VMC 311: Systematic Veterinary Bacteriology and Mycology (2+1)

DIAGNOSIS

Clinical symptoms

• Blood films from dead animals made by puncturing the superficial vein of the ear or in the region of the foot.
• Care should be taken to seal the injection site by placing cotton soaked in alcohol and ignited.
• The smears are heat fixed and stained by Wright's or Giemsa’s stain to reveal B. anthracis as large blue rods with characteristic dark pink or purple coloured capsules.
• In case of horses and pigs since peripheral blood contains fewer organisms, smears should be made from the edematous fluid or LN’s.

Cultural examination

• Swabs from blood are inoculated on to blood agar plates and incubated at 37°C for 24 hrs and examined for their typical growth.
• Swabs are inoculated in agar enriched with blood or serum and incubated for 6 hrs at 37°C and examined by stained smears.

Bacteriological examination of hair, wool, hide, bone, bone meal & others

• Samples are added to cold saline and shaken intermittently for 3 hours.
• The supernatant fluid is then heated to 70°C for 10 minutes.
• Then they are filtered through two layers of muslin cloth, added to melted agar, and poured into petridishes, allowed to set and incubated at 37°C.
• After 12 hours incubation, plates are to be examined for characteristic colony morphology.

Ascoli’s Precipitation test: (Ascoli 1911)

• Grind up the organ or blood of suspected animal and suspend in 5-10 parts of saline and boil for 15 minutes.
• Filter through filter paper and allow it to cool. Place 0.5 ml of anti-anthrax serum (1:50) in a small test tube and overlay with 0.5 ml of clear filtrate.
• Stand at room temperature for 15 minutes. A white ring of precipitation indicates a positive reaction.

String of Pearl’s test: (Charlton,1980)

• B.anthracis produces swollen round cells in chains (string of pearls) when incubated for 3-6 hrs on tryptose agar containing 0.05 –0.5 I.U of penicillin/ml.
• To 100ml of molten nutrient agar, add required Sodium beznyl penicillin and mix carefully.
• Pour into petridishes and allow to set. With a scalpel cut a block about 1.6 cm² from the penicillin agar plate and place it on a microscopic slide in a petridish containing a small piece of moistened absorbent cotton wool to prevent the agar drying out.
• Use a young colony to streak the center of the agar block.
• Place a clean cover slip on the agar block and incubate the petridish at 370C.
• After 2 hrs, remove the slide and examine the inoculum microscopically by oil immersion for the string of pearls growth.
• Differentiation from non-pathogenic Bacillus sp. It is considered reliable but needs experience.
• Cherry gamma phage is used. It can be propagated on B. anthracis strain 14, which makes a suitable control.
• On one half of blood agar plate, streak B. anthracis strain 14 culture as control and on the other half streak the culture to be identified.
• Add drops of phage preparation (1:10 dilution) on both halves. Incubate the plates in the upright position for 8-12 hrs.
• Zones of clearing will be seen on control culture and on the suspected half if it is B. anthracis.

Animal Inoculation

• Guinea pigs & mice are highly susceptible. Materials are inoculated by dermal scarification, subcutaneous or intramuscular.
• Death occurs in 2-3 days and the organisms will be readily identified in the blood & tissues.
• Apart from this laser pyrolysis, gas-liquid chromatography and mass spectrometry are used for detection of anthrax toxin productions.