Biosafety in Microbiological and Biomedical Laboratories*

 

U.S. Department of Health and Human Services 

Public Health Service 

Centers for Disease Control and Prevention 

and 

National Institutes of Health 

Fifth Edition 

2007

U. S. Government Printing Office 

Washington: 2007 

* See update note on second page
This manual is an electronic version of the 

Biosafety in Microbiological and Biomedical 

Laboratories (BMBL), 5th Edition. 

The print publication will have minor adjustments 

to formatting and figures but the overall content 

will remain the same. 

When printed copies are available, you will be able 

to request a copy via the U.S. Government Printing 

Office (http://www.gpo.gov/you). 

Electronic versions of the manual including the 

minor format and figure adjustments will then be 

posted on http://www.cdc.gov/od/ohs/. 
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 in 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. 

During the latest revision of the BMBL additional sections were added; sections on the 

principles and practices of biosafety and on risk assessment were expanded; all agent 

summary statements and appendices were revised; and effort was made to harmonize 

recommendations with guidance issued and regulations promulgated by other federal 

agencies. Wherever possible, an attempt was made to clarify 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 draw into focus the need for inclusion of 

additional information in the BMBL. In an attempt to better serve the needs of our 

community in this new era, information on the following topics has been added in the 5th

edition: 

• 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 on 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 want to 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 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. 

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 Biosafety in Microbiological and Biomedical Laboratories, 5th edition 

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

understanding during the long and comprehensive revision process. We know you will 

find it was well worth the wait. 

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

Director Director 

Division of Occupational Health and Safety Office of Health and Safety 

National Institutes of Health Centers for Disease Control and Prevention 

Bethesda, Maryland Atlanta, Georgia 

 January 2007 
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 

Director of Laboratory Safety 

Office of Laboratory Safety 

Howard Hughes Medical Institute 

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 Disease 

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 

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 

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 

Amy Patterson, M.D. 

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 

Deborah E. Wilson, DrPH, CBSP 

Director 

Division of Occupational Health and Safety 

Office of the Director 

National Institutes of Health 

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 

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 

Jennifer Gaudioso 

Mahmoud Ghannoum 

Mark Gibson 

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 

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 

Yvonne M. Reid 

Eric Resultan 

Greg Raymond 

Eric Resultan 

Judith Rhodes 

Robert L. Rice 

Jonathan Richmond 

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 

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 sp., 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 
Introduction 

laboratories chose not to 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 was 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 sp., Salmonella 

sp., Shigella sp., hepatitis C virus, and Cryptosporidium sp. 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 breech in 

containment practices resulted in eight LAIs with B. melitensis among 26 laboratory 

members, an attack rate of 31%. Reports of LAIs can serve as lessons in the importance 

of maintaining safe conditions in biological research. 
Introduction 

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 

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, occupational physicians, epidemiologists, public health officials and health and 

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

expanded the technical content of the 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. 
Introduction 

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 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. 

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: 
Introduction 

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; 

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. 

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 
Introduction 

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. 

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 
Introduction 

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 laboratory-acquired 

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 Institute; 1974. (DHEW pub; 78-790). 

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

relation to the cancer research program). JABSA. 1997;2:12-29. 

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. 
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. 
Biological Risk Assessment 

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 2004* 

 Risk Group 1 Agents that are not associated with 

disease in healthy adult humans. 

(No or low individual and community risk) 

A microorganism that is unlikely to cause 

human or animal disease. 

Risk Group 2 Agents that are associated with human 

disease which 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 that are 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 that are 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 that 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 

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 

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 
Biological Risk Assessment 

saliva, urine, or feces is an important indicator of hazard. The death of a primate center 

laboratory worker from Cercopithecine herpesvirus 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 F. 

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, 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 
Biological Risk Assessment 

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. They 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 outlines 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 human cell lines.17 

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 
Biological Risk Assessment 

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. 

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 assuring 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. 
Biological Risk Assessment 

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. The 

containment capability of a BSC is compromised by poor location, room air currents, 

decreased airflow, leaking filters, raised sashes, crowded work surfaces, and poor user 

technique. 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 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. 
Biological Risk Assessment 

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. 

There are several excellent resources that provide information and guidance for making 

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

agents 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 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 
Biological Risk Assessment 

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. 

Third, make a final determination of the appropriate biosafety level and select additional 

precautions indicated by the risk assessment. The final 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 3, 4 and 5 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. 
Biological Risk Assessment 

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

susceptibility to disease. Preexisting 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. 

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 experiences 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 
Biological Risk Assessment 

mirror progress in science and technology and serve as the basis for future revisions of 

BMBL. 

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Biological Risk Assessment 

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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. 

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;2. 

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. 
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 PRACTICE 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. 

Laboratory personnel, safety practices, and techniques must be supplemented by 

appropriate facility design and engineering features, safety equipment, and management 

practices. 
Principles of Biosafety 

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 infectious splashes 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. Openfronted 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. 
Principles of Biosafety 

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 ensure directional air flow, air treatment systems to 

decontaminate or remove agents from exhaust air, controlled access zones, airlocks as 

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, to be 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 8 (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 8 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 2. 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 

in time, 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 8. 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 practices may be specified. 
Principles of Biosafety 

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, Naegleria 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 handwashing. 

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 salmonellae, and Toxoplasma spp. 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 

human-derived materials should refer to the OSHA Bloodborne Pathogen Standard 2 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. 

Biosafety Level 3 practices, safety equipment, and facility design and construction are 

applicable to clinical, diagnostic, teaching, research, or production facilities in which 
Principles of Biosafety 

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 Congo-Crimean hemorrhagic fever are manipulated at BSL4. 

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 

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).4,5Additionally, 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 

 *

 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 website (www.cdc.gov). 
Principles of Biosafety 

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. 

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 which 

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. 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). 

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. 
Principles of Biosafety 

4. 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 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. 

Pennsylvania: Clinical and Laboratory Standards Institute; 2005. 
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 1 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 Biosafety Level Criteria – Biosafety Level 1 

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. Plasticware 

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. 
Laboratory Biosafety Level Criteria – Biosafety Level 1 

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 

child-bearing 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, integrity has been 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 – Biosafety Level 1 

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 
Laboratory Biosafety Level Criteria – Biosafety Level 2 

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. Plasticware 

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. 
Laboratory Biosafety Level Criteria – Biosafety Level 2 

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 

child-bearing 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. When appropriate, a baseline serum sample should be stored. 

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. 
Laboratory Biosafety Level Criteria – Biosafety Level 2 

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 safety 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 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 (preferably Class II), 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., 
Laboratory Biosafety Level Criteria – Biosafety Level 2 

cafeteria, library, 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 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, integrity has been 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 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. 
Laboratory Biosafety Level Criteria – Biosafety Level 2 

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. 

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 High Efficiency Particulate Air 

(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. 

9. There are no specific requirements on 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 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 a direct (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 inhalation route 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. 
Laboratory Biosafety Level Criteria – Biosafety Level 3 

All procedures involving the manipulation of infectious materials must be conducted 

within BSCs, other physical containment devices, or by personnel wearing appropriate 

personal protective equipment. 

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. 

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. 
Laboratory Biosafety Level Criteria – Biosafety Level 3 

d. Broken glassware must not be handled directly. Instead, it must be 

removed using a brush and dustpan, tongs, or forceps. Plasticware 

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: 

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 

child-bearing 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. 
Laboratory Biosafety Level Criteria – Biosafety Level 3 

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. 

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 safety 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 
Laboratory Biosafety Level Criteria – Biosafety Level 3 

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. Protective laboratory clothing with a solid-front such as tie-back or wraparound gowns, scrub suits, or coveralls are worn by workers when in the 

laboratory. Protective clothing is not worn outside of the laboratory. Reusable 

clothing is decontaminated with appropriate disinfectant before being 

laundered. Clothing is changed when contaminated. 

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. 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 should: 

a. Change gloves when contaminated, integrity has been 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. 
Laboratory Biosafety Level Criteria – Biosafety Level 3 

The laboratory must be separated from areas that are open to unrestricted 

traffic flow within the building. 

Access to the laboratory is restricted to entry by a series of 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. 

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 of the biological agents in use. 

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. 
Laboratory Biosafety Level Criteria – Biosafety Level 3 

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 air flow. A 

visual monitoring device which confirms directional air flow must be 

provided at the laboratory entry. Audible alarms should be considered 

to notify personnel of air flow disruption. 

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. 

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 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 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. 
Laboratory Biosafety Level Criteria – Biosafety Level 3 

11. 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). 

12. 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 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. 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. 

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 life-threatening disease, aerosol transmission, or 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. Access to the laboratory 

is controlled by the laboratory supervisor in accordance with institutional policies. 

There are two models for BSL-4 laboratories: 
Laboratory Biosafety Level Criteria – Biosafety Level 4 

(1) A Cabinet Laboratory where all handling of agents must be performed in a 

Class III BSC. 

(2) A Suit Laboratory where personnel must wear a positive pressure 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. All persons leaving the laboratory must be 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. 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. Plasticware 

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. 
Laboratory Biosafety Level Criteria – Biosafety Level 4 

d. Whenever practical, laboratory supervisors should adopt improved 

engineering and work practice controls that reduce risk of sharps 

injuries. 

6. Perform all procedures to minimize the creation of splashes and/or aerosols. 

7. Decontaminate work surfaces with appropriate disinfectant after completion 

of work and after any spill or splash of potentially infectious material. 

8. Decontaminate all wastes before removal from the laboratory by an effective 

and validated method. 

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 

child-bearing 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 in accordance with institutional 

policies. 

Only persons whose presence in the facility or individual laboratory rooms is 

required for scientific or support purposes should be 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. 
Laboratory Biosafety Level Criteria – Biosafety Level 4 

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. Laboratory clothing, including undergarments, pants, shirts, 

jumpsuits, shoes, and gloves, must be used by all personnel entering the 

laboratory. 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, 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 service 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 laboratoryacquired infections.

3. Each institution must establish policies and procedures describing the 

collection and storage of serum samples from at-risk personnel.

 

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 or 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 
Laboratory Biosafety Level Criteria – Biosafety Level 4 

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 must 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 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 are interlocked in a manner that prevents opening of the outer 

door unless the autoclave has been operated through a decontamination cycle. 

The doors of a fumigation chamber must be secured in a manner that prevents 

opening of the outer door unless the fumigation chamber has been operated 

through a fumigation cycle. 
Laboratory Biosafety Level Criteria – Biosafety Level 4 

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 facility. 

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 facility 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. 

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. 
Laboratory Biosafety Level Criteria – Biosafety Level 4 

Class III cabinet gloves must be inspected for leaks periodically and changed 

if necessary. Gloves should be replaced annually during cabinet recertification. 

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. Protective laboratory clothing with a solid-front such as tie-back or wraparound gowns, scrub suits, or coveralls must be worn by workers when in the 

laboratory. 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 before being laundered. 

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. 

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 laboratory waste.

Suit Laboratory 

1. All procedures must be conducted by personnel wearing a one-piece positive 

pressure suit ventilated with a life support system. 

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 devices that 

exhaust air through HEPA filtration before being discharged into the 

laboratory. These HEPA filters should be tested annually and replaced as 

needed.
Laboratory Biosafety Level Criteria – Biosafety Level 4 

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. Protective laboratory clothing such as scrub suits must be worn by workers 

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 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 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. 

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 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). 

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 door of the cabinet room(s) and 

the inner change room. A sink must be provided in the outer change room. All 
Laboratory Biosafety Level Criteria – Biosafety Level 4 

sinks in the room(s) containing the Class III BSC and the inner (dirty) change 

room 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. 

Drains in the laboratory floor (if present) must be connected directly to the 

liquid waste decontamination system. 

Services, plumbing or otherwise that penetrate the laboratory walls, floors, 

ceiling, plumbing or otherwise, must 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. 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. 

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. 
Laboratory Biosafety Level Criteria – Biosafety Level 4 

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 each individual 

laboratory system is isolated by gas tight dampers and HEPA filters.

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/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, 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. 

All HEPA filters should be located as near as practicable to the cabinet or 

laboratory in order to minimize the length of potentially contaminated 

ductwork. All HEPA filters must to 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. 

BSCs can also be connected to the laboratory exhaust system by either a 

thimble (canopy) connection or a direct (hard) connection. Provisions to 
Laboratory Biosafety Level Criteria – Biosafety Level 4 

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 primary wall. 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.

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 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. 
Laboratory Biosafety Level Criteria – Biosafety Level 4 

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 must be considered. 

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 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 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. 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 suit laboratory should be placed near procedure areas and 

contain traps 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 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. 
Laboratory Biosafety Level Criteria – Biosafety Level 4 

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, plumbing or otherwise that penetrate the laboratory walls, floors, 

ceiling, plumbing or otherwise, must 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. Sharp edges and corners should be avoided. 

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. 

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 inline 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 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 each individual 

laboratory system is isolated by gas tight dampers and HEPA filters.

The supply and exhaust components of the ventilation system must be 

designed to maintain the laboratory at negative pressure to surrounding areas 
Laboratory Biosafety Level Criteria – Biosafety Level 4 

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 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 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 

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. 

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. 
Laboratory Biosafety Level Criteria – Biosafety Level 4 

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 primary wall. 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. 

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 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 access/egress should be considered.
Laboratory Biosafety Level Criteria 

TABLE I 

SUMMARY OF RECOMMENDED BIOSAFETY LEVELS FOR INFECTIOUS AGENTS

BSL AGENTS PRACTICES PRIMARY BARRIERS AND 

SAFETY EQUIPMENT 

FAC

(SECONDA

1 Not known to consistently cause 

diseases in healthy adults 

Standard Microbiological Practices None required Laboratory ben

required 

2 • Agents associated with human 

disease 

• Routes of transmission include 

percutaneous injury, ingestion, mucous membrane exposure 

BSL-1 practice plus: • Limited access • Biohazard warning signs • “Sharps” precautions • Biosafety manual defining any 

needed waste decontamination or medical surveillance policies 

Primary barriers: • Class I or II BSCs or other 

physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials 

PPEs*: 

• Laboratory coats; gloves; 

face protection as needed 

BSL-1 plus: • Autoclave a

3 • Indigenous or exotic agents 

with potential for aerosol transmission 

• Disease may have serious or 

lethal consequences 

BSL-2 practice plus: • Controlled access • Decontamination of all waste • Decontamination of laboratory 

clothing before laundering 

• Baseline serum 

Primary barriers: • Class I or II BSCs or other 

physical containment devices used for all open manipulation of agents 

PPEs: • Protective laboratory 

clothing; gloves; respiratory protection as needed 

BSL-2 plus: • Physical sep

corridors 

• Self-closing

access 

• Exhaust air 

• Negative ai

4 • Dangerous/exotic agents which 

pose high risk of lifethreatening disease 

• Aerosol-transmitted laboratory 

infections have occurred; or related 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 personnel suit 

BSL-3 plus: • Separate bu

zone 

• Dedicated s

vacuum, ansystems 

• Other requi

 the text 

* PPE – Personal Protective Equipment 
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 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 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 applicable to work with animals infected with agents assigned to 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 1 

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 

(ABSL-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 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). 

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 emergency situations. 

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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 1 

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. 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. 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 child-bearing 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 – Animal Biosafety Level 1 

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 use should only be done in designated areas and are not 

permitted in animal or procedure rooms. 

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. Needles and syringes or other sharp instruments are limited to use in 

the animal facility when there is no alternative for such procedures as 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 1 

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. Plasticware 

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 – Animal Biosafety Level 1 

2. Special containment devices or equipment may be required as determined by 

appropriate risk assessment. 

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. 

3. 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 the NHP should assess risk of mucous membrane 

exposure and wear appropriate protective equipment (e.g., masks, goggles, 

faceshields, etc.) as needed. 

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. 

 Change gloves when contaminated, integrity has been 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 

prohibits 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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 1 

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. 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. 

It is recommended that penetrations in floors, walls and ceiling surfaces are 

sealed, to include openings around ducts, doors and door frames, to facilitate 

pest control and proper cleaning. 

Floors must be slip resistant, impervious to liquids, and resistant to chemicals. 

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 should 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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 1 

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.

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.

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) procedures involving the manipulation of infectious 

materials, or where aerosols or splashes may be created, should 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. 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 emergency situations. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 2 

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 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 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. 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. 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 child-bearing 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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 2 

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 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 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 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 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 use should only be done in designated areas and are 

not permitted in animal or procedure rooms. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 2 

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. Needles and syringes or other sharp instruments are limited to use in 

the animal facility when there is no alternative 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; it should be removed 

using a brush and dustpan, tongs, or forceps. Plasticware 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 containers for 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 2 

appropriate disposal in compliance with applicable institutional, local and 

state requirements. 

Decontaminate of 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 baseline serum sample should be stored. 

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. 

Consideration should be given to the use of restraint devices and practices that 

reduce the risk of exposure during animal manipulations (e.g., physical 

restraint devices, chemical restraint medications, etc). 

3. Decontamination is recommended for all potentially infectious materials and 

animal waste before movement outside the areas where infectious materials 

and/or animals are housed or are manipulated by an appropriate method (e.g. 

autoclave, chemical disinfection, or other approved decontamination 

methods). This includes potentially infectious animal tissues, carcasses, 

contaminated bedding, unused feed, sharps, and other refuse. 

Consideration should be given to means for decontaminating routine 

husbandry equipment, sensitive electronic and medical equipment. 

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 contain 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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 2 

4. Equipment, cages, and racks should be handled in 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. 

5. 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, 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 or splashes. 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 larger cages 

placed in inward flow ventilated enclosures 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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 2 

3. Eye and face protection (mask, goggles, face shield or other splatter guard) are 

used for anticipated splashes/ 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 the NHP should assess risk of mucous membrane 

exposure and wear appropriate protective equipment (e.g., masks, goggles, 

faceshields, etc.) as needed. 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. 

Gloves are changed when contaminated, integrity has been 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 

prohibits 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. 

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. Doors to cubicles inside an animal 

room may open outward or slide horizontally or vertically. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 2 

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 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. 

Penetrations in floors, walls and ceiling surfaces are sealed, to include 

openings around ducts, doors and door frames, to facilitate pest control and 

proper cleaning. 

Floors must be slip resistant, impervious to liquids, and resistant to chemicals. 

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 should 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 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 should 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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 2 

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. 

