1. ANTHRAX The anthrax bacillus, Bacillus anthracis, was the first
bacterium shown to be the cause of a disease- Koch’s
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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2. 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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3. Bacillus anthracis Strain, non-hemolytic on sheep blood agar. B
Bacillus anthracis Strain, non-hemolytic on sheep blood agar. B. anthracis colony characteristics:2-5mm overnight at 35 degrees centigrade without carbon dioxide Non-hemolytic, non-pigmented, dry ground glass surface, edge irregular with comma projections, “Medusa Head”.

4. Robert Koch's original micrographs of the anthrax bacillus
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5. Bacillus anthracis. Gram stain
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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6. 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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7. 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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8. B. cereus and B. thuringiensis
Differential Characteristics of B. anthracis B. cereus and B. thuringiensis
Characteristic
B. anthracis
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
Differential Characteristics of B. anthracis B. cereus and B. thuringiensis
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10. Physical properties ( methods for decontamination)
SPORES SURVIVE FOR MANY YEARS ( DRY STATE AND SOIL )
Moist heat kills – Vegetative cells C X 30 minutes
Spores C X 10 minutes
4% Formaldehyde kills spores
Hypochlorite ( 0.5%) commercially available kills spores
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11. Distribution worldwide
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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12. (Abrasion, inhalation, ingestion)
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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13. Clinically three forms of Human anthrax occur
Cutaneous anthrax
Pulmonary anthrax
Intestinal anthrax
Broadly can be classified into
Non Industrial/Agricultural ( Through infected animals):
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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14. Mainly in professionals( Veterinarian, butcher, Zoo keeper
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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16. Require very high infective dose ( > 10,000 spores)
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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17. Anthrax Toxin – Complex of proteins ( all the components thermolabile)
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
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18. ( 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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20. Enzyme function of LF and EFOnce in the cytosol, the EF and LF then carry out their respective damage-inducing processes.[9]
EF acts as a Ca2+ and calmodulin dependent adenylate cyclase that greatly increases the level of cAMP in the cell. This increase in cAMP upsets water homeostasis, severely throws the intracellular signaling pathways off balance, and impairs macrophage function, allowing the bacteria to further evade the immune system.
LF also helps the bacteria evade the immune system through killing macrophages. Once in these cells, LF acts as a Zn2+-dependent endoprotease that snips off the N-terminus ofmitogen-activated protein kinase kinases (MAPKK). This inhibits these kinases by not allowing them to efficiently bind to their substrates, which leads to altered signaling pathways and ultimately to apoptosis.
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21. Virulence of Anthrax bacillus is due to presence of two plasmids
px01 – Toxin encoding plasmid
- 110 megadalton
- temperature-sensitive plasmid
px 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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22. Anthrax is not highly contagious
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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23. Sudden death of animal in areas where anthrax was reported earlier
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 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)
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24. Resistance against anthrax vary from species to species
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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25. Resistance to Anthrax vary from species to species
Animal model 
Infectious 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
Rat
106 cells
15 units/kg
105
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26. Antibiotic treatment is effective in cutaneous anthrax
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 Penicillin G 4 mu iv qdsX60days
( 400mg iv BDX60days) 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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27. 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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28. 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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