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MEDICAL ASPECTS OF BIOLOGICAL WARFARE 






The Coat of Arms 
1818 

Medical Department of the Army 


iii 


A 1976 etching by Vassil Ekimov of an 
original color print that appeared in 
The Military Surgeon, Vol XLI, No 2,1917 




















Textbooks of Military Medicine 


Published by the 


Office of The Surgeon General 
Borden Institute 

US Army Medical Department Center and School 
Health Readiness Center of Excellence 
Fort Sam Houston, Texas 


Editor in Chief 


John H. Garr, MD, MSE, FACEP 
Colonel, MC, US Army 
Director, Borden Institute 


The TMM Series 


Published Textbooks 

Medical Consequences of Nuclear Warfare (1989) 

Conventional Warfare: Ballistic, Blast, and Burn Injuries 
(1991) 

Occupational Health: The Soldier and the Industrial Base 
(1993) 

Military Dermatology (1994) 

Military Psychiatry: Preparing in Peace for War (1994) 

Anesthesia and Perioperative Care of the Combat 
Casualty (1995) 

War Psychiatry (1995) 

Medical Aspects of Chemical and Biological Warfare 
(1997) 

Rehabilitation of the Injured Soldier, Volume 1 (1998) 
Rehabilitation of the Injured Soldier, Volume 2 (1999) 
Medical Aspects of Harsh Environments, Volume 1 (2002) 
Medical Aspects of Harsh Environments, Volume 2 (2002) 
Ophthalmic Care of the Combat Casualty (2003) 

Military Medical Ethics, Volume 1 (2003) 

Military Medical Ethics, Volume 2 (2003) 

Military Preventive Medicine, Volume 1 (2003) 

Military Preventive Medicine, Volume 2 (2005) 

Recruit Medicine (2006) 

Medical Aspects of Biological Warfare (2007) 

Medical Aspects of Chemical Warfare (2008) 

Care of the Combat Amputee (2009) 

Combat and Operational Behavioral Health (2011) 

Military Quantitative Physiology: Problems and Concepts 
in Military Operational Medicine (2012) 

Medical Consequences of Radiological and Nuclear 
Weapons (2013) 

Forensic and Ethical Issues in Military Behavioral Health 
(2014) 

Combat Anesthesia: The First 24 Hours (2015) 

Otolaryngology/Head and Neck Surgery Combat Casu¬ 
alty Care in Operation Iraqi Freedom and Operation 
Enduring Freedom (2015) 

Medical Aspects of Biological Warfare (2018) 















The bacteria that cause anthrax (green ) are being enveloped by an immune system phagocytic cell (purple). These bacteria live 
in soil and form dormant spores that can survive for decades. When spores enter humans through the respiratory or gastro¬ 
intestinal tracts or the skin, they germinate to bacilli and rapidly increase in number. Phagocytic cells of the host immune 
system are essential for ingesting and killing the bacteria, and this is enhanced after vaccination. This is but one example to 
illustrate the important interactions between pathogens and the infected host's immune system. 

Photograph: Courtesy of Sarah Guilman, Camenzind G. Robinson, and Arthur M. Friedlander, US Army Medical Research 
Institute of Infectious Diseases, Fort Detrick, Maryland. 



MEDICAL ASPECTS of 
BIOLOGICAL WARFARE 


Senior Editors 


Joel Bozue, PhD 
Division of Bacteriology 

US Army Medical Research Institute of Infectious Diseases 

Christopher K. Cote, PhD 
Division of Bacteriology 

US Army Medical Research Institute of Infectious Diseases 

Pamela J. Glass, PhD 
Viral Biology Department, Virology Division 
US Army Medical Research Institute of Infectious Diseases 


Office of The Surgeon General 
Borden Institute 

US Army Medical Department Center and School 
Health Readiness Center of Excellence 
Fort Sam Houston, Texas 


2018 




Editorial Staff: Joan Redding 

Senior Production Editor 


Marcia Metzgar 
Volume Editor 


Vivian Mason 
Volume Editor 


Ronda Lindsay 
Technical Editor 


Douglas Wise 
Senior Layout Editor 


This volume was prepared for military medical educational use. The focus of the information is 
to foster discussion that may form the basis of doctrine and policy. The opinions or assertions 
contained herein are the private views of the authors and are not to be construed as official or as 
reflecting the views of the Department of the Army or the Department of Defense. 

Dosage Selection: 

The authors and publisher have made every effort to ensure the accuracy of dosages cited herein. 
However, it is the responsibility of every practitioner to consult appropriate information sources 
to ascertain correct dosages for each clinical situation, especially for new or unfamiliar drugs 
and procedures. The authors, editors, publisher, and the Department of Defense cannot be held 
responsible for any errors found in this book. 

Use of Trade or Brand Names: 

Use of trade or brand names in this publication is for illustrative purposes only and does not 
imply endorsement by the Department of Defense. 

Neutral Language: 

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


CERTAIN PARTS OF THIS PUBLICATION PERTAIN TO COPYRIGHT RESTRICTIONS. 

ALL RIGHTS RESERVED. 

NO COPYRIGHTED PARTS OF THIS PUBLICATION MAY BE REPRODUCED OR 
TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL (IN¬ 
CLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL 
SYSTEM), WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER OR COPYRIGHT 
OWNER. 

Published by the Office of The Surgeon General 
Borden Institute 

US Army Medical Department Center and School 
Health Readiness Center of Excellence 
Fort Sam Houston, Texas 


Library of Congress Cataloging-in-Publication Data 


Names: Bozue, Joel, editor. I Cote, Christopher K., editor. I Glass, Pamela 
J., editor. I Borden Institute (U.S.), issuing body. 

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

Christopher K. Cote, Pamela J. Glass. 

Other titles: Textbooks of military medicine. 

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

Borden Institute, US Army Medical Department Center and School, Health 
Readiness Center of Excellence, 2018. I Series: Textbooks of military 
medicine I Includes bibliographical references and index. 

Identifiers: LCCN 2017057681 I ISBN 9780160941597 

Subjects: I MESH: Bioterrorism—prevention & control I Communicable Disease 
Control—methods I Military Medicine—methods 

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

lccn.loc.gov/2017057681 

PRINTED IN THE UNITED STATES OF AMERICA 

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




Contents 


Contributors xi 

Peer Reviewers xvii 

Foreword by The Surgeon General xxi 

Preface xxiii 

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

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

2. Epidemiology of Biowarfare and Bioterrorism 37 

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

3. Food, Waterborne, and Agricultural Diseases 71 

Zygmunt F. Dembek, and Edwin L. Anderson 

4. Consequence Management: The Local and National Response 93 

Neal E. Woollen, and Gary W. Carter 

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

Theodore J. Cieslak 

6. Anthrax 129 

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

7. Brucellosis 159 

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

8. Glanders 177 

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

9. Melioidosis 223 

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

10. Plague 247 

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

11. Tularemia 285 

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

Zygmunt F. Dembek 

12. Q Fever 305 

Christina M. Farris and James E. Samuel 

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

Acinetobacter Infections as a Case Study 

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

14. Botulinum Toxin 337 

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

15. Clostridium Perfringens Epsilon Toxin 361 

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


IX 


373 


16. Ricin 

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

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

Streptococcus Pyogenes 

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

18. Toxins from Venoms and Poisons 415 

Scott A. Weinstein and Julian White 

19. Marine Algal Toxins of Concern as Intentional Contaminants 461 

Mark A. Poli and Mark R. Withers 

20. Alpha virus Encephalitides 479 

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

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

21. Hemorrhagic Fever-Causing Mammarenaviruses 517 

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

22. Henipaviruses 547 

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

23. Filoviruses 569 

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

24. Smallpox and Related Orthopoxvirus Infections 615 

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

James V. Lawler, and James W. Martin 

25. Emerging Infectious Diseases and Future Threats 645 

Chris A. Whitehouse and Brett Beitzel 

26. Laboratory Identification of Biological Threats 701 

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

27. Medical Countermeasures 751 

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

28. Future Prospects of Vaccines and Antibodies in Biodefense 823 

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

29. Aerobiology: History, Development, and Programs 855 

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

30. Biosafety 869 

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

31. Biological Surety 895 

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

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

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

Abbreviations and Acronyms xxvii 

Index xxxiii 


x 


Contributors 


ARTHUR O. ANDERSON, MD 

Colonel (Retired), Medical Corps, US Army; Director, Office of 
Human Use and Ethics, US Army Medical Research Institute of 
Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 
21702 

EDWIN L. ANDERSON, MD 

Colonel (Retired), Medical Corps, US Army; Research Professor, 
Department of Internal Medicine, Division of Infectious Diseases, 
Allergy and Immunology, Saint Louis University School of Medi¬ 
cine, 1100 South Grand Boulevard, St. Louis, Missouri 63104 

GILLIAN BARTH, BS 

Veterinary Technician, Veterinary Department, Wilson College, 
1015 Philadelphia Avenue, Chambersburg, Pennsylvania 17201 

SINA BAVARI, PhD 

Science Director, US Army Medical Research Institute of Infec¬ 
tious Diseases, 1425 Porter Street, Room 900, Fort Detrick, 
Maryland 21702; formerly. Division Chief, Toxicology Division, 
US Army Medical Research Institute of Infectious Diseases, 1425 
Porter Street, Room 900, Fort Detrick, Maryland 

BRETT BEITZEL, PhD 

Principal Investigator, Center for Genomic Sciences, US Army 
Medical Research Institute of Infectious Diseases, 1425 Porter 
Street, Fort Detrick, Maryland 21702 

JOEL BOZUE, PhD 

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

PAUL J. BRETT, PhD 

Associate Professor, Department of Microbiology and Immunol¬ 
ogy, University of South Alabama, 610 Clinic Drive, Laboratory of 
Infectious Diseases Building, Mobile, Alabama 36688 

ELIZABETH S. BROWN, PhD 

Medical-Technical Writer and Editor, Department of Clinical Re¬ 
search, Division of Medicine, US Army Medical Research Institute 
of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 
21702 

CRYSTAL W. BURKE, PhD 

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

MARY N. BURTNICK, PhD 

Assistant Professor, Department of Microbiology and Immunolo¬ 
gy, University of South Alabama, 5851 USA Drive North, Medical 
Science Building, Mobile, Alabama 36688 

GARY W. CARTER 

Director, Field Operations and Training, National Strategic Re¬ 
search Institute, 3925 Dewey Avenue, Omaha, Nebraska 68198 


AMERICA M. CERALDE, MBA, MT(AMT) 

Alternate Surety Officer and Chief, Personnel Reliability Program 
Branch, Biosurety Division, US Army Medical Research Institute 
of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 
21702 

MATTHEW S. CHAMBERS, MD, MPH 

Lieutenant Colonel, Medical Corps, US Army; Chief, Preventive 
Medicine, Fort Wainwright, 4077 Neely Road, Fort Wainwright, 
Alaska 99703; formerly. Chief, Field Studies, Division of Medi¬ 
cine, US Army Medical Research Institute of Infectious Diseases, 
1425 Porter Street, Fort Detrick, Maryland 

GEORGE W. CHRISTOPHER, MD, FACP 

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

THEODORE J. CIESLAK, MD 

Colonel, Medical Corps, US Army; Pediatric Infectious Diseases 
Physician, San Antonio Military Medical Center, Department of 
Pediatrics, 3551 Roger Brooke Drive, Fort Sam Houston, Texas, 
78234 

CHRISTOPHER K. COTE, PhD 

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

JON M. DAVIS, PhD 

Lieutenant Colonel, Medical Service Corps, US Army; Depart¬ 
ment of Defense Liaison to the ASPR, Department of Health 
and Human Services, 200 C Street, SW, Washington, DC 20024; 
formerly. Deputy Medical Coordinator, Office of the Assistant 
Secretary of Defense for Nuclear, Chemical, and Biological De¬ 
fense, 3050 Defense Pentagon, Washington, DC 

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

Colonel (Retired), Medical Service Corps, US Army Reserve; As¬ 
sociate Professor, Department of Military and Emergency Medi¬ 
cine, Uniformed Services University of the Health Sciences, 4301 
Jones Bridge Road, Bethesda, Maryland 20814; formerly. Chief 
Biodefense Epidemiology and Education & Training Programs, 
Division of Medicine, US Army Medical Research Institute of 
Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

DAVID DeSHAZER, PhD 

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

LESLEY C. DUPUY, PhD 

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

SAMUEL S. EDWIN, PhD 

Responsible Official and Surety Officer; Chief, Biosurety Division, 
US Army Medical Research Institute of Infectious Diseases, 1425 
Porter Street, Fort Detrick, Maryland 21702 


xi 


EDWARD M. EITZEN, MD, MPH 

Colonel (Retired), Medical Corps, US Army; Senior Partner, 
Biodefense and Public Health Programs, Martin-Blanck and As¬ 
sociates, 2034 Eisenhower Avenue, Suite 270, Alexandria, Virginia 
22314-4678; formerly. Commander, US Army Medical Research 
Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 
Maryland 

CHRISTINA M. FARRIS, PhD 

Lieutenant, Medical Service Corps, US Navy; Viral and Rickettsial 
Diseases Department, Naval Medical Research Center, 503 Robert 
Grant Avenue, Silver Spring, Maryland 20910 

ARTHUR M. FRIEDLANDER, MD 

Colonel (Retired), Medical Corps, US Army; Senior Scientist, US 
Army Medical Research Institute of Infectious Diseases, 1425 Por¬ 
ter Street, Fort Detrick, Maryland 21702, and Adjunct Professor of 
Medicine, Uniformed Services University of the Health Sciences, 
4301 Jones Bridge Road, Bethesda, Maryland 20814 

JEFFREY FROUDE II, PhD 

Clinical Pharmacology Fellow, Experimental Therapeutics Divi¬ 
sion, Walter Reed Institute of Research, 503 Robert Grant Avenue, 
Silver Spring, Maryland 21702 

DANIEL M. GERSTEIN, PhD 

Colonel (Retired), US Army; Adjunct Professor, School of Interna¬ 
tional Studies, American University, 4400 Massachusetts Avenue, 
NW, Washington, DC 20016; formerly. Undersecretary (Acting) 
and Deputy Undersecretary, Science and Technology Directorate, 
Department of Homeland Security, Washington DC 

PAMELA J. GLASS, PhD 

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

ARTHUR J. GOFF, PhD 

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

JOHN C. GORBET, PhD 

Captain, Medical Service Corps, US Army; Chief, Department of 
Aerosol Services, US Army Medical Research Institute of Infec¬ 
tious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702 

BRIDGET CARR GREGORY, DVM, MPH 

Colonel, US Air Force, Biomedical Sciences Corps; Deputy Chief 
of Staff, Defense Threat Reduction Agency/Strategic Command 
Center for Combating Weapons of Mass Destruction, 8725 John 
J. Kingman Road, Fort Belvoir, Virginia 22060; formerly. Chief, 
Education and Training, Division of Medicine, US Army Medical 
Research Institute of Infectious Diseases, 1425 Porter Street, Fort 
Detrick, Maryland 

MARTHA L. HALE, PhD 

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

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

Biosafety Officer, Office of Safety, Radiation, and Environment, 
US Army Medical Research Institute of Infectious Diseases, 1425 
Porter Street, Fort Detrick, Maryland 21702; formerly. Fellow, Na¬ 
tional Biosafety and Biocontainment Training Program, National 
Institutes of Health, 13 South Drive, Bethesda, Maryland 


MARY KATE HART, PhD 

Chief Scientific Officer, Dynport Vaccine Company, 64 Thomas 
Johnson Drive, Frederick, Maryland 21702; formerly. Chief, Virol¬ 
ogy Division, US Army Medical Research Institute of Infectious 
Diseases, 1425 Porter Street, Fort Detrick, Maryland 

ROBERT J. HAWLEY, PhD, RBP, CBSP 

Consultant, Biological Safety and Security, Alliance Biosci¬ 
ences, 6810 Deerpath Road, Suite 315, Elkridge, Maryland 21075; 
formerly Senior Advisor, Science, Midwest Research Institute, 

110 Thomas Johnson Drive, Suite 170, Frederick, Maryland; for¬ 
merly, Chief, Safety and Radiation Protection, US Army Medical 
Research Institute of Infectious Diseases, 1425 Porter Street, Fort 
Detrick, Maryland 

MATTHEW J. HEPBURN, MD 

Colonel, MC, US Army; Program Manager, Biological Technolo¬ 
gies Office, Defense Advanced Research Projects Agency (DAR- 
PA), 675 North Randolph Street, Room 08-140, Arlington, Virginia 
22203-2114; formerly. Clinical Research Unit Lead, US Army Medi¬ 
cal Research Institute of Infectious Diseases, 1425 Porter Street, 

Fort Detrick, Maryland 

SHELLEY P. HONNOLD, DVM, PhD 

Lieutenant Colonel, Veterinary Corps, US Army; Director, Re¬ 
search Support and Chief, Pathology Division, US Army Medi¬ 
cal Research Institute of Infectious Diseases, 1425 Porter Street, 
Fort Detrick, Maryland 21702; formerly. Biodefense Research 
Pathologist, Pathology Division, US Army Medical Research 
Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 
Maryland 

DAVID L. HOOVER, MD 

Colonel (Retired), Medical Corps, US Army; Senior Scientific Ad¬ 
visor, Clinical Research Management, 1265 Ridge Road, Hinckley, 
Ohio 44233; formerly Medical Director, Dynport Vaccine Com¬ 
pany LLC, a CSC Company, 64 Thomas Johnson Drive, Fred¬ 
erick, Maryland, and Scientific Coordinator, Brucella Program, 
Department of Bacterial Diseases, Walter Reed Army Institute of 
Research, Silver Spring, Maryland 

JOHN W. HUGGINS, PhD 

Retired; formerly. Chief, Viral Therapeutics Branch, US Army 
Medical Research Institute of Infectious Diseases, 1425 Porter 
Street, Fort Detrick, Maryland 21702 

M. SOFI IBRAHIM, MSC, PhD 

Lieutenant Colonel, Medical Service Corps, US Army Reserve; 
Research Microbiologist, Edgewood Chemical Biological Center, 
Ricketts Point Road, Gunpowder, Maryland 21010; formerly. 
Microbiologist, Division of Virology, US Army Medical Research 
Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 
Maryland 

PETER B. JAHRLING, PhD 

Captain (Retired), Medical Service Corps, US Army; Director, Na¬ 
tional Institutes of Health/Division of Clinical Research/National 
Institute of Allergy and Infectious Diseases/Integrated Research 
Facility, 8200 Research Plaza, Room 1A-111A, Fort Detrick, 
Maryland 21702; formerly. Principal Scientific Advisor (Senior 
Research Scientist), US Army Medical Research Institute of Infec¬ 
tious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

SARA C. JOHNSTON, PhD 

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


xii 


SHELLEY C. JORGENSEN, PhD, MT(ASCP) 

Lieutenant Colonel, Medical Services, US Army; Chief, Surety 
Branch, US Army Office of The Surgeon General/US Army Medi¬ 
cal Command, G-34 Protection Division, DHHQ, 7700 Arlington 
Boulevard, Falls Church, Virginia 22042 

TODD M. KIJEK 

Major, Medical Service Corps, US Army; PhD candidate and 
Chief, Molecular and Translational Sciences Department, US 
Army Medical Research Institute of Infectious Diseases, 1425 
Porter Street, Fort Detrick, Maryland 21702; formerly. Research 
Microbiologist, Bacteriology Division, US Army Medical Research 
Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 
Maryland 

JASON KINDRACHUK, PhD 

Staff Scientist, Critical Care Medicine Department, Clinical 
Center, National Institutes of Health, 10 Center Drive, Bethesda, 
Maryland 20814 

MARK G. KORTEPETER, MD, MPH 

Colonel, Medical Corps, US Army; Director, Infectious Disease 
Clinical Research Program, Department of Preventive Medicine, 
Associate Dean for Research, Associate Professor of Preventive 
Medicine and Medicine, Consultant to the Army Surgeon General 
for Biodefense, Uniformed Services University of the Health 
Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814; for¬ 
merly, Deputy Commander, US Army Medical Research Institute 
of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

TERESA KRAKAUER, PhD 

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

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

Virology Lead, National Institutes of Health/National Institute of 
Allergy and Infectious Diseases/Division of Clinical Research/In¬ 
tegrated Research Facility, B-8200 Research Plaza, Room 1A-132, 
Fort Detrick, Maryland 21702; formerly. Research Scholar, Har¬ 
vard Medical School, Department of Microbiology and Molecular 
Genetics, New England Primate Research Center, 1 Pine Hill 
Drive, Southborough, Massachusetts 

JAMES V. LAWLER, MPH, MD 

Commander, Medical Corps, US Navy Reserve; ACESO Director 
and Chief, Clinical Research Department, Biodefense Research Di¬ 
rectorate, Naval Medical Research Center, Fort Detrick, Maryland 
21702; formerly. Chief Medical Officer, Integrated Research Facil¬ 
ity, Fort Detrick, Maryland 

FRANK J. LEBEDA, PhD 

Acting Director and Chief Data Officer, Systems Biology Col¬ 
laboration Center for Environmental Health Research, US Army 
Medical Research and Materiel Command, 568 Doughten Drive, 
Fort Detrick, Maryland 21702; formerly. Civilian Deputy Director, 
Combat Casualty Care Research Program, US Army Medical 
Research and Materiel Command, 504 Scott Street, Fort Detrick, 
Maryland 

BENHUR LEE, MD 

Professor, Microbiology Department, Icahn School of Medicine 
at Mount Sinai, One Gustave L. Levy Place, Annenberg Building, 
Room 17-24, New York, New York 10029 


PATRICIA M. LEGLER, PhD 

Research Biologist, US Naval Research Laboratory, 4555 Overlook 
Avenue, Washington, DC 20375 

EMIL LESHO, DO 

Director, Multidrug-Resistant Organism Repository and Surveil¬ 
lance Network, Walter Reed Army Institute of Research, 503 
Walter Grant Avenue, 1W102, Silver Spring, Maryland 20910 

KENNY L. LIN, MS 

GLP Study Coordinator, Nonclinical Development Division, US 
Army Medical Research Institute of Infectious Diseases, 1425 
Porter Street, Fort Detrick, Maryland 21702; formerly. Regulatory 
Scientist, Department of Virology, US Army Medical Research 
Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 
Maryland 

LUTHER E. LINDLER, PhD 

Biosurveillance Liaison, Armed Forces Health Surveillance Cen¬ 
ter, 11800 Tech Road, Silver Spring, Maryland 20904 

GEORGE V. LUDWIG, PhD 

Deputy Principal Assistant for Research and Technology, US 
Army Medical Research and Materiel Command, 810 Schreider 
Street, Fort Detrick, Maryland 21702; formerly. Science Director, 
US Army Medical Research Institute of Infectious Diseases, 1425 
Porter Street, Fort Detrick, Maryland 

JAMES W. MARTIN, MD, FA CP 

Colonel (Retired), Medical Corps, US Army; Chief of Internal 
Medicine, US Army Healthcare Clinic, Vicenza, APO AE 09630- 
0040; formerly. Chief, Operational Medicine Department, US 
Army Medical Research Institute of Infectious Diseases, 1425 
Porter Street, Fort Detrick, Maryland 

SHANNON S. MARTIN, PhD 

Vaccine Program Manager, Clinical Research Management, 
Business Plans and Programs Office, US Army Medical Research 
Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 
Maryland 21702; formerly. Senior Scientist/Technical Develop¬ 
ment Manager, Nonclinical Research Department, Dynport Vac¬ 
cine Company, 64 Thomas Johnson Drive, Frederick, Maryland 

thomas w. McGovern, md, faad, facms, khs 

Major, Medical Corps, US Army (former); Dermatologist/Mohs 
Surgeon, Fort Wayne Dermatology Consultants, 7881 Carnegie 
Boulevard, Fort Wayne, Indiana 46804; formerly. Ward Officer, 

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

TIMOTHY D. MINOGUE, PhD 

Chief, Molecular Diagnostics Department, Diagnostic Systems 
Division, US Army Medical Research Institute of Infectious Dis¬ 
eases, 1425 Porter Street, Fort Detrick, Maryland 21702 

ELAINE M. MORAZZANI, PhD 

Oak Ridge Institute for Science and Education Fellow, Viral Biol¬ 
ogy Department, Virology Division, US Army Medical Research 
Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 
Maryland 21702; formerly, NRC Postdoctoral Fellow, Virology 
Division, US Army Medical Research Institute of Infectious Dis¬ 
eases, 1425 Porter Street, Fort Detrick, Maryland 


xiii 


ERIC C. MOSSEL, PhD 

Major, Medical Service Corps, US Army Reserve; Microbiolo¬ 
gist, Division of Virology, US Army Medical Research Institute 
of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 
21702; formerly. Science and Technology Advisor, Detachment 
8, Research Development and Engineering Command, 5183 Black- 
hawk Road, Aberdeen Proving Ground, Maryland 

AYSEGUL NALCA, MD, PhD 

Chief, Department of Animal Studies, Center for Aerobiological 
Sciences, US Army Medical Research Institute of Infectious Dis¬ 
eases, 1425 Porter Street, Fort Detrick, Maryland 21702 

DAVID A. NORWOOD, PhD 

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

ARNOLD PARK, PhD 

Postdoctoral Fellow, Microbiology Department, Icahn School of 
Medicine at Mount Sinai, One Gustave L. Levy Place, Annenberg 
Building, Room 17-24, New York, New York 10029 

MICHAEL D. PARKER, PhD 

Retired; formerly. Chief, Viral Biology Department, Virology Divi¬ 
sion, US Army Medical Research Institute of Infectious Diseases, 
1425 Porter Street, Fort Detrick, Maryland 

JULIE A. PAVLIN, MD, PhD, MPH 

Colonel (Retired), Medical Corps, US Army; Deputy Director, 
Armed Forces Health Surveillance Center, 11800 Tech Road, 

Silver Spring, Maryland 20904 

BRUNO PETRUCCELLI, MD 

Colonel (Retired), Medical Corps, US Army; formerly. Director, 
Epidemiology and Disease Surveillance, US Army Center for 
Health Promotion and Preventive Medicine, Aberdeen Proving 
Ground, Maryland; currently. Senior Physician/Epidemiologist, 
US Military HIV Research Program, Henry M. Jackson Founda¬ 
tion for the Advancement of Military Medicine, 3720-A Rockledge 
Drive, Suite 400, Bethesda, Maryland 20817 

PHILLIP R. PITTMAN, MD, MPH 

Colonel (Retired), US Army; Chief, Department of Clinical Re¬ 
search, Division of Medicine, US Army Medical Research Institute 
of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 
21702 

MARK A. POLI, PhD, DABT 

Research Chemist, Department of Applied Diagnostics, Diagnostic 
Systems Division, US Army Medical Research Institute of Infec¬ 
tious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702 

MICHEL R. POPOFF, PhD, DVM 

Chief, Anaerobic Bacteria and Toxins, Institut Pasteur, 28 Rue du 
Dr Roux, 75724 Paris, France 

BRET K. PURCELL, PhD, MD 

Colonel, Medical Corps, US Army; Deputy Chief, Bacteriology 
Division, US Army Medical Research Institute of Infectious Dis¬ 
eases, 1425 Porter Street, Fort Detrick, Maryland 21702 


SHELI R. RADOSHITZKY, PhD 

Principal Investigator, Molecular and Translational Sciences Divi¬ 
sion, US Army Medical Research Institute of Infectious Diseases, 
1425 Porter Street, Fort Detrick, Maryland 21702; formerly. Post¬ 
doctoral Fellow, Toxicology Division, US Army Medical Research 
Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 
Maryland 

DOUGLAS S. REED, PhD 

Associate Professor, Aerobiological Manager, RBL, Department 
of Immunology, University of Pittsburgh, 3501 Fifth Avenue, 
Pittsburgh, Pennsylvania 15261; formerly. Microbiologist, Center 
for Aerobiological Sciences, US Army Medical Research Institute 
of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

R. MARTIN ROOP II, PhD 

Professor, Microbiology and Immunology, Brody School of Medi¬ 
cine, East Carolina University, 600 Moye Boulevard, Room 118, 
Biotechnology Building, Greenville, North Carolina 27834 

VIRGINIA I. ROXAS-DUNCAN, PhD 

Alternate Responsible Official and Chief, Select Agent Manage¬ 
ment Branch, Biosurety Division, US Army Medical Research 
Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 
Maryland 21702 

CHAD J. ROY, PhD 

Associate Professor, Department of Microbiology and Immunol¬ 
ogy, Tulane School of Medicine, 1430 Tulane Avenue, New Or¬ 
leans, Louisiana 70112; and Director, Infectious Disease Aerobiol¬ 
ogy, Division of Microbiology, Tulane National Primate Research 
Center, 18703 Three Rivers Road, Covington, Louisiana 70433 

JANICE M. RUSNAK, MD 

Lieutenant Colonel (Retired), US Air Force; Medical Director, 
Battelle, 1564 Freedman Drive, Fort Detrick, Maryland 21702; 
formerly. Deputy Director of Special Immunizations Program, 

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

KAMAL U. SAIKH, PhD 

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

WENDY SAMMONS-JACKSON, PhD 

Lieutenant Colonel, Medical Service Corps, US Army; Military 
Deputy, Science and Technology Department, US Army Medical 
Research and Materiel Command, 810 Schreider Street, Suite 103, 
Fort Detrick, Maryland 21702; formerly. Deputy Director, Military 
Infectious Diseases Research Program, Fort Detrick, Maryland 

JAMES E. SAMUEL, PhD 

Professor and Chair, Department of Microbial Pathogenesis and 
Immunology, College of Medicine, Texas A&M Health Science 
Center, 8447 State Highway 47, Medical Research and Education 
Building, Building 3112, Bryan, Texas 77807 

ALAN L. SCHMALJOHN, PhD 

Professor, Department of Microbiology and Immunology, Univer¬ 
sity of Maryland School of Medicine, 655 West Baltimore Street, 
HSF-I, 322B, Baltimore, Maryland 21201 


xiv 


RANDAL J. SCHOEPP, PhD 

Chief, Immunodiagnostics and Biologies Department, Diagnostic 
Systems Division, US Army Medical Research Institute of Infec¬ 
tious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702 

MARTINA SIWEK, PhD 

Chief Scientist, Cherokee Nation Technology Solutions in Support 
of Global Emerging Infections Surveillance and Response System, 
Armed Forces Health Surveillance Center, 11800 Tech Road, 

Silver Spring, Maryland 20904; formerly. Science Manager/Liai¬ 
son, Biosurveillance Management Office, Joint Program Executive 
Office, Aberdeen Proving Ground, Maryland 

JONATHAN F. SMITH, PhD 

Executive Vice President and Chief Scientific Officer, PaxVax, 

Inc, 3985 Sorrento Valley Boulevard, San Diego, California 92121; 
formerly. Chief, Viral Biology Department, Virology Division, US 
Army Medical Research Institute of Infectious Diseases, 1425 
Porter Street, Fort Detrick, Maryland 

LEONARD A. SMITH, PhD 

Senior Research Scientist (ST) for Medical Countermeasures Tech¬ 
nology, Office of the Chief Scientist, US Army Medical Research 
Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 
Maryland 21702 

JEFFREY E. STEPHENSON, PhD 

Regulatory Compliance Specialist, US Army Medical Research 
and Materiel Command, Telemedicine and Advanced Technol¬ 
ogy Research Center, 1054 Patchel Street, Fort Detrick, Maryland 
21702; formerly. Institutional Review Board Administrator, US 
Army Medical Research Institute of Infectious Diseases, 1425 
Porter Street, Fort Detrick, Maryland 

BRADLEY G. STILES, PhD 

Adjunct Professor of Biology, Biology Department, Wilson Col¬ 
lege, 1015 Philadelphia Avenue, Chambersburg, Pennsylvania 
17201 

JEAN-NICOLAS TOURNIER, MD, PhD 

Colonel, French Armed Forces Health Service, Department of 
Infectious Diseases, Unite Biotherapies anti-infectieuses et im- 
munite, Institut de recherche biomedicale des armees, 1 place du 
General Valerie Andre, Bretigny-sur-Orge, 91220 France; Ecole du 
Val-de-Grace, Paris, France; Unite Genomique virale et vaccina¬ 
tion, Institut Pasteur, Paris, France 

ROBERT G. ULRICH, PhD 

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

NICHOLAS J. VIETRI, MD 

Colonel, Medical Corps, US Army; Infectious Diseases Officer, 
Bacteriology Division, US Army Medical Research Institute of 
Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 
21702; and Assistant Professor of Medicine, Uniformed Services 
University of the Health Sciences, 4301 Jones Bridge Road, 
Bethesda, Maryland 20814 

DAVID M. WAAG, PhD 

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


SCOTT A. WEINSTEIN, PhD, MD 

Physician, Toxinologist, Toxinology Division, Women's & Chil¬ 
dren's Hospital, 72 King William Road, North Adelaide, South 
Australia 5006, Australia 

SUSAN L. WELKOS, PhD 

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

JULIAN WHITE, MD 

Head of Toxinology, Toxinology Division, Women's & Children's 
Hospital, 72 King William Road, North Adelaide, South Australia 
5006, Australia 

CHRIS A. WHITEHOUSE, PhD 

Principal Investigator, Molecular and Translational Sciences 
Department, US Army Medical Research Institute of Infectious 
Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702; for¬ 
merly, Chief, Disease Investigation Branch, US Geological Survey, 
National Wildlife Health Center, 6006 Schroeder Road, Madison, 
Wisconsin 

CATHERINE L. WILHELMSEN, DVM, PhD, CBSP 

Lieutenant Colonel (Retired), Veterinary Corps, US Army; Veteri¬ 
nary Medical Officer, Division of Pathology; formerly. Biosafety 
Officer, Office of Safety, Radiation Protection, and Environmental 
Health, US Army Medical Research Institute of Infectious Dis¬ 
eases, 1425 Porter Street, Fort Detrick, Maryland; formerly. Chief, 
Division of Toxicology, US Army Medical Research Institute of 
Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

MARK R. WITHERS, MD, MPH 

Colonel, Medical Corps, US Army; Clinical Director, Office of 
Medical Support and Oversight, US Army Research Institute of 
Environmental Medicine, 15 Kansas Street, Natick, Massachusetts 
01760; formerly Deputy Chief, Division of Medicine, US Army 
Medical Research Institute of Infectious Diseases, 1425 Porter 
Street, Fort Detrick, Maryland 

MARK J. WOLCOTT, PhD 

Chief, Transitional and Operational Diagnostics Department, 
Diagnostic Systems Division, US Army Medical Research Institute 
of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 
21702 

NEAL E. WOOLLEN, DVM, MSS, PhD 

Colonel, Veterinary Corps, US Army; Director, Department of 
Biological Select Agent and Toxin Biosafety Program Office, 1546 
Porter Street, Fort Detrick, Maryland 21702 

PATRICIA L. WORSHAM, PhD 

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

GLENN WORTMANN, MD 

Colonel (Retired), Medical Corps, US Army; formerly. Chief 
of Infectious Diseases, Walter Reed National Military Medical 
Center, Bethesda, Maryland; currently. Assistant Director, Infec¬ 
tious Diseases Section, MedStar Washington Hospital Center, 110 
Irving Street, NW, Washington, DC 20010 


xv 


KRISTIE M. YEAKLE, RBP 

Alternate Biosafety Officer, Office of Safety, Radiation, and 
Environment, US Army Medical Research Institute of Infectious 
Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702 


xvi 


Peer Reviewers 


AMESH A. ADALJA, MD, FACP, FACEP 

Senior Associate, UPMC Center for Health Security, 621 East 
Pratt Street, Suite 210, Baltimore, Maryland 21202; and Clinical 
Assistant Professor, Department of Critical Care Medicine Clini¬ 
cal Assistant Professor, and Department of Emergency Medicine 
Adjunct Instructor, Division of Infectious Diseases, Department 
of Medicine, University of Pittsburgh School of Medicine, M240 
Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261 

GERARD ANDREWS, PhD 

Associate Professor, Department of Veterinary Sciences, Univer¬ 
sity of Wyoming, College of Agriculture and Natural Resources, 
1174 Snowy Range Road, Laramie, Wyoming 82070 

ROY BARNEWELL, PhD 

Manager and Research Leader, Battelle Memorial Institute, 505 
King Avenue, Columbus, Ohio 43201 

HOLGER BARTH, PhD 

Professor, Institute of Pharmacology and Toxicology, University 
of Ulm Medical Center, Albert-Einstein-Allee 11, 89081 Ulm, 
Germany 

KENNETH BRADLEY, PhD 

Associate Professor, Department of Microbiology, Immunology, 
and Molecular Genetics Director, Molecular Screening Shared Re¬ 
source, University of California, 609 Charles E. Young Drive East, 
Los Angeles, California 90095 

BARBARA A. BROOKMYER, MD, MPH 

Health Officer, Frederick County Health Department, 350 Monte- 
vue Lane, Frederick, Maryland 21702 

LAURA RUSE BROSCH, RN, PhD 

Director, Office of Research Protections and Director, Office of 
Research Protections Human Research Protection Office, Head¬ 
quarters, US Army Medical Research and Materiel Command, 810 
Schreider Street, Fort Detrick, Maryland 21702 

MICHAEL BUCHMEIER, PhD 

Professor, Department of Molecular Biology and Biochemistry, 
Division of Infectious Disease, Department of Medicine, Depart¬ 
ment of Microbiology and Molecular Genetics, and Department of 
Molecular Biology and Biochemistry, University of Califomia-Ir- 
vine, 2400 Biological Sciences III, Irvine, California 92697-3900 

R. MARK BULLER, PhD 

Professor, Department of Molecular Microbiology and Immunol¬ 
ogy, Saint Louis University, 1100 South Grand Boulevard, St. 
Louis, Missouri 63104 

KATHLEEN W. CARR, DVM, PhD, MS 

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

BRIAN W. COOPER, MD 

Global Medical Affairs Medical Director of Early Viral/Bacterial 
Vaccines, Novartis Vaccines, 350 Massachusetts Avenue, Cam¬ 
bridge, Massachusetts 02139; and Professor of Clinical Medicine, 
University of Connecticut School of Medicine, 263 Farmington 
Avenue, Farmington, Connecticut 06030 


PAUL DABISCH, PhD 

Senior Principal Investigator and Aerobiology Group Leader, 
National Biodefense Analysis and Countermeasures Center (oper¬ 
ated by BNBI for the US Department of Homeland Security), 8300 
Research Plaza, Fort Detrick, Maryland 21702 

ROBERT G. DARLING, MD, FACEP 

Captain (Retired), Medical Corps, US Navy; Chief Medical Of¬ 
ficer, Patronus Medical Corporation, Washington, DC; and Assis¬ 
tant Professor Military and Emergency Medicine, The Uniformed 
Services University of the Health Sciences F. Edward Hebert 
School of Medicine, 4301 Jones Bridge Road, Bethesda, Maryland 
20814 

STACY DeGRASSE, PhD 

Science Advisor, Division of Seafood Safety, Office of Food Safety, 
US Food and Drug Administration, 

Center for Food Safety and Applied Nutrition, 5100 Paint Branch 
Parkway, College Park, Maryland 20740 

JUAN CARLOS De La TORRE, PhD 

Professor, Department of Immunology and Microbial Science, 

The Scripps Research Institute, 10550 North Torrey Pines Road, 

La Jolla, California 92037 

DAVID DeSHAZER, PhD 

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

DMITRY DMITROFF, PhD 

Head, Protein Interactions Section, Center for Cancer Research, 
National Cancer Institute, Building 567, Room 152, Frederick, 
Maryland 21702 

ADAM DRIKS, PhD 

Professor, Department of Microbiology and Immunology, Stritch 
School of Medicine, Loyola University Chicago, 2160 South First 
Avenue, Maywood, Illinois 60153 

CAROL EISENHAUER, DVM, Dipl ACLAM 

Colonel (Retired) 

EDWARD EITZEN, MD, MPH 

Colonel (Retired), US Army; Senior Partner, Biodefense and 
Public Health Programs, Martin-Blanck and Associates, 2034 
Eisenhower Avenue, Suite 270, Alexandria, Virginia 22314-4678 

KENNETH L. GAGE, PhD 

Chief, Entomology and Ecology Activity, Bacterial Diseases 
Branch, Division of Vector-Borne Diseases, National Center for 
Emerging and Zoonotic Infectious Diseases, Centers for Disease 
Control and Prevention, 3156 Rampart Road, Fort Collins, Colo¬ 
rado 80521 

DAVE HARBOURT, PhD 

Biosafety Officer, US Army Medical Research Institute of Infec¬ 
tious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702- 
5102 


XVII 


DAN E. HARMS, MS, MT(ASCP) 

Program Manager, Operational Laboratory Policy & Programs 
Executive Secretariat, Defense Health Headquarters, 7700 Arling¬ 
ton Boulevard, Suite 5101, Falls Church, Virginia 22042-5190 

M. DANA HARRIGER, PhD 

Assistant Dean and Professor, Department of Biology, Wilson 
College, 1015 Philadelphia Avenue, Chambersburg, Pennsylvania 
17201 

JOHN HUNGERFORD, PhD 

Research Chemist, Pacific Laboratory Northwest, US Food and 
Drug Administration, 22201 23rd Drive, SE, Bothell, Washington 
98021 

DEBRA L. HUNT, DrPH, CBSP 

Assistant Professor and Director, Biological Safety, Occupational, 
and Environmental Safety Office, Duke University/Duke Uni¬ 
versity Health System, Hock Plaza I, 2424 Erwin Road, Durham, 
North Carolina 27705 

BRADLEY D. JONES, PhD 

Professor, Department of Microbiology, University of Iowa, 
Carver College of Medicine, 375 Newton Road, Iowa City, Iowa 
52242 

HEIDI KASSENBORG, DVM, MPH 

Director, Dairy and Food Inspection Division, Minnesota Depart¬ 
ment of Agriculture, 625 Robert Street North, St. Paul, Minnesota 
55155 

JEREMIAH J. KELLY, ESQ, MPP 

Attorney for Medical Product Development and Regulation, 

Office of the Staff Judge Advocate (JAG), US Army Medical 
Research and Materiel Command, 521 Fraim Street, Fort Detrick, 
Maryland 21701 

ERIC LAFONTAINE, PhD 

Associate Professor, Department of Infectious Diseases, College of 
Veterinary Medicine, The University of Georgia, 501 D. W. Brooks 
Drive, Athens, Georgia 30602-7387 

DIREK LIMMATHUROTSAKUL, MD, PhD 

Assistant Professor, Department of Tropical Hygiene and Mahi- 
dol, Oxford Tropical Medicine Research Unit, Faculty of Tropical 
Medicine, Mahidol University, 999 Phuttamonthon 4 Road, 

Salaya 73170, Thailand 

MARK LITTLE, MD, MBBS, FACEM, MPH&TM, 
DTM&H, IDHA 

Associate Professor, School of Public Health, Tropical Medicine 
and Rehabilitation Sciences, James Cook University, 1 James 
Cook Drive, Townsville City, Queensland 4811, Australia; and 
Emergency Physician and Clinical Toxicologist, Department of 
Emergency Medicine, 165 The Esplanade, Cairns Hospital, Cairns 
City, Queensland 4870, Australia 

STEPHEN LUBY, MD 

Professor, Department of Medicine-Infectious Diseases and Senior 
Fellow, Stanford Woods Institute for the Environment and Free¬ 
man Spogli Institute for International Studies, 473 Via Ortega, 
Stanford, California 94305; and Director of Research, Stanford 
Center for Innovation in Global Health, 473 Via Ortega, Stanford, 
California 94305 


NICHOLAS MANTIS, PhD 

Associate Professor, Division of Infectious Disease, Wadsworth 
Center, New York State Department of Health, Empire State 
Plaza, PO Box 509, Albany, New York 12201; and Department 
of Biomedical Sciences, School of Public Health, University at 
Albany, One University Place, Albany, New York 12144 

GLENN MARSH, PhD 

Senior Research Scientist, Molecular Virology, Emerging Zoonotic 
Diseases Stream, CSIRO Biosecurity Flagship, Australian Animal 
Health Laboratory, 5 Portarlington Road, Geelong, Victoria, 
Australia 3220 

BRUCE McCLANE, PhD 

Professor, Department of Microbiology and Molecular Genetics, 
University of Pittsburgh School of Medicine, 420 Bridgeside Point 
II, 450 Technology Drive, Pittsburgh, Pennsylvania 15219 

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

Director, Division of Medical Toxicology, Department of Emer¬ 
gency Medicine, Hartford Hospital and University of Connecticut 
School of Medicine, 80 Seymour Street, Hartford, Connecticut 
06102 

DIETRICH MEBS, PhD 

Professor, Goethe-University Frankfurt, Forensic Toxicology, 
Institute of Legal Medicine, Kennedyallee 104, D-60596 Frankfurt, 
Germany 

MICHAEL F. MINNICK, PhD 

Professor, Division of Biological Sciences, University of Montana, 
32 Campus Drive, HS 104, Missoula, Montana 59812 

STEPHEN S. MORSE, PhD 

Professor, Department of Epidemiology, Columbia University, 
Mailman School of Public Health, 722 West 168th Street, New 
York, New York 10032 

ERIC MOSSEL, PhD 

Major, Medical Service Corps, US Army Reserve; Microbiologist, 
Division of Virology, US Army Medical Research Institute of Infec¬ 
tious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702 

ROBERT MUNSON, PhD 

Professor, Department of Pediatrics, Department of Molecular 
Virology, Immunology, and Molecular Genetics, and Department 
of Microbiology, The Research Institute at Nationwide Children's 
Hospital and The Ohio State University, 700 Children's Drive, 
Columbus, Ohio 43205 

BILL NAUSCHUETZ, PhD 

Lab Biopreparedness Coordinator, US Army Medical Command, 
2748 Worth Road, Suite 10, Fort Sam Houston, Texas 78234-6039 

DAVID O'CALLAGHAN, PhD 

Director, INSERM U1047, Universite Montpellier 1, Place Eugene 
Bataillon, 34090 Montpellier, France 

GENE OLINGER, PhD, MBA 

High Containment Coordinator, National Institute of Allergy 
and Infectious Diseases, Integrated Research Facility, Division of 
Clinical Research, 8200 Research Plaza, Fort Detrick, Maryland 
21702 


xviii 


GEOFFREY K. PHIFFIPS, MPH 

Safety and Occupational Health Manager, US Army Medical Re¬ 
search and Materiel Command, MCMR-SS, 810 Schreider Street, 
Fort Detrick, Maryland 21702 

ANN POWERS, PhD 

Chief, Alphavirus Laboratory, Division of Vector-Borne Diseases, 
Centers for Disease Control and Prevention, 3156 Rampart Road, 
Fort Collins, Colorado 80521 

JON ROBERTUS, PhD 

Professor Emeritus, The University of Texas at Austin, Molecular 
Biosciences, College of Natural Sciences, 2506 Speedway, Austin, 
Texas 78712 

JANICE M. RUSNAK, MD, FA CP, FIDSA 

Contractor, Battelle, Medical Countermeasure Systems-Joint 
Vaccine Acquisition Program (MCS-JVAP), 1564 Freedman Drive, 
Fort Detrick, Maryland 21702 

JOHN M. SCHERER, PhD, MT(ASCP) 

Colonel, Medical Corps, US Army; Medical Research and Materiel 
Command, 810 Schreider Street, Fort Detrick, Maryland 21702 

LARRY S. SCHLESINGER, MD 

Professor and Chair, Department of Microbial Infection and 
Immunity Director, Center for Microbial Interface Biology, The 
Ohio State University, 798 Biomedical Research Tower, 460W 12th 
Avenue, Columbus, Ohio 43210 

MICHAEL SCHUIT, MS 

Research Scientist, Aerobiology Group, National Biodefense 
Analysis and Countermeasures Center (operated by BNBI for the 
US Department of Homeland Security), 8300 Research Plaza, Fort 
Detrick, Maryland 21702 

HARALD SEIFERT, MD 

Professor of Medical Microbiology and Hygiene, Institute for 
Medical Microbiology, Immunology, and Hygiene, University of 
Cologne, Goldenfelsstrasse 19-21, 50935 Koln, Germany 

EDWARD SHAW, PhD 

Associate Professor, Department of Microbiology and Molecu¬ 
lar Genetics, Oklahoma State University, 307 Life Sciences East, 
Stillwater, Oklahoma 74078 

PETER M. SILVERA, PhD 

Chief, Non-Clinical Development Division, US Army Medical 
Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, 
Maryland 21702 

BAL RAM SINGH, PhD 

Professor and Director, Botulinum Research Center, Institute of 
Advanced Sciences, 78-540 Faunce Corner Mall Road, Dartmouth, 
Massachusetts 02747 

SHAWN J. SKERRETT, MD 

Professor, Division of Pulmonary and Critical Care Medicine, 
University of Washington, Harborview Medical Center, 325 Ninth 
Avenue, Seattle, Washington 98104 

BRAD STILES, PhD 

Adjunct Professor, Department of Biology, Wilson College, 1015 
Philadelphia Avenue, Chambersburg, Pennsylvania 17201 


CHRISTINE TRAVISS, PhD 

Microbiologist, Division of Select Agents and Toxins, Office of 
Public Health Preparedness and Response, Centers for Disease 
Control and Prevention, 1600 Clifton Road, NE, MS A-46, Atlanta, 
Georgia 30333 

RENEE M. TSOLIS, PhD 

Professor, Department of Medical Microbiology and Immunol¬ 
ogy, University of California-Davis, 3146 Tupper Hall, 1 Shields 
Avenue, Davis, California 95616 

BERNARD VERRIER, PhD 

Director, Institut de Biologie et Chimie des Proteines, Federa¬ 
tion de Recherche, 3302 SFR BioSciences (Unite Mixte de Service 
3444/US8), Gerland-Lyon Sud, Universite de Lyon 1, 69007 Lyon, 
France; and Laboratoire de Biologie Tissulaire et dTngenierie 
Therapeutique, Centre National de la Recherche Scientifique, 
Unite Mixte de Recherche 5305, 69007 Lyon, France 

NICHOLAS J. VIETRI, MD 

Colonel, Medical Corps, US Army; Chief, Occupational Medicine, 
US Army Medical Research Institute of Infectious Diseases, 1425 
Porter Street, Fort Detrick, Maryland 21702; and Assistant Profes¬ 
sor of Medicine, Uniformed Services University of the Health 
Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814 

SCOTT C. WEAVER, PhD 

John Sealy Distinguished University Chair in Human Infections 
and Immunity Director, Institute for Human Infections and 
Immunity Scientific Director, Galveston National Laboratory Pro¬ 
fessor, Departments of Pathology, Microbiology, and Immunol¬ 
ogy, Galveston National Laboratory, 301 University Boulevard, 
Galveston, Texas 77555-0610 

JUDITH WHITE, PhD 

Professor, Department of Cell Biology and Developmental Biol¬ 
ogy, University of Virginia, 1340 Jefferson Park Avenue, Charlot¬ 
tesville, Virginia 22908-0732 


xix 



Foreword 


The concept of national defense has been undeniably shaped by the events of September 11, 2001. The 
US anthrax postal attacks immediately following 9/11 forever changed our perspective of biodefense related 
research. More recently, the continued threat of state-sponsored events or individual extremist groups has 
only compounded the severity of this facet of national security. As we focus our medical efforts to succeed at 
the point of injury, and to optimize the success of the operating forces, the identification and preparation for 
biological threats has become a synergistic force multiplier. 

The US Department of Defense (DoD) continues to identify potential threats and prepare for possible 
biological attacks by maintaining a knowledge base and by actively developing and testing novel medical 
countermeasures. For example, scientists at the US Army Medical Research Institute of Infectious Diseases 
(Fort Detrick, MD) have developed vaccines against the causative agents of anthrax, plague, ricin intoxication, 
botulinum intoxication, Ebola virus, and encephalitic alphaviruses. Importantly, DoD scientists partner with 
other federal agencies, academic institutions, and pharmaceutical companies to test and evaluate vaccines and 
therapeutics against many other biological threats. There is no better example of this consortium approach 
then the DoD's efforts during the recent Ebola outbreak in West Africa. DoD scientists and military person¬ 
nel were on the ground diagnosing samples, sequencing viral genomes, and administering supportive care. 
Concurrently, some of the very products tested and evaluated by the DoD were deployed during the medical 
emergency. If it was not for DoD intervention, this outbreak had the potential to be substantially worse and 
spread further across Africa and around the world. Taken together, these brief examples demonstrate exactly 
why Medical Aspects of Biological Warfare needs to be maintained as an up-to-date information source. 

The first edition of Medical Aspects of Chemical and Biological Warfare was published in 1997. A decade later, 
the chemical and biological aspects of this text warranted separate volumes. Thus, in 2007, Medical Aspects of 
Biological Warfare was released as a stand-alone textbook. Because of the fast-paced nature of microbiological 
research, new and emerging threats, and the changing policy, the authors pursued an updated version. In this 
third edition of the Textbooks of Military Medicine's Medical Aspects of Biological Warfare, the authors have gone 
to great lengths to address many facets of biodefense research, preparedness, and consequence management. 
Individual chapters are devoted to understanding the pathogenesis and disease progression associated with 
bacterial and viral biothreat agents, such as Bacillus anthracis and Ebola virus. Additionally, intoxications by 
toxins such as ricin are also described in detail. These chapters highlight the current state of science for these 
agents and toxins: they clearly underscore the importance of pursuing basic science interests in these arenas, 
and the importance of maintaining a core pool of subject matter experts. Without basic science efforts, our 
continued understanding of these threats would suffer, and knowledge gaps would grow. Accordingly, cur¬ 
rent clinical treatment protocols and regimens are also discussed throughout the textbook and offer a bridge 
from the basic research to the applied clinical "real-world" applications. 

This textbook also examines other less apparent biodefense-related topics. Acinetobacter baumii is used as an 
example of how a drug-resistant bacterium can impact the DoD, and further demonstrates how the institutional 
structure and strategic planning can be used to address such threats. Additional chapters discuss Medical 
Management and Consequence Management, and give current perspectives on patient care and federal and 
local response scenarios in the event of a biological attack. This edition also describes current laboratory bio¬ 
safety and biosurety philosophies that have tremendous impacts on the execution of biodefense strategies that 
are constantly evolving. Finally, this version of the textbook gives a nod to the history of biodefense research 
while also addressing new and emerging biological threats, be they natural or engineered. 

The authors, subject matter expert reviewers, and editors have produced a comprehensive and thought¬ 
ful reference source for the DoD, and I am proud of the scientists, physicians, and other professionals who 
contributed their time and efforts to produce the final product. 


Lieutenant General Nadja Y. West, MD 
The Surgeon General 
Commanding General, U.S. Army Medical Command 

Washington, DC 
March 2017 



Preface 


In an ever-changing and complex world, medical defense against biological pathogens must be a central 
pillar of our national defense strategy. Although biological warfare has been a legitimate concern for centuries, 
our current requirements and future operations emphasize the need for a continuing holistic approach to medi¬ 
cal biological defense against these threats. From antiquity to the present day, agents such as Bacillus anthracis 
(etiological agent of anthrax), Francisella tularensis (etiological agent of tularemia), Burkholderia mallei (etiological 
agent of glanders). Yersinia pestis (etiological agent of plague), and Variola (etiological agent of smallpox) have 
been on the forefront of biowarfare and biodefense. With increased uncertainty associated with terrorist groups, 
rogue nations, and "lone wolf" individuals, the threat of biological weapons is even more relevant today. 

Subject matter experts who wrote and reviewed these chapters focused on the most current data available 
at the time to create the most comprehensive reference source available for the US Department of Defense. 
Revising this textbook is important, not only to highlight the current state-of-the-art application for medical 
countermeasures, but also to discuss myriad current and future threats. Some of these evolving issues include 
the ongoing ramifications of the world's largest-ever Ebola virus disease outbreak and the impact of emerging 
antibiotic resistance from select bacterial pathogens. Of recent note is the emerging B cereus biovar anthracis 
strains isolated in Africa from fatal anthrax-like infections in chimpanzees and western lowland gorillas. These 
strains of B cereus were shown to harbor plasmids highly similar to both B anthracis virulence plasmids and, ac¬ 
cordingly, were included on the US Department of Health and Human Services select agent list in 2016. These 
are just a few of the examples that underscore the complexities of biodefense research. Although we must remain 
vigilant in anticipating state-sponsored or terrorist activities, new threats are evolving in the natural world that 
could prove equally catastrophic to our military personnel and national interests. Preparation, cooperation, and 
rehearsal in accordance with the latest methodologies are the key ingredients to success in these current contexts. 

I am deeply grateful for the contributions of the scientists and physicians who collaborated in this endeavor. 
They are nationally and internationally recognized experts in their specialties, and their dedication to updating 
this textbook has been invaluable. I am pleased to introduce the latest edition of Medical Aspects of Biological Warfare. 


Colonel Thomas S. Bundt 
Medical Service Corps, US Army 
Commander, US Army Medical Research Institute of Infectious Diseases 


Fort Detrick, Maryland 
October 2016 


xxiii 



Chapter 1 


HISTORICAL OVERVIEW:FROM 
POISONED DARTS TO PAN-HAZARD 
PREPAREDNESS 

GEORGE W. CHRISTOPHER, MD, FACP*; DANIEL M. GERSTEIN, PhD 1 ; EDWARD M. EITZEN, MD, MPH*; and 
JAMES W. MARTIN, MD, FACP § 


INTRODUCTION 

EARLY USE 

THE WORLD WARS 

THE US PROGRAM 

THE SOVIET PROGRAM 

THE SPECIAL CASE OF IRAQ 

OTHER NATIONAL PROGRAMS 

BIOCRIMES 

BIOLOGICAL TERRORISM 

SOLUTIONS: TOWARD PAN-HAZARD PREPAREDNESS 
Disarmament: The Biological Weapons Convention 
Smallpox Preparedness 
Dual Use Research of Concern 
Toward Pan-Hazard Preparedness 

SUMMARY 


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

f Colonel (Retired), US Army; Adjunct Professor, School of International Studies, American University, 4400 Massachusetts Avenue, NW, Washington, 
DC 20016; formerly, Undersecretary (Acting) and Deputy Undersecretary, Science and Technology Directorate, Department of Homeland Security, 
Washington, DC 

*Colonel (Retired), Medical Corps, US Army; Senior Partner, Biodefense and Public Health Programs, Martin-Blanck and Associates, 2034 Eisenhower 
Avenue, Suite 270, Alexandria, Virginia 22314-4678; formerly, Commander, US Army Medical Research Institute of Infectious Diseases, 1425 Porter 
Street, Fort Detrick, Maryland 

5 Colonel (Retired), Medical Corps, US Army; Chief of Internal Medicine, US Army Healthcare Clinic, Vicenza, APO AE 09630-0040; formerly, Chief, 
Operational Medicine Department, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 


1 



Medical Aspects of Biological Warfare 


INTRODUCTION 


Humans have used technology for destructive as 
well as beneficial purposes since prehistory. Aboriginal 
use of curare and amphibian-derived toxins as arrow 
poisons anticipated modern attempts to weaponize 
biological toxins such as botulinum and ricin. The 
derivation of the modem term "toxin" from the ancient 
Greek term for arrow poison, tgjElicov (paopaicov 
(toxicon pharmicon; toxon = bow, arrow) 1,2 underscores 
the historical link between weaponry and biological 
agents. 

Multiple factors confound the study of the history of 
biological weapons, including secrecy surrounding 
biological warfare programs, difficulties confirming 
allegations of biological attack, the lack of reliable 
microbiological and epidemiological data regard¬ 
ing alleged or attempted attacks, and the use of 
allegations of biological attack for propaganda and 
hoaxes. A review of historical sources and recent 
events in Iraq, Afghanistan, Great Britain, and the 
United States demonstrates that interest in biological 
weapons by state-sponsored programs, terrorists, 
and criminal elements is likely to continue. Human¬ 
kind is witnessing a "democratization in the life sci¬ 
ences," in which the field is becoming industrialized 
and therefore making biotechnology available to an 
ever increasing number of people, some of whom 
will undoubtedly have ill intent. In addition, there 
are growing concerns that well-intentioned life sci¬ 
ences research to advance medical defenses against 
biological weapons agents and other highly virulent 


pathogens may inadvertently provide information 
that could be deliberately misused for biological 
weapons proliferation. 3 

Numerous historical examples exist of military 
disasters resulting from failures to adapt policy, strat¬ 
egy, and doctrine to offset the impact of revolutionary 
advances in weapons technology. 4 Biological medical 
defense programs, begun as narrowly focused efforts to 
counter a limited number of biological weapons agents, 
are being expanded as versatile capabilities, with a 
shift in emphasis from pathogen-specific approaches to 
capabilities-based programs to enable rapid responses 
to novel, potentially genetically engineered biological 
weapons agents. The response to biological weapons 
has fueled robust enterprises in basic and applied 
medical research, product development, manufactur¬ 
ing, stockpiling, infrastructure, public health policy, 
planning, and response capacities at local, national, 
and international levels. 3 Medical capabilities and bio¬ 
medical research are being linked to diplomacy, com¬ 
merce, education, ethics, law enforcement, and other 
activities to enable pan-societal sector responses to both 
biological weapons and the inevitable and dynamic 
challenges of naturally occurring emerging infectious 
diseases. 3 Integration of biological defense and public 
health programs and their mutual development must 
be continuous to optimize outcomes and maximize 
efficient utilization of limited resources, because the 
challenges posed by both biological weapons agents 
and naturally emerging pathogens are open-ended. 5 


EARLY USE 


The impact of infectious diseases on military forces 
has been recognized since ancient times. 6 '' The use of 
disease as a weapon was used long before microbial 
pathogenesis was understood. Military leaders only 
knew that a cause and effect relationship existed 
between certain activities, locations, or exposures to 
victims of disease that resulted in the spread of infec¬ 
tions that ultimately provided a military advantage. 
For example, an early tactic was to allow an enemy to 
take sanctuary in locations endemic for infectious dis¬ 
eases in anticipation that its troops would be afflicted, 
thus allowing unimpeded access of opposing armies to 
areas where transmission of malaria was highly likely. 

Numerous anecdotal accounts exist of the attempted 
use of cadavers, animal carcasses, plant-derived 
toxins, and filth to transmit disease during antiquity 
through the Napoleonic era into modem times. Several 
examples illustrate the complex epidemiologic issues 
raised by biological warfare, the difficulty in differenti¬ 
ating epidemics resulting from biological attacks from 


outbreaks of disease that occur due to disruptions of 
war, and the adverse psychological impact of biological 
attacks on military operations. 

During a naval battle against King Eumenes of 
Pergamum in 184 BCE, Hannibal ordered earthen pots 
filled with snakes to be hurled onto the decks of enemy 
ships. The pots shattered on impact, releasing live 
serpents among the enemy sailors. The Carthaginian 
victory is attributed to the ensuing panic rather than 
envenomation 8 ; this illustrates that the psychological 
contagion of biological weapons may amplify their 
impact beyond their potential to cause organic disease. 

One of the most notorious early biological warfare 
attacks was the hurling of cadavers over the walls 
of the besieged city of Caffa, a Genoese colony in 
the Crimea, in 1346. 9,10 After war broke out between 
the Genoese and the Mongols in 1343 for control of 
the lucrative caravan trade route between the Black 
Sea and the Orient, the Mongols laid siege to Caffa. 
The plague, later known as the Black Death, was 


2 


Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness 


spreading from the Far East and reached the Crimea in 
1346. The Mongols were severely afflicted and forced 
to abandon their siege. As a parting shot, they hurled 
"mountains of dead" over the city wall, probably with 
the use of a trebuchet, in the hope that "the intoler¬ 
able stench would kill everyone inside." An outbreak 
of plague in the city followed. A review by Wheelis 10 
suggests that the introduction of plague into the city 
by the cadavers —as a result of a tactically successful 
biological attack—may be the most biologically plau¬ 
sible of several competing hypotheses on the source 
of the outbreak. Although the predominant mode of 
plague transmission has been attributed to bites from 
infected fleas (which leave cadavers and carcasses to 
parasitize living hosts), modern experience (United 
States 1970-1995) 11 has implicated transmission from 
contact with infected animal carcasses in 20% of 
instances in which the source of the infection could 
be attributed. Contact with tissue and blood would 
have been inevitable during the disposal of hundreds 
or possibly thousands of cadavers. Alternatively, 
plague could have been introduced by imported hu¬ 
man cases or infected rodents brought into the city 
through maritime trade, which was maintained during 
the siege. The importation of plague by a rodent-flea 
transmission cycle across the city wall is considered 
less likely because rats are sedentary and rarely ven¬ 
ture far from their nests; it is unlikely that they would 

THE WO 

The birth of scientific bacteriology during the 19th 
century provided the scientific and technical basis for 
modem biological weapons programs. The Hague Con¬ 
ventions of 1899 and 1904 outlawed the use of "poison 
or poisoned arms," although bacteriological weapons 
were not specifically addressed. 19-20 During World War 
I, German espionage agents reportedly infected draft 
animals intended for military use with Burkholderia 
[Pseudomonas] mallei and Bacillus anthracis . 21-23 Covert 
operations were reportedly conducted in Argentina, 
Norway, Mesopotamia, Romania, Russia, and the 
United States. Unsuccessful attempts were also made to 
cripple grain production in Spain using wheat fungus. 21 

The German bio warfare program of World War I is 
of special interest because it was the first program with 
a scientific basis; it conducted a large-scale (strategic) 
biological campaign, which targeted neutral nations 
as well as belligerents, and it targeted crops and ani¬ 
mals instead of humans. Although German operatives 
thought the program was successful, confirmatory data 
are not available. 21 

In response to chemical warfare during World War 
I, the 1925 Geneva Protocol, an international protocol 
(for the Prohibition of the Use in War of Asphyxiating, 


have traversed an open distance of several hundred 
meters between the Mongol encampment and the city 
walls. 10 Transmission from sylvatic to urban rodents 
is infrequent, at least under current ecological condi¬ 
tions. 12 Regardless of the portal of entry, the epidemic 
may have been amplified under siege conditions due 
to deteriorating sanitation and hygiene resulting in 
expansions of rats and fleas. 

Smallpox was particularly devastating to Native 
Americans. The unintentional yet catastrophic in¬ 
troduction of smallpox to the Aztec empire during 
1520, and its subsequent spread to Peru in advance of 
Pizarro's invasion of the Inca empire, played a major 
role in the conquest of both empires. 13 During the 
French and Indian Wars (1754-1763), British forces 
provided Native Americans with handkerchiefs and 
blankets contaminated with scabs from smallpox pa¬ 
tients to transmit disease. 14-18 An epidemic of smallpox 
followed among the Native Americans of the Ohio 
River Valley. It is difficult to evaluate the tactical suc¬ 
cess of these biological attacks in retrospect because 
smallpox may have been transmitted during other 
contacts with colonists, as had previously occurred 
in New England and the South. Smallpox scabs are 
thought to have low infectivity due to the binding of 
virions in a fibrin matrix, and transmission by fomites 
has been considered less efficient than respiratory 
droplet transmission. 13 


Poisonous or Other Gases, and of Bacteriological 
Methods of Warfare), was formulated by the League 
of Nations' Conference for the Supervision of the 
International Trade in Arms and Ammunition. It had 
no verification mechanism and relied on voluntary 
compliance. Many of the original signatory states 
reserved the right to retaliatory use, making it effec¬ 
tively a no first-use protocol. Signatories that began 
research programs to develop biological weapons be¬ 
tween World War I and II included Belgium, Canada, 
France, Great Britain, Italy, The Netherlands, Poland, 
and the Soviet Union. 24 

After defeating Russia in the 1905 Russo-Japanese 
War, Japan became the dominant foreign power in 
Manchuria, and seized full military control between 
September 1931 and the end of 1932. Major Shiro Ishii, 
a Japanese army physician, established a biological 
weapons laboratory in Harbin, but soon realized that 
his controversial involuntary human research could 
not be conducted freely there. Ishii moved to a secret 
facility at Beiyinhe, 100 km south of Harbin, and be¬ 
gan large-scale experimentation. All research study 
subjects died of either experimental infection or live 
vivisection. These studies continued until a prisoner 


3 


Medical Aspects of Biological Warfare 


riot and escape, which resulted in the closing of the 
facility in 1937. However, larger and more extensive 
facilities were subsequently built. 24 

In 1936 Ishii built Unit 731, a massive research 
facility 24 km south of Harbin, where a census of 200 
prisoners was kept as expendable subjects of experi¬ 
mentation. Ultimately, more than 3,000 Chinese pris¬ 
oners were killed during these experiments. Most of 
the evidence was destroyed at the end of the war, and 
in all likelihood the actual number was much greater. 24 
Additional facilities included Unit 100 at Changchun, 
and Unit Ei 1644 in Nanking. Unit 100 was primar¬ 
ily a veterinary and agricultural biowarfare research 
unit for developing biological weapons for sabotage. 
Although animals and crops were the focus of most 
of the research, numerous human studies were also 
conducted, similar to those conducted by Unit 731. In 
addition to conducting human experimentation. Unit 
Ei 1644 supported Unit 731's research efforts with 
bacterial agent production and flea cultivation. 24 

Eleven Chinese cities were allegedly attacked dur¬ 
ing "field trials" using agents including Yersinia pestis, 
Vibrio cholerae, and Shigella spp. These attacks may 
have backfired because up to 10,000 Japanese soldiers 
reportedly contracted cholera after a biological attack 
on Changde in 1941. 25 The field trials were terminated 
in 1943, yet basic research and human experimenta¬ 
tion continued until the end of the war. 24-26 Despite 
the enormously expensive program (both in terms of 
national treasure and human lives) and the weaponiza- 
tion of many agents, Japan never developed a credible 
biowarfare capability, mainly because of the failure to 
develop an effective delivery system. 1 ' 

In contrast to Japanese efforts during World War 
II, a German offensive biological weapons program 
never materialized. Hitler reportedly issued orders 
prohibiting biological weapons development. Un¬ 
ethical experimental infections of prisoners were done 
primarily to study pathogenesis and develop vaccines 
and sulfonamides, rather than to develop biological 
weapons. With the support of high-ranking Nazi party 
officials, however, scientists began biological weapons 
research, but their results lagged far behind those of 
other countries. 27 

Polish physicians used a vaccine and a serologic test 
in a brilliant example of "biological defense." Knowing 
that inoculation with killed Proteus OX-19 would cause 
false-positive Weil-Felix typhus test results, physicians 


The US military recognized biological warfare as 
a potential threat after World War I. Major Leon Fox 
of the Army Medical Corps wrote an extensive report 


inoculated local populations with formalin-killed 
Proteus OX-19 to create serologic pseudoepidemics of 
typhus. Using serologic surveillance, the German army 
avoided areas with epidemic typhus; consequently, 
residents of these areas were spared deportation to 
concentration camps. 28 Unconfirmed allegations in¬ 
dicate that Polish resistance fighters used letters con¬ 
taminated with B anthracis to cause cutaneous anthrax 
among Gestapo officials 21,29 and used typhus against 
German soldiers. 21 Czechoslovakian agents reportedly 
used a grenade contaminated with botulinum toxin, 
supplied by British Special Operations, to assassinate 
Reinhard Heydrich, the Nazi governor of occupied 
Czechoslovakia 30,31 ; however, the veracity of this claim 
has been challenged. 23 

The perceived threat of biological warfare before 
World War II prompted Great Britain to stockpile 
vaccines and antisera, establish an emergency public 
health laboratory system, and develop biological 
weapons. "Cattle cakes" consisting of cattle feed con¬ 
taminated with B anthracis spores were designed to 
be dropped from aircraft into Axis-occupied Europe 
to cause epizootic anthrax among livestock, 32,33 which 
would in turn induce famine. The cattle cakes were 
intended as a strategic economic weapon rather than 
as a direct cause of human anthrax. In addition, explo¬ 
sive munitions designed to aerosolize and disperse B 
anthracis spores as antipersonnel weapons were tested 
on Gruinard Island near the coast of Scotland in 1942. 
These experiments successfully caused anthrax in 
targeted sheep. 34 The antipersonnel weapons were 
not mass produced, and neither the cattle cakes nor 
the explosive munitions were used. 21 Great Britain 
continued its offensive biological warfare program 
during the early Cold War era in conjunction with the 
United States and Canada, and it performed secret 
open-air tests using pathogens in off-shore ocean 
sites near the Bahamas and Scotland. 21 Great Britain's 
offensive program was terminated between 1955 and 
1956 35 because of budgetary constraints and reliance 
on nuclear deterrence. 32,33 Gruinard Island, which had 
been quarantined because of focal soil contamination 
by B anthracis spores following munitions testing, was 
decontaminated in 1986 using 2,000 tons of seawater 
and 280 tons of formaldehyde. 36 The United Kingdom 
conducts research to develop medical countermeasures 
at the Defence Science and Technologies Laboratories 
at Porton Down. 


concluding that improvements in health and sanitation 
made biological weapons ineffective. In 1941, before 
the US entry into World War II, opinions differed 


4 


Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness 


about the threat of biological warfare. Consequently, 
the Secretary of War asked the National Academy of 
Sciences to appoint a committee to study the issue. The 
committee concluded in February 1942 that biowarfare 
was feasible and the United States should reduce its 
vulnerability. 

President Franklin D Roosevelt established the 
War Reserve Service (with George W Merck as direc¬ 
tor) to develop defensive measures against biologi¬ 
cal weapons. By November 1942 the War Reserve 
Service asked the Army's Chemical Warfare Service 
to assume responsibility for a secret large-scale 
research and development program, including the 
construction and operation of laboratories and pilot 
plants. The Army selected a small National Guard 
airfield at Camp Detrick in Frederick, Maryland, 
for the new facilities in April 1943. By summer of 
1944 the Army had testing facilities in Florn Island, 
Mississippi (later moved to Dugway, Utah), and 
a production facility in Terre Flaute, Indiana. No 
agents were produced at the Terre Flaute plant be¬ 
cause of safety concerns; simulant tests disclosed 
contamination after trial runs. In the only reported 
US offensive use of a biological weapon, the Office of 
Strategic Services (predecessor of the Central Intel¬ 
ligence Agency) used staphylococcal enterotoxin in 
a food-borne attack to cause an acute but self-limited 
illness in a Nazi party official. 37,38 Cattle cakes using 
B anthracis spores were produced at Camp Detrick 
and shipped to Great Britain, but were never used. 
The War Reserve Service was disbanded after the war 
and the Terre Flaute plant was leased for commercial 
pharmaceutical production. 31 In January 1946 Merck 
reported to the Secretary of War that the United 
States needed a credible capability to retaliate if at¬ 
tacked with biological weapons. Basic research and 
development continued at Camp Detrick. 

The United States learned of the extent of Japanese 
biological weapons research after World War II. In 
an action that has become controversial, Ishii and his 


fellow scientists were given amnesty for providing 
information derived from years of biological warfare 
research. 24 

When war broke out on the Korean peninsula in 
June 1950, concerns about Soviet biological weapons 
development and the possibility that the North Ko¬ 
reans, Chinese, or Soviets might resort to biological 
warfare resulted in an expansion of the US program. 
A large-scale production facility in Pine Bluff, Arkan¬ 
sas, was established. The plant featured advanced 
laboratory safety and engineering measures enabling 
large-scale fermentation, concentration, storage, and 
weaponization of microorganisms. In 1951 the first 
biological weapons, anticrop bombs, were produced. 
The first antipersonnel munitions were produced in 
1954 using Brucella suis. The United States weapon- 
ized seven antipersonnel agents and stockpiled three 
anticrop agents (Table 1-1) over 26 years. 39 

Field tests using surrogate agents were conducted 
in the United States between 1949 and 1968, in which 
the general public and test subjects were uninformed. 
At least 239 open-air tests were conducted at several 
locations including the Dugway Proving Ground, 
Utah; remote Pacific Ocean sites; and populated areas 
including Minneapolis, St. Louis, New York City, San 
Francisco, and Eglin Air Force Base, Florida. These 
studies tainted the history of the offensive biological 
warfare program. The Special Operations Division 
at Camp Detrick conducted most of the field tests to 
study possible methods of covert attack and to examine 
aerosolization methods, the behavior of aerosols over 
large geographic areas, and the infectivity and rates 
of decay of aerosolized microbes subjected to solar 
irradiation and climatic conditions. Most tests used 
simulants thought to be nonpathogenic, including 
Bacillus globigii, Serratia marcescens, and particulates 
of zinc cadmium sulfide. 39,40 

In conjunction with the US Department of Agricul¬ 
ture (USDA), several open-air tests were conducted 
using anticrop agents at sites selected for safety. 


TABLE 1-1 

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


Lethal Agents 

Incapacitating Agents 

Anticrop Agents 

Bacillus anthracis 

Brucella suis 

Rice blast 

Francisella tularensis 

Coxiella burnetii 

Rye stem rust 

Botulinum toxin 

Venezuelan equine encephalitis virus 

Wheat stem rust 


Staphylococcal enterotoxin B 



"’Lethal and incapacitating agents were produced and weaponized. Anticrop agents were produced but not weaponized. 


5 





Medical Aspects of Biological Warfare 


Open-air releases of human pathogens ( Coxiella bur¬ 
netii, Francisella [Pasturella] tularensis) were performed 
at the Dugway Proving Ground, Eglin Air Force Base, 
and remote Pacific Ocean sites to study viability 
and infectivity using animal challenge models. 21,39,40 
Controversial studies included environmental tests 
to determine whether African Americans were more 
susceptible to Aspergillus fumigatus, as had been 
observed with Coccidioides immitis. These studies in¬ 
cluded the 1951 exposure of uninformed workers at 
Norfolk Supply Center in Norfolk, Virginia, to crates 
contaminated with Aspergillus spores. In 1966 the US 
Army conducted covert experiments in the New York 
City subways. Light bulbs filled with Bacillus subtilis 
var niger were dropped from subway platforms onto 
the tracks to study the distribution of the simulant 
through the subway system. 39-41 Similar tests were 
conducted using the ventilation system of the New 
York City subways and the Pentagon. 

The first large-scale aerosol vulnerability test 
conducted in San Francisco Bay in September 1950 
using B globigii and S marcescens demonstrated the 
public health issues of such testing. 41 An outbreak 
of 11 cases of nosocomial S marcescens ( Chromobac¬ 
terium prodigiosum) urinary tract infection occurred 
at the nearby Stanford University Hospital; one case 
was complicated by fatal endocarditis. Risk factors 
included urinary tract instrumentation and antibiotic 
exposures. 42 No similar outbreaks were reported by 
other San Francisco area hospitals. A panel of civilian 
and academic public health experts secretly convened 
by the Army in 1952 failed to reach a conclusion 
regarding the possible link between the Stanford 
outbreak and the testing program, but recommended 
that other microbes be used as simulants. 41 Public 
disclosure of the testing program in the Washington 
Post on December 22,1976, and in US Senate hearings 
in 1977 43 resulted in harsh criticism of the continued 
use of S marcescens as a simulant after the Stanford 
epidemic. However, a 1977 report from the Centers 
for Disease Control and Prevention (CDC) concluded 
that in 100 outbreaks of S marcescens infection, none 
was caused by the 8UK strain (biotype A6, serotype 
08:H3, phage type 678) used by the Army testing 
program. 44 Other reports from the 1970s postulated 
a link between S marcescens infection and the testing 
program; however, all clinical isolates available for 
strain typing were antigenically distinct from the 
Army test strain. In all likelihood, the 1950 Stanford 
S marcescens epidemic represents an early example 
of a nosocomial outbreak caused by opportunistic 
pathogens of low virulence complicating the use of 
medical devices and surgical procedures in the setting 
of antibiotic selection pressure. 44 


The US program developed modern biosafety tech¬ 
nologies and procedures including protective equip¬ 
ment, engineering and safety measures, and medical 
countermeasures, including new vaccines. There were 
456 occupational infections and three fatalities (two 
cases of anthrax in 1951 and 1958 and a case of viral 
encephalitis in 1964) reported at Fort Detrick during 
the offensive program (1943-1969). 39 The infection rate 
of fewer than 10 infections per million hours of work 
was within the contemporary National Safety Council 
standards; the morbidity and mortality rates were 
lower than those reported by other laboratories. There 
were 48 infections and no fatalities at the production 
and testing sites. 39 

In 1954 the newly formed Medical Research Unit 
at Fort Detrick began studies to develop vaccines and 
therapy to protect against biological agents. Research¬ 
ers began using human volunteers in 1956 as part of a 
congressionally approved program called "Operation 
Whitecoat." This use of volunteers set the standard 
for ethics and human use in research. Active duty 
soldiers with conscientious objector status served as 
research subjects, and participation was voluntary 
with informed consent. The program concluded with 
the end of conscription in 1973. 