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 a direct (hard) connection. Provisions to 

assure proper safety cabinet performance and air system operation must be 

verified. Correct performance of the BSCs should be recertified at least once a 

year. 

All BSCs should be used according to manufacturer’s recommendation, to 

protect the worker and avoid creating a hazardous environment from volatile 

chemical 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 considered 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. 

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. 

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 emergency situations. 

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 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 should be given to specific biohazards unique to the animal 

species and protocol in use. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 3 

3. 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. 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 child-bearing 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. 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 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 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 3 

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/ and 

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 

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 use should only be done in designated areas and are 

not permitted in animal or procedure rooms. 

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. Needles and syringes or other sharp instruments are limited to use in 

the animal facility when there is no alternative for such procedures as 

parenteral injection, blood collection, or aspiration of fluids from 

laboratory animals and diaphragm bottles. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 3 

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. Plasticware 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 containers for 

appropriate disposal in compliance with applicable institutional, local and 

state requirements. 

Decontaminate of 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. 

Each institution should consider the need for collection and storage of serum 

samples from at-risk personnel. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 3 

2. All procedures involving the manipulation of infectious materials, handling 

infected animals or the generations 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. 

Consideration should be given to the use of restraint devices and practices that 

reduce the risk of exposure during animal manipulations (e.g., physical 

restraint devices, chemical restraint medications, etc). 

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 when operational malfunctions occur. 

5. A method for decontaminating all infectious materials must be available 

within the facility, preferably within the areas where infectious materials 

and/or animals are housed or are manipulated (e.g. autoclave, chemical 

disinfection, or other approved decontamination methods). 

Consideration should 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) 

before removal from the areas where infectious materials and/or animals are 

housed or are manipulated by an appropriate method. 

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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 3 

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.

6. Equipment, cages, and racks should be handled in 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 through the use of primary barrier systems. These systems may 

include solid 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. 

Protective clothing such as uniforms or scrub suits is worn by personnel 

within the animal facility. 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 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 3 

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. Appropriate eye, face and respiratory protection are worn by all personnel 

entering areas where infectious materials and/or animals are housed or are 

manipulated. 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, integrity has been 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 

prohibits 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) 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 3 

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. 

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. Doors to cubicles inside an animal 

room may open outward or slide horizontally or vertically. 

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, to include 

openings around ducts, doors and door frames, to facilitate pest control, 

proper cleaning and decontamination. Walls, floors and ceilings should form a 

sealed and sanitizable surface. 

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 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 3 

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 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, 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 to 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 should 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. 

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, or the exhaust must be HEPA-filtered. 

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 conditions the airflow 

will not be reversed. Audible 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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 3 

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 its 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 

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 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’ recommendations. 

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 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 designated alternate location/s within the facility. 

13. Vacuum lines must be protected with HEPA filters, or their equivalent. Filters 

must e replaced as needed. Liquid disinfectant traps may be required. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 3 

14. Emergency eyewash and shower are readily available; location is determined 

by risk assessment. 

15. 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. 

16. Additional environmental protection (e.g., personnel showers, HEPA filtration 

of exhaust air, containment of other piped services, and the provision or 

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 life-threatening disease, 

aerosol transmission, or 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. Access to the animal facility within the 

ABSL-4 laboratory is controlled by the animal facility director and/or laboratory 

supervisor in accordance with institutional policies. 

There are two models for ABSL-4 laboratories: 

(1) A Cabinet Laboratory where all handling of agents, infected animals and 

housing of infected animals must be performed in Class III BSCs (See Appendix A). 

(2) A Suit Laboratory where 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; 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 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

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 emergency situations 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 laboratoryacquired infections. Facility supervisors should ensure that medical staffs are 

informed of potential occupational hazards within the animal facility 

including those associated with 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. Protective 

clothing such as uniforms or scrub suits are worn by personnel within the 

animal facility. 

 All persons leaving the BSL-4/ABSL-4 laboratory are required to take a 

personal body shower. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

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. Needles and syringes or other sharp instruments are limited for use in 

the animal facility when there is no alternative 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 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. Plasticware 

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 can not be performed in a BSC, alternate 

containment equipment should be used. 

7. Decontaminate work surfaces after completion of work and after any spill or 

splash of potentially infectious material with appropriate disinfectant. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

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 should 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. 

Security sensitive agent information and occupational health requirements 

should be posted in accordance with the institutional policy. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

Advance consideration should 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 

child-bearing 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 should be 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. Laboratory clothing, including undergarments, pants, shirts, 

jumpsuits, shoes, and gloves, must be used by all personnel entering the 

laboratory. 

All persons leaving the ABSL-4 laboratory are required to 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 or disposal. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

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 child-bearing 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, 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, 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, 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

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 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 its established parameters. 

9. 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 ABSL-4 laboratory, and 

other potential emergencies. Training in emergency response procedures must 

be provided to emergency response personnel according to institutional 

policies. 

10. Based on site-specific risk assessment, personnel assigned to work with 

infected animals may be required to work in pairs. Procedures to reduce 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

possible worker exposure must be instituted, such as use of squeeze cages, 

working only with anesthetized animals, or other appropriate practices. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

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 recertification. 

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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

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 should be used where practicable (e.g., physical restraint 

devices, chemical restraint medications, mesh or Kevlar gloves, etc.). 

2. Protective laboratory clothing such as solid-front or wrap-around gowns, 

scrub suits, or coveralls must be worn by workers when in the laboratory. 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 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). 

All procedures must be conducted by personnel wearing a one-piece positive 

pressure suit ventilated with a life support system. 

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. 

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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

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. Protective laboratory clothing such as scrub suits must be worn by workers 

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 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). 

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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

All sinks in the room(s) containing the Class III BSC and the inner (dirty) 

change room 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 that penetrate the walls, floors or ceiling, plumbing or otherwise, 

must 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. 

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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

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 each individual 

laboratory system is isolated by gas tight dampers and HEPA filters. 

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, 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. 

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. 

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. 

Biological safety cabinets can also be connected to the laboratory exhaust 

system by either a thimble (canopy) connection or a direct (hard) connection. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

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. 

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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

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. 

Rooms in the facility must be arranged to ensure exit sequential passage 

through the chemical shower, inner (dirty) change room, personal shower, and 

outer (clean) changing area. 

Entry to this area must be through an airlock fitted with airtight doors. 

Personnel who enter this area 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 emergency 

backup system. 

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 and 

contain traps and be connected to the wastewater decontamination system. 

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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

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, plumbing or otherwise that penetrate the laboratory walls, floors, 

ceiling, plumbing or otherwise, must 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. 

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 each individual 

laboratory system is isolated by gas tight dampers and HEPA filters. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

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 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 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 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. 

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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

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. 

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. 
Vertebrate Animal Biosafety Level Criteria – Animal Biosafety Level 4 

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 C.F.R. Subchapter A, Parts 1, 2, 3 

(1976). 

3. National Research Council; Institute for Laboratory Animal Research. 

Occupational health and safety in the care of research animals. Washington, DC: 

National Academy Press; 1996. 

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 

TABLE 1 

SUMMARY OF RECOMMENDED BIOSAFETY LEVELS FOR ACTIVITIES IN WHICH EXPERIMENTALLY OR NAT

VERTBRATE ANIMALS ARE USED

* PPE – Personal Protective Equipment 

ABSL AGENTS PRACTICES PRIMARY BARRIERS AND 

SAFETY EQUIPMENT 

FA

(SECONDA

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 

Standard animal fa• No recirculatio

• Directional air 

 recommended 

• Hand washing 

2 • Associated with human disease 

• Hazard: percutaneous exposure, 

ingestion, mucous membrane exposure. 

ABSL-1 practice plus: • Limited access 

• Biohazard warning signs • “Sharps” precautions • Biosafety manual 

• Decontamination of all infectious 

wastes and of animal cages prior to washing 

ABSL-1 equipment plus primary barriers: • Containment equipment appropriate 

for animal species 

PPEs*: 

• Laboratory coats, gloves, face and 

respiratory protection as needed 

ABSL-1 plus: • Autoclave ava• Hand washing • Mechanical ca

3 • Indigenous or exotic agents with 

potential for aerosol transmission 

• Disease may have serious health effects 

ABSL-2 practice plus: • Controlled access 

• Decontamination of clothing before 

laundering 

• Cages decontaminated before bedding 

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 aerosols. 

PPEs: • Appropriate respiratory  protection 

ABSL-2 facility p• Physical separ

corridors 

• Self-closing, d• Sealed penetra• Sealed window• Autoclave ava

4 • Dangerous/exotic agents that pose high 

risk of life threatening disease 

• Aerosol transmission, or related 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 exiting 

• All 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, airsupplied positive-pressure personnel suit) used for all procedures and activities 

ABSL-3 facility p• Separate build• Dedicated sup

and decontami

• Other requirem the text 
Section VI 

Principles of Laboratory Biosecurity 

Since the publication of the 4th edition of BMBL manual 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 rereleased as a security and emergency response guidance for laboratories working with 

Select Agents.1

 A further updated and revised Appendix F is included in the 5th edition of 

BMBL and is focused exclusively on Select Agent Laboratories and includes current 

references for the USDA and CDC Select Agent Programs. 

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 BSL3 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.1,2,3 

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 microbiological laboratories do not have select agents 
Biosecurity 

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 occupationallyacquired 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 

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 
Biosecurity 

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. 

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 be limited to: senior management, scientific staff, human resource 

officials, information technology staff, and safety, security and engineering officials. The 
Biosecurity 

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; 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). 

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. 
Biosecurity 

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: 

o access to the agent within your laboratory; 

o how the undesired event could occur; 

o protective measures in place to prevent occurrence; 

o 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: 

o although a wide range of threats are possible, certain threats are more 

probable than others; 

o all agents/assets are not equally attractive to an adversary; 

o 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. 
Biosecurity 

• 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. 

• Management develops a biosecurity plan to describe how the institution will 

mitigate those unacceptable risks including: 

o a written security plan, standard operating procedures, and incident 

response plans; 

o 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: Reevaluate the Institution’s Risk Posture and Protection Objectives

• Management regularly reevaluates and makes necessary modifications to the: 

 

o biosecurity risk statement; 

o biosecurity risk assessment process; 

o the institution’s biosecurity program/plan; 

o 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. 
Biosecurity 

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. 

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 on the levels of access should consider all 

facets of the laboratory’s operations and programs (e.g., laboratory entrance 

requirements, freezer access, etc.). 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 
Biosecurity 

procedures associated with inventory maintenance (e.g., how material is identified, where 

it can be stored, used, etc.); 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 accountable individual who is knowledgeable about the materials in use and 

responsible for security of the materials under his or her control. 

 

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, etc.) 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, etc.) 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. 
Biosecurity 

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 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 “chain-ofnotification” 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, etc.). 

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 in an emergency situation. 

Security Updates and Reevaluations 

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 
Biosecurity 

and implement corrective actions as needed. Audit results and corrective actions should 

be documented. Records should be maintained by the appropriate program officials. 

Select Agents 

If an entity possesses, uses or transfer select agents, it must comply with all requirements 

of the National Select Agent Program. See Appendix F for 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 C.F.R. Part 73 (2005). 

5. Possession, use and transfer of biological agents and toxins, 7 C.F.R. Part 331 

(2005). 

6. Possession, use and transfer of biological agents and toxins, 9 C.F.R. Part 121 

(2005). 

7. Casadevall A, Pirofski L. The weapon potential of a microbe. Trends in 

Microbiology. Vol.12.No.6.2004. 
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 
Occupational Health and Immunoprophylaxis 

information should be reinforced 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 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 
Occupational Health and Immunoprophylaxis 

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. They are available on the CDC 

website (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 of 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 quantities and administered onsite 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 
Occupational Health and Immunoprophylaxis 

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. 

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. 
Occupational Health and Immunoprophylaxis 

• 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 postexposure 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 postexposure prophylaxis may have to be made quickly and in the absence of confirmatory 

laboratory testing. Post-exposure 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 postexposure 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 upon knowledge of 

similar agents, exposure circumstances, and advice received from knowledgeable experts. 

Appropriate post-exposure prophylactic response is always pathogen- and exposuredependent, may be host-factor dependent, and may also be influenced by immediate postexposure 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. 
Occupational Health and Immunoprophylaxis 

The applicable workers’ compensation claim form should be provided with appropriate 

explanations for its completion.19 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 postexposure 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. 

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 and Immunoprophylaxis 

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 [http://www.osha.gov]. 

Washington, DC: The Administration. Applicable OSHA Laws, Regulations and 

Interpretations; [about 2 screens]. Available from: http://www.osha.gov/complinks.html 

4. American Medical Association [http://www.ama-assn.org/]. Chicago: The 

Association; c1995-2006 [updated 2005 Mar 7]. Patient Confidentiality; [about 6 

screens]. Available from: http://www.ama-assn.org/ama/pub/category/4610.html 

5. United States Department of Justice [http://www.usdoj.gov/]. Washington, DC: 

The Department; [updated 2006 July 28]. American Disabilities Act; [about 7 

screens] Available from: 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 C.F.R. 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. 
Occupational Health and Immunoprophylaxis 

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. 

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 from: 

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 [about3 screens]. 

Available from: http://www.fda.gov/cber/ind/ind.htm 

19. Social Security Administration [www.ssa.gov]. Baltimore: The Administration; 

[updated on 2006 Jul 24]. Reports and Studies. Committee on Economic Security 

(CES); [about 22 screens]. Available from: 

http://www.ssa.gov/history/reports/ces/ces6kjaer.html 
SECTION VIII 

Agent Summary Statements 

• Section VIII-A: Bacterial Agents 

Agent: 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 of time 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 also is route-dependent. 

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 lethal dose50 (LD) 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 
Agent Summary Statements- Bacterial Agents 

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 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 

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. 

Containment Recommendations 

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. anthracisspore-contaminated areas after a terrorist attack; 5) work in other settings in which 
Agent Summary Statements- Bacterial Agents 

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. 

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. 

Agent: 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 

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. 
Agent Summary Statements- Bacterial Agents 

Containment Recommendations 

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 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 website 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. 

Agent: 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 
Agent Summary Statements- Bacterial Agents 

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. 

LABORATORY SAFETY 

Brucella infects the blood and a wide variety of body tissues, including cerebral spinal 

fluid, semen, pulmonary excretions, placenta, and occasionally urine. Most laboratoryassociated 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 

Containment Recommendations 

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. 

Agent: Burkholderia mallei 

Burkholderia mallei (formerly Pseudomonas mallei) is a non-motile gram-negative rod 

associated with glanders, a rare disease of equine species and humans. While endemic 
Agent Summary Statements- Bacterial Agents 

foci of infection exist in some areas of the world, glanders due to natural infection is 

extremely rare in the United States. 

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 MODES 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 

B. mallei can be very hazardous in a laboratory setting. In a pre-biosafety era report, onehalf 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 exposure37,38 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. 

Containment Recommendations 

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. 
Agent Summary Statements- Bacterial Agents 

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 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: Burkholderia pseudomallei 

Burkholderia pseudomallei (formerly Pseudomonas pseudomallei) is a motile gramnegative, 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 

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. 

Containment Recommendations 

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 
Agent Summary Statements- Bacterial Agents 

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 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. 

Agent: 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 

through the use of 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 
Agent Summary Statements- Bacterial Agents 

LABORATORY SAFETY 

Pathogenic C. 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. 

Containment Recommendations 

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.

Agent: 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 1955,4

 the majority 

of infections were psittacosis, and these had the highest case fatality rate of laboratoryacquired infectious agents. The major sources of laboratory-associated 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 
Agent Summary Statements- Bacterial Agents 

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 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 

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 

Containment Recommendations 

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 detergent-disinfectant 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 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. 
Agent Summary Statements- Bacterial Agents 

Agent: 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 food-borne 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 naturallyoccurring 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 

LABORATORY SAFETY 

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. 

Containment Recommendations 

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. 
Agent Summary Statements- Bacterial Agents 

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. 

Agent: 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, that 

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 

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 

Containment Recommendations 

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 diphtheria-tetanus 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. 

Agent: 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 wide-spread bacterial diseases in the prevaccine 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 grayish-white 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. 
Agent Summary Statements- Bacterial Agents 

LABORATORY SAFETY 

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 ACIP. 67 While the risk of laboratory-associated diphtheria is 

low, the administration of an adult diphtheria-tetanus toxoid at 10-year intervals may 

further reduce the risk of illness to laboratory and animal care personnel.67 

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: 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-to-person 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 1-14 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 
Agent Summary Statements- Bacterial Agents 

transmission modes in nature. Occasionally infections have occurred from bites or 

scratches by carnivores with contaminated mouth parts or claws. 

LABORATORY SAFETY 

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 

Containment Recommendations 

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 ABSL3 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. 

Agent: 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 that have been 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 
Agent Summary Statements- Bacterial Agents 

as well as proctitis, proctocolitis, enteritis and extraintestinal infections in humans.71,72 H. 

cinaedi has been isolated from dogs, cats and Syrian hamsters. 

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 

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. 

Containment Recommendations 

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. 

Agent: 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 
Agent Summary Statements- Bacterial Agents 

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 

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. Animal-to-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 person-to-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 

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. 

Containment Recommendations 

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. 
Agent Summary Statements- Bacterial Agents 

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. 

Agent: 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 

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 

Containment Recommendations 

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. 

Agent: 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 

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 personnel and other animals. 
Agent Summary Statements- Bacterial Agents 

While ingestion is the most common route of exposure, Listeria can also cause eye and 

skin infections following direct contact with the organism. 

Containment Recommendations 

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. 

Agent: 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 leprosylike 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-to-human 

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 ON INFECTION 

Leprosy is transmitted from person-to-person following prolonged exposure, presumably 

via contact with secretions from infected individuals. 

LABORATORY SAFETY 

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 inoculation are 
Agent Summary Statements- Bacterial Agents 

the primary laboratory hazards associated with handling infectious clinical materials. See 

Appendix B for appropriate tuberculocidal disinfectant. 

Containment Recommendations 

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. 

Agent: 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 MODE 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 
Agent Summary Statements- Bacterial Agents 

within months of infection or can remain latently infected and develop 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. Tuberculosis is 

spread by infectious aerosols produced by coughing. M. bovis is primarily found in 

animals but also can produce tuberculosis in humans. It is spread to humans, primarily 

children, by consumption of non-pasteurized milk and milk products, by handling of 

infected carcasses, and by inhalation. Human-to-human transmission via aerosols also is 

possible. 

