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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

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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

BImal K Das, Microbiology, AIIMS


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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

BImal K Das, Microbiology, AIIMS



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Bacillus anthracis. Gram stain. The cells have characteristic squared ends.The endospores are ellipsoidal shaped and locatedcentrally in the sporangium. The spores are highly refractile tolight and resistant to staining.

BImal K Das, Microbiology, AIIMS


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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 Photomicrographof vegetative cells, intracellular spores (light) andparasporal crystals (dark).

BImal K Das, Microbiology, AIIMS


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McFadyean's reaction showing short chains of Bacillusanthracis cells lying among amorphous,disintegratedcapsular material. White blood cells can also be seen.

BImal K Das, Microbiology, AIIMS


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

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

BImal K Das, Microbiology, AIIMS


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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

BImal K Das, Microbiology, AIIMS


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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

BImal K Das, Microbiology, AIIMS


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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

BImal K Das, Microbiology, AIIMS


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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 LymphaticsGerminate inside Macrophages

Release

Vegetative Forms

BImal K Das, Microbiology, AIIMS


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  • Clinically three forms of Human anthrax occur

  • Cutaneous anthrax

  • Pulmonary anthrax

  • 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)

BImal K Das, Microbiology, AIIMS


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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

BImal K Das, Microbiology, AIIMS


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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%

BImal K Das, Microbiology, AIIMS


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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

BImal K Das, Microbiology, AIIMS


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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

BImal K Das, Microbiology, AIIMS


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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.

BImal K Das, Microbiology, AIIMS


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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

BImal K Das, Microbiology, AIIMS


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  • LABORATORY DIAGNOSIS plasmids

  • 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

BImal K Das, Microbiology, AIIMS


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SAMPLES FROM ANIMAL plasmids

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)

BImal K Das, Microbiology, AIIMS


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IMMUNITY TO ANTHRAX plasmids

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

BImal K Das, Microbiology, AIIMS


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Resistance to Anthrax vary from species to species plasmids

BImal K Das, Microbiology, AIIMS


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TREATMENT plasmids

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

BImal K Das, Microbiology, AIIMS


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  • Vaccine against Anthrax plasmids

  • 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

BImal K Das, Microbiology, AIIMS


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Anthrax and Biological Warfare plasmids

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

BImal K Das, Microbiology, AIIMS