Numerous unsubstantiated allegations were made 
during the Cold War era. During the Korean War 
(1950-1953), North Korean, Chinese, and Soviet of¬ 
ficials made numerous accusations of US biowarfare 
attacks. Many allegations appear to be based on Chi¬ 
nese experiences during World War II field testing 
conducted by the Japanese Unit 731. Polish medical 
personnel were sent to China to support the com¬ 
munist war effort, accompanied by eastern European 
correspondents, who made numerous accusations 
based on anecdotal accounts of patients. These al¬ 
legations, however, were not supported by scientific 
evidence. Some stories, such as the use of insect vectors 
to spread cholera, had dubious scientific plausibility. 
The North Korean and Chinese governments ignored 
or dismissed offers from the International Committee 
of the Red Cross and the World Health Organization 
(WHO) to conduct impartial investigations. The Soviet 
Union thwarted a proposal from the United States and 
15 other nations to the United Nations (UN) request¬ 
ing the establishment of a neutral commission for 
investigation. The United States admitted to having 
biological weapons but denied using them. The cred¬ 
ibility of the United States may have been undermined 
by the knowledge of its biological weapons program 
and its failure to ratify the 1925 Geneva Protocol until 
1975. Although unsubstantiated, these accusations 
resulted in a loss of international goodwill toward 
the United States and demonstrated the propaganda 


6 


Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness 


value of biological warfare allegations, regardless of 
veracity. 4 ’ Reviews of documents from former Soviet 
archives provide evidence that the allegations were 
fictitious propaganda. 45-47 

The Soviet Union accused the United States of 
testing biological weapons on Canadian Eskimos, 
resulting in a plague epidemic, 48 and of collaborating 
with Colombia in a biological attack on Colombian 
and Bolivian peasants. 49 The United States was also 
accused of planning to initiate an epidemic of cholera 
in southeastern China 50 and of the covert release of 
dengue in Cuba. 51 Similarly, the US allegations that 
Soviet armed forces and their proxies had used "yellow 
rain," aerosolized trichothecene mycotoxins (inhibitors 
of DNA and protein synthesis derived from fungi of 
the genus Fusarium) in Laos (1975-1981), Kampuchea 
(1979-1981), and Afghanistan (1979-1981), are widely 
regarded as unsubstantiated. The remote locations of 
the alleged attacks made intelligence investigations dif¬ 
ficult. Western intelligence operatives never witnessed 
these alleged attacks, and no samples of the aerosols 
were recovered. Confounding factors included: 

• contradictory testimonies from survivors of 
alleged attacks; 

• discrepancies in reported symptoms; 

• low disease rates in the allegedly attacked 
populations; 

• the recovery of mycotoxin in fewer than 10% 
of the clinical and environmental samples 
submitted; 

• the presence of Fusarium organisms as envi¬ 
ronmental commensals; 

• the possible decay of toxin under prevailing 
environmental conditions; 

• conflicting results of toxin assays from differ¬ 
ent laboratories; 

• the similarity of alleged yellow rain deposits 
recovered from environmental surfaces to bee 
feces in ultrastructural appearance and pollen 
and mold content; and 

• the natural occurrence of showers of bee feces 
from swarms of honey bees in the rain forests 
of southeast Asia. 52 

The US offensive program resulted in an under¬ 
standing of the strategic nature of biological weapons. 
By the late 1950s assessments of the potential utility of 
biological weapons were mixed. In a letter from one of 
Dwight D Eisenhower's President's Science Advisory 
Council members, George Kistiakowsky, to James 
Killian, the chair of the council, the author made it clear 
that developing highly concentrated formulations of 
biological agents, proper handling of pathogens, and 


appropriate weaponization would result in cases that 
did not act as "normal" disease. 53 At high concentra¬ 
tions and in a dried formulation, biological agents had 
the potential for causing high mortality and morbidity. 
Still, questions remained about the potential to suc¬ 
cessfully use biological weapons in a controlled and 
reliable manner. The follow-on testing authorized by 
President John F Kennedy under the umbrella program 
of Project 112 was designed to fill in these knowledge 
gaps. 40,54 In the Bay of Pigs operation of 1961, military 
planners had developed enough interest in biological 
weapons that their use was contemplated. The code- 
named "Marshall Plan" called for releasing incapacitat¬ 
ing agents to attack defenders on the beach. Ultimately, 
the plan was scrapped and biological weapons were 
not used. 55 

By the late 1960s domestic and international pres¬ 
sures were calling for the elimination of the US offen¬ 
sive biological warfare program. At Dugway Proving 
Ground, an incident involving chemical weapons 
testing caused the death of 3,000 sheep. Debates about 
chemical and biological weapons, both for and against 
the development of offensive capabilities, ensued 
between Congress, the administration, industry, and 
even private citizens. In Europe draft texts of what 
would later become the Convention on the Prohibi¬ 
tion of the Development, Production and Stockpiling 
of Bacteriological and Toxin Weapons and on their 
Destruction (1972 Biological Weapons Convention 
[BWC]) were being developed by Great Britain, Swe¬ 
den, and the Soviet Union. 

In May 1969 US President Richard Nixon called for 
an interagency review of chemical-biological warfare 
policies. The review was authorized as part of Na¬ 
tional Security Study Memorandum 59. The findings 
resulted in recommendations to President Nixon to 
eliminate the US offensive program and retain a de¬ 
fensive program. 

To this end, on November 25, 1969, when visiting 
Fort Detrick, President Nixon announced a new US 
policy on biological warfare, unilaterally renouncing 
the development, production, stockpiling, and use 
of biological weapons. In explaining his decision. 
President Nixon stated, "Biological weapons have 
massive, unpredictable, and potentially uncontrollable 
consequences. They may produce global epidemics 
and impair the health of future generations." 56 Almost 
immediately after the statement, confusion and a po¬ 
tential loophole caused by the ambiguity concerning 
biologically derived toxins that were technically ex¬ 
cluded from the renunciation were corrected through 
National Security Study Memorandum-85, "Review 
of Toxins Policy," which was issued on December 31, 
1969. 


7 


Medical Aspects of Biological Warfare 


The US Army Medical Unit was closed, and Fort 
Detrick and other installations in the offensive 
weapons program were redirected to solely de¬ 
velop defensive measures such as vaccines, drugs, 
and diagnostics. The US Army Medical Research 
Institute of Infectious Diseases (USAMRIID) was 
created with biosafety level 3 and 4 laboratories 
dedicated to developing medical defensive coun¬ 
termeasures. By May 1972 all antipersonnel agents 
had been destroyed, and the production facility at 
Pine Bluff, Arkansas, was converted into a research 
facility. By February 1973 all agriculture-targeted 
biological agents had been destroyed. Although 
staphylococcal entertoxin was used during World 
War II by Office of Strategic Services' espionage 
agents, 37,38 biological weapons have never been used 
by the US Armed Forces. 39 The Central Intelligence 
Agency developed weapons containing cobra ven¬ 
om and saxitoxin for covert operations; all records 
regarding their development and deployment were 
destroyed in 1972; all remaining toxin samples were 
destroyed per presidential orders after a US Sen¬ 
ate investigation. 37 The United States signed and 
ratified both the 1925 Geneva Convention and the 
1972 BWC, which outlaws all offensive biological 

THE SOVII 

Although a signatory to the 1925 Geneva Conven¬ 
tion, the Soviet Union began a weapons development 
program in 1928 58 Linder the control of the state security 
apparatus, GPU (the Unified State Political Administra¬ 
tion of the Committee of People's Commissars of the 
USSR). Work was initially done with typhus, reportedly 
with experimentation on political prisoners at Slovetsky 
Island in the Baltic Sea and nearby concentration camps. 
The program subsequently expanded to include work 
with the agents of Q fever, glanders, and melioidosis, 
and possibly tularemia and plague. Outbreaks of Q 
fever and tularemia among German troops are two 
suggested, but unconfirmed, Soviet uses of biological 
warfare during World War II. 59 However, the origin of 
epidemic tularemia during the battle of Stalingrad as a 
consequence of biological warfare has been challenged 
and attributed to natural causes and a breakdown of 
public health. 60 Similar outbreaks of Q fever in Axis 
troops in Italy, Greece, Bulgaria, and the Ukraine 61 ; in 
Allied troops in the Mediterranean Theater 62-64 ; and 
more recently, among Czech peacekeepers in Bosnia- 
Herzegovina 65 and tularemia among civilians during the 
Kosovo conflict 66 have been attributed to amplification 
of natural transmission cycles during wartime. 

Stalin was forced to move his biological warfare 
operations out of the path of advancing German 
forces. Laboratories were moved to Kirov in eastern 


weapons research, production, and possession, in 
1975 (see Disarmament: The Biological Weapons 
Convention). 

Although many welcomed the termination of the US 
offensive program for moral reasons, the decision was 
partly motivated by pragmatic considerations. Biologi¬ 
cal weapons were unnecessary for national security 
because of a formidable arsenal of conventional, chemi¬ 
cal, and nuclear weapons. Although open-air simulant 
studies suggested that biological weapons would be 
effective, the potential effects of aerosols of virulent 
agents on targeted populations were still conjectural 
and could not be empirically validated for ethical and 
public health reasons. Despite evidence to the contrary 
from information obtained through the US offensive 
program, some still considered biological weapons to 
be untried, unpredictable, and potentially hazardous 
for the users. Field commanders and troops were un¬ 
familiar with their use. Most importantly, the United 
States and allied countries had a strategic interest in 
outlawing biological weapons programs to prevent the 
proliferation of relatively low-cost weapons of mass 
destruction. Outlawing biological weapons made the 
arms race for weapons of mass destruction prohibi¬ 
tively expensive, given the cost of nuclear programs. 21,57 


European Russia, and testing facilities were eventu¬ 
ally established on Vozrozhdeniya Island on the Aral 
Sea between the Soviet Republics of Kazakhstan and 
Uzbekistan. At the conclusion of the war, Soviet troops 
invading Manchuria captured many Unit 731 Japa¬ 
nese scientists and learned of their extensive human 
experimentation through captured documents and 
prisoner interrogations. Emboldened by the Japanese 
findings, Stalin put KGB (Committee of State Security) 
chief Lavrenty Beria in charge of a new biowarfare 
program. The production facility at Sverdlovsk was 
constructed using Japanese plans. After Stalin died in 
1953, Beria was executed, and Nikita Khrushchev, the 
new Kremlin leader, transferred the biological warfare 
program to the Fifteenth Directorate of the Red Army. 
Colonel General Yefim Smirnov, a strong advocate of 
biological weapons who had been the chief of army 
medical services during the war, became the director. 67 

In 1956 Defense Minister Marshall Georgy Zhukov 
announced that the Soviet Union would be capable 
of deploying biological and chemical weapons in the 
next war. By 1960 numerous research facilities existed 
in the Soviet Union. Although the Soviet Union signed 
the 1972 BWC, it doubted US compliance, and subse¬ 
quently expanded its program. 58,59,67 Various institu¬ 
tions under different ministries and production facili¬ 
ties were incorporated into an organization known as 


8 


Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness 


Biopreparat to carry out offensive research, develop¬ 
ment, and production under the label of legitimate 
civil biotechnology research. Biopreparat conducted 
clandestine activities at 52 sites and employed more 
than 50,000 people. Production capacity for weapon- 
ized smallpox was 90 to 100 tons annually. 59 

The Soviet Union was an active participant in 
WHO's 1964 to 1979 smallpox eradication program. 
Soviet physicians participating in the program sent 
specimens to Soviet research facilities. For the Soviets, 
the program presented an opportunity not only to rid 
the world of naturally occurring smallpox, but also — 
reportedly—to obtain virulent strains of smallpox 
virus that could be used to develop biological weapons. 
WHO announced the eradication of smallpox in 1980, 
and the world rejoiced at this public health break¬ 
through. The bioweapon developers in the former 
Soviet Union had a more cynical reaction. Smallpox 
eradication would result in the termination of vaccina¬ 
tion; eventually the world's population would again 
become vulnerable. It was this vulnerability that would 
inspire the former Soviet Union to develop smallpox 
as part of a strategic weapons system, with production 
of the virus on a massive scale and plans for delivery 
using intercontinental missiles. 59 

In addition to military biological weapons pro¬ 
grams, the Soviets developed toxin weapons for use 
by Warsaw Pact intelligence services. An assassination 
using a biological weapon was executed in September 
1978 when a Bulgarian secret service member attacked 
Georgi Markov, a Bulgarian exile living in London. 
A device concealed in an umbrella discharged a tiny 
pellet into the subcutaneous tissue of his leg. He died 
several days later. The pellet, which had been drilled 
to hold a toxic material, was found at autopsy. No 
toxin was identified, but ricin was postulated as the 
only toxin with the potency to kill with such a small 
dose. 68 Vladimir Rostov, a Bulgarian defector living in 
Paris, had been attacked in a similar manner a month 
earlier. He experienced fever and pain and bleeding at 
the wound site, yet had no further complications. After 
learning of Markov's death, he sought medical evalu¬ 
ation; radiographs disclosed a small metallic pellet in 
subcutaneous fat. The pellet was surgically removed. 
Rostov then tested positive for anti-ricin antibodies, 
supporting the probable use of ricin in these attacks. 23 

In October 1979 a Russian emigrant newspaper 
published in Frankfurt, Germany, reported a sketchy 
story of a mysterious anthrax epidemic in the Russian 
city of Sverdlovsk (now Yekaterinburg). The military 
reportedly took control of hospitals in Sverdlovsk to 
care for thousands of patients with a highly fatal form 
of anthrax. Soviet officials attributed the epidemic to 
cutaneous and gastrointestinal anthrax contracted from 
contaminated meat. However, US intelligence agencies 


suspected that the outbreak resulted from inhalational 
anthrax following a release of B anthracis spores from 
Compound 17, a Soviet military microbiology facility. 69-71 
The Central Intelligence Agency sought the opinion of 
Matthew Meselson, a Harvard biologist who had been a 
strong proponent of the Nixon ban of the US biological 
warfare program. He initially doubted the Soviet weapon 
release hypothesis. Other observers reviewing the same 
evidence reached different conclusions, however, and 
satellite imagery from the late spring of 1979 showed 
a flurry of activity at and around the Sverdlovsk in¬ 
stallation consistent with a massive decontamination 
effort. The incident generated enough concern within 
the Reagan administration and the Department of 
Defense (DoD) to increase military biopreparedness. 

Debate of the incident raged for the next 12 years. 
Meselson testified before the US Senate that the bur¬ 
den of evidence supported the claim that the outbreak 
resulted from the Soviets' failure to keep B anthracis- 
infected animals out of the civilian meat supply. In 
1992, after the fall of the Soviet Union, Meselson was 
allowed to take a team of scientists to review autopsy 
material and other evidence from the Sverdlovsk inci¬ 
dent. The team's attempts to review hospital records of 
cases from the outbreak were unsuccessful because the 
RGB had confiscated the records. However, the team 
performed the following: 

• acquired an administrative list of 68 of the 
deceased; 

• obtained information from grave markers in 
a cemetery designated for the anthrax casual¬ 
ties; 

• obtained epidemiological data by interview¬ 
ing nine survivors and relatives and friends 
of 43 deceased; and 

• determined that the cases occurred among 
people who had either lived or worked in a 
narrow zone southeast of Compound 17 dur¬ 
ing the first week of April 1979. 

Archived weather reports at the city's airport 
disclosed that the wind direction on April 2, 1979, 
correlated with the geographic distribution of cases. 
Meselson and his team concluded that the outbreak 
resulted from the escape of aerosolized spores from 
the facility on April 2, 1979, with downwind trans¬ 
mission. 69 Furthermore, Russian pathologists who 
had conducted autopsies on 42 fatalities, and had 
courageously preserved tissue specimens and autopsy 
records at great personal risk, shared their findings 
with Meselson's team and published their results 
confirming inhalational anthrax, 72 described the Soviet 
cover-up of the outbreak, and postulated a release of 
spores from Compound 17. 71 


9 


Medical Aspects of Biological Warfare 


In 1992 Russian leader Boris Yeltsin admitted in pri¬ 
vate conversations with President George H Bush that 
the KGB and military had misrepresented the anthrax 
deaths. Subsequently, in a press release, Yeltsin admit¬ 
ted to the offensive program and the origin of the Sverd¬ 
lovsk biological weapons accident. Additionally, retired 
Soviet general Andrey Mironyuk disclosed that safety 
filters had not been activated on the fateful morning in 
early April 1979, resulting in the escape of aerosolized 
B anthracis and the ensuing epidemic. 73 Soviet defectors, 
including Ken Alibek, first deputy chief of Biopreparat 
from 1988 to 1992, confirmed that not only was the 
Sverdlovsk epidemic caused by an accidental release 
of spores from a biological weapons production plant, 
but also that the Soviet biological warfare program 
had been massive. 59 In September 1992 Russia entered 
an agreement with the United States and the United 
Kingdom that acknowledged a biological weapons 
program inherited from the Soviet Union, committed 
to its termination, and agreed to onsite inspections. 
The United States assisted the Russian Federation and 
other former Soviet republics through the Nunn-Lugar 
Biological Threat Reduction Program (later called the 
Cooperative Biological Engagement Program) to: 

• dismantle biological weapons research, devel¬ 
opment, and production infrastructure; 

• secure dangerous pathogens into central refer¬ 
ence laboratories; 

THE SPECIA1 

The most ominous biological warfare threat that 
US military forces have faced came during Operations 
Desert Shield and Desert Storm in 1990 and 1991. Intel¬ 
ligence reports suggested that Iraq had developed and 
operated a biological weapons program during the 
1980s. Coalition troops trained in protective gear were 
issued ciprofloxacin in theater for use as postexposure 
prophylaxis against an Iraqi anthrax attack. Before the 
hostilities, approximately 150,000 US troops received 
the Food and Drug Administration-licensed anthrax 
vaccine, and 8,000 received a botulinum toxoid vaccine 
approved by the Food and Drug Administration as an 
investigational new drug. Postwar inspections by the 
multinational UN Special Commission (UNSCOM) on 
Iraq were repeatedly confounded by Iraqi misinforma¬ 
tion and obfuscation. After General Hussein Kamal 
defected in 1995, the Iraqi government disclosed that 
it had operated a robust biological weapons pro¬ 
gram at six major sites since the 1980s, contrary to 
its obligations as a state party to the BWC. The Iraqi 
program conducted basic research on B anthracis, ro¬ 
tavirus, camelpox virus, aflatoxin, botulinum toxins. 


• upgrade laboratory safety and security; 

• enhance capacities for diagnosis, surveillance, 
and public health response; and 

• engage scientists with biological weapons 
expertise in projects directed to modeling, 
medical countermeasure development, and 
other peaceful purposes. 74,75 

This led to the dismantlement or conversion 
of three large production facilities and dozens of 
institutes that supported the biological weapons 
program, the destruction of 150 tons of B anthra¬ 
cis weapons agent on Vozrozhdeniya Island, and 
unprecedented transparency at potential dual-use 
facilities that had previously been closed to foreign¬ 
ers. 76 However, in 1999 President Vladimir Putin, 
proposed the development of weapons based on 
new genetic technology. Although this directive was 
promptly dropped from publicly available docu¬ 
ments, he retracted the 1992 disclosures of President 
Yeltsin.'' The Russian government currently denies 
that the former Soviet offensive program had ever 
existed, claiming that it had only conducted defen¬ 
sive research. 58,77 According a 2013 US Department 
of State report, it is still unclear if the Russian Fed¬ 
eration has completed the destruction or diversion 
of the offensive program to peaceful purposes, or if 
it continues to conduct activities inconsistent with 
the BWC. 78 


mycotoxins, and an anticrop agent (wheat cover 
rust); and it tested several delivery systems including 
aerial spray tanks and drone aircraft. Furthermore, 
the Iraqi government had weaponized 6,000 L of B 
anthracis spores and 12,000 L of botulinum toxin in 
aerial bombs, rockets, and missile warheads before 
the 1991 Persian Gulf War (Table 1-2 and Table 1-3). 
Although these weapons were deployed, they were 
not used. 79,80 The reasons behind Saddam Hussein's 
decision not to use these weapons are unclear; perhaps 


TABLE 1-2 

BIOLOGICAL AGENTS PRODUCED BY IRAQ* 


Agent 

Produced (L) 

Weaponized (L) 

Botulinum 

19,000 

10,000 

Bacillus anthracis 

8,500 

6,500 

Aflatoxin 

2,200 

1,580 


"Disclosed by the Iraq government in 1995. 
L: liter 


10 





Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness 


TABLE 1-3 

DELIVERY SYSTEMS FOR BIOLOGICAL 
AGENTS DEVELOPED BY IRAQ* 


Aerial Bombs 


Missile Warheads 


Botulinum 

100 

Botulinum 

13 

Bacillus anthracis 

50 

Bacillus anthracis 

10 

Aflatoxin 

16 

Aflatoxin 

2 


"'Disclosed by the Iraq government in 1995. 


he was concerned about provoking massive retalia¬ 
tion. Alternately, decisive factors may have included 
the possible ineffectiveness of untested delivery and 
dispersal systems, the probable ineffectiveness of liq¬ 
uid slurries resulting from poor aerosolization, and 
the potential hazards to Iraqi troops, who lacked the 
protective equipment and training available to coali¬ 
tion forces. 81 The Iraqis claimed to have destroyed 
their biological arsenal immediately after the war 
but were unable to provide confirmatory evidence. A 
covert military research and development program 
continued for another 4 years, with the intent of re¬ 
suming agent production and weapons manufacture 
after the end of UN sanctions. Infrastructure was 
preserved, and research on producing dried agent 
was conducted under the guise of biopesticide pro¬ 
duction at the A1 Hakam Single Cell Protein Plant 
until its destruction by UNSCOM inspectors in 1996. 
Despite their obvious successes, the UNSCOM inspec¬ 
tors never received full cooperation from the Saddam 
Hussein regime, and were ejected from Iraq in 1998. 

The Iraqi regime continued to promote an air of 
uncertainty after 1998 as to whether it had an active 
ongoing biological weapons program. Amy Smithson, 
in her very detailed account of the Iraqi biological 
weapons program and the UNSCOM inspections, 
suggests three possible reasons why Saddam Hussein 
may have wanted to maintain the perception that his 
biological weapons program was still active 82 : 


South Africa is alleged to have operated a small-scale 
biological weapons program between 1981 and 1993, 
after becoming state-party to both the 1925 Geneva 
Convention (1960) and the BWC (1975). The South 
African biological weapons program, code-named 
Operation Coast, reportedly conducted research on B 
anthracis, V cholerae, ricin, botulinum toxin, and other 
agents, and intended to use genetic engineering to 
develop biological agents that would selectively target 
people of black African ancestry. Although Operation 


1. To deter attacks by regional rivals, especially 
Iran; 

2. To promote his image internally as a strong 
and unassailable leader and thus preserve his 
own internal stranglehold over Iraq; and 

3. To maintain his own outsized vision of his 
ultimate dream and legacy. 

Regardless of his strategic motives, the uncertainty 
about his biological weapons program ultimately 
contributed greatly to the Hussein government's fall 
and his own demise. The breakdown of the inspec¬ 
tions, lack of firsthand information, misinformation 
provided by an informant (Rafid Ahmed Alwan al- 
Janabi, an Iraqi defector code named "Curveball" by 
the Central Intelligence Agency), and the 2001 anthrax 
mailings contributed to growing uncertainties, am¬ 
biguities, and apprehension, culminating in the 2002 
US National Intelligence Estimate and assessments 
by the intelligence services of France, Germany, and 
the United Kingdom, that postulated a robust Iraqi 
biological weapons program. 83,84 International concern 
led to renewed inspections in 2002 under UN Security 
Council Resolution 1441. The Iraqi government failed 
to cooperate fully, and coalition forces invaded Iraq in 
2003, believing at the time that Iraq's regime still posed 
a significant biological weapons threat. In 2005 the 
Iraq Survey Group (an international group composed 
of civilian and military members) concluded that the 
Iraqi military biological weapons program had been 
abandoned from 1995 through 1996 because the poten¬ 
tial discovery of continued activity would risk severe 
political repercussions including the extension of UN 
sanctions. However, Saddam Hussein had perpetuated 
ambiguity regarding a possible program as a strategic 
deterrent against Iran. 85 The Iraqi Intelligence Service 
continued to investigate toxins as tools of assassination, 
concealed its program from UNSCOM inspectors after 
the 1991 Persian Gulf War, and reportedly conducted 
lethal human experimentation until 1994. Small-scale 
covert laboratories were maintained until 2003. 86 


Coast acquired a collection of pathogens, it was not 
successful in developing large-scale delivery systems. 
V cholerae was reportedly used in 1989, but the attack 
failed because of the targeted water supply's chlorine 
content. After diplomatic interventions by the United 
States and Great Britain, the program was closed in 1993, 
coincident with the demise of the apartheid regime. 87-89 

V cholerae was allegedy used by Rhodesian forces 
with South African assistance during the civil war 
of the 1970s to contaminate rivers used as water 


11 





Medical Aspects of Biological Warfare 


sources by rebel forces; these attacks are thought to 
have failed because of dilution. Rhodesian forces 
reportedly used B anthracis against livestock; the 
role of these attacks in an anthrax epizootic dur¬ 
ing 1979-1980 was investigated but could not be 
determined. 88 

Libya allegedly launched a clandestine biological 
weapons effort during the 1990s (while a state-party 
to the BWC), and sought assistance from Iraq, North 
Korea, and South Africa. However, in contrast to its 
chemical weapons program, the effort was limited 
to small-scale research, and according to one official 
never progressed beyond initial planning. 90 Colonel 
Muammar al-Qaddafi, an authoritarian dictator 
who ruled Libya for 42 years, formally renounced 
all weapons of mass destruction in 2003; inspectors 
from the United States and the United Kingdom 
found no evidence of an offensive biological weap¬ 
ons program. 90 

An unclassified 2013 US State Department report 
noted that North Korea may still consider the use of 
biological weapons as a military option, and that it is 
unclear if Iran is conducting activities prohibited by 
the BWC. 78 


Biocrime is the malevolent use of biological agents 
when the perpetrator's motivation is personal, as op¬ 
posed to a broader ideological, political, or religious 
objective. Although biocrimes constitute only a small 
fraction of criminal assaults and are usually unsuc¬ 
cessful, 95 a well-executed attempt may be deadly; the 
resulting disease may pose clinical and forensic chal¬ 
lenges. Biocrimes have generally been more successful 
than bioterrorist attacks; 8 of 66 biocrimes reviewed by 
Tucker produced 29 deaths and 31 injuries. 96 

Perpetrators with scientific or medical expertise 
or those who have recruited trained accomplices 
typically attempt biocrimes. Criminals without a 
technical background have successfully extracted 
ricin from castor beans but have generally been un¬ 
able to obtain or produce other agents. In a review 
of 14 episodes in which agent was used, biological 
agents were usually obtained from a legitimate 
source or stolen; the perpetrators produced agent 
in only two cases. 21,95 Preferred agents have been 
bacteria and toxins (eg, ricin). Food contamination 
has been preferred over direct injection or topical 
application as a means of attack. 

One of the most striking examples of foodbome 
biocrime occurred in Japan between 1964 and 1966. 
Dr Mitsuru Suzuki allegedly contaminated food items, 
medications, barium contrast, and a tongue depressor 


The US Director of National Intelligence reported 
in an open US Senate hearing in 2013 that Syria (a sig¬ 
natory, but not a state-party to the BWC) maintains a 
biological weapons program capable of limited agent 
production; and although Syria is not known to have 
loaded biological agents in effective delivery systems, 
it possesses conventional and chemical weapons 
devices that could be adapted to launch biological 
attacks. 91,92 In the context of the ongoing Syrian civil 
war in 2014, there are concerns regarding potential 
deployment 93 and that further disintegration of the 
Assad regime could enable A1 Qaeda and Hezbollah 
to seize Syrian unconventional weapons. 94 

Some 20 nations are thought to have engaged in 
offensive biological weapons efforts. The total number 
of nations and the extent of their efforts are difficult 
to establish because several have engaged in research 
and development, but not taken their efforts to testing, 
deployment, and use. Although the list of states ap¬ 
pears to be down from the 20 or so that were thought 
to have biological weapons programs in the assess¬ 
ments in the 1980 and 1990s, several states including 
North Korea, Syria, and Iran are still thought to have 
biological weapons programs. 54(p68) 


with Salmonella typhi and agents of dysentery on nu¬ 
merous occasions resulting in more than 120 cases and 
four deaths. 23 A variation on the Suzuki crime occurred 
in 1996 when Diane Thompson, a hospital microbiolo¬ 
gist, deliberately infected 12 coworkers with Shigella 
dysenteriae. She sent an email to her coworkers inviting 
them to eat pastries she had left in the laboratory break 
room. Eight of the 12 casualties and an uneaten muffin 
tested positive for S dysenteriae type 2, identical to the 
laboratory's stock strain by pulsed-field electrophore¬ 
sis. 97 Police learned that her boyfriend had previously 
suffered similar symptoms and had been hospitalized 
at the same facility, and that Thompson had falsified 
his laboratory test results. Thompson was sentenced 
to 20 years in prison. 23 

Murders by direct injection included the use of 
diphtheria toxin in Russia in 1910. The director of a 
Norwegian nursing home was convicted in 1983 of 
murdering 22 patients by injecting a curare deriva¬ 
tive. There have been at least four murder attempts 
by injecting victims with human immunodeficiency 
virus-infected blood. 23 

Numerous and highly varied biocrimes have been 
reported; only several representative examples are 
included in this chapter. The works of Carus, 2 Leiten- 
berg, 21 and Tucker 96 provide comprehensive descrip¬ 
tions and analysis. 


12 


Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness 


BIOLOGICAL TERRORISM 


Bioterrorism is the use of biological agents by an 
individual or group not acting as official agents of a 
government to achieve a political or ideological objec¬ 
tive. Bioterrorist incidents increased markedly after 
1985, with two peaks in 1998 and 2001. The 1998 peak 
followed publicity of the anthrax threat posed by Larry 
Wayne Harris; the 2001 peak followed the Septem¬ 
ber through October anthrax mailings. Successfully 
executed attacks have been few but high in impact; 
the 1984 Rajneeshee Salmonella attack resulted in 751 
cases of infection; the 2001 anthrax mailings resulted 
in 22 cases of infection, five deaths, and approximately 
10,000 individuals being offered postexposure prophy¬ 
laxis. The vast majority of incidents (at least 98% during 
2000-2002) have been hoaxes, which have nonetheless 
produced considerable social disruption. 98,99 

The first large-scale bioterrorism attack in the 
United States occurred in 1984. In the 1960s an Indian 
guru named Bhagwan Shree Rajneesh founded the 
Rajneeshee cult. Rajneesh succeeded in attracting 
followers from the upper middle class and collecting 
significant donations and proceeds from book and 
tape sales. Rajneesh acquired the Big Muddy Ranch 
near The Dalles, Oregon, and built a community for 
his followers named Rajneeshpuram, which became an 
incorporated community. Within a few years, the Raj- 
neeshees came into conflict with the local population 
regarding development and land use. The Rajneeshees 
attempted to gain control of the Wasco County gov¬ 
ernment by bringing in thousands of homeless people 
from cities around the country, counting on their 
votes in the upcoming elections. The Rajneeshees also 
plotted to sicken the local population to prevent them 
from voting. 21 

Two Wasco County commissioners visiting Ra¬ 
jneeshpuram on August 29,1984, were given drinking 
water contaminated with Salmonella typhimurium; both 
became ill and one was hospitalized. In trial runs in 
the months leading up to the November 1984 elections, 
several attempts at environmental, public water, and 
supermarket food contamination were unsuccessful. In 
September, Rajneeshees began contaminating food at 
local restaurants by pouring slurries of S typhimurium 
into salad bars, salad dressing, and coffee creamers at 
10 restaurants. This attack caused 751 cases of enteritis 
and at least 45 hospitalizations. 23,100 

In 1995 in Japan, the Aum Shinrikyo cult released 
sarin gas in the Tokyo subway system, resulting in 
12 deaths and thousands seeking emergency care. 
The cult, founded by Shoko Asahara, had amassed 
approximately 10,000 members and $300 million in 
financial assets. Aum Shinrikyo mimicked the orga¬ 


nization of the Japanese government with "ministries 
and departments." Seiichi Endo, who headed "health 
and welfare," had worked in genetic engineering 
at Kyoto University's viral research center. Hideo 
Murai, who headed "science and technology," had 
an advanced degree in astrophysics and had worked 
in research and development for Kobe Steel Corpora¬ 
tion. Endo attempted to derive botulinum toxin from 
environmental isolates of Clostridium botulinum at the 
cult's Mount Fuji property. A production facility was 
built and horses were stabled for developing a horse 
serum antitoxin. It is uncertain whether Endo success¬ 
fully produced potent botulinum toxin. 23 

In 1993 Aum Shinrikyo built a new research facility 
on the eighth floor of an office building owned by the 
cult in eastern Tokyo. The cult grew B anthracis and 
installed a large industrial sprayer for dissemination. 
The cult is also believed to have worked with C bur¬ 
netii and poisonous mushrooms, and it sent a team to 
Zaire in the midst of an Ebola epidemic to acquire the 
Ebola virus. According to press accounts from 1990 to 
1995, the cult attempted to use aerosolized biological 
agents against nine targets. Three attacks were at¬ 
tempted with B anthracis and six with botulinum toxin. 
In April 1990 the cult equipped three vehicles with 
sprayers containing botulinum toxin targeting Japan's 
parliamentary Diet Building in central Tokyo, the 
city of Yokahama, Yosuka US Navy Base, and Nairta 
International Airport. In June 1993 the cult targeted 
the wedding of Japan's crown prince by spraying 
botulinum toxin from a vehicle in downtown Tokyo. 
Later that month, the cult spread B anthracis using the 
roof-mounted sprayer on its eight-story building. In 
July 1993 the cult targeted the Diet in central Tokyo 
again by using a truck spraying B anthracis, and later 
that month it targeted the Imperial Palace in Tokyo. 
On March 15, 1995, the cult planted three briefcases 
designed to release botulinum toxin in the Tokyo 
subway. Explanations for the cult's failure include the 
possible use of a nontoxin-producing (or low yield) 
strain of C botulinum, use of a low-virulence veterinary 
vaccine strain of B anthracis, ineffective spraying equip¬ 
ment, and perhaps subversion on the part of some cult 
members who were reluctant to execute the planned 
operation. 19 Ultimately, Aum Shinrikyo gave up on its 
biological weapons and released sarin in the Tokyo 
subway on March 20, 1995. 23 

Meanwhile in the United States, two members 
of the Minnesota Patriots Council, an antigovern¬ 
ment extremist group, were arrested for producing 
ricin and planning to attack federal agents by con¬ 
taminating doorknobs. Larry Wayne Harris, a clinical 


13 


Medical Aspects of Biological Warfare 


microbiologist with ties to racist groups, was arrested 
in 1995 for using fraudulent information to obtain a 
culture of Y pestis from the American Type Culture 
Collection. He was arrested a second time in 1998 after 
making threatening remarks to US federal officials and 
violating his parole. Harris had constructed a covert 
laboratory in Nevada and was conducting experiments 
with the Sterne strain of B anthracis, a nonencapsulated 
but toxigenic live attenuated veterinary vaccine, and 
he threatened to attack Las Vegas with B anthracis. 68 
His case led to the establishment of the Select Agent 
Program (42 CFR Part 73, Possession, Use, and Transfer 
of Select Agents and Toxins) that included the develop¬ 
ment of stringent regulations for the procurement and 
shipping of select microbes. 

On October 4, 2001, just 3 weeks after the Septem¬ 
ber 11th attacks on the World Trade Center and the 
Pentagon had made the nation acutely aware of its 
vulnerability to international terrorism, health officials 
in Florida reported a case of inhalational anthrax. Dur¬ 
ing the first week of September, American Media, Inc, 
received a letter addressed to Jennifer Lopez contain¬ 
ing a fan letter and a "powdery substance." The letter 
was passed among its employees, including Robert 
Stevens. Retrospectively, investigators would consider 
not this letter, but perhaps a subsequent letter, as the 
source of his infection. 101 

Stevens was admitted to a Palm Beach, Florida, 
hospital with high fever and disorientation on October 
2, 2001. By October 5, he was dead from inhalational 
anthrax, the first such case in the United States in more 
than 20 years. 

Soon afterward anthrax mailings were received at 
civilian news media operations in New York City and 
in the Hart Senate Office Building in Washington, DC. 

At least five (four recovered) letters containing B 
anthracis spores had been mailed on September 18, 
2001, and October 9, 2001. Twenty-two people con¬ 
tracted anthrax, with 11 inhalational cases resulting 
in five deaths. Thirty-five postal facilities and com¬ 
mercial mailrooms were contaminated. Screening and 
postexposure prophylaxis disrupted operations at the 
Hart US Senate Office Building. Decontamination of 
postal facilities cost more than $1.2 billion and resulted 
in the closure of heavily contaminated facilities in 
Washington, DC (October 2001-December 2003), and 
Trenton, New Jersey (October 2001-March 2005). 102 
More than $27 million was spent on decontaminating 
Capitol Hill facilities. 102 Public alarm was compounded 
by numerous "white powder" hoaxes. 

Farsighted emergency planning and training, in ad¬ 
dition to the integration of federal and local medical, 
public health, and law enforcement agencies in New 
York City and other cities, enabled an unprecedented 


public health response. The Laboratory Response 
Network and military laboratories such as USAMRIID 
processed more than 125,000 clinical specimens and 1 
million environmental samples. USAMRIID ran more 
than 260,000 assays on more than 30,000 samples in 
9 months. Prophylaxis supplied from the national 
stockpile was offered to nearly 10,000 individuals at 
risk. No cases were found among prophylaxis recipi¬ 
ents. 103,104 Treatment guidelines advocating multidrug 
antibiotic combinations and aggressive intensive care 
were disseminated, 105 and the case fatality rate for 
inhalational anthrax—historically exceeding 90%— 
reduced to 45%. 106,107 

The attacks provoked an unprecedented criminal 
investigation that coupled traditional law enforce¬ 
ment with the development and validation of novel 
emerging genetic sequencing techniques. The Federal 
Bureau of Investigation (FBI) special agents and US 
Postal Service Inspectors conducted the investiga¬ 
tion for 7 years, and 29 government, academic, and 
commercial laboratories supported it. Investigators 
conducted more than 10,000 witness interviews on 
six continents and 80 searches, and they also collected 
more than 6,000 items of potential evidence and 5,730 
environmental samples from 60 locations both within 
the United States and in foreign countries, with the co¬ 
operation of the respective host nation governments. 102 

US Attorney General John Aschroft named Dr 
Steven J Hatfill, a USAMRIID scientist between 1997 
and 1999, a "person of interest" during a television 
interview in 2002. Dr Hatfill vehemently denied 
involvement, and sued the federal government, 
claiming that law enforcement officials had leaked 
information to the media in violation of the Privacy 
Act, and had ruined his reputation and career. The 
FBI exonerated him in 2008, and he received $5.82 
million in restitution. 102,108,109 

Forensic analysis was confounded by the highly 
conserved B anthracis genome, which features more 
than 99.99% nucleotide sequence identity among the 
most genetically divergent strains. Investigators went 
beyond the contemporary standard of genetic typing 
by sequencing small DNA segments to advance the 
technique of whole genome sequencing. Comparison 
of the whole genomes of the index case isolate and a 
reference Ames strain disclosed that they were essen¬ 
tially identical, and it could not pinpoint the origin of 
the letter contents. However, a breakthrough followed 
the observation of four phenotypic colony morphology 
variants constituting less than 1% of colonies cultured 
from spore samples taken from three of the anthrax 
letters. Each colony morphology variant was associ¬ 
ated with a distinct mutation restricted to four genetic 
loci. These mutations were absent in environmental 


14 


Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness 


isolates taken during the investigation. 110 Specimens 
were obtained from every culture of B anthracis Ames 
strain (1,071 samples) from all 15 US and three for¬ 
eign laboratories known to possess it. One or more 
of the mutants was detected in 71 of 947 samples that 
could be evaluated; all four mutants were present in 
eight samples. The probability of samples to contain 
all four mutants was calculated to be 0.4383 x 10" 6 or 
0.0004 samples in the 947 sample collection, if the 
samples were unrelated; these eight samples consisted 
of a specimen from RMR-1029, a flask containing a 
liquid spore preparation in the laboratory of anthrax 
researcher Dr Bruce E Ivins at USAMRIID, and seven 
specimens from another laboratory that were descen¬ 
ders of RMR-1029. 111 ' 112 

The FBI concluded that Dr Ivins was the sole per¬ 
petrator based on the following: 

• the genetic analysis results; 

• inconsistencies during interviews; 

• erratic conduct that included irregular labora¬ 
tory hours before each mailing and an unau¬ 
thorized and unreported decontamination of 
his office and laboratory during the investiga¬ 
tion; 

• deteriorating behavior as the investigation 
progressed; and 

• exclusion of other individuals with access to 
RMR-1029 and its descendants. 

The purported motive was to ensure continued sup¬ 
port for the anthrax vaccine research in which Dr Ivins 
was personally heavily invested and was under criti¬ 
cism from multiple sectors. The US Attorney's Office for 
the District of Columbia prepared an indictment charg¬ 
ing him with Use of a Weapon of Mass Destruction, in 
violation of Title 18, United States Code, Section 2332a, 
and related charges. Dr Ivins, aware of the indictment, 
took an overdose of over-the-counter medications and 
died on July 29, 2008. 102 

Lingering doubts were expressed during a plenary 
session at the 2009 American Society for Microbiology 
Biodefense and Emerging Diseases Research Confer¬ 
ence. 113 Evidence was considered circumstantial. No 
evidence of B anthracis contamination was found in 
Dr Ivin's home or vehicles. Unexplained aspects of the 
case included the contamination of the September 18 
mailing with a B subtilis strain that could not be traced 
to USAMRIID and the use of dry spore preparations 
(the production of which is prohibited in the US biode¬ 
fense program), for which there was no direct evidence 
within USAMRIID. A National Academy of Sciences 
review concluded that the genetic typing results were 
consistent with—but not definitive proof of—the deri¬ 


vation of the letter isolates from RMR-1029. Although 
generally supportive of the FBI's efforts, the reviewers 
criticized the FBI's statistical methods and stated that 
an alternative source could not be excluded because of 
possible sharing and mixing of samples among labora¬ 
tories, and because the possibility of identical mutations 
arising through parallel evolution independently in 
unrelated cultures had not—in their opinion—been 
adequately explored. 112 Abnormally high concentra¬ 
tions of silicon 114 and tin existed in the spores that were 
absent in spores from RMR-1029; this raised contro¬ 
versies regarding potential production at the Dugway 
Proving Ground or at a civilian contractor laboratory, 
where work with silicon and surrogate spores had 
previously been done. 115 Finally, Department of Justice 
lawyers used the argument that Dr Ivin's lab had no 
equipment to produce dry spore preparations to defend 
the government against a wrongful death lawsuit filed 
by Robert Stevens' widow. 116 

However, the investigation spurred the advance¬ 
ment of whole genome sequencing, accelerating the 
time required to sequence a bacterial genome from 4 
months to several days, 117 and advanced the emerging 
science of microbial forensics. The investigation raised 
issues regarding laboratory programs for physical se¬ 
curity, personal reliability, and mental health screening 
that—while not directly incriminating Dr Ivins—un¬ 
derscored the importance of re-evaluating laboratory 
security measures and the value of robust employee 
occupational health programs to screen and monitor 
the mental health of researchers working with highly 
virulent pathogens. These issues were addressed by 
strengthening the federal regulations that direct CDC 
oversight of research on dangerous pathogens (see 
discussion of the Federal Experts on Security Advisory 
Panel in Toward Pan-hazard Preparedness). 118 ' 119 

The threat of bioterrorism did not end with the US 
anthrax experience. A1 Qaeda initiated a biological 
weapons program in Afghanistan before the overthrow 
of the Taliban regime. Investigations after the US 
military intervention of 2001 uncovered two A1 Qaeda 
laboratories for biological weapons development, sup¬ 
plied with commercially acquired microbiology equip¬ 
ment and staffed by trained personnel. Fortunately, a 
deployable weapon had not been constructed. 120 US 
forces operating in northern Iraq in 2003 seized a camp 
linked to A1 Qaeda reportedly containing instructions 
and equipment for ricin extraction. 121 

During the period that followed the US anthrax 
attacks, ricin became the bioweapon of choice for a 
number of misanthropes intent on nefarious use of 
biological agents, perhaps because of its relative ease 
of access. The castor beans (ricin source) are available 
worldwide because the oil is extracted for lubricant in 


15 


Medical Aspects of Biological Warfare 


many countries. The toxin extraction techniques have 
been published in many forums to include many an¬ 
archist and terrorist websites. Examples are provided 
of confirmed cases, but many more incidents have oc¬ 
curred worldwide, and most have proven to be hoaxes. 

In January 2003 British authorities uncovered the 
Wood Green ricin plot. A police raid on a London 
apartment yielded a copy of a protocol for ricin pro¬ 
duction, toxin source materials (castor beans), and a 
suitable solvent (acetone) for its extraction. Although 
tests for ricin were negative, 122 one of the tenants, an A1 
Qaeda-trained operative, was convicted of plotting a 
ricin attack. He had planned to contaminate handrails 
in the railway system connecting London and Heath¬ 
row Airport. 123 In March 2003 two flasks containing 
ricin were discovered in a railway station in Paris. 124 

In 2003 US Postal Service employees discovered 
two letters directed to the US Department of Trans¬ 
portation containing vials of ricin. The first letter 
was found on October 15, 2003, at the mail sorting 
center in Greenville, South Carolina. 125 The second 
was discovered at the White House mail processing 
facility in Washington, DC. Both letters were from an 
antagonist who identified himself as "Fallen Angel" 
and was angry about the Department of Transporta¬ 
tion's new limitations being placed on truck drivers' 
daily work hours. 126 In February 2004 ricin was found 
in the sorting machine of Senate Majority Leader Bill 
Frist's office in the Congressional Office Building. No 
evidence was ever found linking the Fallen Angel and 
Frist cases and perpetrators are still at large. On June 
23, 2004, Michael Crooker, a resident of the Boston 
suburb of Agawam, Massachusetts, had his house 
searched by law enforcement officials after attempting 
to mail a firearm. Agents discovered a weapons lab 
that contained castor and abrus seeds (sources of ricin 
and abrin toxins, repectively) as well as the materials 
needed for toxin extraction. Crooker sent a letter to 
the prosecuting attorney threatening to cripple the US 
Postal System by sending toxin-laden letters through 
the mail. He also notified local news journalists that he 
would provide toxins to felons he had met in prison 
who had previously engaged in terrorist activities. He 
pled guilty to possession of ricin and threatening a gov¬ 
ernment official and was sentenced in June 2011. 127 In 
February 2008 Roger Bergendorff, an anarchist living 
in an extended stay hotel in Las Vegas, Nevada, devel¬ 
oped a mysterious illness that puzzled his healthcare 
providers. He was hospitalized and while investigating 
the cause of his illness, officials discovered evidence 
of a ricin extraction operation in his room. He and 
his cousin were both eventually convicted of charges 
related to ricin production. The specifics of intended 
use—if known—have not been disclosed. 128 In March 


2011 four men who were members of a militia orga¬ 
nization began having clandestine meetings in which 
they allegedly planned numerous criminal activities 
to include acquisition of illegal weapons, manufacture 
of toxic agents, theft, and assassination. During these 
meetings they allegedly discussed use of weapons to 
include biologic agents to attack government facilities 
and government employees to include law enforce¬ 
ment officials. One of their plans included producing 
10 pounds of ricin and dispersing it from a moving 
vehicle in the Atlanta area. An FBI informant alerted 
authorities and the operation was disrupted without 
incident in November 2011. 129 

Attacks against government officials resumed after a 
nearly 10-year hiatus with the discovery of an envelope 
testing positive for ricin intercepted at the US Capitol's 
mail facility in April 2013. The letter was addressed to 
Senator Roger Wicker, and a day later an envelope ad¬ 
dressed to President Obama was discovered that also 
contained ricin. A third letter containing ricin was mailed 
to the Lee County Mississippi Court Judge Sadie Holland. 
Within a few weeks the FBI arrested Everett Dutschke 
for producing a toxin weapon and using the mail to 
threaten President Barack Obama, Senator Wicker, and 
Judge Holland. These mailings appear to be acts of 
reprisal in the settlement of personal grudge(s). 130 Less 
than 2 months later, in May 2013 three letters intended 
for New York Mayor Michael Bloomberg were inter¬ 
cepted containing a suspicious oily substance that turned 
out to contain ricin. Similar letters were also mailed to 
President Obama, according to a Secret Service press 
release. Gun control opponents purportedly sent the 
letters, and Shannon Richardson notified the FBI that 
her estranged husband was responsible for the mailings. 
When the allegations failed to withstand police scrutiny 
she was arrested, and received an 18-year prison sen¬ 
tence, having falsely implicated her husband. 131 Despite 
numerous ricin mailings by many diverse individuals, 
the mail delivery of ricin toxin has been ineffective as 
an instrument of harm or assassination—these mailings 
appear to have little impact beyond their psychological 
"scare" effect. Although ricin is a toxin of very high le¬ 
thal potency, its effectiveness is limited by the delivery 
method. No illness or significant environmental con¬ 
tamination has resulted from any of the ricin mailings. 

Many of the bioterrorist incidents have been small 
scale, not well perpetrated, and not particularly suc¬ 
cessful in terms of mortality and morbidity. Still, 
it is clear that several terrorist groups aspire to use 
biological weapons. For example, A1 Qaeda radical 
cleric Anwar al-Awlaki in an article stated that "the 
killing of women and children and the use of chemi¬ 
cal and biological weapons in addition to bombings 
and gun attacks" is acceptable and even encouraged. 54 


16 


Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness 


In Inspire, an online A1 Qaeda magazine, the authors 
called for "chemists and microbiologists" to develop 
weapons and attack the West. These programs con¬ 
tinue to be aspirational, rather than well-established 


developmental efforts. However, with the prolifera¬ 
tion and industrialization of biotechnology described 
previously, the threat of bioterrorism continues to 
increase. 54(p60) 


SOLUTIONS:TOWARD PAN-HAZARD PREPAREDNESS 


Disarmament: The Biological Weapons Convention 

In July 1969 Great Britain issued a statement to the 
UN Conference of the Committee on Disarmament call¬ 
ing for the prohibition of the development, production, 
and stockpiling of bacteriological and toxin weapons. 
In September 1969 (the same year) the Soviet Union 
unexpectedly recommended a disarmament convention 
to the UN General Assembly. In November 1969 WHO 
issued a report on biological weapons, after an earlier 
report by the 18-nation Committee on Disarmament, 
describing the unpredictable nature, lack of control, 
and other attendant risks of biological weapons use. 
The United Nations then developed the 1972 Conven¬ 
tion on the Prohibition of the Development, Produc¬ 
tion and Stockpiling of Bacteriological (Biological) and 
Toxin Weapons and on their Destruction (1972 BWC), 
which prohibited any malicious research, production, 
or possession of biological agents. Among the 103 initial 
cosignatory nations, agreement was reached to "never 
develop, produce, stockpile, or otherwise acquire or 
retain microbiological agents or toxins, whatever their 
origin or method of production, of types and in quanti¬ 
ties that have no justification for prophylactic, protective 
or other peaceful purposes; and weapons, equipment 
or means of delivery designed to use such agents or 
toxins for hostile purposes or in armed conflict." 132 The 
United States ratified both the 1925 Geneva Conven¬ 
tion and the BWC in 1975. Signatory states suspecting 
others of treaty violations may file a complaint with 
the UN Security Council, which, in turn, may order an 
investigation. However, mandatory measures for verifi¬ 
cation and enforcement are lacking; numerous attempts 
to formulate such measures have been unsuccessful 
because of political, security, and proprietary issues. 21 

Since the BWC entered into force in 1975, seven 
review conferences have taken place; these "RevCons" 
(as they are called) constitute the only decision-making 
forums for the BWC and are held every 5 years in Ge¬ 
neva. RevCons are 3-week international meetings that 
allow member nations to reinforce the norm against 
the prohibition of biological weapons, discuss interna¬ 
tional collaboration on biotechnological issues, assess 
the continued relevance of the BWC given changes in 
biotechnology, and make proposals for revitalizing 
the BWC. Unfortunately, RevCons have not pro¬ 
duced many tangible results and have demonstrated 


an inability to deal with difficult issues. The most 
noteworthy accomplishment was development of 
confidence-building measures for annual reporting by 
member state parties. Only 70 or so of the 170 mem¬ 
ber nations actually submit annual reports on their 
activities. On questions such as the relationship of the 
Sverdlovsk anthrax epidemic to the Soviet biological 
weapons program, the Iraqi weapons program, and 
the smallpox retention versus destruction issue, the 
BWC has remained unengaged. 

Several RevCons have dealt directly with the 
potential for developing a verification protocol. The 
1991, 1996, and 2001 RevCons saw the establishment 
of the Ad Hoc Group, the progress made in the Re¬ 
view Conference Final Declaration, and the disaster 
of the United States walking out of the RevCon, 54(pll7) 
respectively. After the 2001 RevCon the BWC saw a 
tumultuous period where its future was questioned. 
The "success" of the 2006 RevCon served to reener¬ 
gize the BWC. The key outcomes were the agreement 
concerning the importance of the BWC forum and the 
development of an intersessional process that would 
include annual member state nations and experts meet¬ 
ings to discuss topical issues. However, neither of these 
two new annual meetings allows for decision-making. 

The lead-up to the 2011 RevCon was anticipated 
by participating nation-states. 54(pll9) The United 
States had released a national strategy for countering 
biological threats at the 2009 meeting of state parties. 
Several pre-BWC conferences were held in which it 
appeared the international community was moving 
toward tangible outcomes in the 2011 RevCon. The 
president of the 2011 meeting, Paul van den Ijssel from 
The Netherlands, had declared the mantra would be 
"ambitious realism." 54(pl22) Unfortunately, it failed to 
live up to expectations. One review of the RevCon 
states, "The December 2011 review conference of the 
Biological Weapons Convention (BWC) demonstrated 
the danger of the bioweapons ban drifting into irrel¬ 
evance. Standstill was the motto of the meeting. Only 
incremental improvements on some procedural issues 
were achieved." 54(pl20) Even modest enhancements, 
such as expanding the implementation support unit's 
three-person organization, were not approved. 

In examining the BWC's future, several tensions 
arise because it is a state-to-state treaty, yet many of 
the current biological threats deal with nonstate issues 


17 


Medical Aspects of Biological Warfare 


such as bioterrorism, biocrimes, and misuse of the 
life sciences. Although member nations allow for 
discussing these issues within the BWC, few have 
demonstrated the desire to make these more topical 
issues the focus of future BWC negotiations, although 
states-parties are obligated under article IV to prohibit 
and prevent proscribed activities within their borders. 
Several other articles of the BWC also create tensions. 
For example, article I establishes the norm against bio¬ 
logical weapons, yet provides no ability to enforce the 
convention. Articles III and X call for not transferring, 
assisting, inducing, acquiring, and retaining biological 
weapons, whereas article X encourages the peaceful ex¬ 
changes of biological science and technology. Although 
the words do not conflict, the interpretation between 
developed and developing nations varies greatly. 

Another area of contention concerns the perennial 
issue of verification. The US position remains as it has 
since 2001 that verification of the BWC is not possible. 
Instead, the United States supports adherence to a policy 
of compliance that begins with national implementa¬ 
tion including ensuring all nations have appropriate 
national laws, regulations, and policies that support 
the BWC, as stipulated in article IV. The US position 
on verification also rests on the assertion that articles 
V and VI that call for bilateral and multilateral consul¬ 
tation and the potential for bringing concerns to the 
UN Security Council, respectively, provide sufficient 
opportunities for voicing concerns about compliance. 
Two other issues that feature prominently in the BWC 
debate are continued concerns about its relevance 
given the pace of biotechnological enhancements and 
the lack of universal adherence to it. On the first issue, 
members continue to profess that the BWC remains 
relevant despite exponential changes in biotechnol¬ 
ogy. With respect to universal adherence, the BWC 
continues to be undersubscribed as compared to other 
treaties dealing with weapons of mass destruction is¬ 
sues, in particular the Nuclear Non-Proliferation Treaty 
and the Chemical Weapons Convention. The BWC 
has 170 member nations, whereas the Nuclear Non- 
Proliferation Treaty and the Chemical Weapons Con¬ 
vention have 189 and 188 member nations, respectively. 

Finally, only one allegation has been formally reg¬ 
istered under the BWC: in June 1997 Cuba accused 
the United States of a biological attack with a crop 
pest insect, Thrips palmi. The allegations were unsub¬ 
stantiated in a BWC consultation that concluded in 
December 1997. 21 Other attempts at biological arms 
control have been conducted outside of the context 
of the BWC; for example, inspections and sanctions 
against Iraq from 1991 to 1998 and 2002 to 2003 were 
accomplished under separate UN Security Council 
Resolutions, 681 and 1441, respectively. 


Smallpox Preparedness 

CDC launched a comprehensive smallpox pre¬ 
paredness program in 2002 because of the potential 
use of variola as a biological weapons agent. WHO, the 
United Kingdom, Germany, and other WHO member 
states initiated similar programs including vaccine 
stockpiles. The US program integrated community, 
regional, state, and federal healthcare and public health 
organizations and featured logistical preparation; 
training and education; risk communication; surveil¬ 
lance; and local preparations for mass vaccination, 
isolation, quarantine, active surveillance, and humane 
treatment of patients in designated facilities. A strategy 
was adopted based on preexposure vaccination of care¬ 
fully screened and trained members of first-response 
teams, epidemiological response teams, clinical teams 
at designated facilities, and military personnel set to 
deploy into the theaters of war. 133 More than 400,000 
selected military personnel and 38,000 civilian emer¬ 
gency responders and healthcare workers in desig¬ 
nated smallpox response teams were vaccinated. Con¬ 
tracts for the production of a new cell culture-derived 
vaccine were awarded in 2000; the Strategic National 
Stockpile has sufficient cell culture-derived vaccine 
for the entire US population, a replication-deficient 
vaccinia (Modified Vaccinia Ankara) for use in im¬ 
munocompromised individuals, and vaccinia immune 
globulin to treat vaccine complications. In addition, the 
US government supported the development of new 
smallpox antiviral therapeutic candidates and funded 
animal model development to enable efficacy testing 
of medical countermeasure candidates. 

The disposition of the remaining WHO-authorized 
variola virus stocks, held in two secure WHO Col¬ 
laborating Centers at CDC in the United States and 
at VECTOR in Koltsovo, Novosibirsk, Russia, was 
debated at the WHO 64th World Health Assembly 
in 2011. Two camps emerged, the destructionists and 
retentionists, and each made arguments to support 
their positions. In the end, the World Health Assembly 
remained committed to its previous position calling for 
the destruction of the viral stocks as a long-term goal, 
but agreed to their retention until the completion of 
research leading to two antiviral drugs with different 
mechanisms of action, a safer and effective vaccine, a 
rapid and accurate diagnostic kit, and the refinement 
of nonhuman primate animal models. The issue was 
also revisited at the 67th World Health Assembly in 
2014. The risks posed by recombinant technology 
were also addressed; a private company in the United 
States that had inserted 63 nucleotides from the variola 
genome into an attenuated but transmissible orthopox 
virus to develop a positive control for a diagnostic test 


18 


Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness 


would be asked to destroy its reagent and to report its 
destruction to WHO. 134 This underscored the need to 
re-evaluate and publicize WHO guidance regarding 
the use of variola genetic sequences in recombinant 
technology. 

Dual Use Research of Concern 

In addition to the threats posed by the deliberate 
release of biological agents, there has been increasing 
recognition of the potential risks posed by legitimate 
scientific research for benevolent medical purposes 
that includes the characterization of, and develop¬ 
ment of medical countermeasures against, highly 
pathogenic microbes. Risks include laboratory ac¬ 
cidents resulting in pathogen release, laboratory 
acquired infections (some of which may be com¬ 
municable to the community), unanticipated results 
of experiments resulting in increased microbial 
virulence or transmissibility, and the deliberate mis¬ 
use of knowledge generated by legitimate scientific 
research for biological weapons proliferation. Dual 
use research of concern (DURC) has been identified 
as biological research with legitimate scientific pur¬ 
pose that may be misused to pose a biologic threat 
to public health and/or national security. Examples 
include, but are not limited to, the following: 

• The genetic modification of mousepox virus 
to express both an ovarian protein and the 
immunomodulator interleukin-4 to induce 
sterility in mice for pest control, reported in 
2001. Immunomodulator interleukin-4 was 
intended to enhance immune responses to 
the ovarian protein. However, the vaccine 
candidate was lethal in small-animal testing; 
immunomodulator interleukin-4 had the 
unanticipated effect of immune suppression, 
resulting in a highly virulent mousepox vi¬ 
rus. 135 

• The in vitro synthesis of wild-strain poliovirus 
type 1 by using synthetic DNA encoding the 
poliovirus genome (with minor mutations 
as genetic markers) in a cell-free extract by 
researchers at the State University of New 
York at Stony Brook in 2002. The researchers 
noted that the knowledge that polioviruses 
can be synthesized using chemical methods 
and reintroduced through bioterrorism may 
inform the closing strategies of WHO's po¬ 
lio eradication campaign. 136,137 It was later 
explained that they had hoped to deliver a 
"wake-up call" regarding the possible misuse 
of viral synthesis for bioterrorism; that WHO's 


polio eradication campaign may be futile 
because of either possible bioterrorism using 
synthetic virus, laboratory accidents, or live 
attenuated oral polio vaccine and circulating 
oral polio vaccine-derived virus-related dis¬ 
ease; and that control may be a more attainable 
outcome. 138 Aside from risking an accidental 
reintroduction to the local community (after 
the elimination of circulating wild-strain po¬ 
lioviruses from the western hemisphere), the 
study raised questions regarding its scientific 
value, 139 whether demonstrating technical 
capabilities to deliver warnings constitutes a 
legitimate scientific purpose, and whether the 
synthesis of a wild-strain poliovirus, which is 
otherwise available to researchers, served any 
benevolent medical purpose. 

• The reconstruction of the 1918 H1N1 influenza 
A pandemic virus, 140 reported in 2005. This 
enabled characterization of a virulent patho¬ 
gen that—in contrast to poliovirus —was oth¬ 
erwise not available for study. This enabled 
insights into pathogenesis, and potentially the 
identification of virulence factors and drug 
targets that could be relevant to counter future 
pandemic strains. 141 Using appropriate bio¬ 
safety and biosecurity measures minimized 
risks to the public. 

• The generation of a mutant of the highly 
pathogenic avian influenza A virus H5N1 
(HPAI H5N1) with enhanced transmissibil¬ 
ity between mammalian hosts (ferrets) that 
was as contagious as seasonal influenza vi¬ 
ruses and retained the virulence of the wild 
strain 142,143 (55%-60% mortality in humans) 
by researchers at Erasmus University (al¬ 
though it was later reported that the mutant 
was attenuated and not as communicable 
as originally claimed), reported during the 
autumn of 2011. Concurrently, researchers 
at the University of Wisconsin developed 
a recombinant 2009 pandemic H1N1 virus 
expressing H5 hemagglutinin receptor bind¬ 
ing proteins that was transmissible between 
ferrets. These announcements stunned many 
in the scientific community and the general 
public as risking a pandemic catastrophe fol¬ 
lowing a laboratory accident or intentional 
release. Policy makers became concerned that 
the publication of these studies would support 
biological weapons proliferation by providing 
information that could be used to produce 
highly communicable and lethal influenza 
viruses. 


19 


Medical Aspects of Biological Warfare 


Within 4 months of the publication of poliovirus 
synthesis, the Center for Strategic and International 
Studies and the National Academy of Sciences held 
a workshop on scientific openness and national se¬ 
curity that involved a wide stakeholder community 
from government, academia, and scientific editorial 
communities that generated voluntary guidelines for 
ensuring the publication of new knowledge while 
safeguarding information that may pose security 
risks. Issues raised by DURC led to the foundation 
of the National Science Advisory Board for Biode¬ 
fense (NSABB) in 2004. NSABB is a federal advisory 
committee within the Office of Science Policy in the 
National Institutes of Health (NIH) that provides ad¬ 
vice, guidance, and leadership regarding biosecurity 
oversight of dual use research, defined as biologi¬ 
cal research with legitimate scientific purpose that 
may be misused to pose a biological threat to public 
health and/or national security. NSABB is chartered 
to recommend strategies and guidance for enhancing 
personnel reliability among individuals with access 
to biological select agents and toxins; provide rec¬ 
ommendations on the development of programs for 
outreach, education, and training in dual use research 
issues for scientists, laboratory workers, students, 
and trainees in relevant disciplines; advise on policies 
governing publication, public communication, and 
dissemination of dual use research methodologies 
and results; recommend strategies for fostering inter¬ 
national engagement on dual use biological research 
issues; advise on the development, utilization, and 
promotion of codes of conduct to interdisciplinary 
life scientists and relevant professional groups; advise 
on policies regarding the conduct, communication, 
and oversight of dual use research and results, as re¬ 
quested; advise on the Federal Select Agent Program, 
as requested; and address any other issues as directed 
by the Secretary of Health and Human Services. 
NSABB concerns include knowledge, products, or 
technologies that may: 

• enhance the harmful consequences of a bio¬ 
logical agent or toxin; 

• disrupt the immunity or the effectiveness 
of an immunization without clinical and/or 
agricultural justification; 

• confer to a biological agent or toxin, resis¬ 
tance to clinically and/or agriculturally use¬ 
ful prophylactic or therapeutic interventions 
or facilitate their ability to evade detection 
methodologies; 

• increase the stability, transmissibility, or the 
ability to disseminate a biological agent or 
toxin; 


• alter the host range or tropism of a biological 
agent or toxin; 

• enhance the susceptibility of a host popula¬ 
tion; and 

• generate a novel pathogenic agent or toxin or 
reconstitute an eradicated or extinct biological 
agent. 

Examples of initiatives coordinated through NSABB 
include Department of Health and Human Services 
(DHHS) guidelines for synthetic biology 144 and guid¬ 
ance for providers of double-stranded DNA to screen 
procurement orders. 145 

In December 2011 NSABB reviewed manuscripts 
of the Erasmus University and University of Wiscon¬ 
sin studies on enhanced transmission of HPAI H5N1 
that were being prepared for publication and made 
the unprecedented, nonbinding recommendation to 
redact methods and experimental details. 146 In addi¬ 
tion, the influenza research community voluntarily 
invoked a moratorium on gain-of-function research 
using HPAI H5N1. 

NSABB members asserted that their recommenda¬ 
tion was an exceptional and adaptive response to a 
special case—a situation generated by the life sciences, 
biodefense, and general public communities being 
caught off-guard—and having limited awareness of 
the research until the manuscripts were being prepared 
(even though NIH had funded both projects), they 
reasoned that: 

• in the future, the value of conducting and 
supporting specific dual use research proj¬ 
ects should be carefully considered a priori 
by a wide stakeholder community including 
experts in life sciences, biosecurity, and mem¬ 
bers of the general public; and 

• decisions to publish results should follow 
the principle of "do no harm," with the best 
interest of public health in mind. 147 

However, supporters of the research and its publica¬ 
tion argued that: 

• medical science must address the most viru¬ 
lent pathogens to be valuable; 

• new knowledge of determinants of transmis¬ 
sibility may be useful to predict the likelihood 
of an epi- or enzootic virus being capable of 
a "species jump" to humans and consequent 
person-to-person transmission; 

• the mutants afforded an opportunity to test 
vaccine and therapeutic candidates against 
potential future emerging viruses; 


20 


Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness 


• methods used in the studies are already well- 
known in the scientific community; 

• persons with malicious intent could use sim¬ 
pler means to inflict disease and injury; and 

• redacting the manuscripts constituted censor¬ 
ship, thus violating long-standing principles 
of academic freedom. 148-151 

As the debate raged, 152-156 WHO concluded that 
such research and its publication is in public health's 
best interest and should be continued in the context 
of rigorous biosafety, biosecurity, and risk communi¬ 
cation. 157 NSABB reconvened in late March 2012 and 
recommended the full publication of the University of 
Wisconsin manuscript and publication of the Erasmus 
University manuscript after appropriate scientific re¬ 
view and revision, with the caveat that the US govern¬ 
ment should develop a mechanism to control access to 
sensitive scientific information. 158 The two manuscripts 
were published later in 2012. 159,160 

The controversy resulted in an updated US Gov¬ 
ernment Policy for Oversight of Life Sciences DURC, 
which was released in March 2012. 161 This policy di¬ 
rected federal departments and agencies that conduct 
or fund life sciences research to do the following: 

• review all current or proposed research proj¬ 
ects to identify those that could potentially 
provide knowledge, information, products, 
or technologies that could be directly misap¬ 
plied to pose a significant threat to public 
health and safety, agricultural crops and other 
plants, animals, the environment, materiel, or 
national security; 

• conduct risk assessments and develop risk 
mitigation plans addressing experimental 
design and methods, biosecurity, biosafety, 
and availability of medical countermeasures; 

• review annual progress reports to determine 
whether DURC results have been generated; 

• request voluntary redaction of research pub¬ 
lications or communications or classification 
of research findings; and 

• coordinate information regarding DURC 
projects with the Assistant to the President for 
Homeland Security and Counterterrorism. 

In addition, the Office of Science and Technology 
Programs is formulating a complementary policy that 
delineates oversight responsibilities for research insti¬ 
tutions receiving federal funds to perform DURC. 162 

In December 2012 NIH hosted a meeting of the in¬ 
fluenza research community to discuss guidelines for 
funding HPAIH5N1 influenza virus gain-of-function 


research, followed by an opportunity for public com¬ 
ment. The resulting guideline was issued on February 
21, 2013, 163,164 and it identified criteria for funding re¬ 
search proposals that may enhance the transmissibility 
of HPAI H5N1 among mammals: 

• the virus anticipated to be generated could 
be produced through a natural evolutionary 
process; 

• the research addresses a scientific question 
with high significance for public health; 

• there are no feasible alternative methods 
to address the same scientific question in a 
manner that poses less risk than the proposed 
approach; 

• biosafety risks to laboratory workers and the 
public can be sufficiently mitigated and man¬ 
aged; 

• biosecurity risks can be sufficiently mitigated 
and managed; 

• the research information is anticipated to be 
broadly shared to realize its potential benefits 
to global health; and 

• the research will be supported through fund¬ 
ing mechanisms that facilitate appropriate 
oversight of the conduct and communication 
of the research. 

The framework also outlined a review process that 
includes department-level scrutiny of proposals con¬ 
sidered for funding by DHHS agencies. 

Five days after the release of the DHHS framework, 
the ethical, societal, scientific, safety, and security 
issues raised by DURC were discussed at the inter¬ 
national level at WHO. There was consensus that 
DURC issues are relevant to all nations and multiple 
stakeholders; management of DURC should take place 
during all phases of research; ethical considerations are 
fundamental; and because management of DURC will 
require a diversity of approaches in different member 
states, an internationally binding agreement would 
be difficult, impractical, and not necessarily effective. 
However, the participants remained open to future 
international guidelines and suggested that existing 
international agreements (eg, the BWC, WHO's In¬ 
ternational Health Regulations [IHR]) could provide 
a basis for overarching principles. WHO will continue 
to engage member states and other stakeholders to 
explore effective approaches. 165 

In the meantime, the influenza research commu¬ 
nity had already ended its moratorium for scientists 
using biosafety and biosecurity measures in compli¬ 
ance with its respective national regulations. 166 The 
subsequent publication of a study completed before 


21 


Medical Aspects of Biological Warfare 


the moratorium using reverse genetics to generate 127 
hybrids of HPAI H5N1 and 2009 pandemic H1N1 
viruses, of which five were communicable among 
guinea pigs, 167 again raised questions regarding the 
medical utility and public health risks of hazardous 
experiments. 168 In August 2013 proponents of gain-of- 
function research publicly announced their intention 
to conduct studies using influenza A H7N9 virus. 169 
Concurrently, DHHS gave assurances that research 
proposals for H7N9 gain-of-function research would 
undergo rigorous scrutiny by experts in multiple 
disciplines including biosafety and ethics and final 
review at the department level, 170 consistent with the 
February DHHS framework. The a priori publication 
of H7N9 research goals was seen as a proactive step 
to enhance transparency and prospective discussion 
and to prevent a recurrence of the 2011-2012 H5N1 
disputes. However, gain-of-function research remains 
a contentious issue 171 because no certainty exists that 
laboratory-generated mutants will emerge in nature. 
The issues generated by potential dual use research 
will continue to fuel discourse regarding relation¬ 
ships among stakeholders, and optimal policy and 
technical solutions. 172-179 

Toward Pan-Hazard Preparedness 

During the late 1990s the US government launched 
an ambitious program to enhance biological pre¬ 
paredness at local, state, and federal levels, including 
measures such as the Presidential Decision Direc- 
tive-39 (1995), Presidential Decision Directive-62 
(1998), and Presidential Decision Directive-63 (1998). 
The Federal Response Plan (now called the National 
Response Plan) coordinates federal agencies respond¬ 
ing to disasters. The Select Agent List was created 
to regulate the purchase, shipment, and research of 
designated microbial agents; lead proponents for the 
Select Agent list were DHHS and USDA. DHHS was 
given oversight of health and medical services, and 
its Office of Emergency Preparedness organized local 
medical response teams in 125 jurisdictions. Prepara¬ 
tions in New York City and other locations included 
plans and exercises for local incident command; co¬ 
ordinated clinical response; surveillance; and massive 
distribution of postexposure prophylaxis at multiple 
distribution centers designed for efficient screening, 
triage, distribution, and documentation. Federal re¬ 
sponse teams were organized, staffed, and deployed 
to large official and public gatherings. CDC estab¬ 
lished a center for bioterrorism response to enhance 
state public health laboratories, improve surveillance 
systems, and improve rapid communication and co¬ 
ordination. The Strategic National Stockpile of key 


pharmaceutical agents and vaccines was prepared. 
The Laboratory Response Network, also managed 
by CDC, provided coordination of testing, sample 
shipment, and communication between designated 
local, regional, and reference laboratories. DoD assets 
integrated into the National Response Plan included 
USAMRIID for emergency medical consultation and 
reference laboratory support; the Naval Medical Re¬ 
search Center for laboratory support; the US Marine 
Corps Chemical and Biological Incident Response 
Force for reconnaissance, initial triage, and the de¬ 
contamination of casualties; and the Army Technical 
Escort Unit for sampling, transport, and disposal of 
dissemination devices. The Army Medical Depart¬ 
ment also fielded six regionally based chemical/ 
biological special medical augmentation response 
teams to deploy within 12 hours to assist local civil¬ 
ian authorities. The National Guard Bureau, under 
legislative direction from Congress, fielded regional 
biological response teams initially called rapid agent 
identification teams, and later renamed civil support 
teams. Many of these new response mechanisms and 
agencies were tested in the autumn of 2001. 

After the anthrax mailings of 2001, bioterrorism 
response was strengthened with additional infrastruc¬ 
ture and linkages among the emergency response, 
public health, clinical, and laboratory sectors. 103,104 
The Office of Public Health Emergency Preparedness 
at DHHS was formed to coordinate civilian medical 
countermeasure development by the National Institute 
of Allergy and Infectious Diseases, CDC, and DoD, 
under the leadership of eminent scientists and physi¬ 
cians such as DA Henderson and Philip K Russell. 

In April 2004 President George W Bush signed 
Homeland Security Presidential Decision Directive-10, 
Biodefense for the 21st Century, which outlined a na¬ 
tional strategy for combating biological terrorism and 
mandated an interagency approach using strengths of 
various executive branch departments, including the 
Department of Homeland Security, DHHS, and DoD. 
Subsequently, the Homeland Security Council and 
the National Security Council formed an interagency 
steering committee called the Weapons of Mass De¬ 
struction Medical Countermeasures Subcommittee, 
whose principals were at the assistant secretary level; 
the group coordinates the various departmental efforts 
to prevent and respond to weapons of mass destruction 
attacks. The Department of Homeland Security took 
the lead on biological threat assessments, and DHHS 
took the lead on medical countermeasures. 

On July 21,2004, Project Bioshield was initiated as a 
$6 billion, 10-year program for acquiring new medical 
countermeasures for the Strategic National Stockpile. 
This legislation provided a significant funding boost to 


22 


Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness 


the Office of Public Health Emergency Preparedness. 
Medical countermeasures added to the Strategic Na¬ 
tional Stockpile include significantly increased doses 
of botulinum antitoxins; antibiotics to treat anthrax, 
tularemia, and plague; anthrax adjunctive therapies; 
and ventilators for respiratory support. 

The potential for the malevolent use of genetic 
engineering to develop novel biological threats with 
enhance virulence 180 resulted in a shift of technical 
emphasis from pathogen-specific projects to a global 
response capability —a threat-agnostic response 
capacity—to enable responses to outbreaks of any 
known or genetically engineered biological agents, 
or novel emerging pathogens. This capability in¬ 
cludes flexible technology platforms to enable rapid 
pathogen identification and characterization, drug 
target identification, and medical countermeasure 
development and mass production. An emphasis 
has been placed on the development of anti-infective 
therapeutics that has a broad spectrum of activity to 
enhance their potential utility against a wide range of 
emerging pathogens. In addition to exploiting highly 
conserved pathogen targets, proposed approaches 
have included host-directed anti-infective therapeu¬ 
tics to upregulate innate immunity, antagonize host 
receptors and processes that are hijacked by patho¬ 
gens to complete their life cycles, and attenuate sepsis 
and other pathogenesis pathways. 

The National Strategy for Countering Biologi¬ 
cal Threats 3 proposed an integrated approach to all 
biological threats, whether from intentional releases 
(biological warfare or terrorism) or accidental releases 
(laboratory accidents or unintended consequences of 
legitimate scientific research) or naturally occurring 
emerging diseases. The strategy is based on the concept 
that all of these challenges require a common set of 
responses (pathogen identification and characteriza¬ 
tion; patient diagnosis; development, mass production, 
and distribution of medical countermeasures; medical 
and public health interventions; risk communication; 
promotion of ethical standards; professional and legal 
codes of conduct; and law enforcement). It proposes a 
pan-sector "all of society" approach that integrates the 
public at large and the scientific, medical, veterinary, 
public health, law enforcement, and diplomatic com¬ 
munities. Initiatives have included reorganization of 
civilian biodefense under the Department of Home¬ 
land Security; strengthening of programs under DoD 
and DHHS (NIH, the Biomedical Advanced Research 
and Development Authority, CDC) that have multipur¬ 
pose utility for biological attacks, naturally occurring 
outbreaks, and other mass casualty disasters; the con¬ 
struction of the Fort Detrick biodefense campus, which 
includes laboratories for the Department of Homeland 


Security and NIH as well as a new USAMRIID facility; 
export controls to regulate exportation of potential 
dual use technologies; the medical countermeasures 
initiative to enhance mass production of medical coun¬ 
termeasures; investments to enhance biosurveillance; 
and federal guidelines for synthetic biology and the 
use of double-stranded DNA. 

The Federal Experts Security Advisory Panel's inter¬ 
agency working group was initiated in 2010 to update 
42 CFR Part 73, Possession, Use, and Transfer of Select 
Agents and Toxins, to prevent intentional or accidental 
releases of highly virulent pathogens without placing 
counterproductive regulatory burdens on laboratories 
that conduct research on CDC select agents. Topics 
that were considered included revising the list of select 
agents, physical security measures, laboratory safety, 
occupational health, and personal reliability. A simpli¬ 
fication of the select agent list was proposed, removing 
or recategorizing agents that are either easy to obtain 
from their natural reservoirs, or that constitute low risk 
due to low virulence, low transmissibility, or the avail¬ 
ability of medical countermeasures. The Federal Experts 
Security Advisory Panel developed a comprehensive 
set of recommendations regarding biosecurity—the 
presence of physical security measures such as labora¬ 
tory access controls, closed circuit visual monitoring, 
etc, and personal reliability—as well as background 
checks of laboratory workers' law enforcement history, 
substance abuse, and mental health, with continuing 
monitoring and periodic reassessments of suitability 
for continued employment. Robust occupational health 
programs, with mandatory reporting of illnesses requir¬ 
ing medical intervention, were emphasized to prevent 
behaviors that could result in accidental or deliberate 
releases of select agents and to promptly recognize and 
treat laboratory-acquired infections and prevent their 
transmission to the general community. The Final Rule 
(October 5, 2012) included a revised select agent list; 
physical security standards for laboratories possessing 
Tier I Select Agents and Toxins; a requirement to con¬ 
duct pre-access assessments and ongoing monitoring 
of personnel with access to Tier I agents and toxins; 
and clarifications of regulatory language concerning 
security, training, biosafety, and incident response. 118,119 

The optimization of biosafety and biosecu¬ 
rity is an iterative process. USDA's Office of 
the Inspector General noted that while there 
had been enhanced compliance with security 
regulations and inspection processes within the 
USDA Select Agent program between 2005 and 
2012, there had been transfers of B anthracis and 
Y pestis samples to unregistered facilities, and access 
to select agents by a person with an expired security 
clearance. USDA concurred with recommendations 


23 


Medical Aspects of Biological Warfare 


to clarify restricted access requirements and estab¬ 
lish policies and procedures for handling requests 
for transferring select agents under special circum¬ 
stances to unregistered facilities. 181 On March 24, 
2013, a vial of Guanarito virus (a Tier I Select Agent) 
was reported missing from the University of Texas 
Medical Branch at Galveston. 182 On the following 
day, the Government Accountability Office issued a 
report concluding that US government interdepart¬ 
ment and interagency biodefense programs using 
high containment laboratories should improve their 
coordination. It also recommended that the Office 
of Science and Technology within the Executive Of¬ 
fice of the President conduct periodic assessments 
of the requirements for, and the number, locations, 
and missions of high-containment laboratories, and 
evaluate the need to establish national standards for 
their design, construction, commissioning, opera¬ 
tion, and maintenance. 183 

International efforts include the following: 


• outreach by DoD and CDC to enhance surveil¬ 
lance with international partners; 

• DoD's Cooperative Biological Engagement 
Program that builds partnerships to convert 
former biological weapons programs to 
peaceful purposes and enhance public health 
capacity; 

• collaborations to strengthen biological defense 
capacities of partner nations (eg, through the 
North Atlantic Treaty Organization and the 
Australia-Canada-United Kingdom-US- 
New Zealand partnership); 

• US government support of BWC confidence¬ 
building measures and international public 
health efforts that may also lead to the early 
identification and containment of biological 
attacks (eg, WHO's IHR); and 

• WHO efforts to enhance implementation of 
the IHR and strengthen ties with the World 
Organization for Animal Health and Interpol. 


SUMMARY 


The use of microbes and toxins to intentionally 
cause harm has been attempted repeatedly throughout 
recorded history. However, military use before the 
development of modem microbiology was limited, 
possibly because of the availability of other weapons 
with more rapid and predictable results. 

Following the inception of modern microbiology, 
several nations began offensive biological warfare 
programs. Information regarding the history of 
state-sponsored biological weapons programs is 
obscured by secrecy, propaganda, and a lack of 
rigorous microbiologic or epidemiologic data to 
confirm allegations of use. Disclosures of former 
national programs underscore the ambitious intent 
and potential realization of covert state-sponsored 
programs. However, military deployment has been 
limited, and never decisive in armed conflict. With the 
exceptions of alleged German sabotage during World 
War I, Japanese field trials during World War II, lim¬ 
ited deployments by South African and Rhodesian 
forces, and small-scale covert operations, there are no 
well-documented biological attacks by nation-states. 
Deterrents may include poor tactical utility related to 
multiple variables during production, storage, and 
delivery; variable incubations and host susceptibili¬ 
ties; availability of medical countermeasures; nuclear 
deterrence; diplomatic efforts; and political vulner¬ 
abilities. The public health disaster at Sverdlovsk, 
the loss of international goodwill toward the United 
States following disclosures during the Cold War, and 
political consequences following the 1996 disclosures 


by Iraq underscore that the attendant liabilities of 
state-sponsored biological weapons programs have 
outweighed potential strategic advantages. 

