Bacterial Pathogens as Biological Weapons and
Agents of Bioterrorism
RONALD A. GREENFIELD, MD; DOUGLAS A. DREVETS, MD; LINDA J. MACHADO, MD;
GENE W. VOSKUHL, MD; PAUL CORNEA, MD; MICHAEL S. BRONZE, MD
ABSTRACT: Bacterial pathogens have been identified as
agents that have been, or could be, used as weapons of
biological warfare and/or biological terrorism. These
agents are relatively easily obtained, prepared, and dis-
persed, either as weapons of mass destruction or for
more limited terrorist attacks. Although phylogenetically
diverse, these agents all have the potential for aerosol
dissemination. Physicians in the United States and most
I n 2000, the United States Centers for Disease
Control and Prevention (CDC) Strategic Planning
Group published a categorization of critical biologi-
cal agents that might be used in a biological attack
and, as such, pose a risk to national security.1 Cat-
egory A agents are high priority because they can
easily be disseminated or transmitted person-to-per-
son, cause high mortality with potential for major
public health impact, might cause public panic and
social disruption, and require special action for pub-
lic health preparedness. Category B agents are sec-
ond highest priority because they are moderately
easy to disseminate, cause moderate morbidity and
low mortality, and require specific enhancements of
CDC’s diagnostic capacity and disease surveillance
activities. Category C agents are third highest pri-
ority and include emerging pathogens that could be
biowarfare or bioterrorist agents because of their
availability, ease of production and dissemination,
potentially high morbidity and mortality, and major
public health impact. Table 1 presents bacterial
pathogens so identified. Table 2 presents a brief
history of the use of bacterial pathogens in biowar-
fare and bioterrorism.2
The purpose of this review is to provide a descrip-
tion of naturally occurring infection (Table 3) and
biological attack potential (Table 4) with these or-
ganisms. Because not all potential scenarios involv-
From the Department of Medicine, Section of Infectious Dis-
eases, University of Oklahoma Health Sciences Center and Veter-
ans Administration Medical Center, Oklahoma City, OK.
Correspondence: Ronald A. Greenfield, M.D., Infectious Dis-
eases Section (111/c), Oklahoma City VA Medical Center, 921 NE
13th Street, Oklahoma City, OK 73104 (E-mail:
ronald-greenfield@ouhsc.edu).
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
of the developed world have never encountered most of
these agents and the diseases they produce. Public
health programs must be prepared, and individual pri-
mary care providers must be able to recognize, diag-
nose, treat, and prevent infection with these agents. KEY
INDEXING TERMS: Anthrax; Bioterrorism; Brucellosis;
Glanders; Melioidosis; Plague; Q fever; Tularemia. [Am
J Med Sci 2002;323(6):299–315.]
ing use of these agents in a biological attack are
predictable, physicians need an understanding of
the spectrum of illness they can produce.
Anthrax
Anthrax (from the Greek anthrakos, meaning
“coal”) is infection with Bacillus anthracis, which is
a large, Gram-positive, aerobic, encapsulated, spore-
forming, nonmotile, rod-shaped bacillus.3 In clinical
specimens, B anthracis typically appears as short
chains of 2 to 3 large Gram-positive bacilli with
irregular staining and are said to have the appear-
ance of “boxcars.” The ultimate reservoir for B an-
thracis is soil. The soil cycle is complex and incom-
pletely understood. However, it is clear that B
anthracis can persist in soil for decades. Spores are
the usual infective form.
Anthrax is a zoonotic disease. It is primarily a
disease of herbivores. Infection in domesticated an-
imals in the United States is largely prevented by
vaccination, but cattle, sheep, goats, and horses are
the most commonly infected domesticated animals,
and epizootics continue to occur.4 Enzootic and
epizootic disease continue to occur in wild animals
(particularly deer) in the United States, primarily in
a band from Texas to North Dakota. Humans con-
tract the disease from contact with contaminated
flesh, blood, excreta, hair, wool, and hides and from
manufactured products, such as bone meal. Cases of
cutaneous and respiratory anthrax occurred in the
textile industry from processing of wool and hides,
leading to such epithets for anthrax as woolsorters’
and ragpickers’ disease.
Worldwide, 20,000 to 100,000 human cases occur
annually, primarily in the Middle East, the Indian
299
Bacterial Agents and Bioterrorism

Table 1. Critical Bacterial Agents with Attack Potential and
the Diseases They Cause
Category A
Bacillus anthracis (anthrax)
Yersinia pestis (plague)
Francisella tularensis (tularemia)
Category B
Coxiella burnetii (Q fever)
Brucella species (brucellosis)
Burkholderia mallei (glanders)
A subset that includes pathogens that are food- or waterborne,
including, but not limited to
Salmonella species (salmonellosis)
Shigella dysenteriae (shigellosis)
Escherichia coli O157:H7 (hemorrhagic colitis)
Vibrio cholerae (cholera)
Cryptosporidium parvuma (cryptosporidiosis)
Category C
Multidrug-resistant Mycobacterium tuberculosis
(tuberculosis)
a
A protozoan, but listed here for convenience.
subcontinent, Asia, Africa, and Latin America.5 The
last epidemics of human anthrax occurred in Zim-
babwe, where ⬎10,000 (⬎95% cutaneous) cases
were reported between 1978 and 1980, and in Par-
aguay in 1987, where 25 cases followed the slaugh-
ter of a single infected cow.6
Anthrax is quite rare in the United States and
Europe. There were 18 cases of naturally occurring
inhalational anthrax in the United States in the
20th century; the last occurred in 1976. The last
naturally occurring reported United States case of
human cutaneous anthrax was in 20004; the preced-
ing case occurred in 1992.
B anthracis is considered the most likely agent for
large-scale biological attack.7 This bacterium has
been relatively easy to acquire, and in vitro growth
and induction of sporulation is also easy. B anthra-
cis spores are quite stable to degradation by drying,
heat, ultraviolet light, gamma radiation, and many
disinfectants. The spore size of 3 to 6 ␮m is suitable
for aerosolization and ideal for human alveolar dep-
osition. Anthrax acquired by the respiratory route
has a high mortality. Early differentiation of inha-
lational anthrax from far more common infections is
difficult.
A World Health Organization (WHO) report esti-
mated that 3 days after the release of 50 kg of B
anthracis spores along a 2-km line upwind of a city
with a population of 500,000, there would be 125,000
infections, producing 95,000 deaths. In another sce-
nario, an aerial spray of B anthracis spores along a
100-km line under ideal meteorological conditions
could produce 50% lethality rates as far as 160 km
downwind.6 Thus, aerosolized release of B anthracis
in a densely populated area could potentially have
an impact on human life equivalent to that of a
nuclear weapon.
Human anthrax caused by intentional exposure
was reported in the United States in October and
November, 2001.8 –14 A total of 22 cases of human
anthrax were reported: 11 confirmed inhalational
anthrax, 7 confirmed cutaneous anthrax, and 4 sus-
pected cutaneous anthrax (case definitions have
been published by the CDC8). Cases were reported
from Florida, New York, New Jersey, the District of
Columbia, and Connecticut.15 The primary, and per-
haps only, vehicle of this epidemic was the mail.
Anthrax is acquired by transdermal penetration,
ingestion, or inhalation of spores.16 The spores are
then phagocytosed by macrophages and carried to
regional lymph nodes. Endospores germinate inside
the macrophages to become vegetative bacteria,
which express the organism’s virulence factors.17
These vegetative bacteria also multiply in the lym-
phatic system, and enter the bloodstream, achieving
densities of up to 107 to 108 bacteria/mL, resulting in
septicemia.16
The virulence of B anthracis requires the expres-
sion of products of 2 virulence plasmids, PX01 [184.5
kilobase pairs (kbp)] and PX02 (95.3 kbp). PX01
encodes genes for the tripartite exotoxin complex.
PX02 encodes 3 genes (capA, capB, and capC) in-

Table 2. A Brief History of Biowarfare or Bioterrorism

14th century Tartar forces catapult plague infected cadavers into besieged Kaffa
1763 British forces during French and Indian war give blankets and kerchiefs from smallpox hospital to Native
Americans
1914–1918 Germany infects animals to be shipped to the Allies or employed by the Allies with Bacillus anthracis and
Burkholderia mallei
1932–42 Japan tests Bacillus anthracis, Neisseria meningitides, Shigella species, Vibrio cholerae, Salmonella species, and
Yersinia pestis in Manchurian prisoners: ⬎10,000 deaths
1940–2 Japan employs Y pestis-infected fleas against China: ⱖ120 deaths
1940–5 Germany tests Rickettsia prowazekii, Rickettsia mooseri, hepatitis A virus, and Plasmodium species in
concentration camp prisoners
1984 Intentional contamination of restaurant salad bars in Oregon by members of a religious cult: 751 cases of
Salmonella species gastroenteritis
1995 Aum Shinrikyo attempts aerosol anthrax attack in Japan without effect
1996 Contamination of Texas hospital laboratory workers’ break room food with Shigella dysenteriae: 45 cases of acute
GI illness
2001 Intentional dissemination of anthrax spores through U.S. mail: 22 cases, 5 deaths

