c


]



 

 

 
   
Anthrax is caused by c
 

 Humans acquire the disease directly from
contact with infected herbivores or indirectly via their products. The clinical forms include
( 1 ) cutaneous anthrax (eschar with edema), from handling infected material (this
accounts for more than 95 percent of cases); (2) intestinal anthrax, from eating infected
meat; and (3) pulmonary anthrax, from inhaling spore -laden dust. Several
other c
 spp, in particular c

and to a lesser extent c  and c


, are periodically associated with bacteremia/septicemia, endocarditis,
meningitis, and infections of wounds, the ears, eyes, respiratory tract, urinary tract, and
gastrointestinal tract. c


 causes two distinct food poisoning syndromes: a
rapid-onset emetic syndrome characterized by nausea and vomiting, and a slower-onset
diarrheal syndrome.




  
 
c
 species are rod-shaped, endospore-forming aerobic or facultatively anaerobic,
Gram-positive bacteria; in some species cultures may turn Gram-negative with age. The
many species of the genus exhibit a wide range of physiologic abilities that allow them to
live in every natural environment. Only one endospore is formed per cell. The spores are
resistant to heat, cold, radiation, desiccation, and disinfectants. c
 

 needs
oxygen to sporulate; this constraint has important consequences for epidemiology and
control. In vivo, c 

 produces a polypeptide (polyglutamic acid) capsule that
protects it from phagocytosis. The genera c
 and 
 constitute the family
Bacillaceae. Species are identified by using morphologic and biochemical criteria.

]  
The virulence factors of c 

 are its capsule and three-component toxin, both
encoded on plasmids. c


 produces numerous enzymes and aggressins. The
principal virulence factors are a necrotizing enterotoxin and a potent
hemolysin(cereolysin). Emetic food poisoning probably results from the release of emetic
factors from specific foods by bacterial enzymes.

• 
The reasons for marked differences in susceptibility to anthrax among different animal
species are not known. The protective actions of the live-spore animal vaccine or the
human chemical vaccines are based on induction of humoral and cell -mediated immunity
to the protective antigen component of anthrax toxin.

 
Individuals at risk for anthrax include those in contact with in fected animals or animal
products. Episodes of c

 food poisoning occur sporadically worldwide and result
from ingestion of contaminated food in which the bacteria have multiplied to high levels
under conditions of improper storage after cooking.

  
6utaneous anthrax is diagnosed on the basis of the characteristic papule (early) or
eschar (later) with extensive surrounding edema, backed by a history of exposure to
animals or their products. Diagnosis is confirmed by observation of characteristic
encapsulated bacilli in polychrome methylene blue-stained smears of blood, exudate,
lymph, cerebrospinal fluid, etc., and/or by culture. Other c
 infections are
diagnosed by culture of the bacteria.

 

Anthrax: 6ontrol in animals is essential for control in humans. In endemic areas, animals
that die suddenly should be handled cautiously and livestock should be vaccinated
annually. A human vaccine is available for individuals in high -risk occupations. Anthrax is
readily treated with antibiotics (e.g., penicillin, tetracycline, chloramphenicol, gentamicin,
or erythromycin).

Other Bacillus Infections: 6ontrol is by good hygiene. Treatment is with non -ß-lactam
antibiotics for Gram-positive bacteria. Food poisoning is controlled by adequate cooking,
avoidance of recontamination of cooked food, and proper storage (efficient refrigeration).
]





  
 
 


Vany of the physiologic properties and specialized metabolites of c
 species are
used in the pharmaceutical, agricultural, and food industries. On the other hand, the
resistance of the spores to sterilization and disinfection makes them problem
contaminants in foods, medical supplies, surgical procedures, etc.

a



The anthrax bacillus, c
 

 was the first bacterium shown to be the cause of a disease. In
1877, Robert Koch grew the organism in pure culture, demonstrated its ability to form endospores, and
produced experimental anthrax by injecting it into animals.

½
  


 



c
 













 

 











 
½



c




! 

