anthrax the anthrax bacillus, bacillus anthracis, was the first bacterium shown to be the cause of a...

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

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


Robert Koch's original micrographs of the anthrax bacillus


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. BImal K Das, Microbiology, AIIMS

Bacillus thuringiensis. Phase Photomicrograph of vegetative cells, intracellular spores (light) and parasporal crystals (dark).

Bacillus cereus

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


McFadyean's reaction showing short chains of Bacillus anthracis cells lying among amorphous,disintegrated capsular material. White blood cells can also be seen.


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 - +

string-of-pearls test + -

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


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


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


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


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 FormsBImal K Das, Microbiology, AIIMS

Clinically three forms of Human anthrax occur

A. Cutaneous anthraxB. Pulmonary anthraxC. 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)


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

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%


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


VIRULENCE FACTORS

Anthrax Toxin – Complex of proteins ( all the components thermolabile)A. Protective antigenB. Edema factorC. 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 & EFBImal K Das, Microbiology, AIIMS

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

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


LABORATORY DIAGNOSISFew 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 maskDisposable 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


SAMPLES FROM ANIMAL

Sudden death of animal in areas where anthrax was reported earlier

Carcasses 1 or 2 day oldAspirate blood - MacFadyean stain for bacilliDirect demonstration by IFADirect plating on blood agar

Putrefying carcassesBlood, tissue and hideCulture on selective mediumSoil 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

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


Animal model

Infectious dose

Toxic dose causing death

Bacteria per ml blood at time death

Mouse 5 cells 1000 units/kg 107

Monkey3000

cells2500 unit/kg 107

Rat 106 cells 15 units/kg 105

Resistance to Anthrax vary from species to species


TREATMENTAntibiotics 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 Ciproflox20-30mg/kgbodywt ivX60days Penicllin G 50,000 u/kg X 60 days

Alternatives – Amox, Tetracycline, Chloramphenicol, Erythromycin, Streptomycin


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 wksB. Followed by three additional doses at 6,12 and 18 monthsC. Annual booster dose

Who are to be vaccinated

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


Anthrax and Biological Warfare

Countries > 10 countries in the worldClouds of spores of Anthrax bacilli – aerosol ( war heads filled with anthrax spores)

- Through dried spores in envelopsSeptember 9/11 WTO attackPostal workers affected – Inhalation anthrax ( 40% mortality)US – Columbia, Florida, New Jersey, N. YorkOther parts of the world


anthrax the anthrax bacillus, bacillus anthracis, was the first bacterium shown to be the cause of a...

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