Non-state groups, lone actors, and even members 
of the medical community have committed bioter¬ 
rorism and biocrimes. The likelihood of amateurs 
using homemade equipment to successfully develop 
and deploy a biological weapon of mass destruction 
is remote. Terrorists still rely on simple yet effective 
explosives as their weapon of choice. However, the 
Aum Shinrikyo program and A1 Qaeda aspirations 
demonstrate intentions to harness modem microbiol¬ 
ogy for malicious purposes. Although most bioterror¬ 
ism incidents and biocrimes have had limited results, 
the 1984 Rajneeshee episode and the 2001 anthrax 
mailings illustrate that even relatively small-scale 
attacks can have enormous public health, economic, 
and social consequences. 

Biological weapons have been renounced by 170 
states-parties to the BWC for numerous political 
and strategic considerations. Counterproliferation 
efforts, including verification of compliance of sig¬ 
natory states, remain challenging. According to an 
unclassified 2013 US Department of State report, 
uncertainties exist about activities in Russia, Iran, 
North Korea, and Syria.' 8 These ambiguities, in addi¬ 
tion to the miscalculations of the 2002 National Intel¬ 
ligence Estimate, underscore the difficulty of assess¬ 
ing biological weapons programs even through the 
rigorous efforts of highly dedicated and skilled pro¬ 
fessionals. These concerns highlight the importance 


24 


Historical Overview: from Poisoned Darts to Pan-Hazard Preparedness 


of strengthening international goodwill and trans¬ 
parency through the B WC and international engage¬ 
ment programs. 

The threats of biological weapons have led to new 
technical strategies: 

• a movement from addressing a static list of a 
limited number of specific pathogens toward 
a threat-agnostic capability-based approach 
using flexible enabling technology platforms 
that can be rapidly adapted to counter novel, 
unanticipated pathogens; 

• broad spectrum therapeutics; and 

• versatile response capacities that can be 
used to counter biological weapons attacks, 
naturally occurring epidemics, or other mass 
casualty disasters. 

The past decade has also seen efforts to integrate 
multidisciplinary societal sectors ranging from re¬ 
search to operational response-surveillance; medical 
care delivery; risk communication; the development, 
mass production, and stockpiling of medical counter¬ 
measures; and planning and exercises at local, regional, 
national, and international levels. The enhancement 
of diagnostic platforms, disease surveillance and 
reporting networks, medical countermeasures, and 
health delivery systems that can be rapidly adapted 
as common solution sets to either biological attacks 
or natural epidemics is essential to cost-effective, eco¬ 
nomically sustainable disease mitigation in an era of 
limited resources. 

Scientific research on highly virulent pathogens is 
essential to biodefense and public health—broadly 
inclusive—to counter biological weapons and novel 
emerging diseases. Such research inevitably carries 
risks, including accidental releases, transmission of 
laboratory-acquired infections to the community, 
unanticipated consequences of well-intended experi¬ 
ments, and the generation of knowledge that could be 
misused to execute biological attacks. Even with effec¬ 
tive risk management, risk never reaches zero, but can 
be decreased to an "irreducible minimum" through 
rigorous biosafety and biosecurity. Steps in the right 
direction include the formulation and enforcement of 
standards and regulations for biosafety, biosecurity, 
and handling of select agents. Risks and benefits should 
be carefully considered a priori, with engagement of a 
broad stakeholder community. Risk management must 
preserve opportunities for scientific creativity and aca¬ 
demic freedom and also must be open to unanticipated 
experimental results that may serendipitously lead to 
valuable new discoveries, such as the reactogenicity 
of tuberculin purified protein derivative, that led to 


the repurposing of a failed therapeutic to a valuable 
diagnostic reagent, and the fungal contamination of a 
bacterial culture that led to the discovery of penicillin. 

Although technical solutions are essential, they are 
not sufficient. An understanding of the history of the 
development and use of biological weapons, as well 
as analyses of risk perception and misperception, and 
appropriate or misguided responses to perceived risks 
requires examination from both technical and socio¬ 
logical points of reference, particularly the sociologies 
of scientific and policy decision-making. Important 
issues include the psycho-social milieus that generate 
biological weapon development and use, and that 
lead either to effective responses to credible threats 
or to misinterpretation and over-reaction to legitimate 
biotechnology. 184 

The late Joshua Lederberg, the 1958 Nobel laure¬ 
ate for medicine or physiology, a pioneer of bacterial 
genetics and recombinant technology, and an expert 
opinion leader in the fields of emerging infectious 
diseases and biological defense, 185 remarked: 

There is no technical solution to the problem of bio¬ 
logical weapons. It needs an ethical, human and mor¬ 
al solution if it's going to happen at all. Don't ask me 
what the odds are for an ethical solution, but there is 
no other solution. 186 

Value-related paradigms of ethical medical research 
directed toward the good of humanity, which underlie 
the preamble of the BWC's appeal "to the conscience 
of mankind," 187 and the National Strategy for Coun¬ 
tering Biological Threats' emphasis that life sciences 
research should be used "solely for peaceful and 
beneficial purposes," 188 proscribe biological weapons, 
and may also inform approaches to dilemmas posed 
by DURC. Proposals to obtain new data, information, 
and knowledge should be evaluated in the context 
of wisdom and in its relevance to the advancement 
of the common good, and be open to the possibili¬ 
ties that human actions may have intrinsic meaning 
and moral value. History demonstrates that when 
ethics and science are decoupled, potential outcomes 
include biological weapons. Ethical considerations 
are as relevant to basic and applied microbiology as 
the principle of beneficence is to medical research 
involving human subjects. Academic freedom must 
be maximized and ethical constructs must be flexible, 
yet circumstances exist in which it is appropriate to 
take principled stands. 

Moral principles lead to codes of professional con¬ 
duct based on a commitment that basic and applied 
sciences must be value-related—purposely directed 
toward the benefit of society as their long-term goal 


25 


Medical Aspects of Biological Warfare 


with a caveat to "do no harm." 179 Professional ethics 
must go deeper than financial disclosures and honest 
reporting of data to address the value and risks of 
proposed experiments. Because an experiment can 
be done—as an achievement outside of a value- and 
goal-related context—does not mean that it should be 
done. It is essential to build a culture of responsibility 
at every level of individual investigators, laboratory 
institutional review boards, funding organizations, 
and national authorities considering the permissibility 
of specific research proposals in the context of purpose, 
methods, potential unintended consequences, and 
value to society. Moral principles underlying the BWC 
and the National Strategy for Countering Biological 
Threats have found expression in the ethical codes of 
the American Society for Microbiology and other pro¬ 
fessional organizations, US government guidelines for 
synthetic biology and DURC, the Cooperative Biologi¬ 
cal Engagement Program, support for implementation 
of the IHR, and NSABB's call for the development and 
dissemination of ethical codes of conduct. 189 

The use of synthetic biology to produce wild-strain 
poliovirus illustrates the relevance of ethics to biologi¬ 
cal weapons proliferation and DURC, and the role of 
coordinated multidisciplinary approaches for risk miti¬ 
gation. An intended outcome was to sound an alarm 
that viruses can be synthetically produced to develop 
biological weapons; a conclusion was that WHO's goal 
of polio eradication may be unrealistic and should be 
reconsidered in view of issues that include the poten¬ 
tial reintroduction of synthetic poliovirus as an act of 
bioterrorism. 136 ' 138 Alternatively, the chemical synthesis 
of the oral polio vaccine would have demonstrated 
an innovative cell-free platform for the production of 
attenuated live viruses for vaccines. This would have 
been an unambiguously benevolent action and would 
have supported the investigators' intention to test 
the hypothesis that live viruses can be synthetically 


produced. The inductive proposition that synthetic 
viruses may pose biological weapons proliferation 
risks would have been obvious. The investigators later 
directed their platform toward novel approaches to 
vaccine development 138,190-193 ; in the context of altruis¬ 
tic medical research, this could have been their stated 
objective and technical approach from the outset. 

During the timeframe when the synthesis of wild- 
strain poliovirus was being conducted and reported, 
WHO was already proposing material and nonmate¬ 
rial solutions for the contingency of a posteradication 
outbreak resulting from either bioterrorism or an ac¬ 
cidental reintroduction. 194-198 A 2013 WHO strategic 
plan for the final phase of polio eradication combines 
multidisciplinary pan-sector approaches including 
the global incorporation of inactivated polio vaccine 
into routine immunization programs, coordinated 
withdrawal of oral polio vaccine, biocontainment of all 
wild and vaccine strains, enhanced surveillance, a vac¬ 
cine stockpile for emergency use, communication, and 
response. 199 The potential abuse of synthetic biology 
for biological weapons proliferation has not derailed 
the polio eradication campaign, 199-202 just as the risk 
of biological warfare using variola did not obviate the 
goal of smallpox eradication. 

Medical capabilities and biomedical research are 
being linked to diplomacy, commerce, education, eth¬ 
ics, law enforcement, and other activities to enable a 
common set of multidisciplinary pan-societal sector 
responses to both biological weapons and the inevi¬ 
table and dynamic challenges of naturally occurring 
emerging infectious diseases. 3 4 5 Integration of biological 
defense and public health programs and their mutual 
development must be continuous to optimize out¬ 
comes and maximize efficient utilization of limited 
resources and because the challenges posed by both 
biological weapons agents and naturally emerging 
pathogens are open ended. 


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179. Reiman DA. "Inconvenient truths" in the pursuit of scientific knowledge and public health. / Infect Dis. 2014;209:170-172. 

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181. US Department of Agriculture Office of the Inspector General. Follow Up on APHIS' Implementation of the Select Agent 
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182. Mulvaney E. Missing virus vial raises concern at UTMB facility. Houston Chronicle. March 24, 2013. http://www.chron. 
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2013. 

183. US Government Accountability Office. High-Containment Laboratories: Assessment of the Nation's Need is Missing. Wash¬ 
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2013. 

184. Vogel KM. Phantom Menace or Looming Danger? A New Framework for Assessing Bioweapons Threats. Baltimore, MD: The 
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185. Lederberg J, ed. Biological Weapons. Limiting the Threat. Cambridge, MA: MIT Press; 1999. 

186. Preston R. The bioweaponeers. The New Yorker. March 9,1998;65. 

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April 11, 2013. http://vaccines.emory.edu/poliodeclarahon/text.pdf. Accessed April 12, 2013. 


36 


Chapter 2 

EPIDEMIOLOGY OF BIOWARFARE 
AND BIOTERRORISM 


ZYGMUNT F. DEMBEK, PhD, MS, MPH, LHD* * * * § ; JULIE A. PAVLIN, MD, PhD, MPH + ; MARTINA SIWEK, PhD*; and 
MARK G. KORTEPETER, MD, MPI L 


INTRODUCTION 

THE EPIDEMIOLOGY OF EPIDEMICS 
Definition 
Recognition 

Potential Epidemiological Clues to an Unnatural Event 
Outbreak Investigation 

EPIDEMIOLOGICAL CASE STUDIES 
Bioterrorism Events 

Accidental Release of Biological Agents 

Studies of Natural Outbreaks for Potential Bioweapon Use 

EPIDEMIOLOGICAL ASSESSMENT TOOL 

IMPROVING RECOGNITION AND SURVEILLANCE OF BIOTERRORISM 

POTENTIAL IMPACT OF ADVANCED MOLECULAR TECHNIQUES ON THE 
EPIDEMIOLOGY OF BIOWARFARE AND BIOTERRORISM 

SUMMARY 


* Colonel (Retired), Medical Service Corps, US Army Reserve; Associate Professor, Department of Military and Emergency Medicine, Uniformed Ser¬ 
vices University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814; formerly, Chief, Biodefense Epidemiology and Education 
& Training Programs, Division of Medicine, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

f Colonel (Retired), Medical Corps, US Army; Deputy Director, Armed Forces Health Surveillance Center, 11800 Tech Road, Silver Spring, Maryland 20904 

*Chief Scientist, Cherokee Nation Technology Solutions in Support of Global Emerging Infections Surveillance and Response System, Armed Forces 
Health Surveillance Center, 11800 Tech Road, Silver Spring, Maryland 20904; formerly, Science Manager/Liaison, Biosurveillance Management Office, 
Joint Program Executive Office, Aberdeen Proving Ground, Maryland 

§ Colonel, Medical Corps, US Army; Director, Infectious Disease Clinical Research Program, Department of Preventive Medicine, Associate Dean for 
Research, Associate Professor of Preventive Medicine and Medicine, Consultant to the Army Surgeon General for Biodefense, Uniformed Services Uni¬ 
versity of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814; formerly, Deputy Commander, US Army Medical Research Institute 
of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

A portion of this chapter has previously been published as: Dembek ZF, Kortepeter MG, Pavlin JA. Discernment between deliberate and 
natural infectious disease outbreaks. Epidemiol Infect. 2007;135:353-371. 


37 



Medical Aspects of Biological Warfare 


INTRODUCTION 


Preparing for and responding to biological warfare 
(BW) or bioterrorism (BT) is a public health issue and 
falls within the purview of public health professionals, 
because preparation for natural disease outbreaks has 
the dual benefit of B W/BT preparation. An understand¬ 
ing of basic epidemiology is needed before, during, 
and after an event to identify populations at risk, target 
preventive measures such as vaccinations, recognize an 
outbreak, track and limit disease spread, and provide 
postexposure treatment or prophylaxis. Many disease- 


specific management needs such as vaccination and 
prophylaxis are discussed elsewhere and are not con¬ 
sidered here. Also, agricultural terrorism is discussed 
in chapter 3. This chapter will focus on detection and 
epidemiological investigation including distinguish¬ 
ing between natural and intentional events. Brief case 
studies will be presented to demonstrate important 
indicators and lessons learned from historical outbreaks. 
Finally, traditional methods of surveillance and ways 
to improve surveillance for BW/BT will be discussed. 


THE EPIDEMIOLOGY OF EPIDEMICS 


Definition 

The word epidemic comes from the Greek "epi" and 
"demos," meaning "upon a mass of people assembled 
in a public place." 1 An epidemic is defined as the occur¬ 
rence in a community or region of an unusually large or 
unexpected number of disease cases for the given place 
and time. 2 Therefore, a critical foundation is knowing 
baseline rates of disease to determine whether an epi¬ 
demic is occurring. This information can be at the local, 
regional, national, or global level, and can be seasonal. 
As an example, thousands of influenza cases in Janu¬ 
ary in the United States may not be unusual; however, 
thousands of cases in the summer may be cause for 
concern, similar to what was seen with an early sum¬ 
mer wave of cases of H1N1 swine variant influenza in 
2009. Also, even a single case of a rare disease can be 
considered an epidemic. With the absence of a woolen 
mill industry in the United States, any inhalational 
anthrax case should be highly suspect. Many of the 
diseases considered as classic BW agents, such as small¬ 
pox (considered to be eradicated), viral hemorrhagic 
fevers, and pneumonic plague are rare, and a single 
case should be investigated. Determining whether an 
outbreak occurs depends, therefore, on the disease, the 
at-risk population, the location, and the time of year. 

For an outbreak to occur, three points of the classic 
epidemiological triangle must be present (Figure 2-1). 
There must be a pathogen or agent, typically a virus, 
bacterium, rickettsia, fungus, or toxin, and a host (in 
this case, a human) who is susceptible to that patho¬ 
gen or agent. The two need to be brought together in 
the right environment to allow infection of the host 
directly by another individual, by a vector, or through 
another vehicle, such as food, water, or contact with 
fomites (inanimate objects). The environment must 
also permit potential transmission to other susceptible 
hosts. Disruption of any of these three points of the 
triangle can limit or disrupt the outbreak; therefore, it is 


important to know and understand the characteristics 
of the three for any specific disease to control an epi¬ 
demic. For example, if potential hosts are vaccinated, 
disease spread would be significantly limited or if the 
environment is modified, spread may also be limited 
(eg, cleaning up garbage around a home limits rat food 
and harborage, and thus minimizes the risk of contact 
with fleas capable of transmitting plague). 3 

Recognition 

Immediate effects on humans and possibly the en¬ 
vironment are evident when an explosion occurs or a 
chemical weapon is released. However, because of the 
incubation periods of infectious pathogens, release of 
a BW/BT agent may be silent and the casualties pro¬ 
duced after a release may be dispersed in time and 
space to primary care clinics and hospital emergency 
departments. Even toxins have latent periods prior to 
symptom onset. Therefore, the success in managing a 
biological event hinges directly on whether and when 
the event is recognized. 


Host 



Figure 2-1. The epidemiological triangle 


38 



Epidemiology of Biowarfare and Bioterrorism 


An example of the ramifications of delayed disease 
outbreak recognition occurred in 1972 in the former 
Yugoslavia. A single unidentified smallpox case led to 
11 secondary cases, also unrecognized. Within a few 
weeks there was an outbreak of 175 smallpox cases and 
35 deaths that led to a massive vaccination effort and 
border closure. 4 Early disease recognition may have 
significantly modified the outcome. Modeling studies 
of a BT-caused smallpox outbreak have shown that 
the more rapidly a postrelease intervention occurred, 
including quarantine and vaccination, the greater 
the chances that intervention would halt the spread 
of disease. 5- ' When medical professionals identify a 
new case, it is unlikely that a BW/BT event would 
be the first cause suspected, especially if the disease 
presents similar to other diseases that might occur si¬ 
multaneously, such as influenza. Clinicians generally 
consider the source to be a common endemic disease 
at first. Alternative considerations might include a new 
or emerging disease, or a laboratory accident before 
considering BW/BT. 8 Therefore, care providers should 
be familiar with the diseases of BW/BT that could be 
spread intentionally and maintain a healthy "index of 
suspicion" to recognize an event early enough to sig¬ 
nificantly modify the outcome. 9 Furthermore, although 
the government has generated lists of potential threat 
agents, public health authorities must be mindful that 
a perpetrator does not necessarily follow any list and 
may choose an organism based on access or some 
other unanticipated reason. Also, a perpetrator might 
listen to government and other media information, and 
respond accordingly, thereby undermining a govern¬ 
ment terrorism response. 

Clinicians, hospital infection control personnel, 
school or healthcare facility nursing staff, laboratory 
personnel, and other public health workers have a 
responsibility to notify public health authorities about 
disease outbreaks. State and local public health officials 
regularly examine and review disease surveillance 
information to detect outbreaks in a timely manner 
and provide information to policymakers on disease 
prevention programs. Time constraints are inherent 
in obtaining case report information because of the 
elapsed time from patient presentation, lab specimen 
collection and submission, and laboratory testing time, 
to final disease or organism reporting. Furthermore, 
the initial BW/BT disease recognition may not come 
from a traditional reporting partner or surveillance 
method. Instead, pharmacists and clinical laboratory 
staff who receive requests or samples from numer¬ 
ous healthcare providers may be the first to note 
an increase in purchases or prescriptions of certain 
medications (eg, antibiotics or antinausea or diar¬ 
rheal agents) or orders for certain laboratory tests (eg. 


sputum or stool cultures), respectively. Also, because 
many of the category A high-threat diseases are zoo¬ 
noses (primarily infect animals), with humans serving 
as accidental hosts, veterinarians may be the first to 
recognize the disease in animals prior to the ensuing 
human disease. Media and law enforcement personnel 
and other nontraditional reporters of outbreaks may 
also provide information on a BT event or potential 
cases. Therefore, it is important for all those different 
types of individuals to maintain the same index of 
suspicion as healthcare providers for unusual events 
in their respective fields. 

Potential Epidemiological Clues to an Unnatural 
Event 

It is often not possible to determine the objectives of a 
BT perpetrator in advance, whether the intent is to kill, 
incapacitate, or obtain visibility. It also may be difficult 
to discern how a biological agent was dispersed, wheth¬ 
er through the air, in contaminated food or water, or by 
direct inoculation. In a biological attack, the number of 
casualties may be small and therefore unrecognized as 
intentionally infected, especially if the agent is a com¬ 
mon cause of disease in the community. In addition, 
given the agent's incubation period, individuals may 
seek care from different care providers or travel to differ¬ 
ent parts of the country before they become ill and seek 
medical care. Despite the potential for these situations 
to occur, it is useful for healthcare providers to be aware 
of potential clues that may be tip-offs or "red flags" of 
something unusual. Although these clues may occur 
with natural outbreaks and do not necessarily signal a 
BW/BT attack, they should at least heighten suspicion 
that something out of the ordinary is occurring. The 
following compilation is an illustrative list; however, 
additional clues may be to Lind elsewhere. 10,11 

Clue 1: A highly unusual event with large num¬ 
bers of casualties. Although the mention of BW or 
BT may elicit images of massive casualties, they may 
not actually occur with a real BW/BT event. Numer¬ 
ous examples of naturally spread illness have caused 
massive casualties and some BW/BT events have few or 
no casualties. Nevertheless, the type of large outbreak 
that should receive particular attention is one in which 
no plausible natural explanation for the cause of the 
infection exists. 

Clue 2: Higher morbidity or mortality than is 
expected. If clinicians are seeing illnesses that are 
causing a higher morbidity or mortality than what is 
typically seen or reported for a specific disease, this 
may indicate an unusual event. A perpetrator may 
have modified an agent to make it more virulent or 
selected antibiotic resistance in an organism usually 


39 


Medical Aspects of Biological Warfare 


sensitive to antibiotics. Individuals could also be ex¬ 
posed to a higher inoculum than they would normally 
receive with natural spread of the agent, thus causing 
higher morbidity or mortality. 

Clue 3: Uncommon disease. Many infectious 
diseases have predictable population and infectivity 
distributions based on environment, host, and vector 
factors; yet unnatural spread may occur if a disease 
outbreak is uncommon for a certain geographical 
area. Concern should be heightened if the naturally 
occurring disease requires a vector for spread and the 
competent vector is missing. For example, if a case 
of yellow fever, which is endemic to parts of South 
America and sub-Saharan Africa, occurred in the 
United States without any known travel, it would be 
a concern. Natural outbreaks have occurred in new 
geographical locations including the West Nile virus 
(WNV) in New York City in 1999. 12 It is important to 
consider whether the occurrence of these uncommon 
diseases is natural. 

Clue 4: Point source outbreak. For any outbreak, it 
is useful to develop an epidemic curve demonstrating 
the timeline of dates when patients developed illness. 
These curves can have different morphologies depend¬ 
ing on whether individuals are exposed at the same 
time from a single source or over time, and whether 
the illness spreads from person to person. In an inten¬ 
tional BT event, a point source outbreak curve would 
most likely be seen 13 when individuals are exposed 
at a similar point in time. The typical point source 
outbreak curve has a relatively quick rise in cases, a 
brief plateau, and then an acute drop, as seen in Figure 
2-2. For example, the epidemic curve might be slightly 
compressed after an aerosol release because infected 
individuals were exposed more closely in time (ie, 
within seconds to minutes of each other) compared 


35-, 


30- 



Onset by Day of Month 


Figure 2-2. Typical point source outbreak epidemic curve 


with individuals becoming ill after eating a common 
food over a period of hours. Or the inoculum may be 
greater than what is typically seen with natural spread, 
thus yielding a shorter incubation than expected. It 
should also be considered that the spread of a bio¬ 
logical agent capable of being transmitted from person 
to person could result in a propagated (secondary 
transmission) outbreak, with a case distribution more 
similar to that depicted in Figure 2-3. 

Clue 5: Multiple epidemics. If a perpetrator can 
obtain and release a single agent, it is also feasible that 
multiple perpetrators could release single or multiple 
agents at different locations. If simultaneous epidemics 
occur at the same or different locations with the same 
or multiple organisms, an unnatural source must be 
considered. It must also be considered that a mixture 
of biological organisms with different disease incuba¬ 
tion periods could be released, and thus would cause 
simultaneous or serial outbreaks of different diseases 
in the same population. 

Clue 6: Lower attack rates in protected individuals. 

This clue is especially important for military personnel. 
If certain military units had some type of respiratory 
protection, such as mission-oriented protective posture 
gear or high-efficiency particulate air-filtered masks, or 
stayed in a high-efficiency particulate air-filtered tent 
and had lower rates of illness than nearby groups that 
were unprotected, this may indicate that a biological 
agent has been released via aerosol. 

Clue 7: Dead animals. Historically, animals have 
been used as sentinels of human disease. The storied 
use of canaries in a coal mine to detect the presence 
of noxious gases is one example. This phenomenon 
was observed during the naturally occurring WNV 
outbreak in New York City in 1999, when many of the 
local crows, along with the exotic birds at the Bronx 
Zoo, developed fatal disease. 14,15 Because many biologi¬ 
cal agents that could be used for BW/BT are zoonoses, 
a local animal die-off may also indicate a biological 
agent release that may also infect humans. 


16- 

14- 

12 - 

10 - 


..lihu. 

e yA v k Mo Mo ul Mb Mb \0 \\ 

^® e ^® c ^® e ^®® ^® c ^® e ^® e ^® e ^® e ^® 

Figure 2-3. Typical continuous common source outbreak 
epidemic curve 


40 







Epidemiology of Biowarfare and Bioterrorism 


Clue 8: Reverse or simultaneous spread. Zoonotic 
illnesses exhibit a typical pattern: an epizootic first oc¬ 
curs among a susceptible animal population, followed 
by cases of human illness. With anthrax, one would 
expect ill animals to be identified before cutaneous 
disease in workers processing the animals or before 
gastrointestinal disease in people who may have eaten 
meat from the infected animals. After the accidental 
release of anthrax spores in Sverdlovsk (see description 
and case review of the 1979 Sverdlovsk anthrax out¬ 
break), an outbreak occurred simultaneously in people 
and animals downwind of the weapons facility. 16 If 
human disease precedes animal disease or human 
and animal disease are simultaneous, then unnatural 
spread should be considered. 

Clue 9: Unusual disease manifestation. More than 
95% of worldwide anthrax cases are cutaneous illness. 
Therefore, a single case of inhalational anthrax should 
be considered highly suspicious for BW/BT until 
proven otherwise. The rare exception is an inhalational 
anthrax case in a woolen mill worker or in someone 
handling animal skins from endemic areas, which 
has recently occurred. 1 ' This logic may be applied to 
cases of a disease such as plague, where the majority 
of naturally occurring cases are the bubonic, not the 
pneumonic form. 

Clue 10: Downwind plume pattern. The geographic 
locations where cases occur can be charted on a geo¬ 
graphic grid or map. If the reported cases appear 
clustered in a downwind pattern, then an aerosol 
release may have occurred. During the investigation 
into the anthrax outbreak in Sverdlovsk in 1979 (as 
examined later in this chapter), mapping out case 
locations helped to determine that the anthrax cases 
were caused by an aerosol release rather than a con¬ 
taminated food source. 16 

Clue 11: Direct evidence. The final clue may be the 
most obvious and the most useful. Determining the 
intentional cause of illnesses is easier if a perpetrator 
leaves a "signature" or direct evidence of a biological 
attack. Such a signature could be a letter filled with 
anthrax spores, 18 a spray device or another vehicle 
for agent spread, or claims by a person or group of 
a biological attack. It would be useful to compare 
samples from any found device with the clinical 
samples obtained from victims to verify that they are 
the same organism. 

Outbreak Investigation 

It is important to understand the basic goals of an 
outbreak investigation, as seen in Exhibit 2-1. Any 
outbreak (a greater than expected number of cases in 
a specific location, group of people, or time period) 


EXHIBIT 2-1 

GOALSOFANOUTBREAKINVESTIGATION 


• Find the source of disease. 

• Rapidly identify cases. 

• Prevent additional cases through implemen¬ 
tation of appropriate control measures. 

• Identify strategies to prevent further out¬ 
breaks. 

• Evaluate existing prevention strategies (in¬ 
cluding control measures immediately put 
into place). 

• Address public concerns. 

• Provide information to leadership to support 
informed decisions. 

• Improve scientific knowledge about the 
disease. 


should be investigated quickly to find the source of 
the disease. If an outbreak is ongoing, the source of 
infection needs to be identified and eliminated quickly. 
Even if the exposure source has dissipated, all cases 
should be identified expeditiously, so that ameliora¬ 
tive care can be offered and case interviews can be 
conducted. Case identification can assist in preventing 
additional cases, especially with a transmissible infec¬ 
tious disease. Providing information to the public and 
to leaders is also key to ensure the best public health 
policies are enacted and followed. With notification of 
any outbreak, whether natural or intentionally caused, 
there are standard steps to follow in an outbreak in¬ 
vestigation (Exhibit 2-2), although these steps may not 
always occur in order. 19 The first step is preparation, 
which involves having the necessary response ele¬ 
ments (personnel, equipment, laboratory capabilities) 
ready and establishing communications in advance 
with partners who may assist in the investigation. Once 
an event is ongoing, the second step is to investigate, 
verify the diagnosis, and decide whether an outbreak 
exists. Early in an outbreak, its significance and scope 
are often not known. Therefore, existing surveillance 
information and heightened targeted surveillance ef¬ 
forts are used to determine whether reported items 
are cause for concern. 

The third step is to define the outbreak and seek a 
definitive diagnosis based on historical, clinical, epide¬ 
miological, and laboratory information. A differential 
diagnosis can then be established. 

The fourth step is to establish a case definition that 
includes the clinical and laboratory features that the ill 
individuals have in common. It is preferable to use a 


41 




Medical Aspects of Biological Warfare 


EXHIBIT 2-2 

TEN STEPS IN AN OUTBREAK 
INVESTIGATION 

1 . 

Prepare for fieldwork (identify resources). 

2. 

Verify the diagnosis. Determine whether an 
outbreak exists. 

3. 

Define the outbreak and seek a diagnosis 
(including specimen collection and testing). 

4. 

Develop a case definition and identify and 
count cases. 

5. 

Develop exposure data with respect of per¬ 
son, place, and time. 

6. 

Implement control measures and continu¬ 
ally evaluate them. 

7. 

Develop the hypothesis. 

8. 

Test and evaluate the hypothesis with ana¬ 
lytical studies and refine the hypothesis. 

9. 

Formulate conclusions. 

10. 

Communicate findings. 


broad case definition at first and avoid excluding any 
potential cases too early. Objective clinical features are 
preferred, such as temperature exceeding 100.4°F, or 
diarrhea defined as greater than three watery bowel 
movements per day, as well as laboratory and patho¬ 
logical reports. The case definition enables the investi¬ 
gator to count cases and compare exposures between 
cases and noncases and compare these with other 
investigators and regions using the same case defini¬ 
tion. To obtain symptom information, it may not be 
sufficient to look at healthcare facilities only, but also 
necessary to interview the ill persons and their family 
members, as well as coworkers, classmates, or others 
with whom they have social contact. It is important 
to maintain a roster of potential cases while obtaining 
this information. Commonly during an investigation, 
there is a risk of double or even triple counting cases 
because they may be reported more than once through 
different means. Key information needed from each 
ill person, besides identifying information to ensure 
accurate case counting and ability to contact the cases 
again if necessary, includes date of illness onset; signs 
and symptoms; recent travel; ill contacts at work, 
home, or school; animal exposures; and treatments 
received. With this information, an epidemic curve 
can be constructed (see Figure 2-2) that may provide 
information as to when a release may have occurred, 
especially if the disease is known, and an expected 
exposure date based on the typical incubation period, 
known ill contacts, or geographic risk factors. 


Different modes of disease spread may have typical 
features that comprise an epidemic curve. If there is 
a common vehicle for disease transmission (such as a 
food or water source) that remains contaminated, it 
might be possible to see a longer illness plateau (a con¬ 
tinuous common source curve [Figure 2-3]) than is seen 
with a point source of infection. If the agent is spread 
person to person, successive waves of illness may be 
seen as one group of individuals infects a follow-on 
group, which in turn infects another, and so on (Figure 
2-4). With time and additional cases, the successive 
waves of illness may overlap with each other. 

The fifth step is to document potential exposure data. 
Cases need to be identified and counted. Once cases 
have been identified, exposures based on person, place, 
and time can be determined. Obtaining information 
from individuals who would likely have had similar 
exposures but are not ill can also help determine the 
potential cause and method of an agent's spread. In¬ 
formation can be obtained either informally or formally 
with a case control study. A case control study is a 
type of study where investigators identify individuals 
with and without disease and compare their potential 
exposures or risk factors for disease. With a known 
exposure, one can also identify exposed and nonex- 
posed populations and determine illness rates with a 
retrospective cohort study to help determine whether 
that particular exposure is a risk factor for disease. 

The sixth step is to implement control measures as 
soon as feasible and continuously evaluate them. If nec¬ 
essary, control measures can be quickly implemented 
and then modified as additional case information 
becomes available. The seventh step is to develop a 
hypothesis. Based on the characteristics of the disease, 
the ill persons, and environmental factors, a hypoth¬ 
esis can usually be generated for how the disease oc¬ 
curred, how it is spreading, and the potential risk to 
the uninfected. The eighth step is to test and evaluate 
the hypothesis using analytical studies and refine the 
hypothesis. 


15 i 



0 2 6 10 14 18 22 26 30 34 38 

Onset by Day of Month 


Figure 2-4. Typical propagated (secondary transmission) 
outbreak epidemic curve 


42 





Epidemiology of Biowarfare and Bioterrorism 


Once developed, it is important to test the hypoth¬ 
esis to ensure it fits with the known facts. Does it 
explain how all the cases were exposed? It is possible 
that some outliers may seem as if they should be ill 
but are not, or some who are ill but have no known 
exposure. These outliers can sometimes be the key 
to determining what happened. With preliminary 
control measures implemented, the hypothesis can 
be tested formally with analytical studies. Further 
modifications in control measures might be needed 
and implemented. 

The ninth step is to formulate a conclusion about 
the nature of the disease and exposure route. Findings 
can then be communicated (the tenth and final step) 
through the media or medical literature, depending on 
the urgency of notification to the public and medical 
community. 

Experience from the anthrax mailings of 2001 indi¬ 
cates that during any BT event, intense pressure will 
be exerted on public health authorities to provide more 
information than is available. 20 As stated earlier, these 
distinct steps may not occur in sequence. It may be nec¬ 
essary to implement control measures with incomplete 
information, especially if an outbreak is fast moving 
or has a high morbidity or mortality rate. Whether the 
control measures appear to limit the disease spread or 
the casualty toll is the ultimate test of the accuracy of 
the original hypothesis. 

Early in an investigation, it will probably not be 
known or suspected that an outbreak was unnaturally 
spread. Therefore, with a few exceptions, the investiga¬ 
tion of an unnaturally spread outbreak will not differ 


The following epidemiological case studies are 
presented to demonstrate the differences between 
naturally occurring and purposefully created epidem¬ 
ics. Biological attacks and some naturally occurring 
epidemics of historical significance are considered in 
the context of BT. Some purposeful BT events have 
not caused illness; however, some naturally occurring 
outbreaks were initially considered as potential BT 
events because of the particular disease or nature of 
clinical case presentation. 

Public health authorities could be held account¬ 
able to make a determination quickly as to whether 
an infectious disease outbreak has been purposefully 
caused, yet they may lack the necessary informa¬ 
tion because there may not be clear evidence or 
responsibility claimed for a BT event. A thorough 
understanding of how to investigate suspect out¬ 
break occurrences may better enable public health 
authorities to make difficult public health policy 
decisions. 


significantly from the investigation of a naturally oc¬ 
curring outbreak. Public health authorities will work 
on both types of outbreaks. The significant difference 
is that, with a purposeful outbreak, a potential crimi¬ 
nal event may have occurred. An additional goal of 
this type of investigation, under the purview of law 
enforcement personnel, is to bring the perpetrator to 
justice. Therefore, law enforcement personnel need to 
partner with public health officials as early as possible 
in any suspected BT case. 21 

Public health authorities must become familiar 
with the use of chain of custody, the process used 
to maintain and document the chronological history 
of the evidence, so that medical evidence/clinical 
samples or environmental samples obtained in the 
investigation will be admissible in a court of law. 
Environmental and biological samples can be cru¬ 
cial in determining whether a deliberate release of 
a pathogen has occurred (see the case study in this 
chapter about the release of Bacillus anthracis in Tokyo 
by the Aum Shinrikyo). 

Although chain of custody is important, public 
safety should be the primary concern. Public health 
authorities must also have an open mind for unusual 
modes of disease spread, being especially careful to 
ensure their personnel's safety if a potential exposure 
risk occurs during the investigation. Public health 
authorities conducting a field investigation should 
have personal protective equipment and be trained 
in its proper use, and they should also have access to 
occupational health resources if pre- or postexposure 
prophylaxis or monitoring is needed. 

L CASE STUDIES 

Bioterrorism Events 

The following section describes BT incidents that 
occurred in the United States and Japan. None of these 
events was immediately recognized as having been 
intentional. The 2001 mail-associated anthrax outbreak 
and mail-associated ricin attack were recognized 
within days to weeks. With new sensors installed in 
mail collection facilities, mailings of ricin in 2013 were 
recognized immediately. However, for previous BT 
incidents (anthrax and glanders in 1915, salmonellosis 
in 1984, and anthrax in 1995), intentionality was not 
recognized for a year or longer after the initial event. 

Anthrax and Glanders—Maryland; New York, New 
York; and Virginia, 1915-1916 

From 1915 through 1918, Germany had a state- 
sponsored offensive BW program to sabotage suppliers 
to the Allies directed at draft, cavalry, and military 


43 


Medical Aspects of Biological Warfare 


livestock. Human disease was neither intended nor 
recorded from these events, although the program 
could have been expanded to spread zoonotic ill¬ 
ness among a target population. Unintended human 
disease may have occurred but was never recorded. 
Countries targeted by Germany included the United 
States, Argentina, Romania, Russia, Norway, and 
Spain. The German army general staff directed and 
implemented the biological sabotage program despite 
official German army doctrine prohibiting such activi¬ 
ties. Germany's plans to spread a wheat fungus and 
contaminate food produced at "meat factories" were 
dropped. 22 One 1916 German plan never carried out 
proposed to drop vats of plague cultures from Zep¬ 
pelins over England. 23 

In April 1915 German-American physician Anton 
Dilger returned to the United States from Germany 
with cultures of Burkholderia mallei and Bacillus anthra- 
cis. His intent was to infect horses and mules being 
shipped from the United States to France and England 
for use in cavalry and transport. These cultures were 
propagated and tested for virulence using guinea pigs 
in the basement of a house (known as "Tony's Lab") 
rented by Anton and his brother, Carl, in Chevy Chase, 
Maryland, near Washington, DC. 24 From the summer of 
1915 through the fall of 1916, the cultures were used to 
infect horses and mules in holding pens in docks at the 
ports of Baltimore, Maryland; Newport News, Virginia; 
Norfolk, Virginia; and New York, New York. Stevedores 
working for German steamships were recruited and 
given 2-inch, cork-stoppered glass vials containing the 
cultures, in which a hollow steel needle had been placed. 
These stevedores were instructed to wear rubber gloves 
while jabbing the animals with the needle. These cul¬ 
tures were also spread to the animals by pouring them 
into the animal feed and drinking water. 25,26 

Case Review of 1915-1916 Anthrax and Glanders 
Incidents 

Biological Agents: B anthracis, gram-positive bacillus; 
B mallei, gram-negative bacillus 

Potential Epidemiological Clues: 2, 7, 8 

Review: A full assessment of the success of this BW 
program 90 years later is not possible. German agents 
claimed that epidemics occurred among the animals shipped 
from the US ports. However, disease observed among ani¬ 
mals might have originated naturally or from stressful holding 
and shipment conditions. 

Few surveillance systems incorporate comprehensive 
veterinary surveillance. This is an important disease detec¬ 
tion vulnerability because many BW agents (ie, B anthracis, 
Brucella suis, B mallei, Burkholderia pseudomallei, Coxiella 
burnetii, Francisella tularensis, Yersinia pestis, encephalitis, 
and hemorrhagic fever viruses) can cause zoonotic illness. 

Lessons Learned: Veterinarians discovering glanders 
or anthrax and other US Department of Agriculture (USDA) 
select agricultural agents in livestock should report these 


diseases to state health and federal authorities as possible 
BT indicators. 2728 

A comprehensive animal surveillance network would 
include reports from veterinary examinations of farm and 
companion animals, and from wildlife examinations by state 
environmental officials and animal rehabilitators. Current 
animal disease surveillance networks that address these 
deficiencies include the National Animal Health Laboratory 
Network 29 and the Centers for Epidemiology and Animal 
Health, 30 both part of the USDA. 

Salmonellosis—The Dalles, Oregon, 1984 

A large outbreak of Salmonella cases occurred in 
and around The Dalles, Oregon, in 1984. This farm¬ 
ing community, with a 1984 population of 10,500, is 
near the Columbia River on the border of Oregon and 
Washington. Salmonellosis is the second most common 
bacterial foodbome illness and is underreported by a 
factor of about 38-fold. 31,32 The average onset period for 
salmonellosis is about 12 to 36 hours, and the disease 
manifests as acute gastroenteritis. Fever occurs, an¬ 
orexia and diarrhea persist for several days, and more 
severe manifestations may at times occur, especially 
in very young or elderly persons. Contaminated food 
(most often poultry) is the principal route of disease 
transmission. 33 

Given its high incidence in the United States, pub¬ 
lic health authorities would not normally consider a 
foodborne salmonellosis outbreak as intentional. It 
has been estimated that 1.4 million salmonellosis in¬ 
fections occur annually in the United States, resulting 
in 15,000 hospitalizations and 400 deaths. 34 Therefore, 
the index of suspicion for an intentional Salmonella 
outbreak was—and remains—low. However, atypical 
events associated with this outbreak eventually led 
officials to realize that this particular disease occur¬ 
rence was historically different. Two cohorts of cases 
occurred: (1) from September 9 through 18,1984, and 
(2) from September 19 through October 10,1984. Public 
health authorities received initial reports of illness on 
September 17, and local and state health officials inter¬ 
viewed the ill persons. Patronizing two restaurants in 
the city of The Dalles and eating salad bar food items 
were commonly cited in these interviews. Salmonella 
typhimurium isolates were then obtained from clinical 
specimens from the ill persons. 35 

The source for this outbreak was puzzling. Epi¬ 
demiological analysis revealed multiple items rather 
than a single suspect item as the cause of the restau¬ 
rant patrons' illness. This finding is not uncommon 
either during the initial stages of an investigation of 
a foodbome disease outbreak (until a suspected food 
item is identified), or when an infected food handler 
is identified as the source of the outbreak. Although 
dozens of food handlers became ill, their time of 


44 


Epidemiology of Biowarfare and Bioterrorism 


symptom onset did not precede those of their custom¬ 
ers. As gastroenteritis cases occurred in increasing 
numbers, health officials imposed a closure of all salad 
bars in The Dalles on September 25. By the end of the 
outbreak, 751 salmonellosis cases were identified, 
with those affected ranging in age from newborns to 
87 years, and most were associated with dining in 10 
area restaurants. At least 45 persons were hospitalized, 
but no fatalities occurred. 

Bhagwan Shree Rajneesh, a charismatic guru, had 
established a community for his followers in 1981 
at a ranch near The Dalles. These cult members, or 
"Rajneeshees," attempted to use Oregon's liberal 
voter registration laws to control zoning and land use 
restrictions to their advantage. Conflict between the 
commune and the neighboring traditional community 
had escalated. To gain political control of the area, 
the Rajneeshees attempted to influence an election 
by making voters too ill to vote. 22 Approximately 12 
individuals were involved in the plot, and up to 8 indi¬ 
viduals distributed S typhimurium cultures to the salad 
bars. After considering the use of several biological 
agents, including Salmonella typhi (the causative agent 
of typhoid fever) and the human immunodeficiency 
virus, the Rajneeshees legally obtained cultures of 
S typhimurium (American Type Culture Collection 
strain 14028) from a commercial supplier and used 
them to grow bacterial stock cultures. The Rajneeshees 
first spread Salmonella by contaminating the com¬ 
mune members' hands to greet outsiders, as well as 
the county courthouse's doorknobs and urinal handles; 
these efforts did not cause illness. The cult also spread 
Salmonella cultures on salad bars in area restaurants. 

Public health authorities conducted an extensive 
investigation in response to the salmonellosis outbreak. 
Authorities identified confirmed cases microbiologi- 
cally by stool culture of S typhimurium, or with the 
clinical criteria of diarrheal illness and at least three 
of the following symptoms: fever, chills, headache, 
nausea, vomiting, abdominal pain, or bloody stools. 
S typhimurium was isolated from 388 patients. In the 4 
years before the outbreak, the local health department 
had collected 16 isolates of Salmonella, 8 of which were 
S typhimurium. No local cases of salmonellosis had been 
reported in 1984 before August. 35 

The 38 restaurants in The Dalles were grouped ac¬ 
cording to the number of culture-confirmed customer 
cases with a single restaurant exposure in the week 
before symptom onset. Additional ill customers were 
located through laboratory reporting of clinical speci¬ 
mens or clinician reporting to public health authorities 
(passive disease surveillance). Press releases were 
issued to encourage disease reporting by patients 
and clinicians. 35 Public health officials interviewed ill 
persons to obtain their symptoms, risk factors, and 


comprehensive food histories, as well as the names of 
all persons who had eaten with them at the restaurant. 
Restaurant employees with the greatest number of cases 
were interviewed twice and required to submit a stool 
sample as a condition of continued employment. The 
state public health laboratory serotyped the Salmonella 
isolates and performed antibiotic-susceptibility test¬ 
ing on a subset. A representative sample of outbreak 
isolates was sent to the Centers for Disease Control 
and Prevention (CDC) for further characterization, 
during which the outbreak strain was compared with 
national surveys of human and veterinary isolates. 
Sanitarians inspected the restaurants, and tap water 
was collected and analyzed. The local health depart¬ 
ment and USDA also investigated the food distribu¬ 
tors and suppliers used in these restaurants. None was 
found to have contaminated food, nor was a common 
supplier found for all of the implicated restaurants. 

Many food items served at the salad bars of the 
restaurants were associated with illness and differed 
among the restaurants. Illness was associated with eat¬ 
ing blue cheese dressing at one of the restaurants. The 
consumption of potato salad had the greatest associa¬ 
tion with illness, followed by lettuce. S typhimurium 
was isolated from the blue cheese dressing collected 
at one restaurant, but not from the dry mix used to 
prepare the dressing. 

The size and nature of the outbreak eventually 
helped to initiate a criminal investigation. The source 
and cause of the outbreak only became known when 
the Federal Bureau of Investigation (FBI) investigated 
the cult for other criminal violations. 36 An Oregon 
public health laboratory official accompanying the 
FBI discovered an open vial containing the original 
culture strain of S typhimurium in the Rajneeshee clinic 
laboratory in October 1985. 22,35 This strain was indis¬ 
tinguishable from the outbreak strain as isolated from 
food items and clinical specimens, and records were 
to Lind documenting its purchase before the outbreak. 35 

Intentional contamination of the salad bars is consis¬ 
tent with the retrospective epidemiology. 35 Eventually 
two cult members were arrested and served federal 
prison terms. Despite the Rajneeshees' success of the 
restaurant-associated BT, the publicity and subsequent 
legal pressure caused them to abandon subsequent 
efforts. 22 

Case Review of 1984 Salmonellosis Outbreak 

Biological Agents: S typhimurium, gram-negative bacillus 

Potential Epidemiological Clues: 1,4, 5, 11 

Review: Public health authorities found no statistical 
association with any single food item. 22 The isolation of S 
typhimurium from the blue cheese dressing, but not from the 
dry mix used in dressing preparation, should have indicated 
to authorities the contamination of the prepared dressing that 
was then served at a salad bar. 


45 


Medical Aspects of Biological Warfare 


The ongoing law enforcement investigation eventually 
revealed purposeful restaurant food contamination by the 
Rajneeshees more than a year after the outbreak occurred. 

Public health and law enforcement authorities lacked co¬ 
operative protocols in 1984; however, law enforcement teams 
in Oregon worked together with public health. 

An outbreak of this magnitude now would initiate a joint 
inquiry and investigation by public health and law enforce¬ 
ment, increasing chances that the outbreak cause would be 
identified in a timelier manner. 

Lessons Learned: These events illustrate the need to 
have joint public health and law enforcement investigations 
and mutual cooperation. 

This outbreak shows t yohe importance of the mode of 
disease spread in discerning the source. 

Although not occurring in this case, when different geo¬ 
graphic locations are affected, there could be a central sup¬ 
plier of a contaminated product shipped to all the locations. 
Since there was not a single supplier in this situation, this 
served as a red flag that multiple contaminations may have 
occurred. 

Anthrax—Tokyo, Japan, 1995 

Sarin is a chemical (nerve) agent that causes block¬ 
ing of the postsynaptic enzyme that degrades acetyl¬ 
choline, thus leading to excessive salivation, lacrima- 
tion, respiratory compromise, and seizures. Many may 
be familiar with it as a result of its use in the Syrian 
civil war in 2014. The notorious sarin attacks in a Tokyo 
suburb, Kameido, in 1994 and 1995, culminated with a 
sarin release in the Tokyo subway system. 37,38 Less well 
known is that before its efforts with chemical weapons, 
the apocalyptic cult Aum Shinrikyo appears to have 
first invested efforts into producing biological agents 
and had attempted to use them. 22 

Shoko Asahara, a charismatic guru, built the Aum 
Shinrikyo cult into a membership of approximately 
10,000 individuals with financial assets exceeding 
$300 million. Aum Shinrikyo's organization mim¬ 
icked a government entity, with various ministries 
and departments, including a ministry of science and 
technology that included graduate-level researchers 
within modern laboratories interested in developing 
biological and chemical weapons. B anthracis cultures 
were also obtained and grown into a slurry for use as 
a biological weapon. This cult may have also inves¬ 
tigated the use of C burnetii (the rickettsial organism 
that causes Q fever) and toxic mushrooms. In 1992 a 
team of 40 cult members, including Asahara, traveled 
to Zaire to attempt to acquire Ebola virus; the success 
of these efforts is unknown. 

The Aum Shinrikyo experimented with the release 
of aerosolized biological agents. In June 1993 the 
cult sprayed B anthracis from the roof of one of its 
buildings in downtown Tokyo. In July 1993 the cult 


sprayed B anthracis from a moving truck onto the Diet 
(Japan's parliament) and also around the Imperial 
Palace in Tokyo. 

Information about the anthrax releases became pub¬ 
lic when, during the arraignment of Asahara on May 
23, 1996, for the Kameido sarin attack, cult members 
testified about their efforts to aerosolize a liquid sus¬ 
pension of B anthracis to cause an inhalational anthrax 
epidemic. Their goal was to have an epidemic trigger 
a world war that would permit Asahara to rule the 
world. 39 In 1999 a retrospective case-detection survey 
was conducted to assess the possibility that some an¬ 
thrax cases may have been unreported. Complaints of 
odors from neighborhood residents were associated 
with the anthrax releases. These complaints were ret¬ 
rospectively mapped to provide the geographic areas 
of the greatest anthrax exposure risk. Physicians at 
39 medical facilities serving this area were surveyed. 
None reported having seen cases of anthrax or rel¬ 
evant syndromes. 39 It is not known whether a similar 
retrospective examination of anthrax-caused animal 
deaths was or could have been performed. Danzig and 
colleagues wrote a comprehensive report that analyzed 
the Aum Shinrikyo's failures and successes in develop¬ 
ing biological and chemical weapons. 40 

Case Review of 1995 Anthrax Releases 

Biological Agents: B anthracis, gram-positive bacillus 

Potential Epidemiological Clues: 11 

Review: Technical errors in either the biological agent 
production or dissemination rendered the attacks harmless. 
In contrast, there were 12 deaths and about 1,000 hospi¬ 
talizations from the sarin releases by the Aum Shinrikyo. 37 

Molecular analysis revealed that the 6 anthracis isolates 
were similar to the Sterne 34F2 strain, the strain of anthrax 
used in animal vaccines. Dispersal of this type of anthrax (re¬ 
garded as nonpathogenic for immunocompetent individuals) 
had little possibility to cause harm to humans. 39 

Even if the strain was pathogenic, the concentration of 
spores in the liquid suspension is significantly less (104 bac- 
teria/mL) than that considered optimal for a biological weapon 
(109-1010 bacteria/mL). The viscosity of the suspension was 
also problematic for successful aerosolization. 39 

The weather on the day of dispersal may have helped 
prevent infection: spore inactivation resulting from solar radia¬ 
tion could have further reduced the anthrax mix’s potency. 39 

Lessons Learned: These experiences show that it is 
difficult to both create a pathogenic biological weapon and 
deploy it successfully. 

Both health and law enforcement officials should be aware 
of the possibility for use of more than one biological agent or 
a combination of agents. 

Environmental sample collection and proper storage are 
important for viability of pathogen cultures. 

The then-emerging discipline of forensic molecular 
biology proved the occurrence of an anthrax release by 
analysis of archived samples 8 years after the incident. 41 The 


46 


Epidemiology of Biowarfare and Bioterrorism 


contributions of advanced molecular techniques to the detec¬ 
tion of BW and BT is examined in the section, Potential Impact 
of Advanced Molecular Techniques on the Epidemiology of 
Biowarfare and Bioterrorism, at the end of this chapter. 

Shigellosis—Dallas, Texas, 1996 

From October 29 through November 1, 1996, 12 
clinical laboratory workers at the St Paul Medical 
Center in Dallas developed severe acute diarrheal 
illness. 22 Shigella dysenteriae type 2 was cultured from 
the stool of eight of these cases. This strain of shigella 
is uncommon and, before this outbreak, had last been 
reported as the source of an outbreak in the United 
States in 1983. A 13th individual became ill after eating 
pastries brought home by one of the laboratory work¬ 
ers; this individual also had stool cultures positive for 
S dysenteriae type 2. Five patients were treated in and 
released from hospital emergency departments and 
four were hospitalized, but no deaths resulted. 42 

During the subsequent epidemiological investiga¬ 
tion, 43 laboratory employees who had worked during 
the first or third shifts, when the ill employees had 
worked, were interviewed. The employees stated 
that an unsigned email sent from a supervisor's 
computer invited recipients to take pastries avail¬ 
able in the laboratory break room. The supervisor 
was away from the office when the email was sent, 
and the break room could only be accessed using 
a numeric security code. The muffins and pastries 
had been commercially prepared, yet no other cases 
in the community occurred outside of the hospital 
laboratory. The ill persons reported eating a pastry 
between 7:15 am and 1:30 pm on October 29. Diar¬ 
rhea onset for the ill laboratory workers occurred 
between 9:00 pm that day and 4:00 am on Novem¬ 
ber 1. The mean incubation period until diarrhea 
onset was 25 hours and was preceded by nausea, 
abdominal discomfort, and bloating. All who ate a 
muffin or doughnut became ill (ie, 100% attack rate). 
No increased risk for illness was found from eating 
food from the break room refrigerator or drinking 
any beverage, eating in the hospital cafeteria, or at¬ 
tending social gatherings during the estimated time 
of exposure to the pathogen. 

An examination of the hospital laboratory storage 
freezer revealed tampering of reference cultures of S 
dysenteriae type 2. The stored reference cultures had 
each contained 25 porous beads that were impregnated 
with microorganisms. The S dysenteriae type 2 vial 
contained at that time only 19 beads, and laboratory 
records indicated that the vial had not been used. S 
dysenteriae type 2 was isolated in virtually pure culture 
from the muffin specimen, and the same organism was 


isolated from the stools of eight laboratory worker 
patients. Pulsed-field gel electrophoresis revealed 
that the reference culture isolates were indistinguish¬ 
able from those obtained from a contaminated muffin 
and the collected stool cultures, but differed from two 
nonoutbreak S dysenteriae type 2 isolates obtained from 
other Texas counties during that time. 

Case Review of 1996 Shigellosis Food Poisonings 

Biological Agents: S dysenteriae type 2, gram-negative 
bacillus 

Potential Epidemiological Clues: 3, 4, 11 

Review: There was a strong epidemiological link among 
the ill persons, the cultured muffin, and the laboratory’s stock 
culture of S dysenteriae type 2. 

The pathogen provided important clues because it was 
known to be uncommon and no research with this micro¬ 
organism had been conducted at the hospital; therefore, 
laboratory technicians were not at risk of infection through 
laboratory error. In addition, no concurrent outbreaks of S 
dysenteriae type 2 were reported nationally at the time. 

Pastry contamination during commercial production was 
unlikely. Shigella contamination by a food service worker dur¬ 
ing food preparation would have had to occur subsequent to 
baking because Shigella bacteria would not have survived 
the heat. 

When the epidemiological report was published, 42 it was 
hypothesized that someone had removed the laboratory 
culture of S dysenteriae type 2 from the freezer, cultured the 
microorganism and inoculated the pastries, and had access 
to the supervisor’s computer and the locked break room. 

On August 28, 1997, a laboratory technician who had ac¬ 
cess to the laboratory culture stocks and a history of purpose¬ 
ful use of biological agents against a boyfriend, was indicted 
on three charges of tampering with a food product, and 
accused of infecting 12 coworkers with S dysenteriae type 
2. She was subsequently sentenced to 20 years in prison. 

Lessons Learned: A match of clinical, food, and labora¬ 
tory isolates helped to prove an epidemiological link among 
them. The knowledge that only postproduction tampering 
of the baked goods could have resulted in their successful 
contamination assisted with the investigation. 

Anthrax—USA, 2001 

On October 4, 2001, an inhalational anthrax case 
was reported in a 63-year-old man in Florida. 44 Public 
health and government authorities initially misunder¬ 
stood the nature of inhalational anthrax exposure and 
assumed that he had contracted the illness by outdoor 
hunting activities. 45 Two other cases were subsequently 
identified in Florida, and a fourth case of anthrax—via 
cutaneous exposure—was identified in a female em¬ 
ployee at NBC News in New York City. 43 Investigators 
then realized that the exposures resulted from anthrax- 
containing letters placed in the mail. On October 15, 
Senate Majority Leader Tom Daschle's office received 


47 


Medical Aspects of Biological Warfare 


a letter that threatened an anthrax attack and also con¬ 
tained anthrax spores. The Hart Senate Office Building 
in Washington, DC, was subsequently closed. 46 By the 
end of the year, anthrax-laden letters placed in the 
mail had caused 22 cases of anthrax-related illness (11 
inhalational [all confirmed], and 11 cutaneous anthrax 
[seven confirmed, four suspected]) and five deaths. 
Almost all anthrax cases were among postal workers 
and those who had handled mail. 47,48 For two cases, 
it was difficult to determine exact exposure risk. A 
12th cutaneous anthrax case related to these mailings 
occurred in March 2002 in a Texas laboratory where 
anthrax samples had been processed. 49,50 

Case Review of 2001 Anthrax Mailings 

Biological Agents: B anthracis, gram-positive bacillus 

Potential Epidemiological Clues: 3, 5, 9, 11 

Review: An unprecedented national response occurred 
involving thousands of investigators from federal, state, and 
local agencies. Close collaboration was required of all agen¬ 
cies, and the CDC and FBI formed partnerships to conduct 
public health and criminal investigations. 9 

Public health surveillance to detect previously unreported 
anthrax cases and determine that no new cases were tak¬ 
ing place severely strained public health capacity. 51,52 This 
outbreak highlighted the importance of containing not only 
the disease but also public panic. 

The Laboratory Response Network, a multilevel network 
connecting local and state public health laboratories with 
national public health and military laboratories, 53 served as 
a lead resource for both identifying and ruling out a poten¬ 
tial biological attack. 54 Molecular subtyping of B anthracis 
strains played an important role in the differentiation and 
identification of B anthracis. High-resolution molecular 
subtyping determined that the anthrax mail-related iso¬ 
lates were indistinguishable and likely came from a single 
source. 55 

Postal workers and others handling mail were shown to 
be at risk from the anthrax-containing letters 56 and contami¬ 
nated postal machinery 57 ; therefore, federal and state health 
officials instituted environmental sampling, 58 cleaning, 59 
and protective measures as well as antibiotic prophylaxis. 60 
Similar protective actions were taken after discovery of the 
anthrax spore-laden envelope opened in the Senate Office 
Building. 45 It was later determined that patients frequently 
did not complete the recommended prophylaxis duration. 61 

As a direct result of the anthrax mailings, on January 31, 
2002, the federal government made $1.1 billion available 
to the states for BT preparedness. 62 Disease detection and 
notification efforts, a cornerstone of BT preparedness, have 
changed dramatically since the incident. Continuing efforts to 
strengthen the public health workforce should help to better 
detect, respond, and manage a future BT crisis. 63 

Lessons Learned: An enhanced index of suspicion is 
necessary for unusual manifestations of BT diseases. Health¬ 
care providers can learn to heighten their index of suspicion 
and diagnosis early if information is available and they are 
aware of a disease in a community. 


Fine particles of a biological agent can become airborne, 
thereby contaminating areas and placing persons at risk 
without direct exposure to the contaminated vehicle. An 
exposure can occur anywhere along the path of the con¬ 
taminant, and increased medical surveillance and possibly 
prophylaxis should be instituted for anyone with potential 
pathogen exposure. 

Risk communication and key messages are important to 
contain potential public unrest. 

Ricin—South Carolina and Washington, DC, 
2003-2004 

After a terrorist plot to use ricin in England in Janu¬ 
ary 2003, 64 this plant-based toxin (a ribosome-inacti¬ 
vating protein) was found in a South Carolina postal 
facility in October 2003. 65 Ricin was also discovered in 
the office of Senator Bill Frist at the Dirksen Senate Of¬ 
fice Building in Washington, DC, on February 3,2004. 66 

On October 15,2003, an envelope containing a note 
threatening to poison water supplies with ricin and a 
sealed container were processed at a mail-processing 
plant and distribution facility in Greenville, South 
Carolina. Laboratory testing at the CDC on October 
21 confirmed the presence of ricin in the container. 
State health authorities interviewed all postal work¬ 
ers at the facility, and statewide surveillance for 
illness consistent with ricin exposure was initiated. 
The postal facility was closed on October 22, and 
epidemiological and environmental investigations 
were conducted. Hospital emergency departments, 
clinicians, health departments, and the postal facility 
were asked to report any cases consistent with ricin 
exposure. State poison control center and intensive 
care unit charts at seven hospitals near the postal 
facility were reviewed daily. A medical toxicologist 
and epidemiologists interviewed all 36 workers at 
the postal facility to determine whether any were ill, 
and no postal employees had illness indicating ricin 
exposure. CDC also conducted environmental test¬ 
ing at the postal facility; all tests were subsequently 
found negative for ricin. 65 

In 2013 ricin poisoning again became a newswor¬ 
thy event when ricin-laced letters were sent to Presi¬ 
dent Barack Obama, New York City Mayor Michael 
Bloomberg, and a gun control lobbyist in Washington, 
DC. A Texas woman. Shannon Guess Richardson, was 
arrested and charged in this case, after her confession 
that she had mailed the letters, and left incriminat¬ 
ing evidence that her husband had committed this 
biocrime. 67 

Case Review of 2003-2004 Ricin Events 

Biological Agents: Ricin communis toxin 

Potential Epidemiological Clues: 3, 11 


48 


Epidemiology of Biowarfare and Bioterrorism 


Review: Ricin is a potent cytotoxin derived from the beans 
of the castor plant (Ft communis). Ricin will likely continue 
to be a threat agent because castor beans are grown and 
used commercially worldwide, and the toxin can be readily 
extracted. 

Ricin is considered to be a more rapidly acting toxin when 
it is ingested or inhaled than when injected. Treatment for 
ricin toxicity is supportive care because no antidote exists, 
and the toxin cannot be removed by dialysis. 

Difficulties inherent in responding to a threat of ricin use 
include the lack of a detection method for locating ricin in 
clinical samples. A mild ricin poisoning may resemble gas¬ 
troenteritis or respiratory illness. Ingestion of higher ricin 
doses leads to severe gastrointestinal symptoms followed 
by vascular collapse and death; inhalation of a small particle 
aerosol may produce severe respiratory symptoms followed 
by acute hypoxic respiratory failure. 68 

Lessons Learned: Any ricin threat should be investigated. 
As no cases resulted from the above exposures, it is likely 
that the material used in these incidents was not processed, 
purified, or dispersed in a manner that would cause human 
illness. 

Biological agents that are readily available in nature 
remain a threat. 

Accidental Release of Biological Agents 

The following case studies document the events 
that transpired after what is understood to be the ac¬ 
cidental release of BW agents, B anthracis 16 and Variola 
major, 69 in the Soviet Union during the 1970s. The 
former Soviet Union had a massive state-sponsored 
biological weapons program, as documented by its 
former deputy director Ken Alibek in his book. Biohaz¬ 
ard. 70 This account provides frightening emphasis on 
the dangers to innocent populations from purposeful 
biological weapon development. 

Anthrax—Sverdlovsk, Soviet Union, 1979 

In April and May 1979, the largest documented 
outbreak of human inhalational anthrax occurred 
in Sverdlovsk in the Soviet Union (now Ekaterin¬ 
burg, Russia), with at least 77 cases of disease and 
66 deaths. Soviet authorities initially reported the 
occurrence of a gastrointestinal anthrax outbreak. 
Gastrointestinal anthrax is an uncharacteristic clini¬ 
cal manifestation from ingesting B anthracis spores, 
although it occasionally occurs in the republics of 
the former Soviet Union. 16,71 When case history and 
autopsy results were reexamined by a joint team 
of Soviet and Western physicians and scientists, it 
became apparent that the Sverdlovsk outbreak and 
subsequent deaths had been caused by inhalational 
anthrax. 16 The geographic distribution of human cases 
coupled with the location of animal cases indicated 


that all anthrax disease occurred within a very nar¬ 
row geographic zone (4 km for the humans, 40 km 
for the animals) from a point of origin in Sverdlovsk. 
Historical meteorological data, when combined with 
this case distribution, demonstrated a point of origin 
at a military microbiological facility. Compound 19. 16 
These data also indicated that the most likely day on 
which this event occurred was April 2, 1979. 16 

Public health authorities established an emergency 
commission that directed public health response 
measures on April 10,1979, which did not include the 
Soviet military. A triage response was established at 
Sverdlovsk city hospital by April 12. Separate areas 
were designated for screening suspected cases and for 
treating nonsystemic cutaneous anthrax cases and for 
intensive care and autopsy. Anthrax illness was not 
believed to be be transmitted from person-to-person. 
Those who had died were placed in coffins contain¬ 
ing chlorinated lime and buried in a separate part 
of the city cemetery. Hospital and factory workers 
were recruited into teams that visited homes of both 
suspected and confirmed cases throughout the city 
to conduct medical interviews, dispense tetracycline 
as a prophylactic antibiotic, disinfect kitchens and 
patient sickrooms, and collect meat and environ¬ 
mental samples for microbiological testing. Local 
fire brigades washed trees and building exteriors 
in the section of the city where most cases were lo¬ 
cated. Some of the control measures that authorities 
enacted likely had little value. Stray dogs were shot, 
and some unpaved streets were paved. Newspaper 
articles were published, and posters were displayed 
that warned residents of the anthrax risk from eating 
uninspected meat or having contact with sick animals. 
Meat shipments entering the city were examined, and 
uninspected meat was embargoed and burned. In 
mid-April a voluntary anthrax vaccination program 
for healthy individuals aged 18 to 55 years was be¬ 
gun in the part of the city where most of the infected 
persons lived. Of the 59,000 people eligible to receive 
anthrax vaccine, about 80% received at least a single 
dose of the vaccine. 16,72 

Case Review of 1979 Sverdlovsk Anthrax Release 

Biological Agents: B anthracis, gram-positive bacillus 

Potential Epidemiological Clues: 1,2, 3, 4, 7, 9, 10 

Review: In the absence of confirmatory information of 
an aerosol anthrax release, the public health response was 
spectacular. Research has estimated that approximately 14% 
more deaths would have occurred in Sverdlovsk in the absence 
of the public health intervention that included distribution of 
antibiotics and vaccination. 72 

The Soviet military’s secrecy hid many facts that would 
have helped physicians to diagnose and treat inhalational 
anthrax exposure. It is possible that many more individuals 


49 


Medical Aspects of Biological Warfare 


than existing medical records indicate may have become ill 
and recovered, or died. 73 Ambulance personnel often made 
an initial case diagnosis of pneumonia. 74 

Government authorities confiscated patient records and 
autopsy reports from the hospital. Some of these records 
could have provided invaluable inhalational anthrax medical 
intervention information from those patients that survived. 
Along with the absence of an epidemiological investigation 
at Sverdlovsk, this was a stunning loss of vital information 
for BW defense purposes. 75 

Former Soviet physicians released important information 
about anthrax prophylaxis and treatment, some of who took 
tissue samples and records home at their own considerable 
personal risk. This information indicated that the incubation 
period for inhalational anthrax may be as long as 2 months 
and that an antibiotic course of 5 days likely prolonged the 
incubation period for illness. 75 

Molecular analysis of tissue samples collected from 11 
victims, and retained by Sverdlovsk physicians, indicate that 
these cases had been exposed to a number of different B 
anthracis strains. 76 

Lessons Learned: Retrospective pathology findings from 
victims, weather patterns, and geographic mapping can help 
to determine the outbreak source and also whether it spread. 

Public health personnel in Sverdlovsk instituted effective 
preventive measures before they knew exactly what the 
exposure was or the cause of the illnesses, and they used in¬ 
formation from cases to determine possible exposure routes. 

Once the disease agent was determined, prophylactic 
antibiotics and vaccination and protective environmental 
measures could be provided. 

Studies of Natural Outbreaks for Potential 
Bioweapon Use 

Although the following accounts are examples 
of naturally occurring outbreaks, some components 
raise suspicion that they were intentionally caused. 
Subsequent to the 1999 WNV outbreak in New York 
City, suggestions were made that Iraqi operatives 
could have covertly released a biological weapon. 
These allegations by Richard Preston in the New 
Yorker magazine were based on documentation 
showing that CDC had provided Iraq with various 
biological agents from 1984 through 1993, including 
Y pestis, dengue, and WNV, 77,78 together with the fact 
that the Iraqi government was known to have had a 
covert biological weapons program. 79 Although never 
shown to be anything other than an imported disease 
outbreak occurring in an opportunistic manner, this 
claim received a lot of political attention. Similar 
allegations of the covert use of a biological weapon 
could have been made with other outbreaks, includ¬ 
ing the 2000 Martha's Vineyard (Massachusetts) 
tularemia outbreak, and they were made during the 
1999 through 2000 Kosovo tularemia outbreak, which 
occurred during wartime. 


West Nile Virus, New York, New York, 1999 

An outbreak of an unusual encephalitis was first rec¬ 
ognized in New York City in late August 1999. On Au¬ 
gust 23 an infectious disease physician from a Queens 
hospital contacted the New York City Department of 
Hygiene and Mental Health to report two patients with 
encephalitis. The health department then conducted 
a citywide investigation that revealed a cluster of six 
patients with encephalitis in which five had profound 
muscle weakness and four required respiratory sup¬ 
port. CDC's initial clinical tests of these patients' cere¬ 
brospinal fluid and serum samples indicated positive 
results for Saint Louis encephalitis on September 3. 
More cases of encephalitis in New York City ensued, 
and because eight of the earliest cases were residents 
of a 2-square-mile area in Queens, aerial and ground 
applications of mosquito pesticides began in northern 
Queens and South Bronx on September 3. 80 

Active encephalitis surveillance began in New York 
City on August 30 and in nearby Nassau and Westches¬ 
ter counties on September 3. A clinical case was defined 
as a presumptive diagnosis of viral encephalitis with 
or without muscle weakness or acute flaccid paralysis, 
Guillain-Barre syndrome, aseptic meningitis, or presence 
of the clinical syndrome as identified in earlier cases. 80 
Before and during this outbreak, an observed increase 
in bird deaths (especially crows) was noted in New 
York City. 14 The USD A National Veterinary Services 
Laboratory in Ames, Iowa, analyzed tissue specimens 
taken from dead birds in the Bronx Zoo for common 
avian pathogens and equine encephalitis. When these 
test results were negative, the samples were forwarded 
to CDC, which revealed on September 23 that the virus 
was similar to WNV in genetic composition. 81 At that time 
WNV had never been isolated in the western hemisphere. 

Concurrently, brain tissue from three New York City 
encephalitis case deaths tested positive for WNV at the 
University of California at Irvine. As of September 28, 
17 confirmed and 20 probable cases had occurred in 
New York City and Nassau and Westchester counties, 
resulting in four deaths. Onset dates were from August 
5 through September 16. The median age of the patients 
was 71 years (range 15-87 years). By October 5 the 
number of laboratory-positive cases had increased to 50 
(27 confirmed and 23 probable). Emergency telephone 
hotlines were established in New York City on Septem¬ 
ber 3, and 130,000 calls were received by September 
28. About 300,000 cans of N,N-diethylmetatoluamide 
(DEET)-based mosquito repellant were distributed 
citywide through local firehouses, and 750,000 pub¬ 
lic health leaflets were distributed with information 
on protection from mosquito bites. Radio, television, 
and the Internet provided public health messages. 80 


50 


Epidemiology of Biowarfare and Bioterrorism 


A seroprevalence survey later determined that ap¬ 
proximately 100 asymptomatic infections and 30 WNV 
fever cases occurred for each WNV encephalitis case 
previously identified in the New York City area. 82 

Case Review of 1999 West Nile Virus Cases 

Biological Agents: WNV, a flavivirus 

Potential Epidemiological Clues: 1,2, 3, 7 

Review: Although some suggestions were made that this 
could have been a bioterrorist attack, the appearance of WNV 
in New York City in 1999 and its subsequent spread to the 
rest of the United States was most likely a natural occurrence. 

Saint Louis encephalitis and WNV are antigenically re¬ 
lated, and cross reactions can occur with some serologic 
testing. 80 Limitations of serologic testing underscore the 
importance of isolation and identification of virus. 80 

Within its normal geographic area of distribution in Africa, 
West Asia, and the Middle East, birds do not normally show 
symptoms when infected with WNV. 83 WNV from this part of 
the world occasionally causes epidemics in Europe that may 
be initiated by migrant birds. 84,85 An epizootic that results in the 
deaths of large numbers of crows may be a clue that either 
a new population is susceptible to the virus or a new, more 
virulent strain of a virus has been introduced. 80 

WNV is transmitted primarily by Culex mosquitoes, 86 
which contributed to its spread in the United States after the 
1999 outbreak. 87 

Genetic testing revealed that the virus was 99% identical 
to a virus isolated in 1999 from a goose in Israel. 88 Potential 
routes for WNV introduction include importation of WNV- 
infected birds, mosquitoes, or ill persons. The New York City 
area where WNV was prevalent includes two large interna¬ 
tional airports. 89 

Before this outbreak, death was rarely associated with 
WNV infection. 90 In patients with WNV encephalitis, computer- 
assisted tomography often revealed preexisting lesions and 
chronic changes in brain tissue, 91 perhaps suggestive of the 
potential for a greater susceptibility to deleterious outcome 
in elderly persons. 

Lessons Learned: This outbreak emphasizes the impor¬ 
tant relationship among veterinarians, physicians, and public 
health authorities in disease surveillance, and the importance 
of considering uncommon pathogens. 90 

The incident is an example of a typical zoonotic disease 
epidemic pattern—a natural epidemic occurred first among 
birds, followed by disease in humans. 

The origin of outbreaks fitting some of the clues for a 
biological attack (a new disease for a geographic region) can¬ 
not be immediately determined without further investigation. 
Emerging diseases, whether new for a particular geographic 
area, like WNV, or a totally new disease (eg, severe acute 
respiratory syndrome or Middle East Respiratory Syndrome 
coronavirus), are not uncommon. 

Tularemia, Martha's Vineyard, Massachusetts, 2000 

During the summer of 2000, an outbreak of primary 
pneumonic tularemia occurred on Martha's Vine¬ 
yard, Massachusetts. 92 In July five cases of primary 


pneumonic tularemia were reported, with onset dates 
between May 30 and June 22. The Massachusetts De¬ 
partment of Public Health and CDC initiated active 
surveillance, and 15 confirmed tularemia cases were 
subsequently identified. A confirmed case was de¬ 
fined as occurring in a visitor or resident to Martha's 
Vineyard who had symptoms suggesting primary 
pneumonic tularemia; was ill between May 15 and 
October 31,2000; and had test results showing a serum 
titer of anti-F tularensis antibody of at least 1:128 on an 
agglutination assay. Of these cases, 11 had the pneu¬ 
monic form of the disease, two had ulceroglandular 
disease, and two had fever and malaise. Fourteen of the 
patients were male, and the median age was 43 years 
(range 13-59). One 43-year-old man died of primary 
pneumonic tularemia. 92 

Control subjects for a case-control study were ob¬ 
tained by random-digit dialing to Martha's Vineyard 
residents, enrolling 100 control subjects at least 18 
years old that had spent at least 15 days on the island 
between May 15 and their September interviews. 
Both ill persons and control subjects were questioned 
about occupation, landscaping activities, animal and 
arthropod exposures, recreational and outdoor activi¬ 
ties, and general health history and status. Information 
was obtained about exposure to risk factors between 
May 15 and the interview, and for 2 weeks before ill¬ 
ness for ill persons and 2 weeks before interview for 
control subjects. 92 

The suspected site of exposure for each patient was 
visited. Activities that may have led to exposure (eg, 
lawn mowing and "weed whacking") were repro¬ 
duced, and environmental and personal air samples 
were taken. Samples from soil, water, grass, wild 
mammals, and dogs were also taken. Epidemiological 
analysis revealed that in the 2 weeks before illness, 
using a lawn mower or brush cutter was significantly 
associated with illness. Of all the environmental and 
animal tissue samples taken, only two were positive for 
F tularensis: (1) a striped skunk and (2) a Norway rat. 92 

Case Review of 2000 Martha’s Vineyard Tularemia 
Outbreak 

Biological Agents: F tularensis, a gram-negative bacillus 

Potential Epidemiological Clues: 1,2, 3, 9 

Review: Caused by a gram-negative bacillus F tularen¬ 
sis, tularemia is a rare infection in the United States. Be¬ 
tween 2001 and 2010, a median number of 126.5 cases per 
year (range: 90-154 cases per year) was reported. 91 More 
than half of all cases reported during these 11 years came 
from Arkansas, Missouri, South Dakota, and Oklahoma, 
and most cases were acquired from tick bites or contact 
with infected rabbits. Higher incidences of the disease 
have been noted in persons ages 5 to 9 and older than 75, 
and incidence was greatest among Native Americans and 
Alaskan natives. 93 


51 


Medical Aspects of Biological Warfare 


The only other previously reported pneumonic tularemia 
outbreak in the United States had occurred on Martha’s 
Vineyard during the summer of 1978. 94 During a single week 
(July 30-August 6) seven persons stayed in a vacation cot¬ 
tage. By August 12, six of these had a fever, headache, and 
myalgia; and the seventh had a low-grade fever by August 
19. A search for additional cases on the island uncovered six 
other tularemia cases, five of which were pneumonic, and 
one was ulceroglandular. No source for the disease exposure 
was discovered, although two rabbits later found dead were 
culture-positive for F tularensis. 

Tularemia had been reported sporadically since rabbits 
had been introduced to Martha’s Vineyard in the 1930s, 93 and 
pneumonic tularemia was first reported in Massachusetts in 
1947. 95 Classic research on human tularemia rates showed 
that very high rabbit populations increase the tularemia 
hazard. 96 

Hospital clinicians on Martha’s Vineyard initially detected 
this outbreak and recognized tularemia caused pneumonic 
summer illness, 97 in part based on the experiences with the 
previous outbreak. 94 

Feldman et al proposed in this outbreak F tularensis was 
shed in animal excreta, persisted in the environment, and 
infected persons after mechanical aerosolization and inhala¬ 
tion. This is a likely exposure scenario, given the principal 
form of primary pneumonic tularemia seen in these cases 
and strong epidemiological association with grass cutting. 92 

A seroprevalence survey conducted in 2001 in Martha’s 
Vineyard demonstrated that landscapers were more likely to 
have an antibody titer to F tularensis than nonlandscapers, 
revealing an occupational risk for tularemia. 92 

Lessons Learned: Naturally occurring disease can 
present in the pneumonic form. However, if tularemia were 
used as a biological weapon, an aerosolized release would 
probably result in multiple simultaneous cases presenting 
with the pneumonic form of the disease. 97 

There may also be disease transmission mechanisms 
(in this example, grass cutting) that are unknown or poorly 
understood. 98 

Tularemia, Kosovo, 1999-2000 

After a decade of political crises and warfare, a 
large outbreak of tularemia occurred in Kosovo from 
1999 through 2000. Tularemia had not been reported 
in Kosovo since 1974." By April 2000, 250 suspected 
cases had been identified and spread nationwide, but 
most cases existed in the western area where ethnic 
Albanians resided. 100 

Unusual outbreaks of zoonoses or vectorborne 
disease may readily occur in war-torn or crisis- 
afflicted regions that have previously been free of 
these diseases. Historically, outbreaks of typhus, 
plague, cholera, dysentery, typhoid fever, and small¬ 
pox have long been observed in war-torn regions. 101 
Among the earliest historic examples is the plague 
of Athens that arose during the second year of the 
Peloponnesian War, as described by Thucydides. 102 


Speculation may arise that these epidemics were 
purposefully caused. Many biological agents are 
zoonotic pathogens," including tularemia, a catego¬ 
ry A BW pathogen. Purposeful use of this pathogen 
merits consideration when such an outbreak occurs 
with a pathogen having the potential to be a biologi¬ 
cal weapon. 103 

Remarks made by the head epidemiologist at the 
Kosovo Institute of Public Health about unidentifiable 
ampoules and white powders discovered near various 
wells could not be verified and added to a perception 
of use of a biological weapon by Serbian forces." F tu¬ 
larensis biovar tularensis (type A) is highly pathogenic 
for humans. It is found mostly in North America and 
has been developed for use as a biological weapon. 
Disease progression often follows an acute and severe 
course, with prominent pneumonitis. F tularensis bi¬ 
ovar holarctica (type B) is less pathogenic and is found 
throughout the northern hemisphere. 104 To further 
complicate matters, a 1998 report documented that 
type A tularemia had been introduced into arthro¬ 
pod populations in the nearby Slovak Republic. 105 
The United Nations mission in Kosovo requested 
that the World Health Organization assist Kosovar 
health authorities in an epidemiological investiga¬ 
tion of the tularemia outbreak. Teams of international 
and Kosovar public health personnel collaborated in 
epidemiological, environmental, and microbiological 
field and laboratory investigations. 106 

Tularemia cases were discovered by both prospec¬ 
tive surveillance and retrospective hospital review of 
a pharyngitis and cervical lymphadenitis syndrome. 
Ill persons were clinically examined and interviewed, 
blood samples were taken from suspected cases, and 
antibiotics were prescribed as appropriate. Rural vil¬ 
lagers reported an increase in mice and rats in the 
summer of 1999. A causal association was suspected 
between the increased population density of rodents 
and human tularemia cases. Tularemia is naturally 
transmitted to humans via small lesions in the skin of 
persons handling diseased rabbits, ingestion of con¬ 
taminated water or food, bites of infectious arthropods, 
or inhalation of infective dusts." 

