Autoclave available ■

Hand washing sink available ■Mechanical cage washer recom- ■mendedNegative airflow into animal and ■procedure rooms recommended

3 Indigenous or exotic agents that may cause 

serious or potentially lethal disease through the inhalation route of exposure

ABSL-2 practice plus:

Controlled access ■

■ Decontamination of clothing before 

launderingCages decontaminated before ■bedding is removed

Disinfectant foot bath as needed ■

ABSL-2 equipment plus:

Containment equipment for housing ■

animals and cage dumping activities

■ Class I, II or III BSCs available for 

manipulative procedures (inoculation, necropsy) that may create infectious aerosolsPPE: Appropriate respiratory protec- ■tion

ABSL-2 facility plus:

Physical separation from access ■

corridorsSelf-closing, double-door access ■Sealed penetrations ■Sealed windows ■Autoclave available in facility ■Entry through ante-room or airlock ■Negative airflow into animal and ■procedure roomsHand washing sink near exit of ■animal or procedure room 

4 Dangerous/exotic agents which post high ■

risk of aerosol transmitted laboratory infections that are frequently fatal, for which there are no vaccines or treatmentsAgents with a close or identical antigenic ■relationship to an agent requiring BSL-4 until data are available to redesignate the levelRelated agents with unknown risk of ■transmission

ABSL-3 practices plus:

Entrance through change room ■

where personal clothing is removed and laboratory clothing is 

put on; shower on exitingAll wastes are decontaminated ■

before removal from the facility

ABSL-3 equipment plus:

Maximum containment equipment ■

(i.e., Class III BSC or partial containment equipment in combination with full body, air-supplied positivepressure suit) used for all procedures and activities

ABSL-3 facility plus:

Separate building or isolated zone ■

Dedicated supply and exhaust, ■vacuum, and decontamination systemsOther requirements outlined in ■the text
104 Biosafety in Microbiological and Biomedical Laboratories

Section VI—Principles of Laboratory Biosecurity

Since the publication of the 4th edition of BMBL in 1999, significant events have 

brought national and international scrutiny to the area of laboratory security. 

These events, including the anthrax attacks on U.S. citizens in October 2001 

and the subsequent expansion of the United States Select Agent regulations in 

December 2003, have led scientists, laboratory managers, security specialists, 

biosafety professionals, and other scientific and institutional leaders to consider 

the need for developing, implementing and/or improving the security of biological 

agents and toxins within their facilities. Appendix F of BMBL 4th edition provided 

a brief outline of issues to consider in developing a security plan for biological 

agents and toxins capable of serious or fatal illness to humans or animals. In 

December 2002, Appendix F was updated and revised as a security and 

emergency response guidance for laboratories working with select agents.1

Section VI replaces the previous appendices. The current Appendix F 

discusses Select Agent and Toxin regulations.

This section describes laboratory biosecurity planning for microbiological 

laboratories. As indicated below, laboratories with good biosafety programs 

already fulfill many of the basic requirements for security of biological materials. 

For laboratories not handling select agents, the access controls and training 

requirements specified for BSL-2 and BSL-3 in BMBL may provide sufficient 

security for the materials being studied. Security assessments and additional 

security measures should be considered when select agents, other agents of 

high public health and agriculture concern, or agents of high commercial value 

such as patented vaccine candidates, are introduced into the laboratory.

The recommendations presented in this section are advisory. Excluding 

the Select Agent regulations, there is no current federal requirement for the 

development of a biosecurity program. However, the application of these 

principles and the assessment process may enhance overall laboratory 

management. Laboratories that fall under the Select Agent regulations should 

consult Appendix F (42 CFR part 73; 7 CFR 331 and 9 CFR 121).4,5,6

The term “biosecurity” has multiple definitions. In the animal industry, the 

term biosecurity relates to the protection of an animal colony from microbial 

contamination. In some countries, the term biosecurity is used in place of the 

term biosafety. For the purposes of this section the term “biosecurity” will refer 

to the protection of microbial agents from loss, theft, diversion or intentional 

misuse. This is consistent with current WHO and American Biological Safety 

Association (ABSA) usage of this term.2,3

Security is not a new concept in biological research and medical 

laboratories. Several of the security measures discussed in this section are 

embedded in the biosafety levels that serve as the foundation for good laboratory 

practices throughout the biological laboratory community. Most biomedical and 
Principles of Laboratory Biosecurity 105

microbiological laboratories do not have select agents or toxins, yet maintain 

control over and account for research materials, protect relevant sensitive 

information, and work in facilities with access controls commensurate with the 

potential public health and economic impact of the biological agents in their 

collections. These measures are in place in most laboratories that apply good 

laboratory management practices and have appropriate biosafety programs.

Biosafety and Biosecurity

Biosafety and biosecurity are related, but not identical, concepts. Biosafety programs 

reduce or eliminate exposure of individuals and the environment to potentially 

hazardous biological agents. Biosafety is achieved by implementing various 

degrees of laboratory control and containment, through laboratory design and 

access restrictions, personnel expertise and training, use of containment equipment, 

and safe methods of managing infectious materials in a laboratory setting.

The objective of biosecurity is to prevent loss, theft or misuse of microorganisms, 

biological materials, and research-related information. This is accomplished by 

limiting access to facilities, research materials and information. While the objectives 

are different, biosafety and biosecurity measures are usually complementary.

Biosafety and biosecurity programs share common components. Both are 

based upon risk assessment and management methodology; personnel expertise 

and responsibility; control and accountability for research materials including 

microorganisms and culture stocks; access control elements; material transfer 

documentation; training; emergency planning; and program management.

Biosafety and biosecurity program risk assessments are performed to 

determine the appropriate levels of controls within each program. Biosafety 

looks at appropriate laboratory procedures and practices necessary to prevent 

exposures and occupationally-acquired infections, while biosecurity addresses 

procedures and practices to ensure that biological materials and relevant 

sensitive information remain secure.

Both programs assess personnel qualifications. The biosafety program 

ensures that staff are qualified to perform their jobs safely through training and 

documentation of technical expertise. Staff must exhibit the appropriate level of 

professional responsibility for management of research materials by adherence 

to appropriate materials management procedures. Biosafety practices require 

laboratory access to be limited when work is in progress. Biosecurity practices 

ensure that access to the laboratory facility and biological materials are limited 

and controlled as necessary. An inventory or material management process for 

control and tracking of biological stocks or other sensitive materials is also a 

component of both programs. For biosafety, the shipment of infectious biological 

materials must adhere to safe packaging, containment and appropriate transport 

procedures, while biosecurity ensures that transfers are controlled, tracked and 
106 Biosafety in Microbiological and Biomedical Laboratories

documented commensurate with the potential risks. Both programs must engage 

laboratory personnel in the development of practices and procedures that fulfill 

the biosafety and biosecurity program objectives but that do not hinder research 

or clinical/diagnostic activities. The success of both of these programs hinges on 

a laboratory culture that understands and accepts the rationale for biosafety and 

biosecurity programs and the corresponding management oversight.

In some cases, biosecurity practices may conflict with biosafety practices, 

requiring personnel and management to devise policies that accommodate both 

sets of objectives. For example, signage may present a conflict between the two 

programs. Standard biosafety practice requires that signage be posted on 

laboratory doors to alert people to the hazards that may be present within the 

laboratory. The biohazard sign normally includes the name of the agent, specific 

hazards associated with the use or handling of the agent and contact information 

for the investigator. These practices may conflict with security objectives. 

Therefore, biosafety and biosecurity considerations must be balanced and 

proportional to the identified risks when developing institutional policies.

Designing a biosecurity program that does not jeopardize laboratory 

operations or interfere with the conduct of research requires a familiarity with 

microbiology and the materials that require protection. Protecting pathogens 

and other sensitive biological materials while preserving the free exchange 

of research materials and information may present significant institutional 

challenges. Therefore, a combination or tiered approach to protecting biological 

materials, commensurate with the identified risks, often provides the best 

resolution to conflicts that may arise. However, in the absence of legal 

requirements for a biosecurity program, the health and safety of laboratory 

personnel and the surrounding environment should take precedence over 

biosecurity concerns.

Risk Management Methodology

A risk management methodology can be used to identify the need for a 

biosecurity program. A risk management approach to laboratory biosecurity 

1) establishes which, if any, agents require biosecurity measures to prevent 

loss, theft, diversion, or intentional misuse, and 2) ensures that the protective 

measures provided, and the costs associated with that protection, are 

proportional to the risk. The need for a biosecurity program should be based 

on the possible impact of the theft, loss, diversion, or intentional misuse of the 

materials, recognizing that different agents and toxins will pose different levels 

of risk. Resources are not infinite. Biosecurity policies and procedures should 

not seek to protect against every conceivable risk. The risks need to be identified, 

prioritized and resources allocated based on that prioritization. Not all institutions 

will rank the same agent at the same risk level. Risk management methodology 

takes into consideration available institutional resources and the risk tolerance 

of the institution.
Principles of Laboratory Biosecurity 107

Developing a Biosecurity Program

Management, researchers and laboratory supervisors must be committed to 

being responsible stewards of infectious agents and toxins. Development of a 

biosecurity program should be a collaborative process involving all stakeholders. 

The stakeholders include but are not limited to: senior management; scientific 

staff; human resource officials; information technology staff; and safety, security 

and engineering officials. The involvement of organizations and/or personnel 

responsible for a facility’s overall security is critical because many potential 

biosecurity measures may already be in place as part of an existing safety or 

security program. This coordinated approach is critical in ensuring that the 

biosecurity program provides reasonable, timely and cost-effective solutions 

addressing the identified security risks without unduly affecting the scientific 

or business enterprise or provision of clinical and/or diagnostic services.

The need for a biosecurity program should reflect sound risk management 

practices based on a site-specific risk assessment. A biosecurity risk assessment 

should analyze the probability and consequences of loss, theft and potential 

misuse of pathogens and toxins.7 Most importantly, the biosecurity risk assessment 

should be used as the basis for making risk management decisions.

Example Guidance: A Biosecurity Risk Assessment and Management Process

Different models exist regarding biosecurity risk assessment. Most models 

share common components such as asset identification, threat, vulnerability and 

mitigation. What follows is one example of how a biosecurity risk assessment may 

be conducted. In this example, the entire risk assessment and risk management 

process may be divided into five main steps, each of which can be further 

subdivided: 1) identify and prioritize biologicals and/or toxins; 2) identify and 

prioritize the adversary/threat to biologicals and/or toxins; 3) analyze the risk of 

specific security scenarios; 4) design and develop an overall risk management 

program; and 5) regularly evaluate the institution’s risk posture and protection 

objectives. Example guidance for these five steps is provided below.

Step 1: Identify and Prioritize Biological Materials

■ Identify the biological materials that exist at the institution, form of the 

material, location and quantities, including non-replicating materials 

(i.e., toxins).

■ Evaluate the potential for misuse of these biologic materials.

■ Evaluate the consequences of misuse of these biologic materials.

■ Prioritize the biologic materials based on the consequences of misuse 

(i.e., risk of malicious use).
108 Biosafety in Microbiological and Biomedical Laboratories

At this point, an institution may find that none of its biologic materials merit 

the development and implementation of a separate biosecurity program or the 

existing security at the facility is adequate. In this event, no additional steps 

would need to be completed.

Step 2: Identify and Prioritize the Threat to Biological Materials

■ Identify the types of “Insiders” who may pose a threat to the biologic 

materials at the institution.

■ Identify the types of “Outsiders” (if any) who may pose a threat to 

the biologic materials at the institution.

■ Evaluate the motive, means, and opportunity of these various 

potential adversaries.

Step 3: Analyze the Risk of Specific Security Scenarios

■ Develop a list of possible biosecurity scenarios, or undesired events 

that could occur at the institution (each scenario is a combination of 

an agent, an adversary, and an action). Consider:

– access to the agent within your laboratory;

– how the undesired event could occur;

– protective measures in place to prevent occurrence;

– how the existing protection measures could be breached 

(i.e., vulnerabilities).

■ Evaluate the probability of each scenario materializing (i.e., the 

likelihood) and its associated consequences. Assumptions include:

– although a wide range of threats are possible, certain threats are 

more probable than others;

– all agents/assets are not equally attractive to an adversary; valid 

and credible threats, existing precautions, and the potential need 

for select enhanced precautions are considered.

■ Prioritize or rank the scenarios by risk for review by management.

Step 4: Develop an Overall Risk Management Program

■ Management commits to oversight, implementation, training and 

maintenance of the biosecurity program.

■ Management develops a biosecurity risk statement, documenting 

which biosecurity scenarios represent an unacceptable risk and must 

be mitigated versus those risks appropriately handled through existing 

protection controls.
Principles of Laboratory Biosecurity 109

■ Management develops a biosecurity plan to describe how the institution 

will mitigate those unacceptable risks including:

– a written security plan, standard operating procedures, and 

incident response plans;

– written protocols for employee training on potential hazards, 

the biosecurity program and incident response plans.

■ Management ensures necessary resources to achieve the protection 

measures documented in the biosecurity plan.

Step 5: Re-evaluate the Institution’s Risk Posture and Protection Objectives

■ Management regularly reevaluates and makes necessary modifications 

to the:

– biosecurity risk statement;

– biosecurity risk assessment process;

– the institution’s biosecurity program/plan;

– the institution’s biosecurity systems.

■ Management assures the daily implementation, training and annual 

re-evaluation of the security program.

Elements of a Biosecurity Program

Many facilities may determine that existing safety and security programs provide 

adequate mitigation for the security concerns identified through biosecurity risk 

assessment. This section offers examples and suggestions for components of a 

biosecurity program should the risk assessment reveal that further protections 

may be warranted. Program components should be site-specific and based upon 

organizational threat/vulnerability assessment and as determined appropriate 

by facility management. Elements discussed below should be implemented, as 

needed, based upon the risk assessment process. They should not be construed 

as “minimum requirements” or “minimum standards” for a biosecurity program.

Program Management

If a biosecurity plan is implemented, institutional management must support the 

biosecurity program. Appropriate authority must be delegated for implementation 

and the necessary resources provided to assure program goals are being met. 

An organizational structure for the biosecurity program that clearly defines the 

chain of command, roles, and responsibilities should be distributed to the staff. 

Program management should ensure that biosecurity plans are created, exercised, 

and revised as needed. The biosecurity program should be integrated into 

relevant institutional policies and plans.
110 Biosafety in Microbiological and Biomedical Laboratories

Physical Security—Access Control and Monitoring

The physical security elements of a laboratory biosecurity program are intended 

to prevent the removal of assets for non-official purposes. An evaluation of the 

physical security measures should include a thorough review of the building 

and premises, the laboratories, and biological material storage areas. Many 

requirements for a biosecurity plan may already exist in a facility’s overall 

security plan.

Access should be limited to authorized and designated employees based on 

the need to enter sensitive areas. Methods for limiting access could be as simple 

as locking doors or having a card key system in place. Evaluations of the levels 

of access should consider all facets of the laboratory’s operations and programs 

(e.g., laboratory entrance requirements, freezer access). The need for entry 

by visitors, laboratory workers, management officials, students, cleaning/

maintenance staff, and emergency response personnel should be considered.

Personnel Management

Personnel management includes identifying the roles and responsibilities for 

employees who handle, use, store and transport dangerous pathogens and/or 

other important assets. The effectiveness of a biosecurity program against 

identified threats depends, first and foremost, on the integrity of those individuals 

who have access to pathogens, toxins, sensitive information and/or other assets. 

Employee screening policies and procedures are used to help evaluate these 

individuals. Policies should be developed for personnel and visitor identification, 

visitor management, access procedures, and reporting of security incidents.

Inventory and Accountability

Material accountability procedures should be established to track the inventory, 

storage, use, transfer and destruction of dangerous biological materials and 

assets when no longer needed. The objective is to know what agents exist at 

a facility, where they are located, and who is responsible for them. To achieve 

this, management should define: 1) the materials (or forms of materials) subject 

to accountability measures; 2) records to be maintained, update intervals and 

timelines for record maintenance; 3) operating procedures associated with 

inventory maintenance (e.g., how material is identified, where it can be used 

and stored); and 4) documentation and reporting requirements.

It is important to emphasize that microbiological agents are capable of 

replication and are often expanded to accommodate the nature of the work 

involving their use. Therefore, knowing the exact “working” quantity of organisms 

at any given time may be impractical. Depending on the risks associated with a 

pathogen or toxin, management can designate an individual who is accountable, 

knowledgeable about the materials in use, and responsible for security of the 

materials under his or her control.
Principles of Laboratory Biosecurity 111

Information Security

Policies should be established for handling sensitive information associated with 

the biosecurity program. For the purpose of these policies, “sensitive information” 

is that which is related to the security of pathogens and toxins, or other critical 

infrastructure information. Examples of sensitive information may include facility 

security plans, access control codes, agent inventories and storage locations. 

Discussion of information security in this section does not pertain to information 

which has been designated “classified” by the United States pursuant to 

Executive Order 12958, as amended, and is governed by United States law 

or to research-related information which is typically unregulated or unrestricted 

through the peer review and approval processes.

The objective of an information security program is to protect information 

from unauthorized release and ensure that the appropriate level of confidentiality 

is preserved. Facilities should develop policies that govern the identification, 

marking and handling of sensitive information. The information security program 

should be tailored to meet the needs of the business environment, support the 

mission of the organization, and mitigate the identified threats. It is critical that 

access to sensitive information be controlled. Policies for properly identifying and 

securing sensitive information including electronic files and removable electronic 

media (e.g., CDs, computer drives) should be developed.

Transport of Biological Agents

Material transport policies should include accountability measures for the 

movement of materials within an institution (e.g., between laboratories, during 

shipping and receiving activities) and outside of the facility (e.g., between 

institutions or locations). Transport policies should address the need for 

appropriate documentation and material accountability and control procedures 

for pathogens in transit between locations. Transport security measures should 

be instituted to ensure that appropriate authorizations have been received and 

that adequate communication between facilities has occurred before, during, 

and after transport of pathogens or other potentially hazardous biological 

materials. Personnel should be adequately trained and familiar with regulatory 

and institutional procedures for proper containment, packaging, labeling, 

documentation and transport of biological materials.

Accident, Injury and Incident Response Plans

Laboratory security policies should consider situations that may require 

emergency responders or public safety personnel to enter the facility in response 

to an accident, injury or other safety issue or security threat. The preservation of 

human life, the safety and health of laboratory employees and the surrounding 

community must take precedence in an emergency over biosecurity concerns. 

Facilities are encouraged to coordinate with medical, fire, police and other 

emergency officials when preparing emergency and security breach response 
112 Biosafety in Microbiological and Biomedical Laboratories

plans. Standard Operation Procedures (SOPs) should be developed that 

minimize the potential exposure of responding personnel to potentially hazardous 

biological materials. Laboratory emergency response plans should be integrated 

with relevant facility-wide or site-specific security plans. These plans should also 

consider such adverse events as bomb threats, natural disasters and severe 

weather, power outages, and other facility emergencies that may introduce 

security threats.

Reporting and Communication

Communication is an important aspect of a biosecurity program. A “chainof-notification” should be established in advance of an actual event. This 

communication chain should include laboratory and program officials, institution 

management, and any relevant regulatory or public authorities. The roles and 

responsibilities of all involved officials and programs should be clearly defined. 

Policies should address the reporting and investigation of potential security 

breaches (e.g., missing biological agents, unusual or threatening phone calls, 

unauthorized personnel in restricted areas).

Training and Practice Drills

Biosecurity training is essential for the successful implementation of a biosecurity 

program. Program management should establish training programs that inform 

and educate individuals regarding their responsibilities within the laboratory 

and the institution. Practice drills should address a variety of scenarios such 

as loss or theft of materials, emergency response to accidents and injuries, 

incident reporting and identification of and response to security breaches. 

These scenarios may be incorporated into existing emergency response drills 

such as fire drills or building evacuation drills associated with bomb threats. 

Incorporating biosecurity measures into existing procedures and response 

plans often provides efficient use of resources, saves time and can minimize 

confusion during emergencies.

Security Updates and Re-evaluations

The biosecurity risk assessment and program should be reviewed and updated 

routinely and following any biosecurity-related incident. Reevaluation is a 

necessary and on-going process in the dynamic environments of today’s 

biomedical and research laboratories. Biosecurity program managers should 

develop and conduct biosecurity program audits and implement corrective 

actions as needed. Audit results and corrective actions should be documented. 

The appropriate program officials should maintain records.

Select Agents

If an entity possesses, uses or transfers select agents, it must comply with 

all requirements of the National Select Agent Program. See Appendix F for 
Principles of Laboratory Biosecurity 113

additional guidance on the CDC and USDA Select Agent Programs (42 CFR Part 

73; 7 CFR 331 and 9 CFR 121).

References

1. Richmond, JY, Nesby-O’Dell, SL. Laboratory security and emergency 

response guidance for laboratories working with select agents. MMWR 

Recomm Rep. 2002;51:(RR-19):1-6.

2. Laboratory biosafety manual. 3rd ed. Geneva: World Health Organization; 2004.

3. American Biological Safety Association. ABSA biosecurity task force white 

paper: understanding biosecurity. Illinois: The Association; 2003.

4. Possession, use and transfer of select agents and toxins, 42 CFR Part 73 

(2005).

5. Possession, use and transfer of biological agents and toxins, 7 CFR Part 

331 (2005).

6. Possession, use and transfer of biological agents and toxins, 9 CFR Part 

121 (2005).

7. Casadevall A, Pirofski L. The weapon potential of a microbe. Bethesda, 

The National Institutes of Health; 2005.
114 Biosafety in Microbiological and Biomedical Laboratories

Section VII—Occupational Health and Immunoprophylaxis

The goal of medical support services in a biomedical research setting is to promote 

a safe and healthy workplace. This is accomplished by limiting opportunities for 

exposure, promptly detecting and treating exposures, and using information 

gained from work injuries to further enhance safety precautions. Occupational 

health and safety in biomedical research settings is a responsibility shared by 

healthcare providers, safety specialists, principal investigators, employers, and 

workplace personnel. Optimal worker protection depends on effective, ongoing 

collaboration among these groups. Supervisors, working with personnel 

representatives, should describe workers’ proposed tasks and responsibilities. 

First line supervisors and safety professionals should identify the potential 

worksite health hazards. Principal investigators may serve as subject matter experts. 

The health provider should design medical support services in consultation with 

representatives from the institutional environmental health and safety program 

and the principal investigators. Workers should be fully informed of the available 

medical support services and encouraged to utilize them. Requisite occupational 

medical services are described below and expanded discussions of the principles 

of effective medical support services are available in authoritative texts.1,2

Services offered by the medical support team should be designed to be in 

compliance with United States Department of Labor (DOL), OSHA regulations, 

patient confidentiality laws, and the Americans with Disabilities Act of 1990.3-8

Medical support services should be based upon detailed risk assessments and 

tailored to meet the organization’s needs. Risk assessments should define 

potential hazards and exposures by job responsibility. They should be provided 

for all personnel regardless of employment status. Contracted workers, students, 

and visitors should be provided occupational medical care by their employer 

or sponsor equivalent to that provided by the host institution for exposures, 

injuries, or other emergencies experienced at the worksite.

Occupational medical services may be provided through a variety of 

arrangements (e.g., in-house or community based) as long as the service is 

readily available and allows timely, appropriate evaluation and treatment. The 

interaction between worker, healthcare provider and employer may be complex, 

such as a contract worker who uses his own medical provider or uses contract 

medical services. Thus, plans for providing medical support for workers should 

be completed before work actually begins. The medical provider must be 

knowledgeable about the nature of potential health risks in the work 

environment and have access to expert consultation.

Prevention is the most effective approach to managing biohazards. 

Prospective workers should be educated about the biohazards to which they 

may be occupationally exposed, the types of exposures that place their health 

at risk, the nature and significance of such risks, as well as the appropriate first 

aid and follow up for potential exposures. That information should be reinforced 
Occupational Health and Immunoprophylaxis 115

annually, at the time of any significant change in job responsibility, and following 

recognized and suspected exposures.9-11

Medical support services for biomedical research facilities should be 

evaluated annually. Joint annual review of occupational injury and illness reports 

by healthcare providers and environmental health and safety representatives 

can assist revision of exposure prevention strategies to minimize occupational 

health hazards that cannot be eliminated.

Occupational Health Support Service Elements

Preplacement Medical Evaluations

Workers who may be exposed to human pathogens should receive a 

preplacement medical evaluation. Healthcare providers should be cognizant of 

potential hazards encountered by the worker. A description of the requirements 

for the position and an understanding of the potential health hazards present in 

the work environment, provided by the worker’s supervisor, should guide the 

evaluation. The healthcare provider should review the worker’s previous and 

ongoing medical problems, current medications, allergies to medicines, animals, 

and other environmental proteins, and prior immunizations. With that information, 

the healthcare provider determines what medical services are indicated to permit 

the individual to safely assume the duties of the position. Occasionally, it may 

be useful to review pre-existing medical records to address specific concerns 

regarding an individual’s medical fitness to perform the duties of a specific position. 

If pre-existing medical records are unavailable or are inadequate, the healthcare 

provider may need to perform a targeted medical exam. Comprehensive physical 

examinations are rarely indicated. During the visit, the healthcare provider should 

inform the worker of potential health hazards in the work area and review steps 

that should be taken in the event of an accidental exposure. This visit also 

establishes a link with the medical support services provider.

When occupational exposure to human pathogens is a risk, employers 

should consider collecting and storing a serum specimen prior to the initiation of 

work with the agent. It can be used to establish baseline sero-reactivity, should 

additional blood samples be collected for serological testing subsequent to a 

recognized or suspected exposure.

Occasionally, it is desirable to determine an individual’s vulnerability to infection 

with specific agents prior to assigning work responsibilities. Some occupational 

exposures present substantially more hazard to identifiable sub-populations of 

workers. Immunodeficient workers or non-immune pregnant female workers may 

experience devastating consequences from exposures that pose a chance of risk 

to pregnant women with prior immunity and other immunocompetent workers 

(e.g., cytomegalovirus or toxoplasmosis). Serologic testing should be used to 

document baseline vulnerability to specific infections to which the worker might 
116 Biosafety in Microbiological and Biomedical Laboratories

be exposed, and non-immune workers should be adequately informed about 

risks. In specific settings, serologic documentation that individual workers 

have pre-existing immunity to specific infections also may be required for 

the protection of research animals.10

Vaccines

Commercial vaccines should be made available to workers to provide protection 

against infectious agents to which they may be occupationally exposed.12-16 The 

Advisory Committee on Immunization Practices (ACIP) provides expert advice 

to the Secretary of the DHHS, the Assistant Secretary for Health, and the CDC 

on the most effective means to prevent vaccine-preventable diseases and to 

increase the safe usage of vaccines and related biological products. The ACIP 

develops recommendations for the routine administration of vaccines to pediatric 

and adult populations, and schedules regarding the appropriate periodicity, 

dosage, and contraindications. The ACIP is the only entity in the federal 

government that makes such recommendations. The ACIP is available at 

the CDC Web site: www.cdc.gov.

If the potential consequences of infection are substantial and the protective 

benefit from immunization is proven, acceptance of such immunization may be 

a condition for employment. Current, applicable vaccine information statements 

must be provided whenever a vaccine is administered. Each worker’s immunization 

history should be evaluated for completeness and currency at the time of 

employment and re-evaluated when the individual is assigned job responsibilities 

with a new biohazard.

When occupational exposure to highly pathogenic agents is possible and 

no commercial vaccine is available, it may be appropriate to immunize workers 

using vaccines or immune serum preparations that are investigational, or for 

which the specific indication constitutes an off-label use. Use of investigational 

products, or of licensed products for off-label indications must be accompanied 

by adequate informed consent outlining the limited availability of information 

on safety and efficacy. Use of investigational products should occur through 

Investigational New Drug (IND) protocols providing safety oversight by both the 

Food and Drug Administration (FDA) and appropriate Institutional Human Subjects 

Research Protection Committees.17,18 Recommendation of investigational 

products, as well as commercial vaccines that are less efficacious, associated 

with high rates of local or systemic reactions, or that produce increasingly severe 

reactions with repeated use, should be considered carefully. Receipt of such 

vaccines is rarely justified as a job requirement.

Investigational vaccines for eastern equine encephalomyelitis (EEE) virus, 

Venezuelan equine encephalitis (VEE) virus, western equine encephalomyelitis 

(WEE) virus, and Rift Valley fever viruses (RVFV), may be available in limited 
Occupational Health and Immunoprophylaxis 117

quantities and administered on-site at the Special Immunization Program, United 

States Army Medical Research Institute of Infectious Diseases (USAMRIID).

Periodic Medical Evaluations

Routine, periodic medical evaluations generally are not recommended; however, 

limited periodic medical evaluations or medical clearances targeted to job 

requirements may occasionally be warranted (e.g., respirator usage).3

 In 

special circumstances, it may be appropriate to offer periodic laboratory 

testing to workers with substantial risk of exposure to infectious agents to 

detect pre-clinical or sub-clinical evidence for an occupationally acquired 

infection. Before asymptomatic workers without specific exposures are tested 

for seroreactivity, the benefit of such testing should be justified, plans for further 

investigation of indeterminate test results should be delineated, and clearly 

defined criteria for interpretation of results should be developed.

Medical Support for Occupational Illnesses and Injuries

Workers should be encouraged to seek medical evaluation for symptoms that 

they suspect may be related to infectious agents in their work area, without fear 

of reprisal. A high index of suspicion for potential occupational exposures should 

be maintained during any unexplained illness among workers or visitors to 

worksites containing biohazards. Modes of transmission, as well as the clinical 

presentation of infections acquired through occupational exposures, may differ 

markedly from naturally acquired infections. Fatal occupational infections have 

resulted from apparently trivial exposures. The healthcare provider should have 

a working understanding of the biohazards present in the workplace and remain 

alert for subtle evidence of infection and atypical presentations. A close working 

relationship with the research or clinical program in which the affected employee 

works is absolutely essential. In the event of injury, consultation between 

healthcare provider, employee, and the employee’s supervisor is required 

for proper medical management and recordkeeping.

All occupational injuries, including exposures to human pathogens, should 

be reported to the medical support services provider. Strategies for responding 

to biohazard exposures should be formulated in advance. Proper post-exposure 

response is facilitated by exposure-specific protocols that define appropriate first 

aid, potential post-exposure prophylaxis options, recommended diagnostic tests, 

and sources of expert medical evaluation. These protocols should address how 

exposures that occur outside of regular work hours are handled and these 

protocols should be distributed to potential healthcare providers (e.g., local 

hospital emergency departments). In exceptional cases, the protocols should 

be reviewed with state and community public health departments. Emergency 

medical support training should be provided on a regular basis for both 

employees and healthcare providers.
118 Biosafety in Microbiological and Biomedical Laboratories

The adequacy and timeliness of wound cleansing or other response after 

an exposure occurs may be the most critical determinant in preventing infection. 

First aid should be defined, widely promulgated, and immediately available to an 

injured worker. Barriers to subsequent medical evaluation and treatment should 

be identified and minimized to facilitate prompt, appropriate care. Laboratory 

SOPs should include a printed summary of the recommended medical response 

to specific exposures that can guide immediate response in the work place and 

that the injured worker can provide to the treating facility. The medical provider’s 

description of the injury should include:

■ The potential infectious agent.

■ The mechanism and route of exposure (percutaneous, splash to 

mucous membranes or skin, aerosol, etc.).

■ Time and place of the incident.

■ Personal protective equipment used at the time of the injury.

■ Prior first aid provided (e.g., nature and duration of cleaning and 

other aid, time that lapsed from exposure to treatment).

■ Aspects of the worker’s personal medical history relevant to risk 

of infection or complications of treatment.

First aid should be repeated if the initial adequacy is in question. Healthcare 

providers must use appropriate barrier precautions to avoid exposure to 

infectious agents and toxins.

In some instances, it may be possible to prevent or ameliorate illness 

through post-exposure prophylaxis. Protocols should be developed in advance 

that clearly identify the situations in which post-exposure prophylaxis are to be 

considered, the appropriate treatment, and the source of products and expert 

consultation. Accurate quantification of risk associated with all exposures is not 

possible, and the decision to administer post-exposure prophylaxis may have 

to be made quickly and in the absence of confirmatory laboratory testing. Postexposure regimens may involve off-label use of licensed products (e.g., use of 

smallpox vaccine for workers exposed to monkeypox) in settings where there is 

insufficient experience to provide exact guidance on the safety or likely protective 

efficacy of the prophylactic regimen. Thus, protocols should exist that delineate 

the circumstances under which it would be appropriate to consider use of each 

product following exposure, as well as the limits of our understanding of the 

value of some post-exposure interventions. In these cases, consultations with 

subject matter experts are especially useful.

Estimating the significance of an exposure may be difficult, despite having 

established protocols. The clinician may need to make a “best-estimate” based 
Occupational Health and Immunoprophylaxis 119

upon knowledge of similar agents, exposure circumstances, and advice received 

from knowledgeable experts. Appropriate post-exposure prophylactic response 

is always pathogen and exposure dependent, and may be host-factor dependent 

and influenced by immediate post-exposure management. Before prophylactic 

treatment is undertaken, confirm the likelihood that an exposure occurred, that 

prophylaxis is indicated and is not contraindicated by past medical history. 

Conveying this information to the injured worker requires clear, honest 

communication. The clinical risk assessment and treatment decision process 

should be carefully explained, the worker’s questions addressed with relevant, 

preprinted educational materials provided. Prompt treatment should be provided, 

with a mutually agreed plan to follow the individual’s clinical course.

The applicable workers compensation claim form should be provided 

with appropriate explanations for its completion. The supervisor must receive a 

description of the accident or incident, confirm the circumstances of the injury 

or exposure and provide relevant advice. The report also should be distributed 

to all other relevant parties, such as the safety professional. Each incident 

should receive prompt reconsideration of the initial risk assessment and 

reevaluation of current strategies to reduce the possibility of future exposures.

Post-exposure serologic testing may be useful, but it is important to determine 

how information obtained from serologic testing will be interpreted. It is also 

essential to collect serum specimens at the appropriate interval for a given 

situation. Assessment of sero-reactivity in exposed workers is most helpful when 

the results of specimens collected over time can be compared. Ideally specimens 

collected prior to, at the time of and several weeks following exposure, should be 

tested simultaneously and results compared to assess changes in the pattern of 

sero-reactivity. Serum collected too early after exposure may fail to react even 

when infection has occurred, because antibodies have not yet been produced in 

detectable quantities. When immediate institution of post-exposure prophylaxis 

may delay seroconversion, or when the agent to which the worker was exposed 

results in seroconversion completed over months (e.g., retroviruses), testing of 

specimens collected late after exposure is particularly important.

Testing of a single serum specimen is generally discouraged and can result 

in misinterpretation of nonspecific sero-reactivity. Evidence of sero-conversion 

or a significant (≥ 4 fold) increase in titer associated with a compatible clinical 

syndrome is highly suggestive of acute infection.

However, the significance of and appropriate response to sero-conversion in 

the absence of illness is not always clear. If sero-reactivity is evident in the earliest 

specimen, it is important to re-test that specimen in tandem with serum specimens 

archived prior to occupational exposure and/or collected serially over time to 

investigate whether a change in titer suggestive of new infection can be identified.
120 Biosafety in Microbiological and Biomedical Laboratories

In some exposure situations, it may be appropriate to store serially collected 

serum samples, and to send them for testing as evidence of seroconversion only 

if symptoms develop that suggest an infection may have occurred (e.g., Monkey 

B virus exposures). Serum collected at the time of employment, and any other 

specimens not immediately tested should be stored frozen at a temperature of 

-20o C or lower in a freezer that does not experience freeze-thaw cycles. An 

inventory system should be established to ensure the accurate and timely 

retrieval of samples, while protecting patient privacy.

When investigational or other non-commercial assays are utilized, the 

importance of appropriate controls and the ability to compare serially collected 

specimens for quantification/characterization of reactivity is increased. The 

availability of aliquoted samples that allow additional testing may be essential 

to assist interpretation of ambiguous results. Caution should be taken to avoid 

placing more confidence in testing outcomes than can be justified by the nature 

of the assays.

Occupational Health in the BSL-4 Setting

Work with BSL-4 agents involves special challenges for occupational health. 

Infections of laboratory staff by such agents may be expected to result in 

serious or lethal disease for which limited treatment options exist. In addition, 

BSL-4 agents are frequently geographically exotic to the areas in which high 

containment labs are located but produce immediate public health concern if 

infections occur in laboratory staff. Potential (if unlikely) transmission from 

infected staff into the human or animal populations in the areas surrounding the 

laboratories may raise such concerns to higher levels. Thus, SOPs for BSL-4 

settings require special attention to management of unexplained worker absence, 

including protocols for monitoring, medical evaluation, work-up, and follow-up 

of workers with unexplained nonspecific illness. Advance planning for the 

provision of medical care to workers potentially infected with BSL-4 agents is a 

fundamental component of an occupational health program for a BSL-4 facility.

References

1. Menckel E, Westerholm P, editors. Evaluation in occupational health 

practice. Oxford: Butterworth-Heinemann; 1999.

2. Levy B, Wegman DH, editors. Occupational health: recognizing and 

preventing work-related disease and injury, 4th ed. Philadelphia: Lippincott 

Williams & Wilkins; 2000.

3. Occupational Safety and Health Administration (www.osha.gov). 

Washington, DC: The Administration. Applicable OSHA Laws, 

Regulations and Interpretations; [about 2 screens]. Available at: 

www.osha.gov/comp-links.html.
Occupational Health and Immunoprophylaxis 121

 4. American Medical Association (www.ama-assn.org). Chicago: The Association; 

1995-2006 [updated 2005 Mar 7]. Patient Confidentiality; [about 6 screens] 

Available at: www.ama-assn.org/ama/pub/category/4610.html.

5. United States Department of Justice (www.usdoj.gov). Washington, DC: The 

Department; [updated 2006 July 28]. American Disabilities Act; [about seven 

screens] Available at: http://www.usdoj.gov/crt/ada/adahom1.htm.

6. Occupational Safety and Health Act of 1970, Pub. L. No. 91-596, 84 Stat. 

1590 (December 29, 1970).

7. Non-mandatory Compliance Guidelines for Hazard Assessment and Personal 

Protective Equipment Selection, 29 CFR Part 1910 Subpart I App B (1994).

8. Multi-Employer Citation Policy, C.P.L. 02-00-124 (1999).

9. National Research Council (US), Committee on Occupational Safety and 

Health in Research Animal Facilities, Institute of Laboratory Animal 

Resources, Commission on Life Sciences. Occupational health and safety 

in the care and use of research animals. Washington, DC: The National 

Academies Press; 1997. 

10. National Research Council (US), Committee on Occupational Health and 

Safety in the Care and Use of Nonhuman Primates, Institute for Laboratory 

Animal Research, Division on Earth and Life Studies. Occupational health 

and safety in the care and use of nonhuman primates. Washington, DC: 

National Academy Press; 2003.

11. Cohen JI, Davenport DS, Stewart JA, et al. Recommendations for prevention 

of and therapy for exposure to B virus (cercopithecine herpesvirus 1). Clin 

Infect Dis 2002;35:1191-203.

12. Atkinson WL, Pickering LK, Schwartz B, et al. General recommendations on 

immunization. Recommendations of the Advisory Committee on 

Immunization Practices (ACIP) and the American Academy of Family 

Physicians (AAFP). MMWR Recomm Rep. 2002;51:1-35.

13. Centers for Disease Control and Prevention. Update on adult immunization. 

Recommendations of the immunization practices advisory committee (ACIP). 

MMWR Recomm Rep. 1991;40:1-94.

14. Centers for Disease Control and Prevention. Immunization of health-care 

workers: recommendations of the Advisory Committee on Immunization 

Practices (ACIP) and the Hospital Infection Control Practices Advisory 

Committee (HICPAC). MMWR Morb Mortal Wkly Rep. 1997;46:1-42.

15. Centers for Disease Control and Prevention. Use of vaccines and immune 

globulins in persons with altered immunocompetence. Advisory Committee 

on Immunization Practices (ACIP). MMWR Morb Mortal Wkly Rep. 1993 Apr 

9;42(RR-04):1-18.

16. Centers for Disease Control and Prevention. Update: vaccine side effects, 

adverse reactions, contraindications, and precautions. Recommendations of 

the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm 

Rep. 1996 Sep 6; 45(RR-12):1-35. Erratum in: MMWR Morb Mortal Wkly 

Rep. 1997;46:227.
122 Biosafety in Microbiological and Biomedical Laboratories

17. United States Department of Health and Human Services (www.dhhs.gov). 

Washington, DC: The Department; [updated 2006 Mar 6]. Office of 

Human Research Protections Guidance; [about two screens]. Available 

at: http://www.hhs.gov/ohrp.

18. United States Food and Drug Administration (www.fda.gov). Rockville, MD; 

The Administration; [updated 2005 June 13]. Information on Submitting an 

Investigational New Drug Application for a Biological Product [about three 

screens] Available at: http://www.fda.gov/cber/ind/ind.htm. 
Agent Summary Statements: Bacterial Agents 123

Section VIII—Agent Summary Statements

Section VIII-A: Bacterial Agents

Bacillus anthracis

Bacillus anthracis, a gram-positive, non-hemolytic, and non-motile bacillus, is 

the etiologic agent of anthrax, an acute bacterial disease of mammals, including 

humans. Like all members of the genus Bacillus, under adverse conditions B. 

anthracis has the ability to produce spores that allow the organism to persist for 

long periods until the return of more favorable conditions. Reports of suspected 

anthrax outbreaks date back to as early as 1250 BC. The study of anthrax and 

B. anthracis in the 1800s contributed greatly to our general understanding of 

infectious diseases. Much of Koch’s postulates were derived from work on 

identifying the etiologic agent of anthrax. Louis Pasteur developed the first 

attenuated live vaccine for anthrax.

Most mammals are susceptible to anthrax; it mostly affects herbivores that 

ingest spores from contaminated soil and, to a lesser extent, carnivores that 

scavenge on the carcasses of diseased animals. Anthrax still occurs frequently 

in parts of central Asia and Africa. In the United States, it occurs sporadically in 

animals in parts of the West, Midwest and Southwest.

The infectious dose varies greatly from species to species and is routedependent. The inhalation anthrax infectious dose (ID) for humans primarily has 

been extrapolated from inhalation challenges of nonhuman primates (NHP) or 

studies done in contaminated mills. Estimates vary greatly but the medium lethal 

dose (LD50) is likely within the range of 2,500-55,000 spores.1

 It is believed that 

very few spores (10 or less) are required for cutaneous anthrax.2

Occupational Infections

Occupational infections are possible when in contact with contaminated animals, 

animal products or pure cultures of B. anthracis, and may include ranchers, 

veterinarians and laboratory workers. Numerous cases of laboratory-associated 

anthrax (primarily cutaneous) have been reported.3,4 Recent cases include 

suspected cutaneous anthrax in a laboratory worker in Texas and a cutaneous 

case in a North Dakota male who disposed of five cows that died of anthrax.5,6

Natural Modes of Infection

The clinical forms of anthrax in humans that result from different routes of 

infection are: 1) cutaneous (via broken skin); 2) gastrointestinal (via ingestion); 

and 3) inhalation anthrax. Cutaneous anthrax is the most common and readily 

treatable form of the disease. Inhalation anthrax used to be known as “Woolsorter 

disease” due to its prevalence in textile mill workers handling wool and other 

contaminated animal products. While naturally occurring disease is no longer a 
124 Biosafety in Microbiological and Biomedical Laboratories

significant public health problem in the United States, anthrax has become a 

bioterrorism concern. In 2001, 22 people were diagnosed with anthrax acquired 

from spores sent through the mail, including 11 cases of inhalation anthrax with 

five deaths and 11 cutaneous cases.7

Laboratory Safety and Containment Recommendations

B. anthracis may be present in blood, skin lesion exudates, cerebrospinal fluid, 

pleural fluid, sputum, and rarely, in urine and feces. The primary hazards to 

laboratory personnel are: direct and indirect contact of broken skin with cultures 

and contaminated laboratory surfaces, accidental parenteral inoculation and, 

rarely, exposure to infectious aerosols. Efforts should be made to avoid production 

of aerosols by working with infectious organisms in a BSC. In addition, all 

centrifugation should be done using aerosol-tight rotors that are opened 

within the BSC after each run.

BSL-2 practices, containment equipment, and facilities are recommended for 

activities using clinical materials and diagnostic quantities of infectious cultures. 

ABSL-2 practices, containment equipment and facilities are recommended for 

studies utilizing experimentally infected laboratory rodents. BSL-3 practices, 

containment equipment, and facilities are recommended for work involving 

production quantities or high concentrations of cultures, screening environmental 

samples (especially powders) from anthrax-contaminated locations, and for 

activities with a high potential for aerosol production. Workers who frequently 

centrifuge B. anthracis suspensions should use autoclavable aerosol-tight rotors. 

In addition, regular routine swabbing specimens for culture should be routinely 

obtained inside the rotor and rotor lid and, if contaminated, rotors should be 

autoclaved before re-use.

Special Issues

Vaccines A licensed vaccine for anthrax is available. Guidelines for its use 

in occupational settings are available from the ACIP.8,9 Worker vaccination is 

recommended for activities that present an increased risk for repeated exposures 

to B. anthracis spores including: 1) work involving production quantities with a high 

potential for aerosol production; 2) handling environmental specimens, especially 

powders associated with anthrax investigations; 3) performing confirmatory testing 

for B. anthracis, with purified cultures; 4) making repeated entries into known 

B. anthracis-spore-contaminated areas after a terrorist attack; 5) work in other 

settings in which repeated exposure to aerosolized B. anthracis spores might 

occur. Vaccination is not recommended for workers involved in routine processing 

of clinical specimens or environmental swabs in general diagnostic laboratories.

Select Agent B. anthracis is a select agent requiring registration with CDC 

and/or USDA for possession, use, storage and/or transfer. See Appendix F 

for additional information.
Agent Summary Statements: Bacterial Agents 125

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A Department of Commerce (DoC) permit may be required for 

the export of this agent to another country. See Appendix C for additional information.

Bordetella pertussis

Bordetella pertussis, an exclusively human respiratory pathogen of worldwide 

distribution, is the etiologic agent of whooping cough or pertussis. The organism 

is a fastidious, small gram-negative coccobacillus that requires highly specialized 

culture and transport media for cultivation in the laboratory. Its natural habitat is 

the human respiratory tract.

Occupational Infections

Occupational transmission of pertussis has been reported, primarily among 

healthcare workers.10-16 Outbreaks, including secondary transmission, among 

workers have been documented in hospitals, long-term care institutions, and 

laboratories. Nosocomial transmissions have been reported in healthcare 

settings. Laboratory-acquired pertussis has been documented.17,18

Natural Modes of Infection

Pertussis is highly communicable, with person-to-person transmission occurring via 

aerosolized respiratory secretions containing the organism. The attack rate among 

susceptible hosts is affected by the frequency, proximity, and time of exposure to 

infected individuals. Although the number of reported pertussis cases declined 

by over 99% following the introduction of vaccination programs in the 1940s, 

the 3- to 4-year cycles of cases have continued into the post-vaccination era.19-21

Laboratory Safety and Containment Recommendations

The agent may be present in high levels in respiratory secretions, and may be 

found in other clinical material, such as blood and lung tissue in its infrequent 

manifestation of septicemia and pneumonia, respectively.22,23 Because the natural 

mode of transmission is via the respiratory route, aerosol generation during the 

manipulation of cultures and contaminated clinical specimens generates the 

greatest potential hazard.

BSL-2 practices, containment equipment, and facilities are recommended 

for all activities involving the use or manipulation of known or potentially 

infectious clinical material and cultures. ABSL-2 practices and containment 

equipment should be employed for housing experimentally infected animals. 

Primary containment devices and equipment, including biological safety cabinets, 

safety centrifuge cups or safety centrifuges should be used for activities likely to 
126 Biosafety in Microbiological and Biomedical Laboratories

generate potentially infectious aerosols. BSL-3 practices, containment 

equipment, and facilities are appropriate for production operations.

Special Issues

Vaccines Pertussis vaccines are available but are not currently approved 

or recommended for use in persons over six years of age. Because this 

recommendation may change in the near future, the reader is advised to 

review the current recommendations of the ACIP published in the Morbidity and 

Mortality Weekly Report (MMWR) and at the CDC Vaccines and Immunizations 

Web site for the latest recommendations for adolescents and adults.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Brucella species

The genus Brucella consists of slow-growing, very small gram-negative 

coccobacilli whose natural hosts are mammals. Seven Brucella species have 

been described using epidemiologic and biological characteristics, although at 

the genetic level all brucellae are closely related. B. melitensis (natural host: 

sheep/goats), B suis (natural host: swine), B. abortus (natural host: cattle), 

B. canis (natural host: dogs), and B. “maris” (natural host: marine mammals) 

have caused illness in humans exposed to the organism including laboratory 

personnel.24,25 Hypersensitivity to Brucella antigens is a potential but rare 

hazard to laboratory personnel. Occasional hypersensitivity reactions to 

Brucella antigens occur in workers exposed to experimentally and naturally 

infected animals or their tissues.

Occupational Infections

Brucellosis has been one of the most frequently reported laboratory infections 

in the past and cases continue to occur.4,26-28 Airborne and mucocutaneous 

exposures can produce LAI. Accidental self-inoculation with vaccine strains 

is an occupational hazard for veterinarians.

Natural Modes of Infection

Brucellosis (Undulant fever, Malta fever, Mediterranean fever) is a zoonotic 

disease of worldwide occurrence. Mammals, particularly cattle, goats, swine, 

and sheep act as reservoirs for brucellae. Multiple routes of transmission have 

been identified, including direct contact with infected animal tissues or products, 

ingestion of contaminated milk, and airborne exposure in pens and stables.
Agent Summary Statements: Bacterial Agents 127

Laboratory Safety and Containment Recommendations

Brucella infects the blood and a wide variety of body tissues, including cerebral 

spinal fluid, semen, pulmonary excretions, placenta, and occasionally urine. Most 

laboratory-associated cases occur in research facilities and involve exposures to 

Brucella organisms grown in large quantities or exposure to placental tissues 

containing Brucella. Cases have occurred in clinical laboratory settings from 

sniffing bacteriological cultures29 or working on open bench tops.30 Aerosols from, 

or direct skin contact with, cultures or with infectious clinical specimens from 

animals (e.g., blood, body fluids, tissues) are commonly implicated in human 

infections. Aerosols generated during laboratory procedures have caused 

multiple cases per exposure.30,31 Mouth pipetting, accidental parenteral 

inoculations, and sprays into eyes, nose and mouth result in infection. The 

infectious dose of Brucella is 10-100 organisms by aerosol route and 

subcutaneous route in laboratory animals.32,33

BSL-2 practices, containment equipment, and facilities are recommended 

for routine clinical specimens of human or animal origin. Products of conception 

containing or believed to contain pathogenic Brucella should be handled with 

BSL-3 practices due to the high concentration of organisms per gram of tissue. 

BSL-3 and ABSL-3 practices, containment equipment, and facilities are 

recommended, for all manipulations of cultures of pathogenic Brucella spp. 

listed in this summary, and for experimental animal studies.

Special Issues

Vaccines Human Brucella vaccines have been developed and tested in 

other countries with limited success. A human vaccine is not available in the 

United States.34

Select Agent Brucella abortus, Brucella melitensis, and Brucella suis are 

select agents requiring registration with CDC and/or USDA for possession, 

use, storage and/or transfer. See Appendix F for additional information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Burkholderia mallei

Burkholderia mallei (formerly Pseudomonas mallei) is a non-motile gramnegative rod associated with glanders, a rare disease of equine species and 

humans. While endemic foci of infection exist in some areas of the world, 

glanders due to natural infection is extremely rare in he United States. 
128 Biosafety in Microbiological and Biomedical Laboratories

Occupational Infections

Glanders occurs almost exclusively among individuals who work with equine 

species and/or handle B. mallei cultures in the laboratory. B. mallei can be very 

infectious in the laboratory setting. The only reported case of human glanders in 

the United States over the past 50 years resulted from a laboratory exposure.35

Modes of transmission may include inhalation and/or mucocutaneous exposure.

Natural Mode of Infection

Glanders is a highly communicable disease of horses, goats, and donkeys. 

Zoonotic transmission occurs to humans, but person-to-person transmission is 

rare. Clinical glanders no longer occurs in the Western Hemisphere or in most 

other areas of the world, although enzootic foci are thought to exist in Asia and 

the eastern Mediterranean.36 Clinical infections in humans are characterized by 

tissue abscesses and tend to be very serious.

Laboratory Safety and Containment Recommendations

B. mallei can be very hazardous in a laboratory setting. In a pre-biosafety era 

report, one-half of the workers in a B. mallei research laboratory were infected 

within a year of working with the organism.37 Laboratory-acquired infections have 

resulted from aerosol and cutaneous exposure.37,38 Laboratory infections usually 

are caused by exposure to bacterial cultures rather than to clinical specimens. 

Workers should take precautions to avoid exposure to aerosols from bacterial 

cultures, and to tissues and purulent drainage from victims of this disease.

Primary isolations from patient fluids or tissues may be performed with BSL-2 

practices, containment equipment, and facilities in a BSC. Procedures must be 

performed under BSL-3 containment whenever infectious aerosols or droplets are 

generated, such as during centrifugation or handling infected animals, or when 

large quantities of the agent are produced. Procedures conducted outside of a 

BSC (centrifugation, animal manipulation, etc.) that generate infectious aerosols 

require respiratory protection. Sealed cups should be used with all centrifuges 

and these should be opened only inside a BSC. Gloves should be worn when 

working with potentially infectious material or animals. Animal work with B. mallei 

should be done with ABSL-3 practices, containment equipment, and facilities.

Special Issues

Select Agent B. mallei is a select agent requiring registration with CDC 

and/or USDA for possession, use, storage and/or transfer. See Appendix F 

for additional information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 
Agent Summary Statements: Bacterial Agents 129

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Burkholderia pseudomallei

Burkholderia pseudomallei (formerly Pseudomonas pseudomallei) is a motile 

gram-negative, oxidase-positive rod that is found in soil and water environments of 

equatorial regions, including Southeast Asia, Northern Australia, Central America 

and South America. This organism is the causative agent of melioidosis, an unusual 

bacterial disease characterized by abscesses in tissues and organs. Victims of the 

disease frequently exhibit recrudescence months or years after the initial infection.

Occupational Infections

Melioidosis is generally considered to be a disease associated with agriculture; 

however, B. pseudomallei can be hazardous for laboratory workers. There are 

two reports of melioidosis in laboratory workers who were infected by aerosols 

or via skin exposure.39,40 Laboratory workers with diabetes are at increased risk 

of contracting melioidosis.41

Natural Modes of Infection

While primarily a disease found in Southeast Asia and Northern Australia, 

melioidosis can occasionally be found in the Americas.42 Natural modes of 

transmission include the exposure of mucous membranes or damaged skin to 

soil or water containing the organism, the aspiration or ingestion of contaminated 

water, or the inhalation of dust from contaminated soil. In endemic areas, 5-20% 

of agricultural workers have antibody titers to B. pseudomallei, in the absence 

of overt disease.43

Laboratory Safety and Containment Recommendations

B. pseudomallei can cause a systemic disease in human patients. Infected 

tissues and purulent drainage from cutaneous or tissue abscesses can be 

sources of infection. Blood and sputum also are potential sources of infection.

Work with clinical specimens from patients suspected of having melioidosis 

and of B. pseudomallei cultures may be performed with BSL-2 practices, 

containment equipment, and facilities. Work should be done in a BSC. Gloves 

always should be worn when manipulating the microorganism. In cases where 

infectious aerosols or droplets could be produced, or where production quantities 

of the organism are generated, these procedures should be confined to BSL-3 

facilities with all pertinent primary containment against escape of aerosols. 

Respiratory protection must be used if the microorganism is manipulated outside 

of a BSC, such as during centrifugation or handling infected animals. Sealed 
130 Biosafety in Microbiological and Biomedical Laboratories

cups should be used in all centrifuges and these should be opened only in a 

BSC. Animal studies with this agent should be done at ABSL-3.

Special Issues

Select Agent B. pseudomallei is a select agent requiring registration with CDC 

and/or USDA for possession, use, storage and/or transfer. See Appendix F for 

additional information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Campylobacter (C. jejuni subsp. jejuni, C. coli, C. fetus subsp. fetus, 

C. upsaliensis)

Campylobacters are curved, S-shaped, or spiral rods associated with 

gastrointestinal infections (primarily C. jejuni subsp. jejuni and C. coli), 

bacteremia, and sepsis (primarily C. fetus subsp. fetus and C. upsaliensis). 

Organisms are isolated from stool specimens using selective media, reduced 

oxygen tension, and elevated incubation temperature (43°C).

Occupational Infections

These organisms rarely cause LAI, although laboratory-associated cases have 

been documented.44-47 Experimentally infected animals also are a potential 

source of infection.48

Natural Modes of Infection

Numerous domestic and wild animals, including poultry, pets, farm animals, 

laboratory animals, and wild birds are known reservoirs and are a potential 

source of infection for laboratory and animal care personnel. While the infective 

dose is not firmly established, ingestion of as few as 500-800 organisms has 

caused symptomatic infection.49-51 Natural transmission usually occurs from 

ingestion of organisms in contaminated food or water and from direct contact 

with infected pets, farm animals, or infants.52

Laboratory Safety and Containment Recommendations

Pathogenic Campylobacter sp. may occur in fecal specimens in large numbers. 

C. fetus subsp. fetus may also be present in blood, exudates from abscesses, 

tissues, and sputa. The primary laboratory hazards are ingestion and parenteral 

inoculation of C. jejuni. The significance of aerosol exposure is not known.
Agent Summary Statements: Bacterial Agents 131

BSL-2 practices, containment equipment, and facilities are recommended 

for activities with cultures or potentially infectious clinical materials. ABSL-2 

practices, containment equipment, and facilities are recommended for activities 

with naturally or experimentally infected animals.

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Chlamydia psittaci (Chlamydophila psittaci), C. trachomatis, C. pneumoniae 

(Chlamydophila pneumoniae)

Chlamydia psittaci, C. pneumoniae (sometimes called Chlamydophila psittaci 

and Chlamydophila pneumoniae) and C. trachomatis are the three species of 

Chlamydia known to infect humans. Chlamydiae are nonmotile, gram-negative 

bacterial pathogens with obligate intracellular life cycles. These three species of 

Chlamydia vary in host spectrum, pathogenicity, and in the clinical spectrum of 

disease. C. psittaci is a zoonotic agent that commonly infects psittacine birds 

and is highly pathogenic for humans. C. trachomatis is historically considered 

an exclusively human pathogen and is the most commonly reported bacterial 

infection in the United States. C. pneumoniae is considered the least pathogenic 

species, often resulting in subclinical or asymptomatic infections in both animals 

and humans.

Occupational Infections

Chlamydial infections caused by C. psittaci and C. trachomatis lymphogranuloma 

venereum (LGV) strains were at one time among the most commonly reported 

laboratory-associated bacterial infections.26 In cases reported before 19554

, the 

majority of infections were psittacosis, and these had the highest case fatality 

rate of laboratory-acquired infectious agents. The major sources of laboratoryassociated psittacosis are contact with and exposure to infectious aerosols in 

the handling, care, or necropsy of naturally or experimentally infected birds. 

Infected mice and eggs also are important sources of C. psittaci. Most reports 

of laboratory-acquired infections with C. trachomatis attribute the infection 

to inhalation of large quantities of aerosolized organisms during purification 

or sonification procedures. Early reports commonly attributed infections to 

exposure to aerosols formed during nasal inoculation of mice or inoculation 

of egg yolk sacs and harvest of chlamydial elementary bodies. Infections 

are associated with fever, chills, malaise, and headache; a dry cough is also 

associated with C. psittaci infection. Some workers exposed to C. trachomatis 
132 Biosafety in Microbiological and Biomedical Laboratories

have developed conditions including mediastinal and supraclavicular 

lymphadenitis, pneumonitis, conjunctivitis, and keratitis.53 Seroconversion to 

chlamydial antigens is common and often striking although early antibiotic 

treatment may prevent an antibody response.

Laboratory-associated infections with C. pneumoniae have been reported.54

Exposed workers were asymptomatic and infection was diagnosed by serology. 

The route of infection was attributed to inhalation of droplet aerosols created during 

procedures associated with culture and harvest of the agent from cell culture.

With all species of Chlamydia, mucosal tissues in the eyes, nose, and 

respiratory tract are most often affected by occupational exposures that can 

lead to infection.

Natural Modes of Infection

C. psittaci is the cause of psittacosis, a respiratory infection that can lead to 

severe pneumonia requiring intensive care support and possible death. Sequelae 

include endocarditis, hepatitis, and neurologic complications. Natural infections 

are acquired by inhaling dried secretions from infected birds. Psittacine birds 

commonly kept as pets (parrots, parakeets, cockatiels, etc.) and poultry are 

most frequently involved in transmission. C. trachomatis can cause a spectrum 

of clinical manifestations including genital tract infections, inclusion conjunctivitis, 

trachoma, pneumonia in infants, and LGV. The LGV strains cause more severe 

and systemic disease than do genital strains. C. trachomatis genital tract infections 

are sexually transmitted and ocular infections (trachoma) are transmitted by 

exposure to secretions from infected persons through contact or fomite 

transmission. C. pneumoniae is a common cause of respiratory infection; up 

to 50% of adults have serologic evidence of previous exposure. Infections with 

C. pneumoniae are transmitted by droplet aerosolization and are most often 

mild or asymptomatic, although there is a body of evidence associating this 

agent with chronic diseases such as atherosclerosis and asthma.

Laboratory Safety and Containment Recommendations

C. psittaci may be present in the tissues, feces, nasal secretions and blood 

of infected birds, and in blood, sputum, and tissues of infected humans. 

C. trachomatis may be present in genital, bubo, and conjunctival fluids of 

infected humans. Exposure to infectious aerosols and droplets, created during 

the handling of infected birds and tissues, are the primary hazards to laboratory 

personnel working with C. psittaci. The primary laboratory hazards of C. trachomatis 

and C. pneumoniae are accidental parenteral inoculation and direct and indirect 

exposure of mucous membranes of the eyes, nose, and mouth to genital, bubo, 

or conjunctival fluids, cell culture materials, and fluids from infected cell cultures 

or eggs. Infectious aerosols, including those that may be created as a result of 

centrifuge malfunctions, also pose a risk for infection.
Agent Summary Statements: Bacterial Agents 133

BSL-2 practices, containment equipment, and facilities are recommended 

for personnel working with clinical specimens and cultures or other materials 

known or suspected to contain the ocular or genital serovars (A through K) of 

C. trachomatis or C. pneumoniae.

BSL-3 practices, containment equipment, and facilities are recommended for 

activities involving the necropsy of infected birds and the diagnostic examination 

of tissues or cultures known to contain or be potentially infected with C. psittaci 

strains of avian origin. Wetting the feathers of infected birds with a detergentdisinfectant prior to necropsy can appreciably reduce the risk of aerosols of 

infected feces and nasal secretions on the feathers and external surfaces of the 

bird. Activities involving non-avian strains of C. psittaci may be performed in a 

BSL-2 facility as long as BSL-3 practices are followed, including but not limited 

to the use of primary containment equipment such as BSCs. ABSL-3 practices, 

containment equipment, and facilities and respiratory protection are recommended 

for personnel working with naturally or experimentally infected caged birds.

BSL-3 practices and containment equipment are recommended for activities 

involving work with culture specimens or clinical isolates known to contain or 

be potentially infected with the LGV serovars (L1

 through L3) of C. trachomatis. 

Laboratory work with the LGV serovars of C. trachomatis can be conducted in a 

BSL-2 facility as long as BSL-3 practices are followed when handling potentially 

infectious materials, including but not limited to use of primary containment 

equipment such as BSCs.

Gloves are recommended for the necropsy of birds and mice, the opening 

of inoculated eggs, and when there is the likelihood of direct skin contact with 

infected tissues, bubo fluids, and other clinical materials.

ABSL-2 practices, containment equipment, and facilities are recommended 

for activities with animals that have been experimentally infected with genital 

serovars of C. trachomatis or C. pneumoniae.

BSL-3 practices, containment equipment, and facilities are indicated for 

activities involving any of these species with high potential for droplet or aerosol 

production and for activities involving large quantities or concentrations of 

infectious materials.

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.
134 Biosafety in Microbiological and Biomedical Laboratories

Neurotoxin-producing Clostridia species

Clostridium botulinum, and rare strains of C. baratii and C. butyricum, are 

anaerobic spore-forming species that cause botulism, a life-threatening foodborne illness. The pathogenicity of these organisms results from the production 

of botulinum toxin, one of the most highly potent neurotoxins currently recognized. 

Purified botulinum neurotoxin is a 150 kDa protein that acts selectively on 

peripheral cholinergic nerve endings to block neurotransmitter release.55

The principal site of action is the neuromuscular junction, where blockade of 

transmission produces muscle weakness or paralysis. The toxin also acts on 

autonomic nerve endings where blockade of transmission can produce a variety 

of adverse effects. The toxin may also contain associated proteins that may 

increase its size to as high as 900 kDA.

Occupational Infections

There has been only one report of botulism associated with handling of the 

toxin in a laboratory setting.56 However, concerns about potential use of the 

toxin as an agent of bioterrorism or biological warfare have led to increased 

handling of the substance by investigators studying mechanism of action 

and/or developing countermeasures to poisoning.57

Natural Modes of Infection

Botulinum toxin occurs in seven different serotypes (A to G), but almost all 

naturally-occurring human illness is due to serotypes A, B, E, and F.58 Botulism 

occurs when botulinum toxin is released into circulation following ingestion of 

preformed toxin. However, animal studies have shown that botulism may occur 

through inhalation of preformed toxin. Use of appropriate personal protective 

equipment should prevent potential exposure through mucus membranes. 

Symptoms and even death are possible by accidental injection of botulinum 

toxin. Risk to toxin exposure is dependent on both route of exposure and toxin 

molecular weight size. Exposure to neurotoxin producing Clostridia species 

does not cause infection; however, in certain rare circumstances (Infant Botulism, 

Wound Botulism, and Adult colonization), the organism can colonize the intestinal 

tract and other sites and produce toxin. In Wound Botulism, exposure to toxin is 

caused by introduction of spores into puncture wounds and in situ production by 

the organism. Infants less than 1 year of age may be susceptible to intestinal 

colonization and develop the syndrome of Infant Botulism as a result of in situ 

production of toxin. Similarly to Infant Botulism, ingestion of spores by adults 

with a compromised gastrointestinal tract (GI), such as following GI surgery or 

long-term administration of antibiotics, may increase risk for intestinal infection 

and in situ production of toxin. See the C. botulinum Toxin Agent Summary 

Statement and Appendix I for additional information.
Agent Summary Statements: Bacterial Agents 135

Laboratory Safety and Containment Recommendations

Neurotoxin producing Clostridia species or its toxin may be present in a variety of 

food products, clinical materials (serum, feces) and environmental samples (soil, 

surface water).59 In addition, bacterial cultures may produce very high levels of 

toxin.60 In healthy adults, it is typically the toxin and not the organism that causes 

disease. Risk of laboratory exposure is due to the presence of the toxin and not 

due to a potential of infection from the organisms that produce the toxin. Although 

spore-forming, there is no known risk to spore exposure except for the potential 

for the presence of residual toxin associated with pure spore preparations. 

Laboratory safety protocols should be developed with the focus on prevention 

of accidental exposure to the toxin produced by these Clostridia species.

BSL-2 practices, containment equipment, and facilities are recommended 

for activities that involve the organism or the toxin61 including the handling of 

potentially contaminated food. Solutions of sodium hypochlorite (0.1%) or 

sodium hydroxide (0.1N) readily inactivate the toxin and are recommended for 

decontamination of work surfaces and for spills. Autoclaving of contaminated 

materials also is appropriate.

Additional primary containment and personnel precautions, such as 

those recommended for BSL-3, should be implemented for activities with a 

high potential for aerosol or droplet production, or for those requiring routine 

handling of larger quantities of the organism or of the toxin. ABSL-2 practices, 

containment equipment, and facilities are recommended for diagnostic studies 

and titration of toxin.

Special Issues

Vaccines A pentavalent (A, B, C, D and E) botulinum toxoid vaccine (PBT) is 

available through the CDC as an Investigational New Drug (IND). Vaccination 

is recommended for all personnel working in direct contact with cultures of 

neurotoxin producing Clostridia species or stock solutions of Botulinum neurotoxin. 

Due to a possible decline in the immunogenicity of available PBT stocks for 

some toxin serotypes, the immunization schedule for the PBT recently has been 

modified to require injections at 0, 2, 12, and 24 weeks, followed by a booster 

at 12 months and annual boosters thereafter. Since there is a possible decline 

in vaccine efficacy, the current vaccine contains toxoid for only 5 of the 7 toxin 

types, this vaccine should not be considered as the sole means of protection 

and should not replace other worker protection measures.

Post-Exposure Treatment An equine antitoxin product is available for 

treatment of patients with symptoms consistent with botulism. However, due 

to the risks inherent in equine products, treatment is not provided as a result 

of exposure unless botulism symptoms are present.
136 Biosafety in Microbiological and Biomedical Laboratories

Select Agent Neurotoxin producing Clostridia species are select agents 

requiring registration with CDC and/or USDA for possession, use, storage and/or 

transfer. See Appendix F for additional information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Clostridium tetani and Tetanus toxin

Clostridium tetani is an anaerobic endospore-forming gram-positive rod found 

in the soil and an intestinal tract commensal. It produces a potent neurotoxin, 

tetanospasmin, which causes tetanus, an acute neurologic condition characterized 

by painful muscular contractions. Tetanospasmin is an exceedingly potent, high 

molecular weight protein toxin, consisting of a heavy chain (100kD) subunit that 

binds the toxin to receptors on neuronal cells and a light chain (50kD) subunit 

that blocks the release of inhibitory neural transmitter molecules within the central 

nervous system. The incidence of tetanus in the United States has declined 

steadily since the introduction of tetanus toxoid vaccines in the 1940’s.62

Occupational Infections

Although the risk of infection to laboratory personnel is low, there have been 

five incidents of laboratory personnel exposure recorded.4

Natural Modes of Infection

Contamination of wounds by soil is the usual mechanism of transmission for 

tetanus. Of the 130 cases of tetanus reported to CDC from 1998 through 2000, 

acute injury (puncture, laceration, abrasion) was the most frequent predisposing 

condition. Elevated incidence rates also were observed for persons aged over 

60 years, diabetics, and intravenous drug users.63 When introduced into a 

suitable anaerobic or microaerophilic environment, C. tetani spores germinate 

and produce tetanospasmin. The incubation period ranges from 3 to 21 days. 

The observed symptoms are primarily associated with the presence of the toxin. 

Wound cultures are not generally useful for diagnosing tetanus.64

Laboratory Safety and Containment Recommendations

The organism may be found in soil, intestinal, or fecal samples. Accidental 

parenteral inoculation of the toxin is the primary hazard to laboratory personnel. 

Because it is uncertain if tetanus toxin can be absorbed through mucous 

membranes, the hazards associated with aerosols and droplets remain unclear.
Agent Summary Statements: Bacterial Agents 137

BSL-2 practices, containment equipment, and facilities are recommended 

for activities involving the manipulation of cultures or toxin. ABSL-2 practices, 

containment equipment, and facilities are recommended for animal studies.

Special Issues

Vaccines The vaccination status of workers should be considered in a risk 

assessment for workers with this organism and/or toxin. While the risk of 

laboratory-associated tetanus is low, the administration of an adult diphtheriatetanus toxoid at 10-year intervals further reduces the risk to laboratory and 

animal care personnel of toxin exposures and wound contamination, and is 

therefore highly recommended.62 The reader is advised to consult the current 

recommendations of the ACIP. 65

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Corynebacterium diphtheriae

Corynebacterium diphtheriae is a pleomorphic gram-positive rod that is isolated 

from the nasopharynx and skin of humans. The organism is easily grown in the 

laboratory on media containing 5% sheep blood. C. diphtheriae produces a 

potent exotoxin and is the causative agent of diphtheria, one of the most widespread bacterial diseases in the pre-vaccine era.

Occupational Infections

Laboratory-associated infections with C. diphtheriae have been documented, 

but laboratory animal-associated infections have not been reported.4,66 Inhalation, 

accidental parenteral inoculation, and ingestion are the primary laboratory hazards.

Natural Modes of Infection

The agent may be present in exudates or secretions of the nose, throat (tonsil), 

pharynx, larynx, wounds, in blood, and on the skin. Travel to endemic areas or 

close contact with persons who have returned recently from such areas, increases 

risk.67 Transmission usually occurs via direct contact with patients or carriers, 

and more rarely, with articles contaminated with secretions from infected people. 

Naturally occurring diphtheria is characterized by the development of grayishwhite membranous lesions involving the tonsils, pharynx, larynx, or nasal mucosa. 

Systemic sequelae are associated with the production of diphtheria toxin. An 

effective vaccine has been developed for diphtheria and this disease has 

become a rarity in countries with vaccination programs.
138 Biosafety in Microbiological and Biomedical Laboratories

Laboratory Safety and Containment Recommendations

BSL-2 practices, containment equipment, and facilities are recommended for all 

activities utilizing known or potentially infected clinical materials or cultures. ABSL-2 

facilities are recommended for studies utilizing infected laboratory animals.

Special Issues

Vaccines A licensed vaccine is available. The reader is advised to consult the 

current recommendations of the CIP.65 While the risk of laboratory-associated 

diphtheriai is low, the administration of an adult diphtheria-tentanus toxoid at 

10-year intervals may further reduce the risk of illness to laboratory and animal 

care personnel.62

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Francisella tularensis

Francisella tularensis is a small gram-negative coccobacillus that is carried 

in numerous animal species, especially rabbits, and is the causal agent of 

tularemia (Rabbit fever, Deer fly fever, Ohara disease, or Francis disease) in 

humans. F. tularensis can be divided into three subspecies, F. tularensis (Type A), 

F. holarctica (Type B) and F. novicida, based on virulence testing, 16S sequence, 

biochemical reactions and epidemiologic features. Type A and Type B strains are 

highly infectious, requiring only 10-50 organisms to cause disease. Subspecies 

F. novicida is infrequently identified as the cause of human disease. Person-toperson transmission of tularemia has not been documented. The incubation 

period varies with the virulence of the strain, dose and route of introduction but 

ranges from 1-4 days with most cases exhibiting symptoms in 3-5 days.68

Occupational Infections

Tularemia has been a commonly reported laboratory-associated bacterial infection.4

Most cases have occurred at facilities involved in tularemia research; however, 

cases have been reported in diagnostic laboratories as well. Occasional cases were 

linked to work with naturally or experimentally infected animals or their ectoparasites.

Natural Modes of Infection

Tick bites, handling or ingesting infectious animal tissues or fluids, ingestion of 

contaminated water or food and inhalation of infective aerosols are the primary 

transmission modes in nature. Occasionally, infections have occurred from bites 

or scratches by carnivores with contaminated mouthparts or claws.
Agent Summary Statements: Bacterial Agents 139

Laboratory Safety and Containment Recommendations

The agent may be present in lesion exudates, respiratory secretions, cerebrospinal 

fluid (CSF), blood, urine, tissues from infected animals, fluids from infected 

animals, and fluids from infected arthropods. Direct contact of skin or mucous 

membranes with infectious materials, accidental parenteral inoculation, ingestion, 

and exposure to aerosols and infectious droplets has resulted in infection. Infection 

has been more commonly associated with cultures than with clinical materials 

and infected animals.69

BSL-2 practices, containment equipment, and facilities are recommended 

for activities involving clinical materials of human or animal origin suspected or 

known to contain F. tularensis. Laboratory personnel should be informed of the 

possibility of tularemia as a differential diagnosis when samples are submitted 

for diagnostic tests. BSL-3 and ABSL-3 practices, containment equipment, and 

facilities are recommended for all manipulations of suspect cultures, animal 

necropsies and for experimental animal studies. Preparatory work on cultures or 

contaminated materials for automated identification systems should be performed 

at BSL-3. Characterized strains of reduced virulence such as F. tularensis Type B 

(strain LVS) and F. tularensis subsp novicida (strain U112) can be manipulated in 

BSL-2. Manipulation of reduced virulence strains at high concentrations should 

be conducted using BSL-3 practices.

Special Issues

Select Agent F. tularensis is a select agent requiring registration with CDC 

and/or USDA for possession, use, storage and/or transfer. See Appendix F for 

additional information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Helicobacter species

Helicobacters are spiral or curved gram-negative rods isolated from gastrointestinal 

and hepatobiliary tracts of mammals and birds. There are currently 20 recognized 

species, including at least nine isolated from humans. Since its discovery in 1982, 

Helicobacter pylori has received increasing attention as an agent of gastritis.70

The main habitat of H. pylori is the human gastric mucosa. Other Helicobacter 

spp. (H. cinaedi, H. canadensis, H. canis, H. pullorum, and H. fennelliae) may 

cause asymptomatic infection as well as proctitis, proctocolitis, enteritis and 

extraintestinal infections in humans.71,72 H. cinaedi has been isolated from dogs, 

cats and Syrian hamsters.
140 Biosafety in Microbiological and Biomedical Laboratories

Occupational Infections

Both experimental and accidental LAI with H. pylori have been reported.73,74

Ingestion is the primary known laboratory hazard. The importance of aerosol 

exposures is unknown.

Natural Modes of Infection

Chronic gastritis and duodenal ulcers are associated with H. pylori infection. 

Epidemiologic associations have also been made with gastric adenocarcinoma. 

Human infection with H. pylori may be long in duration with few or no symptoms, 

or may present as an acute gastric illness. Transmission, while incompletely 

understood, is thought to be by the fecal-oral or oral-oral route.

Laboratory Safety and Containment Recommendations

H. pylori may be present in gastric and oral secretions and stool.75 The 

enterohepatic helicobacters (e.g., H. canadensis, H. canis, H, cinaedi, H. fennelliae, 

H. pullorum, and H. winghamensis) may be isolated from stool specimens, rectal 

swabs, and blood cultures.72 Protocols involving homogenization or vortexing of 

gastric specimens have been described for the isolation of H. pylori.

76 Containment 

of potential aerosols or droplets should be incorporated in these procedures.

BSL-2 practices, containment equipment, and facilities are recommended 

for activities with clinical materials and cultures known to contain or potentially 

contain the agents. ABSL-2 practices, containment equipment, and facilities are 

recommended for activities with experimentally or naturally infected animals.

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Legionella pneumophila and other Legionella-like Agents

Legionella are small, faintly staining gram-negative bacteria. They are obligately 

aerobic, slow-growing, nonfermentative organisms that have a unique 

requirement for L-cysteine and iron salts for in vitro growth. Legionellae are 

readily found in natural aquatic bodies and some species (L. longbeachae) 

have been recovered from soil.77,78 They are able to colonize hot-water tanks at 

a temperature range from 40 to 50°C. There are currently 48 known Legionella 

species, 20 of which have been associated with human disease. L. pneumophila 

is the species most frequently encountered in human infections.79-81
Agent Summary Statements: Bacterial Agents 141

Occupational Infections

Although laboratory-associated cases of legionellosis have not been reported in 

the literature, at least one case, due to presumed aerosol or droplet exposure 

during animal challenge studies with L. pneumophila, has been recorded.82

Experimental infections have been produced in guinea pigs, mice, rats, embryonated 

chicken eggs, and human or animal cell lines.83 A fatal case of pneumonia due to 

L. pneumophila was diagnosed in a calf, but only 1.7% (2/112) of the other cattle 

in the herd had serological evidence of exposure to Legionella.