LABORATORY SAFETY 

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. 

Containment Recommendations 

BSL-2 practices and procedures, containment equipment, and facilities are required for 

non-aerosol-producing manipulations of clinical specimens such as preparation of acidfast smears. All aerosol-generating activities must be conducted in a BSC. Use of a slidewarming 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 

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. 

Agent: 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. 
Agent Summary Statements- Bacterial Agents 

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 

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. 

Containment Recommendations 

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. 

Agent: 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 

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 

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. 

Containment Recommendations 

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. 
Agent Summary Statements- Bacterial Agents 

Agent: 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 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 personto-person and is usually mediated by direct contact with respiratory droplets from 

infected individuals. 

LABORATORY SAFETY 

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. 

Containment Recommendations 
Agent Summary Statements- Bacterial Agents 

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 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 

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.

Agent: Salmonella serotypes, other than S. Typhi 

Salmonellae are gram-negative enteric bacteria associated with diarrheal illness 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, 
Agent Summary Statements- Bacterial Agents 

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 foodborne 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, 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 

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. 

Containment Recommendations 

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. 
Agent Summary Statements- Bacterial Agents 

Agent: 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. 

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 

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. 

Containment Recommendations 

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 
Agent Summary Statements- Bacterial Agents 

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 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. 

Agent: 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
Agent Summary Statements- Bacterial Agents 

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 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 

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. 

Containment Recommendations 

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. 
Agent Summary Statements- Bacterial Agents 

Agent: 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

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 

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. 

Containment Recommendations 

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 
Agent Summary Statements- Bacterial Agents 

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 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: 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 ssp. pallidum (associated with venereal 

syphilis), T. pallidum ssp. endemicum (associated with endemic syphilis), and T. pallidum

ssp. 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 
Agent Summary Statements- Bacterial Agents 

America, the Caribbean, and Indonesia. Endemic syphilis is limited to arid areas of 

Africa and the Middle East.145 

LABORATORY SAFETY 

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. 

Containment Recommendations 

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 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. 

Agent: 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. 
Agent Summary Statements- Bacterial Agents 

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 non-achlorhydric 

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 

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, eye, ear, and gall 

bladder.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. 

Containment Recommendations 

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. 

Agent: 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. 
Agent Summary Statements- Bacterial Agents 

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 laboratory-acquired 

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. 

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 person-to-person transmission. 

LABORATORY SAFETY 

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. 

Containment Recommendations 

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 
Agent Summary Statements- Bacterial Agents 

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.158 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. 

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62. Centers for Disease Control and Prevention. Diphtheria, tetanus, and pertussis: 

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98. Walsh GP, Storrs EE, Burchfield HP, et al. Leprosy-like disease occurring 

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100. Meyers WM, Walsh GP, Brown HL, et al. Leprosy in a mangabey monkey--

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101. Marchoux PE. Un cas d’inoculation accidentelle du bacilli de Hanson en pays non 

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102. Parritt RJ, Olsen RE. Two simultaneous cases of leprosy developing in tattoos. 

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103. Grist NR, Emslie JA. Infections in British clinical laboratories, 1982-3. J Clin 

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104. Muller HE. Laboratory-acquired mycobacterial infection. Lancet. 1988;2:331. 

105. Pike RM, Sulkin SE, Schulze ML. Continuing importance of laboratory-acquired 

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106. Reid DD. Incidence of tuberculosis among workers in medical laboratories. Br 

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107. Kaufmann AF, Andersone DC. Tuberculosis control in nonhuman primates. In: 

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108. Allen BW. Survival of tubercle bacilli in heat-fixed sputum smears. J Clin Pathol. 

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109. Smithwick RW, Stratigos CB. Preparation of acid-fast microscopy smears for 

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110. Oliver J, Reusser TR. Rapid method for the concentration of tubercle bacilli. Am 

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112. Janda WM, Knapp JS. Neisseria and Moraxella catarrhalis. In: Murray PR, 

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113. Diena BB, Wallace R, Ashton FE, et al. Gonococcal conjunctivitis: accidental 

infection. Can Med Assoc J. 1976;115:609-12. 

114. Malhotra R, Karim QN, Acheson JF. Hospital-acquired adult gonococcal 

conjunctivitis. J Infect. 1998;37:305. 

115. Bruins SC, Tight RR. Laboratory acquired gonococcal conjunctivitis. JAMA. 

1979;241:274. 

116. Zajdowicz TR, Kerbs SB, Berg SW, et al. Laboratory-acquired gonococcal 

conjunctivitis: successful treatment with single-dose ceftriaxone. Sex Transm Dis. 

1984;11:28-9. 

117. Centers for Disease Control and Prevention [www.cdc.gov]. Atlanta: The Centers; 

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118. Centers for Disease Control and Prevention. Prevention and control of 

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119. Boutet R, Stuart JM, Kaczmarski EB, et al. Risk of laboratory-acquired 

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121. Centers for Disease Control and Prevention. Immunization of health-care 

workers: recommendations of the Advisory Committee on Immunization 

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122. Centers for Disease Control and Prevention. Laboratory-acquired 

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123. Bopp CA, Brenner FW, Fields PI, et al. Escherichia, Shigella, and Salmonella. In: 

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124. Grist NR, Emslie JA. Infections in British clinical laboratories, 1984-5. J Clin 

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125. Nicklas W. Introduction of salmonellae into a centralized laboratory animal 

facility by infected day-old chicks. Lab Anim. 1987;21:161-3. 

126. Steckelberg JM, Terrell CL, Edson RS. Laboratory-acquired Salmonella 

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127. Baumberg S, Freeman R. Salmonella typhimurium strain LT-2 is still pathogenic 

for man. J Gen Microbiol. 1971;65:99-100. 

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131. Sewell DL. Laboratory-associated infections and biosafety. Clin Microbiol Rev. 

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137. Rao GG, Saunders BP, Masterton RG. Laboratory-acquired verotoxin producing 

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• Section VIII-B: Fungal Agents 

Agent: 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 warmblooded 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 

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. 

Containment Recommendations 

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 
Agent Summary Statements- Fungal Agents 

sporulating mold-form cultures already identified as B. dermatitidis and soil or other 

environmental samples known or likely to contain infectious conidia. 

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. 

Agent: 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%) laboratoryassociated 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 
Agent Summary Statements- Fungal Agents 

infection is much more likely in persons with cellular immunodeficiencies (AIDS, organ 

transplant recipient, lymphoma). 

LABORATORY SAFETY 

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. 

Containment Recommendations 

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. 

Agent: 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 hyphal formation with 

basidiospores. Both basidiospores and asexual yeasts are infectious. 
Agent Summary Statements- Fungal Agents 

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 

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 

Containment Recommendations 

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. 

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. 

Agent: 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 
Agent Summary Statements- Fungal Agents 

(conidia); these are the infectious particles that convert to small budding yeasts under the 

appropriate culture conditions in vitro at 37°C 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 

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. 

Containment Recommendations 

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 

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. 

Agent: 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 

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 
Agent Summary Statements- Fungal Agents 

Containment Recommendations 

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 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. 

Agent: 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 commonest 

infectious agents of humans. This fungal group encompasses members of three genera: 

Epidermophyton, Microsporum, and Trichophyton. The severity of infection depends on 
Agent Summary Statements- Fungal Agents 

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. 

LABORATORY SAFETY 

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). 

Containment Recommendations 

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. 

Agent: 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 laboratory-acquired disseminated infection 
Agent Summary Statements- Fungal Agents 

following presumed inhalation when an undiagnosed HIV-positive individual visited a 

laboratory where students were handling cultures on the open bench.53 

NATURAL MODES OF INFECTION 

The natural mode of infection varies by specific species; most are poorly characterized. 

LABORATORY SAFETY 

Inhalation of conidia from sporulating mold cultures or accidental injection into the skin 

during infection or experimental animals is a potential risk to laboratory personnel. 

Containment Recommendations 

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. 

REFERENCES 

1. DiSalvo AF. The epidemiology of blastomycosis. In: Al-Doory Y, Di Salvo AF, 

editors. Blastomycosis. New York: Plenum Medical Book Company; 1992. p. 75-

104. 

2. Evans N. A clinical report of a case of blastomycosis of the skin from accidental 

inoculation. JAMA. 1903;40:1172-5. 

3. Harrekkm ER. The known and the unknown of the occupational mycoses. In: 

Occupational diseases acquired from animals. Continued education series no. 124. 

Ann Arbor: University of Michigan School of Public Health; 1964. p. 176-8. 

4. Larsh HW, Schwarz J. Accidental inoculation blastomycosis. Cutis. 1977;19:334-

6. 

5. Larson DM, Eckman MR, Alber RL, et al. Primary cutaneous (inoculation) 

blastomycosis: an occupational hazard to pathologists. Amer J Clin Pathol. 

1983;79:253-5. 

6. Wilson JW, Cawley EP, Weidman FD, et al. Primary cutaneous North American 

blastomycosis. AMA Arch Dermatol. 1955;71:39-45. 
Agent Summary Statements- Fungal Agents 

7. Graham WR, Callaway JL. Primary inoculation blastomycosis in a veterinarian. J 

Am Acad Dermatol.1982;7:785-6. 

8. Schwarz J, Kauffman CA. Occupational hazards from deep mycoses. Arch 

Dermatol. 1977;113:1270-5. 

9. Baum GL, Lerner PI. Primary pulmonary blastomycosis: a laboratory acquired 

infection. Ann Intern Med. 1970;73:263-5. 

10. Denton JF, Di Salvo AF, Hirsch ML. Laboratory-acquired North American 

blastomycosis. JAMA. 1967;199:935-6. 

11. Sugar AM, Lyman CA. A practical guide to medically important fungi and the 

diseases they cause. Philadelphia: Lippincott-Raven; 1997. 

12. Fisher MC, Koenig GL, White TJ, et al. Molecular and phenotypic description of 

Coccidioides posadasii sp nov, previously recognized as the non-California 

population of Coccidioides immitis. Mycologia. 2002;94:73-84. 

13. Pappagianis D. Coccidioidomycosis (San Joaquin or Valley Fever). In: DiSalvo 

A, editor. Occupational mycoses. Philadelphia: Lea and Febiger; 1983. p. 13-28. 

14. Nabarro JDN. Primary pulmonary coccidioidomycosis: case of laboratory 

infection in England. Lancet. 1948;1:982-4. 

15. Smith CE. The hazard of acquiring mycotic infections in the laboratory. Presented 

at 78th Annual Meeting American Public Health Association; 1950; St. Louis, 

MO; 1950. 

16. Werner SB, Pappagianis D, Heindl I, et al. An epidemic of coccidioidomycosis 

among archeology students in northern California. N Engl J Med. 1972;286:507-

12. 

17. Crum N, Lamb C, Utz G, et al. Coccidioidomycosis outbreak among United 

States Navy SEALs training in a Coccidioides immitis-endemic area-Coalinga, 

California. J Infect Dis. 2002;186:865-8. 

18. Wilson JW, Smith CE, Plunkett OA. Primary cutaneous coccidioidomycosis: the 

criteria for diagnosis and a report of a case. Calif Med. 1953;79:233-9. 

19. Tomlinson CC, Bancroft P. Granuloma Coccidioides: report of a case responding 

favorably to antimony and potassium tartrate. JAMA. 1928; 91:947-51. 

20. Halde C. Percutaneous Cryptococcus neoformans inoculation without infection. 

Arch Dermatol. 1964; 89:545. 

21. Casadevall A, Mukherjee J, Yuan R, et al. Management of injuries caused by 

Cryptococcus neoformans-contaminated needles. Clin Infect Dis. 1994;19:951-3. 

22. Nosanchuk JD, Mednick A, Shi L, et al. Experimental murine cryptococcal 

infection results in contamination of bedding with Cryptococcus neoformans. 

Contemp Top Lab Anim Sci. 2003;42;9-412. 

23. Bohne T, Sander A, Pfister-Wartha A, et al. Primary cutaneous cryptococcosis 

following trauma of the right forearm. Mycoses. 1996;39:457-9. 

24. Furcolow ML. Airborne histoplasmosis. Bacteriol Rev. 1961;25:301-9. 

25. Pike RM. Past and present hazards of working with infectious agents. Arch Path 

Lab Med. 1978;102:333-6. 

26. Pike RM. Laboratory-associated infections: Summary and analysis of 3,921 cases. 

Hlth Lab Sci. 1976;13:105-14. 

27. Schwarz J, Kauffman CA. Occupational hazards from deep mycoses. Arch 

Dermatol. 1977;113:1270-5. 
Agent Summary Statements- Fungal Agents 

28. Murray JF, Howard D. Laboratory-acquired histoplasmosis. Am Rev Respir Dis. 

1964;89:631-40. 

29. Sewell DL. Laboratory-associated infections and biosafety. Clin Micro Rev. 

1995;8:389-405. 

30. Tosh FE, Balhuizen J, Yates JL, et al. Primary cutaneous histoplasmosis: report of 

a case. Arch Intern Med. 1964;114:118-9. 

31. Tesh RB, Schneidau JD. Primary cutaneous histoplasmosis. N Engl J Med. 

1966;275:597-9. 

32. Spicknall CG, Ryan RW, Cain A. Laboratory-acquired histoplasmosis. N Eng J 

Med. 1956;254:210-4. 

33. Vanselow NA, Davey WN, Bocobo FC. Acute pulmonary histoplasmosis in 

laboratory workers: report of two cases. J Lab Clin Med. 1962;59:236-43. 

34. Ishizaki H, Ikeda M, Kurata Y. Lymphocutaneous sporotrichosis caused by 

accidental inoculation. J Dermatol. 1979;6:321-3. 

35. Fava A. Un cas de sporotrichose conjonctivale et palpebrale primitives. Ann Ocul 

(Paris). 1909;141:338-43. 

36. Wilder WH, McCullough CP. Sporotrichosis of the eye. JAMA. 1914;62:1156-

60. 

37. Carougeau, M. Premier cas Africain de sporotrichose de deBeurmann: 

transmission de la sporotrichose du mulet a l'homme. Bull Mem Soc Med Hop 

(Paris). 1909;28:507-10. 

38. Thompson DW, Kaplan W. Laboratory-acquired sporotrichosis. Sabouraudia. 

1977;15:167-70. 

39. Jeanselme E, Chevallier P. Chancres sporotrichosiques des doigts produits par la 

morsure d'un rat inocule de sporotrichose. Bull Mem Soc Med Hop (Paris). 

1910;30:176-78. 

40. Jeanselme E, Chevallier P. Transmission de la sporotrichose a l'homme par les 

morsures d'un rat blanc inocule avec une nouvelle variete de Sporotrichum: 

Lymphangite gommeuse ascendante. Bull Mem Soc Med Hop (Paris). 

1911;31:287-301. 

41. Meyer KF. The relationship of animal to human sporotrichosis: studies on 

American sporotrichosis III. JAMA. 1915;65:579-85. 

42. Norden A. Sporotrichosis: clinical and laboratory features and a serologic study in 

experimental animals and humans. Acta Pathol Microbiol Scand. 1951;89:3-119. 

43. Cooper CR, Dixon DM, Salkin IF. Laboratory-acquired sporotrichosis. J Med Vet 

Mycol. 1992;30:169-71. 

44. Fielitz H. Ueber eine Laboratoriumsinfektion mit dem Sporotrichum de 

Beurmanni. Centralbl Bakteriol Parasitenk Abt I Orig. 1910;55:361-70. 

45. Sugar AM, Lyman CA. A practical guide to medically important fungi and the 

diseases they cause. Philadelphia: Lippincott-Raven; 1997. p. 86-7. 

46. Voss A, Nulens E. Prevention and control of laboratory-acquired infections. 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 

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. 
• 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 laboratory-acquired 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. 

Agent: 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. 

Occupational Infections 
Agent Summary Statements- Parasitic Agents 

Laboratory-acquired infections with Leishmania spp., Plasmodium spp., Toxoplasma 

gondii, and Trypanosoma spp. have been reported; the majority of these involved needlestick 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 principle vectors. 

Although no laboratory infections with Babesia have been 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. 
Agent Summary Statements- Parasitic Agents 

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 and they 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 

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 Leishmania, Toxoplasma gondii, or T. 

cruzi, or with tissue homogenates or blood containing hemoflagellates. 

Immunocompromised 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, 
Agent Summary Statements- Parasitic Agents 

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 needle-sticks, or through exposure to mucous 

membranes or microabrasions of the skin should be considered. 

Containment Recommendations 

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. 

Agent: 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. 
Agent Summary Statements- Parasitic Agents 

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 

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 
Agent Summary Statements- Parasitic Agents 

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. 

Containment Recommendations 

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. 

Affected surfaces should be flooded (i.e., completely covered) with hydrogen peroxide. 

If surfaces are contaminated by a large volume of liquid, 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 50oC; thereafter, wash them 

in a detergent/disinfectant solution. 
Agent Summary Statements- Parasitic Agents 

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. 

Agent: 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 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 sheep-raising 

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 
Agent Summary Statements- Parasitic Agents 

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 

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). 

Containment Recommendations 

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 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. 
Agent Summary Statements- Parasitic Agents 

Agent: 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 principle 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, 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 

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 
Agent Summary Statements- Parasitic Agents 

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. Laboratoryacquired 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. 

Containment Recommendations 

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. 

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. 

Agent: 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 
Agent Summary Statements- Parasitic Agents 

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 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 in 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. 
Agent Summary Statements- Parasitic Agents 

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 

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 immunosuppressed 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 were 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. 

Containment Recommendations 

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. 
Agent Summary Statements- Parasitic Agents 

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. 
• Section VIII-D: Rickettsial Agents 

Agent: 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 for laboratory and animal care 

personnel. Exposure to naturally infected, often asymptomatic, 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 

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 and in the blood, urine, feces, milk, and 
Agent Summary Statements- Rickettsial Agents 

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 

Containment Recommendations 

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 7). 

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. 

Agents: 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 
Agent Summary Statements- Rickettsial Agents 

conorii, Rickettsia akari, Rickettsia australis, Rickettsia siberica, and 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 as a result 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 

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 
Agent Summary Statements- Rickettsial Agents 

ectoparasites, and their infected tissues are potential sources of human infection. The 

organisms are relatively unstable under ambient environmental conditions. 