A matched case-control study was conducted with 
paired households in villages in regions with the 
greatest number of reported cases. Case households 
had one or more family members with a laboratory- 
confirmed case of tularemia as of November 1, 1999. 
Control households were the two households closest 
to a suspected case household, having no individuals 
with the disease, and the person who prepared the 
family's food was serologically negative for tularemia. 
Blood specimens were also drawn from all suspected 
cases. Questionnaires were completed on household 


52 


Epidemiology of Biowarfare and Bioterrorism 


food consumption, water supply, presence of rodents, 
and condition of wells and food preparation and 
storage areas. The study period began a month before 
symptom onset of the first case in the suspected case 
household. Well water sampling and rodent collection 
and analysis were performed. 

By June 30, 2000, more than 900 suspected tulare¬ 
mia cases had been discovered. From these, 327 were 
confirmed as serologically positive. The earliest onset 
of reported symptoms in the confirmed cases was 
October 1999, with an epidemic peak in January 2000. 
Confirmed cases were identified in 21 of 29 Kosovo 
municipalities. Cases were equally distributed by 
sex, and all age groups were equally affected. Case 
households were more likely to have nonrodent-proof 
water sources, and members in these households were 
less likely to have eaten fresh vegetables. Risk factors 
for case households included rodent feces in food 
preparation and storage areas and large numbers of 
field mice observed outside the house. Of the field 
samples collected, positive antigen for F tularensis 
was detected in striped field mouse and black rat 
fecal specimens. 

Case Review of 2000 Kosovo Tularemia Outbreak 

Biological Agents: F tularensis, a gram-negative bacillus 

Potential Epidemiological Clues: 1, 3, 5, 9 

Review: Clinical and serologic evidence indicate that a 
tularemia outbreak occurred in Kosovo from October 1999 
through May 2000. The case-control study indicated that 
transmission of tularemia was foodborne based on the 
associations of illness and large numbers of rodents in 
the household environment, rodent contamination of food 
storage and preparation areas, and consumption of certain 
uncooked foods. Unprotected water that was not boiled likely 
contributed to the outbreak. 

Initial field investigations rapidly demonstrated that 
a widespread natural event was occurring and likely re¬ 
sulted from the unusual environmental conditions existing 
in war-torn Kosovo. The principal populations affected 
by the tularemia outbreak were ethnic Albanians in rural 
farming villages with limited economic resources. These 
people had fled during North Atlantic Treaty Organiza¬ 
tion bombing and Serbian reprisals during the spring 
of 1999. Refugees discovered bombed and ransacked 
homes, unprotected food storage areas, unharvested 
crops, damaged wells, and a rodent population explosion 
when they returned to their cottages. Both ignorance of 
infection and lack of hygienic measures contributed to a 
foodborne infection in the population." 

F tularensis can survive for prolonged periods in cold, 
moist conditions. 

A natural decrease in rodent population resulting from the 
cold winter, food shortages, and the disease itself likely all 
helped to end the zoonoses." 

Although tularemia was not recognized endemically 
or enzootically in Kosovo before the 1999 through 2000 


outbreak, it became well established in a host reservoir. A 
second outbreak occurred there in 2003, causing more than 
300 cases of oropharyngeal tularemia. 107 

Historically, war in Europe caused tularemia outbreaks. 
During World War II, an outbreak of more than 100,000 cases 
of tularemia occurred in the Soviet Union, 108 and outbreaks 
with hundreds of cases following the war occurred in Austria 
and France. 107 

Lessons Learned: War provides a fertile ground for 
the reemergence of diseases and potential cover for BW 
agent use that is plausible and may go unrecognized as a 
BW event. An extensive epidemiological investigation must 
be conducted to conclude or disprove that a BW event has 
occurred. 

Q Fever, Iraq 2005 

Q fever is a zoonotic disease caused by C burnetii, a 
bacteria found worldwide. Human cases occur from 
inhalation of aerosols or windbome dust contaminated 
with C burnetii from birth products, milk, urine, and 
feces of infected animals —most frequently cattle, 
camel, goats, and sheep. Infections can also occur 
from ingesting raw milk or eggs as well as tick bites or 
human-to-human transmission. 109 Due to the bacteria's 
ability to survive in harsh environmental climates and 
its high infectivity, there is concern of its use as a bio¬ 
logical weapon. The United States developed Q fever 
as a biological weapon before ratifying the Biological 
Weapons Convention. The CDC classifies C burnetii as 
a Category B agent. 

From June 18 to July 10, 2005, 22 of 38 Marines 
(58%) from a single platoon in A1 Asad, Iraq, expe¬ 
rienced a febrile illness. 110 All patients had a rapid 
onset of fever and chills, and the majority had head¬ 
ache, respiratory, and gastrointestinal symptoms. 
The patients were diagnosed with upper respiratory 
infection or atypical pneumonia because there was no 
diagnostic capability. Subsequent testing was nega¬ 
tive for multiple respiratory pathogens. Follow-up 
serologic testing 6 weeks later on 9 of the affected 
patients revealed positive Q fever immunoglobulin 
for all 9, with 10 unaffected persons from the same 
unit negative for antibody. 110 

After confirmation of Q fever, the researchers 
distributed follow-up questionnaires to the company 
that included the affected platoon. They found an as¬ 
sociation between infection and exposure to ticks and a 
trend toward association with exposure to camels and 
the birth of both sheep and dogs. Although the authors 
did not have a sufficient sample size to confirm all risk 
factors, they hypothesize that this particular platoon 
may have sought shelter in an area that was heavily 
infected secondary to recent animal inhabitation and 
birthing or ticks. 110 


53 


Medical Aspects of Biological Warfare 


Before this outbreak, Q fever cases had been re¬ 
ported in US service members deployed to Iraq. An 
evaluation of 62 cases of pneumonia in 2003 found 
eight had seroconverted with Q fever antibody, 111 
and an additional four diagnosed cases in 2003 and 
2004 were reported. 112,113 Three cases of Q fever oc¬ 
curred in US forces in Iraq during the first Persian 
Gulf War (1990-1991). 114 Since the 2005 outbreak in 
the Marines, more cases have been reported, and two 
serosurveys have been performed. One serosurvey 
revealed 10% of 909 military personnel hospitalized 
during deployment in 2003-2004 with symptoms 
compatible with Q fever seroconverted, 115 and another 
serosurvey studying the same company affected in 
the outbreak in 2005 found seroconversion in 7.2% of 
279 tested. 116 The British military has also published 
occurrences of Q fever in deployed forces, including 
26% of "Helmand Fever" cases caused by Q fever in 
Afghanistan. 117 

Surveillance of deployed military working dogs 
in Iraq revealed no seroconversions in 2007-2008, 
compared to a 5.5% seroconversion in feral dogs. 118 
This lack of infection is probably secondary to tick 
control and doxycycline prophylaxis for the military 
working dogs. 


It is useful for public health authorities to de¬ 
termine whether an infectious disease outbreak is 
intentional. Grunow and Finke developed an epide¬ 
miological assessment tool to rule out biological agent 
use during infectious disease outbreaks. 98 This assess¬ 
ment tool's relevance was demonstrated by analysis 
of the 1999-2000 Kosovo tularemia outbreak. 99 In their 
evaluation scheme, each assessment criterion can be 
given a varying number of points dependent on its 
presence and characteristics. There are two types of 
evaluation criteria: (1) nonconclusive and (2) con¬ 
clusive. The most significant nonconclusive criteria 
include a biological threat or risk, special aspects of 
a biological agent, a high concentration of biological 
agent in the environment, and epidemic characteris¬ 
tics. Conclusive criteria include the unquestionable 
identification of the cause of illness as a BW agent 
(eg, demonstrating modifications that make the agent 
different from its naturally occurring equivalent, such 
as stabilizers or physical modifications) or proof of 
the release of such an agent as a biological weapon. 
With conclusive criteria, additional confirmatory 
information is unnecessary. 99 

According to Grunow and Finke's nonconclusive 
criteria, a biological risk may be considered if a political 
or terrorist environment exists from which a biological 
attack could originate: 


Case Review of 2005 Q fever cases 

Biological Agent: C burnetii, gram-negative, facultative, 
intracellular coccobacillus 

Potential Epidemiological Clues: 1,4 

Review: An attack rate of 58% occurred in one platoon. 
Although the research team was unable to determine exact 
movements of the platoon, it is likely they had an exposure 
different from the other platoons. 

A relatively short epidemic curve, especially with a long 
and variable incubation period for the pathogen, suggests a 
point source. This outbreak probably resulted from an isolated 
exposure over a short time period. 

It is a disease of relatively high severity, had an unknown 
cause at time of outbreak, and can raise concern about 
potential intentional cause. 

Q fever is considered a potential bioweapon and a cause 
for concern. 

Lessons Learned: All medical personnel should know 
what diseases are endemic in the area and previous history 
in deployed forces. 

Cases should be reported immediately to allow dissemina¬ 
tion of recommended diagnostics and treatment. In this case, 
the Armed Forces Infectious Disease Society published a set 
of practice guidelines for diagnosis and management of Q 
fever to assist deployed medical personnel. 119 

Investigate outbreaks of disease, even after resolution. 
Knowledge obtained will assist in preventing, recognizing, 
and rapidly treating future cases. 

SSESSMENT TOOL 

• Biorisk. Are BW agents available, with the 
means for distribution, and the will to use 
them? Or can an outbreak be explained by 
natural biological hazards, or the changes 
incurred by military conflict? 

• Biothreat. Does a biological threat exist by 
virtue of a group having a BW agent and 
credibly threatening to use it? 

• Special aspects. Is there plausible evidence of 
purposeful manipulation of a pathogen? 

• Geographic distribution. Is the disease's 
geographic distribution likely given its locale? 
With the advent of a nonendemic pathogen, 
a thorough evaluation should include epide¬ 
miological, epizootic, ecological, microbio¬ 
logical, and forensic analysis. 

• Environmental concentration. Is there a high 
environmental concentration of the pathogen? 

• Epidemic intensity. Is the course of illness 
relative to disease intensity and spread in the 
population expected in naturally occurring 
illness? 

• Transmission mode. Was the path of disease 
transmission considered naturally occurring? 
The appearance of a naturally occurring epi¬ 
demic in itself does not rule out the purposeful 
use of a BW agent. 


54 


Epidemiology of Biowarfare and Bioterrorism 


• Time. Was the seasonal timing of the epidemic 
unusual? 

• Unusually rapid spread. Was the spread of 
the epidemic unusually rapid? 

• Population limitation. Was the epidemic 
limited to a specific (target) population? If 
certain persons were given prior warning of a 
BW attack, then they may protect themselves, 
as compared to naive target populations. 

• Clinical. Were the clinical manifestations of 
the disease to be expected? 


The Grunow-Finke epidemiological assessment 
procedure (Table 2-1) was used to evaluate the case 
studies presented in this chapter. To use the assess¬ 
ment tool uniformly for all the events described in this 
chapter, some artificial constraints were placed on the 
analysis. For this exercise, only nonconclusive criteria 
were used because the use of conclusive criteria may 
have excluded many of the case studies with a retro¬ 
spective assessment. During an outbreak investigation, 
however, epidemiological investigators would also 
initially use the nonconclusive evaluation criteria. With 


TABLE 2-1 


EPIDEMIOLOGICAL ASSESSMENT AND EVALUATION OF CASE STUDY OUTBREAKS 






1915 







Assessment 


Maximum 

Anthrax 

1971 

1979 

1984 

1995 

1996 


(possible 

Weighting 

No. of 

Eastern 

Smallpox 

Anthrax 

Salmonella 

Anthrax 

Shigella 

Nonconclusive Criteria 

points) 

Factor 

Points 

USA 

Aralsk 

Sverdlovsk 

Oregon 

Tokyo 

Texas 

Biorisk 

0-3 

2 

6 

4 

4 

4 

6 

6 

0 

Biothreat 

0-3 

3 

9 

0 

0 

0 

0 

6 

0 

Special aspects 

0-3 

3 

9 

6 

6 

6 

3 

0 

6 

Geographic distribution 
Environmental 

0-3 

1 

3 

3 

3 

3 

2 

3 

2 

concentration 

0-3 

2 

6 

6 

0 

6 

0 

6 

0 

Epidemic intensity 

0-3 

1 

3 

3 

3 

3 

3 

0 

3 

Transmission mode 

0-3 

2 

6 

6 

2 

6 

4 

0 

0 

Time 

0-3 

1 

3 

3 

3 

3 

1 

0 

1 

Unusually rapid spread 

0-3 

1 

3 

3 

1 

3 

3 

0 

3 

Population limitation 

0-3 

1 

3 

1 

0 

1 

0 

0 

3 

Clinical 

0-3 

1 

3 

3 

3 

3 

0 

0 

1 

Score 



54 

38 

25 

38 

22 

21 

19 





2000 








1999 

1999 

Tularemia 

2001 

2003 






WNV 

Tularemia 

Martha's 

Anthrax 

Ricin 

2005 



Nonconclusive Criteria 


NYC 

Kosovo 

Vineyard 

USA 

USA 

Q Fever 



Biorisk 


6 

2 

0 

6 

6 

2 



Biothreat 


6 

3 

0 

6 

9 

6 



Special aspects 


0 

0 

0 

9 

0 

0 



Geographic distribution 
Environmental 


3 

3 

3 

3 

3 

0 



concentration 


4 

4 

4 

6 

6 

0 



Epidemic intensity 


3 

3 

3 

3 

0 

1 



Transmission mode 


2 

2 

6 

6 

0 

0 



Time 


1 

0 

3 

3 

0 

0 



Unusually rapid spread 


3 

1 

3 

3 

0 

1 



Population limitation 


0 

0 

2 

3 

0 

3 



Clinical 


1 

1 

3 

3 

0 

0 



Score 


29 

19 

27 

51 

24 

13 




NYC: New York City 
USA: United States of America 
WNV: West Nile Virus 


55 









Medical Aspects of Biological Warfare 


the exception of the 2001 anthrax and 2003 ricin events, 
none of the outbreaks described had been positively 
identified as having been caused by a biological agent 
until sometime after the events had occurred. 

Grunow and Finke provide the following cut-off 
scores for nonconclusive criteria with respect to the 
likelihood of biological weapon use: 

• unlikely (0%-33% confidence): 0 to 17 points; 

• doubtful (18%-35% confidence): 18 to 35 
points; 

• likely (67%-94% confidence): 36 to 50 points; and 

• highly likely (95%-100% confidence): 51 to 54 
points. 

Based on this scoring, only the 2001 anthrax mail¬ 
ings would be considered as highly likely to have been 

IMPROVING RECOGNITION AND 

Existing disease surveillance systems may not 
be sensitive enough to detect a few cases of illness, 
unless they are legally reportable diseases that have 
confirmed laboratory diagnoses. However, even before 
confirmed diagnoses, disease reporting can be initi¬ 
ated upon patient presentation to healthcare provid¬ 
ers with initial diagnoses, laboratory testing, and the 
reason provided by the patient for the hospital visit. 
Clinicians, laboratories, hospitals, ancillary healthcare 
professionals, veterinarians, medical examiners, morti¬ 
cians, and others may be partners in reporting diseases 
to public health authorities. 

If a medical surveillance system first detects a bio¬ 
logical attack, there may already be a significant num¬ 
ber of cases, and the available time to prevent further 
illness is short or perhaps already over. The point of 
release is the earliest detection point of a biological 
event. Some disease exposures could be prevented 
through publicized avoidance of the area at risk, 
prophylactic medication use, or vaccination of those 
exposed, coupled with immediate disease recognition 
and patient treatment. The Department of Homeland 
Security's Bio Watch program has deployed biological 
detectors in major urban centers nationwide to detect 
trace amounts of airborne biological materials 120,121 to 
help determine the presence and geographic extent of 
a biological release to focus emergency public health 
response and consequence management. Such detec¬ 
tors could be of great utility when pre-positioned 
at large well-publicized gatherings or in cities that 
may be the greatest targets for terrorist activity. 

Although deployed sensors may detect an agent's 
release, the infinite number of venues coupled with 
limited resources to position sensors and analyze air 


caused by a BW agent. The 1915 and 1979 anthrax 
events qualify as likely to have been caused by a BW 
agent. All of the other case study scenarios are either 
doubtful or unlikely to have been caused by a B W agent. 

The authors conducted this evaluative exercise by 
consensus of opinion. Although subjective, the exercise 
underscores the challenges facing epidemiologists to 
determine whether a BT/BW event has occurred, un¬ 
less direct evidence indicates a purposeful event, or 
someone credibly claims responsibility. The basic epi¬ 
demiological principles described earlier in this chap¬ 
ter (including those needed for disease recognition) to 
determine the occurrence of an unnatural event, and 
for basic outbreak investigation, are the foundation of 
infectious disease response and control. Public health 
authorities must remain vigilant to quickly and ap¬ 
propriately respond to any infectious disease event. 

SURVEILLANCE OF BIOTERRORISM 

samples minimizes the chances that an agent release 
will be detected. In most instances, the earliest op¬ 
portunity to detect an attack will be by recognizing 
ill patients. Depending on the agent, the mode of dis¬ 
semination, and the number exposed, initial cases will 
present in different ways. If the disease is severe, such 
as is possible with category A biological agents, one 
properly diagnosed case will launch an investigation, 
as seen during the 2001 anthrax attacks. 47 

Even if the cause is initially unknown, extremely 
severe or rapidly fatal cases of illness in previously 
healthy individuals should be reported to public 
health authorities. If many people are exposed, as 
would be expected with a large aerosol release of a 
biological agent, an overwhelming number of people 
may eventually visit hospital emergency departments 
and outpatient clinics. Even with less severe disease, 
such cases should be recognized and quickly reported. 

However, in the absence of confirmed labora¬ 
tory diagnoses or high attack rates, infectious disease 
outbreaks are often not reported. If the disease is not 
rapidly fatal or cases are distributed among a variety 
of healthcare practitioners, it may not be readily ap¬ 
parent that a disease outbreak is Linder way. Therefore, 
there is a need for better awareness of the health of 
communities—a way to quickly detect shifts in poten¬ 
tially infectious diseases, whether of bioterrorist origin 
or not. This need has been recognized and has resulted 
in the proliferation of what are commonly known as 
syndromic surveillance systems. 

Syndromic surveillance has been defined as the 
ongoing, systematic collection, analysis, and interpre¬ 
tation of data that precede diagnosis and can indicate 
a potential disease outbreak earlier than when public 


56 


Epidemiology of Biowarfare and Bioterrorism 


health authorities would usually be notified. 122 The 
data used in syndromic surveillance systems are usu¬ 
ally nonspecific potential signs and symptoms of an 
illness spectrum indicating that disease may be higher 
than expected in a community. These data can be from 
new or existing sources. 123 For syndrome surveillance 
of BT, the emphasis is on timeliness, with automated 
analysis and visualization tools such as Web-based 
graphs and maps. These tools provide information 
that initiates a public health investigation as soon as 
possible. 124 

Numerous regional and national syndromic surveil¬ 
lance systems have been developed, including programs 
that rely on data collected specifically for the surveil¬ 
lance system and those that use existing medical data 
(eg, diagnostic codes, chief complaints, nurse advice 
calls, ambulance runs) and other information (eg, phar¬ 
macy sales, absenteeism, calls to poison control centers, 
Internet searches for specific symptoms or pathogens, 
participatory epidemiology where people voluntarily 
provide information to a system like Flu Near You 125 or 
even scanning Twitter feeds and other social media sites 
for the use of terms related to illness) to detect changes 
in population health. Systems that use active data col¬ 
lection can be "drop-in" (those instituted for a specific 
high-threat time) such as those performed immediately 
after September 11, 2001, 126-128 or during large gather¬ 
ings for sports (eg, the Olympics) or other events, 129 or 
they can be sustained systems for continuous surveil¬ 
lance. 69,130 Systems that require new data entry benefit 
from greater specificity in the type of syndromes and 
illnesses reported, but they require extra work and are 
difficult to maintain. Systems that use existing data 
can be less specific, especially with information taken 
from behaviors early in the disease, such as over-the- 
counter pharmacy sales, absenteeism, Internet searches, 
and social media use. However, these programs have 
the large advantage of continuous data streams that 
are not dependent on provider input or influenced by 
news reports of disease rates. Such systems (examples 
of which are described below) have become standard 
in many health departments, the military, and CDC. 

In the US Department of Defense, the Electronic 
Surveillance System for the Early Notification of 
Community-based Epidemics uses outpatient diagnos¬ 
tic International Classification of Diseases, Ninth Revision 
codes, chief complaints, radiology and laboratory tests, 
and pharmacy prescriptions to track disease groups 
in military beneficiaries. Temporal and spatial data 
are presented through a Web-based interface, and 
statistical algorithms are run to detect any aberra¬ 
tions that could indicate a disease outbreak. 131 This 
system is available for all permanent US military 
treatment facilities worldwide. Some local and state 


health departments use civilian versions of the Elec¬ 
tronic Surveillance System for the Early Notification of 
Community-based Epidemics. Other civilian systems, 
such as the North Carolina Disease Event Tracking and 
Epidemiologic Collection Tool 132 and various software 
packages made available by the Real-time Outbreak 
and Disease Surveillance Laboratory at the Depart¬ 
ment of Biomedical Informatics at the University of 
Pittsburgh, 133 and the EpiCenter application 134 also use 
syndromic information from emergency departments, 
911 calls, ambulance runs, and poison control center 
calls to monitor the health of populations. 

CDC has developed the BioSense 2.0 program using 
national data sources such as the US Department of 
Defense and Department of Veterans Affairs outpatient 
diagnostic codes, state and local emergency depart¬ 
ment visits, and laboratory test orders from com¬ 
mercial vendors to track disease patterns nationwide. 
The information is provided in a Web-based format to 
health departments. 135 Algorithms are run on the data 
and send out an alert when levels of medical visits or 
laboratory test orders exceed those expected. The in¬ 
formation is presented in temporal and spatial format, 
allowing the health department to track disease based 
on the patient's home zip code. The BioSense 2.0 goal 
is to facilitate sharing of automated detection and visu¬ 
alization algorithms and promote national standards. 

Despite the proliferation of systems, there are defi¬ 
nite limitations in the ability to detect bioterrorist at¬ 
tacks using syndromic surveillance. Some have argued 
that even if syndromic surveillance could detect an 
outbreak faster than traditional methods, the advanced 
warning may not assist with disease mitigation.' 3 The 
warning may not be early enough or effective counter¬ 
measures may not be available. In addition, although 
nonspecific data such as absenteeism and social media 
may provide some early warning, it is very difficult to 
institute preventive measures without more specific 
information. However, nonspecific data can still serve 
as an early indicator, prompting authorities to monitor 
specific data sources more carefully. 

Most importantly, because a BT attack can present 
in a variety of ways depending on the agent, population, 
method of dispersal, and environment, it is impossible 
to predict how any individual surveillance system will 
perform. It is generally agreed that most syndromic 
surveillance systems will not detect a few cases of dis¬ 
ease, but they can assist in detecting more widespread 
disease increases and assessing the population impact, 
an outbreak's spread, and the success of mitigation 
efforts. The coverage area of the surveillance system is 
crucial in determining outbreak detection sensitivity 
in any part of a community. In the future, syndromic 
surveillance will probably be based on national models 


57 


Medical Aspects of Biological Warfare 


such as BioSense 2.0 and use readily available electronic 
databases. Local health departments could then build 
on a national system using local data that can improve 
population coverage. Future disease monitoring and 
reporting systems need to be seamlessly integrated 


with other traditional disease surveillance systems. 
Ideally, these systems should also help to educate 
clinicians on the importance of maintaining a high 
index of suspicion and to promptly report unusual 
diseases or disease clusters to public health authorities. 


POTENTIAL IMPACT OF ADVANCED MOLECULAR TECHNIQUES ON THE 
EPIDEMIOLOGY OF BIOWARFARE AND BIOTERRORISM 


In addition to the use and application of syndromic 
surveillance for the detection of shifts in potentially 
infectious diseases, advances in technologies used for 
both disease diagnosis and surveillance are helping 
scientists and healthcare and public health profes¬ 
sionals more quickly determine what is causing or 
has the potential to cause illness. 136-139 These techno¬ 
logical advances, which include multiplex polymerase 
chain reaction, immunoassays, arrays, and even 
next-generation sequencing, allow a more accurate 
determination of not only the pathogen, 138,139-142 but 
also the presence of mutations or other factors that 
distinguish the organism(s) from previous outbreaks 
or near neighbors 143 and have the potential to result in 
more severe disease. These techniques have identified 
several emerging infectious diseases. 144-146 

Many of the technologies listed have been avail¬ 
able for 30 years or more 147,148 ; however, the increased 
speed and multiplex capability, lower cost, and 
greater application of the technologies as surveil¬ 
lance tools, combined with enhanced surveillance 
reporting systems, create a more likely environment 
for the detection of a possible natural or intentional 
biological event. 149-150 Specifically, the more routine 
use of sequencing has significantly affected biological 
sciences and has the potential to be influential in the 
arena of the epidemiology of biowarfare. Ten years 
ago the cost and sample-to-result time of sequencing 
were prohibitive for routine use. However, the cost 
and processing time continues to decrease, making 
the accessibility to sequencing more universal and 
easily adaptable for inclusion in pathogen identifi¬ 
cation and characterization. 151 In 1990 the National 
Institutes of Health and Department of Energy initi¬ 
ated the human genome project, which required 10 
years to publish a working draft and cost millions 
of dollars. 152,153 A viral or bacterial genome can be 
sequenced in a few hours and can cost as little as 
$100 per isolate. 151,153-157 The use of sequence tech¬ 
nology has been instrumental in not only pathogen 
detection and characterization, including mutations 
that increase morbidity and mortality, but also in 
the development of detection and diagnostic assays 
and therapeutic and prophylactic solutions and/or 
countermeasures. 155 


Most recently, sequencing was used in the Middle 
East Respiratory Syndrome coronavirus outbreak 
to identify the source of the disease, determine the 
distinction from severe acute respiratory syndrome 
corona virus, 143 and develop polymerase chain reac¬ 
tion detection and diagnostic capabilities. 23 Sequenc¬ 
ing was also used in the H7N9 and H1N1 influenza 
outbreaks, 158,159 and in the Escherichia coli O104:H4 
in Germany in 2011 160-162 to assist with identifying 
the causative agent and developing possible coun¬ 
termeasures. Although it appears as though these 
events have all been naturally occurring, the addi¬ 
tion of characterization information in the form of 
sequence has allowed researchers to go back and look 
for possible index cases and the source or reservoir 
for the outbreak in humans. Rapid sequencing may 
also facilitate a more rapid vaccine development, 
as demonstrated in the use of novel techniques for 
influenza vaccine production. 163 The use of sequenc¬ 
ing will continue to assist scientists and public health 
professionals in their search for not only the reservoir, 
point of exposure, possible nefarious intention, and 
comparison with currently known and well charac¬ 
terized diseases, but will also assist in limiting the 
spread of the disease and possible prevention of 
future outbreaks by identifying potential zoonotic 
crossover before it even occurs. 164-167 

Many organizations are conducting surveillance 
globally with the goal of predicting and preventing 
the next outbreak or pandemic, often in zoonotic 
sources. 164,165 The US Agency for International Develop¬ 
ment, 168 the US Department of Defense, and both for- 
profit and nonprofit, nongovernmental organizations 
are all engaged in surveillance efforts using some of 
these technological advancements to identify the next 
potential source of an outbreak and develop detection 
and prophylactic or therapeutic solutions and other 
nonmedical countermeasures to prevent such an event, 
or at the very least, to be well prepared to respond 
robustly and quickly. 

However, not all uses of advanced technologies 
have been without controversy. One recent example 
of the use of sequencing in the creation of a potential 
BW agent came in late 2011 and continues today. 169-171 
Flu researchers Ron Fouchier, of the Erasmus Medical 


58 


Epidemiology of Biowarfare and Bioterrorism 


Center in The Netherlands, and Yoshihiro Kawaoka, 
of the University of Wisconsin-Madison, engineered 
more transmissible strains of H5N1, and, more re¬ 
cently, have focused on H7N9. 172,173 They believe ge¬ 
netic engineering can be used to determine which—if 
any—mutations accelerate the spread of influenza 
between mammals. 173-175 Additionally, scientists 
claim genetically modifying the H7N9 virus in the 
lab will help drive efforts to develop pandemic drugs 
and vaccines, and result in better preparedness and 
response. 175 However, not all scientists agree with the 
type of research being conducted, including infectious 
disease specialist Adel A F Mahmoud, of Princeton 
University. 171 Some scientists worry that these strains 
could escape the laboratory and possibly kill millions, 
or get in the hands of the wrong people. 173 Even the 
US National Science Advisory Board for Biosecurity 
became involved in this debate and has issued several 
rulings and restrictions on publication of information 
from this type of research. Dual use research consid¬ 
erations are also being carefully evaluated in some of 
these instances to ensure that the global populace is 
protected from potential harm. 

There are other limitations with this type of in¬ 
formation gathering and sharing. As seen during 
the E coli outbreak in Germany, when an initial er¬ 
ror is made in the suspected source of the outbreak 
(in this case erroneously stated to be from Spanish 
cucumbers) 176 the information can seriously and 
detrimentally affect a nation, manufacturing or pro¬ 
cessing group, or product identified as the source. 177 
Although the initial source of the outbreak was 
suspected based on epidemiological investigation 
and early molecular testing, the desire to release the 
information superseded molecular validation of the 
suspected outbreak source information 1, s ; it was not 
until the results obtained using advanced molecular 
techniques 160,179,180 combined with further epidemio¬ 
logical investigation identified the more likely out¬ 
break source. 175,181 Additionally, some nations may 
not approve the release of information regarding an 
outbreak or may not allow other scientists to continue 


Because management of BT and B W events depends 
on the disease surveillance, laboratory, and outbreak 
investigation capabilities of public health authorities, 
the science of epidemiology will always be the foun¬ 
dation for a response to these events. An enhanced 
index of suspicion, awareness of potential red flags. 


surveillance or investigations into the source if they 
feel their economy or other factors such as national 
security may be threatened. The existence, or lack 
thereof, of surveillance efforts, systems, and software 
solutions may also hinder the transfer of information 
regarding a potential outbreak or emerging infectious 
disease. 150 

The use of high throughput screening and sequenc¬ 
ing technologies can also be instrumental in detection 
of anomalies indicative of not only natural mutation 
and resistance, but also engineered and intentional 
activities. 180,181 The addition of virulence factors such 
as plasmids that are not typical to given organisms, 
but convey greater morbidity, communicability, and 
so forth can be a potential sign of human manipulation. 
Phylogenetic comparison with known pathogens can 
not only narrow prevention and treatment options, but 
also can highlight a possible unnatural combination 
of strains. Sequence information can even be used to 
generate a pathogen of interest de-novo, without the 
pathogenic element, allowing for possible manipula¬ 
tion of once pathogenic organisms in a lower class 
safety environment and additional options for assay 
and countermeasure development. 182-184 However, 
this capability also allows for generation of dangerous 
pathogens with the proper authorization. 185 Although 
the knowledge obtained from sequencing can be very 
beneficial, it has the potential to cause harm if it falls 
into the wrong hands or is not accurate and does not get 
reported to the appropriate public health professionals. 

However, as evidenced in the last few years when 
several anthrax and plague cases were detected in pa¬ 
tients in the United States, advanced technologies can 
rapidly assist an epidemiologic investigation. Public 
health and laboratory officials moved quickly to in¬ 
vestigate and determine the source of these infections; 
and using a combination of molecular techniques and 
epidemiological outbreak investigation, they found 
none were suspected to be intentionally caused. 186-193 
The addition of advanced molecular techniques can 
lead to faster diagnosis, treatment, and determination 
of intent or origin of infection(s). 


open lines of communication between local healthcare 
providers and law enforcement authorities, knowledge 
of historical outbreak investigation information, robust 
disease surveillance systems, and the use of advanced 
molecular techniques will improve the ability to re¬ 
spond to any future BT or BW event. 


59 


Medical Aspects of Biological Warfare 


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69 



Chapter 3 

FOOD,WATERBORNE,AND 
AGRICULTURAL DISEASES 


ZYGMUNT F. DEMBEK, PhD, MS, MPH, LHD,* and EDWIN L. ANDERSON, MD + 


INTRODUCTION 

FOODBORNE AND WATERBORNE PATHOGENS AND DISEASES 

Bacillus anthracis 
Clostridium botulinum 
Campylobacter jejuni 
Salmonella 

Listeria monocytogenes 
Escherichia coli 
Shigella 

Cryptosporidium 
Hepatitis A 
Mycotoxins 
Parasites 

Threat Potential Summary 
WATER SUPPLY CONCERNS 
AGRICULTURAL TERRORISM 
SMUGGLING AND INVASIVE SPECIES 
FOOD AND WATER SECURITY 
SUMMARY 


*Colonel (Retired), Medical Service Corps, US Army Reserve; Associate Professor, Department of Military and Emergency Medicine, Uniformed Ser¬ 
vices University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814; formerly, Chief, Biodefense Epidemiology and Education 
& Training Programs, Division of Medicine, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland. 
f Colonel (Retired), Medical Corps, US Army; Research Professor, Department of Internal Medicine, Division of Infectious Diseases, Allergy and Im¬ 
munology, Saint Louis University School of Medicine, 1100 South Grand Boulevard, St Louis, Missouri 63104 


71 



Medical Aspects of Biological Warfare 


INTRODUCTION 


Food and waterborne pathogens cause a consider¬ 
able amount of disease in the United States. The eco¬ 
nomic impact from foodbome diseases is estimated at 
about $78 billion per year. 1 The top five pathogens that 
contribute to domestic foodbome illness are, Novovi- 
rus, Salmonella species, Clostridium perfringens, Campy¬ 
lobacter species, and Staphylococcus aureus. 2,3 Many of 
the common foodbome pathogens, whether bacteria, 
viruses, parasites, or toxins, can cause disease if pur¬ 
posefully introduced into water or food sources. These 
pathogens characteristically have the potential to cause 
significant morbidity or mortality, have low infective 
dose and high virulence, are universally available, 
and can be stable in food products or potable water. 
These agents include Clostridium botulinum toxin, the 
hepatitis A virus. Salmonella, Shigella, enterohemor- 
rhagic Escherichia coli species, Cryptosporidium parvum, 


Campylobacter jejuni, Listeria monocytogenes, and Vibrio 
cholerae, among others. Pathogens in the Centers for 
Disease Control and Prevention (CDC) list of biological 
threat agents that also may cause food or waterborne 
disease are Bacillus anthracis, Brucella species, staphy¬ 
lococcal enterotoxin B, and ricin. 4 The potential for 
nonlisted biological agents such as mycotoxins and 
parasites (eg. Taenia species) to be used in a bioterrorist 
event should also be considered. 

This chapter provides an introduction to the far- 
reaching subjects of food and waterborne diseases, 
the potential for terrorist attacks on the food and 
water supply, and terrorism directed at sources of 
the nation's farm-to-food continuum (agricultural ter¬ 
rorism). For a more extensive review of these topics, 
readers may consult more specialized texts on food 5 
and waterborne 6 diseases and agricultural terrorism. 7,8 


FOODBORNE AND WATERBORNE PATHOGENS AND DISEASES 


Bacillus anthracis 

B anthracis is the causative agent of two forms of 
foodbome anthrax: oropharyngeal and gastrointes¬ 
tinal. Although spores of B anthracis would cause the 
most potential harm via an aerosol release, anthrax 
disease is not normally perceived as having bioter¬ 
rorism potential as a foodbome bacterial contaminant 
because the infective dose required for such an attack 
would be high. 9 However, given that the early diagno¬ 
sis of gastrointestinal anthrax is difficult for clinicians 
who have never treated cases of this disease, a higher 
mortality rate than expected may result from a natural 
or purposeful outbreak. Anthrax spores are resistant to 
disinfection by contact chlorination as used by water 
treatment facilities, although higher levels of chlorina¬ 
tion (> 100 ppm) for longer contact times (5 minutes) 
will kill Bacillus spores. 10 

Clostridium botulinum 

C botulinum is the causative agent of botulism in¬ 
toxication, of which there are three natural manifesta¬ 
tions: classic, wound, and infant botulism. Bioterrorism 
use of botulinum toxin could possibly occur through 
inhalational intoxication, as was considered by the 
Aum Shinrikyo cult in Japan in 1990. 11,12 C botulinum 
produces the most potent natural toxin known; the 
human lethal dose of type A toxin is approximately 
1.0 pg/kg. 13 There are seven antigenic types of botu¬ 
linum toxin, denoted by the letters A through G. 
Most human disease is caused by types A, B, and E. 


Botulinum toxins A and B are often associated with 
home food preparation 14 and home canning 13 and 
pickling. 16 Botulism-contaminated food cannot be 
distinguished by visual examination, and the cook is 
often the first to show the toxin's effects (via sampling 
the food during cooking). A 12- to 36-hour incubation 
period is common. The incubation period is followed 
by blurred vision, speech and swallowing difficulties, 
and descending flaccid paralysis. 17 

The current mortality rate associated with botulism 
intoxication is less than 10%. Foodbome botulism mor¬ 
tality during the 1950s (before the advent of modem 
clinical therapies) was approximately 25%. 18 Little 
evidence of acquired immunity from botulinum intoxi¬ 
cation exists, even after a severe infection. Successful 
treatment consists of aggressive trivalent (A, B, E) 
botulinum antitoxin therapy and ventilatory support. 
Early diagnosis is critical for patient survival. Toxin 
can be found in food, stool, and serum samples, which 
may all be used in the standard mouse model assay to 
test for the presence of botulism toxin. 19 

A controversial paper published in 2005 20 explored 
the potential for botulinum toxin contamination of the 
milk supply. A nine-stage dairy cows-to-consumer 
supply chain was examined, which accurately mod¬ 
eled a single milk-processing facility. The release of 
botulinum toxin was assumed to have occurred either 
at a holding tank at the dairy farm, in a tanker truck 
transporting milk from the farm to the processing 
plant, or at a raw milk silo at the plant. By the use of this 
model, it was predicted that 100,000 individuals could 
be poisoned with over 1 gram of toxin, and 10 grams 


72 


Food, Waterborne, and Agricultural Diseases 


would affect about 568,000 milk consumers. 20 The 
National Academy of Sciences published this informa¬ 
tion to foster further discussion and alert authorities to 
dangers to the milk supply from purposeful contami¬ 
nation. 21 The paper also describes interventions that 
the government and the dairy industry could take to 
prevent this scenario. Officials at the US Department 
of Health and Human Services requested that the 
paper not be published, but the National Academy of 
Sciences published it anyway, convinced that the in¬ 
formation would not enable bioterrorists to conduct an 
attack, and that the paper would stimulate biodefense 
efforts. Whether the paper's information presents a 
"roadmap for terrorists" by exposing vulnerabilities 
in food processing remains to be determined 22 ; how¬ 
ever, the hypothetical use of botulinum toxin placed 
at various points into the food supply was proposed 
in a fictional novel over 35 years ago. 23 

Campylobacter jejuni 

Campylobacter, Salmonella, Listeria, and E coli 
0157:H7 can be transmitted zoonotically from con¬ 
taminated animal food sources. These bacteria species 
are ubiquitous and cannot be completely eliminated 
from the food supply. C jejuni is the most commonly 
reported bacterial cause of foodborne infection in the 
United States. 24 Chronic sequelae associated with C 
jejuni infections include Guillain-Barre syndrome 23 and 
arthritis. 26 Infants have the highest age-specific isola¬ 
tion rate for this pathogen in the United States, which 
is attributed to a greater susceptibility upon initial 
exposure and a lower threshold of seeking medical 
treatment for infants. 24 Reservoirs for C jejuni include 
wild fowl and rodents. 27 The intestines of poultry are 
easily colonized with C jejuni, 28 and it is a commensal 
inhabitant of the intestinal tract of cattle. 29 Antibiotic 
resistance of Campylobacter is a growing concern for 
poultry farmers. 30 The infective dose for Campylobacter 
is 100 to 1,000 cells, with poultry the primary source of 
infection in the United States. 31 Insect transmission by 
several fly species has also been documented. 32 There 
is a 3- to 5-day illness onset for campylobacteriosis and 
a 1-week recovery time. Immunity is conferred upon 
recovery, which accounts for a significantly higher 
incidence rate among individuals younger than 2 years 
of age in developing countries. 33 

Salmonella 

Salmonellosis is the second most common food- 
borne illness, 34 and contaminated food is the principal 
route of disease transmission. 35 There are over 2,400 
Salmonella serotypes, many of which can cause gas¬ 


troenteritis, manifested as diarrhea, abdominal pain, 
vomiting, fever, chills, headache, and dehydration. 34,35 
Other diseases from Salmonella infections include enter¬ 
ic fever, septicemia, and localized infections. Poultry is 
a principal reservoir of the salmonellae. 35 Water, shell¬ 
fish, raw salads, and milk are also commonly implicat¬ 
ed as vehicles for this pathogen. In humans, the most 
highly pathogenic Salmonella species is S typhi. 35 This 
bacterium is the causative agent of typhoid fever, which 
comprises about 2.5% of salmonellosis in the United 
States. 35 The symptoms of typhoid include septicemia, 
high fever, headache, and gastrointestinal illness. 35 

During World War II, the Japanese developed 
biological weapons, poisoning prisoners with S ty- 
phimurium and many other bacteria and viruses during 
their experimentation, and contaminating wells with S 
typhimurium along the Russian border of Mongolia. 36 In 
September and October 1984, two large groups of sal¬ 
monellosis cases occurred in The Dalles, Oregon. Case 
interviews by health officials associated patronage of 
two restaurants in The Dalles with illness, especially 
with food items eaten from salad bars. S typhimurium 
isolates were then obtained from clinical specimens. 37 
The size and nature of this outbreak helped to initiate 
a criminal investigation, which was rarely done in 
conjunction with a foodborne disease outbreak. The 
cause of the epidemic became known when the Fed¬ 
eral Bureau of Investigation investigated a nearby cult 
(the Rajneeshees) for additional criminal violations. 38 
In October 1985 authorities found an opened vial that 
contained the original culture type of S typhimurium 
inside a refrigerator within the Rajneeshee clinic 
laboratory. 

A large multistate outbreak of milk-borne salmo¬ 
nellosis from Salmonella enteritica serovar typhimurium 
occurred in northern Illinois in 1985, with more than 
14,000 people reported ill and five deaths. 39,40 The 
cause of the outbreak was the accidental comingling 
of raw milk into the pasteurized product in a milk 
plant. 41 The contaminated milk was distributed via 
supermarket distribution systems, and cases were also 
reported in the neighboring states of Indiana, Iowa, 
and Michigan. 42 Medical treatment was complicated 
because the strain of S typhimurium involved was 
found to be resistant to antibiotics. Such inadvertent 
milk-borne contamination reinforces the potential for a 
ready-made vehicle for transmission of disease among 
a population by deliberate means. 20 

Listeria monocytogenes 

L monocytogenes is ubiquitous in the environment 
and often found in silage, water, and the environs of 
animal fodder. 43 Soft cheeses, 44 raw or contaminated 


73 


Medical Aspects of Biological Warfare 


milk, 45 and contaminated refrigerated foods 46 are of¬ 
ten sources of this organism. Listeriosis can result in 
meningo-encephalitis and septicemia in neonates and 
adults, and fever and abortion in pregnant women. 47 
Fetuses, newborns, 48 the elderly, 49 and immunocom¬ 
promised persons 50 are at greatest risk for serious ill¬ 
ness. Listeriosis case investigations can be problematic 
because of the variable incubation period for illness (3 
to >90 days). Large outbreaks of foodbome listeriosis 
have occurred, including a 1983 Massachusetts epi¬ 
demic where improperly pasteurized milk was the 
source of the infection. 51 Of the 49 infections associated 
with this outbreak, 14 patients died. 51 

Escherichia coli 

E coli 0157:H7 infections often originate from con¬ 
tamination due to a bovine reservoir. 52 This organism 
produces two verotoxins and is a significant cause 
of serious pediatric illness. 53 It can result in bloody 
diarrhea and hemolytic uremic syndrome, which is 
defined as the demonstration of three clinical condi¬ 
tions: (1) microangiopathic hemolytic anemia, (2) acute 
renal failure, and (3) thrombocytopenia. 53 Children 
younger than 5 are at greatest risk for hemolytic ure¬ 
mic syndrome when infected with E coli 0157:H7 or 
other enterohemorrhagic E coli (EHEC) species, and 
deaths from these infections occur most often in the 
age ranges of 1 to 4 years and 61 to 91 years. 52 A major 
source of EHEC exposure is from consumption of and 
contact with beef cattle. 54 About 20% of the ground beef 
consumed in the United States is derived from culled 
dairy cattle, which may be an important contributor 
to this bacterial contamination of the food supply. 55 
For example, during July 2002, the Colorado Depart¬ 
ment of Public Health and Environment identified 
an outbreak of E coli 0157:H7 infections that linked 
28 illnesses in Colorado and six other states to the 
consumption of contaminated ground beef products. 
Seven patients were hospitalized, and five developed 
hemolytic uremic syndrome. 56 

E coli- contaminated food items commonly result 
from use of cattle waste as fertilizer or other contact 
with cattle products. Outbreaks have occurred from 
exposure to various £ cob-tain ted food items, includ¬ 
ing alfalfa 57 and radish 58 sprouts, parsley, 59 lettuce, 60,61 
hazlenuts, 62 apple cider, 63 unpasteurized gouda 
cheese, 64,65 raw milk, 66 recontaminated pasteurized 
milk, 67 prepackaged cookie dough, 68 and salami, 69 
as well as through petting zoos 70 and environmen¬ 
tal transmission.' 1,72 Waterborne outbreaks of E coli 
0157:H7have also occurred. From mid-December 1989 
to mid-January 1990, 243 cases of gastrointestinal ill¬ 
ness from antibiotic-resistant E coli 0157:H7 occurred 


in a rural Missouri township as a result of an unchlori¬ 
nated water supply. 73 Swimming-associated outbreaks 
of E coli 0157:H7 have also occurred. 74,75 

Other enterohemorrhagic E coli strains containing 
Shiga toxins (Shiga toxin-producing E coli [STEC] 
infections) have appeared as public health concerns, 
including STEC 0121, 76 STEC 026, 77 STEC 0145, 78 
and STEC O104:H4. 79 Novel STEC strains can easily 
develop due to opportunistic microbial growth and 
spread in the food supply chain and distribution sys¬ 
tems. The CDC E coli investigation page 80 lists current 
and past identified outbreaks. A recent 10-year study 
in Connecticut of 663 reported STEC infections dem¬ 
onstrated that both 0157 and non-0157 STEC infection 
incidence decreased from 2000 through 2009, and also 
that 0157 was the most common and clinically severe 
type of STEC infection. 81 However, in this and other 
studies, non-E coli 0157 accounted for a minority of all 
clinically significant STEC infections. 82,83 Importantly, 
STEC 0104 and 0157:H7 infections are more likely 
to lead to hospitalization and death than other STEC 
serogroups, as shown by a recent 8-year retrospective 
cohort study of 8,400 patients in Germany. 84 Another 
recent German study demonstrated that cattle density 
is a risk for exposure to E coli 0157:H7 and other STEC 
strains, including all major disease-causing groups 
(026, 0103, Olll, 0128, 0145), but not 091. 85 STEC 
strains therefore appear to be a diverse group of or¬ 
ganisms that demonstrate differences as well as many 
commonalities in exposure and epidemiology. 

Shigella 

Humans are the major reservoir for Shigella and 
the primary source of subsequent infections. 86 It is 
thought that worldwide Shigella-associated illness 
causes about 165 million cases per year, of which 
fewer than 1% occur in industrialized nations. 86 Shigella 
dysenteriae produces severe disease, may be associated 
with life-threatening complications, and causes about 
25,000 cases of illness each year in the United States. 86 
Although not an environmentally hardy organism. 
Shigella is highly infectious and can be very persistent 
in a close community environment. 87 Four serogroups 
(A through D) cause approximately 80% of shigellosis 
cases in the United States. Immunity is serotype-specif¬ 
ic. 88 Vaccine development has been problematic, 89 and 
the species can easily become resistant to antibiotics. 90 
Infants and young children are most susceptible to 
shigellosis, attributable in part to toiletry behaviors 
and child care practices. The infectious dose for Shigella 
is 10 to 100 organisms, and Shigella contamination can 
cause outbreaks associated with food, water, and milk. 
Shigellosis has also been associated with recreational 


74 


Food, Waterborne, and Agricultural Diseases 


swimming. 91 Shigellosis is readily transferred from 
person-to-person contact and through fomites 92 ; it can 
also be transmitted by insect vectors (primarily flies). 93 
There is a 1- to 3-day incubation period for shigello¬ 
sis. Shigella organisms are shed for 3 to 5 weeks after 
symptoms cease, ultimately contributing to a greater 
person-to-person spread than with other enteric patho¬ 
gens such as Salmonella and V cholerae. 

Cryptosporidium 

Cryptosporidium, a protozoan and an obligate intracel¬ 
lular parasite, can cause food and waterborne illness and 
can also be acquired from exposure to contaminated 
recreational water. 94-98 Seroprevalence surveys indicate 
that about 20% of the US population have been infected 
with Cryptosporidium by adulthood. 99 The severity and 
course of infection can vary considerably, dependent 
upon the immune status of the individual. Intestinal 
cryptosporidiosis is often characterized by severe watery 
diarrhea but may also be asymptomatic. Pulmonary 
and tracheal cryptosporidiosis in humans is associated 
with coughing and low-grade fever; these symptoms 
are often accompanied by severe intestinal distress. The 
duration of illness in one study of 50 healthy individuals 
varied from 2 to 26 days, with a mean of 12 days. 100 

The precise infectious dose is unknown; research 
indicates that a range of 9 to 1,024 oocysts will initiate 
infection. 101 The pathobiology is not completely known 
either; however, the intracellular stages of the parasite 
can cause severe tissue alteration. Infected food han¬ 
dlers are a major contributor to disease transmission. 
Consequently, cryptosporidiosis incidence is higher in 
facilities that serve uncooked foods, such as restaurants 
with salad bars. Child care centers can be a problematic 
source of Cryptosporidium infection because diarrhea 
in children in diapers can be difficult to contain. 102 
A significant reservoir worldwide for Cryptosporidium 
parvum is domestic livestock, predominately cattle. 103 
Drinking water outbreaks have affected as many as 
403,000 individuals (in a 1993 outbreak in Milwau¬ 
kee). 94 In the Milwaukee incident, the water was both 
filtered and chlorinated. 104 The organism's resistance to 
chlorine treatment ensures that it will remain a concern 
in treated potable water, 105 and therefore a risk to immu¬ 
nocompromised individuals, in whom it causes severe 
and chronic life-threatening gastroenteritis. 106 

Hepatitis A 

Humans are the source of the Hepatovirus hepatitis 
A virus. 33 Illness caused by hepatitis A is characterized 
by sudden onset of fever, malaise, nausea, anorexia, 
and abdominal discomfort, followed by jaundice. 35 The 


infectious dose is not precisely known but is thought 
to be 10 to 100 virus particles. 35 The virus is hardy, 
and it survives on hands and fomites. Because viral 
particles are excreted in the feces during clinical illness, 
stringent personal hygiene is crucial to prevent disease 
transmission. Hepatitis A is commonly transmitted via 
personal contact, and fewer than 5% of all hepatitis A 
cases are demonstrated to have been caused by food 
or waterborne transmission. 107 Permanent immunity 
to hepatitis A is assumed subsequent to infection 108 or 
immunization completion. 109 The advent of nationwide 
hepatitis A vaccination programs is gradually causing 
a decrease in disease incidence and the susceptible 
population. 110 As a result, hepatitis A may in time cease 
to be a public health concern. 111 

The potential for hepatitis A virus transmission 
in drinking water was demonstrated in an outbreak 
among members of the varsity football team at the Col¬ 
lege of the Holy Cross in Worcester, Massachusetts, in 
1969. The same water supply was used for both irriga¬ 
tion and potable water. Water used by firefighters to 
battle a blaze nearby caused a drop in water pressure, 
and back-siphonage brought groundwater into the 
football practice field's irrigation system. The ground- 
water had been contaminated by children infected with 
hepatitis A in a building immediately adjacent to the 
playing field. The football team members became ill 
after consuming the water from a faucet hooked up to 
this contaminated water source. 112,113 Although 90 of 97 
players and coaches on the team became ill (93% attack 
rate), serologic testing performed years later revealed 
that only 33 had IgM anti-hepatitis A virus in serum 
(34% attack rate). 114 Because of this discrepancy, the 
illness may have been caused by another pathogen 
also present in the water. 

Mycotoxins 

Fungi are plant pathogens that can cause both 
mycoses (infections) and mycotoxicoses (exposures to 
toxic fungal metabolites that may be dietary, dermal, 
or respiratory). Mycotoxins are ubiquitous worldwide 
toxic fungal metabolites and contaminants of stored 
cereal grains. 115,116 Although mycotoxins are not on the 
CDC threat list, individuals with chronic exposure to 
mycotoxins (including aflatoxin Bl, ochratoxin, T-2 
toxin, deoxynivalenol [DON], nivalenol [NIV], and 
others), often exhibit oncogenic symptoms, includ¬ 
ing liver damage, liver cancer, hemorrhaging, mental 
impairment, abdominal pain, vomiting, convulsions, 
and edema. The fact that these toxins are found natu¬ 
rally in commercially available cereal-based foods, 
including bread and related products, noodles, break¬ 
fast cereals, baby and infant foods, and rice, indicate 


75 



Medical Aspects of Biological Warfare 


that a ready substrate for growth is available, and 
deliberate contamination of these foodstuffs may be 
possible. Mycotoxicoses are often undiagnosed and 
hence unrecognized by public health authorities, 
except when large numbers of people are affected. 117 
The symptoms of mycotoxicosis depend on the type 
of mycotoxin; the amount and duration of exposure; 
the age, health, and sex of the exposed individual; 
and many unknown synergistic effects including 
genetics, dietary status, and interactions with other 
toxic insults. 118 

Large naturally occurring outbreaks of trichothe- 
cene intoxications have occurred, including a large 
exposure of trichothecene mycotoxin from moldy 
grain and bread in Orenburg, Russia, in 1944, which 
caused alimentary toxic aleukia and subsequent 
mortality in at least 10% of the population. 119 A 1991 
outbreak caused by moldy wheat and barley affected 
130,000 people in the Anhui province in China. 115 
Fusarium mycotoxins including DON and NIV have 
also been discovered in corn samples in Linxian, 
China, in positive correlation with the incidence 
of esophageal cancer. 119,120 Although outbreaks of 
mycotoxicoses have decreased greatly as a result 
of increases in hygiene measures, they still occur 


in developing countries, 121 are considered a serious 
international health problem, 122 and also pose a risk 
to domestic animals. 122-126 

The history of mycotoxin use as a biological weapon 
includes efforts by Iraq to develop and use aflatoxins 
during the 1980s. 127 ' 128 Iraq's biological weapons program 
cultured strains of Aspergillus flavus and A parasiticus 
and extracted 2,300 liters of concentrated toxin. 127,128 This 
aflatoxin was used mostly to fill missile warheads, and 
the remainder was kept stockpiled. 127,128 The Soviet Union 
is suspected of deploying trichothecene toxins (NIV, 
DON, and T-2) in the "yellow rain" incidents in Laos and 
Cambodia during the 1980s. Whether the toxin exposures 
that occurred at that time were the result of purpose¬ 
ful 129 or natural 130 events has never been completely 
resolved. These events indicate the potential for myco¬ 
toxin use as a biological weapon or bioterrorism agent. 

Parasites 

Parasites such as tapeworms (eg. Taenia species) 
may have potential for use as bioterrorism agents. It 
is conceivable, for example, that a culture of Taenia 
solium eggs could be poured onto a salad bar or into 
water, be ingested, and cause illness. Symptoms of 


TABLE 3-1 

PROPERTIES RELATED TO THREAT POTENTIAL OF COMMON FOOD AND WATERBORNE 
DISEASE PATHOGENS 


Pathogen 

Incubation Period 

Infective or Toxic 
Dose* 

Mortality in United 
States 

Bloody Diarrhea 

Enterohemorrhagic Esch¬ 
erichia coli 

3-4 d 

10-10 2 

rare 

yes 

Salmonella typhi 

8-14 d 

10-10 2 

low 

yes 

Salmonella species 

6-72 h 

10 2 -10 3 

low 

yes 

Shigella dysenteriae 

1-7 d 

10-10 2 

rare 

yes 

Campylobacter jejuni 

2-5 d 

> 5 x 10 2 

rare 

no 

Clostridium botulinum 
toxin 

12-72 h 

70 pg + 

5%-10% 

no 

Vibrio cholera 

2-3 d 

10 6 

rare 

no 

Cryptosporidium species 

7 d 

9-1,024 

rare 

no 

Listeria monocytogenes 

3 > 90 d 

unknown 

high 

no 

Hepatovirus hepatitis A 

30 d 

10-10 2 

low 

no 

Noro virus 

1-2 d 

< 10 2 

rare 

no 

Mycotoxins 

Minutes to months* 

4 mg/kg § 

rare 

yes 


*The number of organisms unless otherwise noted. 
+ Oral lactate dehydrogenase for a 70-kg human. 
*Dose-dependent. 

s Oral lactate dehydrogenase for laboratory rat. 


76 





Food, Waterborne, and Agricultural Diseases 


taeniasis from ingestion of the eggs would include 
cysticercosis (parasite tissue infection), which would 
not appear for weeks to years following infection. 
However, this infection timeline should not eliminate 
parasites from consideration as potential bioterrorism 
agents; such a scenario has been proposed. 131 T solium 
can be transmitted person to person by food handlers 
with poor personal hygiene, adding to the spread of 
the outbreak. 132 Such an outbreak may go undiagnosed 
for an additional period, during which ill persons are 
seen by healthcare providers unfamiliar with tape¬ 
worm infections. A purposeful outbreak of giardiasis 
that occurred in Edinburgh, Scotland, in 1990 dem¬ 
onstrates that parasites can be used for bioterrorism. 
Nine individuals living in the same apartment complex 
developed giardiasis subsequent to the purposeful 
fecal contamination of an unsecured water supply. 133 

Threat Potential Summary 

Table 3-1 provides information about various patho¬ 
gens related to their potential threat as purposeful 
food contaminants. Both bacterial and viral enteric 
pathogens were considered for this compilation. This 
taxonomic approach may prove useful in stimulating 
further discussion of pathogenicity and potential for 

WATER SUPI 

Poisoning water supplies is one of the oldest meth¬ 
ods of biological warfare. 137 The earliest documented 
poisoned drinking water occurred in Greece in 590 
bce, when the Amphictyonic League used hellebore 
to poison the city of Kirrha's water source, causing 
the inhabitants to become violently sick and unable 
to move. 138 In current developed countries, it is more 
difficult for a terrorist to contaminate water because of 
the large volumes of water and the extensive purifica¬ 
tion processes used in modern water treatment facili¬ 
ties, including aeration, coagulation and flocculation, 
clarification, filtration, and chlorination. 138 All of these 
methods remove contaminants and pathogens in the 
water, whether purposefully added or not. 

However, the risk to the US water supply has been 
known for some time. Federal Bureau of Investigation 
Director J Edgar Hoover noted in 1941, "It has long 
been recognized that among public utilities, water 
supply facilities offer a particularly vulnerable point of 
attack to the foreign agent." 139 A terrorist might bypass 
the purification process and introduce a pathogen later 
in the distribution system. A private well water sup¬ 
ply system with a smaller volume of water and a less 
extensive purification system may be more vulnerable. 
Another potential avenue for deliberate waterborne 


misuse. For example. Salmonella was not considered 
a threat agent before its use in the salad bar contami¬ 
nation in 1984. The prior view may have been based 
on factors inherent in Salmonella infection—a high 
dose of Salmonella is required to cause illness. If the 
infectious or toxic dose required for illness from an 
organism is the sole consideration for its classification 
as a bioweapon, then salmonellae should not even be 
considered as a threat agent. However, the use of S 
typhimurium to sicken many hundreds of people in 
the Dalles incident demonstrated a reality of biologi¬ 
cal agents: those that can be cultured and dispersed to 
cause illness will prove effective. Although no deaths 
occurred, the incident involved a rapid-onset illness 
with gastrointestinal effects that spread through 10 
restaurants, causing widespread fear of food poison¬ 
ing and long-lasting economic consequences in the 
community. 134 Given suitable circumstances, almost 
any pathogen could be used to make a target popula¬ 
tion ill. The severity of illness, including symptoms 
such as bloody diarrhea, also should be considered. 
For example, an outbreak of bloody diarrhea could 
have strong psychological effects upon those directly 
affected and perhaps lead to widespread psychological 
effects in the general public 135 if exacerbated by media 
coverage of the outbreak. 136 

CONCERNS 

contamination is the addition of a pathogen to a build¬ 
ing's water supply (ie, an enclosed system), with likely 
little or no subsequent water treatment processes and 
a specific target community. 

Waterborne pathogens included on the CDC threat 
list are V cholerae and C parvum. The Milwaukee 
outbreak of C parvum demonstrates the potential of 
public water supply contamination to affect great 
numbers of people. Another example of an extensive 
waterborne disease outbreak resulting from con¬ 
taminated well water was the 1999 £ coli 0157:H7 
and Campylobacter outbreak involving more than 900 
illnesses and 2 deaths among attendees of a New York 
county fair. 140 According to a comprehensive review 
of potable water threats by Burrows and Renner, 
potential water threat agents also include B anthracis, 
Brucella, V cholera, C perfringens, Yersinia pestis, Chla¬ 
mydia psittaci, Coxiella burnetii, Salmonella, Shigella, 
Francisella tularensis, enteric viruses, smallpox virus, 
aflatoxin, C botulinum toxin, microcystins, ricin, saxi- 
toxin, staphylococcal enterotoxins, T-2 mycotoxin, 
and tetradotoxin. 141 The 1969 hepatitis A outbreak 
at the College of the Holy Cross demonstrates the 
potential for this pathogen to cause illness when 
distributed in a water supply. 


77 


Medical Aspects of Biological Warfare 


Communitywide outbreaks of gastroenteritis, 
caused by Giardia lamblia, Cryptosporidium, various E 
coli serotypes, Torovirus, and other infectious agents, 
have occurred from recreational water use, including 
swimming pools, water slides, and wave pools. 142 
Nongastroenteritis recreational water outbreaks of¬ 
ten include those caused by Pseudomonas aeruginosa, 
Naegleria fowleri, and Legionella. 142 A recent naturally 
occurring outbreak of gastroenteritis associated with a 
contaminated recreational water fountain at a Florida 
beachside park demonstrates the potential for disease 
transmission. 143 In this incident, 44% of the interviewed 
park visitors who used an interactive water fountain 
became ill. Both C parvum and Shigella sonnei were 


subsequently isolated from clinical specimens ob¬ 
tained from these ill persons. The median age of the 
ill persons was 8 years. One can imagine the effect of 
a powerful biological agent such as C botulinum toxin 
covertly added to a recreational public water fountain 
in similar circumstances. 144 

The water utility industry and federal public health 
agencies have carried out plans to improve the ability 
to prevent as well as detect deliberate contamination 
of water systems. 145 An example of a new program 
to detect purposeful contamination of the water sup¬ 
ply is the Water Sentinel program. 146 However, much 
work remains to attain full biosecurity of the US water 
supply. 147 ' 148 


AGRICULTURAL TERRORISM 


Agricultural terrorism (agroterrorism) may be 
directed at stored or processed food, but some of the 
greatest vulnerabilities may exist close to the farm end 
of the farm-to-food continuum (Figure 3-1). Many of the 
potential bioterrorist agents are endemic, and therefore 
cannot easily be controlled. As with processed food and 
water terrorism, agroterrorism concerns are not recent 
developments. The historical use of biological agents to 
affect livestock includes the attempt to interrupt supply 
lines by infecting cavalry and transport animals with 
anthrax and glanders during World War I. In April 
1915, German-American physician Anton Dilger (who 
had served in the German Army) returned to the United 
States from Germany along with cultures of Burkholderia 
mallei and B anthracis. His intent was to infect animals 
(horses and mules) that were shipped from the United 
States to France and England for use in cavalry and 
transport to support their war with Germany. Dilger 
propagated the bacterial cultures and tested them for 
virulence using guinea pigs in the basement of a house 
(known as "Tony's Lab") he and his brother Carl 
rented in Chevy Chase, Maryland, near Washington, 
DC. 149 Over the next 2 years, Dilger's bacterial cultures 
were used to infect horses and mules in holding pens 
in docks at the ports of Baltimore, Maryland; Newport 
News, Virginia; Norfolk, Virginia; and in New York 
City. Stevedores working for German steamships were 
recruited and provided with cork-stoppered glass vials 
containing the bacterial cultures, in which a hollow steel 
needle had been placed. The stevedores were instructed 
to wear rubber gloves while jabbing the animals with 
the needles. These cultures were also spread among the 
animals by pouring them directly into the animal feed 
and drinking water. 150,151 

The significance of foot and mouth disease (FMD) 
as a biological weapon has been known for some time, 
and it is perhaps the greatest agroterrorism threat for 


livestock. Field trials of FMD virus dissemination were 
conducted in Nazi Germany's offensive biological war¬ 
fare program. FMD is thought to be inherently spread 
through airborne virus transmission, a problematic 



Figure 3-1. Some of the greatest vulnerabilities from agricul¬ 
tural terrorism may exist at the farm end of the farm-to-food 
continuum. 

Photograph courtesy of the US Department of Agriculture, 
Washington, DC. 


78 


Food, Waterborne, and Agricultural Diseases 


issue for outbreak containment, 152 and Germany con¬ 
sidered aerial dissemination and dispersal of the FMD 
virus through contaminated hay and grass. 153(pll4) 

Another attack on livestock occurred during the 
Mau-Mau uprising in British-controlled Kenya (1952- 
1960), when the Mau-Mau used the indigenous poison¬ 
ous African milk bush (Synadenium compaction) to kill 
33 cows at a mission station in 1952. 154,155 This use of 
locally obtained poisonous plants could be replicated 
anywhere lacking constant monitoring of animal feed. 

Anticrop terrorism has also been suspected on nu¬ 
merous occasions. The Colorado potato beetle ( Leptino - 
tarsa decemlineata ) is a crop pest of plants of the genus 
Solanum, which includes potatoes, tomatoes, and egg¬ 
plants. During World War II, Germany initiated large- 
scale breeding and field trial dispersals of the insects 
in Germany, when Dr Martin Schwartz conducted an 
offensive research program at the Kruft Potato Beetle 
Research Station near Koblenz. 153(pll0) This program 
may have backfired by initiating local crop infesta¬ 
tions; however, outbreaks of the pest also occurred in 
England and the United States, which were suspected 
to be caused by a German release of the insects. 156,157 
Perhaps because of this research conducted in Nazi 
Germany, in 1950 Soviet-occupied East Germany ac¬ 
cused the United States of releasing the beetle during 
in infestation. 158 Herbicides have also been used for 
wartime missions, such as the large-scale use of the 
defoliant Agent Orange by the United States to both 
defoliate and destroy crops used by North Vietnamese 
forces. 159 In 1989 a group known as "the Breeders" an¬ 
nounced that it had released Mediterranean fruit flies 
in southern California to protest the use of pesticides 
in that region. 160 

State-sponsored agricultural terrorism remains a 
global concern today, given vulnerabilities inherent in 
modem farming practices. In the United States, live¬ 
stock may be susceptible to agroterrorism (Figure 3-2). 
Because US disease eradication efforts among livestock 
herds have been so successful, much of the nation's 
livestock is either vaccinated or monitored for disease 
by farmers and veterinarians. However, the risk of harm 
from agroterrorism to large numbers of livestock has 
been increased through the widespread use of modem 
livestock farming, such as concentrated animal feed¬ 
ing operations (CAFOs). The pervasiveness of CAFOs 
in the US agriculture industry is all-encompassing. 
For example, in the US between 1997 and 2007: 161 

• hog factory farms added 4,600 hogs every day; 

• factory farm dairies added nearly 650 cows 
every day; 

• factory farms added 5,800 broiler chickens 
every hour; 



Figure 3-2. Livestock may be more susceptible to agroter¬ 
rorism than crops. 

Photograph courtesy of the US Department of Agriculture, 
Washington, DC. 


• factory farmed broiler chickens doubled to 1.1 
billion; 

• hog factory farms grew by 42%, to 5,144; 

• the average size of egg factory farms increased 
by half to 614,000 hens; 

• the number of cows on factory-farm dairies 
nearly doubled, to 4.9 million; 

• the number of hogs on factory farms grew by 
more than a third, to 62.9 million; 

• the number of factory farm egg-laying hens 
increased by 24%, to 266.5 million; 

• the number of US beef cattle on industrial 
feedlots grew by 17%, to 13.5 million; and 

• nearly half of factory-farm egg-laying hens 
were located in just five states: Iowa, Ohio, 
Indiana, California, and Pennsylvania. 

Altogether, CAFO aggregation has had the great¬ 
est effect on livestock operations with the greatest 
numbers of animals. From 1982 to 1997, livestock 
operations with 1,000 or more animals increased by 
47%. In comparison, farms with less than 25 animals 
(a family farm) decreased by 28%. 162 While such ag¬ 
gregation has enabled economic viability and success 
for the farming industry, it also can provide a single¬ 
source opportunity for foodbome contamination or 
adulteration for the would-be bioterrorist desiring to 
affect the food supply. 

Upon infection, livestock may become a vector 163 
or reservoir 164 for disease transmission. This potential 
was plainly demonstrated in the 2001 outbreak of 


79 



Medical Aspects of Biological Warfare 


FMD in the United Kingdom. 165 This outbreak was the 
single largest FMD epidemic ever experienced in the 
world. 166 Agricultural and food losses to the United 
Kingdom exceeded $4.6 billion, 167 and psychological 
effects in residents of the worst affected areas were 
extensive and long-lasting. 168 The United States has 
not had an outbreak of this disease since 1929, 169 
and the US Department of Agriculture (USDA) has 
developed national protective measures to prevent a 
reintroduction. 170 

Perhaps the greatest national risks from agroter- 
rorism involve the potential for widespread economic 
consequences. Not only would immediate loss to a 
crop occur from such an event, but incidental costs 
would also result from lost production, the destruc¬ 
tion of potentially diseased products, and containment 
(including quarantine, drugs, and diagnostic and vet¬ 
erinary services). The costs of these programs would be 
borne by farmers as well as federal and state govern¬ 
ments. 171 Export markets would be rapidly lost as other 
nations close their borders to imports from a country 


with diseased livestock. As an example, a single case 
of mad cow disease (bovine spongiform encephalopa¬ 
thy) was found in Washington state on December 23, 
2003; by December 26, Japan had banned all US beef 
imports, and beef prices dropped by as much as 20% 
in the following week. 172 Additionally, multiplier 
economic effects would occur from decreased sales 
by agriculturally dependent businesses and tourism. 
Other animal pathogens besides FMD and bovine 
spongiform encephalopathy that could have severe 
economic consequences if uncontrolled include highly 
pathogenic avian influenza, 173 rinderpest, 174 and Afri¬ 
can 173 and classical swine fever. 176 

The USDA's Animal and Plant Health Inspection 
Service has developed a select agent and toxin list of 
pathogens and toxins that endanger agriculture in the 
United States 177 (some of these zoonotic pathogens also 
endanger humans and appear on the CDC Category 
A list 4 ; these pathogens are listed separately by the 
USD A as overlap agents and toxins). Another USD A 
list enumerates harmful plant pathogens. 177 


SMUGGLING AND INVASIVE SPECIES 


The problem of smuggling and the unintentional 
introduction of invasive species has grown in re¬ 
cent years. Increased and more rapid international 
trade, increased trade in fresh commodities, new 
travel and trading routes, and increased difficulties 
in enforcing quarantines have all contributed to this 
problem. The potential consequences of smuggling 
and unintentional introduction of invasive species 
may go far beyond the direct damages or costs of 
control. The economic costs of all invasive species in 
the United States is estimated at between $120 billion 
and $138 billion per year. 178,179 These invasive species 
consist of microbes (30.1%), mammals (27.2%), plants 
(25.0%), and arthropods (15.4%). The full range of 


On December 3, 2004, the former secretary of the 
Department of Health and Human Services, Tommy 
Thompson, warned of a possible terrorist attack on 
the nation's food supply: "For the life of me, I cannot 
understand why the terrorists have not attacked our 
food supply, because it is so easy to do . . . We are 
importing a lot of food from the Middle East, and it 
would be easy to tamper with that." 182 In American 
society, the farm-to-food continuum, which includes 
production, processing, distribution, and preparation, 
has myriad potential vulnerabilities for natural and 
intentional contamination. 182 Centralized food pro¬ 
duction and widened product distribution systems 


economic costs of biological species invasions reaches 
far beyond the immediate impacts on the affected 
producers and often include consequences to local, 
national and global markets. In 1995, member na¬ 
tions of the World Trade Organization signed the 
Uruguay Round Agreement on the Application of 
Sanitary and Phytosanitary Measures, which set out 
basic rules for food safety and animal and plant health 
standards and increased the transparency of sanitary 
and phytosanitary measures. 180 The agreement cov¬ 
ers all measures to protect human or animal health 
from foodborne risks, human health from animal- or 
plant-carried diseases, and animals and plants from 
pests or diseases. 180 

ER SECURITY 

present increased opportunities for the intentional 
contamination of food. 183 As covered in the discus¬ 
sions above, many opportunities exist along the food 
and water production continuum to accidentally or 
intentionally introduce various pathogens, many 
of which are not categorized as threat agents. 184 
Strategies to counter these threats should focus on 
enhancing knowledge of all raw material inputs to 
the system; identifying and addressing the most likely 
points of vulnerability; disposing of end products 
after they leave the systems; and accounting for em¬ 
ployees, visitors, computers, and physical security 
throughout the continuum. 


80 


Food, Waterborne, aiid Agricultural Diseases 


Knowledge of the various processes involved in 
food production will help to determine potential vul¬ 
nerabilities for agricultural terrorism. The typical food 
distribution system includes agricultural production 
and harvesting, storage and transport of raw com¬ 
modities, processing and manufacture, storage and 
transport of processed and manufactured products, 
wholesale and retail distribution, and the food service 
sector. 185 The responsibility for food safety and security 
throughout the food distribution network is shared by 
the producers and suppliers as well as many different 
state and federal agencies. Typically, a state's health 
and agricultural agencies ensure that the food comes 
from safe sources and is served with safeguards to 
prevent foodbome disease transmission. Equivalent 
federal agencies share these responsibilities, including 
the US Food and Drug Administration, USD A, Depart¬ 
ment of Health and Human Services, US Public Health 
Service, CDC, and other partner agencies now part of 
the Department of Homeland Security, including the 
Federal Bureau of Investigation and the US Customs 
Service. 

One prevention strategy is to anticipate inten¬ 
tions or motivations that could result in an attack 
using a particular product or organization. These 
motivations could include religion or ideology; 
personal grievances (real or perceived); and conten¬ 
tious issues such as animal rights, environmental 
protection, and abortion. Research facilities, food 
processors, and food retailers could be targets of ter¬ 
rorism and should take extra preventive measures. 
Knowledge of terrorism trends can be an indicator 
for the need to change security measures to meet 
the threat. However, because the US food industry 
is highly competitive on a price basis, additional 
preventive measures may only be an option if they 
are government subsidized. 

From an attacker's standpoint, the choice of meth¬ 
ods and weapons is determined by the target and the 
delivery medium. It is rare that someone would at¬ 
tempt to cause harm without consideration of whom 
or how many people are affected. The target popula¬ 
tion may then define the vulnerabilities. For example, 
animal feed could be contaminated if the goal was to 
affect a CAFO. 

Strategies also can be implemented to address spe¬ 
cific vulnerabilities. The first task is to define produc¬ 
tion processes in terms of the inputs and outputs at all 
potential nodes of vulnerability. For example, foods 
that are either eaten uncooked or that can be contami¬ 
nated after cooking should receive special quality con¬ 
trol attention. Also, knowledge of where raw materials 
including water are obtained can help identify needs 
for enhanced security and accountability. 


A thorough knowledge of the existing hazard 
analysis critical control points (HACCPs) for each food 
item considered to be a potential vehicle for foodbome 
disease is essential to understanding and preventing 
illness. A comprehensive HACCP analysis will provide 
a systematic method of documenting that food safety 
hazards have been addressed. 186 Hazard analysis in¬ 
volves food safety issues only, including storage and 
holding temperatures, pH, sanitary conditions, physi¬ 
cal storage security, and any other factor that could 
impact the safety and integrity of a food item during 
manufacture, storage, delivery, or food preparation. 186 
General guidance to conducting an HACCP program 
would necessarily include: 186 

• Hazard analysis: what are the food safety 
hazards that can be controlled? 

• Establish critical control points (CCP): where 
can things go wrong, and how can they be 
controlled? 

• Establish critical limits: what physical values 
(temperature, pH, etc) indicate that the pro¬ 
cess is in control? 

• Establish monitoring procedures: how will 
the CCPs be monitored? 

• Establish corrective actions: what happens if 
a critical limit is exceeded? 

• Establish a record keeping system: "If it isn't 
written down, it didn't occur." 

• Establish verification procedures: how can 
you know if the system works? 

HACCP principles have been successfully applied 
to the production, storage, and serving of many types 
of food items. 187-189 However, there remain many chal¬ 
lenges to providing a safe and wholesome food supply, 
and resolution of issues through the use of HACCP may 
also provide solutions that could prevent bioterrorism. 
For example, food items that are common sources of 
foodbome infections may also present opportunities 
to a potential bioterrorist, by virtue of a lack of proper 
temperature use and monitoring. It has been demon¬ 
strated that salsa and guacamole are frequent vehicles 
of foodbome disease outbreaks in the United States. 190 
Unsurprisingly, fresh serrano and jalapeno peppers 
used in these food items have caused huge multistate 
Salmonella outbreaks. 191,192 Fresh salsa and guacamole 
require careful preparation and storage, and food pre¬ 
vention strategies based upon the HACCP principles can 
greatly help to reduce the incidence of foodbome disease, 
as well as to maintain monitoring of these food items. 

Focus is often targeted on the inputs to food, 
water, or agricultural production, and when a 
product leaves the plant, that attention may be dis- 


81 


Medical Aspects of Biological Warfare 


continued. The time and route of delivery, as well as 
the security of the transportation, may be the most 
vulnerable points in the continuum and should not 
be overlooked when planning security. Studying 
incidents of nonpurposeful foodborne pathogen 
contamination, such as the 1985 Minnesota salmonel¬ 
losis outbreak, 193 may reveal potential avenues for 
purposeful outbreak scenarios. This outbreak and 
many others demonstrate that foodborne bioter¬ 
rorism might have greater chances of success when 
pathogens are introduced after processing and as 
close to consumption as possible, thus circumvent¬ 
ing opportunities for dilution and destruction by 
cooking or pasteurization. 

Implementing rational employee hiring and ac¬ 
countability procedures may also effectively mitigate 
food, water, or agricultural vulnerabilities. 186 Addi¬ 
tional strategies include implementing procedures 
for laboratory testing and monitoring, reporting and 
investigating inspection discrepancies, and ensuring 
computer and information security. 186 

Various disease surveillance systems (covered in 
greater details in other chapters) are in place, including 


local, state health agency, and CDC programs to track 
and identify trends in foodborne illness, including 
FoodNet, 194 PulseNet, 195 CalciNet, 196 WBDOSS, 197 ' 198 
and syndromic surveillance systems such as RODS 199 
and BioSense. 200 Additional methods to inspect and 
protect food and water supply chains, and rapidly 
integrate disease surveillance, are being actively ex¬ 
amined and implemented. 201 

Furthermore, under the Food Safety and Modern¬ 
ization Act of 2010 the Food and Drug Administration 
has proposed a rule that would require the largest 
food businesses in the United States and overseas to 
take measures to prevent food facilities from being 
targeted by intentional attempts to contaminate the 
food supply. 202 Under the proposed rule, food facili¬ 
ties would be required to have a written food defense 
plan addressing significant vulnerabilities in their 
food production process, and to take measures to 
address these vulnerabilities, establish monitoring 
measures and corrective actions, confirm that the 
system is working, and ensure that workers assigned 
to vulnerable areas receive suitable training and 
maintain records. 202 


SUMMARY 


Any biological pathogen, whether bacteria, virus, 
toxin, or parasite, has the potential to be used in a ter¬ 
rorism context. Historical examination of both pur¬ 
poseful and inadvertent food and waterborne disease 
outbreaks can greatly assist in understanding how 
such events occur and how they may be prevented. 
A comprehensive understanding of animal produc¬ 


tion and crop farming, as well as food production 
and distribution, is required to ensure protection 
for the agricultural industry from terrorism events. 
Absolute safety of the food supply is perhaps an 
unattainable goal, but should be the benchmark for 
which all food protection and agricultural efforts 
are directed. 