84 The disease 

was linked to exposure to a hot water system colonized with Legionella. Animalto-animal transmission has not been demonstrated.

Natural Modes of Infection

Legionella is commonly found in environmental sources, typically in man-made 

warm water systems. The mode of transmission from these reservoirs is 

aerosolization, aspiration or direct inoculation into the airway.85 Direct personto-person transmission does not occur. The spectrum of illness caused by 

Legionella species ranges from a mild, self-limited flu-like illness (Pontiac fever) 

to a disseminated and often fatal disease characterized by pneumonia and 

respiratory failure (Legionnaires disease). Although rare, Legionella has been 

implicated in cases of sinusitis, cellulitis, pericarditis, and endocarditis.86

Legionellosis may be either community-acquired or nosocomial. Risk factors 

include smoking, chronic lung disease, and immunosuppression. Surgery, 

especially involving transplantation, has been implicated as a risk factor for 

nosocomial transmission.

Laboratory Safety and Containment Recommendations

The agent may be present in respiratory tract specimens (sputum, pleural fluid, 

bronchoscopy specimens, lung tissue), and in extrapulmonary sites. A potential 

hazard may exist for generation of aerosols containing high concentrations of 

the agent.

BSL-2 practices, containment equipment, and facilities are recommended 

for all activities involving the use or manipulation of potentially infectious 

materials, including minimizing the potential for dissemination of the organism 

from cultures of organisms known to cause disease. ABSL-2 practices, 

containment equipment and facilities are recommended for activities with 

experimentally-infected animals. Routine processing of environmental water 

samples for Legionella may be performed with standard BSL-2 practices. 

For activities likely to produce extensive aerosols and when large quantities 

of the pathogenic organisms are manipulated, BSL-2 with BSL-3 practices 

is recommended.
142 Biosafety in Microbiological and Biomedical Laboratories

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Leptospira

The genus Leptospira is composed of spiral-shaped bacteria with hooked ends. 

Leptospires are ubiquitous in nature, either free-living in fresh water or associated 

with renal infection in animals. Historically, these organisms have been classified 

into pathogenic (L. interrogans) and saprophytic (L. biflexa) groups, but recent 

studies have identified more than 12 species based on genetic analysis. These 

organisms also have been characterized serologically, with more than 200 

pathogenic and 60 saprophytic serovars identified as of 2003.87 These organisms 

are the cause of leptospirosis, a zoonotic disease of worldwide distribution. 

Growth of leptospires in the laboratory requires specialized media and culture 

techniques, and cases of leptospirosis are usually diagnosed by serology.

Occupational Infections

Leptospirosis is a well-documented laboratory hazard. Approximately, 70 LAI 

and 10 deaths have been reported.4,26 Direct and indirect contact with fluids and 

tissues of experimentally or naturally infected mammals during handling, care, or 

necropsy are potential sources of infection.88-90 It is important to remember that 

rodents are natural carriers of leptospires. Animals with chronic renal infection shed 

large numbers of leptospires in the urine continuously or intermittently, for long 

periods of time. Rarely, infection may be transmitted by bites of infected animals.88

Natural Modes of Infection

Human leptospirosis typically results from direct contact with infected animals, 

contaminated animal products, or contaminated water sources. Common routes 

of infection include abrasions, cuts in the skin or via the conjunctiva. Higher rates 

of infection observed in agricultural workers and other occupations associated 

with animal contact.

Laboratory Safety and Containment Recommendations

The organism may be present in urine, blood, and tissues of infected animals 

and humans. Ingestion, accidental parenteral inoculation, and direct and indirect 

contact of skin or mucous membranes, particularly the conjunctiva, with cultures 

or infected tissues or body fluids are the primary laboratory hazards. The 

importance of aerosol exposure is not known.
Agent Summary Statements: Bacterial Agents 143

BSL-2 practices, containment equipment, and facilities are recommended for 

all activities involving the use or manipulation of known or potentially infective 

tissues, body fluids, and cultures. The housing and manipulation of infected animals 

should be performed at ABSL-2. Gloves should be worn to handle and necropsy 

infected animals and to handle infectious materials and cultures in the laboratory.

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Listeria monocytogenes

Listeria monocytogenes is a gram-positive, non-spore-forming, aerobic bacillus; 

that is weakly beta-hemolytic on sheep blood agar and catalase-positive.91 The 

organism has been isolated from soil, animal feed (silage) and a wide range of 

human foods and food processing environments. It may also be isolated from 

symptomatic/asymptomatic animals (particularly ruminants) and humans.91,92

This organism is the causative agent of listeriosis, a food-borne disease of 

humans and animals.

Occupational Infections

Cutaneous listeriosis, characterized by pustular or papular lesions on the arms 

and hands, has been described in veterinarians and farmers.93 Asymptomatic 

carriage has been reported in laboratorians.94

Natural Modes of Infection

Most human cases of listeriosis result from eating contaminated foods, notably 

soft cheeses, ready-to-eat meat products (hot dogs, luncheon meats), paté and 

smoked fish/seafood.95 Listeriosis can present in healthy adults with symptoms 

of fever and gastroenteritis, pregnant women and their fetuses, newborns, and 

persons with impaired immune function are at greatest risk of developing severe 

infections including sepsis, meningitis, and fetal demise. In pregnant women, 

Listeria monocytogenes infections occur most often in the third trimester and 

may precipitate labor. Transplacental transmission of L. monocytogenes poses 

a grave risk to the fetus.92

Laboratory Safety and Containment Recommendations

Listeria monocytogenes may be found in feces, CSF, and blood, as well as 

numerous food and environmental samples.91,92,96,97 Naturally or experimentally 

infected animals are a source of exposure to laboratory workers, animal care 
144 Biosafety in Microbiological and Biomedical Laboratories

personnel and other animals. While ingestion is the most common route of 

exposure, Listeria can also cause eye and skin infections following direct contact 

with the organism.

BSL-2 practices, containment equipment, and facilities are recommended 

when working with clinical specimens and cultures known or suspected to contain 

the agent. Gloves and eye protection should be worn while handling infected or 

potentially infected materials. ABSL-2 practices, containment equipment and 

facilities are recommended for activities involving experimentally or naturally 

infected animals. Due to potential risks to the fetus, pregnant women should 

be advised of the risk of exposure to L. monocytogenes.

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Mycobacterium leprae

Mycobacterium leprae is the causative agent of leprosy (Hansen disease). The 

organism has not been cultivated in laboratory medium but can be maintained in a 

metabolically active state for some period. Organisms are recovered from infected 

tissue and can be propagated in laboratory animals, specifically armadillos and the 

footpads of mice. The infectious dose in humans is unknown. Although naturally 

occurring leprosy or leprosy-like diseases have been reported in armadillos98 and 

in NHP,99,100 humans are the only known important reservoir of this disease.

Occupational Infections

There are no cases reported as a result of working in a laboratory with biopsy or 

other clinical materials of human or animal origin. However, inadvertent human-tohuman transmissions following an accidental needle stick by a surgeon and after 

use of a presumably contaminated tattoo needle were reported prior to 1950.101,102

Natural Modes of Infection

Leprosy is transmitted from person-to-person following prolonged exposure, 

presumably via contact with secretions from infected individuals.

Laboratory Safety and Containment Recommendations

The infectious agent may be present in tissues and exudates from lesions of 

infected humans and experimentally or naturally infected animals. Direct contact of 

the skin and mucous membranes with infectious materials and accidental parenteral 
Agent Summary Statements: Bacterial Agents 145

inoculation are the primary laboratory hazards associated with handling infectious 

clinical materials. See Appendix B for appropriate tuberculocidal disinfectant.

BSL-2 practices, containment equipment, and facilities are recommended 

for all activities with known or potentially infectious materials from humans and 

animals. Extraordinary care should be taken to avoid accidental parenteral 

inoculation with contaminated sharp instruments. ABSL-2 practices, containment 

equipment, and facilities are recommended for animal studies utilizing rodents, 

armadillos, and NHP, because coughing with dissemination of infectious droplets 

does not occur in these species.

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Mycobacterium tuberculosis complex

The Mycobacterium tuberculosis complex includes M. tuberculosis, M. bovis, 

M. africanum, and M. microti that cause tuberculosis in humans, and more recently 

recognized M. caprae and M. pinnipedii that have been isolated from animals. 

M. tuberculosis grows slowly, requiring three weeks for formation of colonies on solid 

media. The organism has a thick, lipid-rich cell wall that renders bacilli resistant to 

harsh treatments including alkali and detergents and allows them to stain acid-fast.

Occupational Infections

M. tuberculosis and M. bovis infections are a proven hazard to laboratory 

personnel as well as others who may be exposed to infectious aerosols in the 

laboratory, autopsy rooms, and other healthcare facilities.4,26,103-105 The incidence 

of tuberculosis in laboratory personnel working with M. tuberculosis has been 

reported to be three times higher than that of those not working with the agent.106

Naturally or experimentally infected NHP are a proven source of human 

infection.107 Experimentally infected guinea pigs or mice do not pose the same 

hazard because droplet nuclei are not produced by coughing in these species; 

however, litter from infected animal cages may become contaminated and serve 

as a source of infectious aerosols.

Natural Modes of Infection

M. tuberculosis is the etiologic agent of tuberculosis, a leading cause of morbidity 

and mortality worldwide. Persons infected with M. tuberculosis can develop active 

disease within months of infection or can remain latently infected and develop 
146 Biosafety in Microbiological and Biomedical Laboratories

disease later in life. The primary focus of infection is the lungs, but most other 

organs can be involved. HIV infection is a serious risk factor for development of 

active disease. Infectious aerosols produced by coughing spread tuberculosis. 

M. bovis is primarily found in animals but also can produce tuberculosis in 

humans. It is spread to humans, primarily children, by consumption of nonpasteurized milk and milk products, by handling of infected carcasses, and 

by inhalation. Human-to-human transmission via aerosols also is possible.

Laboratory Safety and Containment Recommendations

Tubercle bacilli may be present in sputum, gastric lavage fluids, CSF, urine, and 

in a variety of tissues. Exposure to laboratory-generated aerosols is the most 

important hazard encountered. Tubercle bacilli may survive in heat-fixed 

smears108 and may be aerosolized in the preparation of frozen sections and 

during manipulation of liquid cultures. Because of the low infective dose of 

M. tuberculosis (i.e., ID50 <10 bacilli), sputa and other clinical specimens from 

suspected or known cases of tuberculosis must be considered potentially 

infectious and handled with appropriate precautions. Accidental needle-sticks 

are also a recognized hazard.

BSL-2 practices and procedures, containment equipment, and facilities 

are required for non-aerosol-producing manipulations of clinical specimens 

such as preparation of acid-fast smears. All aerosol-generating activities must 

be conducted in a BSC. Use of a slide-warming tray, rather than a flame, is 

recommended for fixation of slides. Liquifaction and concentration of sputa for 

acid-fast staining may be conducted safely on the open bench by first treating the 

specimen in a BSC with an equal volume of 5% sodium hypochlorite solution 

(undiluted household bleach) and waiting 15 minutes before processing.109,110

BSL-3 practices, containment equipment, and facilities are required for 

laboratory activities in the propagation and manipulation of cultures of any of 

the subspecies of the M. tuberculosis complex and for animal studies using 

experimentally or naturally infected NHP. Animal studies using guinea pigs or 

mice can be conducted at ABSL-2.111 BSL-3 practices should include the use of 

respiratory protection and the implementation of specific procedures and use of 

specialized equipment to prevent and contain aerosols. Disinfectants proven to 

be tuberculocidal should be used. See Appendix B for additional information.

Manipulation of small quantities of the attenuated vaccine strain M. bovis 

Bacillus Calmette-Guérin (BCG) can be performed at BSL-2 in laboratories 

that do not culture M. tuberculosis and do not have BSL-3 facilities. However, 

considerable care must be exercised to verify the identity of the strain and to 

ensure that cultures are not contaminated with virulent M. tuberculosis or other 

M. bovis strains. Selection of an appropriate tuberculocidal disinfectant is an 

important consideration for laboratories working with mycobacteria. See 

Appendix B for additional information.
Agent Summary Statements: Bacterial Agents 147

Special Issues

Surveillance Annual or semi-annual skin testing with purified protein 

derivative (PPD) of previously skin-test-negative personnel can be used 

as a surveillance procedure.

Vaccines The attenuated live BCG, is available and used in other countries 

but is not used in the United States for immunization.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Mycobacterium spp. other than M. tuberculosis complex and M. leprae

More than 100 species of mycobacteria are recognized. These include both 

slowly growing and rapidly growing species. In the past, mycobacterial isolates 

that were not identified as M. tuberculosis complex were often called atypical 

mycobacteria, but these are now more commonly referred to as nontuberculous 

mycobacteria or mycobacteria other than tuberculosis. Many of the species are 

common environmental organisms, and approximately 25 of them are associated 

with infections in humans. A number of additional species are associated with 

infections in immunocompromised persons, especially HIV-infected individuals. 

All of these species are considered opportunistic pathogens in humans and none 

are considered communicable. Mycobacteria are frequently isolated from clinical 

samples but may not be associated with disease. The most common types of 

infections and causes are:

1. pulmonary disease with a clinical presentation resembling tuberculosis 

caused by M. kansasii, M. avium, and M. intracellulare;

2. lymphadenitis associated with M. avium and M. scrofulaceum;

3. disseminated infections in immunocompromised individuals caused 

by M. avium;

4. skin ulcers and soft tissue wound infections including Buruli ulcer 

caused by M. ulcerans, swimming pool granuloma caused by 

M. marinum associated with exposure to organisms in fresh and 

salt water and fish tanks, and tissue infections resulting from trauma, 

surgical procedures, or injection of contaminated materials caused 

by M. fortuitum, M. chelonei, and M. abscesens.
148 Biosafety in Microbiological and Biomedical Laboratories

Occupational Infections

Laboratory-acquired infections with Mycobacterium spp. other than 

M. tuberculosis complex have not been reported.

Natural Modes of Infection

Person-to-person transmission has not been demonstrated. Presumably, 

pulmonary infections are the result of inhalation of aerosolized bacilli, most likely 

from the surface of contaminated water. Mycobacteria are widely distributed 

in the environment and in animals. They are also common in potable water 

supplies, perhaps as the result of the formation of biofilms. The source of 

M. avium infections in immunocompromised persons has not been established.

Laboratory Safety and Containment Recommendations

Various species of mycobacteria may be present in sputa, exudates from 

lesions, tissues, and in environmental samples. Direct contact of skin or mucous 

membranes with infectious materials, ingestion, and accidental parenteral 

inoculation are the primary laboratory hazards associated with clinical materials 

and cultures. Aerosols created during the manipulation of broth cultures or 

tissue homogenates of these organisms also pose a potential infection hazard.

BSL-2 practices, containment equipment, and facilities are recommended 

for activities with clinical materials and cultures of Mycobacteria spp. other than 

M. tuberculosis complex. Clinical specimens may also contain M. tuberculosis 

and care must be exercised to ensure the correct identification of cultures. Special 

caution should be exercised in handling M. ulcerans to avoid skin exposure. 

ABSL-2 practices, containment equipment, and facilities are recommended for 

animal studies. Selection of an appropriate tuberculocidal disinfectant is an 

important consideration for laboratories working with mycobacteria. See 

Appendix B for additional information.

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Neisseria gonorrhoeae

Neisseria gonorrhoeae is a gram-negative, oxidase-positive diplococcus associated 

with gonorrhea, a sexually transmitted disease of humans. The organism may be 

isolated from clinical specimens and cultivated in the laboratory using specialized 

growth media.112
Agent Summary Statements: Bacterial Agents 149

Occupational Infections

Laboratory-associated gonococcal infections have been reported in the United 

States and elsewhere.113-116 These infections have presented as conjunctivitis, with 

either direct finger-to-eye contact or exposure to splashes of either liquid cultures 

or contaminated solutions proposed as the most likely means of transmission.

Natural Modes of Infection

Gonorrhea is a sexually transmitted disease of worldwide importance. The 2004 

rate of reported infections for this disease in the United States was 112 per 

100,000 population.117 The natural mode of infection is through direct contact with 

exudates from mucous membranes of infected individuals. This usually occurs 

by sexual activity, although newborns may also become infected during birth.112

Laboratory Safety and Containment Recommendations

The agent may be present in conjunctival, urethral and cervical exudates, synovial 

fluid, urine, feces, and CSF. Accidental parenteral inoculation and direct or indirect 

contact of mucous membranes with infectious clinical materials are known primary 

laboratory hazards. Laboratory-acquired illness due to aerosol transmission has 

not been documented.

BSL-2 practices, containment equipment, and facilities are recommended 

for all activities involving the use or manipulation of clinical materials or cultures. 

Gloves should be worn when handling infected laboratory animals and when 

there is the likelihood of direct skin contact with infectious materials. Additional 

primary containment and personnel precautions such as those described for 

BSL-3 may be indicated when there is high risk of aerosol or droplet production, 

and for activities involving production quantities or high concentrations of 

infectious materials. Animal studies may be performed at ABSL-2.

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Neisseria meningitidis

Neisseria meningitidis is a gram-negative coccus responsible for serious acute 

meningitis and septicemia in humans. Virulence is associated with the expression 

of a polysaccharide capsule. Thirteen different capsular serotypes have been 

identified, with types A, B, C, Y, and W135 associated with the highest incidence 
150 Biosafety in Microbiological and Biomedical Laboratories

of disease. The handling of invasive N. meningitidis isolates from blood or CSF 

represents an increased risk to microbiologists.118,119

Occupational Infections

Recent studies of LAI and exposures have indicated that manipulating suspensions 

of N. meningitidis outside a BSC is associated with a high risk for contracting 

meningococcal disease.119 Investigations of potential laboratory-acquired cases of 

meningococcal diseases in the United States showed a many-fold higher attack 

rate for microbiologists compared to that of the United States general population 

age 30-59 years, and a case fatality rate of 50%, substantially higher than the 

12-15% associated with disease among the general population. Almost all the 

microbiologists had manipulated sterile site isolates on an open laboratory bench.120

While isolates obtained from respiratory sources are generally less pathogenic 

and consequently represent lower risk for microbiologists, rigorous protection 

from droplets or aerosols is mandated when microbiological procedures are 

performed on all N. meningitidis isolates, especially on those from sterile sites.

Natural Modes of Infection

The human upper respiratory tract is the natural reservoir for N. meningitidis. 

Invasion of organisms from the respiratory mucosa into the circulatory system 

causes infection that can range in severity from subclinical to fulminant fatal 

disease. Transmission is person-to-person and is usually mediated by direct 

contact with respiratory droplets from infected individuals.

Laboratory Safety and Containment Recommendations

N. meningitidis may be present in pharyngeal exudates, CSF, blood, and saliva. 

Parenteral inoculation, droplet exposure of mucous membranes, infectious 

aerosol and ingestion are the primary hazards to laboratory personnel. Based 

on the mechanism of natural infection and the risk associated with handling 

of isolates on an open laboratory bench, exposure to droplets or aerosols of 

N. meningitidis is the most likely risk for infection in the laboratory.

Specimens for N. meningitidis analysis and cultures of N. meningitidis not 

associated with invasive disease may be handled in BSL-2 facilities with rigorous 

application of BSL-2 standard practices, special practices, and safety equipment. 

All sterile-site isolates of N. meningitidis should be manipulated within a BSC. 

Isolates of unknown source should be treated as sterile-site isolates.

If a BSC is unavailable, manipulation of these isolates should be minimized, 

primarily focused on serogroup identification using phenolized saline solution 

while wearing a laboratory coat, gloves, and safety glasses or full-face splash 

shield. BSL-3 practices and procedures are indicated for activities with a high 

potential for droplet or aerosol production and for activities involving production 
Agent Summary Statements: Bacterial Agents 151

quantities or high concentrations of infectious materials. Animal studies should 

be performed under ABSL-2 conditions.

Special Issues

Vaccines The quadrivalent meningococcal polysaccharide vaccine, which 

includes serogroups A, C, Y, and W-135, will decrease but not eliminate the 

risk of infection, because it is less than 100% effective and does not provide 

protection against serogroup B, which caused one-half of the laboratory-acquired 

cases in the United States in 2000.118,120 Laboratorians who are exposed routinely 

to potential aerosols of N. meningitidis should consider vaccination.118,121,122

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Salmonella serotypes, other than S. Typhi

Salmonellae are gram-negative enteric bacteria associated with diarrheal illness 

in humans. They are motile oxidase-negative organisms that are easily cultivated 

on standard bacteriologic media, although enrichment and selective media may 

be required for isolation from clinical materials. Recent taxonomic studies have 

organized this genus into two species, S. enterica and S. bongori, containing 

more than 2500 antigenically distinct subtypes or serotypes.123 S. enterica 

contains the vast majority of serotypes associated with human disease. S. enterica 

serotypes Typhimurium and Enteritidis (commonly designated S. Typhimurium

and S. Enteritidis) are the serotypes most frequently encountered in the United 

States. This summary statement covers all pathogenic serotypes except S. Typhi.

Occupational Infections

Salmonellosis is a documented hazard to laboratory personnel.4,26,124-125 Primary 

reservoir hosts include a broad spectrum of domestic and wild animals, including 

birds, mammals, and reptiles, all of which may serve as a source of infection to 

laboratory personnel. Case reports of laboratory-acquired infections indicate a 

presentation of symptoms (fever, severe diarrhea, abdominal cramping) similar 

to those of naturally-acquired infections, although one case also developed 

erythema nodosum and reactive arthritis.126,127

Natural Modes of Infection

Salmonellosis is a food borne disease of worldwide distribution. An estimated 

5 million cases of salmonellosis occur annually in the United States. A wide range 

of domestic and feral animals (poultry, swine, rodents, cattle, iguanas, turtles, 
152 Biosafety in Microbiological and Biomedical Laboratories

chicks, dogs, cats) may serve as reservoirs for this disease, as well as humans.128

The most common mode of transmission is by ingestion of food from contaminated 

animals or contaminated during processing. The disease usually presents as an 

acute enterocolitis, with an incubation period ranging from 6 to 72 hours.

Laboratory Safety and Containment Recommendations

The agent may be present in feces, blood, urine, and in food, feed, and 

environmental materials. Ingestion or parenteral inoculation are the primary 

laboratory hazards. The importance of aerosol exposure is not known. Naturally 

or experimentally infected animals are a potential source of infection for 

laboratory and animal care personnel, and for other animals

Strict compliance with BSL-2 practices, containment equipment, and facilities 

are recommended for all activities utilizing known or potentially infectious clinical 

materials or cultures. This includes conducting procedures with aerosol or high 

splash potential in primary containment devices such as a BSCs or safety centrifuge 

cups. Personal protective equipment should be used in accordance with a risk 

assessment, including splash shields, face protection, gowns, and gloves. The 

importance of proper gloving techniques and frequent and thorough hand washing 

is emphasized. Care in manipulating faucet handles to prevent contamination of 

cleaned hands or the use of sinks equipped with remote water control devices, 

such as foot pedals, is highly recommended. Special attention to the timely and 

appropriate decontamination of work surfaces, including potentially contaminated 

equipment and laboratory fixtures, is strongly advised. ABSL-2 facilities and 

practices are recommended for activities with experimentally infected animals.

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Salmonella Typhi

Recent taxonomic studies have organized the genus Salmonella into two species, 

S. enterica and S. bongori, containing more than 2500 antigenically distinct 

subtypes or serotypes.123 S. enterica contains the vast majority of serotypes 

associated with human disease. S. enterica serotype Typhi, commonly designated 

S. Typhi, is the causative agent of typhoid fever. S. Typhi is a motile gram-negative 

enteric bacterium that is easily cultivated on standard bacteriologic media, although 

enrichment and selective media may be required for isolation of this organism 

from clinical materials.
Agent Summary Statements: Bacterial Agents 153

Occupational Infections

Typhoid fever is a demonstrated hazard to laboratory personnel.4,129,130 Ingestion 

and less frequently, parenteral inoculation are the most significant modes of 

transmission in the laboratory. Secondary transmission to other individuals 

outside of the laboratory is also a concern.131 Laboratory-acquired S. Typhi

infections usually present with symptoms of septicemia, headache, abdominal 

pain, and high fever.129

Natural Modes of Infection

Typhoid fever is a serious, potentially lethal bloodstream infection of worldwide 

distribution. Humans are the sole reservoir and asymptomatic carriers may occur. 

The infectious dose is low (<103 organisms) and the incubation period may vary 

from one to six weeks, depending upon the dose of the organism. The natural 

mode of transmission is by ingestion of food or water contaminated by feces or 

urine of patients or asymptomatic carriers.123

Laboratory Safety and Containment Recommendations

The agent may be present in feces, blood, gallbladder (bile), and urine. Humans 

are the only known reservoir of infection. Ingestion and parenteral inoculation 

of the organism represent the primary laboratory hazards. The importance of 

aerosol exposure is not known.

Strict compliance with BSL-2 practices, containment equipment, and facilities 

are recommended for all activities utilizing known or potentially infectious clinical 

materials or cultures. This includes conducting procedures with aerosol or high 

splash potential in primary containment devices such as BSCs or safety centrifuge 

cups. Personal protective equipment should be used in accordance with a risk 

assessment, including splash shields, face protection, gowns, and gloves. The 

importance of proper gloving techniques and frequent and thorough hand washing 

is emphasized. Care in manipulating faucet handles to prevent contamination of 

cleaned hands or the use of sinks equipped with remote water control devices, 

such as foot pedals, is highly recommended. Special attention to the timely and 

appropriate decontamination of work surfaces, including potentially contaminated 

equipment and laboratory fixtures, is strongly advised. BSL-3 practices and 

equipment are recommended for activities likely to produce significant aerosols or 

for activities involving production quantities of organisms. ABSL-2 facilities, practices 

and equipment are recommended for activities with experimentally infected 

animals. ABSL-3 conditions may be considered for protocols involving aerosols.

Special Issues

Vaccines Vaccines for S. Typhi are available and should be considered for 

personnel regularly working with potentially infectious materials. The reader is 

advised to consult the current recommendations of the Advisory Committee on 
154 Biosafety in Microbiological and Biomedical Laboratories

Immunization Practices (ACIP) published in the CDC Morbidity and Mortality 

Weekly Report for recommendations for vaccination against S. Typhi.

132

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Shiga toxin (Verocytotoxin)-producing Escherichia coli

Escherichi coli is one of five species in the gram-negative genus Escherichia. 

This organism is a common inhabitant of the bowel flora of healthy humans and 

other mammals and is one of the most intensively studied prokaryotes. An extensive 

serotyping system has been developed for E. coli based the O (somatic) and H 

(flagellar) antigens expressed by these organisms. Certain pathogenic clones of 

E. coli may cause urinary tract infections, bacteremia, meningitis, and diarrheal 

disease in humans, and these clones are associated with specific serotypes.

The diarrheagenic E. coli strains have been characterized into at least four 

basic pathogenicity groups: Shiga toxin (Verocytotoxin)-producing E. coli (a subset 

of which are referred to as enterohemorrhagic E. coli), enterotoxigenic E. coli, 

enteropathogenic E. coli, and enteroinvasive E. coli.123 In addition to clinical 

significance, E. coli strains are commonly-used hosts for cloning experiments 

and other genetic manipulations in the laboratory. This summary statement 

provides recommendations for safe manipulation of Shiga toxin-producing 

E. coli strains. Procedures for safely handling laboratory derivatives of E. coli or 

other pathotypes of E. coli should be based upon a thorough risk assessment.

Occupational Infections

Shiga toxin-producing E. coli strains, including strains of serotype O157:H7, 

are a demonstrated hazard to laboratory personnel.133-138 The infectious dose is 

estimated to be low—similar to that reported for Shigella spp., 10-100 organisms.136

Domestic farm animals (particularly bovines) are significant reservoirs of the 

organisms; however, experimentally infected small animals are also sources of 

infection in the laboratory.139 Verocytotoxin-producing Escherichia coli have also 

been in wild birds and rodents in close proximity to farms.140

Natural Modes of Infection

Cattle represent the most common natural reservoir of Shiga-toxin producing 

E. coli. Transmission usually occurs by ingestion of contaminated food, including 

raw milk, fruits, vegetables, and particularly ground beef. Human-to-human 

transmission has been observed in families, day care centers, and custodial 

institutions. Water-borne transmission has been reported from outbreaks 
Agent Summary Statements: Bacterial Agents 155

associated with swimming in a crowded lake and drinking unchlorinated municipal 

water.139 In a small proportion of patients (usually children) infected with these 

organisms, the disease progresses to hemolytic uremic syndrome or death.

Laboratory Safety and Containment Recommendations

Shiga toxin-producing E. coli are usually isolated from feces. However, a variety 

of food specimens contaminated with the organisms including uncooked ground 

beef, unpasteurized dairy products and contaminated produce may present 

laboratory hazards. This agent may be found in blood or urine specimens from 

infected humans or animals. Accidental ingestion is the primary laboratory 

hazard. The importance of aerosol exposure is not known.

Strict compliance with BSL-2 practices, containment equipment, and facilities 

are recommended for all activities utilizing known or potentially infectious clinical 

materials or cultures. Procedures with aerosol or high splash potential should be 

conducted with primary containment equipment or in devices such as a BSC or 

safety centrifuge cups. Personal protective equipment, such as splash shields, 

face protection, gowns, and gloves should be used in accordance with a risk 

assessment. The importance of proper gloving techniques and frequent and 

thorough hand washing is emphasized. Care in manipulating faucet handles 

to prevent contamination of cleaned hands or the use of sinks equipped with 

remote water control devices, such as foot pedals, is highly recommended. 

Special attention to the timely and appropriate decontamination of work surfaces, 

including potentially contaminated equipment and laboratory fixtures, is strongly 

advised. ABSL-2 practices and facilities are recommended for activities with 

experimentally or naturally infected animals.

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Shigella

The genus Shigella is composed of nonmotile gram-negative bacteria in the 

family Enterobacteriaceae. There are four subgroups that have been historically 

treated as separate species, even though more recent genetic analysis indicates 

that they are members of the same species. These include subgroup A (Shigella 

dysenteriae), subgroup B (S. flexneri), subgroup C (S. boydii), and subgroup D 

(S. sonnei). Members of the genus Shigella have been recognized since the 

late 19th century as causative agents of bacillary dysentery, or shigellosis.123
156 Biosafety in Microbiological and Biomedical Laboratories

Occupational Infections

Shigellosis is one of the most frequently reported laboratory-acquired infections 

in the United States.131,141 A survey of 397 laboratories in the United Kingdom 

revealed that in 1994-1995, four of nine reported laboratory-acquired infections 

were caused by Shigella.

142 Experimentally infected guinea pigs, other rodents, 

and NHP are proven sources of laboratory-acquired infection.143,144

Natural Modes of Infection

Humans and other large primates are the only natural reservoirs of Shigella 

bacteria. Most transmission is by fecal-oral route; infection also is caused by 

ingestion of contaminated food or water.123 Infection with Shigella dysenteriae 

type 1 causes more severe, prolonged, and frequently fatal illness than does 

infection with other Shigella. Complications of shigellosis include hemolytic 

uremic syndrome, which is associated with S. dysenteriae 1 infection, and 

Reiter chronic arthritis syndrome, which is associated with S. flexneri infection.

Laboratory Safety and Containment Recommendations

The agent may be present in feces and, rarely, in the blood of infected humans 

or animals. Accidental ingestion and parenteral inoculation of the agent are the 

primary laboratory hazards. The 50% infectious dose (oral) of Shigella for humans is 

only a few hundred organisms.143 The importance of aerosol exposure is not known.

Strict compliance with BSL-2 practices, containment equipment, and 

facilities are recommended for all activities utilizing known or potentially 

infectious clinical materials or cultures. Procedures with aerosol or high splash 

potential should be conducted with primary containment equipment such as a 

BSC or safety centrifuge cups. Personal protective equipment should be used 

in accordance with a risk assessment, including splash shields, face protection, 

gowns, and gloves. The importance of proper gloving techniques and frequent 

and thorough hand washing is emphasized. Care in manipulating faucet handles 

to prevent contamination of cleaned hands or the use of sinks equipped with 

remote water control devices, such as foot pedals, is highly recommended. 

Special attention to the timely and appropriate decontamination of work surfaces, 

including potentially contaminated equipment and laboratory fixtures, is strongly 

advised. ABSL-2 facilities and practices are recommended for activities with 

experimentally or naturally infected animals.

Special Issues

Vaccines Vaccines are currently not available for use in humans.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 
Agent Summary Statements: Bacterial Agents 157

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Treponema pallidum

Treponema pallidum is a species of extremely fastidious spirochetes that die 

readily upon desiccation or exposure to atmospheric levels of oxygen, and have 

not been cultured continuously in vitro.

145 T. pallidum cells have lipid-rich outer 

membranes and are highly susceptible to disinfection with common alcohols (i.e., 

70% isopropanol). This species contains three subspecies including T. pallidum 

spp. pallidum (associated with venereal syphilis), T. pallidum spp. endemicum 

(associated with endemic syphilis), and T. pallidum spp. pertenue (associated 

with Yaws). These organisms are obligate human pathogens.

Occupational Infections

T. pallidum is a documented hazard to laboratory personnel. Pike lists 20 cases 

of LAI.4 Syphilis has been transmitted to personnel working with a concentrated 

suspension of T. pallidum obtained from an experimental rabbit orchitis.146 T. pallidum 

is present in the circulation during primary and secondary syphilis. The ID50 of 

T. pallidum needed to infect rabbits by subcutaneous injection has been reported 

to be as low as 23 organisms.147 The concentration of T. pallidum in patients’ 

blood during early syphilis, however, has not been determined. No cases of 

laboratory animal-associated infections are reported; however, rabbit-adapted 

T. pallidum (Nichols strain and possibly others) retains virulence for humans.

Natural Modes of Infection

Humans are the only known natural reservoir of T. pallidum and transmission 

occurs via direct sexual contact (venereal syphilis), direct skin contact (Yaws), 

or direct mucous contact (endemic syphilis). Venereal syphilis is a sexually 

transmitted disease that occurs in many areas of the world, whereas Yaws 

occurs in tropical areas of Africa, South America, the Caribbean, and Indonesia. 

Endemic syphilis is limited to arid areas of Africa and the Middle East.145

Laboratory Safety and Containment Recommendations

The agent may be present in materials collected from cutaneous and mucosal 

lesions and in blood. Accidental parenteral inoculation, contact with mucous 

membranes or broken skin with infectious clinical materials are the primary 

hazards to laboratory personnel.

BSL-2 practices, containment equipment, and facilities are recommended for 

all activities involving the use or manipulation of blood or other clinical samples 

from humans or infected rabbits. Gloves should be worn when there is a likelihood 
158 Biosafety in Microbiological and Biomedical Laboratories

of direct skin contact with infective materials. Periodic serological monitoring 

should be considered in personnel regularly working with these materials. ABSL-2 

practices, containment equipment, and facilities are recommended for work with 

infected animals.

Special Issues

Vaccines Vaccines are currently not available for use in humans.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Vibrio enteritis species (V. cholerae, V. parahaemolyticus)

Vibrio species are straight or curved motile gram-negative rods. Growth of Vibrio 

species is stimulated by sodium and the natural habitats of these organisms are 

primarily aquatic environments. Although 12 different Vibrio species have been 

isolated from clinical specimens, V. cholerae and V. parahaemolyticus are the 

best-documented causes of human disease.148 Vibrios may cause either diarrhea 

or extraintestinal infections.

Occupational Infections

Rare cases of bacterial enteritis due to LAI with either V. cholerae or 

V. parahaemolyticus have been reported from around the world.4

 Naturally 

and experimentally infected animals149 and shellfish150,151 are potential sources 

for such illnesses.

Natural Modes of Infection

The most common natural mode of infection is the ingestion of contaminated 

food or water. The human oral infecting dose of V. cholerae in healthy nonachlorhydric individuals is approximately 106

-1011 colony forming units,152 while 

that of V. parahaemolyticus ranges from 105

-107 cells.153 The importance of 

aerosol exposure is unknown although it has been implicated in at least one 

instance.149 The risk of infection following oral exposure is increased in persons 

with abnormal gastrointestinal physiology including individuals on antacids, with 

achlorhydria, or with partial or complete gastrectomies.154

Laboratory Safety and Containment Recommendations

Pathogenic vibrios can be present in human fecal samples, or in the meats and 

the exterior surfaces of marine invertebrates such as shellfish. Other clinical 

specimens from which vibrios may be isolated include blood, arm or leg wounds, 
Agent Summary Statements: Bacterial Agents 159

eye, ear, and gallbladder.148 Accidental oral ingestion of V. cholerae or V. 

parahaemolyticus principally results from hands contaminated from the use of 

syringes or the handling of naturally contaminated marine samples without gloves.

BSL-2 practices, containment equipment, and facilities are recommended for 

activities with cultures or potentially infectious clinical materials. ABSL-2 practices, 

containment equipment, and facilities are recommended for activities with 

naturally or experimentally infected animals.

Special Issues

Vaccines The reader is advised to consult the current recommendations of 

the ACIP published in the MMWR for vaccination recommendations against 

V. cholera. There are currently no human vaccines against V. parahaemolyticus.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Yersinia pestis

Yersinia pestis, the causative agent of plague, is a gram-negative, microaerophilic 

coccobacillus frequently characterized by a “safety pin” appearance on stained 

preparations from specimens. It is nonmotile and nonsporulating. There are three 

biotypes of Y. pestis, differentiated by their ability to ferment glycerol and reduce 

nitrate. All three biotypes are virulent. The incubation period for bubonic plague 

ranges from two to six days while the incubation period for pneumonic plague is 

one to six days. Pneumonic plague is transmissible person-to-person;155 whereas 

bubonic plague is not. Laboratory animal studies have shown the lethal and 

infectious doses of Y. pestis to be quite low (less than 100 colony forming units).156

Occupational Infections

Y. pestis is a documented laboratory hazard. Prior to 1950, at least 10 laboratoryacquired cases were reported in the United States, four of which were fatal.4,157

Veterinary staff and pet owners have become infected when handling domestic 

cats with oropharyngeal or pneumonic plague.

Natural Modes of Infection

Infective fleabites are the most common mode of transmission, but direct human 

contact with infected tissues or body fluids of animals and humans also may 

serve as sources of infection.
160 Biosafety in Microbiological and Biomedical Laboratories

Primary pneumonic plague arises from the inhalation of infectious respiratory 

droplets or other airborne materials from infected animals or humans. This form 

of plague has a high case fatality rate if not treated and poses the risk of personto-person transmission.

Laboratory Safety and Containment Recommendations

The agent has been isolated, in order of frequency of recovery, from bubo 

aspirate, blood, liver, spleen, sputum, lung, bone marrow, CSF, and infrequently 

from feces and urine, depending on the clinical form and stage of the disease. 

Primary hazards to laboratory personnel include direct contact with cultures and 

infectious materials from humans or animal hosts and inhalation of infectious 

aerosols or droplets generated during their manipulation. Laboratory and field 

personnel should be counseled on methods to avoid fleabites and accidental 

autoinoculation when handling potentially infected live or dead animals.

BSL-2 practices, containment equipment, and facilities are recommended for 

all activities involving the handling of potentially infectious clinical materials and 

cultures. In addition, because the infectious dose is so small, all work, including 

necropsies of potentially infected animals should be performed in a BSC. Special 

care should be taken to avoid generating aerosols or airborne droplets while 

handling infectious materials or when performing necropsies on naturally or 

experimentally infected animals. Gloves should be worn when handling potentially 

infectious materials including field or laboratory infected animals. BSL-3 is 

recommended for activities with high potential for droplet or aerosol production, 

and for activities involving large-scale production or high concentrations of 

infectious materials. Resistance of Y. pestis strains to antibiotics used in the 

treatment of plague should be considered in a thorough risk assessment and 

may require additional containment for personal protective equipment. For animal 

studies, a risk assessment that takes into account the animal species, infective 

strain, and proposed procedures should be performed in order to determine if 

ABSL-2 or ABSL-3 practices, containment equipment, and facilities should be 

employed. BSL-3 facilities and arthropod containment level 3 practices are 

recommended for all laboratory work involving infected arthropods.157 See 

Appendix G for additional information on arthropod containment guidelines.

Special Issues

Select Agent Yersinia pestis is an HHS select agent requiring registration 

with CDC for the possession, use, storage and transfer. See Appendix F for 

further information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.
Agent Summary Statements: Bacterial Agents-References 161

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137.Spina N, Zansky S, Dumas N, et al. Four laboratory-associated cases of 

infection with Escherichia coli O157:H7. J Clin Microbiol. 2005;43:2938-9.

138.Chin J. Diarrhea caused by Escherichia coli. In: Chin J, ed. Control of 

Communicable Diseases. 17th ed. Washington, DC: American Public Health 

Association; 2000. p. 155-65.

139.Nielsen EM, Skov MN, Madsen JJ, et.al. Verocytotoxin-producing 

Escherichia coli in wild birds and rodents in close proximity to farms. 

Appl Envrion Microbiol. 2004;70:6944-7.

140.Mermel LA, Josephson SL, Dempsey J, et al. Outbreak of Shigella sonnei 

in a clinical microbiology laboratory. J Clin Microbiol. 1997;35:3163-5.

141.Walker D, Campbell D. A survey of infections in United Kingdom laboratories, 

1994-1995. J Clin Pathol. 1999;52:415-8.

142.Parsot C, Sansonetti PJ. Invasion and the pathogenesis of Shigella infection. 

Curr Top Microbiol Immunol. 1996;209:25-42.

143.National Research Council. Zoonoses. In: Occupational health and safety in 

the care and use of research animals. Washington, DC: National Academy 

Press. 1997. p. 65-106.

144.Norris SJ, Pope V, Johnson RE, et al. Treponema and other human hostassociated spirochetes. In: Murray PR, Barron EJ, Jorgensen JH, et al, 

editors. Manual of clinical microbiology. 8th ed. Washington, DC: ASM 

Press. 2003. p. 955-71.

145.Fitzgerald JJ, Johnson RC, Smith M. Accidental laboratory infection with 

Treponema pallidum, Nichols strain. J Am Vener Dis Assoc. 1976;3:76-8.
Agent Summary Statements: Bacterial Agents-References 169

146.Magnuson HJ, Thomas EW, Olansky S, et al. Inoculation syphilis in human 

volunteers. Medicine. 1956;35:33-82.

147.Farmer JJ, Janda JM, Birkhead K. Vibrio. In: Murray PR, Baron EJ, 

Jorgensen JH, et al, editors. Manual of clinical microbiology. 8th ed. 

Washington DC: ASM Press. 2003. p. 706-18.

148.Sheehy TW, Sprinz H, Augerson WS, et al. Laboratory Vibrio cholerae 

infection in the United States. JAMA. 1966;197:321-6.

149.Lee KK, Liu PC, Huang CY. Vibrio parahaemolyticus infectious for both 

humans and edible mollusk abalone. Microbes Infect. 2003;5:481-5.

150.Morris JG Jr. Cholerae and other types of vibriosis: a story of human 

pandemics and oysters on the half shell. Clin Infect Dis. 2003;37:272-80.

151.Reidl J, Klose KE. Vibrio cholerae and cholera: out of the water and into 

the host. FEMS Microbiol Rev. 2002;26:125-39.

152.Daniels NA, Ray B, Easton A, et al. Emergence of a new Vibrio 

parahaemolyticus serotype in raw oysters. JAMA. 2000;284:1541-5. 

Erratum in: JAMA 2001;285:169.

153.American Public Health Association. Cholera and other vibrioses. In: 

Heymann HL, editor. Control of communicable diseases. 18th ed. Baltimore 

(MD): United Book Press, Inc. 2004. p. 103-15.

154.Dennis DT, Gage, KL. Plague. In: Cohen J, Powderly WG, editors. Infectious 

diseases. Vol II. 2nd ed. Edinburgh: Mosby; 2004. p. 1641-8.

155.Russell P, Eley SM, Bell DL, et al. 1996. Doxycycline or ciprofloxacin 

prophylaxis and therapy against Yersinia pestis infection in mice. J 

Antimicrobial Chemother. 1996;37:769-74.

156.Burmeister RW, Tigertt WD, Overholt EL. Laboratory-acquired pneumonic 

plague. Report of a case and review of previous cases. Ann Intern Med. 

1962;56:789-800.

157.Higgs S. Arthropod containment guidelines. Vector-Borne and Zoonotic 

Diseases. 2003;3:57-98.
170 Biosafety in Microbiological and Biomedical Laboratories

Section VIII-B: Fungal Agents

Blastomyces dermatitidis

Blastomyces dermatitidis is a dimorphic fungal pathogen existing in nature and 

in laboratory cultures at room temperature as a filamentous mold with asexual 

spores (conidia) that are the infectious particles; these convert to large budding 

yeasts under the appropriate culture conditions in vitro at 37°C and in the 

parasitic phase in vivo in warm-blooded animals. The sexual stage is an 

Ascomycete with infectious ascospores.

Occupational Infections

Three groups are at greatest risk of laboratory-acquired infection: microbiologists, 

veterinarians and pathologists.1

 Laboratory-associated local infections have 

been reported following accidental parenteral inoculation with infected tissues or 

cultures containing yeast forms of B. dermatitidis.

2-8 Pulmonary infections have 

occurred following the presumed inhalation of conidia from mold-form cultures; 

two persons developed pneumonia and one had an osteolytic lesion from which 

B. dermatitidis was cultured.9,10 Presumably, pulmonary infections are associated 

only with sporulating mold forms.

Natural Modes of Infection

The fungus has been reported from multiple geographically separated countries, 

but is best known as a fungus endemic to North America and in association with 

plant material in the environment. Infections are not communicable, but require 

common exposure from a point source. Although presumed to dwell within the soil 

of endemic areas, B. dermatitidis is extremely difficult to isolate from soil. Outbreaks 

associated with the exposure of people to decaying wood have been reported.11

Laboratory Safety and Containment Recommendations

Yeast forms may be present in the tissues of infected animals and in clinical 

specimens. Parenteral (subcutaneous) inoculation of these materials may 

cause local skin infection and granulomas. Mold form cultures of B. dermatitidis 

containing infectious conidia, and processing of soil or other environmental 

samples, may pose a hazard of aerosol exposure.

BSL-2 and ABSL-2 practices, containment equipment, and facilities are 

recommended for activities with clinical materials, animal tissues, yeast-form 

cultures, and infected animals. BSL-3 practices, containment equipment, and 

facilities are required for handling sporulating mold-form cultures already 

identified as B. dermatitidis and soil or other environmental samples known 

or likely to contain infectious conidia.
Agent Summary Statements: Fungal Agents 171

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Coccidioides immitis and Coccidioides posadasii

Coccidioides spp. is endemic to lower sonoran deserts of the western 

hemisphere including northern Mexico, southern Arizona, central and southern 

California, and west Texas. The original species (C. immitis) has been divided 

into C. immitis and C. posadasii.

12 These species are dimorphic fungal pathogens 

existing in nature and in laboratory cultures at room temperature as filamentous 

molds with asexual spores (single-cell arthroconidia three to five microns in size) 

that are the infectious particles that convert to spherules under the appropriate 

culture conditions in vitro at 37°C and in vivo in warm-blooded animals.

Occupational Infections

Laboratory-associated coccidioidomycosis is a documented hazard of working 

with sporulating cultures of Coccidioides spp.13-15 Occupational exposure has also 

been associated in endemic regions with archeology16 and high dust exposure.17

Attack rates for laboratory and occupational exposure are higher than for ambient 

exposure when large numbers of spores are inhaled. Smith reported that 28 of 31 

(90%) laboratory-associated infections in his institution resulted in clinical disease, 

whereas more than half of infections acquired in nature were asymptomatic.18

Risk of respiratory infection from exposure to infected tissue or aerosols of 

infected secretions is very low. Accidental percutaneous inoculation has 

typically resulted in local granuloma formation.19

Natural Modes of Infection

Single spores can produce ambient infections by the respiratory route. Peak 

exposures occur during arid seasons. Coccidioides spp. grow in infected tissue 

as larger multicellular spherules, up to 70 microns in diameter and pose little or 

no risk of infection from direct exposure.

The majority of ambient infections is subclinical and results in life-long 

protection from subsequent exposures. The incubation period is one to three 

weeks and manifests as a community-acquired pneumonia with immunologically 

mediated fatigue, skin rashes, and joint pain. One of the synonyms for 

coccidioidomycosis is desert rheumatism. A small proportion of infections is 

complicated by hematogenous dissemination from the lungs to other organs, 

most frequently skin, the skeleton, and the meninges. Disseminated infection is 
172 Biosafety in Microbiological and Biomedical Laboratories

much more likely in persons with cellular immunodeficiencies (AIDS, organ 

transplant recipient, lymphoma).

Laboratory Safety and Containment Recommendations

Because of their size, the arthroconidia are conducive to ready dispersal in 

air and retention in the deep pulmonary spaces. The much larger size of the 

spherule considerably reduces the effectiveness of this form of the fungus as 

an airborne pathogen.

Spherules of the fungus may be present in clinical specimens and animal 

tissues, and infectious arthroconidia in mold cultures and soil or other samples 

from natural sites. Inhalation of arthroconidia from environmental samples or 

cultures of the mold form is a serious laboratory hazard. Personnel should be 

aware that infected animal or human clinical specimens or tissues stored or 

shipped in such a manner as to promote germination of arthroconidia pose a 

theoretical laboratory hazard.

BSL-2 practices, containment equipment, and facilities are recommended for 

handling and processing clinical specimens, identifying isolates, and processing 

animal tissues. ABSL-2 practices, containment equipment, and facilities are 

recommended for experimental animal studies when the route of challenge 

is parenteral.

BSL-3 practices, containment equipment, and facilities are recommended 

for propagating and manipulating sporulating cultures already identified as 

Coccidioides spp. and for processing soil or other environmental materials 

known to contain infectious arthroconidia. Experimental animal studies should 

be done at BSL-3 when challenge is via the intranasal or pulmonary route.

Special Issues

Select Agent Some Coccidioides spp. are select agents requiring registration 

with CDC and/or USDA for possession, use, storage and/or transfer. See 

Appendix F for additional information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Cryptococcus Neoformans

Cryptococcus neoformans is a monomorphic fungal pathogen existing in nature, 

in laboratory cultures at room temperature and in vivo as a budding yeast. The 

sexual stage is grouped with the Basidiomycetes and is characterized by sparse 
Agent Summary Statements: Fungal Agents 173

hyphal formation with basidiospores. Both basidiospores and asexual yeasts 

are infectious.

Occupational Infections

Accidental inoculation of a heavy inoculum of C. neoformans into the hands 

of laboratory workers has occurred during injection or necropsy of laboratory 

animals.20,21 Either a local granuloma or no lesion was reported, suggesting low 

pathogenicity by this route. Respiratory infections as a consequence of laboratory 

exposure have not been recorded.

Natural Modes of Infection

The fungus is distributed worldwide in the environment and is associated with 

pigeon feces. Infections are not transmissible from person-to-person, but require 

common exposure via the respiratory route to a point source.

Laboratory Safety and Containment Recommendations

Accidental parenteral inoculation of cultures or other infectious materials represents 

a potential hazard to laboratory personnel, particularly to those who may be 

immunocompromised. Bites by experimentally infected mice and manipulations 

of infectious environmental materials (e.g., pigeon feces) may also represent a 

potential hazard to laboratory personnel. C. neoformans has been isolated from 

bedding of cages housing mice with pulmonary infection indicating the potential 

for contamination of cages and animal facilities by infected animals.22 Reports 

of cutaneous cryptococcal infection following minor skin injuries suggests that 

localized infection may complicate skin injuries incurred in laboratories that 

handle C. neoformans.

23

BSL-2 and ABSL-2 practices, containment equipment, and facilities are 

recommended for activities with known or potentially infectious clinical, environmental, 

or culture materials and with experimentally infected animals. This agent and any 

samples that may contain this agent should also be handled in a Class II BSC. 

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Histoplasma capsulatum

Histoplasma capsulatum is a dimorphic fungal pathogen existing in nature 

and in laboratory cultures at room temperature as a filamentous mold with 

asexual spores (conidia); these are the infectious particles that convert to 

small budding yeasts under the appropriate culture conditions in vitro at 37°C 
174 Biosafety in Microbiological and Biomedical Laboratories

and in the parasitic phase in vivo. The sexual stage is an Ascomycete with 

infectious ascospores.

Occupational Infections

Laboratory-associated histoplasmosis is a documented hazard in facilities 

conducting diagnostic or investigative work.24-27 Pulmonary infections have 

resulted from handling mold form cultures.28,29 Local infection has resulted from 

skin puncture during autopsy of an infected human,30 from accidental needle 

inoculation of a viable culture,31 and from spray from a needle into the eye.32

Collecting and processing soil samples from endemic areas has caused 

pulmonary infections in laboratory workers.33 Conidia are resistant to drying 

and may remain viable for long periods of time. The small size of the infective 

conidia (less than 5 microns) is conducive to airborne dispersal and 

intrapulmonary retention. Work with experimental animals suggests that 

hyphal fragments are capable of serving as viable inocula.24

Natural Modes of Infection

The fungus is distributed worldwide in the environment and is associated 

with starling and bat feces. It has been isolated from soil, often in river valleys, 

between latitudes 45°N and 45°S. Histoplasmosis is naturally acquired by 

the inhalation of infectious particles, usually microconidia.24 Infections are 

not transmissible from person-to-person, but require common exposure to 

a point source.

Laboratory Safety and Containment Recommendations

The infective stage of this dimorphic fungus (conidia) is present in sporulating 

mold form cultures and in soil from endemic areas. The yeast form in tissues or 

fluids from infected animals may produce local infection following parenteral 

inoculation or splash onto mucous membranes.

BSL-2 and ABSL-2 practices, containment equipment, and facilities are 

recommended for handling and processing clinical specimens, identifying 

isolates, animal tissues and mold cultures, identifying cultures in routine 

diagnostic laboratories, and for inoculating experimental animals, regardless 

of route. Any culture identifying dimorphic fungi should be handled in a 

Class II BSC.

BSL-3 practices, containment equipment, and facilities are recommended 

for propagating sporulating cultures of H. capsulatum in the mold form, as well 

as processing soil or other environmental materials known or likely to contain 

infectious conidia.
Agent Summary Statements: Fungal Agents 175

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Sporothrix schenckii

Sporothrix schenckii is a dimorphic fungal pathogen existing in nature and in 

laboratory cultures at room temperature as a filamentous mold with asexual 

spores (conidia); these are the infectious particles that convert to small budding 

yeasts in the parasitic phase in vivo. The sexual stage is unknown.

Occupational Infections

Most cases of sporotrichosis are reported sporadically following accidental 

inoculation with contaminated material. Large outbreaks have been documented 

in persons occupationally or recreationally exposed to soil or plant material 

containing the fungus. However, S. schenckii has caused a substantial number 

of local skin or eye infections in laboratory personnel.34 Most occupational 

cases have been associated with accidents and have involved splashing culture 

material into the eye,35,36 scratching,37 or injecting38 infected material into the skin 

or being bitten by an experimentally infected animal.39,40 Skin infections in the 

absence of trauma have resulted also from handling cultures41-43 or necropsy 

of animals44 without any apparent trauma.

Natural Modes of Infection

The fungus is distributed worldwide in the environment and is associated with 

sphagnum moss and gardening, often involving sphagnum moss and traumatic 

implantation. Infections are not transmissible from person-to-person, but require 

common exposure to a point source. Rare respiratory and zoonotic infections 

occur. It is thought that naturally occurring lung disease results from inhalation.

Laboratory Safety and Containment Recommendations

Although localized skin and eye infections have occurred in an occupational 

setting, no pulmonary infections have been reported as a result from laboratory 

exposure. It should be noted that serious disseminated infections have been 

reported in immunocompromised persons.45

BSL-2 and ABSL-2 practices, containment equipment, and facilities are 

recommended for laboratory handling of suspected clinical specimens, soil and 

vegetation, and experimental animal activities with S. schenckii. Gloves should 
176 Biosafety in Microbiological and Biomedical Laboratories

be worn during manipulation of S. schenckii and when handling experimentally 

infected animals. Any culture identifying dimorphic fungi should be handled in a 

Class II BSC.

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Dermatophytes (Epidermophyton, Microsporum, and Trichophyton)

The dermatophytes are biologically related species of the genera, 

Epidermophyton, Microsporum, and Trichophyton that exist as monomorphic 

pathogens in nature, in laboratory cultures at room temperature and in vivo as 

filamentous molds. The sexual stages, when known, are Ascomycetes with 

infectious ascospores. These fungi are distributed worldwide, with particular 

species being endemic in particular regions. The species are grouped by natural 

environment habitat as being primarily associated with humans (anthrophilic), 

other animals (zoophilic), or soil (geophilic).

Occupational Infections

Although skin, hair, and nail infections by these molds are among the most 

prevalent of human infections, the processing of clinical material has not been 

associated with laboratory infections. Infections have been acquired through 

contacts with naturally or experimentally infected laboratory animals (mice, 

rabbits, guinea pigs, etc.) and, occasionally, with handling cultures.26,29,45,46

Systemic dermatophytosis is a rare condition. Superficial chronic infections 

occur frequently among immunocompromised individuals as well as elderly and 

diabetic persons. Susceptible individuals should use extra caution.47-50

Natural Modes of Infection

Infections can be transmissible from person-to-person, or acquired from 

common exposure to a point source. The dermatophytes cause infection 

(dermatophytosis) by invading the keratinized tissues of living animals and 

are among the most common infectious agents of humans. This fungal group 

encompasses members of three genera: Epidermophyton, Microsporum, and 

Trichophyton. The severity of infection depends on the infective species or 

strain, the anatomic site and other host factors. One of the most severe 

dermatophytoses is favus, a disfiguring disease of the scalp caused by 

Trychophyton schoenleinii.
Agent Summary Statements: Fungal Agents 177

Laboratory Safety and Containment Recommendations

Dermatophytes pose a moderate potential hazard to individuals with normal 

immune status. In the clinical laboratory setting, the inappropriate handling of 

cultures is the most common source of infection for laboratory personnel. The 

most common laboratory procedure for detection of the infective dermatophyte is 

the direct microscopic examination of contaminated skin, hair, and nails, followed 

by its isolation and identification on appropriated culture media. Direct contact 

with contaminated skin, hair, and nails of humans could be another source of 

infection.48,49 In research laboratories, dermatophytosis can be acquired by 

contact with contaminated soil (source of infection: geophilic species) or animal 

hosts (source of infection: zoophilic species).

BSL-2 and ABSL-2 practices, containment equipment, and facilities are 

recommended for handling cultures and soil samples. Any culture identifying 

dimorphic fungi should be handled in a Class II BSC.

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Miscellaneous Molds

Several molds have caused serious infection in immunocompetent hosts 

following presumed inhalation or accidental subcutaneous inoculation from 

environmental sources. These agents include the dimorphic mold, Penicillium 

marneffei, and the dematiaceous (brown-pigmented) molds, Bipolaris species, 

Cladophialophora bantiana, Exophiala (Wangiella) dermatitidis, Exserohilum 

species, Fonsecaea pedrosoi, Ochroconis gallopava (Dactylaria gallopava), 

Ramichloridium mackenziei (Ramichloridium obovoideum), Rhinocladiella 

atrovirens, and Scedosporium prolificans.

51

Occupational Infections

Even though no laboratory-acquired infections appear to have been reported 

with most of these agents, the gravity of naturally-acquired illness is sufficient to 

merit special precautions in the laboratory. Penicillium marneffei has caused a 

localized infection in a laboratory worker.52 It also caused a case of laboratoryacquired disseminated infection following presumed inhalation when an 

undiagnosed HIV-positive individual visited a laboratory where students 

were handling cultures on the open bench.53
178 Biosafety in Microbiological and Biomedical Laboratories

Natural Modes of Infection

The natural mode of infection varies by specific species; most are 

poorly characterized.

Laboratory Safety and Containment Recommendations

Inhalation of conidia from sporulating mold cultures or accidental injection 

into the skin during infection of experimental animals are potential risks to 

laboratory personnel.

BSL-2 practices, containment equipment, and facilities are recommended 

for propagating and manipulating cultures known to contain these agents. 

Any culture identifying dimorphic fungi should be handled in a Class II BSC.

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

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In: Murray PR, Baron EJ, Jorgensen JH et al, editors. Manual of Clinical 

Microbiology. 8th edition. Washington, DC: ASM Press; 2003. p. 109-20.

47. Kamalam A, Thambiah AS. Trichophyton simii infection due to laboratory 

accident. Dermatologica. 1979; 159:180-1.
Agent Summary Statements: Fungal Agents-References 181

48. Scher RK, Baran R. Onychomycosis in clinical practice: factors contributing 

to recurrence. Br J Dermatol. 2003;149 Suppl 65:5-9.

49. Gupta AK. Onychomycosis in the elderly. Drugs Aging. 2000;16:397-407.

50. Romano C, Massai L, Asta F, et al. Prevalence of dermatophytic skin and 

nail infections in diabetic patients. Mycoses. 2001;44:83-6.

51. Brandt ME, Warnock DW. Epidemiology, clinical manifestations, and therapy 

of infections caused by dematiaceous fungi. J Chemother. 2003;1536-47.

52. Segretain G. Penicillium marneffii, n. sp., agent of a mycosis of the 

reticuloendothelial system. Mycopathologia. 1959;11:327-53. [French]

53. Hilmarsdottir I, Coutell A, Elbaz J, et al. A French case of laboratory-acquired 

disseminated Penicillium marneffei infection in a patient with AIDS. Clin 

Infect Dis. 1994;19:357-58.
182 Biosafety in Microbiological and Biomedical Laboratories

Section VIII-C: Parasitic Agents

General Issues

Additional details about occupationally-acquired cases of parasitic infections, 

as well as recommendations for post exposure management, are provided 

elsewhere.1-3 Effective antimicrobial treatment is available for most parasitic 

infections.4

 Immunocompromised persons should receive individualized 

counseling (specific to host and parasite factors) from their personal healthcare 

provider and their employer about the potential risks associated with working 

with live organisms.

BSL-2 and ABSL-2 practices,5

 containment equipment, and facilities are 

recommended for activities with infective stages of the parasites discussed 

in this chapter.

Microsporidia, historically considered parasites, are now recognized by most 

experts to be fungi; however, microsporidia are maintained in the parasitic agent 

section is this edition. These organisms are discussed here because a laboratoryacquired case of infection has been reported,6

 and most persons currently still 

look for microsporidia associated with discussion of parasitic agents.

Importation of parasitic agents may require CDC and/or USDA importation 

permits. Domestic transport of this agent may require a permit from USDA/ 

APHIS/VS.

Blood and Tissue Protozoal Parasites

Blood and tissue protozoal parasites that pose greatest occupational risk include 

Babesia, Leishmania, Plasmodium, Toxoplasma, and Trypanosoma. Other 

tissue protozoa of potential concern include free-living ameba (Acanthamoeba, 

Balamuthia mandrillaris, Naegleria fowleri) and some species of microsporidia 

including Encephalitozoon cuniculi that commonly cause extraintestinal infection.

Leishmania spp. cause human leishmaniasis; Plasmodium spp. cause 

human malaria, or some, such as P. cynomolgi cause nonhuman primate malaria; 

Toxoplasma gondii causes toxoplasmosis; Trypanosoma cruzi causes American 

trypanosomiasis or Chagas disease; and Trypanosoma brucei gambiense and 

T. b. rhodesiense cause African trypanosomiasis or (African) sleeping sickness. 

With the exception of Leishmania and Toxoplasma, these agents are classically 

thought of as bloodborne and have stages that circulate in the blood. Although 

not always recognized, both Leishmania and Toxoplasma may have stages that 

circulate in the blood. Some, such as Plasmodium and Trypanosoma cruzi, also 

have tissue stages. Leishmania spp. are well recognized to have skin and deep 

tissue stages and Toxoplasma gondii forms tissue cysts, including in the central 

nervous system.
Agent Summary Statements: Parasitic Agents 183

Occupational Infections

Laboratory-acquired infections with Leishmania spp., Plasmodium spp., Toxoplasma 

gondii, and Trypanosoma spp. have been reported; the majority of these involved 

needle-stick or other cutaneous exposure to infectious stages of the organisms 

through abraded skin, including microabrasions.1,2

Laboratory-acquired infections may be asymptomatic. If clinically manifest, 

they may exhibit features similar to those seen in naturally acquired infections, 

although bypassing natural modes of infection could result in atypical signs and 

symptoms. Cutaneous leishmaniasis could manifest as various types of skin 

lesions (e.g., nodules, ulcers, plaques), while visceral leishmaniasis may result 

in fever, hepatosplenomegaly, and pancytopenia. However, only one of the 

laboratorians known to have become infected with L. (L.) donovani, an organism 

typically associated with visceral leishmaniasis, developed clinical manifestations 

of visceral involvement (e.g., fever, splenomegaly, leukopenia).1

 The other 

laboratorians developed skin lesions. Laboratory-acquired malaria infections 

may result in fever and chills, fatigue, and hemolytic anemia. Laboratorians 

can become infected with T. gondii through accidental ingestion of sporulated 

oocysts, but also may become infected through skin or mucous membrane 

contact with either tachyzoites or bradyzoites in human or animal tissue or 

culture. Symptoms in laboratory-acquired T. gondii infections may be restricted 

to flu-like conditions with enlarged lymph nodes, although rash may be present. 

Trypanosoma cruzi infection could manifest initially as swelling and redness at the 

inoculation site, fever, rash, and adenopathy. Myocarditis and electrocardiographic 

changes may develop. Infection with T. b. rhodesiense and T. b. gambiense also 

may cause initial swelling and redness at the inoculation site, followed by fever, 

rash, adenopathy, headache, fatigue and neurologic signs.

Blood and tissue protozoal infections associated with exposure to laboratory 

animals are not common. Potential direct sources of infection for laboratory 

personnel include accidental needle-stick while inoculating or bleeding animals, 

contact with lesion material from cutaneous leishmaniasis, and contact with 

blood of experimentally or naturally infected animals. In the case of rodents 

experimentally inoculated with Toxoplasma gondii via the intraperitoneal route, 

contact with peritoneal fluid could result in exposure to infectious organisms. 

Mosquito-transmitted malaria infections can occur under laboratory conditions 

as nearly half of the occupationally acquired malaria infections were reported 

to be vector borne, and contact with body fluids (including feces) of reduviids 

(triatomines) experimentally or naturally infected with T. cruzi poses a risk to 

laboratory personnel.

Babesia microti and other Babesia spp. can cause human babesiosis or 

piroplasmosis. Under natural conditions, Babesia is transmitted by the bite of an 

infected tick, or by blood transfusion; in the United States, hard ticks (Ixodes) are 

the principal vectors. Although no laboratory infections with Babesia have been 
184 Biosafety in Microbiological and Biomedical Laboratories

reported, they could easily result from accidental needle-stick or other cutaneous 

exposure of abraded skin to blood containing parasites. Persons who are asplenic, 

immunocompromised, or elderly have increased risk for severe illness if infected.

Natural Modes of Infection

Leishmaniasis is endemic in parts of the tropics, subtropics, and southern 

Europe, while malaria is widely distributed throughout the tropics. However, the 

prevalence of these diseases varies widely among endemic areas; the diseases 

can be very focal in nature. The four species of malaria that infect humans 

have no animal reservoir hosts. Some Leishmania spp. may have a number of 

important mammalian reservoir hosts, including rodents and dogs. Only cats 

and other felines can serve as definitive hosts for Toxoplasma gondii, which is 

distributed worldwide. Birds and mammals, including sheep, pigs, rodents, cattle, 

deer, and humans can be infected from ingestion of tissue cysts or fecal oocysts 

and subsequently develop tissue cysts throughout the body. Chagas disease 

occurs from Mexico southward throughout most of Central and South America, 

with the exception of the southern-most tip of South America. It has been 

characterized in some accounts as a zoonotic infection, yet the role of animals 

in maintaining human infection is unclear. A variety of domestic and wild animals 

are found naturally infected with T. cruzi, but human infection undoubtedly serves 

as the major source of infection for other humans. African trypanosomiasis 

is endemic in sub-Saharan Africa but is extremely focal in its distribution. 

Generally, T. b. gambiense occurs in West and Central Africa while T. b. 

rhodesiense occurs in East and Southeast Africa. T. b. rhodesiense is a 

zoonotic infection with cattle or, in a more limited role, game animals serving 

as reservoir hosts, whereas humans are the only epidemiologically important 

hosts for T. b. gambiense.

Leishmania, Plasmodium, and both American and African trypanosomes 

are all transmitted in nature by blood-sucking insects. Sandflies in the genera 

Phlebotomus and Lutzomyia transmit Leishmania; mosquitoes in the genus 

Anopheles transmit Plasmodium; reduviid (triatomine) bugs such as Triatoma, 

Rhodnius, and Panstrongylus transmit T. cruzi (in the feces rather than the saliva 

of the bug), and tsetse flies in the genus Glossina transmit African trypanosomes.

Laboratory Safety and Containment Recommendations

Infective stages may be present in blood, CSF, bone marrow, or other biopsy 

tissue, lesion exudates, and infected arthropods. Depending on the parasite, 

the primary laboratory hazards are skin penetration through wounds or 

microabrasions, accidental parenteral inoculation, and transmission by arthropod 

vectors. Aerosol or droplet exposure of organisms to the mucous membranes of 

the eyes, nose, or mouth are potential hazards when working with cultures of 
Agent Summary Statements: Parasitic Agents 185

Leishmania, Toxoplasma gondii, or T. cruzi, or with tissue homogenates or blood 

containing hemoflagellates. Immuno-compromised persons should avoid working 

with live organisms.

Because of the potential for grave consequences of toxoplasmosis in the 

developing fetus, women who are or might become pregnant and who are at 

risk for infection with T. gondii should receive counseling from their personal 

physician and employer regarding appropriate means of mitigating the risk 

(including alternate work assignments, additional PPE, etc.). Working with 

infectious oocysts poses the greatest risk of acquiring infection; needle-sticks 

with material containing tachyzoites or bradyzoites also pose a significant risk. 

Infection with tachyzoites or bradyzoites through mucous membranes or skin 

abrasions is also possible. Kittens and cats that might be naturally infected with 

Toxoplasma pose some risk to personnel.5

 Good hygiene and use of personal 

protection measures would reduce the risk.

One laboratory infection with microsporidia has been reported, associated 

with conjunctival exposure to spores leading to the development of 

keratoconjunctivitis. Infection could also result from ingestion of spores in feces, 

urine, sputum, CSF, or culture. No laboratory-acquired infections have been 

reported with Acanthamoeba spp., Balamuthia mandrillaris or Naegleria fowleri; 

however, the possibility of becoming infected by inhalation, by accidental needlesticks, or through exposure to mucous membranes or microabrasions of the skin 

should be considered.

BSL-2 and ABSL-2 practices, containment equipment, and facilities are 

recommended for activities with infective stages of the parasites listed.5

 Infected 

arthropods should be maintained in facilities that reasonably preclude the 

exposure of personnel or the escape of insects. (See Appendix E.) Personal 

protection (e.g., lab coat, gloves, face shield), in conjunction with containment in 

a BSC, is indicated when working with cultures, tissue homogenates, or blood 

containing organisms.

Special Issues

Treatment Highly effective medical treatment for most protozoal infections 

exists.4

 An importation or domestic transfer permit for this agent can be obtained 

from USDA/APHIS/VS.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.
186 Biosafety in Microbiological and Biomedical Laboratories

Intestinal Protozoal Parasites

Intestinal protozoal parasites that pose greatest occupational risk include 

Cryptosporidium, Isospora, Entamoeba histolytica, and Giardia. Other intestinal 

pathogens of concern are some species of microsporidia, specifically Septata 

intestinalis and Enterocytozoon bieneusi. Cryptosporidium parvum, C. hominis, 

and Isospora belli cause intestinal coccidiosis, most often referred to as 

cryptosporidiosis and isosporiasis, respectively. Entamoeba histolytica can 

cause both intestinal and extraintestinal infection (e.g., liver abscess) called 

amebiasis, and Giardia intestinalis causes giardiasis.

Occupational Infections

Laboratory-acquired infections with Cryptosporidium spp., E. histolytica, 

G. intestinalis, and I. belli have been reported.1-3 The mode of exposure in 

laboratory-acquired infections in this group of agents mimics the natural infection 

routes for the most part, and consequently, clinical symptoms are typically very 

similar to those seen in naturally acquired infections. For Cryptosporidium, 

E. histolytica, G. intestinalis, and I. belli, the common clinical manifestations 

are symptoms of gastroenteritis (e.g., diarrhea, abdominal pain and cramping, 

loss of appetite). Infection with E. histolytica may result in bloody stools.

Laboratory animal-associated infections with this group of organisms have 

been reported and provide a direct source of infection for laboratory personnel 

who are exposed to feces of experimentally or naturally infected animals.3

Handling Cryptosporidium oocysts requires special care, as laboratory-acquired 

infections have occurred commonly in personnel working with this agent, 

especially if calves are used as the source of oocysts. Other experimentally 

infected animals pose potential risks as well. Circumstantial evidence suggests 

that airborne transmission of oocysts of this small organism (i.e., 4-6 μm 

diameter) may occur. Rigid adherence to protocol should reduce the occurrence 

of laboratory-acquired infection in laboratory and animal care personnel.

Natural Modes of Infection

All of these intestinal protozoa have a cosmopolitan distribution, and in some 

settings, including developed countries, the prevalence of infection can be high. 

The natural mode of infection for this group of organisms is typically ingestion of 

an environmentally hardy oocyst (for the coccidia) or cyst (for E. histolytica and 

G. intestinalis). The ID50, best established for Cryptosporidium, has been shown 

for some strains to be 5-10 oocysts.7

 This suggests that even a single oocyst 

might pose a risk for infection in an exposed laboratorian. The infectious dose 

for other parasites in this group is not as well established, but is probably in the 

same range. Further, because these protozoa multiply in the host, ingestion of 

even small inocula can cause infection and illness. The role for animal reservoir 

hosts is diverse in this group of organisms. In the case of C. hominis, principally 

humans are infected, whereas for C. parvum, humans, cattle, and other 
Agent Summary Statements: Parasitic Agents 187

mammals can be infected and serve as reservoir hosts for human infection. 

In the case of E. histolytica, humans serve as the only significant source of 

infection, and there is no convincing evidence that any animal serves as reservoir 

host for I. belli. The extent to which Giardia spp. parasitizing animals can infect 

humans is only now becoming better understood, but most human infection 

seems to be acquired from human-to-human transmission. The organisms in 

this group do not require more than one host to complete their life cycle because 

they infect, develop, and result in shedding of infectious stages all in a single 

host. Ingestion of contaminated drinking or recreational water has also been a 

common source of cryptosporidiosis and giardiasis.

Laboratory Safety and Containment Recommendations

Infective stages may be present in the feces or other body fluids and tissues. 

Depending on the parasite, ingestion is the primary laboratory hazard. 

Immunocompromised persons should avoid working with live organisms. 

Laboratorians who work only with killed or inactivated parasite materials, 

or parasite fractions, are not at significant risk.

Similarly, no accidental laboratory infection with Sarcocystis has been 

reported, although care should be exercised when working with infected meat 

products to avoid accidental ingestion. It is not known if laboratorians could be 

accidentally infected through parenteral inoculation of Sarcocystis; nevertheless 

caution should be exercised when working with cultures, homogenates, etc.

BSL-2 and ABSL-2 practices, containment equipment, and facilities are 

recommended for activities with infective stages of the parasites listed.5 Primary 

containment (e.g., BSC) or personal protection (e.g., face shield) is especially 

important when working with Cryptosporidium. Oocysts are infectious when shed 

(i.e., are already sporulated and do not require further development time outside the 

host), often are present in stool in high numbers, and are environmentally hardy.

Commercially available iodine-containing disinfectants are effective 

against E. histolytica and G. intestinalis, when used as directed, as are high 

concentrations of chlorine (1 cup of full-strength commercial bleach [~5% 

chlorine] per gallon of water [1:16, vol/vol]).1,2

If a laboratory spill contains Cryptosporidium oocysts, the following approach 

is recommended.2

 A conventional laboratory detergent/cleaner should be used to 

remove contaminating matter from surfaces (e.g., of bench tops and equipment). 

After organic material has been removed, 3% hydrogen peroxide (i.e., undiluted, 

commercial hydrogen peroxide, identified on the bottle as 3% or “10 vol” hydrogen 

peroxide) can be used to disinfect surfaces; dispensing bottles that contain 

undiluted hydrogen peroxide should be readily available in laboratories in 

which surfaces could become contaminated.
188 Biosafety in Microbiological and Biomedical Laboratories

Affected surfaces should be flooded (i.e., completely covered) with hydrogen 

peroxide. If a large volume of liquid contaminates surfaces, to avoid diluting the 

hydrogen peroxide, absorb the bulk of the spill with disposable paper towels. 

Dispense hydrogen peroxide repeatedly, as needed, to keep affected surfaces 

covered (i.e., wet/moist) for ~30 minutes. Absorb residual hydrogen peroxide with 

disposable paper towels and allow surfaces to dry thoroughly (10 to 30 minutes) 

before use. All paper towel litter and other disposable materials should be 

autoclaved or similarly disinfected before disposal. Reusable laboratory items 

can be disinfected and washed in a laboratory dishwasher by using the “sanitize” 

cycle and a detergent containing chlorine. Alternatively, immerse contaminated 

items for ~1 hour in a water bath preheated to 50o C; thereafter, wash them in a 

detergent/disinfectant solution.

Special Issues

Treatment Highly effective medical treatment exists for most protozoal infections; 

treatment with nitazoxanide for Cryptosporidium is now available, but efficacy 

has not been proven.4

Transfer of Agent Importation of these agents may require CDC and/or USDA 

importation permits. Domestic transport of these agents may require a permit 

from USDA/APHIS/VS. A DoC permit may be required for the export of this 

agent to another country. See Appendix C for additional information.

Trematode Parasites

Trematode parasites that pose greatest occupational risk are the Schistosoma 

spp., although others including Fasciola are of concern. Schistosoma mansoni 

causes intestinal schistosomiasis or bilharziasis, also known as Manson’s blood 

fluke, in which the adult flukes reside in the venules of the bowel and rectum. 

Fasciola hepatica, the sheep liver fluke, causes fascioliasis, where the adult 

flukes live in the common and hepatic bile ducts of the human or animal host.

Occupational Infections

Laboratory-acquired infections with S. mansoni and F. hepatica have been 

reported, but accidental infections with other Schistosoma spp. could also 

occur.1,2 By nature of the infection, none have been directly associated with 

laboratory animals, with the exception of infected mollusk intermediate hosts.

Laboratory-acquired infections with F. hepatica may be asymptomatic, 

but could have clinical manifestations such as right upper quadrant pain, biliary 

colic, obstructive jaundice, elevated transaminase levels, and other pathology 

associated with hepatic damage resulting from migration of the fluke through the 

liver en route to the bile duct. Most laboratory exposures to schistosomes would 
Agent Summary Statements: Parasitic Agents 189

result in predictably low worm burdens with minimal disease potential. However, 

clinical manifestations of infection with S. mansoni could include dermatitis, fever, 

cough, hepatosplenomegaly, and adenopathy.

Natural Modes of Infection

Fasciola hepatica has a cosmopolitan distribution and is most common in sheepraising areas, although other natural hosts include goats, cattle, hogs, deer, and 

rodents. Snails in the family Lymnaeidae, primarily species of Lymnaea, are 

intermediate hosts for F. hepatica, and release cercariae that encyst on vegetation. 