Containment Recommendations 

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. 
Agent Summary Statements- Rickettsial Agents 

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. 

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. 
Agent Summary Statements- Rickettsial Agents 

19. Saslaw S, Carlisle HN. Aerosol infection of monkeys with Rickettsia rickettsii. 

Bacteriol Rev. 1966;30:636-45. 
• Section VIII-E: Viral Agents 

Agent: 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.). 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 

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. 

Containment Recommendations 

BSL-2 practices, containment equipment, and facilities are recommended for laboratory 

handling of sera from persons potentially infected with hantaviruses. The use of a 
Agent Summary Statements- Viral Agents 

certified BSC is recommended for all handling of human body fluids when potential 

exists for splatter or aerosol. 

Potentially infected tissue samples should be handled in BSL-2 facilities following BSL3 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. 

Agent: 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 as a result 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 of the genus, 
Agent Summary Statements- Viral Agents 

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 

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 

Containment Recommendations 

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. 

Agent: 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 singlestranded RNA virus, the type species of the genus Hepevirus, a floating genus not 

assigned to any family. 
Agent Summary Statements- Viral Agents 

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 enterically–transmitted cases of hepatitis, 

mostly waterborne. In Asia, epidemics involving thousands of cases have occurred. 

LABORATORY SAFETY 

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. 

Containment Recommendations 

 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 experimentallyinfected nonhuman primates or other animal models that may shed the virus. 

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. 
Agent Summary Statements- Viral Agents 

Agent: 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 ear piercing with inadequately sterilized instruments. Non-parenteral routes 

are also important, and cases may result from domestic and sexual contact, especially 

homosexual practices. 

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 INFECTION 

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 

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. 

Containment Recommendations 

BSL-2 practices, containment equipment, and facilities are recommended for all activities 

utilizing known or potentially infectious body fluids and tissues. Additional primary 
Agent Summary Statements- Viral Agents 

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 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. 

Agent: 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 in situations 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, since 

at this time they were unavailable. 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 
Agent Summary Statements- Viral Agents 

in the eye while transporting a caged macaque. In part on this basis, the eye splash was 

considered low risk. However, 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 

 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. 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 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 
Agent Summary Statements- Viral Agents 

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, etc. 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. 

Containment Recommendations 

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 of 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. 

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 
Agent Summary Statements- Viral Agents 

case management. Barrier precautions should be observed with confirmed cases. B virus 

infection, as with all alphaherpesviruses, is lifelong in macaques40 and there are no 

effective vaccines available at present. 

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. 

Agent: 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), Epstein-Barr 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 the pathogenesis of several lymphomas and nasopharyngeal cancer.42 EBV is 

serologically distinct from the other herpesviruses; it infects and transforms Blymphocytes. 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. 

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 
Agent Summary Statements- Viral Agents 

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 cycles (HHV-6).41 Transmission of 

human herpesviruses in nature are, in general, associated with contact 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 

Clinical materials and isolates of herpesviruses may pose a risk of infection following 

ingestion, accidental parenteral inoculation, and droplet exposure of the mucous 
Agent Summary Statements- Viral Agents 

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. 

Containment Recommendations 

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 separately in another agent summary statement in this section. 

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. 

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: 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
Agent Summary Statements- Viral Agents 

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. 

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 

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 
Agent Summary Statements- Viral Agents 

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. 

Containment Recommendations 

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, HEPA-filtered 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. 

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 at the present time, 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. 
Agent Summary Statements- Viral Agents 

 

• 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 airpurifying 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. 

 

• Clear evidence of reduced virus replication in the respiratory tract of 

appropriate animal models, compared with the level of replication of the wildtype 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. 
Agent Summary Statements- Viral Agents 

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: 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. 
Agent Summary Statements- Viral Agents 

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 

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, subsequent infection of the host and 

virus excretion may ensue. Pregnant women infected with LCMV have transmitted the 

virus to their fetus with death or serious central nervous system malformation as a 

consequence.65 

Containment Recommendations 

BSL-2 practices, containment equipment, and facilities are suitable for activities utilizing 

known or potentially infectious body fluids, and for cell culture passage of laboratoryadapted 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 nonhuman primates should be handled at BSL-3. ABSL-2 practices, containment equipment, 
Agent Summary Statements- Viral Agents 

and facilities are suitable for studies in adult mice with 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. 

Agent: 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 

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 inapparent 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. 
Agent Summary Statements- Viral Agents 

LABORATORY SAFETY 

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 non-immunized 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. 

Containment Recommendations 

 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. 

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. 
Agent Summary Statements- Viral Agents 

Agent: 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 Laboratoryacquired 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 reasons, including chronic 

illness, AIDS, other infections, medications, cancer and cancer therapies, or pregnancy.70

LABORATORY SAFETY 

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. 
Agent Summary Statements- Viral Agents 

Containment Recommendations 

Worldwide, all live variola virus work is to be done only within WHO approved BSL4/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 cow pox 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

Vaccination is not required for individuals working only in laboratories where no other 

orthopoxviruses or recombinants are handled.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 other work with monkeypox virus is performed by 

vaccinated personnel. 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. BSL-2 and ABSL-2 plus vaccination, are 

recommended for work with most other poxviruses. 

SPECIAL ISSUES 

Other Considerations The CDC internet site www.cdc.gov provides information on 

poxviruses, especially variola and monkeypox viruses, and on smallpox vaccination, and 

for 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 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. 
Agent Summary Statements- Viral Agents 

Agent: 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 lyssavirus 1, European bat 

lyssavirus 2, Lagos bat virus, and Mokola virus. 

OCCUPATIONAL INFECTIONS 

 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 

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. 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, are the most 

likely sources for exposure of laboratory and animal care personnel. Infectious aerosols 

have not been a demonstrated hazard to personnel working with routine clinical materials 

and 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. 

Containment Recommendations 

BSL-2 and/or ABSL-2 practices, containment equipment, and facilities are recommended 

for all activities utilizing known or potentially infectious materials or animals. Preexposure 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. 
Agent Summary Statements- Viral Agents 

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. 

Agent: 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. And the Spumaretrovirinae with one genus 

Spumavirus containing a variety of NHP viruses (foamy viruses) which can occasionally 

infect humans in close contact with NHP. 

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) HIVinfected 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 
Agent Summary Statements- Viral Agents 

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 blood-contaminated needle after bleeding an SIVinfected 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 

HIV has been isolated from blood, semen, saliva, tears, urine, CSF, amniotic fluid, breast 

milk, cervical secretion, and tissue 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 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. Whether 

infection can occur via the respiratory tract is unknown. The need for using sharps in the 
Agent Summary Statements- Viral Agents 

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 

Containment Recommendations 

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 researchlaboratory-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 7). 

The risk associated with retroviral vector systems can vary significantly; especially 

lentiviral vectors. Because each gene transfer system can vary significantly, no specific 

guideline can be offered other than to have all gene transfer protocols reviewed by an 

IBC. 

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: 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 
Agent Summary Statements- Viral Agents 

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 high-frequency 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, to date, 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. 

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 at this time. 

LABORATORY SAFETY 

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 

Containment Recommendations 

n 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 
Agent Summary Statements- Viral Agents 

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 that may 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. 

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). 
Agent Summary Statements- Viral Agents 

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.; 

• 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. 

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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• 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 3, 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 1 provides a key 

for the SALS basis for assignment of viruses listed in Table 3. 

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; 

• 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). 

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 

2). The agents in these Tables may require permits from USDA/DOC/DHHS. 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

Table 1 

EXPLANATION OF SYMBOLS USED IN TABLE 3 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. 

Table 2 

VACCINE STRAINS OF BSL-3 AND -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 

Yellow fever 17-D 

Japanese encephalitis 14-14-2 

Based on the recommendations listed within tables, the following guidelines should be adhered to where 

applicable. 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

VIRUSES WITH BSL-2 CONTAINMENT RECOMMENDED 

The recommendation for conducting work with the viruses listed in Table 3 at BSL-2 was 

based on the existence of adequate historical laboratory experience 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 Hazards 

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 procedure with a potential for 

creating infectious aerosols or splashes are conducted. 

Containment Recommendations 

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. 

VIRUSES WITH BSL-3 CONTAINMENT RECOMMENDED 

The recommendations for viruses listed in Table 3 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. 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

Laboratory Hazards 

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. 

Containment Recommendations 

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). 

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 onsite at the Special Immunization Program, USAMRIID. 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 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

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, located at Ft. Detrick, Frederick MD. As 

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. An example would be 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 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; 3) personal body shower. Additional appropriate training for all animal care 

personnel should be considered. 

VIRUSES WITH BSL-4 CONTAINMENT RECOMMENDATIONS 

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 their retention at this level or 

movement to work at a lower level. 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

Laboratory Hazards 

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 NHP. 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 

Containment Recommendations 

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 widely-distributed surveys evaluated 

numerous criteria for each particular virus including 1) past occurrence of laboratoryacquired infections correlated with facilities and practices used; 2) volume of work 

performed as a measure of 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. 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

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 (See 

Table 3). 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 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 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

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 rigorous containment 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 Recombinant DNA Advisory 

Committee may be necessary. 

Agent: 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, 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 INFECTION 

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. 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

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 

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. 

Containment Recommendations 

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. 

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. 

Agent: 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
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

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. 

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 U.S. 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 

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. 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

Containment Recommendations 

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 nonimmune 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 maybe 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. 

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. 

Agent: 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 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

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, other tissues including aborted fetal 

tissues of sick animals. 

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 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

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 

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.

Containment Recommendations 

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, for research conducted in non-endemic areas, the 

USDA may require full BSL-3Ag containment (See Appendix D). 

SPECIAL ISSUES 

Vaccines Two apparently effective vaccines have been developed by 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 or ABSL-3 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. 

REFERENCES 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

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. 

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 Thelper 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. 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

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. 

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. 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

TABLE 3 

ALPHABETIC LISTING OF 597 ARBOVIRUSES AND HEMORRHAGIC FEVER VIRUSES* 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Abras ABRV Orthobunyavirus 2 A7 Patois Absettarov ABSV Flavivirus 4 A4 Bf Abu Hammad AHV Nairovirus 2 S Dera Ghazi Khan Acado ACDV Orbivirus 2 S Corriparta Acara ACAV Orthobunyavirus 2 S Capim Adelaide River ARV Lyssavirus 2 IE 

Bovine Ephemeral Fever 

African Horsesickness AHSV Orbivirus 3c A1 

African Horsesickness 

African Swine Fever ASFV Asfivirus 3c IE Asfivirus Aguacate AGUV Phlebovirus 2 S Phlebotomus Fever Aino AINOV Orthobunyavirus 2 S Simbu Akabane AKAV Orthobunyavirus 3c S Simbu Alenquer ALEV Phlebovirus 2 IE Phlebotomus Fever Alfuy ALFV Flavivirus 2 S Bf Alkhumra ALKV Flavivirus 4 A4 Bf Allpahuayo ALLPV Arenavirus 3 IE Tacaribe Almeirim ALMV Orbivirus 2 IE Changuinola Almpiwar ALMV Rhabdoviridae 2 S Altamira ALTV Orbivirus 2 IE Changuinola Amapari AMAV Arenavirus 2 A5 Tacaribe Ambe AMBEV Phlebovirus 2 IE Ananindeua ANUV Orthobunyavirus 2 A7 Guama 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Andasibe ANDV Orbivirus 2 A7 Andes ANDV Hantavirus 3a IE Hantaan Anhanga ANHV Phlebovirus 2 S Phlebotomus Fever Anhembi AMBV Orthobunyavirus 2 S Bunyamwera Anopheles A ANAV Orthobunyavirus 2 S Anopheles A Anopheles B ANBV Orthobunyavirus 2 S Anopheles B Antequera ANTV Bunyaviridae 2 IE Resistencia Apeu APEUV Orthobunyavirus 2 S Cf Apoi APOIV Flavivirus 2 S Bf Araguari ARAV Unassigned 3 IE Aransas Bay ABV Bunyaviridae 2 IE UPOLU Arbia ARBV Phlebovirus 2 IE Phlebotomus Fever Arboledas ADSV Phlebovirus 2 A7 Phlebotomus Fever Aride ARIV Unassigned 2 S Ariquemes ARQV Phlebovirus 2 A7 Phlebotomus Fever Arkonam ARKV Orbivirus 2 S Ieri Armero ARMV Phlebovirus 2 A7 Phlebotomus Fever Aroa AROAV Flavivirus 2 S Bf Aruac ARUV Rhabdoviridae 2 S Arumateua ARMTV Orthobunyavirus 2 A7 Arumowot AMTV Phlebovirus 2 S Phlebotomus Fever Aura AURAV Alphavirus 2 S Af Avalon AVAV Nairovirus 2 S Sakhalin Babahoyo BABV Orthobunyavirus 2 A7 Patois Babanki BBKV Alphavirus 2 A7 Af Bagaza BAGV Flavivirus 2 S Bf Bahig BAHV Orthobunyavirus 2 S Tete 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Bakau BAKV Orthobunyavirus 2 S Bakau Baku BAKUV Orbivirus 2 S Kemerovo Bandia BDAV Nairovirus 2 S Qalyub Bangoran BGNV Rhabdoviridae 2 S Bangui BGIV Bunyaviridae 2 S Banzi BANV Flavivirus 2 S Bf Barmah Forest BFV Alphavirus 2 A7 Af Barranqueras BQSV Bunyaviridae 2 IE Resistencia Barur BARV Rhabdoviridae 2 S Kern Canyon Batai BATV Orthobunyavirus 2 S Bunyamwera Batama BMAV Orthobunyavirus 2 A7 Tete Batken BKNV Thogotovirus 2 IE Bauline BAUV Orbivirus 2 S Kemerovo Bear Canyon BRCV Arenavirus 3 A7 Bebaru BEBV Alphavirus 2 S Af Belem BLMV Bunyaviridae 2 IE Belmont BELV Bunyaviridae 2 S Belterra BELTV Phlebovirus 2 A7 Phlebotomus Fever Benevides BENV Orthobunyavirus 2 A7 Capim Benfica BENV Orthobunyavirus 2 A7 Capim Bermejo BMJV Hantavirus 3 IE Hantaan Berrimah BRMV Lyssavirus 2 IE 

Bovine Ephemeral Fever 

Beritoga BERV Orthobunyavirus 2 S Guama Bhanja BHAV Bunyaviridae 3 S Bhanja Bimbo BBOV Rhabdoviridae 2 IE Bimitti BIMV Orthobunyavirus 2 S Guama 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Birao BIRV Orthobunyavirus 2 S Bunyamwera Bluetoungue (exotic serotypes) BTV Orbivirus 3c S Bluetongue Bluetoungue (non-exotic) BTV Orbivirus 2c S Bluetongue Bobaya BOBV Bunyaviridae 2 IE Bobia BIAV Orthobunyavirus 2 IE Olifantsylei Boraceia BORV Orthobunyavirus 2 S Anopheles B Botambi BOTV Orthobunyavirus 2 S Olifantsylei Boteke BTKV Vesiculovirus 2 S Vesicular Stomatitis Bouboui BOUV Flavivirus 2 S Bf Bovine Ephemeral Fever BEFV Lyssavirus 3c A1 

Bovine Ephemeral Fever 

Bozo BOZOV Orthobunyavirus 2 A7 Bunyamwera Breu Branco BRBV Orbivirus 2 A7 Buenaventura BUEV Phlebovirus 2 IE Phlebotomus Fever Bujaru BUJV Phlebovirus 2 S Phlebotomus Fever Bunyamwera BUNV Orthobunyavirus 2 S Bunyamwera Bunyip Creek BCV Orbivirus 2 S Palyam Burg El Arab BEAV Rhabdoviridae 2 S Matariva Bushbush BSBV Orthobunyavirus 2 S Capim Bussuquara BSQV Flavivirus 2 S Bf Buttonwillow BUTV Orthobunyavirus 2 S Simbu Bwamba BWAV Orthobunyavirus 2 S Bwamba Cabassou CABV Alphavirus 3 IE Af Cacao CACV Phlebovirus 2 S Phlebotomus Fever Cache Valley CVV Orthobunyavirus 2 S Bunyamwera Cacipacore CPCV Flavivirus 2 IE Bf Caimito CAIV Phlebovirus 2 S Phlebotomus Fever 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Calchaqui CQIV Vesiculovirus 2 A7 Vesicular Stomatitis California Encephalitis CEV Orthobunyavirus 2 S California Calovo CVOV Orthobunyavirus 2 S Bunyamwera Cananeia CNAV Orthobunyavirus 2 IE GUAMA Candiru CDUV Phlebovirus 2 S Phlebotomus Fever Caninde CANV Orbivirus 2 IE Changuinola Cano Delgadito CADV Hantavirus 3a IE Hantaan Cape Wrath CWV Orbivirus 2 S Kemerovo Capim CAPV Orthobunyavirus 2 S Capim Caraipe CRPV Orthobunyavirus 2 A7 Carajas CRJV Vesiculovirus 2 A7 Vesicular Stomatitis Caraparu CARV Orthobunyavirus 2 S Cf Carey Island CIV Flavivirus 2 S Bf Catu CATUV Orthobunyavirus 2 S Guama Chaco CHOV Rhabdoviridae 2 S Timbo Chagres CHGV Phlebovirus 2 S Phlebotomus Fever Chandipura CHPV Vesiculovirus 2 S Vesicular Stomatitis Changuinola CGLV Orbivirus 2 S Changuinola Charleville CHVV Lyssavirus 2 S Rab Chenuda CNUV Orbivirus 2 S Kemerovo Chikungunya CHIKV Alphavirus 3 S Af Chilibre CHIV Phlebovirus 2 S Phlebotomus Fever Chim CHIMV Bunyaviridae 2 IE Chobar Gorge CGV Orbivirus 2 S Chobar Gorge Clo Mor CMV Nairovirus 2 S Sakhalin Coastal Plains CPV Lyssavirus 2 IE Tibrogargan Cocal COCV Vesiculovirus 2 A3 Vesicular Stomatitis 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Codajas CDJV Orbivirus 2 A7 Colorado Tick Fever CTFV Coltivirus 2 S Colorado Tick Fever Congo-Crimean Hemorrhagic Fever CCHFV Nairovirus 4 A6 CCHF Connecticut CNTV Rhabdoviridae 2 IE Sawgrass Corfou CFUV Phlebovirus 2 A7 Phlebotomus Fever Corriparta CORV Orbivirus 2 S Corriaparta Cotia CPV Poxviridae 2 S Cowbone Ridge CRV Flavivirus 2 S Bf Csiro Village CVGV Orbivirus 2 S Palyam Cuiaba CUIV Rhabdoviridae 2 S Curionopolis CRNPV Rhabdoviridae 2 A7 Dabakala DABV Orthobunyavirus 2 A7 Olifantsylei D’Aguilar DAGV Orbivirus 2 S Palyam Dakar Bat Virus DBV Flavivirus 2 S Bf Deer Tick Virus DRTV Flavivirus 3 A7 Dengue Virus Type 1 DENV-1 Flavivirus 2 S Bf Dengue Virus Type 2 DENV-2 Flavivirus 2 S Bf Dengue Virus Type 3 DENV-3 Flavivirus 2 S Bf Dengue Virus Type 4 DENV-4 Flavivirus 2 S Bf Dera Ghazi Khan DGKV Nairovirus 2 S Dera Ghazi Khan Dobrava-Belgrade DOBV Hantavirus 3a IE Dhori DHOV Orthomyxoviridae 2 S Douglas DOUV Orthobunyavirus 3 IE Simbu Durania DURV Phlebovirus 2 A7 Phlebotomus Fever Dugbe DUGV Nairovirus 3 S 