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92 


Chapter 4 


CONSEQUENCE MANAGEMENT: 
THE LOCAL AND NATIONAL 
RESPONSE 


NEAL E. WOOLLEN, DVM, MSS, PhD/ and GARY W. CARTER + 


INTRODUCTION 

CONSEQUENCE MANAGEMENT 

CONSEQUENCES OF A BIOLOGICAL INCIDENT 

LOCAL AND NATIONAL RESPONSE 

RECOVERY 

SUMMARY 


*Colonel, Veterinary Corps, US Army; Director, Department of Defense Biological Select Agent and Toxin Biosafety Program Office, 1546 Porter Street, Fort 
Detrick, Maryland 21702 

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


93 



Medical Aspects of Biological Warfare 


INTRODUCTION 


Consequence management is critical to minimizing 
the long-term impact from any natural or manmade 
disaster. A common aim of organizations that seek 
to induce terror is to cause maximum disruption to 
societies with no regard for the impact on human life. 
Effective consequence management will minimize 
a disaster's impact on a society, its people, its infra¬ 
structure, and its economy. It will deliver the right 
solutions at the right time to the right locations in a co¬ 
ordinated and controlled response. Many, but not all, 
consequences of a catastrophic event are predictable, 
and many catastrophic events share common types of 
consequences. An effective consequence management 
plan focuses on the critical functions and capabilities 
necessary to span multiple types of disasters that ef¬ 
fectively minimize the disaster's impact. The plan must 
be comprehensive, flexible, and scalable. It must be 
adaptable to variable outcomes of varying magnitude 
and potential cascading effects of catastrophic events 
that may require a rapid transition from the initial plan 
to a more comprehensive one. 

Constructing an effective consequence management 
plan starts with understanding the threats to a populace, 
geographic location, or specific entity and the potential 
impact of those threats on all facets of the affected area. 

It must identify local response capabilities and functions 
and develop an activation strategy for timely implemen¬ 
tation. It should also demonstrate an understanding of 

CONSEQUENCE 

The definition of consequence management has 
evolved over time. At one time a clear separation 
existed between crisis management and consequence 
management. In a November 20, 2003 hearing be¬ 
fore the House of Representatives, Subcommittee 
on Crime, Terrorism, and Homeland Security, Mr. 
Howard Coble defined crisis management as actions 
taken to anticipate, prevent, or resolve a threat, with 
consequence management fulfilling the cleanup and 
restoration functions after an attack. 1 To truly mitigate 
the short-term and long-term impacts of catastrophic 
events, one must think and plan over a continuum 
of prevention, protection, and mitigation through 
response and recovery. 

The NDRF emphasizes that community recovery 
can be accelerated through a community's efforts in 
predisaster preparedness, mitigation, and recovery 
capacity building. 2 Recovery efforts must not interfere 
with immediate response efforts to preserve life and 
health and maintain critical infrastructure. However, 
immediate response efforts can mitigate long-term 


regional, state, and federal response capabilities and 
functions and outline a mechanism for requesting as¬ 
sistance. Effective consequence management starts at 
the local level, but can rapidly escalate with the need 
to coordinate higher-level supportive response with 
ongoing local response and recovery efforts. 

Federal response and recovery key planning con¬ 
siderations and responsibilities are identified in the 
National Response Framework (NRF) and the National 
Disaster Recovery Framework (NDRF), available for 
download from the Department of Homeland Secu¬ 
rity (DHS) and the Federal Emergency Management 
Agency (FEMA) digital libraries. They identify es¬ 
sential support functions and recovery support func¬ 
tions that guide local, state, and interagency federal 
all-hazards planning for response and recovery. The 
Biological Incident Annex to the NRF outlines the 
actions, roles, and responsibilities associated with a 
human disease outbreak of known or unknown origin 
requiring federal assistance. These documents empha¬ 
size a common theme: response and recovery will start 
at the local level and local involvement throughout is 
critical to success. Robust local planning to reduce an 
entity's vulnerability to the threats of biological inci¬ 
dents, with a well-thought plan for disaster response 
and recovery that meshes well with state and federal 
assistance plans, can save lives, minimize impact, and 
defeat terrorist objectives. 

MANAGEMENT 

effects and the overall impact of the disaster with sig¬ 
nificant impact on postdisaster recovery—positively 
or negatively—and the two should be planned and 
executed in harmony. 

The NDRF emphasizes that recovery encompasses 
far more than the restoration of a community's physical 
structures; it must also provide a continuum of care to 
meet the needs of the affected community members 
who have experienced financial, emotional, or physical 
impacts. 2 Good communication and coordination early 
and throughout a disaster response will help inform 
long-term recovery planning as well as prevent selec¬ 
tion of short-term solutions that may result in negative 
long-term impact. Integration among the frameworks 
is considered one of the key themes of the National 
Preparedness Goal (NPG). 3 

Planning must be risk based and risk assessments 
must be comprehensive and standardized. Planners, 
regardless of their affiliation, must understand what 
threats have the greatest potential impact on their area 
of responsibility. Threat and hazard identification and 


94 


Consequence Management: The Local and National Response 


risk assessment guidance is provided in the Compre¬ 
hensive Preparedness Guide 201. 4 The guide describes 
a five-step process for threat and hazard identification 
and risk assessment: 

1. identifying threats and hazards of concern; 

2. giving them context; 

3. examining core capabilities; 

4. establishing capability targets; and 

5. applying results. 

The Army Techniques Publication No. 5-19 outlines 
a risk management approach to analyzing and mitigat¬ 
ing hazardous operations. Core components of risk 
management are conducting a hazard analysis based 
on source, mechanism, and outcome of each hazard; 
an assessment of risk based on the probability the 
hazard will be experienced; and the severity of the 
outcome, followed by development of a strategy to 
mitigate risk that focuses on lowering the probability 
or lessening the severity resulting in an acceptable level 
of residual risk. Risk assessment must be continuous 
and updated throughout the course of a response to 
hazardous conditions, and it must be specific to the 
environment, local infrastructure, and population. 
Severity of outcomes will likely vary significantly be¬ 
tween heavily populated areas and sparsely populated 
areas and could vary considerably within the same 
city depending on what part of the city is affected, 
time of day, or whether the incident occurs during a 
workweek, weekend, or special event. 

Both risk management systems stress the impor¬ 
tance of identifying hazards through lessons learned 
from past events and subject matter expert opinion, 
understanding how they may affect an entity's op¬ 
erations, identifying capabilities that can mitigate the 
impact of the hazard, and proper resourcing to imple¬ 
ment the necessary control measures. These systems 
can assist planners with selecting controls to mitigate 
outcomes, inform predisaster resourcing to enhance 
preparedness (eg, establishment of memoranda of 
agreement), decrease the residual risk, and assist with 
comprehensive consequence management planning. 
An example of where this can assist consequence 
management following a population's exposure to a 
biological agent or toxin is to factor in the mechanism 
by which the resultant disease can spread. 

Some aspects of consequence management follow¬ 
ing exposure to a biological agent that causes conta¬ 
gious disease could significantly differ from one that 
is not. An overreaction for a disease-causing agent that 
is not contagious could cause unnecessary negative 
impact to the local infrastructure and economy and 
further complicate long-term recovery. Conversely, 


a lack of planning or inability to control the spread 
of a contagious disease could result in uncontrolled 
spread beyond the contamination zone with severe to 
catastrophic impact on life, health, infrastructure, and 
economy over a broad geographic region. A contagion 
will likely require implementation of quarantine and/ 
or isolation as a control measure, but quarantine and/ 
or isolation may not be appropriate for an agent that 
does not cause a contagious disease. The mechanism 
by which a hazard produces negative outcomes is 
important. When local planners do not have subject 
matter experts available to assist with planning, they 
should seek assistance from county, state, and federal 
public health professionals. 

The general population does not understand 
the unique differences among the various potential 
pathogens, their mechanisms of transmission, and the 
differences in risk created by each. Misinformation dis¬ 
seminated through rumors or poorly informed news 
outlets can create additional challenges to the response 
and recovery effort. It can lead to confusion as well 
as a loss of confidence in those leading the response 
and recovery effort. Timely accurate information dis¬ 
semination to the community is important, whenever 
a threat to public safety occurs. 

An analysis of the human response to the cata¬ 
strophic events of September 11, 2001 reveals that 
fear during a crisis situation does not automatically 
result in panic, and the negative impact of fear may 
manifest more significantly during the consequence 
management period when considerable uncertainty 
exists. 5 It revealed a tremendous spirit of cooperation 
and compassion among the affected population during 
the crisis period. Timely release of accurate informa¬ 
tion can mitigate the effects of misinformation and 
facilitate a spirit of teamwork throughout response 
and recovery. Close coordination of press releases 
with public affairs professionals is critical to accurate 
information dissemination. 

One intending to induce terror will rely on a haz¬ 
ard's natural and intended consequences but will 
also benefit from unintended consequences that are 
a product of poor or narrowly focused planning, that 
is not flexible and scalable, resulting in an inefficient 
response with poor communication. Similarly, the 
magnitude of impact from a naturally occurring 
outbreak of infectious disease can be minimized or 
magnified by the quality of response planning and 
efficiency of execution. 

Selecting the appropriate medical countermeasures 
and understanding the potential effects will rely on ac¬ 
curate agent identification at the time of the incident, 
but detailed planning for specified agents would be an 
inefficient approach to general consequence manage- 


95 


Medical Aspects of Biological Warfare 


merit and would risk not having an actionable plan in 
place when needed. Local, state, and federal response 
and recovery planning can leverage common mecha- 

CONSEQUENCES OF A 

Biological incidents may be one of the most challeng¬ 
ing threat conditions for planning consequence manage¬ 
ment. Response and recovery for most disasters follow 
a major catastrophic event that is relatively rapidly de¬ 
finable in scope and magnitude of impact. Conversely, 
it is unlikely that a biological incident will present as 
a well defined major catastrophic event. However, it 
carries the potential for significant casualties and major 
disruption to a community, region, or nation's infra¬ 
structure and economy, with a significant fear factor 
and potential for panic among a nation's population. If 
it involves a contagion, it also has the potential to spread 
well beyond the original target area in a short period 
of time. If it involves a zoonotic disease-causing agent, 
or is equally infectious to a local animal population, 
secondary spread from human-animal contact may 
persist and human health efforts must be synchronized 
with that of the animal health and feral animal control 
industries. With the exception of an emerging infec¬ 
tious disease with pandemic potential, where initial 
cases have already been identified, no warning or op¬ 
portunity will likely occur for leaders to proactively 
surge or position resources. A scalable response must be 
part of a community's and nation's normal framework. 

A series of framework documents can guide plan¬ 
ners through consequence management planning and 
execution: 

• National Prevention Framework, 

• National Mitigation Framework, 

• NRF, and 

• NDRF. 

All disasters have the potential for cascading effects, 
especially for a biological incident. Those intending to 
induce terror will attempt to leverage this potential to 
achieve their objectives and the response and recovery 
efforts must control them to minimize the impact on 
a society. Whether a biological incident is intentional, 
accidental, or results from a natural disease outbreak, 
it is important to rapidly identify the potentially af¬ 
fected area and population and control movement. 
Without these controls rapidly implemented, the zone 
of potential contamination will grow. Concurrent with 
controlling movement into and out of the affected area, 
the agent or agents involved must be rapidly identified. 
Differing biological agents have differing incubation 
periods, environmental survivability, and modes of 
transmission. Appropriate medical countermeasure 


nisms and common potential outcomes to be prepared 
for many potential threats rather than constructing 
plans for every possible disease causing agent. 

BIOLOGICAL INCIDENT 

and decontamination procedure identification and 
implementation timelines will rely on accurate agent 
identification. If animals and insect vectors can be 
potential sources of residual infection and sources for 
spreading the disease causing agents, the appropriate 
subject matter experts must be included in response 
and recovery planning. 

Sick people with relatively generic conditions show 
up in emergency departments, urgent care clinics, and 
primary care clinics on a daily basis. It is likely that 
a disaster will not be declared until their condition is 
identified as part of a natural disease with pandemic 
potential or tied to an act of bioterrorism. By this time 
the consequences of the causative event are being 
experienced, and the challenge of determining where 
it started and what caused it exists. The immediate 
consequences are obvious and rely on availability of 
the appropriate diagnostic assays and medical counter¬ 
measures. Some critical questions must be immediately 
answered: 

• Is this disease typical of the human population 
or geographic location? 

• Is it contagious? 

• Are animals also affected? 

• Is it zoonotic? 

• Does the pattern of disease in the population 
suggest a natural outbreak or an intentional 
release? 

Answers to these questions will inform immediate 
response and identify potential cascading effects and 
consequences that must be managed. If the incident is 
determined to be suspicious for an intentional release, 
a criminal investigation must immediately accompany 
the public health and epidemiologic investigations. 
These efforts must be synchronized to ensure no dis¬ 
ruption to response efforts intended to preserve life 
and health while also preserving evidence to facilitate 
the criminal investigation. 

A biological incident will present a significant 
amount of uncertainty for professionals responding 
to the incident and this will be magnified throughout 
the community. Timely accurate dissemination of 
information can mitigate a public reaction that may 
compound the problem if not appropriately managed. 

The National Incident Management System, which 
calls for a unified approach to multiagency coordi¬ 
nation during response and recovery operations. 


96 


Consequence Management: The Local and National Response 


emphasizes the unified approach concept based on 
chain of command, unity of command, unity of effort, 
and when implemented, unified command. 6 Unity of 
effort is critical to disseminating consistent informa¬ 
tive messages at the right time to assist with manag¬ 
ing public fear and reaction. Conflicting information 
from multiple agencies creates confusion, a lack of 
confidence in the response and recovery effort, and 
potential panic. 

The anthrax (Bacillus anthracis) attacks of 2001 
provide lessons on the value of effective risk com¬ 
munications and public reaction. Confusion follow¬ 
ing the initial reports was widespread on a national 
scale. Examples of communication failures include 
the following: 

• 46% of the population thought anthrax was a 
contagious disease. 

• 70% of the New Jersey population were con¬ 
cerned that they or someone close to them had 
been exposed. 

• Agencies were overwhelmed by requests for 
information. 

• Reports of discontent with both quality and 
timeliness of information were heard.' 

An example of risk communication success came 
from a multiagency task force in New Jersey that 
was reported as beneficial for enhancing cooperation 
between law enforcement and health organizations 
and reducing tensions. Law enforcement and health 
organizations approach communications differently, 
with law enforcement leaning toward secrecy and 
public health valuing openness. 7 By managing dis¬ 
parate philosophies through a task force or unified 
command element, messaging can be less confusing 
for the recipients, with greater efficiency in providing 
informative messages in a timely manner that do not 
compromise criminal investigations and serve to pal¬ 
liate public fear and reaction. 

Preserving the life and health of an affected popula¬ 
tion must be the primary concern during response and 
recovery, but consequence management planning and 
execution for a biological incident cannot be narrowly 
focused on individual patient care. If a biological agent 
is intentionally dispersed, the dispersion method must 
be rapidly characterized to determine the extent of 
contamination and identify the affected human and/or 
animal population(s). If agent dispersion is covert and 
the first indication of an act of bioterrorism or criminal 
act involving a biological agent is one or more patients 
presenting with clinical disease, determining the extent 
of contamination and the affected population will be 
challenging but critical to effectively managing the 
consequences. Once the affected area is determined. 


access to and egress from the area must be controlled, 
but controls must be tailored to the type of agent(s) 
identified. 

A detailed assessment of the affected area that 
includes personnel and equipment movement, wild 
and domestic animal populations and movement, 
and surface water flow patterns must be conducted 
to determine the potential for contamination spread 
and other potentially affected populations. If the af¬ 
fected area involves significant community services, 
planners will need to determine how to continue 
providing those services to the unaffected community 
to minimize the incident's overall impact, especially 
if it involved any services critical to the response and 
recovery effort. The initial assessment of causative 
agent, method of dispersion, and extent of contamina¬ 
tion will also assist health providers and planners on 
selection of medical countermeasures and the potential 
number of expected human casualties. If the extent of 
contamination is large or multifocal, or if the causative 
agent is a contagion, patient care facilities and medical 
countermeasures could be rapidly overwhelmed, ne¬ 
cessitating the need for rapid coordination of external 
support. Plans and support agreements to manage this 
potential consequence must be in place before an inci¬ 
dent. These agreements are an area where neighboring 
communities and neighboring states can seek mutual 
aid and support when one or the other is affected by 
a catastrophic event. 

The impact of a biological incident will likely extend 
well beyond the primary concern of human health 
considerations. Whether secondary hazards from a 
biological incident are real or perceived, they will have 
significant impact on the affected entity and surround¬ 
ing population. Biological incidents have the potential 
to overwhelm local healthcare resources and render 
any business or public service provider inoperable. 
If a community's healthcare facility and emergency 
response capabilities are contaminated, it is imperative 
that a backup plan for care and emergency response is 
in place. How effectively a community conducts waste 
management will either create or mitigate secondary 
concerns and the impact of accumulating waste. Waste 
may also require special treatment that is not part of 
a community's standard operating procedures, and 
special assistance may be required. 

Communities should develop continuity of opera¬ 
tions plans to identify backup resourcing for critical 
services. Businesses should also develop continuity 
of operations plans to protect their livelihood in case 
they are directly or indirectly affected by an emergent 
situation and unable to occupy their normal place of 
business. The former $3.8 million American Media Inc. 
building in Boca Raton, Florida, was quarantined on 
October 10, 2001, sold for $40,000 in April 2003, and 


97 


Medical Aspects of Biological Warfare 


was not reported to be clear of Bacillus anthracis until 
February 7, 2007. 8 The Hart Senate Office Building 
reopened after it cost $27 million to decontaminate 


it. Economic impact on an affected business can be 
catastrophic and a protracted disruption to services 
can occur when a public service facility is affected. 


LOCAL AND NATIONAL RESPONSE 


The NPG suggests that successful consequence 
management of a biological incident will result from 
capabilities being available across the whole commu¬ 
nity to prevent, protect against, mitigate, respond to, 
and recover from the threats and hazards that pose 
the greatest risk. 9 The NPG and supporting frame¬ 
work documents emphasize that an effective response 
starts at the local level with individuals, community 
organizations, the private and nonprofit sector, faith- 
based organizations, and local governments all having 
a critical role with support from the state and federal 
governments. 

The NPG identifies core capabilities for five mission 
areas: (1) prevention, (2) protection, (3) mitigation, 
(4) response, and (5) recovery. It recognizes that the 
core capabilities listed are ambitious and will require 
a national effort involving the whole community to 
be effective, with three core capabilities spanning all 
five mission areas: (1) planning, (2) public informa¬ 
tion and warning, and (3) operational coordination. 9 
The NPG lists five key findings from the Strategic 
National Risk Assessment; two key risk areas correlate 
with the subject of this chapter: a virulent strain of 
pandemic influenza and terrorist use of weapons of 
mass destruction. 9 

Planners at all levels should assess their level of 
readiness to manage consequences associated with 
these risks, but not limit their planning to just these 
risks. Every community and state may have differing 
priority lists for planning. Military planners routinely 
assess two types of risks to operations: most likely 
and most dangerous. This approach can also prove 
valuable for planners as they develop local and state 
core capabilities for effectively managing risks posed 
by various biological threats. 

Although planners should be familiar with several 
framework documents, the remainder of this chapter 
will primarily focus on guidance from the NRF, the 
NRF Biological Incident Annex, the NDRF, and other 
supportive documents. Framework documents em¬ 
phasize the significance of local readiness and response 
and provide federal agency level guidance that can be 
tailored to all levels of government planning. With a 
few exceptions, federal assets will not be mobilized 
until local and state capabilities have been or likely 
will be exceeded and a state's governor requests federal 
assistance under the Stafford Act. These documents 
provide valuable guidance for planning at all levels. 


The NRF lists 14 core capabilities, 15 emergency 
support functions, and four priorities for the response 
mission area that local and state planners can use as a 
template for their planning activities. Core capabilities 
include the following: 

1. planning; 

2. public information and warning; 

3. operational coordination; 

4. critical transportation; 

5. environmental response/health and safety; 

6. fatality management services; 

7. infrastructure systems; 

8. mass care services; 

9. mass search and rescue operations; 

10. on-scene security and protection; 

11. operational communications; 

12. public and private services and resources; 

13. public health and medical services; and 

14. situational assessment. 10 

Emergency support functions (ESF) include the 
following: 

1. ESF1 transportation; 

2. ESF2 communications; 

3. ESF3 public works and engineering; 

4. ESF4 firefighting; 

5. ESF5 information and planning; 

6. ESF6 mass care, emergency assistance, tem¬ 
porary housing and human services; 

7. ESF7 logistics; 

8. ESF8 public health and medical services; 

9. ESF9 search and rescue; 

10. ESF10 oil and hazardous materials response; 

11. ESF11 agriculture and natural resources; 

12. ESF12 energy; 

13. ESF13 public safety and security; 

14. ESF14 (replaced by the NDRF); and 

15. ESF15 external affairs. 

Response mission priorities include the following: 

1. save lives; 

2. protect property and the environment; 

3. stabilize the incident; and 

4. provide for basic human needs. 10 


98 


Consequence Management: The Local and National Response 


Coordinating agencies are identified for each of the 
emergency support functions at the federal level; local 
and state planners should identify assets for planning 
at their respective levels. Objectives for each of the 
core capabilities listed in the NRF are summarized in 
Table 4-1. Local planners will likely not have resources 
available to meet all of these objectives, but the list can 
assist them with determining what local assets need 
to be factored into a local response plan and what 
support requirements are needed to coordinate with 


neighboring communities or the private sector through 
support agreements, or requests from county, state, or 
federal partners. 

The effective response to a catastrophic event will 
require meeting many of the core capability objectives 
through local assets, at least for initial response and 
later to augment state or federal response efforts. Lo¬ 
cal assets may include individuals, the private sector, 
nongovernmental organizations, and neighboring 
communities. 


TABLE 4-1 

CORE CAPABILITIES AND OBJECTIVES 


Capability 


Objectives 


Planning Conduct a systematic process engaging the whole community in the development of execut¬ 

able strategic, operational, and/or community-based approaches to meet defined objectives. 

Public Information and Warning Deliver coordinated, prompt, reliable, and actionable information to the whole community 

through the use of clear, consistent, accessible, and culturally and linguistically appropri¬ 
ate methods to effectively relay information regarding any threat or hazard and the actions 
being taken and the assistance being made available. 

Operational Coordination Establish and maintain a unified and coordinated operational structure and process that ap¬ 

propriately integrates all critical stakeholders and supports the execution of core capabilities. 

Critical Transportation Provide transportation (including infrastructure access and accessible transportation services) 

for response priority objectives, including the evacuation of people and animals, and the 
delivery of vital response personnel, equipment, and services to the affected areas. 

Environmental Response/Health and Safety Ensure the availability of guidance and resources to address all hazards, including hazardous 

materials, acts of terrorism, and natural disasters, in support of the responder operations 
and the affected communities. 


Fatality Management Services 

Infrastructure Systems 

Mass Care Services 

Mass Search and Rescue Operations 

On-scene Security and Protection 

Operational Communications 

Public and Private Services and Resources 

Public Health and Medical Services 

Situational Assessment 


Provide fatality management services, including body recovery and victim identification to 
provide temporary mortuary solutions, sharing information with Mass Care Services for 
the purpose of reunifying family members and caregivers with missing persons/remains, 
and providing counseling to the bereaved. 

Stabilize critical infrastructure functions, minimize health and safety threats, and efficiently 
restore and revitalize systems and services to support a viable resilient community. 

Provide life-sustaining services to the affected population with a focus on hydration, feeding, 
and sheltering to those with the most need, as well as support for reunifying families. 

Deliver traditional and atypical search and rescue capabilities, including personnel services, 
animals, and assets to survivors in need, with the goal of saving the greatest number of 
endangered lives in the shortest time possible. 

Ensure a safe and secure environment through law enforcement and related security and 
protection operations for people and communities located within affected areas and for 
all traditional and atypical response personnel engaged in lifesaving and life-sustaining 
operations. 

Ensure the capacity for timely communications in support of security, situational awareness, 
and operations by any and all means available between affected communities in the impact 
area and all response forces. 

Provide essential public and private services and resources to the affected population and 
surrounding communities to include emergency power to critical facilities, fuel support for 
emergency responders, and access to community staples (eg, grocery stores, pharmacies, 
and banks) and fire and other first response services. 

Provide lifesaving medical treatment via emergency medical services and related operations, 
and avoid additional disease and injury by providing targeted public health and medical 
support and products to all people in need within the affected area. 

Provide all decision makers with decision-relevant information regarding the nature and 
extent of the hazard, any cascading effects, and the status of the response. 


Data source: Department of Homeland Security. National Response Framework. 2nd ed. Washington, DC: DHS; May 2013. 


99 





Medical Aspects of Biological Warfare 


Individuals within a community possess talents 
and experience that can be organized for response 
through community organizations. Individuals 
should participate in community preparedness plan¬ 
ning activities and develop household emergency 
plans. 10 Individuals can also participate in FEMA's 
Community Emergency Response Team Program, 
which educates people on how to prepare for haz¬ 
ards that may affect their area and trains them in 
basic disaster response skills, such as fire safety, light 
search and rescue, team organization, and disaster 
medical operations. 11 Community Emergency Re¬ 
sponse Team Program trained individuals can play 
a critical role in assisting with local planning and 
response. Private sector entities can support local 
emergency management and should also participate 
in community preparedness planning activities. 10 
Private sector entities should also conduct continuity 
of operations planning within their own organiza¬ 
tion to establish a plan that will foster their contin¬ 
ued support or service to the community while also 
preserving their livelihood. 

Nongovernmental organizations may factor into 
any level: local, state, or federal response. They manage 
volunteers and resources to support incident response 
through collaboration with responders, all levels of 
government, and other agencies and organizations. 10 

Neighboring communities can play a critical role 
in consequence management for a biological incident. 
Community dynamics can be significantly disrupted 
within the contamination zone of a biological incident, 
but life outside the contamination zone will continue 
with no change in requirement for services and support. 
If a community's critical services are located within 
the contamination zone and are rendered inoperable 
because of real or perceived contamination, then mutual 
aid agreements among communities can fill critical gaps 
in the response effort as well as continuation of services 
to the unaffected parts of the community. 

When local resources are exhausted or prove to 
be inadequate, local authorities may seek county or 
state assistance; in some situations, local authorities 
may seek assistance directly from the federal govern¬ 
ment for non-Stafford Act incidents. 10 Some federal 
departments or agencies, using funding sources other 
than the President's Disaster Relief Fund, can conduct 
or lead federal response actions under their own 
authorities. 10 Examples include immediate lifesav¬ 
ing assistance, wild-land firefighting, response to an 
agricultural disease, cybersecurity incidents, and oil 
and hazardous substance response operations. 10 The 
Secretary of the Department of Health and Human 
Services (DHHS) has the authority to take actions to 
protect the public health and welfare and declare a 
public health emergency. 10 


State governors are responsible for the public safety 
and welfare of their state's residents. Their responsi¬ 
bilities and authorities include making, amending, 
or suspending orders or regulations associated with 
response; communicating to the public; coordinating 
with tribal governments; commanding the state mili¬ 
tary force (National Guard personnel not in federal 
service); coordinating assistance from other states; and 
requesting federal assistance. 10 A state's response to 
emergency situations is coordinated through the state 
emergency management agency. 

Numerous state departments and agencies have a 
role in response and recovery, but the National Guard 
is one of the governor's key assets for a biological 
incident. National Guard members can be valuable to 
consequence management because of their expertise 
in emergency medical response; communications; 
logistics; search and rescue; civil engineering; and 
chemical, biological, radiological, and nuclear re¬ 
sponse, planning, and decontamination. 10 Weapons 
of mass destruction/civil support teams are highly 
specialized National Guard units designed to provide 
unique capabilities for response to chemical, biological, 
radiological, or nuclear incidents, primarily in a Title 32 
operational status within Washington, DC, the United 
States, its territories, and its possessions. 12 

Federal financial aid or other support to response, 
recovery, and mitigation efforts are authorized fol¬ 
lowing a Stafford Act emergency or major disaster 
declaration by the president. An emergency declara¬ 
tion is more limited in scope, provides fewer federal 
programs, and is not normally associated with recov¬ 
ery programs, but it may be used before an incident 
to mitigate the threat of a potential catastrophe. 10 Most 
of the president's authority under the Stafford Act has 
been delegated to the FEMA administrator through the 
Secretary of Homeland Security. 10 A state's governor 
may request federal assistance through the FEMA re¬ 
gional administrator when the situation is considered 
beyond the capabilities of the state and affected local 
government. 10 

The DHHS is the coordinating agency for the bio¬ 
logical incident annex to the NRF. Federal government 
objectives for response to a biological incident—natu¬ 
rally occurring or as an act of terrorism—are as follows: 

• detect the event through disease surveillance 
and environmental monitoring; 

• identify and protect the population(s) at risk; 

• determine the source of the disease; 

• assess the public health, law enforcement, and 
international implications; 

• control and contain any possible epidemic; 

• augment and surge public health and medical 
services; 


100 


Consequence Management: The Local and National Response 


• identify the cause and prevent the recurrence 
of any potential resurgence, additional out¬ 
breaks, or further spread of disease; and 

• assess the extent of residual biological con¬ 
tamination and conduct response, restoration, 
and recovery actions. 13 

Detection of a biological incident may be by the 
presentation of disease in humans or animals or envi¬ 
ronmental surveillance systems, or by acts of bioter¬ 
rorism detected though the normal operations of other 
cooperating departments and agencies. 13 The National 
Biosurveillance Integration System is a tool that sup¬ 
ports detecting disease outbreaks by leveraging data 
from multiple surveillance systems that monitor human 
health, animal health, plant health, and food and water. 13 
Monitoring for dangerous pathogens in some heavily 
populated places is accomplished through the Bio Watch 
program, which serves as an early detection and warning 
system. 14 This program has been criticized for creating 
false alarms, but those criticisms probably come from 
individuals who do not fully understand the technologies 
used, intent of the program, and confirmatory process 
that follows an alert. DHS partners with public health 
laboratories through the Laboratory Response Network 
to rapidly confirm any alerts from BioWatch systems. 

Detection technologies need to be rapid and sensi¬ 
tive; they need to ensure that no false negatives occur. 
Specificity is ensured through the laboratory confirma¬ 
tory process that follows. People will live with a few 
false alarms, but they will become sick and potentially 
die from false negative results at the detection level. It 
is unlikely that samples will be forwarded for confir¬ 
matory analysis if results are negative at the detection 
level, so these systems should be judged more on their 
potential to prevent false negatives than false positives. 
These systems are value added if they are not over in¬ 
terpreted before confirmatory analysis. Claims of false 
positives have been characterized as unsubstantiated, 
with more than 7 million tests performed by public 
health laboratories and no false positives. 14 

DHHS convenes a meeting of ESF #8 partners, after 
notification of a credible threat or disease outbreak, to 
assess the situation and determine appropriate public 
health and medical actions. 13 If the threat or disease 
outbreak is suspected to be tied to a criminal or ter¬ 
rorist act, the Federal Bureau of Investigation will 
lead a concurrent criminal investigation and possibly 
establish a joint operations center. 13 Joint operations 
centers are valuable to establishing unity of effort. 
Agencies with disparate primary objectives will be 
working simultaneously toward a common outcome, 
but do not always fully understand each other's mis¬ 
sion priorities; synchronization of efforts is critical to 
mission success. 


It is important for first responders to understand 
that they may be working within a crime scene 
and that all materials may have evidentiary value, 
but this fact cannot compromise mitigating the 
immediate threats to life and health. If a criminal 
or terrorist incident initially presents as disease in 
humans or animals, criminal intent may not be ap¬ 
parent for some time and evidence may already be 
compromised. The Laboratory Response Network 
is used to test samples whenever a credible threat 
of a biological crime or act of terrorism exists. 13 If 
contamination of food is suspected the Food Emer¬ 
gency Response Network, a complementary system 
to the Laboratory Response Network, may be used 
for food sample analysis. 13 

Other federal agencies will support DHHS during 
a biological incident response. The DHS will serve 
as the incident coordinator. The Environmental 
Protection Agency will develop and implement 
sampling strategies when a potential for environ¬ 
mental contamination exists. The Department of 
Agriculture will provide support for an outbreak 
of an agriculturally significant zoonotic disease 
or human foodborne pathogen. Federal public an¬ 
nouncements, statements, or press releases will be 
coordinated with the DHS Office of Public Affairs, 
consistent with ESF #15. 13 

An epidemic resulting from the introduction of a 
contagious biological agent into a population is one 
of the most significant—and likely the most danger¬ 
ous-potential consequences of a biological incident. 
Effectively managing this potential consequence relies 
on the following: 

• rapid detection, and identification and confir¬ 
mation of the biological agent; 

• identification of the population at risk; 

• determination of how the agent is transmitted; 

• determination of appropriate medical coun¬ 
termeasures; 

• administration of countermeasures; 

• rapid dissemination of safety information to 
the public; and 

• control and containment strategies. 

Planning must include worst-case scenario branches 
for mass casualties if early control measures are not 
effective and containment is not achieved, requiring 
augmentation and surging of health and medical 
resources in order to track and prevent additional 
disease outbreaks. 

DHHS assists partner public health and medical au¬ 
thorities with epidemic surveillance and coordination, 
and it will assess the need for increased surveillance. 
DHS, with partner organizations, coordinates timely. 


101 


Medical Aspects of Biological Warfare 


consistent, accurate, and actionable information dis¬ 
semination. The public health system, starting at the 
local level, initiates appropriate protective measures 
for the affected population, including all workers 
involved in incident response. DHHS, with partner or¬ 
ganizations involved, evaluates the need for isolation, 
quarantine, or shelter-in-place measures to prevent 
spread of disease. If isolation and/or social distancing 
are recommended, the affected state's governor may 
implement these measures under state or local legal 
authorities. Tribal leaders also possess this authority 
under tribal legal authority. 

DHHS may take appropriate federal actions to 
prevent the import or interstate spread of disease. 
If the source of the disease outbreak is identified as 
originating outside the United States, DHHS works 
with DHS and other agencies to identify and isolate 
persons, cargo, mail, or conveyances that may be 
contaminated. If it is determined that food, animals, 
and other agricultural products need to be quar¬ 
antined, livestock or poultry need to be vaccinated 
or depopulated, and/or movement of animals and 
equipment need to be restricted, DHHS will work 
with the Department of Agriculture and other partner 
organizations. DHHS works through the Depart¬ 
ment of State to notify affected foreign governments 
if foreign nationals are subjected to isolation and/or 
quarantine. 13 

The ability to care for sick and/or potentially 
exposed people is one of the most critical response 
requirements that must be incorporated into pre¬ 
disaster response planning. The Strategic National 
Stockpile is a national repository of medical coun¬ 
termeasures, vaccines, and medical supplies stored 
in strategic locations. 15 Division of Strategic National 
Stockpile personnel, from the Centers for Disease 
Control and Prevention's Office of Public Health 
Preparedness and Response, will assist as local and 
state health departments prepare for receipt, distri¬ 
bution, and dispensing of medical countermeasures 
from the Strategic National Stockpile. 15 These medi¬ 
cal countermeasures, vaccines, and medical supplies 
are free to the public, and states have plans to receive 
and distribute them once federal and local authori¬ 
ties agree that they are needed. 16 Strategic National 
Stockpile supplies include 12-hour push packages, 
CHEMPACKs (program that provides antidotes to 
nerve agents [three countermeasures used concomi¬ 
tantly] for prepositioning by state, local, and tribal 
officials), and federal medical stations. The 12-hour 
push packages contain 50 tons of a broad spectrum 
of medical assets and can be delivered to any state in 
the continental United States within 12 hours from 


the decision to deploy; if the incident requires ad¬ 
ditional or different supplies, they can be delivered 
within 24 to 36 hours. 15 Federal medical stations 
are rapidly deployable and modular, stocked with 
beds and supplies to care for up to 250 patients for 
up to 3 days. 15 

As with other aspects of the integrated local, state, 
and federal response effort, local technical expertise 
and local planning will be critical to efficient and suc¬ 
cessful delivery of medical care to those who need it. 
Centers for Disease Control and Prevention developed 
the public health preparedness capabilities/national 
standards for state and local planning that can assist 
state and local public health officials with planning 
for this and other critical public health planning con¬ 
siderations. It identifies 15 public health preparedness 
capabilities under six domains 1 ': 

1. biosurveillance; 

2. community resilience; 

3. countermeasures and mitigation; 

4. incident management; 

5. information management; and 

6. surge management. 

This downloadable document is an excellent plan¬ 
ning guide that links planning and execution activities 
back to NRF emergency support functions and pro¬ 
vides links to additional resources. 

Beyond the challenge of medical countermeasure 
availability and distribution, a biological incident 
may challenge the ability of healthcare systems to 
adequately care for large numbers of patients that 
exceed local capabilities and capacities, and it will 
likely affect their ability to continue providing a 
standard of care to the local community for routine 
health issues. Maintaining medical system resiliency 
may require regional, state, or federal coordination 
and medical surge capacity and capability. The 
medical surge capacity and capability management 
system was developed to provide a systems-based 
approach for managing the complexity of mass ca¬ 
sualty or complex incidents. 18 Surge capacity is the 
ability to respond to a markedly increased number 
of patients. Surge capability is the ability to address 
unusual or very specialized medical needs. The 
medical surge capacity and capability management 
system is consistent with the National Incident Man¬ 
agement System and guides public health and medi¬ 
cal response through a six-tier approach, escalating 
from management of individual healthcare assets 
to federal support to state, tribal, and jurisdictional 
management. 


102 


Consequence Management: The Local and National Response 


RECOVERY 


Recovery operations focus on returning the affected 
region or entity, as closely as possible, to predisaster 
conditions. Initiation of recovery efforts does not re¬ 
quire full completion of response operations, but the 
transition process must be well synchronized with any 
continuing response efforts. Many components of the 
response effort will also influence recovery activities. 

As healthcare transitions from emergent and tempo¬ 
rary medical care, activities must ensure continuity of 
care and reestablishment of any disrupted healthcare 
capabilities. Continuity of care may need to be estab¬ 
lished through temporary facilities until services are 
fully restored; candidate facilities should be identified 
during consequence management planning activities. 
Surveillance should be initiated during response, as dis¬ 
cussed in the previous section, and continued through 
recovery until health officials determine that the discov¬ 
ery of new cases has met criteria for discontinuation. 

Effective messaging by public health professionals 
can serve to mitigate public fear and prevent panic. 
Several people will be identified, throughout response 
and recovery, who may benefit from counseling and 
behavioral health services. These services should be 
restored or made available as soon as possible. Lessons 
learned from previous disasters suggest that during 
the transition and recovery period, public fear can 
increase. During this period people will have gained 
awareness of the morbidity and mortality associated 
with the infectious agent or toxin and will have con¬ 
tinued—possibly escalating—fear about exposure to 
this invisible threat. 

Ineffective messaging may have contributed to 
public fear and panic during the 2001 Amerithrax 
incident. Many people thought anthrax was a conta¬ 
gious disease, and because the infectious agent was 
delivered in a powdered form there was widespread 
fear of powders in general. People were more aware 
of powders and powder-appearing residues after the 
incident. Powders associated with many normal activi¬ 
ties that went unnoticed or created no concern before 
the incident suddenly created concern, fear, and panic. 
The US Army Medical Research Institute of Infectious 
Diseases and many other laboratories involved in the 
recovery effort received thousands of samples for 
analysis that normally have an innocuous powder as¬ 
sociated with them. Effective accurate communications 
may have mitigated some of these concerns and will 
remain important throughout any recovery. 

Exposed populations and contaminated buildings, 
equipment, and environments will likely be identified 
during the response effort, but continued surveillance 


will remain important to identify additional cases of 
human or animal disease and potential contamination 
spread that will need to be included in decontamina¬ 
tion efforts. Decontamination can and will likely be 
challenging. Its effectiveness will depend on accurate 
identification of the contaminating infectious agent 
or toxin; assessment of primary and secondary areas 
of contamination; and selection of suitable decon¬ 
tamination reagents, equipment, and methods that 
factor in effectiveness for the contaminating agent 
and the environment. Appropriate subject matter 
experts should be included in planning and executing 
decontamination. 

Personnel involved in recovery operations will not 
have the benefit of established clearance strategies for 
reoccupation of contaminated facilities or resumed 
use of contaminated equipment for all potential 
biological agents. Members of the Environmental 
Protection Agency and the Centers for Disease Con¬ 
trol and Prevention published an interim clearance 
strategy for a building or an outdoor environment 
after an incident involving Bacillus anthracis in July 
2012. It was determined that no detection of viable 
spores is the best practicable clearance goal, 19 which 
is a sound goal for B anthracis as well as many other 
potential biological agents. It infers that the agent 
identification technology used will identify viability 
as well as continued presence of the pathogen on or 
in the sampled item. 

Some of the more sensitive agent identification 
technologies (nucleic acid amplification and anti¬ 
gen detection) will not demonstrate agent viability. 
Agents killed or neutralized during decontamina¬ 
tion may still be detected by these technologies and 
not properly inform clearance decisions. Cleanup 
procedures could be unnecessarily prolonged with 
no added benefit if decisions are being made based 
on technologies that do not aid the risk assessment 
procedure by demonstrating agent viability. Local 
or state public health officials or property owners 
will likely make the final decision on clearance. 19 
However, the lack of established standards and 
complexity of this decision process will likely 
necessitate the support of external subject matter 
experts. 

Establishing transportation routes becomes criti¬ 
cal during both response and recovery to facilitate 
response and recovery mitigation activities as well 
as continue providing critical services and support 
inside the contaminated area. Once a biological inci¬ 
dent has occurred, containment becomes important 


103 


Medical Aspects of Biological Warfare 


to all aspects of the management strategy. Factor¬ 
ing containment considerations into all subsequent 
planning will prevent the incident from growing in 
scale and magnitude, minimizing impact on human 
health, infrastructure, and economy. Strategies will 
vary depending on the situation and conditions, but 
some basic principles can be applied to all. Whenever 
one is dealing with biological contamination, it is 
beneficial to establish at least three zones: 

1. known or high probability to be contaminated 
(hot zone); 

2. not expected or low probability to be con¬ 
taminated (warm zone); and 

3. expected to be clean (cold zone). 

Operational procedures should be established 
for each zone that facilitate the movement of neces¬ 
sary supplies, personnel, and equipment to sustain 
operations and facilitate recovery without spreading 
contamination. If a clean corridor cannot be estab¬ 
lished through the warm zone to the hot zone, handoff 
procedures will need to be established for cross-zone 
movement. Decontamination procedures at each hand¬ 
off point will need to be established for any movement 
from hot zone to warm zone and from the warm zone 
to the cold zone. One strategy may be to have dedicated 
equipment in each zone that will facilitate the move¬ 
ment of personnel and supplies from the cold zone to 
the hot zone and sustain operations in the warm zone 
and hot zone. Personal protective equipment require¬ 
ments must also be established for each zone to prevent 
secondary contamination to workers. 

The NDRF promotes nine core principles for re¬ 
covery success: 

1. individual and family empowerment; 

2. leadership and local primacy; 

3. predisaster recovery planning; 

4. partnerships and inclusiveness; 

5. public information; 

6. unity of effort; 

7. timeliness and flexibility; 

8. resilience and sustainability; and 

9. psychological and emotional recovery. 2 

It promotes a concept of all-community involve¬ 
ment in recovery efforts to ensure that no groups 
of people and their unique interests are excluded 
during the recovery effort and that services are made 
equally available to everyone as all affected members 
of a community attempt to rebound from their losses. 
It affirms that local leaders and local governments 
maintain a primary role even when their response 


capabilities have been overwhelmed and state or 
federal assistance is required. It recognizes that 
partnerships and collaborations with unity of effort 
are essential to successful recovery and emphasizes 
that compliance with the principles of equal op¬ 
portunity and civil rights must be upheld. It further 
emphasizes the importance of clear, consistent, cul¬ 
turally appropriate, and frequent communications 
to the affected public. Timeliness and flexibility are 
emphasized to minimize missed opportunities and 
foster the ability to adapt to changing conditions. 
It recognizes that recovery can be negatively af¬ 
fected by cascading effects and additional hazards, 
emphasizing the significance of risk management 
to enhance resilience and sustainability practices 
to reconstruct the environment and revitalize the 
economic, social, and natural environments. Psy¬ 
chological and emotional recovery is recognized 
as vital to individuals, families, and communities. 

Local governments are responsible for planning and 
managing a community's recovery from all disasters. 2 
They shoulder the burden of preparing hazard miti¬ 
gation and recovery plans, raising hazard awareness, 
and educating their people on resources available to 
enhance resilience. Even though state and federal 
standards exist, the local government decides whether 
to adopt, codify, and enforce mitigation measures. 
Individuals, families, and businesses will look to local 
leaders for support during disasters, and local gov¬ 
ernments should establish continuity of government 
and continuity of operations plans. They are at risk of 
becoming overwhelmed and will likely need assistance 
from state and federal offices for critical staffing and 
recovery expertise. A critical local asset during any 
biological incident is the local or county public health 
agency, which will have established contingency plans 
and can assist with coordinating medical surge when 
needed. 

States lead, manage, and drive the overall recov¬ 
ery process; they coordinate recovery activities that 
include providing financial and technical support. 2 
They serve as a conduit to local and tribal govern¬ 
ments for federal recovery assistance programs, and 
they may develop programs or secure funding to 
finance or implement recovery projects. States can 
also reassign existing resources to facilitate recovery, 
and they play a critical role in strategic messag¬ 
ing to enhance public awareness. The state public 
health agency will play a critical role in messaging 
and coordinating medical assistance to the affected 
community. It is critical that state offices remain mis¬ 
sion capable during a disaster, and they should also 
develop and maintain continuity of government and 
continuity of operations plans. 


104 


Consequence Management: The Local and National Response 


The federal government may use the NDRF to en¬ 
gage necessary and available department and agency 
capabilities to support local recovery efforts when a 
disaster exceeds the capacity of the state and tribal 
resources or affects federal property or national se¬ 
curity interests. 2 Federal support is important when 
local and state resources are overwhelmed, especially 
during the early weeks following a large-scale disas¬ 
ter or catastrophic incident; the duration and extent 
of federal support will be partially determined by 
scale and enduring impact of the disaster. The federal 
government also plays a critical role in messaging to 
enhance public awareness about the threat and to 
inform stakeholders about federal grants and loans 
that can assist recovery efforts. The lead federal agency 
for coordination of health and social services during 
recovery is the DHHS. 


Similar to ESFs in the NRF outlining federal as¬ 
sistance for disaster response, the NDRF identifies 
multiagency coordinated recovery support functions 
in the following areas: 

• community planning and capacity building; 

• economics; 

• health and social services; 

• housing; 

• infrastructure systems; and 

• natural and cultural resources. 

Each annex outlines pre- and postdisaster activities 
as well as a list of objectives. The recovery support 
functions develop guidance and standard operating 
procedures for rapid activation to support community 
recovery. 2 


SUMMARY 


Consequence management has historically 
received the least amount of planning emphasis 
and has not been adequately tested through ro¬ 
bust exercises. By the nature of the problem it is 
complex, involves multiple agencies, and spans a 
considerable amount of time to exercise through 
response and recovery operations. The national 
framework documents offer a template for core 
capability development that can lead to readiness 
across a broad spectrum of potential disasters, 
and they can facilitate robust planning at the local 
level. These documents are already being used to 
develop plans at the state and federal levels. Unity 
of effort, which is critical to both effective response 
and recovery, can be developed through mul¬ 
tiagency exercises. Historically, response without 
continuation through full recovery has been exer¬ 
cised. Emergency response exercises can be time 
compressed, but should span the full spectrum of 
response and recovery, so agencies will be prepared 
to work together when a disaster strikes. 


Biological incidents are unique challenges. It is un¬ 
likely to know when a biological agent is dispersed or 
when the index case of a pandemic crosses a nation's 
border. Buildings will not be flattened, but they may 
be unsuitable for human occupation for an extended 
time, compromising critical services to the community, 
state, or nation. Economic impact could be significant 
and devastating to individuals and industries. A small 
focal dispersion of a biological agent could lead to 
broad impact with significant morbidity, mortality, 
and public fear if response and recovery efforts are 
not efficiently implemented to identify, contain, treat, 
protect, and clean. 

Consequence management is critical to mitigating 
the magnitude of impact a disaster has on a com¬ 
munity, state, and nation. History has proven that 
all disasters or terrorist acts cannot be prevented, but 
through effective consequence management the impact 
of both can be minimized and the terrorist's aim of 
maximal disruption can be defeated. Most importantly, 
lives can be saved. 


REFERENCES 


1. Serial No. 65: Homeland Security: The Balance Between Crisis and Consequence Management Through Training and Assistattce: 
Hearing before the Subcommittee on Crime, Terrorism, and Homeland Security of the Committee on the Judiciary House of Repre¬ 
sentatives, One Hundred Eighth Congress, First Session on HR 2512, HR 3266, and HR 3158. Washington, DC: Government 
Printing Office; November 20, 2003. https://www.hsdl.org/?view&did=446489. Accessed October 30, 2015. 

2. Department of Homeland Security. National Disaster Recovery Framework: Strengthening Disaster Recovery for the Nation. 
Washington, DC: DHS; September 2011. https://www.hsdl.org/?view&did=687785. Accessed October 30, 2015. 


105 


Medical Aspects of Biological Warfare 


3. Department of Homeland Security. Overview of the National Planning Frameworks. Washington, DC: DHS; May 2013:1. 
https://www.hsdl.org/?view&did=736071. Accessed October 30, 2015. 

4. Department of Homeland Security. Threat and Hazard Identification and Risk Assessment Guide: Comprehensive Prepared¬ 
ness Guide (CPG) 201, Second Edition. Washington, DC: DHS; August 2013. https://www.fema.gov/medialibrary/assets/ 
documents/26335. Accessed October 30, 2015. 

5. Department of Health and Human Services. Terrorism and Other Public Health Emergencies: A Reference Guide for Media. 
Washington, DC: DHHS; September 2005.http://permanent.access.gpo.gov/lps65017/HHSMedisReferenceGuideFinal. 
pdf. Accessed October 30, 2015. 

6. Department of Homeland Security. National Incident Management System. Washington, DC: DHS; December 2008:9. 
https://www.hsdl.org/?view&did=232899. Accessed October 30, 2015. 

7. Chess C, Clarke L. Facilitation of risk communication during the anthrax attacks of 2001: the organizational backstory. 
Am J Public Health. 2007;97:1578-1583. 

8. Sarmiento G. Former AMI building declared free of anthrax contamination. Palm Beach Post. February 8, 2007. http:// 
www.ph.ucla.edu/epi/bioter/formeramibldganthraxfree.html. Accessed October 30, 2015. 

9. Department of Homeland Security. National Preparedness Goal. 1st ed. Washington, DC: DHS; September 2011:1. https:// 
www.hsdl.org/?view&did=689207. Accessed October 30, 2015. 

10. Department of Homeland Security. National Response Framework. 2nd ed. Washington, DC: DHS; May 2013. http:// 
www.dhsem.state.co.us/sites/default/files/2013%20National%20Response%20Framework.pdf. Accessed October 30, 
2015. 

11. Department of Homeland Security. Community Emergency Response Teams (CERT). Washington, DC: DHS; January 16, 
2014. http://www.fema.gov/community-emergency-response-teams. Accessed October 30, 2015. 

12. Weapons of Mass Destruction Civil S upport Team Management. Arlington, VA: Departments of the Army and the Air Force, 
National Guard Bureau; January 12, 2006. NGR 500-3/ANGI 10-2503. http://www.ngbpdc.ngb.army.mil/pubs/10/ 
angil0_2503old.pdf. Accessed October 30, 2015. 

13. Department of Health and Human Services. Biological Incident Annex. Washington, DC: DHHS; 2008. https://www. 
hsdl.org/?view&did=236117. Accessed October 30, 2015. 

14. Garza A. The Truth About BioWatch: The Importance of Early Detection of a Potential Biological Attack. Washington, DC: 
Department of Homeland Security, July 12,2012. http://www.dhs.gov/blog/2012/07/12/truth-about-biowatch. Accessed 
October 30, 2015. 

15. Centers for Disease Control and Prevention. Division of Strategic National Stockpile Fact Sheet. Atlanta, GA: CDC, 
Office of Public Health Preparedness and Response; June 2014. www.cdc.gov/phpr/documents/DSNS_fact_sheet.pdf. 
Accessed October 30, 2015. 

16. Centers for Disease Control and Prevention. Strategic National Stockpile. Atlanta, GA: CDC, Office of Public Health 
Preparedness and Response; July 10, 2014. http://cdc.gov/phpr/stockpile/stockpile.htm. Accessed October 30, 2015. 

17. Centers for Disease Control and Prevention, Department of Health and Human Services. Public Health Preparedness 
Capabilities: National Standards for State and Local Planning. Washington, DC: CDC, DHHS; March 2011:2. http://www. 
cdc.gov/phpr/capabilities/DSLR_capabilities_July.pdf. Accessed October 30, 2015. 

18. Department of Health and Human Services. Medical Surge Capacity and Capability: A Management System for Integrat¬ 
ing Medical and Health Resources During Large-Scale Emergencies, Second Edition. Washington, DC: DHHS; September 
2007:1-3. http://www.phe.gov/Preparedness/planning/mscc/handbook/Documents/mscc080626.pdf. Accessed October 
30, 2015. 


106 


Consequence Management: The Local and National Response 


19. Centers for Disease Control and Prevention and Environmental Protection Agency. Interim Clearance Strategy for 
Environments Contaminated with Bacillus anthracis. Washington, DC: CDC and EPA; July 2012:4. http://www.epa.gov/ 
osweroel/docs/misc/cdc-epa-interim-clearance-strategy.pdf. Accessed October 30, 2015. 


107 



Chapter 5 

MEDICAL MANAGEMENT OF 
POTENTIAL BIOLOGICAL CASUALTIES: 
A STEPWISE APPROACH 

THEODORE J. CIESLAK, MD* 


INTRODUCTION 

10-STEP APPROACH TO CASUALTY MANAGEMENT 
Step 1: Maintain a Healthy Index of Suspicion 
Step 2: Protect Yourself 

Step 3: Save the Patient's Life (Primary Assessment) 

Step 4. Disinfect or Decontaminate as Appropriate 
Step 5: Establish a Diagnosis (Secondary Assessment) 

Step 6: Provide Prompt Therapy 

Step 7: Institute Proper Infection Control Measures 

Step 8: Alert the Proper Authorities 

Step 9: Conduct an Epidemiological Investigation and Manage the Psychological 

Aftermath of a Biological Attack 

Step 10: Maintain a Level of Proficiency 

SUMMARY 


*Colonel, Medical Corps, US Army; Pediatric Infectious Diseases Physician, San Antonio Military Medical Center, Department of Pediatrics, 3551 Roger 
Brooke Drive, Fort Sam Houston, Texas, 78234 


109 



Medical Aspects of Biological Warfare 


INTRODUCTION 


Response to a biological attack is relatively straight¬ 
forward when the etiologic agent employed is known. 
A larger problem arises, however, in the context of 
diagnostic uncertainty. In some cases, an attack may 
be threatened or suspected, but whether such an attack 
has, in fact, occurred can remain unclear. Moreover, 
it may be uncertain whether casualties in certain 
situations arise from exposure to a biological agent, a 
chemical or radiological agent, a naturally occurring 
infectious disease process, or toxic industrial exposure, 
or may simply reflect a heightened awareness of back¬ 
ground disease within a community or population. Ex¬ 
perience with West Nile virus, 1 severe acute respiratory 
syndrome, 2 pneumonic tularemia, 3-4 and monkeypox 5 
highlight this dilemma. In each of these cases, the pos¬ 
sibility of bioterrorism was properly raised, although 
each outbreak ultimately proved to have a natural 
origin. In some instances, proof of such an origin may 
be difficult or impossible to attain, providing "plausible 
deniability," precisely the reason some belligerents may 
opt to employ biological agents. This chapter provides 
a structured framework for dealing with outbreaks 
of unknown origin and etiology on the battlefield, as 
well as in a potential bioterrorism scenario involving 
military support installations or the civilian populace. 

In responding to the unknown, it is helpful in many 
situations to employ a standardized, stepwise ap¬ 
proach. This is especially true in the setting of a medical 
mass casualty event (MASCAL), where the use of such 
an approach (as advocated by the Advanced Trauma 
Life Support [ATLS] model sponsored by the American 
College of Surgeons 6 ) is already well accepted and 
practiced. It is also especially true under austere or 
battlefield conditions. Although major theater-level 
and continental United States-based military medi¬ 
cal centers (and research institutions, such as the US 
Army Medical Research Institute of Infectious Diseases 
[USAMRIID] and US Army Medical Research Insti¬ 
tute of Chemical Defense) may possess sophisticated 
diagnostic and response capabilities, providers on the 
battlefield and at lower-role medical treatment facili¬ 
ties are typically required to make rapid therapeutic 
decisions based on incomplete information and with 


little immediate support. Civilian clinicians, first re¬ 
sponders, and public health personnel practicing in 
rural or remote areas during a terrorist attack would 
face similar decision-making challenges. In the setting 
of a biological (or chemical or radiological) attack, 
similar to the setting of a MASCAL trauma event, 
such decisions may have life-and-death implications. 
In such situations, a stepwise or algorithmic approach 
becomes invaluable. 

USAMRIID has developed a 10-step approach to 
managing casualties that might result from biological 
warfare or terrorism. Many facets of this approach 
may be helpful in dealing with potential chemical 
or radiological casualties as well. In today's complex 
world, it is no longer adequate for most clinicians 
and medical personnel to simply understand disease 
processes. Rather, these personnel, whether military or 
civilian, must have tactical, operational, and strategic 
knowledge of threat response—and, in fact, of disaster 
response in general—as it applies to weapons of mass 
destruction. Tactical response concerns those elements 
of diagnosis and treatment of specific diseases that 
traditionally have been the realm of the individual 
practitioner. Operational response can be thought of 
as involving the mechanisms by which the provider 
interacts with his or her institution (hospital, clinic, 
medical unit) to provide mass care during a disaster. 
Strategic response involves system-wide disaster 
preparedness and response. In a civilian setting, this 
includes mechanisms by which state and federal 
disaster response elements might become involved. 
Medical personnel today need to have at least a basic 
understanding of operational and strategic response 
in addition to a firm grounding in tactical medical and 
public health intervention. The first 7 steps of this 10- 
step approach deal predominately with tactical issues 
(ie, at the level of the individual provider). Steps 8 and 
9 transition into operational and strategic response 
(ie, at the level of the institution and of the system, 
as a whole). The derivation of the 10-step approach is 
reported elsewhere, 7-10 and a condensed version ap¬ 
pears in recent editions of USAMRIID's Blue Book. 11 It 
is expanded upon here. 


10-STEP APPROACH TO CASUALTY MANAGEMENT 


Step 1: Maintain a Healthy Index of Suspicion 

In the case of chemical warfare or terrorism, the 
intentional nature of an attack is often evident. In this 
case, victims would likely be tightly clustered in time 
and space; they would succumb in close proximity 


(both temporally and geographically) to a dispersal 
device. Complicating the discovery of the intentional 
nature of a biological attack, however, is the fact that 
biological agents possess inherent incubation periods, 
while conventional, chemical, and nuclear weapons do 
not. These incubation periods, typically of several days 


110 


Medical Management of Potential Biological Casualties: A Stepwise Approach 


(but up to several weeks in the case of agents such as 
Coxiella burnetii and the Brucellae), allow for the wide 
dispersion of victims in time and space. Additionally, 
they make it likely that the first responder to a biologi¬ 
cal attack would not be the firefighter, police officer, 
paramedic, or other traditional first responder, but 
rather primary care providers, hospital emergency 
departments, and public health officials. In such cir¬ 
cumstances, maintaining a healthy index of suspicion 
is imperative. 

In some instances, maintaining an index of sus¬ 
picion might be simplified by the fact that diseases 
caused by biological agents may present with specific 
characteristic clinical findings, which allow for a very 
limited differential diagnosis. The hallmark of inha- 
lational anthrax is a widened mediastinum, a clinical 
finding seen in few naturally occurring conditions. 
With botulism, the hallmark presentation is that of a 
descending, symmetric, flaccid paralysis. Whereas an 
individual patient with flaccid paralysis might prompt 
consideration of disorders such as Guillain-Barre syn¬ 
drome, Eaton-Lambert syndrome, poliomyelitis, and 
myasthenia gravis, the near-simultaneous presenta¬ 
tion of multiple patients with flaccid paralysis should 
quickly lead one to a diagnosis of botulism. Similarly, 
patients with plague and melioidosis may exhibit he¬ 
moptysis in the later stages of illness. Such a finding 
is uncommon among previously healthy individuals, 
but can be caused by tuberculosis, staphylococcal and 
Klebsiella pneumonia, carcinoma, and trauma. Multiple 
patients with hemoptysis, however, should prompt 
consideration of a plague or melioidosis diagnosis. 
Smallpox is characterized by a very unique exanthem, 
perhaps evocative of Varicella or syphilis in its earliest 
stages, but readily distinguishable from these entities 
as it progresses. 

Yet, by the time each of these characteristic findings 
develops, treatment is less likely to be effective. Thera¬ 
py is thus best instituted during the incubation or pro¬ 
dromal phases of these diseases if it is to be beneficial. 
Unfortunately, during their prodromes, these diseases 
are likely to appear as undifferentiated febrile illnesses, 
difficult, if not impossible, to distinguish from myriad 
other common infectious diseases. Similarly, many 
other diseases potentially arising from a biological 
attack (such as tularemia, brucellosis, melioidosis, Q 
fever, and Venezuelan equine encephalitis) may appear 
simply as undifferentiated febrile illnesses throughout 
their course. Prompt diagnosis and targeted therapy is 
thus possible only with a very high index of suspicion. 

Epidemiological clues can lead a clinician to suspect 
that a disease outbreak may have been intentional (Ex¬ 
hibit 5-1). 12 Large numbers of victims tightly clustered 
in time and space, or limited to a discrete population. 


EXHIBIT 5-1 

EPIDEMIOLOGICAL CLUES TO A 
BIOTERRORIST ATTACK 

• 

Presence of an unusually large epidemic 

• 

High infection rate 

• 

Disease limited to a discrete population 

• 

Unexpected severity of disease 

• 

Evidence of an unusual route of expo¬ 
sure 

• 

Disease in an atypical geographic locale 

• 

Disease occurring outside normal trans¬ 
mission seasons 

• 

Disease occurring in the absence of usual 
vector 

• 

Simultaneous outbreaks of multiple 
diseases 

• 

Simultaneous occurrence of human and 
zoonotic disease 

• 

Unusual organism strains 

• 

Unusual antimicrobial sensitivity pat¬ 
terns 

• 

Disparity in attack rates among persons 
indoors and outdoors 

• 

Terrorist claims 

• 

Intelligence reports 

• 

Discovery of unusual munitions 

Data source: Pavlin JA. Epidemiology of bioterrorism. 

Emerg Infect Dis. 1999;5:528-30. 


should raise suspicion. Similarly, unexpected deaths 
and cases of unexpectedly severe illness merit concern. 
An outbreak of a disease not typically seen in a specific 
geographic location, in a given age group, or during a 
certain season, likewise warrants further investigation. 
Simultaneous outbreaks of a disease in noncontiguous 
areas should prompt one to consider an intentional 
release, as should simultaneous or sequential out¬ 
breaks of different diseases in the same locale. Even a 
single case of rare disorders, such as anthrax or certain 
viral hemorrhagic fevers (Ebola, Marburg, Lassa, and 
many others) would be suspicious, and a single case of 
smallpox, because it no longer occurs naturally, would 
almost certainly represent an intentional release. The 
presence of dying animals (or the simultaneous occur¬ 
rence of zoonotic disease outbreaks among humans 
and animals) might provide evidence of an unnatural 
aerosol release. Evidence of a disparate attack rate 
between those known to be indoors and outdoors at 
a given time should also be sought and evaluated. 


Ill 






Medical Aspects of Biological Warfare 


Finally, intelligence reports, terrorist claims, and the 
discovery of aerosol spray devices would obviously 
lend credence to the theory that a disease outbreak 
was of sinister origin. 

On the modern battlefield, an array of developing 
technology is available to assist clinicians, preventive 
medicine and chemical corps personnel, operators, 
and commanders in maintaining their index of sus¬ 
picion through early "stand-off" detection of bio¬ 
logical threats. The Portal Shield is the Department 
of Defense's (DoD's) first automated biological detec¬ 
tion system, and was designed to provide fixed-site 
protection to air and port facilities. Portal Shield is 
equipped with modular sensors capable of simultane¬ 
ously assaying for eight different agents and providing 
presumptive identification within about 25 minutes. 
The Biological Integrated Detection System, a system 
mounted on a high-mobility multipurpose wheeled 
vehicle, is equipped with samplers, an aerodynamic 
particle sizer, a flow cytometer, and a chemical bio¬ 
logical mass spectrometer. The Joint Biological Point 
Detection System integrates into the M31A2 Biological 
Integrated Detection System platform (Figure 5-1) to 
permit rapid, real-time detection of 10 separate bio¬ 
logical threat agents on the battlefield; the system is 
capable of definitively identifying biowarfare threat 
agents within 18 minutes. The Joint Biological Agent 
Identification and Diagnostic Systems (JBAIDS) is a 
reusable, portable, and modifiable biological agent 
identification and diagnostic system capable of rapid, 
reliable, and simultaneous identification of multiple 
biological agents and other pathogens of operational 
concern. The JBAIDS anthrax, tularemia, plague, and 



Figure 5-1. The Biological Integrated Detection System 
(BIDS) is a semi-automated biological agent detection/ 
identification suite mounted on a dedicated heavy high 
mobility multipurpose wheeled vehicle. The system uses 
multicomplimentary bio-detection technologies. 


Q fever detection systems are cleared by the Food and 
Drug Administration (FDA) for diagnostic use. Until 
these technologies are refined, validated, and made 
widely available, though, those tasked with respond¬ 
ing to an attack must rely on clinical, epidemiologi¬ 
cal, and intelligence clues to maintain their index of 
suspicion. 

Step 2: Protect Yourself 

Providers who themselves become casualties are of 
little use to their patients. Before approaching casual¬ 
ties of biological or chemical warfare or victims of a 
potential terrorist attack, clinicians should be familiar 
with basic means of self-protection. Such protective 
measures generally fall into one of three categories: 
(1) physical protection, (2) chemical protection, and 
(3) immunologic protection. Under a given set of cir¬ 
cumstances, clinicians and laboratory personnel might 
appropriately avail themselves of one or more of these 
forms of protection. 

Physical Protection 

Since the beginning of modem gas warfare on the 
battlefields near Ypres, Belgium, in 1915, physical 
protection during military operations has involved 
gas masks and, more recently, charcoal-impregnated 
chemical protective overgarments. Although military- 
style protective clothing and masks were designed with 
chemical agent protection in mind, they are capable of 
offering protection against biological agents as well. 
Although some countries have advocated the issuance 
of military-style protective masks and ensembles to 
civilians (eg, the Israeli government has issued masks 
to its general populace), such items, even if offered, 
would likely be unavailable to civilians at the precise 
moment of agent release; the unannounced release of 
odorless and colorless biological agents by belliger¬ 
ents or terrorists would afford no opportunity to don 
protective gear, even if it were available. Furthermore, 
misuse of protective equipment in the past has led to 
fatalities, including cases of infants and adults suf¬ 
focating in protective ensembles. 13,14 Although mili¬ 
tary masks such as the M40/42, M45, and M50 series 
provide ample protection against inhalation hazards 
posed by chemical and biological weapons as well as 
against radioactive dust particles, they add heat stress 
and are potentially mission-degrading. Moreover, a 
simple surgical mask will usually afford adequate 
protection against inhalation of infectious aerosols 
of virtually any of the biological agents typically men¬ 
tioned in a terrorism context. An important exception 
might be smallpox, in which case a high-efficiency 


112 






Medical Management of Potential Biological Casualties: A Stepwise Approach 


particulate air (HEPA) filter mask would be ideal. With 
the exception of smallpox, pneumonic plague, and cer¬ 
tain viral hemorrhagic fevers, the agents in the Centers 
for Disease Control and Prevention's (CDC's) categories 
A and B (Exhibit 5-2) are not contagious via the respi¬ 
ratory route. Respiratory protection is thus necessary 
when operating in an area of primary release, but would 
not be required in most patient-care settings (see step 7). 

Chemical Protection 

During Operations Desert Shield and Desert Storm, 
tens of thousands of US troops were given pyridostig¬ 
mine under an emergency-use authorization, and in 
early 2003, the FDA gave its final approval for the use 
of pyridostigmine bromide as preexposure prophylaxis 
against intoxication with soman, an organophosphate- 
based chemical nerve agent. It is conceivable, given 
credible and specific intelligence, that similar strate¬ 
gies might be employed against biological weapons. 
For example, if a specific terrorist group possessing a 
specific weaponized agent were known to be operating 
in a given locale, public health authorities might con¬ 
ceivably contemplate the widespread distribution of 
an appropriate prophylactic antibiotic. Obviously, the 
opportunities to employ such a strategy are likely to 
remain few and far between, and the logistics of doing 
so would be exceedingly difficult in a civilian setting. 


Immunologic Protection 

For the near future, active vaccination is likely 
to provide one of the most practical methods for 
administering preexposure prophylaxis against 
biological attack. In the military, decisions regard¬ 
ing vaccination policy are typically made through 
the office of the Assistant Secretary of Defense for 
Health Affairs, with input from high-level military 
medical, public health, and intelligence sources. 
The decision to offer a specific vaccine in a specific 
circumstance is a complex one that must take into 
account a careful risk-benefit calculation. During 
Operations Desert Shield and Desert Storm, some 
150,000 service members received at least one dose 
of anthrax vaccine, while about 8,000 received a 
botulinum toxoid vaccine. Since 1998, the US mili¬ 
tary has intermittently employed force-wide an¬ 
thrax vaccination, and since 2003 has administered 
smallpox vaccine to deploying troops and certain 
medical response teams. 

In a civilian counter-terrorism context, the deci¬ 
sion to employ a specific vaccine is even more dif¬ 
ficult and complex. Factors that would influence a 
decision by public health officials to recommend 
vaccination include intelligence (eg, how likely or 
plausible is an attack? How imminent is the threat? 
How specific is the threat?), vaccine safety, vaccine 


EXHIBIT 5-2 

CRITICAL AGENTS FOR HEALTH PREPAREDNESS 


Category A* 

Category B + 

Category C* 

• Variola virus 

• Coxiella burnetii 

Other biological agents that may 

• Bacillus anthracis 

• Brucellae 

emerge as future threats to public 

• Yersinia pestis 

• Burkholderia mallei 

health, such as: 

• Botulinum toxin 

• Burkholderia pseudomallei 

• Nipah virus 

• Francisella tularensis 

• Alphaviruses 

• Hantaviruses 

• Filoviruses and arenaviruses 

• Certain toxins (ricin, staphylo¬ 
coccal enterotoxin B, trichothe- 
cenes) 

• Food safety threat agents 
(Salmonellae, Escherichia coli 
0157:H7) 

• Water safety threat agents ( Vib¬ 
rio cholerae, etc) 

• Yellow fever virus 

• Drug-resistant tuberculosis 

• Tick-borne encephalitis 

‘Agents with high public health impact requiring intensive public health preparedness and intervention. 

+ Agents with a somewhat lesser need for public health preparedness. 

*Other biological agents that may emerge as future threats to public health. 

Data source: Centers for Disease Control and Prevention. Biological and chemical terrorism: strategic plan for preparedness and 
response. MMWR. 2000;49(KR-04):1-14. 


113 






Medical Aspects of Biological Warfare 


availability, disease consequences (ie, is the threat 
from a lethal agent or from an incapacitant?), and the 
availability of postexposure prophylaxis or therapy. 
Recently, civilian public health and policy planners 
have given extensive consideration to the widespread 
distribution of anthrax and smallpox vaccines. 

Anthrax. Anthrax Vaccine, Adsorbed (AVA, Bio- 
Thrax; Emergent BioSolutions, Lansing MI) is a fully 
licensed product, approved by the FDA in 1970. The 
vaccine consists of a purified preparation of protec¬ 
tive antigen, a potent immunogen necessary for entry 
of key anthrax toxin components (lethal and edema 
factors) into mammalian cells. Administered alone, 
protective antigen is nontoxic. In a large controlled 
trial, AVA was effective in preventing cutaneous an¬ 
thrax among textile workers. 15 Based on an increasing 
amount of animal data, there is every reason to be¬ 
lieve that this vaccine is quite effective at preventing 
inhalational anthrax as well. 16 Moreover, well over 
20 clinical studies, surveys, and reports now attest to 
the safety of AVA, 17,18 and the FDA has reaffirmed the 
vaccine as being safe and effective in light of those 
studies. 19 Nonetheless, although widespread use of 
AVA has occurred within the US military (as of Janu¬ 
ary 2014, more than 12.1 million doses of AVA had 
been given to more than 2.4 million service members), 
logistical and other considerations make large-scale 
civilian vaccination impractical at present. The vac¬ 
cine is licensed as a five-dose series, given at 0 and 4 
weeks, and at 6,12, and 18 months. Yearly boosters are 
recommended for those at ongoing risk of exposure. 
Further complicating any potential civilian anthrax 
vaccination strategy is the fact that AVA is approved 
by the FDA only for individuals 18 to 65 years old. 
Although a large-scale preexposure offering of AVA 
to the general public might thus be problematic, some 
have recommended that a three-dose series of AVA 
(given at time zero and at 2 and 4 weeks after the 
initial dose), combined with 60 days of antibiotics 
under an investigational new drug (IND) protocol or 
emergency use authorization, might be an acceptable 
alternative to longer (60-100 days) antibiotic courses 
alone for postexposure prophylaxis against inhala¬ 
tional anthrax. 20 This recommendation was based 
on nonhuman primate challenge studies; no human 
studies currently exist to support such a strategy, 
and AVA is not licensed by the FDA for postexposure 
prophylaxis or therapy. 

Smallpox. Widespread vaccination against small¬ 
pox is equally controversial and problematic. None¬ 
theless, in 2002, President George W. Bush announced 
a plan to vaccinate selected American healthcare 
workers and military personnel. Within the DoD, 
service members deploying to locations thought at 


risk for biological attack and members of designated 
smallpox epidemiological and clinical response teams 
were selected for vaccination. The program includes 
prevaccination screening to exclude members with 
vaccine contraindications or household contacts at 
risk, instruction on vaccine site care and potential 
complications, and mandatory follow-up. As of Janu¬ 
ary 10, 2014, over 2.4 million military response team 
members, hospital workers, and operational forces 
had been vaccinated, with one death occurring due to 
a lupus-like illness. Although the emergence of myo- 
pericarditis (there were 161 confirmed, suspected, 
or probable cases among 1.4 million vaccinees as of 
January 2008) as a complication of vaccination 21 led 
to a revision of prevaccine screening (candidates with 
multiple cardiac risk factors are now excluded), rates 
of other adverse reactions were low. Cases of auto¬ 
inoculation or transmission to household and other 
contacts have been rare. 22-24 One case of progressive 
vaccinia occurred in a primary vaccine recipient, 23 and 
three cases of eczema vaccinatum occurred among 
contacts of vaccinees. 26,27 No cases of fetal vaccinia 
have been reported. Vaccinia immune globulin was 
required on only seven occasions, to treat ocular 
vaccinia, 28 progressive vaccinia, 26 eczema vaccina¬ 
tum, 27,28 and as prophylaxis for a vaccinated patient 
who sustained large burn wounds. The success of 
this smallpox immunization program suggests that 
mass vaccination can be accomplished with greater 
safety than previously thought possible. 29 

Although universal civilian vaccination was not 
recommended under President Bush's plan, the pos¬ 
sibility of a future strategy calling for such recom¬ 
mendations was allowed for, and provisions were 
made to provide smallpox vaccine to those members 
of the general public who specifically requested it. The 
wisdom of widespread civilian vaccination is difficult 
to assess. Most medical decisions involve a risk-benefit 
analysis on the part of the responsible clinician. In the 
case of smallpox vaccination, the risks are well known, 
and they are significant. 30,31 The benefits, however, 
are far less certain; although the global eradication of 
smallpox surely ranks among the greatest public health 
accomplishments of recent history and the wisdom 
of vaccination with live vaccines went unquestioned 
during the era of endemic smallpox, the likelihood 
of contracting smallpox today via a terrorist attack is 
unknown and likely miniscule for the average civilian. 
In this regard, the risk-benefit calculation is not based 
on medical considerations, but rather on intelligence 
estimates to which few are privy. 

Despite these concerns, a prerelease mass vaccina¬ 
tion program for the general population may be the 
most effective countermeasure to the terror threat 


114 


Medical Management of Potential Biological Casualties: A Stepwise Approach 


posed by smallpox. By inducing individual and herd 
immunity and by obviating the extreme difficulty of 
conducting postrelease vaccine and quarantine efforts, 
a program involving the resumption of universal 
smallpox vaccination possesses distinct advantages 
over other response plans. However, such an approach 
is hampered not only by the unknown risk of a small¬ 
pox release, but also by safety and logistics issues. 32,33 

A large number of persons are at risk for severe 
vaccine reactions today compared to the previous era 
of routine civilian smallpox vaccination, which ended 
in 1972. This increase in risk is due to the presence 
in the population of a large number of persons with 
compromised immunity associated with human im¬ 
munodeficiency virus and with advances in immu¬ 
nosuppressive therapy and bone marrow and solid 
organ transplantation. This phenomenon raises concern 
about the safety and risk-benefit ratio of any preexpo¬ 
sure vaccination program. 34 Similarly, the occurrence 
of rare but severe smallpox vaccine complications in 
otherwise healthy recipients could result in morbidity 
and mortality that would be unacceptable in times of 
low risk. Risk analysis favors prerelease mass vaccina¬ 
tion of the general population only if the probability of 
a large-scale attack is high. Prerelease mass vaccination 
of healthcare workers might again be contemplated in 
the future, owing to the risk of exposure while caring for 
patients, and the value of keeping healthcare workers 
healthy and functioning in the setting of an epidemic. 33 

The smallpox vaccine currently employed in the 
United States is ACAM2000 (Acambis Inc, Cambridge, 
MA), which uses modem cell-culture-based produc¬ 
tion of vaccinia, an orthopoxvirus closely related to 
variola. ACAM2000 was licensed by the FDA in 2007, 
and replaced Dryvax (Wyeth Laboratories, Marietta, 
PA), a preparation derived from the harvested lymph 
of inoculated calves, in 2008. It is unlikely that this will 
significantly diminish the risk of adverse reactions, how¬ 
ever, as the new vaccine employs the same live strain of 
vaccinia vims. The vast majority of adverse reactions to 
current vaccinia-containing vaccines derive from the live 
nature of the vims rather than the method of preparation. 

The CDC controls release of civilian ACAM2000 
stocks and conditions for release have been estab¬ 
lished. 36 The current CDC smallpox response strat¬ 
egy is based on preexposure vaccination of carefully 
screened first responders and members of epidemio¬ 
logical and clinical response teams. CDC plans also 
provide for a program to treat certain severe complica¬ 
tions of vaccination using vaccinia immune globulin 
under an IND protocol, as well as for compensation of 
persons experiencing such complications, through the 
establishment of a smallpox vaccine injury compensa¬ 
tion program. 37 


The CDC's response plan calls for "ring vaccina¬ 
tion" after a smallpox release: identification and isola¬ 
tion of cases, with vaccination and active surveillance 
of contacts. Mass vaccination would be reserved for 
those instances when the number or location of cases 
renders the ring strategy inefficient, or when the risk 
of additional virus release is high. 38 Although ring 
vaccination was successful historically (in the setting 
of herd immunity), mathematical models predict that 
this strategy may be problematic when applied to 
large or multifocal epidemics today. 39 Furthermore, 
there is controversy among experts regarding the pre¬ 
dicted benefit of postrelease mass vaccination due to 
lack of herd immunity, a highly mobile population, a 
relatively long incubation period, and the difficulties 
associated with prompt implementation of quarantine 
and mass vaccination. 40,41 Finally, it should be kept 
in mind that vaccination is but one component of a 
multifaceted response, which should also include 
farsighted planning and logistical preparation, risk 
communication, surveillance, treatment, isolation, 
and quarantine. 

Other Agents. Few authorities, either military or 
civilian, have advocated widespread vaccination 
against potential agents of bioterrorism other than 
anthrax and smallpox, and the implementation of any 
such strategy would currently be problematic. A vac¬ 
cine against plague, previously licensed in the United 
States, is currently out of production. It required a 
three-dose primary series followed by annual boost¬ 
ers. Moreover, it was licensed only for persons 18 to 
61 years old. Finally, although reasonably effective 
against bubonic plague and widely employed by the 
DoD to protect against endemic disease, it probably 
afforded little protection against pneumonic plague, 
the form of disease likely to be associated with war¬ 
fare or terrorism. A vaccine against one specific viral 
hemorrhagic fever, namely yellow fever, is widely 
available, although its causative virus is not regarded 
as a significant weaponization threat by most poli¬ 
cymakers and health officials. Again, while the US 
military has administered yellow fever vaccine to 
large numbers of troops, it does so to guard against 
endemic disease, rather than a bioweapon threat. Ad¬ 
ditionally, a vaccine against Q fever (Q Vax, CSL Ltd, 
Victoria, Australia) is licensed in Australia. Although 
this vaccine might conceivably prove a useful addi¬ 
tion to the military biodefense armamentarium, the 
self-limited nature of Q fever makes it unlikely that 
widespread use of this vaccine would be contemplat¬ 
ed for the general public. Numerous research efforts 
are aimed at developing improved next-generation 
vaccines against anthrax, smallpox, and plague. 
Similarly, vaccines effective against tularemia. 


115 


Medical Aspects of Biological Warfare 


brucellosis, botulism, the equine encephalitides, 
staphylococcal enterotoxins, ricin, and several viral 
hemorrhagic fevers, as well as other potential agents 
of bioterrorism, are in various stages of development. 42 
Investigational vaccines against tularemia, botulism, the 
equine encephalitides (especially Venezuelan equine 
encephalitis), staphylococcal enterotoxin B, Q fever, 
and other agents, have been used under IND protocols 
to protect scientists studying these agents. 

