BW-ch04.pdf

Anthrax

Chapter 4

ANTHRAX

BRET K. PURCELL, P h D, MD * ; PATRICIA L. WORSHAM, P h D † ; and ARTHUR M. FRIEDLANDER, MD ‡

INTRODUCTION AND HISTORY

THE ORGANISM

EPIDEMIOLOGY

PATHOGENESIS

CLINICAL DISEASE

Cutaneous Anthrax

Inhalational Anthrax

Oropharyngeal and Gastrointestinal Anthrax

Meningitis

DIAGNOSIS

TREATMENT

PROPHYLAXIS

Prophylactic Treatment After Exposure

Active Immunization

Side Effects

SUMMARY

* LieutenantColonel,MedicalCorps,USArmy;Chief,BacterialTherapeutics,DivisionofBacteriology,USArmyMedicalResearchInstituteofInfectious

Diseases,1425PorterStreet,FortDetrick,Maryland21702

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

21702

‡ Colonel,MedicalCorps,USArmy(Ret);SeniorScientist,DivisionofBacteriology,USArmyMedicalResearchInstituteofInfectiousDiseases,1425

PorterStreet,FortDetrick,Maryland21702;andAdjunctProfessorofMedicine,UniformedServicesUniversityoftheHealthSciences,4301Jones

BridgeRoad,Bethesda,Maryland20814

MedicalAspectsofBiologicalWarfare

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. 1-4 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. 5,6 Anthrax has a long association with

human history. The fifth and sixth plagues described

in Exodus may have been anthrax in domesticated

animals followed by cutaneous anthrax in humans.

Virgil described anthrax in domestic and wild animals

in his Georgics , and anthrax was an economically im-

portant agricultural disease during the 16th through

18th centuries in Europe. 7,8

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. 9,10 Anthrax also was the first

disease for which an effective live bacterial vaccine

was developed; Louis Pasteur developed that vaccine

in 1881. 11 Additionally, anthrax represents the first

described occupational respiratory infectious disease.

During the latter half of the 19th century, inhalational

anthrax, 12 a previously unrecognized form, occurred

among woolsorters in England as a result of the gen-

eration of infectious aerosols of anthrax spores under

industrial conditions from the processing of contami-

nated goat hair and alpaca wool. 13

The military has long been concerned about anthrax

as a potential biological weapon because anthrax

spores are infectious by the aerosol route, and a high

mortality rate is associated with untreated inhala-

tional anthrax. In 1979 the largest inhalational anthrax

epidemic of the 20th century occurred in Sverdlovsk,

Russia. Anthrax spores were accidentally released from

a military research facility located upwind from where

the cases occurred. According to the accounts provided

by two Soviet physicians, 96 human anthrax cases

were reported, of which 79 were gastrointestinal and

17 cutaneous. The 79 gastrointestinal cases resulted in

64 deaths. Although the initial report of this event at-

tributed the infections to a gastrointestinal source, later

evidence indicated that an aerosol release of weapon-

ized anthrax spores from a military production facility

had occurred, and thus, inhalational anthrax may have

been 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. 14

Cases were also reported in animals located more than

50 km from the site. 15,16 Polymerase chain reaction ex-

amination of tissue samples collected from 11 of the

Fig. 4-1. ( 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.

Photograph: Courtesy of Susan Welkos, PhD, Division of Bacteriology, US Army Medical Research Institute of Infectious Dis-

eases, Fort Detrick, Maryland.

Photograph: Courtesy of John Ezzell, PhD, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland.

Anthrax

victims demonstrated that virulent B anthracis DNA

was present in all these patients, and at least five dif-

ferent strains of virulent anthrax were detected based

on variable number tandem repeat analysis. 17

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 Banthracis spores. The spore powder,

which was sealed in letters addressed to members of

the press and of Congress, was mailed through the

US Postal Service. 18-20 According to the Centers for

Disease Control and Prevention, 22 people contracted

anthrax from the letters. 18,21-25 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. 26-29 This incident

profoundly affected the law enforcement, scientific,

and medical communities within the United States.