Persons become infected with F. hepatica by eating raw or poorly cooked 

vegetation, especially green leafy plants such as watercress, on which 

metacercariae have encysted.

Schistosoma mansoni is widely distributed in Africa, South America, and 

the Caribbean; the prevalence of infection has been rapidly changing in some 

areas. Infection occurs when persons are exposed to free-swimming cercariae 

in contaminated bodies of water; cercariae can penetrate intact skin. The natural 

snail hosts capable of supporting development of S. mansoni are various species 

of Biomphalaria.

Laboratory Safety and Containment Recommendations

Infective stages of F. hepatica (metacercariae) and S. mansoni (cercariae) may 

be found, respectively, encysted on aquatic plants or in the water in laboratory 

aquaria used to maintain snail intermediate hosts. Ingestion of fluke metacercariae 

and skin penetration by schistosome cercariae are the primary laboratory 

hazards. Dissection or crushing of schistosome-infected snails may also result in 

exposure of skin or mucous membrane to cercariae-containing droplets. Additionally, 

metacercariae may be inadvertently transferred from hand to mouth by fingers 

or gloves, following contact with contaminated aquatic vegetation or aquaria.

All reported cases of laboratory-acquired schistosomiasis have been caused 

by S. mansoni, which probably reflects the fact that many more laboratories work 

with S. mansoni than with other Schistosoma spp. However, accidental infection 

with S. haematobium, S. japonicum, and S. mekongi could easily occur in the 

same manner as described for S. mansoni.

Exposure to cercariae of non-human species of schistosomes (e.g., avian 

species) may cause mild to severe dermatitis (swimmer’s itch).

BSL-2 and ABSL-2 practices, containment equipment and facilities are 

recommended for laboratory work with infective stages of the parasites listed.5

Gloves should be worn when there may be direct contact with water containing 

cercariae or vegetation with encysted metacercariae from naturally or 

experimentally infected snail intermediate hosts. Long-sleeved laboratory coats 

or other protective garb should be worn when working in the immediate area of 
190 Biosafety in Microbiological and Biomedical Laboratories

aquaria or other water sources that may contain schistosome cercariae. Water 

from laboratory aquaria containing snails and cercariae should be decontaminated 

(e.g., ethanol, hypochlorite, iodine, or heat) before discharged to sanitary sewers.

Special Issues

Treatment Highly effective medical treatment for most trematode infections exists.4

Transfer of Agent Importation of these agents may require CDC and/or USDA 

importation permits. Domestic transport of these agents may require a permit 

from USDA/APHIS/VS. A DoC permit may be required for the export of this 

agent to another country. See Appendix C for additional information.

Cestode Parasites

Cestode parasites of potential risk for laboratorians include Echinococcus spp.,

Hymenolepis nana, and Taenia solium. Echinococcosis is an infection caused by 

cestodes in the genus Echinococcus; E. granulosus causes cystic echinococcosis, 

E. multilocularis causes alveolar echinococcosis, and E. vogeli and E. oligarthrus 

cause polycystic echinococcosis. Humans serve as intermediate hosts and harbor 

the metacestode or larval stage, which produces a hydatid cyst. Hymenolepis 

nana, the dwarf tapeworm, is cosmopolitan in distribution and produces 

hymenolepiasis, or intestinal infection with the adult tapeworm. Taenia solium, 

the pork tapeworm, causes both taeniasis (infection of the intestinal tract with 

the adult worm), and cysticercosis (infection of subcutaneous, intermuscular, 

and central nervous system with the metacestode stage or cysticercus).

Occupational Infections

No laboratory-acquired infections have been reported with any cestode parasite.

Natural Modes of Infection

The infectious stage of Echinococcus, Hymenolepis, and Taenia is the 

oncosphere contained within the egg. Hymenolepis nana is a one-host parasite 

and does not require an intermediate host; it is directly transmissible by ingestion 

of feces of infected humans or rodents. The life cycles of Echinococcus and 

Taenia require two hosts. Canids, including dogs, wolves, foxes, coyotes, and 

jackals, are the definitive hosts for E. granulosus, and various herbivores such as 

sheep, cattle, deer, and horses are the intermediate hosts. Foxes and coyotes 

are the principal definitive hosts for E. multilocularis, although dogs and cats also 

can become infected and rodents serve as the intermediate hosts. Bush dogs 

and pacas serve as the definitive and intermediate hosts, respectively, for E. 

vogeli. Dogs also may be infected. Echinococcus oligarthrus uses wild felines, 
Agent Summary Statements: Parasitic Agents 191

including cougar, jaguarondi, jaguar, ocelot, and pampas cat, as definitive hosts 

and various rodents such as agoutis, pacas, spiny rats, and rabbits serve as 

intermediate hosts. People become infected when eggs shed by the definitive 

host are accidentally ingested. For T. solium, people can serve both as definitive 

host (harbor the adult tapeworm), and as accidental intermediate host (harbor the 

larval stages cysticerci). Pigs are the usual intermediate host, becoming infected 

as they scavenge human feces containing eggs.

Laboratory Safety and Containment Recommendations

Infective eggs of Echinococcus spp. may be present in the feces of carnivore 

definitive hosts.3 Echinococcus granulosus poses the greatest risk because it is 

the most common and widely distributed species, and because dogs are the primary 

definitive hosts. For T. solium, infective eggs in the feces of humans serve as the 

source of infection. Accidental ingestion of infective eggs from these sources is 

the primary laboratory hazard. Ingestion of cysticerci of T. solium (Cysticercus 

cellulosae) leads to human infection with the adult tapeworm. For those cestodes 

listed, the ingestion of a single infective egg from the feces of the definitive host 

could potentially result in serious disease. Ingestion of the eggs of H. nana in the 

feces of definitive hosts (humans or rodents) could result in intestinal infection.

Although no laboratory-acquired infections with either Echinococcus spp. 

or T. solium have been reported, the consequences of such infections could be 

serious. Laboratory-acquired infections with cestodes could result in various 

clinical manifestations, depending upon the type of cestode. Human infection 

with Echinococcus spp. could range from asymptomatic to severe. The severity 

and nature of the signs and symptoms depends upon the location of the cysts, 

their size, and condition (alive versus dead). Clinical manifestations of a liver 

cyst could include hepatosplenomegaly, right epigastric pain, and nausea, while 

a lung cyst may cause chest pain, dyspnea, and hemoptysis. For T. solium, 

ingestion of eggs from human feces can result in cysticercosis, with cysts located 

in subcutaneous and intermuscular tissues, where they may be asymptomatic. 

Cysts in the central nervous system may cause seizures and other neurologic 

symptoms. Ingestion of tissue cysts of T. solium can lead to development of adult 

worms in the intestine of humans. Immunocompromised persons working with 

these cestodes must take special care as the asexual multiplication of the larval 

stages of these parasites makes them especially dangerous to such persons.

BSL-2 and ABSL-2 practices, containment equipment, and facilities are 

recommended for work with infective stages of these parasites.5

 Special attention 

should be given to personal hygiene (e.g., hand washing) and laboratory practices 

that would reduce the risk of accidental ingestion of infective eggs. Gloves are 

recommended when there may be direct contact with feces or with surfaces 

contaminated with fresh feces of carnivores infected with Echinococcus spp., 

humans infected with T. solium, or humans or rodents infected with H. nana.
192 Biosafety in Microbiological and Biomedical Laboratories

Special Issues

Treatment Highly effective medical treatment for most cestode infections exists.4

Transfer of Agent Importation of these agents may require CDC and/or USDA 

importation permits. Domestic transport of these agents may require a permit 

from USDA/APHIS/VS. A DoC permit may be required for the export of this agent 

to another country. See Appendix C for additional information.

Nematode Parasites

Nematode parasites that pose greatest occupational risk include the ascarids, 

especially Ascaris and Baylisascaris; hookworms, both human and animal; 

Strongyloides, both human and animal; Enterobius; and the human filariae, 

primarily Wuchereria and Brugia. Ancylostoma braziliense and A. caninum cause 

hookworm infection in cats and dogs, respectively. Ascaris lumbricoides causes 

ascariasis and is known as the large intestinal roundworm of humans. Enterobius 

vermicularis, known as the human pinworm or seatworm, causes enterobiasis or 

oxyuriasis. Strongyloides, the threadworm, causes strongyloidiasis. Ancylostoma, 

Ascaris, and Strongyloides reside as adults in the small intestine of their natural 

hosts, whereas E. vermicularis colonizes the cecum and appendix.

Occupational Infections

Laboratory-associated infections with Ancylostoma spp., A. lumbricoides, 

E. vermicularis, and Strongyloides spp. have been reported.1-3 Laboratory 

infections with hookworms and Strongyloides presumptively acquired from 

infected animals have been reported. Allergic reactions to various antigenic 

components of human and animal ascarids (e.g., aerosolized antigens) may 

pose risk to sensitized persons.

Laboratory-acquired infections with these nematodes can be asymptomatic, 

or can present with a range of clinical manifestations dependent upon the 

species and their location in host. Infection with hookworm of animal origin can 

result in cutaneous larva migrans or creeping eruption of the skin. Infection with 

A. lumbricoides may produce cough, fever, and pneumonitis as larvae migrate 

through the lung, followed by abdominal cramps and diarrhea or constipation 

from adult worms in the intestine. Infection with E. vermicularis usually causes 

perianal pruritis, with intense itching. Infection with animal Strongyloides spp. 

may induce cutaneous larva migrans.

Natural Modes of Infection

Ancylostoma infection in dogs and cats is endemic worldwide. Human infection 

occurs through penetration of the skin. Cutaneous larva migrans or creeping 

eruption occurs when infective larvae of animal hookworms, typically dog and 
Agent Summary Statements: Parasitic Agents 193

cat hookworms, penetrate the skin and begin wandering. Ancylostoma larvae 

can also cause infection if ingested. These larvae do not typically reach the 

intestinal tract, although A. caninum has on rare occasions developed into 

non-gravid adult worms in the human gut.

Ascaris lumbricoides infection is endemic in tropical and subtropical regions 

of the world. Infection occurs following accidental ingestion of infective eggs. 

Unembryonated eggs passed in the stool require two to three weeks to become 

infectious, and Ascaris eggs are very hardy in the environment.

Enterobius vermicularis occurs worldwide, although infection tends to be 

more common in school-age children than adults, and in temperate than tropical 

regions. Pinworm infection is acquired by ingestion of infective eggs, most often 

on contaminated fingers following scratching of the perianal skin. Eggs passed 

by female worms are not immediately infective, but only require several hours’

incubation to become fully infectious. Infection with this worm is relatively short 

(60 days on average), and reinfection is required to maintain an infection.

Strongyloides infection in animals is endemic worldwide. People become 

infected with animal Strongyloides when infective, filariform larvae penetrate 

the skin, and can develop cutaneous creeping eruption (larva currens).

Laboratory Safety and Containment Recommendations

Eggs and larvae of most nematodes are not infective in freshly passed feces; 

development to the infective stages may require from one day to several weeks. 

Ingestion of the infective eggs or skin penetration by infective larvae are the 

primary hazards to laboratory staff and animal care personnel. Development of 

hypersensitivity is common in laboratory personnel with frequent exposure to 

aerosolized antigens of ascarids.

Ascarid eggs are sticky, and special care should be taken to ensure thorough 

cleaning of contaminated surfaces and equipment. Caution should be used even 

when working with formalin-fixed stool samples because ascarid eggs can 

remain viable and continue to develop to the infective stage in formalin.8

Working with infective eggs of other ascarids, such as Toxocara and 

Baylisascaris, poses significant risk because of the potential for visceral 

migration of larvae, including invasion of the eyes and central nervous system. 

Strongyloides stercoralis is of particular concern to immuno-suppressed persons 

because potentially life-threatening systemic hyperinfection can occur. Lugol’s 

iodine kills infective larvae and should be sprayed onto skin or laboratory 

surfaces that are contaminated accidentally. The larvae of Trichinella in fresh or 

digested tissue could cause infection if accidentally ingested. Arthropods infected 

with filarial parasites pose a potential hazard to laboratory personnel.
194 Biosafety in Microbiological and Biomedical Laboratories

BSL-2 and ABSL-2 practices, containment equipment, and facilities are 

recommended for activities with infective stages of the nematodes listed here.5

Exposure to aerosolized sensitizing antigens of ascarids should be avoided. 

Primary containment (e.g., BSC) is recommended for work that may result in 

aerosolization of sensitization from occurring.

Special Issues

Treatment Highly effective medical treatment for most nematode infections exists.4

Transfer of Agent Importation of these agents may require CDC and/or USDA 

importation permits. Domestic transport of these agents may require a permit 

from USDA/APHIS/VS. A DoC permit may be required for the export of this 

agent to another country. See Appendix C for additional information.

References

1. Herwaldt BL. Laboratory-acquired parasitic infections from accidental 

exposures. Clin Microbiol Rev. 2001:14:659-88.

2. Herwaldt BL. Protozoa and helminths. In Fleming DO and Hunt DL (eds). 

4th ed. Biological Safety: Principles and Practices. Washington, DC, ASM 

Press, 2006.

3. Pike RM. Laboratory-associated infections: summary and analysis of 3921 

cases. Health Lab Sci 1976;13:105-14.

4. The Medical Letter [www.medicalletter.org] New York: The Medical Letter; 

[cited 2007]. Drugs for Parasitic Infections; [about 24 screens].

5. Hankenson FC, Johnston NA, Weigler BJ, et al. Zoonoses of occupational 

health importance in contemporary laboratory animal research. Comp Med. 

2003;53:579-601.

6. van Gool T, Biderre C, Delbac F, Wentink-Bonnema E, Peek R, Vivares CP. 

Serodiagnostic studies in an immunocompetent individual infected with 

Encephalitozoon cuniculi. J Infect Dis 2004;189:2243-9.

7. Messner MJ, Chappel CL, Okhuysen PC. Risk assessment for 

Cryptosporidium: a hierarchical Bayesian analysis of human dose response 

data. Water Res 2001;35:3934-40.

8. Ash LR, Orihel TC. Parasites, a guide to laboratory procedures and 

identification. Chicago: ASCP Press; 1991.
Agent Summary Statements: Rickettsial Agents 195

Section VIII-D: Rickettsial Agents

Coxiella burnetii

Coxiella burnetii is the etiologic agent of Q fever. C. burnetii is a bacterial obligate 

intracellular pathogen that undergoes its developmental cycle within an acidic 

vacuolar compartment exhibiting many characteristics of a phagolysosome. The 

developmental cycle consists of a large (approximately 1 μm in length) cell variant 

that is believed to be the more metabolically active, replicative cell type and a 

smaller, more structurally stable cell variant that is highly infectious and quite 

resistant to drying and environmental conditions.1-4 The organism undergoes a 

virulent (Phase I) to avirulent (Phase II) transition upon serial laboratory passage 

in eggs or tissue culture.

The infectious dose of virulent Phase I organisms in laboratory animals 

has been calculated to be as small as a single organism.5

 The estimated human 

infectious dose for Q fever by inhalation is approximately 10 organisms.6

 Typically, 

the disease manifests with flu-like symptoms including fever, headache, and 

myalgia but can also cause pneumonia and hepatomegaly. Infections range from 

sub-clinical to severe although primary infections respond readily to antibiotic 

treatment. Although rare, C. burnetii is known to cause chronic infections such 

as endocarditis or granulomatous hepatitis.7

Occupational Infections

Q fever is the second most commonly reported LAI in Pike’s compilation. Outbreaks 

involving 15 or more persons were recorded in several institutions.8,9 Infectious 

aerosols are the most likely route of laboratory-acquired infections. Experimentally 

infected animals also may serve as potential sources of infection or laboratory 

and animal care personnel. Exposure to naturally infected, often fasymptomatic 

sheep and their birth products is a documented hazard to personnel.10,11

Natural Modes of Infection

Q fever (Q for query) occurs worldwide. Broad ranges of domestic and wild 

mammals are natural hosts for Q fever and sources of human infection. Parturient 

animals and their birth products are common sources of infection. The placenta 

of infected sheep may contain as many as 109 organisms per gram of tissue12

and milk may contain 105 organisms per gram. The resistance of the organism to 

drying and its low infectious dose can lead to dispersal from contaminated sites.

Laboratory Safety and Containment Recommendations

The necessity of using embryonated eggs or cell culture techniques for the 

propagation of C. burnetii leads to extensive purification procedures. Exposure to 

infectious aerosols and parenteral inoculation cause most infections in laboratory 

and animal care personnel.8,9 The agent may be present in infected arthropods 
196 Biosafety in Microbiological and Biomedical Laboratories

and in the blood, urine, feces, milk, and tissues of infected animals or human 

hosts. Exposure to naturally infected, often asymptomatic, sheep and their birth 

products is a documented hazard to personnel.10,11 Recommended precautions 

for facilities using sheep as experimental animals are described elsewhere.10,13

BSL-2 practices and facilities are recommended for nonpropagative laboratory 

procedures, including serological examinations and staining of impression smears. 

BSL-3 practices and facilities are recommended for activities involving the inoculation, 

incubation, and harvesting of embryonated eggs or cell cultures, the necropsy of 

infected animals and the manipulation of infected tissues. Experimentally infected 

animals should be maintained under ABSL-3 because infected rodents may shed the 

organisms in urine or feces.8

 A specific plaque-purified clonal isolate of an avirulent 

(Phase II) strain (Nine Mile) may be safely handled under BSL-2 conditions.14

Special Issues

Vaccines An investigational Phase I, Q fever vaccine (IND) is available on a 

limited basis from the Special Immunizations Program (301-619-4653) of the 

USAMRIID, Fort Detrick, Maryland, for at-risk personnel under a cooperative 

agreement with the individual’s requesting institution. The use of this vaccine 

should be restricted to those who are at high risk of exposure and who have no 

demonstrated sensitivity to Q fever antigen. The vaccine can be reactogenic in 

those with prior immunity, thus requires skin testing before administration. The 

vaccine is only administered at USAMRIID and requires enrollment in their Q 

fever IND Immunization Program. For at-risk laboratory workers to participate in 

this program, fees are applicable. Individuals with valvular heart disease should 

not work with C. burnetii. (See Section VII.)

Select Agent C. burnetii is a select agent requiring registration with CDC 

and/or USDA for possession, use, storage and/or transfer. See Appendix F 

for additional information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Rickettsia prowazekii; Rickettsia typhi (R. mooseri); Orientia (Rickettsia) 

tsutsugamushi and Spotted Fever Group agents of human disease; 

Rickettsia rickettsii, Rickettsia conorii, Rickettsia akari, Rickettsia australis, 

Rickettsia siberica, and Rickettsia japonicum

Rickettsia prowazekii, Rickettsia typhi (R. mooseri), Orientia (Rickettsia) tsutsugamushi 

and the Spotted Fever Group agents of human disease (Rickettsia rickettsii, 

Rickettsia conorii, Rickettsia akari, Rickettsia australis, Rickettsia siberica, and 
Agent Summary Statements: Rickettsial Agents 197

Rickettsia japonicum) are the etiologic agents of epidemic typhus, endemic (murine) 

typhus), scrub typhus, Rocky Mountain spotted fever, Mediterranean spotted fever, 

rickettsialpox, Queensland tick typhus, and North Asian spotted fever, respectively.

Rickettsia spp. are bacterial obligate intracellular pathogens that are 

transmitted by arthropod vectors and replicate within the cytoplasm of eukaryotic 

host cells. Two groups are recognized within the genus, the typhus group and 

the spotted fever group. The more distantly related scrub typhus group is now 

considered a distinct genus, Orientia. Rickettsiae are primarily associated with 

arthropod vectors in which they may exist as endosymbionts that infect mammals, 

including humans, through the bite of infected ticks, lice, or fleas.15

Occupational Infections

Pike reported 57 cases of laboratory-associated typhus (type not specified), 56 

cases of epidemic typhus with three deaths, and 68 cases of murine typhus.8

Three cases of murine typhus have been reported from a research facility.16 Two 

were associated with handling of infectious materials on the open bench; the third 

case resulted from an accidental parenteral inoculation. These three cases 

represented an attack rate of 20% in personnel working with infectious materials. 

Rocky Mountain spotted fever is a documented hazard to laboratory personnel. 

Pike reported 63 laboratory-associated cases, 11 of which were fatal.8

 Oster 

reported nine cases occurring over a six-year period in one laboratory. All were 

believed to have been acquired because of exposure to infectious aerosols.17

Natural Modes of Infection

The epidemiology of rickettsial infections reflects the prevalence of rickettsiae 

in the vector population and the interactions of arthropod vectors with humans. 

Epidemic typhus is unusual among rickettsiae in that humans are considered the 

primary host. Transmission is by the human body louse; thus, outbreaks are now 

associated with breakdowns of social conditions. Endemic typhus is maintained 

in rodents and transmitted to humans by fleas. The various spotted fever group 

rickettsiae are limited geographically, probably by the distribution of the arthropod 

vector, although specific spotted fever group rickettsiae are found on all continents.15

Laboratory Safety and Containment Recommendations

The necessity of using embryonated eggs or cell culture techniques for the 

propagation of Rickettsia spp. incorporates extensive purification procedures. 

Accidental parenteral inoculation and exposure to infectious aerosols are the 

most likely sources of LAI.18 Aerosol transmission of R. rickettsii has been 

experimentally documented in nonhuman primates.19 Five cases of rickettsialpox 

recorded by Pike were associated with exposure to bites of infected mites.8

Naturally and experimentally infected mammals, their ectoparasites, and their 

infected tissues are potential sources of human infection. The organisms are 

relatively unstable under ambient environmental conditions.
198 Biosafety in Microbiological and Biomedical Laboratories

BSL-2 practices, containment equipment, and facilities are recommended 

for nonpropagative laboratory procedures, including serological and fluorescent 

antibody procedures, and for the staining of impression smears. BSL-3 practices, 

containment equipment, and facilities are recommended for all other manipulations 

of known or potentially infectious materials, including necropsy of experimentally 

infected animals and trituration of their tissues, and inoculation, incubation, and 

harvesting of embryonated eggs or cell cultures. ABSL-2 practices, containment 

equipment, and facilities are recommended for the holding of experimentally 

infected mammals other than arthropods. BSL-3 practices, containment equipment, 

and facilities are recommended for animal studies with arthropods naturally or 

experimentally infected with rickettsial agents of human disease. (See Appendix E.)

Several species, including R. montana, R. rhipicephali, R. belli, and R. canada, 

are not known to cause human disease and may be handled under BSL-2 conditions. 

New species are being described frequently and should be evaluated for appropriate 

containment on a case-by-case basis. Because of the proven value of antibiotic 

therapy in the early stages of ricketsial infection, it is essential that laboratories 

have an effective system for reporting febrile illnesses in laboratory personnel, 

medical evaluation of potential cases and, when indicated, institution of 

appropriate antibiotic therapy.

Special Issues

Medical Response Under natural circumstances, the severity of disease caused 

by rickettsial agents varies considerably. In the laboratory, very large inocula are 

possible, which might produce unusual and perhaps very serious responses. 

Surveillance of personnel for laboratory-associated infections with rickettsial 

agents can dramatically reduce the risk of serious consequences of disease. 

Experience indicates that infections adequately treated with specific anti-rickettsial 

chemotherapy on the first day of disease do not generally present serious 

problems. However, delay in instituting appropriate chemotherapy may result 

in debilitating or severe acute disease ranging from increased periods of 

convalescence in typhus and scrub typhus to death in R. rickettsii infections. 

The key to reducing the severity of disease from laboratory-associated infections 

is a reliable medical response which includes: 1) round-the-clock availability of 

an experienced medical officer; 2) indoctrination of all personnel on the potential 

hazards of working with rickettsial agents and advantages of early therapy; 3) a 

reporting system for all recognized overt exposures and accidents; 4) the reporting 

of all febrile illnesses, especially those associated with headache, malaise, and 

prostration when no other certain cause exists; and 5) an open and non-punitive 

atmosphere that encourages reporting of any febrile illness.

Select Agent R. prowazekii and R. rickettsii are select agents requiring 

registration with CDC and/or USDA for possession, use, storage and/or transfer. 

See Appendix F for additional information.
Agent Summary Statements: Rickettsial Agents-References 199

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS.

References

1. Babudieri B. Q fever: a zoonosis. Adv Vet Sci. 1959;5:81-182.

2. Ignatovich VF. The course of inactivation of Rickettsia burnetii in fluid media. 

Zh Mikrobiol Epidemiol Immunobiol. 1959;30:134-41.

3. Sawyer LA, Fishbein DB, McDade JE. Q fever: current concepts. Rev Infect 

Dis. 1987;9:935-46.

4. Heinzen RA, Hackstadt T, Samuel JE. Developmental biology of Coxiella 

burnetii. Trends Microbiol. 1999;7:149-54.

5. Ormsbee R, Peacock M, Gerloff R, et al. Limits of rickettsial infectivity. Infect 

Immun. 1978;19:239-45.

6. Wedum AG, Barkley WE, Hellman A. Handling of infectious agents. J Am Vet 

Med Assoc. 1972;161:1557-67.

7. Maurin M, Raoult D. Q fever. Clin Microbiol Rev. 1999;12:518-53.

8. Pike RM. Laboratory-associated infections: Summary and analysis of 3,921 

cases. Health Lab Sci. 1976;13:105-14.

9. Johnson JE, Kadull PJ. Laboratory-acquired Q fever. A report of fifty cases. 

Am J Med. 1966;41:391-403.

10. Spinelli JS, Ascher MS, Brooks DL, et al. Q fever crisis in San Francisco: 

controlling a sheep zoonosis in a lab animal facility. Lab Anim. 1981;10:24-7.

11. Meiklejohn G, Reimer LG, Graves PS, et al. Cryptic epidemic of Q fever in 

a medical school. J Infect Dis. 1981;144:107-13.

12. Welsh HH, Lennette EH, Abinanti FR, et al. Q fever in California IV. 

Occurrence of Coxiella burnetii in the placenta of naturally infected sheep. 

Public Health Rep. 1951;66:1473-7.

13. Bernard KW, Helmick CG, Kaplan JE, et al. Q fever control measures: 

Recommendations for research facilities using sheep. Infect Control. 

1982;3:461-5.

14. Hackstadt T. Biosafety concerns and Coxiella burnetii. Trends Microbiol. 

1996;4:341-2.

15. Hackstadt T. The biology of rickettsiae. Infect Agents Dis. 1996;5:127-43.

16. Centers for Disease Control and Prevention. Laboratory-acquired endemic 

typhus. MMWR Morb Mortal Wkly Rep. 1978;27:215-6.

17. Oster CN, Burke DS, Kenyon RH, et al. Laboratory-acquired Rocky 

Mountain spotted fever. The hazard of aerosol transmission. N Engl J Med. 

1977;297:859-63.

18. Hattwick MA, O’Brien RJ, Hanson BF. Rocky Mountain spotted fever: 

epidemiology of an increasing problem. Ann Intern Med. 1976;84:732-9.

19. Saslaw S, Carlisle HN. Aerosol infection of monkeys with Rickettsia rickettsii. 

Bacteriol Rev. 1966;30:636-45.
200 Biosafety in Microbiological and Biomedical Laboratories

Section VIII-E: Viral Agents

Hantaviruses

Hantaviruses are negative sense RNA viruses belonging to the genus Hantavirus 

within the family Bunyaviridae. The natural hosts of hantaviruses are rodent 

species and they occur worldwide. Hantavirus pulmonary syndrome (HPS) is 

a severe disease caused by hantaviruses such as Sin Nombre virus or Andes 

virus whose hosts are rodents in the subfamily Sigmodontinae. This subfamily 

only occurs in the New World, so HPS is not seen outside North and South 

America. Hantaviruses in Europe and Asia frequently cause kidney disease, 

called nephropathica epidemica in Europe, and hemorrhagic fever with renal 

syndrome (HFRS) in Asia.

Occupational Infections

Documented laboratory-acquired infections have occurred in individuals working 

with hantaviruses.1-4 Extreme caution must be used in performing any laboratory 

operation that may create aerosols (centrifugation, vortex-mixing, etc.). Operations 

involving rats, voles, and other laboratory rodents, should be conducted with 

special caution because of the extreme hazard of aerosol infection, especially 

from infected rodent urine.

Natural Modes of Infection

HPS is a severe, often fatal disease that is caused by Sin Nombre and Andes or 

related viruses.5,6 Most cases of human illness have resulted from exposures to 

naturally infected wild rodents or to their excreta. Person-to-person transmission 

does not occur, with the exception of a few rare instances documented for Andes 

virus.7

 Arthropod vectors are not known to transmit hantaviruses.

Laboratory Safety and Containment Recommendations

Laboratory transmission of hantaviruses from rodents to humans via the aerosol 

route is well documented.4-7 Exposures to rodent excreta, especially aerosolized 

infectious urine, fresh necropsy material, and animal bedding are presumed to 

be associated with risk. Other potential routes of laboratory infection include 

ingestion, contact of infectious materials with mucous membranes or broken 

skin and, in particular, animal bites. Viral RNA has been detected in necropsy 

specimens and in patient blood and plasma obtained early in the course of 

HPS;8,9 however, the infectivity of blood or tissues is unknown.

BSL-2 practices, containment equipment, and facilities are recommended for 

laboratory handling of sera from persons potentially infected with hantaviruses. 

The use of a certified BSC is recommended for all handling of human body fluids 

when potential exists for splatter or aerosol.
Agent Summary Statements: Viral Agents 201

Potentially infected tissue samples should be handled in BSL-2 facilities 

following BSL-3 practices and procedures. Cell-culture virus propagation and 

purification should be carried out in a BSL-3 facility using BSL-3 practices, 

containment equipment and procedures.

Experimentally infected rodent species known not to excrete the virus can 

be housed in ABSL-2 facilities using ABSL-2 practices and procedures. Primary 

physical containment devices including BSCs should be used whenever procedures 

with potential for generating aerosols are conducted. Serum or tissue samples 

from potentially infected rodents should be handled at BSL-2 using BSL-3 

practices, containment equipment and procedures. All work involving inoculation 

of virus-containing samples into rodent species permissive for chronic infection 

should be conducted at ABSL-4.

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit 

from USDA/APHIS/VS.

Hendra Virus (formerly known as Equine Morbillivirus) and Nipah Virus

Hendra virus and Nipah virus are members of a newly recognized genus called 

Henipavirus, within the family Paramyxoviridae. Outbreaks of a previously 

unrecognized paramyxovirus, at first called equine morbillivirus, later named 

Hendra virus, occurred in horses in Australia in 1994 and 1995. During 1998-

1999, an outbreak of illness caused by a similar but distinct virus, now known as 

Nipah virus, occurred in Malaysia and Singapore. Human illness, characterized 

by fever, severe headache, myalgia and signs of encephalitis occurred in 

individuals in close contact with pigs (i.e., pig farmers and abattoir workers).10-14

A few patients developed a respiratory disease. Approximately 40% of patients 

with encephalitis died. Recently, cases of Nipah virus infection were described 

in Bangladesh, apparently the result of close contact with infected fruit bats 

without an intermediate (e.g., pig) host.

Occupational Infections

No laboratory-acquired infections are known to have occurred because of Hendra 

or Nipah virus exposure; however, three people in close contact with ill horses 

developed encephalitis or respiratory disease and two died.15-20

Natural Modes of Infection

The natural reservoir hosts for the Hendra and Nipah viruses appear to be fruit 

bats of the genus Pteropus.

21-23 Studies suggest that a locally occurring member 
202 Biosafety in Microbiological and Biomedical Laboratories

of the genus, Pteropus giganteus, is the reservoir for the virus in Bangladesh.24

Individuals who had regular contact with bats had no evidence of infection 

(antibody) in one study in Australia.25

Laboratory Safety and Containment Recommendations

The exact mode of transmission of these viruses has not been established. Most 

clinical cases to date have been associated with close contact with horses, their 

blood or body fluids (Australia) or pigs (Malaysia/Singapore) but presumed direct 

transmission from Pteropus bats has been recorded in Bangladesh. Hendra 

and Nipah viruses have been isolated from tissues of infected animals. In the 

outbreaks in Malaysia and Singapore, viral antigen was found in central nervous 

system, kidney and lung tissues of fatal human cases26 and virus was present in 

secretions of patients, albeit at low levels.27 Active surveillance for infection of 

healthcare workers in Malaysia has not detected evidence of occupationally 

acquired infections in this setting.28

Because of the unknown risks to laboratory workers and the potential 

impact on indigenous livestock should the virus escape a diagnostic or research 

laboratory, health officials and laboratory managers should evaluate the need 

to work with the virus and the containment capability of the facility before 

undertaking any work with Hendra, Nipah or suspected related viruses. BSL-4 

is required for all work with these viruses. Once a diagnosis of Nipah or Hendra 

virus is suspected, all diagnostic specimens also must be handled at BSL-4. 

ABSL-4 is required for any work with infected animals.

Special Issues

Select Agent Hendra and Nipah virus are select agents requiring registration 

with CDC and/or USDA for possession, use, storage and/or transfer. See 

Appendix F for additional information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Hepatitis A Virus, Hepatitis E Virus

Hepatitis A virus is a positive single-stranded RNA virus, the type species of the 

Hepatovirus genus in the family Picornaviridae. Hepatitis E virus is a positive 

single-stranded RNA virus, the type species of the genus Hepevirus, a floating 

genus not assigned to any family.
Agent Summary Statements: Viral Agents 203

Occupational Infections

Laboratory-associated infections with hepatitis A or E viruses do not appear to be 

an important occupational risk among laboratory personnel. However, hepatitis A 

is a documented hazard in animal handlers and others working with naturally or 

experimentally infected chimpanzees and other nonhuman primates.29 Workers 

handling other recently captured, susceptible primates (owl monkeys, marmosets) 

also may be at risk for hepatitis A infection. Hepatitis E virus appears to be less 

of a risk to personnel than hepatitis A virus, except during pregnancy, when 

infection can result in severe or fatal disease.

Natural Modes of Infection

Most infections with hepatitis A are foodborne and occasionally water-borne. The 

virus is present in feces during the prodromal phase of the disease and usually 

disappears once jaundice occurs. Hepatitis E virus causes acute entericallytransmitted cases of hepatitis, mostly waterborne. In Asia, epidemics involving 

thousands of cases have occurred.

Laboratory Safety and Containment Recommendations

The agents may be present in feces and blood of infected humans and 

nonhuman primates. Feces, stool suspensions, and other contaminated materials 

are the primary hazards to laboratory personnel. Care should be taken to avoid 

puncture wounds when handling contaminated blood from humans or nonhuman 

primates. There is no evidence that aerosol exposure results in infection.

BSL-2 practices, containment equipment, and facilities are recommended for 

the manipulation of hepatitis A and E virus, infected feces, blood or other tissues. 

ABSL-2 practices and facilities are recommended for activities using naturally or 

experimentally-infected nonhuman primates or other animal models that may 

shed the virus.

Special Issues

Vaccines A licensed inactivated vaccine against hepatitis A is available. 

Vaccines against hepatitis E are not currently available.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.
204 Biosafety in Microbiological and Biomedical Laboratories

Hepatitis B Virus, Hepatitis C Virus (formerly known as nonA nonB Virus), 

Hepatitis D Virus

Hepatitis B virus (HBV) is the type species of the Orthohepadnavirus genus in 

the family Hepadnaviridae. Hepatitis C virus (HCV) is the type species of the 

Hepacivirus genus in the family Flaviviridae. Hepatitis D virus (HDV) is the only 

member of the genus Deltavirus.

These viruses are naturally acquired from a carrier during blood transfusion, 

vaccination, tattooing, or body piercing with inadequately sterilized instruments. 

Non-parenteral routes, such as domestic contact and unprotected (heterosexual 

and homosexual) intercourse, are also major modes of transmission.

Individuals who are infected with the HBV are at risk of infection with HDV, 

a defective RNA virus that requires the presence of HBV virus for replication. 

Infection with HDV usually exacerbates the symptoms caused by HBV infection.

Occupational Infections

Hepatitis B has been one of the most frequently occurring laboratory-associated 

infections, and laboratory workers are recognized as a high-risk group for 

acquiring such infections.30

Hepatitis C virus infection can occur in the laboratory situation as well.31

The prevalence of antibody to hepatitis C (anti-HCV) is slightly higher in medical 

care workers than in the general population. Epidemiologic evidence indicates 

that HCV is spread predominantly by the parenteral route.32

Laboratory Safety and Containment Recommendations

HBV may be present in blood and blood products of human origin, in urine, 

semen, CSF and saliva. Parenteral inoculation, droplet exposure of mucous 

membranes, and contact exposure of broken skin are the primary laboratory 

hazards.33 The virus may be stable in dried blood or blood components for 

several days. Attenuated or avirulent strains have not been identified.

HCV has been detected primarily in blood and serum, less frequently in 

saliva and rarely or not at all in urine or semen. It appears to be relatively 

unstable to storage at room temperature and repeated freezing and thawing.

BSL-2 practices, containment equipment, and facilities are recommended 

for all activities utilizing known or potentially infectious body fluids and tissues. 

Additional primary containment and personnel precautions, such as those 

described for BSL-3, may be indicated for activities with potential for droplet 

or aerosol production and for activities involving production quantities or 

concentrations of infectious materials. ABSL-2 practices, containment equipment 

and facilities are recommended for activities utilizing naturally or experimentally 

infected chimpanzees or other NHP. Gloves should be worn when working with 
Agent Summary Statements: Viral Agents 205

infected animals and when there is the likelihood of skin contact with infectious 

materials. In addition to these recommended precautions, persons working with 

HBV, HCV, or other bloodborne pathogens should consult the OSHA Bloodborne 

Pathogen Standard.34 Questions related to interpretation of this Standard should 

be directed to federal, regional or state OSHA offices.

Special Issues

Vaccines Licensed recombinant vaccines against hepatitis B are available and 

are highly recommended for and offered to laboratory personnel.35 Vaccines 

against hepatitis C and D are not yet available for use in humans, but vaccination 

against HBV will also prevent HDV infection.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS.

Herpesvirus Simiae (Cerocopithecine Herpesvirus I, Herpes B Virus)

B virus is a member of the alphaherpesvirus genus (simplexvirus) in the family 

Herpesviridae. It occurs naturally in macaque monkeys, of which there are nine 

distinct species. Macaques may have primary, recurrent, or latent infections often 

with no apparent symptoms or lesions. B virus is the only member of the family of 

simplex herpesviruses that can cause zoonotic infections. Human infections have 

been identified in at least 50 instances, with approximately 80% mortality when 

untreated. There remains an approximate 20% mortality in the absence of timely 

treatment with antiviral agents.36 There have been no reported cases where 

prompt first aid with wound or exposure site cleansing was performed, and no 

cases where cleaning and post exposure prophylaxis were done. Cases prior 

to 1970 were not treated with antiviral agents because none were available. 

Morbidity and mortality associated with zoonotic infection results from invasion 

of the central nervous system, resulting in ascending paralysis ultimately with 

loss of ability to sustain respiration in the absence of mechanical ventilation. 

From 1987-2004, five additional fatal infections bring the number of lethal 

infections to 29 since the discovery of B virus in 1933.

Occupational Infections

B virus is a hazard in facilities where macaque monkeys are present. Mucosal 

secretions (saliva, genital secretions, and conjunctival secretions) are the primary 

body fluids associated with risk of B virus transmission. However, it is possible 

for other materials to become contaminated. For instance, a research assistant 

at the Yerkes Primate Center who died following mucosal splash without injury 

in 1997 was splashed with something in the eye while transporting a caged 

macaque. In part on this basis, the eye splash was considered low risk. However, 
206 Biosafety in Microbiological and Biomedical Laboratories

feces, urine or other fluids may be contaminated with virus shed from mucosal 

fluids. Zoonoses have been reported following virus transmission through a bite, 

scratch, or splash accident. Cases of B virus have also been reported after 

exposure to monkey cell cultures and to central nervous system tissue. There is 

often no apparent evidence of B virus infection in the animals or their cells and 

tissues, making it imperative that all suspect exposures be treated according to 

recommended standards.36 The risks associated with this hazard are, however, 

readily reduced by practicing barrier precautions and by rapid and thorough 

cleansing immediately following a possible site contamination. Precautions should 

be followed when work requires the use of any macaque species, even antibody 

negative animals. In most documented cases of B virus zoonosis, virus was not 

recovered from potential sources except in four cases, making speculations that 

some macaque species may be safer than others unfounded. The loss of five 

lives in the past two decades underscores that B virus infections have a low 

probability of occurrence, but when they do occur it is with high consequences.

Specific, regular training in risk assessments for B virus hazards including 

understanding the modes of exposure and transmission should be provided to 

individuals encountering B virus hazards. This training should include proper use 

of personal protective equipment, which is essential to prevention. Immediate 

and thorough cleansing following bites, scratches, splashes, or contact with 

potential fomites in high-risk areas appears to be helpful in prevention of B virus 

infections.37 First aid and emergency medical assistance procedures are most 

effective when institutions set the standard to be practiced by all individuals 

encountering B virus hazards.

Natural Modes of Infection

B virus occurs as a natural infection of Asiatic macaque monkeys, and some 

10% of newly caught rhesus monkeys have antibodies against the virus, which 

is frequently present in kidney cell cultures of this animal.

Reservoir species include Macaca mulatta, M. fascicularis, M.fusata, 

M. arctoides, M. cyclopsis and M. radiata. In these species the virus causes 

vesicular lesions on the tongue and lips, and sometimes of the skin. B virus is 

not present in blood or serum in infected macaques. Transmission of B virus 

appears to increase when macaques reach sexual maturity.

Laboratory Safety and Containment Recommendations

The National Academies Press has recently published ILAR’s guidelines for 

working with nonhuman primates.38 Additional resources are provided in the 

references following this agent summary statement. Asymptomatic B virus 

shedding accounts for most transmission among monkeys and human workers, 

but those working in the laboratory with potentially infected cells or tissues from 

macaques are also at risk. Exposure of mucous membranes or through skin 
Agent Summary Statements: Viral Agents 207

breaks provides this agent access to a new host, whether the virus is being shed 

from a macaque or human, or present in or on contaminated cells, tissues, or 

surfaces.36 B virus is not generally found in serum or blood, but these products 

obtained through venipuncture should be handled carefully because contamination 

of needles via skin can occur. When working with macaques directly, virus can 

be transmitted through bites, scratches, or splashes only when the animal is 

shedding virus from mucosal sites. Fomites, or contaminated surfaces (e.g., 

cages, surgical equipment, tables), should always be considered sources of B 

virus unless verified as decontaminated or sterilized. Zoonotically infected 

humans should be cautioned about autoinoculation of other susceptible sites 

when shedding virus during acute infection.

BSL-2 practices and facilities are suitable for all activities involving the use 

or manipulation of tissues, cells, blood, or serum from macaques with appropriate 

personal protective equipment. BSL-3 practices are recommended for handling 

materials from which B virus is being cultured using appropriate personal 

protective equipment, and BSL-4 facilities are recommended for propagation 

of virus obtained from diagnostic samples or stocks. Experimental infections 

of macaques as well as small animal models with B virus are recommended 

to be restricted to BSL-4 containment.

All macaques regardless of their origin should be considered potentially 

infected. Animals with no detectable antibody are not necessarily B virus-free. 

Macaques should be handled with strict barrier precaution protocols and injuries 

should be tended immediately according to the recommendations of the B Virus 

Working Group led by NIH and CDC.36

Barrier precautions and appropriate first aid are the keys to prevention of 

severe morbidity and mortality often associated with B virus zoonoses. These 

prevention tools were not implemented in each of the five B virus fatalities during 

the past two decades. Guidelines are available for safely working with macaques 

and should be consulted.36,39 The correct use of gloves, masks, and protective 

coats, gowns, aprons, or overalls is recommended for all personnel while working 

with non-human primates, especially macaques and other Old World species, 

including for all persons entering animal rooms where non-human primates are 

housed. To minimize the potential for mucous membrane exposure, some form 

of barrier is required to prevent droplet splashes to eyes, mouth, and nasal 

passages. Types and use of personal protective equipment (e.g., goggles or 

glasses with solid side shields and masks, or wrap-around face shields) should 

be determined with reference to the institutional risk assessment. Specifications 

of protective equipment must be balanced with the work to be performed so that 

the barriers selected do not increase work place risk by obscuring vision and 

contributing to increased risk of bites, needle sticks, scratches, or splashes.
208 Biosafety in Microbiological and Biomedical Laboratories

Special Issues

Post-exposure prophylaxis with oral acyclovir or valacyclovir should be 

considered for significant exposures to B virus. Therapy with intravenous 

acyclovir and/or ganciclovir in documented B virus infections is also important 

in reduction of morbidity following B virus zoonotic infection.36 In selected cases, 

IND permission has been granted for therapy with experimental antiviral drugs. 

Because of the seriousness of B virus infection, experienced medical and 

laboratory personnel should be consulted to develop individual case 

management. Barrier precautions should be observed with confirmed cases. 

B virus infection, as with all alphaherpesviruses, is lifelong in macaques.40

There are no effective vaccines available.

Select Agent B virus is a select agent requiring registration with CDC and/or 

USDA for possession, use, storage and/or transfer. See Appendix F for additional 

information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS.

Human Herpes Virus

The herpesviruses are ubiquitous human pathogens and are commonly present 

in a variety of clinical materials submitted for virus isolation. Thus far, nine 

herpesviruses have been isolated from humans: herpes simplex virus-1(HSV-1), 

HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), EpsteinBarr virus (EBV), and human herpesviruses (HHV) 6A, 6B, 7, and 8.41

HSV infection is characterized by a localized primary lesion. Primary 

infection with HSV-1 may be mild and unapparent occurring in early childhood. 

In approximately 10% of infections, overt illness marked by fever and malaise 

occurs. HSV-1 is a common cause of meningoencephalitis. Genital infections, 

usually caused by HSV-2, generally occur in adults and are sexually transmissible. 

Neonatal infections are most frequently caused by HSV-2 but HSV-1 infections 

are also common. In the neonate, disseminated disease and encephalitis are 

often fatal. EBV is the cause of infectious mononucleosis. It is also associated 

with the pathogenesis of several lymphomas and nasopharyngeal cancer.42 EBV 

is serologically distinct from the other herpesviruses; it infects and transforms 

B-lymphocytes. HCMV infection is common and often undiagnosed presenting 

as a nonspecific febrile illness. HCMV causes up to 10% of all cases of 

mononucleosis in young adults. The most severe form of the disease is seen 

in infants infected in utero. Children surviving infection may evidence mental 

retardation, microencephaly, motor disabilities and chronic liver disease.42

HCMV is one of the most common congenital diseases.
Agent Summary Statements: Viral Agents 209

VZV is the causative agent of chickenpox and herpes zoster. Chickenpox 

usually occurs in childhood and zoster occurs more commonly in adults. HHV-6 

is the causative agent of exanthema subitum (roseola), a common childhood 

exanthem.43 Nonspecific febrile illness and febrile seizures are also clinical 

manifestations of disease. HHV-6 may reactivate in immunocompetent 

individuals during pregnancy or during critical illness. Two distinct variants, 

HHV-6A and HHV-6B, exist, the latter causing roseola. HHV-7 is a constitutive 

inhabitant of adult human saliva.44 Clinical manifestations are less well understood 

but the virus has also been associated with roseola. HHV-8, also known as 

Kaposi’s sarcoma-associated virus, was first identified by Chang and co-workers 

in 1994.42 HHV-8 is believed to be the causative agent of Kaposi’s sarcoma and 

has been associated with primary effusion lymphoma.45 The natural history of 

HHV-8 has not been completely elucidated. High risk groups for HHV-8 include 

HIV-infected men who have sex with men and individuals from areas of high 

endemicity, such as Africa or the Mediterranean.45 The prevalence of HHV-8 is 

also higher among intravenous drug users than in the general population.45 At 

least one report has provided evidence that in African children, HHV-8 infection 

may be transmitted from mother to child.46 While few of the human herpesviruses 

have been demonstrated to cause laboratory-acquired infections, they are both 

primary and opportunistic pathogens, especially in immunocompromised hosts. 

Herpesvirus simiae (B-virus, Monkey B virus) is discussed separately in another 

agent summary statement in this section.

Occupational Infections

Few of the human herpesviruses have been documented as sources of 

laboratory acquired infections.

In a limited study, Gartner and co-workers have investigated the HHV-8 

immunoglobulin G (IgG) seroprevalence rates for healthcare workers caring for 

patients with a high risk for HHV-8 infection in a non-endemic area. Healthcare 

workers in contact with risk group patients were infected more frequently than 

healthcare workers without contact with risk groups. Workers without contact with 

risk group patients were infected no more frequently than the control group.53

Although this diverse group of indigenous viral agents has not demonstrated 

a high potential hazard for laboratory-associated infection, frequent presence in 

clinical materials and common use in research warrant the application of 

appropriate laboratory containment and safe practices.

Natural Modes of Infection

Given the wide array of viruses included in this family, the natural modes of 

infection vary greatly, as does the pathogenesis of the various viruses. Some 

have wide host ranges, multiply effectively, and rapidly destroy the cells they 

infect (HSV-1, HSV-2). Others have restricted host ranges or long replicative 
210 Biosafety in Microbiological and Biomedical Laboratories

cycles (HHV-6).41 Transmission of human herpesviruses in nature are, in general, 

associated with close, intimate contact with a person excreting the virus in their 

saliva, urine, or other bodily fluids.47 VZV is transmitted person-to-person through 

direct contact, through aerosolized vesicular fluids and respiratory secretions, and 

indirectly transmitted by fomites. Latency is a trait common to most herpesviruses, 

although the site and duration vary greatly. For example, EBV will persist in an 

asymptomatic, latent form in the host immune system, primarily in EBV-specific 

cytotoxic T cells42 while latent HSV has been detected only in sensory neurons.48,49

HHV-8 has been transmitted through organ transplantation50 and blood 

transfusion;51 some evidence suggests non-sexual horizontal transmission.52

Laboratory Safety and Containment Recommendations

Clinical materials and isolates of herpesviruses may pose a risk of infection 

following ingestion, accidental parenteral inoculation, and droplet exposure of the 

mucous membranes of the eyes, nose, or mouth, or inhalation of concentrated 

aerosolized materials. HHV-8 may be present in human blood or blood products 

and tissues or saliva. Aerosol transmission cannot be excluded as a potential 

route of transmission. Clinical specimens containing the more virulent 

Herpesvirus simiae (B-virus) may be inadvertently submitted for diagnosis of 

suspected herpes simplex infection. HCMV may pose a special risk during 

pregnancy because of potential infection of the fetus. All human herpesviruses 

pose an increased risk to persons who are immunocompromised.

BSL-2 practices, containment equipment, and facilities are recommended for 

activities utilizing known or potentially infectious clinical materials or cultures of 

indigenous viral agents that are associated or identified as a primary pathogen of 

human disease. Although there is little evidence that infectious aerosols are a 

significant source of LAI, it is prudent to avoid the generation of aerosols during 

the handling of clinical materials or isolates, or during the necropsy of animals. 

Primary containment devices (e.g., BSC) should be utilized to prevent exposure 

of workers to infectious aerosols. Additional containment and procedures, such 

as those described for BSL-3, should be considered when producing, purifying, 

and concentrating human herpesviruses, based on risk assessment.

Containment recommendations for herpesvirus simiae (B-virus, Monkey B virus) 

are described in the preceding agent summary statement.

Special Issues

Vaccine A live, attenuated vaccine for varicella zoster is licensed and available 

in the United States. In the event of a laboratory exposure to a non-immune 

individual, varicella vaccine is likely to prevent or at least modify disease.47

Treatment Antiviral medications are available for treatment of several of 

the herpesviruses.
Agent Summary Statements: Viral Agents 211

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Influenza

Influenza is an acute viral disease of the respiratory tract. The most common 

clinical manifestations are fever, headache, malaise, sore throat and cough. 

GI tract manifestations (nausea, vomiting and diarrhea) are rare but may 

accompany the respiratory phase in children. The two most important features 

of influenza are the epidemic nature of illness and the mortality that arises 

from pulmonary complications of the disease.54

The influenza viruses are enveloped RNA viruses belonging to the 

Orthomyxoviridae. There are three serotypes of influenza viruses, A, B and C. 

Influenza A is further classified into subtypes by the surface glycoproteins that 

possess either hemagglutinin (H) or neuraminidase (N) activity. Emergence of 

completely new subtypes (antigenic shift) occurs at irregular intervals with Type A 

viruses. New subtypes are responsible for pandemics and can result from 

reassortment of human and avian influenza virus genes. Antigenic changes 

within a type or subtype (antigenic drift) of A and B viruses are ongoing 

processes that are responsible for frequent epidemics and regional outbreaks 

and make the annual reformulation of influenza vaccine necessary.

Influenza viral infections, with different antigenic subtypes, occur naturally 

in swine, horses, mink, seals and in many domestic and wild avian species. 

Interspecies transmission and reassortment of influenza A viruses have been 

reported to occur among humans and wild and domestic fowl. The human influenza 

viruses responsible for the 1918, 1957 and 1968 pandemics contained gene 

segments closely related to those of avian influenza viruses.55 Swine influenza 

has also been isolated in human outbreaks.56

Control of influenza is a continuing human and veterinary public health concern.

Occupational Infections

LAI have not been routinely documented in the literature, but informal accounts 

and published reports indicate that such infections are known to have occurred, 

particularly when new strains showing antigenic shift or drift are introduced 

into a laboratory for diagnostic/research purposes.56 Occupationally-acquired, 

nosocomial infections are documented.57,58 Laboratory animal-associated 

infections have not been reported; however, there is possibility of human 

infection acquired from infected ferrets and vice versa.
212 Biosafety in Microbiological and Biomedical Laboratories

Natural Modes of Infection

Airborne spread is the predominant mode of transmission especially in crowded, 

enclosed spaces. Transmission may also occur through direct contact since 

influenza viruses may persist for hours on surfaces particularly in the cold and 

under conditions of low humidity.55 The incubation period is from one to three days. 

Recommendations for treatment and prophylaxis of influenza are available.59

Laboratory Safety and Containment Recommendations

The agent may be present in respiratory tissues or secretions of humans and 

most infected animals and birds. In addition, the agent may be present in the 

intestines and cloacae of many infected avian species. Influenza viruses may be 

disseminated in multiple organs in some infected animal species. The primary 

laboratory hazard is inhalation of virus from aerosols generated by infecting 

animals or by aspirating, dispensing, mixing, centrifuging or otherwise manipulating 

virus-infected samples. In addition, laboratory infection can result from direct 

inoculation of mucus membranes through virus-contaminated gloves following 

handling of tissues, feces or secretions from infected animals. Genetic manipulation 

has the potential for altering the host range, pathogenicity, and antigenic 

composition of influenza viruses. The potential for introducing influenza 

viruses with novel genetic composition into humans is unknown.

BSL-2 facilities, practices and procedures are recommended for diagnostic, 

research and production activities utilizing contemporary, circulating human 

influenza strains (e.g., H1/H3/B) and low pathogenicity avian influenza (LPAI) 

strains (e.g., H1-4, H6, H8-16), and equine and swine influenza viruses. ABSL-2 

is appropriate for work with these viruses in animal models. All avian and swine 

influenza viruses require an APHIS permit. Based on economic ramifications and 

source of the virus, LPAI H5 and H7 and swine influenza viruses may have 

additional APHIS permit-driven containment requirements and personnel 

practices and/or restrictions.

Non-Contemporary Human Influenza (H2N2) Strains

Non-contemporary, wild-type human influenza (H2N2) strains should be handled 

with increased caution. Important considerations in working with these strains are 

the number of years since an antigenically related virus last circulated and the 

potential for presence of a susceptible population. BSL-3 and ABSL-3 practices, 

procedures and facilities are recommended with rigorous adherence to additional 

respiratory protection and clothing change protocols. Negative pressure, HEPAfiltered respirators or positive air-purifying respirators (PAPRs) are recommended 

for use. Cold-adapted, live attenuated H2N2 vaccine strains may continue to be 

worked with at BSL-2.
Agent Summary Statements: Viral Agents 213

1918 Influenza Strain

Any research involving reverse genetics of the 1918 influenza strain should 

proceed with extreme caution. The risk to laboratory workers is unknown, but 

the pandemic potential is thought to be significant. Until further risk assessment 

data are available, the following practices and conditions are recommended for 

manipulation of reconstructed 1918 influenza viruses and laboratory animals 

infected with the viruses. These practices and procedures are considered 

minimum standards for work with the fully reconstructed virus.

■ BSL-3 and ABSL-3 practices, procedures and facilities.

■ Large laboratory animals such as NHP should be housed in primary 

barrier systems in ABSL-3 facilities.

■ Rigorous adherence to additional respiratory protection and clothing 

change protocols.

■ Use of negative pressure, HEPA-filtered respirators or PAPRs.

■ Use of HEPA filtration for treatment of exhaust air.

■ Amendment of personnel practices to include personal showers prior 

to exiting the laboratory.

Highly Pathogenic Avian Influenza (HPAI)

Manipulating HPAI viruses in biomedical research laboratories requires similar 

caution because some strains may pose increased risk to laboratory workers 

and have significant agricultural and economic implications. BSL-3 and ABSL-3 

practices, procedures and facilities are recommended along with clothing change 

and personal showering protocols. Loose-housed animals infected with HPAI 

strains must be contained within BSL-3-Ag facilities. (See Appendix D.) Negative 

pressure, HEPA-filtered respirators or positive air-purifying respirators are 

recommended for HPAI viruses with potential to infect humans. The HPAI are 

agricultural select agents requiring registration of personnel and facilities with 

the lead agency for the institution (CDC or USDA-APHIS). An APHIS permit is 

also required. Additional containment requirements and personnel practices 

and/or restrictions may be added as conditions of the permit.

Other Influenza Recombinant or Reassortant Viruses

When considering the biocontainment level and attendant practices and 

procedures for work with other influenza recombinant or reassortant viruses, 

the local IBC should consider but not limit consideration to the following in the 

conduct of protocol-driven risk assessment.

■ The gene constellation used.
214 Biosafety in Microbiological and Biomedical Laboratories

■ Clear evidence of reduced virus replication in the respiratory tract of 

appropriate animal models, compared with the level of replication of the 

wild-type parent virus from which it was derived.

■ Evidence of clonal purity and phenotypic stability.

■ The number of years since a virus that was antigenically related to the 

donor of the hemagglutinin and neuraminidase genes last circulated.

If adequate risk assessment data are not available, a more cautious approach 

utilizing elevated biocontainment levels and practices is warranted. There may be 

specific requirements regarding the setting of containment levels if your institution 

is subject to the NIH Guidelines.

Special Issues

Occupational Health Considerations Institutions performing work with HPAI 

and avian viruses that have infected humans; non-contemporary wild-type human 

influenza strains, including recombinants and reassortants; and viruses created 

by reverse genetics of the 1918 pandemic strain should develop and implement 

a specific medical surveillance and response plan. At the minimum these plans 

should: 1) require storage of baseline serum samples from individuals working 

with these influenza strains; 2) strongly recommend annual vaccination with the 

currently licensed influenza vaccine for such individuals; 3) provide employee 

counseling regarding disease symptoms including fever, conjunctivitis and 

respiratory symptoms; 4) establish a protocol for monitoring personnel for these 

symptoms; and 5) establish a clear medical protocol for responding to suspected 

laboratory-acquired infections. Antiviral drugs (e.g., oseltamivir, amantadine, 

rimantadine, zanamivir) should be available for treatment and prophylaxis, as 

necessary.59 It is recommended that the sensitivities of the virus being studied to 

the antivirals be ascertained. All personnel should be enrolled in an appropriately 

constituted respiratory protection program.

Influenza viruses may require USDA and/or USPHS import permits 

depending on the host range and pathogenicity of the virus in question. 

Select Agent Strains of HPAI and 1918 influenza virus are select agents 

requiring registration with CDC and/or USDA for possession, use, storage 

and/or transfer. See Appendix F for additional information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.
Agent Summary Statements: Viral Agents 215

Lymphocytic Choriomeningitis Virus

Lymphocytic choriomeningitis (LCM) is a rodent-borne viral infectious disease 

that presents as aseptic meningitis, encephalitis, or meningoencephalitis. The 

causative agent is the LCM virus (LCMV) that was initially isolated in 1933. 

The virus is the protypical member of the family Arenaviridae.

Occupational Infections

LAI with LCM virus are well documented. Most infections occur when chronic 

viral infection exists in laboratory rodents, especially mice, hamsters and guinea 

pigs.60-62 Nude and severe combined immune deficient (SCID) mice may pose a 

special risk of harboring silent chronic infections. Inadvertently infected cell cultures 

also represent a potential source of infection and dissemination of the agent.

Natural Modes of Infection

LCM and milder LCMV infections have been reported in Europe, the Americas, 

Australia, and Japan, and may occur wherever infected rodent hosts of the virus 

are found. Several serologic studies conducted in urban areas have shown 

that the prevalence of LCMV infection among humans ranges from 2% to 10%. 

Seroprevalence of 37.5% has been reported in humans in the Slovak Republic.63

The common house mouse, Mus musculus, naturally spreads LCMV. Once 

infected, these mice can become chronically infected as demonstrated by the 

presence of virus in blood and/or by persistently shedding virus in urine. Infections 

have also occurred in NHP in zoos, including macaques and marmosets. 

(Callitrichid hepatitis virus is a LCMV.)

Humans become infected by inhaling infectious aerosolized particles of 

rodent urine, feces, or saliva; by ingesting food contaminated with virus; by 

contamination of mucous membranes with infected body fluids; or by directly 

exposing cuts or other open wounds to virus-infected blood. Four recipients 

of organs from a donor who had unrecognized disseminated LCMV infection 

sustained severe disease and three succumbed. The source of donor infection 

was traced to a pet hamster that was not overtly ill.64

Laboratory Safety and Containment Recommendations

The agent may be present in blood, CSF, urine, secretions of the nasopharynx, 

feces and tissues of infected animal hosts and humans. Parenteral inoculation, 

inhalation, contamination of mucous membranes or broken skin with infectious 

tissues or fluids from infected animals are common hazards. Aerosol 

transmission is well documented.60

Of special note, tumors may acquire LCMV as an adventitious virus without 

obvious effects on the tumor. Virus may survive freezing and storage in liquid 

nitrogen for long periods. When infected tumor cells are transplanted, 
216 Biosafety in Microbiological and Biomedical Laboratories

subsequent infection of the host and virus excretion may ensue. Pregnant 

women infected with LCMV have transmitted the virus to their fetuses with death 

or serious central nervous system malformation as a consequence.65

BSL-2 practices, containment equipment, and facilities are suitable for 

activities utilizing known or potentially infectious body fluids, and for cell culture 

passage of laboratory-adapted strains. BSL-3 is required for activities with high 

potential for aerosol production, work with production quantities or high 

concentrations of infectious materials, and for manipulation of infected 

transplantable tumors, field isolates and clinical materials from human cases. 

Strains of LCMV that are shown to be lethal in non-human primates should be 

handled at BSL-3. ABSL-2 practices, containment equipment, and facilities are 

suitable for studies in adult mice with mouse brain-passaged strains requiring 

BSL-2 containment. Work with infected hamsters also should be done at ABSL-3.

Special Issues

Vaccines Vaccines are not available for use in humans.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Poliovirus

Poliovirus is the type species of the Enterovirus genus in the family 

Picornaviridae. Enteroviruses are transient inhabitants of the gastrointestinal 

tract, and are stable at acid pH. Picornaviruses are small, ether-insensitive 

viruses with an RNA genome.

There are three poliovirus serotypes (P1, P2, and P3). Immunity to one 

serotype does not produce significant immunity to the other serotypes.

Occupational Infections

Laboratory-associated poliomyelitis is uncommon. Twelve cases, including two 

deaths, were reported between 1941 and 1976.62,66 No laboratory-associated 

poliomyelitis has been reported for nearly 30 years. Both inactivated poliovirus 

vaccine (IPV) and oral poliovirus vaccine (OPV) are highly effective in preventing 

disease, but neither vaccine provides complete protection against infection. 

Poliovirus infections among immunized laboratory workers are uncommon but 

remain undetermined in the absence of laboratory confirmation. An immunized 

laboratory worker may unknowingly be a source of poliovirus transmission to 

unvaccinated persons in the community.67
Agent Summary Statements: Viral Agents 217

Natural Modes of Infection

At one time poliovirus infection occurred throughout the world. Transmission of wild 

poliovirus ceased in the United States in 1979, or possibly earlier. A polio eradication 

program conducted by the Pan American Health Organization led to elimination 

of polio from the Western Hemisphere in 1991. The Global Polio Eradication 

Program has dramatically reduced poliovirus transmission throughout the world.

Humans are the only known reservoir of poliovirus, which is transmitted most 

frequently by persons with unapparent infections. Person-to-person spread of 

poliovirus via the fecal-oral route is the most important route of transmission, 

although the oral-oral route may account for some cases.

Laboratory Safety and Containment Recommendations

The agent is present in the feces and in throat secretions of infected persons 

and in lymph nodes, brain tissue, and spinal cord tissue in fatal cases. For nonimmunized persons in the laboratory, ingestion or parenteral inoculation are the 

primary routes of infection. For immunized persons, the primary risks are the 

same, except for parenteral inoculation, which likely presents a lower risk. The 

importance of aerosol exposure is unknown. Laboratory animal-associated 

infections have not been reported, but infected nonhuman primates should be 

considered to present a risk.

BSL-2 practices, containment equipment, and facilities are recommended 

for all activities utilizing wild poliovirus infectious culture fluids, environmental 

samples, and clinical materials. In addition, potentially infectious materials 

collected for any purpose should be handled at BSL-2. Laboratory personnel 

working with such materials must have documented polio vaccination. Persons 

who have had a primary series of OPV or IPV and who are at an increased risk 

can receive another dose of IPV, but available data do not indicate the need for 

more than a single lifetime IPV booster dose for adults.68 ABSL-2 practices, 

containment equipment, and facilities are recommended for studies of virulent 

viruses in animals. Laboratories should use authentic Sabin OPV attenuated 

strains unless there are strong scientific reasons for working with wild polioviruses.

In anticipation of polio eradication, the WHO recommends destruction of 

all poliovirus stocks and potential infectious materials if there is no longer a 

programmatic or research need for such materials.69 Institutions/laboratories 

in the United States that currently retain wild poliovirus infectious or potential 

infectious material should be on the United States National Inventory maintained 

by CDC. When one year has elapsed after detection of the last wild poliovirus 

worldwide, CDC will inform relevant institutions/laboratories about additional 

containment procedures. Safety recommendations are subject to change based 

on international polio eradication activities.
218 Biosafety in Microbiological and Biomedical Laboratories

Special Issues

When OPV immunization stops, global control and biosafety requirements for 

wild as well as attenuated (Sabin) poliovirus materials are expected to become 

more stringent, consistent with the increased consequences of inadvertent 

transmission to a growing susceptible community.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Poxviruses

Four genera of the subfamily Chordopoxvirinae, family Poxviridae, 

(Orthopoxvirus, Parapoxvirus, Yatapoxvirus, and Molluscipoxvirus) contain 

species that can cause lesions on human skin or mucous membranes with mild 

to severe systemic rash illness in laboratorians. Species within the first three 

genera mostly arise as zoonotic agents.70,71 Laboratory-acquired poxvirus 

infections of most concern are from the orthopoxviruses that infect humans: 

variola virus (causes smallpox; human-specific), monkeypox virus (causes 

smallpox-like disease), cowpox virus (causes skin pustule, generalized rash), 

and vaccinia virus (causes skin pustule, systemic illness).70-75

Occupational Infections

Vaccinia virus, the leading agent of laboratory-acquired poxvirus infections, is 

used to make the current smallpox vaccine and may occur as a rare zoonosis.70,71

Laboratory-acquired infections with standard, mutant, or bioengineered forms of 

vaccinia virus have occurred, even in previously vaccinated laboratorians. In 

addition, vaccination with live vaccinia virus sometimes has side effects, which 

range from mild events (e.g., fever, fatigue, swollen lymph nodes) to rare, severe, 

and at times fatal outcomes (e.g., generalized vaccinia, encephalitis, vaccinia 

necrosum, eczema vaccinatum, ocular keratitis, corneal infection, fetal infection 

of pregnancy, and possibly myocardial infarction, myopericarditis, or angina), 

thus vaccination contraindications should be carefully followed.70,73-75

Natural Modes of Infection

Smallpox has been eradicated from the world since 1980, but monkey pox virus 

is endemic in rodents in parts of Africa. Importation of African rodents into North 

America in 2003 resulted in an outbreak of monkeypox in humans.72 Molluscum 

contagiosum, a disease due to Molluscipoxvirus infection, results in pearly white 

lesions that may persist for months in persons immunocompromised for various 
Agent Summary Statements: Viral Agents 219

reasons, including chronic illness, AIDS, other infections, medications, cancer 

and cancer therapies, or pregnancy.70

Laboratory Safety and Containment Recommendations

Poxviruses are stable in a wide range of environmental temperatures and 

humidity and may be transmitted by fomites.70 Virus may enter the body through 

mucous membranes, broken skin, or by ingestion, parenteral inoculation or 

droplet or fine-particle aerosol inhalation. Sources of laboratory-acquired infection 

include exposure to aerosols, environmental samples, naturally or experimentally 

infected animals, infectious cultures, or clinical samples, including 

vesiculopustular rash lesion fluid or crusted scabs, various tissue specimens, 

excretions and respiratory secretions.

Worldwide, all live variola virus work is to be done only within WHO approved 

BSL-4/ABSL-4 facilities; one is at the CDC in Atlanta and the other is at the State 

Research Center of Virology and Biotechnology (VECTOR) in Koltsovo, Russia.76

In general, all persons working in or entering laboratory or animal care 

areas where activities with vaccinia, monkey pox, or cowpox viruses are being 

conducted should have evidence of satisfactory vaccination. Vaccination is 

advised every three years for work with monkeypox virus and every 10 years for 

cowpox and vaccinia viruses (neither vaccination nor vaccinia immunoglobulin 

protect against poxviruses of other genera).73-75

ABSL-3 practices, containment equipment, and facilities are recommended 

for monkeypox work in experimentally or naturally infected animals. BSL-2 

facilities with BSL-3 practices are advised if vaccinated personnel perform other 

work with monkeypox virus. These practices include the use of Class I or II BSCs 

and barriers, such as safety cups or sealed rotors, for all centrifugations. The NIH 

Guidelines have assessed the risk of manipulating attenuated vaccinia strains 

(modified virus Ankara [MVA], NYVAC, TROVAC, and ALVAC) in areas where no 

other human orthopoxviruses are being used and have recommended BSL-1.76

However, higher levels of containment are recommended if these strains are 

used in work areas where other orthopoxviruses are manipulated. Vaccination is 

not required for individuals working only in laboratories where no other 

orthopoxviruses or recombinants are handled.75 BSL-2 and ABSL-2 plus 

vaccination are recommended for work with most other poxviruses.

Special Issues

Other Considerations The CDC Web site www.cdc.gov provides information 

on poxviruses, especially variola and monkeypox viruses, smallpox vaccination, 

and reporting vaccination adverse events. Clinical and other laboratories using 

poxviruses and clinicians can phone the CDC Clinician Information Line (877-

554-4625) and/or the CDC public information hotline (888-246-2675) concerning 

variola and other human poxvirus infections, smallpox vaccine, vaccinia 
220 Biosafety in Microbiological and Biomedical Laboratories

immunoglobulin, poxvirus antiviral drugs, or other treatments or quarantine 

issues. Contact CDC regarding applications to transfer monkeypox viruses.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Rabies Virus (and related lyssaviruses)

Rabies is an acute, progressive, fatal encephalitis caused by negative-stranded 

RNA viruses in the genus Lyssavirus, family Rhabdoviridae.

77 Rabies virus is the 

representative member (type species) of the genus. Members of the group 

include Australian bat lyssavirus, Duvenhage virus, European bat lyssavirus1

, 

European bat lyssavirus2

, Lagos bat virus, and Mokola virus.

Occupational Infections

Rabies LAI are extremely rare; two have been documented. Both resulted from 

presumed exposure to high concentrations of infectious aerosols, one generated 

in a vaccine production facility,78 and the other in a research facility.79 Naturally or 

experimentally infected animals, their tissues, and their excretions are a potential 

source of exposure for laboratory and animal care personnel.

Natural Modes of Infection

The natural hosts of rabies are many bat species and terrestrial carnivores, 

but most mammals can be infected. The saliva of infected animals is highly 

infectious, and bites are the usual means of transmission, although infection 

through superficial skin lesions or mucosa is possible.

Laboratory Safety and Containment Recommendations

When working with infected animals, the highest viral concentrations are present 

in central nervous system (CNS) tissue, salivary glands, and saliva, but rabies 

viral antigens may be detected in all innervated tissues. The most likely sources 

for exposure of laboratory and animal care personnel are accidental parenteral 

inoculation, cuts, or needle sticks with contaminated laboratory equipment, bites 

by infected animals, and exposure of mucous membranes or broken skin to 

infectious tissue or fluids. Infectious aerosols have not been a demonstrated 

hazard to personnel working with routine clinical materials or conducting 

diagnostic examinations. Fixed and attenuated strains of virus are presumed to 

be less hazardous, but the two recorded cases of laboratory-associated rabies 

resulted from presumed exposure to the fixed Challenge Virus Standard and 

Street Alabama Dufferin strains, respectively.
Agent Summary Statements: Viral Agents 221

BSL-2 and/or ABSL-2 practices, containment equipment, and facilities are 

recommended for all activities utilizing known or potentially infectious materials 

or animals. Pre-exposure rabies vaccination is recommended for all individuals 

prior to working with lyssaviruses or infected animals, or engaging in diagnostic, 

production, or research activities with these viruses.80 Rabies vaccination also 

is recommended for all individuals entering or working in the same room where 

lyssaviruses or infected animals are used. Prompt administration of postexposure 

booster vaccinations is recommended following recognized exposures in 

previously vaccinated individuals per current guidelines.81 For routine diagnostic 

activities, it is not always feasible to open the skull or remove the brain of an 

infected animal within a BSC, but it is pertinent to use appropriate methods 

and personal protection equipment, including dedicated laboratory clothing, 

heavy protective gloves to avoid cuts or sticks from cutting instruments or 

bone fragments, and a face shield or PAPR to protect the skin and mucous 

membranes of the eyes, nose, and mouth from exposure to tissue fragments 

or infectious droplets.

If a Stryker saw is used to open the skull, avoid contacting brain tissue with 

the blade of the saw. Additional primary containment and personnel precautions, 

such as those described for BSL-3, are indicated for activities with a high 

potential for droplet or aerosol production, and for activities involving large 

production quantities or high concentrations of infectious materials.

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Retroviruses, including Human and Simian Immunodeficiency Viruses 

(HIV and SIV)

The family Retroviridae is divided into two subfamilies, the Orthoretrovirinae with 

six genera including the Lentivirus genus, which includes HIV-1 and HIV-2. Other 

important human pathogens are human T-lymphotropic viruses 1 and 2 (HTLV-1 

and HTLV-2), members of the Deltaretrovirus genus. The Spumaretrovirinae, 

with one genus, Spumavirus, contains a variety of NHP viruses (foamy viruses) 

that can occasionally infect humans in close contact with NHPs.
222 Biosafety in Microbiological and Biomedical Laboratories

Occupational Infections

Data on occupational HIV transmission in laboratory workers are collected 

through two CDC-supported national surveillance systems: surveillance for 

1) AIDS, and 2) HIV-infected persons who may have acquired their infection 

through occupational exposures. For surveillance purposes, laboratory workers 

are defined as those persons, including students and trainees, who have worked 

in a clinical or HIV laboratory setting anytime since 1978. Cases reported in 

these two systems are classified as either documented or possible occupational 

transmission. Those classified as documented occupational transmission had 

evidence of HIV seroconversion (a negative HIV-antibody test at the time of the 

exposure which converted to positive) following a discrete percutaneous or 

mucocutaneous occupational exposure to blood, body fluids, or other clinical or 

laboratory specimens. As of June 1998, CDC had reports of 16 laboratory workers 

(all clinical) in the United States with documented occupational transmission.82

Workers have been reported to develop antibodies to simian 

immunodeficiency virus (SIV) following exposures. One case was associated 

with a needle-stick that occurred while the worker was manipulating a bloodcontaminated needle after bleeding an SIV-infected macaque monkey.83 Another 

case involved a laboratory worker who handled macaque SIV-infected blood 

specimens without gloves. Though no specific incident was recalled, this worker 

had dermatitis on the forearms and hands while working with the infected blood 

specimens.84 A third worker85 was exposed to SIV-infected primate blood through 

a needle-stick and subsequently developed antibodies to SIV. To date there is no 

evidence of illness or immunological incompetence in any of these workers.

Natural Modes of Infection

Retroviruses are widely distributed as infectious agents of vertebrates. Within 

the human population, spread is by close sexual contact or parenteral exposure 

through blood or blood products.

Laboratory Safety and Containment Recommendations

HIV has been isolated from blood, semen, saliva, tears, urine, CSF, amniotic 

fluid, breast milk, cervical secretion, and tissues of infected persons and 

experimentally infected nonhuman primates.86

Although the risk of occupationally-acquired HIV is primarily through 

exposure to infected blood, it is also prudent to wear gloves when manipulating 

other body fluids such as feces, saliva, urine, tears, sweat, vomitus, and human 

breast milk. This also reduces the potential for exposure to other microorganisms 

that may cause other types of infections. 

In the laboratory, virus should be presumed to be present in all blood or 

clinical specimens contaminated with blood, in any unfixed tissue or organ (other 
Agent Summary Statements: Viral Agents 223

than intact skin) from a human (living or dead), in HIV cultures, in all materials 

derived from HIV cultures, and in/on all equipment and devices coming into 

direct contact with any of these materials.

SIV has been isolated from blood, CSF, and a variety of tissues of infected 

nonhuman primates. Limited data exist on the concentration of virus in semen, 

saliva, cervical secretions, urine, breast milk, and amniotic fluid. Virus should be 

presumed to be present in all SIV cultures, in animals experimentally infected 

or inoculated with SIV, in all materials derived from SIV cultures, and in/on all 

equipment and devices coming into direct contact with any of these materials.87

The skin (especially when scratches, cuts, abrasions, dermatitis, or other 

lesions are present) and mucous membranes of the eye, nose, and mouth 

should be considered as potential pathways for entry of these retroviruses during 

laboratory activities. It is unknown whether infection can occur via the respiratory 

tract. The need for using sharps in the laboratory should be evaluated. Needles, 

sharp instruments, broken glass, and other sharp objects must be carefully 

handled and properly discarded. Care must be taken to avoid spilling and 

splashing infected cell-culture liquid and other potentially infected materials.85

BSL-2 practices, containment equipment, and facilities are recommended 

for activities involving blood-contaminated clinical specimens, body fluids and 

tissues. HTLV-1 and HTLV-2 should also be handled at this level. Activities such 

as producing research-laboratory-scale quantities of HIV or SIV, manipulating 

concentrated virus preparations, and conducting procedures that may produce 

droplets or aerosols, are performed in a BSL-2 facility, using BSL-3 practices. 

Activities involving large-scale volumes or preparation of concentrated HIV or 

SIV are conducted at BSL-3. ABSL-2 is appropriate for NHP and other animals 

infected with HIV or SIV. Human serum from any source that is used as a control 

or reagent in a test procedure should be handled at BSL-2.