Step 3: Save the Patient's Life (Primary Assessment) 

Once self-protective measures are implemented, 
the clinician can approach the MASCAL scenario and 
begin assessing patients (the "primary survey," in 
keeping with ATLS guidelines 6 ). This initial assessment 
is intended to be brief and its purpose limited to the 
discovery and treatment of those conditions present¬ 
ing an immediate threat to life or limb. Biological (or 
chemical) warfare victims may also have conventional 
injuries; attention should thus be focused at this point on 
maintaining a patent airway and providing for adequate 
breathing and circulation. The need for decontamina¬ 
tion and administration of antidotes for rapid-acting 
chemical agents (nerve agents and cyanide) should be 
determined at this time. An "ABCDE" algorithm aids 
the clinician in recalling the specifics of the primary 
assessment. "A" stands for airway, which should be 
evaluated for the presence of conventional injury, but 
should also be examined because exposure to certain 
chemical agents (such as mustard, lewisite, or phos¬ 
gene) can damage the airway. "B" denotes breathing; 
many agents of biological (and chemical) terrorism may 
cause the patient to experience respiratory difficulty. 
Examples include anthrax, plague, tularemia, botulism, 
Q fever, the staphylococcal enterotoxins, and ricin, as 
well as cyanide, nerve agents, and phosgene. "C" de¬ 
notes circulation, which may be compromised due to 
conventional or traumatic injuries sustained during a 
MASCAL event, but may also be involved in the septic 
shock associated with plague and in the circulatory 
collapse associated with the viral hemorrhagic fevers. 
"D" refers to disability, specifically, neuromuscular dis¬ 
ability. Note that botulism and nerve agent exposures 
are likely to present with a preponderance of neuromus¬ 
cular symptomatology. Finally, "E" refers to exposure. 
In a MASCAL setting, this serves as a reminder to re¬ 
move the victim's clothing to perform a more thorough 
secondary assessment. It is here that one considers the 
need for decontamination and disinfection. 

Step 4: Disinfect or Decontaminate as Appropriate 

Once patients have been stabilized, decontami¬ 


nation can be accomplished, where appropriate. 
On the battlefield, considerable mature military 
doctrine drives decontamination efforts, which are 
carried out by unit personnel, guided or assisted by 
specific, highly trained Chemical Corps decontami¬ 
nation units. It should be pointed out, however, that 
decontamination, in the classical sense, may not be 
necessary after a biological attack (the same cannot 
always be said after a chemical attack). This is due, 
again, to the inherent incubation periods of biologi¬ 
cal agents. Because victims will not typically become 
symptomatic until several days after exposure to such 
agents, they are likely to have bathed and changed 
clothing several times before presenting for medical 
care, thus effectively accomplishing self-decontami¬ 
nation. Exceptions might include personnel directly 
exposed to an observed attack or persons encounter¬ 
ing a substance in a threatening letter, where common 
sense might dictate topical disinfection. Even in these 
situations, bathing with soap and water and conven¬ 
tional laundry measures would likely be adequate. 
Moreover, it should be kept in mind that situations 
such as the case of the threatening letter represent 
crime scenes. Any medical interest in disinfection 
must be weighed against law enforcement concerns 
regarding preservation of vital evidence, which can be 
destroyed through hasty and ill-considered attempts 
at decontamination. Furthermore, significant psycho¬ 
logical stress has been caused by unnecessary, costly, 
and resource-intensive attempts at decontamination 
in the past. 43 Some of these attempts have involved 
forced disrobing and showering in public streets; 
to avoid such problems, the following measured 
responses should be considered. 44 

The Announced Threat (or Presumed Hoax). The 
need to preserve evidence, and maintain a chain-of- 
custody when handling that evidence, is an important 
consideration at any crime scene. Although human and 
environmental health protection concerns take prece¬ 
dence over law enforcement procedures, threat and 
hoax scenarios nonetheless require the early involve¬ 
ment of law enforcement personnel and a respect for the 
need to maintain an uncompromised crime scene. De¬ 
contamination or disinfection is not typically necessary. 

The Telephoned Threat or the "Empty Letter." In 
the majority of cases involving a telephoned threat, 
no delivery device or package is located. If a device 
is found or a threat is subsequently deemed credible, 
public health authorities should contact potentially 
exposed individuals, obtain appropriate information, 
and consider instituting prophylaxis or therapy. An 
envelope containing nothing other than a written 
threat poses little risk and should be handled in the 
same manner as a telephoned threat. Because the 


116 


Medical Management of Potential Biological Casualties: A Stepwise Approach 


envelope constitutes evidence in a crime, however, 
further handling should be left to law enforcement 
professionals. In these cases, no decontamination is 
typically necessary, pending results of the legal and 
public health investigation. 

The Suspicious Package. When a package is dis¬ 
covered and found to contain powder, liquid, or other 
physical material, response should be individualized. 
In most cases, the package should not be disturbed fur¬ 
ther, the room should be vacated, additional untrained 
persons should be prohibited from approaching the 
scene and from handling the package or its contents, 
and law enforcement and public health officials should 
again be promptly notified. Persons who have come 
in contact with contents should remove clothing as 
soon as practical and seal it in a plastic bag. Victims 
should then wash with soap and water 45 and, in most 
cases, may be sent home after adequate instructions 
for follow-up are provided and contact information 
obtained. In general, antibiotic prophylaxis would 
not be necessary before the preliminary identifica¬ 
tion of package contents by a competent laboratory, 
although decisions to provide or withhold postexpo¬ 
sure prophylaxis are best made after consultation with 
public health authorities. Floors, walls, and furniture 
would not require decontamination before labora¬ 
tory analysis is completed. Nonporous contaminated 
personal items, such as eyeglasses and jewelry, may 
be washed with soap and water or immersed in 0.5% 
hypochlorite (household bleach diluted tenfold) if a 
foreign substance has contacted the items. 

The Delivery Device. If an aerosol delivery de¬ 
vice or other evidence of a credible aerosol threat is 
discovered, the room (and potentially the building) 
should be evacuated. Law enforcement and public 
health personnel should be notified immediately 
and further handling of the device left to personnel 
with highly specialized training, such as the Army's 
22nd and 110th Chemical Battalions (Technical Escort 
Units), the Marine Corps Chemical-Biological Incident 
Response Force (CBIRF), or the Federal Bureau of 
Investigation's Hazardous Materials Response Unit. 
Contact information should be obtained from potential 
victims and detailed instructions provided. Clothing 
removal, soap and water showering, and decontami¬ 
nation of personal effects should be accomplished as 
above (the CBIRF brings with it extensive decontami¬ 
nation capabilities). Decisions regarding institution of 
empiric postexposure prophylaxis pending determi¬ 
nation of the nature of the threat and identification of 
the involved biological agents should again be left to 
local and state public health authorities. In providing 
a reasoned and measured response to each situation, 
public health and law enforcement personnel can as¬ 


sist in minimizing the disruption and cost associated 
with large-scale decontamination, costly hazardous 
materials unit involvement, and broad institution of 
therapeutic interventions, and can help avoid wide¬ 
spread public panic. 

Step 5: Establish a Diagnosis (Secondary Assessment) 

Once decontamination has been considered, and 
accomplished as warranted, the clinician may perform 
a more thorough and targeted assessment aimed at 
establishing a diagnosis (the ATLS "secondary sur¬ 
vey"). The thoroughness and accuracy with which one 
establishes this diagnosis will vary depending upon 
the circumstances the clinician finds him- or herself in. 
At robust roles of care (Role 4), the clinician may well 
have access to infectious disease and microbiology 
professionals, as well as to sophisticated diagnostic 
assays. Under such circumstances, it may be possible 
to arrive at a definitive microbiologic diagnosis fairly 
promptly. On the other hand, it is equally conceivable 
that the primary care provider, practicing at lower 
roles of care (Roles 1 to 3) or in more austere circum¬ 
stances, may need to intervene promptly based on 
limited information and without immediate access to 
subspecialty consultation. Even in such cases, however, 
reasonable care can be instituted based simply on a 
syndromic diagnosis. An "AMPLE" (A: allergies, ar¬ 
thropod exposures; M: medications [as well as military 
occupational specialty and mission-oriented protective 
posture status]; P: past illnesses and vaccinations; L: 
last meal; E: environment) history may aid in estab¬ 
lishing this diagnosis. A brief but focused physical 
examination, even one performed by inexperienced 
practitioners, can, at a minimum, reveal whether a 
victim of a biological or chemical attack exhibits pri¬ 
marily respiratory, neuromuscular, or dermatologic 
signs, or suffers simply from an undifferentiated febrile 
illness. By placing patients into one of these broad 
syndromic categories, empiric therapy can be initiated 
(see step 6); such empiric therapy can be refined and 
tailored once more information becomes available. 46,47 

When the situation permits, laboratory studies 
should be obtained to aid in later definitive diagnosis 
(Exhibit 5-3). On the battlefield, samples obtained at 
lower echelons would normally be submitted to the lo¬ 
cal clinical laboratory and, from there, through clinical 
laboratory channels to the 1st Area Medical Laboratory 
(AML). The AML is a theater-level tactical laboratory 
with very robust scientific capabilities, including the 
ability to rapidly identify biological, chemical, and 
radiological threat agents, as well as endemic, oc¬ 
cupational, and environmental health hazards. The 
AML also has "reach-back" ability and works closely 


117 


Medical Aspects of Biological Warfare 


EXHIBIT 5-3 

SAMPLES TO CONSIDER OBTAINING 
FROM POTENTIAL BIOWARFARE OR 
BIOTERRORISM VICTIMS’ 


• Complete blood count 

• Arterial blood gas 

• Nasal swabs for culture and PCR 

• Blood for bacterial culture and PCR 

• Serum for serologic studies 

• Sputum for bacterial culture 

• Blood and urine for toxin assay 

• Throat swab for viral culture, PCR, and 
ELISA 

• Environmental samples 

This list is not all-inclusive, nor is it meant to imply 
that every sample should be obtained from every 
patient. In general, laboratory sampling should be 
guided by clinical judgment and the specifics of the 
situation. This is a list of samples to consider obtaining 
in situations where the nature of an incident is unclear 
and empiric therapy must be started before definitive 
diagnosis. 

ELISA: enzyme-linked immunosorbent assay 
PCR: polymerase chain reaction 


with national laboratories at USAMRIID and the US 
Army Medical Research Institute of Chemical Defense 
in Maryland. 

Step 6: Provide Prompt Therapy 

Once a diagnosis (whether definitive or syndromic) 
is established, prompt therapy must be provided. 
In the cases of anthrax and plague, in particular, 
survival is directly linked to the speed with which 
appropriate therapy is instituted. A delay of more 
than 24 hours in the treatment of either disease leads 
to a uniformly grim prognosis. When the identity of 
a bioterrorist agent is known, the provision of proper 
therapy is straightforward (Table 5-1). When a clini¬ 
cian is faced with multiple victims and the nature of 
the illness is not known, however, empiric therapy 
must be instituted. Guidelines for providing empiric 
therapy in such situations have been published, and 
an algorithmic approach to syndromic diagnosis and 
empiric therapy has been developed (Figure 5-2). 
Doxycycline, ciprofloxacin, or levofloxacin should be 
administered empirically to patients with significant 
respiratory symptoms when exposure to a biological 
attack is considered a possibility. 


Step 7: Institute Proper Infection Control Measures 

The clinician must practice proper infection control 
procedures to ensure that contagious diseases are not 
propagated among patients. The majority of biological 
threat agents are not contagious. Among these are the 
causative agents of anthrax, tularemia, brucellosis, Q 
fever, the alphaviral equine encephalitides, glanders, 
melioidosis, and many others, including all of the 
toxins. Standard precautions alone suffice, in most 
cases, when caring for victims of such diseases. 48 More 
stringent transmission-based precautions should be 
applied in certain circumstances. Three subcategories 
of transmission-based precautions exist. Droplet pre¬ 
cautions are required to manage victims of pneumonic 
plague. Ordinary surgical masks are a component of 
proper droplet precautions and constitute adequate 
protection against acquisition of plague bacilli by 
the aerosol route. Contact precautions should be 
employed when managing certain viral hemorrhagic 
fever patients. In theory, these would be adequate for 
managing even Ebola victims given the transmission 
of this disease through infected blood and body fluids. 
Recent experience with Ebola in West Africa, however, 
illustrates the ease with which such precautions might 
be compromised. Given the prodigious amounts of 
body fluids (emesis and diarrhea) produced by these 
patients, the very low infectious inoculum of Ebola, and 
the propensity for hemorrhagic sputum to be aerosol¬ 
ized during coughing, the CDC now recommends that 
both contact and droplet precautions be employed when 
managing Ebola victims. Airborne precautions, ideally 
including an N-95 HEPA-filter mask, should be used 
when caring for smallpox victims. A summary of hos¬ 
pital infection control precautions as they apply to vic¬ 
tims of biological terrorism is presented in Exhibit 5-4. 

Step 8: Alert the Proper Authorities 

As soon as it is suspected that a case of disease might 
be the result of exposure to biological or chemical 
agents, the proper authorities must be alerted so that 
appropriate warnings may be issued and outbreak- 
control measures implemented. On the battlefield 
and in other military settings, the command must be 
notified immediately. It is similarly important to no¬ 
tify preventive medicine officials, as well as chemical 
corps and laboratory personnel. Early involvement 
of preventive medicine personnel ensures that an 
epidemiological investigation is begun promptly (see 
step 9) and that potential victims (beyond the index 
cases) are identified and treated early, when such 
treatment is most likely to be beneficial. Similarly, 
early notification of Army chemical corps personnel 
allows for battlefield surveillance, detection, and 


118 





Medical Management of Potential Biological Casualties: A Stepwise Approach 


TABLE 5-1 

RECOMMENDED THERAPY OF AND PROPHYLAXIS AGAINST DISEASES CAUSED BY 
CATEGORY A BIOTHREAT AGENTS 

Condition Adults Children 


Anthrax, inhalational, 
therapy* * (patients who are 
clinically stable after 14 days 
can be switched to a single 
oral agent [ciprofloxacin or 
doxycycline] to complete a 
60-day course 1 ) 


Anthrax, inhalational, postex¬ 
posure prophylaxis (60-day 
course 1 ) 

Anthrax, cutaneous in setting 
of terrorism, therapy ¥ 

Plague, therapy 


Plague, prophylaxis 
Tularemia, therapy 

Tularemia, prophylaxis 

Smallpox, therapy 
Smallpox, prophylaxis 


Botulism, therapy 


Viral hemorrhagic fevers, 
therapy 


Ciprofloxacin 400 mg IV ql2h OR 
Levofloxacin 500 mg IV q24h OR 
Doxycycline 100 mg IV ql2h 

AND 

Clindamycin* 900 mg IV q8h 

AND 

Penicillin G s 4 mil U IV q4h 
AND CONSIDER 
Raxibacumab 40 mg/kg IV 

Ciprofloxacin 500 mg PO ql2h OR 
Levofloxacin 500 mg PO q24h OR 
Doxycycline 100 mg PO ql2h 

Ciprofloxacin 500 mg PO ql2h OR 
Levofloxacin 500 mg PO q24h OR 
Doxycycline 100 mg PO ql2h 

Gentamicin 5 mg/kg IV qd OR 
Doxycycline 100 mg IV ql2h OR 
Ciprofloxacin 400 mg IV ql2h OR 
Levofloxacin 500 mg IV q24h 

Doxycycline 100 mg PO ql2h OR 
Ciprofloxacin 500 mg PO ql2h OR 
Levofloxacin 500 mg PO q24h 

Gentamicin 5 mg/kg IV qd OR 
Doxycycline 100 mg IV ql2h OR 
Ciprofloxacin 400 mg IV ql2h 

Doxycycline 100 mg PO ql2h OR 
Ciprofloxacin 500 mg PO ql2h 

Supportive care 

Vaccination may be effective if given 
within the first several days after 
exposure. 

Supportive care; antitoxin may halt 
the progression of symptoms but is 
unlikely to reverse them. 

Supportive care; ribavirin may be ben¬ 
eficial in select cases. 


Ciprofloxacin 10-15 mg/kg IV ql2h OR 
Levofloxacin 8 mg/kg IV ql2h OR 
Doxycycline 2.2 mg/kg IV ql2h 

AND 

Clindamycin* 10-15 mg/kg IV q8h 

AND 

Penicillin G § 400-600 k U/kg/d IV x q 4h 

AND CONSIDER 

Raxibacumab IV (> 50 kg: 40 mg/kg; 15-50 kg: 60 
mg/kg; < 15 kg: 80 mg/kg) 

Ciprofloxacin 10-15 mg/kg PO ql2h OR 
Levofloxacin 8 mg/kg PO ql2h OR 
Doxycycline 2.2 mg/kg PO ql2h 

Ciprofloxacin 10-15 mg/kg PO ql2h OR 
Levofloxacin 8 mg/kg PO ql2h OR 
Doxycycline 2.2 mg/kg PO ql2h 

Gentamicin 2.5 mg/kg IV q8h OR 
Doxycycline 2.2 mg/kg IV ql2h OR 
Ciprofloxacin 15 mg/kg IV ql2h OR 
Levofloxacin 8 mg/kg IV ql2h 

Doxycycline 2.2 mg/kg PO ql2h OR Ciprofloxacin 
20 mg/kg PO ql2h OR 
Levofloxacin 8 mg/kg PO ql2h 

Gentamicin 2.5 mg/kg IV q8h OR 
Doxycycline 2.2 mg/kg IV ql2h OR 
Ciprofloxacin 15 mg/kg IV ql2h 

Doxycycline 2.2 mg/kg PO ql2h OR Ciprofloxacin 
20 mg/kg PO ql2h 

Supportive care 

Vaccination may be effective if given within the first 
several days after exposure. 

Supportive care; antitoxin may halt the progression 
of symptoms but is unlikely to reverse them. 

Supportive care; ribavirin may be beneficial in 
select cases. 


*In a mass casualty setting, where resources are severely constrained, oral therapy may need to be substituted for the preferred parenteral option. 
Assuming the organism is sensitive, children may be switched to oral amoxicillin (80 mg/kg/d * q8h) to complete a 60-day course. We recom¬ 
mend that the first 14 days of therapy or postexposure prophylaxis, however, include ciprofloxacin, levofloxacin, or doxycycline regardless 
of age. A three-dose series of Anthrax Vaccine Adsorbed may permit shortening of the antibiotic course to 30 days. 

*Rifampin or clarithromycin may be acceptable alternatives to clindamycin as drugs that target bacterial protein synthesis. If ciprofloxacin 
or another quinolone is employed, doxycycline may be used as a second agent, as it also targets protein synthesis. 

§ Ampicillin, imipenem, meropenem, or chloramphenicol may be acceptable alternatives to penicillin as drugs with good central nervous 
system penetration. 

¥ 10 days of therapy may be adequate for endemic cutaneous disease. A full 60-day course is recommended in the setting of terrorism, how¬ 
ever, because of the possibility of a concomitant inhalational exposure. 

IV: intravenous; PO: per os (by mouth) 


119 





Medical Aspects of Biological Warfare 



Figure 5-2. An empiric and algorithmic approach to the diagnosis and management of potential biological casualties, 
cipro: ciprofloxacin; CXR: chest X-ray; doxy: doxycycline; JE: Japanese encephalitis; nl: normal limits; prn: as needed; Rx: 
prescription; VEE: Venezuelan equine encephalitis; VHF: viral hemorrhagic fever; +: positive finding; ++: strongly positive 
finding; +/-: with or without finding 

Adapted with permission from Henretig FM, Cieslak TJ, Kortepeter MG, Fleisher GR. Medical management of the suspected 
victim of bioterrorism: an algorithmic approach to the undifferentiated patient. Emerg Med Clin North Am. 2002;20:351-364. 



Figure 5-3. The M93 "Fox" nuclear, biological, and chemical 
reconnaissance vehicle. 


delineation of the limits of contamination. Using M93 
"Fox" or M1135 Stryker (Figure 5-3) nuclear, biological, 
chemical reconnaissance vehicles, these personnel can 
collect soil, water, and vegetation samples, mark areas 
of contamination, and transmit data to commanders in 
real time. Finally, notifying laboratory personnel not 
only permits them to focus their efforts at diagnosis, 
but also allows them to take necessary precautions. 

In a civilian terrorism response scenario, notifica¬ 
tion of a suspected biological, chemical, or radiologi¬ 
cal attack would typically be made through local or 
regional health department channels. In the United 
States, a few larger cities have their own health depart¬ 
ments. In most areas, though, the county represents 
the lowest jurisdiction at which an independent health 
department exists. In some rural areas lacking county 


120 
















































































Medical Management of Potential Biological Casualties: A Stepwise Approach 


EXHIBIT 54 

CONVENTIONAL INFECTIOUS DISEASES AND DISEASES POTENTIALLY RESULTING FROM 
AN ACT OF BIOTERRORISM:REQUIRED HOSPITAL INFECTION CONTROL PRECAUTIONS’ 

Standard (handwashing) Contact (gloves and Droplet (private room* and Airborne (private room,* 

gown*) surgical mask®) negative pressure room, 

HEPA filter mask) 


All patients 

MRSA, VRE 

Meningococcal disease 

Pulmonary TB 

Anthrax 

Enteric infections 

Resistant pneumococci 

Measles 

Botulism 

Skin infections 

Pertussis 

Varicella 

Tularemia 

Lice 

Group A streptococci 

Smallpox 

Brucellosis 

Scabies 

Mycoplasma 


Q Fever 

Clostridium difficile disease 

Adenovirus 


Glanders 

RSV, parainfluenza 

Influenza 


Melioidosis 

Ricin intoxication 

SEB intoxication 

T-2 intoxication 

VEE, EEE, WEE 

Certain VHFs 

• Ebola* 

• Marburg* 

• Lassa Fever 

Smallpox 

Melioidosis (with cutane¬ 
ous lesions) 

Pneumonic plague 



Thorough guidelines for hospital infection control can be found in: Cole LA. Bioterrorism threats: learning from inappropriate 
responses. ] Puhl Hlth Manage Pract. 2000;6:8-18. 

*Gloves and/or gown should also be worn as a part of standard precautions (and other forms of precaution) when contact with blood, 
body fluids, and other contaminated substances is likely. 

*Mixing patients with the same disease is an acceptable alternative to a private room. 

^Surgical masks should also be employed as a part of standard and contact precautions (along with eye protection and a face shield) 
if procedures are likely to generate splashes or sprays of infectious material. 

¥ While Ebola is transmitted primarily via infected blood and body fluids, the voluminous emesis and diarrhea produced by Ebola 
patients, the very low infectious inoculum of the virus, and the ease with which hemorrhagic respiratory secretions can be aerosol¬ 
ized during coughing, the CDC now recommends that both contact and droplet precautions be employed when managing Ebola 
victims; similar caution would likely apply to Marburg (and perhaps other VHF) patients as well. 

EEE: eastern equine encephalomyelitis; HEPA: high-efficiency particulate air; MRSA: methicillin-resistant Staphylococcus aureus ; RSV: 
respiratory syntactical virus; SEB: staphylococcal enterotoxin B; TB: tuberculosis; VEE: Venezuelan equine encephalitis; VHF: viral 
hemorrhagic fever; VRE: vancomycin-resistant enterococci; WEE: western equine encephalomyelitis 


health departments, practitioners would access the 
state health department directly. Once alerted, local 
and regional health authorities know how to request 
additional support from health officials at higher 
jurisdictions. Each practitioner should have a point of 
contact with such agencies and should be familiar with 
mechanisms for contacting them before a crisis arises. 

If an outbreak proves to be the result of terror¬ 
ism, or if the scope of the outbreak overwhelms local 
resources, a regional or national response becomes 
imperative. Under such circumstances, an extensive 
panoply of supporting assets and capabilities may 
be summoned. The National Incident Management 
System and its component Incident Command System 
(ICS) provide a standardized approach to command 
and control at an incident scene. 49 Local officials use 
the ICS when responding to both natural and human- 


made disasters, and ICS would be equally applicable 
in responding to a biological attack. Under the ICS, a 
designated official, typically the fire chief or the chief 
of police, serves as local incident commander. The 
incident commander may be able to summon groups of 
volunteer medical personnel through the Metropolitan 
Medical Response System, which includes medical 
strike teams in 124 local jurisdictions. These teams, 
under contract with mayors of the 124 municipalities, 
are organized under the Department of Homeland 
Security's Office of Domestic Preparedness. 

In any incident or disaster, whether natural or 
human-made, the local incident commander may 
request assistance from the state through the state 
coordinating officer if it appears that local resources 
or capabilities will be exceeded. The state coordinating 
officer works with the governor and other state of- 


121 






Medical Aspects of Biological Warfare 


EXHIBIT 5-5 

THE LABORATORY RESPONSE NETWORK 

Sentinel laboratories. These laboratories, found in many hospitals and local public health facilities, have the ability 
to "rule-out" specific bioterrorism threat agents, to handle specimens safely, and to forward specimens on to higher 
echelon laboratories within the network. 

Reference laboratories. These laboratories, typically found at state health departments, and at military, veterinary, 
agricultural, and water-testing facilities, can employ BSL-3 practices, and can often conduct nucleic acid amplifica¬ 
tion and molecular typing studies. The more than 100 reference laboratories can confirm ("rule-in") the presence of 
the various biological threat agents. 

National laboratories. These laboratories, including those at the CDC and USAMRIID, can employ BSL-4 practices, 
and serve as the final authority in the work-up of bioterrorism specimens. These laboratories provide specialized 
reagents to lower level laboratories and have the ability to bank specimens, perform serotyping, and detect genetic 
recombinants and chimeras. 


BSL: biosafety level 

CDC: Centers for Disease Control and Prevention 

USAMRIID: US Army Medical Research Institute of Infectious Disease 


EXHIBIT 5-6 

BIOSAFETY LEVELS 

Biosafety Level 1: includes practices employed by a microbiology laboratory that deals only with well-character¬ 
ized organisms that do not typically produce disease in humans. Work is conducted on open bench tops using 
standard microbiologic practices. Example: high school biology laboratory 

Biosafety Level 2: includes practices employed by laboratories that deal with most human pathogens of moderate 
potential hazard. Laboratory coats and gloves are typically worn, access to the laboratory is restricted to trained 
personnel, and safety cabinets are often employed. Example: clinical hospital laboratory 

Biosafety Level 3: Includes practices employed by laboratories that work with agents with the potential to cause 
serious and lethal disease by the inhalational route of exposure. Work is generally conducted in safety cabinets, 
workers are often vaccinated against the agents in question, and respiratory protection is worn. Clothing (such 
as scrub suits) is exchanged upon exiting the laboratory. Laboratories are negatively pressurized. Example: state 
health department laboratory 

Biosafety Level 4: Also includes practices employed by laboratories working with highly hazardous human patho¬ 
gens infectious via the inhalational route. BSL-4 organisms differ from those requiring BSL-3 precautions in that 
no vaccine or antibiotic therapy is available. Personnel may only enter the laboratory through a series of changing 
and shower rooms. Equipment and supplies enter via a double-door autoclave. Strict and sophisticated engineering 
controls are employed and personnel wear sealed positive-pressure space suits with supplied air. Laboratories are 
negatively pressurized. Examples: laboratories at the CDC, USAMRIID, the Canadian Science Center for Human 
and Animal Health, and a few other research facilities 

BSL: biosafety level 

CDC: Centers for Disease Control and Prevention 

USAMRIID: US Army Medical Research Institute of Infectious Disease 


ficials to make state-level assets (such as state health 
departments, state public health laboratories, and state 
police assets) available. Most state public health labo¬ 
ratories participate as "reference" laboratories in the 
Association of Public Health Laboratories and CDC's 


Laboratory Response Network. These facilities support 
hundreds of "sentinel" laboratories in local hospitals 
throughout the nation, and can provide sophisti¬ 
cated confirmatory diagnosis and typing of biological 
agents 50 (an overview of public health laboratory capa- 


122 








Medical Management of Potential Biological Casualties: A Stepwise Approach 


bilities is provided in Exhibit 5-5; the biosafety-level 51 
precautions they employ are outlined in Exhibit 5-6). 
State police can provide law enforcement assistance 
and state police laboratories can assist with forensic 
analysis. Finally, governors can access military assets 
at the state level through National Guard units un¬ 
der their direct control. These units can provide law 
enforcement, public works assistance, mobile field 
hospital bed capacity, and other support. Every state 
governor now has, at his or her disposal, one of some 57 
military Weapons of Mass Destruction-Civil Support 
Teams (WMD-CSTs). These 22-person advisory teams 
can offer expertise and provide liaison to additional 
military assets at the federal level. 

When state capabilities are overwhelmed or insuf¬ 
ficient, the state coordinating officer may alert the 
federal coordinating officer, who can, in turn, assist 
in activating the national response framework. The 
national response framework guides delivery of fed¬ 
eral assets and provides for a coordinated multiagency 
federal response. Federal response and support to state 
and local jurisdictions, according to the framework, is 
organized into 15 emergency support functions (ESFs). 
ESF 8 provides for health and medical services. While 
a specific agency is assigned primary responsibility 
for each of the 15 ESFs, more than two dozen federal 
agencies, as well as the American Red Cross, can, 
under federal law, be tasked to provide assistance. 
Federal disaster medical support is primarily the re¬ 
sponsibility of the Department of Health and Human 
Services which, through its Office of Emergency Re¬ 
sponse, oversees the National Disaster Medical System 
(NDMS). 52 A principal component of the NDMS is its 
network of disaster medical assistance teams, each 
of which consists of trained medical volunteers with 
the ability to arrive at a disaster site within 8 to 16 
hours. Another important component of the NDMS 
is its excess hospital bed capacity, held at numerous 
Department of Veterans Affairs, military, and civilian 
hospitals throughout the nation. 

Finally, several other federal agencies may play an 
important role in the response to disasters, includ¬ 
ing, in particular, those resulting from a biological 
attack. The CDC and USAMRIID provide national 
laboratories, which support the reference labs at the 
state level and are capable of dealing with virtually 
all potential biological threat agents. 53 Expert consul¬ 
tation and epidemiological investigative assistance 
is also available through the CDC, and bioweapons 
threat evaluation and medical consultation is like¬ 
wise available through USAMRIID. Additionally, the 
military can provide expert advice and assistance to 
civilian authorities through Army National Guard's 
CBRNE Enhanced Response Force Package Teams, 
which can arrive at a disaster site within a few hours 


of notification, as well as through the aforementioned 
CBIRF, which is capable of providing reconnaissance, 
decontamination, and field treatment. Military sup¬ 
port, when provided, would be subordinate to civilian 
authorities and would be provided and tailored by the 
Joint Task Force for Civil Support, a component of US 
Army Northern Command that provides a command- 
and-control element for all military assets involved 
in disaster response missions and other contingen¬ 
cies within the United States. Finally, the CDC has 
developed the Strategic National Stockpile, whereby 
critical drugs and vaccines necessary to combat a large 
disaster or terrorist attack are stockpiled at several 
locations throughout the country, available for rapid 
deployment to an affected area. 54 Release of stockpile 
components is currently controlled by the Department 
of Health and Human Services. 

Step 9: Conduct an Epidemiological Investigation 
and Manage the Psychological Aftermath of a 
Biological Attack 

Clinicians must be versed in the basic principles 
of epidemiology and be prepared to assist in the 
epidemiological investigation, which will be of para¬ 
mount importance after a suspected terrorist attack. 
Although preventive medicine officers, environmental 
science officers, veterinarians, preventive medicine 
technicians (68S in US Army organizations), and 
field sanitation personnel may be invaluable in the 
course of such an investigation, the clinician should, 
nonetheless, have a working knowledge of the steps, 
known as the epidemiological sequence, 55 involved 
in the conduct of an epidemiological investigation 


EXHIBIT 5-7 

THE EPIDEMIOLOGICAL SEQUENCE 

1. Make an observation 

2. Count cases 

3. Relate cases to population 

4. Make comparisons 

5. Develop the hypothesis 

6. Test the hypothesis 

7. Make scientific inferences 

8. Conduct studies 

9. Intervene and evaluate 

Data source: Centers for Disease Control and Prevention. 
Investigating an outbreak. In: Principles of Epidemiology: Self 
Study Course SS3030. 2nd ed. Atlanta, GA: CDC; 1998:347-A24. 


123 





Medical Aspects of Biological Warfare 


(Exhibit 5-7). Although the well-prepared clinician 
may positively impact the health and well-being of 
individual patients, it is only through the rapid con¬ 
duct of a competent epidemiological investigation 
that large numbers of exposed persons are likely to 
be reached, and successful medical and psychologi¬ 
cal prophylaxis implemented, before the widespread 
outbreak of disease or panic. 

In addition to the instigation of an epidemiological 
investigation and the institution of specific medical 
countermeasures against biological agent exposures, 
the clinician should be prepared to address the psycho¬ 
logical effects of known, suspected, or feared exposure 
to threat agents. 56 An announced or threatened biologi¬ 
cal attack can provoke fear, uncertainty, and anxiety 
in the population, and can result in an overwhelming 
number of patients seeking evaluation and demanding 
therapy for feared exposure. Such a scenario might also 
follow the covert release of an agent once the resulting 
epidemic is characterized as being the consequence of 
a biological (or chemical or radiological) attack. Symp¬ 
toms due to anxiety and autonomic arousal, as well 
as side effects from postexposure prophylactic drugs, 
may mimic prodromal disease due to biological agent 
exposure and pose dilemmas in differential diagnosis. 
Persons with symptoms arising from naturally occur¬ 
ring infectious diseases may likewise pose significant 
challenges to healthcare providers and public health 
officials. 

Public panic and behavioral contagion are best pre¬ 
vented by timely, accurate, well-coordinated, and real¬ 
istic risk communication from health and government 
authorities. Such communication should include an 
assessment of the risk of exposure, information regard¬ 
ing the resulting disease, and a recommended course 
of action for suspected exposure. As the epidemic sub¬ 
sides and public knowledge increases, public anxiety 
will decrease to realistic and manageable levels. This 
cycle of uncertainty, panic, response, and resolution 
occurred during the October 2001 anthrax bioterror 
event. 57 Readily accessible, biological, chemical, and 
radiological agent-specific information packages for 
local public health authorities and the general public 
are available through the CDC website, and can be of 
valuable assistance in risk communication. 

Effective risk communication is possible only in 
the presence of well-conceived risk communica¬ 
tion plans and tactics, worked out well in advance 
of an actual event. Similar advanced planning must 
take into account the need to rapidly establish local 
centers for the initial evaluation and administration of 


postexposure prophylaxis. Finally, the development 
of patient and contact tracing mechanisms and vac¬ 
cine screening tools, the mechanisms for accession of 
stockpiled vaccines and medications, and the means 
by which to identify and prepare local facilities and 
healthcare teams for the care of mass casualties must 
be clearly elucidated in advance. The CDC's smallpox 
response plan 40 provides a useful template for such a 
coordinated, multifaceted approach, and the wisdom 
of farsighted planning and coordination was amply 
demonstrated by the efficient mass prophylaxis of over 
10,000 individuals in New York City during the events 
surrounding the discovery of anthrax-contaminated 
mail in 2001. 58 

Step 10: Maintain a Level of Proficiency 

Once response plans have been developed, they 
must be exercised. Military commanders and their 
units are typically well versed in planning and execut¬ 
ing conventional field-training and command-post 
exercises. In the future, such exercises must account 
for the real possibility that military units may encoun¬ 
ter biological weapons on the battlefield. Similarly, 
planning and exercises must account for the tandem 
threat posed by bioterrorist attacks against garrison 
activities. Local civilian exercises (which can often 
include military participants) are likewise a necessary 
component of disaster preparation. Such exercises 
should be designed so as to test incident command 
and control, communications, logistics, laboratory 
coordination, and clinical capabilities. These exercises 
may involve only the leadership of an organization 
and focus on planning and decision making (the 
command-post exercise), they may involve notional 
play around a tabletop exercise, or they may involve 
actual hands-on training and evaluation in a disaster 
drill or field-training exercise. In fact, the CDC ex¬ 
pended considerable effort prior to the 2009 H1N1 
influenza pandemic preparing for just such an event, 
conducting numerous tabletop and full-scale exercises 
involving CDC personnel as well as state public health 
participants. The Joint Commission requires hospitals 
to conduct a hazard vulnerability analysis, develop 
an emergency operations plan, and evaluate this plan 
twice yearly; one of these evaluations must include 
a community-wide drill. 59 Moreover, the Joint Com¬ 
mission specifically mandates that hospitals provide 
facilities (and training in the use of such facilities) for 
radioactive, biological, and chemical isolation and 
decontamination. 


124 


Medical Management of Potential Biological Casualties: A Stepwise Approach 


SUMMARY 


Many resources, including this text, are now avail¬ 
able to assist both military and civilian clinicians 
and public health professionals in planning for, and 
maintaining proficiency in, the management of real 
or threatened terror attacks. Finally, as discussed 
under step 8 above, numerous governmental, mili¬ 
tary, and civilian organizations have now been orga¬ 


nized, trained, and equipped to provide assistance 
and consultation to clinicians, first responders, and 
public health officials faced with planning for and 
treating the victims of a potential terrorist attack. It 
is assistance that, if incorporated into thorough plan¬ 
ning efforts, will hopefully never be needed for actual 
patient care purposes. 


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12. Pavlin JA. Epidemiology of bioterrorism. Emerg Infect Dis. 1999;5:528-530. 

13. Hiss J, Arensburg B. Suffocation from misuse of gas masks during the Gulf war. Br Med J. 1992;304:92. 

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29. Grabenstein JD, Winkenwerder W Jr. US military smallpox vaccination experience. JAMA. 2003;289;3278-3282. 

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40. Mortimer PP. Can postexposure vaccination against smallpox succeed? Clin Infect Dis. 2003;36:622-629. 

41. Mack T. A different view of smallpox and vaccination. N Engl J Med. 2003;348:460-463. 

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53. Centers for Disease Control and Prevention. Biological and chemical terrorism: strategic plan for preparedness and 
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EM 1-24. 


128 


Chapter 6 
ANTHRAX 


BRET K. PURCELL, PhD, MD* * * * § ; CHRISTOPHER K. COTE, PhD + ; PATRICIA L. WORSHAM, PhD*; and 
ARTHUR M. FRIEDLANDER, MD 5 


INTRODUCTION AND HISTORY 

THE ORGANISM 

EPIDEMIOLOGY 

PATHOGENESIS 

CLINICAL DISEASE 
Cutaneous Anthrax 
Inhalational Anthrax 
Meningitis 

Oropharyngeal and Gastrointestinal Anthrax 
DIAGNOSIS 
TREATMENT 
PROPHYLAXIS 

Prophylactic Treatment After Exposure 
Active Immunization 
Side Effects 

SUMMARY 


*Colonel, Medical Corps, US Army; Deputy Chief, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter 
Street, Fort Detrick, Maryland 21702 

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

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

§ Colonel (Retired), Medical Corps, US Army; Senior Scientist, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort 
Detrick, Maryland 21702, and Adjunct Professor of Medicine, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, 
Maryland 20814 


129 



Medical Aspects of Biological Warfare 


INTRODUCTION AND HISTORY 


Anthrax, a zoonotic disease caused by Bacillus 
anthracis, occurs in domesticated and wild animals, 
primarily herbivores, including goats, sheep, cattle, 
horses, and swine. 1-3 Humans usually become infected 
by contact with infected animals or contaminated 
animal products, most commonly via the cutaneous 
route and only rarely via the respiratory or gastroin¬ 
testinal routes. 6,7 Anthrax has a long association with 
human history. The fifth and sixth plagues described 
in Exodus may have been anthrax in domesticated 
animals followed by cutaneous anthrax in humans. 
Virgil described anthrax in domestic and wild animals 
in his Georgies, and anthrax was an economically im¬ 
portant agricultural disease during the 16th through 
18th centuries in Europe. 8,9 

Anthrax, which is intimately associated with 
the origins of microbiology and immunology, was 
the first disease for which a microbial origin was 
definitively established. Robert Koch established 
the microbial origin for anthrax in 1876. 10,11 Anthrax 
also was the first disease for which an effective live 
bacterial vaccine was developed; Louis Pasteur de¬ 
veloped that vaccine in 1881. 12 Additionally, anthrax 
represents the first described occupational respira¬ 
tory infectious disease. During the latter half of the 
19th century, inhalational anthrax, 13 a previously 
unrecognized form, occurred among wool-sorters 
in England as a result of the generation of infectious 
aerosols of anthrax spores under industrial condi¬ 
tions from the processing of contaminated goat hair 
and alpaca wool. 14 

The military has long been concerned about B 
anthracis as a potential biological weapon because 
anthrax spores are infectious by the aerosol route, 
and a high mortality rate is associated with untreated 
inhalational anthrax. In 1979 the largest inhalational 
anthrax epidemic of the 20th century occurred in 
Sverdlovsk, Russia. B anthracis spores were acciden¬ 
tally released from a military research facility located 
upwind from where the cases occurred. According 
to the accounts provided by two Soviet physicians, 
96 human anthrax cases were reported, of which 79 
were gastrointestinal and 17 cutaneous. The 79 gastro¬ 
intestinal cases resulted in 64 deaths. 13 Although the 
initial report of this event attributed the infections to 
a gastrointestinal source, later evidence indicated that 
an aerosol release of weaponized anthrax spores from 
a military production facility had occurred, and thus, 
inhalational anthrax was the predominant cause of 
these civilian casualties. Retrospective analysis using 
administrative name lists of compensated families. 


household interviews, grave markers, pathologists' 
notes, various hospital lists, and clinical case histories 
of five survivors yielded evidence of 77 anthrax cases, 
with 66 deaths and 11 survivors. 15 Cases were also 
reported in animals located more than 50 km from 
the site. 16,17 Polymerase chain reaction examination of 
tissue samples collected from 11 of the victims dem¬ 
onstrated that virulent B anthracis DNA was present 
in all these patients, and at least five different strains 
of virulent B anthracis were detected based on variable 
number tandem repeat analysis. 18 

The retrospective data associated with the Sverd¬ 
lovsk incident as well as studies performed for the 
Department of Homeland Security have been used 
by several computer modeling efforts to better un¬ 
derstand the human infectious dose. 19,20 Under the 
direction of the Department of Health and Human 
Services, the Office of the Assistant Secretary of 
Preparedness and Response, Public Health Emer¬ 
gency Medical Countermeasures Enterprise, and 
Biomedical Advanced Research and Development, 
these agencies have developed a variety of computer 
dissemination models for a wide variety of potential 
scenarios. 

Although the Sverdlovsk incident is not well 
known among US civilians, most people are familiar 
with the 2001 bioterrorist attack in the United States 
in letters containing dried B anthracis spores. The 
spore powder, which was sealed in letters addressed 
to members of Congress and the press, was mailed 
through the US Postal Service. 21-24 According to the 
Centers for Disease Control and Prevention, 22 people 
contracted anthrax from the letters. 21,25-29 Of the 11 
individuals who developed inhalational anthrax, five 
died and six survived after intensive antimicrobial 
therapy. Eleven other people contracted cutaneous 
anthrax; all survived after treatment. Thousands of 
other persons received prophylaxis with antibiotics 
and, in some cases, postexposure vaccination. 30-33 

Considerable research has been devoted to biode¬ 
fense research and modeling since this event. 34-45 It has 
been estimated that the 2001 anthrax attacks cost the 
United States more than $1 billion in medical planning, 
response, and remediation costs. 4M9 Additionally, this 
incident profoundly affected the law enforcement, 
scientific, and medical communities within the United 
States and throughout the world. Although the source 
of these letters has never been definitively identified, 
the impact on biodefense research establishments has 
been a transformational event for researchers and 
institutes. 


130 


Anthrax 



Figure 6-1. (a) Gram stain of a blood smear from an infected guinea pig demonstrating intracellular bacilli chains within a 
polymorphonuclear leukocyte, (b) Gram stain of peripheral blood smear from a nonhuman primate infected with Bacillus 
anthracis, Ames strain. 

Photographs: (a) Courtesy of Susan Welkos, PhD, Division of Bacteriology, US Army Medical Research Institute of Infectious 
Diseases, Fort Detrick, Maryland, (b) Courtesy of John Ezzell, PhD, US Army Medical Research Institute of Infectious Dis¬ 
eases, Fort Detrick, Maryland. 


THE ORGANISM 


B anthracis is a large, gram-positive, spore-forming, 
nonmotile bacillus (1-1.5 pm x 3-10 pm) that is closely 
related to Bacillus cereus and Bacillus thuringiensis. The 
organism grows readily on sheep blood agar aerobi¬ 
cally and is nonhemolytic under these conditions. 
The colonies are large, rough, and grayish white, with 
irregular, curving outgrowths from the margin. The 
organism forms a prominent capsule both in vitro in 
the presence of bicarbonate in the culture media and 
elevated levels of carbon dioxide in the bacterial plate 
incubator and in tissue in vivo. In tissue, the encap¬ 
sulated bacteria occur singly or in chains of two or 
three bacilli (Figure 6-1). The organism does not form 
spores in living tissue; sporulation occurs only after 
the infected carcass tissues are exposed to oxygen. The 
spores, which cause no swelling of the bacilli, are oval 
and they occur centrally or paracentrally (Figure 6-2). B 
anthracis spores are composed of dozens of spore coat 
proteins that—in part—protect the genomic material 
housed in the core. 50,51 The spores are surrounded by 
a loose fitting membrane referred to as the exospo- 
rium. The exosporium has been shown to impact how 
the spore interacts with certain types of mammalian 
cells. 52,53 The spores, which are resistant to environ¬ 
mental stressors, may survive for decades in certain 
soil conditions. Bacterial identification is confirmed 
by demonstration of the protective antigen (PA) toxin 
component, lysis by a specific bacteriophage, detec¬ 


tion of capsule by fluorescent antibody, and virulence 
for mice and guinea pigs. 54,55 Additional confirmatory 
tests to identify toxin and capsule genes by polymerase 
chain reaction, developed as research tools, have been 



Figure 6-2. Scanning electron micrograph of a preparation 
of Bacillus anthracis spores. Two elongated bacilli are also 
presented among the oval-shaped spores. Original magni¬ 
fication x 2,620. 

Photograph: Courtesy of John Ezzell, PhD, US Army Medi¬ 
cal Research Institute of Infectious Diseases, Fort Detrick, 
Maryland. 


131 


Medical Aspects of Biological Warfare 


incorporated into the Laboratory Response Network 
established by the Centers for Disease Control and 
Prevention. 56-59 

The diagnosis of anthrax has been complicated by 
the identification of strains of B cereus, which produce 
anthrax-like disease. Because B cereus is hemolytic and 
resistant to the anthrax-specific gamma bacteriophage, 
such isolates would not typically be tested for the 


presence of genes encoding anthrax toxin, especially 
because B cereus is often regarded as an environmental 
contaminant. 60 Continued reports of bacterial strains 
harboring anthrax toxin genes have demonstrated 
not only the importance of appropriate detection 
strategies, but also the possibility of emerging risks 
associated with the possible transfer of B anthracis 
characteristics to other organisms. 61,62 


EPIDEMIOLOGY 


B anthracis, an organism that exists in the soil as a 
spore, occurs worldwide. Whether its persistence in 
the soil results from significant multiplication of the 
organism, or from cycles of bacterial amplification in 
infected animals whose carcasses then contaminate the 
soil, remains unsettled. 63-67 The form of the organism 
in infected animals is the bacillus. 

Domestic or wild animals become infected when 
they ingest spores while grazing on contaminated 
land or eating contaminated feed. Pasteur origi¬ 
nally reported that environmental conditions such as 
drought, which may promote trauma in the oral cav¬ 
ity on grazing, may increase the chances of acquiring 
anthrax. 68 Spread from animal to animal by mechanical 
means—by biting flies and from one environmental 
site to another by nonbiting flies and by vultures—has 
been suggested to occur. 64,69 

Anthrax in humans is associated with agricul¬ 
tural, horticultural, or industrial exposure to infected 
animals or contaminated animal products. In less 
developed countries, primarily Africa, Asia, and the 
Middle East, disease occurs from contact with infected 
domesticated animals or contaminated animal prod¬ 
ucts. Contact may include handling contaminated 
carcasses, hides, wool, hair, and bones or ingesting 
contaminated meat. Cases associated with industrial 
exposure—rarely seen—occur in workers processing 
contaminated hair, wool, hides, and bones. Direct 
contact with contaminated material leads to cutane¬ 
ous disease, and ingestion of infected meat leads to 
oropharyngeal or gastrointestinal forms of anthrax. 
It has been well documented that intravenous drug 
users can become infected with B anthracis, resulting 
in a septicemic form of anthrax. 70- 8 Inhalation of a 
sufficient quantity of spores, usually seen only during 
generation of aerosols in an enclosed space associated 
with processing contaminated wool or hair, leads to 
inhalational anthrax. Military research facilities have 


B anthracis produces two protein exotoxins, known 
as the lethal toxin (LT) and the edema toxin (ET); an 
antiphagocytic capsule; and other known and puta¬ 


played a major role in studying and defining anthrax, 
as well as many other zoonotic diseases in wild and 
domestic animals and the subsequent infections in 
humans. 79 

Unreliable reporting makes it difficult to estimate 
with accuracy the true incidence of human anthrax. It 
was estimated in 1958 that between 20,000 and 100,000 
cases occurred annually worldwide. 80 In more recent 
years, anthrax in animals has been reported in 82 
countries, and human cases continue to be reported 
from Africa, Asia, Europe, and the Americas. 81-85 In the 
1996-1997 global anthrax report, a general decrease ap¬ 
peared in anthrax cases worldwide; however, anthrax 
remains underdiagnosed and underreported. 86 

In the United States the annual incidence of human 
anthrax has steadily declined from about 127 cases in 
the early part of the 20th century to about 1 per year for 
the past 10 years. 87 The vast majority of these cases have 
been cutaneous. Under natural conditions, inhalational 
anthrax is rare; before the anthrax bioterrorism event 
in 2001, only 18 cases had been reported in the United 
States in the 20th century. 88,89 In the early part of the 
20th century, inhalational anthrax cases were reported 
in rural villagers in Russia who worked with contami¬ 
nated sheep wool inside their homes. 90 However, in 
recent years a significant decrease occurred in anthrax 
cases in domestic animals in east Russia. Five inhala¬ 
tional anthrax cases occurred in woolen mill workers 
in New Hampshire in the 1950s. 91 During economic 
hardship and disruption of veterinary and human 
public health practices (eg, during wartime), large 
anthrax epidemics have occurred. The largest reported 
human anthrax epidemic occurred in Zimbabwe from 
1978 through 1980, with an estimated 10,000 cases. 92 

Essentially all cases were cutaneous, including rare 
gastrointestinal disease cases and eight inhalational 
anthrax cases, although no autopsy confirmation was 
reported. 93 


tive virulence factors. 94 The role of the capsule in 
pathogenesis was demonstrated in the early 1900s, 
when anthrax strains lacking a capsule were shown 


132 


Anthrax 


to be attenuated. 95 In more recent years, the genes 
encoding synthesis of the capsule were identified on 
the 96-kilobase plasmid known as pX02. Molecular 
analysis revealed that strains cured of this plasmid no 
longer produced the capsule and were attenuated, thus 
confirming the critical role of the capsule in virulence. 96 
The capsule is composed of a polymer of D-glutamic 
acid, which confers resistance to phagocytosis and 
may contribute to the resistance of anthrax to lysis 
by serum cationic proteins. 97-102 Capsule production 
is necessary for dissemination to the spleen in a mu¬ 
rine inhalational anthrax model. 103 The capsule has 
also been the focus of several efforts to develop new 
generation anthrax vaccines. 104-106 Evidence indicates 
that the capsule may enhance the protection afforded 
by PA-based vaccines against anthrax if opsonizing 
antibodies are produced. 106 

Koch first suggested the importance of toxins 
in his initial studies on anthrax. In 1954 Smith and 
Keppie 107 demonstrated a toxic factor in the serum of 
infected animals that was lethal when injected into 
other animals. The role of toxins in virulence and im¬ 
munity was firmly established by many researchers in 
the ensuing years. 108,109 Advances in molecular biology 
have produced a more complete understanding of the 
biochemical mechanisms of action of the toxins, and 
they have begun to provide a more definitive picture 
of their role in the pathogenesis of the disease. 

Two protein exotoxins, known as the LT and the 
ET, are encoded on a 182-kb plasmid (pXOl), distinct 
from that coding for the capsule. In an environment 
of increased bicarbonate in the growth media, at¬ 
mospheric carbon dioxide within the plate incuba¬ 
tion chamber, and increased temperature, such as is 
found in the infected host, transcription of the genes 
encoding these and other virulence-associated gene 
products is enhanced. 94,110-113 A complex regulatory 
cascade controlled in large part by the atxA and acpA 
genes encoded on the toxin plasmid pXOl and pX02, 
respectively, directs the production of virulence factors 
in response to these environmental signals. 114,115 The 
anthrax toxins, like many bacterial and plant toxins, 
possess two components: (1) a cell binding, pore¬ 
forming, or B, domain; and (2) an active, or A, domain 
that has the toxic and—usually—the enzymatic activity 
(Figure 6-3). The B and A anthrax toxin components, 
which are synthesized from different genes, are se¬ 
creted as noncovalently linked proteins. The anthrax 
toxins are unusual because both toxins share the B 
protein, PA. Thus, the LT is composed of the PA63 
(MW [molecular weight] 63,000 after cleavage from a 
MW 83,000 protein) heptamer or octamer combined 
with a second protein, which is known as lethal fac¬ 
tor (LF [MW 90,000]), and the ET is composed of PA 
complexed with the edema factor (EF [MW 89,000]). 


Each of these three toxin proteins—the B protein and 
both A proteins—individually is without biological 
activity. The critical role of the toxins in pathogenesis 
was established when it was shown that deletion 
of the toxin-encoding plasmid pXOl 96,116 or the PA 
gene alone 117 attenuates the organism. Crude toxin 
preparations have been shown to impair neutrophil 
chemotaxis 118,119 and phagocytosis. 97 

The ET, which causes edema when injected into the 
skin of experimental animals, is likely responsible for 
the marked edema often present at bacterial replication 
sites. 120,121 This toxin is a calmodulin-dependent adenyl¬ 
ate cyclase that impairs phagocytosis and priming for 
the respiratory burst in neutrophils; it also inhibits 
the production of interleukin-6 and tumor necrosis 
factor by monocytes, which may further weaken host 
resistance. 122-124 ET also impairs dendritic cell function 
and appears to act with LT to suppress the innate im- 

125 

mune response. 



Figure 6-3. Composition of anthrax lethal protein toxin. 
Molecular models of the protective antigen (PA) 63 heptamer 
and the PA 63 heptamer-lethal factor (LF) complex, (a, b) Side 
and top views of PA 63 heptamer (green ) bound to three LF 
molecules (yellow), (c, d) The surface renderings are colored 
according to the negative (red) and positive (blue) electrostatic 
surface potential, (c) Top view of the PA 63 heptamer. The 
yellow box highlights the protomer-protomer interface and 
where LF binds to heptameric PA. (d) A hypothetical PA 63 
heptamer-LF interface. 

Photographs: Courtesy of Kelly Halverson, PhD, US Army 
Medical Research Institute of Infectious Diseases, Fort Detrick, 
Maryland. 


133 




Medical Aspects of Biological Warfare 


The LT is a zinc metalloprotease that is lethal for 
experimental animals 120,121,126 and is directly cytolytic 
for rodent macrophages, causing release of the poten¬ 
tially toxic cytokines interleukin-1 and tumor necrosis 
factor. 127 In in vitro models, LT cleaves members of the 
mitogen-activated protein kinase (MAPK) kinase fam¬ 
ily, which are an integral part of a phosphorelay system 
that links surface receptors to transcription of specific 
genes within the nucleus. Thus, LT interferes with 
the MAPK signaling pathways necessary for many 
normal cell functions. 128 In macrophage and dendritic 
cell models, LT leads to inhibition of proinflammatory 
cytokines, downregulation of costimulatory molecules, 
and ineffective T-cell priming. 128-131 In vitro it also 
appears to promote apoptosis of endothelial cells lin¬ 
ing the vascular system, leading to speculation that 
LT-induced barrier dysfunction leads to the vascular 
permeability changes accompanying systemic anthrax 
infection. 132 Effects on hormone receptors, including 
glucocorticoids, have also been reported. Although 
much of the information regarding LT activity has been 
obtained from animal-derived cell culture models. 
Fang et al reported that—in vitro—LT inhibits MAPK 
kinase dependent interleukin-2 production and pro¬ 
liferative responses in human CD4+ T cells. 133 Studies 
using tissue-specific CMG2 knockout mice strongly 
indicate that LTs/ETs target myeloid-derived cells to 
promote bacterial survival early in infection. 134 In ad¬ 
dition, the data suggest that elevated levels of toxin 
specifically target host organs and are responsible 
for the significant morbidity and mortality caused by 
anthrax infection. 135 

Studies in cell culture models have provided a 
clearer understanding of the molecular interactions 
of the toxin proteins. 128 PA first binds, most likely by 
a domain at its carboxy-terminus, to a specific cell re¬ 
ceptor. 136-138 Two proteins have been proposed as the 
PA receptor: (1) Tumor endothelial marker 8 TEM8, 
(ANTX1); and (2) capillary morphogenesis protein, 
CMG2 (ANTX2). 139-141 Both receptors have a von Wil- 
librand factor type A domain that appears to interact 
with PA. Once bound, PA is cleaved by a furin-like 
protease, resulting in retention of a 63-kilodalton 
fragment of PA on the cell surface. 142,143 This cleavage 
promotes formation of PA heptamers and creates a 
binding site on PA to which up to three molecules 
of the LF and the EF can bind with high affinity. 129 
Heptamerization 144 and octamerization 141-144 stimulates 
endocytosis of PA (or PA EF or PA LF complexes), 
which are then delivered into early endosomes. The 
mildly acidic pH of the endosome is hypothesized to 
trigger membrane insertion of the heptameric PA into 
intraluminal vesicles. 145 EF and LF are translocated into 
the lumen of the vesicle and are thereby protected from 


lysosomal proteases. 145 The toxins are then translocated 
via endosomal carrier vesicles to the cell cytosol, where 
they express their toxic activity. 145 In addition, studies 
have also suggested that the formation of octamers 
provides stability to these toxin products and permits 
active LT to travel freely in the circulatory system. 146 

The processes leading to toxin activity in the in¬ 
fected animal may be more complicated because the 
toxin proteins appear to exist in the serum as a complex 
of PA and EF/LF. 147 The proteolytic activation of PA 
necessary to form LT or ET may occur in interstitial 
fluid or serum rather than on the cell surface. 147 The 
LT or ET may then bind to target cells and be internal¬ 
ized. This theory was bolstered by Panchal et al who 
demonstrated that purified LF complexed with the PA 
heptamer cleaved both a synthetic peptide substrate 
and endogenous MAPK kinase substrates and killed 
susceptible macrophage cells. 148 In addition, com¬ 
plexes of the heptameric PA-LF found in the plasma 
of infected animals showed functional activity. 148 Ter¬ 
minally, toxin is present in very high concentrations 
in the blood, which probably accounts for the sudden 
death observations in infected experimental animals. 

Although these toxins were once thought to be 
exclusively found in B anthracis, recent cases of inha- 
lational disease have been identified that possess the 
hallmarks of anthrax disease; however, the bacteria 
recovered were not B anthracis but did possess anthrax 
toxin genes. 149-152 Studies have identified isolates of B 
cereus that carried a plasmid homologous to the anthrax 
toxin plasmid pXOl. The poly glutamate capsule was 
not produced by this B cereus isolate. However, gene 
sequences encoding a polysaccharide capsule were 
present on a smaller plasmid. 149 Capsule-producing 
strains of B cereus have caused severe pneumonia. 150 
Consequently, a possibility of false positives exists in 
diagnostic tests that rely on toxin-based identification 
of genes or gene products. Subsequent investigations 
of these strains determined that the virulence of these 
strains in mice, guinea pigs, and rabbits was signifi¬ 
cantly attenuated when compared to fully virulent B 
anthracis. 153454 It was also shown that vaccines that are 
effective against fully virulent B anthracis can protect 
mice and guinea pigs from infection with the anthrax¬ 
like B cereus strain. 154 

Infection begins when the spores are introduced 
through the skin or mucosa. Spores are then ingested 
at the local site by macrophages. Phagocytosed spores 
can have multiple fates depending on the stage of 
infection and the spore burden of individual phago¬ 
cytes. 155,156 Within the lungs, spores are translocated by 
pulmonary macrophages and dendritic cells. Phago¬ 
cytes have a dual role; they can transport spores to the 
lymphatic system 137-160 but also are bactericidal toward 


134 


Anthrax 


germinating spores under certain conditions. 158,161-163 
Another hypothesis has been proposed that may ex¬ 
plain the toxins' effects early in the infectious process. 
Banks, Ward, and Bradley 164 have hypothesized that 
intoxication may occur after spores have been engulfed 
by phagocytic cells. The anthrax toxin receptors have 
been located on the inside of the phagolysosome, and 
the germinating spore may secrete toxins that interact 
with these receptors within the phagolysosome. The 
effector molecules (EF and/or LF) can then be translo¬ 
cated into the cytoplasm. 155,164 

Once a spore becomes vegetative, it can produce a 
robust capsule and large amounts of toxins. At these 
sites, the bacteria proliferate and produce the ETs and 
LTs that impair host leukocyte function and lead to the 
distinctive pathological findings: edema, hemorrhage, 
tissue necrosis, and a relative lack of leukocytes. Once 
the vegetative cells emerge from the phagolysome, 
they replicate within the cell and finally exit through 
the host cell plasma membrane. 160 In inhalational an¬ 
thrax, the spores are ingested by alveolar phagocytes, 
which transport them to the regional tracheobronchial 
lymph nodes, where germination occurs. 165 

Anthrolysin O (ALO) and phospholipases may also 
play critical roles as virulence factors for B anthracis 166 
and mediate the toxicity of B anthracis to lung epithe¬ 
lial cells under microaerobic conditions. 167 ALO has 
been found to cause lysis of human phagocytes and 
epithelial cells. The mechanism of action appears to 
be from ALO pore-forming alterations of the cellular 
membrane, resulting in acute primary membrane per- 
meabilization followed by a burst of reactive radicals 
released from the mitochondria. 


The military seeks to defend against anthrax used 
as an inhalational biological weapon. However, other 
anthrax forms are more likely to be seen by medical 
officers—particularly when deployed to third world 
countries—and are therefore included for completeness. 

Cutaneous Anthrax 

More than 95% of anthrax cases are cutaneous. 179-181 
After inoculation, the incubation period is 1 to 5 days. 
The disease first appears as a small papule that pro¬ 
gresses over a day or two to a vesicle containing sero- 
sanguineous fluid with many organisms and a pau¬ 
city of leukocytes. Histopathology findings consist of 
varying degrees of ulceration, vasculitis, perivascular 
inflammation, coagulative necrosis, hemorrhage, and 
edema. 182 The vesicle—which may be 1 to 2 cm in di¬ 
ameter-ruptures, leaving a necrotic ulcer (Figure 6-4). 


The evidence reported from animal studies over¬ 
whelmingly suggests that the alveolar spaces are not 
permissive for significant levels of spore germination. 
Rather, spores begin to germinate once phagocytosed 
during translocation to and upon deposition within 
lymph nodes. 165,168-171 However, several studies have 
suggested that small amounts of germination may oc¬ 
cur within the alveolar spaces. 171,172 Additionally, the 
nasal-associated lymphoid tissue has been explored 
as another area from which infection may be initi¬ 
ated. 171,173,174 These data, largely collected through in- 
vivo imaging technologies, suggest that other scenarios 
may lead to spore germination after inhalation. 159 
Once in the tracheobronchial lymph nodes, the local 
production of toxins by extracellular bacilli generates 
the characteristic pathology picture: massive hemor¬ 
rhagic, edematous, and necrotizing lymphadenitis; 
and mediastinitis (the latter is almost pathognomonic 
of this disease). 175 

These findings in human disease have been repli¬ 
cated in various animal disease models. 176,177 The bacilli 
can then spread to the blood, leading to septicemia 
with seeding of other organs and frequently caus¬ 
ing hemorrhagic meningitis. Death is most likely the 
result of systemic inflammatory response syndrome 
triggered by the release of endogenous cellular con¬ 
tents from damaged or dying cells, termed damage- 
associated molecular patterns and in combination with 
exogenous microbial exposure or pathogen-associated 
molecular patterns, 178 resulting in respiratory failure 
associated with pulmonary edema, direct cardiac tis¬ 
sue damage, overwhelming bacteremia, accompanied 
frequently with meningitis. 

DISEASE 

Satellite vesicles may also be present. The lesion is 
usually painless, and varying degrees of edema may 
be present around it. 183 The edema may occasionally 
be massive, encompassing the entire face or limb, 
which is described as "malignant edema." Patients 
usually have fever, malaise, and headache, which may 
be severe in those with extensive edema. There may 
also be local lymphadenitis. The ulcer base develops a 
characteristic black eschar, and after 2 to 3 weeks the 
eschar separates, often leaving a scar and sometimes 
requiring surgical reconstruction. 184,185 Debridement 
has been shown to improve survival rates in a mouse 
model of subcutaneous anthrax 159 ; however, no clinical 
studies have been conducted to validate this procedure 
in human clinical disease. Septicemia is rare, and with 
treatment, mortality should be less than 1 %. 184,186-188 j n 
addition, no age-related risk factor appears to be as¬ 
sociated with cutaneous human anthrax. 189 


135 


Medical Aspects of Biological Warfare 



Figure 6-4. Cutaneous lesions of anthrax, (a) Ulcer with 
vesicle ring, (b) Black eschar with surrounding erythema, 
(c) Marked edema of extremity secondary to anthrax edema 
toxin with multiple black eschar. 

Photographs: Courtesy of the Centers for Disease Control 
and Prevention, Atlanta, Georgia, www.bt.cdc.gov/agent/ 
anthrax/anthrax-images/cutaneous.asp. 




Of recent interest has been the identification of an¬ 
thrax cases among intravenous drug users in western 
Europe. 70-75 In 2000 a case of cutaneous anthrax was 
identified in a Norwegian patient who participated in 
subdermal drug injection, commonly known as "skin 
popper." 76 The first reported case of intravenous drug 
user-associated anthrax was in Scotland with subse¬ 
quent 47 confirmed cases and 13 fatalities. These num¬ 
bers increased to a total of 119 cases from December 
2009 to December 2010. 74 This disease is thought to be 
initiated by direct injection of spore-contaminated her¬ 
oin, which led to clinical presentations ranging from 
subcutaneous disease to septicemic anthrax. 70-78,190-192 

Inhalational Anthrax 

Inhalational anthrax begins after an incubation period 
of 1 to 6 days with nonspecific symptoms of malaise, 
fatigue, myalgia, and fever. 193-195 A nonproductive cough 
and mild chest discomfort may also occur. These symp¬ 
toms usually persist for 2 or 3 days, and in some cases 
there may be a short period of improvement. Then a sud¬ 
den onset of increasing respiratory distress with dyspnea, 
stridor, cyanosis, increased chest pain, and diaphoresis 
occurs. Associated edema of the chest and neck may also 


be present. Chest radiograph examination usually shows 
the characteristic widening of the mediastinum from 
necrosis and hemorrhage of the lymph nodes and sur¬ 
rounding tissues, often with associated pleural effusions 
(Figure 6-5). In the 2001 bioterrorist event, the pleural 
effusions were initially small but rapidly progressed 
and persisted despite effective antibiotic therapy. 195,196 
The effusions were predominantly serosanguineous, 
and immunohistochemistry revealed the presence of 
B anthracis cell wall and capsule antigens. Effusion 
fluid from deceased patients who had received fewer 
than 55 hours of antibiotic therapy revealed bacilli. 11 ' 

Polymerase chain reaction analysis of the pleural 
fluid was also positive for B anthracis DNA. 198 Pneu¬ 
monia has not been a consistent finding but can occur 
in some patients and may be attributed to vascular 
permeability, intra-alveolar edema, and hyaline mem¬ 
brane formation. 197 Although inhalational anthrax cas¬ 
es have been rare in this century, except for the 11 cases 
arising from the anthrax letters in 2001, several cases 
have occurred in patients with underlying pulmonary 
disease, suggesting that this condition may increase 
susceptibility to the disease. 68 Meningitis is present 
in up to 50% of cases, and some patients may present 
with seizures. The onset of respiratory distress is fol- 


136 



Anthrax 



Figure 6-5. (a) Frontal chest radiograph reveals mediastinal 
and hilar widening, bilateral pleural effusions, and decreased 
lung volumes, (b) Chest axial computed tomography (CT) 
(mediastinal window) shows enlarged, hyperdense sub- 
carinal (arrow) and left hilar (arrowhead) lymph nodes, 
compatible with intranodal hemorrhage, (c) On lung win¬ 
dow CT, peribronchial consolidation (curved arrow) reflects 
lymphatic spread of anthrax infection. 

Radiologic Images: Courtesy of JR Galvin, MD and AA 
Frazier, MD, Department of Radiologic Pathology, Armed 
Forces Institute of Pathology, Washington, DC. 




b 


c 


lowed by the rapid onset of shock and death within 
24 to 36 hours. Mortality had been essentially 100% in 
the absence of appropriate treatment; however, during 
2001 the mortality rate was 45%. 195,196 

An inhalational pulmonary disease thought initially 
to be anthrax has been identified to be caused by B 
cereus strains. 152,199 These cases were found in metal 
welders, and susceptibility of these patients to this 
unusual pathogen may be related to inhalation of 
heavy metals during welding. Heavy metal exposure 
produces immunosuppression and an increased sus¬ 
ceptibility to infection. 

Meningitis 

Meningitis may occur after bacteremia as a complica¬ 
tion of any of the disease's clinical forms. 190-192 Meningitis 
may also occur—rarely—without a clinically apparent 
primary focus, and it is often hemorrhagic, which is im¬ 
portant diagnostically, and almost always fatal (Figure 
6-6). Shi dies have suggested that LF, EF, and protease 
InhA inhibit neutrophil signaling pathways in brain 
endothelium, thus promoting anthrax meningitis. 193-195 



Figure 6-6. Meningitis with subarachnoid hemorrhage in 
a man from Thailand who died 5 days after eating under¬ 
cooked carabao (water buffalo). 

Reproduced from: Binford CH, Connor DH, eds. Pathology 
of Tropical and Extraordinary Diseases. Vol 1. Washington, 
DC: Armed Forces Institute of Pathology; 1976: 121. AFIP 
Negative 75-12374-3. 


137 




Medical Aspects of Biological Warfare 


Oropharyngeal and Gastrointestinal Anthrax 

Oropharyngeal and gastrointestinal anthrax result 
from ingesting infected meat that has not been suf¬ 
ficiently cooked or by ingesting anthrax spores either 
directly or from swallowing contaminated respiratory 
secretions. 178,200:201 After an incubation period of 2 to 
5 days, patients with oropharyngeal disease present 
with severe sore throat or a local oral or tonsillar ulcer, 
usually associated with fever, toxicity, and swelling 


The most critical aspect in making an anthrax diagno¬ 
sis is a high index of suspicion associated with a compat¬ 
ible history of exposure. Cutaneous anthrax should be 
considered after a painless pruritic papule, vesicle, or 
ulcer develops — often with surrounding edema—and 
then becomes a black eschar. With extensive or mas¬ 
sive edema, such a lesion is almost pathognomonic. 
Gram stain or culture of the lesion usually confirms 
the diagnosis. Bacterial culture tests include colony 
morphology on sheep blood agar plates incubated at 
35°C to 37°C for 15 to 24 hours. B anthracis colonies are 2 
to 5 mm in diameter, flat or slightly convex, irregularly 
round with possible comma-shaped ("Medusa-head") 
projections with a ground-glass appearance (Figure 6-7). 
The colonies tend to have tenacious consistency when 
moved with a bacterial loop and are not |3-hemolytic. 


of the neck resulting from cervical or submandibular 
lymphadenitis and edema. Dysphagia and respiratory 
distress may also be present. Gastrointestinal anthrax 
begins with nonspecific symptoms of nausea, vomit¬ 
ing, and fever; in most cases severe abdominal pain 
follows. The presenting sign may be an acute abdomen, 
which may be associated with hematemesis, massive 
ascites, and bloody diarrhea. Mortality in both forms 
may be as high as 50%, especially in the gastrointes¬ 
tinal form. 


The bacteria appear as gram-positive, 1 to 8 pm long 
and 1 to 1.5 pm wide bacilli. India ink staining re¬ 
veals capsulated bacteria. A motility test should be 
performed either by wet mount or motility media; B 
anthracis is nonmotile. Gamma bacteriophage lysis 
and direct fluorescent antibody tests are performed at 
Level D laboratories as confirmatory tests (Figures 6-7 
and 6-8). Commercial polymerase chain reaction kits 
specific for the B anthracis pXOl and pX02 plasmids 
are also available to assist in identifying this organism. 
The differential diagnosis should include tularemia, 
staphylococcal or streptococcal disease, and orf (a viral 
disease of sheep and goats transmissible to humans). 

The diagnosis of inhalational anthrax is difficult, 
but the disease should be suspected with a history of 
exposure to a B an ffzraczs-contain ing aerosol. The early 



Figure 6-7. (a) Isolated colonies of Bacillus anthracis on sheep blood agar plate, (b) Detection of B anthracis using specific 
gamma-phage mediated cell-lysis. 

Photographs: Courtesy of Bret K Purcell, PhD, MD, Division of Bacteriology, US Army Medical Research Institute of Infec¬ 
tious Diseases and the Defense Threat Reduction Agency/Threat Agent Detection and Response Program, National Center 
for Disease Control, Tbilisi, Georgia, 2005. 


138 











Anthrax 



Figure 6-8. Direct fluorescent antibody stain of Bacillus 
anthracis capsule. 

Photograph: Courtesy of David Heath, PhD, Division of Bac¬ 
teriology, US Army Medical Research Institute of Infectious 
Diseases and the Defense Threat Reduction Agency/Threat 
Agent Detection and Response Program, National Center 
for Disease Control, Tbilisi, Georgia, 2005. 

symptoms are nonspecific 194,202-204 and include fever, 
chills, dyspnea, cough, headache, vomiting, weak¬ 
ness, myalgias, abdominal pain, and chest or pleuritic 
pain. This stage of the disease may last from hours to 
a few days. However, the development of respiratory 
distress in association with radiographic evidence of 
a widened mediastinum resulting from hemorrhagic 
mediastinitis and the presence of hemorrhagic pleural 
effusion or hemorrhagic meningitis should suggest the 
diagnosis. Contrast-enhanced computer tomography 
images reveal diffuse hemorrhagic mediastinal and 
hilar adenopathy with edema, perihilar infiltrates, 
bronchial mucosal thickening, and hemorrhagic 
pleural, and pericardial effusions. 205 During the later 
stages of the disease patients develop sudden fever, 
dyspnea, diaphoresis, cyanosis, hypotension, shock, 
and death. 202 Blood culture should demonstrate growth 


Cutaneous anthrax without toxicity or systemic 
symptoms may be treated with oral penicillin if the 
infection did not originate with a potential aerosol ex¬ 
posure. However, if an inhalational exposure is also sus¬ 
pected, ciprofloxacin or doxycycline is recommended 
as first-line therapy 202,203,215 Effective therapy reduces 


in 6 to 24 hours if the patient has not received antibiot¬ 
ics before collection, and a Gram stain of peripheral 
blood smears often reveals large bacilli in later disease 
stages. Sputum examination is not helpful in making 
the diagnosis because pneumonia is usually not a 
feature of inhalational anthrax. 

Gastrointestinal anthrax is difficult to diagnose 
because of its rarity and nonspecific symptoms in¬ 
cluding nausea, vomiting, anorexia, and fever. As the 
disease progresses, patients often develop acute, severe 
abdominal pain, hematemesis, and bloody diarrhea. 
Diagnosis is usually considered only with a history 
of ingesting contaminated meat in the setting of an 
outbreak. Microbiological cultures do not help confirm 
the diagnosis. The diagnosis of oropharyngeal anthrax 
can be made from the clinical and physical findings 
in a patient with the appropriate epidemiological 
history. Sore throat, dysphagia, hoarseness, cervical 
lymphadenopathy, and edema as well as fever are 
often presenting symptoms. 194,206,207 

Meningitis resulting from anthrax is clinically in¬ 
distinguishable from meningitis attributable to other 
etiologies. An important distinguishing feature is that 
the cerebral spinal fluid is hemorrhagic in as many 
as 50% of cases. The diagnosis can be confirmed by 
identifying the organism in cerebral spinal fluid by 
microscopy, culture, or both. 

Serology is generally only useful in making a ret¬ 
rospective diagnosis. Antibody to PA or the capsule 
develops in 68% to 93% 208-211 of reported cutaneous 
anthrax cases and 67% to 94% 210,211 of reported oropha¬ 
ryngeal anthrax cases. A positive skin test to anthraxin 
(an undefined antigen derived from acid hydrolysis 
of the bacillus that was developed and evaluated in 
the former Soviet Union) has also been reported 212 to 
help with the retrospective anthrax diagnosis. Western 
countries have limited experience with this test. 213 The 
Food and Drug Administration (FDA) has recently 
approved two tests: (1) the QuickEFISA Anthrax-PA 
Kit (Immunetics, Boston, MA) for identification of PA 
toxin in blood from infected human casualties, and (2) 
the PCR Joint Biological Agent Identification and Di¬ 
agnostic System (Idaho Technology Inc, Salt Fake City, 
UT) anthrax test for rapid identification of bacteria in 
blood and blood culture samples. 214 


edema and systemic symptoms but does not change 
the evolution of the skin lesion. Treatment should be 
continued for 7 to 10 days, unless inhalational exposure 
is suspected; then treatment should be continued for 60 
days. However, recent studies of the 2001 bioterrorism 
event have identified problems associated with pro- 


139 



Medical Aspects of Biological Warfare 


longed treatment, mass prophylaxis, and medication 
compliance. 216-221 Amoxicillin is recommended for pa¬ 
tients who cannot take fluoroquinolones or tetracycline- 
class drugs; however, increasing evidence shows that B 
anthracis possesses [3-lactamase genes that may reduce 
the efficacy of this treatment. 222-227 In addition, if a bioter¬ 
rorism event occurs, the bacterial strains used may be 
naturally antibiotic resistant or genetically modified to 
confer resistance to one or more antibiotics. 

Tetracycline, erythromycin, and chloramphenicol 
have also been used successfully 228 for treating rare 
cases caused by naturally occurring penicillin-resistant 
organisms. Additional antibiotics shown to be active 
in vitro include gentamicin, cefazolin, cephalothin, 
vancomycin, clindamycin, and imipenem. 229-231 These 
drugs should be effective in vivo, but no reported 
clinical experience exists. Experimental infections us¬ 
ing the inhalational mouse model have demonstrated 
significant efficacy using these additional antibiotics. 

Inhalational, oropharyngeal, and gastrointestinal 
anthrax should be treated with intravenous therapy 
using two or more antibiotics. The therapy should 
initially include a fluoroquinolone or doxycycline with 
one or more of the following antibiotics: clindamy¬ 
cin, rifampin, penicillin, ampicillin, vancomycin, 
amino-glycosides, chloramphenicol, imipenem, and/ 
or clarithromycin. 202,215 Tactical Combat Casualty Care 
guidelines have been established for medical manage¬ 
ment of patients in chemical, biological, radiological, 
nuclear, or high-yield explosives environments. 232 The 
Centers for Disease Control and Prevention issued 
guidelines for the treatment and management of hu¬ 
man anthrax disease. 205,206 New guidelines published 
in 2014 recommend linezolid over clindamycin—when 
appropriate—to prevent toxin formation and the use 
of adjunctive corticosteroids when indicated. 233-236 The 
World Health Organization has also issued guidelines 
for the surveillance and control of anthrax in humans 
and animals and can be accessed at the following web¬ 
site: http://www.who.int/csr/resources/publications/ 
anthrax/WHO_EMC_ZDI_98_6/en/. 


Prophylactic Treatment After Exposure 

Experimental evidence 242 has demonstrated that 
treatment with antibiotics (including ciprofloxacin, 
doxycycline, and penicillin) beginning 1 day after 
exposure to a lethal aerosol challenge with anthrax 
spores can significantly protect against death. Com¬ 
bining antibiotics with active vaccination provides 
the optimal protection. Recent analysis has suggested 
postexposure vaccination may shorten the duration of 


Patients often require intensive care unit support, 
including appropriate vasopressors, oxygen, and 
other supportive therapy, because of the disease's 
severity and rapid onset. Recommendations for treat¬ 
ment during pregnancy and for pediatric populations 
follow similar guidelines. 234-236 The development of 
severe bacterial sepsis has been well documented for 
anthrax in both human clinical disease and experi¬ 
mental animal models. The expression of LT and ET as 
well as other virulence factors such as ALO promote 
the development of systemic inflammatory response 
syndrome by both damage-associated molecular pat¬ 
terns and pathogen-associated molecular patterns. 178 
This immunologic stimulation, if unregulated or lim¬ 
ited, results in the formation of a cytokine cascade and 
eventual storm resulting in multiorgan system failure 
and rapid death of humans exposed to inhalational 
anthrax as well as other select agents. This immu¬ 
nologic over-response has prompted the evaluation 
of various augmentation therapies to mitigate these 
events. One such therapy that received FDA approval 
in 2012 is raxibacumab (GlaxoSmithKline, Brentford, 
Middlesex, United Kingdom), a human IgGl mono¬ 
clonal antibody directed against the PA antigen of B 
anthracis. 237,238 This product was the first monoclonal 
antibody approved for use in the treatment of se¬ 
vere inhalational anthrax under the FDA's Animal 
Efficacy Rule. 237-239 The study found that 64% of 
Cynomolgus macaque monkeys and 44% of rabbits with 
inhalational anthrax survived, whereas all placebo 
control animals died from both groups. 239 An addi¬ 
tional study comparing antibiotics and raxibacumab 
against antibiotics demonstrated a 82% survival for 
combination therapy versus 65% for antibiotics only. 
When rabbits were treated with levofloxacin plus 
raxibacumab verses levofloxacin alone, the absolute 
difference in survival rates between the groups was 
not statistically significant; however, clinically there 
was only an 18% death rate in the levofloxacin plus 
raxibacumab group and a 35% death rate in the le¬ 
vofloxacin only group. 240,241 


antibiotic prophylaxis, providing the least expensive 
and most effective strategy to counter a bioterrorism 
event. 243-245 

Active Immunization 

Emergent BioSolutions (Rockville, MD) produces 
the only licensed human vaccine against anthrax, 
anthrax vaccine adsorbed (BioThrax). This vaccine is 
made from sterile filtrates of microaerophilic cultures 


140 


Anthrax 


of an attenuated, unencapsulated, nonproteolytic 
strain (V770-NP1-R) of B anthracis. The filtrate, contain¬ 
ing predominantly 83-kDa PA, is adsorbed to 1.2 mg/ 
mL of aluminum hydroxide in 0.85% sodium chloride. 
The final product also contains 100 pg/mL of formal¬ 
dehyde and 25 pg/mL of benzethonium chloride as 
preservatives. Some vaccine lots contain small amounts 
of LF and lesser amounts of EF, as determined by anti¬ 
body responses in vaccinated animals. 246,247 Low levels 
of antibody to LF and EF by Western blot have been 
reported in some vaccines, but these did not contribute 
significantly during toxin neutralization assays. 248 The 
vaccine is stored at 2°C to 8°C. The vaccine should be 
given to industrial workers exposed to potentially 
contaminated animal products imported from coun¬ 
tries in which animal anthrax remains uncontrolled. 
These products include wool, goat hair, hides, and 
bones. People in direct contact with potentially infected 
animals and laboratory workers should also be vac¬ 
cinated. Vaccination is also indicated for protection 
against anthrax use in biological warfare. 