 
 
  
 

c 







"
  










 
 
 

#$


 


%

 

&



 


 
c
 



c
 

is very large, Gram-positive, sporeforming rod, 1 - 1.2µm in width x 3 - 5µm in
length. The bacterium can be cultivated in ordinary nutrient medium under aerobic or anaerobic
conditions. Genotypically and phenotypically it is very similar to c


 which is found in soil
habitats around the world, and to c
 
 the pathogen for larvae of 
 The
three species have the same cellular size and morphology and form oval spores located centrally in a
nonswollen sporangium.
½
c
 



 
'(()

 
*



% 



 


 
 







 

 


 





 
 





  

c
 
  is distinguished from c

 or c 

 by its pathogenicity for
Lepidopteran insects (moths and caterpillars) and by production of an intracellular 


 in association with spore formation. The bacteria and protein crystals are sold as "Bt" insecticide,
which is used for the biological control of certain garden and crop pests.

½
+
c
 
 
,
,


* 

*
 "

 
 


 




$
((()

c


 is a normal inhabitant of the soil, but it can be regularly isolated from foods such as
grains and spices. c

causes two types of food-borne intoxications (as opposed to infections).
One type is characterized by nausea and vomiting and abdominal cramps and has an incubation period
of 1 to 6 hours. It resembles   


 
 food poisoning in its symptoms and incubation
period. This is the "short-incubation" or emetic form of the disease. The second type is manifested
primarily by abdominal cramps and diarrhea with an incubation period of 8 to 16 hours. Diarrhea may be
a small volume or profuse and watery. This type is referred to as the "long-incubation" or diarrheal form
of the disease, and it resembles food poisoning caused by 


 In either type, the
illness usually lasts less than 24 hours after onset.

The short-incubation form is caused by a preformed, heat-stable




 "
- -. The mechanism
and site of action of this toxin are unknown, although the small molecule forms ion channels and holes
in membranes. The long-incubation form of illness is mediated by the heat-labile
diarrheagenic


. and/or  




/!#, which cause intestinal fluid
secretion, probably by several mechanisms, including pore formation and activation of adenylatecyclase
enzymes.
½
0c




 
0'()
!



"





 

 
 * 





 
1

2 
* 1



 




 
 



 
 












Anthrax is primarily a disease of domesticated and wild animals, particularly herbivorous animals, such
as cattle, sheep, horses, mules and goats. Humans become infected incidentally when brought into
contact with diseased animals, which includes their flesh, bones, hides, hair and excrement.

The natural history of c
 

 is obscure. Although the spores have been found naturally in soil
samples from around the world, the organisms cannot be regularly cultivated from soils where there is
an absence of endemic anthrax. In the United States there are recognized areas of infection in South
Dakota, Nebraska, Arkansas, Texas, Louisiana, Vississippi, 6alifornia and small areas that exist in other
states. Even in endemic areas, anthrax occurs irregularly, often with many years between occurrences.

In the United States, the incidence of naturally-acquired anthrax is extremely rare (1-2 cases of
cutaneous disease per year). Worldwide, the incidence is unknown, although c 

 is present in
most of the world. Unreliable reporting makes it difficult to estimate the true incidence of human
anthrax worldwide. However, in fall 2001, 22 cases of anthrax (11 inhalation, 11 cutaneous) were
identified in the United States following intentional contamination of the mail.

The most common form of the disease in humans is 
 


, which is usually acquired via
injured skin or mucous membranes. A minor scratch or abrasion, usually on an exposed area of the face
or neck or arms, is inoculated by spores from the soil or a contaminated animal or carcass. The spores
germinate, vegetative cells multiply, and a characteristic  
 
 develops at the site. This
develops into  within 12-36 hours after infection. The papule changes rapidly to a *  , then a
pustule  


 ), and finally into a 

  from which infection may disseminate,
giving rise to  
 . Lymphatic swelling also occurs within seven days. In severe cases, where the
blood stream is eventually invaded, the disease is frequently fatal.

Another form of the disease,  




(woolsorters' disease), results most commonly from
inhalation of spore-containing dust where animal hair or hides are being handled. The disease begins
abruptly with high fever and chest pain. It progresses rapidly to a systemic hemorrhagic pathology and
is often fatal if treatment cannot stop the invasive aspect of the infection.




 

 is analogous to cutaneous anthrax but occurs on the intestinal mucosa. As in
cutaneous anthrax, the organisms probably invade the mucosa through a preexisting lesion. The
bacteria spread from the mucosal lesion to the lymphatic system. Intestinal anthrax results from the
ingestion of poorly cooked meat from infected animals. Gastrointestinal anthrax is rare but may occur as
explosive outbreaks associated with ingestion of infected animals. Intestinal anthrax has an extremely
high mortality rate.