In addition to the aforementioned recommendations, persons working with 

HIV, SIV, or other bloodborne pathogens should consult the OSHA Bloodborne 

Pathogen Standard.88 Questions related to interpretation of this Standard should 

be directed to federal, regional or state OSHA offices.

Special Issues

It is recommended that all institutions establish written policies regarding the 

management of laboratory exposure to HIV and SIV, including treatment and 

prophylaxis protocols. (See Section VII.)

The risk associated with retroviral vector systems can vary significantly, 

especially lentiviral vectors. Because the risk associated with each gene transfer 

system can vary, no specific guideline can be offered other than to have all gene 

transfer protocols reviewed by an IBC.
224 Biosafety in Microbiological and Biomedical Laboratories

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Severe Acute Respiratory Syndrome (SARS) Coronavirus

SARS is a viral respiratory illness caused by a previously undescribed 

coronavirus, SARS-associated coronavirus (SARS-CoV) within the family 

Coronaviridae. SARS was retrospectively recognized in China in November 

2002. Over the next few months, the illness spread to other south-east.

Asian countries, North America, South America, and Europe following major 

airline routes. The majority of disease spread occurred in hospitals, among family 

members and contacts of hospital workers. From November 2002 through July 

2003, when the global outbreak was contained, a total of 8,098 probable cases 

of SARS were reported to the WHO from 29 countries.89

In general, SARS patients present with fever (temperature greater than 

100.4°F [>38.0°C]), malaise and myalgias quickly followed by respiratory 

symptoms including shortness of breath and cough. Ten to 20 percent of patients 

may have diarrhea. Review of probable cases indicates that the shortness of 

breath sometimes rapidly progresses to respiratory failure requiring ventilation. 

The case fatality rate is about 11%.

Occupational Infections

Healthcare workers are at increased risk of acquiring SARS from an infected 

patient especially if involved in pulmonary/respiratory procedures such as 

endotracheal intubation, aerosolization or nebulization of medications, diagnostic 

sputum induction, airway suctioning, positive pressure ventilation and highfrequency oscillatory ventilation.

Two confirmed episodes of SARS-CoV transmission to laboratory workers 

occurred in research laboratories in Singapore and Taiwan.89,90 Both occurrences 

were linked to breaches in laboratory practices. Laboratory-acquired infections 

in China during 2004 demonstrated secondary and tertiary spread of the disease 

to close contacts and healthcare providers of one of the employees involved.91

Although no laboratory-acquired cases have been associated with the routine 

processing of diagnostic specimens, SARS coronavirus represents an emerging 

infectious disease for which risk to the medical and laboratory community is not 

fully understood.
Agent Summary Statements: Viral Agents 225

Natural Modes of Infection

The mode of transmission in nature is not well understood. It appears that SARS 

is transmitted from person-to-person through close contact such as caring for, 

living with, or having direct contact with respiratory secretions or body fluids of a 

suspect or probable case.92 SARS is thought to be spread primarily through droplets, 

aerosols and possibly fomites. The natural reservoir for SARS CoV is unknown.

Laboratory Safety and Containment Recommendations

SARS-CoV may be detected in respiratory, blood, or stool specimens. The exact 

mode of transmission of SARS-CoV laboratory-acquired infection has not been 

established, but in clinical settings the primary mode of transmission appears 

through direct or indirect contact of mucous membranes with infectious 

respiratory droplets.93,94

In clinical laboratories, whole blood, serum, plasma and urine specimens 

should be handled using Standard Precautions, which includes use of gloves, 

gown, mask, and eye protection. Any procedure with the potential to generate 

aerosols (e.g., vortexing or sonication of specimens in an open tube) should be 

performed in a BSC. Use sealed centrifuge rotors or gasketed safety carriers for 

centrifugation. Rotors and safety carriers should be loaded and unloaded in a 

BSC. Procedures conducted outside a BSC must be performed in a manner 

that minimizes the risk of personnel exposure and environmental release.

The following procedures may be conducted in the BSL-2 setting: pathologic 

examination and processing of formalin-fixed or otherwise inactivated tissues, 

molecular analysis of extracted nucleic acid preparations, electron microscopic 

studies with glutaraldehyde-fixed grids, routine examination of bacterial and fungal 

cultures, routine staining and microscopic analysis of fixed smears, and final 

packaging of specimens for transport to diagnostic laboratories for additional testing 

(specimens should already be in a sealed, decontaminated primary container).

Activities involving manipulation of untreated specimens should be 

performed in BSL-2 facilities following BSL-3 practices. In the rare event that 

a procedure or process involving untreated specimens cannot be conducted 

in a BSC, gloves, gown, eye protection, and respiratory protection (acceptable 

methods of respiratory protection include: a properly fit-tested, National Institute 

for Occupational Safety and Health [NIOSH]-approved filter respirator [N-95 or 

higher level] or a PAPR equipped with HEPA filters) should be used. All personnel 

who use respiratory protective devices should be enrolled in an appropriately 

constituted respiratory protection program.

Work surfaces should be decontaminated upon completion of work with 

appropriate disinfectants. All waste must be decontaminated prior to disposal.
226 Biosafety in Microbiological and Biomedical Laboratories

SARS-CoV propagation in cell culture and the initial characterization of viral 

agents recovered in cultures of SARS specimens must be performed in a BSL-3 

facility using BSL-3 practices and procedures. Risk assessment may dictate the 

additional use of respiratory protection.

Inoculation of animals for potential recovery of SARS-CoV from SARS samples, 

research studies, and protocols involving animal inoculation for characterization of 

putative SARS agents must be performed in ABSL-3 facilities using ABSL-3 work 

practices. Respiratory protection should be used as warranted by risk assessment.

In the event of any break in laboratory procedure or accidents (e.g., accidental 

spillage of material suspected of containing SARS-CoV), procedures for emergency 

exposure management and/or environmental decontamination should be 

immediately implemented and the supervisor should be notified. The worker 

and the supervisor, in consultation with occupational health or infection control 

personnel, should evaluate the break in procedure to determine if an exposure 

occurred (see Special Issues, below).

Special Issues

Occupational Health Considerations Institutions performing work with SARS 

coronavirus should require storage of a baseline serum sample from individuals 

who work with the virus or virus-containing specimens. Personnel working with 

the virus or samples containing or potentially containing the virus should be 

trained regarding the symptoms of SARS-CoV infection and counseled to report 

any fever or respiratory symptoms to their supervisor immediately. They should 

be evaluated for possible exposure and the clinical features and course of 

their illness should be closely monitored. Institutions performing work with the 

SARS-CoV or handling specimens likely to contain the agent should develop 

and implement a specific occupational medical plan with respect to this agent. 

The plan, at a minimum, should contain procedures for managing:

■ identifiable breaks in laboratory procedures;

■ exposed workers without symptoms;

■ exposed workers who develop symptoms within ten days of an 

exposure; and

■ symptomatic laboratory workers with no recognized exposure.

Further information and guidance regarding the development of a personnel 

exposure response plan is available from the CDC.95 Laboratory workers who are 

believed to have had a laboratory exposure to SARS-CoV should be evaluated, 

counseled about the risk of SARS-CoV transmission to others, and monitored 

for fever or lower respiratory symptoms as well as for any of the following: sore 

throat, rhinorrhea, chills, rigors, myalgia, headache, and diarrhea.
Agent Summary Statements: Viral Agents-References 227

Local and/or state public health departments should be promptly notified of 

laboratory exposures and illness in exposed laboratory workers.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS.

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58. Horcajada JP, Pumarola T, Martinez JA, et al. A nosocomial outbreak of 

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59. Centers for Disease Control and Prevention. Guidelines for preventing 

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Healthcare Infection Control Practices Advisory Committee. MMWR 

Recomm Rep. 2004;53(RR-3):1-36.

60. Bowen GS, Calisher CH, Winkler WG, et al. Laboratory studies of a 

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Am J Epidemiol. 1975;102:233-40.

61. Jahrling PB, Peters CJ. Lymphocytic choriomeningitis virus: a neglected 

pathogen of man. Arch Pathol Lab Med. 1992;116:486-8.

62. Pike RM. Laboratory-associated infections: summary and analysis of 3,921 

cases. Hlth Lab Sci. 1976;13:105-14.

63. Reiserova L, Kaluzova M, Kaluz S, et al. Identification of MaTu-MX agent as 

a new strain of lymphocytic choriomeningitis virus (LCMV) and serological 

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1999;257:73-83.

64. Centers for Disease Control and Prevention. Lymphocytic choriomeningitis 

virus infection in organ transplant recipients: Massachusetts, Rhode Island, 

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65. Wright R, Johnson D, Neumann M, et al. Congenital lymphocytic 

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Agent Summary Statements: Viral Agents-References 231

66. Dowdle WR, Gary HE, Sanders R, et al. Can post-eradication laboratory 

containment of wild polioviruses be achieved? Bull World Health Organ. 

2002;80:311-6.

67. Mulders MN, Reimerink JHJ, Koopmans MPG, et al. Genetic analysis of wild 

type poliovirus importation into The Netherlands (1979-1995). J Infect Dis. 

1997;176:617-24.

68. Centers for Disease Control and Prevention. Poliomyelitis prevention in the 

United States. Updated recommendations of the Advisory Committee on 

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69. Uirusu. WHO global action plan for laboratory containment of wild poliovirus. 

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70. Esposito JJ, Fenner F. Poxviruses. In: Knipe DM, Howley PM, Griffin DE, 

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Philadelphia. 2002. p. 2885-921.

71. Lewis-Jones S. Zoonotic poxvirus infections in humans. Curr Opin Infect Dis. 

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72. Reed KD, Melski JW, Graham MB, et al. The detection of monkeypox in 

humans in the Western Hemisphere. N Engl J Med. 2004;350:342-50.

73. Wharton M, Strikas RA, Harpaz R, et al. Recommendations for using 

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74. Centers for Disease Control and Prevention. Supplemental 

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75. Centers for Disease Control and Prevention. Vaccinia (smallpox) vaccine: 

recommendations of the Advisory Committee on Immunization Practices 

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76. NIH Guidelines for Research Involving Recombinant DNA Molecules. 

Bethesda: The National Institutes of Health (US), Office of Biotechnology 

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77. Rupprecht CE, Hanlon CA, Hemachudha T. Rabies re-examined. Lancet 

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78. Winkler WG, Fashinell TR, Leffingwell L, et al. Airborne rabies transmission 

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79. Centers for Disease Control and Prevention. Rabies in a laboratory worker, 

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81. Rupprecht CE, Gibbons RV. Prophylaxis against rabies. New Engl J Med. 

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232 Biosafety in Microbiological and Biomedical Laboratories

82. Centers for Disease Control and Prevention. HIV/AIDS surveillance report, 

June 1998. Atlanta, GA; 1998.

83. Khabbaz RF, Rowe T, Murphey-Corb M, et al. Simian immunodeficiency 

virus needlestick accident in a laboratory worker. Lancet. 1992;340:271-3.

84. Centers for Disease Control and Prevention. Seroconversion to simian 

immunodeficiency virus in two laboratory workers. MMWR Morb Mortal Wkly 

Rep. 1992;41:678-81.

85. Sotir M, Switzer W, Schable C, et al. Risk of occupational exposure to 

potentially infectious nonhuman primate materials and to simian 

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86. Schochetman G, George JR. AIDS testing: methodology and management 

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87. Centers for Disease Control and Prevention. Update: universal precautions 

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88. Occupational exposure to bloodborne pathogens. Final Rule. Fed. Register 

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89. Center for Disease Control, Taiwan. A Report on the Laboratory-Acquired 

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90. Singapore Ministry of Health. Singapore: Singapore Ministry of Health. 

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93. Chow PK, Ooi, EE, Tan, HK, et al. Healthcare worker seroconversion in 

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Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 233

Section VIII-F: Arboviruses and Related Zoonotic Viruses

In 1979, the American Committee on Arthropod-Borne Viruses (ACAV) 

Subcommittee on Arbovirus Laboratory Safety (SALS) first provided biosafety 

recommendations for each of the 424 viruses then registered in the International 

Catalogue of Arboviruses, including Certain Other Viruses of Vertebrates.1

Working together, SALS, the CDC and the NIH have periodically updated the 

catalogue by providing recommended biosafety practices and containment for 

arboviruses registered since 1979. These recommendations are based, in part, 

on risk assessments derived from information provided by a worldwide survey of 

laboratories working with arboviruses, new published reports on the viruses, as 

well as discussions with scientists working with each virus.

Table 6, located at the end of this Section, provides an alphabetical listing of 

597 viruses and includes common name, virus family or genus, acronym, BSL 

recommendation, the basis for the rating, the antigenic group2

 (if known), HEPA 

filtration requirements, and regulatory requirements (i.e., import/export permits 

from either the CDC or the USDA). In addition, many of the organisms are 

classified as select agents and require special security measures to possess, 

use, or transport. (See Appendix F.) Table 4 provides a key for the SALS basis 

for assignment of viruses listed in Table 6.

Agent summary statements have been included for certain arboviruses. They 

were submitted by a panel of experts for more detailed consideration due to one 

or more of the following factors:

■ at the time of writing this edition, the organism represented an emerging 

public health threat in the United States;

■ the organism presented unique biocontainment challenge(s) that required 

further detail; and

■ the organism presented a significant risk of laboratory-acquired infection.

These recommendations were made in August 2005; requirements for biosafety, 

shipping, and select agent registration can change. Please be sure to confirm the 

requirements with the appropriate Federal agency. If the pathogen of interest is 

one listed in Appendix D, contact the USDA for additional biosafety requirements. 

USDA guidance may supersede the information found in this Chapter.

Recommendations for the containment of infected arthropod vectors were 

drafted by a subcommittee of the American Committee on Medical Entomology 

(ACME), and circulated widely among medical entomology professionals. (See 

Appendix E.)
234 Biosafety in Microbiological and Biomedical Laboratories

Some commonly used vaccine strains for which attenuation has been firmly 

established are recognized by SALS. These vaccine strains may be handled 

safely at BSL-2 (Table 5). The agents in Table 4 and 5 may require permits from 

USDA/DOC/DHHS.

Table 4. Explanation of Symbols Used in Table 6 to Define Basis for 

Assignment of Viruses to Biosafety Levels

Symbol Definition

S Results of SALS survey and information from the Catalog.1

IE Insufficient experience with virus in laboratory facilities with low biocontainment.

A Additional criteria.

A1 Disease in sheep, cattle or horses.

A2 Fatal human laboratory infection—probably aerosol.

A3 Extensive laboratory experience and mild nature of aerosol laboratory infections 

justifies BSL-2.

A4 Placed in BSL-4 based on the close antigenic relationship with a known BSL-4 agent 

plus insufficient experience.

A5 BSL-2 arenaviruses are not known to cause serious acute disease in humans and 

are not acutely pathogenic for laboratory animals including primates. In view of 

reported high frequency of laboratory aerosol infection in workers manipulating high 

concentrations of Pichinde virus, it is strongly recommended that work with high 

concentrations of BSL-2 arenaviruses be done at BSL-3.

A6 Level assigned to prototype or wild-type virus. A lower level may be recommended for 

variants with well-defined reduced virulence characteristics.

A7 Placed at this biosafety level based on close antigenic or genetic relationship to other 

viruses in a group of 3 or more viruses, all of which are classified at this level.

A8 BSL-2 hantaviruses are not known to cause laboratory infections, overt disease 

in humans, or severe disease in experimental primates. Because of antigenic and 

biologic relationships to highly pathogenic hantaviruses and the likelihood that 

experimentally infected rodents may shed large amounts of virus, it is recommended 

that work with high concentrations or experimentally infected rodents be conducted at 

BSL-3.
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 235

Table 5. Vaccine Strains of BSL-3 and BSL-4 Viruses that May Be Handled 

as BSL-2

Virus Vaccine Strain

Chikungunya 181/25

Junin Candid #1

Rift Valley fever MP-12

Venezuelan equine encephalomyelitis TC83 & V3526

Yellow fever 17-D

Japanese encephalitis 14-14-2

Based on the recommendations listed with the tables, the following guidelines 

should be adhered to where applicable.

Viruses with BSL-2 Containment Recommended

The recommendation for conducting work with the viruses listed in Table 6 at 

BSL-2 are based on the existence of historical laboratory experience adequate 

to assess the risks when working with this group of viruses. This indicates a) no 

overt laboratory-associated infections are reported, b) infections resulted from 

exposures other than by infectious aerosols, or c) if disease from aerosol 

exposure is documented, it is uncommon.

Laboratory Safety and Containment Recommendations

Agents listed in this group may be present in blood, CSF, various tissues, and/or 

infected arthropods, depending on the agent and the stage of infection. The 

primary laboratory hazards comprise accidental parenteral inoculation, contact of 

the virus with broken skin or mucous membranes, and bites of infected laboratory 

rodents or arthropods. Properly maintained BSCs, preferable Class II, or other 

appropriate personal protective equipment or physical containment devices are 

used whenever procedures with a potential for creating infectious aerosols or 

splashes are conducted.

BSL-2 practices, containment equipment, and facilities are recommended 

for activities with potentially infectious clinical materials and arthropods and for 

manipulations of infected tissue cultures, embryonate hen’s eggs, and rodents.

Large quantities and/or high concentrations of any virus have the potential 

to overwhelm both innate immune mechanisms and vaccine-induced immunity. 

When a BSL-2 virus is being produced in large quantities or in high concentrations, 

additional risk assessment is required. This might indicate BSL-3 practices, 

including additional respiratory protection, based on the risk assessment of 

the proposed experiment.
236 Biosafety in Microbiological and Biomedical Laboratories

Viruses with BSL-3 Containment Recommended

The recommendations for viruses listed in Table 6 that require BSL-3 

containment are based on multiple criteria. SALS considered the laboratory 

experience for some viruses to be inadequate to assess risk, regardless of the 

available information regarding disease severity. In some cases, SALS recorded 

overt LAI transmitted by the aerosol route in the absence or non-use of protective 

vaccines, and considered that the natural disease in humans is potentially 

severe, life threatening, or causes residual damage.1 Arboviruses also were 

classified as requiring BSL-3 containment if they caused diseases in domestic 

animals in countries outside of the United States.

Laboratory Safety and Containment Recommendations

The agents listed in this group may be present in blood, CSF, urine, and exudates, 

depending on the specific agent and stage of disease. The primary laboratory 

hazards are exposure to aerosols of infectious solutions and animal bedding, 

accidental parenteral inoculation, and contact with broken skin. Some of these 

agents (e.g., VEE virus) may be relatively stable in dried blood or exudates.

BSL-3 practices, containment equipment, and facilities are recommended for 

activities using potentially infectious clinical materials and infected tissue 

cultures, animals, or arthropods.

A licensed attenuated live virus is available for immunization against yellow 

fever. It is recommended for all personnel who work with this agent or with 

infected animals, and those entering rooms where the agents or infected 

animals are present.

Junin virus has been reclassified to BSL-3, provided that all at-risk personnel 

are immunized and the laboratory is equipped with HEPA-filtered exhaust. 

SALS also has reclassified Central European tick-borne encephalitis (CETBE) 

viruses to BSL-3, provided all at-risk personnel are immunized. CETBE is not a 

registered name in The International Catalogue of Arboviruses (1985). Until the 

registration issue is resolved taxonomically, CETBE refers to the following group 

of very closely related, if not essentially identical, tick-borne flaviviruses isolated 

from Czechoslovakia, Finland and Russia: Absettarov, Hanzalova, Hypr, and 

Kumlinge viruses. While there is a vaccine available that confers immunity to the 

CETBE group of genetically (>98%) homogeneous viruses, the efficacy of this 

vaccine against Russian spring-summer encephalitis (RSSE) virus infections has 

not been established. Thus, the CETBE group of viruses has been reclassified 

as BSL-3 when personnel are immunized with CETBE vaccine, while RSSE 

remains classified as BSL-4. It should be noted that CETBE viruses are currently 

listed as select agents and require special security and permitting considerations. 

(See Appendix F.)
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 237

Investigational vaccines for eastern equine encephalomyelitis (EEE) virus, 

Venezuelan equine encephalitis (VEE), western equine encephalomyelitis (WEE) 

virus, and Rift Valley fever viruses (RVFV), may be available in limited quantities 

and administered on-site at the Special Immunization Program of USAMRIID, 

located at Ft. Detrick, Frederick, MD. Details are available at the end of this section.

The use of investigational vaccines for laboratory personnel should be 

considered if the vaccine is available. Initial studies have shown the vaccine 

to be effective in producing an appropriate immunologic response, and the 

adverse effects of vaccination are within acceptable parameters. The decision to 

recommend vaccines for laboratory personnel must be carefully considered and 

based on an risk assessment which includes a review of the characteristics of the 

agent and the disease, benefits versus the risk of vaccination, the experience of 

the laboratory personnel, laboratory procedures to be used with the agent, and 

the contraindications for vaccination including the health status of the employee.

If the investigational vaccine is contraindicated, does not provide acceptable 

reliability for producing an immune response, or laboratory personnel refuse 

vaccination, the use of appropriate personal protective equipment may provide 

an alternative. Respiratory protection, such as use of a PAPR, should be 

considered in areas using organisms with a well-established risk of aerosol 

infections in the laboratory, such as VEE viruses.

Any respiratory protection equipment must be provided in accordance with 

the institution’s respiratory protection program. Other degrees of respiratory 

protection may be warranted based on an assessment of risk as defined in 

Chapter 2 of this manual. All personnel in a laboratory with the infectious agent 

must use comparable personal protective equipment that meets or exceeds the 

requirements, even if they are not working with the organism. Sharps precautions 

as described under BSL-2 and BSL-3 requirements must be continually and 

strictly reinforced, regardless of whether investigational vaccines are used.

Non-licensed vaccines are available in limited quantities and administered 

on-site at the Special Immunization Program of USAMRIID. IND vaccines are 

administered under a cooperative agreement between the U.S. Army and the 

individual’s requesting organization. Contact the Special Immunization Program 

by telephone at (301) 619-4653.

Enhanced BSL-3 Containment

Situations may arise for which enhancements to BSL-3 practices and equipment 

are required; for example, when a BSL-3 laboratory performs diagnostic testing 

on specimens from patients with hemorrhagic fevers thought to be due to dengue 

or yellow fever viruses. When the origin of these specimens is Africa, the Middle 

East, or South America, such specimens might contain etiologic agents, such as 

arenaviruses, filoviruses, or other viruses that are usually manipulated in a BSL-4 
238 Biosafety in Microbiological and Biomedical Laboratories

laboratory. Examples of enhancements to BSL-3 laboratories might include: 

1) enhanced respiratory protection of personnel against aerosols; 2) HEPA 

filtration of dedicated exhaust air from the laboratory; and 3) personal body 

shower. Additional appropriate training for all animal care personnel should 

be considered.

Viruses with BSL-4 Containment Recommended

The recommendations for viruses assigned to BSL-4 containment are based 

on documented cases of severe and frequently fatal naturally occurring human 

infections and aerosol-transmitted laboratory infections. SALS recommends that 

certain agents with a close antigenic relationship to agents assigned to BSL-4 

also be provisionally handled at this level until sufficient laboratory data indicates 

that work with the agent may be assigned to a lower biosafety level.

Laboratory Safety and Containment Recommendations 

The infectious agents may be present in blood, urine, respiratory and throat 

secretions, semen, and other fluids and tissues from human or animal hosts, and 

in arthropods, rodents, and NHPs. Respiratory exposure to infectious aerosols, 

mucous membrane exposure to infectious droplets, and accidental parenteral 

inoculation are the primary hazards to laboratory or animal care personnel.3,4

BSL-4 practices, containment equipment, and facilities are recommended 

for all activities utilizing known or potentially infectious materials of human, 

animal, or arthropod origin. Clinical specimens from persons suspected of 

being infected with one of the agents listed in this summary should be 

submitted to a laboratory with a BSL-4 maximum containment facility.5

Dealing with Unknown Arboviruses

The ACAV has published reports documenting laboratory workers who acquired 

arbovirus infections during the course of their duties.6

 In the first such document, 

it was recognized that these laboratory infections typically occurred by unnatural 

routes such as percutaneous or aerosol exposure, that “lab adapted” strains were 

still pathogenic for humans, and that as more laboratories worked with newly 

identified agents, the frequency of laboratory-acquired infections was increasing. 

Therefore, to assess the risk of these viruses and provide safety guidelines to 

those working with them, ACAV appointed SALS to evaluate the hazards of 

working with arboviruses in the laboratory setting.7,8

The SALS committee made a series of recommendations, published in 1980, 

describing four levels of laboratory practices and containment guidelines that 

were progressively more restrictive. These levels were determined after widelydistributed surveys evaluated numerous criteria for each particular virus 

including: 1) past occurrence of laboratory-acquired infections correlated with 

facilities and practices used; 2) volume of work performed as a measure of 
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 239

potential exposure risk; 3) immune status of laboratory personnel; 4) incidence 

and severity of naturally-acquired infections in adults; and 5) incidence of disease 

in animals outside the United States (to assess import risk).

While these criteria are still important factors to consider in any risk 

assessment for manipulating arboviruses in the laboratory, it is important to 

note that there have been many modifications to personal laboratory practices 

(e.g., working in BSC while wearing extensive personal protective equipment in 

contrast to working with viruses on an open bench top) and significant changes 

in laboratory equipment and facilities (e.g., BSC, PAPR) available since the initial 

SALS evaluation. Clearly, when dealing with a newly recognized arbovirus, there 

is insufficient previous experience with it; thus, the virus should be assigned a 

higher biosafety level. However, with increased ability to safely characterize 

viruses, the relationship to other disease-causing arboviruses can be established 

with reduced exposure to the investigators. Therefore, in addition to those 

established by SALS, additional assessment criteria should be considered.

One criterion for a newly identified arbovirus is a thorough description of how 

the virus will be handled and investigated. For example, experiments involving 

pure genetic analysis could be handled differently than those where the virus will 

be put into animals or arthropods.9

 Additionally, an individual risk assessment 

should consider the fact that not all strains of a particular virus exhibit the same 

degree of pathogenicity or transmissibility. While variable pathogenicity occurs 

frequently with naturally identified strains, it is of particular note for strains that 

are modified in the laboratory. It may be tempting to assign biosafety levels to 

hybrid or chimeric strains based on the parental types but due to possible altered 

biohazard potential, assignment to a different biosafety level may be justified.10 A 

clear description of the strains involved should accompany any risk assessment.

Most of the identified arboviruses have been assigned biosafety levels; 

however, a number of those that are infrequently studied, newly identified, or 

have only single isolation events may not have been evaluated by SALS, ACAV, 

CDC, or the NIH (Table 6). Thorough risk assessment is important for all arboviral 

research and it is of particular importance for work involving unclassified viruses. 

A careful assessment by the laboratory director, institutional biosafety officer and 

safety committee, and as necessary, outside experts is necessary to minimize 

the risk of human, animal, and environmental exposure while allowing research 

to progress.

Chimeric Viruses

The ability to construct cDNA clones encoding a complete RNA viral genome 

has led to the generation of recombinant viruses containing a mixture of genes 

from two or more different viruses. Chimeric, full-length viruses and truncated 

replicons have been constructed from numerous alphaviruses and flaviviruses. 

For example, alphavirus replicons encoding foreign genes have been used 
240 Biosafety in Microbiological and Biomedical Laboratories

widely as immunogens against bunyavirus, filovirus, arenavirus, and other 

antigens. These replicons have been safe and usually immunogenic in rodent 

hosts leading to their development as candidate human vaccines against several 

virus groups including retroviruses.11-14

Because chimeric viruses contain portions of multiple viruses, the IBC, in 

conjunction with the biosafety officer and the researchers, must conduct a risk 

assessment that, in addition to standard criteria, includes specific elements 

that need to be considered before assigning appropriate biosafety levels and 

containment practices. These elements include: 1) the ability of the chimeric 

virus to replicate in cell culture and animal model systems in comparison with its 

parental strains;15 2) altered virulence characteristics or attenuation compared 

with the parental viruses in animal models;16 3) virulence or attenuation patterns 

by intracranial routes using large doses for agents affecting the CNS;17,18 and 

4) demonstration of lack of reversion to virulence or parental phenotype.

Many patterns of attenuation have been observed with chimeric flaviviruses 

and alphaviruses using the criteria described above. Additionally, some of these 

chimeras are in phase II testing as human vaccines.19

Chimeric viruses may have some safety features not associated with 

parental viruses. For example, they are generated from genetically stable cDNA 

clones without the need for animal or cell culture passage. This minimizes the 

possibility of mutations that could alter virulence properties. Because some 

chimeric strains incorporate genomic segments lacking gene regions or genetic 

elements critical for virulence, there may be limited possibility of laboratory 

recombination to generate strains exhibiting wild-type virulence.

Ongoing surveillance and laboratory studies suggest that many arboviruses 

continue to be a risk to human and animal populations. The attenuation of 

all chimeric strains should be verified using the most rigorouscontainment 

requirements of the parental strains. The local IBC should evaluate containment 

recommendations for each chimeric virus on a case-by-case basis, using 

virulence data from an appropriate animal model. Additional guidance from 

the NIH Office of Biotechnology Activities and/or the Recombinant DNA 

Advisory Committee (RAC) may be necessary.

West Nile Virus (WNV)

WNV has emerged in recent years in temperate regions of Europe and North 

America, presenting a threat to public and animal health. This virus belongs to 

the family Flaviviridae and the genus Flavivirus, Japanese encephalitis virus 

antigenic complex. The complex currently includes Alfuy, Cacipacore, Japanese 

encephalitis, Koutango, Kunjin, Murray Valley encephalitis, St. Louis encephalitis, 
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 241

Rocio, Stratford, Usutu, West Nile, and Yaounde viruses. Flaviviruses share a 

common size (40-60nm), symmetry (enveloped, icosahedral nucleocapsid), 

nucleic acid (positive-sense, single stranded RNA approximately 10,000-11,000 

bases) and virus morphology. The virus was first isolated from a febrile adult 

woman in the West Nile District of Uganda in 1937.20 The ecology was characterized 

in Egypt in the 1950s; equine disease was first noted in Egypt and France in 

the early 1960s.21,22 It first appeared in North America in 1999 as encephalitis 

reported in humans and horses.23 The virus has been detected in Africa, Europe, 

the Middle East, west and central Asia, Oceania (subtype Kunjin virus), and 

most recently, North America.

Occupational Infections

LAI with WNV have been reported in the literature. SALS reported 15 human 

infections from laboratory accidents in 1980. One of these infections was attributed 

to aerosol exposure. Two parenteral inoculations have been reported recently 

during work with animals.24

Natural Modes of Infections

In the United States, infected mosquitoes, primarily members of the Culex genus, 

transmit WNV. Virus amplification occurs during periods of adult mosquito bloodfeeding by continuous transmission between mosquito vectors and bird reservoir 

hosts. People, horses, and most other mammals are not known to develop infectious 

viremias very often, and thus are probably “dead-end” or incidental hosts.

Laboratory Safety and Containment Recommendations

WNV may be present in blood, serum, tissues, and CSF of infected humans, 

birds, mammals, and reptiles. The virus has been found in oral fluids and feces 

of birds. Parenteral inoculation with contaminated materials poses the greatest 

hazard; contact exposure of broken skin is a possible risk. Sharps precautions 

should be strictly adhered to when handling potentially infectious materials. Workers 

performing necropsies on infected animals may be at higher risk of infection.

BSL-2 practices, containment equipment, and facilities are recommended 

for activities with human diagnostic specimens, although it is unusual to recover 

virus from specimens obtained from clinically ill patients. BSL-2 is recommended 

for processing field collected mosquito pools whereas BSL-3 and ABSL-3 practices, 

containment equipment, and facilities are recommended for all manipulations of 

WNV cultures and for experimental animal and vector studies, respectively.

Dissection of field collected dead birds for histopathology and culture is 

recommended at BSL-3 containment due to the potentially high levels of virus 

found in such samples. Non-invasive procedures performed on dead birds (such 

as oropharyngeal or cloacal swabs) can be conducted at BSL-2.
242 Biosafety in Microbiological and Biomedical Laboratories

Special Issues

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Eastern Equine Encephalitis (EEE) Virus, Venezuelan Equine Encephalitis 

(VEE) Virus, and Western Equine Encephalitis (WEE) Virus

VEE, EEE, and WEE viruses are members of the genus Alphavirus in the family 

Togaviridae. They are small, enveloped viruses with a genome consisting of a 

single strand of positive-sense RNA. All three viruses can cause encephalitis 

often accompanied by long-term neurological sequelae. Incubation period 

ranges from 1-10 days and the duration of acute illness is typically days to 

weeks depending upon severity of illness. Although not the natural route of 

transmission, the viruses are highly infectious by the aerosol route; laboratory 

acquired infections have been documented.25

Occupational Infections

These alphaviruses, especially VEE virus, are infectious by aerosol in laboratory 

studies and more than 160 EEE virus, VEE virus, or WEE virus laboratory-acquired 

infections have been documented. Many infections were due to procedures 

involving high virus concentrations and aerosol-generating activities such as 

centrifugation and mouth pipetting. Procedures involving animals (e.g., infection 

of newly hatched chicks with EEE virus and WEE virus) and mosquitoes also 

are particularly hazardous.

Natural Modes of Infection

Alphaviruses are zoonoses maintained and amplified in natural transmission 

cycles involving a variety of mosquito species and either small rodents or birds. 

Humans and equines are accidental hosts with naturally acquired alphavirus 

infections resulting from the bites of infected mosquitoes.

EEE virus occurs in focal locations along the eastern seaboard, the Gulf 

Coast and some inland Midwestern locations of the United States, in Canada, 

some Caribbean Islands, and Central and South America.26 Small outbreaks of 

human disease have occurred in the United States, the Dominican Republic, 

Cuba, and Jamaica. In the United States, equine epizootics are common 

occurrences during the summer in coastal regions bordering the Atlantic and 

Gulf of Mexico, in other eastern and Midwestern states, and as far north as 

Quebec, Ontario, and Alberta in Canada.
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 243

In Central and South America, focal outbreaks due to VEE virus occur 

periodically with rare large regional epizootics involving thousands of equine 

cases and deaths in predominantly rural settings. These epizootic/epidemic 

viruses are theorized to emerge periodically from mutations occurring in the 

continuously circulating enzootic VEE viruses in northern South America. The 

classical epizootic varieties of the virus are not present in the United States. An 

enzootic subtype, Everglades virus (VEE antigenic complex subtype II virus), 

exists naturally in southern Florida, while endemic foci of Bijou Bridge virus 

(VEE antigenic complex subtype III-B virus), have been described in the 

western United States.27

The WEE virus is found mainly in western parts of the United States and 

Canada. Sporadic infections also occur in Central and South America.

Laboratory Safety and Containment Recommendations

Alphaviruses may be present in blood, CSF, other tissues (e.g., brain), or throat 

washings. The primary laboratory hazards are parenteral inoculation, contact of 

the virus with broken skin or mucus membranes, bites of infected animals or 

arthropods, or aerosol inhalation.

Diagnostic and research activities involving clinical material, infectious 

cultures, and infected animals or arthropods should be performed under BSL-3 

practices, containment equipment, and facilities. Due to the high risk of aerosol 

infection, additional personal protective equipment, including respiratory protection, 

should be considered for non-immune personnel. Animal work with VEE virus, 

EEE virus and WEE virus should be performed under ABSL-3 conditions. HEPA 

filtration is required on the exhaust system of laboratory and animal facilities 

using VEE virus.

Special Issues

Vaccines Two strains of VEE virus (TC-83 and V3526) are highly attenuated in 

vertebrate studies and have been either exempted (strain TC-83) or excluded 

(strain V3526) from select agent regulations. Because of the low level of 

pathogenicity, these strains may be safely handled under BSL-2 conditions 

without vaccination or additional personal protective equipment.

Investigational vaccine protocols have been developed to immunize at-risk 

laboratory or field personnel against these alphaviruses, however, the vaccines 

are available only on a limited basis and may be contraindicated for some 

personnel. Therefore, additional personal protective equipment may be warranted 

in lieu of vaccination. For personnel who have no neutralizing antibody titer 

(either by previous vaccination or natural infection), additional respiratory 

protection is recommended for all procedures.
244 Biosafety in Microbiological and Biomedical Laboratories

Select Agent VEE virus and EEE virus are select agents requiring registration 

with CDC and/or USDA for possession, use, storage and/or transfer. See 

Appendix F for additional information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS.

Rift Valley Fever Virus (RVFV)

RVFV was first isolated in Kenya in 1936 and subsequently shown to be 

endemically present in almost all areas of sub-Saharan Africa.28 In periods of 

heavy rainfall, large epizootics occur involving primarily sheep, cattle, and human 

disease, although many other species are infected. The primordial vertebrate 

reservoir is unknown, but the introduction of large herds of highly susceptible 

domestic breeds in the last few decades has provided a substrate for massive 

virus amplification. The virus has been introduced into Egypt, Saudi Arabia, and 

Yemen and caused epizootics and epidemics in those countries. The largest of 

these was in 1977 to 1979 in Egypt with many thousands of human cases and 

610 reported deaths.29

Most human infections are symptomatic and the most common syndrome 

consists of fever, myalgia, malaise, anorexia, and other non-specific symptoms. 

Recovery within one to two weeks is usual but hemorrhagic fever, encephalitis, or 

retinitis also occurs. Hemorrhagic fever develops as the primary illness proceeds 

and is characterized by disseminated intravascular coagulation and hepatitis. 

Perhaps 2% of cases will develop this complication and the mortality is high. 

Encephalitis follows an apparent recovery in <1% of cases and results in a 

substantial mortality and sequelae. Retinal vasculitis occurs in convalescence of 

a substantial but not precisely known proportion of cases. The retinal lesions are 

often macular and permanent, leading to substantial loss of visual acuity.

Infected sheep and cattle suffer a mortality rate of 10-35%, and spontaneous 

abortion occurs virtually in all pregnant females. Other animals studied have 

lower viremia and lesser mortality but may abort. This virus is an OIE List A 

disease and triggers export sanctions.

Occupational Infections

The potential for infection of humans by routes other than arthropod transmission 

was first recognized in veterinarians performing necropsies. Subsequently, it 

became apparent that contact with infected animal tissues and infectious aerosols 

were dangerous; many infections were documented in herders, slaughterhouse 

workers, and veterinarians. Most of these infections resulted from exposure to 

blood and other tissues including aborted fetal tissues of sick animals.
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 245

There have been 47 reported laboratory infections; before modern containment 

and vaccination became available virtually every laboratory that began work with 

the virus suffered infections suggestive of aerosol transmission.30,31

Natural Modes of Infection

Field studies show RVFV to be transmitted predominantly by mosquitoes, although 

other arthropods may be infected and transmit. Mechanical transmission also has 

been documented in the laboratory. Floodwater Aedes species are the primary 

vector and transovarial transmission is an important part of the maintenance 

cycle.32 However, many different mosquito species are implicated in horizontal 

transmission in field studies, and laboratory studies have shown a large number 

of mosquito species worldwide to be competent vectors, including North 

American mosquitoes.

It is currently believed that the virus passes dry seasons in the ova of 

flood-water Aedes mosquitoes. Rain allows infectious mosquitoes to emerge 

and feed on vertebrates. Several mosquito species can be responsible for 

horizontal spread, particularly in epizootic/epidemic situations. The vertebrate 

amplifiers are usually sheep and cattle, with two caveats; as yet undefined 

native African vertebrate amplifier is thought to exist and very high viremias 

in humans are thought to play some role in viral amplifications.33

Transmission of diseases occurs between infected animals but is of low 

efficiency and virus titers in throat swabs are low. Nosocomial infection rarely 

if ever occurs. There are no examples of latency with RVFV, although virus 

may be isolated from lymphoid organs of mice and sheep for four to six weeks 

post-infection.

Laboratory Safety and Containment Recommendations

Concentrations of RVFV in blood and tissues of sick animals are often very high. 

Placenta, amniotic fluid, and fetuses from aborted domestic animals are highly 

infectious. Large numbers of infectious virus also are generated in cell cultures 

and laboratory animals.

BSL-3 practices, containment equipment and facilities are recommended for 

processing human or animal material in endemic zones or in non-endemic areas 

in emergency circumstances. Particular care should be given to stringent aerosol 

containment practices, autoclaving waste, decontamination of work areas, and 

control of egress of material from the laboratory. Other cultures, cells, or similar 

biological material that could potentially harbor RVFV should not be used in a 

RVFV laboratory and subsequently removed.

Diagnostic or research studies outside endemic areas should be performed 

in a BSL-3 laboratory. Personnel also must have additional respiratory protection 

(such as a PAPR) or be vaccinated for RVFV. In addition, the USDA may require 
246 Biosafety in Microbiological and Biomedical Laboratories

full BSL-3-Ag containment for research conducted in non-endemic areas in 

loose-housed animals. (See Appendix D.)

Special Issues

Vaccines Two apparently effective vaccines have been developed by the 

Department of Defense (DoD) and have been used in volunteers, laboratory 

staff, and field workers under investigational protocols, but neither vaccine is 

available at this time.

Select Agent RVFV is a select agent requiring registration with CDC and/or 

USDA for possession, use, storage and/or transfer. See Appendix F for 

additional information.

The live-attenuated MP-12 vaccine strain is specifically exempted from the 

Select Agent rules. In general, BSL-2 containment is recommended for working 

with this strain.

The USDA may require enhanced ABSL-3, ABSL-3, or BSL-3-Ag facilities 

and practices for working with RVFV in the United States. (See Appendix D.) 

Investigators should contact the USDA for further guidance before initiating research.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS.

Table 6. Alphabetic Listing of 597 Arboviruses and Hemorrhagic 

Fever Viruses*

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Abras ABRV Orthobunvavarus 2 A7 Patois No

Absettarov ABSV Flavivirus 4 A4 Bf Yes

Abu Hammad AHV Nairovirus 2 S Dera Ghazi

Khan No

Acado ACDV Orbivirus 2 S Corriparta No

Acara ACAV Orthobunyavirus 2 S Capim No

Adelaide River ARV Lyssavirus 2 IE Bovine Ephemeral Fever No

African Horse 

sickness AHSV Orbivirus 3c A1 African

Horsesickness Yes

African Swine

Fever ASFV Asfivirus 3c IE Asfivirus Yes
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 247

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Aguacate AGUV Phlebovirus 2 S Phlebotomus

Fever No

Aino AINOV Orthobunyavirus 2 S Simbu No

Akabane AKAV Orthobunyavirus 3c S Simbu Yes

Alenquer ALEV Phlebovirus 2 IE Phlebotomus

Fever No

Alfuy ALFV Flavivirus 2 S Bf No

Alkhumra ALKV Flavivirus 4 A4 Bf Yes

Allpahuayo ALLPV Arenavirus 3 IE Tacaribe No

Almeirim ALMV Orbivirus 2 IE Changuinola No

Almpiwar ALMV Rhabdoviridae 2 S No

Altamira ALTV Orbivirus 2 IE Changuinola No

Amapari AMAV Arenavirus 2 A5 Tacaribe No

Ambe AMBEV Phlebovirus 2 IE No

Ananindeua ANUV Orthobunyavirus 2 A7 Guama No

Andasibe ANDV Orbivirus 2 A7 No

Andes ANDV Hantavirus 3a IE Hantaan No

Anhanga ANHV Phlebovirus 2 S Phlebotomus

Fever No

Anhembi AMBV Orthobunyavirus 2 S Bunyamwera No

Anopheles A ANAV Orthobunyavirus 2 S Anopheles A No

Anopheles B ANBV Orthobunyavirus 2 S Anopheles B No

Antequera ANTV Bunyaviridae 2 IE Resistencia No

Apeu APEUV Orthobunyavirus 2 S Cf No

Apoi APOIV Flavivirus 2 S Bf No

Araguari ARAV Unassigned 3 IE No

Aransas Bay ABV Bunyaviridae 2 IE UPOLU No

Arbia ARBV Phlebovirus 2 IE Phlebotomus

Fever No

Arboledas ADSV Phlebovirus 2 A7 Phlebotomus

Fever No

Aride ARIV Unassigned 2 S No

Ariquemes ARQV Phlebovirus 2 A7 Phlebotomus

Fever No

Arkonam ARKV Orbivirus 2 S leri No

Armero ARMV Phlebovirus 2 A7 Phlebotomus

Fever No

Aroa AROAV Flavivirus 2 S Bf No

Aruac ARUV Rhabdoviridae 2 S No
248 Biosafety in Microbiological and Biomedical Laboratories

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Arumateua ARMTV Orthobunyavirus 2 A7 No

Arumowot AMTV Phlebovirus 2 S Phlebotomus

Fever No

Aura AURAV Alphavirus 2 S Af No

Avalon AVAV Nairovirus 2 S Sakhalin No

Babahoyo BABV Orthobunyavirus 2 A7 Patois No

Babanki BBKV Alphavirus 2 A7 Af No

Bagaza BAGV Flavivirus 2 S Bf No

Bahig BAHV Orthobunyavirus 2 S Tete No

Bakau BAKV Orthobunyavirus 2 S Bakau No

Baku BAKUV Orbivirus 2 S Kemerovo No

Bandia BDAV Nairovirus 2 S Qalyub No

Bangoran BGNV Rhabdoviridae 2 S No

Bangui BGIV Bunyaviridae 2 S No

Banzi BANV Flavivirus 2 S Bf No

Barmah Forest BFV Alphavirus 2 A7 Af No

Barranqueras BQSV Bunyaviridae 2 IE Resistencia No

Barur BARV Rhabdoviridae 2 S Kern Canyon No

Batai BATV Orthobunyavirus 2 S Bunyamwera No

Batama BMAV Orthobunyavirus 2 A7 Tete No

Batken BKNV Thogotovirus 2 IE No

Bauline BAUV Orbivirus 2 S Kemerovo No

Bear Canyon BRCV Arenavirus 3 A7 No

Bebaru BEBV Alphavirus 2 S Af No

Belem BLMV Bunyaviridae 2 IE No

Belmont ELV Bunyaviridae 2 S No

Belterra BELTV Phlebovirus 2 A7 Phlebotomus

Fever No

Benevides BENV Orthobunyavirus 2 A7 Capim No

Benfica BENV Orthobunyavirus 2 A7 Capim No

Bermejo BMJV Hantavirus 3 IE Hantaan No

Berrimah BRMV Lyssavirus 2 IE

Bovine Ephemeral

Fever

No

Beritoga BERV Orthobunyavirus 2 S Guama No

Bhanja BHAV Bunyaviridae 3 S Bhanja No

Bimbo BBOV Rhabdoviridae 2 IE No
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 249

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Bimitti BIMV Orthobunyavirus 2 S Guama No

Birao BIRV Orthobunyavirus 2 S Bunyamwera No

Bluetoungue

(exotic serotypes) BTV Orbivirus 3c S Bluetongue No

Bluetoungue

(non-exotic) BTV Orbivirus 2c S Bluetongue No

Bobaya BOBV Bunyaviridae 2 IE No

Bobia BIAV Orthobunyavirus 2 IE Olifantsylei No

Boraceia BORV Orthobunyavirus 2 S Anopheles B No

Botambi BOTV Orthobunyavirus 2 S Olifantsylei No

Boteke BTKV Vesiculovirus 2 S Vesicular

Stomatitis No

Bouboui BOUV Flavivirus 2 S Bf No

Bovine

Ephemeral Fever BEFV Lyssavirus 3c A1

Bovine Ephemeral

Fever

No

Bozo BOZOV Orthobunyavirus 2 A7 Bunyamwera No

Breu Branco BRBV Orbivirus 2 A7 No

Buenaventura BUEV Phlebovirus 2 IE Phlebotomus

Fever No

Bujaru BUJV Phlebovirus 2 S Phlebotomus

Fever No

Bunyamwera BUNV Orthobunyavirus 2 S Bunyamwera No

Bunyip Creek BCV Orbivirus 2 S Palyam No

Burg El Arab BEAV Rhabdoviridae 2 S Matariva No

Bushbush BSBV Orthobunyavirus 2 S Capim No

Bussuquara BSQV Flavivirus 2 S Bf No

Buttonwillow BUTV Orthobunyavirus 2 S Simbu No

Bwamba BWAV Orthobunyavirus 2 S Bwamba No

Cabassou CABV Alphavirus 3 IE Af Yes

Cacao CACV Phlebovirus 2 S Phlebotomus

Fever No

Cache Valley CVV Orthobunyavirus 2 S Bunyamwera No

Cacipacore CPCV Flavivirus 2 IE Bf No

Caimito CAIV Phlebovirus 2 S Phlebotomus

Fever No

Calchaqui CQIV Vesiculovirus 2 A7 Vesicular

Stomatitis No

California

Encephalitis CEV Orthobunyavirus 2 S California No

Calovo CVOV Orthobunyavirus 2 S Bunyamwera No

Cananeia CNAV Orthobunyavirus 2 IE GUAMA No
250 Biosafety in Microbiological and Biomedical Laboratories

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Candiru CDUV Phlebovirus 2 S Phlebotomus

Fever No

Caninde CANV Orbivirus 2 IE Changuinola No

Cano Delgadito CADV Hantavirus 3a IE Hantaan No

Cape Wrath CWV Orbivirus 2 S Kemerovo No

Capim CAPV Orthobunyavirus 2 S Capim No

Caraipe CRPV Orthobunyavirus 2 A7 No

Carajas CRJV Vesiculovirus 2 A7 Vesicular

Stomatitis No

Caraparu CARV Orthobunyavirus 2 S Cf No

Carey Island CIV Flavivirus 2 S Bf No

Catu CATUV Orthobunyavirus 2 S Guama No

Chaco CHOV Rhabdoviridae 2 S Timbo No

Chagres CHGV Phlebovirus 2 S Phlebotomus

Fever No

Chandipura CHPV Vesiculovirus 2 S Vesicular

Stomatitis No

Changuinola CGLV Orbivirus 2 S Changuinola No

Charleville CHVV Lyssavirus 2 S Rab No

Chenuda CNUV Orbivirus 2 S Kmerovo No

Chikungunya CHIKV Alphavirus 3 S Af Yes

Chilibre CHIV Phlebovirus 2 S Phlebotomus

Fever No

Chim CHIMV Bunyaviridae 2 IE No

Chobar Gorge CGV Orbivirus 2 S Chobar Gorge No

Clo Mor CMV Nairovirus 2 S Sakhalin No

Coastal Plains CPV Lyssavirus 2 IE Tibrogargan No

Cocal COCV Vesiculovirus 2 A3 Vesicular

Stomatitis No

Codajas CDJV Orbivirus 2 A7 No

Colorado Tick

Fever CTFV Coltivirus 2 S Colorado Tick

Fever No

Congo-Crimean

Hemorrhagic

Fever

CCHFV Nairovirus 4 A6 CCHF Yes

Connecticut CNTV Rhabdoviridae 2 IE Sawgrass No

Corfou CFUV Phlebovirus 2 A7 Phlebotomus

Fever No

Corriparta CORV Orbivirus 2 S Corriaparta No

Cotia CPV Poxviridae 2 S No

Cowbone Ridge CRV Flavivirus 2 S Bf No
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 251

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Csiro Village CVGV Orbivirus 2 S Palyam No

Cuiaba CUIV Rhabdoviridae 2 S No

Curionopolis CRNPV Rhabdoviridae 2 A7 No

Dabakala DABV Orthobunyavirus 2 A7 Olifantsylei No

D’Aguilar DAGV Orbivirus 2 S Palyam No

Dakar Bat Virus DBV Flavivirus 2 S Bf No

Deer Tick Virus DRTV Flavivirus 3 A7 No

Dengue Virus

Type 1 DENV-1 Flavivirus 2 S Bf No

Dengue Virus

Type 2 DENV-2 Flavivirus 2 S Bf No

Dengue Virus

Type 3 DENV-3 Flavivirus 2 S Bf No

Dengue Virus

Type 4 DENV-4 Flavivirus 2 S Bf No

Dera Ghazi Khan DGKV Nairovirus 2 S Dera Ghazi 

Khan No

DobravaBelgrade DOBV Hantavirus 3a IE No

Dhori DHOV Orthomyxoviridae 2 S No

Douglas DOUV Orthobunyavirus 3 IE Simbu No

Durania DURV Phlebovirus 2 A7 Phlebotomus

Fever No

Dugbe DUGV Nairovirus 3 S Nairobi Sheep

Disease No

Eastern Equine

Encephalitis EEEV Alphavirus 3c S Af No

Ebola (Including

Reston) EBOV Filovirus 4 S EBO Yes

Edge Hill EHV Flavivirus 2 S Bf No

Enseada ENSV Bunyaviridae 3 IE No

Entebbe Bat ENTV Flavivirus 2 S Bf No

Epizootic

Hemorrhagic

Disease

EHDV Orbivirus 2 S

Epizootic

Hemorrhagic

Disease

No

Erve ERVEV Bunyaviridae 2 S Thiafora No

Estero Real ERV Orthobunyavirus 2 IE Patois No

Eubenangee EUBV Orbivirus 2 S Eubenangee No

Everglades EVEV Alphavirus 3 S Af Yes

Eyach EYAV Coltivirus 2 S Colorado Tick

Fever No

Farmington FRMV Vesiculovirus 2 A7 No

Flanders FLAV Rhabdoviridae 2 S Hart Park No
252 Biosafety in Microbiological and Biomedical Laboratories

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Flexal FLEV Arenavirus 3 S Tacaribe No

Fomede FV Orbivirus 2 A7 Chobar Gorge No

Forecariah FORV Bunyaviridae 2 A7 Bhanja No

Fort Morgan FMV Alphavirus 2 S Af No

Fort Sherman FSV Orthobunyavirus 2 A7 Bunyamwera No

Frijoles FRIV Phlebovirus 2 S Phlebotomus

Fever No

Gabek Forest GFV Phlebovirus 2 A7 Phlebotomus

Fever No

Gadgets Gully GGYV Flavivirus 2 IE Bf No

Gamboa GAMV Orthobunyavirus 2 S Gamboa No

Gan Gan GGV Bunyaviridae 2 A7 Mapputta No

Garba GARV Rhabdoviridae 2 IE Matariva No

Garissa GRSV Orthobunyavirus 3 A7 Bunyamwera No

Germiston GERV Orthobunyavirus 3 Bunyamwera Yes

Getah GETV Alphavirus 2 A1 Af No

Gomoka GOMV Orbivirus 2 S Ieri No

Gordil GORV Phlebovirus 2 IE Phlebotomus

Fever No

Gossas GOSV Rhabdoviridae 2 S No

Grand Arbaud GAV Phlebovirus 2 S Uukuniemi No

Gray Lodge GLOV Vesiculovirus 2 IE Vesicular

Stomatitis No

Great Island GIV Orbivirus 2 S Kemerovo No

Guajara GJAV Orthobunyavirus 2 S Capim No

Guama GMAV Orthobunyavirus 2 S Guama No

Guanarito GTOV Arenavirus 4 A4 Tacaribe Yes

Guaratuba GTBV Orthobunyavirus 2 A7 Guama No

Guaroa GROV Orthobunyavirus 2 S California No

Gumbo Limbo GLV Orthobunyavirus 2 S Cf No

Gurupi GURV Orbivirus 2 IE Changuinola No

Hantaan HTNV Hantavirus 3a S Hantaan No

Hanzalova HANV Flavivirus 4 A4 Bf Yes

Hart Park HPV Rhabdoviridae 2 S Hart Park No

Hazara HAZV Nairovirus 2 S CHF-Congo No

Highlands J HJV Alphavirus 2 S Af No

Huacho HUAV Orbivirus 2 S Kemerovo No
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 253

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Hughes HUGV Nairovirus 2 S Hughes No

Hypr HYPRV Flavivirus 4 S Bf Yes

Iaco IACOV Orthobunyavirus 2 IE Bunyamwera No

Ibaraki IBAV Orbivirus 2 IE

Epizootic

Hemorrhagic

Disease

Yes

Icoaraci ICOV Phlebovirus 2 S Phlebotomus

Fever No

Ieri IERIV Orbivirus 2 S Ieri No

Ife IFEV Orbivirus b 2 IE No

Iguape IGUV Flavivirus 2 A7 Bf No

Ilesha ILEV Orthobunyavirus 2 S Bunyamwera No

Ilheus ILHV Flavivirus 2 S Bf No

Ingwavuma INGV Orthobunyavirus 2 S Simbu No

Inhangapi INHV Rhabdoviridae 2 IE No

Inini INIV Orthobunyavirus 2 IE Simbu No

Inkoo INKV Orthobunyavirus 2 S California No

Ippy IPPYV Arenavirus 2 S Tacaribe No

Iriri IRRV Rhabdoviridae 2 A7 No

Irituia IRIV Orbivirus 2 S Changuinola No

Isfahan ISFV Vesiculovirus 2 S Vesicular

Stomatitis No

Israel Turkey

Meningitis ITV Flavivirus 2 with 3 

practices S Bf No

Issyk-Kul ISKV Bunyaviridae 3 IE No

Itacaiunas ITCNV Rhabdoviridae 2 A7 No

Itaituba ITAV Phlebovirus 2 IE Phlebotomus

Fever No

Itaporanga ITPV Phlebovirus 2 S Phlebotomus

Fever No

Itaqui ITQV Orthobunyavirus 2 S Cf No

Itimirim ITIV Orthobunyavirus 2 IE Guama No

Itupiranga ITUV Orbivirus b 2 IE No

Ixcanal IXCV Phlebovirus 2 A7 Phlebotomus

Fever No

Jacareacanga JACV Orbivirus 2 IE Corriparta No

Jacunda JCNV Phlebovirus 2 A7 Phlebotomus

Fever No

Jamanxi JAMV Orbivirus 2 IE Changuinola No

Jamestown

Canyon JCV Orthobunyavirus 2 S California No
254 Biosafety in Microbiological and Biomedical Laboratories

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Japanaut JAPV Orbivirus b 2 S No

Japanese

Encephalitis JEV Flavivirus 3c S Bf No

Jari JARIV Orbivirus 2 IE Changuinola No

Jatobal JTBV Orthobunyavirus 2 A7 No

Jerry Slough JSV Orthobunyavirus 2 S California No

Joa JOAV Phlebovirus 2 A7 No

Johnston Atoll JAV Unassigned 2 S Quaranfil No

Joinjakaka JOIV Rhabdoviridae 2 S No

Juan Diaz JDV Orthobunyavirus 2 S Capim No

Jugra JUGV Flavivirus 2 S Bf No

Junin JUNV Arenavirus 4 A6 Tacaribe Yes

Jurona JURV Vesiculovirus 2 S Vesicular

Stomatitis No

Juruaca JRCV Picornavirus b 2 A7 No

Jutiapa JUTV Flavivirus 2 S Bf No

Kadam KADV Flavivirus 2 S Bf No

Kaeng Khoi KKV Orthobunyavirus b 2 S No

Kaikalur KAIV Orthobunyavirus 2 S Simbu No

Kairi KRIV Orthobunyavirus 2 A1 Bunyamwera No

Kaisodi KSOV Bunyaviridae 2 S Kaisodi No

Kamese KAMV Rhabdoviridae 2 S Hart Park No

Kamiti River KRV Flavivirus 2 A7 No

Kammavanpettai KMPV Orbivirus 2 S No

Kannamangalam KANV Rhabdoviridae 2 S No

Kao Shuan KSV Nairovirus 2 S Dera Ghazi 

Khan No

Karimabad KARV Phlebovirus 2 S Phlebotomus

Fever No

Karshi KSIV Flavivirus 2 S Bf No

Kasba KASV Orbivirus 2 S Palyam No

Kedougou KEDV Flavivirus 2 A7 Bf No

Kemerovo KEMV Orbivirus 2 S Kemerovo No

Kern Canyon KCV Rhabdoviridae 2 S Kern Canyon No

Ketapang KETV Orthobunyavirus 2 S Bakau No

Keterah KTRV Bunyaviridae 2 S No

Keuraliba KEUV Rhabdoviridae 2 S Le Dantec No
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 255

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Keystone KEYV Orthobunyavirus 2 S California No

Khabarovsk KHAV Hantavirus 3a IE Hantaan No

Khasan KHAV Nairovirus 2 IE CCHF No

Kimberley KIMV Lyssavirus 2 A7

Bovine Ephemeral

Fever

No

Kindia KINV Orbivirus 2 A7 Palyam No

Kismayo KISV Bunyaviridae 2 S Bhanja No

Klamath KLAV Vesiculovirus 2 S Vesicular

Stomatitis No

Kokobera KOKV Flavivirus 2 S Bf No

Kolongo KOLV Lyssavirus 2 S Rab No

Koongol KOOV Orthobunyavirus 2 S Koongol No

Kotonkan KOTV Lyssavirus 2 S Rab No

Koutango KOUV Flavivirus 3 S Bf No

Kowanyama KOWV Bunyaviridae 2 S No

Kumlinge KUMV Flavivirus 4 A4 Bf Yes

Kunjin KUNV Flavivirus 2 S Bf No

Kununurra KNAV Rhabdoviridae 2 S No

Kwatta KWAV Vesiculovirus 2 S Vesicular

Stomatitis No

Kyasanur Forest

Disease KFDV Flavivirus 4 S Bf Yes

Kyzylagach KYZV Alphavirus 2 IE Af No

La Crosse LACV Orthobunyavirus 2 S California No

Lagos Bat LBV Lyssavirus 2 S Rab No

Laguna Negra LANV Hantavirus 3a IE No

La Joya LJV Vesiculovirus 2 S Vesicular

Stomatitis No

Lake Clarendon LCV Orbivirus b 2 IE No

Landjia LJAV Rhabdoviridae 2 S No

Langat LGTV Flavivirus 2 S Bf No

Lanjan LJNV Bunyaviridae 2 S Kaisodi No

Las Maloyas LMV Orthobunyavirus 2 A7 Anopheles A No

Lassa LASV Arenavirus 4 S Tacaribe Yes

Latino LATV Arenavirus 2 A5 Tacaribe No

Lebombo LEBV Orbivirus 2 S No

Lechiguanas LECHV Hantavirus 3a IE Hantaan No
256 Biosafety in Microbiological and Biomedical Laboratories

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Le Dantec LDV Rhabdoviridae 2 S Le Dantec No

Lednice LEDV Orthobunyavirus 2 A7 Turlock No

Lipovnik LIPV Orbivirus 2 S Kemerovo No

Llano Seco LLSV Orbivirus 2 IE Umatilla No

Lokern LOKV Orthobunyavirus 2 S Bunyamwera No

Lone Star LSV Bunyaviridae 2 S No

Louping Ill LIV Flavivirus 3c S Bf Yes

Lukuni LUKV Orthobunyavirus 2 S Anopheles A No

Macaua MCAV Orthobunyavirus 2 IE Bunyamwera No

Machupo MACV Arenavirus 4 S Tacaribe Yes

Madrid MADV Orthobunyavirus 2 S Cf No

Maguari MAGV Orthobunyavirus 2 S Bunyamwera No

Mahogany

Hammock MHV Orthobunyavirus 2 S Guama No

Main Drain MDV Orthobunyavirus 2 S Bunyamwera No

Malakal MALV Lyssavirus 2 S Bovine Ephemeral No

Manawa MWAV Phlebovirus 2 S Uukumiemi No

Manitoba MNTBV Rhabdoviridae 2 A7 No

Manzanilla MANV Orthobunyavirus 2 S Simbu No

Mapputta MAPV Bunyaviridae 2 S Mapputta No

Maporal MPRLV Hantavirus 3a IE Hantaan No

Maprik MPKV Bunyaviridae 2 S Mapputta No

Maraba MARAV Vesiculovirus 2 A7 No

Marajo MRJV Unassigned 2 IE No

Marburg MARV Filovirus 4 S Marburg Yes

Marco MCOV Rhabdoviridae 2 S No

Mariquita MRQV Phlebovirus 2 A7 Phlebotomus

Fever No

Marituba MTBV Orthobunyavirus 2 S Cf No

Marrakai MARV Orbivirus 2 S Palyam No

Matariya MTYV Rhabdoviridae 2 S Matariva No

Matruh MTRV Orthobunyavirus 2 S Tete No

Matucare MATV Orbivirus 2 S No

Mayaro MAYV Alphavirus 2 S Af No

Mboke MBOV Orthobunyavirus 2 A7 Bunyamwera No

Meaban MEAV Flavivirus 2 IE Bf No
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 257

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Melao MELV Orthobunyavirus 2 S California No

Mermet MERV Orthobunyavirus 2 S Simbu No

Middelburg MIDV Alphavirus 2 A1 Af No

Minatitlan MNTV Orthobunyavirus 2 S Minatitlan No

Minnal MINV Orbivirus 2 S Umatilla No

Mirim MIRV Orthobunyavirus 2 S Guama No

Mitchell River MRV Orbivirus 2 S No

Mobala MOBV Arenavirus 3 A7 Tacaribe No

Modoc MODV Flavivirus 2 S Bf No

Moju MOJUV Orthobunyavirus 2 S Guama No

Mojui Dos

Campos MDCV Orthobunyavirus 2 IE No

Mono Lake MLV Orbivirus 2 S Kemerovo No

Mont. Myotis

Leukemia MMLV Flavivirus 2 S Bf No

Monte Dourado MDOV Orbivirus 2 IE Changuinola No

Mopeia MOPV Arenavirus 3 A7 No

Moriche MORV Orthobunyavirus 2 S Capim No

Morro Bay MBV Orthobunyavrius 2 IE California No

Morumbi MRMBV Phlebovirus 2 A7 Phlebotomus

Fever No

Mosqueiro MQOV Rhabdoviridae 2 A7 Hart Park No

Mossuril MOSV Rhabdoviridae 2 S Hart Park No

Mount Elgon Bat MEBV Vesiculovirus 2 S Vesicular

Stomatitis No

M’Poko MPOV Orthobunyavirus 2 S Turlock No

Mucambo MUCV Alphavirus 3 S Af Yes

Mucura MCRV Phlebovirus 2 A7 Phlebotomus

Fever No

Munguba MUNV Phlebovirus 2 IE Phlebotomus

Fever No

Murray Valley

Encephalitis MVEV Flavivirus 3 S Bf No

Murutucu MURV Orthobunyavirus 2 S Cf No

Mykines MYKV Orbivirus 2 A7 Kemerovo No

Nairobi Sheep

Disease NSDV Nairovirus 3c A1 Nairobi Sheep

Disease No

Naranjal NJLV Flavivirus 2 IE Bf No

Nariva NARV Paramyxoviridae 2 IE No

Nasoule NASV Lyssavirus 2 A7 Rab No
258 Biosafety in Microbiological and Biomedical Laboratories

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Navarro NAVV Rhabdoviridae 2 S No

Ndelle NDEV Orthoreovirus 2 A7 Ndelle No

Ndumu NDUV Alphavirus 2 A1 Af No

Negishi NEGV Flavivirus 3 S Bf No

Nepuyo NEPV Orthobunyavirus 2 S Cf No

Netivot NETV Orbivirus 2 A7 No

New Minto NMV Rhabdoviridae 2 IE Sawgrass No

Ngaingan NGAV Lyssavirus 2 S Tibrogargan No

Ngari d NRIV Orthobunyavirus 3 A7 Bunyamera No

Ngoupe NGOV Orbivirus 2 A7 Eubenangee No

Nique NIQV Phlebovirus 2 S Phlebotomus

Fever No

Nkolbisson NKOV Rhabdoviridae 2 S Kern Canyon No

Nodamura NOV Alphanodavirus 2 IE No

Nola NOLAV Orthobunyavirus 2 S Bakau No

Northway NORV Orthobunyavirus 2 IE Bunyamwera No

Ntaya NTAV Flavivirus 2 S Bf No

Nugget NUGV Orbivirus 2 S Kemerovo No

Nyamanini NYMV Unassigned 2 S Nyamanini No

Nyando NDV Orthobunyavirus 2 S Nyando No

Oak Vale OVV Rhabdoviridae 2 A7 No

Odrenisrou ODRV Phlebovirus 2 A7 Phlebotomus

Fever No

Okhotskiy OKHV Orbivirus 2 S Kemerovo No

Okola OKOV Bunyaviridae 2 S Tanga No

Olifantsvlei OLIV Orthobunyavirus 2 S Olifantsylei No

Omo OMOV Nairovirus 2 A7 Qalyub No

Omsk

Hemorrhagic OHFV Flavivirus 4 S Bf Yes

O’Nyong-Nyong ONNV Alphavirus 2 S Af Yes

Oran ORANV Hantavirus 3a IE Hantaan No

Oriboca ORIV Orthobunyavirus 2 S Cf No

Oriximina ORXV Phlebovirus 2 IE Phlebotomus

Fever No

Oropouche OROV Orthobunyavirus 3 S Simbu Yes

Orungo ORUV Orbivirus 2 S Orungo No

Ossa OSSAV Orthobunyavirus 2 S Cf No
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 259

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Ouango OUAV Rhabdoviridae 2 IE No

Oubangui OUBV Poxviridae 2 IE No

Oubi OUBIV Orthobunyavirus 2 A7 Olifantsylei No

Ourem OURV Orbivirus 2 IE Changuinola No

Pacora PCAV Bunyaviridae 2 S No

Pacui PACV Phlebovirus 2 S Phlebotomus

Fever No

Pahayokee PAHV Orthobunyavirus 2 S Patois No

Palma PMAV Bunyaviridae 2 IE Bhanja No

Palestina PLSV Orthobunyavirus 2 IE Minatitlan No

Palyam PALV Orbivirus 2 S Palyam No

Para PARAV Orthobunyavirus 2 IE Simbu No

Paramushir PMRV Nairovirus 2 IE Sakhalin No

Parana PARV Arenavirus 2 A5 Tacaribe No

Paroo River PRV Orbivirus 2 IE No

Pata PATAV Orbivirus 2 S No

Pathum Thani PTHV Nairovirus 2 S Dera Ghazi 

Khan No

Patois PATV Orthobunyavirus 2 S Patois No

Peaton PEAV Orthobunyavirus 2 A1 Simbu No

Pergamino PRGV Hantavirus 3a IE No

Perinet PERV Vesiculovirus 2 A7 Vesicular 

Stomatitis No

Petevo PETV Orbivirus 2 A7 Palyam No

Phnom-Penh Bat PPBV Flavivirus 2 S Bf No

Pichinde PICV Arenavirus 2 A5 Tacaribe No

Picola PIAV Orbivirus 2 IE Wongorr No

Pirital PIRV Arenavirus 3 IE No

Piry PIRYV Vesiculovirus 3 S Vesicular

Stomatitis No

Pixuna PIXV Alphavirus 2 S Af No

Playas PLAV Orthobunyavirus 2 IE Bunyamwera No

Pongola PGAV Orthobunyavirus 2 S Bwamba No

Ponteves PTVV Phlebovirus 2 A7 Uukuniemi No

Potosi POTV Orthobunyavirus 2 IE Bunyamwera No

Powassan POWV Flavivirus 3 S Bf No

Precarious Point PPV Phlebovirus 2 A7 Uukuniemi No
260 Biosafety in Microbiological and Biomedical Laboratories

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Pretoria PREV Nairovirus 2 S Dera Ghazi 

Khan No

Prospect Hill PHV Hantavirus 2 A8 Hantaan No

Puchong PUCV Lyssavirus 2 S

Bovine Ephemeral

ever

No

Pueblo Viejo PVV Orthobunyavirus 2 IE Gamboa No

Punta Salinas PSV Nairovirus 2 S Hughes No

Punta Toro PTV Phlebovirus 2 S Phlebotomus

Fever No

Purus PURV Orbivirus 2 IE Changuinola No

Puumala PUUV Hantavirus 3a IE Hantaan No

Qalyub QYBV Nairovirus 2 S Qalyub No

Quaranfil QRFV Unassigned 2 S Quaranfil No

Radi RADIV Vesiculovirus 2 A7 Vesicular

Stomatitis No

Razdan RAZV Bunyaviridae 2 IE No

Resistencia RTAV Bunyaviridae 2 IE Resistencia No

Restan RESV Orthobunyavirus 2 S Cf No

Rhode Island RHIV Rhabdoviridae 2 A7 No

Rift Valley Fever RVFV Phlebovirus 3c S Phlebotomus

Fever Yes

Rio Bravo RBV Flavivirus 2 S Bf No

Rio Grande RGV Phlebovirus 2 S Phlebotomus

Fever No

Rio Preto RIOPV Unassigned 2 IE No

Rochambeau RBUV Lyssavirus 2 IE Rab No

Rocio ROCV Flavivirus 3 S Bf Yes

Ross River RRV Alphavirus 2 S Af No

Royal Farm RFV Flavivirus 2 S Bf No

Russian

Spring-Summer

Encephalitis

RSSEV Flavivirus 4 S Bf Yes

Saaremaa SAAV Hantavirus 3a IE Hantaan No

Sabia SABV Arenavirus 4 A4 Yes

Sabo SABOV Orthobunyavirus 2 S Simbu No

Saboya SABV Flavivirus 2 S Bf No

Sagiyama SAGV Alphavirus 2 A1 Af No

Saint-Floris SAFV Phlebovirus 2 S Phlebotomus

Fever No

Sakhalin SAKV Nairovirus 2 S Sakhalin No
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 261

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Salanga SGAV Poxviridae 2 IE SGA No

Salehabad SALV Phlebovirus 2 S Phlebotomus

Fever No

Salmon River SAVV Coltivirus 2 IE Colorado Tick

Fever No

Sal Vieja SVV Flavivirus 2 A7 Bf No

San Angelo SAV Orthobunyavirus 2 S California No

Sandfly Fever,

Naples SFNV Phlebovirus 2 S Phlebotomus

Fever No

Sandfly Fever,

Sicilian SFSV Phlebovirus 2 S Phlebotomus

Fever No

Sandjimba SJAV Lyssavirus 2 S Rab No

Sango SANV Orthobunyavirus 2 S Simbu No

San Juan SJV Orthobunyavirus 2 IE Gamboa No

San Perlita SPV Flavivirus 2 A7 Bf No

Santarem STMV Bunyaviridae 2 IE No

Santa Rosa SARV Orthobunyavirus 2 IE Bunyamwera No

Saraca SRAV Orbivirus 2 IE Changuinola No

Sathuperi SATV Orthobunyavirus 2 S Simbu No

Saumarez Reef SREV Flavivirus 2 IE Bf No

Sawgrass SAWV Rhabdoviridae 2 S Sawgrass No

Sebokele SEBV Unassigned 2 S No

Sedlec SEDV Bunyaviridae 2 A7 No

Seletar SELV Orbivirus 2 S Kemerovo No

Sembalam SEMV Unassigned 2 S No

Semliki Forest SFV Alphavirus 3 A2 Af No

Sena Madureira SMV Rhabdoviridae 2 IE Timbo No

Seoul SEOV Hantavirus 3a IE Hantaan No

Sepik SEPV Flavivirus 2 IE Bf No

Serra Do Navio SDNV Orthobunyavirus 2 A7 California No

Serra Norte SRNV Phlebovirus 2 A7 No

Shamonda SHAV Orthobunyavirus 2 S Simbu No

Shark River SRV Orthobunyavirus 2 S Patois No

Shokwe SHOV Orthobunyavirus 2 IE Bunyamwera No

Shuni SHUV Orthobunyavirus 2 S Simbu No

Silverwater SILV Bunyaviridae 2 S Kaisodi No

Simbu SIMV Orthobunyavirus 2 S Simbu No
262 Biosafety in Microbiological and Biomedical Laboratories

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Simian

Hemorrhagic

Fever

SHFV Arterivirus 2 A2

Simian

Hemorrhagic

Fever

No

Sindbis SINV Alphavirus 2 S Af No

Sin Nombre SNV Hantavirus 3a IE Hantaan No

Sixgun City SCV Orbivirus 2 S Kemerovo No

Slovakia SLOV Unassigned 3 IE No

Snowshoe Hare SSHV Orthobunyavirus 2 S California No

Sokoluk SOKV Flavivirus 2 S Bf No

Soldado SOLV Nairovirus 2 S Hughes No

Somone SOMV Unassigned 3 IE Somone No

Sororoca SORV Orthobunyavirus 2 S Bunyamwera No

Spondweni SPOV Flavivirus 2 S Bf No

Sripur SRIV Rhabdoviridae 3 IE No

St. Louis

Encephalitis SLEV Flavivirus 3 S Bf No

Stratford STRV Flavivirus 2 S Bf No

Sunday Canyon SCAV Bunyaviridae 2 S No

Tacaiuma TCMV Orthobunyavirus 2 S Anopheles A No

Tacaribe TCRV Arenavirus 2 A5 Tacaribe No

Taggert TAGV Nairovirus 2 S Sakhalin No

Tahyna TAHV Orthobunyavirus 2 S California No

Tai TAIV Bunyaviridae 2 A7 Bunyamwera No

Tamdy TDYV Bunyaviridae 2 IE No

Tamiami TAMV Arenavirus 2 A5 Tacaribe No

Tanga TANV Bunyaviridae 2 S Tanga No

Tanjong Rabok TRV Orthobunyavirus 2 S Bakau No

Tapara TAPV Phlebovirus 2 A7 No

Tataguine TATV Bunyaviridae 2 S No

Tehran THEV Phlebovirus 2 A7 Phlebotomus

Fever No

Telok Forest TFV Orthobunyavirus 2 IE Bakau No

Tembe TMEV Orbivirus b 2 S No

Tembusu TMUV Flavivirus 2 S Bf No

Tensaw TENV Orthobunyavirus 2 S Bunyamwera No

Termeil TERV Bunyavirus b 2 IE No

Tete TETEV Orthobunyavirus 2 S Tete No
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses 263

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Thiafora TFAV Bunyaviridae 2 A7 Thiafora No

Thimiri THIV Orthobunyavirus 2 S Simbu No

Thogoto THOV Orthomyxoviridae 2 S Thogoto No

Thottapalayam TPMV Hantavirus 2 S Hantaan No

Tibrogargan TIBV Lyssavirus 2 S Tibrogargan No

Tilligerry TILV Orbivirus 2 IE Eubenangee No

Timbo TIMV Rhabdoviridae 2 S Timbo No

Timboteua TBTV Orthobunyavirus 2 A7 Guama No

Tinaroo TINV Orthobunyavirus 2 IE Simbu No

Tindholmur TDMV Orbivirus 2 A7 Kemerovo No

Tlacotalpan TLAV Orthobunyavirus 2 IE Bunyamwera No

Tonate TONV Alphavirus 3 IE Af Yes

Topografov TOPV Hantavirus 3a IE Hantaan No

Toscana TOSV Phlebovirus 2 S Phlebotomus

Fever No

Toure TOUV Unassigned 2 S No

Tracambe TRCV Orbivirus 2 A7 No

Tribec TRBV Orbivirus 2 S Kemerovo No

Triniti TNTV Togaviridae 2 S No

Trivittatus TVTV Orthobunyavirus 2 S California No

Trocara TROCV Alphavirus 2 IE Af No

Trombetas TRMV Orthobunyavirus 2 A7 No

Trubanaman TRUV Bunyaviridae 2 S Mapputta No

Tsuruse TSUV Orthobunyavirus 2 S Tete No

Tucurui TUCRV Orthobunyavirus 2 A7 No

Tula TULV Hantavirus 2 A8 No

Tunis TUNV Phlebovirus 2 A7 Phlebotomus

Fever No

Turlock TURV Orthobunyavirus 2 S Turlock No

Turuna TUAV Phlebovirus 2 IE Phlebotomus

Fever No

Tyuleniy TYUV Flavivirus 2 S Bf No

Uganda S UGSV Flavivirus 2 S Bf No

Umatilla UMAV Orbivirus 2 S Umatilla No

Umbre UMBV Orthobunyavirus 2 S Turlock No

Una UNAV Alphavirus 2 S Af No

Upolu UPOV Bunyaviridae 2 S Upolu No
264 Biosafety in Microbiological and Biomedical Laboratories

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Uriurana UURV Phlebovirus 2 A7 Phlebotomus

Fever No

Urucuri URUV Phlebovirus 2 S Phlebotomus

Fever No

Usutu USUV Flavivirus 2 S Bf No

Utinga UTIV Orthobunyavirus 2 IE Simbu No

Uukuniemi UUKV Phlebovirus 2 S Uukuniemi No

Vellore VELV Orbivirus 2 S Palyam No

Venezuelan

Equine

Encephalitis

VEEV Alphavirus 3c S Af Yes

Venkatapuram VKTV Unassigned 2 S No

Vinces VINV Orthobunyavirus 2 A7 Cf No

Virgin River VRV Orthobunyavirus 2 A7 Anopheles A No

Vesicular

StomatitisAlagoasVSAV Vesiculovirus 2c S Vesicular

Stomatitis No

Vesicular

StomatitisIndianaVSIV Vesiculovirus 2c A3 Vesicular

Stomatitis No

Vesicular

Stomatitis-New 

Jersey

VSNJV Vesiculovirus 2c A3 Vesicular

Stomatitis No

Wad Medani WMV Orbivirus 2 S Kemerovo No

Wallal WALV Orbivirus 2 S Wallal No

Wanowrie WANV Bunyaviridae 2 S No

Warrego WARV Orbivirus 2 S Warrego No

Wesselsbron WESSV Flavivirus 3c S Bf Yes

Western Equine

Encephalitis WEEV Alphavirus 3 S Af No

West Nile WNV Flavivirus 3 S Bf No

Whataroa WHAV Alphavirus 2 S Af No

Whitewater

Arroyo WWAV Arenavirus 3 IE Tacaribe No

Witwatersrand WITV Bunyaviridae 2 S No

Wongal WONV Orthobunyavirus 2 S Koongol No

Wongorr WGRV Orbivirus 2 S Wongorr No

Wyeomyia WYOV Orthobunyavirus 2 S Bunyamwera No

Xiburema XIBV Rhabdoviridae 2 IE No

Xingu XINV Orthobunyavirus 3 No

Yacaaba YACV Bunyaviridae 2 IE No

Yaounde YAOV Flavivirus 2 A7 Bf No
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses-References 265

Name Acronym

Taxonomic

Status 

(Family or

Genus)

Recommended 

Biosafety 

Level

Basis of 

Rating

Antigenic

Group

HEPA 

Filtration

on Lab 

Exhaust

Yaquina Head YHV Orbivirus 2 S Kemerovo No

Yata YATAV Rhabdoviridae 2 S No

Yellow Fever YFV Flavivirus 3 S Bf Yes

Yogue YOGV Bunyaviridae 2 S Yogue No

Yoka YOKA Poxviridae 2 IE No

Yug Bogdanovac YBV Vesiculovirus 2 IE Vesicular

Stomatitis No

Zaliv Terpeniya ZTV Phlebovirus 2 S Uukuniemi No

Zegla ZEGV Orthobunyavirus 2 S Patois No

Zika ZIKV Flavivirus 2 S Bf No

Zirqa ZIRV Nairovirus 2 S Hughes No

* Federal regulations, import/export requirements, and taxonomic status are subject to 

changes. Check with the appropriate federal agency to confirm regulations. a Containment requirements will vary based on virus concentration, animal species, or 

virus type. See the Hantavirus agent summary statement in the viral agent chapter. b Tentative placement in the genus. c These organisms are considered pathogens of significant agricultural importance by the 

USDA (see Appendix D) and may require additional containment (up to and including 

BSL-3-Ag containment). Not all strains of each organism are necessarily of concern to 

the USDA. Contact USDA for more information regarding exact containment/permit 

requirements before initiating work. d Alternate name for Ganjam virus. e Garissa virus is considered an isolate of this virus, so same containment requirements apply. f Antigenic groups designated A, B, and C refer to the original comprehensive and unifying 

serogroups established by Casals, Brown, and Whitman based on cross-reactivity among 

known arboviruses (2,21). Group A viruses are members of the genus Alphavirus, group B 

belong to the family Flaviviridae, and Group C viruses are members of the family Bunyaviridae.