300 June 2002 Volume 323 Number 6

 Table 3. Selected features of naturally occurring disease due to bacteria classified as critical potential biological weapons and agents of bioterrorism

Annual Incidence Mortality (%)

Incubation
Disease Forms Reservoir/Transmission U.S. World (Days) Symptoms/Signs Isolation Treatment Untreated Treated

Anthrax Cutaneous Soil, herbivores, wool 0 20,000–100,000 3–5 Papule then vesicle with edema Contact Ciprofloxacin or doxycycline 16 ⬍1
then ulcer with eschar
Gastrointestinal 1–2 Ulcers, bleeding Standard Ciprofloxacin 100 Unknown

THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES

Inhalational 4–6 Hemorrhagic mediastinitis, Standard Ciprofloxacin plus other agents 100 45
pleural effusions, shock
Plague Bubonic Flea bites, rodents, human- 10 2,500 2–8 Febrile lymphadenitis Droplet Streptomycin or gentamicin 50 ⬍10
to-human
Primary septicemic 2–4 Septicemia, purpura, acral 100 60
cyanosis
Pneumonic 2–4 Fulminant pneumonia 100 60
Tularemia Ulceroglandular Arthropods, various 100 Unknown 3–5 Necrotic ulcer with Contact Streptomycin, gentamicin, or 5–35 ⬍1
mammals lymphadenopathy ciprofloxacin
Glandular Lymphadenopathy Standard
Oculoglandular Conjunctivitis with Contact
lymphadenopathy
Oropharyngeal Severe sore throat Standard
Pneumonic Fever, chills, dyspnea,
nonproductive cough
Typhoidal Fever, chills, no localizing
findings
Brucellosis Disseminated, local Animal secretions 100 Unknown 13–28 Nonspecific febrile illness, Standard Doxycycline plus rifampin ⬍5 ⬍5
localized complications
Q fever Acute febrile illness Cattle, sheep, goats, etc. 20 Unknown 10–40 Self-limited, acute febrile illness Standard Tetracycline or doxycycline 1–2 ⬍1
Pneumonia Atypical pneumonia
Hepatitis Mild acute hepatitis
Complications Endocarditis, etc.
Glanders Acute, local Horses, donkeys, mules 0 Unknown 1–5 Local suppuration Standard Sulfonamides, doxycycline, 50 20
ciprofloxacin
Pneumonic Acute pneumonia 95 20
Septicemic Fever, chills, rash, shock 95 ⬎50

301
Greenfield et al
Bacterial Agents and Bioterrorism

volved in synthesis of the poly(D-glutamyl) capsule.

2% formaldehyde, 5% H2O2
0.5% Hypochlorite, 1% Lysol,
The capsule provides antiphagocytic protection for
Decontamination the vegetative cells. However, the products of PXO1
are responsible for the primary pathophysiology of
0.5% Hypochlorite

0.5% Hypochlorite
0.5% Hypochlorite

0.5% Hypochlorite

0.5% Hypochlorite
anthrax. These act in concert to generate toxins that
Detergent water produce both the local and systemic manifestations
of anthrax. The 3 components are protective antigen
(PA, 83 kd), edema factor (89 kd), and lethal factor
Table 4. Selected Salient Features Concerning Use of Critical Biological Agents as Biological Weapons and Agents of Bioterrorism

(90 kd).
PA binds to a specific host cell surface receptor,
ATR (anthrax toxin receptor), which is a type I
membrane protein with an extracellular von Wille-
Restricted availability

brand factor A domain that binds directly to PA.18

No human vaccine

PA then undergoes proteolytic cleavage at the se-

Vaccine

quence RKKR by a furin-like protease. The active

Not available

Not available

cleavage product is the cell-bound heptameric C-

available

terminal 63-kD protein (PA63). PA63 possesses a

high-affinity binding site for edema factor and lethal
b
IND

IND

factor. After PA63 binds one of these factors, the

complex is internalized by receptor-mediated endo-
cytosis. After acidification of the vesicle, PA63 is
Doxycycline or ciprofloxacin

Doxycycline or ciprofloxacin

Doxycycline or ciprofloxacin
Postexposure Prophylaxisa

Tetracycline or doxycycline
Doxycycline plus rifampin

inserted into the lysosomal membrane and the fac-

tor is transported into the cytosol, where it exerts its
sulfamethoxazole

effects, as edema toxin (ET) or lethal toxin (LT).

? Trimethoprim-

ET, as it name implies, is responsible for the

prominent edema at the site(s) of infection. ET is a
calcium- and calmodulin-dependent adenylyl cy-
clase.19 It also inhibits polymorphonuclear leukocyte
(PMNL) function and inhibits monocyte production
of tumor necrosis factor-␣ and interleukin-6 at
site(s) of infection. LT, as its name implies, is re-
first 48 hrs of therapy
Droplet pre-cautions for

sponsible for the toxemia of anthrax. Animal studies

Isolation of Cases

suggest a “point of no return” in the course of an-

Droplet precautions

thrax, at which even complete eradication of the

pathogen does not prevent mortality. LT is a zinc
metalloprotease that cleaves members of the mito-
Standard

Standard

Standard

Standard

gen-activated protein kinase kinase family.20 This

results in inhibition of mitogen-activated signal
transduction pathways, which causes macrophage
cell death with release of their mediators. LT is also
required for activation of capsule production.
By direct contact with

The infective dose for 50% of humans (ID50) by

Person-to-Person

highly contagious
Transmission

inhalation is estimated as 8,000 to 40,0000 spores

Droplet aerosols,

(about 10⫺6 g), but the minimal infecting dose is not

skin lesions

known. Spores are deposited in alveoli but do not

Investigational new drug status in U.S.
Reported

generate much inflammatory response. Rather, they

are transported in macrophages to hilar and medi-
None

Rare

Rare

astinal lymph nodes, where they germinate, produce

toxins, and cause edema and hemorrhagic necrosis
of the mediastinum. The hemorrhagic necrosis fre-
Likely Attack

Disseminated
pneumonic
Inhalational,
cutaneous

For nonpregnant adults.

quently extends to the pleurae, resulting in bloody

Pneumonic

Pneumonic

Pneumonic
Typhoidal,
Form

pleural effusions. The hemorrhagic necrosis may

also extend to the trachea and neck, resulting in
neck edema, dry cough, and stridor.
The 3 major clinical forms of anthrax are cutane-
Brucellosis