3  
 due to c 

 is a very rare complication that may result from a primary infection
elsewhere.
,
 


c
 



c
 

 clearly owes its pathogenicity to two major determinants of virulence: the formation of
a &4&

 , which mediates the invasive stage of the infection, and the production of
the multicomponent


 which mediates the toxigenic stage.

,&4&



c
 

 forms a single antigenic type of  consisting of a poly-D-glutamate polypeptide.
All virulent strains of c 

 form this capsule. Production of capsular material is associated with
the formation of a characteristic mucoid or "smooth" colony type. "Smooth" (S) to "rough" (R) colonial
variants occur, which is correlated with ability to produce the capsule. R variants are relatively avirulent.
6apsule production depends on a 60 megadalton "
)(5 its transfer to nonencapsulated c


via transduction produces the encapsulated phenotype.

½
6 


 % 


  




  



&4&




c
 


# 

a 
 $



((()



 
&  



 


 








  



 

&
½


((()

The poly-D-glutamyl capsule is itself nontoxic, but functions to protect the organism against complement
and the bactericidal components of serum and phagocytes, and against phagocytic engulfment and
destruction. The capsule plays its most important role during the establishment of the infection, and a
less significant role in the terminal phases of the disease, which are mediated by the anthrax toxin.

The poly-D-glutamyl capsule is formed in vivo or in the laboratory when the bacterium is grown on
serum plates in a 5% 6O2 atmosphere. The capsular material can be detected by the VcFadyean
reaction which involves staining with polychrome methylene blue. Blue rods in a background of
purple/pink-stained capsular material is a positive test (Figure 9). Neither c

 nor c
 
synthesizes this capsular polymer, so the detection of capsular material can be used to
distinguish c 

 from its closest relatives.
½
73½  
 

 


 



c
 


 
 
 
"







8


 



 





The toxigenic properties of c
 

 were not recognized until 1954. Prior to that time, because
of the tremendous number of anthrax bacilli observed in the blood of animals dying of the disease
9
(10 bacteria/ml), it was assumed that death was due to blockage of the capillaries, popularly known as
6
the "log-jam" theory. But experimentally it was shown that only about 3 x 10 cells/ml are necessary to
cause death of the animal. Furthermore, the cell-free plasma of animals dying of anthrax infection
contained a toxin which causes symptoms of anthrax when injected into normal guinea pigs. These
observations left little doubt that a diffusible exotoxin plays a major role in the pathogenesis of anthrax.

One component of the 


 has a lethal mode of the action that is not entirely understood at
this time. Death is apparently due to oxygen depletion, secondary shock, increased vascular
permeability, respiratory failure and cardiac failure. Death from anthrax in humans or animals frequently
occurs suddenly and unexpectedly. The level of the lethal toxin in the circulation increases rapidly quite
late in the disease, and it closely parallels the concentration of organisms in the blood.

Production of the anthrax toxin is mediated by a temperature-sensitive"

)(, of 110
megadaltons. The toxin consists of three distinct antigenic components. Each component of the toxin is
a thermolabile protein with a mw of approximately 80kDa.

Factor I is the  


-½ which is necessary for the edema producing activity of the toxin. EF
is known to be an   

 
 , similar to the c
 
 adenylatecyclase
toxin.

Factor II is the 

*



,, because it induces protective antitoxic antibodies in guinea
pigs. PA is the   
!
 of the anthrax toxin which has two active (A) domains, EF (above)
and LF (below).

Factor III is known as the 




#½ because it is essential for the

 

of the anthrax
toxin. Apart from their antigenicity, each of the three factors exhibits no significant biological activity in
an animal. However, combinations of two or three of the toxin components yield the following results in
experimental animals.

PA+LF combine to produce lethal activity

EF+PA produce edema
EF+LF is inactive
PA+LF+EF produces edema and necrosis and is lethal

These experiments suggest that the anthrax toxin has the familiar A-B enzymatic-binding structure of
bacterial exotoxins with PA acting as the B fragment and either EF or LF acting as the active A fragment.
EF+PA has been shown to elevate cyclic AVP to extraordinary levels in susceptible cells. 6hanges in
intracellular cAVP are known to affect changes in membrane permeability and may account for edema.
In macrophages and neutrophils an additional effect is the depletion of ATP reserves which are needed
for the engulfment process. Hence, one effect of the toxin may be to impair the activity of regional
phagocytes during the infectious process.