Nairobi Sheep Disease 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Eastern Equine Encephalitis EEEV Alphavirus 3c S Af Ebola (Including Reston) EBOV Filovirus 4 S EBO Edge Hill EHV Flavivirus 2 S Bf Enseada ENSV Bunyaviridae 3 IE Entebbe Bat ENTV Flavivirus 2 S Bf Epizootic Hemorrhagic Disease EHDV Orbivirus 2 S 

Epizootic Hemorrhagic Disease 

Erve ERVEV Bunyaviridae 2 S Thiafora Estero Real ERV Orthobunyavirus 2 IE Patois Eubenangee EUBV Orbivirus 2 S Eubenangee Everglades EVEV Alphavirus 3 S Af Eyach EYAV Coltivirus 2 S Colorado Tick Fever Farmington FRMV Vesiculovirus 2 A7 Flanders FLAV Rhabdoviridae 2 S Hart Park Flexal FLEV Arenavirus 3 S Tacaribe Fomede FV Orbivirus 2 A7 Chobar Gorge Forecariah FORV Bunyaviridae 2 A7 Bhanja Fort Morgan FMV Alphavirus 2 S Af Fort Sherman FSV Orthobunyavirus 2 A7 Bunyamwera Frijoles FRIV Phlebovirus 2 S Phlebotomus Fever Gabek Forest GFV Phlebovirus 2 A7 Phlebotomus Fever Gadgets Gully GGYV Flavivirus 2 IE Bf Gamboa GAMV Orthobunyavirus 2 S Gamboa Gan Gan GGV Bunyaviridae 2 A7 Mapputta Garba GARV Rhabdoviridae 2 IE Matariva Garissa GRSV Orthobunyavirus 3 A7 Bunyamwera Germiston GERV Orthobunyavirus 3 S Bunyamwera 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Getah GETV Alphavirus 2 A1 Af Gomoka GOMV Orbivirus 2 S Ieri Gordil GORV Phlebovirus 2 IE Phlebotomus Fever Gossas GOSV Rhabdoviridae 2 S Grand Arbaud GAV Phlebovirus 2 S Uukuniemi Gray Lodge GLOV Vesiculovirus 2 IE Vesicular Stomatitis Great Island GIV Orbivirus 2 S Kemerovo Guajara GJAV Orthobunyavirus 2 S Capim Guama GMAV Orthobunyavirus 2 S Guama Guanarito GTOV Arenavirus 4 A4 Tacaribe Guaratuba GTBV Orthobunyavirus 2 A7 Guama Guaroa GROV Orthobunyavirus 2 S California Gumbo Limbo GLV Orthobunyavirus 2 S Cf Gurupi GURV Orbivirus 2 IE Changuinola Hantaan HTNV Hantavirus 3a S Hantaan Hanzalova HANV Flavivirus 4 A4 Bf Hart Park HPV Rhabdoviridae 2 S Hart Park Hazara HAZV Nairovirus 2 S CHF-Congo Highlands J HJV Alphavirus 2 S Af Huacho HUAV Orbivirus 2 S Kemerovo Hughes HUGV Nairovirus 2 S Hughes Hypr HYPRV Flavivirus 4 S Bf Iaco IACOV Orthobunyavirus 2 IE Bunyamwera Ibaraki IBAV Orbivirus 2 IE 

Epizootic Hemorrhagic Disease 

Icoaraci ICOV Phlebovirus 2 S Phlebotomus Fever Ieri IERIV Orbivirus 2 S Ieri 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Ife IFEV Orbivirus b 2 IE Iguape IGUV Flavivirus 2 A7 Bf Ilesha ILEV Orthobunyavirus 2 S Bunyamwera Ilheus ILHV Flavivirus 2 S Bf Ingwavuma INGV Orthobunyavirus 2 S Simbu Inhangapi INHV Rhabdoviridae 2 IE Inini INIV Orthobunyavirus 2 IE Simbu Inkoo INKV Orthobunyavirus 2 S California Ippy IPPYV Arenavirus 2 S Tacaribe Iriri IRRV Rhabdoviridae 2 A7 Irituia IRIV Orbivirus 2 S Changuinola Isfahan ISFV Vesiculovirus 2 S Vesicular Stomatitis Israel Turkey Meningitis ITV Flavivirus 2 with 3 practices S Bf Issyk-Kul ISKV Bunyaviridae 3 IE Itacaiunas ITCNV Rhabdoviridae 2 A7 Itaituba ITAV Phlebovirus 2 IE Phlebotomus Fever Itaporanga ITPV Phlebovirus 2 S Phlebotomus Fever Itaqui ITQV Orthobunyavirus 2 S Cf Itimirim ITIV Orthobunyavirus 2 IE Guama Itupiranga ITUV Orbivirus b 2 IE Ixcanal IXCV Phlebovirus 2 A7 Phlebotomus Fever Jacareacanga JACV Orbivirus 2 IE Corriparta Jacunda JCNV Phlebovirus 2 A7 Phlebotomus Fever Jamanxi JAMV Orbivirus 2 IE Changuinola Jamestown Canyon JCV Orthobunyavirus 2 S California Japanaut JAPV Orbivirus b 2 S Japanese Encephalitis JEV Flavivirus 3c S Bf 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Jari JARIV Orbivirus 2 IE Changuinola Jatobal JTBV Orthobunyavirus 2 A7 Jerry Slough JSV Orthobunyavirus 2 S California Joa JOAV Phlebovirus 2 A7 Johnston Atoll JAV Unassigned 2 S Quaranfil Joinjakaka JOIV Rhabdoviridae 2 S Juan Diaz JDV Orthobunyavirus 2 S Capim Jugra JUGV Flavivirus 2 S Bf Junin JUNV Arenavirus 4 A6 Tacaribe Jurona JURV Vesiculovirus 2 S Vesicular Stomatitis Juruaca JRCV Picornavirus\b 2 A7 Jutiapa JUTV Flavivirus 2 S Bf Kadam KADV Flavivirus 2 S Bf Kaeng Khoi KKV Orthobunyavirus b 2 S Kaikalur KAIV Orthobunyavirus 2 S Simbu Kairi KRIV Orthobunyavirus 2 A1 Bunyamwera Kaisodi KSOV Bunyaviridae 2 S Kaisodi Kamese KAMV Rhabdoviridae 2 S Hart Park Kamiti River KRV Flavivirus 2 A7 Kammavanpettai KMPV Orbivirus 2 S Kannamangalam KANV Rhabdoviridae 2 S Kao Shuan KSV Nairovirus 2 S Dera Ghazi Khan Karimabad KARV Phlebovirus 2 S Phlebotomus Fever Karshi KSIV Flavivirus 2 S Bf Kasba KASV Orbivirus 2 S Palyam Kedougou KEDV Flavivirus 2 A7 Bf Kemerovo KEMV Orbivirus 2 S Kemerovo 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Kern Canyon KCV Rhabdoviridae 2 S Kern Canyon Ketapang KETV Orthobunyavirus 2 S Bakau Keterah KTRV Bunyaviridae 2 S Keuraliba KEUV Rhabdoviridae 2 S Le Dantec Keystone KEYV Orthobunyavirus 2 S California Khabarovsk KHAV Hantavirus 3a IE Hantaan Khasan KHAV Nairovirus 2 IE CCHF Kimberley KIMV Lyssavirus 2 A7 

Bovine Ephemeral Fever 

Kindia KINV Orbivirus 2 A7 Palyam Kismayo KISV Bunyaviridae 2 S Bhanja Klamath KLAV Vesiculovirus 2 S Vesicular Stomatitis Kokobera KOKV Flavivirus 2 S Bf Kolongo KOLV Lyssavirus 2 S Rab Koongol KOOV Orthobunyavirus 2 S Koongol Kotonkan KOTV Lyssavirus 2 S Rab Koutango KOUV Flavivirus 3 S Bf Kowanyama KOWV Bunyaviridae 2 S Kumlinge KUMV Flavivirus 4 A4 Bf Kunjin KUNV Flavivirus 2 S Bf Kununurra KNAV Rhabdoviridae 2 S Kwatta KWAV Vesiculovirus 2 S Vesicular Stomatitis Kyasanur Forest Disease KFDV Flavivirus 4 S Bf Kyzylagach KYZV Alphavirus 2 IE Af La Crosse LACV Orthobunyavirus 2 S California Lagos Bat LBV Lyssavirus 2 S Rab Laguna Negra LANV Hantavirus 3a IE 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

La Joya LJV Vesiculovirus 2 S Vesicular Stomatitis Lake Clarendon LCV Orbivirus b 2 IE Landjia LJAV Rhabdoviridae 2 S Langat LGTV Flavivirus 2 S Bf Lanjan LJNV Bunyaviridae 2 S Kaisodi Las Maloyas LMV Orthobunyavirus 2 A7 Anopheles A Lassa LASV Arenavirus 4 S Tacaribe Latino LATV Arenavirus 2 A5 Tacaribe Lebombo LEBV Orbivirus 2 S Lechiguanas LECHV Hantavirus 3a IE Hantaan Le Dantec LDV Rhabdoviridae 2 S Le Dantec Lednice LEDV Orthobunyavirus 2 A7 Turlock Lipovnik LIPV Orbivirus 2 S Kemerovo Llano Seco LLSV Orbivirus 2 IE Umatilla Lokern LOKV Orthobunyavirus 2 S Bunyamwera Lone Star LSV Bunyaviridae 2 S Louping Ill LIV Flavivirus 3c S Bf Lukuni LUKV Orthobunyavirus 2 S Anopheles A Macaua MCAV Orthobunyavirus 2 IE Bunyamwera Machupo MACV Arenavirus 4 S Tacaribe Madrid MADV Orthobunyavirus 2 S Cf Maguari MAGV Orthobunyavirus 2 S Bunyamwera Mahogany Hammock MHV Orthobunyavirus 2 S Guama Main Drain MDV Orthobunyavirus 2 S Bunyamwera Malakal MALV Lyssavirus 2 S 

Bovine Ephemeral Fever 

Manawa MWAV Phlebovirus 2 S Uukumiemi 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Manitoba MNTBV Rhabdoviridae 2 A7 Manzanilla MANV Orthobunyavirus 2 S Simbu Mapputta MAPV Bunyaviridae 2 S Mapputta Maporal MPRLV Hantavirus 3a IE Hantaan Maprik MPKV Bunyaviridae 2 S Mapputta Maraba MARAV Vesiculovirus 2 A7 Marajo MRJV Unassigned 2 IE Marburg MARV Filovirus 4 S Marburg Marco MCOV Rhabdoviridae 2 S Mariquita MRQV Phlebovirus 2 A7 Phlebotomus Fever Marituba MTBV Orthobunyavirus 2 S Cf Marrakai MARV Orbivirus 2 S Palyam Matariya MTYV Rhabdoviridae 2 S Matariva Matruh MTRV Orthobunyavirus 2 S Tete Matucare MATV Orbivirus 2 S Mayaro MAYV Alphavirus 2 S Af Mboke MBOV Orthobunyavirus 2 A7 Bunyamwera Meaban MEAV Flavivirus 2 IE Bf Melao MELV Orthobunyavirus 2 S California Mermet MERV Orthobunyavirus 2 S Simbu Middelburg MIDV Alphavirus 2 A1 Af Minatitlan MNTV Orthobunyavirus 2 S Minatitlan Minnal MINV Orbivirus 2 S Umatilla Mirim MIRV Orthobunyavirus 2 S Guama Mitchell River MRV Orbivirus 2 S Mobala MOBV Arenavirus 3 A7 Tacaribe Modoc MODV Flavivirus 2 S Bf 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Moju MOJUV Orthobunyavirus 2 S Guama Mojui Dos Campos MDCV Orthobunyavirus 2 IE Mono Lake MLV Orbivirus 2 S Kemerovo Mont. Myotis Leukemia MMLV Flavivirus 2 S Bf Monte Dourado MDOV Orbivirus 2 IE Changuinola Mopeia MOPV Arenavirus 3 A7 Moriche MORV Orthobunyavirus 2 S Capim Morro Bay MBV Orthobunyavrius 2 IE California Morumbi MRMBV Phlebovirus 2 A7 Phlebotomus Fever Mosqueiro MQOV Rhabdoviridae 2 A7 Hart Park Mossuril MOSV Rhabdoviridae 2 S Hart Park Mount Elgon Bat MEBV Vesiculovirus 2 S Vesicular Stomatitis M’Poko MPOV Orthobunyavirus 2 S Turlock Mucambo MUCV Alphavirus 3 S Af Mucura MCRV Phlebovirus 2 A7 Phlebotomus Fever Munguba MUNV Phlebovirus 2 IE Phlebotomus Fever Murray Valley Encephalitis MVEV Flavivirus 3 S Bf Murutucu MURV Orthobunyavirus 2 S Cf Mykines MYKV Orbivirus 2 A7 Kemerovo Nairobi Sheep Disease e NSDV Nairovirus 3c A1 

Nairobi Sheep Disease 

Naranjal NJLV Flavivirus 2 IE Bf Nariva NARV Paramyxoviridae 2 IE Nasoule NASV Lyssavirus 2 A7 Rab Navarro NAVV Rhabdoviridae 2 S Ndelle NDEV Orthoreovirus 2 A7 Ndelle Ndumu NDUV Alphavirus 2 A1 Af 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Negishi NEGV Flavivirus 3 S Bf Nepuyo NEPV Orthobunyavirus 2 S Cf Netivot NETV Orbivirus 2 A7 New Minto NMV Rhabdoviridae 2 IE Sawgrass Ngaingan NGAV Lyssavirus 2 S Tibrogargan Ngari d NRIV Orthobunyavirus 3 A7 Bunyamera Ngoupe NGOV Orbivirus 2 A7 Eubenangee Nique NIQV Phlebovirus 2 S Phlebotomus Fever Nkolbisson NKOV Rhabdoviridae 2 S Kern Canyon Nodamura NOV Alphanodavirus 2 IE Nola NOLAV Orthobunyavirus 2 S Bakau Northway NORV Orthobunyavirus 2 IE Bunyamwera Ntaya NTAV Flavivirus 2 S Bf Nugget NUGV Orbivirus 2 S Kemerovo Nyamanini NYMV Unassigned 2 S Nyamanini Nyando NDV Orthobunyavirus 2 S Nyando Oak Vale OVV Rhabdoviridae 2 A7 Odrenisrou ODRV Phlebovirus 2 A7 Phlebotomus Fever Okhotskiy OKHV Orbivirus 2 S Kemerovo Okola OKOV Bunyaviridae 2 S Tanga Olifantsvlei OLIV Orthobunyavirus 2 S Olifantsylei Omo OMOV Nairovirus 2 A7 Qalyub Omsk Hemorrhagic Fever OHFV Flavivirus 4 S Bf O’Nyong-Nyong ONNV Alphavirus 2 S Af Oran ORANV Hantavirus 3a IE Hantaan Oriboca ORIV Orthobunyavirus 2 S Cf Oriximina ORXV Phlebovirus 2 IE Phlebotomus Fever 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Oropouche OROV Orthobunyavirus 3 S Simbu Orungo ORUV Orbivirus 2 S Orungo Ossa OSSAV Orthobunyavirus 2 S Cf Ouango OUAV Rhabdoviridae 2 IE Oubangui OUBV Poxviridae 2 IE Oubi OUBIV Orthobunyavirus 2 A7 Olifantsylei Ourem OURV Orbivirus 2 IE Changuinola Pacora PCAV Bunyaviridae 2 S Pacui PACV Phlebovirus 2 S Phlebotomus Fever Pahayokee PAHV Orthobunyavirus 2 S Patois Palma PMAV Bunyaviridae 2 IE Bhanja Palestina PLSV Orthobunyavirus 2 IE Minatitlan Palyam PALV Orbivirus 2 S Palyam Para PARAV Orthobunyavirus 2 IE Simbu Paramushir PMRV Nairovirus 2 IE Sakhalin Parana PARV Arenavirus 2 A5 Tacaribe Paroo River PRV Orbivirus 2 IE Pata PATAV Orbivirus 2 S Pathum Thani PTHV Nairovirus 2 S Dera Ghazi Khan Patois PATV Orthobunyavirus 2 S Patois Peaton PEAV Orthobunyavirus 2 A1 Simbu Pergamino PRGV Hantavirus 3a IE Perinet PERV Vesiculovirus 2 A7 Vesicular Stomatitis Petevo PETV Orbivirus 2 A7 Palyam Phnom-Penh Bat PPBV Flavivirus 2 S Bf Pichinde PICV Arenavirus 2 A5 Tacaribe Picola PIAV Orbivirus 2 IE Wongorr 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Pirital PIRV Arenavirus 3 IE Piry PIRYV Vesiculovirus 3 S Vesicular Stomatitis Pixuna PIXV Alphavirus 2 S Af Playas PLAV Orthobunyavirus 2 IE Bunyamwera Pongola PGAV Orthobunyavirus 2 S Bwamba Ponteves PTVV Phlebovirus 2 A7 Uukuniemi Potosi POTV Orthobunyavirus 2 IE Bunyamwera Powassan POWV Flavivirus 3 S Bf Precarious Point PPV Phlebovirus 2 A7 Uukuniemi Pretoria PREV Nairovirus 2 S Dera Ghazi Khan Prospect Hill PHV Hantavirus 2 A8 Hantaan Puchong PUCV Lyssavirus 2 S 