References

1. American Committee on Arthropod-borne Viruses. Subcommittee on 

Information Exchange International catalogue of arboviruses including 

certain other viruses of vertebrates. 3rd ed. San Antonio (TX): American 

Society of Tropical Medicine and Hygiene; 1985.

2. Casals J, Brown LV. Hemagglutinations with arthropod-borne viruses. J Exp 

Med. 1954;99:429-49.

3. Leifer E, Gocke DJ, Bourne H. Lassa fever, a new virus disease of man from 

West Africa. II. Report of a laboratory acquired infection treated with plasma 

from a person recently recovered from the disease. Am J Trop Med Hyg. 

1970;19:667-9.

4. Weissenbacher MC, Grela ME, Sabattini MS, et al. Inapparent infections 

with Junin virus among laboratory workers. J Infect Dis. 1978;137:309-13.
266 Biosafety in Microbiological and Biomedical Laboratories

5. Centers for Disease Control and Prevention, Office of Biosafety. Classification 

of etiologic agents on the basis of hazard. 4th edition. US Department of 

Health, Education and Welfare; US Public Health Service. 1979.

6. Hanson RP, Sulkin SE, Beuscher EL, et al. Arbovirus infections of laboratory 

workers. Extent of problem emphasizes the need for more effective 

measures to reduce hazards. Science. 1967;158:1283-86.

7. Karabatsos N. Supplement to international catalogue of arboviruses, 

including certain other viruses of vertebrates. Am J Trop Med Hyg. 

1978;27:372-440.

8. Department of Health, Education, and Welfare. International catalogue of 

arboviruses including certain other viruses of vertebrates. Berge TO, editor. 

Washington, DC. 1975. Pub No. (CDC) 75-8301.

9. Hunt GJ, Tabachnick WJ. Handling small arbovirus vectors safely during 

biosafety level 3 containment: Culicoides variipennis sonorensis (Diptera: 

Ceratopogonidae) and exotic bluetongue viruses. J Med Entomol. 

1996;33:271-7.

10. Warne SR. The safety of work with genetically modified viruses. In: Ring 

CJA, Blair ED, editors. Genetically engineered viruses: development and 

applications. Oxford: BIOS Scientific Publishers; 2001. p. 255-73.

11. Berglund P, Quesada-Rolander M, Putkonen P, et al. Outcome of 

immunization of cynomolgus monkeys with recombinant Semliki Forest 

virus encoding human immunodeficiency virus type 1 envelope protein and 

challenge with a high dose of SHIV-4 virus. AIDS Res Hum Retroviruses. 

1997;13:1487-95.

12. Davis NL, Caley IJ, Brown KW, et al. Vaccination of macaques against 

pathogenic simian immunodeficiency virus with Venezuelan equine 

encephalitis virus replicon particles. J Virol. 2000;74:371-8.

13. Fernandez IM, Golding H, Benaissa-Trouw BJ, et al. Induction of HIV-1 IIIb 

neutralizing antibodies in BALB/c mice by a chimaeric peptide consisting 

of a T-helper cell epitope of Semliki Forest virus and a B-cell epitope of HIV. 

Vaccine. 1998;16:1936-40.

14. Notka F, Stahl-Hennig C, Dittmer U, et al. Construction and characterization 

of recombinant VLPs and Semliki-Forest virus live vectors for comparative 

evaluation in the SHIV monkey model. Biol Chem. 1999;380:341-52.

15. Kuhn RJ, Griffin DE, Owen KE, et al. Chimeric Sindbis-Ross River viruses 

to study interactions between alphavirus nonstructural and structural regions. 

J Virol. 1996;70:7900-9.

16. Schoepp RJ, Smith JF, Parker MD. Recombinant chimeric western and 

eastern equine encephalitis viruses as potential vaccine candidates. 

Virology. 2002;302:299-309.

17. Paessler S, Fayzulin RZ, Anishchenko M, et al. Recombinant Sindbis/ 

Venezuelan equine encephalitis virus is highly attenuated and immunogenic. 

J Virol. 2003;77:9278-86.
Agent Summary Statements: Arboviruses and Related Zoonotic Viruses-References 267

18. Arroyo J, Miller CA, Catalan J, et al. Yellow fever vector live-virus vaccines: 

West Nile virus vaccine development. Trends Mol Med. 2001;7:350-4.

19. Monath TP, McCarthy K, Bedford P, et al. Clinical proof of principle for 

ChimeriVax: recombinant live attenuated vaccines against flavivirus 

infections. Vaccine. 2002;20:1004-18.

20. Smithburn KC, Hughes TP, Burke AW, et al. A neurotropic virus isolated from 

the blood of a native of Uganda. Am J Trop Med Hyg. 1940;20:471-92.

21. Melnick JL, Paul JR, Riordan JT, et al. Isolation from human sera in Egypt of 

a virus apparently identical to West Nile virus. Proc Soc Exp Biol Med. 

1951;77:661-5.

22. Taylor RM, Work TH, Hurlbut HS, Rizk F. A study of the ecology of West Nile 

virus in Egypt. Am J Trop Med Hyg. 1956;5:579-620.

23. Gerhardt R. West Nile virus in the United States (1999-2005). J Am Anim 

Hosp Assoc. 2006;42:170-7.

24. Centers for Disease Control and Prevention. Laboratory-acquired West Nile 

virus infections—United States, 2002. MMWR Morb Mortal Wkly Rep. 

2002;51:1133-5.

25. Rusnak JM, Kortepeter MG, Hawley RJ, et al. Risk of occupationally 

acquired illnesses from biological threat agents in unvaccinated laboratory 

workers. Biosecur Bioterror. 2004;2:281-93.

26. Morris CD. Eastern equine encephalitis. In: Monath TP editor. The 

arboviruses: epidemiology and ecology. Vol III. Boca Raton: CRC Press; 

1988. p. 2-20.

27. Kinney RM, Trent DW, France JK. Comparative immunological and 

biochemical analyses of viruses in the Venezuelan equine encephalitis 

complex. J Gen Virol. 1983;64:135-47.

28. Flick R, Bouloy M. Rift Valley fever virus. Curr Mol Med. 2005;5:827-34

29. Imam IZE, Darwish MA. A preliminary report on an epidemic of Rift Valley 

fever (RVF) in Egypt. J Egypt Public Health Assoc. 1977;52:417-8. 

30. Francis T Jr., Magill TP. Rift valley fever: a report of three cases of laboratory 

infection and the experimental transmission of the disease to ferrets. J Exp 

Med. 1935;62:433-48.

31. Smithburn KC, Haddow AJ, Mahaffy AF. Rift valley fever: accidental 

infections among laboratory workers. J Immunol. 1949;62:213-27.

32. Linthicum KJ, Anyamba A, Tucker CJ, et al. Climate and satellite indicators 

to forecast Rift Valley epidemics in Kenya. Science. 1999;285:397-400.

33. Weaver SC. Host range, amplification and arboviral disease emergence. 

Arch Virol Suppl. 2005;19:33-44.
268 Biosafety in Microbiological and Biomedical Laboratories

Section VIII-G: Toxin Agents

Botulinum Neurotoxin

Seven immunologically distinct serotypes of Botulinum neurotoxin (BoNT) have 

been isolated (A, B, C1, D, E, F and G). Each BoNT holotoxin is a disulfide-bonded 

heterodimer composed of a zinc metallo-protease “light chain” (approximately 

50 kD) and a receptor binding “heavy chain” (approximately 100 kD). The heavy 

chain enhances cell binding and translocation of the catalytic light chain across the 

vesicular membrane.1

 There are also a number of important accessory proteins 

that can stabilize the natural toxin complex in biological systems or in buffer.

Four of the serotypes (A, B, E and, less commonly, F) are responsible for most 

human poisoning through contaminated food, wound infection, or infant botulism, 

whereas livestock may be at greater risk for poisoning with serotypes B, C1 and 

D.2,3 It is important to recognize, however, that all BoNT serotypes are highly toxic 

and lethal by injection or aerosol delivery. BoNT is one of the most toxic proteins 

known; absorption of less than one microgram (μg) of BoNT can cause severe 

incapacitation or death, depending upon the serotype and the route of exposure.

Diagnosis of Laboratory Exposures

Botulism is primarily clinically diagnosed through physician observations of signs 

and symptoms that are similar for all serotypes and all routes of intoxication.4

 There 

typically is a latency of several hours to days, depending upon the amount of toxin 

absorbed, before the signs and symptoms of BoNT poisoning occur. The first 

symptoms of exposure generally include blurred vision, dry mouth and difficulty 

swallowing and speaking. This is followed by a descending, symmetrical flaccid 

paralysis, which can progress to generalized muscle weakness and respiratory 

failure. Sophisticated tests such as nerve conduction studies and single-fiber 

electromyography can support the diagnosis and distinguish it from similar 

neuromuscular conditions. Routine laboratory tests are of limited value because of the 

low levels of BoNT required to intoxicate, as well as the delay in onset of symptoms.

Laboratory Safety and Containment Recommendations

Solutions of sodium hypochlorite (0.1%) or sodium hydroxide (0.1N) readily 

inactivate the toxin and are recommended for decontamination of work surfaces 

and for spills. Additional considerations for the safe use and inactivation of 

toxins of biological origin are found in Appendix I. Because neurotoxin producing 

Clostridia species requires an anaerobic environment for growth and it is 

essentially not transmissible among individuals, exposure to pre-formed BoNT is 

the primary concern for laboratory workers. Two of the most significant hazards 

in working with BoNT or growing neurotoxin producing Clostridia species 

cultures are unintentional aerosol generation, especially during centrifugation, 

and accidental needle-stick. Although BoNT does not penetrate intact skin, 
Agent Summary Statements: Toxin Agents 269

proteins can be absorbed through broken or lacerated skin and, therefore, BoNT 

samples or contaminated material should be handled with gloves.

Workers in diagnostic laboratories should be aware that neurotoxin producing 

Clostridia species or its spores can be stable for weeks or longer in a variety of 

food products, clinical samples (e.g., serum, feces) and environmental samples 

(e.g., soil). Stability of the toxin itself will depend upon the sterility, temperature, 

pH and ionic strength of the sample matrix, but useful comparative data are 

available from the food industry. BoNT retains its activity for long periods (at least 

6-12 months) in a variety of frozen foods, especially under acidic conditions (pH 

4.5-5.0) and/or high ionic strength, but the toxin is readily inactivated by heating.5

A documented incident of laboratory intoxication with BoNT occurred in 

workers who were performing necropsies on animals that had been exposed 

24 h earlier to aerosolized BoNT serotype A; the laboratory workers presumably 

inhaled aerosols generated from the animal fur. The intoxications were relatively 

mild, and all affected individuals recovered after a week of hospitalization.6

Despite the low incidence of laboratory-associated botulism, the remarkable 

toxicity of BoNT necessitates that laboratory workers exercise caution during 

all experimental procedures.

BSL-2 practices, containment equipment, and facilities are recommended 

for routine dilutions, titrations or diagnostic studies with materials known to 

contain or have the potential to contain BoNT. Additional primary containment 

and personnel precautions, such as those recommended for BSL-3, should be 

implemented for activities with a high potential for aerosol or droplet production, 

or for those requiring routine handling of larger quantities of toxin.

Personnel not directly involved in laboratory studies involving botulinum 

toxin, such as maintenance personnel, should be discouraged from entering the 

laboratory when BoNT is in use until after the toxin and all work surfaces have 

been decontaminated. Purified preparations of toxin components, e.g. isolated 

BoNT “light chains” or “heavy chains,” should be handled as if contaminated 

with holotoxin unless proven otherwise by toxicity bioassays.

Special Issues

Vaccines A pentavalent (A, B, C, D and E) botulinum toxoid vaccine (PBT) is 

available through the CDC as an IND. Vaccination is recommended for all personnel 

working in direct contact with cultures of neurotoxin producing Clostridia species 

or stock solutions of BoNT. Due to a possible decline in the immunogenicity of 

available PBT stocks for some toxin serotypes, the immunization schedule for the 

PBT recently has been modified to require injections at 0, 2, 12, and 24 weeks, 

followed by a booster at 12 months and annual boosters thereafter. Since there is 

a possible decline in vaccine efficacy, the current vaccine contains toxoid for only 

5 of the 7 toxin types, this vaccine should not be considered as the sole means of 

protection and should not replace other worker protection measures.
270 Biosafety in Microbiological and Biomedical Laboratories

Select Agent Botulinum toxin is a select agent requiring registration with CDC 

and/or USDA for possession, use, storage and/or transfer if quantities are above 

the minimum exemption level. See Appendix F for additional information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Staphylococcal Enterotoxins (SE)

SE are a group of closely related extracellular protein toxins of 23 to 29 kD 

molecular weight that are produced by distinct gene clusters found in a wide 

variety of S. aureus strains.8,9 SE belong to a large family of homologous 

pyrogenic exotoxins from staphylococci, streptococci and mycoplasma which are 

capable of causing a range of illnesses in man through pathological amplification 

of the normal T-cell receptor response, cytokine/lymphokine release, 

immunosuppression and endotoxic shock.9,10

SE serotype A (SEA) is a common cause of severe gastroenteritis in 

humans.11 It has been estimated from accidental food poisoning that exposure to 

as little as 0.05 to 1 μg SEA by the gastric route causes incapacitating illness.12-15

Comparative human toxicity for different serotypes of SE is largely unknown, but 

human volunteers exposed to 20-25 μg SE serotype B (SEB) in distilled water 

experienced enteritis similar to that caused by SEA.16

SE are highly toxic by intravenous and inhalation routes of exposure. 

By inference from accidental exposure of laboratory workers and controlled 

experiments with NHP, it has been estimated that inhalation of less than 1 ng/kg 

SEB can incapacitate more than 50% of exposed humans, and that the inhalation 

LD50 in humans may be as low as 20 ng/kg SEB.17

Exposure of mucous membranes to SE in a laboratory setting has been 

reported to cause incapacitating gastrointestinal symptoms, conjunctivitis and 

localized cutaneous swelling.18

Diagnosis of Laboratory Exposures

Diagnosis of SE intoxication is based on clinical and epidemiologic features. 

Gastric intoxication with SE begins rapidly after exposure (1-4 h) and is 

characterized by severe vomiting, sometimes accompanied by diarrhea, but 

without a high fever. At higher exposure levels, intoxication progresses to 

hypovolemia, dehydration, vasodilatation in the kidneys, and lethal shock.11
Agent Summary Statements: Toxin Agents 271

While fever is uncommon after oral ingestion, inhalation of SE causes a 

marked fever and respiratory distress. Inhalation of SEB causes a severe, 

incapacitating illness of rapid onset (3-4 h) lasting 3 to 4 days characterized by 

high fever, headache, and a nonproductive cough; swallowing small amounts 

of SE during an inhalation exposure may result in gastric symptoms as well.19

Differential diagnosis of SE inhalation may be unclear initially because the 

symptoms are similar to those caused by several respiratory pathogens such 

as influenza, adenovirus, and mycoplasma. Naturally occurring pneumonias 

or influenza, however, would typically involve patients presenting over a more 

prolonged interval of time, whereas SE intoxication tends to plateau rapidly, 

within a few hours. Nonspecific laboratory findings of SE inhalation include a 

neutrophilic leukocytosis, an elevated erythrocyte sedimentation rate, and chest 

X-ray abnormalities consistent with pulmonary edema.19

Laboratory confirmation of intoxication includes SE detection by immunoassay 

of environmental and clinical samples, and gene amplification to detect staphylococcal 

genes in environmental samples. SE may be undetectable in the serum at the 

time symptoms occur; nevertheless, a serum specimen should be drawn as early 

as possible after exposure. Data from animal studies suggest the presence of SE 

in the serum or urine is transient. Respiratory secretions and nasal swabs may 

demonstrate the toxin early (within 24 h of inhalation exposure). Evaluation of 

neutralizing antibody titers in acute and convalescent sera of exposed individuals 

can be undertaken, but may yield false positives resulting from pre-existing 

antibodies produced in response to natural SE exposure.

Laboratory Safety and Containment Recommendations

General considerations for the safe use and inactivation of toxins of biological 

origin are found in Appendix I. Accidental ingestion, parenteral inoculation, and 

droplet or aerosol exposure of mucous membranes are believed to be the 

primary hazards of SE for laboratory and animal-care personnel. SE are relatively 

stable, monomeric proteins, readily soluble in water, and resistant to proteolytic 

degradation and temperature fluctuations. The physical/chemical stability of SE 

suggests that additional care must be taken by laboratory workers to avoid 

exposure to residual toxin that may persist in the environment.

Active SE toxins may be present in clinical samples, lesion fluids, respiratory 

secretions, or tissues of exposed animals. Additional care should be taken during 

necropsy of exposed animals or in handling clinical stool samples because SE 

toxins retain toxic activity throughout the digestive tract.

Accidental laboratory exposures to SE serotype B have been reviewed.18

Documented accidents included inhalation of SE aerosols generated from 

pressurized equipment failure, as well as re-aerosolization of residual toxin from 

the fur of exposed animals. The most common cause of laboratory intoxication 
272 Biosafety in Microbiological and Biomedical Laboratories

with SE is expected to result from accidental self-exposure via the mucous 

membranes by touching contaminated hands to the face or eyes.

BSL-2 practices and containment equipment and facilities should be used 

when handling SE or potentially contaminated material. Because SE is highly 

active by the oral or ocular exposure route, the use of a laboratory coat, gloves 

and safety glasses is mandatory when handling toxin or toxin-contaminated 

solutions. Frequent and careful hand-washing and laboratory decontamination 

should be strictly enforced when working with SE. Depending upon a risk 

assessment of the laboratory operation, the use of a disposable face mask 

may be required to avoid accidental ingestion.

BSL-3 facilities, equipment, and practices are indicated for activities with 

a high potential for aerosol or droplet production and those involving the use 

of large quantities of SE.

Special Issues

Vaccines No approved vaccine or specific antidote is currently available for 

human use, but experimental, recombinant vaccines are under development.

Select Agent SE is a select agent requiring registration with CDC and/or USDA 

for possession, use, storage and/or transfer. See Appendix F for additional 

information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Ricin Toxin

Ricin is produced in maturing seeds of the castor bean, Ricinus communis, which 

has been recognized for centuries as a highly poisonous plant for humans and 

livestock.20 Ricin belongs to a family of ribosome inactivating proteins from plants, 

including abrin, modeccin, and viscumin, that share a similar overall structure 

and mechanism of action.21 The ricin holotoxin is a disulfide-bonded heterodimer 

composed of an A-chain (approximately 34 kD polypeptide) and a B-chain 

(approximately 32 kD). The A-chain is an N-glycosidase enzyme and a potent 

inhibitor of protein synthesis, whereas the B-chain is a relatively non-toxic lectin 

that facilitates toxin binding and internalization to target cells.20

Ricin is much less toxic by weight than is BoNT or SE, and published case 

reports suggest that intramuscular or gastric ingestion of ricin is rarely fatal in 

adults.22 Animal studies and human poisonings suggest that the effects of ricin 
Agent Summary Statements: Toxin Agents 273

depend upon the route of exposure, with inhalation and intravenous exposure 

being the most toxic. In laboratory mice, for example, the LD50 by intravenous 

injection is about 5 μg/kg, whereas it is 20 mg/kg by intragasteric route.23,24 The 

ricin aerosol LD50 for NHP is estimated to be 10-15 μg/kg.17 The human lethal 

dose has not been established rigorously, but may be as low as 1-5 mg of ricin 

by injection 25 or by the aerosol route (extropolation from two species of NGP).

Diagnosis of Laboratory Exposures

The primary diagnosis is through clinical manifestations that vary greatly 

depending upon the route of exposure. Following inhalation exposure of NHP, 

there is typically a latency period of 24-72 h that may be characterized by loss of 

appetite and listlessness. The latency period progresses rapidly to severe pulmonary 

distress, depending upon the exposure level. Most of the pathology occurs in 

the lung and upper respiratory tract, including inflammation, bloody sputum, and 

pulmonary edema. Toxicity from ricin inhalation would be expected to progress 

despite treatment with antibiotics, as opposed to an infectious process. There 

would be no mediastinitis as seen with inhalation anthrax. Ricin patients would 

not be expected to plateau clinically as occurs after inhalation of SEB.

Gastric ingestion of ricin causes nausea, vomiting, diarrhea, abdominal 

cramps and dehydration. Initial symptoms may appear more rapidly following 

gastric ingestion (1-5 h), but generally require exposure to much higher levels 

of toxin compared with the inhalation route. Following intramuscular injection of 

ricin, symptoms may persist for days and include nausea, vomiting, anorexia, and 

high fever. The site of ricin injection typically shows signs of inflammation with 

marked swelling and induration. One case of poisoning by ricin injection resulted 

in fever, vomiting, irregular blood pressure, and death by vascular collapse after 

a period of several days; it is unclear in this case if the toxin was deposited 

intramuscularly or in the bloodstream.25

Specific immunoassay of serum and respiratory secretions or 

immunohistochemical stains of tissue may be used where available to confirm 

a diagnosis. Ricin is an extremely immunogenic toxin, and paired acute and 

convalescent sera should be obtained from survivors for measurement of 

antibody response. Polymerase chain reaction (PCR) can detect residual castor 

bean DNA in most ricin preparations. Additional supportive clinical or diagnostic 

features, after aerosol exposure to ricin, may include the following: bilateral 

infiltrates on chest radiographs, arterial hypoxemia, neutrophilic leukocytosis, 

and a bronchial aspirate rich in protein.24

Laboratory Safety and Containment Recommendations

General considerations for the safe use and inactivation of toxins of biological 

origin are found in Appendix I. Precautions should be extended to handling 

potentially contaminated clinical, diagnostic and post-mortem samples because 
274 Biosafety in Microbiological and Biomedical Laboratories

ricin may retain toxicity in the lesion fluids, respiratory secretions, or unfixed 

tissues of exposed animals.

When the ricin A-chain is separated from the B-chain and administered 

parenterally to animals, its toxicity is diminished by >1,000-fold compared 

with ricin holotoxin.26 However, purified preparations of natural ricin A-chain 

or B-chain, as well as crude extracts from castor beans, should be handled 

as if contaminated by ricin until proven otherwise by bioassay.

BSL-2 practices, containment equipment and facilities are recommended, 

especially a laboratory coat, gloves, and respiratory protection, when handling 

ricin toxin or potentially contaminated materials.

Ricin is a relatively non-specific cytotoxin and irritant that should be handled 

in the laboratory as a non-volatile toxic chemical. A BSC (Class II, Type B1 or B2) 

or a chemical fume hood equipped with an exhaust HEPA filter and charcoal filter 

are indicated for activities with a high potential for aerosol, such as powder samples, 

and the use of large quantities of toxin. Laboratory coat, gloves, and full-face 

respirator should be worn if there is a potential for creating a toxin aerosol.

Special Issues

Vaccines No approved vaccine or specific antidote is currently available for 

human use, but experimental, recombinant vaccines are under development.

Select Agent Ricin toxin is a select agent requiring registration with CDC 

and/or USDA for possession, use, storage and/or transfer. See Appendix F 

for additional information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

Selected Low Molecular Weight (LMW) Toxins

LMW toxins comprise a structurally and functionally diverse class of natural 

poisons, ranging in size from several hundred to a few thousand daltons, that 

includes complex organic structures, as well as disulfide cross-linked and cyclic 

polypeptides. Tremendous structural diversity may occur within a particular type 

of LMW toxin, often resulting in incomplete toxicological or pharmacological 

characterization of minor isoforms. Grouping LMW toxins together has primarily 

been a means of distinguishing them from protein toxins with respect to key 

biophysical characteristics. Compared with proteins, the LMW toxins are of 

smaller size, which alters their filtration and biodistribution properties, are 
Agent Summary Statements: Toxin Agents 275

generally more stable and persistent in the environment, and may exhibit poor 

water-solubility necessitating the use of organic solvent; these characteristics 

pose special challenges for safe handling, containment, and decontamination 

of LMW toxins within the laboratory.

The set of LMW toxins selected for discussion herein are employed routinely 

as laboratory reagents, and/or have been designated as potential public health 

threats by the CDC, including: T-2 mycotoxin produced by Fusarium fungi;27,28

saxitoxin and related paralytic shellfish poisons produced by dinoflagellates 

of the Gonyaulax family;29 tetrodotoxin from a number of marine animals,30

brevetoxin from the dinoflagellate Ptychodiscus brevis;

31 palytoxin from marine 

coelenterates belonging to the genus Palythoa,

32 polypeptide conotoxins α-GI 

(includes GIA) and α-MI from the Conus genus of gastropod mollusks;33 and 

the monocyclic polypeptide, microcystin-LR from freshwater cyanobacteria 

Microcystis aeruginosa.

34

Trichothecene mycotoxins comprise a broad class of structurally complex, 

non-volatile sesquiterpene compounds that are potent inhibitors of protein 

synthesis.27,28 Mycotoxin exposure occurs by consumption of moldy grains, and 

at least one of these toxins, designated “T-2,” has been implicated as a potential 

biological warfare agent.27 T-2 is a lipid-soluble molecule that can be absorbed 

into the body rapidly through exposed mucosal surfaces.35 Toxic effects are most 

pronounced in metabolically active target organs and include emesis, diarrhea, 

weight loss, nervous disorder, cardiovascular alterations, immunodepression, 

hemostatic derangement, bone marrow damage, skin toxicity, decreased 

reproductive capacity, and death.27 The LD50 for T-2 in laboratory animals ranges 

from 0.2 to 10 mg/kg, depending on the route of exposure, with aerosol toxicity 

estimated to be 20 to 50 times greater than parenteral exposure.17,27 Of special 

note, T-2 is a potent vesicant capable of directly damaging skin or corneas. Skin 

lesions, including frank blisters, have been observed in animals with local, topical 

application of 50 to 100 ng of toxin.27,35

Saxitoxin and tetrodotoxin are paralytic marine toxins that interfere with normal 

function of the sodium channel in excitable cells of heart, muscle and neuronal 

tissue.36 Animals exposed to 1-10 μg/kg toxin by parenteral routes typically 

develop a rapid onset of excitability, muscle spasm, and respiratory distress; 

death may occur within 10-15 minutes from respiratory paralysis.29,37 Humans 

ingesting seafood contaminated with saxitoxin or tetrodotoxin show similar signs 

of toxicity, typically preceded by paresthesias of the lips, face and extremities.36,38

Brevetoxins are cyclic-polyether, paralytic shellfish neurotoxins produced by 

marine dinoflagellates that accumulate in filter-feeding mollusks and may cause 

human intoxication from ingestion of contaminated seafood, or by irritation from 

sea spray containing the toxin.36 The toxin depolarizes and opens voltage-gated 

sodium ion channels, effectively making the sodium channel of affected nerve or 

muscle cells hyper-excitable. Symptoms of human ingestion are expected to 
276 Biosafety in Microbiological and Biomedical Laboratories

include paresthesias of the face, throat and fingers or toes, followed by dizziness, 

chills, muscle pains, nausea, gastroenteritis, and reduced heart rate. Brevetoxin 

has a parenteral LD50 of 200 μg/kg in mice and guinea pigs.31 Guinea pigs 

exposed to a slow infusion of brevetoxin develop fatal respiratory failure within 

30 minutes of exposure to 20 μg/kg toxin.37

Palytoxin is a structurally complex, articulated fatty acid associated with soft 

coral Palythoa vestitus that is capable of binding and converting the essential 

cellular Na+/K+ pump into a non-selective cation channel.32,39 Palytoxin is among 

the most potent coronary vasoconstrictors known, killing animals within minutes by 

cutting off oxygen to the myocardium.40 The LD50 for intravenous administration 

ranges from 0.025 to 0.45 μg/kg in different species of laboratory animals.40

Palytoxin is lethal by several parenteral routes, but is about 200-fold less toxic if 

administered to the alimentary tract (oral or rectal) compared with intravenous 

administration.40 Palytoxin disrupts normal corneal function and causes 

irreversible blindness at topically applied levels of approximately 400 ng/kg, 

despite extensive rinsing after ocular instillation.40

Conotoxins are polypeptides, typically 10-30 amino acids long and stabilized 

by distinct patterns of disulfide bonds, that have been isolated from the toxic 

venom of marine snails and shown to be neurologically active or toxic in 

mammals.33 Of the estimated >105 different polypeptides (conopeptides) present 

in venom of over 500 known species of Conus, only a few have been rigorously 

tested for animal toxicity. Of the isolated conotoxin subtypes that have been 

analyzed, at least two post-synaptic paralytic toxins, designated α-GI (includes 

GIA) and α-MI, have been reported to be toxic in laboratory mice with LD50 values 

in the range of 10-100 μg/kg depending upon the species and route of exposure.

Workers should be aware, however, that human toxicity of whole or 

partially fractionated Conus venom, as well as synthetic combinations of 

isolated conotoxins, may exceed that of individual components. For example, 

untreated cases of human poisoning with venom of C. geographus result in an 

approximately 70% fatality rate, probably as a result of the presence of mixtures 

of various α- and μ-conotoxins with common or synergistic biological targets.33,41

The α-conotoxins act as potent nicotinic antagonists and the μ-conotoxins block 

the sodium channel.33 Symptoms of envenomation depend upon the Conus 

species involved, generally occur rapidly after exposure (minutes), and range 

from severe pain to spreading numbness.42 Severe intoxication results in muscle 

paralysis, blurred or double vision, difficulty breathing and swallowing, and 

respiratory or cardiovascular collapse.42

Microcystins (also called cyanoginosins) are monocyclic heptapeptides 

composed of specific combinations of L-, and D-amino acids, some with 

uncommon side chain structures, that are produced by various freshwater 

cyanobacteria.43 The toxins are potent inhibitors of liver protein phosphatase 

type 1 and are capable of causing massive hepatic hemorrhage and death.43
Agent Summary Statements: Toxin Agents 277

One of the more potent toxins in this family, microcystin-LR, has a parenteral 

LD50 of 30 to 200 μg/kg in rodents.34 Exposure to microcystin-LR causes animals 

to become listless and prone in the cage; death occurs in 16 to 24 h. The toxic 

effects of microcystin vary depending upon the route of exposure and may 

include hypotension and cardiogenic shock, in addition to hepatotoxicity.34,44

Diagnosis of Laboratory Exposures

LMW toxins are a diverse set of molecules with a correspondingly wide range of 

signs and symptoms of laboratory exposure, as discussed above for each toxin. 

Common symptoms can be expected for LMW toxins with common mechanisms 

of action. For example, several paralytic marine toxins that interfere with normal 

sodium channel function cause rapid paresthesias of the lips, face and digits after 

ingestion. The rapid onset of illness or injury (minutes to hours) generally supports 

a diagnosis of chemical or LMW toxin exposure. Painful skin lesions may occur 

almost immediately after contact with T-2 mycotoxin, and ocular irritation or 

lesions will occur in minutes to hours after contact with T-2 or palytoxin.

Specific diagnosis of LMW toxins in the form of a rapid diagnostic test is not 

presently available in the field. Serum and urine should be collected for testing 

at specialized reference laboratories by methods including antigen detection, 

receptor-binding assays, or liquid chromatographic analyses of metabolites. 

Metabolites of several marine toxins, including saxitoxin, tetrodotoxin, and 

brevetoxins, are well-studied as part of routine regulation of food supplies.36

Likewise, T-2 mycotoxin absorption and biodistribution has been studied, and 

its metabolites can be detected as late as 28 days after exposure.27 Pathologic 

specimens include blood, urine, lung, liver, and stomach contents. Environmental 

and clinical samples can be tested using a gas liquid chromatography-mass 

spectrometry technique.

Laboratory Safety and Containment Recommendations

General considerations for the safe use and inactivation of toxins of biological 

origin are found in Appendix I. Ingestion, parenteral inoculation, skin and eye 

contamination, and droplet or aerosol exposure of mucous membranes are the 

primary hazards to laboratory and animal care personnel. LMW toxins also can 

contaminate food sources or small-volume water supplies. Additionally, the T-2 

mycotoxin is a potent vesicant and requires additional safety precautions to 

prevent contact with exposed skin or eyes. Palytoxin also is highly toxic by the 

ocular route of exposure.

In addition to their high toxicity, the physical/chemical stability of the LMW 

toxins contribute to the risks involved in handling them in the laboratory 

environment. Unlike many protein toxins, the LMW toxins can contaminate 

surfaces as a stable, dry film that may pose an essentially indefinite contact 
278 Biosafety in Microbiological and Biomedical Laboratories

threat to laboratory workers. Special emphasis, therefore, must be placed upon 

proper decontamination of work surfaces and equipment.45

When handling LMW toxins or potentially contaminated material, BSL-2 

practices, containment, equipment and facilities are recommended, especially 

the wearing of a laboratory coat, safety glasses and disposable gloves; the 

gloves must be impervious to organic solvents or other diluents employed with 

the toxin.

A BSC (Class II, Type B1 or B2) or a chemical fume hood equipped with 

exhaust HEPA filters and a charcoal filter are indicated for activities with a high 

potential for aerosol, such as powder samples, and the use of large quantities of 

toxin. Laboratory coat and gloves should be worn if potential skin contact exists. 

The use of respiratory protection should be considered if potential aerosolization 

of toxin exists.

For LMW toxins that are not easily decontaminated with bleach solutions, it 

is recommended to use pre-positioned, disposable liners for laboratory bench 

surfaces to facilitate clean up and decontamination.

Special Issues

Vaccines No approved vaccines are currently available for human use. 

Experimental therapeutics for LMW toxins have been reviewed.46

Select Agent Some LMW toxins are a select agent requiring registration with 

CDC and/or USDA for possession, use, storage and/or transfer. See Appendix F 

for additional information.

Transfer of Agent Importation of this agent may require CDC and/or USDA 

importation permits. Domestic transport of this agent may require a permit from 

USDA/APHIS/VS. A DoC permit may be required for the export of this agent to 

another country. See Appendix C for additional information.

References

1. Simpson LL. Identification of the major steps in botulinum toxin action. 

Annu Rev Pharmacol Toxicol. 2004;44:167-93.

2. Gangarosa EJ, Donadio JA, Armstrong RW, et al. Botulism in the United 

States, 1899-1969. Am J Epidemiol. 1971;93:93-101.

3. Hatheway C. Botulism. In: Balows A, Hausler W, Ohashi M, et al., editors. 

Laboratory diagnosis of infectious diseases: principles and practice. Vol. 1. 

New York: Springer-Verlag; 1988. p. 111-33.

4. Shapiro RL, Hatheway C, Swerdlow DL. Botulism in the United States: a 

clinical and epidemiologic review. Ann Intern Med. 1998;129:221-8.
Agent Summary Statements: Toxin Agents-References 279

5. Woolford A, Schantz EJ, Woodburn M. Heat inactivation of botulinum toxin 

type A in some convenience foods after frozen storage. J Food Sci. 

1978;43:622-4.

6. Holzer E. Botulismus durch inhalation. Med Klin. 1962;57:1735-8.

7. Franz DR, Pitt LM, Clayton MA, et al. Efficacy of prophylactic and 

therapeutic administration of antitoxin for inhalation botulism. In: DasGupta 

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biomedical aspects. New York: Plenum Press; 1993. p. 473-6.

8. Llewelyn M, Cohen J. Superantigens: microbial agents that corrupt immunity. 

Lancet Infect Dis. 2002;2:156-62.

9. Jarraud S, Peyrat MA, Lim A, et al. egc, a highly prevalent operon of 

enterotoxin gene, forms a putative nursery of superantigens in 

Staphylococcus aureus. J Immunol. 2001;166:669-77. Erratum in: J 

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10. Marrack P, Kappler J. The Staphylococcal enterotoxins and their relatives. 

Science. 1990;248:705-11. Erratum in: Science. 1990;248:1066.

11. Jett M, Ionin B, Das R, et al. The Staphylococcal enterotoxins. In: Sussman 

M, editor. Molecular medical microbiology. Vol. 2. San Diego: Academic 

Press; 2001. p. 1089-1116.

12. Bergdoll MS. Enterotoxins. In: Montie TC, Kadis S, Ajl SJ, editors. Microbial 

toxins: bacterial protein toxins. Vol. 3. New York: Academic Press;1970. 

p. 265-326.

13. Evenson ML, Hinds MW, Bernstein RS, et al. Estimation of human dose of 

staphylococcal enterotoxin A from a large outbreak of staphylococcal food 

poisoning involving chocolate milk. Int J Food Microbiol. 1988;7:311-16.

14. Asao T, Kumeda Y, Kawai T, et al. An extensive outbreak of staphylococcal 

food poisoning due to low-fat milk in Japan: estimation of enterotoxin A in the 

incriminated milk and powdered skim milk. Epidemiol Infect. 2003;130:33-40.

15. Do Carmo LS, Cummings C, Linardi VR, et al. A case study of a massive 

staphylococcal food poisoning incident. Foodborne Pathog Dis. 2004;1:241-46.

16. Raj HD, Bergdoll MS. Effect of enterotoxin B on human volunteers. J 

Bacteriol. 1969;98:833-34.

17. LeClaire RD, Pitt MLM. Biological weapons defense: effect levels. In: Lindler 

LE, Lebeda FJ, Korch GW, editors. Biological weapons defense: infectious 

diseases and counter bioterrorism. Totowa, New Jersey: Humana Press, 

Inc.; 2005. p. 41-61.

18. Rusnak JM, Kortepeter M, Ulrich R, et al. Laboratory exposures to 

Staphylococcal enterotoxin B. Emerg Infect Dis. 2004;10:1544-9.

19. Ulrich R, Sidell S, Taylor T, et al. Staphylococcal enterotoxin B and related 

pyrogenic toxins. In: Sidell FR, Takafuji ET, Franz DR, editors. Medical 

aspects of chemical and biological warfare. Vol. 6. Textbook of military 

medicine, part 1: warfare, weaponry, and the casualty. Washington, DC: 

Office of the Surgeon General at TMM Publications, Borden Institute, Walter 

Reed Army Medical Center; 1997. p. 621-30.
280 Biosafety in Microbiological and Biomedical Laboratories

20. Olsnes S. The history of ricin, abrin and related toxins. Toxicon. 

2004;44:361-70.

21. Hartley MR, Lord JM. Cytotoxic ribosome-inactivating lectins from plants. 

Biochim Biophys Acta. 2004;1701:1-14.

22. Doan LG. Ricin: mechanism of toxicity, clinical manifestations, and vaccine 

development. A review. J Toxicol Clin Toxicol. 2004;42:201-8.

23. Cumber AJ, Forrester JA, Foxwell BM, et al. Preparation of antibody-toxin 

conjugates. Methods Enzymol. 1985;112:207-25.

24. Franz D, Jaax N. Ricin toxin. In: Sidell FR, Takafuji ET, Franz DR, editors. 

Medical aspects of chemical and biological warfare. Vol. 6. Textbook of 

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DC. Office of the Surgeon General at TMM Publications, Borden Institute, 

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26. Soler-Rodriguez AM, Uhr JW, Richardson J, et al. The toxicity of chemically 

deglycosylated ricin A-chain in mice. Int J Immunopharmacol. 1992;14:281-91.

27. Wannemacher R, Wiener SL. Trichothecene mycotoxins. In: Sidell FR, 

Takafuji ET, Franz DR, editors. Medical aspects of chemical and biological 

warfare. Vol. 6. Textbook of military medicine, part 1: warfare, weaponry, 

and the casualty. Washington, DC: Office of the Surgeon General at TMM 

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28. Bamburg J. Chemical and biochemical studies of the trichothecene 

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29. Schantz EJ. Chemistry and biology of saxitoxin and related toxins. 

Ann N Y Acad Sci. 1986;479:15-23.

30. Yasumoto T, Nagai H, Yasumura D, et al. Interspecies distribution and 

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31. Baden DG, Mende TJ, Lichter W, et al. Crystallization and toxicology of 

T34: a major toxin from Florida’s red tide organism (Ptychodiscus brevis). 

Toxicon. 1981;19:455-62.

32. Moore RE, Scheuer PJ. Palytoxin: a new marine toxin from a coelenterate. 

Science. 1971;172:495-8.

33. Olivera BM, Cruz LJ. Conotoxins, in retrospect. Toxicon. 2001;39:7-14.

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35. Bunner B, Wannemacher RW Jr, Dinterman RE, et al. Cutaneous absorption 

and decontamination of [3H]T-2 toxin in the rat model. J Toxicol Environ 

Health. 1989;26:413-423.
Agent Summary Statements: Toxin Agents-References 281

36. Poli M. Foodborne Marine biotoxins. In: Miliotis MD, Bier JW, editors. 

International handbook of foodborne pathogens. New York: Marcel Dekker; 

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37. Franz DR, LeClaire RD. Respiratory effects of brevetoxin and saxitoxin in 

awake guinea pigs. Toxicon. 1989;27:647-54.

38. Kao CY. Tetrodotoxin, saxitoxin and their significance in the study of 

excitation phenomena. Pharmacol Rev. 1966;18:997-1049.

39. Artigas P, Gadsby DC. Na+/K+-pump ligands modulate gating of palytoxininduced ion channels. Proc Natl Acad Sci USA. 2003;100:501-5.

40. Wiles JS, Vick JA, Christensen MK. Toxicological evaluation of palytoxin in 

several animal species. Toxicon. 1974;12:427-33.

41. Cruz LJ, White J. Clinical Toxicology of Conus Snail Stings. In: Meier J, 

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poisons. Boca Raton: CRC Press; 1995. p. 117-28.

42. McIntosh JM, Jones RM. Cone venom-from accidental stings to deliberate 

injection. Toxicon. 2001;39:1447-51.

43. Dawson RM. The toxicology of microcystins. Toxicon. 1998;36:953-62. 

44. LeClaire RD, Parker GW, Franz DR. Hemodynamic and calorimetric changes 

induced by microcystin-LR in the rat. J Appl Toxicol. 1995;15:303-11.

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282 Biosafety in Microbiological and Biomedical Laboratories

Section VIII-H: Prion Diseases

Transmissible spongiform encephalopathies (TSE) or prion diseases are 

neurodegenerative diseases which affect humans and a variety of domestic and 

wild animal species (Tables 7 and 8).1,2 A central biochemical feature of prion 

diseases is the conversion of normal prion protein (PrP) to an abnormal, 

misfolded, pathogenic isoform designated PrPSc (named for “scrapie,” the 

prototypic prion disease). The infectious agents that transmit prion diseases are 

resistant to inactivation by heat and chemicals and thus require special biosafety 

precautions. Prion diseases are transmissible by inoculation or ingestion of 

infected tissues or homogenates, and infectivity is present at high levels in brain 

or other central nervous system tissues, and at slightly lower levels in lymphoid 

tissues including spleen, lymph nodes, gut, bone marrow, and blood. Although 

the biochemical nature of the infectious TSE agent, or prion, is not yet proven, 

the infectivity is strongly associated with the presence of PrPSc, suggesting that 

this material may be a major component of the infectious agent.

A chromosomal gene encodes PrPC (the cellular isoform of PrP) and no PrP 

genes are found in purified preparations of prions. PrPSc is derived from PrPC by 

a posttranslational process whereby PrPSc acquires a high beta-sheet content 

and a resistance to inactivation by normal disinfection processes. The PrPSc is 

less soluble in aqueous buffers and, when incubated with protease (proteinase 

K), the PrPC is completely digested (sometimes indicated by the “sensitive” 

superscript, PrPsen) while PrPSc is resistant to protease (PrPres). Neither PrPspecific nucleic acids nor virus-like particles have been detected in purified, 

infectious preparations.

Occupational Infections

No occupational infections have been recorded from working with prions. 

No increased incidence of Creutzfeldt-Jakob disease (CJD) has been found 

amongst pathologists who encounter cases of the disease post-mortem.

Natural Modes of Infection

The recognized diseases caused by prions are listed under Table 7 (human 

diseases) and Table 8 (animal diseases). The only clear risk factor for disease 

transmission is the consumption of infected tissues such as human brain in 

the case of kuru, and meat including nervous tissue in the case of bovine 

spongiform encephalopathy and related diseases such as feline spongiform 

encephalopathy. It is also possible to acquire certain diseases such as familial 

CJD by inheritance through the germ line.
Agent Summary Statements: Prion Diseases 283

Most TSE agents, or prions, have a preference for infection of the 

homologous species, but cross-species infection with a reduced efficiency is 

also possible. After cross-species infection there is often a gradual adaptation 

of specificity for the new host; however, infectivity for the original host may also 

be propagated for several passages over a time-span of years. The process of 

cross-species adaptation can also vary among individuals in the same species 

and the rate of adaptation and the final species specificity is difficult to predict 

with accuracy. Such considerations help to form the basis for the biosafety 

classification of different prions.

Table 7. The Human Prion Diseases

Disease Abbreviation Mechanism of Pathogenesis

Kuru Infection through ritualistic cannibalism

Creutzfeldt-Jakob disease CJD Unknown mechanism

Sporadic CJD sCJD Unknown mechanism; possibly somatic 

mutation or spontaneous conversion of 

PrPc

 to PrPSc

Variant CJD vCJD Infection presumably from consumption 

of BSE-contaminated cattle products and 

secondary bloodborne transmission

Familial CJD fCJD Germline mutations in PrP gene

Latrogenic CJD iCJD Infection from contaminated corneal 

and dural grafts, pituitary hormone, or 

neurosurgical equipment

Gerstmann-SträusslerScheinker syndromeGSS Germline mutations in PrP gene

Fatal familial insomnia FFI Germline mutations in PrP gene
284 Biosafety in Microbiological and Biomedical Laboratories

Table 8. The Animal Prion Diseases

Disease Abbreviation Natural Host Mechanism of Pathogenesis

Scrapie Sheep, goats, 

mouflon

Infection in genetically susceptible 

sheep

Bovine spongiform 

encephalopathy

BSE Cattle Infection with prion-contaminated 

feedstuffs

Chronic wasting 

disease

CWD Mule, deer, 

white-tailed 

deer, Rocky 

Mountain elk

Unknown mechanism; possibly from 

direct animal contact or indirectly 

from contaminated feed and water 

sources

Exotic ungulate 

encephalopathy

EUE Nyala, greater 

kudu and oryx

Infection with BSE-contaminated 

feedstuffs

Feline spongiform 

encephalopathy

FSE Domestic and 

wild cats in 

captivity

Infection with BSE-contaminated 

feedstuffs

Transmissible 

mink encephalopathy

TME Mink (farm 

raised

Infection with prion-contaminated 

feedstuffs

Laboratory Safety and Containment Recommendations

In the laboratory setting prions from human tissue and human prions propagated 

in animals should be manipulated at BSL-2. BSE prions can likewise be 

manipulated at BSL-2. Due to the high probability that BSE prions have been 

transmitted to humans, certain circumstances may require the use of BSL-3 

facilities and practices. All other animal prions are manipulated at BSL-2.

However, when a prion from one species is inoculated into another the resultant 

infected animal should be treated according to the guidelines applying to the 

source of the inoculum. Contact APHIS National Center for Import and Export 

at (301) 734-5960 for specific guidance.

Although the exact mechanism of spread of scrapie among sheep and goats 

developing natural scrapie is unknown, there is considerable evidence that one 

of the primary sources is oral inoculation with placental membranes from infected 

ewes. There has been no evidence for transmission of scrapie to humans, even 

though the disease was recognized in sheep for over 200 years. The diseases 

TME, BSE, FSE, and EUE are all thought to occur after the consumption of 

prion-infected foods.1,2 The exact mechanism of CWD spread among mule 

deer, white-tailed deer and Rocky Mountain elk is unknown. There is strong 

evidence that CWD is laterally transmitted and environmental contamination 

may play an important role in local maintenance of the disease.2

In the care of patients diagnosed with human prion disease, Standard 

Precautions are adequate. However, the human prion diseases in this setting 
Agent Summary Statements: Prion Diseases 285

are not communicable or contagious.3 There is no evidence of contact or aerosol 

transmission of prions from one human to another. However, they are infectious 

under some circumstances, such as ritualistic cannibalism in New Guinea 

causing kuru, the administration of prion-contaminated growth hormone causing 

iatrogenic CJD, and the transplantation of prion-contaminated dura mater and 

corneal grafts. It is highly suspected that variant CJD can also be transmitted by 

blood transfusion.4

 However, there is no evidence for bloodborne transmission 

of non-variant forms of CJD. Familial CJD, GSS, and FFI are all dominantly 

inherited prion diseases; many different mutations of the PrP gene have been 

shown to be genetically linked to the development of inherited prion disease. 

Prions from many cases of inherited prion disease have been transmitted to 

apes, monkeys, and mice, especially those carrying human PrP transgenes.

Special Issues

Inactivation of Prions Prions are characterized by resistance to conventional 

inactivation procedures including irradiation, boiling, dry heat, and chemicals 

(formalin, betapropiolactone, alcohols). While prion infectivity in purified samples 

is diminished by prolonged digestion with proteases, results from boiling in 

sodium dodecyl sulfate and urea are variable. Likewise, denaturing organic 

solvents such as phenol or chaotropic reagents such as guanidine isothiocyanate 

have also resulted in greatly reduced but not complete inactivation. The use of 

conventional autoclaves as the sole treatment has not resulted in complete 

inactivation of prions.5

 Formalin-fixed and paraffin-embedded tissues, especially 

of the brain, remain infectious. Some investigators recommend that formalin-fixed 

tissues from suspected cases of prion disease be immersed for 30 min in 96% 

formic acid or phenol before histopathologic processing (Table 9), but such 

treatment may severely distort the microscopic neuropathology.

The safest and most unambiguous method for ensuring that there is no 

risk of residual infectivity on contaminated instruments and other materials 

is to discard and destroy them by incineration.6

 Current recommendations for 

inactivation of prions on instruments and other materials are based on the use 

of sodium hypochlorite, NaOH, Environ LpH and the moist heat of autoclaving 

with combinations of heat and chemical being most effective (Table 9).5,6

Surgical Procedures Precautions for surgical procedures on patients 

diagnosed with prion disease are outlined in an infection control guideline 

for transmissible spongiform encephalopathies developed by a consultation 

convened by the WHO in 1999.6 Sterilization of reusable surgical instruments 

and decontamination of surfaces should be performed in accordance with 

recommendations described by the CDC (www.cdc.gov) and the WHO 

infection control guidelines.6

 Table 9 summarizes the key recommendations 

for decontamination of reusable instruments and surfaces. Contaminated 

disposable instruments or materials should be incinerated at 1000° C or greater.7
286 Biosafety in Microbiological and Biomedical Laboratories

Autopsies Routine autopsies and the processing of small amounts of formalinfixed tissues containing human prions can safely be done using Standard 

Precautions.8

 The absence of any known effective treatment for prion disease 

demands caution. The highest concentrations of prions are in the central 

nervous system and its coverings. Based on animal studies, it is likely that 

prions are also found in spleen, thymus, lymph nodes, and intestine. The 

main precaution to be taken by laboratorians working with prion-infected or 

contaminated material is to avoid accidental puncture of the skin.3

 Persons 

handling contaminated specimens should wear cut-resistant gloves if possible. 

If accidental contamination of unbroken skin occurs, the area should be washed 

with detergent and abundant quantities of warm water (avoid scrubbing); brief 

exposure (1 minute to 1N NaOH or a 1:10 dilution of bleach) can be considered 

for maximum safety.6

 Additional guidance related to occupational injury are 

provided in the WHO infection control guidelines.6

 Unfixed samples of brain, 

spinal cord, and other tissues containing human prions should be processed 

with extreme care in a BSL-2 facility utilizing BSL-3 practices.

Bovine Spongiform Encephalopathy Although the eventual total number 

of variant CJD cases resulting from BSE transmission to humans is unknown, 

a review of the epidemiological data from the United Kingdom indicates that 

BSE transmission to humans is not efficient.9

 The most prudent approach is to 

study BSE prions at a minimum in a BSL-2 facility utilizing BSL-3 practices. 

When performing necropsies on large animals where there is an opportunity 

that the worker may be accidentally splashed or have contact with high-risk 

materials (e.g., spinal column, brain) personnel should wear full body coverage 

personal protective equipment (e.g., gloves, rear closing gown and face shield). 

Disposable plasticware, which can be discarded as a dry regulated medical 

waste, is highly recommended. Because the paraformaldehyde vaporization 

procedure does not diminish prion titers, BSCs must be decontaminated with 1N 

NaOH and rinsed with water. HEPA filters should be bagged out and incinerated. 

Although there is no evidence to suggest that aerosol transmission occurs in 

the natural disease, it is prudent to avoid the generation of aerosols or droplets 

during the manipulation of tissues or fluids and during the necropsy of 

experimental animals. It is further strongly recommended that impervious 

gloves be worn for activities that provide the opportunity for skin contact 

with infectious tissues and fluids.

Animal carcasses and other tissue waste can be disposed by incineration with 

a minimum secondary temperature of 1000°C (1832°F).6

 Pathological incinerators 

should maintain a primary chamber temperature in compliance with design and 

applicable state regulations, and employ good combustion practices. Medical 

waste incinerators should comply with applicable state and federal regulations.
Agent Summary Statements: Prion Diseases 287

The alkaline hydrolysis process, using a pressurized vessel that exposes 

the carcass or tissues to 1 N NaOH or KOH heated to 150°C, can be used as 

an alternative to incineration for the disposal of carcasses and tissue.5,10 The 

process has been shown to completely inactive TSEs (301v agent used) when 

used for the recommended period.

Table 9. Tissue Preparation for Human CJD and Related Diseases

1. Histology technicians wear gloves, apron, laboratory coat, and face protection.

2. Adequate fixation of small tissue samples (e.g., biopsies) from a patient with suspected prion 

disease can be followed by post-fixation in 96% absolute formic acid for 30 minutes, followed by 

45 hours in fresh 10% formalin.

3. Liquid waste is collected in a 4L waste bottle initially containing 600 ml 6N NaOH.

4. Gloves, embedding molds, and all handling materials are disposed s regulated medical waste.

5. Tissue cassettes are processed manually to prevent contamination of tissue processors.

6. Tissues are embedded in a disposable embedding mold. If used, forceps are decontaminated as 

in Table 10.

7. In preparing sections, gloves are worn, section waste is collected and disposed in a regulated medical waste receptacle. The knife stage is wiped with 2N NaOH, and the knife used is 

discarded immediately in a “regulated medical waste sharps” receptacle. Slides are labeled with 

“CJD Precautions.” The sectioned block is sealed with paraffin.

8. Routine staining:

a. slides are processed by hand;

b. reagents are prepared in 100 ml disposable specimen cups;

c. after placing the cover slip on, slides are decontaminated by soaking them for 1 hour in 

2N NaOH;

d. slides are labeled as “Infectious-CJD.”

9. Other suggestions:

a. disposable specimen cups or slide mailers may be used for reagents;

b. slides for immunocytochemistry may be processed in disposable Petri dishes;

c. equipment is decontaminated as described above or disposed as regulated medical 

waste.

Handling and processing of tissues from patients with suspected prion 

disease The special characteristics of work with prions require particular attention 

to the facilities, equipment, policies, and procedures involved.10 The related 

considerations outlined in Table 9 should be incorporated into the laboratory’s 

risk management for this work.
288 Biosafety in Microbiological and Biomedical Laboratories

Table 10. Prion Inactivation Methods for Reusable Instruments 

and Surfaces

1. Immerse in 1 N NaOH, heat in a gravity displacement autoclave at 121oC for 30 minutes. Clean 

and sterilize by conventional means.

2. Immerse in 1 N NaOH or sodium hypochlorite (20,000 ppm) for 1 hours. Transfer into water and 

autoclave (gravity displacement) at 121oC for 1 hour. Clean and sterilize by conventional means.

3. Immerse in 1N NaOH or sodium hypochlorite (20,000) for 1 hour. Rinse instruments with water, 

transfer to open pan and autoclave at 121oC (gravity displacement) or 134oC (porous load) for 1 

hour. Clean and sterilize by conventional means.

4. Surfaces or heat-sensitive instruments can be treated with 2N NaOH or sodium hypochlorite 

(20,000 ppm) for 1 hour. Ensure surfaces remain wet for entire period, then rinse well with water. 

Before chemical treatment, it is strongly recommended that gross contamination of surfaces 

be reduced because the presence of excess organic material will reduce the strength of either 

NaOH or sodium hypochlorite solutions.

5. Environ LpH (EPA Reg. No. 1043-118) may be used on washable, hard, non-porous surfaces 

(such as floors, tables, equipment, and counters), items (such as non-disposable instruments, 

sharps, and sharp containers), and/or laboratory waste solutions (such as formalin or other 

liquids). This product is currently being used under FIFRA Section 18 exemptions in a number of 

states. Users should consult with the state environmental protection office prior to use.

(Adapted from www.cdc.gov 11,12)

Working Solutions 1 N NaOH equals 40 grams of NaOH per liter of water. 

Solution should be prepared daily. A stock solution of 10 N NaOH can be prepared 

and fresh 1:10 dilutions (1 part 10 N NaOH plus 9 parts water) used daily.

20,000 ppm sodium hypochlorite equals a 2% solution. Most commercial 

household bleach contains 5.25% sodium hypochlorite, therefore, make a 1:2.5 

dilution (1 part 5.25% bleach plus 1.5 parts water) to produce a 20,000 ppm 

solution. This ratio can also be stated as two parts 5.25% bleach to three parts 

water. Working solutions should be prepared daily.

CAUTION: Above solutions are corrosive and require suitable personal 

protective equipment and proper secondary containment. These strong corrosive 

solutions require careful disposal in accordance with local regulations.

Precautions in using NaOH or sodium hypochlorite solutions in autoclaves:

NaOH spills or gas may damage the autoclave if proper containers are not used. 

The use of containers with a rim and lid designed for condensation to collect and 

drip back into the pan is recommended. Persons who use this procedure should 

be cautious in handling hot NaOH solution (post-autoclave) and in avoiding 

potential exposure to gaseous NaOH; exercise caution during all sterilization 

steps; and allow the autoclave, instruments, and solutions to cool down before 

removal. Immersion in sodium hypochlorite bleach can cause severe damage to 

some instruments.
Agent Summary Statements: Prion Diseases-References 289

References

1. Prusiner SB. Prion diseases and the BSE crisis. Science. 1997;278:245-51.

2. Williams ES, Miller MW. Transmissible spongiform encephalopathies in 

non-domestic animals: origin, transmission and risk factors. Rev Sci Tech 

Off Int Epiz. 2003;22:145-56.

3. Ridley RM, Baker HF. Occupational risk of Creutzfeldt-Jakob disease. 

Lancet. 1993;341:641-2.

4. Llewelyn CA, Hewitt PE, Knight RS, et al. Possible transmission of variant 

Creutzfeldt-Jakob disease by blood transfusion. Lancet. 2004;7;363:417-21.

5. Taylor DM, Woodgate SL. Rendering practices and inactivation of 

transmissible spongiform encephalopathy agents. Rev Sci Tech Off Int Epiz. 

2003;22:297-310.

6. World Health Organization. [http://www.who.int/en/]. Geneva (Switzerland): 

The Organization; [updated 2006 Sept 21; cited 2006 Sept 21]. 2000. WHO 

Infection Control Guidelines for Transmissible Spongiform Encephalopathies. 

Report of a WHO Consultation, Geneva, Switzerland, 23-26 March 1999. 

Available from: http://www.who.int/csr/resources/publications/bse/WHO_

CDS_CSR_APH_2000_3/en/

7. Brown P, Rau EH, Johnson BK, Bacote AE, Gibbs CJ, Jr, Gajdusek DC. New 

studies on the heat resistance of hamster-adapted scrapie agent: threshold 

survival after ashing at 600 degrees C suggests an inorganic template of 

replication. Proc Natl Acad Sci USA. 2000;97:3418–3421. doi: 10.1073/

pnas.050566797.

8. Ironside JW and JE Bell. The ‘high-risk’ neuropathological autopsy in AIDS 

and Creutzfeldt-Jakob disease: principles and practice. Neuropathol Appl 

Neurobiol. 1996;22:388-393.

9. Hilton DA. Pathogenesis and prevalence of variant Creutzfeldt-Jakob 

disease. Pathol J. 2006;208:134-41.

10. Richmond JY, Hill RH, Weyant RS, et al. What’s hot in animal biosafety? 

ILAR J. 2003;44:20-7.

11. Ernst DR, Race RE. Comparative analysis of scrapie agent inactivation 

methods. J. Virol. Methods. 1994; 41:193-202.

12. Race RE, Raymond GJ. Inactivation of transmissible spongiform 

encephalopathy (prion) agents by Environ LpH. J Virol. 2004;78:2164-5.
290 Biosafety in Microbiological and Biomedical Laboratories

Appendix A – Primary Containment for Biohazards: 

Selection, Installation and Use of Biological Safety Cabinets

Section I—Introduction

This document presents information on the design, selection, function and use of 

Biological Safety Cabinets (BSCs), which are the primary means of containment 

developed for working safely with infectious microorganisms. Brief descriptions of 

the facility and engineering concepts for the conduct of microbiological research 

are also provided. BSCs are only one part of an overall biosafety program, which 

requires consistent use of good microbiological practices, use of primary containment 

equipment and proper containment facility design. Detailed descriptions of 

acceptable work practices, procedures and facilities, known as Biosafety Levels 

1 through 4, are presented in the CDC/NIH publication Biosafety in Microbiological 

and Biomedical Laboratories (BMBL).1

BSCs are designed to provide personnel, environmental and product 

protection when appropriate practices and procedures are followed. Three kinds 

of biological safety cabinets, designated as Class I, II and III, have been 

developed to meet varying research and clinical needs.

Most BSCs use high efficiency particulate air (HEPA) filters in the exhaust and 

supply systems. The exception is a Class I BSC, which does not have HEPA filtered 

supply air. These filters and their use in BSCs are briefly described in Section II. 

Section III presents a general description of the special features of BSCs that 

provide varying degrees of personnel, environmental, and product protection.

Laboratory hazards and risk assessment are discussed in Section IV. 

Section V presents work practices, procedures and practical tips to maximize 

information regarding the protection afforded by the most commonly used BSCs. 

Facility and engineering requirements needed for the operation of each type of 

BSC are presented in Section VI. Section VII reviews requirements for routine 

annual certification of cabinet operation and integrity.

These sections are not meant to be definitive or all encompassing. Rather, 

an overview is provided to clarify the expectations, functions and performance of 

these critical primary barriers. This document has been written for the biosafety 

officer, laboratorian, engineer or manager who desires a better understanding 

of each type of cabinet; factors considered for the selection of a BSC to meet 

specific operational needs; and the services required to maintain the operational 

integrity of the cabinet.

Proper maintenance of cabinets used for work at all biosafety levels cannot 

be over emphasized. Biosafety Officers (BSOs) should understand that an active 

cabinet is a primary containment device. A BSC must be routinely inspected and 

tested by training personnel, following strict protocols, to verify that it is working 
Appendix A: HEPA Filters and Biological Containment Devices 291

properly. This process is referred to as certification of the cabinet and should be 

performed annually.

Section II—The High Efficiency Particulate Air (HEPA) Filter and the 

Development of Biological Containment Devices

From the earliest laboratory-acquired typhoid infections to the hazards posed by 

bioterrorism, antibiotic-resistant bacteria and rapidly mutating viruses, threats to 

worker safety have stimulated the development and refinement of workstations in 

which infectious microorganisms could be safely handled. The needs to work with 

tissue cultures, maintain sterility of cell lines, and minimize cross-contamination 

have contributed to concerns regarding product integrity.

The use of proper procedures and equipment (as described in BMBL)1

 cannot 

be overemphasized in providing primary personnel and environmental protection. 

For example, high-speed blenders designed to reduce aerosol generation, 

needle-locking syringes, micro burners and safety centrifuge cups or sealed 

rotors are among the engineered devices that protect laboratory workers from 

biological hazards. An important piece of safety equipment is the biological safety 

cabinet in which manipulations of infectious microorganisms are performed.

Background

Early prototype clean air cubicles were designed to protect the materials being 

manipulated from environmental or worker-generated contamination rather than 

to protect the worker from the risks associated with the manipulation of potentially 

hazardous materials. Filtered air was blown across the work surface directly at 

the worker. Therefore, these cubicles could not be used for handling infectious 

agents because the worker was in a contaminated air stream.

To protect the worker during manipulations of infectious agents, a small 

workstation was needed that could be installed in existing laboratories with minimum 

modification to the room. The earliest designs for primary containment devices 

were essentially non-ventilated “boxes” built of wood and later of stainless steel, 

within which simple operations such as weighing materials could be accomplished.2

Early versions of ventilated cabinets did not have adequate or controlled 

directional air movement. They were characterized by mass airflow into the cabinets 

albeit with widely varying air volumes across openings. Mass airflow into cabinet 

drew “contaminated” air away from the laboratory worker. This was the forerunner of 

the Class I BSC. However, since the air was unfiltered, the cabinet was contaminated 

with environmental microorganisms and other undesirable particulate matter.

Control of airborne particulate materials became possible with the development 

of filters, which efficiently removed microscopic contaminants from the air. The 

HEPA filter was developed to create dust-free work environments (e.g., “clean 

rooms” and “clean benches”) in the 1940s.2
292 Biosafety in Microbiological and Biomedical Laboratories

HEPA filters remove the most penetrating particle size (MPPS) of 0.3 μm 

with an efficiency of at least 99.97%. Particles both larger and smaller than the 

MPPS are removed with greater efficiency. Bacteria, spores and viruses are 

removed from the air by these filters. HEPA filter efficiency and the mechanics 

of particle collection by these filters have been studied and well-documented 3,4

therefore only a brief description is included here.

The typical HEPA filter medium is a single sheet of borosilicate fibers treated 

with a wet-strength water-repellant binder. The filter medium is pleated to increase 

the overall surface area inside the filter frames and the pleats are often divided 

by corrugated aluminum separators (Figure 1). The separators prevent the pleats 

from collapsing in the air stream and provide a path for airflow. Alternate designs 

providing substitutions for the aluminum separators may also be used. The filter 

is glued into a wood, metal or plastic frame. Careless handling of the filter (e.g., 

improper storage or dropping) can damage the medium at the glue joint and 

cause tears or shifting of the filter resulting in leaks in the medium. This is the 

primary reason why filter integrity must be tested when a BSC is installed initially 

and each time it is moved or relocated. (See Section VII.)

Various types of containment and clean air devices incorporate the use of 

HEPA filters in the exhaust and/or supply air system to remove airborne particulate 

material. Depending on the configuration of these filters and the direction of the 

airflow, varying degrees of personnel, environmental and product protection can be 

achieved.5

 Section V describes the proper practices and procedures necessary 

to maximize the protection afforded by the device.

Section III—Biological Safety Cabinets

The similarities and differences in protection offered by the various classes of 

BSCs are reflected in Table 1. Please also refer to Table 2 and Section IV for 

further considerations pertinent to BSC selection and risk assessment.

The Class I BSC

The Class I BSC provides personnel and environmental protection, but no 

product protection. It is similar in terms of air movement to a chemical fume hood, 

but has a HEPA filter in the exhaust system to protect the environment (Figure 2). 

In the Class I BSC, unfiltered room air is drawn in through the work opening and 

across the work surface. Personnel protection is provided by this inward airflow 

as long as a minimum velocity of 75 linear feet per minute (lfm) is maintained6

through the front opening. Because product protection is provided by the Class II 

BSCs, general usage of the Class I BSC has declined. However, in many cases, 

Class I BSCs are used specifically to enclose equipment (e.g., centrifuges, 

harvesting equipment or small fermenters), or procedures with potential to 

generate aerosols (e.g., cage dumping, culture aeration or tissue homogenation).
Appendix A: Biological Safety Cabinets 293

The classical Class I BSC is hard-ducted (i.e., direct connection) to the building 

exhaust system and the building exhaust fan provides the negative pressure 

necessary to draw room air into the cabinet. Cabinet air is drawn through a HEPA 

filter as it enters the cabinet exhaust plenum. A second HEPA filter may be installed 

at the terminal end of the building exhaust system prior to the exhaust fan.

Some Class I BSCs are equipped with an integral exhaust fan. The cabinet 

exhaust fan must be interlocked with the building exhaust fan. In the event that 

the building exhaust fan fails, the cabinet exhaust fan must turn off so that the 

building exhaust ducts are not pressurized. If the ducts are pressurized and the 

HEPA filter has developed a leak, contaminated air could be discharged into 

other parts of the building or the environment. The use of two filters in the 

cabinet increases the static pressure on the fan.

A panel with openings to allow access for the hands and arms to the work 

surface can be added to the Class I cabinet. The restricted opening results in 

increased inward air velocity, increasing worker protection. For added safety, 

arm-length gloves can be attached to the panel. Makeup air is then drawn through 

an auxiliary air supply opening (which may contain a filter) and/or around a 

loose-fitting front panel.

Some Class I models used for animal cage changing are designed to allow 

recirculation of air into the room after HEPA filtration and may require more 

frequent filter replacement due to filter loading and odor from organic materials 

captured on the filter. This type of Class I BSC should be certified annually for 

sufficient airflow and filter integrity.

The Class II BSC

As biomedical researchers began to use sterile animal tissue and cell culture 

systems, particularly for the propagation of viruses, cabinets were needed that also 

provided product protection. In the early 1960s, the “laminar flow” principle evolved. 

Unidirectional air moving at a fixed velocity along parallel lines was demonstrated 

to reduce turbulence resulting in predictable particle behavior. Biocontainment 

technology also incorporated this laminar flow principle with the use of the HEPA 

filter to aid in the capture and removal of airborne contaminants from the air stream.7

This combination of technologies serves to help protect the laboratory worker 

from potentially infectious aerosols4

 generated within the cabinet and provides 

necessary product protection, as well. Class II BSCs are partial barrier systems 

that rely on the directional movement of air to provide containment. As the air 

curtain is disrupted (e.g., movement of materials in and out of a cabinet, rapid or 

sweeping movement of the arms) the potential for contaminant release into the 

laboratory work environment is increased, as is the risk of product contamination.

The Class II (Types A1, A2, B1 and B2)8

 BSCs provide personnel, 

environmental and product protection. Airflow is drawn into the front grille of 
294 Biosafety in Microbiological and Biomedical Laboratories

the cabinet, providing personnel protection. In addition, the downward flow of 

HEPA-filtered air provides product protection by minimizing the chance of crosscontamination across the work surface of the cabinet. Because cabinet exhaust 

air is passed through a certified HEPA filter, it is particulate-free (environmental 

protection), and may be recirculated to the laboratory (Type A1 and A2 BSCs) or 

discharged from the building via a canopy or “thimble” connected to the building 

exhaust. Exhaust air from Types B1 and B2 BSCs must be discharged directly to 

the outdoors via a hard connection.

HEPA filters are effective at trapping particulates and thus infectious agents 

but do not capture volatile chemicals or gases. Only Type A2-exhausted or Types 

B1and B2 BSCs exhausting to the outside should be used when working with 

volatile, toxic chemicals, but amounts must be limited (Table 2).

All Class II cabinets are designed for work involving microorganisms 

assigned to biosafety levels 1, 2, 3 and 4.1 Class II BSCs provide the microbefree work environment necessary for cell culture propagation and also may be 

used for the formulation of nonvolatile antineoplastic or chemotherapeutic drugs.9

Class II BSCs may be used with organisms requiring BSL-4 containment in a 

BSL-4 suit laboratory by a worker wearing a positive pressure protective suit.

1. The Class II, Type A1 BSC: An internal fan (Figure 3) draws sufficient 

room air through the front grille to maintain a minimum calculated or 

measured average inflow velocity of at least 75 lfm at the face opening 

of the cabinet. The supply air flows through a HEPA filter and provides 

particulate-free air to the work surface. Airflow provided in this manner 

reduces turbulence in the work zone and minimizes the potential for 

cross-contamination.

The downward moving air “splits” as it approaches the work surface; the 

fan6

 draws part of the air to the front grille and the remainder to the rear 

grille. Although there are variations among different cabinets, this split 

generally occurs about halfway between the front and rear grilles and 

two to six inches above the work surface.

The air is drawn through the front and rear grilles by a fan pushed into 

the space between the supply and exhaust filters. Due to the relative 

size of these two filters, approximately 30% of the air passes through 

the exhaust HEPA filter and 70% recirculates through the supply HEPA 

filter back into the work zone of the cabinet. Most Class II, Type A1 

and A2 cabinets have dampers to modulate this division of airflow.

A Class II Type A1 BSC is not to be used for work involving volatile 

toxic chemicals. The buildup of chemical vapors in the cabinet (by 

recirculated air) and in the laboratory (from exhaust air) could create 

health and safety hazards (See Section IV).
Appendix A: Biological Safety Cabinets 295

It is possible to exhaust the air from a Type A1 or A2 cabinet outside 

of the building. However, it must be done in a manner that does not 

alter the balance of the cabinet exhaust system, thereby disturbing the 

internal cabinet airflow. The proper method of connecting a Type A1 or 

A2 cabinet to the building exhaust system is through use of a canopy 

hood,8,10 which provides a small opening or air gap (usually 1 inch) 

around the cabinet exhaust filter housing (Figure 4). The airflow of the 

building exhaust must be sufficient to maintain the flow of room air into 

the gap between the canopy unit and the filter housing. The canopy 

must be removable or be designed to allow for operational testing of the 

cabinet. (See Section VI.) Class II Type A1 or A2 cabinets should never be 

hard-ducted to the building exhaust system.8

 Fluctuations in air volume 

and pressure that are common to all building exhaust systems sometimes 

make it difficult to match the airflow requirements of the cabinet. 

2. The Class II, Type B1 BSC: Some biomedical research requires the use 

of small quantities of hazardous chemicals, such as organic solvents or 

carcinogens. Carcinogens used in cell culture or microbial systems 

require both biological and chemical containment.11

The Class II, Type B cabinet originated with the National Cancer 

Institute (NCI)-designed Type 212 (later called Type B) BSC (Figure 5A), 

and was designed for manipulations of minute quantities of hazardous 

chemicals with in vitro biological systems. The NSF International NSF/

ANSI Standard 49—2007 definition of Type B1 cabinets8

 includes this 

classic NCI design Type B, and cabinets without supply HEPA filters 

located immediately below the work surface (Figure 5B), and/or those 

with exhaust/recirculation down flow splits other than exactly 70/30%.

The cabinet supply blowers draw room air (plus a portion of the cabinet’s 

recirculated air) through the front grille and through the supply HEPA 

filters located immediately below the work surface. This particulate-free 

air flows upward through a plenum at each side of the cabinet and then 

downward to the work area through a backpressure plate. In some 

cabinets, there is an additional supply HEPA filter to remove particulates 

that may be generated by the blower-motor system.

Room air is drawn through the face opening of the cabinet at a minimum 

measured inflow velocity of 100 lfm. As with the Type A1 and A2 cabinets, 

there is a split in the down-flowing air stream just above the work surface. 

In the Type B1 cabinet, approximately 70 percent of the down flow air 

exits through the rear grille, passes through the exhaust HEPA filter, and 

is discharged from the building. The remaining 30 percent of the down 

flow air is drawn through the front grille. Since the air that flows to the 

rear grille is discharged into the exhaust system, activities that may 
296 Biosafety in Microbiological and Biomedical Laboratories

generate hazardous chemical vapors or particulates should be 

conducted toward the rear of the cabinetwork area.13

Type B1 cabinets must be hard-ducted, preferably to a dedicated, 

independent exhaust system. As indicated earlier, fans for laboratory 

exhaust systems should be located at the terminal end of the ductwork 

to avoid pressuring the exhaust ducts. A failure in the building exhaust 

system may not be apparent to the user, as the supply blowers in the 

cabinet will continue to operate. A pressure-independent monitor and 

alarm should be installed to provide warning and shut off the BSC 

supply fan, should failure in exhaust airflow occur. Since this feature is 

not supplied by all cabinet manufacturers, it is prudent to install a sensor 

such as a flow monitor and alarm in the exhaust system as necessary. 