As a result of the attacks, there has been increased

governmental and public awareness of the threat posed

by Banthracis and other pathogens, particularly those

with a potential for aerosol-mediated infection. 30-43 The

amount of funding budgeted to prepare and protect

the nation from a bioterror attack has rapidly increased

since 2001, and a significant amount of this funding has

supported anthrax studies. Some of the new anthrax

studies have focused on improved sample collection,

Fig. 4-2. 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 2620.

Photograph: Courtesy of John Ezzell, PhD, US Army Medi-

cal Research Institute of Infectious Diseases, Fort Detrick,

Maryland.

rapid detection/diagnosis, decontamination, and

microbial forensics. Because of the ongoing terrorism

threat, there has been a particular sense of urgency

regarding the development and improvement of medi-

cal countermeasures, such as therapeutics, vaccines,

diagnostics, and devices.

THE ORGANISM

B anthracis is a large, gram-positive, spore-forming,

nonmotile bacillus (1–1.5 µm x 3–10 µm) that is closely

related to B cereus and B thuringiensis. The organism

grows readily on sheep blood agar aerobically and is

nonhemolytic under these conditions. The colonies are

large, rough, and grayish white, with irregular, curving

outgrowths from the margin. The organism forms a

prominent capsule both in vitro in the presence of

bicarbonate and carbon dioxide and in tissue in vivo.

In tissue, the encapsulated bacteria occur singly or in

chains of two or three bacilli (Figure 4-1). The organ-

ism does not form spores in living tissue; sporulation

occurs only after the infected body has been opened

and exposed to oxygen. The spores, which cause no

swelling of the bacilli, are oval and occur centrally or

paracentrally (Figure 4-2). The spores are very resistant

and 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, detection of capsule by fluo-

rescent antibody, and virulence for mice and guinea

pigs. 44,45 Additional confirmatory tests to identify toxin

and capsule genes by polymerase chain reaction, de-

veloped as research tools, have been incorporated into

the Laboratory Response Network established by the

Centers for Disease Control and Prevention. 46-49

EPIDEMIOLOGY

Anthrax, 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. 50,51 The form of the organism

in infected animals is the bacillus. Sporulation occurs

only when the organism in the carcass is exposed to air.

Domestic or wild animals become infected when

they ingest spores while grazing on contaminated

MedicalAspectsofBiologicalWarfare

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. 52 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. 51,53

Anthrax in humans is associated with agricultural,

horticultural, or industrial exposure to infected animals

or contaminated animal products. In less developed

countries, primarily Africa, Asia, and the Middle East,

disease occurs from contact with infected domesticated

animals or contaminated animal products. Contact

may include handling contaminated carcasses, hides,

wool, hair, and bones and ingesting contaminated

meat. Cases associated with industrial exposure, rarely

seen now, occur in workers processing contaminated

hair, wool, hides, and bones. Direct contact with con-

taminated material leads to cutaneous disease, and

ingestion of infected meat leads to oropharyngeal or

gastrointestinal forms of anthrax. Inhalation of a suf-

ficient quantity of spores, usually seen only during

generation of aerosols in an enclosed space associated

with processing contaminated wool or hair, leads to

inhalational anthrax. Military research facilities have

played a major role in studying and defining anthrax,

as well as many other zoonotic diseases in wild and

domestic animals and the subsequent infections in

humans. 54

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. 55 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. 56-60 In the

1996–1997 global anthrax report, there appeared to be a

general decrease in anthrax cases worldwide; however,

anthrax remains underdiagnosed and underreported. 61

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. 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. 62,63 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. 64 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. 65 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.

Essentially all cases were cutaneous, including rare

gastrointestinal disease cases and eight inhalational

anthrax cases, although no autopsy confirmation was

reported. 66

PATHOGENESIS

Banthracis possesses two protein exotoxins, known

as the lethal toxin and the edema toxin; an antiphago-

cytic capsule; and other known and putative virulence

factors. 67 The role of the capsule in pathogenesis was

demonstrated in the early 1900s, when anthrax strains

lacking a capsule were shown to be avirulent. 68 In

more recent years, the genes encoding synthesis of

the capsule were encoded on the 96-kilobase (kb)