ous (⬎95% of all cases worldwide), gastrointestinal

Tularemia
Disease

Glanders
Anthrax

(GI), and inhalational (respiratory) anthrax. Each

Q fever
Plague

may result in septicemic anthrax with massive bac-

teremia, disseminated infection, toxemia, and
a

302 June 2002 Volume 323 Number 6

 Greenfield et al

death. Hematogenous dissemination of B anthracis Table 5. Initial Findings in Patients with Inhalational Anthrax
during the 2001 U.S. Epidemic25
frequently results in lesions of the gastrointestinal
(GI) tract submucosa, causing upper and lower GI Symptoms
bleeding. Hemorrhagic, purulent meningitis also
frequently occurs. Fever, chills 10/10
Cutaneous anthrax occurs on exposed skin, most Fatigue, malaise, lethargy 10/10
frequently the arms and hands, but also the head Cough (minimal or nonproductive) 9/10
and neck. Infection begins as a small red macule Nausea or vomiting 9/10
Dyspnea 8/10
that progresses to a papule, resembling an insect Sweats, often drenching 7/10
bite, that appears 3 to 5 days (range, 12 hours to 12 Chest discomfort or pleuritic pain 7/10
days) after transdermal exposure to spores.21 Over Myalgias 6/10
the ensuing 1 to 2 days, the papule vesiculates, often Headache 5/10
with local hemorrhage, and then develops into a Confusion 4/10
Abdominal pain 3/10
painless ulcer that is frequently surrounded by ves- Sore throat 2/10
icles. In the next 1 to 2 days, the ulcer enlarges to 1 Rhinorrhea 1/10
to 3 cm and develops the characteristic black eschar Initial physical findings
with surrounding edema. Cutaneous anthrax may Fever (⬎37.8°C) 7/10
Tachycardia (heart rate ⬎100/min) 8/10
be accompanied by regional lymphadenitis and mod- Hypotension (systolic BP ⬍110 mm Hg) 1/10
est systemic symptoms (fever, malaise, and head- Initial laboratory results
ache). The ulcer ultimately heals, usually with min- WBC, median 9.8 ⫻ 103/mm3
imal scarring. Although antibiotic therapy reduces Neutrophilia (⬎70%) 7/10
Elevated transaminases (AST or ALT 9/10
edema and systemic symptoms and the risk of dis- ⬎40)
semination, it does not have substantial impact on Hypoxemiaa 6/10
the evolution of the skin lesion. Microangiopathic Metabolic acidosis 2/10
hemolytic anemia and coagulopathy associated with Elevated serum creatinine (⬎1.5 mg/dL) 1/10
cutaneous anthrax was recently described.14 Other Initial chest radiograph findings
Mediastinal widening 7/10
rare complications include “malignant edema,” mul- Infiltrates/consolidation 7/10
tiple bullae, stridor from the edema of neck or tho- Pleural effusion(s) 8/10
racic anthrax, and septicemia. Images of anthrax Any abnormality 10/10
can be viewed by searching at http://phil.cdc.gov/ Initial chest computed tomography findings
Mediastinal lymphadenopathy/widening 7/8
phil/default.asp. Pleural effusion(s) 8/8
GI anthrax is distinctly rare and has never been Infiltrates/consolidation 5/8
encountered in the United States. It is acquired by
consumption of inadequately cooked contaminated a
Alveolar-arterial oxygen gradient ⬎30 mm Hg or room air O2
meat. Oropharyngeal anthrax involves a lesion in saturation ⬍94%.
the oral cavity, usually the posterior wall, hard WBC, white blood cell; AST, aspartate aminotransferase; ALT,
alanine aminotransferase.
palate, or tonsils, that resembles the cutaneous le-
sion. It is complicated by cervical edema and local
lymphadenitis resulting in dysphagia and stridor.
The primary lesions of abdominal anthrax occur Patients develop abdominal pain, hematemesis and
most frequently in the cecum and adjacent areas but melena, and suppurative and hemorrhagic meningi-
may occur anywhere along the GI tract. Initially, tis. Delirium and coma occur frequently. If un-
patients have nonspecific symptoms of nausea, vom- treated, cardiovascular collapse and death ensue.
iting, anorexia, and fever. These are followed by The differential diagnosis of cutaneous anthrax
abdominal pain, hematemesis, and bloody diarrhea. includes brown recluse spider bite, ecthyma, ulcer-
Subsequently, massive infected ascites develops. oglandular tularemia, accidental vaccinia, and ne-
Septicemia and death ensue in 2 to 5 days from the crotic herpes simplex.15 Gram stain and culture of
onset of illness. vesicular fluid or the ulcer base are recommended
Inhalational anthrax is typically a biphasic ill- for diagnosis, however, antimicrobial therapy
ness. In the recent United States epidemic, the onset quickly renders the lesion negative. Serologic test-
of illness was 4 to 6 days after known exposure. The ing and punch biopsy at the edge of the lesion,
initial illness lasts 1 to 3 days and is nonspecific, examined by silver staining and immunohistochem-
with malaise, low-grade fever, GI complaints, and ical testing, are useful in diagnosing cutaneous an-
nonproductive cough (Table 5).22–25 Then, some- thrax in patients who have received antibiotic
times after a brief clinical improvement, a second therapy.
phase of illness begins, with dyspnea at rest, stridor, The differential diagnosis of inhalational anthrax
dry cough, tachypnea, high fever, chills, and pro- is broad and includes influenza and a myriad of viral
found diaphoresis. Initial laboratory data, and ra- influenza-like illnesses. Unlike patients with inha-
diographic findings are also shown in Table 5.22–25 lational anthrax, adults with influenza or other viral

THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES 303

Bacterial Agents and Bioterrorism
respiratory illnesses do not usually have early dys-
pnea or vomiting and are more likely to have sore
throat and/or rhinorrhea.26
The diagnosis of respiratory anthrax can be estab-
lished by several means.15 Although sputum cul-
tures are generally not diagnostic, blood cultures
have been positive in all patients in whom they were
obtained before initiation of antimicrobial therapy.
Immunohistochemical examination of pleural fluid
or transbronchial biopsy specimens, using antibod-
ies to capsular and cell wall antigens of B anthracis,
is useful. B anthracis-specific nucleic acid amplifica-
tion and detection by polymerase chain reaction of
sterile fluids (blood, pleural fluid) is also useful. An
enzyme-linked immunosorbent assay (ELISA) to de-
tect IgG response to PA is highly sensitive (detects
98.6% of true positives) after about 10 days of ill-
ness, but it is only 80% specific. Specificity is im-
proved by a confirmatory competitive-inhibition as-
say. Nasal swab cultures remain of uncertain
diagnostic value.
B anthracis isolates (including all from the 2001
United States epidemic) are sensitive to the fluoro-
quinolones, rifampin, tetracycline, vancomycin, imi-
penem and meropenem, chloramphenicol, clindamy-
cin, and the aminoglycosides. Penicillins have
historically been used for treatment and prophylaxis
of anthrax. However, modern techniques have dem-
onstrated a penicillinase gene and low-level expres-
sion of its product.15 This has led to concern about
the potential of penicillin therapy to induce high-
level penicillinase production, particularly when the
infecting burden is high. Concern has also been
raised about the known poor penetration of penicil-
lins into macrophages, where germination of spores
occurs. Thus, penicillin and its congeners cannot be
recommended as monotherapy for any form of an-
thrax. There is also concern based on reports that
suggest that the former Soviet Union program had
engineered penicillin- and tetracycline-resistant B
anthracis.
Ciprofloxacin is the preferred fluoroquinolone for
anthrax because it is the only fluoroquinolone
proven effective in animal models, which led to US
Food and Drug Administration approval for this
indication. Theoretically, however, all modern fluo-
roquinolones should be equally efficacious. In
addition, clindamycin may be useful because of
rapid inhibition of toxin production, as has been
demonstrated in staphylococcal and streptococcal
toxic shock syndrome and Clostridium species
infections.15
Ciprofloxacin [500 mg for adults (10 –15 mg/kg for
children ⱕ8 years or ⱕ45 kg) by mouth every 12
hours] and doxycycline [100 mg for adults (2.2 mg/kg
for children ⱕ8 years or ⱕ45 kg) by mouth every 12
hours] are the preferred agents for treatment of
cutaneous anthrax. If susceptibility of an epidemic
strain is not known, ciprofloxacin is preferred. Nat-
304
urally occurring disease can be treated for 7 to 10
days. Bioterrorism-related disease should be treated
for at least 60 days because of potential concurrent
respiratory exposure.
For inhalational anthrax, ciprofloxacin (400 mg,
intravenously, every 12 hours for adults) is pre-
ferred treatment. Most would add additional agents.
Penicillin may be useful because of known penetra-
tion into cerebrospinal fluid. Clindamycin might be
added for reasons detailed above. Details for treat-
ing special populations are reported elsewhere.7
Aggressive supportive care is critical in manage-
ment of patients with respiratory anthrax. Chest-
tube drainage of pleural effusions seems benefi-
cial.23 Glucocorticosteroids have been advocated for
treatment of anthrax meningitis or substantial me-
diastinal or neck edema.16 Other antitoxin agents
investigated in vitro include angiotensin-converting
enzyme inhibitors, calcium channel blockers, and
tumor necrosis factor inhibitors.15 Specific anthrax
IgG antiserum (collected from vaccinees) or the ad-
ministration of vaccine may prove useful adjuncts.15
In the preantibiotic era (1933–1938), mortality
from cutaneous anthrax was 16%, but death after
appropriate antibiotic therapy is rare (⬍1%). GI
anthrax is uniformly fatal. In the preantibiotic era,
inhalational anthrax was universally fatal. In the
middle 20th century, mortality was reduced to 86%.
In the 2001 US epidemic, mortality from inhala-
tional anthrax was 5 of 11 (45%). Treatment before
the onset of shock resulted in patient survival.
A vaccine (Anthrax Vaccine Adsorbed) is currently
licensed in the United States but is available only to
military personnel.27 It is an aluminum hydroxide-
precipitated sterile filtrate of cultures of an aviru-
lent unencapsulated strain (Sterne strain) that elab-
orates PA. Anthrax Vaccine Adsorbed protects
rabbits and nonhuman primates against a lethal
aerosol challenge with all strains tested.28 The vac-
cine schedule is 0, 2, and 4 weeks and 6, 12, and 18
months, followed by yearly boosters. Reactogenicity
is mild to moderate.29
Postexposure prophylaxis (PEP) is recommended
for persons with a known exposure and those with a
credible, epidemiologically assessed risk of potential
exposure. Until antimicrobial susceptibility of the
specific or epidemic organism is known, ciprofloxa-
cin (as dosed for treatment) is recommended. If
susceptibility is established, doxycycline (as dosed
for treatment) and amoxicillin (500 mg by mouth
every 8 hours for adults) are probably equally as
effective as ciprofloxacin. More than 32,000 persons
received some prophylaxis with ciprofloxacin and
⬎5,000 were advised to complete at least a 60-day
course related to the 2001 United States attack.9 No
one who received PEP developed disease. Prelimi-
nary surveillance indicated that approximately 20%
reported some adverse event potentially attribut-
able to ciprofloxacin.9 Recent recommendations,
June 2002 Volume 323 Number 6