To maintain critical operations, laboratories using Type B1 BSCs should 

connect the exhaust blower to the emergency power supply.

3. The Class II, Type B2 BSC: This BSC is a total-exhaust cabinet; no air is 

recirculated within it (Figure 6). This cabinet provides simultaneous primary 

biological and chemical (small quantity) containment. Consideration 

must be given to the chemicals used in BSCs as some chemicals can 

destroy the filter medium, housings and/or gaskets causing loss of 

containment. The supply blower draws either room or outside air in at 

the top of the cabinet, passes it through a HEPA filter and down into the 

work area of the cabinet. The building exhaust system draws air through 

both the rear and front grills, capturing the supply air plus the additional 

amount of room air needed to produce a minimum calculated or measured 

inflow face velocity of 100 lfm. All air entering this cabinet is exhausted, 

and passes through a HEPA filter (and perhaps some other air-cleaning 

device such as a carbon filter if required for the work being performed) 

prior to discharge to the outside. This cabinet exhausts as much as 

1200 cubic feet per minute of conditioned room air making this cabinet 

expensive to operate. The higher static air pressure required to operate 

this cabinet also results in additional costs associated with heavier gauge 

ductwork and higher capacity exhaust fan. Therefore, the need for the 

Class II, Type B2 should be justified by the research to be conducted.

Should the building exhaust system fail, the cabinet will be pressurized, 

resulting in a flow of air from the work area back into the laboratory. 

Cabinets built since the early 1980’s usually have an interlock system, 

installed by the manufacturer, to prevent the supply blower from 

operating whenever the exhaust flow is insufficient; systems can be 

retrofitted if necessary. Exhaust air movement should be monitored 

by a pressure-independent device, such as a flow monitor.

4. The Class II, Type A2 BSC (Formerly called A/B3): Only when this BSC 

(Figure 7) is ducted to the outdoors does it meet the requirements of the 
Appendix A: Biological Safety Cabinets 297

former Class II Type B3.8 The Type A2 cabinet has a minimum calculated 

or measured inflow velocity of 100 lfm. All positive pressure contaminated 

plenums within the cabinet are surrounded by a negative air pressure 

plenum thus ensuring that any leakage from a contaminated plenum will 

be drawn into the cabinet and not released to the environment. Minute 

quantities of volatile toxic chemicals or radionuclides can be used in a 

Type A2 cabinet only if it exhausts to the outside via a properly 

functioning canopy connection.8

5. Special Applications: Class II BSCs can be modified to accommodate 

special tasks. For example, the front sash can be modified by the 

manufacturer to accommodate the eyepieces of a microscope. The 

work surface can be designed to accept a carboy, a centrifuge or other 

equipment that may require containment. A rigid plate with openings for 

the arms can be added if needed. Good cabinet design, microbiological 

aerosol tracer testing of the modification and appropriate certification 

(see Section VII) are required to ensure that the basic systems operate 

properly after modification. Maximum containment potential is achieved 

only through strict adherence to proper practices and procedures 

(see Section V).

The Class III BSC

The Class III BSC (Figure 8) was designed for work with highly infectious 

microbiological agents and for the conduct of hazardous operations and provides 

maximum protection for the environment and the worker. It is a gas-tight (no leak 

greater than 1x10-7 cc/sec with 1% test gas at 3 inches pressure Water Gauge14) 

enclosure with a non-opening view window. Access for passage of materials into 

the cabinet is through a dunk tank, that is accessible through the cabinet floor, or 

double-door pass-through box (e.g., an autoclave) that can be decontaminated 

between uses. Reversing that process allows materials to be removed from the 

Class III BSC safely. Both supply and exhaust air are HEPA filtered on a Class III 

cabinet. Exhaust air must pass through two HEPA filters, or a HEPA filter and an 

air incinerator, before discharge directly to the outdoors. Class III cabinets are not 

exhausted through the general laboratory exhaust system. Airflow is maintained 

by an exhaust system exterior to the cabinet, which keeps the cabinet under 

negative pressure (minimum of 0.5 inches of water gauge.) 

Long, heavy-duty rubber gloves are attached in a gas-tight manner to ports 

in the cabinet to allow direct manipulation of the materials isolated inside. Although 

these gloves restrict movement, they prevent the user’s direct contact with the 

hazardous materials. The trade-off is clearly on the side of maximizing personal 

safety. Depending on the design of the cabinet, the supply HEPA filter provides 

particulate-free, albeit somewhat turbulent, airflow within the work environment. 

Laminar airflow is not a characteristic of a Class III cabinet.
298 Biosafety in Microbiological and Biomedical Laboratories

Several Class III BSCs can be joined together in a “line” to provide a larger 

work area. Such cabinet lines are custom-built; the equipment installed in the 

cabinet line (e.g., refrigerators, small elevators, shelves to hold small animal cage 

racks, microscopes, centrifuges, incubators) is generally custom-built as well.

Horizontal Laminar Flow “Clean Bench”

Horizontal laminar flow “clean benches” (Figure 9A) are not BSCs. These pieces 

of equipment discharge HEPA-filtered air from the back of the cabinet across the 

work surface and toward the user. These devices only provide product protection. 

They can be used for certain clean activities, such as the dust-free assembly of 

sterile equipment or electronic devices. Clean benches should never be used 

when handling cell culture materials, drug formulations, potentially infectious 

materials, or any other potentially hazardous materials. The worker will be 

exposed to the materials being manipulated on the clean bench potentially 

resulting in hypersensitivity, toxicity or infection depending on the materials being 

handled. Horizontal airflow “clean benches” must never be used as a substitute 

for a biological safety cabinet. Users must be aware of the differences between 

these two devices.

Vertical Flow “Clean Bench”

Vertical flow clean benches (Figure 9B) also are not BSCs. They may be useful, 

for example, in hospital pharmacies when a clean area is needed for preparation 

of intravenous solutions. While these units generally have a sash, the air is usually 

discharged into the room under the sash, resulting in the same potential problems 

presented by the horizontal laminar flow clean benches. These benches should 

never be used for the manipulation of potentially infectious or toxic materials or 

for preparation of antineoplastic agents.

Section IV—Other Laboratory Hazards and Risk Assessment

Primary containment is an important strategy in minimizing exposure to the many 

chemical, radiological and biological hazards encountered in the laboratory. An 

overview is provided, in Table 2, of the various classes of BSCs, the level of 

containment afforded by each and the appropriate risk assessment considerations. 

Microbiological risk assessment is addressed in depth in BMBL.1

Working with Chemicals in BSCs

Work with infectious microorganisms often requires the use of various chemical 

agents, and many commonly used chemicals vaporize easily. Therefore, 

evaluation of the inherent hazards of the chemicals must be part of the risk 

assessment when selecting a BSC. Flammable chemicals should not be used 

in Class II, Type A1 or A2 cabinets since vapor buildup inside the cabinet presents 

a fire hazard. In order to determine the greatest chemical concentration, which 

might be entrained in the air stream following an accident or spill, it is necessary 
Appendix A: Other Laboratory Hazards and Risk Assessment 299

to evaluate the quantities to be used. Mathematical models are available to assist 

in these determinations.13 For more information regarding the risks associated 

with exposure to chemicals, the reader should consult the Threshold Limit Values 

(TLVs) for various chemical substances established by the American Conference 

of Governmental Industrial Hygienists.15

The electrical systems of Class II BSCs are not spark-proof. Therefore, a 

chemical concentration approaching the lower explosive limits of the compound 

must be prohibited. Furthermore, since non-exhausted Class II, Type A1 and A2 

cabinets return chemical vapors to the cabinetwork space and the room, they 

may expose the operator and other room occupants to toxic chemical vapors.

A chemical fume hood should be used for procedures using volatile chemicals 

instead of a BSC. Chemical fume hoods are connected to an independent exhaust 

system and operate with single-pass air discharged, directly or through a manifold, 

outside the building. They may also be used when manipulating chemical 

carcinogens.11 When manipulating small quantities of volatile toxic chemicals 

required for use in microbiological studies, Class I and Class II (Type B2) BSCs, 

exhausted to the outdoors, can be used. The Class II, Type B1 and A2 canopyexhausted cabinets may be used with minute or tracer quantities of nonvolatile 

toxic chemicals.8

Many liquid chemicals, including nonvolatile antineoplastic agents, 

chemotherapeutic drugs and low-level radionuclides, can be safely handled 

inside Class II, Type A cabinets.9 Class II BSCs should not be used for labeling 

of biohazardous materials with radioactive iodine. Hard-ducted, ventilated 

containment devices incorporating both HEPA and charcoal filters in the 

exhaust systems are necessary for the conduct of this type of work (Figure 10).

Many virology and cell culture laboratories use diluted preparations of 

chemical carcinogens11,16 and other toxic substances. Prior to maintenance, 

careful evaluation must be made of potential problems associated with 

decontaminating the cabinet and the exhaust system. Air treatment systems, 

such as a charcoal filter in a bag-in/bag-out housing,17 (Figure 13) may be 

required so that discharged air meets applicable emission regulations.

National Sanitation Foundation (NSF)/ANSI Standard 49—20078

 requires 

biologically-contaminated ducts and plenums of Class II, Type A2 and B cabinets 

be maintained under negative air pressure, or surrounded by negative pressure 

ducts and plenums.

Radiological Hazards in the BSC

As indicated above, volatile radionuclides such as I125 should not be used within 

Class II BSCs. When using nonvolatile radionuclides inside a BSC, the same 

hazards exist as if working with radioactive materials on the bench top. Work that 

has the potential for splatter or creation of aerosols can be done within the BSC. 
300 Biosafety in Microbiological and Biomedical Laboratories

Radiologic monitoring must be performed. A straight, vertical (not sloping) beta 

shield may be used inside the BSC to provide worker protection. A sloping shield 

can disrupt the air curtain and increase the possibility of contaminated air being 

released from the cabinet. A radiation safety professional should be contacted for 

specific guidance.

Risk Assessment

The potential for adverse events must be evaluated to eliminate or reduce to the 

greatest extent possible worker exposure to infectious organisms and to prevent 

release to the environment. Agent summary statements detailed in BMBL1

 provide 

data for microorganisms known to have caused laboratory-associated infections 

that may be used in protocol-driven risk assessment. Through the process of risk 

assessment, the laboratory environment and the work to be conducted are 

evaluated to identify hazards and develop interventions to ameliorate risks.

A properly certified and operational BSC is an effective engineering control 

(see Section VI) that must be used in concert with the appropriate practices, 

procedures and other administrative controls to further reduce the risk of exposure 

to potentially infectious microorganisms. Suggested work practices and procedures 

for minimizing risks when working in a BSC are detailed in the next section.

Section V — BSC Use by the Investigator: Work Practices and Procedures

Preparing for Work Within a Class II BSC

Preparing a written checklist of materials necessary for a particular activity and 

placing necessary materials in the BSC before beginning work serves to minimize 

the number and extent of air curtain disruptions compromising the fragile air 

barrier of the cabinet. The rapid movement of a worker’s arms in a sweeping 

motion into and out of the cabinet will disrupt the air curtain and compromise the 

partial containment barrier provided by the BSC. Moving arms in and out slowly, 

perpendicular to the face opening of the cabinet will reduce this risk. Other 

personnel activities in the room (e.g., rapid movements near the face of the 

cabinet, walking traffic, room fans, open/closing room doors) may also disrupt 

the cabinet air barrier.6

Laboratory coats should be worn buttoned over street clothing; latex, vinyl, 

nitrile or other suitable gloves are worn to provide hand protection. Increasing 

levels of PPE may be warranted as determined by an individual risk assessment. 

For example, a solid front, back-closing laboratory gown provides better 

protection of personal clothing than a traditional laboratory coat and is a 

recommended practice at BSL-3.

Before beginning work, the investigator should adjust the stool height so 

that his/her face is above the front opening. Manipulation of materials should be 

delayed for approximately one minute after placing the hands/arms inside the 
Appendix A: BSC Use by the Investigator: Work Practices and Procedures 301

cabinet. This allows the cabinet to stabilize, to “air sweep” the hands and arms, 

and to allow time for turbulence reduction. When the user’s arms rest flatly 

across the front grille, occluding the grille opening, room air laden with particles 

may flow directly into the work area, rather than being drawn down through the 

front grille. Raising the arms slightly will alleviate this problem. The front grille 

must not be blocked with toweling, research notes, discarded plastic wrappers, 

pipetting devices, etc. All operations should be performed on the work surface at 

least four inches in from the front grille. If there is a drain valve under the work 

surface, it should be closed prior to beginning work in the BSC.

Materials or equipment placed inside the cabinet may cause disruption of 

the airflow, resulting in turbulence, possible cross-contamination and/or breach 

of containment. Extra supplies (e.g., additional gloves, culture plates or flasks, 

culture media) should be stored outside the cabinet. Only the materials and 

equipment required for the immediate work should be placed in the BSC.

BSCs are designed for 24-hour per day operation and some investigators 

find that continuous operation helps to control the laboratory’s level of dust and 

other airborne particulates. Although energy conservation may suggest BSC 

operation only when needed, especially if the cabinet is not used routinely, room 

air balance is an overriding consideration. Air discharged through ducted BSCs 

must be considered in the overall air balance of the laboratory.

If the cabinet has been shut down, the blowers should be operated at least 

four minutes before beginning work to allow the cabinet to “purge.” This purge will 

remove any suspended particulates in the cabinet. The work surface, the interior 

walls (except the supply filter diffuser), and the interior surface of the window 

should be wiped with 70% ethanol (EtOH), a 1:100 dilution of household bleach 

(i.e., 0.05% sodium hypochlorite), or other disinfectant as determined by the 

investigator to meet the requirements of the particular activity. When bleach 

is used, a second wiping with sterile water is needed to remove the residual 

chlorine, which may eventually corrode stainless steel surfaces. Wiping with 

non-sterile water may recontaminate cabinet surfaces, a critical issue when 

sterility is essential (e.g., maintenance of cell cultures).

Similarly, the surfaces of all materials and containers placed into the cabinet 

should be wiped with 70% EtOH to reduce the introduction of contaminants to the 

cabinet environment. This simple step will reduce introduction of mold spores and 

thereby minimize contamination of cultures. Further reduction of microbial load 

on materials to be placed or used in BSCs may be achieved by periodic 

decontamination of incubators and refrigerators.

Material Placement Inside the BSC

Plastic-backed absorbent toweling can be placed on the work surface but not on 

the front or rear grille openings. The use of toweling facilitates routine cleanup 
302 Biosafety in Microbiological and Biomedical Laboratories

and reduces splatter and aerosol generation19 during an overt spill. It can be 

folded and placed in a biohazard bag or other appropriate receptacle when 

work is completed.

All materials should be placed as far back in the cabinet as practical, toward 

the rear edge of the work surface and away from the front grille of the cabinet 

(Figure 11). Similarly, aerosol-generating equipment (e.g., vortex mixers, tabletop 

centrifuges) should be placed toward the rear of the cabinet to take advantage of 

the air split described in Section III. Bulky items such as biohazard bags, discard 

pipette trays and vacuum collection flasks should be placed to one side of the 

interior of the cabinet. If placing those items in the cabinet requires opening the 

sash, make sure that the sash is returned to its original position before work is 

initiated. The correct sash position (usually 8 ̋ or 10 ̋ above the base of the opening) 

should be indicated on the front of the cabinet. On most BSCs, an audible alarm 

will sound if the sash is in the wrong position while the fan is operating.

Certain common practices interfere with the operation of the BSC. The 

biohazard collection bag should not be taped to the outside of the cabinet. 

Upright pipette collection containers should not be used in BSCs nor placed on 

the floor outside the cabinet. The frequent inward/outward movement needed 

to place objects in these containers is disruptive to the integrity of the cabinet 

air barrier and can compromise both personnel and product protection. Only 

horizontal pipette discard trays containing an appropriate chemical disinfectant 

should be used within the cabinet. Furthermore, potentially contaminated 

materials should not be brought out of the cabinet until they have been surface 

decontaminated. Alternatively, contaminated materials can be placed into a closable 

container for transfer to an incubator, autoclave or another part of the laboratory.

Operations Within a Class II BSC

Laboratory Hazards

Many procedures conducted in BSCs may create splatter or aerosols. Good 

microbiological techniques should always be used when working in a BSC. For 

example, techniques used to reduce splatter and aerosol generation will also 

minimize the potential for personnel exposure to infectious materials manipulated 

within the cabinet. Class II cabinets are designed so that horizontally nebulized 

spores introduced into the cabinet will be captured by the downward flowing 

cabinet air within fourteen inches8

 of travel. Therefore, as a general rule of 

thumb, keeping clean materials at least one foot away from aerosol-generating 

activities will minimize the potential for cross-contamination.

The workflow should be from “clean to dirty” (Figure 11). Materials and 

supplies should be placed in the cabinet in such a way as to limit the movement 

of “dirty” items over “clean” ones.
Appendix A: BSC Use by the Investigator: Work Practices and Procedures 303

Several measures can be taken to reduce the chance for cross-contamination 

of materials when working in a BSC. Opened tubes or bottles should not be held 

in a vertical position. Investigators working with Petri dishes and tissue culture 

plates should hold the lid above the open sterile surface to minimize direct 

impaction of downward air. Bottle or tube caps should not be placed on the 

toweling. Items should be recapped or covered as soon as possible.

Open flames are not required in the near microbe-free environment of a 

biological safety cabinet. On an open bench, flaming the neck of a culture vessel 

will create an upward air current that prevents microorganisms from falling into 

the tube or flask. An open flame in a BSC, however, creates turbulence that 

disrupts the pattern of HEPA-filtered air being supplied to the work surface. When 

deemed absolutely necessary, touch-plate micro burners equipped with a pilot 

light to provide a flame on demand may be used. Internal cabinet air disturbance 

and heat buildup will be minimized. The burner must be turned off when work 

is completed. Small electric “furnaces” are available for decontaminating 

bacteriological loops and needles and are preferable to an open flame inside the 

BSC. Disposable or recyclable sterile loops should be used whenever possible.

Aspirator bottles or suction flasks should be connected to an overflow 

collection flask containing appropriate disinfectant, and to an in-line HEPA or 

equivalent filter (Figure 12). This combination will provide protection to the 

central building vacuum system or vacuum pump, as well as to the personnel 

who service this equipment. Inactivation of aspirated materials can be accomplished 

by placing sufficient chemical decontamination solution into the flask to inactivate 

the microorganisms as they are collected. Once inactivation occurs, liquid 

materials can be disposed of as noninfectious waste.

Investigators must determine the appropriate method of decontaminating 

materials that will be removed from the BSC at the conclusion of the work. When 

chemical means are appropriate, suitable liquid disinfectant should be placed into 

the discard pan before work begins. Items should be introduced into the pan with 

minimum splatter and allowed appropriate contact time as per manufacturer’s 

instructions. Alternatively, liquids can be autoclaved prior to disposal. Contaminated 

items should be placed into a biohazard bag, discard tray, or other suitable 

container prior to removal from the BSC.

When a steam autoclave is used, contaminated materials should be placed 

into a biohazard bag or discard pan containing enough water to ensure steam 

generation during the autoclave cycle. The bag should be taped shut or the 

discard pan should be covered in the BSC prior to transfer to the autoclave. 

The bag should be transported and autoclaved in a leak proof tray or pan. 

It is a prudent practice to decontaminate the exterior surface of bags and 

pans just prior to removal from the cabinet.
304 Biosafety in Microbiological and Biomedical Laboratories

Decontamination

Cabinet Surface Decontamination

With the cabinet blower running, all containers and equipment should be surface 

decontaminated and removed from the cabinet when work is completed. At the 

end of the workday, the final surface decontamination of the cabinet should 

include a wipe-down of the work surface, the cabinet’s sides and back and the 

interior of the glass. If necessary, the cabinet should also be monitored for 

radioactivity and decontaminated when necessary. Investigators should remove 

their gloves and gowns in a manner to prevent contamination of unprotected 

skin and aerosol generation and wash their hands as the final step in safe 

microbiological practices. The cabinet blower may be turned off after these 

operations are completed, or left on.

Small spills within the operating BSC can be handled immediately by 

removing the contaminated absorbent paper toweling and placing it into the 

biohazard bag or receptacle. Any splatter onto items within the cabinet, as well 

as the cabinet interior, should be immediately cleaned up with a towel dampened 

with an appropriate decontaminating solution. Gloves should be changed after the 

work surface is decontaminated and before placing clean absorbent toweling in 

the cabinet. Hands should be washed whenever gloves are changed or removed.

Spills large enough to result in liquids flowing through the front or rear grilles 

require decontamination that is more extensive. All items within the cabinet 

should be surface decontaminated and removed. After ensuring that the drain 

valve is closed, decontaminating solution can be poured onto the work surface 

and through the grille(s) into the drain pan.

Twenty to 30 minutes is generally considered an appropriate contact time for 

decontamination, but this varies with the disinfectant and the microbiological 

agent. Manufacturer’s directions should be followed. The spilled fluid and 

disinfectant solution on the work surface should be absorbed with paper towels 

and discarded into a biohazard bag. The drain pan should be emptied into a 

collection vessel containing disinfectant. A hose barb and flexible tube should be 

attached to the drain valve and be of sufficient length to allow the open end to be 

submerged in the disinfectant within the collection vessel. This procedure serves 

to minimize aerosol generation. The drain pan should be flushed with water and 

the drain tube removed.

Should the spilled liquid contain radioactive material, a similar procedure can be 

followed. Radiation safety personnel should be contacted for specific instructions.

Periodic removal of the cabinetwork surface and/or grilles after the completion 

of drain pan decontamination may be justified because of dirty drain pan surfaces 

and grilles, which ultimately could occlude the drain valve or block airflow. However, 

extreme caution should be observed on wiping these surfaces to avoid injury 
Appendix A: Facility and Engineering Requirements 305

from broken glass that may be present and sharp metal edges. Always use 

disposable paper toweling and avoid applying harsh force. Wipe dirty surfaces 

gently. Never leave toweling on the drain pan because the paper could block 

the drain valve or the air passages in the cabinet.

Gas Decontamination

BSCs that have been used for work involving infectious materials must be 

decontaminated before HEPA filters are changed or internal repair work is 

done.20-23 Before a BSC is relocated, a risk assessment considering the agents 

manipulated within the BSC must be performed to determine the need and 

method for decontamination. The most common decontamination method uses 

formaldehyde gas, although more recently, hydrogen peroxide vapor21 and 

chlorine dioxide gas have been used successfully.

Section VI—Facility and Engineering Requirements

Secondary Barriers

Whereas BSCs are considered to be the primary containment barrier for 

manipulation of infectious materials, the laboratory room itself is considered to 

be the secondary containment barrier.24 Inward directional airflow is established25

by exhausting a greater volume of air than is supplied to a given laboratory and 

by drawing makeup air from the adjacent space. This is optional at BSL-2 but 

must be maintained at BSL-3 and BSL-4.1,26 The air balance for the entire facility 

should be established and maintained to ensure that airflow is from areas of 

least to greater potential contamination.

Building Exhaust

At BSL-3 and BSL-4, exhaust laboratory air must be directly exhausted to the 

outside since it is considered potentially contaminated. This concept is referred 

to as a dedicated, single-pass exhaust system. The exhausted room air can be 

HEPA-filtered when a high level of aerosol containment is needed, which is 

always true at BSL-4 and may be optional at BSL-3. When the building exhaust 

system is used to vent a ducted BSC, the system must have sufficient capacity 

to maintain the exhaust flow if changes in the static pressure within the system 

should occur. The connection to a BSC must be constant air volume (CAV). 

The HVAC exhaust system must be sized to handle both the room exhaust and 

the exhaust requirements of all containment devices that may be present. Adequate 

supply air must be provided to ensure appropriate function of the exhaust system. 

Right angle bends changing duct diameters and transitional connections within 

the systems will add to the demand on the exhaust fan. The building exhaust air 

should be discharged away from supply air intakes, to prevent re-entrainment of 

laboratory exhaust air into the building air supply system. Refer to recognized 

design guides for locating the exhaust terminus relative to nearby air intakes.27
306 Biosafety in Microbiological and Biomedical Laboratories

Utility Services

Utility services needed within a BSC must be planned carefully. Protection of vacuum 

systems must be addressed (Figure 12). Electrical outlets inside the cabinet must 

be protected by ground fault circuit interrupters and should be supplied by an 

independent circuit. When propane or natural gas is provided, a clearly marked 

emergency gas shut-off valve outside the cabinet must be installed for fire safety. 

All non-electrical utility services should have exposed, accessible shut-off valves. 

The use of compressed air within a BSC must be carefully considered and controlled 

to prevent aerosol production and reduce the potential for vessel pressurization.

Ultraviolet Lamps

Ultraviolet (UV) lamps are not recommended in BSCs8

 nor are they necessary. 

If installed, UV lamps must be cleaned weekly to remove any dust and dirt that 

may block the germicidal effectiveness of the ultraviolet light. The lamps should be 

checked weekly with a UV meter to ensure that the appropriate intensity of UV light 

is being emitted. UV lamps must be turned off when the room is occupied to protect 

eyes and skin from UV exposure, which can burn the cornea and cause skin cancer. 

If the cabinet has a sliding sash, close the sash when operating the UV lamp.

BSC Placement

BSCs were developed (see Section I) as workstations to provide personnel, 

environmental and product protection during the manipulation of infectious 

microorganisms. Certain considerations must be met to ensure maximum 

effectiveness of these primary barriers. Whenever possible, adequate clearance 

should be provided behind and on each side of the cabinet to allow easy access 

for maintenance and to ensure that the cabinet air re-circulated to the laboratory 

is not hindered. A 12 to 14 inch clearance above the cabinet may be required 

to provide for accurate air velocity measurement across the exhaust filter 

surface28,29 and for exhaust filter changes. When the BSC is hard-ducted or 

connected by a canopy unit to the ventilation system, adequate space must be 

provided so that the configuration of the ductwork will not interfere with airflow. 

The canopy unit must provide adequate access to the exhaust HEPA filter for testing.

The ideal location for the biological safety cabinet is remote from the entry 

(i.e., the rear of the laboratory away from traffic), since people walking parallel to the 

face of a BSC can disrupt the air curtain.16,20,30 The air curtain created at the front 

of the cabinet is quite fragile, amounting to a nominal inward and downward 

velocity of 1 mph. Open windows, air supply registers, portable fans or laboratory 

equipment that creates air movement (e.g., centrifuges, vacuum pumps) should 

not be located near the BSC. Similarly, chemical fume hoods must not be located 

close to BSCs.
Appendix A: Certification of BSCs 307

HEPA Filters

HEPA filters, whether part of a building exhaust system or part of a cabinet, will 

require replacement when they become loaded to the extent that sufficient airflow 

can no longer be maintained. In most instances, filters must be decontaminated 

before removal. To contain the formaldehyde gas typically used for microbiological 

decontamination, exhaust systems containing HEPA filters require airtight dampers 

to be installed on both the inlet and discharge side of the filter housing. This 

ensures containment of the gas inside the filter housing during decontamination. 

Access panel ports in the filter housing also allow for performance testing of the 

HEPA filter. (See Section VII.)

A bag-in/bag-out filter assembly3,17 (Figure 13) can be used in situations 

where HEPA filtration is necessary for operations involving biohazardous 

materials and hazardous or toxic chemicals. The bag-in/bag-out system is used 

when it is not possible to gas or vapor decontaminate the HEPA filters, or when 

hazardous chemicals or radionuclides have been used in the BSC, and provides 

protection against exposure for the maintenance personnel and the environment. 

Note, however, that this requirement must be identified at the time of purchase 

and installation; a bag-in/bag-out assembly cannot be added to a cabinet afterthe-fact without an extensive engineering evaluation.

Section VII — Certification of BSCs

Development of Containment Standards

The evolution of containment equipment for varied research and diagnostic 

applications created the need for consistency in construction and performance. 

Federal Standard 209a,32,33 was developed to establish classes of air cleanliness 

and methods for monitoring clean workstations and clean rooms where HEPA 

filters are used to control airborne particulates.

The first “standard” to be developed specifically for BSCs12 served as a 

Federal procurement specification for the NIH Class II, Type 1 (now called Type A1) 

BSC, which had a fixed or hinged front window or a vertical sliding sash, vertical 

downward airflow and HEPA-filtered supply and exhaust air. This specification 

described design criteria and defined prototype tests for microbiological aerosol 

challenge, velocity profiles, and leak testing of the HEPA filters. A similar 

procurement specification was generated31 when the Class II, Type 2 (now 

called Type B1) BSC was developed.

a Federal Standard No. 209E9 has been replaced by ISO 14644. This standard does not apply 

to BSCs and should not be considered a basis for their performance or integrity certification. 

However, the methodology of ISO 14644 can be used to quantify the particle count within 

the work area of a BSC. ISO 14644 defines how to classify a clean room/clean zone. 

Performance tests and procedures needed to achieve a specified cleanliness classification 

are outlined by the Institute of Environmental Sciences and Technology’s IEST-RP-CC-006.
308 Biosafety in Microbiological and Biomedical Laboratories

National Sanitation Foundation (NSF) Standard #49 for Class II BSCs 

was first published in 1976, providing the first independent standard for design, 

manufacture and testing of BSCs. This standard “replaced” the NIH specifications, 

which were being used by other institutions and organizations purchasing BSCs. 

NSF/ANSI Standard 49—2007b,8 incorporates current specifications regarding 

design, materials, construction, and testing. This Standard for BSCs establishes 

performance criteria and provides the minimum testing requirements that are 

accepted in the United States. Cabinets, which meet the Standard and are 

certified by NSF bear an NSF Mark.

NSF/ANSI Standard 49—2007 pertains to all models of Class II cabinets 

(Type A1, A2, B1, B2) and provides a series of specifications regarding:

■ Design/construction

■ Performance

■ Installation recommendations

■ Recommended microbiological decontamination procedure

■ References and specifications pertinent to Class II Biosafety Cabinetry

Annex F of NSF/ANSI Standard 49—2007, which covers field-testing of 

BSCs, is now a normative part of the Standard. This Standard is reviewed 

periodically by a committee of experts to ensure that it remains consistent 

with developing technologies.

The operational integrity of a BSC must be validated before it is placed into 

service and after it has been repaired or relocated. Relocation may break the 

HEPA filter seals or otherwise damage the filters or the cabinet. Each BSC should 

be tested and certified at least annually to ensure continued, proper operation.

On-site field-testing (NSF/ANSI Standard 49—2007 Annex F plus Addendum 

#1) must be performed by experienced, qualified personnel. Some basic information 

is included in the Standard to assist in understanding the frequency and kinds of 

tests to be performed. In 1993, NSF began a program for accreditation of certifiers 

based on written and practical examinations. Education and training programs for 

persons seeking accreditation as qualified to perform all field certification tests 

are offered by a variety of organizations. Selecting competent individuals to 

perform testing and certification is important. It is suggested that the institutional 

BSO be consulted when identifying companies qualified to conduct the necessary 

field performance tests.

b The standard can be ordered from the NSF for a nominal fee at NSF International, 789 

North Dixboro Road, P.O. Box 130140, Ann Arbor, Michigan, 48113-0140; Telephone: 

734-769-8010; Fax: 734-769-0190; e-mail: info@nsf.org; Telex 753215 NSF INTL.
Appendix A: Certification of BSCs 309

It is strongly recommended that, whenever possible, accredited field certifiers 

are used to test and certify BSCs. If in-house personnel are performing the 

certifications, then these individuals should become accredited.

The annual tests applicable to each of the three classes of BSCs are listed 

in Table 3. Table 4 indicates where to find information regarding the conduct of 

selected tests. BSCs consistently perform well when proper annual certification 

procedures are followed; cabinet or filter failures tend to occur infrequently.

Performance Testing BSCs in the Field

Class II BSCs are the primary containment devices that protect the worker, product 

and environment from exposure to microbiological agents. BSC operation, as 

specified by NSF/ANSI Standard 49—2007, Annex F plus Addendum #1 needs 

to be verified at the time of installation and, as a minimum, annually thereafter. 

The purpose and acceptance level of the operational tests (Table 3) ensure the 

balance of inflow and exhaust air, the distribution of air onto the work surface, 

and the integrity of the cabinet and the filters. Other tests check electrical and 

physical features of the BSC.

A. Down flow Velocity Profile Test: This test is performed to measure the 

velocity of air moving through the cabinet workspace, and is to be 

performed on all Class II BSCs.

B. Inflow Velocity Test: This test is performed to determine the calculated 

or directly measured velocity through the work access opening, to verify 

the nominal set point average inflow velocity and to calculate the 

exhaust airflow volume rate.

C. Airflow Smoke Patterns Test: This test is performed to determine if: 

1) the airflow along the entire perimeter of the work access opening is 

inward; 2) if airflow within the work area is downward with no dead spots 

or refluxing; 3) if ambient air passes onto or over the work surface; and 

4) if there is no escape to the outside of the cabinet at the sides and top of 

the window. The smoke test is an indicator of airflow direction, not velocity.

D. HEPA Filter Leak Test: This test is performed to determine the integrity 

of supply and exhaust HEPA filters, filter housing and filter mounting 

frames while the cabinet is operated at the nominal set point velocities. 

An aerosol in the form of generated particulates of dioctylphthalate (DOP) 

or an accepted alternative (e.g., poly alpha olefin (PAO), di(2-ethylhexyl) 

sebecate, polyethylene glycol and medical grade light mineral oil) is 

required for leak-testing HEPA filters and their seals. The aerosol is 

generated on the intake side of the filter and particles passing through the 

filter or around the seal are measured with a photometer on the discharge 

side. This test is suitable for ascertaining the integrity of all HEPA filters.
310 Biosafety in Microbiological and Biomedical Laboratories

E. Cabinet Integrity Test (A1 Cabinets only): This pressure holding test is 

performed to determine if exterior surfaces of all plenums, welds, gaskets 

and plenum penetrations or seals are free of leaks. In the field, it need 

only be performed on Type A1 cabinets at the time of initial installation 

when the BSC is in a free-standing position (all four sides are easily 

accessible) in the room in which it will be used, after a cabinet has been 

relocated to a new location, and again after removal of access panels to 

plenums for repairs or a filter change. This test may also be performed 

on fully installed cabinets. Cabinet integrity can also be checked using 

the bubble test; liquid soap can be spread along welds, gaskets and 

penetrations to visualize air leaks that may occur.

F. Electrical Leakage and Ground Circuit Resistance and Polarity Tests:

Electrical testing has been taken out of NSF/ANSI 49 Standard—2007 

for new cabinets certified under the this Standard. This responsibility 

has been turned over to UL. All new cabinets must meet UL 61010A-1 

in order to be certified by NSF. These safety tests are performed to 

determine if a potential shock hazard exists by measuring the electrical 

leakage, polarity, ground fault interrupter function and ground circuit 

resistance to the cabinet connection. An electrical technician other than 

the field certification personnel may perform the tests at the same time 

the other field certification tests are conducted. The polarity of electrical 

outlets is checked (Table 3, E). The ground fault circuit interrupter 

should trip when approximately five milliamperes (mA) is applied.

G. Lighting Intensity Test: This test is performed to measure the light 

intensity on the work surface of the cabinet as an aid in minimizing 

cabinet operator fatigue.

H. Vibration Test: This test is performed to determine the amount of 

vibration in an operating cabinet as a guide to satisfactory mechanical 

performance, as an aid in minimizing cabinet operator fatigue and to 

prevent damage to delicate tissue culture specimens.

I. Noise Level Test: This test is performed to measure the noise levels 

produced by the cabinets, as a guide to satisfactory mechanical 

performance and an aid in minimizing cabinet operator fatigue.

J. UV Lamp Test: A few BSCs have UV lamps. When used, they must be 

tested periodically to ensure that their energy output is sufficient to kill 

microorganisms. The surface on the bulb should be cleaned with 70% 

ethanol prior to performing this test. Five minutes after the lamp has 

been turned on, the sensor of the UV meter is placed in the center of the 

work surface. The radiation output should not be less than 40 microwatts 

per square centimeter at a wavelength of 254 nanometers (nm).
Appendix A: Tables 311

Finally, accurate test results can only be assured when the testing equipment 

is properly maintained and calibrated. It is appropriate to request the calibration 

information for the test equipment being used by the certifier.

Table 1. Selection of a Safety Cabinet through Risk Assessment

Biological Risk 

Assessed

Protection Provided

Personnel Product Environmental BSC Class

BSL 1 – 3 Yes No Yes I

BSL 1 – 3 Yes Yes Yes II (A1, A2, B1, B2)

BSL – 4 Yes Yes Yes III; II—When used in 

suit room with suit

Table 2. Comparison of Biosafety Cabinet Characteristics

BSC 

Class

Face 

Velocity Airflow Pattern

Applications

Nonvolatile 

Toxic 

Chemicals and 

Radionuclides

Volatile Toxic 

Chemicals and 

Radionuclides

I 75 In at front through HEPA to the 

outside or into the room through 

HEPA (Figure 2)

Yes When exhausted 

outdoors1,2

II, A1 75 70% recirculated to the cabinet 

work area through HEPA; 30% 

balance can be exhausted 

through HEPA back into the 

room or to outside through a 

canopy unit (Figure 3)

Yes

(minute 

amounts)

No

II, B1 100 30% recirculated, 70% 

exhausted. Exhaust cabinet air 

must pass through a dedicated 

duct to the outside through a 

HEPA filter (Figures 5A, 5B)

Yes Yes

(minute 

amounts)1,2

I, B2 100 No recirculation; total exhaust to 

the outside through a HEPA filter 

(Figure 6)

Yes Yes

(small amounts)1,2

II, A2 100 Similar to II, A1, but has 100 Ifm 

intake air velocity and plenums 

are under negative pressure to 

room; exhaust air can be ducted 

to the outside through a canopy 

unit (Figure 7)

Yes When exhausted 

outdoors 

(FORMALLY 

“B3”) (minute 

amounts)1,2
312 Biosafety in Microbiological and Biomedical Laboratories

BSC 

Class

Face 

Velocity Airflow Pattern

Applications

Nonvolatile 

Toxic 

Chemicals and 

Radionuclides

Volatile Toxic 

Chemicals and 

Radionuclides

III N/A Supply air is HELP filtered. 

Exhaust air passes through two 

HEPA filters in series and is 

exhausted to the outside via a 

hard connection (Figure 8)

Yes Yes

(small amounts)1,2

1 Installation requires a special duct to the outside, an in-line charcoal filter, and a spark proof 

(explosion proof) motor and other electrical components in the cabinet. Discharge of a Class I 

or Class II, Type A2 cabinet into a room should not occur if volatile chemicals are used. 2 In no instance should the chemical concentration approach the lower explosion limits of 

the compounds.

Table 3. Field Performance Tests Applied to the Three Classes of Biological 

Safety Cabinets

Test Performed for

Biosafety Cabinet

Class I Class II Class III

Primary Containment

Cabinet Integrity N/A A (A1 Only) A

HEPA Filter Leak Required Required Required

Down flow Velocity N/A Required N/A

Face Velocity Required Required N/A

Negative Pressure / Ventilation Rate B N/A Required

Airflow Smoke Patterns Required Required E, F

Alarms and Interlocks C, D C, D Required

Electrical Safety

Electrical Leakage, etc. E, D E, D E, D

Ground Fault Interrupter D D D
Appendix A: Tables 313

Test Performed for

Biosafety Cabinet

Class I Class II Class III

Other

Lighting Intensity E E E

UV Intensity C, E C, E C, E

Noise Level E E E

Vibration E E E

Required Required during certification.

A Required for proper certification if the cabinet is new, has been moved or panels 

have been removed for maintenance.

B If used with gloves.

C If present.

D Encouraged for electrical safety.

E Optional, at the discretion of the user.

F Used to determine air distribution within cabinet for clean to dirty procedures.

N/A Not applicable.

Table 4. Reference for Applicable Containment Test

Test

Biosafety Cabinet Type

Class I Class II Class III

HEPA Filter Leak (F.5)1 (F.5) (F.5)

Airflow Smoke Pattern No smoke shall 

reflux out of BSC 

once drawn in 

(F.4) N/A

Cabinet Integrity N/A (F.6) [p.138 – 141]2

Face Velocity Open Front [75-125 lfm] 75 lfm—type A1; 

100 lfm type A2, 

B1 & B2: (F.3)

N/A

Face VelocityGloves Ports / 

No Gloves

150 lfm N/A N/A

Water Gauge Pressure Glove 

Ports and Gloves

N/A N/A (-0.5 “w.c.”) [p. 

145]

Down flow Velocity N/A (F.2) N/A

1 Parenthetical references are to the NSF/ANSI Standard 49—2007; letters and numerals 

indicate specific sections and subsections. 2 Bracketed reference [ ] is to the Laboratory Safety Monograph; page numbers are indicated.
314 Biosafety in Microbiological and Biomedical Laboratories

Figure 1. HEPA filters are typically constructed of paper-thin sheets of 

borosilicate medium, pleated to increase surface area, and affixed to a frame. 

Aluminum separators are often added for stability.
Appendix A: Figures 315

Figure 2. The Class I BSC (A) front opening; (B) sash; (C) exhaust HEPA filter; 

(D) exhaust plenum. Note: The cabinet needs to be hard connected to the 

building exhaust system if toxic vapors are to be used.

Figure 3. The Class II, Type A1 BSC (A) front opening; (B) sash; (C) exhaust 

HEPA filter; (D) supply HEPA filter; (E) common plenum; (F) blower.
316 Biosafety in Microbiological and Biomedical Laboratories

Figure 4. Canopy (thimble) unit for ducting a Class II, Type A BSC (A) balancing 

damper; (B) flexible connector to exhaust system; (C) cabinet exhaust HEPA filter 

housing; (D) canopy unit; (E) BSC. Note: There is a 1” gap between the canopy 

unit (D) and the exhaust filter housing (C), through which room air is exhausted.

Figure 5A. The Class II, Type B1 BSC (classic design) (A) front opening; (B) sash; 

(C) exhaust HEPA filter; (D) supply HEPA filter; (E) negative pressure dedicated 

exhaust plenum; (F) blower; (G) additional HEPA filter for supply air. Note: The 

cabinet exhaust needs to be hard connected to the building exhaust system.
Appendix A: Figures 317

Figure 5B. The Class II, Type B1 BSC (bench top design) (A) front opening; 

(B) sash; (C) exhaust HEPA filter; (D) supply plenum; (E) supply HEPA filter; 

(F) blower; (G) negative pressure exhaust plenum. Note: The cabinet exhaust 

needs to be hard connected to the building exhaust system.

Connection to the building exhaust system is required.
318 Biosafety in Microbiological and Biomedical Laboratories

Figure 6. The Class II, Type B2 BSC (A) front opening; (B) sash; (C) exhaust 

HEPA filter; (D) supply HEPA filter; (E) negative pressure exhaust plenum. 

Note: The carbon filter in the exhaust system is not shown. The cabinet needs 

to be hard connected to the building exhaust system.

Figure 7. The tabletop model of a Class II, Type A2 BSC (A) front opening; 

(B) sash; (C) exhaust HEPA filter; (D) supply HEPA filter; (E) positive pressure 

common plenum; (F) negative pressure plenum. The Class II Type A2 BSC is 

not equivalent to what was formerly called a Class II Type B3 unless it is 

connected to the laboratory exhaust system. Note: The A2 BSC should be 

canopy connected to the exhaust system.
Appendix A: Figures 319

Figure 8. The Class III BSC (A) glove ports with O-ring for attaching arm-length 

gloves to cabinet; (B) sash; (C) exhaust HEPA filter; (D) supply HEPA filter; (E) 

double-ended autoclave or pass-through box. Note: A chemical dunk tank may 

be installed which would be located beneath the work surface of the BSC with 

access from above. The cabinet exhaust needs to be hard connected to an 

exhaust system where the fan is generally separate from the exhaust fans of 

the facility ventilation system. The exhaust air must be double HEPA-filtered 

or HEPA-filtered and incinerated.

Figure 9A. The horizontal laminar flow “clean bench” (A) front opening; 

(B) supply grille; (C) supply HEPA filter; (D) supply plenum; (E) blower. 
320 Biosafety in Microbiological and Biomedical Laboratories

Figure 9B. The vertical laminar flow “clean bench” (A) front opening; (B) sash; 

(C) supply HEPA filter; (D) blower. Note: Some vertical flow clean benches have 

recirculated air through front and/or rear perforated grilles.
Appendix A: Figures 321

Figure 10. A modified containment cabinet or Class I BSC can be used for 

labeling infectious microorganisms with I125. (A) arm holes; (B) LexanR hinged 

doors; (C) exhaust charcoal filter; (D) exhaust HEPA filter; (E) filter housing 

with required connection to building exhaust (see also Figure 13).
322 Biosafety in Microbiological and Biomedical Laboratories

Figure 11. A typical layout for working “clean to dirty” within a Class II BSC. 

Clean cultures (left) can be inoculated (center); contaminated pipettes can be 

discarded in the shallow pan and other contaminated materials can be placed in 

the biohazard bag (right). This arrangement is reversed for left-handed persons.

Figure 12. One method to protect a house vacuum system during aspiration 

of infectious fluids. The left suction flask (A) is used to collect the contaminated 

fluids into a suitable decontamination solution; the right flask (B) serves as a 

fluid overflow collection vessel. An in-line HEPA filter (C) is used to protect the 

vacuum system (D) from aerosolized microorganisms.
Appendix A: References 323

Figure 13. A bag-in-bag-out filter enclosure allows for the removal of the 

contaminated filter without worker exposure. (A) filters; (B) bags; (C) safety 

straps; (D) cinching straps; (E) shock cord located in the mouth of the PVC 

bag restricts the bag around the second rib of the housing lip.

Acknowledgements

We gratefully acknowledge the Baker Company; Filtration Group, Inc.; Flanders 

Filters; and Forma Scientific, Inc., for use of some drawings and figures 

reproduced herein.

Referrences

1. Centers for Disease Control and Prevention; The National Institutes of 

Health. Biosafety in microbiological and biomedical laboratories. 4th ed. 

Washington, DC. 1999.

2. Kruse RH, Puckett WH, Richardson JH. Biological safety cabinetry. 

Clin Microbiol Rev. 1991;4:207-541.

3. First MW. Filters, high capacity filters and high efficiency filters: review 

and production. In-Place Filter Testing Workshop; 1971; Boston, 

Massachusetts. 1971.

4. The National Institutes of Health (NIH); Rockville Bio-Engineering Services, 

Dow Chemical USA. A Workshop for Certification of Biological Safety 

Cabinets. Bethesda: The Institute, Office of Biohazards and Environmental 

Control; 1974.

5. Richmond, JY. Safe practices and procedures for working with human specimens 

in biomedical research laboratories. J Clin Immunoassay. 1988;13:115-9.

6. Barbeito MS, Taylor LA. Containment of microbial aerosols in a 

microbiological safety cabinet. Appl Microbiol. 1968;16:1255-29.

7. Whitfield WJ. A new approach to clean room design. Final report. 

Albuquerque (NM): Sandia Corporation; 1962. Report No.: SC-4673 (RR).

8. NSF International (NSF); American National Standards Institute (ANSI). 

NSF/ANSI Standard 49-2007. Class II (laminar flow) biosafety cabinetry. 

Ann Arbor (MI); 2004.
324 Biosafety in Microbiological and Biomedical Laboratories

9. Occupational Safety and Health Administration, Office of Occupational 

Medicine. Guidelines for cytotoxic (antineoplastic) drugs. Appendix A. 

Standard 01-23-001. 1986.

10. Jones RL Jr., Tepper B, Greenier TG, et al. Effects of Thimble Connections 

of Biological Safety Cabinets. Abstracts of 32nd Biological Safety 

Conference; 1989; New Orleans, LA: 1989. 

11. National Institutes of Health (NIH). NIH guidelines for the laboratory use 

of chemical carcinogens. Washington, DC: U. S. Government Printing 

Office; 1981.

12. Class II. Type 1 safety cabinet specification the National Institute of 

Health-030112. The Institute; 1973, May.

13. Stuart DG, First MW, Jones RL Jr., et al. Comparison of chemical vapor 

handling by three types of class II biological safety cabinets. Particulate 

and Microbial Control. 1983;2:18-24.

14. Stuart D, Kiley M, Ghidoni D, et al. The class III biological safety cabinet. 

In: Richmond JY, editor. Anthology of biosafety vol. VII. biosafety level 3. 

Mundelein, IL: American Biological Safety Association; 2004.

15. American Conference of Government Industrial Hygienists (ACGIH). 

Threshold limit values for chemical substances and physical agents 

and biological exposure indices. Cincinnati, OH: ACGIH; 2006.

16. National Institutes of Health NIH, National Cancer Institute (NCI), Office 

of Research Safety and the Special Committee of Safety and Health 

Experts. Laboratory safety monograph: a supplement to the NIH guidelines 

for recombinant DNA research. Bethesda (MD): The Institute; 1979.

17. Barbeito M, West, D, editors. Laboratory ventilation for hazard control. 

Proceedings of a Cancer Research Safety Symposium; l976 Oct 21-22; 

Frederick, MD: Litton Bionetics, Inc.; 1976.

18. National Cancer Institute Safety Standards for Research Involving Chemical 

Carcinogens. Bethesda: The National Institutes of Health (US), National 

Cancer Institute; 1975.

19. Anderson RE, Stein L, Moss ML, et al. Potential infectious hazards of 

bacteriological techniques. J Bacteriol. 1952;64:473-81.

20. Baker Company. Factors to consider before selecting a laminar flow 

biological safety cabinet. Sanford (ME): Baker Company; 1993.

21. Jones R, Drake J, Eagleson D. 1993. US hydrogen peroxide vapor 

to decontaminate biological cabinets. Acumen. 1993;1:1.

22. Jones R, Stuart D, Large S, et al. 1993. Cycle parameters for 

decontaminating a biological safety cabinet using H2O2 vapor. 

Acumen, 1993;1:2.

23. Jones R, Stuart D, PhD, Large S, et al. Decontamination of a HEPA 

filter using hydrogen peroxide vapor. Acumen. 1993;1:3.

24. Fox D, editor. Proceedings of the National Cancer Institute symposium 

on design of biomedical research facilities. Monograph series. Volume 4; 

l979 Oct 18-19; Frederick, MD: Litton Bionetics, Inc.; 1979.
Appendix A: References 325

25. American Society of Heating, Refrigeration and Air Conditioning Engineers, 

Inc. (ASHRAE). Laboratories. In: ASHRAE Handbook, 1982 Applications. 

Atlanta (GA): ASHRAE; 1982.

26. US Department of Agriculture (USDA), Agricultural Research Services 

(ARS). ARS construction project design standard manual, 242.1. Beltsville 

(MD): USDA; 1991.

27. American Conference of Governmental Industrial Hygienists (ACGIH). 

Industrial ventilation, a manual of recommended practice. Cincinnati (OH): 

ACGIH; 1998.

28. Jones RL, Jr., Stuart DG, Eagleson D, et al. The effects of changing intake 

and supply air flow on biological safety performance. Appl Occup Environ 

Hyg. 1990;5:370.

29. Jones RL, Jr., Stuart DG, Eagleson, D, et al. Effects of ceiling height on 

determining calculated intake air velocities for biological safety cabinets. 

Appl Occup Environ Hyg. 1991;6:683.

30. Rake BW. Influence of cross drafts on the performance of a biological 

safety cabinet. Appl Env Microbiol. 1978;36:278-83.

31. Specifications for general purpose clean air biological safety cabinet. Bethesda: 

The National Institutes of Health (US), National Cancer Institute; 1973.

32. Airborne Particulate Cleanliness Classes in Clean rooms and Clean Zones, 

Federal Standard No. 209E. (1992).

33. Clean Room and Work Station Requirements, Controlled Environment, 

Federal Standard No. 209B. (1973).
326 Biosafety in Microbiological and Biomedical Laboratories

Appendix B—Decontamination and Disinfection

This section describes basic strategies for decontaminating surfaces, items, 

and areas in laboratories to eliminate the possibility of transmission of infectious 

agents to laboratory workers, the general public, and the environment. Factors 

necessary for environmentally mediated infection transmission are reviewed as 

well as methods for sterilization and disinfection and the levels of antimicrobial 

activity associated with liquid chemical germicides. General approaches are 

emphasized, not detailed protocols and methods. The principles of sterilization 

and disinfection are stated and compared.

Environmentally Mediated Infection Transmission

Environmentally associated laboratory infections can be transmitted directly 

or indirectly from environmental sources (e.g., air, contaminated fomites and 

laboratory instruments, and aerosols) to laboratory staff. Fortunately, LAI are 

rare events1

 because there are a number of requirements necessary for 

environmental transmission to occur.2

 Commonly referred to as the “chain of 

infection” they include: presence of a pathogen of sufficient virulence, relatively 

high concentration of the pathogen (i.e., infectious dose), and a mechanism of 

transmission of the pathogen from environment to the host, a correct portal of 

entry to a susceptible host.

To accomplish successful transmission from an environmental source, all 

of these requirements for the “chain of infection” must be present. The absence 

of any one element will prevent transmission. Additionally, the pathogen in 

question must overcome environmental stresses to retain viability, virulence, 

and the capability to initiate infection in the host. In the laboratory setting, high 

concentrations of pathogens can be common. Reduction of environmental 

microbial contamination by conventional cleaning procedures is often enough 

to prevent environmentally mediated transmission. However, it is the general 

practice in laboratories to use sterilization methods to remove the potential for 

infection transmission.

Principles of Sterilization and Disinfection

In order to implement a laboratory biosafety program it is important to understand 

the principles of decontamination, cleaning, sterilization, and disinfection. We 

review here the definitions of sterilization, disinfection, antisepsis, decontamination, 

and sanitization to avoid misuse and confusion. The definitions and implied 

capabilities of each inactivation procedure are discussed with an emphasis 

on achievement and in some cases, monitoring of each state. 
Appendix B: Decontamination and Disinfection 327

Sterilization

Any item, device, or solution is considered to be sterile when it is completely 

free of all living microorganisms and viruses. The definition is categorical and 

absolute (i.e., an item is either sterile or it is not). A sterilization procedure is one 

that kills all microorganisms, including high numbers of bacterial endospores. 

Sterilization can be accomplished by heat, ethylene oxide gas, hydrogen 

peroxide gas, plasma, ozone, and radiation (in industry). From an operational 

standpoint, a sterilization procedure cannot be categorically defined. Rather, the 

procedure is defined as a process, after which the probability of a microorganism 

surviving on an item subjected to treatment is less than one in one million (10-6). 

This is referred to as the “sterility assurance level.” 3,4

Disinfection

Disinfection is generally a less lethal process than sterilization. It eliminates 

nearly all recognized pathogenic microorganisms but not necessarily all microbial 

forms (e.g., bacterial spores) on inanimate objects. Disinfection does not ensure 

an “overkill’’ and therefore lacks the margin of safety achieved by sterilization 

procedures. The effectiveness of a disinfection procedure is controlled significantly 

by a number of factors, each one of which may have a pronounced effect on the 

end result. Among these are:

■ the nature and number of contaminating microorganisms (especially 

the presence of bacterial spores);

■ the amount of organic matter present (e.g., soil, feces, and blood);

■ the type and condition of instruments, devices, and materials to 

be disinfected;

■ the temperature.

Disinfection is a procedure that reduces the level of microbial contamination, 

but there is a broad range of activity that extends from sterility at one extreme to a 

minimal reduction in the number of microbial contaminants at the other. By definition, 

chemical disinfection and in particular, high-level disinfection differs from chemical 

sterilization by its lack of sporicidal power. This is an over simplification of the 

actual situation because a few chemical germicides used as disinfectants do, in 

fact, kill large numbers of spores even though high concentrations and several 

hours of exposure may be required. Non-sporicidal disinfectants may differ in 

their capacity to accomplish disinfection or decontamination. Some germicides 

rapidly kill only the ordinary vegetative forms of bacteria such as staphylococci 

and streptococci, some forms of fungi, and lipid-containing viruses, whereas 

others are effective against such relatively resistant organisms as Mycobacterium 

tuberculosis var. bovis, non-lipid viruses, and most forms of fungi.
328 Biosafety in Microbiological and Biomedical Laboratories

Spaulding Classification

In 1972, Dr. Earl Spaulding5 proposed a system for classifying liquid chemical 

germicides and inanimate surfaces that has been used subsequently by CDC, 

FDA, and opinion leaders in the United States. This system, as it applies to 

device surfaces, is divided into three general categories based on the theoretical 

risk of infection if the surfaces are contaminated at time of use. From the 

laboratory perspective, these categories are:

■ critical—instruments or devices that are exposed to normally sterile 

areas of the body require sterilization;

■ semi-critical—instruments or devices that touch mucous membranes 

may be either sterilized or disinfected;

■ non-critical—instruments or devices that touch skin or come into 

contact with persons only indirectly can be either cleaned and then 

disinfected with an intermediate-level disinfectant, sanitized with a 

low-level disinfectant, or simply cleaned with soap and water.

In 1991, microbiologists at CDC proposed an additional category, 

environmental surfaces (e.g., floors, walls, and other “housekeeping surfaces”) 

that do not make direct contact with a person’s skin.6

 Spaulding also classified 

chemical germicides by activity level:

High-level Disinfection

This procedure kills vegetative microorganisms and inactivates viruses, but not 

necessarily high numbers of bacterial spores. Such disinfectants are capable 

of sterilization when the contact time is relatively long (e.g., 6 to 10 hours). As 

high-level disinfectants, they are used for relatively short periods of time (e.g., 

10 to 30 minutes). These chemical germicides are potent sporicides and, in 

the United States, are classified by the FDA as sterilant/disinfectants. They 

are formulated for use on medical devices, but not on environmental surfaces 

such as laboratory benches or floors.7

Intermediate-level Disinfection

This procedure kills vegetative microorganisms, including Mycobacterium 

tuberculosis, all fungi, and inactivates most viruses. Chemical germicides 

used in this procedure often correspond to Environmental Protection Agency 

(EPA)-approved “hospital disinfectants” that are also “tuberculocidal.” They 

are used commonly in laboratories for disinfection of laboratory benches 

and as part of detergent germicides used for housekeeping purposes.
Appendix B: Decontamination and Disinfection 329

Low-level Disinfection

This procedure kills most vegetative bacteria except M. tuberculosis, some fungi, 

and inactivates some viruses. The EPA approves chemical germicides used in 

this procedure in the US as “hospital disinfectants” or “sanitizers.”

Decontamination in the Microbiology Laboratory

Decontamination in the microbiology laboratory must be carried out with great 

care. In this arena, decontamination may entail disinfection of work surfaces, 

decontamination of equipment so it is safe to handle, or may require sterilization. 

Regardless of the method, the purpose of decontamination is to protect the 

laboratory worker, the environment, and anyone who enters the laboratory or 

handles laboratory products away from the laboratory. Reduction of crosscontamination in the laboratory is an added benefit.

Decontamination and Cleaning

Decontamination renders an area, device, item, or material safe to handle (i.e., 

safe in the context of being reasonably free from a risk of disease transmission). 

The primary objective is to reduce the level of microbial contamination so that 

infection transmission is eliminated. The decontamination process may be 

ordinary soap and water cleaning of an instrument, device, or area. In laboratory 

settings, decontamination of items, spent laboratory materials, and regulated 

laboratory wastes is often accomplished by a sterilization procedure such as 

steam autoclaving, perhaps the most cost-effective way of decontaminating a 

device or an item.

The presence of any organic matter necessitates longer contact time with 

a decontamination method if the item or area is not pre-cleaned. For example, 

a steam cycle used to sterilize pre-cleaned items is 20 minutes at 121°C. 

When steam sterilization is used to decontaminate items that have a high 

bio-burden and there is no pre-cleaning (i.e., infectious waste) the cycle is 

longer. Decontamination in laboratory settings often requires longer exposure 

times because pathogenic microorganisms may be protected from contact 

with the decontaminating agents.
330 Biosafety in Microbiological and Biomedical Laboratories

Table 1. Descending Order of Resistance to Germicidal Chemicals

Bacterial Spores

Bacillus subtilis, Clostridium sporogenes

▼

Mycobacteria

Mycobacterium tuberculosis var. bovis, Nontuberculous mycobacteria

▼

Nonlipid or Small Viruses

Poliovirus, Coxsackievirus, Rhinovirus

▼

Fungi

Trichophyton spp., Cryptococcus spp., Candida spp.

▼

Vegetative Bacteria

Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella choleraesuis, 

Enterococci

▼

Lipid or Medium-size Viruses

Herpes simplex virus, CMV, Respiratory syncytial virus, HBV, HCV, HIV, Hantavirus, 

Ebola virus

Note: There are exceptions to this list. Pseudomonas spp are sensitive to 

high-level disinfectants, but if they grow in water and form biofilms on surfaces, 

the protected cells can approach the resistance of bacterial spores to the same 

disinfectant. The same is true for the resistance to glutaraldehyde by some 

nontuberculous mycobacteria, some fungal ascospores of Microascus cinereus 

and Cheatomium globosum, and the pink pigmented Methylobacteria. Prions 

are also resistant to most liquid chemical germicides and are discussed in the 

last part of this section.

Chemical germicides used for decontamination range in activity from highlevel disinfectants (i.e., high concentrations of sodium hypochlorite [chlorine 

bleach]), which might be used to decontaminate spills of cultured or concentrated 

infectious agents in research or clinical laboratories, to low-level disinfectants 

or sanitizers for general housekeeping purposes or spot decontamination of 

environmental surfaces in healthcare settings. Resistance of selected organisms 

to decontamination is presented in descending order in Table 1. If dangerous 

and highly infectious agents are present in a laboratory, the methods for 

decontamination of spills, laboratory equipment, BSC, or infectious waste 

are very significant and may include prolonged autoclave cycles, incineration 

or gaseous treatment of surfaces.
Appendix B: Decontamination and Disinfection 331

Decontamination of Large Spaces

Space decontamination is a specialized activity and should be performed by 

specialists with proper training and protective equipment.8 Decontamination 

requirements for BSL-3 and BSL-4 laboratory space have an impact on the 

design of these facilities. The interior surfaces of BSL-3 laboratories must be 

water resistant in order for them to be easily cleaned and decontaminated. 

Penetrations in these surfaces should be sealed or capable of being sealed 

for decontamination purposes. Thus, in the BSL-3 laboratory, surface 

decontamination, not fumigation, is the primary means of decontaminating 

space. Care should be taken that penetrations in the walls, floors and ceilings 

are kept to a minimum and are “sight sealed.” Verification of the seals is usually 

not required for most BSL-3 laboratories. The BSL-4 laboratory design requires 

interior surfaces that are water resistant AND sealed to facilitate fumigation. 

These seals must be tested and verified to ensure containment in order to permit 

both liquid disinfection and fumigation. Periodic fumigation is required in the 

BSL-4 suit laboratory to allow routine maintenance and certification of equipment. 

Procedures for decontamination of large spaces such as incubators or rooms 

are varied and influenced significantly by the type of etiologic agent involved, the 

characteristics of the structure containing the space, and the materials present 

in the space. The primary methods for space decontamination follow.

Formaldehyde—Paraformaldehyde

Formaldehyde gas at a concentration of 0.3 grams/cubic foot for four hours is 

often used for space decontamination. Gaseous formaldehyde can be generated 

by heating flake paraformaldehyde (0.3 grams per cubic foot) in a frying pan, 

thereby converting it to formaldehyde gas. The humidity must be controlled and 

the system works optimally at 80% relative humidity. This method is effective in 

killing microorganisms but toxicity issues are present.1,9 Additional information on 

environmental and safety issues related to paraformaldehyde is available from 

the EPA Web site: www.epa.gov/pesticides.

Hydrogen Peroxide Vapor

Hydrogen peroxide can be vaporized and used for the decontamination of glove 

boxes as well as small room areas. Vapor phase hydrogen peroxide has been 

shown to be an effective sporicide at concentrations ranging from 0.5 mg/L to 

<10 mg/L. The optimal concentration of this agent is about 2.4 mg/L with a contact 

time of at least one hour. This system can be used to decontaminate glove boxes, 

walk in incubators and small rooms. An advantage of this system is that the end 

products (i.e., water) are not toxic. Low relative humidity can be used.10-14

Chlorine Dioxide Gas

Chlorine dioxide gas sterilization can be used for decontamination of laboratory 

rooms, equipment, glove boxes, and incubators. The concentration of gas at the 
332 Biosafety in Microbiological and Biomedical Laboratories

site of decontamination should be approximately 10 mg/L with contact time of 

one to two hours.

Chlorine dioxide possesses the bactericidal, virucidal and sporicidal 

properties of chlorine, but unlike chlorine, does not lead to the formation of 

trihalomethanes or combine with ammonia to form chlorinated organic products 

(chloramines). The gas cannot be compressed and stored in high-pressure 

cylinders, but is generated upon demand using a column-based solid phase 

generation system. Gas is diluted to the use concentration, usually between 

10 and 30 mg/L. Within reasonable limits, a chlorine dioxide gas generation 

system is unaffected by the size or location of the ultimate destination for the gas. 

Relative humidity does need to be controlled and high humidities are optimal. 

Although most often used in closed sterilizers, the destination enclosure for the 

chlorine dioxide gas does not, in fact, need to be such a chamber. Because 

chlorine dioxide gas exits the generator at a modest positive pressure and flow 

rate, the enclosure also need not be evacuated and could be a sterility-testing 

isolator, a glove box or sealed BSC, or even a small room that could be sealed 

to prevent gas egress.15 Chlorine dioxide gas is rapidly broken down by light; 

care must be taken to eliminate light sources in spaces to be decontaminated.

Decontamination of Surfaces

Liquid chemical germicides formulated as disinfectants may be used for 

decontamination of large areas. The usual procedure is to flood the area with 

a disinfectant for periods up to several hours. This approach is messy and with 

some of the disinfectants used represents a toxic hazard to laboratory staff. For 

example, most of the “high-level” disinfectants on the United States market are 

formulated to use on instruments and medical devices and not on environmental 

surfaces. Intermediate and low-level disinfectants are formulated to use on fomites 

and environmental surfaces but lack the potency of a high-level disinfectant. For 

the most part intermediate and low level disinfectants can be safely used and, as 

with all disinfectants, the manufacturer’s instructions should be closely followed.7

Disinfectants that have been used for decontamination include sodium hypochlorite 

solutions at concentrations of 500 to 6000 parts per million (ppm), oxidative 

disinfectants such as hydrogen peroxide and peracetic acid, phenols, and iodophors.

Concentrations and exposure times vary depending on the formulation and 

the manufacturer’s instructions for use.6,16 See Table 2 for a list of chemical 

germicides and their activity levels. A spill control plan must be available in the 

laboratory. This plan should include the rationale for selection of the disinfecting 

agent, the approach to its application, contact time and other parameters. Agents 

requiring BSL-3 and BSL-4 containment pose a high risk to workers and possibly 

to the environment and should be managed by well-informed professional staff 

trained and equipped to work with concentrated material.
Appendix B: Decontamination and Disinfection 333

Table 2. Activity Levels of Selected Liquid Germicidesa

Procedure / Product Aqueous Concentration Activity Level

Sterilization

glutaraldehyde variable

hydrogen peroxide 6 – 30%

formaldehyde 6 – 8%

chlorine dioxide variable

peracetic acid

Disinfection

glutaraldehyde variable high to intermediate

ortho-phthalaldehyde 0.5% high

hydrogen peroxide 3 – 6% high to intermediate

formaldehyde b 1 – 8% high to low

chlorine dioxide variable high

peracetic acid variable high

chlorine compounds c 500 to 5000 ml/L free/available 

chlorine

Intermediate

alcohols (ethyl, isopropyl) d 70% Intermediate

phenolic compounds 0.5 to 3% intermediate to low

iodophor compounds e 30 – 50 mg/L free iodine up to 

10,000 mg/L available iodine 

0.1 – 0.2%

intermediate to low

quaternary ammonium 

compounds

low

a This list of chemical germicides centers on generic formulations. A large number of 

commercial products based on these generic components can be considered for use. 

Users should ensure that commercial formulations are registered with EPA or by the FDA. b Because of the ongoing controversy of the role of formaldehyde as a potential occupational 

carcinogen, the use of formaldehyde is limited to certain specific circumstances under 

carefully controlled conditions (e.g., for the disinfection of certain hemodialysis equipment). 

There are no FDA cleared liquid chemical sterilant/disinfectants that contain formaldehyde. c Generic disinfectants containing chlorine are available in liquid or solid form (e.g., sodium or 

calcium hypochlorite). Although the indicated concentrations are rapid acting and broadspectrum (tuberculocidal, bactericidal, fungicidal, and virucidal), no proprietary hypochlorite 

formulations are formally registered with EPA or cleared by FDA. Common household 

bleach is an excellent and inexpensive source of sodium hypochlorite. Concentrations 

between 500 and 1000 mg/L chlorine are appropriate for the vast majority of uses 

requiring an intermediate level of germicidal activity; higher concentrations are extremely 

corrosive as well as irritating to personnel, and their use should be limited to situations 

where there is an excessive amount of organic material or unusually high concentrations 

of microorganisms (e.g., spills of cultured material in the laboratory).
334 Biosafety in Microbiological and Biomedical Laboratories

d The effectiveness of alcohols as intermediate level germicides is limited because they 

evaporate rapidly, resulting in short contact times, and also lack the ability to penetrate 

residual organic material. They are rapidly tuberculocidal, bactericidal and fungicidal, but 

may vary in spectrum of virucidal activity (see text). Items to be disinfected with alcohols 

should be carefully pre-cleaned then totally submerged for an appropriate exposure time 

(e.g., 10 minutes). e Only those iodophors registered with EPA as hard-surface disinfectants should be used, 

closely following the manufacturer’s instructions regarding proper dilution and product 

stability. Antiseptic iodophors are not suitable to disinfect devices, environmental 

surfaces, or medical instruments.

Special Infectious Agent Issues

Transmissible Spongiform Encephalopathy Agents (Prions)

The major exception to the rule in the previous discussion of microbial 

inactivation and decontamination is the causative agent of CJD or other prion 

agents responsible for transmissible spongiform encephalopathies of the central 

nervous system in humans or animals. Studies show that prions are resistant to 

conventional uses of heat and/or chemical germicides for the sterilization of 

instruments and devices. (See Section IX.)

References

1. Vesley D, Lauer J, Hawley R. Decontamination, sterilization, disinfection, and 

antisepsis. In: Fleming DO, Hunt DL, editors. Laboratory safety: principles 

and practices. 3rd ed. Washington, DC: ASM Press; 2001. p. 383-402.

2. Rhame FS. The inanimate environment. In: Bennett JV, Brachmann PS, editors. 

Hospital infections. 4th ed. Philadelphia: Lippincott-Raven; 1998. p. 299-324.

3. Favero M. Developing indicators for sterilization. In: Rutala W, editor. 

Disinfection, sterilization and antisepsis in health care. Washington, DC: 

Association for Professionals in Infection Control and Epidemiology, Inc.; 

1998. p. 119-132.

4. Favero M. Sterility assurance: concepts for patient safety. In: Rutala W, 

editor. Disinfection, sterilization and antisepsis: principles and practices in 

healthcare facilities. Washington, DC: Association for Professionals in 

Infection Control and Epidemiology, Inc.; 2001. p. 110-9.

5. Spaulding EH. Chemical disinfection and antisepsis in the hospital. J Hosp 

Res. 1972:9;5-31.

6. Favero M, Bond W. Chemical disinfection of medical surgical material. In: 

Block S, editor. Disinfection, sterilization, and preservation. 5th edition. 

Philadelphia: Lippencott, Williams and Wilkens; 2001. p. 881-917.

7. Centers for Disease Control and Prevention [www.cdc.gov]. Atlanta: The 

Centers for Disease Control and Prevention; [updated 2006 Sept 21]. Guidelines 

for Environmental Infection Control in Health-Care Facilities, 2003; [about 2 

screens] Available from: http://www.cdc.gov/hicpac/pubs.html

8. Tearle P. Decontamination by fumigation. Commun Dis Public Health. 

2003;6:166-8.
Appendix B: Decontamination and Disinfection 335

9. Fink R, Liberman DF, Murphy K, et al. Biological safety cabinets, 

decontamination or sterilization with paraformaldehyde. Am Ind Hyg Assoc 

J. 1988;49:277-9.

10. Klapes NA, Vesley D. Vapor-phase hydrogen peroxide as a surface 

decontaminant and sterilant. Appl Environ Microbiol. 1990;56:503-6.

11. Graham GS, Rickloff JR. Development of VHP sterilization technology. 

J Healthc Mater Manage. 1992;54:56-8.

12. Johnson JW, Arnold JF, Nail SL, et al. Vaporized hydrogen peroxide 

sterilization of freeze dryers. J Parenter Sci Technol. 1992;46:215-25.

13. Heckert RA, Best M, Jordan LT, et al. Efficacy of vaporized hydrogen 

peroxide against exotic animal viruses. Appl Environ Microbiol. 

1997;63:3916-8.

14. Krause J, McDonnell G, Riedesel H. Biodecontamination of animal rooms 

and heat-sensitive equipment with vaporized hydrogen peroxide. Contemp 

Top Lab Anim Sci. 2001;40:18-21.

15, Knapp JE, Battisti DL. Chlorine Dioxide. In: Block S, editor. Disinfection, 

sterilization, and preservation. 5th ed. Philadelphia: Lippencott, Williams 

and Wilkens; 2001. p. 215-27.

16. Weber DJ, Rutala WA. Occupational risks associated with the use of 

selected disinfectants and sterilants. In: Rutala WA, editor. Disinfection, 

sterilization and antisepsis in health care. Champlain (NY): Polyscience 

Publications: 1998 p. 211-26.
336 Biosafety in Microbiological and Biomedical Laboratories

Appendix C—Transportation of Infectious Substances

An infectious substance is a material known to contain or reasonably expected to 

contain a pathogen. A pathogen is a microorganism (including bacteria, viruses, 

rickettsiae, parasites, fungi) or other agent, such as a proteinaceous infectious 

particle (prion), that can cause disease in humans or animals. Infectious substances 

may exist as purified and concentrated cultures, but may also be present in a 

variety of materials, such as body fluids or tissues. Transportation of infectious 

substances and materials that are known or suspected to contain them are 

regulated as hazardous materials by the United State Department of Transportation 

(DOT), foreign governments, and the International Civil Aviation Organization, and 

their transportation is subject to regulatory controls. For transport purposes, the 

term “infectious substance” is understood to include the term “etiologic agent.”

Transportation Regulations

International and domestic transport regulations for infectious substances are 

designed to prevent the release of these materials in transit to protect the public, 

workers, property, and the environment from the harmful effects that may occur 

from exposure to these materials. Protection is achieved through rigorous 

packaging requirements and hazard communication. Packages must be designed 

to withstand rough handling and other forces experienced in transportation, such 

as changes in air pressure and temperature, vibration, stacking, and moisture. 

Hazard communication includes shipping papers, labels, markings on the outside 

of packagings, and other information necessary to enable transport workers and 

emergency response personnel to correctly identify the material and respond 

efficiently in an emergency situation. In addition, shippers and carriers must be 

trained on these regulations so they can properly prepare shipments and 

recognize and respond to the risks posed by these materials.

Select agents include infectious substances that have been identified by 

the CDC and the USDA as having the potential to pose a severe threat to public 

health and safety. Persons who offer for transportation or transport select agents 

in commerce in the United States must develop and implement security plans for 

such transportation. A security plan must include an assessment of the possible 

transportation security risks for materials covered by the security plan and specific 

measures to reduce or eliminate the assessed risks. At a minimum, a security 

plan must include measures to address those risks associated with personnel 

security, en route security, and unauthorized access.

Regulations

Department of Transportation. 49 CFR Part 171-180, Hazardous Materials 

Regulations. Applies to the shipment of infectious substances in commercial 

transportation within the United States. Information on these regulations is 

available at: http://www.phmsa.dot.gov/hazmat.
Appendix C: Transportation of Infectious Substances 337

United States Postal Service (USPS). 39 CFR Part 20, International Postal 

Service (International Mail Manual), and Part 111, General Information on Postal 

Service (Domestic Mail Manual). Regulations on transporting infectious substances 

through the USPS are codified in Section 601.10.17 of the Domestic Mail Manual 

and Section 135 of the International Mail Manual. A copy of the Domestic and 

International Mail Manuals may be obtained from the U.S. Government Printing 

Office by calling Monday through Friday, 7:30 a.m. – 9:00 p.m. EST: (202) 512-1800; 

toll free (866) 512-1800; or at the USPS Web site: http://bookstore.gpo.gov/.

Occupational Health and Safety Administration (OSHA). 29 CFR Part 

1910.1030, Occupational Exposure to Bloodborne Pathogens. These regulations 

provide minimal packaging and labeling for blood and body fluids when transported 

within a laboratory or outside of it. Information may be obtained from your local 

OSHA office or at the OSHA Web site: http://www.osha.gov.

Technical Instructions for the Safe Transport of Dangerous Goods by Air 

(Technical Instructions). International Civil Aviation Organization (ICAO). Applies 

to the shipment of infectious substances by air and is recognized in the United 

States and by most countries worldwide. A copy of these regulations may be obtained 

from the ICAO Document Sales Unit at (514) 954-8022, fax: (514) 954-6769; 

e-mail: sales_unit@icao.int; or from the ICAO Web site: http://www.icao.int.

Dangerous Goods Regulations. International Air Transport Association 

(IATA). These regulations are issued by an airline association, are based on the 

ICAO Technical Instructions, and are followed by most airline carriers. A copy of 

these regulations is available at: http://www.iata.org/index.htm or 

http://www.who.int/en/ ; or by contacting the IATA Customer Care office at: 

telephone: +1 (514) 390 6726; fax: +1 (514) 874 9659; for Canada and USA 

(800) 716-6326 (toll free); Europe, Africa and Middle East +41 (22) 770 2751; fax: 

+41 (22) 770 2674; TTY: YMQTPXB, or e-mail: custserv@iata.org.

Transfers

Regulations governing the transfer of biological agents are designed to ensure 

that possession of these agents is in the best interest of the public and the nation. 

These regulations require documentation of personnel, facilities, justification of 

need and pre-approval of the transfer by a federal authority. The following 

regulations apply to this category:

Importation of Etiologic Agents of Human Disease. 42 CFR Part 71 Foreign 

Quarantine. Part 71.54 Etiological Agents, Hosts and Vectors. This regulation 

requires an import permit from the CDC for importation of etiologic agents, hosts 

or vectors of human disease. The regulation, application form, and additional 

guidance is available at the CDC Web site: http://www.cdc.gov/od/eaipp. 
338 Biosafety in Microbiological and Biomedical Laboratories

Completed application forms may be submitted to the CDC Etiologic Agent 

Import Permit Program by fax: (404) 718-2093, or by mail:

Centers for Disease Control and Prevention

Etiologic Agent Import Permit Program

1600 Clifton Road, N.E., Mailstop A-46

Atlanta, GA 30333

Importation of select agents or toxins into the U.S. also requires the intended 

recipient to be registered with the Select Agent Program and submit an APHIS/ 

CDC Form 2 to obtain approval to import the select agent or toxin prior to each 

importation event (see 42 CFR 73 and/or 9 CFR 121). More information 

regarding select agents and toxins is available at: www.selectagents.gov.

