ANTHRAX

The anthrax bacillus, Bacillus anthracis, was the first

bacterium shown to be the cause of a disease- Kochs
Postulate
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.
Anthrax is a disease of domesticated and wild animals

Men suffer from anthrax occasionally due to close

contact with infected animal or animal products
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Bacillus anthracis
Gram positive rods
Capsulated ( Protein) Capsule form in animal tissue and in special
laboratory condition ( 5% CO2)
Forms endospore, centrally located, do not form in animal tissues
MacFadyean ( Polychrome methylene blue) stain blue bacilli
with purple capsule
Aerobic/ Facultative anerobe
Grows on all ordinary medium (Medusa head appearance-uneven
wavy margin)
Inverted fur tree appearance in liquid medium
Biochemicals : Catalase +, reduces nitrate to nitrite, lecithinase+,
glucose, maltose, sucrose, trehalose fermented
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Robert Koch's original micrographs of the anthrax bacillus

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Bacillus anthracis. Gram stain. The cells have characteristic

squared ends. The endospores are ellipsoidal shaped and located
centrally in the sporangium. The spores are highly refractile to
light and resistant to staining.
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Bacillus cereus

Genotypically and
phenotypically it is very similar
to Bacillus cereus, which is
found in soil habitats around
the world

Bacillus thuringiensis.
Phase Photomicrograph
of vegetative cells,
intracellular spores (light)
and
parasporal crystals (dark).
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McFadyean's reaction showing short chains of Bacillus

anthracis cells lying among amorphous,disintegrated
capsular material. White blood cells can also be seen.
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Differential Characteristics of B. anthracis B. cereus and B. thuringiensis

Characteristic

B. anthracis

Differential Characteristics of B. anthracis B. cereus and B. thuringiensis

B. cereus and
B. thuringiensis

growth requirement for thiamin

hemolysis on sheep blood agar

glutamyl-polypeptide capsule

lysis by gamma phage

motility

growth on chloralhydrate agar

string-of-pearls test

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Physical properties ( methods for decontamination)

SPORES SURVIVE FOR MANY YEARS ( DRY STATE AND SOIL )
Moist heat kills Vegetative cells 60 0 C X 30 minutes
Spores 100 0 C X 10 minutes
4% Formaldehyde kills spores
4% KMnO4 kills spores
Hypochlorite ( 0.5%) commercially available kills spores

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Epiedemiology

Distribution worldwide
Not common in West. Common in Africa ( Zimbabwe),
S.E. Asia, China, South America, Turkey, Pakistan, India
Human to human or animal to animal transmission is rare
( not contagious)

Grazing animals become infected through ingestion of

spores in the soil ( Carcasses become the source)
Epidemic : A. Spread to contiguous geographic areas by
infected animal
B. Non contiguous geographic areas by
- biting flies ( Zimbabwe)
- Vultures
- Contaminated surface water pool
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INDIA
Largest live stock population in the world
Incidence is not accurately known ( Sporadic cases reported)
Pondicherry ( JIPMER) - 30 human cases reported ( Mostly Cutaneous,
Septicemic or Meningeal)
Vellore ( CMC)- 49 human cases
Chittor ( Rajasthan)- 30 human cases
Tirupati ( Andhrapradesh)- 25 human cases

Midnapur ( WB)- 22 human cases

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Pathogenesis
Endospores
(Abrasion, inhalation, ingestion)

Death

Introduced

Septicemia

Phagocytosed by Macrophages

10 7 to 10 8/ml

Regional LNs

Blood stream

Multiply in Lymphatics

Germinate inside Macrophages

Release
Vegetative Forms

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Clinically three forms of Human anthrax occur

A. Cutaneous anthrax
B. Pulmonary anthrax
C. Intestinal anthrax
Broadly can be classified into
Non Industrial/Agricultural ( Through infected animals):
Cutaneous anthrax
Rarely intestinal anthrax
Industrial Anthrax ( Through animal products):
Mostly through animal products( wools, hair, hides, bones)
Likely to develop Cutaneous and pulmonary anthrax ( inhalation)
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Cutaneous Anthrax
Mainly in professionals( Veterinarian, butcher, Zoo keeper

Spores infect skin- a characteristic gelatinous edema develops at the

site (Papule- Vesicle-Malignant Pustule- Necrotic ulcer)
80-90% heal spontaneously ( 2-6wks)

0-20% progressive disease develop septicemia

95-99% of all human anthrax occur as cutaneous anthrax
Intestinal Anthrax
Due to in ingestion of infected carcasses
Mucosal lesion to the lymphatic system
Rare in developed countries
Extremely high mortality rate

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PULMONARY ANTHRAX
Require very high infective dose ( > 10,000 spores)
Acquired through inhalation of spores ( Bioterrorism - aerosol)

Present with symptoms of severe respiratory infection( High fever &

Chest pain)
Haemorrhagic mediastinitis

Progress to septicemia very rapidly

10 7 to 10 9 bacilli/ ml of blood at the time of death
Mortality rate is very high > 95%
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DIFFERENTIAL DIAGNOSIS OF ANTHRAX