based on theoretical concerns, have added optionally
a total of 100 days of chemotherapy with or without
anthrax vaccine (dosed at days 60, 74, and 88) as
investigational PEP.15
Plague
Yersinia pestis, the etiologic agent of plague, is a
Gram-negative, nonmotile, facultatively anaerobic,
non–spore-forming coccobacillus. It is a member of
the Enterobacteriaceae family. It exhibits bipolar
staining with common laboratory stains (Wright,
Giemsa, and Wayson) producing an appearance in
clinical specimens described as a “safety-pin.” The Y
pestis genome, composed of the chromosome and 3
plasmids, has been sequenced.30 The genome has
evidence of frequent intragenomic recombination
and contains many genes that seem to have been
acquired from other bacteria and viruses (including
adhesins, secretion systems, and insecticidal tox-
ins). There is evidence that the genome has under-
gone large-scale genetic flux.
Like anthrax, plague is a zoonosis. The plague life
cycle is a complex interaction between rodents and
fleas, with human infection occurring occasionally.
The most important enzootic and epizootic reser-
voirs of plague are rodents, including rats, mice,
gerbils, voles, ground squirrels, chipmunks, prairie
dogs, marmots, and tarabagans.31 Cats may become
infected by ingestion of infected rodents. The plague
bacillus is transmitted among rodents and from ro-
dents to humans by fleas. When a flea ingests a
blood meal from a bacteremic animal, ingested bac-
teria multiply in the insect mid-gut. Replicating
bacteria cause obstruction of the proventriculus,
which stimulates more feeding activity. When the
infected flea attempts to ingest a blood meal, bacte-
ria are regurgitated into the host.
Human infection can also be acquired by contact
with fluids from infected animals (eg, during field
dressing of hunted animals). Additionally, transmis-
sion by respiratory droplets from index cases with
plague pneumonia clearly occurs between humans,
as well as between animals (usually cats) and
humans.32
Plague occurs worldwide, primarily in developing
countries of Africa (76.2%) and Asia.33 In the United
States, approximately 10 cases of human plague are
reported annually, primarily from the southwestern
states.34
Plague fulfills criteria for a high-risk potential
weapon of bioterrorism: humans are easily infected
via the respiratory tract, aerosolized bacteria are
easily propagated person-to-person, pneumonic
plague has a high attack rate and produces severe
clinical disease, and plague has a high psychological
impact factor. Several of these attributes were re-
cently modeled on a large scale during an exercise
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
Greenfield et al
directed by the Department of Justice entitled
“TOPOFF.”35
When Y pestis bacilli are inoculated into a host,
they are phagocytosed and killed by PMNL but are
able to escape intracellular killing by macro-
phages.31 There is rapid temperature-dependent
up-regulation of plasmid genes encoding virulence
factors such as iron scavenging proteins, an antiph-
agocytic capsule, and a variety of proteins that in-
terfere with the normal functioning of mammalian
phagocytes.36 Bacteria migrate from the site of inoc-
ulation to regional lymph nodes, where they repli-
cate and produce an inflammatory, necrotizing ade-
nitis responsible for the characteristic buboes of
bubonic plague (from the Greek bubon, meaning
“groin”).31 Lastly, bacteria gain access to the blood-
stream, achieving densities as high as 107 organ-
isms/mL, establish secondary buboes, and dissemi-
nate to other organs, including lungs, spleen, and
the central nervous system.37 Hypotension, oliguria,
altered mental status, and disseminated intravascu-
lar coagulation (DIC) may develop and are attribut-
able to Gram-negative endotoxin. Plague images
can be viewed at http://www.cdc.gov/ncidod/dvbid/
plague/pics.htm.
The 3 major forms of human Y pestis infection in
humans are “classic” bubonic plague, primary septi-
cemic plague, and pneumonic plague, accounting for
84, 13, and 2% of cases reported in the United States
from 1947 to 1996, respectively.38 Less common
manifestations include meningitis, pharyngitis, and
GI symptoms.38
Bubonic plague is an acute febrile lymphadenitis.
The hallmark is the bubo, a swollen, warm, ery-
thematous, and exquisitely tender lymph node or
group of lymph nodes in the groin, axilla, or cervical
region that develops 2 to 8 days after transdermal
exposure. Abrupt onset of fever, chills, headache,
and malaise may have onset up to 24 hours before,
or have onset simultaneously with the appearance of
the bubo.39 Nausea, vomiting, diarrhea, and abdom-
inal pain are often present.39,40 Patients are typi-
cally prostrate and lethargic with episodic restless-
ness and agitation. They are febrile, tachycardic,
frequently hypotensive, and often have tender
hepatosplenomegaly.
Secondary bacteremia/septicemia frequently de-
velops in bubonic plague and is associated with
metastatic infection, DIC, purpuric skin lesions (due
to vasculitis and occlusion by fibrin thrombi), and
acral gangrene (the likely basis of the epithet “The
Black Death”). Primary septicemic plague is distin-
guished from secondary septicemia by the absence of
buboes.
Secondary pneumonic plague results from bacte-
remia, whereas primary pneumonic plague is estab-
lished by inhalation of respiratory droplets contain-
ing bacilli. Symptoms of secondary pneumonic
plague include shortness of breath, cough, chest
305
Bacterial Agents and Bioterrorism
pain, and bloody sputum in addition to those attrib-
uted to the bubo. Primary pneumonic plague mani-
fests as the abrupt onset of fever and flu-like symp-
toms 2 to 4 days after exposure and rapidly
progresses to fulminating pneumonia and respira-
tory failure.38 Both forms of pneumonic plague are
readily transmissible by airborne droplets.
The principal differential diagnosis of bubonic
plague is streptococcal lymphadenitis with bactere-
mia. The differential diagnosis of pulmonic plague
includes influenza, inhalation anthrax, and over-
whelming community-acquired pneumonia. Septice-
mic plague must be differentiated from septicemia
from other causes, in particular that caused by
Neisseria meningitides or Haemophilus influenzae
with DIC, and from thrombotic thrombocytopenic
purpura.
Plague is diagnosed by demonstrating Y pestis in
blood or body fluids such as a lymph node aspirate,
sputum, or cerebrospinal fluid. If plague is sus-
pected clinically, duplicate cultures should be made
for incubation at 25° C, which encourages rapid
growth, and 37°C for identification of the capsular
F1 antigen.31,39 A rapid diagnosis of bubonic plague
can be made tentatively by Gram stain of fluid
aspirated from the bubo, sputum, blood, or cerebro-
spinal fluid showing compatible Gram-negative coc-
cobacilli or by fluorescent antibody testing. Serologic
studies are useful for retrospective diagnosis or ep-
idemiological studies.
Streptomycin (15 mg/kg, up to 1 g injected intra-
muscularly every 12 hours) has been considered the
drug of choice for severe infection; however, genta-
micin (5 mg/kg/day intravenously/intramuscularly
as a single dose or in divided doses) has similar
efficacy and wider availability in the United States.
Alternative drugs that can be administered paren-
terally or orally depending upon the clinical situa-
tion include doxycycline (dosed as for anthrax), cip-
rofloxacin (dosed as for anthrax), or chloramphenicol
(25 mg/kg 4 times a day). Treatment should be
continued for a total of 10 days.31,38,39 Most naturally
occurring isolates of Y pestis are susceptible to these
antibiotics; however, a multidrug resistant organ-
ism was identified in a case of human bubonic
plague in Madagascar.41 Therefore, treatment
should be guided by susceptibility testing of the
specific or epidemic organism.
Untreated bubonic plague has a case fatality rate
of 40 to 60%, whereas the pneumonic and septicemic
forms are typically fatal. Treatment of plague has
reduced the case fatality rates of bubonic plague to
less than 10%, but case fatality rates of 60% are still
common with septicemic and pneumonic plague.31,40
A vaccine consisting of formaldehyde-killed bacilli
had been licensed in the United States and was
reserved for military and laboratory personnel and
others with a high occupational risk of exposure.
Although effective for preventing or attenuating bu-
306
bonic plague, the vaccine was not effective in pneu-
monic plague, and production was discontinued in
1999. An F1-V (fusion protein) vaccine is in devel-
opment; it protected mice for a year against an
inhalation challenge and is now being tested in
primates.42 Thus, the key to plague prevention in
humans is reducing human exposure to rodents and
fleas.
Hospitalized patients that pose a risk for respira-
tory spread should remain in strict isolation for at
least 48 hours after appropriate antibiotics are ini-
tiated. A disposable surgical mask is deemed ade-
quate for preventing transmission of plague by re-
spiratory droplet.38
PEP is recommended for persons with close con-
tact (⬍2 m) to an infectious person, or those who
have had a potential attack exposure.31,38,39 The
recommended adult PEP regimens are doxycycline
or ciprofloxacin in full therapeutic doses. Chloram-
phenicol (in therapeutic dose) is an alternative. Pro-
phylaxis should be continued for 7 days after
exposure.
Tularemia
Francisella tularensis is a small, aerobic, nonmo-
tile, Gram-negative coccobacillus. F tularensis bio-
var tularensis (Jellison type A) is the most virulent
biotype and is found predominantly in North Amer-
ica. F tularensis biovar palearctica (Jellison type B)
is found in the United States and other temperate
climates and causes less severe disease. F tularensis
is fastidious and will not grow on routine solid me-
dia. Colonies grow after 2 to 4 days’ incubation at 35
to 37°C. The organism is killed by heat but can
survive for prolonged periods in cold or freezing
conditions. Almost all strains produce ␤-lactamase.
Tularemia (named for bacteremia in Tulare
County, California) has a worldwide distribution,
although the majority of cases occur in the United
States.43 In 1994, the incidence in the United States
was 0.04 cases per 100,000 persons. Arkansas, Mis-
souri, and Oklahoma accounted for 53% of United
States cases reported between 1990 and 1994.
Like anthrax and plague, tularemia is a zoonosis.
Like plague, arthropod vectors transmit the disease
among natural reservoirs, including rabbits, deer,
squirrels, mice, and beaver. Human infection is in-
cidental and occurs most often by insect bite or
contact with contaminated animal products. Arthro-
pod-borne disease (primarily from tick and deer fly
bites) accounts for most summertime cases. A sec-
ond smaller incidence peak is seen in December and
results from hunting-associated acquisition. Occu-
pational exposure occurs in farmers, hunters, trap-
pers, veterinarians, and laboratorians.
F tularensis is a category A agent because of its
ease of dissemination, high infectivity, and capacity
to cause serious illness and death. In 1969, a WHO
June 2002 Volume 323 Number 6