Importation of Etiologic Agents of Livestock, Poultry and Other Animal Diseases 

and Other Materials Derived from Livestock, Poultry or Other Animal. 9 CFR Parts 

122. Organisms and Vectors. The USDA, APHIS, Veterinary Services (VS) requires 

that a permit be issued prior to the importation or domestic transfer (interstate 

movement) of etiologic disease agents of livestock, poultry, other animals. Information 

may be obtained at (301) 734-5960, or from the USDA Web site: http://www.

aphis.usda.gov/animal_health. Completed permit applications may be submitted 

electronically at: http://www.aphis.usda.gov/permits/learn_epermits.shtml; 

or by fax to (301) 734-3652; or by mail to:

USDA APHIS VS

National Center for Import and Export

4700 River Road

Unit 2, Mailstop 22, Cubicle 1A07

Riverdale, MD 20737

Importation of select agents into the United States also requires the intended 

recipient to be registered with the Select Agent Program and submit an APHIS/ 

CDC Form 2 to obtain approval to import the select agent or toxin prior to each 

importation event (see 42 CFR 73 and/or 9 CFR 121). More information 

regarding select agents and toxins is available at: http://www.aphis.usda.gov/

programs/ag_selectagent/index.shtml.

Importation of Plant Pests 7 CFR Part 330. Federal Plant Pest Regulations; 

General; Plant Pests; Soil; Stone and Quarry Products; Garbage. This regulation 

requires a permit for movement into or through the United States, or interstate 

any plant pest or a regulated product, article, or means of conveyance in 

accordance with this part. Information can be obtained by calling (877) 770-5990 

or at the USDA Web site: http://www.aphis.usda.gov/permits.

Export of Etiologic Agents of Humans, Animals, Plants and Related Materials; 

Department of Commerce (DoC); 5 CFR Parts 730 to 799. This regulation requires 

that exporters of a wide variety of etiologic agents of human, plant and animal 
Appendix C: Transportation of Infectious Substances 339

diseases, including genetic material, and products which might be used for culture 

of large amounts of agents, will require an export license. Information may be 

obtained by calling the DoC Bureau of Export Administration at (202) 482-4811, 

or at the DoC Web site: http://www.ntis.gov/products/export-regs.aspx; or at 

http://www.access.gpo.gov/bis/index.html; and http://www.bis.doc.gov.

Transfer of CDC Select Agents and Toxins. 42 CFR Part 73 Possession, Use, 

and Transfer of Select Agents and Toxins. The CDC regulates the possession, 

use, and transfer of select agents and toxins that have the potential to pose a 

severe threat to public health and safety. The CDC Select Agent Program registers 

all laboratories and other entities in the United States that possess, use, or transfer 

a select agent or toxin. Entities transferring or receiving select agents and toxins 

must be registered with the Select Agent Program and submit an APHIS/CDC 

Form 2 (see 42 CFR 73 and/or 9 CFR 121) to obtain approval prior to transfer 

of a select agent or toxin. The regulations, Select Agent Program forms, and 

additional guidance is available at the CDC Web site: www.selectagents.gov.

Transfer of USDA Select Agents and Toxins. 9 CFR Part 121 Possession, Use, 

and Transfer of Select Agents and Toxins. The USDA, APHIS, VS regulates the 

possession, use, and transfer of select agents and toxins that have the potential 

to pose a severe threat to animal health or animal products. The VS Select Agent 

Program oversees these activities and registers all laboratories and other entities 

in the U.S. that possess, use, or transfer a VS select agent or toxin. Entities 

transferring or receiving select agents and toxins must be registered with either 

the CDC or APHIS Select Agent Program, and submit an APHIS/CDC Form 2 

(see 42 CFR 73 and/or 9 CFR 121) to obtain approval prior to transfer of a select 

agent or toxin. The regulations, Select Agent Program forms, and additional 

guidance is available at the APHIS Web site: http://www.aphis.usda.gov/

programs/ag_selectagent/index.shtml.

Transfer of USDA Plant Pests

The movement of Plant Pests is regulated under two distinct and separate 

regulations: (1) 7 CFR Part 331. Agricultural Bioterrorism Protection Act of 2002; 

Possession, Use, and Transfer of Biological Agents and Toxins; and (2) 7 CFR 

Part 330 Federal Plant Pest Regulations; General; Plant Pests; Soil; Stone and 

Quarry Products; Garbage. The regulation found at 7 CFR Part 331 requires an 

approved Transfer Form (APHIS/CDC Form 2) prior to importation, interstate, or 

intrastate movement of a Select Agent Plant Pest. In addition, under 7 CFR Part 

330, the movement of a Plant Pest also requires a permit for movement into or 

through the United States, or interstate any plant pest or a regulated product, 

article, or means of conveyance in accordance with this part. Information can be 

obtained by calling (301) 734-5960 or at the USDA Web site: http://www.aphis.

usda.gov/programs/ag_selectagent/index.shtml.
340 Biosafety in Microbiological and Biomedical Laboratories

General DOT Packaging Requirements for Transport of Infectious 

Substances by Aircraft

The DOT packagings for transporting infectious substances by aircraft are required 

by domestic and international aircraft carriers, and are the basis for infectious 

substance packagings for motor vehicle, railcar, and vessel transport. The following 

is a summary of each packaging type and related transportation requirements.

Category A Infectious Substance (UN 2814 and UN 2900): Figure 1. A Category 

A material is an infectious substance that is transported in a form that is capable 

of causing permanent disability or life-threatening or fatal disease to otherwise 

healthy humans or animals when exposure to it occurs. An exposure occurs 

when an infectious substance is released outside of its protective packaging, 

resulting in physical contact with humans or animals. Category A infectious 

substances are assigned to identification number “UN 2814” for substances 

that cause disease in humans or in both humans and animals, or “UN 2900” 

for substances that cause disease in animals only.

Figure 1 shows an example of the UN standard triple packaging system for 

materials known or suspected of being a Category A infectious substance. The 

package consists of a watertight primary receptacle or receptacles; a watertight 

secondary packaging; for liquid materials, the secondary packaging must contain 

absorbent material in sufficient quantities to absorb the entire contents of all primary 

receptacles; and a rigid outer packaging of adequate strength for its capacity, 

mass, and intended use. Each surface of the external dimension of the packaging 

must be 100 mm (3.9 inches) or more. The completed package must pass specific 

performance tests, including a drop test and a water-spray test, and must be 

capable of withstanding, without leakage, an internal pressure producing a pressure 

differential of not less than 95 kPa (0.95 bar, 14 psi). The completed package 

must also be capable of withstanding, without leakage, temperatures in the range 

of -40oC to +55oC (-40oF to 131oF). The completed package must be marked 

“Infectious substances, affecting humans, UN 2814” or “Infectious substances, 

affecting animals, UN 2900” and labeled with a Division 6.2 (infectious substance) 

label. In addition, the package must be accompanied by appropriate shipping 

documentation, including a shipping paper and emergency response information.
Appendix C: Transportation of Infectious Substances 341

Figure 1. A Category A UN Standard Triple Packaging

Biological specimen, Category B (UN 3373): Figure 2. (previously known as 

Clinical specimen and Diagnostic Specimen). A Category B infectious substance 

is one that does not meet the criteria for inclusion in Category A. A Category B 

infectious substance does not cause permanent disability or life-threatening or 

fatal disease to humans or animals when exposure to it occurs. The proper 

shipping name for a Category B infectious substance, “Biological specimen, 

Category B,” is assigned to identification number “UN 3373.” The proper 

shipping names “Diagnostic specimen” and “Clinical specimen” may no 

longer be used (as of January 1, 2007).
342 Biosafety in Microbiological and Biomedical Laboratories

Figure 2 shows an example of the triple packaging system for materials 

known or suspected of containing a Category B infectious substance. A Category 

B infectious substance must be placed in a packaging consisting of a leak proof 

primary receptacle, leak proof secondary packaging, and rigid outer packaging. 

At least one surface of the outer packaging must have a minimum dimension of 

100 mm by 100 mm (3.9 inches). The packaging must be of good quality and 

strong enough to withstand the shocks and loadings normally encountered during 

transportation. For liquid materials, the secondary packaging must contain 

absorbent material in sufficient quantities to absorb the entire contents of all 

primary receptacles. The primary or secondary packaging must be capable of 

withstanding, without leakage, an internal pressure producing a pressure 

differential of 95 kPa. The package must be constructed and closed to prevent 

any loss of contents that might be caused under normal transportation conditions 

by vibration or changes in temperature, humidity, or pressure. The completed 

package must be capable of passing a 1.2-meter (3.9 feet) drop test. The 

package must be marked with a diamond-shaped marking containing the 

identification number “UN 3373” and with the proper shipping name “Biological 

substance, Category B.” In addition, the name, address, and telephone number 

of a person knowledgeable about the material must be provided on a written 

document, such as an air waybill, or on the package itself.

Figure 2. A Category B Non-specification Triple Packaging
Appendix D: Agriculture Pathogen Biosafety 343

Appendix D—Agriculture Pathogen Biosafety

The contents of this Appendix were provided by USDA. All questions regarding 

its contents should be forwarded to the USDA.

Contents

I. Introduction

II. BSL-3-Ag

III. BSL-3, Enhanced

IV. Pathogens of Veterinary Significance

V. Summaries of Selected Agriculture Agents

VI. Additional information

I. Introduction

Risk assessment and management guidelines for agriculture differ from 

human public health standards. Risk management for agriculture research is 

based on the potential economic impact of animal and plant morbidity, and 

mortality, and the trade implications of disease. Agricultural guidelines take 

this difference into account. Worker protection is important but great emphasis 

is placed on reducing the risk of agent escape into the environment. This 

Appendix describes the facility parameters and work practices of what has 

come to be known as BSL-3-Ag. BSL-3-Ag is unique to agriculture because 

of the necessity to protect the environment from an economic, high risk 

pathogen in a situation where studies are conducted employing large 

agricultural animals or other similar situations in which the facility barriers 

now serve as primary containment. Also described are some of the 

enhancements beyond BSL-3 that may be required by USDA/APHIS when 

working in the laboratory or vivarium with veterinary agents of concern. This 

Appendix provides guidance and is not regulatory nor is it meant to describe 

policy. Conditions for approval to work with specific agricultural agents are 

provided at the time USDA/APHIS permits a location to work with an agent.

II. BSL-3-Ag for Work with Loose-housed Animals

In agriculture, special biocontainment features are required for certain types 

of research involving high consequence livestock pathogens in animal 

species or other research where the room provides the primary containment. 

To support such research, USDA has developed a special facility designed, 

constructed and operated at a unique animal containment level called BSL3-Ag. Using the containment features of the standard ABSL-3 facility as a 

starting point, BSL-3-Ag facilities are specifically designed to protect the 

environment by including almost all of the features ordinarily used for BSL-4 

facilities as enhancements. All BSL-3-Ag containment spaces must be 

designed, constructed and certified as primary containment barriers.
344 Biosafety in Microbiological and Biomedical Laboratories

The BSL-3-Ag facility can be a separate building, but more often, it is an 

isolated zone within a facility operating at a lower biosafety level, usually at 

BSL-3. This isolated zone has strictly controlled access with special physical 

security measures and functions on the “box within a box” principle. All BSL3-Ag facilities that cannot readily house animals in primary containment 

devices require the features for an ABSL-3 facility with the following 

enhancements typical of BSL-4 facilities:

1. Personnel change and shower rooms that provide for the separation 

of laboratory clothing from animal facility clothing and that control 

access to the containment spaces. The facility is arranged so that 

personnel ingress and egress are only through a series of rooms 

consisting of: a ventilated vestibule with compressible gaskets on the 

two doors, a “clean” change room outside containment, a shower room 

at the non-containment/containment boundary, and a “dirty” change 

room within containment. Complete animal facility clothing (including 

undergarments, pants and shirts or jump suits, and shoes and gloves)

is typically provided in the “dirty” change room, and put on by personnel 

before entering the research areas. In some facilities, complete animal 

facility clothing and personal protective equipment are provided in the 

“clean” change room, where they can be stored and stowed for use 

without entry into containment. When leaving a BSL-3-Ag animal space 

that acts as the primary barrier and that contains large volumes of 

aerosols containing highly infectious agents (an animal room, necropsy 

room, carcass disposal area, contaminated corridor, etc.), personnel 

usually would be required to remove “dirty” lab clothing, take a shower, 

and put on “clean” lab clothing immediately after leaving this high 

risk animal space and before going to any other part of the BSL-Ag 

facility. When leaving the facility, these personnel would take another 

shower at the access control shower and put on their street clothing. 

Soiled clothing worn in a BSL-3-Ag space is autoclaved before being 

laundered. Personnel moving from one space within containment 

to another will follow the practices and procedures described in the 

biosafety manual specifically developed for the particular facility and 

adopted by the laboratory director.

2. Access doors are self closing and lockable. Emergency exit doors 

are provided, but are locked on the outside against unauthorized use. 

The architect or engineer shall consider the practicality of providing 

vestibules at emergency exits.

3. Supplies, materials and equipment enter the BSL-3-Ag space only 

through an airlock, fumigation chamber, an interlocked and double-door 

autoclave or shower.
Appendix D: Agriculture Pathogen Biosafety 345

4. Double-door autoclaves engineered with bioseals are provided to 

decontaminate laboratory waste passing out of the containment area. 

The double doors of the autoclaves must be interlocked so that the 

outer door can be opened only after the completion of the sterilizing 

cycle, and to prevent the simultaneous opening of both doors. All double 

door autoclaves are situated through an exterior wall of the containment 

area, with the autoclave unit forming an airtight seal with the barrier wall 

and the bulk of the autoclave situated outside the containment space 

so that autoclave maintenance can be performed conveniently. A gas 

sterilizer, a pass-through liquid dunk tank, or a cold gas decontamination 

chamber must be provided for the safe removal of materials and 

equipment that are steam or heat sensitive. Disposable materials 

must be decontaminated through autoclaving or other validated 

decontamination method followed by incineration.

5. Dedicated, single pass, directional, and pressure gradient ventilation 

systems must be used. All BSL-3-Ag facilities have independent air supply 

and exhaust systems that are operated to provide directional airflow and 

a negative air pressure within the containment space. The directional 

airflow within the containment spaces moves from areas of least hazard 

potential toward areas of greatest hazard potential. A visible means of 

displaying pressure differentials is provided. The pressure differential 

display/gauge can be seen inside and outside of the containment space, 

and an alarm sounds when the preset pressure differential is not 

maintained. The air supply and exhaust systems must be interlocked 

to prevent reversal of the directional airflow and positive pressurization 

of containment spaces in the event of an exhaust system failure.

6. Supply and exhaust air to and from the containment space is HEPA 

filtered. Exhaust air is discharged in such a manner that it cannot be 

drawn into outside air intake systems. The HEPA filters are outside of 

containment but are located as near as possible to the containment space 

to minimize the length of potentially contaminated air ducts. The HEPA 

filter housings are fabricated to permit scan testing of the filters in place 

after installation, and to permit filter decontamination before removal. 

Backup HEPA filter units are strongly recommended to allow filter changes 

without disrupting research. The most severe requirements for these 

modern, high level biocontainment facilities include HEPA filters arranged 

both in series and in parallel on the exhaust side, and parallel HEPA 

filters on the supply side of the HVAC systems serving “high risk” areas 

where large amounts of aerosols containing BSL-3-Ag agents could 

be expected (e.g., animal rooms, contaminated corridors, necropsy 

areas, carcass disposal facilities). For these high-risk areas, redundant 

supply and exhaust fans are recommended. The supply and exhaust air 

systems should be equipped with pre-filters (80-90% efficient) to prolong 
346 Biosafety in Microbiological and Biomedical Laboratories

the life of the HEPA filters. Air handling systems must provide 100% 

outside conditioned air to the containment spaces.

7. Liquid effluents from BSL-3-Ag areas must be collected and 

decontaminated in a central liquid waste sterilization system before 

disposal into the sanitary sewers. Typically, a heat decontamination 

system is utilized in these facilities and equipment must be provided to 

process, heat and hold the contaminated liquid effluents to temperatures, 

pressures and times sufficient to inactivate all biohazardous materials 

that reasonably can be expected to be studied at the facility in the future. 

The system may need to operate at a wide range of temperatures and 

holding times to process effluents economically and efficiently. Double 

containment piping systems with leak alarms and annular space 

decontaminating capability should be considered for these wastes. 

Effluents from laboratory sinks, cabinets, floors and autoclave chambers 

are sterilized by heat treatment. Under certain conditions, liquid wastes 

from shower rooms and toilets may be decontaminated by chemical 

treatment systems. Facilities must be constructed with appropriate 

basements or piping tunnels to allow for inspection of plumbing systems.

8. Each BSL-3-Ag containment space shall have its interior surfaces (walls, 

floors, and ceilings) and penetrations sealed to create a functional area 

capable of being certified as airtight. It is recommended that a pressure 

decay test be used (new construction only). Information on how to conduct 

a pressure decay test may be found within Appendix 9B of the ARS Facilities 

Design Manual (Policy and Procedure 242.1M-ARS; http://www.afm.

ars.usda.gov/). This requirement includes all interior surfaces of all 

animal BSL-3-Ag spaces, not just the surfaces making up the external 

containment boundary. All walls are constructed slab to slab, and all 

penetrations, of whatever type, are sealed airtight to prevent escape 

of contained agents and to allow gaseous fumigation for biological 

decontamination. This requirement prevents cross contamination 

between individual BSL-3-Ag spaces and allows gaseous fumigation in 

one space without affecting other spaces. Exterior windows and vision 

panels, if required, are breakage-resistant and sealed. Greenhouses 

constructed to meet the BSL-3-Ag containment level will undergo the 

following tests, or the latest subsequent standards: (a) an air infiltration 

test conducted according to ASTM E 283-91; (b) a static pressure water 

resistance test conducted according to ASTM E 331-93; and (c) a dynamic 

pressure water resistance test conducted according to AAMA 501.1-94.

9. All ductwork serving BSL-3-Ag spaces shall be airtight (pressure testedconsult your facility engineer for testing and certification details).

10. The hinges and latch/knob areas of all passage doors shall be sealed to 

airtight requirements (pressure decay testing).
Appendix D: Agriculture Pathogen Biosafety 347

11. All airlock doors shall have air inflated or compressible gaskets. The 

compressed air lines to the air inflated gaskets shall be provided with 

HEPA filters and check valves.

12. Restraining devices shall be provided in large animal rooms.

13. Necropsy rooms shall be sized and equipped to accommodate large 

farm animals.

14. Pathological incinerators, or other approved means, must be provided for 

the safe disposal of the large carcasses of infected animals. Redundancy 

and the use of multiple technologies need to be considered and evaluated.

15. HEPA filters must be installed on all atmospheric vents serving plumbing 

traps, as near as possible to the point of use, or to the service cock, of 

central or local vacuum systems, and on the return lines of compressed 

air systems. All HEPA filters are installed to allow in-place decontamination 

and replacement. All traps are filled with liquid disinfectant.

16. If BSCs are installed, they should be located such that their operation is 

not adversely affected by air circulation and foot traffic. Class II BSCs 

use HEPA filters to treat their supply and exhaust air. Selection of the 

appropriate type of Class II BSCs will be dependent upon the proposed 

procedures and type of reagents utilized. BSC selection should be made 

with input from a knowledgeable safety professional well versed on the 

operational limitations of class II biohazard cabinetry. Supply air to a 

Class III cabinet is HEPA filtered, and the exhaust air must be double 

filtered (through a cabinet HEPA and then through a HEPA in a dedicated 

building exhaust system) before being discharged to the atmosphere.

III. BSL-3 and ABSL-3 Plus Potential Facility Enhancements for 

Agriculture Agent Permitting

The descriptions and requirements listed above for BSL-3-Ag studies are 

based on the use of high-risk organisms in animal systems or other types of 

agriculture research where the facility barriers, usually considered secondary 

barriers, now act as primary barriers. Certain agents that typically require a 

BSL-3-Ag facility for research that utilizes large agricultural animals may be 

studied in small animals in an enhanced BSL-3 laboratory or enhanced 

ABSL-3 when the research is done within primary containment devices. In 

these situations, the facility no longer serves as the primary barrier as with 

the large animal rooms. Therefore, when manipulating high consequence 

livestock pathogens in the laboratory or small animal facility, facility design 

and work procedures must meet the requirements of BSL-3 or ABSL-3 with 

additional enhancements unique to agriculture. Agriculture enhancements 

are agent, site and protocol dependent. The facility may have personnel 
348 Biosafety in Microbiological and Biomedical Laboratories

enter and exit through a clothing change and shower room, have a doubledoor autoclave and/or fumigation chamber, HEPA filter supply and exhaust 

air, and a validated or approved system in place to decontaminate research 

materials and waste. Surfaces must be smooth to support wipe-down 

decontamination and penetrations should be sealed and the room capable 

of sealing in case gaseous decontamination is required. Because all work 

with infectious material is conducted within primary containment, there 

is no requirement for pressure decay testing the room itself.

The need for any potential agriculture enhancements is dependant upon a 

risk assessment. Therefore, after an assessment and in consultation with 

USDA/ APHIS, the required agriculture enhancement(s) may include:

1. Personnel change and shower rooms that provide for the separation 

of street clothing from laboratory clothing and that control access to the 

containment spaces. The facility is arranged so that personnel ingress 

and egress are only through a series of rooms (usually one series for 

men and one for women) consisting of: a ventilated vestibule with a 

“clean” change room outside containment, a shower room at the noncontainment/containment boundary, and a “dirty” change room within 

containment. Complete laboratory clothing (including undergarments, 

pants and shirts or jump suits, and shoes and gloves) is provided in 

the “dirty” change room, and put on by personnel before entering the 

research areas. In some facilities, complete laboratory clothing and 

personal protective equipment are provided in the “clean” change 

room, where they can be stored and stowed for use without entry into 

containment. When leaving a BSL-3 enhanced space, personnel usually 

would be required to remove their “dirty” laboratory clothing, take a 

shower, and put on “clean” laboratory clothing immediately after leaving 

the BSL-3 enhanced space and before going to any other part of the 

facility. Soiled clothing worn in a BSL-3 enhanced space should be 

autoclaved before being laundered outside of the containment space. 

Personnel moving from one space within containment to another will 

follow the practices and procedures described in the biosafety manual 

specifically developed for the particular facility and adopted by the 

laboratory director.

2. Access doors to these facilities are self closing and lockable. 

Emergency exit doors are provided but are locked on the outside 

against unauthorized use. The architect or engineer shall consider 

the practicality of providing vestibules at emergency exits.

3. Supplies, materials and equipment enter the BSL-3 enhanced space 

only through the double-door ventilated vestibule, fumigation chamber 

or an interlocked and double-door autoclave.
Appendix D: Agriculture Pathogen Biosafety 349

4. Double-door autoclaves engineered with bioseals are provided to 

decontaminate laboratory waste passing out of the containment area. 

The double doors of the autoclaves must be interlocked so that the outer 

door can be opened only after the completion of the sterilizing cycle, 

and to prevent the simultaneous opening of both doors. All double door 

autoclaves are situated through an exterior wall of the containment area, 

with the autoclave unit forming an airtight seal with the barrier wall and 

the bulk of the autoclave situated outside the containment space so that 

autoclave maintenance can be performed conveniently. A gas sterilizer, 

a pass-through liquid dunk tank, or a cold gas decontamination chamber 

must be provided for the safe removal of materials and equipment that 

are steam or heat sensitive. All other materials must be autoclaved or 

otherwise decontaminated by a method validated to inactivate the agent 

before being removed from the BSL-3 enhanced space. Wastes and 

other materials being removed from the BSL-3 enhanced space must 

be disposed of through incineration or other approved process.

5. Dedicated, single pass, directional, and pressure gradient ventilation 

systems must be used. All BSL-3 enhanced facilities have independent 

air supply and exhaust systems operated to provide directional airflow 

and a negative air pressure within the containment space. The directional 

airflow within the containment spaces moves from areas of least hazard 

potential toward areas of greatest hazard potential. A visible means of 

displaying pressure differentials is provided. The pressure differential 

display/gauge can be seen inside and outside of the containment 

space, and an alarm sounds when the preset pressure differential is 

not maintained. Supply and exhaust air to and from the containment 

space is HEPA filtered, with special electrical interlocks to prevent 

positive pressurization during electrical or mechanical breakdowns.

6. The exhaust air is discharged in such a manner that it cannot be drawn 

into outside air intake systems. HEPA filters located outside of the 

containment barrier are located as near as possible to the containment 

space to minimize the length of potentially contaminated air ducts. The 

HEPA filter housings are fabricated to permit scan testing of the filters 

in place after installation, and to permit filter decontamination before 

removal. Backup parallel HEPA filter units are strongly recommended 

to allow filter changes without disrupting research. Air handling systems 

must provide 100% outside conditioned air to the containment spaces.

7. Contaminated liquid wastes from BSL-3 enhanced areas must be 

collected and decontaminated by a method validated to inactivate the 

agent being used before disposal into the sanitary sewers. Treatment 

requirement will be determined by a site-specific, agent-specific risk 

assessment. Floor drains are discouraged in ABSL-3 and BSL-3 
350 Biosafety in Microbiological and Biomedical Laboratories

agriculture enhanced laboratories lacking a liquid waste central 

sterilization system. If floor drains are present, they should be capped 

and sealed. Facilities should be constructed with appropriate basements 

or piping tunnels to allow for inspection of plumbing systems, if a central 

liquid waste sterilization system is used.

8. Each BSL-3 enhanced containment space shall have its interior surfaces 

(walls, floors, and ceilings) and penetrations sealed to create a functional 

area capable of being decontaminated using a gaseous or vapor 

phase method. All walls are contiguous with the floor and ceiling, and 

all penetrations, of whatever type, are sealed. Construction materials 

should be appropriate for the intended end use. Exterior windows and 

vision panels, if required, are breakage-resistant and sealed.

9. All exhaust ductwork prior to the HEPA exhaust filter serving BSL-3 

enhanced spaces shall be subjected to pressure decay testing before 

acceptance of the facility for use. Consult your facility engineer for 

testing and commissioning details.

IV. Pathogens of Veterinary Significance

Some pathogens of livestock, poultry and fish may require special laboratory 

design, operation, and containment features. This may be BSL-3, BSL-3 plus 

enhancements or BSL-4 and for animals ABSL-2, ABSL-3 or BSL-3-Ag. The 

importation, possession, or use of the following agents is prohibited or restricted 

by law or by USDA regulations or administrative policies.

This Appendix does not cover manipulation of diagnostic samples; however, 

if a foreign animal disease agent is suspected, samples should be immediately 

forwarded to a USDA diagnostic laboratory (The National Veterinary Services 

Laboratories, Ames, IA or the Foreign Animal Disease Diagnostic Laboratory, 

Plum Island, NY). A list of agents and their requirements follows.

African horse sickness virus a, b Louping ill virus a

African swine fever virus a, b, c Lumpy skin disease virus a, b, c

Akabane virus b Malignant catarrhal fever virus 

(exotic strains or alcelaphine herpesvirus type 1) b

Avian influenza virus (highly pathogenic) a, b, c Menangle virus b

Bacillus anthracis a, b Mycobacterium bovis

Besnoitia besnoiti Mycoplasma agalactiae

Bluetongue virus (exotic) a, b Mycoplasma mycoides subsp. mycoides (small 

colony type) a, b, c
Appendix D: Agriculture Pathogen Biosafety 351

Borna disease virus Mycoplasma capricolum a, b, c

Bovine infectious petechial fever agent Nairobi sheep disease virus (Ganjam virus)

Bovine spongiform encephalopathy prion b Newcastle disease virus (velogenic strains) a, b, c

Brucella abortus a, b Nipah virus a, b, d

Brucella melitensis a, b Peste des petits ruminants virus 

(plague of small ruminants) a, b, c

Brucella suis a, b Rift Valley fever virus a, b, c

Burkholderia mallei/Pseudomonas mallei

(Glanders) a, b

Rinderpest virus a, b, c

Burkholderia pseudomallei a, b Sheep pox virus a, b

Camelpox virus b Spring Viremia of Carp virus

Classical swine fever virus a, b, c Swine vesicular disease virus b

Coccidioides immitis b Teschen disease virus a

Cochliomyia hominivorax (Screwworm) Theileria annulata

Coxiella burnetti (Q fever) b Theileria lawrencei

Ephemeral fever virus Theileria bovis

Ehrlichia (Cowdria) ruminantium (heartwater) b Theileria hirci

Eastern equine encephalitis virus a, b Trypanosoma brucei

Foot and mouth disease virus a, b, c Trypanosoma congolense

Francisella tularensis b Trypanosoma equiperdum (dourine)

Goat pox a, b Trypanosoma evansi

Hemorrhagic disease of rabbits virus Trypanosoma vivax

Hendra virus a, b, d Venezuelan equine encephalomyelitis virus a, b

Histoplasma (Zymonema) farciminosum Vesicular exanthema virus

Infectious salmon anemia virus Vesicular stomatitis virus (exotic) a, b

Japanese encephalitis virus a, b Wesselsbron disease virus

Notes:

a Export license required by Department of Commerce (See: http://www.bis.doc.gov/index.htm). b Agents regulated as Select Agents under the Bioterrorism Act of 2002. Possession of these agents 

requires registration with either the CDC or APHIS and a permit issued for interstate movement or 

importation by APHIS-VS. Most require BSL-3/ABSL-3 or higher containment (enhancements as 

described in this Appendix or on a case-by-case basis as determined by APHIS-VS). c Requires BSL-3-Ag containment for all work with the agent in loose-housed animals. d Requires BSL-4 containment for all work with the agent.
352 Biosafety in Microbiological and Biomedical Laboratories

A USDA/APHIS import or interstate movement permit is required to obtain 

any infectious agent of animals or plants that is regulated by USDA/APHIS. An 

import permit is also required to import any livestock or poultry product such as 

blood, serum, or other tissues.

V. Summaries of Selected Agriculture Agents

African Swine Fever Virus (ASFV)

ASF is a tick-borne and contagious, febrile, systemic viral disease of swine.1,2,3

The ASF virus (ASFV) is a large (about 200 nm) lipoprotein-enveloped, 

icosahedral, double-stranded DNA virus in the family Asfarviridae, genus 

Asfivirus. This virus is quite stable and will survive over a wide range of pH. The 

virus will survive for 15 weeks in putrefied blood, three hours at 50oC, 70 days in 

blood on wooden boards, 11 days in feces held at room temperature, 18 months 

in pig blood held at 4oC, 150 days in boned meat held at 39oF, and 140 days in 

salted dried hams. Initially, domestic and wild pigs (Africa: warthog, bush pig, and 

giant forest hog; Europe: feral pig) were thought to be the only hosts of ASFV. 

Subsequently, researchers showed that ASFV replicates in Ornithodoros ticks 

and that there is transstadial, transovarial, and sexual transmission. ASF in wild 

pigs in Africa is now believed to cycle between soft ticks living in warthog burrows 

and newborn warthogs. Ornithodoros ticks collected from Haiti, the Dominican 

Republic, and southern California have been shown to be capable vectors of 

ASFV, but in contrast to the African ticks, many of the ticks from California died 

after being infected with ASFV. Because ASFV-infected ticks can infect pigs, 

ASFV is the only DNA virus that can qualify as an arbovirus.

Even though the soft tick has been shown to be a vector (and in Africa 

probably the reservoir of ASFV), the primary method of spread from country to 

country has been through the feeding of uncooked garbage containing ASFVinfected pork scraps to pigs.

Aerosol transmission is not important in the spread of ASF. Because ASFV 

does not replicate in epithelial cells, the amount of virus shed by an ASF-infected 

pig is much less than the amount of virus shed by a hog-cholera-infected pig. 

The blood of a recently infected pig contains a very high ASFV titer.

Laboratory Safety and Containment Recommendations

Humans are not susceptible to ASFV infection. The greatest risk of working with the 

virus is the escape of the organism into a susceptible pig population, which would 

necessitate USDA emergency procedures to contain and eradicate the disease.

ASF is considered a foreign animal disease in the United States. Due to the 

highly contagious nature of the agent and the severe economic consequences of 

disease in the United States, this organism should only be handled in vitro in a 

BSL-3 laboratory with enhancements as required by the USDA and in vivo in a 
Appendix D: Agriculture Pathogen Biosafety 353

USDA-approved BSL-3-Ag facility for loosely housed animals. Special 

consideration should be given to infected vector control.

Special Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.

African Horse Sickness Virus (AHSV)

AHSV is a member of genus Orbivirus in the family Reoviridae. Nine serotypes, 

numbers 1 – 9, are recognized. AHSV grows readily in embryonated chicken 

eggs, suckling mice, and a variety of standard cell cultures. AHSV infects and 

causes viremia in equids. Most horses die from the disease, about half of 

donkeys and most mules survive, but zebras show no disease. Viremias may 

last up to one month despite the rapid development of neutralizing antibodies. 

AHSV may cause disease in dogs, but these are not thought to be important 

in the natural history of the disease.4,5

AHSV has been recognized in central Africa and periodically spreads to 

naive populations in South and North Africa, the Iberian Peninsula, the Middle 

East, Pakistan, Afghanistan, and India. AHSV is vectored by Culicoides species 

and perhaps by mosquitoes, biting flies, and ticks limiting viral spread to climates 

and seasons favorable to the vectors. At least one North American Culicoides 

species transmits AHSV. AHSV may infect carnivores that consume infected 

animals but these are not thought to be relevant to natural transmission to equids.

Occupational Infections

Encephalitis and uveochorioretinitis were observed in four laboratory workers 

accidentally exposed to freeze-dried modified live vaccine preparations. Although 

AHSV could not be conclusively linked to disease, all four had neutralizing 

antibodies. Encephalitis was documented in experimentally infected monkeys.

Laboratory Safety and Containment Recommendations

Virus may be present in virtually any sample taken from an infected animal, but 

the highest concentrations are found in spleen, lung, and lymph nodes. The only 

documented risk to laboratory workers involves aerosol exposure to large amounts 

of vaccine virus. AHSV is unusually stable in blood or serum stored at 4°C.

AHS is considered a foreign animal disease in the United States. Due to the 

severe economic consequences of disease presence in the United States, this 

organism should only be handled in vitro in a BSL-3 laboratory with 
354 Biosafety in Microbiological and Biomedical Laboratories

enhancements as required by the USDA and in vivo in a USDA-approved ABSL3 animal facility with enhancements. Blood, serum, or tissues taken from equids 

in areas where AHSV exists are potential means of transmitting the agent long 

distances. Special consideration should be given to infected vector containment.

Special Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.

Akabane Virus (AKAV)

AKAV is a member of the genus Orthobunyavirus in the Simbu serogroup of 

the family Bunyaviridae. The Simbu serogroup also includes Aino, Peaton, 

and Tinaroo viruses that can cause similar disease. Experimental infection of 

pregnant hamsters leads to death of the fetus. This virus grows and causes 

disease in chick embryos. Isolated in suckling mice and hamster lung cell 

cultures, AKAV is an important cause of disease in ruminants. The virus does not 

cause overt disease in adults but infects the placenta and fetal tissues in cattle, 

sheep, and goats to cause abortions, stillbirths, and congenital malformations. 

The broad range of clinical signs in the fetus is related primarily to central 

nervous system damage that occurs during the first trimester of pregnancy.

6,7

AKAV is not known to infect or cause disease in humans; concern focuses 

only on effects to agriculture and wildlife. Common names of disease include 

congenital arthrogryposis-hydranencephaly syndrome, Akabane disease, acorn 

calves, silly calves, curly lamb disease, curly calf disease, and dummy calf disease. 

The host range of naturally occurring Akabane disease appears limited to cattle, 

sheep, swine, and goats but other animals including swine and numerous wildlife 

species become infected. AKAV is an Old World virus, being found in Africa, Asia, 

and Australia. Disease is unusual in areas where the virus is common because 

animals generally become immune before pregnancy. AKAV spreads naturally 

only in gnat and mosquito insect vectors that become infected after feeding on 

viremic animals.

Laboratory Safety and Containment Recommendations

AKAV may be present in blood, sera, and tissues from infected animals, as well 

as vectors from endemic regions. Parenteral injection of these materials into 

naive animals and vector-borne spread to other animals represents a significant 

risk to agricultural interests.
Appendix D: Agriculture Pathogen Biosafety 355

Akabane disease is considered a foreign animal disease in the United 

States. Due to the severe economic consequences of disease presence in the 

United States, this organism should only be handled in vitro in a BSL-3 laboratory 

with enhancements as required by the USDA and in vivo in a USDA-approved 

ABSL-3 animal facility with enhancements. Special consideration should be given 

to infected vector containment.

Specials Issues

Although it is virtually certain AKAV will grow and cause disease in New World 

livestock, it is not known if it will cause viremias in New World wildlife high enough 

to infect vectors, if it can be vectored by New World insects, or if it will cause 

disease in New World wildlife. Because fetal disease may not become evident 

until months after virus transmission, an introduction into a new ecosystem may 

not be recognized before the virus has become firmly entrenched.

The importation, possession, or use of this agent is prohibited or restricted 

by law or by USDA regulations or administrative policies. A USDA/APHIS import 

or interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.

Bluetongue Virus (BTV)

BTV is a member of the family Reoviridae, genus Orbivirus. There are 24 

recognized serotypes numbered 1 through 24. BTV is notable for causing 

disease in sheep and cattle and is very similar to other orbiviruses that cause 

disease in deer (epizootic hemorrhagic disease of deer virus) and horses (AHSV), 

and a few that cause disease in man (Colorado tick fever virus and others). These 

viruses have dsRNA genomes distributed amongst 10 segments, enabling efficient 

reassortment. Growth on a wide variety of cultured cells is usually cytocidal. 

Growth in animals results in viremia within three to four days that endures as 

long as 50 days despite the presence of high levels of neutralizing antibodies.

8,9

BTV infects all ruminants, but bluetongue disease is unusual except in sheep 

and is unpredictable even in sheep. Disease is evidenced by fever, hyperemia, 

swelling, and rarely erosions and ulceration of the buccal and nasal mucosa. 

Hyperemia of the coronary bands of the hooves may cause lameness. In the 

worst cases, the disease progresses through weakness, depression, rapid 

weight loss, prostration, and death. Maternal transmission to the fetus may 

cause abortion or fetal abnormalities in the first trimester. Bluetongue disease 

also occurs in cattle but is rarely diagnosed. BTV may infect fetal calves and result 

in abortion or fetal brain damage. The full host range of BTV is still unknown but 

includes wild ruminants, neonatal mice, dogs, and chicken embryos.
356 Biosafety in Microbiological and Biomedical Laboratories

BTV infection occurs in tropical, subtropical, and temperate climates where 

the Culicoides vectors exist. Global warming may be expanding the geographic 

range of Culicoides, and therefore BTV, into higher latitudes. Most countries have 

a unique assortment of the 24 serotypes. For example, BTV serotypes 2, 10, 11, 

13, and 17 are currently active in the United States, but serotypes 1, 3, 4, 6, 8, 

12, and 17 were present in the Caribbean basin when last surveyed. Concern 

over the spread of individual serotypes by trade in animals and animal products 

has engendered costly worldwide trade barriers.

The primary natural mode of transmission is by Culicoides midges. Only a 

few of more than 1,000 species of Culicoides transmit BTV. A strong correlation 

between the vector species and the associated BTV suggests these viruses may 

have adapted to their local vector. Thus, BTV does not exist in areas such as the 

Northeast United States where the local Culicoides fails to transmit BTV. Virus is 

present in semen at peak of viremia, but this is not considered a major route of 

transmission. Because of the prolonged viremia, iatrogenic transmission is 

possible. Only modified-live (attenuated) virus vaccines are available and a 

vaccine for only one serotype is currently available in the United States.

Laboratory Safety and Containment Recommendations

BTV is not known to cause disease in humans under any conditions. BTV 

commonly enters the laboratory in blood samples. The virus is stable at -70°C and 

in blood or washed blood cells held at 4°C. Sera prepared from viremic animals 

may represent some risk if introduced parenterally into naive animals. Blood, 

sera, and bovine semen can carry BTV across disease control boundaries.

The most significant threat from BTV occurs when virus is inoculated 

parenterally into naive animals. If appropriate Culicoides are present, virus can be 

transmitted to other hosts. Therefore, BTV-infected animals must be controlled for 

the two-month period of viremia and protected against Culicoides by physical means 

and/ or performing experiments at least two months before local Culicoides emerge. 

Thus, BTV exotic to the United States should only be handled in vitro in a BSL-3 

laboratory with enhancements as required by the USDA and in vivo in a USDAapproved ABSL-3 with enhancements. Special consideration should be given to 

infected vector containment. Special containment is only needed when working 

with serotypes of BTV that are exotic to the country or locality. BTV on laboratory 

surfaces is susceptible to 95% ethanol and 0.5% sodium hypochlorite solution.

Special Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.
Appendix D: Agriculture Pathogen Biosafety 357

Classical Swine Fever Virus (Hog Cholera)

Classical swine fever is a highly contagious viral disease of swine that occurs 

worldwide in an acute, a subacute, a chronic, or a persistent form.10-12 In the acute 

form, the disease is characterized by high fever, severe depression, multiple 

superficial and internal hemorrhages, and high morbidity and mortality. In the chronic 

form, the signs of depression, anorexia, and fever are less severe than in the 

acute form, and recovery is occasionally seen in mature animals. Transplacental 

infection with viral strains of low virulence often results in persistently infected 

piglets, which constitute a major cause of virus dissemination to noninfected 

farms. Although minor antigenic variants of classical swine fever virus (CSFV) 

have been reported, there is only one serotype. Hog cholera virus is a lipidenveloped pathogen belonging to the family Flaviviridae, genus Pestivirus. The 

organism has a close antigenic relationship with the bovine viral diarrhea virus 

(BVDV) and the border disease virus (BDV). In a protein-rich environment, hog 

cholera virus is very stable and can survive for months in refrigerated meat and 

for years in frozen meat. The virus is sensitive to drying (desiccation) and is 

rapidly inactivated by a pH of less than 3 and greater than 11.

The pig is the only natural reservoir of CSFV. Blood, tissues, secretions and 

excretions from an infected animal contain virus. Transmission occurs mostly 

by the oral route, though infection can occur through the conjunctiva, mucous 

membrane, skin abrasion, insemination, and percutaneous blood transfer (e.g., 

common needle, contaminated instruments). Airborne transmission is not thought 

to be important in the epizootiology of classical swine fever. Introduction of 

infected pigs is the principal source of infection in classical swine fever-free 

herds. Farming activities such as auction sales, livestock shows, visits by feed 

dealers, and rendering trucks also are potential sources of contagion. Feeding 

of raw or insufficiently cooked garbage is a potent source of hog cholera virus. 

During the warm season, insect vectors common to the farm environment may 

spread hog cholera virus mechanically. There is no evidence, however, that hog 

cholera virus replicates in invertebrate vectors.

Laboratory Safety and Containment Recommendations

Humans being are not susceptible to infection by CSFV. The greatest risk of 

working with these viruses is the escape of the organism into susceptible 

domestic or feral pig populations, which would necessitate USDA emergency 

procedures to contain and eradicate the diseases.

The virus is considered cause of a foreign animal disease in the United 

States. Due to the highly contagious nature of the agent and the severe economic 

consequences of disease presence in the United States, this organism should 

only be handled in vitro in a BSL-3 laboratory with enhancements as required by 

the USDA and in vivo in a USDA-approved BSL-3-Ag facility for loosely housed 
358 Biosafety in Microbiological and Biomedical Laboratories

animals. Laboratory workers should have no contact with susceptible hosts for 

five days after working with the agent.

Specials Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.

Contagious Bovine Pleuropneumonia Agent (CBPP)

CBPP is a highly infectious acute, subacute, or chronic disease, primarily of 

cattle, affecting the lungs and occasionally the joints, caused by Mycoplasma 

mycoides mycoides.

13-15 Contagious bovine pleuropneumonia is caused by 

M. mycoides mycoides small-colony type (SC type). M. mycoides mycoides 

large-colony type is pathogenic for sheep and goats but not for cattle. M. mycoides 

mycoides (SC type) survives well only in vivo and is quickly inactivated when 

exposed to normal external environmental conditions. The pathogen does not 

survive in meat or meat products and does not survive outside the animal in 

nature for more than a few days. Many of the routinely used disinfectants will 

effectively inactivate the organism.

CBPP is predominantly a disease of the genus Bos; both bovine and zebu 

cattle are naturally infected. There are many reported breed differences with respect 

to susceptibility. In general, European breeds tend to be more susceptible than 

indigenous African breeds. In zoos, the infection has been recorded in bison and 

yak. Although it has been reported that the domestic buffalo (Bubalus bubalis) is 

susceptible, the disease is difficult to produce experimentally in this species.

CBPP is endemic in most of Africa. It is a problem in parts of Asia, especially 

India and China. Periodically, CBPP occurs in Europe, and outbreaks within the 

last decade have occurred in Spain, Portugal, and Italy. The disease was eradicated 

from the United States in the nineteenth century, and it is not present currently in 

the Western hemisphere.

CBPP is spread by inhalation of droplets from an infected, coughing animal. 

Consequently, relatively close contact is required for transmission to occur. 

Outbreaks usually begin as the result of movement of an infected animal into 

a naive herd. There are limited anecdotal reports of fomite transmission, but 

fomites are not generally thought to be a problem.
Appendix D: Agriculture Pathogen Biosafety 359

Laboratory Safety and Containment Recommendations

Humans are not susceptible to infection by CBPP. The greatest risk of working 

with these mycoplasma is the escape of the organism into susceptible domestic 

bovine populations, which would necessitate USDA emergency procedures to 

contain and eradicate the diseases.

CBPP is considered a foreign animal disease in the United States. Due to the 

highly contagious nature of the agent and the severe economic consequences 

of disease presence in the United States, this organism should only be handled 

in vitro in a BSL-3 laboratory with enhancements as required by the USDA and 

in vivo in a USDA-approved BSL-3-Ag facility for loosely housed animals.

Special Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.

Contagious Caprine Pleuropneumonia Agent (CCPP)

CCPP is an acute highly contagious disease of goats caused by a mycoplasma 

and characterized by fever, coughing, severe respiratory distress, and high 

mortality.16-18 The principal lesion at necropsy is fibrinous pleuropneumonia. 

The causative agent of CCPP is considered to be M. mycoides capri (type strain 

PG-3) or a new mycoplasma M. capricolum subsp. capripneumoniae (designated 

F-38).19-21 Neither of these agents occurs in North America.

M. mycoides mycoides has also been isolated from goats with pneumonia. 

This agent (the so-called large colony or LC variant of M. mycoides mycoides) 

usually produces septicemia, polyarthritis, mastitis, encephalitis, conjunctivitis, 

hepatitis, or pneumonia in goats. Some strains of this agent (LC variant) will cause 

pneumonia closely resembling CCPP, but the agent is not highly contagious and 

is not considered to cause CCPP. It does occur in North America. M. capricolum 

capricolum, a goat pathogen commonly associated with mastitis and polyarthritis 

in goats, can also produce pneumonia resembling CCPP, but it usually causes 

severe septicemia and polyarthritis. This agent (which does occur in the United 

States) is closely related to mycoplasma F-38 but can be differentiated from it 

using monoclonal antibodies.

CCPP is a disease of goats, and where the classical disease has been 

described, only goats were involved in spite of the presence of sheep and cattle. 

Mycoplasma F-38, the probable cause of the classic disease, does not cause 

disease in sheep or cattle. M. mycoides capri, the other agent considered a 
360 Biosafety in Microbiological and Biomedical Laboratories

cause of CCPP, will result in a fatal disease in experimentally inoculated sheep 

and can spread from goats to sheep. It is however, not recognized as a cause 

of natural disease in sheep.

CCPP has been described in many countries of Africa, the Middle East, 

Eastern Europe, the former Soviet Union, and the Far East. It is a major scourge 

in many of the most important goat-producing countries in the world and is 

considered by many to be the world’s most devastating goat disease.

CCPP is transmitted by direct contact through inhalation of infective aerosols. 

Of the two known causative agents, F-38 is far more contagious. Outbreaks of 

the disease often occur after heavy rains (e.g., after the monsoons in India) and 

after cold spells. This is probably because recovered carrier animals start 

shedding the mycoplasmas after the stress of sudden climatic change. It is 

believed that a long-term carrier state may exist.

Laboratory Safety and Containment Recommendations

Humans are not susceptible to infection by the agent that causes CCPP. The 

greatest risk of working with this mycoplasma is the escape of the organism into 

susceptible domestic caprine populations, which would necessitate USDA 

emergency procedures to contain and eradicate the diseases.

CCPP is considered a foreign animal disease in the United States. Due to the 

highly contagious nature of the agent and the severe economic consequences of 

disease in the United States, this organism should only be handled in vitro in a 

BSL-3 laboratory with enhancements as required by the USDA and in vivo in a 

USDA-approved BSL-3-Ag facility for loosely housed animals.

Special Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.

Foot and Mouth Disease Virus (FMD)

FMD is a severe, highly communicable viral disease of cloven-hoofed animals 

(cattle, swine, sheep, and goats), causing fever, malaise, vesicular lesions in 

affected livestock and in some cases death in young animals due to myocardial 

lesions.22 It can also affect a variety of wild ruminants (e.g., deer, bison). FMD is 

one of the most devastating diseases of livestock, causing large economic losses 

when introduced to FMD-free countries. The etiologic agent, FMD virus (FMDV), 

is a member of the aphtovirus genus, family picornaviridae with seven serotypes 
Appendix D: Agriculture Pathogen Biosafety 361

(A, O, C, Asia1, SAT1, SAT2 and SAT3).23 Humans are considered accidental 

hosts for FMDV and rarely become infected or develop clinical disease. Historically, 

humans have been exposed to large quantities of FMDV both during natural 

outbreaks among large herds of animals and in laboratory settings. Despite this, 

there has been an extremely low incidence of human infections reported and 

many have been anecdotal. Reports of fever, headaches and vesicles in the skin 

(especially at an accidental inoculation site) and oral mucosa have been associated 

with documented FMDV infections. The symptoms can be easily mistaken with 

those of Hand, Foot and Mouth Disease caused by coxsackie A viruses. On the 

other hand, humans have been shown to carry virus in their throats for up to 

three days after exposure to aerosols from infected animals, potentially making 

them carriers of FMDV. Humans and their clothing and footwear have been 

implicated as fomites for transmission of FMDV during outbreaks. Therefore, 

most FMDV laboratories impose a five day period of contact avoidance with 

susceptible species for personnel working with the viruses.

Laboratory Safety and Containment Recommendations

Laboratory practices for FMDV are principally designed to prevent transmission to 

susceptible livestock, but also to protect workers. The greatest risk of working with 

FMD is the escape of the organism into susceptible animal populations, which would 

necessitate USDA emergency procedures to contain and eradicate the disease.

The virus is considered a cause of a foreign animal disease in the United 

States. Due to the highly contagious nature and the severe economic consequences 

of disease presence in the United States, this virus should only be handled in vitro 

in a BSL-3 laboratory with enhancements as required by the USDA (see Section 

IV of this Appendix) and in vivo in USDA-approved BSL-3-Ag animal facilities. 

Infected animals are handled with standard protection (gloves, protective 

clothing). Change of clothing, personal showers and clearing of the throat and 

nose are required upon exiting contaminated areas in order to minimize virus 

transmission to susceptible species. Laboratory workers should have no contact 

with susceptible hosts for five days after working with the agent. In the United 

States, the Plum Island Animal Disease Center in New York is the only laboratory 

authorized to possess and work with this agent.

Special Issues

FMDV is a select agent. Possession, transfer and use of this agent requires 

application of procedures as detailed in the Agricultural Bioterrorism Protection 

Act of 2002 and codified in 9 CFR Part 121. All rules concerning the possession, 

storage, use, and transfer of select agents apply. Please review Appendix F of 

this document for further instructions regarding select agents. Law prohibits 

research with FMD on the United States mainland.
362 Biosafety in Microbiological and Biomedical Laboratories

Heartwater Disease Agent (HD)

HD is a non-contagious disease of domestic and wild ruminants caused by Ehrlichia 

ruminantium.

24 E. ruminantium (formerly Cowdria ruminantium) is a member of the 

family Rickettsiaceae characterized by organisms that are obligate intracellular 

parasites. These organisms often persist in the face of an immune response due 

to their protected intracellular status. Rickettsias in natural conditions are found 

in mammals and blood-sucking arthropods. Ticks of the genus Amblyomma 

transmit E. ruminantium. HD occurs primarily in Africa, but has been recognized 

in the West Indies since the 1980’s. The pathogen is transmitted by ticks of the 

genus Amblyomma, most importantly A. variegatum (tropical bont tick). This tick 

has wide distribution in Africa and is present on several Caribbean islands. Three 

North American tick species, A. maculatum (Gulf Coast tick), A. cajennese, and 

A. dissimile, can transmit the organism, causing concern that competent vectors 

could transmit E. ruminantium in the United States.

Severe HD comprises fever, depression, rapid breathing, and convulsions 

in cattle, sheep, goats and water buffalo. Whitetail deer also are susceptible to 

E. ruminantium infection and develop severe clinical disease. HD has not been 

diagnosed in the United States but occurs in numerous Caribbean islands, as 

well as in most countries of Africa, south of the Sahara Desert.

Laboratory Safety and Containment Recommendations

E. ruminantium can be found in whole blood, brain and experimentally in liver 

and kidney. It is not a human pathogen. Humans are not susceptible to infection 

with the agent that causes HD. The greatest risk of working with this agent is the 

escape of this organism (or infected ticks) into a susceptible domestic bovine 

population, which would necessitate USDA emergency procedures to contain 

and eradicate the disease.

HD is considered a foreign animal disease in the United States. E. ruminantium 

should be handled in vitro in BSL-3 laboratory facilities. Animal work should be 

conducted in ABSL-3 animal facilities or in ABSL-2 animal facilities with special 

modifications such as tick dams (where applicable).

Special Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.
Appendix D: Agriculture Pathogen Biosafety 363

Infectious Salmon Anemia (ISA) Virus

ISA is a disease of Atlantic salmon (Salmo salar) caused by an orthomyxovirus 

in the family Orthomyxoviridae, genus Isavirus. Both wild and cultured Atlantic 

salmon are susceptible to infection, as are brown trout (Salmo trutta), rainbow 

trout (Oncorhynchus mykiss) and herring. The first clinical cases of ISA in Atlantic 

salmon were reported from Norway in 1984. Since then, ISA has been observed 

in Canada (1996), Scotland (1998), Chile (1999), Faroe Islands (2000) and the 

U.S. (2001).25,26 There is significant molecular difference between virus isolates 

(i.e., “Norwegian”, “Scottish” and “North American”).27 Clinical signs of ISA include 

severe anemia, swelling and hemorrhaging in the kidney and other organs, pale 

gills, protruding eyes, darkening of the posterior gut, fluid in the body cavity and 

lethargy. The infection is systemic and most noted in blood and mucus, muscle, 

internal organs and feces. The principal target organ for ISA virus (ISAV) is the 

liver. Signs usually appear two to four weeks after the initial infection.

Reservoirs of ISAV infection are unknown, but the spread of infection may 

occur due to the purchase of subclinically infected smolts, from farm to farm, 

and from fish slaughterhouses or industries where organic material (especially 

blood and processing water) from ISAV-infected fish is discharged without 

necessary treatment.28

Laboratory Safety and Containment Recommendations

Humans are not susceptible to ISAV infection. The greatest risk of working with this 

virus is the escape of the organism into a susceptible fish population, which would 

necessitate USDA emergency procedures to contain and eradicate the disease.

ISA is considered a reportable disease in the United States. ISAV should be 

handled in vitro in BSL-2 laboratory facilities with enhancements as required by 

USDA. Animal inoculations should be handled in ABSL-3 animal facilities with 

special modifications as required. Recommended precautions include incineration of 

fish, tissues, blood and materials (gloves, laboratory coats, etc.) used in the collection 

and processing of fish samples. All surfaces exposed to potentially infected fish 

should be disinfected with 0.04 to 0.13% acetic acid, chlorine dioxide at 100 

parts/million for five minutes or sodium hypochlorite 30 mg available chlorine/liter 

for two days or neutralized with sodium thiosulfate after three hours. General 

principles of laboratory safety should be practiced in handling and processing 

fish samples for diagnostic or investigative studies. Laboratory managers should 

evaluate the need to work with ISAV and the containment capability of the facility 

before undertaking work with the virus or suspected ISAV-infected fish.

Special Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 
364 Biosafety in Microbiological and Biomedical Laboratories

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.

Lumpy Skin Disease (LSD )Virus

LSD is an acute to chronic viral disease of cattle characterized by skin nodules 

that may have inverted conical necrosis (sit fast) with lymphadenitis accompanied 

by a persistent fever.29-31 The causative agent of LSD is a capripoxvirus in the 

family Poxviridae, genus Capripoxvirus. The prototype strain of LSD virus (LSDV) 

is the Neethling virus. LSDV is one of the largest viruses (170-260 nm by 

300-450 nm) and there is only one serotype. The LSDV is very closely related 

serologically to the virus of sheep and goat pox (SGP) from which it cannot be 

distinguished by routine virus neutralization or other serological tests. The virus 

is very resistant to physical and chemical agents, persists in necrotic skin for 

at least 33 days and remains viable in lesions in air-dried hides for at least 

18 days at ambient temperature.

LSD is a disorder of cattle. Other wild ungulates have not been infected 

during epizootics in Africa. Lumpy skin disease was first described in Northern 

Rhodesia in 1929. Since then, the disease has spread over most of Africa in a 

series of epizootics and most recently into the Middle East. Biting insects play the 

major (mechanical) role in the transmission of LSDV. Direct contact seems to 

play a minor role in the spread of LSD.

Laboratory Safety and Containment Recommendations

Human beings are not susceptible to infection by LSDV. The greatest risk of 

working with this virus is the escape of the organism into susceptible domestic 

animal populations, which would necessitate USDA emergency procedures to 

contain and eradicate the diseases.

Lumpy skin disease is considered a foreign animal disease in the United 

States. Due to the highly contagious nature of the agent and the severe economic 

consequences of disease in the United States, this organism should only be 

handled in vitro in a BSL-3 laboratory with enhancements as required by the USDA 

and in vivo in a USDA-approved BSL-3-Ag facility for loosely housed animals.

Special Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.
Appendix D: Agriculture Pathogen Biosafety 365

Malignant Catarrhal Fever Virus (MCFV) (Exotic Strains)

Alcelaphine herpesvirus 1 (AlHV-1) is a herpesvirus of the Rhadinovirus genus 

in the Gammaherpesvirinae subfamily.32 Common names for AlHV-1 include 

wildebeest-associated malignant catarrhal fever virus (MCFV), African form MCFV, 

and exotic MCFV. It also was previously called bovine herpesvirus3

. As a typical 

herpesvirus, AlHV-1 is a linear double-stranded DNA, enveloped virus. The virus 

can be propagated in certain primary or secondary cell cultures such as bovine 

thyroid and testis cells. The isolation of AlHV-1 requires the use of viable lymphoid 

cells from the diseased animal or cell-free virus in ocular/nasal secretions from 

wildebeest calves during a viral shedding period. Like other herpesviruses, 

AlHV-1 is fragile and quickly inactivated in harsh environments (for example, 

desiccation, high temperatures, and UV/sunlight), and by common disinfectants.

Wildebeest-associated MCF caused by AlHV-1 is also known as the African 

form of MCF, malignant catarrh, or snotsiekte (snotting sickness). The disease 

primarily affects many poorly adapted species of Artiodactyla that suffer very high 

case mortality (>95%) but low case morbidity (<7%). Wildebeest are the reservoir 

for AlHV-1 and the virus does not cause any significant disease in its natural host. 

Wildebeest-associated MCF primarily occurs in domestic cattle in Africa and in a 

variety of clinically susceptible ruminant species in zoological collections where 

wildebeest are present. Virtually all free-living wildebeest are infected with the 

virus and calves less than four months of age serve as the source of virus for 

transmission. The disease can be experimentally transmitted between cattle only 

by injection with infected viable cells from lymphoid tissues of affected animals. 

The disease cannot be transmitted by natural means from one clinically 

susceptible host to another, because there is essentially no cell-free virus in 

tissues or secretions of diseased animals. MCF is not a contagious disease.

Laboratory Safety and Containment Recommendations

There is no evidence that AlHV-1 can infect humans. Virus can be grown in

several bovine cell lines at relatively low titers (ranging from 103 to 105 TCID50). 

Infectivity in blood and tissues of affected animals is generally associated with 

viable lymphoid cells. The virus can be easily inactivated by wiping down 

surfaces with common disinfectants (such as bleach and sodium hypochlorite) 

and by autoclaving virus-contaminated materials.

This organism should only be handled in vitro in a BSL-3 laboratory with 

enhancements as required by the USDA and in vivo in a USDA-approved 

ABSL-3 animal facility with enhancements.

Special Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 
366 Biosafety in Microbiological and Biomedical Laboratories

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.

Menangle Virus (MenV)

MenV caused a single outbreak of reproductive disease in an Australian swine 

operation. Clinical signs included stillborn, deformed, mummified piglets and a 

drop in the farrowing rate. Transmission between pigs has been postulated to 

be of a fecal-oral nature. A serological survey of fruit bats living near the swine 

operation revealed the presence of antibodies to MenV. Fruit bats are considered 

to be the natural host of the virus and their proximity to the affected premises led 

to an incidental infection in the pig population.33,34

MenV is a member of the family Paramyxoviridae, subfamily Paramyxovirinae. 

Other members of this family include Hendra virus, Nipah virus and Tioman virus

of which Hendra and Nipah have been found to be fruit bat-associated. This virus 

was isolated from stillborn piglets from a single outbreak of reproductive disease 

in a commercial swine operation in New South Wales, Australia in 1997.

Occupational Infections

There was serological evidence of MenV infection in two people that had close 

contact with infected pigs on the affected premises. They demonstrated clinical 

signs similar to those seen with influenza such as chills, fever, drenching sweats, 

headache and rash. Both workers recovered fully from their illness.

Laboratory Safety and Containment Recommendations

Laboratory practices for MenV are principally designed to prevent transmission to 

susceptible livestock, but also to protect workers. The greatest risk of working with 

MenV is the escape of the organism into susceptible animal populations, which 

would necessitate USDA emergency procedures to contain and eradicate the disease.

MenV is considered cause of a foreign animal disease in the United States 

and is a human pathogen. Due to the severe economic consequences of disease 

presence in the United States, this organism should only be handled in vitro in a 

BSL-3 laboratory with enhancements as required by the USDA and in vivo in a 

USDA-approved ABSL-3 animal facility with enhancements.

Special Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.
Appendix D: Agriculture Pathogen Biosafety 367

Newcastle Disease (ND) Virus

ND is one of the most serious infectious diseases of poultry worldwide. It is 

primarily a respiratory disease, but nervous and enteric forms occur. All bird 

species are probably susceptible to infection with ND virus (NDV). The severity 

of the disease caused by any given NDV strain can vary from an unapparent 

infection to 100% mortality. The chicken is the most susceptible species. The biocontainment requirements for working with a particular strain are based on the 

virulence of the virus as determined by chicken inoculation and more recently 

by determination of amino acid sequence of the fusion protein cleavage site 

(as defined by the World Organization for Animal Health).35 The virus is shed in 

respiratory secretions and in feces. Natural transmission among birds occurs 

by aerosol inhalation or by consumption of contaminated feed or water.36,37

NDV is classified in the Avulavirus genus within the family Paramyxoviridae, 

subfamily Paramyxovirinae, in the order Mononegavirales. All NDV isolates are 

of a single serotype avian paramyxovirus type 1 (APMV-1) that includes the 

antigenic variants isolated from pigeons called pigeon paramyxovirus1

. All strains 

are readily propagated in embryonated chicken eggs and a variety of avian and 

mammalian cell cultures although special additives may be required to propagate 

the low virulence (lentogenic) viruses in some cell types.35-37

Occupational Infections

The most common infection is a self-limiting conjunctivitis with tearing and pain 

that develops within 24 hours of an eye exposure by aerosol, splash of infective 

fluids, or eye contact with contaminated hands. The occurrence of upper 

respiratory or generalized symptoms is rare.38

Laboratory Safety and Containment Recommendations

NDV isolates may be recovered from any infected bird, but on occasion may be 

recovered from humans infected by contact with infected poultry. Humans treated 

with live NDV in experimental cancer therapies, or those who are exposed by 

laboratory contamination also are sources of the virus.38 The greatest risk is for 

susceptible birds that may be exposed to NDV. If isolates of moderate to high 

virulence for chickens are used for human cancer therapies, those isolates are 

probably of greater risk for inadvertent exposure of birds and poultry than they 

are to the humans handling or being treated with those viruses.

ND (produced by moderate or highly virulent forms of the virus) is 

considered a foreign animal disease in the United States. Due to the highly 

contagious nature of the agent and the severe economic consequences of 

disease presence in the United States, this organism should only be handled 

in vitro in a BSL-3 laboratory with enhancements as required by the USDA 

and in vivo in a USDA-approved BSL-3-Ag facility for loosely housed animals. 

Laboratory workers should have no contact with susceptible hosts for five days 
368 Biosafety in Microbiological and Biomedical Laboratories

after working with the agent. Laboratory and animal studies with low virulence 

viruses or diagnostic accessions should be handled at BSL-2.

Special Issues

Velogenic strains of NDV are USDA select agents. Possession, transfer and use 

of this agent requires application of procedures as detailed in 9 CFR Part 121, 

Agricultural Bioterrorism Protection Act of 2002; Possession, Use and Transfer of 

Biological Agents and Toxins. All rules concerning the possession, storage, use, 

and transfer of select agents apply. Please review Appendix F of this document 

for further instructions regarding select agents. An importation or interstate 

movement permit for NDV must be obtained from USDA/APHIS/VS.

Peste Des Petits Ruminants Virus (PPRV)

PPRV causes disease variously termed stomatitis pneumoenteritis complex, 

kata, goat plague and pseudorinderpest. The virus affects sheep and especially 

goats, and is regarded as the most important disease of goats and possibly 

sheep in West Africa where they are a major source of animal protein. The 

disease is reported from sub-Saharan Africa north of the equator, the Arabian 

Peninsula, the Middle East, and the Indian Subcontinent. The virus has particular 

affinity for lymphoid tissues and epithelial tissue of the gastrointestinal and 

respiratory tracts, causing high fever, diphtheritic oral plaques, proliferative lip 

lesions, diarrhea, dehydration, pneumonia and death. In susceptible populations 

morbidity is commonly 90% and mortality 50-80%, but can reach 100%.39

PPRV is a member of the family Paramyxoviridae, subfamily 

Paramyxovirinae, genus Morbillivirus, and species peste-des-petits-ruminants 

virus. Other important morbilliviruses include measles virus, rinderpest virus and 

canine distemper virus. As in all morbilliviruses, it is pleomorphic, enveloped, 

about 150 nm in diameter and contains a single molecule of linear, noninfectious, negative sense ssRNA.40

The virus is environmentally fragile and requires close direct contact for 

transmission. Outbreaks typically occur after animal movement and commingling 

during seasonal migrations or religious festivals. Sources of virus include tears, 

nasal discharge, coughed secretions, and all secretions and excretions of 

incubating and sick animals. There is no carrier state, and animals recovering 

from natural infection have lifetime immunity.

Laboratory Safety and Containment Recommendations

PPRV is not known to infect humans in either laboratory or field settings. The 

greatest risk of working with PPRV is the escape of the organism into a 
Appendix D: Agriculture Pathogen Biosafety 369

susceptible sheep or goat population, which would necessitate USDA emergency 

procedures to contain and eradicate the disease.

The virus is considered cause of a foreign animal disease in the United 

States. Due to the highly contagious nature of the agent and the severe economic 

consequences of disease presence in the United States, this organism should 

only be handled in vitro in a BSL-3 laboratory with enhancements as required by 

the USDA and in vivo in a USDA-approved BSL-3-Ag facility for loosely housed 

animals. Laboratory workers should have no contact with susceptible hosts for 

five days after working with the agent.

Special Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.

Rinderpest Virus (RPV)

Rinderpest (RP) is a highly contagious viral disease of domestic cattle, buffaloes, 

sheep, goats and some breeds of pigs and a large variety of wildlife species.41

It is characterized by fever, oral erosions, diarrhea, lymphoid necrosis and high 

mortality. The disease is present in the Indian subcontinent, Near East and subSaharan Africa including Kenya and Somalia.

RPV is a single stranded RNA virus in the family Paramyxoviridae, genus 

Morbillivirus. It is immunologically related to canine distemper virus, human measles 

virus, peste des petits ruminants virus, and marine mammal morbilliviruses. There 

is only one serotype of RPV including several strains with a wide range of virulence.42

RPV is a relatively fragile virus. The virus is sensitive to sunlight, heat, and 

most disinfectants. It rapidly inactivates at pH 2 and 12. Optimal pH for survival 

is 6.5 – 7.0. Glycerol and lipid solvents inactivate this virus.

Spread of RPV is by direct and indirect contact with infected animals. Aerosol 

transmission is not a significant means of transmission. Incubation period varies 

with strain of virus, dosage, and route of exposure. Following natural exposure, 

the incubation period ranges from 3 to 15 days but is usually 4 to 5 days.

Laboratory Safety and Containment Recommendations

There are no reports of RPV being a health hazard to humans. The greatest 

risk of working with RPV is the escape of the organism into susceptible animal 

populations, which would necessitate USDA emergency procedures to contain 

and eradicate the disease.
370 Biosafety in Microbiological and Biomedical Laboratories

The virus is considered cause of a foreign animal disease in the United 

States. Due to the highly contagious nature of the agent and the severe 

economic consequences of disease presence in the United States, this organism 

should only be handled in vitro in a BSL-3 laboratory with enhancements as 

required by the USDA and in vivo in a USDA-approved BSL-3-Ag facility for 

loosely housed animals. Laboratory workers should have no contact with 

susceptible hosts for five days after working with the agent.

Special Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.

Sheep and Goat Pox Virus (SGPV)

Sheep and goat pox (SGP) is an acute to chronic disease of sheep and goats 

characterized by generalized pox lesions throughout the skin and mucous 

membranes, a persistent fever, lymphadenitis, and often a focal viral pneumonia 

with lesions distributed uniformly throughout the lungs. Subclinical cases may 

occur. The virus that causes SGP is a capripoxvirus (SGPV), one of the largest 

viruses (170 – 260 nm by 300 – 450 nm) in the Poxviridae family, genus 

Capripoxvirus. It is closely related to the virus that causes lumpy skin disease. 

The SGPV is very resistant to physical and chemical agents.43-45

SGPV causes clinical disease in sheep and goats. The virus replicates in 

cattle but does not cause clinical disease. The disease has not been detected in 

wild ungulate populations. It is endemic in Africa, the Middle East, the Indian 

subcontinent, and much of Asia.

Contact is the main means of transmission of SGPV. Inhalation of aerosols 

from acutely affected animals, aerosols generated from dust contaminated from 

pox scabs in barns and night holding areas, and contact through skin abrasions 

either by fomites or by direct contact are the natural means of transmitting SGPV. 

Insect transmission (mechanical) is possible. The virus can cause infection 

experimentally by intravenous, intradermal, intranasal, or subcutaneous 

inoculation.

Laboratory Safety and Containment Recommendations

Humans are not susceptible to infection by these poxviruses. The greatest 

risk of working with these agents is the escape of the organism into susceptible 

domestic animal populations, which would necessitate USDA emergency 

procedures to contain and eradicate the diseases.46
Appendix D: Agriculture Pathogen Biosafety 371

These viruses are considered cause of a foreign animal disease in the 

United States. Due to the highly contagious nature of the agent and the severe 

economic consequences of disease presence in the United States, this organism 

should only be handled in vitro in a BSL-3 laboratory with enhancements as 

required by the USDA and in vivo in a USDA-approved ABSL-3 animal facility 

with enhancements.

Special Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.

Spring Viremia of Carp Virus (SVCV)

Spring Viremia of Carp virus (SVCV) is a rhabdovirus in the family 

Rhabdoviridae, genus Vesiculovirus that infects a broad range of fish species 

and causes high mortality in susceptible hosts in cold water. It is a World 

Organization for Animal Health Office International des Épizooties (OIE) 

reportable disease. Infections have occurred in common and koi carp (Cyprinus 

carpio), grass carp (Crenopharyngodon idellus), silver carp (Hypophthalmichthys 

molitix), bighead (Aristichthys nobilis), cruian carp (Carassius carassius), goldfish 

(C. auratus), roach (Rutilus rutilus), ide (Leuciscus idus), tench (Tinca tinca) and 

sheatfish (Silurus glanis). Long indigenous to Europe, the Middle East and Asia, 

the disease was reported recently in South and North America. In the spring of 

2002, SVCV was isolated from koi carp farmed in North Carolina. That year the 

virus was detected in fish in several lakes and rivers in Wisconsin, including the 

Mississippi River. SVCV causes impairment in salt-water balance in fish resulting 

in edema and hemorrhages.

Reservoirs of SVCV are infected fish and carriers from either cultured, feral 

or wild fish populations.47 Virulent virus is shed via feces, urine, and gill, skin 

and mucus exudates. Liver, kidney, spleen, gill and brain are the primary organs 

containing the virus during infection.48 It is surmised that horizontal transmission 

occurs when waterborne virus enters through the gills. Vertical transmission may 

be possible, especially via ovarian fluids. This virus may remain infective for long 

periods of time in water or mud. Once the virus is established in a pond or farm, 

it may be difficult to eradicate without destruction of all fish at the farm.25,28,49

Laboratory Safety and Containment Recommendations

Human beings are not susceptible to SVCV infection. The greatest risk of 

working with SVCV is the escape of the organism into a susceptible fish 
372 Biosafety in Microbiological and Biomedical Laboratories

population, which would necessitate USDA emergency procedures to contain 

and eradicate the disease.

SVC is considered a reportable disease in the United States. SVCV should 

be handled in vitro in BSL-2 laboratory facilities with enhancements as required 

by USDA. Animal inoculations should be handled in ABSL-3 animal facilities with 

special modifications as required. The OIE Diagnostic Manual for Aquatic Animal 

Disease has specifications for surveillance programs to achieve and maintain 

health status of aquaculture facilities.48 Recommendations for preventing the 

disease and spread of disease include the use of a water source free of virus, 

disinfection of eggs and equipment, and proper disposal of dead fish.

Special Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.

Swine Vesicular Disease Virus (SVDV)

Swine vesicular disease virus (SVDV) is classified in the genus Enterovirus, 

the family Picornaviridae, and is closely related to the human enterovirus 

coxsackievirus B5.50 The virus is the causative agent of SVD, a contagious 

disease of pigs characterized by fever and vesicles with subsequent erosion in 

the mouth and on the snout, feet, and teats.51,52 The major importance of SVD 

is that it clinically resembles FMD, and any outbreaks of vesicular disease in 

pigs must be assumed to be FMD until proven otherwise by laboratory tests.

Occupational Infections

SVDV can cause an “influenza-like” illness in man1

 and human infection has 

been reported in laboratory personnel working with the virus.53,54 The virus may 

be present in blood, vesicular fluid, and tissues of infected pigs. Direct and 

indirect contacts of infected materials, contaminated laboratory surfaces, and 

accidental autoinoculation, are the primary hazards to laboratory personnel.

Laboratory Safety and Containment Recommendations

Laboratory practices for SVDV are principally designed to prevent transmission 

to susceptible livestock, but also to protect workers. Gloves are recommended 

for the necropsy and handling of infected animals and cell cultures. The greatest 

risk of working with SVD is the escape of the organism into susceptible animal 

populations, which would necessitate USDA emergency procedures to contain 

and eradicate the disease.55
Appendix D: References 373

SVD is considered a foreign animal disease in the United States. Due to the 

severe economic consequences of disease presence in the United States, SVDV 

should only be handled in vitro in a BSL-3 laboratory with enhancements as 

required by the USDA and in vivo in a USDA-approved ABSL-3 animal facility 

with enhancements.

Special Issues

The importation, possession, or use of this agent is prohibited or restricted by 

law or by USDA regulations or administrative policies. A USDA/APHIS import or 

interstate movement permit is required to obtain this agent or any livestock or 

poultry product, such as blood, serum, or other tissues containing the agent.

References

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374 Biosafety in Microbiological and Biomedical Laboratories

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animal diseases. Richmond (VA): U.S. Animal Health Association; 1998. 

p.311-321.

33. Mackenzie JS, Chua KB, Daniels PW, et al. Emerging viral disease of 

Southeast Asia and the Western Pacific. Emerg Infect Dis. 2001;7:497-504.

34. Kirkland PD, Daniels PW, Nor MN, et al. Menangel and Nipah virus 

infections of pigs. Vet Clin North Am Food Anim Pract. 2002;18:557-71.

35. Alexander DJ. Newcastle disease. 2004. In: Manual of standards for 

diagnostic tests and vaccines for terrestrial animals: mammals, birds and 

bees. Paris: Office International des Épizooties; 2004. p. 270-82.

36. Alexander DJ. Newcastle disease. In: Swayne DE, Glisson JR, Jackwood 

MW, et al, editors. A laboratory manual for the isolation and identification of 

avian pathogens. 4th ed. Kennett Square (PA): American Association of 

Avian Pathologists; 1998. p. 156-63.

37. Alexander DJ. Newcastle disease, other paramyxoviruses and pneumovirus 

infections. In: Saif YM, Barnes YM, Glisson HJ, et al, editors. Diseases of 

poultry. 11th ed. Ames (IA): Iowa State Press: 2003. p. 63-87.

38. Swayne DE, King DJ. Avian Influenza and Newcastle disease. J Am Vet Med 

Assoc. 2003;222;1534-40.

39. Saliki JT. Peste des petits ruminants. In: Manual of standards for diagnostic 

tests and vaccines for terrestrial animals: mammals, birds and bees. Paris: 

Office International des Épizooties; 2004. p. 344-52.

40. Diallo A. Peste des petits ruminants. In: Manual of standards for diagnostic 

tests and vaccines for terrestrial animals: mammals, birds and bees. Paris: 

Office International des Épizooties; 2004. p. 153-62.

41. Mebus CA. Rinderpest. In: Mebus CA, editor. Foreign animal diseases. 

Richmond (VA): U.S. Animal Health Association; 1998. p. 362-71.

42. Taylor WP, Roeder P. Rinderpest. In: Manual of standards for diagnostic 

tests and vaccines for terrestrial animals: mammals, birds and bees. Paris: 

Office International des Épizooties; 2004. p. 142-52.

43. Davies FG. Characteristics of a virus causing a pox disease in sheep and 

goats in Kenya, with observations on the epidemiology and control. J Hyg 

(Lond). 1976;76:163-71.

44. Davies FG. Sheep and goat pox. In: Gibbs EPK, editor. Virus diseases of 

food animals. Vol 2. London: Academic Press; 1981. p. 733-48.

45. House JA. Sheep and goat pox. In: Mebus CA, editor. Foreign animal 

diseases. Richmond (VA): U.S. Animal Health Association; 1998. p. 384-91.

46. Kitching RP, Carn V. Sheep and goat pox. In: Manual of standards for 

diagnostic tests and vaccines for terrestrial animals: mammals, birds and 

bees. Paris: Office International des Épizooties; 2004. p. 211-20. 