CUTANEOUS ANTHRAX
Boils, Erysipelas, Cutaneous TB, Leprosy, Plague, Vaccinia, Rickettsial pox,
tularemia
INTESTINAL ANTHRAX
Typhoid fever, Acute Gastroenteritis, Tularemia, Peritonitis, Peptic ulcer,
Mechanical obstruction
PULMONARY ANTHRAX
Viral pneumonia, Mycoplasma. Psittacosis, Legionnaires disease, Q fever,
Histoplasmosis, Coccidiodomycosis, Silicosis, Sarcoidosis
Meningeal Anthrax : Sometime manifest as meningitis
D/D : Bacterial meningitis
Aseptic meningitis
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VIRULENCE FACTORS
Anthrax Toxin Complex of proteins ( all the components thermolabile)
A. Protective antigen
B. Edema factor
C. Lethal Factor
Protein capsule Poly D Glutamic acid capsule
- Inhibits phagocytosis ( Unencapsulated strains
nonpathogenic)
Anthrax Toxin
Protective antigen : Binds plasma membrane of target cells
Cleaved to 2 fragments ( cellular trypsin or proteases)
Larger fragment is attached to cell surface binding domain for LF & EF
Specific receptor mediated endocytosis of LF & EF

EDEMA FACTOR
( Edema Factor + Protective Ag = Edema toxin)
Calmodulin dependent adenyl cyclase
Increased cellular cAMP

Edema

Impaired Neutrophil function

Depletes ATP from Macrophages

LETHAL FACTOR
( Lethal Factor + Protective Ag = Lethal toxin)
Zinc metallo proteases that inactivates protein kinases
Stimulates Macrophages TNF alpha and IL 1 beta Shock & Death

Death due to oxygen depletion, secondary shock, increased vascular

permeability, respiratory failure and cardiac failure.
Sudden and unexpected.
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Virulence of Anthrax bacillus is due to presence of two plasmids

px01 Toxin encoding plasmid

- 110 megadalton
- temperature-sensitive plasmid
px02 - Capsule encoding plasmid ( 3 genes - cap A, cap B, cap C)
- 60 megadalton plasmid
- synthesis of poly glutamic acid capsule
Both plasmids are required for virulence
- loss of either - attenuation
- genes expressed only in vegetative state
Pasteur strain - Encapsulated
Sterne strain Non encapsulated
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LABORATORY DIAGNOSIS
Few points to remember
Anthrax is not highly contagious
Cutaneous anthrax is not lethal and is readily treated with
common antibiotics
ID for human pulmonary / intestinal infection is > 10,000 spores
SPECIMEN TO COLLECT ( HUMAN ANTHRAX)
Disposable gloves, masks, overalls, boots, head gear and dust mask
Disposable items Autoclave and incinerate
Cutaneous anthrax: Vesicular exudate swabs and capillary tube aspirate
Intestinal anthrax: - Stool sample - isolate guinea pig inoculation
- Blood( venipuncture) smear examination for bacilli
- Peritoneal fluid for culture
- Paired sera for Ab
Pulmonary anthrax: If mild disease ( No sample)
Severely ill Blood , sputum, serum samples for Ab
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SAMPLES FROM ANIMAL

Sudden death of animal in areas where anthrax was reported earlier

Carcasses 1 or 2 day old
Aspirate blood - MacFadyean stain for bacilli
Direct demonstration by IFA
Direct plating on blood agar
Putrefying carcasses
Blood, tissue and hide
Culture on selective medium
Soil sample from the areas where the carcass as lying
Serological assay
ELISA: based on anthrax toxin ( PA, LF and EF) for routine confirmation and
vaccine response)
Molecular techniques ( Only in the referral laboratories):
- RFLP
- PCR Fingerprinting
Animal Inoculation: Guinea pig and mice inoculation

Culture is confirmed by gamma phage lysis ( PlyG lysin enzyme- g phage)

IMMUNITY TO ANTHRAX
Resistance against anthrax vary from species to species
- Human are partially immune to anthrax
Resistance can be of two types
- Resistance to the establishment of infection but sensitive to toxin
- Resistance to toxin but susceptible to infection
Animals surviving naturally acquired anthrax are immune to reinfection
Protective antibodies against the anthrax toxin and against the capsule

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Resistance to Anthrax vary from species to species

Animal Infectio
mod
us
el
dose

Toxic dose
causing
death

Bacteria per ml
blood at time
death

Mouse

5 cells

1000 units/kg

107

Monkey

3000
cells

2500 unit/kg

107

Rat

106 cells

15 units/kg

105

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TREATMENT
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
Antibiotic treatment is effective in cutaneous anthrax

Inhalation anthrax can be effectively treated with antibiotics administered prior to

lymphatic spread or septicemia

INITIAL THERAPY

OPTIMAL THERAPY

Adults

Ciproflox
( 400mg iv BDX60days)

Penicillin G 4 mu iv qdsX60days
Doxycycline 100mg iv BDX60 days

Children

Ciproflox
20-30mg/kgbodywt ivX60days

Penicllin G 50,000 u/kg X 60 days

Alternatives Amox, Tetracycline, Chloramphenicol, Erythromycin, Streptomycin

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Vaccine against Anthrax

Killed bacilli and/or capsular antigens produce no significant immunity.
A nonencapsulated toxigenic strain (Sterne Strain) has been used effectively in
livestock.

Vaccine for humans: ( avirulent and nonencapsulated) sublethal amounts of the toxin
produced
Licensed in the U.S. is a preparation of the protective antigen (PA)
Dose:

A. 3 doses subcutaneously at the interval of 2 wks

B. Followed by three additional doses at 6,12 and 18 months
C. Annual booster dose
Who are to be vaccinated

- Professionals ( Veternarians, butcher, Zoo keeper, Wild life workers, Forest guards)
- Military personnels

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Anthrax and Biological Warfare

Countries > 10 countries in the world
Clouds of spores of Anthrax bacilli aerosol ( war heads filled with anthrax spores)
- Through dried spores in envelops
September 9/11 WTO attack
Postal workers affected Inhalation anthrax ( 40% mortality)
US Columbia, Florida, New Jersey, N. York
Other parts of the world
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