INVESTIGATION OF ANIMAL BOTULISM OUTBREAKS BY PCR AND STANDARD METHODS

A double PCR procedure is proposed for identification of Clostridium b otulinum C and D. This method consists of a first PCR amplification wi th a degenerate primer pair able to amplify a 340 bp common DNA fragme nt from botulinum neurotoxin (BoNT) C1 and D genes, followed by two su bsequent PCR amplifications with two primer pairs specific for BoNT/Cl and D respectively (198 bp DNA fragment). This method was found to be specific for C. botulinum C and D, amongst 81 strains of C. botulinum and 21 different species of other Clostridium and bacteria tested. Th e detection limit ranged from 10 to 10(3) bacteria in the reaction vol ume according to the C. botulinum C and D strains. In 160 naturally co ntaminated animal and food samples submitted to a 48 h enrichment cult ure, the double PCR showed an 89.4% correlation rate with the standard mouse bioassay. A clear distinction between botulism type C and D was obtained. The double PCR provides a reliable alternative for detectio n and identification of C. botulinum C and D in clinical and food samp les.