committee projected that 50 kg of F tularensis dis-
persed among a population of 5 million persons
would result in 19,000 deaths and 250,000 injuries,
with an economic impact of $5.4 billion per 100,000
persons exposed.44 Pneumonic or typhoidal tulare-
mia would be the most likely presentations after
intentional exposure.
Infection by F tularensis can occur through skin,
lungs, GI tract, or mucous membranes. Person-to-
person transmission does not occur. The infectious
dose is dependent on the portal of entry: as few as 10
organisms are needed to cause disease by inhalation
or intradermal injection, whereas 108 organisms are
required for enteral infection.
A papule forms at the site of cutaneous inocula-
tion 3 to 5 days after exposure. Bacteria multiply
locally, spread to regional lymph nodes, and then
disseminate via the lymphatic/hematogenous route.
Bacteremia may be present during this early phase.
Histologic examination shows suppurative necrosis
with or without caseating or noncaseating
granulomata.
Clinical manifestations range from asymptomatic
infection to a rapidly progressive, fulminant, and
fatal disease. Disease presentation is dependent on
inoculum size, portal of entry, host immune status,
and virulence of the strain. The incubation period
generally is 3 to 5 days (range, 1–21 days).43 Abrupt
onset of fever, chills, headache, malaise, and an-
orexia is characteristic. A nonspecific secondary
rash (diffuse maculopapular or vesiculopapular
rash, erythema nodosum, erythema multiforme, ac-
neiform lesions, and urticaria) develops in up to 35%
of cases. The clinical course of untreated nonfulmi-
nant disease has been described as having 3 phases:
31 days of fever, 31 days of bed rest, and 31 days of
disability.45
Six forms of disease have been described, often
with significant overlap: ulceroglandular (21–78% of
United States cases), glandular (3–20%), oculoglan-
dular (⬍5%), oropharyngeal (⬍12%), pneumonic (7–
20%), and typhoidal (5–30%).43 In ulceroglandular
tularemia, a painful, indurated, erythematous pap-
ule, typically on the fingers or hand, appears 3 to 5
days after exposure. Two days later, the papule
becomes necrotic and ulcerates, resulting in a tender
punched-out lesion with raised borders. Multiple
lesions can occur. The ulcer is accompanied by local-
ized, painful lymphadenopathy. Untreated lesions
may persist for weeks to months. An image of ulcer-
oglandular tularemia can be viewed by searching at
http://phil.cdc.gov/phil/default.asp. In glandular tu-
laremia, there is painful regional lymphadenopathy,
but skin lesions are absent or minor and overlooked.
Oculoglandular tularemia results from conjuncti-
val exposure to F tularensis by contact with contam-
inated fingers, aerosol, or splash. The disease is
unilateral in 88% of cases. Photophobia, excessive
lacrimation, lid edema, conjunctivitis, and preauric-
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
Greenfield et al
ular, submaxillary, and cervical lymphadenopathy
are common. Complications include corneal ulcer-
ation and dacryocystitis.
Oropharyngeal tularemia results from oral expo-
sure from contaminated foods, fluids, or droplets. It
is more common in children, and multiple family
members may be simultaneously ill. Severe sore
throat pain, exudative or membranous pharyngitis
or tonsillitis, and oropharyngeal ulceration are
characteristic.
In pneumonic tularemia, the initial presentation
is marked by respiratory complaints. This form re-
sults from inhalation (primary) or hematogenous
spread (secondary). Primary pneumonic tularemia
occurs most notably in laboratory workers, farmers,
and sheep shearers. Symptoms include dyspnea,
nonproductive or minimally productive cough, and
pleuritic chest pain. Hemoptysis is uncommon. High
fever, shaking chills, and profuse sweating are usu-
ally present. Chest radiographic findings can in-
clude hilar adenopathy, lobar or miliary infiltrates,
and/or pleural effusions. Pleural fluid is exudative
with a lymphocyte predominance. Pleural biopsy
may show granulomas and therefore be confused
with tuberculosis. Secondary pneumonia may com-
plicate other forms of tularemia, notably typhoidal
and ulceroglandular.
Typhoidal tularemia is acquired from any of the
aforementioned exposures but does not present with
prominent lymphadenopathy or other localizing
symptoms or signs. Many patients have serious un-
derlying medical conditions and present with acute
prostration, fulminant disease, and rapid death. The
disease may also take a protracted course. Symp-
toms may include fever, chills, sore throat, head-
ache, myalgias, nausea, vomiting, and diarrhea.
The differential diagnosis of ulceroglandular tula-
remia includes anthrax, sporotrichosis, and nontu-
berculous mycobacterial infection. The differential
diagnosis of oculoglandular tularemia includes pyo-
genic bacterial infection, syphilis, herpes zoster, ad-
enoviral infection, and cat-scratch disease. Oropha-
ryngeal tularemia must be differentiated from acute
streptococcal pharyngitis, Epstein-Barr virus mono-
nucleosis, diphtheria, and viral pharyngitis. It
should be considered in any patient from an endemic
area with exudative pharyngitis unresponsive to
penicillin therapy. The differential diagnosis of
pneumonic tularemia includes Q fever and psittaco-
sis, and atypical pneumonia due to Mycoplasma
pneumoniae, Chlamydia pneumoniae, and Legio-
nella pneumophilia. Pheumonic tularemia can
present as an atypical community-acquired pneumo-
nia that does not respond to ␤-lactam or macrolide
therapy. The differential diagnosis of typhoidal tu-
laremia includes typhoid fever, brucellosis, Legio-
nella infection, Q fever, disseminated mycobacterial
or fungal infection, rickettsioses, infective endocar-
307
Bacterial Agents and Bioterrorism
ditis, and any other cause of prolonged fever without
localizing signs.43
In all forms of tularemia, a history of exposure
and a high clinical suspicion are essential to diag-
nosis. Nonspecific laboratory findings typically in-
clude leukocytosis and elevated sedimentation rate.
Sterile pyuria occurs in 20 to 35% of cases. Throm-
bocytopenia, hyponatremia, elevated aspartate ami-
notransferase/alanine aminotransferase, and evi-
dence of rhabdomyolysis occasionally occur.
The organism may be cultured from blood, lymph
nodes, sputum, and gastric aspirates. The mainstay
of diagnosis is serology: ELISA, microagglutinin,
hemoagglutinin, and tube agglutinin assays are
available. Tube agglutinins develop by day 10 to 14
in 50 to 70% of patients and peak at 4 to 8 weeks.42
Historically, the treatment of tularemia is strep-
tomycin (7.5 to 10 mg/kg injected intramuscularly
every 12 hours for adults), because it seems to result
in the lowest relapse rate. There is concern about
this treatment in the event of an attack, however,
because a fully virulent streptomycin-resistant
strain exists, and streptomycin availability is limit-
ed.42 Gentamicin (3–5 mg/kg intravenously daily for
adults) and ciprofloxacin (750 mg by mouth or 400
mg intravenously every 12 hours for adults) are
effective primary alternative regimens.42 Tetracy-
cline, doxycycline, and chloramphenicol are less de-
sirable alternatives because of higher relapse rates.
Chloramphenicol plus streptomycin has been used
to treat meningitis. Treatment should be continued
for 10 to 14 days.
The untreated mortality of tularemia ranges from
5 to 35%. Prompt diagnosis and treatment can re-
duce mortality to 1%. Recovery and debilitation can
be prolonged. Recovery from tularemia conveys life-
long immunity.
Optimal prevention is avoidance of exposure by
use of personal protective measures, early tick re-
moval, and disinfection of potentially contaminated
water. Because of its extreme infectivity, laboratory
personnel must be informed of the possibility of
tularemia, and Biosafety Level 3 (BSL3) precautions
are warranted. A live attenuated vaccine is recom-
mended for laboratorians routinely working with F
tularensis. The vaccine confers incomplete protec-
tion, but disease course is milder. The use of vaccine
or pre-emptive antibiotics is not recommended after
a potential natural exposure. Isolation of tularemia
patients or PEP for contacts of tularemia patients is
not recommended because person-to-person trans-
mission does not occur.
In the event that F tularensis is used as a biologic
weapon, exposed persons (adults and children)
should receive a 14-day course of PEP with oral
doxycycline or ciprofloxacin, at doses recommended
above for PEP for anthrax and plague.46 If the or-
ganism has been covertly dispersed and identified
only after persons become ill, those exposed should
308
begin therapy if an unexplained fever or influenza-
like illness develops within 14 days of exposure.46
Brucellosis
Brucellae are small, Gram-negative, aerobic, non-
motile, non–spore-forming, unencapsulated coccoba-
cilli. Six species with numerous biotypes exist, but
only 4 cause human disease: Brucella abortus, B
melitensis, B suis, and, rarely, B canis.47 In vitro
growth is slow, so cultures must be incubated at
least 4 weeks before they can be declared negative.
Brucellosis is a worldwide zoonosis that princi-
pally occurs in the Mediterranean basin, the Ara-
bian gulf, the Indian subcontinent, and parts of
Mexico and Central and South America. In the
United States, the annual reported incidence is ap-
proximately 100 cases per year. Brucellae mainly
infect domesticated animals (cattle by B abortus,
goats and sheep by B melitensis, swine by B suis,
and dogs by B canis), but wild rodents, caribou,
hares, bison, camels, and yaks are also infected. In
animals, brucellosis is a lifelong infection. The bac-
terium is localized in the reproductive tract (due to
presence of erythritol), leading to abortions and ste-
rility. Bacteria are present in milk, urine, and pla-
cental products of infected animals.
Transmission to humans occurs through opened
skin exposed to animal secretions, infected aerosols,
inoculation into the conjunctival sac, or ingestion of
unpasteurized dairy products, particularly goat
milk and its derivatives. Rare modes of transmission
include ingestion of blood, bone marrow, or raw
meat. The disease has long been associated with
occupational exposure in workers in the livestock
industry, particularly abattoir workers. Laboratori-
ans have acquired brucellosis by inhalation. The
attenuated animal vaccine strains (B abortus strain
19 and B melitensis strain Rev-1) can cause disease
after inadvertent human inoculation. Human-to-hu-
man transmission is unusual. Brucellosis acquired
from a contaminated food source can concurrently
affect multiple family members.
In 1954, B suis became the first agent weaponized
by the United States.42 It is a category B agent
because only 10 to 100 aerosolized organisms are
needed to cause disease, the nonspecific disease
symptoms might not raise immediate suspicion, di-
agnosis is difficult and delayed, and treatment is not
always effective. However, the long incubation pe-
riod and low mortality may limit its usefulness as an
attack agent.
The brucellae are facultative intracellular patho-
gens and are able to survive and even multiply in
PMNL and monocyte-derived host cells. The latter
probably explains the propensity to localize to the
lymphatic, hepatosplenic, and bone marrow sys-
tems. The principal virulence factor seems to be
smooth cell-wall lipopolysaccharide, which enables
June 2002 Volume 323 Number 6