Bovine Ephemeral Fever 

Pueblo Viejo PVV Orthobunyavirus 2 IE Gamboa Punta Salinas PSV Nairovirus 2 S Hughes Punta Toro PTV Phlebovirus 2 S Phlebotomus Fever Purus PURV Orbivirus 2 IE Changuinola Puumala PUUV Hantavirus 3a IE Hantaan Qalyub QYBV Nairovirus 2 S Qalyub Quaranfil QRFV Unassigned 2 S Quaranfil Radi RADIV Vesiculovirus 2 A7 Vesicular Stomatitis Razdan RAZV Bunyaviridae 2 IE Resistencia RTAV Bunyaviridae 2 IE Resistencia Restan RESV Orthobunyavirus 2 S Cf Rhode Island RHIV Rhabdoviridae 2 A7 Rift Valley Fever RVFV Phlebovirus 3c S Phlebotomus Fever Rio Bravo RBV Flavivirus 2 S Bf 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Rio Grande RGV Phlebovirus 2 S Phlebotomus Fever Rio Preto RIOPV Unassigned 2 IE Rochambeau RBUV Lyssavirus 2 IE Rab Rocio ROCV Flavivirus 3 S Bf Ross River RRV Alphavirus 2 S Af Royal Farm RFV Flavivirus 2 S Bf Russian Spring-Summer Encephalitis RSSEV Flavivirus 4 S Bf Saaremaa SAAV Hantavirus 3a IE Hantaan Sabia SABV Arenavirus 4 A4 Sabo SABOV Orthobunyavirus 2 S Simbu Saboya SABV Flavivirus 2 S Bf Sagiyama SAGV Alphavirus 2 A1 Af Saint-Floris SAFV Phlebovirus 2 S Phlebotomus Fever Sakhalin SAKV Nairovirus 2 S Sakhalin Salanga SGAV Poxviridae 2 IE SGA Salehabad SALV Phlebovirus 2 S Phlebotomus Fever Salmon River SAVV Coltivirus 2 IE Colorado Tick Fever Sal Vieja SVV Flavivirus 2 A7 Bf San Angelo SAV Orthobunyavirus 2 S California Sandfly Fever, Naples SFNV Phlebovirus 2 S Phlebotomus Fever Sandfly Fever, Sicilian SFSV Phlebovirus 2 S Phlebotomus Fever Sandjimba SJAV Lyssavirus 2 S Rab Sango SANV Orthobunyavirus 2 S Simbu San Juan SJV Orthobunyavirus 2 IE Gamboa San Perlita SPV Flavivirus 2 A7 Bf Santarem STMV Bunyaviridae 2 IE 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Santa Rosa SARV Orthobunyavirus 2 IE Bunyamwera Saraca SRAV Orbivirus 2 IE Changuinola Sathuperi SATV Orthobunyavirus 2 S Simbu Saumarez Reef SREV Flavivirus 2 IE Bf Sawgrass SAWV Rhabdoviridae 2 S Sawgrass Sebokele SEBV Unassigned 2 S Sedlec SEDV Bunyaviridae 2 A7 Seletar SELV Orbivirus 2 S Kemerovo Sembalam SEMV Unassigned 2 S Semliki Forest SFV Alphavirus 3 A2 Af Sena Madureira SMV Rhabdoviridae 2 IE Timbo Seoul SEOV Hantavirus 3a IE Hantaan Sepik SEPV Flavivirus 2 IE Bf Serra Do Navio SDNV Orthobunyavirus 2 A7 California Serra Norte SRNV Phlebovirus 2 A7 Shamonda SHAV Orthobunyavirus 2 S Simbu Shark River SRV Orthobunyavirus 2 S Patois Shokwe SHOV Orthobunyavirus 2 IE Bunyamwera Shuni SHUV Orthobunyavirus 2 S Simbu Silverwater SILV Bunyaviridae 2 S Kaisodi Simbu SIMV Orthobunyavirus 2 S Simbu Simian Hemorrhagic Fever SHFV Arterivirus 2 2 

Simian Hemorrhagic Fever 

Sindbis SINV Alphavirus 2 S Af Sin Nombre SNV Hantavirus 3a IE Hantaan Sixgun City SCV Orbivirus 2 S Kemerovo Slovakia SLOV Unassigned 3 IE 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Snowshoe Hare SSHV Orthobunyavirus 2 S California Sokoluk SOKV Flavivirus 2 S Bf Soldado SOLV Nairovirus 2 S Hughes Somone SOMV Unassigned 3 IE Somone Sororoca SORV Orthobunyavirus 2 S Bunyamwera Spondweni SPOV Flavivirus 2 S Bf Sripur SRIV Rhabdoviridae 3 IE St. Louis Encephalitis SLEV Flavivirus 3 S Bf Stratford STRV Flavivirus 2 S Bf Sunday Canyon SCAV Bunyaviridae 2 S Tacaiuma TCMV Orthobunyavirus 2 S Anopheles A Tacaribe TCRV Arenavirus 2 A5 Tacaribe Taggert TAGV Nairovirus 2 S Sakhalin Tahyna TAHV Orthobunyavirus 2 S California Tai TAIV Bunyaviridae 2 A7 Bunyamwera Tamdy TDYV Bunyaviridae 2 IE Tamiami TAMV Arenavirus 2 A5 Tacaribe Tanga TANV Bunyaviridae 2 S Tanga Tanjong Rabok TRV Orthobunyavirus 2 S Bakau Tapara TAPV Phlebovirus 2 A7 Tataguine TATV Bunyaviridae 2 S Tehran THEV Phlebovirus 2 A7 Phlebotomus Fever Telok Forest TFV Orthobunyavirus 2 IE Bakau Tembe TMEV Orbivirus b 2 S Tembusu TMUV Flavivirus 2 S Bf Tensaw TENV Orthobunyavirus 2 S Bunyamwera Termeil TERV Bunyavirus b 2 IE 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Tete TETEV Orthobunyavirus 2 S Tete Thiafora TFAV Bunyaviridae 2 A7 Thiafora Thimiri THIV Orthobunyavirus 2 S Simbu Thogoto THOV Orthomyxoviridae 2 S Thogoto Thottapalayam TPMV Hantavirus 2 S Hantaan Tibrogargan TIBV Lyssavirus 2 S Tibrogargan Tilligerry TILV Orbivirus 2 IE Eubenangee Timbo TIMV Rhabdoviridae 2 S Timbo Timboteua TBTV Orthobunyavirus 2 A7 Guama Tinaroo TINV Orthobunyavirus 2 IE Simbu Tindholmur TDMV Orbivirus 2 A7 Kemerovo Tlacotalpan TLAV Orthobunyavirus 2 IE Bunyamwera Tonate TONV Alphavirus 3 IE Af Topografov TOPV Hantavirus 3a IE Hantaan Toscana TOSV Phlebovirus 2 S Phlebotomus Fever Toure TOUV Unassigned 2 S Tracambe TRCV Orbivirus 2 A7 Tribec TRBV Orbivirus 2 S Kemerovo Triniti TNTV Togaviridae 2 S Trivittatus TVTV Orthobunyavirus 2 S California Trocara TROCV Alphavirus 2 IE Af Trombetas TRMV Orthobunyavirus 2 A7 Trubanaman TRUV Bunyaviridae 2 S Mapputta Tsuruse TSUV Orthobunyavirus 2 S Tete Tucurui TUCRV Orthobunyavirus 2 A7 Tula TULV Hantavirus 2 A8 Tunis TUNV Phlebovirus 2 A7 Phlebotomus Fever 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Turlock TURV Orthobunyavirus 2 S Turlock Turuna TUAV Phlebovirus 2 IE Phlebotomus Fever Tyuleniy TYUV Flavivirus 2 S Bf Uganda S UGSV Flavivirus 2 S Bf Umatilla UMAV Orbivirus 2 S Umatilla Umbre UMBV Orthobunyavirus 2 S Turlock Una UNAV Alphavirus 2 S Af Upolu UPOV Bunyaviridae 2 S Upolu Uriurana UURV Phlebovirus 2 A7 Phlebotomus Fever Urucuri URUV Phlebovirus 2 S Phlebotomus Fever Usutu USUV Flavivirus 2 S Bf Utinga UTIV Orthobunyavirus 2 IE Simbu Uukuniemi UUKV Phlebovirus 2 S Uukuniemi Vellore VELV Orbivirus 2 S Palyam Venezuelan Equine Encephalitis VEEV Alphavirus 3c S Af Venkatapuram VKTV Unassigned 2 S Vinces VINV Orthobunyavirus 2 A7 Cf Virgin River VRV Orthobunyavirus 2 A7 Anopheles A Vesicular Stomatitis-Alagoas VSAV Vesiculovirus 2c S Vesicular Stomatitis Vesicular Stomatitis-Indiana VSIV Vesiculovirus 2c A3 Vesicular Stomatitis Vesicular Stomatitis-New Jersey VSNJV Vesiculovirus 2c A3 Vesicular Stomatitis Wad Medani WMV Orbivirus 2 S Kemerovo Wallal WALV Orbivirus 2 S Wallal Wanowrie WANV Bunyaviridae 2 S Warrego WARV Orbivirus 2 S Warrego 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

NAME ACRONYM 

TAXONOMIC STATUS (FAMILY OR GENUS) 

RECOMMENDED BIOSAFETY LEVEL 

BASIS OF RATING 

ANTIGENIC GROUP 

Wesselsbron WESSV Flavivirus 3c S Bf Western Equine Encephalitis WEEV Alphavirus 3 S Af West Nile WNV Flavivirus 3 S Bf Whataroa WHAV Alphavirus 2 S Af Whitewater Arroyo WWAV Arenavirus 3 IE Tacaribe Witwatersrand WITV Bunyaviridae 2 S Wongal WONV Orthobunyavirus 2 S Koongol Wongorr WGRV Orbivirus 2 S Wongorr Wyeomyia WYOV Orthobunyavirus 2 S Bunyamwera Xiburema XIBV Rhabdoviridae 2 IE Xingu XINV Orthobunyavirus 3 Yacaaba YACV Bunyaviridae 2 IE Yaounde YAOV Flavivirus 2 A7 Bf Yaquina Head YHV Orbivirus 2 S Kemerovo Yata YATAV Rhabdoviridae 2 S Yellow Fever YFV Flavivirus 3 S Bf Yogue YOGV Bunyaviridae 2 S Yogue Yoka YOKA Poxviridae 2 IE Yug Bogdanovac YBV Vesiculovirus 2 IE Vesicular Stomatitis Zaliv Terpeniya ZTV Phlebovirus 2 S Uukuniemi Zegla ZEGV Orthobunyavirus 2 S Patois Zika ZIKV Flavivirus 2 S Bf Zirqa ZIRV Nairovirus 2 S Hughes 

* Federal regulations, import/export requirements, and taxonomic status are subject to changes. Check with the appropriate fede

regulations. 
Agent Summary Statements- Arboviruses and Related Zoonotic Viruses 

a Containment requirements will vary based on virus concentration, animal species, or virus type. See the Hantavirus agent sum

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 req

containment (up to and including BSL-3-Ag containment). Not all strains of each organism are necessarily of concern to the U

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, B

based on cross-reactivity among known arboviruses (2,21). Group A viruses are members of the genus Alphavirus, group B b

Flaviviridae, and Group C viruses are members of the family Bunyaviridae. 
Agent Summary Statements – Toxin Agents 

• Section VIII-G: Toxin Agents 

Agent: 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 

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, proteins can be absorbed through broken or 

lacerated skin and, therefore, BoNT samples or contaminated material should be handled with 

gloves. 
Agent Summary Statements – Toxin Agents 

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 

of time (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. 

Containment Recommendations 

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. 

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. 
Agent Summary Statements – Toxin Agents 

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: 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 

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 
Agent Summary Statements – Toxin Agents 

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 

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 reaerosolization of residual toxin from the fur of exposed animals. The most common cause of 

laboratory intoxication with SE is expected to result from accidental self-exposure via the 

mucous membranes by touching contaminated hands to the face or eyes. 

Containment Recommendations 

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. 
Agent Summary Statements – Toxin Agents 

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. 

Agent: 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 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 intragastric 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 injection25 or by the aerosol route 

(extrapolation from two species of NHP). 

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. 
Agent Summary Statements – Toxin Agents 

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 

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 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. 

Containment Recommendations 

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. 
Agent Summary Statements – Toxin Agents 

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. 

Agent: 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 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 
Agent Summary Statements – Toxin Agents 

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 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 nonselective 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 it 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 

to date, 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 
Agent Summary Statements – Toxin Agents 

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 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 

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 threat to laboratory workers. Special emphasis, therefore, must be placed upon 

proper decontamination of work surfaces and equipment.45 
Agent Summary Statements – Toxin Agents 

Containment Recommendations 

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. 

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7. Franz DR, Pitt LM, Clayton MA, et al. Efficacy of prophylactic and therapeutic 

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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 

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15. Do Carmo LS, Cummings C, Linardi VR, et al. A case study of a massive staphylococcal 

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17. LeClaire RD, Pitt MLM. Biological weapons defense: effect levels. In: Lindler LE, 

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18. Rusnak JM, Kortepeter M, Ulrich R, et al. Laboratory exposures to Staphylococcal 

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19. Ulrich R, Sidell S, Taylor T, et al. Staphylococcal enterotoxin B and related pyrogenic 

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the casualty. Washington, DC: Office of the Surgeon General at TMM Publications, 

Borden Institute, Walter Reed Army Medical Center; 1997. p. 621-30. 

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21. Hartley MR, Lord JM. Cytotoxic ribosome-inactivating lectins from plants. Biochim 

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22. Doan LG. Ricin: mechanism of toxicity, clinical manifestations, and vaccine 

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warfare, weaponry, and the casualty. Washington, DC. Office of the Surgeon General at 

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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: 

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28. Bamburg J. Chemical and biochemical studies of the trichothecene mycotoxins. In: JV 

Rodricks, editor. Mycotoxins and other fungal related food problems. Vol. 149. Advances 

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and Food Chemistry; 1976. p. 144-162. 

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 possible origin of 

tetrodotoxin. Ann N Y Acad Sci 1986;479:44-51. 

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. 

34. Carmichael W. Algal toxins. In: Callow J, editor. Advances in botanical research. Vol. 

12. London: Academic Press; 1986. p. 47-101. 

35. Bunner B, Wannemacher RW Jr, Dinterman RE, et al. Cutaneous absorption and 

decontamination of [3

H]T-2 toxin in the rat model. J Toxicol Environ Health. 

1989;26:413-423. 

36. Poli M. Foodborne Marine biotoxins. In: Miliotis MD, Bier JW, editors. International 

handbook of foodborne pathogens. New York: Marcel Dekker; 2003;445-58. 

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 palytoxin-induced 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, White J, 

editors. Handbook of clinical toxicology of animal venoms and 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. 
Agent Summary Statements – Toxin Agents 

45. Wannemacher RW. Procedures for inactivation and safety containment of toxins. In: 

Proceedings for the symposium on agents of biological origin. 1989; Aberdeen Proving 

Ground, MD. Aberdeen, Maryland: U.S. Army Chemical Research, Development and 

Engineering Center; 1989. p. 115-22 

46. Paddle BM. Therapy and prophylaxis of inhaled biological toxins. J Appl Toxicol. 

2003;23:139-70. 
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 1 and 

2).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 PrP-specific 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 1 (human diseases) and Table 2 

(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 germline. 
Agent Summary Statements – Prion Diseases 

TABLE 1 

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 

Iatrogenic CJD iCJD Infection from contaminated corneal and dural grafts, pituitary 

hormone, or neurosurgical equipment 

Gerstmann-SträusslerScheinker syndrome GSS Germline mutations in PrP gene 

Fatal familial insomnia FFI Germline mutations in PrP gene 

 TABLE 2 

THE ANIMAL PRION DISEASES 

DISEASE ABBREVIATION NATURAL 

HOST 

MECHANISM OF PATHOGENESIS 

Scrapie Sheep, goats and 

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 

and 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 
Agent Summary Statements – Prion Diseases 

Species-specificity of prions. 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 difficulty to predict 

with accuracy. Such considerations help to form the basis for the biosafety classification of 

different prions. 

LABORATORY SAFETY 

Biosafety level classification 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. All other animal prions 

are considered BSL-2 pathogens. 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

 

Human prion diseases In the care of patients diagnosed with human prion disease, Standard 

Precautions are adequate. However, the human prion diseases in this setting 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 
Agent Summary Statements – Prion Diseases 

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 3), 

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 (See Table 4).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 4 summarizes the key recommendations for decontamination 

of reusable instruments and surfaces. Contaminated disposable instruments or materials should 

be incinerated. 

Autopsies Routine autopsies and the processing of small amounts of formalin-fixed tissues 

containing human prions can safely be done using BSL-2 precautions.7

 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 cutresistant 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 at least in a BSL-2 facility. 

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.8

 The most 

prudent approach is to study BSE prions at a minimum in a BSL-2 facility. 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, etc.) personnel 
Agent Summary Statements – Prion Diseases 

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 1000o

C (1832oF).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 be in compliance with applicable 

state and federal regulations. 

The alkaline hydrolysis process, using a pressurized vessel that exposes the carcass or tissues to 

1 N NaOH or KOH heated to 150oC, can be used as an alternative to incineration for the disposal 

of carcasses and tissue.5,9 The process has been shown to completely inactive TSEs (301v agent 

used) when used for the recommended period of time. 

TABLE 3 

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 48 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 as 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 4. 

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 coverslip 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. 
Agent Summary Statements – Prion Diseases 

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.9

 The related considerations outlined in Table 3 should be 

incorporated into the laboratory's risk management for this work. 

TABLE 4 

PRION INACTIVATION METHODS FOR REUSABLE INSTRUMENTS AND SURFACES 

1. Immerse in 1 N NaOH, and 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 hour. Transfer into water and autoclave 

(gravity displacement) at 121o

C for 1 hour. Clean and sterilize by conventional means. 