47. Goodwin AE, Peterson JE, Meyers TR, et al. Transmission of exotic fish 

viruses: the relative risks of wild and cultured bait. Fisheries. 2004;29:19-23
376 Biosafety in Microbiological and Biomedical Laboratories

48. Office International des Épizooties. Spring viremia of carp. In: Manual of 

diagnostic tests for aquatic animals. 4th ed. Paris: Office International des 

Épizooties; 2003. p. 108-14.

49. Schlotfeldt HJ, Alderman DJ. What should I do? A practical guide for the 

freshwater fish farmer. Dorset (England): European Association of Fish 

Pathologists; 1995.

50. Fenner FJ, Gibbs EPJ, Murphy FA, et al. Picornaviridae. In: Fenner FJ, 

editor. Veterinary virology. 2nd ed. San Diego, CA: Academic Press; 1993. 

p. 403-23.

51. Mebus CA. Swine vesicular disease. In: Mebus CA, editor. Foreign animal 

diseases. Richmond (VA): U.S. Animal Health Association; 1998. p. 392-95.

52. Office International des Épizooties. Swine vesicular disease. In: Manual of 

diagnostic tests for aquatic animals. 4th ed. Paris: Office International des 

Épizooties; 2003.

53. Brown F, Goodridge D, Burrows R. Infection of man by swine vesicular 

disease virus. J Comp Path. 1976;86:409-14.

54. Graves JH. Serological relationship of swine vesicular disease virus and 

Coxsackie B5 virus. Nature. 1973;245:314-5.

55. Kitching RP, Mackay DKJ, Donaldson AI. Swine Vesicular Disease. In: 

Manual of standards for diagnostic tests and vaccines for terrestrial animals: 

mammals, birds and bees. Paris: Office International des Épizooties; 2004. 

p. 136-41.

VI. Additional Information:

U.S. Department of Agriculture

Animal and Plant Health Inspection Service

Veterinary Services, National Center for Import and Export

4700 River Road, Unit 133

Riverdale, Maryland 20737-1231

Telephone: (301) 734-5960

Fax: (301) 734-3256

Internet: http://www.aphis.usda.gov/animal_health/permits

Further information on Plant Select Agents, or permits for field release of 

genetically engineered organisms may be obtained from:

U.S. Department of Agriculture Animal

and Plant Health Inspection Service

Plant Protection and Quarantine, Permits, Agricultural Bioterrorism

4700 River Road, Unit 2

Riverdale, Maryland 20737-1231

Telephone: (301) 734-5960

Internet: http://www.aphis.usda.gov/programs/ag_selectagent/index.shtml
Appendix E: Arthropod Containment Guidelines 377

Appendix E—Arthropod Containment Guidelines (ACG)

An ad hoc committee of concerned vector biologists including members of the 

American Committee Medical Entomology (ACME), a subcommittee of the 

American Society of Tropical Medicine and Hygiene (ASTMH), and other 

interested persons drafted the “Arthropod Containment Guidelines.” The ACG 

provide principles of risk assessment for arthropods of public health importance. 

The risk assessment and practices are designed to be consistent with the NIH 

Guidelines for recombinant DNA research and BMBL. Arthropods included are 

those that transmit pathogens; however, those arthropods that cause myiasis, 

infestation, biting, and stinging are not included. The ACG also specifically 

exclude most uses of Drosophila spp.

The ACG were published in Vector Borne and Zoonotic Diseases.1 They are 

freely downloadable from www.liebertonline.com and at the AMCE Web site: 

www.astmh.org.

The ACG recommend biosafety measures specific for arthropods of public 

health importance considering that:

■ Arthropods present unique containment challenges not 

encountered with microbial pathogens.

■ Arthropod containment has not been covered specifically in 

BMBL or the NIH Guidelines.

The ACG contain two sections of greatest interest to most researchers:

1. The Principles of Risk Assessment that discusses arthropods in the 

usual context (e.g., those known to contain a pathogenic agent, those 

with uncertain pathogens, and those with no agent).

2. They also consider the following:

■ Biological containment is a significant factor that reduces the 

hazards associated with accidental escape of arthropods.

■ Epidemiological context alters the risks of an escape and its 

impact on the location or site in which the work is performed.

■ The phenotype of the vector, such as insecticide resistance; and 

■ genetically modified arthropods with an emphasis on 

phenotypic change.
378 Biosafety in Microbiological and Biomedical Laboratories

Four Arthropod Containment Levels (ACL 1 – 4) add increasingly stringent 

measures and are similar to biosafety levels. The most flexible level is ACL-2 that 

covers most exotic and transgenic arthropods and those infected with pathogens 

requiring BSL-2 containment. Like BMBL, each level has the following form:

■ standard practices;

■ special practices;

■ equipment (primary barriers);

■ facilities (secondary barriers).

The ACG does not reflect a formal endorsement by ACME or ASTMH. The 

guidelines are subject to change based on further consideration of the 

requirements for containment of arthropods and vectors.

References

1. American Committee of Medical Entomology; American Society of Tropical 

Medicine and Hygiene. Arthropod containment guidelines. A project of the 

American Committee of Medical Entomology and American Society of 

Tropical Medicine and Hygiene. Vector Borne Zoonotic Dis. 2003;3:61-98.
Appendix F: Select Agents and Toxins 379

Appendix F—Select Agents and Toxins

The Public Health Security and Bioterrorism Preparedness and Response Act 

of 2002, Subtitle A of Public Law 107-188 (42 U.S.C. 262a), requires DHHS to 

regulate the possession, use, and transfer of biological agents or toxins (i.e., 

select agents and toxins) that could pose a severe threat to public health and 

safety. The Agricultural Bioterrorism Protection Act of 2002, Subtitle B of Public 

Law 107-188 (7 U.S.C. 8401), requires the USDA to regulate the possession, 

use, and transfer of biological agents or toxins (i.e., select agents and toxins) 

that could pose a severe threat to animal or plant health, or animal or plant 

products. These Acts require the establishment of a national database of registered 

entities, and set criminal penalties for failing to comply with the requirements of 

the Acts. In accordance with these Acts, DHHS and USDA promulgated regulations 

requiring entities to register with the CDC or the APHIS if they possess, use, or 

transfer a select agent or toxin (42 CFR Part 73, 7 CFR Part 331, and 9 CFR Part 

121). CDC and APHIS coordinate regulatory activities for those agents that would 

be regulated by both agencies (“overlap” select agents).

The Attorney General has the authority and responsibility to conduct electronic 

database checks (i.e., the security risk assessments) on entities that apply to 

possess, use, or transfer select agents, as well as personnel that require access to 

select agents and toxins. The FBI, Criminal Justice Information Services Division 

(CJIS), has been delegated authority for conducting these security risk assessments.

The regulations provide that, unless exempted, entities must register with 

CDC or APHIS if they possess, use, or transfer select agents or toxins. The 

current list of select agents and toxins is available on the CDC and APHIS Web 

sites (see below). The regulations set out a procedure for excluding an attenuated 

strain of a select agent or toxin and exemptions for certain products and for 

select agents or toxins identified in specimens presented for diagnosis, 

verification, or proficiency testing.

The regulations also contain requirements to ensure that the select agents 

and toxins are handled safely and secured against unauthorized access, theft, 

loss, or release. For example, entities and their personnel must undergo a 

security risk assessment by CJIS as part of their registration; entities must limit 

access to select agents and toxins and develop and implement biosafety, 

security, and incident response plans. In addition, all select agents or toxins must 

be transferred in accordance with the regulations and any theft, loss, or release 

of a select agent or toxin must be reported to CDC or APHIS.

For additional information concerning the select agent regulations, contact 

CDC or APHIS. Information is also available at the following Web sites: www.

selectagents.gov; http://www.aphis.usda.gov/programs/ag_selectagent/index.

shtml.
380 Biosafety in Microbiological and Biomedical Laboratories

Appendix G—Integrated Pest Management (IPM)

IPM is an important part of managing a research facility. Many pests, such 

as flies and cockroaches, can mechanically transmit disease pathogens and 

compromise the research environment. Even the presence of innocuous insects 

can contribute to the perception of unsanitary conditions.

The most common approach to pest control has been the application of 

pesticides, either as a preventive or remedial measure. Pesticides can be effective 

and may be necessary as a corrective measure, but they have limited long-term 

effect when used alone. Pesticides also can contaminate the research 

environment through pesticide drift and volatilization.

To control pests and minimize the use of pesticides, it is necessary to employ 

a comprehensive program approach that integrates housekeeping, maintenance, 

and pest control services. This method of pest control is often referred to as IPM. 

The primary goal of an IPM program is to prevent pest problems by managing 

the facility environment to make it less conducive to pest infestation. Along with 

limited applications of pesticides, pest control is achieved through proactive 

operational and administrative intervention strategies to correct conditions that 

foster pest problems.

Prior to developing any type of IPM program, it is important to define an 

operational framework for IPM services that helps promote collaboration between 

IPM specialists and facility personnel. This framework should incorporate facility 

restrictions and operational and procedural issues into the IPM program. An 

effective IPM program is an integral part of the facility’s management. An IPM 

policy statement should be included in the facility’s standard operating 

procedures to increase awareness of the program.

Training sources for the principles and practices of structural (indoor) IPM 

programs are available through university entomology departments, county 

extension offices, the Entomological Society of America, state departments of 

agriculture, state pest control associations, the National Pest Control Association, 

suppliers of pest control equipment, and IPM consultants and firms. Several 

universities offer correspondence courses, short courses, and training 

conferences on structural pest management.

IPM is a strategy-based service that considers not only the cost of the 

services, but also the effectiveness of the program’s components. Each IPM 

program is site-specific, tailored to the environment where applied.

Laboratory IPM services will be different from those in an office building or 

an animal care facility. Interrelated components of “Environmental pest 

management” follow.
Appendix G: Integrated Pest Management 381

Facility Design

IPM issues and requirements should be addressed in a research facility’s 

planning, design, and construction. This provides an opportunity to incorporate 

features that help exclude pests, minimize pest habitat, and promote proper 

sanitation in order to reduce future corrections that can disrupt research 

operations.

Monitoring

Monitoring is the central activity of an IPM program and is used to minimize 

pesticide use. Traps, visual inspections, and staff interviews identify areas and 

conditions that may foster pest activity.

Sanitation and Facility Maintenance

Many pest problems can be prevented or corrected by ensuring proper 

sanitation, reducing clutter and pest habitat, and by performing repairs that 

exclude pests. Records of structural deficiencies and housekeeping conditions 

should be maintained to track problems and determine if corrective actions were 

completed and in a timely manner.

Communication

A staff member should be designated to meet with IPM personnel to assist in 

resolving facility issues that impact on pest management. Reports communicated 

verbally and in writing concerning pest activity and improvement 

recommendations for personnel, practices and facility conditions should be 

provided to the designated personnel. Facility personnel should receive training 

on pest identification, biology, and sanitation, which can promote understanding 

and cooperation with the goals of the IPM program.

Recordkeeping

A logbook should be used to record pest activity and conditions pertinent to the 

IPM program. It may contain protocols and procedures for IPM services in that 

facility, Material Safety Data Sheets on pesticides, pesticide labels, treatment 

records, floor plans, survey reports, etc.

Non-pesticide Pest Control

Pest control methods such as trapping, exclusion, caulking, washing, and 

freezing can be applied safely and effectively when used in conjunction with 

proper sanitation and structural repair.
382 Biosafety in Microbiological and Biomedical Laboratories

Pest Control with Pesticides

Preventive applications of pesticides should be discouraged, and treatments 

should be restricted to areas of known pest activity. When pesticides are applied, 

the least toxic product(s) available should be used and applied in the most 

effective and safe manner.

Program Evaluation and Quality Assurance

Quality assurance and program review should be performed to provide an 

objective, ongoing evaluation of IPM activities and effectiveness to ensure that 

the program does, in fact, control pests and meet the specific needs of the facility 

program(s) and its occupants. Based upon this review, current IPM protocols can 

be modified and new procedures implemented.

Technical Expertise

A qualified entomologist can provide helpful technical guidance to develop and 

implement an IPM program. Pest management personnel should be licensed and 

certified by the appropriate regulatory agency.

Safety

IPM minimizes the potential of pesticide exposure to the research environment 

and the staff by limiting the scope of pesticide treatments.

References

1. Robinson WH. Urban entomology: insect and mite pests in the human 

environment. New York: Chapman and Hall; 1996.

2. Bennett GW, Owens JM, editors. Advances in urban pest management. 

New York: Van Nostrand Reinhold Company; 1986.

3. Olkowski W, Daar S, Olkowski H. Common sense pest control: least-toxic 

solutions for your home, garden, pests and community. Newton (CT): The 

Taunton Press, Inc.; 1991.

4. National Pest Control Association [http://www.pestworld.org]. Fairfax (VA): The 

Association; [cited 2006 Sept 25]. Available from: http://www.pestworld.org.

5. Biocontrol Network [http://www.biconet.com]. Brentwood (TN): Biocontrol 

Network; c1995-2006 [updated 2006 Sept; cited 2006 Sept 25]. Available 

from: http://www.biconet.com.
Appendix H: Working with Human, NHP 383

Appendix H—Working with Human, NHP and Other 

Mammalian Cells and Tissues

Although risk of laboratory infection from working with cell cultures in general is 

low, risk increases when working with human and other primate cells, and 

primary cells from other mammalian species. There are reports of infection of 

laboratory workers handling primary rhesus monkey kidney cells,1

 and the 

bloodborne pathogen risks from working with primary human cells, tissues and 

body fluids are widely recognized.2,3 OSHA has developed a bloodborne 

pathogens standard that should be applied to all work in the laboratory with 

human blood, tissues, body fluids and primary cell lines.4

 Procedures have also 

been published to reduce contamination of cell cultures with microorganisms.5,6

Potential Laboratory Hazards

Potential laboratory hazards associated with human cells and tissues include the 

bloodborne pathogens HBV, HIV, HCV, HTLV, EBV, HPV and CMV as well as 

agents such as Mycobacterium tuberculosis that may be present in human lung 

tissue. Other primate cells and tissues also present risks to laboratory workers.7

Cells immortalized with viral agents such as SV-40, EBV adenovirus or HPV, as 

well as cells carrying viral genomic material also present potential hazards to 

laboratory workers. Tumorigenic human cells also are potential hazards as a 

result of self-inoculation.8

 There has been one reported case of development of a 

tumor from an accidental needle-stick.9

 Laboratory workers should never handle 

autologous cells or tissues.1

 NHP cells, blood, lymphoid and neural tissues 

should always be considered potentially hazardous.

Recommended Practices

Each institution should conduct a risk assessment based on the origin of the cells 

or tissues (species and tissue type), as well as the source (recently isolated or 

well-characterized). Human and other primate cells should be handled using 

BSL-2 practices and containment. All work should be performed in a BSC, and all 

material decontaminated by autoclaving or disinfection before discarding.6,10,11,12

BSL-2 recommendations for personnel protective equipment such as laboratory 

coats, gloves and eye protection should be rigorously followed. All laboratory staff 

working with human cells and tissues should be enrolled in an occupational 

medicine program specific for bloodborne pathogens and should work under the 

policies and guidelines established by the institution’s Exposure Control Plan.4

Laboratory staff working with human cells and tissues should provide a baseline 

serum sample, be offered hepatitis B immunization, and be evaluated by a health 

care professional following an exposure incident. Similar programs should be 

considered for work with NHP blood, body fluids, and other tissues.
384 Biosafety in Microbiological and Biomedical Laboratories

References

1. Doblhoff-Dier O, Stacey G. Cell lines: applications and biosafety. In: Fleming 

D, Hunt D, editors. Biological safety: principles and practices. Washington, 

DC: ASM Press; 2000. p. 221-39.

2. Centers for Disease Control and Prevention. Update: universal precautions 

for prevention of transmission of human immunodeficiency virus, hepatitis B 

virus and other bloodborne pathogens in healthcare settings. MMWR Morb 

Mortal Wkly Rep. 1988;37:377-82, 387-8.

3. Centers for Disease Control and Prevention. Guidelines for prevention of 

transmission of human immunodeficiency virus and hepatitis B virus to 

healthcare and public safety workers. MMWR Morb Mortal Wkly Rep. 

1989;38;No.SU-06.

4. Occupational exposure to bloodborne pathogens. Final Rule. Standard 

interpretations: applicability of 1910.1030 to established human cell lines, 29 

C.F.R. Sect. 1910.1030 (1991).

5. McGarrity GJ, Coriell LL. Procedures to reduce contamination of cell 

cultures. In Vitro. 1971;6:257-65.

6. McGarrity GJ. Spread and control of mycoplasmal infection of cell culture. In 

Vitro. 1976;12:643-8.

7. Caputo JL. Safety procedures. In: Freshney RI Freshney MG, editors. 

Culture of immortalized cells. New York: Wiley-Liss; 1996.

8. Weiss RA. Why cell biologists should be aware of genetically transmitted 

viruses. Natl Cancer Inst Monogr.1978;48:183-9.

9. Gugel EA, Sanders ME. Needle-stick transmission of human colonic 

adenocarcinoma (letter). N Engl J Med. 1986;315:1487.

10. Barkley WE. Safety considerations in the cell culture laboratory. Methods 

Enzymol. 1979;58:36-43.

11. Grizzle WE, Polt S. Guidelines to avoid personnel contamination by infective 

agents in research laboratories that use human tissues. J Tissue Cult 

Methods. 1988;11:191-9.

12. Caputo JL. Biosafety procedures in cell culture. J of Tissue Cult Methods. 

1988;11:233-7.
Appendix I: Guidelines for Work with Toxins of Biological Origin 385

Appendix I—Guidelines for Work with Toxins of 

Biological Origin

Biological toxins comprise a broad range of poisons, predominantly of natural 

origin but increasingly accessible by modern synthetic methods, which may 

cause death or severe incapacitation at relatively low exposure levels.1,2

Laboratory safety principles are summarized herein for several toxins currently 

regulated as “Select Agent Toxins,” including BoNT, SE, ricin and selected 

LMW toxins. Additional details are provided in the agent summary statements.

General Considerations for Toxin Use

Laboratory work with most toxins, in amounts routinely employed in the 

biomedical sciences, can be performed safely with minimal risk to the worker 

and negligible risk to the surrounding community. Toxins do not replicate, are 

not infectious, and are difficult to transmit mechanically or manually from 

person to person. Many commonly employed toxins have very low volatility 

and, especially in the case of protein toxins, are relatively unstable in the 

environment; these characteristics further limit the spread of toxins.

Toxins can be handled using established general guidelines for toxic or 

highly-toxic chemicals with the incorporation of additional safety and security 

measures based upon a risk assessment for each specific laboratory operation.3,4

The main laboratory risks are accidental exposure by direct contamination of 

mouth, eyes or other mucous membranes; by inadvertent aerosol generation; 

and by needle-sticks or other accidents that may compromise the normal 

barrier of the skin.

Training and Laboratory Planning

Each laboratory worker must be trained in the theory and practice of the toxins to 

be used, with special emphasis on the nature of the practical hazards associated 

with laboratory operations. This includes how to handle transfers of liquids containing 

toxin, where to place waste solutions and contaminated materials or equipment, 

and how to decontaminate work areas after routine operations, as well as after 

accidental spills. The worker must be reliable and sufficiently adept at all required 

manipulations before being provided with toxin.

A risk assessment should be conducted to develop safe operating 

procedures before undertaking laboratory operations with toxins; suggested 

“pre-operational checklists” for working with toxins are available.4

 For complex 

operations, it is recommended that new workers undergo supervised practice 

runs in which the exact laboratory procedures to be undertaken are rehearsed 

without active toxin. If toxins and infectious agents are used together, then 

both must be considered when containment equipment is selected and safety 

procedures are developed. Likewise, animal safety practices must be considered 

for toxin work involving animals.
386 Biosafety in Microbiological and Biomedical Laboratories

Each laboratory that uses toxins should develop a specific chemical 

hygiene plan. The National Research Council has provided a review of prudent 

laboratory practices when handling toxic and highly toxic chemicals, including 

the development of chemical hygiene plans and guidelines for compliance with 

regulations governing occupational safety and health, hazard communication, 

and environmental protection.5

An inventory control system should be in place to account for toxin use and 

disposition. If toxins are stored in the laboratory, containers should be sealed, 

labeled, and secured to ensure restricted access; refrigerators and other storage 

containers should be clearly labeled and provide contact information for trained, 

responsible laboratory staff.

Laboratory work with toxins should be done only in designated rooms with 

controlled access and at pre-determined bench areas. When toxins are in use, 

the room should be clearly posted: “Toxins in Use—Authorized Personnel Only.” 

Unrelated and nonessential work should be restricted from areas where stock 

solutions of toxin or organisms producing toxin are used. Visitors or other 

untrained personnel granted laboratory access must be monitored and 

protected from inadvertently handling laboratory equipment used to 

manipulate the toxin or organism.

Safety Equipment and Containment

Routine operations with dilute toxin solutions are conducted under BSL-2 

conditions with the aid of personal protective equipment and a well-maintained 

BSC or comparable engineering controls.6

 Engineering controls should be 

selected according to the risk assessment for each specific toxin operation. 

A certified BSC or chemical fume hood will suffice for routine operations with 

most protein toxins. Low molecular weight toxin solutions, or work involving 

volatile chemicals or radionucleotides combined with toxin solutions, may 

require the use of a charcoal-based hood filter in addition to HEPA filtration.

All work with toxins should be conducted within the operationally effective 

zone of the hood or BSC, and each user should verify the inward airflow before 

initiating work. When using an open-fronted fume hood or BSC, workers should 

wear suitable laboratory PPE to protect the hands and arms, such as laboratory 

coats, smocks, or coveralls and disposable gloves. When working with toxins that 

pose direct percutaneous hazards, special care must be taken to select gloves 

that are impervious to the toxin and the diluents or solvents employed. When 

conducting liquid transfers and other operations that pose a potential splash or 

droplet hazard in an open-fronted hood or BSC, workers should wear safety 

glasses and disposable facemask, or a face shield.
Appendix I: Guidelines for Work with Toxins of Biological Origin 387

Toxin should be removed from the hood or BSC only after the exterior of the 

closed primary container has been decontaminated and placed in a clean secondary 

container. Toxin solutions, especially concentrated stock solutions, should be 

transported in leak/spill-proof secondary containers. The interior of the hood or BSC 

should be decontaminated periodically, for example, at the end of a series of related 

experiments. Until thoroughly decontaminated, the hood or BSC should be posted 

to indicate that toxins remain in use, and access should remain restricted.

Selected operations with toxins may require modified BSL-3 practices and 

procedures. The determination to use BSL-3 is made in consultation with available 

safety staff and is based upon a risk assessment that considers the variables of 

each specific laboratory operation, especially the toxin under study, the physical 

state of the toxin (solution or dry form), the total amount of toxin used relative to 

the estimated human lethal dose, the volume of the material manipulated, the 

methodology, and any human or equipment performance limitations.

Inadvertent Toxin Aerosols

Emphasis must be placed on evaluating and modifying experimental procedures 

to eliminate the possibility of inadvertent generation of toxin aerosols. Pressurized 

tubes or other containers holding toxins should be opened in a BSC, chemical 

fume hood, or other ventilated enclosure. Operations that expose toxin solutions 

to vacuum or pressure, for example sterilization of toxin solutions by membrane 

filtration, should always be handled in this manner, and the operator should also 

use appropriate respiratory protection. If vacuum lines are used with toxin, they 

should be protected with a HEPA filter to prevent entry of toxins into the line.

Centrifugation of cultures or materials potentially containing toxins should 

only be performed using sealed, thick-walled tubes in safety centrifuge cups or 

sealed rotors. The outside surfaces of containers and rotors should be routinely 

cleaned before each use to prevent contamination that may generate an aerosol. 

After centrifugation, the entire rotor assembly is taken from the centrifuge to a 

BSC to open it and remove its tubes.

Mechanical Injuries

Accidental needle-sticks or mechanical injury from “sharps” such as glass or 

metal implements pose a well-known risk to laboratory workers, and the 

consequences may be catastrophic for operations involving toxins in amounts 

that exceed a human lethal dose.

Only workers trained and experienced in handling animals should be 

permitted to conduct operations involving injection of toxin solutions using hollowbore needles. Discarded needles/syringes and other sharps should be placed 

directly into properly labeled, puncture-resistant sharps containers, and 

decontaminated as soon as is practical.
388 Biosafety in Microbiological and Biomedical Laboratories

Glassware should be replaced with plastic for handling toxin solutions 

wherever practical to minimize the risk of cuts or abrasions from contaminated 

surfaces. Thin-walled glass equipment should be completely avoided. Glass 

Pasteur pipettes are particularly dangerous for transferring toxin solutions and 

should be replaced with disposable plastic pipettes. Glass chromatography 

columns under pressure must be enclosed within a plastic water jacket or other 

secondary container.

Additional Precautions

Experiments should be planned to eliminate or minimize work with dry toxin (e.g., 

freeze-dried preparations). Unavoidable operations with dry toxin should only be 

undertaken with appropriate respiratory protection and engineering controls. Dry 

toxin can be manipulated using a Class III BSC, or with the use of secondary 

containment such as a disposable glove bag or glove box within a hood or Class 

II BSC. “Static-free” disposable gloves should be worn when working with dry 

forms of toxins that are subject to spread by electrostatic dispersal.

In specialized laboratories, the intentional, controlled generation of aerosols 

from toxin solutions may be undertaken to test antidotes or vaccines in 

experimental animals. These are extremely hazardous operations that should 

only be conducted after extensive validation of equipment and personnel, using 

non-toxic simulants. Aerosol exposure of animals should be done in a certified 

Class III BSC or hoodline. While removing exposed animals from the hoodline, 

and for required animal handling during the first 24 h after exposure, workers 

should take additional precautions, including wearing protective clothing (e.g., 

disposable Tyvek suit) and appropriate respiratory protection. To minimize the 

risk of dry toxin generating a secondary aerosol, areas of animal skin or fur 

exposed to aerosols should be gently wiped with a damp cloth containing water 

or buffered cleaning solution before the animals are returned to holding areas.

For high-risk operations involving dry forms of toxins, intentional aerosol 

formation, or the use of hollow-bore needles in conjunction with amounts of toxin 

estimated to be lethal for humans, consideration should be given to requiring the 

presence of at least two knowledgeable individuals at all times in the laboratory.7

Decontamination and Spills

Toxin stability varies considerably outside of physiological conditions depending 

upon the temperature, pH, ionic strength, availability of co-factors and other 

characteristics of the surrounding matrix. Literature values for dry heat 

inactivation of toxins can be misleading due to variations in experimental 

conditions, matrix composition, and experimental criteria for assessing toxin 

activity. Moreover, inactivation is not always a linear function of heating time; 

some protein toxins possess a capacity to re-fold and partially reverse 
Appendix I: Guidelines for Work with Toxins of Biological Origin 389

inactivation caused by heating. In addition, the conditions for denaturizing toxins 

in aqueous solutions are not necessarily applicable for inactivating dry, powdered 

toxin preparations.

General guidelines for laboratory decontamination of selected toxins 

are summarized in Tables 1 and 2, but inactivation procedures should not be 

assumed to be 100% effective without validation using specific toxin bioassays. 

Many toxins are susceptible to inactivation with dilute sodium hydroxide (NaOH) 

at concentrations of 0.1-0.25N, and/or sodium hypochlorite (NaOCl) bleach 

solutions at concentrations of 0.1-0.5% (w/v). Use freshly prepared bleach 

solutions for decontamination; undiluted, commercially available bleach 

solutions typically contain 3-6% (w/v) NaOCl.

Depending upon the toxin, contaminated materials and toxin waste solutions 

can be inactivated by incineration or extensive autoclaving, or by soaking in 

suitable decontamination solutions (Table 2). All disposable material used for 

toxin work should be placed in secondary containers, autoclaved and disposed 

of as toxic waste. Contaminated or potentially contaminated protective clothing 

and equipment should be decontaminated using suitable chemical methods or 

autoclaving before removal from the laboratory for disposal, cleaning or repair. If 

decontamination is impracticable, materials should be disposed of as toxic waste.

In the event of a spill, avoid splashes or generating aerosols during cleanup 

by covering the spill with paper towels or other disposable, absorbent material. 

Apply an appropriate decontamination solution to the spill, beginning at the 

perimeter and working towards the center, and allow sufficient contact time to 

completely inactivate the toxin (Table 2).

Decontamination of buildings or offices containing sensitive equipment or 

documents poses special challenges. Large-scale decontamination is not covered 

explicitly here, but careful extrapolation from the basic principles may inform 

more extensive clean-up efforts.

Select Agent Toxins

Due diligence should be taken in shipment or storage of any amount of toxin. 

There are specific regulatory requirements for working with toxins designated 

as a “Select Agent” by the DHHS and/or the USDA. Select agents require 

registration with CDC and/or USDA for possession, use, storage and/or transfer. 

Importation of this agent may require CDC and/or USDA importation permits. 

Domestic transport of the agent may require a permit from USDA/ APHIS/VS. 

A DoC permit may be required for the export of the agent to another country. 

See Appendix C for additional information.
390 Biosafety in Microbiological and Biomedical Laboratories

Table 1. Physical Inactivation of Selected Toxins

Toxin Steam 

Autoclave

Dry Heat

(10 min) Freeze-thaw Gamma Irradiation

Botulinum 

neurotoxin

Yes a > 100o C b No c Incomplete d

Staphylococcal 

Enterotoxin

Yes e > 100o C; 

refolds f

No g Incomplete h

Ricin Yes i > 100o C i No j Incomplete k

Microcystin No l > 260o C m No n ND

Saxitoxin No l > 260o C m No n ND

Palytoxin No l > 260o C m No n ND

Tetrodotoxin No l > 260o C m No n ND

T-2 mycotoxin No l > 815o C m No n ND

Brevetoxin (PbTx-2) No l > 815o C m No n ND

Notes:

ND indicates “not determined” from available decontamination literature. a Steam autoclaving should be at >121°C for 1 h. For volumes larger than 1 liter, especially those 

containing Clostridium botulinum spores, autoclave at >121°C for 2 h to ensure that sufficient heat 

has penetrated to kill all spores.8,9

b Exposure to 100°C for 10 min. inactivates BoNT. Heat denaturation of BoNT as a function of time is 

biphasic with most of the activity destroyed relatively rapidly, but with some residual toxin (e.g., 1-5%) 

inactivated much more slowly.10 

c Measured using BoNT serotype A at -20°C in food matrices at pH 4.1 – 6.2 over a period of 180 days.11

d Measured using BoNT serotypes A and B with gamma irradiation from a 60Co source.12,13

e Protracted steam autoclaving, similar to that described for BoNT, followed by incineration is 

recommended for disposal of SE-contaminated materials. f Inactivation may not be complete depending upon the extent of toxin re-folding after denaturation. 

Biological activity of SE can be retained despite heat and pressure treatment routinely used in canned 

food product processing.14

g SE toxins are resistant to degradation from freezing, chilling or storage at ambient temperature.15

Active SEB in the freeze-dried state can be stored for years. h References 15,16

i Dry heat of >100oC for 60 min in an ashing oven or steam autoclave treatment at >121oC for 1 h reduced 

the activity of pure ricin by >99%.17 Heat inactivation of impure toxin preparations (e.g., crude ricin plant 

extracts) may vary. Heat-denatured ricin can undergo limited refolding (<1%) to yield active toxin. j Ricin holotoxin is not inactivated significantly by freezing, chilling or storage at ambient temperature. In the 

liquid state with a preservative (sodium azide), ricin can be stored at 4oC for years with little loss in potency. k Irradiation causes a dose-dependent loss of activity for aqueous solutions of ricin, but complete 

inactivation is difficult to achieve; 75 MRad reduced activity 90%, but complete inactivation was not 

achieved even at 100 MRad.18 Gamma irradiation from a laboratory 60Co source can be used to 

partially inactivate aqueous solutions of ricin, but dried ricin powders are significantly resistant to 

inactivation by this method. l Autoclaving with 17 lb pressure (121-132o C) for 30 min failed to inactivate LMW toxins.17,19 All 

burnable waste from LMW toxins should be incinerated at temperatures in excess of 815°C (1,500o F). m Toxin solutions were dried at 150o C in a crucible, placed in an ashing oven at various temperatures 

for either 10 or 30 min, reconstituted and tested for concentration and/or activity; tabulated values are 

temperatures exceeding those required to achieve 99% toxin inactivation.17

n LMW toxins are generally very resistant to temperature fluctuations and can be stored in the freezedried state for years and retain toxicity.
Appendix I: Guidelines for Work with Toxins of Biological Origin 391

Table 2. Chemical Inactivation of Selected Toxins

Toxin NaOCl 

(30 min)

NaOH

(30 min)

NaCOl + NaOH (30 

min)

Ozone 

Treatment

Botulinum 

neurotoxin

> 0.1% a > 0/25 N ND Yes b

Staphylococcal 

Enterotoxin

> 0.5% c > 0.25 N ND ND

Ricin > 1.0% d ND > 0.1% + 0.25N e ND

Saxitoxin ≥ 0.1% e ND 0.25% + 0.25N e ND

Palytoxin ≥ 0.1% e ND 0.25% + 0.25N e ND

Microcystin ≥ 0.5% e ND 0.25% + 0.25N e ND

Tetrodotoxin ≥ 0.5% e ND 0.25% + 0.25N e ND

T-2 mycotoxin ≥ 2.5% e, f ND 0.25% + 0.25N e ND

Brevetoxin 

(PbTx-2)

≥ 2.5% e, f ND 0.25% + 0.25N e ND

Notes: 

ND indicates “not determined” from available decontamination literature. a Solutions of NaOCl (#0.1%) or NaOH (> 0.25 N) for 30 min inactivate BoNT and are recommended 

for decontaminating work surfaces and spills of C. botulinum or BoNT. Chlorine at a concentration of 

0.3-0.5 mg/L as a solution of hypochlorite rapidly inactivates BoNT (serotypes B or E tested) in 

water.20 Chlorine dioxide inactivates BoNT, but chloramine is less effective.21

b Ozone (> 2 mg/L) or powdered activated charcoal treatment also completely inactivate BoNT 

(serotypes A, B tested) in water under defined condition.20,22

c SEB is inactivated with 0.5% hypochlorite for 10-15 mi.23

d Ricin is inactivated by a 30 min exposure to concentrations of NaOCl ranging from 0.1-2.5%, or by a 

mixture of 0.25% NaOCl plus 0.25 N NaOH.17 In general, solutions of 1.0% NaOCl are effective for 

decontamination of ricin from laboratory surfaces, equipment, animal cages, or small spills. e The minimal effective concentration of NaOCl was dependent on toxin and contact time; all LMW 

toxins tested were inactivated at least 99% by treatment with 2.5% NaOCl, or with a combination of 

0.25% NaOCl and 0.25N NaOH.17

f For T-2 mycotoxin and brevetoxin, liquid samples, accidental spills, and nonburnable waste should be 

soaked in 2.5% NaOCl with 0.25% N NaOH for 4 h. Cages and bedding from animals exposed to T-2 

mycotoxin or brevetoxin should be treated with 0.25% NaOCl and 0.025 N NaOH for 4 h. Exposure 

for 30 min to 1.0% NaOCl is an effective procedure for the laboratory (working solutions, equipment, 

animal cages, working area and spills) for the inactivation of saxitoxin or tetrodotoxin. 

Decontamination of equipment and waste contaminated with select brevetoxins has been reviewed.19

Alternate methods of chemical decontamination: 1 N sulfuric or hydrochloric 

acid did not inactivate T-2 mycotoxin and only partially inactivated microcystin-LR, 

saxitoxin, and brevetoxin (PbTx-2). Tetrodotoxin and palytoxin were inactivated 

by hydrochloric acid, but only at relatively high molar concentrations. T2 was not 

inactivated by exposure to 18% formaldehyde plus methanol (16 h), 90% freon-113 

+ 10% acetic acid, calcium hypochlorite, sodium bisulfate, or mild oxidizing.17 

Hydrogen peroxide was ineffective in inactivating T-2 mycotoxin. This agent did 

cause some inactivation of saxitoxin and tetrodotoxin, but required a 16 h contact 

time in the presence of ultraviolet light.
392 Biosafety in Microbiological and Biomedical Laboratories

References

1. Franz DR. Defense against toxin weapons. In: Sidell FR, Takafuji ET, Franz 

DR, editors. Medical aspects of chemical and biological warfare. Vol 6. 

Textbook of military medicine, part 1: warfare, weaponry, and the casualty. 

Washington, DC: Office of the Surgeon General at TMM Publications, 

Borden Institute, Walter Reed Army Medical Center; 1997. p. 603-19.

2. Millard CB. Medical defense against protein toxin weapons: review and 

perspective. In: Lindler LE, Lebeda FJ, Korch GW, editors. Biological 

weapons defense: infectious diseases and counterbioterrorism. Totowa, NJ: 

Humana Press; 2005. p. 255-84.

3. Hamilton MH. The biological defense safety program--technical safety 

requirements. In: Series The Biological Defense Safety Program--Technical 

Safety Requirements. Department of Defense--Department of Army, 32CFR 

Part 627; 1993. p. 647-95.

4. Johnson B, Mastnjak R, Resnick IG. Safety and health considerations for 

conducting work with biological toxins. In: Richmond J, editor. Anthology of 

biosafety II: facility design considerations. Vol. 2. Mundelein, IL: American 

Biological Safety Association; 2000. p. 88-111.

5. Committee on Prudent Practices for Handling, Storage, and Disposal of 

Chemicals in Laboratories; Board on Chemical Sciences and Technology; 

Commission on Physical Sciences, Mathematics, and Applications; National 

Research Council. Prudent practices in the laboratory: handling and disposal 

of chemicals. Washington, DC: National Academy Press; 1995. p. xv:427.

6. Kruse RH, Puckett WH, Richardson JH. Biological safety cabinetry. Clin 

Microbiol Rev. 1991;4:207-41.

7. Morin R, Kozlovac J. Biological toxins. In: Fleming DO, Hunt DL, editors. 

Biological safety principles and practice. 3rd editon. Washington, DC: 

American Society for Microbiology; 2000. p. 261-72.

8. Balows A. Laboratory diagnosis of infectious diseases: principles and 

practice. New York: Springer-Verlag: 1988.

9. Hatheway C. Botulism. In: Balows A, Hausler W, Ohashi M, et al, editors. 

Laboratory diagnosis of infectious diseases: principles and practice. Vol 1. 

New York: Springer-Verlag; 1988. p. 111-33.

10. Siegel LS. Destruction of botulinum toxins in food and water. In: Hauschild 

AHW, Dodds KL, editors. Clostridium botulinum: ecology and control in 

foods. New York: Marcel Dekker, Inc.; 1993. p. 323-41.

11. Woolford A, Schantz EJ, Woodburn M. Heat inactivation of botulinum toxin 

type A in some convenience foods after frozen storage. J Food Sci. 

1978;43:622-4.

12. Dack GM. Effect of irradiation on Clostridium botulinum toxin subjected to 

ultra centrifugation. Report No. 7. Natick, MA: Quartermaster Food and 

Container Institute for the Armed Forces; 1956.
Appendix I: References 393

13. Wagenaar R, Dack GM. Effect in surface ripened cheese of irradiation on 

spores and toxin of Clostridium botulinum types A and B. Food Res. 

1956;21:226-34.

14. Bennett R, Berry M. Serological reactivity and in vivo toxicity of 

Staphylococcus aureus enterotoxin A and D in select canned foods. J Food 

Sci. 1987;52:416-8.

15. Concon J. Bacterial food contaminants: bacterial toxins. In: Food toxicology. 

Vol. B. Food science and technology. New York: Marcel Dekker, Inc.; 1988. 

p. 771-841.

16. Modi NK, Rose SA, Tranter HS. The effects of irradiation and temperature on 

the immunological activity of staphylococcal enterotoxin A. Int J Food 

Microbiol. 1990;11:85-92.

17. Wannemacher R, Bunner D, Dinterman R. Inactivation of low molecular 

weight agents of biological origin. In: Symposium on agents of biological 

origin. Aberdeen Proving Grounds, MD: US Army Chemical Research, 

Development and Engineering Center; 1989. p. 115-22.

18. Haigler HT, Woodbury DJ, Kempner ES. Radiation inactivation of ricin occurs 

with transfer of destructive energy across a disulfide bridge. Proc Natl Acad 

Sci USA. 1985;82:5357-9.

19. Poli MA. Laboratory procedures for detoxification of equipment and waste 

contaminated with brevetoxins PbTx-2 and PbTx-3. J Assoc Off Anal Chem. 

1988;71:1000-2.

20. Notermans S, Havelaar A. Removal and inactivation of botulinum toxins 

during production of drinking water from surface water. Antonie Van 

Leeuwenhoek. 1980;46:511-14.

21. Brazis A, Bryant A, Leslie J, et al. Effectiveness of halogens or halogen 

compounds in detoxifying Clostridium botulinum toxins. J Am Waterworks 

Assoc. 1959;51:902-12.

22. Graikoski J, Blogoslawski W, Choromanski J. Ozone inactivation of 

botulinum type E toxin. Ozone: Sci Eng. 1985;6:229-34.

23. Robinson JP. Annex 2. Toxins. In: Public health response to biological and 

chemical weapons: WHO guidance. 2nd edition. Geneva: World Health 

Organization; 2004. p. 214-28.
394 Biosafety in Microbiological and Biomedical Laboratories

Appendix J—NIH Oversight of Research Involving 

Recombinant Biosafety Issues

The NIH locus for oversight of recombinant DNA research is the Office of 

Biotechnology Activities (OBA), which is located within the Office of Science 

Policy, in the Office of the Director of the NIH. The OBA implements and 

manages the various oversight tools and information resources that NIH uses to 

promote the science, safety and ethics of recombinant DNA research. The key 

tools of biosafety oversight are the NIH Guidelines, IBCs, and the Recombinant 

DNA Advisory Committee (RAC). The NIH also undertakes special initiatives to 

promote the analysis and dissemination of information key to our understanding 

of recombinant DNA, including human gene transfer research. These initiatives 

include a query-capable database and conferences and symposia on timely 

scientific, safety, and policy issues. The NIH system of oversight is predicated 

on ethical and scientific responsibilities, with goals to promote the exchange 

of important scientific information, enable high-quality research, and help 

advance all fields of science employing recombinant DNA.

The NIH Guidelines promote safe conduct of research involving recombinant 

DNA by specifying appropriate biosafety practices and procedures for research 

involving the construction and handling of either recombinant DNA molecules 

or organisms and viruses that contain recombinant DNA. Recombinant DNA 

molecules are defined in the NIH Guidelines as those constructed outside of a 

living cell by joining natural or synthetic DNA segments to DNA molecules that 

can replicate in a living cell. The NIH Guidelines are applicable to all recombinant 

DNA work at an institution that receives any funding from the NIH for recombinant 

DNA research. Compliance with the NIH Guidelines is mandatory for investigators 

conducting recombinant DNA research funded by the NIH or performed at or 

sponsored by any public or private entity that receives any NIH funding for 

recombinant DNA research. This broad reach of the NIH Guidelines is intended 

to instill biosafety practices throughout the institution, which is necessary if the 

practices are to be effective.

The NIH Guidelines were first published in 1976 and are revised as 

technological, scientific, and policy developments warrant. They outline the 

roles and responsibilities of various entities associated with recombinant DNA 

research, including institutions, investigators, biological safety officers, and 

the NIH (see Section IV of the NIH Guidelines). They describe four levels of 

biosafety and containment practices that correspond to the potential risk of 

experimentation and require different levels of review for recombinant DNA 

research, based on the nature and risks of the activity. These include:

1. Review by the RAC, and approval by the NIH Director and the IBC.

2. Review by the NIH OBA and approval by the IBC.
Appendix J: NIH Oversight of Research Involving Recombinant Biosafety Issues 395

3. Review by the RAC and approvals by the IBC and Institutional 

Review Board.

4. Approval by the IBC prior to initiation of the research.

5. Notification of the IBC simultaneous with initiation of the work.

See Section III of the NIH Guidelines for additional details. In all instances, it 

is important to note that review by an IBC is required.

The federally mandated responsibilities for an IBC are articulated solely in 

the NIH Guidelines. Their membership, procedures, and functions are outlined in 

Section IV-B-2. Institutions, ultimately responsible for the effectiveness of IBCs, 

may define additional roles and responsibilities for these committees in addition 

to those specified in the Guidelines. To access the NIH Guidelines see the 

following Web site: http://oba.od.nih.gov/rdna/nih_guidelines_oba.html.

The Recombinant DNA Advisory Committee is a panel of national experts in 

various fields of science, medicine, genetics, and ethics. It includes individuals 

who represent patient perspectives. The RAC considers the current state of 

knowledge and technology regarding recombinant DNA research and advises 

the NIH Director and OBA on basic and clinical research involving recombinant 

DNA and on the need for changes to the NIH Guidelines.

Additional information on OBA, the NIH Guidelines, and the NIH RAC can 

be found at: http://oba.od.nih.gov.
396 Biosafety in Microbiological and Biomedical Laboratories

Appendix K—Resources

Resources for information, consultation, and advice on biohazard control, 

decontamination procedures, and other aspects of laboratory and animal safety 

management include:

AAALAC International

Association for Assessment and Accreditation

of Laboratory Animal Care International

5283 Corporate Drive

Suite 203

Fredrick, MD 21703-2879

Telephone: (301) 696-9626

Fax: (301) 696-9627

Web site: http://www.aaalac.org

American Biological Safety Association (ABSA)

American Biological Safety Association

1200 Allanson Road

Mundelein, IL 60060-3808

Telephone: (847) 949-1517

Fax: (847) 566-4580

Web site: http://www.absa.org

CDC Etiologic Agent Import Permit Program

Centers for Disease Control and Prevention

Etiologic Agent Import Permit Program

1600 Clifton Road, NE Mailstop: F-46

Atlanta, GA 30333

Telephone: (404) 718-2077

Fax: (404) 718-2093

Web site: http://www.cdc.gov/od/eaipp

CDC Office of Health and Safety

Centers for Disease Control and Prevention

Office of Health and Safety

Mailstop: F-05

1600 Clifton Road, NE

Atlanta, GA 30333

Telephone: (404) 639-7233

Fax: (404) 639-2294

Web site: www.cdc.gov/biosafety
Appendix K: Resources 397

CDC Select Agent Program

Centers for Disease Control and Prevention

Division of Select Agents and Toxins

Mailstop: A-46

1600 Clifton Road, NE

Atlanta, GA 30333

Telephone: (404) 718-2000

Fax: (404) 718-2096

Web site: www.selectagents.gov

Clinical and Laboratory Standards Institute

940 West Valley Road, Suite 1400

Wayne, PA 19087

Telephone: (610) 688-0100

Fax: (610) 688-0700

Web site: http://www.clsi.org

College of American Pathologists

1350 I St. N.W. Suite 590

Washington, DC 20005-3305

Telephone: (800) 392-9994

Telephone: (202) 354-7100

Fax: (202) 354-7155

Web site: http://www.cap.org

Department of the Army

Biological Defense Safety Program

Department of Defense

32 CFR Parts 626, 627

Web site: www.gpo.gov

National B-Virus Resource Laboratory

National B Virus Resource Laboratory

Attention: Dr. Julia Hillard

Viral Immunology Center

Georgia State University

50 Decatur Street

Atlanta, GA 30303

Telephone: (404) 651-0808

Fax: (404) 651-0814

Web site: http://www2.gsu.edu/~wwwvir
398 Biosafety in Microbiological and Biomedical Laboratories

NIH Division of Occupational Health and Safety

National Institutes of Health

Division of Occupational Health and Safety

Building 13, Room 3K04

13 South Drive, MSC 5760

Bethesda, MD 20892

Telephone: (301) 496-2960

Fax: (301) 402-0313

Web site: http://dohs.ors.od.nih.gov/index.htm

NIH Office of Biotechnology Activities

National Institutes of Health

Office of Biotechnology Activities

6705 Rockledge Drive

Suite 750, MSC 7985

Bethesda, MD 20892

Telephone: (301) 496-9838

Fax: (301) 496-9839

Web site: http://oba.od.nih.gov

NIH Office of Laboratory Animal Welfare (OLAW)

National Institutes of Health

Office of Laboratory Animal Welfare (OLAW)

RKL 1, Suite 360, MSC 7982

6705 Rockledge Drive

Bethesda, MD 20892-7982

Telephone: (301) 496-7163

Web site: http://grants.nih.gov/grants/olaw/olaw.htm

Occupational Safety and Health Administration

U.S. Department of Labor

200 Constitution Avenue NW

Washington, DC 20210

Telephone: (800) 321-6742

Web site: http://www.osha.gov/index.html

USDA-APHIS National Center for Import / Export

USDA Animal and Plant Health Inspection Service Veterinary Services National 

Center for Import and Export

4700 River Road, Unit 40

Riverdale, MD 20737

Web site: http://www.aphis.usda.gov/import_export/index.shtml
Appendix K: Resources 399

USDA Agriculture Select Agent Program

USDA Agriculture Select Agent Program

Animal and Plant Health Inspection Service

U.S. Department of Agriculture

4700 River Road, Unit 2, Mailstop 22

Riverdale, MD 20737

Web site: http://www.aphis.usda.gov/programs/ag_selectagent/index.shtml

USDA National Animal Disease Center

U.S. Department of Agriculture

National Animal Disease Center

P.O. Box 70

2300 Dayton Road

Ames, IA 50010

Telephone: (515) 663-7200

Fax: (515) 663-7458

Web site: http://www.ars.usda.gov/main/site_main.htm?modecode=36-25-30-00

USDA Plant Select Agents & Plant Protection and Quarantine

Animal and Plant Health Inspection Service

Plant Protection and Quarantine, Permits, Agricultural Bioterrorism

U.S. Department of Agriculture

4700 River Road, Unit 133

Riverdale, MD 20737

Telephone: (877) 770-5990

Fax: (301) 734-5786

Web site: http://www.aphis.usda.gov/programs/ag_selectagent/index.shtml;

and http://www.aphis.usda.gov/permits/index.shtml

US Department of Transportation

Hazardous Materials Center

Pipeline & Hazardous Materials Center

U.S. Department of Transportation

400 7th Street, S.W.

Washington, DC 20590

Telephone: (800) 467-4922

Web site: http://www.phmsa.dot.gov/hazmat
400 Biosafety in Microbiological and Biomedical Laboratories

US Department of Commerce

Export Administration Program

Bureau of Industry and Security (BIS)

Export Administration Regulations (EAR)

U.S. Department of Commerce

14th Street and Constitution Avenue, N.W.

Washington, DC 20230

Telephone: (202) 482-4811

Web site: http://www.access.gpo.gov/bis/index.html

World Health Organization Biosafety Program

World Health Organization Biosafety Program

Avenue Appia 20

1211 Geneva 27

Switzerland

Telephone: (+ 41 22) 791 21 11

Fax: (+ 41 22) 791 3111

Web site: http://www.who.int/ihr/biosafety/en
Appendix L: Acronyms 401

Appendix L—Acronyms

A1HV-1 Alcelaphine Herpesvirus-1

ABSA American Biological Safety Association

ABSL Animal Biosafety Level

ACAV American Committee on Arthropod-Borne Viruses

ACIP Advisory Committee on Immunization Practices 

ACG Arthropod Containment Guidelines

ACL Arthropod Containment Levels

ACME American Committee of Medical Entomology

AHS African Horse Sickness 

AHSV African Horse Sickness Virus 

AKAV Akabane Virus

APHIS Animal and Plant Health Inspection Service

APMV-1 Avian Paramyxovirus Type 1

ASF African Swine Fever

ASFV African Swine Fever Virus

ASHRAE American Society of Heating, Refrigerating, and Air-Conditioning 

Engineers

ASTMH American Society of Tropical Medicine and Hygiene

BCG Bacillus Calmette-Guérin

BDV Border Disease Virus

BMBL Biosafety in Microbiological and Biomedical Laboratories

BoNT Botulinium neurotoxin

BSC Biological Safety Cabinet

BSE Bovine Spongiform Encephalopathy

BSL Biosafety Level

BSL-3-Ag BSL-3-Agriculture

BSO Biological Safety Officer

BTV Bluetongue Virus

BVDL Bovine Viral Diarrhea Virus

CAV Constant Air Volume

CBPP Contagious Bovine Pleuropneumonia

CCPP Contagious Caprine Pleuropneumonia

CETBE Central European Tick-Borne Encephalitis

CDC Centers for Disease Control and Prevention

CHV-1 Cercopithecine Herpesvirus-1

CJD Creutzfeldt-Jakob Disease

CJIS Criminal Justice Information Services Division

CNS Central Nervous System

CSF Cerebrospinal Fluid

CSFV Classical Swine Fever Virus

DHHS Department of Health and Human Services

DoC Department of Commerce

DOD Department of Defense

DOL Department of Labor
402 Biosafety in Microbiological and Biomedical Laboratories

DOT Department of Transportation

EBV Epstein-Barr Virus

EEE Eastern Equine Encephalomyelitis

EPA Environmental Protection Agency

EtOH Ethanol

FDA Food and Drug Administration

FFI Fatal Familial Insomnia

FMD Foot and Mouth Disease

FMDV Foot and Mouth Disease Virus

GI Gastrointestinal Tract

GSS Gerstmann-Straussler-Scheinker Syndrome

HEPA High Efficiency Particulate Air

HBV Hepatitis B Virus

HCMV Human Cytomegalovirus

HCV Hepatitis C Virus

HD Heartwater Disease

HDV Hepatitis D Virus

HFRS Hemorrhagic Fever with Renal Syndrome

HHV Human Herpes Virus 

HHV-6A Human Herpes Virus -6A

HHV-6B Human Herpes Virus -6B

HHV-7 Human Herpes Virus -7

HHV-8 Human Herpes Virus -8

HIV Human Immunodeficiency Virus

HPAI Highly Pathogenic Avian Influenza

HPAIV Highly Pathogenic Avian Influenza Virus

HPS Hantavirus Pulmonary Syndrome

HSV-1 Herpes Simplex Virus-1

HSV-2 Herpes Simplex Virus-2

HTLV Human T-Lymphotropic Viruses

HVAC Heating, Ventilation, and Air Conditioning

IACUC Institutional Animal Care and Use Committee

IATA International Air Transport Association

IBC Institutional Biosafety Committee

ICAO International Civil Aviation Organization

ID Infectious Dose

ID50 Number of organisms necessary to infect 50% of a group of 

animals

IgG Immunoglobulin

ILAR Institute for Laboratory Animal Research

IND Investigational New Drug

IPM Integrated Pest Management

IPV Inactivated Poliovirus Vaccine

ISA Infectious Salmon Anemia
Appendix L: Acronyms 403

ISAV Infectious Salmon Anemia Virus

LAI Laboratory-Associated Infections

LCM Lymphocytic Choriomeningitis

LCMV Lymphocytic Choriomeningitis Virus

LD Lethal Dose

lfm Linear Feet Per Minute

LGV Lymphogranuloma Venereum

LMW Low Molecular Weight

LSD Lumpy Skin Disease

LSDV Lumpy Skin Disease Virus

MCF Malignant Catarrhal Fever

MenV Menangle Virus

MMWR Morbidity and Mortality Weekly Report

MPPS Most Penetrating Particle Size

NaOCl Sodium Hypochlorite

NaOH Sodium Hydroxide

NBL National Biocontainment Laboratory

NCI National Cancer Institute

ND Newcastle Disease

NDV Newcastle Disease Virus

NHP Nonhuman Primate

NIH National Institutes of Health

NIOSH National Institute for Occupational Safety and Health

OBA NIH Office of Biotechnology Activities

OIE World Organization for Animal Health

OPV Oral Poliovirus Vaccine

OSHA Occupational Safety and Health Administration

PAPR Positive Air-Purifying Respirator

PBT Pentavalent Botulinum Toxoid Vaccine

PPD Purified Protein Derivative

PPM Parts Per Million

PPRV Pest des Petits Ruminants Virus

Prp Prion Protein

RAC Recombinant DNA Advisory Committee

RBL Regional Biocontainment Laboratory

RP Rinderpest

RPV Rinderpest Virus

RVF Rift Valley Fever

RVFV Rift Valley Fever Virus

SALS Subcommittee on Arbovirus Laboratory Safety

SARS Severe Acute Respiratory Syndrome

SARS-CoV SARS-Associated Coronavirus

SCID Severe Combined Immune Deficient

SC type Small-Colony type
404 Biosafety in Microbiological and Biomedical Laboratories

SE Staphylococcal Enterotoxins

SEA SE Serotype A

SEB SE Serotype B

SIV Simian Immunodeficiency Virus

SGP Sheep and Goat Pox

SGPV Sheep and Goat Pox Virus

SOP Standard Operating Procedure

SVCV Spring Viremia of Carp Virus

SVD Swine Vesicular Disease

SVDV Swine Vesicular Disease Virus

TLV Threshold Limit Values

TME Transmissible Mink Encephalopathy

TSE Transmissible Spongiform Encephalopathy

UV Ultraviolet

USAMRIID U.S. Army Medical Research Institute of Infectious Diseases

USDA U.S. Department of Agriculture

USPS United States Postal Service

UPS Uninterrupted Power Supply

VAV Variable Air Volume

VEE Venezuelan Equine Encephalitis

VS Veterinary Services

VZV Varicella-Zoster Virus

WEE Western Equine Encephalomyelitis

WHO World Health Organization

WNV West Nile Virus
Index 405

Index

A

African horse sickness (AHSV) 246, 350, 353-55, 373, 401

African swine fever virus (ASFV) 246, 350, 352, 373, 401

Agriculture Agents 343, 352

Agriculture pathogen biosafety 343

Akabane virus 350, 354, 401

Allergic reactions 192

animal biosafety levels 61

Animal Biosafety Levels (ABSL) 60

Animal Biosafety Level 1 61

Animal Biosafety Level 2 67

Animal Biosafety Level 3 61, 75

Animal Biosafety Level 4 85

Animal facilities 27, 60, 91, 173, 243, 361-63, 372

Anthrax iii, 104, 123-24, 161, 273

Arboviruses 2, 5, 233, 236, 238-40, 246, 265-267

arenaviruses 234, 237

armadillos 144-45, 166

Arthropod containment 61, 160, 169, 377-78, 401

Ascaris 192-93

B

B virus (herpesvirus simiae) 1, 12, 19, 25, 29, 120-21, 204-10, 229, 232, 243, 

384, 397, 402

Bacillus anthracis 123, 350

Bacillus subtilis 25, 330

Bacterial agents 123

Besnoitia besnoiti 350

Biological safety cabinets (BSC) 15, 18, 22, 25-27, 36, 38, 41, 44-45, 49, 51-52, 

54, 57, 72, 75, 84, 86-87, 92-95, 97, 100, 103, 124-25, 128-30, 146, 150, 

155-56, 160, 173-74, 176-78, 185, 187, 194, 200, 210, 221, 225, 239, 274, 

278, 290-313, 315-19, 321-25, 330, 332, 335, 347, 383, 386-88, 401

Class I 23, 27, 59, 103, 219, 290-93, 299, 311-13, 315, 321

Class II 23, 27, 38, 41, 44, 51, 53-54, 57, 59, 75, 84, 86, 93-94, 96-97, 100, 

103, 173-74, 176-78, 219, 235, 274, 278, 290, 292-300, 302, 307-09, 

311-13, 315-18, 322-24, 347, 388

Class III 23, 26, 41, 44-45, 48-50, 52, 54, 59, 84-86, 92-95, 97, 103, 290, 

297-98, 311-13, 319, 324, 347, 388

Positive-pressure personnel suit 26

Biosafety levels (BSL) 4, 9-10, 24, 27, 30, 59, 60-61, 103-04, 234, 239-40, 290, 

294, 378, 401

Biosafety Level 1 (BSL-1) 4-5, 23, 25, 30, 32-33, 59, 219
406 Biosafety in Microbiological and Biomedical Laboratories

Biosafety Level 2 (BSL-2) 4, 7, 17, 23, 25, 27, 33, 35, 37, 59, 67, 104, 

124-25, 127-29, 131, 133, 135, 137-41, 143-46, 148-50, 152-53, 155-57, 

159-60, 170, 172-75, 177-78, 182, 185, 187, 189, 191, 194, 196, 198, 

200-01, 203-04, 207, 210, 212, 216-17, 219, 221, 223, 225, 234-35, 237, 

241, 243, 246, 269, 272, 274, 278, 284, 286, 305, 311, 363, 368, 372, 

378, 383, 386

Biosafety Level 3 (BSL-3) 4, 7, 16, 24, 26-27, 38, 41-42, 45, 53, 57, 59, 

104, 124, 126-29, 133, 135, 139, 141, 146, 149-50, 153, 160, 170, 172, 

174, 196, 198, 201, 204, 207, 210, 212-13, 216, 219, 221, 223, 225-26, 

234-38, 241, 243, 245, 269, 272, 284, 286, 300, 305, 311, 331-32, 

343-44, 347-53, 355-57, 359-62, 364-67, 369-71, 373, 387

Biosafety Level 4 (BSL-4) 4, 16, 26, 45, 48-49, 51, 53-59, 87, 96, 100, 103, 

120, 202, 207, 219, 234-38, 294, 305, 311, 331-32, 343-44, 350-51

Biosecurity iii, 6, 28, 86, 104-13, 374

Bioterrorism 1, 124, 134, 161, 279, 291, 339, 351, 361, 368, 376, 379, 399

Blastomyces dermatitidis 1, 170

Bloodborne pathogens 13, 20, 25, 27-29, 205, 223, 232, 383-84

Bluetongue virus 350

BMBL iii-iv, ix, 1, 3-7, 9, 11, 16, 19, 104, 290-91, 298, 377-78, 401

Bordetella pertussis 125, 162

Borna disease virus 351

Botulinum neurotoxin 134-35, 268, 390-91

Botulism 134-35, 164, 268-69, 278-79, 392

Bovine pleuropneumonia agent 358

Bovine spongiform encephalopathy 282, 284, 286, 351, 401

Brucella 1, 2, 7, 126-27, 162, 351

abortus 126-27, 162, 351

canis 126, 162

maris 126

melitensis 2, 7, 126-27, 162, 351

suis 126-27, 351

Brucellosis 126, 162

BSL-3-Ag xix, 53, 61, 96, 100, 213, 246, 265, 343-47, 350-51, 353, 357, 359, 

360-61, 364, 367, 369, 370, 401

Burkholderia mallei 127, 351

Burkholderia pseudomallei 129, 163, 351

C

C virus 2, 13, 204, 228, 402

Cabinet laboratory 45, 49, 51, 53-54, 58, 85-86, 92, 94, 96, 101

Camelpox virus 351

Campylobacter 130, 163, 165

coli 130

fetus 130

jejuni 130, 163

upsaliensis 130
Index 407

Caprine Pleuropneumonia Agent 359

Category A UN Standard Triple Packaging 341

Category B Non-specification Triple Packaging 342

Cestode parasites 190

Taenia solium 190

Cestode Parasites 190

Chemical Inactivation of Selected Toxins 391

Chimeric viruses 239-40

chimpanzees 203-04

Chlamydia 131-32

pneumoniae 131-33, 164

psittaci 1, 12, 131-33

trachomatis 131-33, 164

Cholera 159, 169, 352, 357, 373

Classical swine fever virus 351, 357, 401

Clean 43-44, 52-53, 55, 57, 83, 94, 96, 98, 100, 278, 288, 291-92, 298, 302, 

304, 307, 312, 319-20, 322-23, 325, 344, 348, 387, 389

Clinical laboratories iii, 2, 4, 27, 60, 163, 166-68, 225, 330

Clostridium

botulinum 134-35, 164, 390-93

tetani 136

Coccidioides immitis 1, 12, 171, 179, 351

Coccidioides posadasii 171, 179

Cochliomyia hominivorax 351

Congo-Crimean hemorrhagic fever 26, 250

Conidia 170, 173-75, 178

Containment

Primary containment 23, 25, 56, 72, 79, 93, 99, 125, 129, 133, 135, 149, 

152-53, 155-56, 187, 194, 204, 210, 221, 269, 290-91, 298, 305, 309, 312, 

343-44, 347-48

Secondary containment 45, 85, 288, 305, 388

Corynebacterium diphtheriae 137

Coxiella burnetii 1, 2, 11-12, 26, 195, 199

Creutzfeldt-Jakob 282-83, 289, 401

Cryptococcus neoformans 172, 179

Cysticercus cellulosae 191

Cytomegalovirus 115, 208, 229-30, 402

D

Decontamination iii, 23, 25, 31, 34, 38-40, 42-44, 46, 48-59, 64, 70-71, 75, 78, 

80, 82-83, 85, 87-88, 91-92, 94-101, 103, 135, 152-53, 155-56, 226, 245, 268, 

272, 275, 278, 280, 285, 301, 303-05, 307-08, 322, 324, 326-27, 329-32, 

334-35, 345-49, 388-91, 396

Dengue 237, 251
408 Biosafety in Microbiological and Biomedical Laboratories

Dermatophytes

Epidermophyton 176

Microsporum 176

Trichophyton 176

Diagnostic specimens 28, 202, 224, 241, 341

diphtheria 137-38, 164

Diphtheria 164

Disinfection 38-39, 44, 71, 80, 157, 282, 326-29, 331, 333-35, 372, 383

DOT Packaging Requirements 340

E

Eastern equine encephalitis virus xviii, 242, 351

Ebola 251, 330

emergency response 49, 91, 104, 110, 112-13, 336, 340

Encephalitis

Russian spring-summer 236

Russian Spring-Summer 260

Epidermophyton 176

equine encephalomyelitis 19, 116, 235, 237, 351

Escherichia coli 154, 168

F

Facility design 17, 22-23, 25-26, 30, 44-45, 55, 58, 60-61, 85, 98, 101, 290, 347, 

381, 392

Fasciola 188-89

Filoviruses 237

Foot and mouth disease 351, 360-61, 374, 402

Francisella tularensis 1, 12, 138, 164, 351

Fungal Agents 170

G

Ganjam virus 265, 351

Germicidal Chemicals 330

Giardia 186-87

Giardia spp. 187

Gloves 4, 15, 23, 25, 32, 37, 42, 47, 50-51, 59, 63, 65, 69, 72-73, 77, 81-82, 89, 

92-94, 103, 128-29, 133, 143-44, 149-50, 152-53, 155-57, 159-60, 175, 185, 

189, 191, 204, 207, 212, 221-22, 225, 269, 272, 274, 278, 286-87, 293, 297, 

300-01, 304, 312-13, 319, 344, 348, 361, 363, 372, 383, 386, 388

gonorrhoeae 148

Guidelines 3-4, 6, 8, 10, 12-13, 17, 19, 24-25, 28-29, 61, 121, 124, 160, 169, 

206-07, 214, 219, 221, 227, 229-31, 235, 238, 284-86, 289, 324, 334, 343, 

377-78, 383-86, 389, 394-95, 401
Index 409

H

Hantaviruses 200, 234

Heartwater Disease Agent 362

Helicobacter pylori 139, 165

Hemorrhagic fever 26-27, 200, 227, 244, 402

Hendra virus 201-02, 228, 351, 366

HEPA filter 44, 50, 53-54, 57, 75, 92, 96, 100, 274, 291-97, 306-09, 311-13, 

315-22, 345, 348-49, 387

Hepatitis

A virus 202-03

B virus (HBV) 1-2, 13, 25, 27, 29, 204-05, 229, 232, 330, 383-84, 402

C virus (HCV) 2, 13, 204-05, 228, 330, 383, 402

D virus (HDV) 204-05, 402

E virus 202-03

Herpesviruses 205, 208-10, 365

Herpesvirus simiae 205, 20-10, 229

Human herpesviruses 208-10

Herpesvirus simiae 205, 209-10, 229

CHV-1 12, 401

Histoplasma 173, 351

capsulatum 173-74

farciminosum 351

Hog cholera 357, 373

Human Herpes Virus 208, 402

Human immunodeficiency virus (HIV) 13, 25, 27-28, 146-47, 177, 209, 221-23, 

232, 266, 330, 383-84, 402

Hypr 236, 253

I

Immunoprophylaxis 114

Importation and interstate shipment 28

infectious canine hepatitis virus 25

Influenza 5, 20, 211-14, 230, 271, 350, 366, 372, 375, 402

International transfer 336

Interstate shipment 28

Investigational New Drug (IND) 116, 122, 135, 196, 208, 237, 269, 402

J

Junin virus 236, 265

K

Kuru 283

Kyasanur Forest disease 255
410 Biosafety in Microbiological and Biomedical Laboratories

L

Laboratory-associated infections (LAI) 1-2, 6-7, 9, 18, 20, 126, 130, 132, 137, 

140, 142, 150, 157-58, 161, 168, 171, 179-80, 192, 194-95, 197-99, 203-04, 

210-11, 215, 220, 228, 230, 235-36, 241, 300, 326, 403

Laboratory coat(s) 32, 36, 59, 63, 65, 69, 72, 77, 81, 103, 150, 189, 272, 274, 

278, 287, 300, 363, 383, 386

Laboratory hazard(s) 11, 17, 130, 132, 137, 140, 142, 145, 148-49, 152-53, 

155-56, 159, 172, 184, 187, 189, 191, 204, 212, 235-36, 243, 290, 298, 302, 383

Laboratory practice(s) 15, 22, 24, 104, 191, 224, 238-39, 361, 366, 372, 386

Laboratory security and emergency response 113

Legionella pneumophila 140, 165

Leishmania spp. 20, 182-85

Leprosy 144, 166

Leptospira interrogans 166

Leptospirosis 142, 166

Listeria monocytogenes 143, 166

Loose-housed animals 27, 213, 246, 343, 351

Louping ill virus 256, 350

Low molecular weight (LMW) toxin(s) 274-75, 277-78, 385-86, 390-91, 403

Lumpy skin disease virus (LSDV) 350, 364, 403

Lymphocytic choriomeningitis 13, 20, 215, 230, 403

Lymphogranuloma venereum (LGV) 131-33, 403

M

Macaque 12, 205-07, 222

Machupo 256

malaria 182-84

Malignant catarrhal fever virus 350, 365

Mammalian Cells and Tissues 383

Marburg 26, 256

mask 15, 36, 42, 72, 225, 272

medical surveillance 35, 40, 47, 59, 62, 68, 70, 76, 79, 86, 90, 103, 214

Menangle virus (MenV) 350, 366, 403

metacercariae 189

mice 13, 20, 131, 133, 141, 144-46, 166, 169, 173, 176, 215-16, 245, 266, 273, 

276, 280, 285, 353-55

Microsporum 176

Molds xvi, 171, 176-77, 287

monkey pox 218-19

Mycobacterium

avium 147-48

bovis 145-46, 327, 330, 350

chelonei 147

fortuitum 147
Index 411

kansasii 147

leprae 144, 147

marinum 147

scrofulaceum 147

tuberculosis 1-2, 20, 26-27, 145-48, 327-30, 383

ulcerans 147-48

Mycoplasma agalactiae 350

Mycoplasma mycoides 350, 358, 374

N

Naegleria

fowleri 182, 185

Nairobi sheep disease 251, 257, 351

needles and syringes 31, 34, 39, 46, 63, 70, 78, 87

Neisseria

gonorrhoeae 148

meningitidis 149-51

Nematode parasites 192

Newcastle disease virus (NDV) 258, 351, 367-68, 403

Nigeria gruberi 25

NIH Guidelines 3, 8, 10, 12-13, 19, 24-25, 214, 219, 231, 324, 377, 394, 395

O

Occupational health v, vii-viii, 13, 60, 62, 67-69, 75, 77, 88-89, 102, 114-15, 

120-21, 168, 194, 214, 226, 229, 337, 398

Omsk Hemorrhagic 258

P

Paramyxovirus 201, 227-28, 367, 401

Parasitic agents

Blood and Tissue Protozoal Parasites 182

Cestode 190

Intestinal 186

Nematode 192

Trematode 188

Pathogens iii, 3, 5, 6, 9, 13, 17, 20, 25, 27-29, 61, 63, 69, 77, 106-07, 110-11, 

115, 117, 131, 147, 157, 171, 176, 186, 197, 205, 208-09, 221, 223, 229, 232, 

265, 271, 281, 326, 337, 343, 347, 350, 374-75, 377-78, 380, 383-84

Penicillium marneffei 177, 181

Performance Testing BSCs 309

Personal protective equipment 15, 22-23, 25, 32, 36, 41, 49, 62-65, 67-69, 

71-73, 75, 77, 79-82, 88, 92, 118, 121, 134, 152-53, 155-56, 160, 206-07, 235, 

237, 239, 243, 286, 288, 344, 348, 386

personnel suit 26

Peste des petits ruminants virus (PPRV) 351, 368-69, 403
412 Biosafety in Microbiological and Biomedical Laboratories

Pest management 31, 35, 40, 46, 64, 70, 78, 89, 380-82, 402

Physical Inactivation of Selected Toxins 390

Physical security 110, 344

Pike and Sulkin 1-2

Pipetting 14-15, 30, 34, 36, 39, 45, 63, 69, 78, 87, 127, 242, 301

Plague 159-60, 169, 351, 368

Plasmodium spp. 182-84

Plastic-backed absorbent toweling 301

Poliovirus 216-18, 231, 330, 402-03

Pontiac fever 141

Poxviruses 218-19, 231, 370

Primary barriers 22-23, 27, 32, 36, 41, 49, 59, 64, 72, 80, 92, 103, 290, 306, 

347, 378

Primates 60, 102, 121, 123, 156, 166, 197, 203, 206-07, 216-17, 222-23, 

229, 234

Prion diseases 282-85, 289

Production quantities 124, 129, 149-50, 153, 204, 216, 221

Protozoal parasites 182, 186

Pseudomonas

pseudomallei 129, 163

psittacosis 131, 132

Q

Q fever 19, 195-96, 199, 351

R

Rabies virus 220

Radiological Hazards 299

Regulations iii, vi, 6, 28, 31, 34, 40, 44, 60, 85, 104, 114, 120, 243, 265, 286, 

288, 299, 336-39, 350, 353-56, 358-60, 362-66, 369-73, 379, 386, 400

Retroviruses 119, 221-23, 240, 266

Ricin toxin 272, 274, 280

Rickettsia

akari 196

australis 196

belli 198

canada 198

conorii 196

Coxiella burnetii 195

montana 198

mooseri 196

Orientia 196, 197

prowazekii 196, 198

rhipicephali 198

rickettsii 196-99

siberica 196
Index 413

spotted fever group 196-97

Surveillance of personnel 198

tsutsugamushi 196

typhi 196

rickettsial agents 198

Rickettsial Agents 195

Rift Valley fever virus 244, 267, 351, 403

Risk assessment iii, 1, 5, 9-12, 14-19, 22, 24, 30, 32, 37, 42-44, 50, 52-53, 

60-65, 67-70, 72, 75-77, 79, 81-83, 85, 92-93, 95, 99, 105, 107, 109, 112, 

119, 137, 152-56, 160, 194, 207, 210, 213-14, 226, 235, 237, 239-40, 272, 

290, 292, 298, 300, 305, 311, 343, 348-49, 377, 379, 383, 385-87

Risk group 10, 24, 204, 209

Rocky Mountain spotted fever 197, 199

S

Safety equipment 1, 3-4, 6, 9, 15, 17, 18, 22-27, 30, 32-33, 36, 41, 49, 59-61, 

64, 67, 72, 76, 80, 92, 103, 150, 291, 386

Salmon anemia virus 351, 403

Salmonella

Salmonellosis 151

typhi 1, 151-54, 168

SALS 233-34, 236, 238-39, 241, 403

SARS-associated coronavirus (SARS-CoV) 224-26, 403

Schistosoma spp. 188-89

Screwworm 351

Secondary barriers 23, 25-26, 33, 37, 42, 51, 59, 65, 73, 82, 94, 103, 305, 

347, 378

Select agents 5, 28, 104-05, 112-13, 127, 136, 172, 198, 202, 213-14, 233, 236, 

244, 336, 338-39, 351, 361, 368, 376, 379, 389, 397, 399

Serum 35, 40, 47, 70, 79, 90, 115-16, 119-20, 135, 201, 204, 206, 207, 214, 223, 

225-26, 241, 269, 271, 273, 277, 352-56, 358-60, 362, 364, 366, 369-73, 383

Severe Acute Respiratory Syndrome (SARS) 224, 232, 403

Sharps 14, 17, 30-31, 34, 39, 45-46, 59, 63-64, 69-71, 78, 80, 87, 103, 223, 237, 

241, 287-88, 387

Sheep and Goat Pox Virus (SGPV) 370, 404

Shiga toxin 154-55

Shigella spp. 2, 154

Shigellosis 155-56

Shipment 28

Simian immunodeficiency virus (SIV) 221-23, 232, 404

Smallpox 118, 218-19, 231

Spaulding Classification 328

Sporothrix schenckii 175

Spotted fever group 196, 197

Spring viremia of carp virus (SVCV) 351, 371, 404
414 Biosafety in Microbiological and Biomedical Laboratories

Staphylococcal Enterotoxins (SE) 270-72, 279, 385, 390-91, 393, 404

Sterilization iii, vi, 285, 288, 326-29, 331, 333-35, 346, 350, 387

Strongyloides spp. 192

Suit laboratory 45, 51, 55-57, 85, 93, 98, 100, 294, 331

Surveillance 2, 35-36, 40-41, 47, 49, 59, 62, 68, 70-71, 76, 79-80, 86, 88, 90, 

103, 147, 164, 198, 202, 214, 222, 232, 240, 372

Swine vesicular disease virus (SVDV) 351, 372-73, 376, 404

Syphilis 157, 169

T

Taenia solium 190

Teschen disease virus 351

Tetanus 136-37, 164, 279

Theileria

annulata 351

bovis 351

hirci 351

lawrencei 351

Tissue preparation 287

Toxin Agents 268

Toxins of biological origin 268, 271, 273, 277, 385

Toxoplasma 25, 182-85

Trachoma 132

Training v, vii, 2, 6-7, 9, 15, 18, 22, 25-26, 30, 32-33, 35, 38, 40, 45-46, 48-49, 

61-62, 67-68, 75-76, 85, 89-91, 104-05, 108-09, 112, 117, 179, 206, 238, 290, 

308, 331, 380-81, 385

Transmissible spongiform encephalopathies (TSE) 282, 285, 289, 334, 404

Trematode parasites

Fasciola 188

Schistosoma 188

Treponema pallidum 157, 168

Trichophyton 176, 180, 330

Trypanosoma

cruzi 182-85

evansi 351

vivax 351

Tuberculosis 1-2, 20, 26-27, 145-48, 166, 327-30, 383

Tularemia 138-39, 164

Typhoid fever 153

Typhus 197-99

U

Ultraviolet (UV) lamps 306, 310

Universal precautions 28-29, 232, 384
Index 415

V

Vaccines 22, 45, 59, 85, 103, 116, 121, 124, 126-27, 135-38, 147, 151, 153, 156, 

158-59, 196, 203, 205, 208, 216, 236-37, 240, 243, 246, 267, 269, 272, 274, 

278, 356, 373-76, 388

Vaccinia 218-19, 231

Variola 218-19

Venezuelan equine encephalomyelitis 19, 235, 351

Vesicular exanthema virus 351

Vesicular stomatitis virus 351

Vibrio

cholerae 158-59, 169

parahaemolyticus 158-59, 169

Viral agent(s) 200, 209-10, 226, 265, 383

W

warning signs 59, 103

Wesselsbron disease virus 351

Western equine encephalomyelitis (WEE) virus 116, 237, 242, 264, 266, 404

West Nile virus (WNV) 240-41, 264, 267, 404

Y

Yellow fever 235-37, 265, 267

Yersinia pestis 159-60, 169

Z

Zoonotic viruses 5, 233