anthrax the anthrax bacillus, bacillus anthracis, was the first bacterium shown to be the cause of a...
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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
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
Robert Koch's original micrographs of the anthrax bacillus
BImal K Das, Microbiology, AIIMS
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
BImal K Das, Microbiology, AIIMS
McFadyean's reaction showing short chains of Bacillus anthracis cells lying among amorphous,disintegrated capsular material. White blood cells can also be seen.
BImal K Das, Microbiology, AIIMS
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
BImal K Das, Microbiology, AIIMS
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
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
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
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)
BImal K Das, Microbiology, AIIMS
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%
BImal K Das, Microbiology, AIIMS
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
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
BImal K Das, Microbiology, AIIMS
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
BImal K Das, Microbiology, AIIMS
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
BImal K Das, Microbiology, AIIMS
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
BImal K Das, Microbiology, AIIMS
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
BImal K Das, Microbiology, AIIMS
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
BImal K Das, Microbiology, AIIMS
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
BImal K Das, Microbiology, AIIMS