3. Immerse in 1N NaOH or sodium hypochlorite (20,000 ppm) for 1 hour. Rinse instruments with water, transfer 

to open pan and autoclave at 121o

C (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 time 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, 10,11) 

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 

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 CreutzfeldtJakob 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. 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. 

8. Hilton DA. Pathogenesis and prevalence of variant Creutzfeldt-Jakob disease. Pathol J. 

2006;208:134-41. 

9. Richmond JY, Hill RH, Weyant RS, et al. What’s hot in animal biosafety? ILAR J. 

2003;44:20-7. 

10. Ernst DR, Race RE. Comparative analysis of scrapie agent inactivation methods. J. Virol. 

Methods. 1994; 41:193-202. 

11. Race RE, Raymond GJ. Inactivation of transmissible spongiform encephalopathy (prion) 

agents by Environ LpH. J Virol. 2004;78:2164-5. 
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 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, described 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 do 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 with 

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. 

BSOs should understand that an active cabinet is a primary safety device. A BSC must be 

routinely inspected and tested by trained personnel, following strict protocols, to verify that it is 
Appendix A 

working 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 bio-terrorism, 

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, microburners and safety 

centrifuge cups or sealed rotors are among the engineered devices that protect laboratory workers 

from biological hazards. The most important piece of containment equipment, however, 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 and were characterized by mass airflow with widely varying air volumes across 

openings. The feature of mass airflow into the cabinet was added to draw "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. 

HEPA Filters. Control of airborne particulate materials became possible with the development of 

filters which would efficiently remove 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 1940's.2 
Appendix A 

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 medium of a typical HEPA filter is a single sheet of borosilicate fibers which has been 

treated with a wet-strength water-repellant binder. The filter medium is pleated to increase the 

overall surface area inside the filter frame, 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 initially installed 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 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 across the work surface. Personnel protection is provided by this inward airflow as 

long as a minimum velocity of 75 linear feet per minute (lfpm) 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). 

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 in the terminal end of the building exhaust prior to the 

exhaust fan. 
Appendix A 

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. Note that a filter should be 

installed on the cabinet air supply intake. 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. The re-circulating Class I BSC 

should be annually certified 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 and aid in 

the capture and removal of airborne contaminants from the air stream.7 Biocontainment 

technology also incorporated this laminar flow principle with the use of the HEPA filter to 

provide a particulate-free work environment. This combination of technologies serves to protect 

the laboratory worker from potentially infectious microorganisms or materials being 

manipulated4 within the cabinet and provides necessary product protection, as well. Class II 

BSCs are partial barrier systems that rely on the laminar movement of air to provide 

containment. If 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 the cabinet, providing personnel protection. 

In addition, the downward laminar flow of HEPA-filtered air provides product protection by 

minimizing the chance of cross-contamination 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 connection. Exhaust air from Types B1 and B2 BSCs must be 

discharged 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 
Appendix A 

the outside should be used when working with volatile, toxic chemicals, but amounts must be 

limited (See Table 2). 

All Class II cabinets are designed for work involving microorganisms assigned to biosafety 

levels 1, 2 and 3.1

 Class II BSCs provide the microbe-free 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 if used in a BSL-4 suit laboratory by a worker wearing a positive pressure 

protective suit. 

1. The Class II, Type A1 BSC. An internal blower (Figure 3) draws sufficient room air 

through the front grille to maintain a minimum calculated or measured average inflow 

velocity of at least 75 lfpm at the face opening of the cabinet. The supply air flows 

through a HEPA filter and provides particulate-free air to the work surface. Laminar 

airflow reduces turbulence in the work zone and minimizes the potential for crosscontamination. 

The downward moving air "splits" as it approaches the work surface; the blower 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 discharged through the front and rear grilles under negative air pressure into a 

blower and 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. 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). 

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, and 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 volume 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.a 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 make it difficult to match the airflow 

requirements of the cabinet. Existing Class II Type A1 or A2 hard ducted exhaust 

connections can be altered and the room air balanced to allow for the recommended air 
Appendix A 

gap inflows to avoid fluctuations in airflow. This may require added building exhaust 

system capacity.*

2. The Class II, Type B1 BSC. Some biomedical research requires the use of small 

quantities of hazardous chemicals, such as carcinogens. The powdered form of these 

carcinogens should be weighed or manipulated in a chemical fume hood or a static-air 

glove box equipped with a double-door airlock. 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 these hazardous chemicals with in vitro biological 

systems. The NSF International NSF/ANSI Standard 49 - 2002 definition of Type B1 

cabinets8

 includes this classic NCI design Type B, as well as cabinets without supply 

HEPA filters located immediately below the work surface (Figure 5B), and/or those with 

exhaust/recirculation downflow 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 back-pressure 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 lfpm. 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 downflow air exits through the rear grille, passes through 

the exhaust HEPA filter, and is discharged from the building. The remaining 30 percent 

of the downflow air is drawn through the front grille. Since the air which flows to the rear 

grille is discharged into the exhaust system, activities that may generate hazardous 

chemical vapors or particulates should be conducted towards the rear of the cabinet work 

area.13

Type B1 cabinets must be hard-ducted, preferably to a dedicated, independent exhaust 

system, or to a properly-designed laboratory building exhaust. As indicated earlier, fans 

for laboratory exhaust systems should be located at the terminal end of the duct work. 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. 

 *

 Contact manufacturers for any additional specifications. 
Appendix A 

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 

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 lfpm. 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 pressureindependent 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 former Class II Type B3.8 The 

Type A2 cabinet has a minimum calculated or measured inflow velocity of 100 lfpm. All 

positive pressure biologically 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 eye 

pieces of a microscope, or 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 
Appendix A 

protection for the environment and the worker. It is a gas-tight (no leak greater than1x10-7 cc/sec 

of SF6 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 to the 

outdoors. Airflow is maintained by a dedicated, independent exhaust system exterior to the 

cabinet, which keeps the cabinet under negative pressure (minimum of 0.5 inches of pressure 

Water Gauge). 

Long, heavy-duty rubber gloves are attached in a gas-tight manner to ports in the cabinet and 

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 air-flow is not a characteristic of a Class III cabinet. 

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, 

etc.) 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 or drug formulations, or when manipulating potentially infectious 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 Laminar Flow “Clean Bench”. Vertical laminar 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. 
Appendix A 

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 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 cabinet work 

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 singlepass 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 canopy-exhausted cabinets 

may be used with minute or tracer quantities of nonvolatile 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. 
Appendix A 

Recommendations from the former Office of Research Safety of the NCI18 stated that work 

involving the use of chemical carcinogens for in vitro procedures can be performed in a Class II 

cabinet which meets the following parameters: 1) exhaust airflow is sufficient to provide a 

minimum inward velocity of 100 lfpm at the face opening of the cabinet; 2) contaminated air 

plenums under positive pressure are leak-tight; and 3) cabinet air is discharged to the outdoors. 

National Sanitation Foundation (NSF)/ANSI 49 - 2002 8 currently recommends that biologicallycontaminated 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 and be exhausted to 

the outdoors. 

Radiological Hazards in the BSC. As indicated above, volatile radionuclides such as I125 should 

not be used within Class II, Type A1 BSCs; or an A2 BSC unless the exhaust air is discharged 

out of doors and appropriate additional filtration techniques are used (See Table 2). 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. 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 untoward 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) 

which 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 
Appendix A 

the cabinet, walking traffic, room fans, open/closing room doors, etc.) 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 can be included 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 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 hours 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. 
Appendix A 

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 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, aerosolgenerating 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 work flow should be from "clean to dirty" (See 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. 

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. 
Appendix A 

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 which 

prevents microorganisms from falling into the tube or flask. An open flame in a BSC, however, 

creates turbulence which disrupts the pattern of HEPA-filtered air being supplied to the work 

surface. When deemed absolutely necessary, touch-plate microburners 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 (See 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 to be 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. 

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 work day, 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 
Appendix A 

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 more 

extensive decontamination. 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 thirty 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 cabinet work 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 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. 
Appendix A 

SECTION VI 

FACILITY AND ENGINEERING REQUIREMENTS 

Secondary Barriers. Whereas BSCs are considered to be the primary safety barrier for 

manipulation of infectious materials, the laboratory room itself is considered to be the secondary 

safety 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-3.1,26 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. Otherwise, each cabinet must have a dedicated exhaust system. The connection to a BSC 

must be constant air volume (CAV). Variable air volume (VAV) on a BSC exhaust must be 

avoided. 

The room exhaust system should be sized to handle both the room and all containment devices 

vented through the system. Adequate supply air must be provided to ensure appropriate function 

of the exhaust system. The facility engineer must be consulted before locating a new cabinet 

requiring connection to the building exhaust system. Right angle bends, long horizontal runs 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 reentrainment 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 

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 required in BSCs 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 
Appendix A 

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 work stations 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 duct work 

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. 

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 bagin/bag-out assembly cannot be added to a cabinet after-the-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 
Appendix A 

performance. Federal Standard 20932,33,†

 was developed to establish classes of air cleanliness and 

methods for monitoring clean work stations 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 laminar airflow and HEPAfiltered supply and exhaust air. This guideline specified 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. 

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 49 – 2002, 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 49 – 2002‡ 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 49 2002, 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. 

 

b

 Federal Standard No. 209E32 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. 

‡ The standard can be ordered from NSF for a nominal charge 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 

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 testing that follows the recommendations for field testing (NSF/ANSI 49 – 2002 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, and it is 

suggested that the institutional BSO be consulted in identifying companies qualified to conduct 

the necessary field performance tests. 

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 49 – 2002, Annex F plus Addendum #1 needs to be 

verified at the time of installation and 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. Downflow Velocity and Volume 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 the airflow along 

the entire perimeter of the work access opening is inward, if airflow within the work 

area is downward with no dead spots or refluxing, if ambient air passes onto or over 

the work surface and if there is refluxing to the outside at the window wiper gasket 

and side seals. The smoke test is an indicator of airflow direction, not velocity. 
Appendix A 

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. 

E. Cabinet Leak Test: 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 just prior to 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 – 2002 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. They may be performed by an electrical technician other than the field 

certification personnel at the same time the other field certification tests are 

conducted. The polarity of electrical outlets is checked (See 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. 
Appendix A 

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). 

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 

Protection Provided Biological Risk 

Assessed 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 suitroom 

with suit 
Appendix A 

Table 2. Comparison of Biosafety Cabinet Characteristics 

Applications 

BSC 

Class Face Velocity Airflow Pattern 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 

outdoors 1,2 

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 

II, 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 

lfpm intake air velocity and 

plenums are under negative 

pressure to room; exhaust air 

can be ducted to outside 

through a canopy unit (figure 

7) 

Yes 

When exhausted 

outdoors 

(Formerly "B3") 

(minute amounts) 

1,2 

III N/A 

Supply air is HEPA 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 may require 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. 
Appendix A 

Table 3. Field Performance Tests to be Applied to the Three 

Classes of Biological Safety Cabinets 

 Biosafety Cabinet 

Test Performed for Class Class Class 

 I II III 

Primary Containment 

Cabinet Integrity N/A A 

A (A1 

Only) 

HEPA Filter Leak Req Req Req 

Downflow Velocity N/A Req N/A 

Face Velocity Req Req N/A 

Negative Pressure/Ventilation Rate B N/A Req 

Airflow Smoke Patterns Req Req E/F 

Alarms and Interlocks C,D C,D Req 

 

Electrical Safety 

Electrical Leakage, Etc. E,D E,D E,D 

Ground Fault Interrupter D D D 

 

Other 

Lighting Intensity E E E 

UV Intensity C,E C,E C,E 

Noise Level E E E 

Vibration E E E 

 

Req 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. 
Appendix A 

Table 4. Reference for Applicable Containment Tests 

Cabinet Type by Class Test 

I II 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 lfpm 

(F.3.3.3.3) 

75 lfpm - type A1; 

100 lfpm type A2, 

B1& B2: 

(F.3) 

N/A 

Face Velocity 

Gloves Ports / 

No Gloves 

150 lfpm 

(F.3.3.3.3) N/A NA 

Water Gauge 

Pressure Glove 

Ports & Gloves 

N/A N/A (- 0.5 "w.c.”) 

[p.145] 

Downflow Velocity N/A (F.2) N/A 

1. Parenthetical references are to the NSF/ANSI Standard 49 2004, letters and numerals indicate specific 

sections and subsections. 

2. Bracketed reference ([ ]) is to the Laboratory Safety Monogragh, Page numbers are indicated. 
Appendix A 

FIGURES 

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 

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. 
Appendix A 

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. 
Appendix A 

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 

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 building exhaust system required. 
Appendix A 

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, F. filter screen. 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 building exhaust system. Note: The A2 BSC 

should be canopy connected to the exhaust system. 
Appendix A 

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 independent dedicated exhaust system. The exhaust air must be double HEPA 

filtered or HEPA filtered and incinerated. 
Appendix A 

Figure 9A. The horizontal laminar flow “clean bench”. A. front opening, B. supply grille, C. 

supply HEPA filter, D. supply plenum, E. blower, F. grille. 
Appendix A 

Figure 9B. The vertical laminar flow “clean bench”. A. front opening, B. sash, C. supply 

HEPA filter, D. blower. 
Appendix A 

Figure 10. A modified containment cabinet or Class I BSC can be used for labeling infectious 

microorganisms with I 125. 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). 
Appendix A 

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 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 

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. 
Appendix A 

ACKNOWLEDGEMENTS 

We gratefully acknowledge Baker Company, Filtration Group, Inc, Flanders Filters and Forma 

Scientific for the use of drawings reproduced herein. 

Th Steering Committee also thanks Dave Stuart, Ph.D., for his technical review and editing 

assistance and Richard Green for his cover design. 
Appendix A 

REFERENCES 

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 49-2004. Class II (laminar flow) biosafety cabinetry. Ann Arbor (MI); 2004. 

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. 
Appendix A 

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 l8-l9; Frederick, 

MD: Litton Bionetics, Inc.; 1979. 

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). 
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. 

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, 
Appendix B 

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. 

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; 
Appendix B 

• 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. 

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. 
Appendix B 

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 precleaned. For example, a steam cycle used to sterilize 

precleaned items is 20 minutes at 121°C. When steam sterilization is used to decontaminate 

items that have a high bioburden 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. 

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. 
Appendix B 

Chemical germicides used for decontamination range in activity from high-level 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 (see below). 

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 are: 

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 at: 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 
Appendix B 

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 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 

TABLE 2 

ACTIVITY LEVELS OF SELECTED LIQUID GERMICIDES a 

PROCEDURE/PRODUCT AQUEOUS CONCENTRATION ACTIVITY LEVEL 

STERILIZATION

glutaraldehyde 

hydrogen peroxide 

formaldehyde 

chlorine dioxide 

peracetic acid 

variable 

6-30% 

6-8%b 

variable 

variable 

DISINFECTION

glutaraldehyde 

ortho-phthalaldehyde 

hydrogen peroxide 

formaldehyde 

chlorine dioxide 

peracetic acid 

chlorine compounds c

alcohols(ethyl,isopropyl) d 

phenolic compounds 

iodophor compounds e

quaternary ammonium 

compounds 

variable 

0.5% 

3-6% 

1-8% 

variable 

variable 

500 to 5000 mg/L free/ 

available chlorine 

70% 

0.5 to 3% 

30-50 mg/L free iodine up to 

10,000 mg/L available iodine 

0.1 - 0.2% 

High to intermediate 

High 

High to intermediate 

High to low 

High 

High 

Intermediate 

Intermediate 

Intermediate to low 

Intermediate to low 

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 broad-spectrum (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). 

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. 
Appendix B 

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 9). 

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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/ncidod/hip/enviro/guide.htm 

8. Tearle P. Decontamination by fumigation. Commun Dis Public Health. 2003;6:166-8. 

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. 
Appendix B 

14. Krause J, McDonnell G, Riedesel H. Biodecontamination of animal rooms and heatsensitive 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. 
Appendix C 

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 may be obtained at the Internet website: http://hazmat.dot.gov. 

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).
Appendix C 

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 Government 

Printing Office by calling Monday through Friday, 7:30 a.m. - 9:00 p.m. EST: 202- 512-1800, 

866-512-1800 (toll free), or from the Internet at: http://bookstore.gpo.gov/http://pe.usps.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 from the Internet website: 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 Internet website: 

http://www.icao.org. 

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 can be obtained from the 

Internet websites: http://www.iata.org/index.htm or http://www.who.int/en/, or by contacting the 

IATA Customer Care office at: Tel: +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 can be found at the CDC website: 

http://www.cdc.gov/od/eaipp/. 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 
Appendix C 

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 can be found at: 

http://www.cdc.gov/od/sap. 

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 

Internet at: http://www.aphis.usda.gov/vs/ncie/. Completed permit applications may be submitted 

electronically through https://web01.aphis.usda.gov/ias.nsf/Mainform?OpenForm; by Fax to 

(301) 734-3652; or by mail: 

USDA APHIS VS 

National Center for Import and Export 

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 can be found at 

http://www.aphis.usda.gov/programs/ag_selectagent/index.html. 

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 through the Internet at: http://www.aphis.usda.gov./ppq/permits. 

Export of Etiologic Agents of Humans, Animals, Plants and Related Materials. Department of 

Commerce (DoC). 15 CFR Parts 730 to 799. This regulation requires that exporters of a wide 

variety of etiologic agents of human, plant and animal 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 through the Internet at: http://bxa.fedworld.gov, 

http://www.access.gpo.gov/bis/index.html, or http://www.bis.doc.gov/.

Transfer of CDC Select Agents and Toxin. 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. 
Appendix C 

The regulations, Select Agent Program forms, and additional guidance can be found at the CDC 

website: http://www.cdc.gov/od/sap. 

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 can be found at the APHIS website: 

http://www.aphis.usda.gov/programs/ag_selectagent/index.html 

TRANSFER OF USDA PLANT PESTS 

The movement of Plant Pests is regulated under two distinct and separate regulations: 7 CFR 

Part 331. Agricultural Bioterrorism Protection Act of 2002; Possession, Use, and Transfer of 

Biological Agents and Toxins and 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 through the Internet at: 

http://www.aphis.usda.gov/programs/ag_selectagent/index.html. 