resistance to intracellular killing by PMNL.47 Other
factors that contribute to intracellular survival in
PMNL include production of adenine and guanine
monophosphate, which suppress the myeloperoxide-
H2O2-halide system, and a copper-zinc superoxide
dismutase, which provides protection from reactive
oxygen intermediates.48 Intracellular survival in
macrophages is facilitated by the inhibition of pha-
gosome-lysosome fusion by soluble brucellar prod-
ucts and the production of a number of stress-in-
duced proteins.48 Although antibodies appear, the
principal mechanism of recovery is Th1 cell-medi-
ated immunity. Cytokines that contribute to acti-
vated macrophage activity against brucellae include
interferon-␥, tumor necrosis factor-␣, and
interleukin-12.31
Disease onset is acute or subacute, within 2 to 4
weeks of exposure. The manifestations are protean
and nonspecific.48 “Undulant” fever, sweats, mal-
aise, anorexia, depression, headache, and backache
predominate. Mild lymphadenopathy, hepatomeg-
aly, and splenomegaly occur. GI symptoms (anorex-
ia, abdominal pain, nausea, vomiting, and diarrhea
or constipation) occur in up to 70% of patients.42
Granulomatous or mononuclear infiltrative hepati-
tis may occur. Lumbar pain and tenderness occur in
up to 60% of cases, often caused by various osteoar-
ticular infections of the axial skeleton. The skeletal
system is frequently affected (20 – 60% of cases)49
with sacroiliitis the most common infection, followed
by infection of large weight-bearing joints.50 Cough
and pleuritic chest pain occur in up to 20% of pa-
tients. Acute pneumonia is unusual, but a variety of
pulmonary syndromes are described.51 Although de-
pression and mental inattention are common, seri-
ous central nervous system disease appears in ⬍5%
of cases. Endocarditis is rare (⬍2%) but causes the
majority of brucella-related fatalities. Orchitis oc-
curs in up to 20% of infected men. The bone marrow
is involved in up to 75% cases. Nonspecific cutane-
ous infections (5%) occur.
Chronic infection is arbitrarily defined as an in-
fection persisting over 12 months.52 This is usually
related to a persistent focus.53 “Delayed convales-
cence” is described after treatment, with nonspecific
symptoms of fatigue and ill-being but without phys-
ical or serologic evidence of persistent infection. A
postinfectious reactive spondyloarthropathy has
been described.
The history may suggest brucellosis when occupa-
tion, travel, exposure to animals, or ingestion of
unprocessed foods is elicited. Biological attack
should be suspected if a case is without contact risk.
The diagnosis is established by isolation of Brucella
species from blood, bone marrow (higher yield than
blood), or other sterile sites. Serologic studies are
often necessary for diagnosis. A serum agglutination
test is readily available: a seroconversion or a titer
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
Greenfield et al
ⱖ1:160 is diagnostic. Investigational ELISA and
polymerase chain reaction techniques are available.
Recommended therapy for most patients with bru-
cellosis is doxycycline (100 mg by mouth twice daily
for adults) plus rifampin (600 mg by mouth per day
for adults) for at least 6 weeks.42,49 This regimen
seems as effective as streptomycin (1 g injected
intramuscularly per day for 2–3 weeks) or gentami-
cin (3–5 mg/kg/day for 2–3 weeks), each combined
with doxycycline (100 mg by mouth twice daily for 6
weeks). Other potentially useful regimens include
trimethoprim/sulfamethoxazole (TMP/SMX) plus
gentamicin and a fluoroquinolone plus rifampin.42
Severe complications, such as meningitis and endo-
carditis, require more aggressive treatment but
there is not consensus. Ceftriaxone should be con-
sidered for meningitis. Medical therapy plus valve
replacement surgery may be required for treatment
of endocarditis. Corticosteroids may have a role in
neurobrucellosis. Relapses (usually within 3– 6
months of end of treatment) are not uncommon even
after prolonged treatment.
Animal vaccines have eliminated most disease
among domesticated animals in the United States,
and are critical to prevention. Endemic disease can
be diminished by avoidance of unpasteurized dairy
products. PEP is not generally recommended after
possible endemic exposure, but persons with high-
risk exposure to animal vaccine, inadvertent expo-
sure in a laboratory, or exposure in a bioterrorism
context should be given PEP. PEP should be a 3- to
6-week course of one of the regimens shown to be
effective for treatment of brucellosis.32
Q Fever
Q fever (originally named Query fever, for fever
due to an unknown infection) is caused by infection
with Coxiella burnetii, which is a pleomorphic coc-
cobacillus with a Gram-negative cell wall. It sur-
vives in (requires) phagolysosomes of the host cells,
and sporulates to resist adverse environment condi-
tions. Spores are resistant to heat and desiccation,
but C burnetii has been recovered from fresh meat in
cold storage for 1 month, from fixed paraffinized
tissues, and from tissues stored in formaldehyde for
4 to 5 months.
C burnetii is a also a worldwide zoonosis that
infects mammals, birds, fish, and arthropods (which
probably spread enzootic disease), and occasionally
humans. The most common animal reservoirs are
cattle, sheep, and goats.54
Human infection follows inhalation of aerosolized
bacteria. A single inhaled organism may produce
clinical illness. The placenta of infected animals
may be heavily contaminated (up to 109 organisms/g
in sheep) and at the time of parturition, bacteria are
released into the environment. Air samples are pos-
itive for up to 2 weeks after parturition and the
309
Bacterial Agents and Bioterrorism
organism is viable in the soil for up to 150 days.55–57
Human-to-human transmission is rare although re-
ported through blood transfusion, performing au-
topsy, and among household members. Transmis-
sion to health care workers has not been described.
Q fever is a category B agent because of its airborne
stability and high infectivity.
After human inhalation of a contaminated aero-
sol, bacteria multiply in the lungs, are released into
the bloodstream, and may cause disseminated infec-
tion. It is likely that the infecting dose, the charac-
teristics of the infecting strain, and the host immune
status are the principal determinants of incubation
time and disease manifestations.54,58
Acute Q fever is either asymptomatic, a self-lim-
ited febrile illness, an atypical pneumonia, or hepa-
titis. In some areas, 11 to 12% of persons have
antibodies to Q fever and no recall of symptomatic
illness. The self-limited febrile illness is probably its
most common form. The symptoms are nonspecific
and include headaches, fatigue, and myalgias last-
ing 2 to 14 days.
Pneumonia occurs in approximately 50% of cases
and is often an incidental radiographic finding asso-
ciated with a febrile illness.59,60 Only half of these
patients have a cough or rales, and 25% experience
pleuritic chest pain. Severe headaches are a useful
clue in diagnosis. Other symptoms include fatigue,
chills, sweats, myalgias, and nausea. Radiographic
findings are variable, but pleural-based opacities
are common.33 Small pleural effusions may be
present.
Approximately 33% of patients with Q fever de-
velop acute hepatitis, often in the absence of clini-
cally apparent pneumonia.42 Chronic manifesta-
tions of Q fever are unusual, but include
endocarditis, chronic granulomatous hepatitis,
aseptic meningitis, and encephalitis.
Q fever is difficult to distinguish from other types
of pneumonia and viral syndromes. C burnetii is
difficult to culture; therefore, serological tests are
needed for diagnosis. Complement fixation, indirect
fluorescent antibody, and ELISA assays are avail-
able and useful. Antibodies may be detectable in
serum as soon as the second week of illness and may
persist for ⬎1 year.
Most cases eventually resolve without treatment,
but suspected cases should be treated to reduce the
risk of complications. Tetracycline (500 mg by
mouth every 6 hours for adults) or doxycycline (100
mg by mouth every 12 hours for adults) given for 5 to
7 days rapidly abates fever and reduces the duration
of illness. Fluoroquinolones should be considered in
patients unable to take tetracycline or doxycycline.42
Agents used for treatment of endocarditis include
doxycycline, fluoroquinolones, TMP/SMX, rifampin,
and hydroxychloroquine, in various combinations.34
Treatment length is usually 1 to 2 years and valve
replacement surgery is frequently necessary.35
310
The overall mortality of Q fever is 1 to 2%, how-
ever the chronic form has a much higher mortality
rate, especially in immunocompromised patients.
Those with acute Q fever may develop the chronic
form 1 to 20 years after the initial infection.
Avoiding consumption of unpasteurized milk, de-
stroying aborted animal material, and controlling
ectoparasites on cattle, sheep, and goats are the
principal prevention strategies. In laboratories,
BSL3 containment practices are needed. An inves-
tigational vaccine is available for laboratory or ani-
mal workers but should be used only in persons with
a negative skin test to avoid serious reactions. Vac-
cine is available as an Investigational New Drug
from USAMRIID (Fort Detrick, MD). A Q fever
vaccine is licensed in Australia.
A 5-day course of PEP with doxycycline (dosed as
for treatment) should begin 8 to 12 days after expo-
sure. Unique among the diseases discussed herein,
PEP is not effective and may only prolong the onset
of disease if given immediately (1–7 days) after
exposure.32
Glanders and Melioidosis
The etiologic agent of glanders, Burkholderia mal-
lei, is a small, Gram-negative bacillus that primarily
infects horses, donkeys, and mules. It has a micro-
scopic “safety-pin” appearance. Like Pseudomonas
species, Burkholderia species posses an array of
exotoxins that are important in pathogenesis, in-
cluding pyocyanin, lecithinase, collagenase, lipase,
and hemolysin.
B mallei exists exclusively in equine hosts. De-
spite frequent contact with equids, humans have
seldom acquired glanders, either because of expo-
sure to only low concentrations of organisms from ill
animals or because strains virulent for equids are
often less virulent for humans.42 Glanders can be
transmitted to humans by invading the nasal, oral,
and conjunctival mucosa, by inhalation, and
through open skin. Naturally occurring human in-
fection has occurred largely in veterinarians, abat-
toir workers, equine caretakers, and laboratorians.
Cases of human-to-human transmission have been
reported, possibly through sexual contact, and in
family members providing care. A single case of
glanders (a laboratorian) has been reported in the
United States since 1938.61 Naturally-occurring dis-
ease has not been encountered in the United States
in over 61 years.62 Sporadic cases continue to occur
in Asia, Africa, the Middle East, and South America.
Aerosolized B mallei has a high attack rate and
causes severe disease with high mortality, leading to
classification as a category B agent. In hamsters, 1
to 10 inhaled organisms are lethal. A case of glan-
ders in the United States should suggest biological
attack.
There are acute, subacute, and chronic forms of
June 2002 Volume 323 Number 6