plasmid known as pXO2. Molecular analysis revealed

that strains cured of this plasmid no longer produced

the capsule and were attenuated, thus confirming the

critical role of the capsule in virulence. 69 The capsule

is composed of a polymer of poly-D-glutamic acid,

which confers resistance to phagocytosis and may

contribute to the resistance of anthrax to lysis by serum

cationic proteins. 70 Capsule production is necessary for

dissemination to the spleen in a murine inhalational

anthrax model. 71 Recently, the capsule has also been

the focus of several efforts to develop new generation

anthrax vaccines. 72-74 Evidence indicates that the cap-

sule may enhance the protection afforded by PA-based

vaccines against anthrax if opsonizing antibodies are

produced. 74

Koch first suggested the importance of toxins in his

initial studies on anthrax. In 1954 Smith and Keppie 75

demonstrated a toxic factor in the serum of infected

animals that was lethal when injected into other ani-

mals. The role of toxins in virulence and immunity

was firmly established by many researchers in the

ensuing years. 76-78 Advances in molecular biology

in the past decade have produced a more complete

understanding of the biochemical mechanisms of ac-

tion of the toxins and have begun to provide a more

definitive picture of their role in the pathogenesis of

the disease.

Two protein exotoxins, known as the lethal toxin

and edema toxin, are encoded on a 182-kb plasmid

(pXO1), distinct from that coding for the capsule. In an

Anthrax

environment of increased bicarbonate, carbon dioxide,

and increased temperature, such as is found in the in-

fected host, transcription of the genes encoding these

and other virulence-associated gene products is en-

hanced. 67,79-82 A complex regulatory cascade controlled

in large part by the atxA and acpA genes encoded on

the toxin plasmid pXO1 and pXO2, respectively, directs

the production of virulence factors in response to these

environmental signals. 83,84

Recently, a retrospective study identified an isolate

of Bcereus that carried a plasmid homologous to the

anthrax toxin plasmid pXO1. This strain was obtained

from a patient with symptoms similar to inhalational

anthrax. 85 This finding led to considerable concern be-

cause “anthrax toxin” sequences are considered unique

to Banthracis . Although a polyglutamate capsule was

not produced, sequences encoding a polysaccharide

capsule were present on a smaller plasmid. The possi-

bility of false positives from toxin-based identification

tests should be considered because many diagnostic

schemes have focused on toxin genes and gene prod-

ucts. The virulence of this isolate has not yet been ex-

tensively studied, and the role of the lethal and edema

toxins in the pathogenesis of this strain is unknown.

Likewise, the incidence of such strains in nature is un-

clear. Because Bcereus 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 Bcereus is

often regarded as an environmental contaminant. 85

Other human cases of anthrax-like Bcereus infections

have been reported. 86,87

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 enzy-

matic activity (Figure 4-3). The B and A anthrax toxin

components are synthesized from different genes

and are secreted as noncovalently linked proteins.

The anthrax toxins are unusual in that the B protein,

PA, is shared by both toxins. Thus, the lethal toxin

is composed of the PA 63 (MW 63,000 after cleavage

from a MW 83,000 protein) heptamer combined with a

second protein, which is known as the lethal factor (LF

[MW 90,000]), and the edema toxin is composed of PA

complexed with the edema factor (EF [MW 89,000]).

Each of the 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 pXO1 69,88 or the PA

gene alone 89 attenuates the organism. The lethal

toxin appears to be more important for virulence in

a mouse model than the edema toxin. 90 Crude toxin

preparations have been shown to impair neutrophil

chemotaxis 91 and phagocytosis. 70

The edema toxin causes edema when injected into

the skin of experimental animals and is likely respon-

sible for the marked edema often present at bacte-

rial replication sites. 92,93 This toxin is a calmodulin-

dependent adenylate cyclase that impairs phagocytosis

and priming for the respiratory burst in neutrophils;

it also inhibits the production of interleukin-6 and tu-

mor necrosis factor by monocytes, which may further

weaken host resistance. 94-96 Edema toxin also impairs

dendritic cell function and appears to act with lethal

toxin to suppress the innate immune response. 97

The lethal toxin is a zinc metalloprotease that is

lethal for experimental animals 92,93,98 and is directly

cytolytic for macrophages, causing release of the

potentially toxic cytokines interleukin-1 and tumor

Fig. 4-3. Composition of anthrax lethal protein toxin. Molecu-

lar 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.

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