DETECTION OF CLOSTRIDIUM-BOTULINUM IN FISH AND ENVIRONMENTAL-SAMPLES USING POLYMERASE CHAIN-REACTION

A test protocol for the detection and enumeration of Clostridium botul inum in fish and sediment samples with specific identification of neur otoxin types A, B, E and F was developed. Specific amplification produ cts generated by polymerase chain reaction (PCR) formed the basis of i dentification of the toxin-producing organism, whereas quantification of the results was achieved with the most probable number (MPN) method . Twenty-six C. botulinum strains studied with PCR assays after enrich ment in trypticase-peptone-glucose-yeast extract (TPGY) broth gave ide ntical results as with the mouse bioassay. The suitability of the dete ction method for food and environmental surveys was assessed by runnin g it on 32 samples of rainbow trout inoculated with spore loads rangin g from 10(2) to 10(6) C. botulinum type E spores per kg. The organism was detected in all samples, and MPN estimates corresponded well to in oculum levels. In order to assess possible natural contamination, 16 f ish and 16 visceral samples of rainbow trout, as well as ten aquatic s ediment samples were tested. Of these, eight (80%) of the sediment sam ples were positive, with estimated spore counts of C. botulinum type E ranging from 95-2710 per kg sample.