The effects of EF and LF on neutrophils have been studied in some detail. Phagocytosis by opsonized or
heat-killed c
 

 cells is not inhibited by either EF or LF, but a combination of EF + LF
inhibits engulfment of the bacteria and the oxidative burst in the pmns. The two toxin components also
increased levels of cAVP in the neutrophils. These studies suggest that the two active components of the
toxin, EF + LF, together increase host susceptibility to infection by suppressing neutrophil function and
impairing host resistance.

LF+PA have combined lethal activity as stated above. The lethal factor is a Zn++dependent protease that
induces cytokine production in macrophages and lymphocytes, and its mechanism of action is slowly
becoming understood. The crystal structure of lethal factor is known to to be a member of the
mitogen-activated protein kinase (VAPKK) family of enzymes that disrupts cellular signaling.
Furthermore, the identity of the human receptor for anthrax PA, named 



  
, has
been demonstrated to be a type I membrane protein that binds directly to PA.

In summary, the virulence of c
 

 is attributable to three bacterial components; 1.
6apsular material composed of poly-D-glutamate polypeptide; 2. EF component of exotoxin; 3.LF
component of exotoxin. Both the capsule and the anthrax toxin may play a role in the early stages of
infection, through their direct effects on phagocytes. Virulent anthrax bacilli multiply at the site of the
lesion. Phagocytes migrate to the area but the encapsulated organisms can resist phagocytic engulfment,
or if engulfed, can resist killing and digestion. A short range effect of the toxin is its further impairment
of phagocytic activity and its lethal effect on leukocytes, including phagocytes, at the site. After the
organisms and their toxin enter the circulation, the systemic pathology, which may be lethal, will result.

c
 

 coordinates the expression of its virulence factors in response to a specific
environmental signal. Anthrax toxin proteins and the antiphagocytic capsule are produced in response to
growth in increased atmospheric 6O2. This 6O2 signal is thought to be of physiological significance for a
pathogen which invades mammalian host tissues.

a 






6onsiderable variation in genetic susceptibility to anthrax exists among animal species. Resistant
animals fall into two groups: (1) resistant to establishment of anthrax but sensitive to the toxin and (2)
resistant to the toxin but susceptible to establishment of disease. This is illustrated in the table below.
Neither the source of the inoculum (spores or vegetative cells or a mixture) nor the route of inoculation
(subcutaneous, gastrointestinal, or inhalational) is stated. The infectious dose of anthrax is expected to
vary widely based on these parameters, as well.




  



c 



 








* 
 



 

  






 





  



 
 

* 

*
 
 
 



 

Animal anfectious Toxic dose causing Bacteria per ml blood at

model p dosep deathp time deathp

p pp  p
 p  p


p  pp  p
 p  p
p  pp p
 p  p

Animals surviving naturally-acquired anthrax are immune to reinfection. Second attacks are extremely
rare. Permanent immunity to anthrax seems to require antibodies to both the toxin and the capsular
polypeptide, but the relative importance of the two kinds of antibodies appears to vary widely in
different animals.

å  composed of killed bacilli and/or capsular antigens produce no significant immunity. A
nonencapsulated toxigenic strain has been used effectively in livestock. The 9

9
 of c



 produces sublethal amounts of the toxin that induces formation of protective antibody.

The 

*

 , which is used in the U.S., is a preparation of the 

*


 recovered from the culture filtrate of an avirulent, nonencapsulated strain of c



 that produces PA during active growth. Anthrax immunization consists of three subcutaneous
injections given two weeks apart followed by three additional subcutaneous injections given at 6, 12,
and 18 months. Annual booster injections of the vaccine are required to maintain a protective level of
immunity.

The vaccine is indicated for individuals who come in contact in the workplace with imported animal hides,
furs, bone, meat, wool, animal hair (especially goat hair) and bristles; and for individuals engaged in
diagnostic or investigational activities which may bring them into contact with anthrax spores. Otherwise,
it has been indicated for the military during the current era of biological warfare.

The vaccine should only be administered to healthy individuals from 18 to 65 years of age, since
investigations to date have been conducted exclusively in that population. It is not known whether the
anthrax vaccine can cause fetal harm, and pregnant women should not be vaccinated.