Recommendations have been made for anthrax vac¬ 
cine use in the United States. 249,250 The current guide¬ 
lines recommend the anthrax vaccine adsorbed vaccine 
should be administered to prime the immune system 
to prevent infection as either a preexposure vaccine 
or after exposure to aerosolized B anthracis pores. For 
preexposure protection the Advisory Committee on 
Immunization Practices recommends intramuscular 
injections starting on day 0 followed by week 4, and 
every 6 months (6, 12, and 18 months) for a total of 
5 doses as the initial vaccination series. Since no in 
vitro correlate of immunity exists for humans, annual 
boosters are recommended if the potential for exposure 
continues. For postexposure to anthrax, those persons 
who have been previously unvaccinated should receive 
the vaccine as a three dose, subcutaneous series (at 0,2, 
and 4 weeks) in addition to the administration of a 60- 
day course of an appropriate antimicrobial therapeutic. 

More than 2.6 million US military personnel have 
received the licensed anthrax vaccine adsorbed vac¬ 
cine, and no unusual rates of serious adverse events 
have been noted. 251 Additional studies also support the 
safety of the anthrax vaccine. 252-260 The next genera¬ 
tion vaccine, recombinant PA, may afford equivalent 
protection with a decrease in reactogenicity. A live 
attenuated, unencapsulated spore vaccine is used 
for humans in the former Soviet Union. The vac¬ 
cine is given by scarification or subcutaneously. Its 
developers claim that it is reasonably well tolerated 
and shows some degree of protective efficacy against 
cutaneous anthrax in clinical field trials. 212 New at¬ 
tenuated vaccines developed in the United States are 
being evaluated for efficacy in inhalational anthrax 


animal models. 261 Recent studies have demonstrated 
a fourfold rise in anti-PA immunoglobulin G (IgG) 
titers of 85% and 100% in adults receiving two and 
three doses, respectively, of either subcutaneous or 
intramuscular AVA. 262-265 

One hundred percent of the vaccinees developed 
a rise in titer in response to the yearly booster dose. 
When tested by an enzyme-linked immunosorbent as¬ 
say, the current serologic test of choice, more than 95% 
of vaccinees seroconvert after the initial three doses. 248 

A rough correlation exists between antibody titer 
to PA and protection of experimental animals from 
infection after vaccination with the human vaccine. 
FFowever, the exact relationship between antibody 
to PA as measured in these assays and immunity to 
infection remains obscure because the live attenuated 
Sterne veterinary vaccine (made from an unencapsu¬ 
lated, toxin-producing strain) protects animals better 
than the human vaccine, yet it induces lower levels of 
antibody to PA. 246-248 

A recent study evaluating the response of mice to 
recombinant PA revealed significant variation of fine 
specificity of humoral response to the antigen even 
among genetically identical mice using the same im¬ 
munogen and environment. 266,267 The authors demon¬ 
strated a heterogeneity of response to the PA antigen 
and identified specific epitopes that correlated to 
seroconversion and LeTx neutralization. Then they 
speculated that this observed stochastic variation in 
humoral immunity was likely a major contributing fac¬ 
tor to the heterogeneity of vaccine response. Although 
these data suggest enhancing immunologic recognition 
of specific epitopes can improve vaccine protective 
response, the current anthrax vaccine adsorbed vaccine 
has demonstrated significant protection to nonhuman 
primates when exposed to inhalational challenge with 
large doses of anthrax spores. 268-274 

The protective efficacy of experimental PA-based 
vaccines produced from sterile culture filtrates of B 
anthracis was clearly demonstrated by various animal 
models and routes of challenge. 273 A placebo-controlled 
clinical trial was conducted with a vaccine similar to 
the currently licensed US vaccine. 276 

This field-tested vaccine was composed of the ster¬ 
ile, cell-free culture supernatant from an attenuated, 
unencapsulated strain of B anthracis, different from that 
used to produce the licensed vaccine and grown under 
aerobic, rather than microaerophilic, conditions. 277 

This vaccine was precipitated with alum rather 
than adsorbed to aluminum hydroxide. The study 
population worked in four mills in the northeastern 
United States where B anthracis-conta minated im¬ 
ported goat hair was used. The vaccinated group, 
compared to a placebo-inoculated control group. 


141 


Medical Aspects of Biological Warfare 


was afforded 92.5% protection against cutaneous 
anthrax, with a lower 95% confidence limit of 65% 
effectiveness. There were insufficient inhalational 
anthrax cases to determine whether the vaccine was 
effective. This same vaccine was previously shown 
to protect rhesus monkeys and other animal models 
against an aerosol exposure to anthrax spores. 277-282 
No controlled clinical trials in humans of the efficacy 
of the currently licensed US vaccine have been con¬ 
ducted. This vaccine has been extensively tested in 
animals and has protected guinea pigs against both an 
intramuscular 247,248,280 and an aerosol challenge. 246 The 
licensed vaccine has also been shown to protect rhesus 
monkeys against an aerosol challenge. 242,270,278,282 The 
Centers for Disease Control and Prevention issued 
recommendations on the use of the anthrax vaccine 
in 2009. 273 

Recombinant PA is undergoing clinical trials and is 
considered the next-generation anthrax vaccine. Ad¬ 
ditionally, other nontoxin based vaccine approaches 
are being explored. These approaches include using 
the B anthracis capsule 270,279,281,283-285 and spore-specific 
proteins. 286-289 Although these novel antigens have 
been promising, it is generally agreed that PA will 
continue to have a prominent role in licensed anthrax 
vaccines. 


Side Effects 

In two different studies, the incidence of significant 
local and systemic reactions to the vaccine used in the 
placebo-controlled field trial was 2.4% to 2.8% 82 and 
0.2% to 1.3%. 277 The vaccine licensed in the United States 
is reported to have a similar incidence of reactions. 290 
Local reactions considered significant include indura¬ 
tion, erythema in an area larger than 5 cm in diameter, 
edema, pruritus, warmth, and tenderness. These reac¬ 
tions peak at 1 to 2 days and usually resolve within 2 to 3 
days afterward. Rare reactions include edema extending 
from the local site to the elbow or forearm, and a small, 
painless nodule that may persist for weeks. A recent 
study indicated that administering the vaccine over the 
deltoid muscle instead of the triceps can significantly 
reduce the frequency of local reactions. 251 

People who have recovered from a cutaneous 
infection with anthrax may have severe local reac¬ 
tions from being vaccinated. 276 Systemic reactions are 
characterized by flu-like symptoms, mild myalgia, 
arthralgia, headache, and mild-to-moderate malaise 
that last for 1 to 2 days. No long-term sequelae of lo¬ 
cal or systemic reactions exist and no suggestion of a 
high frequency or unusual pattern of serious adverse 
events exists. 251,256,257,291,292 


SUMMARY 


Anthrax is a zoonotic disease that occurs in domes¬ 
ticated and wild animals. Humans become infected 
by contact with infected animals or contaminated 
products. Under natural circumstances, infection oc¬ 
curs by the cutaneous route and only rarely by the 
inhalational or gastrointestinal routes. An aerosol 
exposure to spores causes inhalational anthrax, which 
is of military concern because of its potential for use 
as a biological warfare agent. Aerosol exposure begins 
with nonspecific symptoms followed in 2 to 3 days by 
the sudden onset of respiratory distress with dyspnea. 


cyanosis, and stridor; it is rapidly fatal. Radiography 
of the chest often reveals characteristic mediastinal 
widening, indicating hemorrhagic mediastinitis. 
Hemorrhagic meningitis frequently coexists. Given 
the rarity of the disease and its rapid progression, it is 
difficult to diagnose inhalational anthrax. Treatment 
consists of massive doses of antibiotics and supportive 
care. Postexposure antibiotic prophylaxis is effective 
in laboratory animals and should be instituted as soon 
as possible after exposure. A licensed, antigen-based, 
nonviable vaccine is available for human use. 


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283. Chabot DJ, Scorpio A, Tobery SA, Little SF, Norris SL, Friedlander AM. Anthrax capsule vaccine protects against 
experimental infection. Vaccine. 2004;23:43M7. 

284. Joyce J, Cook J, Chabot D, et al. Immunogenicity and protective efficacy of Bacillus anthracis poly-gamma-D-glutamic 
acid capsule covalently coupled to a protein carrier using a novel triazine-based conjugation strategy. } Biol Chem. 
2006;281:4831-4843. 

285. Chabot DJ, Joyce J, Caulfield M, et al. Efficacy of a capsule conjugate vaccine against inhalational anthrax in rabbits 
and monkeys. Vaccine. 2012;30:846-852. 

286. Brossier F, Weber-Levy M, Mock M, Sirard JC. Protective antigen-mediated antibody response against a heterologous 
protein produced in vivo by Bacillus anthracis. Infect Immun. 2000;68:5731-5734. 

287. Gauthier YP, Toumier JN, Paucod JC, et al. Efficacy of a vaccine based on protective antigen and killed spores against 
experimental inhalational anthrax. Infect Immun. 2009;77:1197-1207. 

288. Cote CK, Kaatz L, Reinhardt J, et al. Characterization of a multi-component anthrax vaccine designed to target the 
initial stages of infection as well as toxaemia. J Med Microbiol. 2012;61(Pt 10):1380-1392. 


156 


Anthrax 


289. Vergis JM, Cote CK, Bozue J, et al. Immunization of mice with formalin-inactivated spores from avirulent Bacillus cereus 
strains provides significant protection from challenge with Bacillus anthracis Ames. Clin Vaccine Immunol. 2013;20:56-65. 

290. Puziss M, Wright GG. Studies on immunity in anthrax. X. Gel-adsorbed protective antigen for immunization of man. 
J Bacterid. 1963;85:230-236. 

291. Sever JL, Brenner AI, Gale AD, et al. Safety of anthrax vaccine: a review by the Anthrax Vaccine Expert Committee 
(AVEC) of adverse events reported to the Vaccine Adverse Event Reporting System (VAERS). Pharmacoepidemiol Drug 
Saf. 2002;11:189-202. 

292. Sulsky SI, Luippold R, Garman P, et al. Disability among US Army veterans vaccinated against anthrax. Vaccine. 
2012;30:6150-6156. 


157 



Chapter 7 

BRUCELLOSIS 


BRET K.PURCELL, PhD, MD* * * * ; R. MARTIN ROOP II, PhD + ; ARTHUR M.FRIEDLANDER, MD } ; and 
DAVID L.HOOVER, MD § 


INTRODUCTION 

THE INFECTIOUS AGENT 

EPIDEMIOLOGY 

PATHOGENESIS 

CLINICAL MANIFESTATIONS 

DIAGNOSIS 

TREATMENT 

PROPHYLAXIS 

SUMMARY 


*Colonel, Medical Corps, US Army; Deputy Chief, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter 
Street, Fort Detrick, Maryland 21702 

*Professor, Microbiology and Immunology, Brody School of Medicine, East Carolina University, 600 Moye Boulevard, Room 118, Biotechnology Build¬ 
ing, Greenville, North Carolina 27834 

t Colonel (Retired), Medical Corps, US Army; Senior Scientist, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort 
Detrick, Maryland 21702 

§ Colonel (Retired), Medical Corps, US Army; Senior Scientific Advisor, Clinical Research Management, 1265 Ridge Road, Hinckley, Ohio 44233; for¬ 
merly Medical Director, Dynport Vaccine Company LLC, a CSC Company, 64 Thomas Johnson Drive, Frederick, Maryland, and Scientific Coordinator, 
Brucella Program, Department of Bacterial Diseases, Walter Reed Army Institute of Research, Silver Spring, Maryland 


159 



Medical Aspects of Biological Warfare 


INTRODUCTION 


Brucellosis is a zoonotic infection of domesticated 
and wild animals caused by bacteria of the genus 
Brucella. Humans become infected by ingesting animal 
food products directly contacting infected animals or 
inhaling infectious aerosols either inadvertently or by 
intentional means through bioterrorism. Brucellosis is 
currently considered to be one of the world's leading 
zoonoses. 1 

Military medicine has played a large role in dis¬ 
covering and defining brucellosis in humans. 2 In 1751 
G Cleghorn, a British army surgeon stationed on the 
Mediterranean island of Minorca, described cases of 
chronic, relapsing febrile illness and cited Hippocrates' 
description of a similar disease more than 2,000 years 
earlier. 3 Three additional British army surgeons work¬ 
ing on the island of Malta during the 1800s were re¬ 
sponsible for important descriptions of the disease. JA 
Marston described clinical characteristics of his own 
infection in 1861. 4 In 1887 David Bruce, for whom the 
genus Brucella is named, isolated the causative organ¬ 
ism from the spleens of five fatal cases and placed this 
bacterium within the genus Micrococcus. 5 Ten years 
later, ML Hughes, who had coined the name "undulant 
fever," published a monograph that detailed clinical 
and pathological findings in 844 patients. 6 

In that same year, Bernhard Bang, a Danish inves¬ 
tigator, identified a bacterium, which he called the 
"bacillus of abortion," in placentas and fetuses of cattle 
suffering from contagious abortion. 7 In 1917 Alice C 
Evans recognized that Bang's organism was identi¬ 
cal to that described by Bruce as the causative agent 
of human brucellosis. The bacterium infects mainly 
cattle, sheep, goats, and other ruminants, in which it 
causes abortion, fetal death, and genital infections. 8,9 
Humans, who are usually infected incidentally by 
contact with infected animals or ingestion of dairy 
foods, may develop numerous symptoms in addition 
to the usual ones of fever, malaise, and muscle pain. 
With the worldwide distribution of brucellosis, in¬ 
ternational travel and military deployments increase 
the risk of exposure to this disease. 10 In particular, the 

THE INFECT 

Brucellae are small, nonmotile, nonsporulating, 
nontoxigenic, nonfermenting, facultatively intracel¬ 
lular, gram-negative bacteria that represent a single 
"genospecies" from a phylogenetic perspective. 27 
However, for epidemiologic purposes and ease and 
accuracy of communication. Brucella strains are clas¬ 
sified as separate "nomenspecies" based on readily 
distinguished phenotypic characteristics that include 


deployment of US military and coalition forces into 
Iraq, Afghanistan, Libya, and other Middle Eastern 
countries has posed particular risk from environmental 
and food source animals. 11 ” 13 The disease frequently 
becomes chronic and may relapse, even with treatment. 
Laboratory-acquired infections have been documented 
as awareness of this disease has increased, 14 ” 17 and 
as biodefense research expands in the academic and 
biotechnology industries, laboratory accidents may 
unfortunately become more frequent and significant. 18 
Strict adherence to proper engineering controls, good 
laboratory and microbiology techniques, and the 
use of personal protective equipment significantly 
reduces the incidence of laboratory-acquired infec¬ 
tions. 19,20 No vaccine is available that can safely be 
used to prevent laboratory-acquired brucellosis. 

The ease of transmission by aerosol underscores 
the concern that Brucella might be used as a biologi¬ 
cal warfare agent. The United States began develop¬ 
ing Brucella suis as a biological weapon in 1942. The 
agent was formulated to maintain long-term viability, 
placed into bombs, and tested in field trials during 
1944-1945 with animal targets. By 1969 the United 
States terminated its offensive program for develop¬ 
ment and deployment of Brucella as a weapon and 
destroyed all of its biological weapon munitions. 
Although the munitions developed were never used 
in combat, studies conducted under the offensive 
program reinforced the concern that Brucella might be 
used against US troops as a biological warfare agent. 21 
Even before the post-September 11, 2001 attacks, 
civilian populations were recognized as potential 
high yield targets. In 1997 a model of aerosol attack 
with Brucella on an urban population estimated an 
economic impact of $477.7 million per 100,000 persons 
exposed. 22 Brucella represents one of many biological 
agents of zoonotic disease that could pose threats 
as terrorist weapons against human or agricultural 
targets. 23 Several reviews that focus on the potential 
use of the brucellae as agents of bioterrorism or bio¬ 
warfare have been published. 24 ” 26 

BUS AGENT 

host specificity. 28 There are presently 10 of these recog¬ 
nized "nomenspecies" (Table 7-1). Brucella melitensis, B 
suis, Brucella abortus, and Brucella canis are the classic 
causative agents of disease in humans. Human infec¬ 
tions with the marine mammal strain Brucella ceti 29,30 
and a strain ( Brucella inopinata) of unknown origin 31 ” 33 
have also recently been described, but prevalence of 
such infections is unclear. 


160 


Brucellosis 


Human infections with Brucella ovis, Brucella neoto- 
mae, Brucella pinnipedialis, and Brucella microti have not 
been described. Brucellae grow best on trypticase soy- 
based media or other enriched media with a typical 
doubling time of 2 hours in liquid culture. Although 
B melitensis bacteremia can be detected within 1 week 
by using automated culture systems, 34 cultures should 
be maintained for at least 4 weeks, with weekly sub¬ 
culture, for diagnostic purposes. Most biovars of B 
abortus require incubation in an atmosphere of 5% 
to 10% carbon dioxide for growth. Brucellae may 
produce urease and may oxidize nitrite to nitrate; 
they are oxidase- and catalase-positive. Species and 
biovars are differentiated by their carbon dioxide 
requirements; ability to use glutamic acid, ornithine, 
lysine, and ribose; hydrogen sulfide production; 
growth in the presence of thionine or basic fuchsin 
dyes; agglutination by antisera directed against 
certain lipopolysaccharide (LPS) epitopes; and by 
susceptibility to lysis by bacteriophage. Brucella can 
grow on blood agar plates and does not require X or 
V factors for growth. 

Serological agglutinating antibodies have been 
used worldwide as the definitive diagnostic test for 
brucellosis infection. The standard tube agglutina¬ 
tion test is the modified Brucella microagglutination 
test. 35 This test uses direct agglutination of bacterial 
antigens by specific antibodies of the immunoglobulin 
(Ig), IgG, and IgA classes. Acute infection is indicated 
by the presence of antigen-specific IgM antibodies. 


TABLE 7-1 

TYPICAL HOST SPECIFICITY OF BRUCELLA 
SPECIES 


Brucella Species 

Animal Host 

Human Pathogenicity 

B melitensis 

Sheep, goats 

High 

B suis 

Swine 

High 

B abortus 

Cattle, bison 

Intermediate 

B canis 

Dogs 

Low 

B ceti 

Dolphins, 

porpoises 

Unknown* 

B inopinata 

Humans 

Unknown* 

B pinnipedialis 

Seals 

Not reported 

B ovis 

Sheep 

Not reported 

B neotomae 

Rodents 

Not reported 

B microti 

Rodents 

Not reported 


*B ceti and B inopinata strains have been isolated from human dis¬ 
ease, but the importance of these strains as human pathogens is 
presently unknown. 


but these antibodies decline rapidly within weeks of 
the onset of infection. Chronic or relapsing disease is 
characterized by elevated or increasing levels of IgG 
and IgA classes. 36 A four-fold or greater rise in Brucella 
agglutination titers demonstrated between acute and 
convalescent serum specimens collected at least 2 
weeks apart in conjunction with clinically compatible 
illness is considered a confirmatory test for brucellosis 
infection. Additional confirmatory tests for infection 
include the isolation of Brucella from clinical speci¬ 
mens or the identification of Brucella bacteria in tissue 
cultures by specific immunohistochemical staining. 37 
Although highly sensitive and specific, occasionally 
false positive tests and cross reactions do occur using 
Brucella antibody tests. The cell wall lipopolysaccha¬ 
ride of the Brucella organism is antigenically similar to 
other gram-negative bacteria. Antibodies to Moraxella 
phenylpyruvica, Yersinia enterocolitica, Escherichia coli 
0157, and specific Salmonella strains are known to 
provide false positive reactions. 38,39 

Analysis of fragment lengths of DNA cut by 
various restriction enzymes has also been used to 
differentiate brucellae groupings. 33 Single nucleotide 
polymorphism analyses using real time polymerase 
chain reaction (PCR) have been used to rapidly 
identify Brucella isolates to the species level. 40 Both 
the multiple loci variable number of tandem repeat 
analysis and the Bruce-ladder multiplex PCR assays 
have been recently used to type a variety of marine 
Brucella isolates and differentiate by biovar typing of 
B suis and B cam's. 41,42 Recent studies using proteomics, 
complete genomic sequencing, and multi-locus analy¬ 
sis of variable number tandem repeats have rapidly 
expanded the information on virulence determinants, 
identification of pathogenicity islands, and evolution¬ 
ary relatedness among the Brucella species. 43-47 Micro¬ 
arrays have now been developed to phylogenetically 
classify and forensically identify unknown pathogens 
as well as genotype Brucella species. 48,49 The LPS com¬ 
ponent of the outer cell membranes of the brucellae is 
different—both structurally and functionally—from 
that of other gram-negative organisms. 31,32 For in¬ 
stance, in addition to its capacity to provide resistance 
to complement and potentially serve as a ligand for 
binding to host cells, experimental evidence indicates 
that the O-chain of LPS of "smooth" (fully expressed 
O-chain versus "rough" strains with substantially 
reduced or absent O-chain) Brucella strains directly 
interferes with the capacity of host macrophages to 
process antigens via the major histocompatibility 
complex class II pathway 50 and influences in the 
intracellular trafficking of the Brucella containing 
vacuoles in host macrophages preventing their fusion 
with lysosomes. 51 The chemical compositions of the 


161 





Medical Aspects of Biological Warfare 


lipid A and core moieties of the Brucella LPS are also 
distinct from those found in the enteric and many 
other gram-negative bacteria, and these differences 
greatly reduce the "recognition" of the brucellae by 
the Toll-like receptors on host macrophages, which 
allows these bacteria to induce a dampened inflam¬ 
matory response and use a "stealthy" approach for 
establishing infections. 52 

One of the unique features of Brucella strains is 
that unlike most pathogenic bacteria, these bacteria 
produce relatively few "classical" virulence factors. 53 
Probably the most widely studied virulence determi¬ 
nants in the Brucella strains are the LPS and the Type 
IV secretion system. 54 The brucellae use this transport 
system to secrete effector proteins into the cytoplasm 
of infected mammalian cells. These effector proteins 
interfere with the activity of the host cell proteins 
that control the intracellular membrane trafficking. 


Animals may transmit Brucella organisms during 
septic abortion, at the time of slaughter, and in their 
milk. For infected patients, no conclusive evidence 
indicates that brucellosis can be transmitted from 
person to person. The incidence of human disease is 
thus closely tied to the prevalence of infection in sheep, 
goats, pigs, and cattle, and to practices that allow 
exposure of humans to potentially infected animals 
or their products. In the United States, where all 50 
states are considered to be "free" of bovine brucellosis 
and dairy products are routinely pasteurized, illness 
occurs primarily in individuals such as veterinarians, 
shepherds, cattlemen, and slaughterhouse workers 
who have occupational exposure to infected animals. 
In many other countries, humans more commonly 
acquire infection by ingesting unpasteurized dairy 
products, especially cheese. 

Less obvious exposures can also lead to infection. In 
the United States and Australia, for example, hunters 
have acquired B suis infection from feral swine. 56,57 It 
was also not uncommon for veterinarians to develop 
brucellosis after accidental exposure to B abortus Strain 
19 in the United States when this strain was being 
used as a live vaccine in cattle. 58 Another bovine vac¬ 
cine strain. Brucella Abortus Vaccine, Strain RB-51, 
has been used to eradicate brucellosis from the US 
livestock herds. 39 Accidental human infections with 
this vaccine cannot be identified using the standard 
LPS-based diagnostics assay. Brucellae are also highly 
infectious in laboratory settings; numerous laboratory 
workers who culture the organism become infected. 
Disease with a relatively high proportion of respiratory 


The net result is that the phagosomes within which 
the brucellae reside in host macrophages avoid 
extensive interactions with lysosomes and eventu¬ 
ally fuse with the host cell endoplasmic reticulum. 
The formation of these so-called replicative Brucella 
containing vacuoles (or rBCVs) is essential for the 
virulence of the naturally occurring smooth Brucella 
strains such as B melitensis, B suis, and B abortus. The 
capacity of Brucella strains to survive and replicate in 
host macrophages is critical for their virulence. Ac¬ 
cordingly, in addition to gene products such as these 
that overtly interfere with biology of the host cell, the 
brucellae also produce numerous proteins that allow 
them to successfully resist the environmental stresses 
they encounter during their intracellular residence in 
host macrophages. These stresses include exposure to 
acidic pH, reactive oxygen species and antimicrobial 
peptides, and nutrient deprivation. 55 


complaints has also been reported in individuals who 
have camped in the desert during the spring lambing 
season. 46 B canis, a naturally rough strain that typi¬ 
cally causes genital infection in dogs, can also infect 
humans. 60 Although B canis infections were once con¬ 
sidered rare, it has become apparent that in some areas 
of the world these infections were probably unrecog¬ 
nized. 60 In the United States the total number of cases 
of brucellosis remains very low (0.02 to 0.09 cases per 
100,000 person-years). 61,62 A major contributing factor 
to this low incidence of brucellosis can be attributed 
to a national eradication campaign to eliminate bru¬ 
cellosis in domestic cattle herds. When implemented 
the human incidence of disease dropped from a high 
of 6,321 cases in 1947 to 136 cases in 2001 (0.48 cases 
per million). These few cases are primarily caused by 
infections with B melitensis and now most human cases 
are distributed in Hispanic populations residing on 
either side of the Mexico border. 61 The endemic regions 
located in Latin America, Europe, Africa, and Asia ac¬ 
count for most of the human cases of brucellosis with 
the highest incidences occurring in the former Yugo¬ 
slav Republic of Macedonia, Algeria, Peru, Iraq, Iran, 
Syria, Turkey, Kyrgyzstan, and Mongolia. 61,62 With the 
improvement of diagnostic methods, ever increasing 
international tourism, and establishment of new eradi¬ 
cation programs, the epidemiology of brucellosis will 
continue to shift and evolve requiring constant vigi¬ 
lance for new foci of disease. Unfortunately, with the 
rapidly changing political, international, and financial 
environments, worldwide eradication of this zoonotic 
disease will be extremely difficult. 


162 


Brucellosis 


PATHOGENESIS 


Brucellae can enter mammalian hosts through 
skin abrasions or cuts, the conjunctiva, and the 
respiratory tract, and, unlike enteric pathogens 
such as Salmonella or Shigella species that infect the 
lower gastrointestinal tract, the most likely site of 
bacterial entry is the mucosae of the upper gastro¬ 
intestinal tract. 63,64 Organisms are rapidly ingested 
by polymorphonuclear leukocytes, which gener¬ 
ally fail to kill them, 65,66 and are also phagocytosed 
by macrophages (Figure 7-1). Bacteria transported 
in macrophages, which traffic to lymphoid tissue 
draining the upper gastrointestinal mucosa, may 
eventually disseminate to lymph nodes, liver, spleen, 
mammary glands, joints, kidneys, and bone marrow. 
As noted previously, the brucellae are resistant to the 
microbicidal activity of macrophages, and it is their 
capacity to survive and replicate for prolonged peri¬ 
ods in these phagocytes that underlies their ability 
to produce chronic infections. 55 Histopathologically, 
the host cellular response may range from abscess 
formation to lymphocytic infiltration to granuloma 
formation with caseous necrosis. 58 

Studies in experimental models have provided 
important insights into host defenses that even¬ 
tually control infection with Brucella organisms. 
Serum complement effectively lyses some rough 
strains (ie, those that lack O-polysaccharide side 
chains on their LPS), but has little effect on smooth 
strains (ie, bacteria with a long O-polysaccharide 



Figure 7-1. Impression tissue smear from a bovine aborted 
fetus infected with Brucella abortus. The bacteria appear as 
lightly stained, gram-negative cells. 

Photograph: Courtesy of John Ezzell, PhD, US Army Medi¬ 
cal Research Institute of Infectious Diseases, Fort Detrick, 
Maryland. 


side chain); B melitensis may be less susceptible 
than B abortus to complement-mediated killing. 67,68 
Administration of antibody to mice before chal¬ 
lenge with rough or smooth strains of brucellae 
reduces the number of organisms that appear in 
liver and spleen. This effect is caused mainly by 
antibodies directed against LPS, with little or no 
contribution of antibodies directed against other 
cellular components. 69 

The intensity of an infection in mice can be reduced 
by transferring from immune to nonimmune animals 
differentiated CD4 + and CD8 + T cells 70 or by the Ig 
fractions of serum. In particular, the T-cell response 
to Brucella appears to play a key role in the develop¬ 
ment of immunity and protection against chronic 
disease. 71,72 Neutralization of B abortus-induced 
host interferon gamma (IFN-y) during infection in 
pregnant mice prevents abortion. 73 Moreover, macro¬ 
phages treated with IFN-y in vitro inhibit intracellular 
bacterial replication. 74 Studies in humans support a 
role for IFN-y in protection; homozygosity for the 
IFN-y +874A allele is associated with about a two-fold 
increase in incidence of brucellosis. 73 In ruminants, 
vaccination with live vaccines is required in order to 
provide protection. 76-78 

These observations suggest that brucellae, like 
other facultative or obligate intramacrophage 
pathogens, are primarily controlled by macrophages 
activated to enhanced microbicidal activity by IFN-y 
and other cytokines produced by immune T lym¬ 
phocytes. It is likely that antibody, complement, 
and macrophage-activating cytokines produced by 
natural killer cells play supportive roles in early 
infection or in controlling growth of extracellular 
bacteria. 

In ruminants. Brucella organisms bypass the most 
effective host defenses by targeting embryonic and 
trophoblastic tissue. In cells of these tissues, the bac¬ 
teria grow not only in the phagosome but also in the 
cytoplasm and the rough endoplasmic reticulum. 79 
In the absence of effective intracellular microbicidal 
mechanisms, these tissues permit exuberant bacterial 
growth, which leads to fetal death and abortion. In 
ruminants, the presence of erythritol in the placenta 
may further enhance growth of brucellae. Products 
of conception at the time of abortion may contain 
up to 10 10 bacteria per gram of tissue. 80 When septic 
abortion occurs, the intense concentration of bacteria 
and aerosolization of infected body fluids during 
parturition often results in infection of other animals 
and people. 


163 


Medical Aspects of Biological Warfare 


CLINICAL MANIFESTATIONS 


Clinical manifestations of brucellosis are diverse 
and the course of the disease is variable. 81 Patients 
with brucellosis may present with an acute, systemic 
febrile illness; an insidious chronic infection; or a 
localized inflammatory process. However, in the 
absence of suspicion for brucellosis, many cases 
seen in the United States are not diagnosed in the 
early stage of disease, but they are discovered once 
a focal complication has developed, such as a joint 
infection. Disease may be abrupt or insidious in 
onset, with an incubation period of 3 days to several 
weeks. Patients usually complain of nonspecific 
symptoms such as fever, sweats, fatigue, anorexia, 
and muscle or joint aches (Table 7-2). Neuropsy¬ 
chiatric symptoms, notably depression, headache, 
and irritability, occur frequently. In addition, focal 
infection of bone, joints, or genitourinary tract may 
cause local pain. Cough, pleuritic chest pain, and 
dyspepsia may also be noted. Symptoms of patients 
infected by aerosol are indistinguishable from those 
of patients infected by other routes. Chronically 
infected patients frequently lose weight. Symp¬ 
toms often last for 3 to 6 months and occasionally 
for a year or more. Physical examination is usually 
normal, although hepatomegaly, splenomegaly, or 
lymphadenopathy may occur. Brucellosis does not 
usually cause leukocytosis, and some patients may 
be moderately neutropenic 82 ; however, cases of 
pancytopenia have been noted. 83 In addition, bone 
marrow hypoplasia, immune thrombocytopenic 


TABLE 7-2 

SYMPTOMS AND SIGNS OF BRUCELLOSIS 


Symptom or Sign 

Patients Affected (%) 

Fever 

90-95 

Malaise 

80-95 

Body aches 

40-70 

Sweats 

40-90 

Arthralgia 

20-40 

Splenomegaly 

10-30 

Hepatomegaly 

10-70 


Data sources: (1) Mousa AR, Elhag KM, Khogali M, Marafie AA. 
The nature of human brucellosis in Kuwait: study of 379 cases. Rev 
Infect D/s. 1988;10:211-217. (2) Buchanan TM, Faber LC, Feldman 
RA. Brucellosis in the United States, 1960-1972: an abattoir-associ¬ 
ated disease, I: clinical features and therapy. Medicine (Baltimore). 
1974;53:403-413. (3) Gotuzzo E, Alarcon GS, Bocanegra TS, et al. 
Articular involvement in human brucellosis: a retrospective analysis 
of 304 cases. Semin Arthritis Rheum. 1982;12:245-255. 


purpura, and erythema nodosum may occur during 
brucellosis infections. 84-86 Disease manifestations 
cannot be strictly related to the infecting species. 

Infection with B melitensis leads to bone or joint 
disease in about 30% of patients; sacroiliitis develops 
in 6% to 15%, particularly in young adults. 87-89 Arthritis 
of large joints occurs with about the same frequency as 
sacroiliitis. In contrast to septic arthritis caused by pyo¬ 
genic organisms, joint inflammation seen in patients 
with B melitensis is mild, and erythema of overlying 
skin is uncommon. Synovial fluid is exudative, but cell 
counts are in the low thousands with predominantly 
mononuclear cells. In both sacroiliitis and peripheral 
joint infections, destruction of bone is unusual. Organ¬ 
isms can be cultured from fluid in about 20% of cases; 
culture of the synovium may increase the yield. Spon¬ 
dylitis, another important osteoarticular manifestation 
of brucellosis, tends to affect middle-aged or elderly 
patients, causing back (usually lumbar) pain, local 
tenderness, and occasionally radicular symptoms. 90 
Radiographic findings, similar to those of tubercu¬ 
lous infection, typically include disk space narrowing 
and epiphysitis, particularly of the antero-superior 
quadrant of the vertebrae, and presence of bridging 
syndesmophytes as repair occurs. Bone scan of spon¬ 
dylitic areas is often negative or only weakly positive. 
Paravertebral abscess occurs rarely. In contrast with 
frequent infection of the axial skeleton, osteomyelitis 
of long bones is rare. 91 

Infection of the genitourinary tract, an important 
target in ruminant animals, also may lead to signs 
and symptoms of disease in humans. 92-94 Pyelonephri¬ 
tis, cystitis, Bartholin's gland abscess and, in males, 
epididymo-orchitis, may occur. Both diseases may 
mimic their tuberculous counterparts, with "sterile" 
pyuria on routine bacteriologic culture. With blad¬ 
der and kidney infection. Brucella organisms can be 
cultured from the urine. Brucellosis in pregnancy can 
lead to placental and fetal infection. 95 Whether abortion 
is more common in brucellosis than in other severe 
bacterial infections, however, is unknown. 

Lung infections have also been described, par¬ 
ticularly before the advent of effective antibiotics. 
Although up to one-quarter of patients may complain 
of respiratory symptoms, mostly cough, dyspnea, 
or pleuritic pain, chest radiograph examinations are 
usually normal. 96 Diffuse or focal infiltrates, pleural 
effusion, abscess, and granulomas may be noted. 

Hepatitis and, rarely, liver abscess also occur. Mild 
elevations of serum lactate dehydrogenase and alkaline 
phosphatase are common. Serum transaminases are 
frequently elevated. 9 ' Biopsy may show well-formed 


164 





Brucellosis 


granulomas or nonspecific hepatitis with collections of 
mononuclear cells. 81 Spontaneous bacterial peritonitis 
has also been reported. 98,99 

Other sites of infection include the heart, central 
nervous system, and skin. Brucella endocarditis, a 
rare, but most feared complication, accounts for 
80% of deaths from brucellosis. 100,101 Central nervous 


system infection usually manifests itself as chronic 
meningoencephalitis, but subarachnoid hemorrhage 
and myelitis also occur. Guillain-Barre syndrome has 
been associated with acute neurobrucellosis and in¬ 
volvement of spinal roots has been noted on magnetic 
resonance imaging. 102,103 A few cases of skin abscesses 
have been reported. 


DIAGNOSIS 


A thorough history that describes details of ap¬ 
propriate exposure (eg, laboratories, animals, animal 
products, or environmental exposure to locations 
inhabited by potentially infected animals) is the most 
important diagnostic tool. The differential diagnosis for 
brucellosis is broad and includes noninfectious causes 
such as vasculitis, sacroiliitis, lumbar disk disorders, 
thrombotic thrombocytopenic purpura, ankylosing 
spondylitis, abortion complications, depression/sui- 
cide, collagen-vascular disease, erythema nodosum, 
pediatric chronic fatigue syndrome, and malignancy. 
The infectious disease differential includes fever of 
unknown origin, rickettsial diseases, bacterial and viral 
pneumonia, bronchitis, cat scratch fever, cryptococcosis, 
acute epididymitis, cystitis in females, gastroenteritis, 
hepatitis, histoplasmosis, infectious mononucleosis, 
infective endocarditis, influenza, leptospirosis, malaria, 
meningitis, osteomyelitis, Epstein-Barr virus infec¬ 
tion, spontaneous bacterial peritonitis, tuberculosis, 
tularemia, typhoid fever, and urinary tract infections 
in men. Brucellosis should also be strongly considered 
in differential diagnosis of febrile illness if troops have 
been exposed to a presumed biological attack. PCR and 
antibody-based, antigen-detection systems may dem¬ 
onstrate the presence of the organism in environmental 
samples collected from the attack area. 

When the disease is considered, diagnosis is based 
on clinical history, bacterial isolation from clinical 
samples, biochemical identification of the organism, 
and by serology. The Centers for Disease Control and 
Prevention's clinical description of brucellosis is "an 
illness characterized by acute or insidious onset of 
fever, night sweats, undue fatigue, anorexia, weight 
loss, headache and arthalgia." 104 Cultivation of Brucella 
poses a significant hazard to clinical laboratory person¬ 
nel. 105-108 Rapid detection of the organism in clinical 
samples using PCR-enzyme-linked immunosorbent 
assays (ELISAs) or real-time PCR assays can be used 
to detect Brucella DNA in clinical specimens as well 
as cultivated bacteria and may eventually prove to be 
the optimal method for identification of these infec¬ 
tions. 109-112 Although PCR may have many advantages, 
a positive PCR is not proof of viable Brucella. Many of 
the assays used are not standardized and have led to 


false "outbreak" investigations in the United States 
and, therefore, these assays require proper validation 
and standardization by the testing laboratory. Typi¬ 
cally, the most reliable and simple PCR identification 
uses a single pair of primers directed against the 16S- 
23S rRNA operon containing the IS711 or BCSP31 
genes. 111 To identify four of the major Brucella species, 
combination primers directed against the BCSP31, 
OMP3B, OMP2A, and OMP31 external membrane 
protein genes are used. 111 Multiplex PCR provides 
a method to identify all known species of Brucella. 
Despite these technical advances, PCR has sensitivity 
and specificity limitations that depend heavily on the 
quality of DNA isolated and potential inhibitors pres¬ 
ent within the clinical samples. 109-111 

According to the Centers for Disease Control and 
Prevention case definition for brucellosis, the infection 
may be diagnosed if any of the following laboratory 
criteria is met: 

• isolation of the organism from a clinical 
specimen; 

• fourfold or greater rise in Brucella agglutina¬ 
tion titer between acute and convalescent- 
phase serum obtained greater than 2 weeks 
apart; and 

• demonstration by immunofluorescence of 
Brucella in a clinical specimen. 104,112 

Although several serologic techniques such as the 
Coombs test have been developed and tested, the tube 
agglutination test remains the standard method. 113 This 
test, which measures the ability of serum to aggluti¬ 
nate killed organisms, reflects the presence of anti-O- 
polysaccharide antibody. Use of the tube agglutination 
test after treating serum with 2-mercaptoethanol or 
dithiothreitol to dissociate IgM immunoglobulin into 
monomers makes these antibodies inactive and per¬ 
mits agglutination by immunoglobulin G antibodies 
that are resistant to dissolution by chemical agents. A 
titer of 1:160 or higher is considered diagnostic. Most 
patients already have high titers at the time of clinical 
presentation, so a fourfold rise in titer may not occur. 
Immunoglobulin M rises early in disease and may 


165 


Medical Aspects of Biological Warfare 


persist at low levels (eg, 1:20) for months or years 
after successful treatment. Persistence or increase of 
2-mercaptoethanol-resistant (essentially immuno¬ 
globulin G) antibody titers has been associated with 
persistent disease or relapse. 114 Serum testing should 
always include dilution to at least 1:320, as inhibition 
of agglutination at lower dilutions may occur. The 
tube agglutination test does not detect antibodies to 
B canis because this rough organism does not have 
O-polysaccharide on its surface. Unfortunately, given 
the need for trained personnel and standardization of 
the test reagents and control sera, only some references 
laboratories, such as the Centers for Disease Control 
and Prevention in Atlanta, Georgia, and the ARUP 
National Reference Laboratory in Utah, perform the 
tube agglutination test. ELISAs have been developed 
for use with B canis, but are not well standardized. 
Although ELISAs developed for other brucellae 
similarly suffer from lack of standardization, recent 
improvements have resulted in greater sensitivity and 
specificity. ELISAs will probably replace the serum ag¬ 
glutination and Coombs tests, thus allowing for screen¬ 
ing and confirmation of brucellosis in one test. 115,116 

In addition to serologic testing, diagnosis should 
be pursued by microbiologic culture of blood or body 
fluid samples. If unautomated systems are used, blood 
cultures should be incubated for 21 days, with blind 
subculturing every 7 days and terminal subculturing 
of negative blood cultures. For automated systems, 
incubation of cultures for 10 days with blind culture 
at 7 days is recommended. 117 Because it is extremely 
infectious for laboratory workers, the organism should 
be subcultured only in a biohazard hood. Appropri¬ 
ate personal protective equipment such as a powered 
air purifying respirator with hood, gown, and gloves 
should be used when working with cultures or prepar¬ 
ing and manipulating bacteria for studies. The reported 
frequency of isolation from blood varies widely, from 
less than 10% to 90%; B melitensis is said to be more 
readily cultured than B abortus. A recent study indi¬ 
cated that BACTEC™ Myco/F lytic medium (Becton 
Dickinson Diagnostic Instrument Systems, Sparks, 
MD), pediatric Peds Plus/F or adult Plus Aerobic/F 
medium in conjunction with BACTEC™ 9240 blood 
culture system yielded detection rates of 80% and 
100%, respectively. 34 Culture of bone marrow may in- 


* *4 

% • 


« 

' * . 
« 

¥ 



Figure 7-2. Direct fluorescent antibody staining of Brucella 
abortus. 

Photograph: Courtesy of Dr John W Ezzell and Terry G 
Abshire, US Army Medical Research Institute of Infectious 
Diseases, Fort Detrick, Maryland. 

crease the yield and is considered superior to blood. 118 
In addition, direct fluorescent antibody tests under 
development may offer a method of rapidly identifying 
these organisms in clinical specimens (Figure 7-2). The 
case classification of "probable" is defined as a clini¬ 
cally compatible case that is epidemiologically linked 
to a confirmed case or that has supportive serology 
(ie. Brucella agglutination titer greater than or equal 
to 160 in one or more serum specimens obtained after 
the onset of symptoms), and a "confirmed" is a clini¬ 
cally compatible case that is laboratory confirmed. 104119 

Future trends on rapid identification may use 
sophisticated protein microarrays to rapidly screen 
clinical samples or bacterial isolates. 111 However, many 
of these state-of-the-art identification methods will re¬ 
main out of reach for resource and fiscally constrained, 
endemic countries, and thus for many of these areas the 
primary methods of identification of Brucella infections 
will remain the clinical presentation and traditional 
diagnostic methods. 


TREATMENT 


Brucellae are sensitive in vitro to a number of oral 
antibiotics and to aminoglycosides. In June 2005 at the 
Clinical Laboratory Standards Institute (CLSI formally 
known as National Committee for Clinical Laboratory 
Standards or NCCLS) meeting, the minimum inhibitory 
concentration breakpoints were established (Table 7-3) 


for Brucella along with the standard procedures for in 
vitro testing. 120 Therapy with a single drug has resulted 
in a high relapse rate, so combined regimens should 
be used whenever possible. 104,121-125 A 6-week regimen 
of doxycydine (200 mg/day administered orally) and 
streptomycin (1 g/day administered intramuscularly for 


166 



Brucellosis 


TABLE 7-3 

BRUCELLOSIS MINIMUM INHIBITORY 
CONCENTRATION BREAKPOINT RANGES 


Antimicrobial 

Minimum Inhibitory Concentration 
range (pg/mL) 

Azithromycin 

0.25->64 

Chloramphenicol 

0.5-4 

Ciprofloxacin 

0.25-8 

Streptomycin* 

<8 

Tetracycline 

0.03-0.5 

Doxycycline 

<1 

Gentamicin 

0.5-4 

Rifampin 

<0.12-2 

Levofloxacin 

<0.06-4 

Trimethoprim - 
Sulfamethoxazole 

<2/38 


*The streptomycin-susceptible breakpoint is > 16 pg/mL when the 
test is incubated in CO, and > 8 pg/mL when incubated in room air. 
Data sources: (1) Jorgensen JH. CLSI M45-A2: Methods for Anti¬ 
microbial Dilution and Disk Susceptibility Testing of Infrequently 
Isolated or Fastidious Bacteria; Approved Guideline-Second Edi¬ 
tion, M45A2. 2010, Clinical and Laboratory Standards Institute, 
ISBN(s):1562387324. (2) Patel J, Heine H, oral personal communica¬ 
tion between these principal investigators at the Clinical and Labora¬ 
tory Standards Institute Guideline Meeting, June 2005. 

the first 2 to 3 weeks) is effective therapy for adults with 
most forms of brucellosis. 125,126 However, a randomized, 
double-blind study using doxycycline plus rifampin or 
doxycycline plus streptomycin demonstrated that 100 
mg twice daily oral doxycycline plus 15 mg/kg body 
weight of oral rifampin once a day for 45 days was as 
effective as the classical doxycycline plus streptomycin 
combination, provided these patients did not have evi¬ 
dence of spondylitis. 127 A 6-week oral regimen of both 
rifampin (900 mg/day) and doxycycline (200 mg/day) 
is an effective therapeutic treatment with a relapse rate 
lower than 10%. 128 Several studies, however, suggest 
that treatment with a combination of streptomycin 


To prevent brucellosis, animal handlers should 
wear appropriate protective clothing when working 
with infected animals. Meat should be well cooked; 
milk should be pasteurized. Laboratory workers 
should culture the organism only with appropriate 
biosafety level 2 or 3 containment, depending on the 
stage of bacterial identification (diagnostic sample 
verses isolated culture). 138 Chemoprophylaxis is not 
generally recommended for possible exposure to 
endemic disease. 

In the event of a biological attack, the M40 mask 
(3M, St Paul, MN) should adequately protect per¬ 


and doxycycline is more successful and may result in 
less frequent relapse than treatment with the combina¬ 
tion of rifampin and doxycycline. 12 ^ 130 Although it is a 
highly effective component of therapy for complicated 
infections, streptomycin has disadvantages of limited 
availability and requirement for intramuscular injection. 
Other aminoglycosides (netilmicin and gentamicin), 
which can be given intravenously and may be more 
readily available, have been substituted for streptomy¬ 
cin with success in a limited number of studies. 9 ' Fluo¬ 
roquinolones in combination with rifampin have dem¬ 
onstrated efficacy similar to the doxycycline-rifampin 
regimen and may replace doxycycline plus rifampin due 
to potential doxycycline-rifampin interactions. 125,131-134 

Endocarditis may best be treated with rifampin, 
streptomycin, and doxycycline for 6 weeks; infected 
valves may need to be replaced early in therapy. 125,135 
However, if patients do not demonstrate congestive 
heart failure, valvular destruction, abscess formation, 
or a prosthetic valve, conservative therapy with three 
antibiotics —(1) doxycycline, fluoroquinolone and 
trimethoprim/sulfamethoxazole, (2) tetracycline or 
doxycycline plus rifampin, and (3) aminoglycoside 
or trimethoprim/sulfamethoxazole—may be effective 
therapy. 136 Patients with spondylitis may require treat¬ 
ment for 3 months or longer. Central nervous system 
disease responds to a combination of rifampin and tri¬ 
methoprim/sulfamethoxazole, but patients may need 
prolonged therapy. The latter antibiotic combination is 
also effective for children younger than 8 years old. 137 
The Joint Food and Agriculture Organization-World 
Health Organization Expert Committee recommends 
treating pregnant women with rifampin. 128 

Organisms used in a biological attack may be resis¬ 
tant to these first-line antimicrobial agents. Medical 
officers should make every effort to obtain tissue and 
environmental samples for bacteriological culture, so 
that the antibiotic susceptibility profile of the infecting 
brucellae may be determined and the therapy adjusted 
accordingly. 


sonnel from airborne brucellae, as the organisms 
are probably unable to penetrate intact skin. After 
personnel have been evacuated from the attack area, 
clothing, skin, and other surfaces can be decontami¬ 
nated with standard disinfectants to minimize risk 
of infection by accidental ingestion, or by conjuncti¬ 
val inoculation of viable organisms. A 3- to 6-week 
course of therapy with one of the treatments listed 
above should be considered after a confirmed bio¬ 
logical attack or an accidental exposure in a research 
laboratory. 138,139 There is no safe and effective vaccine 
currently available to use in humans. 


167 





Medical Aspects of Biological Warfare 


SUMMARY 


Brucellosis is a naturally occurring disease in a 
wide variety of wild and domestic mammals. Al¬ 
though humans are not natural hosts for Brucella 
strains, they can be infected by ingesting contami¬ 
nated foods (oral route) or slaughtering infected 
animals (percutaneous route). The brucellae are 
highly infectious by the airborne route, and this is 
the route of infection that is presumed to be of the 
biggest threat to military personnel. Laboratory 
workers can easily become infected when Brucella 
cultures are handled outside of a biosafety cabinet. 
Individuals presumably infected by aerosol have 
symptoms indistinguishable from patients infected 
by other routes: fever, chills, and myalgia are most 
common. 

Because the brucellae disseminate throughout the 
reticuloendothelial system, they may cause disease 
in virtually any organ system. Large joints and the 
axial skeleton are favored targets; arthritis appears in 
approximately one-third of patients. Fatalities occur 
rarely, usually in association with central nervous 
system or endocardial infection. 


Serologic diagnosis uses an agglutination test that 
detects antibodies to LPS. This test, however, is not use¬ 
ful to diagnose infection caused by B canis, a naturally 
O-polysaccharide-deficient strain. Although ELISAs 
can more easily be standardized and performed in 
most clinical laboratories, these tests tend to have a 
higher degree of false-positive results, 139 and there¬ 
fore the Rose Bengal (slide-type) agglutination test 140 
or Brucella microagglutination test 141 continue to be 
considered the gold standards for diagnosis. Infection 
can be most reliably confirmed by culture of blood, 
bone marrow, or other infected body fluids, but the 
sensitivity of culture varies widely. 

Nearly all patients respond to a 6-week course 
of oral therapy with a combination of rifampin and 
doxycycline; fewer than 10% of patients relapse. Al¬ 
ternatively, doxycycline plus fluoroquinolone may 
be as effective for treating this disease. Six weeks of 
doxycycline plus streptomycin for the first 3 weeks is 
also effective therapy; the limited availability of strep¬ 
tomycin may be overcome by substitution of netilmicin 
or gentamicin. No vaccine is available for humans. 


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91. Rotes-Querol J. Osteo-articular sites of brucellosis. Ann Rheum Dis. 1957;16:63-68. 

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95. Lubani MM, Dudin KI, Sharda DC, et al. Neonatal brucellosis. Eur ] Pediatr. 1988;147:520-522. 

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106. Martin-Mazuelos E, Nogales MC, Florez C, Gomez-Mateos JM, Lozano F, Sanchez A. Outbreak of Brucella melitensis 
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108. Noviello S, Gallo R, Kelly M, et al. Laboratory-acquired brucellosis. Emerg Infect Dis. 2004;10:1848-1850. 

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110. Christopher S, Umapathy BL, Ravikumar KL. Brucellosis: review on the recent trends in pathogenicity and laboratory 
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111. Al Dahouk S, Sprague LD, Neubauer H. New developments in the diagnostic procedures for zoonotic brucellosis in 
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113. Young EJ. Serologic diagnosis of human brucellosis: analysis of 214 cases by agglutination tests and review of the 
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115. Al Dahouk S, Tomaso H, Nockler K, Neubauer H, Frangoulidis D. Laboratory-based diagnosis of brucellosis — a review 
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121. Hall WH. Modern chemotherapy for brucellosis in humans. Rev Infect Dis. 1990;12:1060-1099. 

122. Falagas, M, Bliziotis IA. Quinolones for treatment of human brucellosis: critical review of the evidence from micro¬ 
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123. Pappas G. Treatment of brucellosis. BMJ. 2008;336:678-679. 

124. Skalsky K, Yahav D, Bishara J, Pitlik S, Leibovici L, Paul M. Treatment of human brucellosis: systematic review and 
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127. Ariza J, Gudiol F, Pallares R, et al. Treatment of human brucellosis with doxycycline plus rifampin or doxycycline 
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129. Montejo JM, Alberola I, Glez-Zarate ZP, et al. Open, randomized therapeutic trial of six antimicrobial regimens in the 
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130. Solera J, Rodriguez-Zapata M, Geijo P, et al. Doxycycline-rifampin versus doxycycline-streptomycin in treatment of 
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131. AkovaM, Uzun O, AkalinHE, Hayran M, Unal S, Gur D. Quinolones in treatment of human brucellosis: comparative 
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135. Chan R, Hardiman RP. Endocarditis caused by Brucella melitensis. Med ] Aust. 1993;158:631-632. 

136. Mert A, Kocak F, Ozaras R, et al. The role of antibiotic treatment alone for the management of Brucella endocarditis in 
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137. Lubani MM, Dudin KI, Sharda DC, et al. A multicenter therapeutic study of 1,100 children with brucellosis. Pediatr 
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141. Brown SL, Klein GC, McKinney FT, Jones WL. Safranin O-stained antigen microagglutination test for detection of 
Brucella antibodies. J Clin Microbiol. 1981;13:398^00. 


175 



Chapter 8 
GLANDERS 


SUSAN L. WELKOS, PhD* *; BRIDGET CARR GREGORY, DVM, MPH + ; DAVID M. WAAG, PhD*; and 
MARY N. BURTNICK, PhD 5 


INTRODUCTION 
MILITARY RELEVANCE 
HISTORY 

INFECTIOUS AGENT 

DISEASE 

Epidemiology 

Transmission 

Pathogenesis 

Clinical Disease in Animals 
Clinical Disease in Humans 
Laboratory Diagnosis 
Treatment 
Prophylaxis 

SUMMARY 


*Microbiologist, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702 
f Colonel, US Air Force, Biomedical Sciences Corps; Deputy Chief of Staff, Defense Threat Reduction Agency/Strategic Commmand Center for Combating 
Weapons of Mass Destruction, 8725 John J. Kingman Road, Fort Belvoir, Virginia 22060;formerly, Chief, Education and Training, Division of Medicine, 
US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 

* Microbiologist, Bacteriology Division, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702 
5 Assistant Professor, Department of Microbiology and Immunology, University of South Alabama, 5851 USA Drive North, Medical Science Building, 
Mobile, Alabama 36688 


177 



Medical Aspects of Biological Warfare 


INTRODUCTION 


Glanders is a debilitating and often fatal zoonotic 
disease of solipeds including horses, mules, and 
donkeys caused by infection with the bacterium 
Burkholderia mallei. It is characterized by ulcerating 
granulomatous lesions of the skin and mucus mem¬ 
branes. Disease progression and pathology in humans 
and horses are similar, yet the clinical presentation 
of any two cases in the same species —even if related 
by direct transmission—may vary significantly. 1-5 
Generalized symptoms include fever, myalgia, head¬ 
ache, fatigue, diarrhea, and weight loss. After infec¬ 
tion, the organism travels through lymph channels 
first to regional lymph nodes often causing irritation 
(lymphangitis, lymphadenitis) en route. Unchecked, 
organisms may enter the bloodstream and be carried 
throughout the body. Without effective treatment, the 
course of disease may range from one that is acute and 
rapidly fatal to one that is very slow and protracted 
with alternating remissions and exacerbations. 

Glanders is an old disease, having been de¬ 
scribed toward the beginning of recorded history. 
It is less commonly known by other names, includ¬ 


ing equinia, malleus, droes, and farcy. Farcy is an 
ancient term given to a particular cutaneous mani¬ 
festation of glanders that at the time (before 1882) 
was believed to be a completely separate disease 
in horses. With this cutaneous manifestation of 
glanders, nodular abscesses (farcy buds) became ul¬ 
cerated, and regional cutaneous lymphatic vessels 
became thickened and indurated (farcy pipes) and 
oozed a glanders-typical yellow-green gelatinous 
pus (farcy oil). 6 Pure farcy without ulceration of the 
mucous membranes is rare, if not just a temporary 
stage, as is vice versa. 3 Humans, goats, dogs, cats, 
rabbits, and carnivorous predators living in close 
proximity to infected equids or carcasses have 
been naturally infected. 1,7 Camels have also been 
infected and are associated with human disease. 7 
Naturally occurring glanders has been eradicated in 
most countries, but is still found in parts of Africa, 
the Middle East, Eastern Europe, Asia, and South 
America. Glanders has drawn interest as a possible 
warfare agent in the biological weapons programs 
of several countries. 


MILITARY RELEVANCE 


B mallei was one of the first biological warfare 
agents used in the 20th century. Germany used an 
ambitious biological sabotage campaign in several 
countries, including the United States, Russia, Roma¬ 
nia, France, and Mesopotamia, on both the western 
and eastern fronts during World War I. Additionally, 
cattle, horses, mules, and other livestock being shipped 
from the United States to the Allies were beleaguered 
and inoculated with cultures of B mallei . 8 In 1914, a 
member of the German army named Anton Dilger, 
an American-educated surgeon, was sent home to live 
with his parents in Virginia after a nervous breakdown. 
He brought strains of Bacillus anthracis and B mallei and 
set up a laboratory with his brother's help to grow the 
organisms in a private home in Chevy Chase, Mary¬ 
land. Organisms were delivered to another contact 
from Germany waiting in Baltimore, who then inocu¬ 
lated horses awaiting shipment to the Allies in Europe. 

German agents also infected 4,500 mules in Meso¬ 
potamia with glanders, a German agent was arrested 
in Russia with similar intentions in 1916, and French 
cavalry horses were also targets for intentional glan¬ 
ders infection. 9 Germany and its allies infected many 
mules and horses on Russia's eastern front, and this 
action successfully impaired artillery movement and 
troop and supply convoys. Concurrent with this rise 
in animal cases during and after the war, human cases 


increased in Russia. Attempts to contaminate animal 
feeds also occurred in the United States. Between 1932 
and 1945 the Japanese used B mallei to deliberately in¬ 
fect horses, civilians, and prisoners of war at the Ping 
Fan Institute, also known as Unit 731, in occupied Man¬ 
churia. Two laboratory workers accidentally exposed 
to B mallei died at the institute in 1937. 10 

In response to perceived biological warfare 
threats from Japan and Germany, the United States 
began work on biological warfare agents at Camp 
Detrick, Maryland (now Fort Detrick) in 1942. B 
mallei was studied for potential use but was not 
weaponized. Between November 1944 and Septem¬ 
ber 1953, seven laboratory-acquired human infec¬ 
tions from Malleomyces mallei (the taxonomic name 
of glanders at that time) occurred in Camp Detrick 
employees. Howe and Miller reported the first six 
of these infections in a case series, which remains 
the largest reported human case series in US medi¬ 
cal literature. 3 Information on the seventh case was 
not published before 2005. All seven original case 
files were thoroughly reviewed for this chapter. 
An eighth laboratory-acquired infection occurred 
in March 2000 during US defensive research on B 
mallei . n Also, the Soviets were alleged to have used 
weaponized B mallei against opposition forces in 
Afghanistan between 1982 and 1984. 12 


178 


Glanders 


The United States signed the Convention on the 
Prohibition of the Development, Production and 
Stockpiling of Bacteriological and Toxin Weapons, 
which banned development, production, stockpiling, 
acquisition, and retention of biological agents, toxins, 
and the weapons to deliver them in 1972. 9 All offensive 
biological warfare work at Fort Detrick had ceased 
by this time; any remaining biological weapons were 
destroyed by 1973. Biodefense related research aimed 
toward the development of countermeasures to combat 
B mallei infections, however, continues to be conducted 
in the United States. A report by the Monterey Institute 
of International Studies states that between 1931 and 
1945 Japan developed B mallei as a biowarfare agent. 
There are no known current attempts for acquisition 
and use by terrorists. 13 

B mallei was considered a potential biothreat agent 
in 1947 because of its high infectivity, high degree 
of incapacitation among those infected, and agent 
availability. 14 It could be a more significant threat 
if weaponized. As exemplified by past clusters of 
laboratory-acquired infections, B mallei is particularly 
infectious by the respiratory route. It is not considered 


to be highly contagious among humans, and reports of 
person-to-person transmission are rare. If a determined 
bioterrorist gained access to the agent, whether from 
an infected animal, laboratory culture, or commercial 
culture, the consequences could be severe. Because 
the clinical symptoms of glanders are protean and 
nonspecific, and most physicians in the west are not 
familiar with the disease, diagnosis and treatment 
may be delayed postattack, even in regions with the 
most advanced medical facilities. Delayed diagnosis 
and treatment would likely result in significant mor¬ 
bidity and mortality. In addition, treatment may be 
complicated by the relative scarcity of knowledge 
and experience in therapy. As equids and some other 
animals are susceptible, further spread from animals 
to humans may continue long after an initial attack. 
Fortunately, glanders is curable and postexposure 
prophylaxis may be an option if an attack was rapidly 
confirmed. As with other agents, genetic engineering 
could be used to produce a strain with unpredictable 
virulence and atypical antibiotic resistance. Thus, if B 
mallei was cultured, concentrated, and delivered as an 
infectious aerosol, significant casualties could result. 15 


HISTORY 


Glanders is one of the oldest documented infectious 
diseases with symptoms being recorded by Hippocrates 
as early as 425 bce. Aristotle described the disease in 
horses in 330 bce and named it "malleus," meaning ham¬ 
mer or mallet. It was associated with clustered horses 
around the globe, particularly army horses and mules. 
The occurrence of glanders in domesticated equids was 
so familiar that horses and their glanders commonly 
appeared together in early literature. By about the 4th 
century, Apsyrtus and Vegetius recognized the conta¬ 
gious nature of the disease and recommended isolation 
of affected animals. Glanders was not studied in a sys¬ 
tematic matter until centuries later. The first veterinary 
school was established in Lyon, France, in the mid-1700s 
to deal with the serious problems of rinderpest and glan¬ 
ders. 16 Many researchers at the school became infected 
and died of glanders during their studies. The first 
account of humans glanders was not published until 
1821. 3 In 1837, Rayer proved the transmissibility of the 
disease by successfully infecting a horse using material 
taken from a pustule of a human glanders patient. 1718 
In 1882, Loeffler and Schutz isolated the causative 
agent, now called Burkholderia mallei, in pure culture 
from the liver and spleen of a glanderous horse. 1,2 

Up until the industrial revolution, horses and mules 
were the primary modes of transportation in all devel¬ 
oping economies. Particularly in urban locations, these 
animals were housed under crowded conditions, and 


glanders was passed from the infected to the uninfected. 
Horses and mules were in high demand during the 
American Civil War. Thousands of animals passed 
through remount stations where glanders was found 
in epidemic proportions. The problem was exacerbated 
after the American Civil War when infected military 
stock was sold to civilians, which facilitated spread 
of the disease to communities. Heavy losses of horses 
and the infrequent but deadly transmission to humans 
in the late 19th century led several countries to con¬ 
sider glanders control and eradication programs. Early 
programs in some countries involved destroying only 
clinically ill equids, with compensation, and meticulous 
disinfection of the premises of such cases. Despite these 
tactics, glanders would reemerge in new or remaining 
animals in stables and bams that once housed infected 
animals and the number of countrywide cases increased. 
The notion of a carrier-state began to be accepted. 
In spite of epidemic disease in equine populations, 
there were no simultaneous epidemics in humans. 

Vaccines and therapeutic agents were developed but 
they did not reduce the glanders burden. By 1890, the 
mallein diagnostic skin test was developed. Control 
and eradication programs would soon incorporate 
the testing of all contact equids, followed by quaran¬ 
tine and a recommendation for slaughter of all skin 
test-positive animals. This program failed in some 
locales at first because of lack of enforcement and lack 


179 


Medical Aspects of Biological Warfare 


of incentive to owners for killing their nonclinically 
ill animals. Some horse owners would deliberately 
hide contact animals to avoid testing, or they would 
sell these and asymptomatic test-positive animals to 
unsuspecting individuals to salvage economic loss. 4 
Inexpensive steam transportation helped the disease 
spread by shipping B mallei -infected animals to other 
regions and countries. The United States was blamed 
for the import of glanderous horses to Cuba in 1872 and 
for the great increase of glanders cases in Canada near 
the turn of the 20th century, where tens of thousands 
of US horses were shipped annually. 3,4 

Once control programs offered indemnity to test¬ 
positive and contact animals, and popular belief 
accepted the existence of a carrier-state, glanders 
eradication progressed more rapidly. Eliminating 
glanders in livestock effectively also eradicated the 
disease in humans in countries with such programs. 
Great Britain's experience with the rise and fall of glan¬ 
ders outbreaks in equids typifies many countries, and 
is shown in Figure 8-1. 19 Eradication of glanders was 
achieved in Great Britain by 1928 and in Canada by 


1938, about 30 years after eradication programs were 
initiated. 20 Glanders was successfully eradicated in 
the United States by 1942; the last naturally occurring 
human case was recorded in 1934. 21,22 

Glanders is a zoonotic disease of concern interna¬ 
tionally and is notifiable to the 164-member Office 
International des Epizooties in accordance with the 
International Animal Health Code. 23 Eradication 
programs still exist for several countries attempting 
to eliminate the disease. In more than 500,000 equids 
tested in Turkey between 2000 and 2001, for example, 
less than 2% tested positive and were destroyed. Only 
one of these, a mule, showed clinical signs of infection. 
Over the past two decades, glanders in livestock was 
reported in Afghanistan, Bahrain, Belarus, Bolivia, 
Brazil, China, Eritrea, Ethiopia, India, Iran, Iraq, Ku¬ 
wait, Latvia, Lebanon, Mongolia, Myanmar, Pakistan, 
Russia, Turkey, and the United Arab Emirates. 21,24-35 
Between 1996 and 2003 glanders in humans was re¬ 
ported in Cameroon, Curacao, Sri Lanka, Turkey, and 
the United States (laboratory acquired). 21 Exhibit 8-1 
depicts the year equine glanders was last reported to 


Equine Glanders (and Farcy) in Great Britain: 1877-1928 



$ $ ftP N 9? N qP N q>° n c£> n c£> ^ ,o> N ,o> v n O) N ^ l c$' & ^ 


Year 


Figure 8-1. Glanders cases and outbreaks reported to the Department for Environment, Food, and Rural Affairs in Great 
Britain, 1877-1928. Glanders was eradicated in Great Britain in 1928. 

Data source: http://www.defra.gov.uk/animalh/diseases/notifiable/glanders/index.htm. 


180 

























Glanders 


EXHIBIT 8-1 

YEAR EQUINE GLANDERS LAST REPORTED TO OIE BEFORE 1996* 


Country or Territory 

Year 

Country or Territory 

Year 

Australia 

1891 

Moldavia 

1957 

Austria 

1952 

Nambia 

1925 

Bulgaria 

1954 

Netherlands 

1957 

Canada 

1938 

Northern Ireland 

1910 

Croatia 

1959 

Norway 

1889 

Denmark 

1928 

Poland 

1957 

Egypt 

1928 

Portugal 

1952 

Estonia 

1945 

Romania 

1960 

Finland 

1943 

Serbia and Montenegro 

1959 

France 

1965 

Slovakia 

1954 

Georgia 

1960 

South Africa 

1945 

Germany 

1955 

Spain 

1956 

Great Britain 

1928 

Sudan 

1989 

Greece 

1965 

Sweden 

1943 

Hungary 

1956 

Switzerland 

1937 

Ireland 

1920 

United States of America 

1942 

Israel 

1951 

Yug Rep of Macedonia (former) 

1957 

Japan 

1935 

Zimbabwe 

1911 


The most recent year evidence of equine glanders was reported to the OIE among countries and territories free of equine glanders for at 
least 5 years (between 1996 and 2013). Included only are territories for which data exist on the reporting of equine glanders to the OIE. 
OIE: Office International des Epizooties 


the Office International des Epizooties among coun¬ 
tries and territories that have been without glanders 
activity (by Office International des Epizooties report) 
since 1996. Given the recent outbreaks in horses, 
donkeys, and dromedaries in some regions of India, 
Bahrain, Brazil, Lebanon, Pakistan, and the United 


Arab Emirates, glanders is currently considered a re- 
emerging infectious disease in these areas. 24-26 ' 28,30-32 ' 34-37 
Bioterrorism should be considered as a possible source 
in the event that confirmed human glanders occurs, in 
the absence of infected animals, in the countries and 
territories listed in Exhibit 8-1. 


INFECTIOUS AGENT 


Glanders is caused by B mallei, a gram-negative 
bacillus that is a close relative to Burkholderia pseu¬ 
domallei, the etiologic agent of melioidosis. Whole- 
genome comparisons of B pseudomallei and B mallei 
in combination with multilocus sequence typing 
(MLST) analyses suggest that B mallei is a clonal de¬ 
scendant of B pseudomallei that has evolved through 
genome downsizing. 38 Unlike B pseudomallei, which 
can be isolated from tropical soil, B mallei is an 
obligate animal pathogen and has not been found 
free-living in the environment. 39 The lack of flagellar- 
based motility is a primary means by which B mallei 
can be differentiated from B pseudomallei. Growth 
requirements are not complex and B mallei can be 
cultivated on basic nutrient medium. However, 
glycerol or glucose can be added to the medium to 
enhance growth. When stained, the cells typically 
exhibit bipolar staining. 


B mallei is well traveled taxonomically. Since its 
discovery, this microorganism has been placed in 
several genera, including Bacillus, Cory neb acterium, 
Mycobacterium, Loefflerella, Pfeifferella, Malleomyces, 
Actinobacillus, and Pseudomonas, and was finally 
assigned to the genus Burkholderia in 1992. 40,41 This 
microorganism is not particularly hardy in the en¬ 
vironment. 40 B mallei is susceptible to drying, heat, 
and sunlight. In warm and moist environments, the 
organism may survive a few months and can survive 
in room temperature water as long as 1 month. 119,42 
Experimentally and under the most favorable tem¬ 
perature and moisture conditions, Loeffler was able to 
extend the viability of B mallei to 100 days. Survival 
of B mallei in distilled water in the laboratory was 
determined to be less than 30 days. 43 In nature, vi¬ 
ability of the organism is unlikely after 90 days, and 
most infectivity is lost within 3 weeks. 


181 






Medical Aspects of Biological Warfare 


Particularly in culture, B mallei is easily aerosolized 
as demonstrated by at least seven of the eight labo¬ 
ratory-acquired infections in the United States since 
1944. Given its high infectivity by aerosol, laboratory 
studies on this Centers for Disease Control and Pre¬ 
vention Tier 1 select agent are performed at biosafety 
level 3 (BSL-3) facilities. Varying degrees of virulence 


among strains have been shown in the laboratory and 
in nature. 4,5 '' The infectious dose is considered to be 
low, depending on the route of infection, susceptibil¬ 
ity, and strain virulence. One to 10 organisms of some 
strains by aerosol are lethal to hamsters. 5 Inhaling only 
a very few organisms may cause disease in humans, 
equids, and other susceptible species. 44-46 


DISEASE 


Epidemiology 

Naturally acquired cases of glanders in humans or 
equines are sporadic and rare; most countries have 
eradicated glanders. Glanders is still infrequently 
reported in northern Africa, the Middle East, South 
America, Asia, and eastern Europe. 21 Serologic cross¬ 
reactivity with B pseudomallei precludes the accurate 
distribution and prevalence of B mallei by serologic 
means alone. However, new reagents and assays 
potentially leading to improved serodiagnosis of hu¬ 
man glanders have been described, as detailed below. 
Although human outbreaks have been reported in 
Austria and Turkey, no human epidemic has been 
recorded. 47 

In nature, chronically infected horses are considered 
to be the reservoir of B mallei, and they may also serve 
as amplifying hosts. A disease of primarily solipeds, 
donkeys are considered most prone to develop acute 
forms of glanders, whereas horses are more prone to 
develop chronic and latent disease. Mules, a crossbred 
animal resulting from the mating of horse and donkey, 
are susceptible to both acute and chronic disease as 
well as latent infections. 40,48,49 A recent report indicates 
that Old World camels (dromedaries) can acquire 
glanders naturally when kept in close proximity to 
infected horses. 35 Clinical disease in dromedaries 
closely resembled that seen in equids. Humans are an 
accidental host. 

Zoonotic transmission of B mallei from equid to 
human is uncommon, even with close and frequent 
contact with infected animals, which may be explained 
by low concentrations of organisms from infection sites 
and a species-specific difference in susceptibility to 
virulent strains. During World War II human glanders 
was rare despite a 30% prevalence in horses in China. 50 
Between 5% and 25% of tested animals in Mongolia 
were reactive, yet no human cases were reported. 
With successful transmission, however, humans are 
susceptible to infection. 

Humans exposed to infected equids have contracted 
glanders in occupational, hobby, and lifestyle settings. 
Naturally infected humans have included veterinar¬ 
ians and veterinary students, farriers, flayers (hide 


workers), transport workers, soldiers, slaughterhouse 
personnel, farmers, horse-fanciers and caretakers, and 
stable hands. Subclinical or inapparent infections in 
horses and mules have posed a hidden risk to humans. 
Infection by ingesting contaminated food and water 
has occurred; however, it does not appear to be a sig¬ 
nificant route of entry for infections in humans. 1,7,51 
Laboratory workers have also been rarely and spo¬ 
radically infected. In contrast to zoonotic transmission, 
culture aerosols are highly infectious to laboratory 
workers. The six infected workers in the Howe and 
Miller case series represented 46% of the personnel 
actually working in the laboratories during the year 
of occurrence. 5 

Different strains of B mallei can now be discrimi¬ 
nated by multiple-locus variable number tandem 
repeat analysis (MLVA), MLST, random amplified 
polymorphic DNA, and other fingerprinting methods. 
Such procedures have been shown to be useful in track¬ 
ing the source and spread of an outbreak strain and in 
geographic/clonal relationship studies, and they have 
been described and summarized elsewhere. 24,52 For 
example, Godoy et al used MLST with a set of seven 
loci in epidemiologic studies analyzing many isolates 
of B mallei, B pseudomallei, and Burkholderia thailandensis 
(a closely related but nonpathogenic environmental 
species) from diverse geographical locations that repre¬ 
sented 71 sequence types; specific clones isolated from 
animals that were associated with disease in humans 
were identified. 35,53 MLST was most useful for distin¬ 
guishing strains of B pseudomallei and B thailandensis, 
which were clearly distinguished by the divergence 
between the alleles of seven loci. However, all the 
geographically diverse isolates of B mallei analyzed 
had identical allelic profiles that clustered within the 
B pseudomallei group of isolates; alleles at six of the loci 
in B mallei were also present in B pseudomallei isolates, 
and the allele at the seventh locus in B mallei differed 
at only a single nucleotide site from B pseudomallei. B 
mallei was considered to cluster within and to be a clone 
of B pseudomallei instead of a separate species. How¬ 
ever, one recent analysis of camel-associated glanders 
used one B mallei- specific sequence type to confirm the 
laboratory identification of glanders. 35 


182 


Glanders 


MLVA has been found to be more useful for subtyp¬ 
ing different strains of B mallei. For example, an MLVA 
analysis of a B mallei strain isolated from a diseased 
camel in Bahrain revealed close genetic proximity to 
a specific strain, which caused an earlier outbreak 
of glanders in horses in the United Arab Emirates in 
2004. 35 The MLVA was based on 23 different loci, as 
reported previously. 28 Similar analyses focused on B 
mallei isolates from the Punjab region of Pakistan dem¬ 
onstrated that these strains were genetically distinct 
from isolates from other countries. 28 In the event of 
a deliberate release of B mallei or a focal outbreak of 
glanders in humans or animals, these types of analyses 
would be critical tools for facilitating investigations 
aimed at determining the source of the organism. 

Transmission 

Transmission is direct by bacterial invasion of the 
nasal, oral, and conjunctival mucous membranes; by 
inhalation into the lungs; and by invasion of abraded 
or lacerated skin. Areas of the body most often exposed 
include the arms, hands, and face. Considering the 
affinity for warm and moist conditions, B mallei may 
survive longest in stable bedding, manure, feed and 
water troughs (particularly if heated), wastewater, and 
in enclosed equine transporters. 1 Transmission has 
occurred via handling contaminated fomites such as 
grooming tools, hoof trimming equipment, harnesses, 
tack, feeding and husbandry equipment, bedding, and 
veterinary equipment. Such equipment stored away 
from any contact with equids for at least 3 months, even 
without disinfection, is not likely to be an infection source. 

Reports of the circumstances surrounding zoonotic 
transmission are diverse. Here are a few examples: 

• equids snorting in the vicinity of humans or 
human food; 

• the wiping of equine nasal exudate off a hu¬ 
man arm with a blade of grass (local infection 
occurred at wipe site); 

• sleeping in the same bam or stall as apparently 
healthy equids; 

• accidental puncture with contaminated 
equipment; 

• wiping an eye or nostril after contact with an 
equid; 

• being licked by a glandered horse; and 

• stall cleaning without any direct equine 
contact. 3 ' 54 ' 55 

The nature of much of the work in horse handling is 
physical, often producing skin abrasions under normal 
circumstances. Although absorption through intact 


skin is believed to be unlikely, patients may insist their 
skin was intact at the time of exposure. Among 105 
chronic human cases associated with equid exposure 
described by Robins, only 40 (38%) reported a wound 
present. 3 In 27 cases (17%) the absence of a wound was 
specifically noted. 3 

Laboratory infections have followed procedures 
that involved washing and aeration of cultures. Air 
samples and swabs from equipment, tables, and 
benches failed to detect residual contamination in 
laboratories after the six US laboratory-acquired events 
that occurred between 1944 and 1945. Seven of the 
eight Fort Detrick laboratory-acquired infections also 
occurred at a time when mouth-pipetting was common 
practice. The first six patients acknowledged using this 
technique to clear blocked pipettes and to blow con¬ 
tents out of pipettes that were calibrated to the tip. The 
eighth case patient involved a microbiologist who had 
2 years of experience of working with B mallei in BSL-3 
containment but did not always wear latex gloves. 11,56 
Based on the clinical manifestation of unilateral axil¬ 
lary lymphadenopathy, transmission in this case was 
believed to be percutaneous, yet a break in the skin or 
a specific exposure-associated laboratory incident was 
not recalled. This is not surprising as most laboratory- 
acquired infections are not associated with injury, or 
a recollection of injury. 57 This patient had a 13-year 
history of diabetes, however, and collected blood via 
finger-stick morning and evening. It is possible that 
a recent finger-stick site may have been a potential 
entry point. Bacterial surveys of the laboratory found 
no contamination, and all engineering controls were 
validated as functional. 

Human-to-human transmission is rare, but it has 
been reported. The majority of documented events 
were in medical practice, at autopsy, in the diagnostic 
laboratory, and in patient care settings before clearer 
understanding of universal precautions existed. 1,3,11 
Transmission also occurred in home settings. Close 
contact while caring for glanders-infected individuals 
at home led to infecting other family members. 3 At least 
one entire family became infected. In this case, two 
children and the wife of a chronically infected stable 
hand contracted glanders. The wife was presumably 
infected sexually; the 4-year-old child was likely in¬ 
fected by close contact with a 2-year-old sibling who 
was presumably infected by one of the parents. Robins 
found that among the 156 chronic infections he studied, 
10% were directly caused by another human case. 3 

Human infection by ingestion has not been de¬ 
finitively reported. Stomach contents were found to 
inactivate B mallei experimentally in 30 minutes. 47 In 
his detailed 1886 report on the etiology of glanders, 
Loeffler describes several accounts of feeding meat 


183 


Medical Aspects of Biological Warfare 


from glanderous horses to humans without causing 
disease. 1 In one account, more than 100 glanderous 
horses were slaughtered and fed to soldiers without 
incident. Although not clear in his report, it is most 
likely that in these cases the meat was cooked just as 
was customary for a military mess at that time. In an¬ 
other case, consumption of raw glanderous meat by a 
veterinarian seeking to answer the ingestion question 
did not produce disease. An 1866 veterinary journal 
report, however, describes two persons who contracted 
glanders after consuming milk from a glanderous mare. 
Because these individuals were also exposed to the 
mare, infection by ingestion could not be determined. 1 

Monogastric animals, including the lion, tiger, 
domestic cat, dog, and bear, became infected with 
B mallei after ingesting raw meat. 1 Regarding wild 
animals, Loeffler posited that crunching bones might 
cause enough oral trauma to introduce the organism 
through defects in the oral mucosa rather than by entry 
through the healthy digestive tract. 1 This explanation, 
however, does not explain infections in dogs, domestic 
cats, and captive wildlife that were fed only boneless 
meat from glanderous horses. In 2010, four lions and 
one tiger at a zoo in Tehran died from glanders. 29 Al¬ 
though not definitively proven, this outbreak was at¬ 
tributed to ingestion of soliped meat that had not been 
screened for B mallei. Based on this limited collection of 
testimonies and the current understanding of glanders 
pathogenesis, one may infer that ingestion of the live 
organism by humans is unlikely to cause disease. 