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 
Appendix C 

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 -40 oC to +55 oC (-40 oF to 131 oF). 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 

FIGURE 1 

A CATEGORY A UN SSTANDARD TRIPLE PACKAGING 
Appendix C 

346

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 after January 1, 2007. 

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 leakproof primary receptacle, leakproof 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. 
Appendix C 

347

FIGURE 2

A CATEGORY B NON-SPECIFICATION TRIPLE PACKAGING
Appemdix D 

348

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 BSL-3-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. 
Appemdix D 

349

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 BSL-3-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. 

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 
Appemdix D 

350

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, etc.). 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 

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 
Appemdix D 

351

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). 

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. 
Appemdix D 

352

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 enter and exit through a clothing change and shower room, have a double-door 

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. 
Appemdix D 

353

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 non-containment/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. 

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 
Appemdix D 

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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 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. 
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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 virusa,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

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 Rinderpest virus a, b, c 
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(Glanders) a, b 

Burkholderia pseudomallei a, b Sheep pox virus a, b 

Camelpox virus b Spring Viremia of Carp virus 

Classical swine fever virusa, 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. 

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. 
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357

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, doublestranded 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 50o

C, 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 ASFV-infected 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 

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. 

CONTAINMENT RECOMMENDATIONS 

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 USDA-approved BSL-3-Ag 

facility for loosely housed animals. Special consideration should be given to infected 

vector control. 
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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 

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. 

Containment Recommendations 

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 enhancements as required by the 

USDA and in vivo in a USDA-approved ABSL-3 animal facility with enhancements. 

Blood, serum, or tissues taken from equids in areas where AHSV exists are potential 
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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 

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. 

Containment Recommendations 

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 
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the USDA and in vivo in a USDA-approved ABSL-3 animal facility with enhancements. 

Special consideration should be given to infected vector containment. 

SPECIAL 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 1-24. 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. 

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. 
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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 

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. 

Containment Recommendations 

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 USDA-approved 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. 

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 
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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 lipid-enveloped 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 

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. 

Containment Recommendations 

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. 
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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. 

LABORATORY SAFETY 

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. 

Containment Recommendations 

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. 
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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 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. 
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LABORATORY SAFETY 

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. 

Containment Recommendations 

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 (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. 
Appemdix D 

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LABORATORY SAFETY 

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. 

Containment Recommendations 

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 9CFR 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. 

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 bloodsucking 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 
Appemdix D 

367

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 

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. 

Containment Recommendations 

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. 

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 

 
Appemdix D 

368

LABORATORY SAFETY 

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. 

Containment Recommendations 

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 ISAVinfected 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. 

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 
Appemdix D 

369

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 

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. 

Containment Recommendations 

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. 

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 wildebeestassociated malignant catarrhal fever virus (MCFV), African form MCFV, and exotic 

MCFV. It also was previously called bovine herpesvirus 3. 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 
Appemdix D 

370

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 

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. 

Containment Recommendations 

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. 

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. 
Appemdix D 

371

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 

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. 

Containment Recommendations 

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. 

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 bio-containment 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 paramyxovirus 1. All strains are readily propagated 
Appemdix D 

372

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 

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. 

Containment Recommendations 

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 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 9CFR 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. 
Appemdix D 

373

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, non-infectious, 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 

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 susceptible sheep or goat 

population, which would necessitate USDA emergency procedures to contain and 

eradicate the disease. 

Containment Recommendations 

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. 
Appemdix D 

374

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 sub-Saharan 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 

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. 

Containment Recommendations 

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. 
Appemdix D 

375

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 

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 

Containment Recommendations 

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 USDAapproved 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. 
Appemdix D 

376

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 

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 population, which would 

necessitate USDA emergency procedures to contain and eradicate the disease. 

Containment Recommendations 

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 
Appemdix D 

377

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 

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 

Containment Recommendations 

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. 
Appemdix D 

378

REFERENCES 

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2. Mebus CA. African swine fever. Adv Virus Res. 1988;35:251-69. 

3. Mebus CA. African swine fever. In: Mebus CA, editor. Foreign animal diseases. 

Richmond (VA): U.S. Animal Health Association; 1998. p. 52-61. 

4. M’Fadyean J. African horse sickness. J Comp Path Ther. 1900;13:1-20.

5. Erasmus BJ. African horse sickness. In: Mebus CA, editor. Foreign animal 

diseases. Richmond (VA): U.S. Animal Health Association; 1998. p. 41-51. 

6. Della-Porta AJ, O'Halloran ML, Parsonson M, et al. Akabane disease: isolation of 

the virus from naturally infected ovine foetuses. Aust Vet J. 1977;53:51-2. 

7. St. George TD. Akabane. In: Mebus CA, editor. Foreign animal diseases. 

Richmond (VA): U.S. Animal Health Association; 1998. p. 62-70. 

8. Bowne JG. Bluetongue disease. Adv Vet Sci Comp Med. 1971;15:1-46. 

9. Stott JL. Bluetongue and epizootic hemorrhagic disease. In: Mebus CA, editor. 

Foreign animal diseases. Richmond (VA): U.S. Animal Health Association; 1998. 

p. 106-17. 

10. Van Oirschot JT, Terpstra C. Hog cholera virus. In: Pensaert MB, editor. Virus 

infections of porcines. New York: Elsever Science Publishers; 1989. p. 113-30. 

11. Van Oirschot JT. Classical swine fever. In: Straw BE, D’Allaire S, Mengelng W, 

et al. Diseases of swine. 8th ed. Ames (IA): Iowa State University Press; 1999. p. 

159-72. 

12. Dulac DC. Hog cholera. In: Mebus CA, editor. Foreign animal diseases. 

Richmond (VA): U.S. Animal Health Association; 1998. p. 273-82. 

13. Thiaucourt F. Contagious bovine pleuropneumonia. In: Manual of standards for 

diagnostic tests and vaccines for terrestrial animals: mammals, birds and bees. 

Paris: Office International des Épizooties; 2004. p. 163-74. 

14. Contagious bovine pleuropneumonia. In: Aiello SE, Mays A, editors. The Merck 

veterinary manual. 8th ed. Whitehouse Station (NJ): Merck and Company; 1998. 

p. 1078-9. 

15. Brown, C. Contagious bovine pleuropneumonia. In: Mebus CA, editor. Foreign 

animal diseases. Richmond (VA): U.S. Animal Health Association; 1998. p. 154-

60. 

16. Cottew GS. Overview of mycoplasmoses in sheep and goats. Isr J Med Sci. 

1984;20:962-4. 

17. DaMassa AJ, Wakenell PS, Brooks DL. Mycoplasmas of goats and sheep. J Vet 

Diagn Invest. 1992;4:101-13. 

18. Mare JC. Contagious caprine pleuropneumonia. In: Mebus CA, editor. Foreign 

animal diseases. Richmond (VA): U.S. Animal Health Association; 1998. p. 161-

9. 

19. Cottew GS, Breard A, DaMassa AJ. Taxonomy of the Mycoplasma mycoides

cluster. Isr. J Med Sci. 1987;23:632-5. 

20. Christiansen C, Erno H. Classification of the F38 group of caprine Mycoplasma 

strains by DNA hybridization. J Gen Microbiol. 1982;128:2523-6. 
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21. DaMassa AJ, Holmberg CA, Brooks DL. Comparison of caprine mycoplasmosis 

caused by Mycoplasma capricolum, M. mycoides subsp. mycoides, and M. 

putrefasciens. Isr J Med Sci. 1987;23:636-40. 

22. House J. Foot and mouth disease. In: Mebus CA, editor. Foreign animal diseases. 

Richmond (VA): U.S. Animal Health Association; 1998. p. 213-24. 

23. Kitching RP, Barnett PV, Mackay DKJ, et al. Foot and Mouth Disease. In: 

Manual of standards for diagnostic tests and vaccines for terrestrial animals: 

mammals, birds and bees. Paris: Office International des Épizooties; 2004. p. 111-

128. 

24. Mare J. Heartwater. In: Mebus CA, editor. Foreign animal diseases. Richmond 

(VA): U.S. Animal Health Association; 1998. p. 253-64. 

25. Lee CS, O’Bryen PJ, editors. Biosecurity in aquaculture production systems: 

exclusion of pathogens and other undesirables. Baton Rouge (LA): World 

Aquaculture Society; 2003. p. 293. 

26. Miller O, Cipriano RC. International response to infectious salmon anemia: 

prevention, control and eradication. Proceedings of a Symposium; 2002 Sep 3-4; 

New Orleans, LA. Washington, DC: US Department of Agriculture, US 

Department of the Interior and the US Department of Commerce; 2003. 

27. Office International des Épizooties. Infectious salmon anaemia. In: Manual of 

diagnostic tests for aquatic animals. 4th ed. Paris: Office International des 

Épizooties; 2003. p. 152-61. 

28. Bruno DW, Alderman DJ, Schlotfeldt HJ. What should I do? A practical guide for 

the marine fish farmer. Dorset (England): European Association of Fish 

Pathologists; 1995. 

29. Kitching RP. Lumpy skin disease. In: Manual of standards for diagnostic tests and 

vaccines for terrestrial animals: mammals, birds and bees. Paris: Office 

International des Épizooties; 2004. p. 175-84. 

30. Lumpy skin disease. In: Aiello SE, Mays A, editors. The Merck veterinary 

manual. 8th ed. Whitehouse Station (NJ): Merck and Company; 1998; p. 621-2. 

31. House JA. Lumpy skin disease. In: Mebus CA, editor. Foreign animal diseases. 

Richmond (VA): U.S. Animal Health Association; 1998. p. 303-10. 

32. Heuschele WP. Malignant catarrhal fever. In: Mebus CA, editor. Foreign 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. 

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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. 
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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 

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. 
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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 

Riverdale, Maryland 20737-1231 

Telephone: (301) 734-8896 

http://www.aphis.usda.gov/ppq/permits/agr_bioterrorism/
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Appendix E 

 Arthropod Containment Guidelines (ACG) 

An ad hoc committee of concerned vector biologists including members of the American 

Committee Medical Entomology, 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 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 are available online from the AMCE 

website at www.astmh.org/SIC/acme.cfm. 

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. 
Appemdix E 

383

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. 
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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 C.F.R. Part 73, 7 C.F.R. Part 331, and 9 C.F.R. 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 websites (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 on the Internet at www.cdc.gov/od/sap and 

www.aphis.usda.gov/programs/ag_selectagent/index.html. Information is available at 

www.selectagents.gov.
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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 is 

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 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" include: 
Appemdix G 

386

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 have been completed in a timely manner 

and were effective. 

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 for subjects such as pest identification, biology, and 

sanitation, which can promote understanding and cooperation with the goals of the IPM 

program. 

RECORD KEEPING 

 

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. 
Appemdix G 

387

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. 
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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 tissuesshould 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 wellcharacterized). 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. 
Appemdix H 

389

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. 
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390

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. 
Appemdix I 

391

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, safety glasses 

and disposable facemask, or a face shield, should be worn. 

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. 
Appemdix I 

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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 hollow-bore 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. 

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. 
Appemdix I 

393

ADDITIONAL PRECAUTIONS 

Experiments should be planned to eliminate or minimize work with dry toxin (e.g. freezedried 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, and some protein toxins possess a capacity to re-fold, and 

partially reverse 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. 
Appemdix I 

394

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 (See 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 (See 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. 
Appemdix I 

395

TABLE 1 

PHYSICAL INACTIVATION OF SELECTED TOXINS 

TOXIN STEAM 

AUTOCLAVE 

DRY HEAT 

(10 MIN) 

FREEZETHAW GAMMA 

IRRADIATION 

Botulinum neurotoxin Yesa > 100oCb Noc Incompleted 

Staphylococcal 

Enterotoxin 

Yese > 100oC; refoldsf Nog Incompleteh

Ricin Yesi > 100oCi Noj Incompletek 

Microcystin Nol > 260oCm Non ND 

Saxitoxin Nol > 260oC m Non ND 

Palytoxin Nol > 260oCm Non ND 

Tetrodotoxin Nol > 260oCm Non ND 

T-2 mycotoxin Nol > 815oCm Non ND 

Brevetoxin (PbTx-2) Nol > 815oCm Non ND 

Table 1 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 freeze-dried 

state for years and retain toxicity. 
Appemdix I 

396

TABLE 2 

CHEMICAL INACTIVATION OF SELECTED TOXINS 

TOXIN NAOCL 

(30 MIN) 

NAOH 

(30 MIN) 

NAOCL + NAOH 

(30 MIN) 

OZONE 

TREATMENT 

Botulinum 

neurotoxin 

> 0.1%a >0.25 N ND Yesb

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 

Table 2 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. 

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 
Appemdix I 

397

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. 

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. 
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398

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. 
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Appendix J 

NIH Oversight of Research Involving Recombinant Biosafety Issues 

The NIH locus for oversight of recombinant DNA research is the 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 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. 
Appemdix J 

400

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 website: 

www4.od.nih.gov/oba/rac/guidelines/guidelines.html 

The RAC 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://www4.od.nih.gov/oba/. 
Appemdix K 

401

Appendix K 

Resources for Information 

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 

Website: http://www.aaalac.org 

American Biological Safety Association 

American Biological Safety Association 

1200 Allanson Road 

Mundelein, IL 60060-3808 

Telephone: (847) 949-1517 

Fax: (847) 566-4580 

Website: http://www.absa.org/ 

CDC Etiologic Agent Import Permit Program 

Centers for Disease Control and Prevention 

Etiologic Agent Import Permit Program (Mailstop: F-46) 

Atlanta, Georgia 30333 

Telephone: (404) 718-2077 

Fax: (404) 718-2093 

Website: 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 

Atlanta, Georgia 30333 

Telephone: (404) 639-7233 

Fax: (404) 639-2294 

Website: http://www.cdc.gov/od/ohs/ 
Appemdix K 

402

CDC Select Agent Program 

Centers for Disease Control and Prevention 

Division of Select Agents and Toxins (Mailstop: A-46) 

1600 Clifton Road 

Atlanta, Georgia 30333 

Telephone: (404) 718-2000 

Fax: (404) 718-2096 

Website: http://www.cdc.gov/od/sap/ 

Clinical and Laboratory Standards Institute (CLIS)

940 West Valley Road, Suite 1400 

Wayne, PA 19087 

Telephone: (610) 688-0100 

Fax: (610) 688-0700 

Website: http://www.clsi.org/ 

College of American Pathologist (CAP)

1350 I St. N.W. Suite 590 

Washington, DC 20005-3305 

Telephone: (800) 392-9994 

Telephone: (202) 354-7100 

Fax: (202) 354-7155 

Website: http://www.cap.org 

Department of the Army

Department of Defense 

32 CFR 

Parts 626, 627

Biological Defense Safety Program 

Website: www.gpo.gov 

National Animal Disease Center 

National Animal Disease Center 

U.S. Department of Agriculture 

PO Box 70 

2300 Dayton Road 

Ames, Iowa 50010 

Telephone: (515) 663-7200 

Fax: (515) 663-7458 

Website: http://www.ars.usda.gov/Main/site_main.htm?modecode=36-25-30-00 
Appemdix K 

403

National Herpes-B Virus Laboratory

National B Virus Resource Laboratory 

Attention: Dr. Julia Hillard 

Viral Immunology Center 

Georgia State University 

50 Decatur Street 

Atlanta, Georgia 30303 

Telephone: (404) 651-0808 

Fax: (404) 651-0814 

E-mail: http://www2.gsu.edu/~wwwvir/ 

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, Maryland 20892 

Telephone: (301) 496-2960 

Fax: (301) 402-0313 

Website: http://dohs.ors.od.nih.gov/index.htm 

NIH Office of Biotechnology / rDNA Guidelines 

National Institutes of Health 

Office of Biotechnology Activities 

6705 Rockledge Drive, Suite 750, MSC 7985 

Bethesda, Maryland 20892 

Telephone: (301) 496-9838 

Fax: (301) 496-9839 

Website: http://www4.od.nih.gov/oba/ 

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 

Website: http://grants.nih.gov/grants/olaw/olaw.htm 

Occupational Safety and Health Administration

Occupational Safety and Health Administration 

U.S. Department of Labor 

200 Constitution Avenue, NW 

Washington, DC 20210 

Telephone: (800) 321-6742 

Website: http://www.osha.gov/index.html 
Appemdix K 

404

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, Maryland 20737 

Website: http://www.aphis.usda.gov/vs/import_export.htm

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 

Riverdale, Maryland 20737 

Website: http://www.aphis.usda.gov/programs/ag_selectagent/ 

USDA Plant Select Agents & Plant Protection and Quarantine 

U.S. Department of Agriculture 

Animal and Plant Health Inspection Service 

Plant Protection and Quarantine, Permits, Agricultural Bioterrorism 

8700 River Road 

Riverdale, Maryland 20737 

Telephone: (877) 770-5990 

Fax: (301) 734-5786 

Website: http://www.aphis.usda.gov/programs/ag_selectagent/index.html 

 and 

 http://www.aphis.usda.gov/ppq/permits 

US Department of Transportation 

U.S. Department of Transportation 

Hazardous Materials Center 

Pipeline & Hazardous Materials Center 

400 7th Street, S.W. 

Washington, DC 20590 

Telephone: (800) 467-4922

Website: http://hazmat.dot.gov 

US DOC Export Administration Program 

U.S. Department Of Commerce 

Bureau of Industry Security (BIS) 

Export Administration Regulations (EAR) 

14th Street and Constitution Avenue, NW 

Washington, DC 20230 

Telephone: (202) 482-4811 

Website: http://www.access.gpo.gov/bis/index.html 
Appemdix K 

405

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 

Websites: http://www.who.int/csr/labepidemiology/projects/biosafetymain/en/ 

and 

http://www.who.int/csr/resources/publications/csrpublications/en/index2.html 
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 
Appemdix L 

407

DOD Department of Defense 

DOL Department of Labor 

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 Hanta 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 
Appemdix L 

408

ISAV Infectious Salmon Anemia Virus 

LAI Laboratory-Associated Infections 

LCM Lymphocytic Choriomeningitis 

LCMV Lymphocytic Choriomeningitis Virus 

LD Lethal Dose 

lfpm 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 

NIH Guidelines NIH Guidelines for Research Involving Recombinant DNA 

Molecules 

NIOSH National Institutes 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 
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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 Encephalopathies 

TSE Transmissible Spongiform Encephalopathies 

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