BIOLOGICAL WARFARE

 Biological weapons of mass destruction BWMD-poor mans

atomic bomb
 Live stocks of small pox-USA, Russia(eradication-1980)
 17 countries 1995,1969-Nixon
 Anthrax-perfect germ for bioterrorism , dry powder, persist
for 36 yr-Gruinard Scotland –british-1942=2 WW=1986-
1990 ,1979-Sverdlovsk-100 death,2001 sept11 US mail
 ANTHRAX

• The anthrax bacillus, Bacillus anthracis, was the first

bacterium shown to be the cause of a disease ,grown in
pure culture- Koch’s Postulate -proved germ theory of
disease.
• 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 Zoonosis- disease of domesticated and wild
animals.
• Men suffer from anthrax occasionally due to close
contact with infected animal or animal products
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.
McFadyean's reaction showing short chains of Bacillus
anthracis cells lying among amorphous,disintegrated
capsular material. White blood cells can also be seen.
 Bacillus anthracis
• Morphology-Gram positive rods, size 3-10 × 1.6 micron.non motile

• Capsulated ( Protein - ve charge resist phagocytosis ; PP- glutamic acid) –

Capsule form in animal tissue and in special laboratory condition ( 5% CO2) or Media
bicarb ,serum , albumin , charcoal , starch.

• Forms endospore at the end of exponential growth phage competence and

sporulation factor -csf - quorum sensing, centrally located, eliptical , oval-bamboo
stick appearance, do not form in live animal tissues - O2,water.
• Mac Fadyean Reaction- ( Polychrome methylene blue) stain bacilli blue.
with purple capsule-amorphous purplish material around bacilli representing
capsular material.
String of pears reaction-in presence of 0.05 U penicillin cell become large , spherical
• cultural characteristics-Aerobic/ Facultative anerobe,35-37°c.
•Grows on all ordinary medium (Medusa head appearance-uneven
wavy margin due to interlacing chain of bacilli)-irregular,2-3 mm,frosted glass ap.
• Inverted fur tree appearance in gelatin stab culture medium-slow.
• Biochemicals : Catalase +, reduces nitrate to nitrite, lecithinase +,
glucose, maltose, sucrose, fermented produce – acid.
Selective media-PLET-HIA
Differential Characteristics of B. anthracis B. cereus and B. thuringiensis

Characteristic B. anthracis Anthracoid Bacilli

fast+
Gelatin hydrolysis slow+

hemolysis on sheep blood agar - +

glutamyl-polypeptide capsule + -

lysis by gamma phage + -

motility - +

growth on chloralhydrate agar - +

string-of-pearls test + -
Resistance

• SPORES SURVIVE FOR MANY YEARS ( DRY STATE AND SOIL )-35
•In bone marrow-1 wk,skin-2 wk

• Moist heat kills – Vegetative cells 60 0 C X 30 minutes

survive- Spores 100 0 C X 10 minutes, 5%phenol,Hgcl2

2% Formaldehyde kills spores-DUCKERING(hair , wool)

4% KMnO4 kills spores

Hypochlorite ( 0.5%) commercially available kills spores

(1 part household bleach to 9 parts water)
 Epiedemiology
• Distribution worldwide, cattle & sheep, horses, swine.
•frog resistant
•India has largest live stock population in the world-south india.
• 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 highly 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 insects flies ( Zimbabwe)-stomoxys spp.
- Vultures
- Contaminated surface water pool
 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
 VIRULENCE FACTORS

Protein capsule – Poly D Glutamic acid capsule

- Inhibits phagocytosis ( Unencapsulated strains –
nonpathogenic)

Anthrax Toxin – Complex of proteins ( all the components thermolabile)

A. Protective antigen
B. Edema factor
C. Lethal Factor

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.

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

 Human 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)

Clinically three forms of Human anthrax occur

A. Cutaneous anthrax
B. Pulmonary anthrax
C. Intestinal anthrax
 Cutaneous Anthrax

• Mainly in professionals( Veterinarian, butcher, Zoo keeper)

• Spores infect skin- a characteristic gelatinous edema develops at the

site (1-3 d Papule- Vesicle-Malignant Pustule- Necrotic ulcer cover with black
eschar)

• 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 ingestion of infected carcasses

• Mucosal lesion to the lymphatic system –violent enteritis

• Rare in developed countries

• Extremely high mortality rate -50%

 Anthrax: Cutaneous
Vesicle Day 6
development
Day 4
Day 2

Day 10

Eschar
formation
 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-pneumonia.

• 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%

Anthrax: Inhalational

Mediastinal widening
 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, 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
 B. anthracis:
Presumptive Identification

Clinical specimen (blood, CSF, etc.)

Gram stain Isolate on SBA

Capsule production
Colony
morphology
Hemolysis
Motility Gram stain
Spores Malachite green
 B. anthracis:
Confirmatory Identification

Isolate

Capsule DFA
Phage
lysis Capsule antigen
Horse Bicarbonate
Cell wall
blood media
(M’Fadyean (M’Fadyean stain
Stain) India ink stain)
 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.
 TREATMENT
Antibiotics should be given to unvaccinated individuals exposed to inhalation
anthrax.