These features of transmission exemplify the re¬ 
quirement for BSL-3 containment and safety practices 
when working with B mallei. Adherence to safety 
procedures and universal barrier precautions is also 
prudent. In the presence of potentially infected equids, 
transmission risk is also reduced by universal precau¬ 
tions as well as procedures that reduce inhalation risk 
of potentially contaminated aerosols. Advances in 
medicine, infection control, and therapeutics make it 
less likely today than 100 years ago for human-to-hu- 
man transmission to occur even in the event of a human 
outbreak, whether related to bioterrorism or not. It is 
also highly unlikely that an equid reservoir would be¬ 
come established. Acute disease is expected to manifest 
in a significant proportion of exposed equids, which 
would allow emergency response, quarantine, trace- 
back, and eradication procedures. Long-term exposure 
to asymptomatic chronically infected equids that evade 
detection and are handled without precautions could 
become a sporadic but perilous risk to humans, and 
less caution may be used around them. 3 

Among equids, transmission is primarily by oro- 
nasal mucous membrane exposure, inhalation, and 
mastication (possibly ingestion) of skin exudates and 


respiratory secretions of infected animals, including 
those with latent and subclinical infection. The sharing 
of feed and water troughs facilitates this, as do com¬ 
mon equid behaviors that include grooming and 
snorting. 40,48,49 Since equids cannot breathe through 
their mouths, simple exhalation and snorting to clear 
nasal passages serve to finely aerosolize infectious 
nasal efflux from an infected equid, which poses a 
transmission risk to susceptible hosts (including hu¬ 
mans) in the vicinity. 

Transmission through ocular mucous membranes 
and abrasions in the skin is also possible. Vertical 
transmission from mare to foal has occurred naturally 
in horses. In utero transmission from sow guinea pig to 
pup has also occurred in housed laboratory animals. 1 
Sexual transmission from stallion or jack to mare or 
jenny has also been observed. The breeding of asymp¬ 
tomatic stallions was responsible for some glanders 
spread near the turn of the 20th century. 4 

Carnivores can become infected after eating con¬ 
taminated carcasses and meat. 29,58 Reported outbreaks 
in captive wild felids suggest that they appear to be 
more susceptible than canids. 40,48 ' 58 ' 59 Glanders has also 
been transmitted to goats housed with infected horses. 1 
Laboratory animals are also susceptible, including mice, 
hamsters, guinea pigs, rabbits, and monkeys. 1 Cattle, 
swine, and chickens appear to be resistant, even after 
experimental injection. 1,59,60 Pigeons were infected ex¬ 
perimentally. 1 A review of experiments on glanders in 
animals led Loeffler to suggest that the field mouse, don¬ 
key, mule, horse, goat, cat, and guinea pig were more 
susceptible to glanders infection and clinical disease 
than humans. 1 Among other susceptible host species, 
the rabbit and dog appeared to be less susceptible to 
disease than humans. Recent reports have described the 
use of invertebrate species including wax moth larvae, 
cockroaches, and nematodes as viable experimental 
models of glanders that are primarily useful for identi¬ 
fying virulence factors. 61-63 

Pathogenesis 

Overview of Pathogenesis 

Although glanders has been largely eradicated 
in humans, and for the most part in animal popula¬ 
tions, B mallei is considered a significant potential 
biothreat agent. 64 Both the acute and chronic forms of 
glanders were described in detail long before effec¬ 
tive treatments were available and when the disease 
was still prevalent. In the 1906 review of 156 chronic 
human glanders cases, Robins stated that distinguish¬ 
ing between chronic and acute disease was difficult 
because chronic disease was often interrupted with 


184 


Glanders 


acute symptoms and acute onset disease may run a 
chronic course. 3 For convenience purposes he defined 
chronic cases as those lasting longer than 6 months. 
Most historical literature attempting to differentiate 
the two classifies a more fulminant and rapidly fatal 
clinical course (within 2 to 4 weeks) as an acute form 
of glanders. An acute course is found more often with 
untreated acute pneumonic and frank septicemic infec¬ 
tion, whether primary or recurrent. 5,47,65 Chronic infec¬ 
tions are most common in horses where they comprise 
the majority of cases, whereas acute disease is more 
common in humans and donkeys. 7 

B mallei most often enters the human body through 
abrasions or openings in the skin, particularly where 
occupationally exposed on the hands and forearms, 
face, and neck. An abrasion is not always present, 
however, at least grossly. Normal, intact skin resists 
penetration of the organism; however, in several hu¬ 
man infections, the affected persons insisted no wound 


or penetration occurred during the likely exposure 
interval. Thus, a patient history in which there is no 
recollection of exposure to horses or of abrasion should 
not preclude glanders as a differential diagnosis. 
Organisms may also enter through oral, nasal, and 
ocular mucous membranes, as well as via inhalation. 
The latter has occurred in several laboratory-acquired 
infections; however, at least one laboratory-acquired 
case most likely occurred through cutaneous exposure. 
When present, the most characteristic feature of the 
disease is glanders nodes—small papular to egg-sized 
abscesses—that are very slow to heal if they open. 

The incubation period for glanders is variable, rang¬ 
ing from less than a day to several weeks. Cutaneous 
and mucous membrane exposure generally leads to 
symptoms in 3 to 5 days; without direct inoculation of 
the organism, however, the duration may be longer. 3 
Inhalational exposure may incur a slightly longer 
range of about 7 to 21 days. 3,5 




Figure 8-2. Interactions of Burkholderia mallei with host cells, (a) Proposed model of the intracellular lifestyle of B mallei in 
phagocytic cells. Following entry in host cells, B mallei rapidly escapes from the phagosome and enters into the cytosol 
where it can grow, polymerize host cell actin [red), spread cell to cell, and induce host cell fusion resulting in the formation 
of multinucleated giant cells. B mallei interacts with various pattern recognition receptors and evades host cell autophagy. 
Genes, proteins, or systems that are known to be important at various points are indicated in blue text. VirAG senses a sig¬ 
nal within the phagosome that activates T6SS-1 gene expression; T3SS AP is required for escape from the phagosome; BimA 
is necessary for actin-based motility and actin tail formation; T6SS-1 is critical for multinucleated giant cell formation, (b) 
Fluorescent micrograph of B mallei infected RAW 264.7 murine macrophages. B mallei was added to RAW 264.7 cells at a 
multiplicity of infection of 10; at 12 hours postinfection cells were fixed and stained for actin and nuclei. B mallei expressing 
green fluorescent protein is shown in green ; actin stained with Alexa Fluor 568 phalloidin is shown in red ; nuclei stained with 
DRAQ5 are shown in blue. MNGC: multinucleated giant cell 

Photograph: Courtesy of Mary N Burtnick, University of South Alabama, Mobile, Alabama. 


185 



Medical Aspects of Biological Warfare 


Intracellular Lifestyle 

B mallei is a host-adapted deletion derivative of B 
pseudomallei and is genetically more uniform and less 
diverse than the latter. 66,67 The severity, clinical course, 
and frequent chronicity of glanders are likely related 
to the capacity of B mallei to survive and persist within 
host cells and thus evade destruction by the immune 
system. As an intracellular pathogen, survival of B mal¬ 
lei involves binding to and invading eukaryotic cells, 
successfully escaping phagosomal compartments, and 
growing in the cytosol. Relatively little is known con¬ 
cerning how the organism binds to cells or the surface 
receptor involved. B mallei adheres poorly to and does 
not invade A549 and LA-4 respiratory epithelial cell 
lines, but readily invades phagocytic cell lines such 
as J774.2 and RAW 264.7 murine macrophages. 68-70 As 
shown in Figure 8-2 and as discussed below, B mallei 
uses a type III secretion system to escape from the 
phagosome into the cytosol, where it can multiply and 
use actin-based motility to move about the cell. 68,69,71,72 
In addition, B mallei can induce cell-to-cell fusion re¬ 
sulting in the formation of multinucleated giant cells 
(MNGCs), which are thought to provide a protective 
niche and metabolic resources for the bacteria, but 
little is known about the bacterial or host factors that 
are required for MNGC formation. Recent evidence 
points to expression of a type VI secretion system as a 
requirement for efficient intra- and intercellular spread 
and host cell fusion/ 3,74 B mallei can also evade innate 
host immune responses when present extracellularly 
due in part to its surface structures, which will be 
described below. However, little is known about the 
specific molecular mechanisms of B mallei virulence, 
and more study is urgently needed. 

Animal Models 

Research on pathogenicity requires the availability 
of relevant, well-characterized animal models. Various 
animal models of glanders have been reported for use 
in studies on pathogenesis and countermeasure evalu¬ 
ation, including guinea pigs, mice, hamsters, nonhu¬ 
man primates (NHPs), and several invertebrates. Major 
models will be described and are summarized in Table 
8-1; several recent reviews are available that provide 
further details of the development of in vivo models 
for glanders. 44,75,76 

Guinea Pigs and Hamsters 

Guinea pigs and hamsters are the laboratory ani¬ 
mals exhibiting the greatest susceptibility to B mallei, 
and guinea pigs were initially used most extensively. 


However, these animals varied in their individual sus¬ 
ceptibility to infection. Syrian hamsters proved to be 
uniformly susceptible to infection and have been used 
more extensively in recent studies on B mallei , 44,60 ' 75,77 l n 
1999, Fritz et al characterized disease in these animals 
bacteriologically and pathologically to include gross, 
histological, immunologic, and electron microscopic 
pathology. 78 The hamster was shown to be much more 
susceptible to B mallei than is presumed for humans, 
with an LD 50 of less than 10 colony-forming units 
(cfu) by the intraperitoneal (IP) route, with mortal¬ 
ity/morbidity monitored for 5 days. 77,78 Nevertheless, 
the course of disease and extensive development of 
glanders-associated pathology in a broad range of 
organs, especially in reticuloendothelial organs (such 
as spleen, lymph nodes, liver, and bone marrow) but 
also in other organs such as lung and brain, are similar 
to those in humans. The changes observed consist of 
infiltrates with an equal mixture of macrophage and 
polymorphonuclear leukocyte inflammatory cells; 
these can become organized into discrete, often bac- 
teria-filled, nodules referred to pyogranulomas. More 
extensive information on the pathology of glanders 
in these susceptible species is described elsewhere. 44 

Mice 

Mice vary by strain in their susceptibility to B mallei, 
a finding that mirrors that observed in studies with B 
pseudomallei . 79-81 However, mice are all considered to 
be moderately resistant to infection, similar to humans. 
Depending on the strain, route, and dose of infection 
selected, they have been used to model a range of 
disease manifestations and states ranging from latent 
to acute or chronic. Since genetic constructs and re¬ 
agents specific to mice are widely available, they are 
common tools for studies on pathogenesis and pro¬ 
tection. In most studies, the C57B1/6 strain was more 
resistant than BALB/c strain mice by the pulmonary 
route. 44,76,82 Studies by Goodyear et al have shown 
that when B mallei was administered intranasally to 
C57B1/6 mice, high dose inocula (5,000 cfu) resulted 
in acute infections that were lethal within 3 to 4 days, 
whereas low dose inocula (500 cfu) resulted in chronic 
infections. 82 In recent as well as previous studies, the 
relatively more sensitive B ALB/c strain has been used 
most often. 44,76,83,84 In acute infection studies with 
BALB/c mice, a lethal IP dose often results in spleno¬ 
megaly with multiple splenic white foci consisting 
of pyogranulomatous inflammation. This pathology 
occurs also in other reticuloendothelial organs, as 
typically described for hamsters; but mice usually 
do not exhibit this pathology in other organs, such as 
the lung, as occurs later in infection of hamsters. 44,83 


186 


Glanders 


TABLE 8-1 

EXPERIMENTAL ANIMAL MODELS OF GLANDERS 


Model 

Route of Infection 

Features 

Horses 

IT 

Physiologically relevant model that mimics the natural development of chronic 
glanders in its reservoir host. Horses typically exhibit a long disease course 
with periods of improvement and relapse. 

Rhesus monkeys 

SC and aerosol 

Development of SC lesions that healed after 3 weeks. No evidence of chronic or 
acute infection. Evidence of potential nonlethal chronic infection after aerosol 



exposure. 

African green 
monkey 

Aerosol 

Development of acute glanders resembling the human infection. 

Syrian golden 
hamsters 
(Mesocricetus 
auratus ) 

IP 

Extremely susceptible infection model (LD 50 <10 cfu) with development of acute 
infection resulting in a rapid disease course. Bacteria are transported to the 
mediastinal lymph nodes and seeded to other tissues, forming lesions in the 
spleen as early as 1 day postinoculation and death occurring around 6 days 
later. 

Guinea pigs 

SC and IP 

Development of acute glanders. Bacteria are transported to inguinal and axil¬ 
lary lymph nodes (SC), or mediastinal and mesenteric lymph nodes (IP), at 
early time points postinoculation. 

BALB/c mice 

Aerosol, IN, and IP 

Good model for acute and potentially chronic glanders. Commonly used model 
due to low cost, susceptibility to B mallei infection, and a well-documented 
disease pathology. Mice are more resistant to B mallei when delivered IP. 

C57B1/6 mice 

Aerosol, IN, and IP 

More resistant to B mallei infection than BALB/c mice by pulmonary routes. 

High doses delivered IN resulted in acute disease, whereas low doses pro¬ 
duced chronic infections. 

Wax moth larvae 
(Galleria 
mellonella) 

Injection into 
hemocoel 

Used to screen putative virulence mutants; between 3 to 200 cfu of wild type 

B mallei leads to >90% killing within 6 days. 

Madagascar 

hissing 

cockroaches 

Injection into dorsal 
abdomen 

Used to screen putative virulence mutants; highly susceptible infection model 
(LD 50 <10 cfu) resulting in death within 5 days. 

Nematodes 
(Caenorhabditis 
elegans) 

Feeding 

Used to screen putative virulence mutants; limited sensitivity resulting from 
high infectious dose. 


cfu: colony forming unit 
IN: intranasal 
IP: intraperitoneal 
IT: intratracheal 
LD: lethal dose 
SC: subcutaneous 


Aerosol exposure to a lethal dose of B mallei produces 
gross and microscopic pathologic changes similar to 
those after IP injection, except that there is more lung 
involvement, that is, focal inflammation and necrosis 
early in infection develop into extensive consolidation 
with chronic inflammatory cell infiltration (David L 
Fritz, David DeShazer, David M Waag, USAMRIID, 
Fort Detrick, MD, unpublished data, 2012). 84 It has 
been observed that aerosol-exposed BALB/c mice 
develop acute inflammation of the nasal passages, 
which later extends to the nasal sinuses and ultimately 
into the brain, especially the olfactory lobe (David M 


Waag, USAMRIID, Fort Detrick, MD, unpublished 
data, 2013). 84 Unlike humans, mice are obligate nose 
breathers, an anatomical difference that may explain 
these findings. Recent models using an intratracheal 
(IT) route of infection address this potentially con¬ 
founding issue. 85 However, the brain infection in mice 
might serve as a potential protected site for bacterial 
survival and provide a model for studying recrudes¬ 
cence of disease. 

Acute infection virulence data obtained with murine 
models often used laboratory strains such as the type 
strain B mallei ATCC 23344 (aka China 7) or the most 


187 





Medical Aspects of Biological Warfare 


recent human clinical isolate of B mallei, referred to as 
the FMH 23344 strain of B mallei ATCC 23344“ The 50% 
lethal doses (LD 50 s) for BALB/c mice have varied by 
route, and the mice were more susceptible by pulmo¬ 
nary routes than by the IP route. The range of LD 50 s, as 
determined 10 to 35 days postchallenge (most 21 or 28 
days), is exemplified as follows for BALB/c mice: IP (7 
x 10 5 cfu), intranasal (IN; 680 cfu), IT (818 cfu), whole- 
body aerosol (913 cfu), and nose-only aerosol (1,859 
cfu). 76,77,83-88 Recently, the virulence of B mallei strain 
FMH 23344 was reevaluated in C57B1/6 and BALB/c 
mice challenged by the whole-body aerosol route. As 
expected, C57B1/6 mice were significantly more resis¬ 
tant than BALB/c mice; the respective LD 50 s (after 21 
days) were 7,665 cfu and 395 cfu, respectively (David 
M Waag, USAMRIID, Fort Detrick, MD, unpublished 
data, 2013). 

No well-defined murine models for long-term 
chronic or latent glanders exist. However, B mallei 
clearly establishes protracted persistent infections 
in mice as it does in horses and humans, as shown 
especially in BALB/c mice. In recent aerosol and IP 
challenge studies, spleen cultures revealed the pres¬ 
ence of long-term residual infection with B mallei in 
surviving mice. Such infections were more consis¬ 
tently detected in BALB/c than in C57B1/6 survivors, 
that is, BALB/c mice surviving greater than or equal 
to 60 days after aerosol challenge with the FMH 
derivative of strain B mallei ATCC 23344 had posi¬ 
tive spleens; whereas C57B1/6 survivors had usually 
cleared the infection (Christopher K Cote and David 
M Waag, USAMRIID, Fort Detrick, MD, unpublished 
data, 2012). Fritz et al conducted a natural history 
study of B mallei ATCC 23344 in mice infected lethally 
and sublethally by the IP route. 83 Pyogranulomatous 
inflammation was observed histologically in mul¬ 
tiple reticuloendothelial organs, and the incidence 
and severity of the changes did not decrease in the 
sublethally infected mice. Barnes et al treated mice 
that had been infected by aerosol with B mallei with a 
postexposure prophylactic regimen of trimethoprim- 
sulfamethoxazole that effectively prevented acute in¬ 
fection; two-thirds of the mice survived to study end 
at day 74. 89 However, the treatment did not eradicate 
the bacteria and a clinical relapse of infection occurred 
by day 30 postchallenge. B mallei was detected in the 
organs of all surviving mice. Similar data have been 
reported, and the results overall suggest that BALB/c 
mice could serve as a useful model for both acute and 
chronic glanders in a relatively resistant host such 
as humans. 89,90 Further research to develop defined 
murine models for persistent forms of B mallei infec¬ 
tion are needed to identify the factors involved in the 
evolution of infection. 


Nonhuman Primates 

Although NHPs are phylogenetically the closest 
animal to humans, few studies have described their 
use as laboratory models. In one study, rhesus mon¬ 
keys were given different doses of a virulent strain 
by the subcutaneous route, and the monkey receiving 
the highest dose developed a cutaneous abscess that 
resolved completely after 3 weeks. 44,60,76 Russian inves¬ 
tigators reported studies with baboons but few details 
are available. 44 Different species of NHPs appear to 
vary significantly in susceptibility but overall there is 
a lack of substantial data characterizing experimental 
infection in these animals. The challenge is to develop a 
model(s) that best mimics the human acute and chronic 
responses to B mallei. Recent studies have shown that 
rhesus macaques are resistant to acute infection (John 
J Yeager and M Louise Pitt, USAMRIID, Fort Det¬ 
rick, MD, unpublished data, 2012); however, Yingst 
et al showed that rhesus monkeys given subclinical 
doses of B mallei exhibited clinical presentations and 
pathological lesions that correlated well with those 
described for human cases of glanders. 91 They sug¬ 
gested that the rhesus macaque is a potentially viable 
model for human disease albeit not acute lethal illness. 
A longer-term study is being done to include LD 50 de¬ 
terminations with extensive clinical, pathological, and 
laboratory evaluations to further analyze three NHPs 
as models of human glanders (Patricia L Worsham, 
David M Waag, and Taylor B Chance, USAMRIID, Fort 
Detrick, MD, unpublished data, 2013). African Green 
monkeys ( Chlorocebus aethiops) were much more sensi¬ 
tive to infection than cynomolgus macaques or rhesus 
macaques and appear to be a potential model for 
acute infection (Patricia L Worsham, David M Waag, 
and Taylor B Chance, USAMRIID, Fort Detrick, MD, 
unpublished data, 2013). 

Invertebrate Models 

Several nonmammalian surrogate models have 
recently been used to study virulence mechanisms 
and host-pathogen interactions of the human patho¬ 
genic species of Burkholderia, including insect larvae, 
cockroaches, a phagocytic amoeba, and soil-dwelling 
nematode. For B mallei, models using wax moth larvae 
(Galleria mellonella ) and the Madagascar hissing (MH) 
cockroach have been reported. 62,63 Insect models are 
useful mammalian surrogates for several reasons. Sig¬ 
nificant similarities exist between the innate immune 
systems. Both hosts harbor Toll receptors (insects) or 
Toll-like receptors (TLRs, mammals) that recognize 
pathogen markers and produce protective responses 
such as antimicrobial peptides; also insects possess 


188 


Glanders 


phagocytic hemocytes that can take up and kill mi¬ 
crobes, in a manner similar to that of neutrophils. Other 
advantages of using insects as models involve ready 
availability, reduced costs and facile housing/mainte¬ 
nance, and exemption from regulatory oversight and 
expense. B mallei was shown to be as virulent for the 
larvae as it was for hamsters and mice, whereas Burk- 
holderia that are nonpathogenic in mammals were not 
pathogenic for the insects. Notably, in tests with mutants 
of B mallei harboring known virulence-associated gene 
defects (eg, in capsule production or in the type three 
secretion system [T3SS] AP ), lethality for wax moth larvae 
corresponded to the extent of reduced virulence of the 
mutants in hamsters and mice. 63 Fisher et al found the 
MH cockroach to be easier to handle than wax moth 
larvae and their ability to grow at 37°C made them 
more amenable than other insects to mutant analysis 
with B mallei, a mammalian host-adapted pathogen. 62 
Thus, MH cockroaches appear to be a valid surrogate 
and alternative to mammals as a model for virulence 
mechanisms of B mallei important in host interactions. 

Virulence Mechanisms 

Surface Polysaccharides. Since B mallei appears to 
be genetically derived from the environmental sapro¬ 
phyte B pseudomallei, it shares many virulence factors 
with the latter. 75,92 However, some of the B pseudomallei 
factors required for its independent lifestyle appear 
to have been lost in B mallei as a consequence to its 
adaptation to the equine hosts. In addition, differences 
in the presence or role of some virulence factors in B 
mallei compared to B pseudomallei have been described 
(possibly related to the increased presence in B mallei 
of insertion sequence elements and genetic rearrange¬ 
ments), as will be illustrated. The factors and activities 
identified as being essential for B mallei virulence and 
host persistence include the following: 

• a capsular polysaccharide (CPS); 

• lipopolysaccharide (LPS); 

• animal pathogen-like T3SS (T3SS AP ); 

• the cluster 1 T6SS (T6SS-1); and 

• the VirAG two-component regulatory 
system. 38,72,74,77,93 

Other putative virulence factors, including various 
autotransporter proteins, adhesins, quorum sensing, 
and iron-binding compounds, have been identified but 
their roles in virulence are unconfirmed (as described 
below). 

Two major polysaccharide (PS) antigens that are 
present on the surface of B mallei, a CPS and LPS, play 
important roles in the pathogenesis of glanders and 


in host responses to the infection. The presence of a 
CPS on the surface of B mallei was shown by immuno- 
electron microscopy. 77 The structure of the CPS antigen 
has recently been characterized and shown to be identi¬ 
cal to the CPS expressed by B pseudomallei, which is a 
homopolymer of -3)-2-0-acetyl-6-deoxy-fi-D-manno- 
heptopyranose-(l-. 77,94 Consistent with this finding, 
anti-CPS monoclonal antibodies (mAbs) that have been 
characterized recognize the CPS of both pathogens. 95-97 
The surface-expressed nature of the LPS is evidenced 
by its availability to the host immune system and its 
ability to activate TLR4 complexes. 97-99 The structure of 
B mallei O-polysaccharide (OPS) has been determined 
to be a repeating unit of -3)-p-D-glucopyranose-(l,3)-6- 
deoxy-a-L-talopyranose-(l- where the talose residues 
contain 2-O-methyl or 2-O-acetyl side groups. 93,100 In 
comparison to B pseudomallei, B mallei OPS lacks 4-0- 
acetyl modifications on the talose residues. 93,100,101 This 
structural difference explains why mAbs specific for 
either B mallei LPS or B pseudomallei can be isolated. 101,102 
The virulence roles of these PS antigens were demon¬ 
strated by the construction of mutant strains lacking 
either CPS or OPS, which proved to be avirulent in 
animal models.' 7,93,103 The precise functions of CPS 
and LPS in pathogenesis are not fully characterized; 
however, the CPS may contribute to survival in serum 
by inhibiting complement deposition, opsonization, 
and phagocytosis, as well as possibly conferring re¬ 
sistance to the harsh environment of the phagosome 
until the bacteria are able to escape. 75 OPS is known 
to be critical for serum resistance since B mallei strains 
lacking OPS moieties are rapidly killed by 30% normal 
human serum. 93 mAbs to both B mallei CPS and LPS 
have been identified that are either bactericidal for the 
organism or have strong opsonic activities. 9 ' The roles 
of these two PS moieties in pathogenesis were further 
confirmed by demonstrating that passive administra¬ 
tion of LPS- or CPS-specific mAbs effectively protected 
mice against lethal pulmonary challenge. 97,104 

Secretion Systems and Secreted Proteins. B mallei, 
a highly successful facultative intracellular pathogen 
that can survive in many eukaryotic cell lines, pos¬ 
sesses a variety of mechanisms to adapt to and alter the 
host environment. 68,69,71 The organism harbors an array 
of specialized secretory systems that are essential to 
this process. Little is known about these systems or the 
specific roles of their components, although a number 
of the genes identified appear to be homologous to 
the more extensively studied species B pseudomallei. 
B mallei- specific studies have focused primarily on 
characterization of T3SS AP and T6SS-1. 69 ' 72 - 75 ' 105 ' 106 Genes 
encoding other secretion systems, including the type 
II and type V systems, are also present in B mallei. 75 
The effector proteins delivered by these systems are 


189 


Medical Aspects of Biological Warfare 


predicted to disable or modulate critical host proteins 
and pathways involved in cell signaling, cytoskeleton 
and ubiquitin function, and cell death pathways, 
thus facilitating pathogen survival and propagation 
in the host. 70,75,107-109 In addition, both B mallei and B 
pseudomallei exhibit actin-based motility and the dis¬ 
tinct ability to induce formation of MNGCs in tissue 
culture models, potential mechanisms that allow the 
pathogen to spread in the host via direct cell-to-cell 
passage (Figure 8-2). :73 ' 75 ' 110 

After B mallei is phagocytosed by the host cell, it 
escapes from the endocytic vacuoles into the host cell 
cytoplasm where it uses actin-based motility to spread 
intra- and intercellularly. Virulence-associated T3SS AI , 
and T6SS-1, as well as other secreted proteins, appear to 
be essential for these various functions. 68 ' 69,71-73 ' 75,103,111 ' 112 
Many gram-negative bacterial pathogens harbor such 
secretion systems and use them to synchronize the 
secretion and delivery of effector proteins directly into 
target host cells via needle-like injection apparatuses. 
B mallei, T3SS AP is required for virulence in animal 
models of infection, as well as for phagosomal escape 
and survival in macrophage tissue culture models of 
infection. 69,72,75 Once free in the cytosol, the microbe 
activates processes for evading host cell killing and 
for polymerization of host cell actin. 1 BimA, a type V 
secreted (T5S) protein, plays a major role in facilitating 
actin-based motility in B mallei. n3,ni 

Although the T3SS AI , is important for phagosomal 
escape and survival within host cells, the exact roles 
of specific effector proteins delivered by this system 
have yet to be clearly identified. Many proteins are 
predicted to be part of the B mallei T3SSs by in silico 
annotation; some have been partially characterized, 
and the potential roles of a few in virulence have been 
studied. 75,92 These proteins include BopA and BopE; 
Bip B, C, and D; and BapB, as described in detail in pre¬ 
viously published reports. 70,75,92 In B mallei a mutation 
in the T3SS-encoded BopA effector protein resulted in 
a slower growth rate in macrophages and an apparent 
reduced ability to escape the cells. This mutation also 
attenuated infection by B mallei in BALB/c mice, sug¬ 
gesting BopA may contribute to survival within— and 
possibly escape from—host alveolar macrophages. 70,115 
In B pseudomallei, BopA appears to enhance survival 
by helping the microbe evade autophagy-induced 
phagocytic vacuole degradation. 116 Bip B, C, and D 
proteins appear to be structural components of the 
T3SS injector apparatus that are involved in contact 
of the tip with host cells. 7 " 

T6SS-1, which is important during the intracellular 
lifestyle of the B mallei, is essential for B mallei virulence 
in a hamster model of glanders. 63,74 In a RAW 264.7 
macrophage model, T6SS-1 expression was shown to 


occur following internalization of B mallei, but before 
escape of the organism from the phagosomal envi¬ 
ronment. 73 Once in the cytosol of host cells, T6SS-1 
mutants displayed defects in actin polymerization and 
an inability to induce MNGC formation. 73,105 T6SSs are 
proposed to resemble inverted bacteriophage tail-like 
structures involved in delivering effector molecules di¬ 
rectly into target cells. These systems are tightly regu¬ 
lated at the genetic level so that they are only expressed 
at the appropriate time and place. Two key components 
of T6SS-1 are the Hcpl and VgrGl proteins, which are 
both secreted and structural components of the T6SS 
apparatus and are considered reliable indicators of 
T6SS function. 74,112,117,118 Various components of T6SS- 
1 are being characterized because these proteins may 
represent potential diagnostic, therapeutic, or vaccine 
targets. 73,74,105,111,112 

The VirAG two-component system is an important 
regulator of virulence gene expression in B mallei and 
is required for virulence in hamsters. 38 Approximately 
60 genes are under the regulatory control of VirAG, 
including the T6SS-1 gene cluster and genes involved 
in actin-based intracellular motility (bimBCADE). 74 
When the VirAG system is overexpressed in vitro, 
Hcpl and VgrGl are secreted into culture superna¬ 
tant by T6SS-1. VirAG also controls the expression 
of fssM, which encodes a putative ubiquitin-specific 
protease (ie, deubiquitinase) that is expressed shortly 
after intracellular uptake and provides the bacteria 
with an enzymatic tool that can potentially regulate 
multiple eukaryotic cell processes. 74,108 Shanks et al 
characterized the expression and regulation of B 
mallei TssM, and demonstrated that it was a potent 
ubiquitin-specific protease. 108 Ubiquitin is a host pro¬ 
tein that attaches to other proteins so as to direct their 
intracellular fate. Bacterial deubiquitinases remove 
the ubiquitin residues, disrupting this process and 
promoting bacterial evasion of host immune responses 
and survival. Although the TssM protease may provide 
B mallei a selective advantage within the cell during 
infection, a role in virulence for hamsters was not 
shown. Interestingly, even though tssM is coregulated 
with and physically linked to the T6SS-1 gene cluster, 
TssM is not secreted by either T6SS-1 or T3SS AI , 108 More 
research is needed to determine the signal sensed by 
VirAG in vivo that ultimately results in the expression 
of this important regulon. 

Autotransported and ATP-binding Cassette Pro¬ 
teins. Several autotransporter (AT) and ATP-binding 
cassette (ABC) transporters that may have roles in the 
infectious process have been described for B mallei. 
ATs are large families of outer membrane proteins 
in gram-negative bacteria that are secreted by the 
T5S pathway, and they include virulence-associated 


190 


Glanders 


invasins, adhesins, proteases, and actin-nucleating fac¬ 
tors. 119,120 AT proteins have three common features: (1) 
an N-terminal signal sequence for periplasmic trans¬ 
location, (2) a central functional domain(s), and (3) an 
outer membrane channel-forming C-terminus needed 
for surface export of the central domain. 119 Eight of 
the 11 AT analogs of B pseudomallei are shared by B 
mallei and include BimA, an AT involved in actin tail 
formation and actin-based motility, and BoaA, an AT 
with a potential role in bacterial adhesion to epithelial 
cells. 74,76,114,121 BimA is expressed by both B pseudomallei 
and B mallei, although their sequences vary. In both 
species BimA is required for actin-based motility and 
MNGC formation in infected tissue culture monolay¬ 
ers. 74,76,114,122 Interestingly, in B mallei, the bimBCADE 
genes were found to be dispensable for virulence in 
hamsters. 74 

A group of immunodominant Burkholderia antigens, 
designated Hep-Hag autotransporter (BuHA) proteins, 
shares structural similarities with hemagglutinins and 
invasins. 123 These proteins were present in 53% of a B 
mallei expression library examined and only 3% of a 
B pseudomallei library. They appear to function as sur¬ 
face proteins that modulate interactions of the bacte¬ 
rial cell with the host and environment; homologs in 
other bacteria have significant roles in virulence, but 
their possible roles in B mallei virulence and immune 
modulation require further study. Finally, several ABC 
protein systems with established roles in the virulence 
and pathogenicity of various gram-negative pathogens 
have been identified in the Burkholderia . 124,125 Although 
their contribution to B mallei pathogenicity has not been 
evaluated, some components (eg, the ABC transporter 
protein LolC) have been shown to be immunogenic 
and to elicit significant partial protection against both 
B mallei and B pseudomallei. These proteins deserve 
further analyses as both putative virulence factors and 
vaccine targets. 

Quorum Sensing Systems. Quorum sensing (QS) 
permits bacteria to monitor their population density 
and modify gene transcription at critical population 
levels. 126 Many host-associated bacteria use small 
amphipathic acyl-homoserine lactone signals for QS; 
and Duerkop et al identified octanoy 1-homo serine 
lactone as the predominant Bmall synthase-produced 
acyl-homo serine lactone signal and activator for the 
B mallei LuxR QS system. 126 Nevertheless, numerous 
animal pathogens lack such systems, yet are viru¬ 
lent. Using mutants in bmal genes of the B mallei QS 
systems, Ulrich et al showed that QS was critical for 
virulence of B mallei in aerosol-exposed BALB/c mice. 127 
However, using constructs with similar mutations in 
bmal, Majerczyk et al recently determined that QS 
was not required for lethal infection of mice exposed 


by aerosol to B mallei.™ These studies do not exclude 
a role for QS in glanders in other animal models or the 
natural equine host. 

Other Potential Virulence Factors. A role for pilin/ 
fimbriae structures in the pathogenesis of glanders 
is poorly defined. 75,129 For instance, type IV pili are 
required for virulence of B pseudomallei, and although 
they are expressed by B mallei in vivo and are highly 
immunogenic, a role for them in adherence and viru¬ 
lence of B mallei has yet to be shown. Neither active 
nor passive immunization with pilin or anti-pilin 
antibodies protected mice against subcutaneous or 
aerosol challenge. Its protective role in a natural host 
or incidentally infected human or after exposure by 
different means remains to be determined. 129 Several 
other virulence mechanisms are being investigated, 
and innovative techniques and combination approach¬ 
es (in silico, in vitro, and in vivo) are being used to 
identify putative novel virulence factors with possible 
roles in pathogenesis of B mallei or in protective im¬ 
munity. 67,92 One of these virulence mechanisms is the 
carboxy-terminal processing protease of B mallei and 
compounds potentially involved in iron acquisition, 
such as the siderophore malleobactin. 130-132 

Clinical Disease in Animals 

B mallei naturally infects horses, donkeys, and 
mules, but other species have occasionally become 
infected. 41,45,58,133 If glanders is suspected as a differ¬ 
ential diagnosis, local and regional animal and public 
health authorities must be immediately notified. The 
incubation period for glanders in equids ranges from 
a few days to many months, with most between 2 to 
6 weeks. The infectious process, disease progression, 
and pathology in equids are similar to those in humans. 
Donkeys are most likely to succumb to acute disease 
and die in a week to 10 days. 1,4 Horses are more likely 
to incur a slowly progressive, chronic disease. Recur¬ 
ring clinical disease and even death in horses may 
manifest months to years after dormancy, particularly 
after any stress that causes a rise in temperature such 
as infectious disease, roundup, transport, overwork, 
poor diet, exercise, immunization, and even mallein 
testing. 1,4,134 Changes in season, from winter to spring 
and from summer to fall, have also been associated 
with recurrent disease. 4 

The primary route of infection in the natural host 
is oral, by chewing or contacting contaminated food 
and water, feeding and husbandry equipment, and 
by direct close contact with infected animals. 135 Tooth 
eruption, irregular tooth wear, coarse feeds, and bri¬ 
dling contribute to oral trauma, a common finding that 
leaves the mucosa and mucocutaneous junctions more 


191 


Medical Aspects of Biological Warfare 


vulnerable to infection. Equids are also gregarious and 
prefer to be in close contact with at least one other. 
Grooming and nibbling behavior also exacerbate the 
potential for exposure from direct contact. Contami¬ 
nated aerosols, such as those produced by snorting or 
coughing, may also easily find their way into the eyes, 
mouth, or skin abrasions of other equids in the vicin¬ 
ity. Tack, such as a harness, can cause skin irritation 
that—if contaminated—may allow easy entry of the or¬ 
ganism. Despite the oral route of infection, significant 
pathology is usually seen in the airways and lungs. 40 

With early infection or resurgence, constitutional 
signs are often the first to manifest. These signs may 
include thirst, fever (low grade to high), shivering, 
head drooping, tachycardia, tachypnea, weight 
loss, rough hair coat, indolence, prostration, and a 
reluctance to move. 136 Swelling of the limbs and joints 
may be seen. The lungs, mucosa of the respiratory tract, 
and lymphatic system are most frequently involved 
wherever the infection originates. Horses experimen¬ 
tally infected by cutaneous flank injection of infectious 
material developed a respiratory tract infection within 
a few weeks. 1 In some cases (or at various stages of 
disease) the lungs may appear to be the only organ 
involved. Regional or diffuse pneumonia and pleuritis 
are common. The lungs and upper respiratory tract are 
also the organs and tissues that show the oldest signs 
of chronic disease. Lung pathology is typically more 
marked and extensive in donkeys than in horses. 

The nasal form of glanders classically described in 
equids is a somewhat local infection of the nasal cavity 
at least characterized by yellowish-green unilateral or 
bilateral nasal discharge, with or without nodules or 
ulcers on the nasal mucosa. Regional lymphadenopa- 
thy and lymphangitis most often accompany nasal 
signs. Laryngeal, tracheal, and lower respiratory tract 
pathology is often present, however, even if micro¬ 
scopically supporting the concept that a local infection 
is more likely just early infection, or rare. Nasal signs 
are common with recurrence of chronic infection. Al¬ 
though the nasal form has been associated with equids, 
similar pathology has been described in humans. 3,54 

With clinical expression of upper respiratory infec¬ 
tion, a highly infectious, sticky, yellow-gray to greenish 
viscid unilateral or bilateral nasal exudate is produced. 
The glottis may be edematous and nasal discharge may 
be so thick as to obstruct nasal passages. The margins 
of the external nares are often swollen and crusted. The 
exudate may be periodically blood-tinged. The muzzle 
and distal forelimbs may be covered with this exudate; 
the latter is due to wiping the nose. The nasal mucosa 
may be nodular and ulcerous with ulcers often rapidly 
spreading. Ulcers may be deep and coalesce forming 
larger ulcers. Mucosal abscesses of the septum and 


nasal conchae may have swollen edges and display 
small yellow and gray nodules. These abscesses may 
invade the turbinates and cartilaginous structures, 
leading to perforation and erosion of the nasal septum. 
Particularly where the larger ulcers heal, white stellate 
or radial scars are left on the mucosa. These scars may 
be seen with an endoscopy, and they are near-hallmark 
signs of prior infection. Visible or palpable regional 
lymphadenopathy (particularly submandibular) and 
lymphangitis are present. 

The equid will frequently snort to clear nasal pas¬ 
sages, effectively showering the immediate area with 
the infectious exudate. The animal may cough, or a 
cough may be easily elicited by placing pressure on 
the throat over the larynx when there is laryngeal 
involvement. The air-exchange produced by a cough 
may exacerbate nasal discharge as equids breathe 
through their nose and not their mouths. Dyspnea, 
particularly inspiratory, may result from swelling in 
the nasal cavity or larynx. Expiratory dyspnea is also 
not uncommon, particularly with chronic involvement 
of the upper and lower respiratory tract. 55 Auscultation 
and diagnostic imaging findings may support localized 
or diffuse lung disease and pleurisy. Clinical signs may 
be mild and transient, or severe and progressive. Death 
may occur within a few days. 

At necropsy, glanders nodes will likely be found 
in the lungs, even if incidentally. Their consistency 
may be caseous to calcified depending on lesion age. 
These nodes may be of any size and occur as just a few 
or as hundreds in a diffuse miliary pattern. Pleuritis 
may also be found at necropsy. The microorganism is 
relatively abundant in the affected tissues. Animals 
may die within 3 to 4 weeks from bronchopneumonia 
and septicemia. 

The progression of cutaneous and mucous mem¬ 
brane infection in the equid is similar to infections in 
humans. An entry wound may not be found. Lymphatic 
involvement may be more visible, however. Subsequent 
to cutaneous or mucosal infection, regional lymphangi¬ 
tis develops within 7 to 10 days. Typically, the lymphat¬ 
ics undergo a visible or palpable "string of pearls" stage 
within 10 days and then turn to more solid, fingerlike 
cords that can be traced to regional lymph nodes. Nod¬ 
ules along the lymphatic vessels may erupt, exuding 
gelatinous pus. Lymph nodes may be enlarged and 
indurated, and less frequently they may rupture and 
suppurate. With disease progression, more eruptions, 
enlargement of eruptions, and coalescence of lesions are 
expected. As a rule these are very slow to heal. Thick 
crusts of wound secretions, hair, bedding, and dirt 
may mat around the lesions. With ocular involvement, 
photophobia, excessive lacrimation, mucopurulent 
ocular discharge, conjunctivitis, and apparent partial 


192 


Glanders 


blindness may occur, and this may result in behavioral 
changes such as avoidance or fear. With disseminated 
disease, cutaneous and mucous membrane lesions 
may appear anywhere, particularly the respiratory 
tract, as previously mentioned, and the limbs. The hind 
limb is more commonly affected than the forelimb. 42,48 

Acute septicemia may occur at any stage of infec¬ 
tion. A septicemic course is typically progressive 
with signs leading to multiple organ failure including 
watery diarrhea, colic, marked dyspnea, prostration, 
cardiovascular collapse, and death. Donkeys are 
particularly susceptible to B mallei septicemia; this 
form manifests in most of those naturally and experi¬ 
mentally infected. Disseminated disease in horses is 
typically more protracted, however. Clinical signs 
vary widely and may include any of those previously 
mentioned. Horses may be asymptomatic. They may 
appear slightly thin, unthrifty, or have an occasional 
or persistent nasal discharge. There may be a tran¬ 
sient mild to moderate fever. Mucous membrane and 
cutaneous lesions as well as lymphadenopathy and 
lymphangitis may also be transient or chronic. Visceral 
abscess is common, and the spleen and the liver are 
frequently involved. Intact males may have orchitis, 
which may not be evident without a reproductive 
examination. 40,137 Remission is unlikely with dissemi¬ 
nated disease particularly if it involves visceral organs. 

In the event an equid presents with clinical or nec¬ 
ropsy signs consistent with glanders, the premises 
should be immediately quarantined and local and 
regional animal health authorities should be notified. 
Treatment should not be attempted. Although a clini¬ 
cal prognosis for various forms of glanders infection 
may be surmised, it is less relevant now because of the 
global interest in eradicating (by test-and-slaughter) 
the disease. 

Chronically infected horses may display cycles of 
worsening disease followed by apparent recovery 
where few symptoms are displayed. Clinical signs 
include intermittent cough, lethargy, and lesions in 
the nasal region, lungs, and skin, just as with acute 
disease. 136 Lungs may develop lesions similar to tu- 
bercules. Nodules may appear in the submucosa of 
the nasal cavity, particularly the nasal septum and 
turbinates. Nodules found in the liver and spleen may 
be up to 1 cm in diameter and have a purulent center 
surrounded by epithelioid and giant cells. 138 Attempts 
to isolate B mallei from chronically infected animals are 
usually unsuccessful. Thrombosis can be found in the 
large venous vessels of nasal mucous membranes. 25 
Nodules in the skin along lymphatics may be seen in 
chronically infected animals as they thicken. Nodules 
may ulcerate and rupture, spewing a thick exudate 
that may be a source of infection. 


Clinical Disease in Humans 

Even during its peak near the turn of the 20th 
century, human glanders was uncommon but well 
documented. The clinical course of glanders is based 
on reports of hundreds of cases published before anti¬ 
biotics were developed and from a small series of cases 
that occurred in the United States since the discovery 
of sulfonamides. The earlier reports describe a nearly 
always fatal disease of short (a few days to weeks) 
to long (months to years) duration that was usually 
acquired from close contact with infected equids. The 
most recent cases were laboratory acquired, and all 
patients survived. 

Glanders manifestations can vary. At least five forms 
of infection have been described, including localized, 
pulmonary, septicemic, disseminated, and the afore¬ 
mentioned chronic infection, but none is exclusive. The 
most important distinction is whether the infection is 
localized, which is unusual except early in the infec¬ 
tious process. The variety of forms is largely explained 
by route of infection, regional lymphatic inflammation 
and drainage, and loci of dissemination and embolism 
via hematogenous or lymphatic spread. With disease 
progression and chronicity, all forms may manifest. 

Localized infections are regionally confined and 
typically characterized by pus-forming nodules and 
abscesses that ulcerate and drain for long periods of 
time. Lymphangitis or regional lymphadenopathy may 
develop in the lymphatic vessels that drain the entry or 
infection site. Increased mucus production from affected 
ocular, nasal, and respiratory mucosa is often present. 
Localized infections typically disseminate, leading 
to pulmonary, septicemic, or disseminated infection. 

Constitutional signs and symptoms typically occur 
early in the disease and some may persist through 
treatment. These signs and symptoms may be severe, 
leaving the patient extremely prostrate. Common 
signs and symptoms include fever or low grade fever 
in the afternoon to evening, chills with or without 
rigors, severe headache, malaise, generalized myal¬ 
gias (particularly of the limbs, joints, neck, and back), 
dizziness, nausea, vomiting, diarrhea, tachypnea, 
diaphoresis (includes night sweats), altered mental 
status, and fatigue. Other nonspecific signs may be 
tender lymph nodes, sore throat, chest pain, blurred 
vision, splenomegaly, abdominal pain, photophobia, 
and marked lacrimation. Any or many of these signs 
may be present. Following constitutional signs, clini¬ 
cal courses are discussed in greater detail as they are 
associated with route of entry and disease spread. 

Cutaneous manifestations include multiple papular 
or pustular lesions that may erupt anywhere on the 
body. Cutaneous or mucosal infections may spread. 


193 


Medical Aspects of Biological Warfare 


leading to disseminated infections. Dissemination to 
internal organs produces abscesses in virtually any 
organ, most commonly the spleen, liver, and lungs. 
Disseminated infections are associated with septic 
shock and high mortality, yet they may also produce 
a more chronic, indolent course of infection. 

With cutaneous entry through an abrasion, an 
inflammatory response of varying degrees (virulence 
dependent) occurs with accompanying pain and swell¬ 
ing. A glanders node may appear usually as a single 
blister, gradually developing into an ulcer that may 
be hemorrhagic. 7,55 Localized infection develops at the 
entry site with a mucopurulent discharge. Inflamma¬ 
tion may extend along regional lymphatics and cause 
lymphangitis perhaps with numerous foci of suppu¬ 
ration along their course. This irritation is caused by 
endotoxins present in some B mallei strains affecting 
the smooth muscle of the lymphatics. Lymphatic 
vessels may be easily palpable as firm, ropy cords. 
Regional lymph nodes become involved and similarly 
inflamed. Infection may remain localized, but more of¬ 
ten spreads, particularly without adequate treatment. 
Further spread occurs via the lymphatics and through 
hematogenous dissemination as thrombi and emboli 
are formed. Local endothelial tissue inflammation and 
suppuration can occur at any place along the route of 
spread, producing abscesses that may drain through 
the skin. Superficially, these abscesses may appear as 
papules or diffuse abscesses in inflamed skin, or larger 
(egg-sized) swellings deeper in the subcutaneous tissue 
and superficial musculature. Published case descrip¬ 
tions have described glanders nodes anywhere includ¬ 
ing the face, neck, shoulders, lumbosacral region, arms, 
and legs. 7,55 When the legs are affected, glanderous 
nodes occur more often on the medial aspect than the 
lateral. At first these glanderous nodes may be hard 
and painful, but eventually they ulcerate and slough. 
These nodes may exude relatively tenacious pus that 
varies in consistency from mucopurulent to gelatinous 
to oily, depending somewhat on chronicity. The nodes 
heal slowly and recur without adequate treatment. At 
any time the patient may become acutely ill and septi¬ 
cemic. Other organs and tissues may also be showered 
with infectious emboli. 

The infectious process through the oral, nasal, or 
ocular mucus membrane is similar to the cutaneous 
process. Weakened or abraded membranes are more 
vulnerable to entry than are intact membranes. Poten¬ 
tial entry may be associated with contaminated hands, 
fingers, objects, and aerosols contacting the eye, nose, 
and mouth. A localized infection typically follows. 
Within 1 to 5 days the affected membranes become 
injected, swell, and weep a serosanguinous to mucopu¬ 
rulent discharge. Papular and ulcerative lesions similar 


in character to those in the skin may appear. Single or 
multiple oral blisters and sores may also appear. Hy¬ 
peremia may be diffuse (entire pharynx, conjunctiva, 
etc) or localized. With ocular involvement, excessive 
lacrimation and photophobia are common. With nasal 
involvement, the nose may become greatly swollen 
and inflamed and copious nasal discharge may occur. 
Infection may invade the nasal septum and bony tis¬ 
sues, causing fistulae and tissue destruction. The entire 
face can become swollen, and regional lymph glands 
may inflame and suppurate. Infection may also extend 
lower in the respiratory tract resulting in tracheitis and 
bronchitis, which can be accompanied by cough and 
the production of mucopurulent sputum. If mucous 
membrane involvement is extensive, constitutional 
signs are also usually severe including high fever, 
severe headache, fatigue, prostration, earache, and 
various neurologic signs. 

Infection of the respiratory tract may be anticipated 
after aerosol exposure or secondarily as a consequence of 
disseminated infection. A pulmonary infection typically 
produces pneumonia, pulmonary abscess, pleuritis, and 
pleural effusion, with associated signs and symptoms 
such as cough, dyspnea, chest pain, and mucopurulent 
sputum. Nasal exudate and cervical lymphadenopathy 
may also be present if the upper respiratory tract is 
involved. Nonspecific signs and symptoms often ac¬ 
company respiratory infections, such as fatigue, fever, 
chills, headache, myalgias, and gastrointestinal signs. 
Pulmonary abscess and pleuritis are common sequelae. 
Symptoms including tender cervical lymph nodes, 
fatigue, lymphangitis, sore throat, pleuritic chest pain, 
cough, fever (often exceeding 102°F), chills, tachypnea, 
dyspnea, and mucopurulent discharge may take 2 to 3 
weeks to develop. Nonspecific signs are also usually 
present including night sweats, rigors, myalgia, severe 
headache, tachycardia, nausea, weight loss, dizziness, 
and mucosal eruptions. Some of the latter symptoms 
may indicate disseminated infection. Imaging studies 
may show diffuse or localized infiltration depending on 
the stage of infection. Miliary to necrotizing nodules or a 
localized (lobar to bilateral) bronchopneumonia are oth¬ 
er potential radiographic signs. Developing abscesses 
may be well circumscribed and circular, later becoming 
cavitated with evidence of central necrosis. Pleural ir¬ 
ritation may also be visible on imaging studies. Acute 
bronchopulmonic or pneumonic disease untreated 
tends to have a rapid onset of symptoms and was once 
said to be almost uniformly fatal within 10 to 30 days. 5 
Most laboratory-acquired infections have resulted from 
inhalational exposure resulting in pulmonary infection. 

Clinical features of eight laboratory-acquired infec¬ 
tions from Camp (Fort) Detrick are summarized in 
Table 8-2. These infections include the six-case series 


194 


Glanders 


published by Howe and Miller in 1945, a previously 
unpublished case that occurred in 1953, and the 2000 
case first presented by the Centers for Disease Control 
and Prevention. 11 The most common symptoms expe¬ 
rienced by at least four of the eight include—in order 
of most common occurrence—afternoon to evening 
low-grade fever, malaise, fatigue, headache, myalgias 
including backache, lymphadenopathy, and chest pain 
(Table 8-2). Shaded elements in the table represent the 
first signs and symptoms according to the medical 
records of the first seven patients, and according to 
the published case description of the eighth patient. 
An important clinical feature that is not reflected in the 
table is that at least half of the patients not only "felt 
better" but also were clinically better for a time after 
the first wave of disease symptoms. This period lasted 
from a few days for patient 7, to 2 months for patient 2. 
Inhalation is suspected as the route of exposure for the 
first seven patients, whereas percutaneous exposure 
probably led to the eighth case. 

Septicemic glanders results from the seeding of 
B mallei into the bloodstream, whether as a primary 
event, secondary to a local or pulmonary infection, or 
as a relapse to chronic or latent infection. Septicemia 
may be passing and lead to protracted disseminated 
infection or be fulminant and rapidly fatal. Septicemic 
glanders may produce numerous signs consistent 
with a highly pathogenic bacterial septicemia. With¬ 
out aggressive treatment, B mallei septicemia runs an 
acute course and may lead to death in 7 to 10 days. 
The thromboembolic process of glanders was well 
described by the early 1900s. 1,3 B mallei causes damage 
and subsequent death of the endothelial cells lining 
the vessels. As the cells detach, the endothelial lining 
is predisposed to thrombosis. Thrombi serve as an 
excellent culture medium and seed the bloodstream 
with bacteria. The embolic process may be realized by 
the patient as sharp stinging pain in the receiving part 
or tissue of the body. Robins describes one protracted 
chronic infection in which the patient was always aware 
of pain before multiple impending dissemination sites. 3 
Bacteremia is transient; however, the more acute or 
sudden the onset of a septicemic course, the more likely 
B mallei may be isolated from the blood. Bacteremia is 
also more likely shortly before and during the appear¬ 
ance of multiple eruptions and pustules, if they occur. 

Century-old accounts of acute septicemic glanders 
suggest that virulent organisms and toxins may be so 
rapidly absorbed that systemic disease is actually pri¬ 
mary, preceding the more patent ulcerative and lym- 
phoglandular manifestations. Death may occur before 
these develop, however. Clinical signs and symptoms 
of the septicemic process may develop immediately or 
up to 2 weeks after initial infection or resurgence. These 


signs and symptoms include any severe constitutional 
sign and any of the cutaneous, mucous membrane, 
nervous, and respiratory signs previously discussed. 
Multiple organs may be involved. Erythroderma, 
jaundice, severe gastrointestinal distress, abdominal 
spasm, and severe respiratory signs may develop. 
Tachycardia, blurred vision, photophobia, excessive 
lacrimation, altered mental status, hepatomegaly, 
splenomegaly, granulomatous or necrotizing lesions, 
and lymphadenopathy may also be present. Death 
usually occurs in 7 to 30 days without adequate treat¬ 
ment. The prognosis for acute B mallei septicemia is 
guarded regardless of treatment. 

Dissemination can also occur in a more benign 
process resulting in a chronic course, which may be 
interrupted with latent periods of up to 10 years. 6 
Dissemination typically occurs without adequate treat¬ 
ment 1 to 4 weeks after B mallei infection of the lymph 
nodes. The organs most often involved in disseminated 
infection are the spleen, liver, and lungs, although 
any can be affected. Other sites include the skeleton, 
brain, meninges, musculature, and any cutaneous or 
mucous membrane locations. The kidneys are rarely 
affected, however. Clinical signs may be absent, limited 
simply to weight loss, or be highly severe and variable 
and include any of the aforementioned. Cutaneous 
eruptions may appear anywhere on the body and 
often originate from deep pockets of infection in the 
musculature. The extremities are often affected. Gen¬ 
eralized lymphadenopathy with induration, enlarge¬ 
ment, and nodularity of regional lymphatic vessels 
may be found on the extremities and in other affected 
areas. Miliary abscesses of organs and tissues may 
resemble tuberculosis. Robins described several cases 
of disseminated chronic infections in which no clini¬ 
cal symptoms were apparent, yet at autopsy patients 
were riddled with abscesses, including in the lungs. 3 
Robins chronicles a patient with the longest known 
infection (15 years, only 5 of which were latent) who 
finally died of disseminated disease. Symptoms of this 
particular disseminated infection included nasal and 
aural discharge, submaxillary adenitis, phlegmon of 
the nose, perforation of the nasal septum, jaundice, 
diarrhea, and amyloid disease. 3 

The amount of infection and pathology in a surviv¬ 
ing patient can be particularly alarming when com¬ 
pared to a usually more rapidly fulminant disease such 
as septicemic anthrax. Protracted disseminated infec¬ 
tions are associated with septic shock and a guarded 
prognosis. Diagnostic imaging studies are indicated 
to identify potential infection. Before antibiotics, dis¬ 
seminated infection was ultimately fatal either by 
resurgence of acute disease or from exhaustion of the 
patient. Based on the few cases treated with antibiotics. 


195 


Medical Aspects of Biological Warfare 


TABLE 8-2 

CLINICAL FEATURES OF EIGHT US LABORATORY-ACQUIRED BURKHOLDERIA MALLEI 
INFECTIONS 


Signs and Symptoms* Patient 1 + Patient 2 + Patient 3 + Patient 4 + Patient 5 + Patient 6 + Patient 7 + Patient 8 + 
November November February April August August July March 

1944 1944 1945 1945 1945 1945 1953 2000 


Fever, pm rise 

99.0-99.4 

99.0-101.2 

101.0-103.4 

99.0-103.8 

99.0-102.8 

- 

99.0-101.4 

99-104.5 

Rigors, chills 



+ 

+ 




+ 

Night sweats 




+ 



+ 

+ 

Pain in chest 

+ 




+ 

+ 

+ 


Myalgia 

+ 

+ 







Malaise 

+ 


+ 

+ 

+ 

+ 

+ 

+ 

Headache 


+ 

+ 

+ 

+ 

+ 



Backache 



+ 

+ 

+ 




Stiff or sore neck 



+ 






Dehydration 

+ 


+ 






Earache 



+ 






Cough 


+ 



- 


+ 


Mucopurulent sputum 


+ 







Oropharyngeal 

Postnasal 

drip 

Blister 

under 

tongue; 

nasal 

obstruction 




Sore 

throat 



Pharynx injected 

+ 

+ 



+ 




Lymphadenopathy 

Cervical 


Cervical 

- 

Cervical 



L axilla 

Neurologic signs 



Stupor 

Carpo¬ 

pedal 

spasm 





Drowsy 



+ 

+ 





Apprehension 



+ 




+ 


Dizziness 




+ 





Fatigue 

+ 

+ 

+ 


+ 


+ 

+ 

Weight loss 

+ 






+ 

+ 

Anorexia 




+ 



+ 


Blurred vision 




+ 





Lacrimation 




+ 





Photophobia 



+ 

+ 





Abdominal signs 




Pain L- 
upper 
quadrant; 
spasm 


Diarrhea 

Indigestion, 

flatulence, 

belching 

Epigastric 

tenderness 

Nausea, vomiting 




+ 





Enlarged spleen 




+ 




+ 

Chest radiographs 

R-upper; 

-Abscess 

R-lower; 

-Abscess 

R-upper, 

-Abscess 

Clear 

L-middle, 

-Abscess 

L-lower, 

pneumo¬ 

nitis 

L-hilum 

-Abscess 

Clear 


(Table 8-2 continues) 


196 





















































Glanders 


Table 8-2 continued 


WBC 

Normal-low; 

neutropenia 

Normal 

High; 

neutro¬ 

philia 

High to 
normal to 
low; 
Neutro¬ 
phils 

Normal 

Normal 
to high- 
normal; 
Neutro¬ 
phils 

Normal, 
L-shift; 
atyp mono, 
lymph 

Normal 
late in 
disease 

Primary site 

Pulmonary 

Pulmonary 

Pulmonary 

Unknown 

Pulmonary 

Pulmonary 

Pulmonary 

Cutaneous 

Disseminated 



Possible 

Likely 

spleen 

Possible 



+ 

Secondary sites 




Unknown 




Liver, 

spleen 

Likely route of entry 

Inhalation 

Inhalation 

Inhalation 

Inhalation 

Inhalation 

Inhalation 

Inhalation 

Percutaneous 

Sputum/throat culture 

- 


- 


- 


+ 

NA 

Blood culture 

- 

- 

- 

- 

- 

- 

- 

+ at 2 mos 

Isolation of organism 

- 

- 

- 

- 

- 

- 

+ 

+ 

CFT positive 6 

Day 50 

Day 50 

Day 12 

Day 40 

- 

- 

- 

NA 

Agglutinin positive* 

Day 50 

Day 50 

Day 5 

Day 23 

Day 22 

Day 23 

Day 19 

NA 

Mallein test positive 

Day 58 

Day 58 

Day 21 

Day 18 

Day 72 

- 

- 

NA 

Successful treatment 

Sulfa¬ 

diazine 

10 days 

Sulfa¬ 

diazine 

10 days 

Sulfa¬ 

diazine 

36 days 

Sulfa¬ 

diazine 

20 days 

Sulfa¬ 

diazine 

20 days 

Sulfa¬ 

diazine 

20 days 

Aureo- 

mycin 

28 days 

Doxycy- 

cline 

6.5 mos 

Onset of antibiotic^ 

Day 60 

Day 60 

Days 2, 
15,115 

Day 18 

Day 16 

Day 9 

Day 21 

~ 5 wks 

Recovery time post trx 

21 days 

Immediate 

188 days 

12 days 

15 days 

Immediate 

Immediate 

> 6.5 mos 


""Shaded elements in the table represent the first signs and symptoms according to the medical records of the first seven patients and ac¬ 
cording to the eighth patient's published case description. 

^Patients 1 through 7: Data from original case files. WBC deviations involved only neutrophils. Absolute lymphocyte counts were all normal. 
Patients 1 and 2: Glanders as a differential diagnosis was delayed. CFTs positive > 10 months, agglutinin titers positive > 10 months, mallein 
positive >16 months. 

Patient 3: First sulfadiazine treatment was halted because of falling sedimentation rate; two more treatments followed at onset days indicated. 
Patient 4: See "Patients 1 through 7" note above. 

Patient 5: Eleven normal complete blood counts except occasional slight relative lymphocytosis; lymphadenopathy also at axillary, epi- 
trochlear, and inguinal. 

Patient 6: Patient did not take temperature but felt feverish. Agglutinin test considered positive due to titers rising from zero to 1:320. 
Patient 7: Previously unpublished case. Early WBC cytology showed transient atypical monocytes and lymphocytes (atyp mono, lymph). 
Patient 8: Initial blood culture was negative; data from Srinivasan A, Kraus CN, DeShazer D, et al. Glanders in a military research micro¬ 
biologist. N Engl ] Med. 2001;345:256-258. 

-t-Temperature ranges represent the span of recordings that exceeded normal. 

§ CFTs were considered positive if >/= 1:20. 

¥ Agglutinin titers were positive if >/= 1:640 because of background titers in healthy patients of up to 1:320. 

\)nset of antibiotic refers to the day of disease that the successful antibiotics were started; Patient 8 received two prior unsuccessful courses. 
+: positive or present 


-: negative or not present 
[blank]: not reported or no mention 
CFT: complement fixation test 
mos: months 


NA: not applicable or not done 
post trx: posttreatment 
WBC: white blood cell 
wks: weeks 


survival is likely if early and long-term effective 
therapy is instituted. Even with treatment, clinical 
symptoms may continue several months before com¬ 
plete resolution, particularly if treatment is delayed. 

Radiographic imaging is useful to monitor pul¬ 
monary infection. Early radiographic signs are typi¬ 
cally infiltrative or support early abscess formation. 


Segmental or lobar infiltrates are common. With 
time, pulmonary abscesses tend to undergo central 
degeneration and necrosis, which radiographically 
resembles cavitation, and these may be single or mul¬ 
tiple. Unilateral or bilateral bronchopneumonia may 
be seen, as well as a smattering of miliary nodules. 
Because of the potential for disseminated disease. 


197 

























Medical Aspects of Biological Warfare 


computed tomography scan is useful for monitoring 
deep tissues and visceral organs. 

Complete blood count and chemistry studies for 
glanders patients vary depending on the disease's 
location of infection and duration, and the degree of 
dissemination or septicemia. Complete blood count 
may be normal early and throughout the pretreatment 
disease course. Based on the laboratory-acquired cases, 
deviations in the white blood cell count typically involve 
only the absolute neutrophil count rather than other cell 
lines (Table 8-2). Neutropenia or neutrophilia, with or 
without a left shift, may be transient findings. Leukco- 
penia with mild to moderate relative lymphocytosis was 
seen in three of the six laboratory-acquired infections 
reported by Howe and Miller, 5 which may be attributed 
to a low absolute neutrophil count. Absolute lympho¬ 
cyte counts were consistently within normal limits. 

Historically, mortality rates have been reported to 
be 95% without treatment and up to 50% with treat¬ 
ment. A more recent analysis estimates the mortality 
rate for localized disease is 20% when treated, and 
the overall mortality rate is 40%. 65 Since the near 
eradication of glanders and the development of ef¬ 
fective antibiotics, even these may be high estimates. 
Successful cure was achieved in 100% of the eight US 
laboratory-acquired cases, despite three of the eight 
cases (37%) experiencing a delay in effective treat¬ 
ment of 2 months. 5,11,56 A brief period of "apparent 
recovery" is a common clinical feature that can easily 
lead to delayed treatment and complications. Four 
of the eight patients were successfully treated with 
sulfadiazine for at least 20 days. The first two who 
received delayed treatment still recovered with only 
10 days of sulfadiazine, yet recovery was protracted. 
The most recent patient (patient 8) had disseminated 
disease, which included abscesses of the spleen and 
liver, and required ventilatory assistance before im¬ 
proving on a prolonged course of several antibiotics. 
These recent cases imply that prognoses range from 
good with localized infection and prompt treatment 
to guarded with septicemic infection. 

Laboratory Diagnosis 

Morphology and Growth Characteristics 

A definitive glanders diagnosis in humans occurs 
when the organism is isolated in culture and correctly 
identified. In endemic regions, phenotypic character¬ 
istics such as colony and cell morphology in combina¬ 
tion with biochemical assays may still be a practical 
means to definitively diagnose glanders. These meth¬ 
ods may take 2 to 7 days to confirm a diagnosis. 139 
Gram stains alone of pus from lesions may be pro¬ 


ductive, but microorganisms are generally difficult to 
find or isolate. B mallei can be cultured and identified 
with standard bacteriological media. 46 In potentially 
contaminated samples, supplements to inhibit the 
growth of gram-positive organisms (eg, crystal violet, 
bacitracin, penicillin) and some gram-negatives (eg, 
polymyxin B) and facilitate selective isolation of B 
mallei can be useful. 140,141 The optimum growth tem¬ 
perature is approximately 37°C. Growth is typically 
slow on nutrient agar, but is more rapid (2 days) when 
enhanced with 1% to 5% glucose and/or glycerol, and 
on most meat infusion nutrient media. 140,142 B mallei 
colonies typically are smooth and about 1 mm in 
width, white (turning yellow with age), semitrans- 
lucent and viscid on Loeffler's serum agar and blood 
agar. After incubating for 3 days on sterile potato 
slices, growth appears as a shiny, moist, yellowish 
transparent film. 24,143 Selective differentiation from the 
related organisms B pseudomallei and Pseudomonas ae¬ 
ruginosa may be achieved by examining the following 
phenotypes. Whereas B mallei does not grow at 42°C 
or at 21 °C or in the presence of 2% sodium chloride, 
B pseudomallei and P aeruginosa do. Also, it has been 
reported that B mallei does not grow on MacConkey 
agar, whereas both B pseudomallei and P aeruginosa 
grow. 7,24 However, others found that B mallei strains 
grew on this agar as nonlactose fermenting colonies. 24 

B mallei is a small, nonsporulating, aerobic gram¬ 
negative bacillus approximately 2 to 4 pm long and 
0.5 to 1 pm wide. It is nonmotile, a characteristic 
that differentiates it from related organisms such 
as B pseudomallei. The presence of a thick polysac¬ 
charide capsule can be demonstrated on the surface 
by immuno-electron microscopy. 77 In the presence 
of nitrogen, the organism can grow as aerobic and 
facultative anaerobe. 22,24,144 Size may vary by strain 
and by environmental factors including temperature, 
growth medium, and age of culture. Organisms from 
young cultures and fresh exudate or tissue samples 
typically stain in a bipolar fashion with Wright stain 
and methylene blue. Organisms from older cultures 
may be pleomorphic. 140 In vivo B mallei is found most 
often to be extracellular. Since the disease is rare, 
samples should be designated as "glanders suspect" 
until confirmed by more extensive testing. Sample 
security to include appropriate chain-of-custody 
documentation is also prudent for all samples. 

Isolation 

The isolation of B mallei in culture is the gold stan¬ 
dard for a glanders diagnosis. However, it can be 
difficult to obtain clinical specimens harboring viable 
bacteria; and invasive techniques, such as aspiration 


198 


Glanders 


and biopsy, may be required. Even then, B mallei bac¬ 
teria are often difficult to find, even in acute abscesses. 
Although isolation from blood has sometimes been 
successful in acute human cases, blood cultures are 
frequently negative until the disease's terminal stages 
and do not generally appear to be a reliable indicator 
of infection, at least in animals such as NHP (Patricia 
L Worsham, David M Waag, and Taylor B Chance, 
USAMRIID, Fort Detrick, MD, unpublished data, 2013; 
Samuel L Yingst and Mark J Wolcott, USAMRIID, 
Fort Detrick, MD, unpublished data, 2013). 24,44,56 To 
amplify the presence of low numbers of bacteria in 
normally sterile sites, animal inoculation methods 
were often used previously. However, such studies are 
impractical and inadvisable now for several reasons: 

• the time required for disease to manifest; 

• logistical requirement for special containment 
facilities; 

• stringent current animal regulatory require¬ 
ments; and 

• adverse public reception of such animal 
work. 

Isolation of the agent in nonendemic regions or 
from potentially contaminated samples may require 
use of selective media. Several media are commer¬ 
cially available for isolation of human Burkholderia 
pathogens, such as BCA ( Burkholderia cepacia agar 
also referred to as PC [Pseudomonas cepacia] agar), 
OFPBL (Oxidative-Fermentative-Polymyxin B- 
Bacitracin-Factose agar), and Burkholderia Cepacia 
Selective Agar. These and other media have been 
described previously. 145-147 For example, BCA was 
originally developed as a selective medium to iso¬ 
late Burkholderia cepacia complex from the sputum 
of individuals with cystic fibrosis. Commercial 
preparations typically contain crystal violet, bile 
salts, ticarcillin, and polymyxin B (Remel, Fenexa, 
KS). It was found to be sensitive and selective for 
both B pseudomallei and B mallei. 146,147 OFPBL agar is 
another commercially available selective medium 
used to isolate B cepacia from cystic fibrosis patients. 
It permitted growth of 80% of strains tested, even 
though the colonies are very small and translucent; 
the growth often causes the agar to change from 
green to yellow due to lactose fermentation. 146 OF¬ 
PBL can be used in conjunction with (but not in place 
of) BCA agar for selective isolation of B mallei or B 
pseudomallei. Although both media are significantly 
discriminating for B mallei and B pseudomallei, they 
are not totally selective and strains of B cepacia, other 
Burkholderia, and some non-Burkholderia organisms 
can be expected to be isolated with them. 146 


Identification 

Biochemical Identification. Automated biochemi¬ 
cal kits are available commercially, such as API 20NE 
(bioMerieux, Durham, NC), RapID NF (Remel, 
Lenexa, KS), VITEK (bioMerieux, Durham, NC), and 
Biolog Inc phenotype microarray systems; however, 
they have often misidentified the Burkholderia. In 
2012, USAMRIID Supervisory Research Microbi¬ 
ologist Mark Wolcott relayed in several written and 
oral communications that B mallei and B pseudomallei 
have been misidentified as nonpathogenic bacteria 
or other pathogens such as the B cepacia complex or 
Pseudomonas. 24,56,148,149 This situation was exemplified 
with the most recent case of human glanders in which 
the infecting B mallei strain was identified by an auto¬ 
mated bacterial identification system as Pseudomonas 
fluorescens or Pseudomonas putida. 56 Another drawback 
is that many of these methods require the organism 
to be cultured in vitro before testing, resulting in a 
delay in the diagnosis. 

The MIDI Sherlock Microbial Identification System 
(Microbial Identification System, version 4; MIDI Inc, 
Newark, DE) can identify isolates of the Burkholderia 
by gas-liquid chromatography of cellular fatty acids. 
Inglis obtained good results using MIDI with B pseu¬ 
domallei; and gas-liquid chromatography was used 
to correctly classify the B mallei from the most recent 
case of human glanders in the Burkholderia. 56,150 How¬ 
ever, the bacteria must first be cultured under specific 
standardized conditions, and sample preparation is 
laborious and time-consuming. It does not usually 
allow speciation of the Burkholderia because of, for 
example, the highly similar cellular fatty acid compo¬ 
sitions. 53 Yet specific fatty acids and derivatives, such 
as methyl esters, are being identified that appear to be 
Burkholderia species-specific. 148,151 

Nucleic Acid-based Identification 

A major obstacle to isolating the pathogenic Burk¬ 
holderia directly from samples is their low concentration 
in tissues and biological fluids of infected hosts. The 
development of methods for the reliable detection of 
glanders that does not rely on isolation of the organ¬ 
ism is especially important for the diagnosis of chronic 
glanders. Therefore, many attempts have been made to 
develop indirect assays, such as nucleic acid analysis. 
The genomes of at least nine strains of B mallei have been 
sequenced, 152 and the data are being used to enhance 
the ability to specifically identify this microorganism 
and increase understanding of how B mallei interacts 
with its host. 38 Several nucleic acid-based diagnostic 
methods can confirm specific identification of B mallei, 


199 


Medical Aspects of Biological Warfare 


often within several hours. Whereas some of the DNA- 
based procedures reported could be performed directly 
on clinical samples, others required preliminary cultur¬ 
ing to isolate the bacteria. Many of the methods include 
polymerase chain reaction (PCR)-based and DNA gene 
sequencing-based assays. 153-155 The latter have included 
the 16S and 23S rRNA-encoding genes, S21 ribosomal 
protein gene loci sequences, and MLST procedures. 
For instance, Frickmann et al showed that the sequence 
comparison of a 120 base pair ribosomal protein S21 
gene fragment was useful as a diagnostic procedure 
for the discrimination of B mallei and B pseudomallei 
from the nonpathogenic B thailandensis and several 
other environmental species of Burkholderia, but it did 
not differentiate between B mallei and B pseudomallei . 156 
Gene fragments and a single nucleotide polymorphism 
in the 16S rRNA gene have been reported to differenti¬ 
ate B mallei from B pseudomallei. 155 ' 157 Analysis of the 
16S ribosomal RNA gene sequence analysis identified 
B mallei from other Burkholderia species in the 2000 US 
laboratory-acquired infection. 56 However, it will be 
necessary to analyze many different species of B mal¬ 
lei and B pseudomallei (and other species) to establish 
the specificity of these and similar single-locus typing 
procedures. For discrimination of the closely related 
species B mallei and B pseudomallei, sequencing of the 
entire 16S rRNA gene, sequencing of the 16S-23S rRNA 
intergenic spacer, or the addition of a specific PCR or 
other genotyping method to a 16S rRNA gene fragment 
test has been recommended. 158 

PCR-based techniques and DNA gene sequencing 
are increasingly used in clinical settings and public 
health laboratories for bacterial identification. 159 Au¬ 
tomation of sequencing and improved reagents have 
also reduced the cost per test and the time required 
for identification. Furthermore, because killed bacteria 
or their templates may be used, these techniques also 
have the advantage of reducing the risk of exposure 
and infection to laboratory personnel compared 
to conventional methods. 153 The current interest in 
biowarfare defense research prompted an increased 
capability based on recent publications. 153-155,157,160-165 
Numerous PCR assays have been described, some of 
which were recently described in detail in a review 
on PCR methods. 166 They target various genetic ele¬ 
ments, such as specific insertion sequences or single 
nucleotide polymorphisms, secretion system genes, 
and flagellar biosynthetic genes. 157,162-165 Most of these 
assays require evaluation using many more diverse 
strains of the pathogenic Burkholderia and related/ 
unrelated species and testing for diagnostic applicabil¬ 
ity in a controlled infection study, such as an animal 
natural history study, for full validation. For instance, 
a real-time PCR assay, BurkDiff, was designed to 


target a unique conserved region in the B mallei and B 
pseudomallei genomes containing a single nucleotide 
polymorphism that differentiates the two species. 157 
Assay sensitivity and specificity were assessed and 
confirmed by screening BurkDiff across 469 isolates of 
B pseudomallei, 49 isolates of B mallei, and 390 nontar¬ 
get isolates. The agreement of results with traditional 
identification methods and lack of cross-reactivity 
prompted the suggestion that BurkDiff may be a robust 
and specific assay for the detection and differentiation 
of B mallei and B pseudomallei; however, test results may 
be difficult to interpret and the assay must be assessed 
for its diagnostic applicability in controlled models for 
B mallei infection. 167 

Several PCR assays have targeted genes of the T3SS 
or other secretion systems; these multigenic systems 
have been shown to be important in the pathogenesis of 
B mallei and B pseudomallei. 165 ' 166 ' 165 - 171 Two assays based 
on the bimA ( Burkholderia intracellular motility A) gene 
of the type V secretion system were developed. 162,163 
It was demonstrated that the N-terminal nucleotide 
sequence of bimA contains a unique B mallei region 
not present in the B pseudomallei and B thailandensis 
bimA genes, as verified in tests with numerous strains 
of these three species. 162,163 The value of the assays for 
early, rapid, and specific diagnosis of glanders in mice 
infected by the aerosol route was shown. 162 However, 
it was reported later that the bimA of B pseudomallei 
strains from Australia contain an N-terminus identical 
to that in B mallei bimA and a B thailandensis-Wkc strain 
was detected in a bimA-based PCR assay. 109,166,172 Fi¬ 
nally, other PCR assays based on flagellar biosynthesis 
proteins (such as fliP) have been shown to be highly 
sensitive in tests with B mallei. 29 ' 166 ' 173,174 

Because of potential problems intrinsic to PCR as¬ 
says such as false positives, gene mutation, and PCR 
inhibitors, some have recommended the use of two 
DNA targets (two PCR assays) in combination with 
sequencing of the amplicons. 52 Several multiplex PCR 
assays that target several and partially alleviate these 
issues have been developed. For example, Lee, Wang, 
and Yap described a sensitive and specific multiplex 
PCR using a short variable copy repetitive sequence, a 
metalloprotease gene fragment, and a sequence unique 
to B thailandensis that could distinguish B mallei, B 
pseudomallei, and B thailandensis. 175 Koh et al separated 
the species B mallei, B pseudomallei, B thailandensis, and 
the Burkholderia cepacia complex by using four specific 
primers: (1) a putative sugar binding protein, (2) a 
hypothetical protein, (3) a putative outer membrane 
protein, and (4) 16S rDNA. 176 

Other DNA-based techniques using pulsed-field 
gel electrophoresis and ribotyping have been used 
to identify strains of B pseudomallei and differentiate 


200 


Glanders 


their virulence; these methods have not been tested 
with B mallei and would likely be more time and labor 
intensive than gene sequencing. 177178 More recently, 
improved in silico probe design based on short unique 
regions of the target genome has aided in the potential 
use of microarray procedures to selectively distinguish 
B mallei and B pseudomallei genetically; more microar¬ 
ray tests of these genetic targets with many bacterial 
strains are needed. 179 

Immunological Detection 

Immunoassays that detect the presence of specific 
microorganisms can be useful in disease diagnosis. 
No such tests specifically for B mallei are established 
despite advancements in immunodetection of B pseu¬ 
domallei. 180 ' 181 Similar efforts to develop B mallei- specific 
immunodetection methods are important because 
antibiotics that are efficacious for one disease might 
not be effective for the other. mAbs elicited by B mal¬ 
lei whole cells and that targeted the LPS have been 
described. 104 They appeared to be specific for B mallei, 
failing to recognize B pseudomallei, and might be use¬ 
ful reagents for a direct immunoassay for B mallei. In 
related studies, two groups developed large panels of 
mAbs to capsule polysaccharide and LPS that were 
specific for B mallei, B pseudomallei, or both and dem¬ 
onstrated strong binding to the bacteria. 99,165 The study 
by Zou et al also revealed additional mAbs of possible 
diagnostic value, specifically, a pathogenicity-linked 
antigen epitope(s) on capsule-like polysaccharides 
found only in the pathogenic species of Burkholderia 
(both B mallei and B pseudomallei), and several B mal¬ 
lei LPS-specific mAbs. 99 It is possible that by using a 
combination of mAbs from different antigen groups, 
different strains of B mallei and B pseudomallei can be 
effectively differentiated from each other and from 
other nonpathogenic Burkholderia species. 

Serologic Diagnosis 

Although serological tests have been developed 
for diagnostic use in equines, no such tests exist to 
identify glanders specifically in humans. The mallein 
skin test has been primarily used to detect glanders 
in horses. 24,182 A human version of the skin test was 
of little diagnostic value because of the multiweek 
delay to obtain a positive result. However, modified 
tests yielded somewhat improved results. In eight 
laboratory-acquired, confirmed cases of human glan¬ 
ders in the United States, the test was negative in two, 
not completed in one, and first positive in five on days 
18 to 72 postinfection. 177 Overall, it appears that this 
diagnostic test for human glanders is minimally useful. 


In vitro tests to include the indirect hemagglutina¬ 
tion assay (IHA) and complement fixation test (CFT) 
have been used for serologic glanders detection. The 
IHA, which is the most frequently used serological test 
for human melioidosis, can also be used to identify 
glanders cases. 183-185 In melioidosis testing, the fail¬ 
ure of the IHA to detect antibody responses despite 
culture-confirmed disease has been observed. 183 The 
CFT is still used universally in veterinary medicine 
as a reasonably reliable and low-cost procedure for 
animal glanders diagnosis. 44,186-188 However, the 
CFT can be nonspecific and may not detect all cases 
or stages of glanders. In addition to occasional false 
negative results, it has also produced frequent false 
positive results (low specificity) and been hampered 
by inhibitory effects on complement of sera. 24 ' 183,1