A new type of *

* to anthrax is currently on the horizon. This was recently announced by
R.G. 6rystal and colleagues from the Vedical 6ollege of 6ornell University, in the February, 2005 issue of
the journal, Volecular Therapy. They demonstrated that mice vaccinated with a human adenovirus
expressing a single-chain antibody directed against protective antigen (PA) became immune to anthrax
within 24 hours of vaccination. This is much quicker than is possible with existing anthrax vaccines,
which are a relatively crude preparation of PA.

6urrently available anthrax vaccines have limited use in a bioterrorism attack because they are active
vaccines in which multiple doses are required over several months to elicit protective immunity against
anthrax. Passive vaccines, on the other hand, introduce fully formed antibodies directly to the body and
immunity is achieved much sooner.

In mice receiving the adenovirus-based anti-PA vaccine, PA-specific serum antibodies were detectable
within 24 hours. These antibodies had neutralizing activity that protected mice from an intravenous
lethal toxin challenge administered 1-14 days post vaccination.

6rystal, et al envision a possible scenario wherein both the passive and active vaccine might be given.
Passive vaccines lose their effectiveness fairly rapidly over time, whereas active vaccines do not. The
passive vaccine could provide protection that would last a couple of weeks, but that would provide a
safety margin for development of more active, long-term immunity stimulated by the active vaccine.

Passive immunotherapy with such adenovirus-based vectors expressing anti-PA antibody, either alone or
in combination with antibiotics, may be a rapid, convenient, and highly effective strategy to protect
against or treat anthrax in a bioterrorism attack.

Also, in cases of anthrax, coadministration of the passive vaccine with antibiotics may maximize the
utility of antibiotic therapy. 6oadministration would counter the effects of lethal toxin, and likely prolong
the time frame for effective antibiotic treatment and/or reduce the amount of antibiotic therapy required.

 






Antibiotics should be given to unvaccinated individuals exposed to inhalation anthrax. Penicillin,

tetracyclines and fluoroquinolones are effective if administered before the onset of lymphatic spread or
septicemia, estimated to be about 24 hours. Antibiotic treatment is also known to lessen the severity of
disease in individuals who acquire anthrax through the skin. Inhalation anthrax was formerly thought to
be nearly 100% fatal despite antibiotic treatment, particularly if treatment is started after symptoms
appear. A recent Army study resulted in successful treatment of monkeys with antibiotic therapy after
being exposed to anthrax spores. The antibiotic therapy was begun one day after exposure.



 
!
8

The inhalation of anthrax spores can lead to infection and disease. The possibility of creating aerosols
containing anthrax spores has made c 

a chosen weapon of bioterrorism. Several powers may
have the ability to load spores of c 

 into weapons. Domestic terrorists may develop means to
distribute spores via mass attacks or small-scale attacks at a local level.

As an agent of biological warfare it is expected that a cloud of anthrax spores would be released at a
strategic location to be inhaled by the individuals under attack. Spores of c 

 can be produced
and stored in a dry form and remain viable for decades in storage or after release.

There is no evidence of person-to-person transmission of anthrax. Quarantine of affected individuals is

not recommended. Anthrax spores may survive in the soil, water and on surfaces for many years.
Spores can only be destroyed by steam sterilization or burning. 6hemical disinfection of buildings is
problematic. The U.S. Navy Vanual on Operational Vedicine and Fleet Support, entitled Biological
Warfare Defense Information Sheet states "Disinfection of contaminated articles may be accomplished
using a 0.05% hypochlorite solution (1 tbs.. bleach per gallon of water). Spore destruction requires
steam sterilization."

o o
Anthrax spores are killed by boiling (100 6 or 212 F) for 30 minutes (the actual reported time is
considerably less). If boiling as a means of disinfection, the spores must be in liquid suspension to
ensure killing, and in a sealed container to avoid aerosolization or vaporization of droplet nuclei
containing spores.

An infection of local animal populations such as sheep and cattle could follow a biological attack with
spores. Infected animals could then transmit the disease to humans through the cutaneous, intestinal or
inhalation route by spores from a contaminated animal, carcass or hide.

A segment of the U.S. military population has been vaccinated against anthrax. Anthrax vaccine consists
of a series of six doses with yearly boosters. The first vaccine of the series must be given at least four
weeks before exposure to the disease. This vaccine protects against anthrax that is acquired through the
skin and it is believed that it would also be effective against inhaled spores in a biowarfare situation. Of
course, a vaccinated military population would be needed to respond to a bioterrorist attack with
anthrax spores.