glanders. Acute disease manifests differently de-
pending on the route of inoculation. Acute localized
suppurative infection occurs at the site of transder-
mal inoculation. Within 1 to 5 days, a nodule forms,
with associated regional lymphangitis. Infection of
the eyes, nose, or respiratory tract causes mucopu-
rulent drainage and later extensive, destructive
granulomatous lesions that may ulcerate and drain.
Acute aerosolized infection presents as pneumonia,
pulmonary abscesses, and/or pleural effusion.
Symptoms include severe cough and pleuritic pain.
The septicemic form of glanders (either primary or
secondary) presents as fever, rigors, sweats, myal-
gias, pleuritic chest pain, diarrhea, photophobia,
and excessive lacrimation. Cervical adenopathy,
mild splenomegaly, and erysipeloid lesions on the
face and limbs followed by a generalized pustular
eruption are frequent. Blood cultures are usually
negative until late in the disease. Untreated, this
form is usually fatal in 7 to 10 days.
Subacute suppurative disease can affect the
lungs, liver, spleen, or subcutaneous tissues. Suppu-
ration is associated with high swinging fevers and
consumption.
The chronic form of glanders is characterized by
cutaneous and intramuscular abscesses on the limbs
associated with regional lymphadenopathy.63 He-
patic and splenic abscesses can occur. Nasal dis-
charge and ulceration are present in 50% of cases.
Rare complications include osteomyelitis, brain ab-
scess, and meningitis. Chronic disease can become
acute at any time.
Gram stain and culture of infected tissue(s) is
diagnostic. Serology may be helpful if cultures are
unrevealing. Agglutination tests may be positive
after 7 to 10 days but are difficult to interpret.64
Complement fixation tests are most specific and
titers ⱖ 1:20 are positive.42
Antibiotics effective in experimental glanders in
hamsters include doxycycline, rifampin, TMP/SMX,
and ciprofloxacin. Sulfadiazine (100 mg/kg/day in
divided doses for 3 weeks) was effective in experi-
mental animals and human subjects.65 Therapy
should be guided by antimicrobial susceptibility
testing of the specific or epidemic organism.
Severe glanders requires intensive and eradica-
tion treatment phases. The intensive phase is 14
days of therapy with ceftazidime (40 mg/kg intrave-
nously every 8 hours) combined with TMP/SMX
(TMP 2 mg/kg PO every 6 hours).42 This phase
should continue until there is a symptomatic re-
sponse. The eradication phase consists of oral anti-
biotics (doxycycline or TMP/SMX) for 3 to 6 months.
If tolerated and compliance is assured, a combina-
tion of chloramphenicol, TMP/SMX, and doxycycline
might be more effective.66
In the septicemic form of glanders, untreated mor-
tality is 95% and remains ⬎50% with treatment.
Mortality is 20% even in treated localized disease.
THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES
Greenfield et al
The main complication of the chronic form is disfig-
uring skin, subcutaneous, and mucous membrane
lesions.
There is no vaccine for glanders. Standard precau-
tions should be observed to prevent person-to-person
transmission. Laboratory work requires BSL3 con-
tainment practices. PEP may be tried with TMP/
SMX.42 Animals infected with glanders should be
euthanized.
B pseudomallei, the causative agent of melioid-
osis, is not categorized as a critical agent, but
sources also consider it a potential agent of biowar-
fare or bioterrorism.42 Melioidosis is endemic in
tropical and subtropical areas of southeast Asia and
northern Australia. B pseudomallei is present in
wetlands and bodies of water, and humans acquire
infection from soil or water either transdermally or
by inhalation. Clinical melioidosis occurs in acute
and chronic forms that are similar to glanders. In
endemic areas, the pulmonary form of the disease
accounts for 50% of cases.67 Diagnosis is made by
direct fluorescent antibody testing and complement
fixation serology. Localized melioidosis is treated
with systemic antibiotics (amoxicillin/clavulanate,
tetracycline, or TMP/SMX) for 60 to 150 days and
the prognosis is good. The overall mortality for pa-
tients hospitalized with melioidosis is 50%.68 There
is no vaccine.
Other Agents
Multidrug-resistant Mycobacterium tuberculosis
(MDR-TB) is a category C agent. Use of such organ-
isms for biological attack would seem to be of dimin-
ished effectiveness because of the prolonged incuba-
tion period and the subacute nature of the resulting
disease, but the fear factor would be high. In immu-
nocompetent hosts, MDR-TB has had an attribut-
able mortality as high as 22% and an overall suc-
cessful treatment response rate as low as 56%,69 but
results may not be so dismal in patients not previ-
ously treated.70,71 In immunocompromised hosts,
however, disease is more aggressive. For example,
case fatality rates from MDR-TB as high as 80%
with median survival reached at 2 months after
exposure have been reported in persons living with
HIV/AIDS.72 Small and Fujiwara have recently re-
viewed treatment of tuberculosis.73
Food- or waterborne pathogens are also classified
as category B agents (Table 1). These agents and the
diseases they cause will be discussed here only
briefly. The threat with any of these agents is acute
debilitating diarrheal disease. With appropriate
fluid and electrolyte therapy and antimicrobial ther-
apy as indicated, mortality would be lower than that
of many of the entities previously discussed. Pa-
tients with compromised cardiovascular status or
immunosuppression could have higher initial mor-
311
Bacterial Agents and Bioterrorism
tality, and may experience recurrent or protracted
disease.
Salmonella typhi and S paratyphi, the causative
agents of typhoid fever and paratyphoid fever, re-
spectively, are exclusively human pathogens; thus,
natural transmission requires human fecal-oral
transmission. Typhoid and paratyphoid fever are
severe systemic illnesses characterized by fever and
abdominal pain. Supportive therapy and antimicro-
bial therapy selected on the basis of in vitro suscep-
tibility testing of the specific or epidemic strain are
the recommended treatment. In the preantibiotic
era, mortality of typhoid fever was approximately
15%. In the United States, current typhoid mortality
is 1%.74
Nontyphoidal salmonellae are most often associ-
ated with contaminated food products and are the
most common cause of food-borne disease outbreaks.
