Systematic Veterinary Bacteriology and Mycology

DIAGNOSIS

• The diagnosis of botulism is based on history, clinical signs and demonstration and identification of toxin in serum of moribund or recently dead animals as well as the detection of toxin and /or C.botulinum in the suspected foodstuff.

Toxin demonstration

• Serum or centrifiuged serum exudates from animals can be directly inoculated i/v ly (0.3ml) or i/p ly (0.5ml) into mice.
• If toxin is present the characteristic wasp waist appearance in the mice will be seen in a few hrs or upto 5 days.
• The appearance is due to abdominal breathing because of paralysis of respiratory muscles.
• Extraction of toxin in foodstuffs is accomplished by grinding the material in saline.
• The suspension is centrifuged and the supernatant is filtered through a 0.45μm filter.
• As the toxin can be in a protoxin form 9 parts of filtrate are treated with one part of 1% trypsin solution and incubated at 37⁰C for 45mts.
• Mice or guinea pigs are inoculated intra-peritoneally.

Toxin identification

• Mouse (or guinea pig) neutralization tests using a polyvalent antitoxin initially, followed by monovalent antitoxin.

Isolation of C.botulinum from foodstuffs

• Several samples of the foodstuffs are macerated in a small amount of physiological saline.
• The suspension is heated at 65-80⁰C for 30mts to kill most of the contaminating organism and to induce the C.botulinum spores to germinate.
• Blood agar plates are inoculated with the suspension and incubated under Co₂ at 35⁰C for up to 5 days.
• To determine whether the isolate is a toxin producing strain, a cooked meat broth is inoculated and incubated at 30⁰C for 5-10 days.
• Filtrates are prepared and lab. animals can be used for demonstration and identification of the toxin.