Antibiotic treatment is effective in cutaneous anthrax.

Inhalation anthrax can be effectively treated with Penicillin, tetracyclines and

fluoroquinolones 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

 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
Immune Protection Against Anthrax
 Live cellular vaccines
 "Sterne" type live spore (toxigenic, noncapsulating)
 Former USSR STI live spore (toxigenic, non-
capsulating)
 "Pasteur" type (attinuated mixed culture, reduced
virulence)
 Sterile, acellular vaccines
 US "anthrax vaccine adsorbed" (AVA)—not licensed
for use in civilian populations
 UK "anthrax vaccine precipitated" (AVP)
 Recombinant PA research vaccines
 AI3+; Freund’s; Saponin, Monophosphoryl lipid A;
Ribi
Bacillus cereus

Essentials of Medical Microbiology by Apurba S

Sastry
© 2018, Jaypee Brothers Medical Publishers
Bacillus cereus
 Normal habitant of soil
 Widely isolated from vegetables, milk, cereals, spices, meat &
poultry
 Food poisoning
 Diarrheal toxin (causes diarrheal type of food poisoning)
 Emetic toxin (causes emetic type of food poisoning)
 Ocular disease - Severe keratitis & panophthalmitis following
trauma to the eye

Essentials of Medical Microbiology by Apurba S

Sastry
© 2018, Jaypee Brothers Medical Publishers
Bacillus cereus Food Poisoning
B.cereus Diarrheal type Emetic type
Incubation 8-16 hours 1-5 hours
period
Toxin Secreted in intestine Preformed toxin
(Similar to (formed in diet,
Clostridium similar to S.aureus
perfringens enterotoxin)
enterotoxin)
Heat labile Heat stable
Food items Meat, vegetables, Rice (Chinese
contaminated dried beans, cereals
Essentials of Medical Microbiology by Apurba S
fried rice)
Clinical
Sastry
Diarrhea, fever , rarely
© 2018, Jaypee Brothers Medical Publishers
Vomiting,
Laboratory Diagnosis & Treatment
 ™
Sample – feces
 Culture isolation
- MYPA (mannitol, egg yolk, polymyxin, phenol red and agar)
- PEMBA (polymyxin B, egg yolk, mannitol, bromothymol blue,
agar)
 ™
Motile, non-capsulated & not susceptible to gamma phage

Essentials of Medical Microbiology by Apurba S

Sastry
© 2018, Jaypee Brothers Medical Publishers
 Treatment of Bacillus cereus
- Susceptible to clindamycin, erythromycin, vancomycin,
aminoglycosides and tetracycline
- Resistant to penicillin (by producing β-lactamase) and
trimethoprim

Essentials of Medical Microbiology by Apurba S

Sastry
© 2018, Jaypee Brothers Medical Publishers
Summary of B. cereus Infections
Summary of B. cereus Infections (cont.)

REVIEW
 Foodborne Diseases of B. cereus

(Intoxication) (Foodborne Infection)

 Other Bacillus spp.
 Bacillus thuringiensis
• Endotoxin-BT corn; Other GMO’s (genetically modified
organisms)

 Bacillus stearothermophilus
• Spores used to test efficiency of killing in
autoclaves,B.pumilus-Radiation,
•B.subtilis-fumigation ,biotin , riboflavin, rope in bread
• Bacillus licheniformis- bacitracin,B.megaterium-
B12,B2,industrial enzyme,
 Anthrax Vaccines
Indication Route Dosing Schedule
Pre-exposure Intramuscular Primary series:
prophylaxis for (0.5 mL/dose) 0,1, and 6
persons months
at high risk of Boosters: at 6 and
exposure 12
month after
primary
series and then
yearly
Post-exposure Subcutaneous 0, 2, and 4 weeks
prophylaxis (0.5 mL/dose)
Essentials of Medical Microbiology by Apurba S Sastry
© 2018, Jaypee Brothers Medical Publishers
postexposure
 Anthrax bacilli v/s Anthracoid bacilli
Anthrax bacilli Anthracoid bacilli
Motility Non motile Motile
Capsule Present Absent
Bacilli In long chain In short chain
Under low Medusa head colony Not seen
power seen
microscope
Blood agar No hemolysis Hemolytic colony
Broth
Essentials of MedicalTurbidity
Sastry
absent
Microbiology by Apurba S Usually turbid
© 2018, Jaypee Brothers Medical Publishers
 Anthrax bacilli v/s Anthracoid bacilli
Anthrax bacilli Anthracoid bacilli
Salicin Not fermented Fermented
Gamma Susceptible Resistant
phage
Gelatin stab Inverted fir tree Not seen
agar appearance seen. Rapid gelatin
Gelatin liquefaction liquefaction
slow
Solid String of pearls No growth
medium
Essentialswith
of Medicalappearance
Microbiology by Apurba S
Sastry
penicillin
© 2018, Jaypee Brothers Medical Publishers