Foods of animal origin, including meat, poultry,
eggs, or dairy products, or others contaminated with
these foods, can be contaminated with Salmonella
species.74 A terrorist attack with S typhimurium in
the United States has been described.75 Infection
with nontyphoidal salmonellae usually produces a
self-limited acute gastroenteritis, indistinguishable
clinically from that caused by many other agents.
Nausea, vomiting, and diarrhea occur within 6 to 48
hours after the ingestion of contaminated food or
water. Fever, abdominal cramping, nausea, vomit-
ing, and chills are frequent. Headache, myalgias,
and other systemic symptoms also occur. The diar-
rhea typically lasts 3 to 7 days. Occasionally, pa-
tients require hospitalization because of dehydra-
tion. Antimicrobial therapy in addition to fluid and
electrolyte management is indicated for those with
disease requiring hospitalization. In the United
States, overall mortality is 0.4%.74
S dysenteriae type 1 (Sd1, the Shiga bacillus) is
the principal cause of epidemic dysentery. Bacillary
dysentery is typically transmitted person-to-person,
but food- and waterborne epidemics are described.
Until the past century, more soldiers were killed by
epidemic dysentery than combat-related injuries.
Intentional food contamination resulting in a small
outbreak of shigellosis in the United States has been
described.76 Transmission requires low numbers of
organisms (ie, hundreds to thousands).77 After an
incubation period of 1 to 3 days, illness begins with
fever and abdominal cramps, followed by watery
diarrhea, then smaller volume bloody mucoid stools
with urgency and tenesmus (dysentery). The illness
is usually self-limited, with an average duration of 7
days, but therapy with antibiotics to which the in-
fecting organism is susceptible limits the course of
illness and probably reduces the occurrence of at
least some complications.77 Complications include
febrile seizures in children, toxic megacolon, and
hemolytic uremic syndrome (HUS). Death is un-
312
usual except in severely malnourished children and
the elderly.
Escherichia coli O157:H7, or enterohemorrhagic E
coli (EHEC) is the cause of hemorrhagic colitis. The
epidemiology has been reviewed.78 The major source
of EHEC is ground beef; other sources include con-
sumption of unpasteurized milk and juice, sprouts,
lettuce, and salami, and contact with cattle. Water-
borne transmission occurs through swimming in
contaminated lakes or pools, or drinking unchlori-
nated water. The organism is easily transmitted
person-to-person. An estimated 73,000 cases occur
annually in the United States. The disease is un-
commonly reported in patients in less developed
countries. Unlike dysentery, there is no enteroinva-
sion. Typically the patient is afebrile and passes few
bloody stools. The disease seems to be mediated
primarily by Shiga toxin(s), apparently acquired by
E coli from Sd1. It is most notable for its principal
complication of HUS, which is more common in
children ⬍5 years old and in the elderly. The mor-
tality from HUS is 3 to 5%.78
Vibrio cholerae is the causative agent of cholera,
which is a feared epidemic disease. The natural
reservoir for V cholerae is aquatic environments. It
occurs in epidemic and endemic patterns with the
modes of transmission being person-to-person fecal-
oral spread, and ingestion of contaminated water
and food, particularly shellfish and fresh vegeta-
bles.79 V cholerae can survive on a variety of food-
stuffs for up to 5 days at ambient temperature and
up to 10 days at refrigerator temperatures.80 There
were 137,071 cases and 4,908 deaths attributed to
cholera worldwide in 2000.81 After adhesion to GI
mucosa, elaboration of cholera toxin is responsible
for the secretory diarrhea.79 Most persons infected
with cholera do not become ill. When illness does
occur, ⬎90% of episodes are mild to moderate, acute,
self-limited, and produce watery diarrhea. Less than
10% of cases develop severe cholera with profuse
watery diarrhea and vomiting, which may lead to
circulatory collapse, shock, and death.80 The key to
treatment is fluid and electrolyte therapy. In severe
cases, effective antibiotic therapy (guided by in vitro
susceptibility testing) reduces the volume and dura-
tion of diarrhea. Without adequate treatment, the
mortality of severe cholera is 25 to 50%; with ther-
apy, mortality can be reduced to ⬍1%.82 Candidate
vaccines are in development.
Cryptosporidium parvum is an intracellular pro-
tozoan, not a bacterial pathogen, but included here
to complete the critical agent list (Table 1). C par-
vum is an emerging pathogen, now considered one of
the most common enteric pathogens in human and
domesticated animals worldwide.83 Human infec-
tion occurs after human-to-human, animal-to-hu-
man, and environmental, particularly waterborne,
transmission. Cryptosporidial oocysts are remark-
ably resistant to adverse environmental conditions
June 2002 Volume 323 Number 6

and many disinfectants and may persist in the en-
vironment for at least many months. Perhaps the
best demonstration of potential terrorist impact of
intentional C parvum contamination of the water
display is demonstrated by the United States epi-
demic of cryptosporidiosis acquired from inadver-
tent contamination of the water supply of Milwau-
kee in 1993.84 An estimated 403,000 persons became
infected in this outbreak. The clinical manifesta-
tions included watery diarrhea (93%), abdominal
cramps (84%), fever (57%), and vomiting (48%). The
median duration of illness was 9 days. Fluid and
electrolyte therapy is the mainstay of treatment.
There is no effective antimicrobial therapy.
Conclusion
Bacterial pathogens have been expected to be the
most likely agents of a modern era biological attack,
and this prediction has recently proven to be correct.
Clinicians should be familiar with the features of the
diverse diseases due to these pathogens to enable
early recognition and reporting and appropriate
management. Because, as the recent US epidemic
has proven, the manifestations of intentional release
are not fully predictable, this review has focused on
all clinical manifestation of infection with these crit-
ical pathogens. Unfortunately, potentially useful
pathogens may extend beyond this list of critical
pathogens, but it is imperative to review disease
manifestations caused by those pathogens that have
been so identified.
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