Studies on Isolation and Identification of Clostridium botulinum Investigating Field Samples Specially from Equine Grass Sickness Cases

Isolation and identification of Clostridium botulinum from field samples obtained from: i) equine grass sickness (EGS) cases (animal and environmental samples) together with control animal samples; and ii) classical botulism-suspected cases, was an objective. Using mouse bioassay, MB-ELISA and PCR to identify the isolates, out of 288 samples investigated, only four isolates were confirmed to be C. botulinum and all from samples other than EGS samples. This result could show that C. botulinum is at least not prevailing in GS samples and its association with the disease remains doubtful. Two of the confirmed C. botulinum isolates were type C and the other two were type D. The two type C isolates were from intestinal contents of two broiler chickens from a farm with botulism outbreak; one of the type D isolates was from intestine of a case of bovine botulism and the other from faeces of a rabbit experimentally infected with C. botulinum type D together with other bacteria. The colonies of the two type C and the bovine type D isolate are not the same and the cells can swarm if the plates are not dry enough. So, these isolates may be different strains and because of their swarming character, which was not found reported, they may require further study to see if they were isolated before or not.Toxins of 53 isolates were neutralized by botulinum antitoxins by mouse bioassay, 27 were from GS samples (24 from GS cases samples and three from control samples). The neutralized isolates were lipase-positive clostridia (23), lecithinase-positive clostridia (23) and E. coli isolates (7). Only four of them (from non-EGS samples) were confirmed to be specific. Ten of the lipase-positive isolates were from GS samples (all from case samples), ten of the lecithinase-positive isolates were from GS samples (nine from case samples and one from control samples) and all the seven E. coli isolates were from GS samples (five from case samples). Fourteen of the 53 isolates were found contaminated with C. tetani, which was proved to be the cause of their false-positive results. Tetanus toxin from several C. tetani isolates was neutralized by different botulinum antitoxins (A-F), but mainly by polyvalent type ABE (AVENTIS BEHRING) and type CD mixture (OVI). The neutralization results showed by E. coli, C. perfringens-like and C. tetani and the association of these species with the GS case samples and not with the control samples was considered to have a direct or indirect role in the aetiology of EGS. Also, it was observed that types and numbers of some other Clostridium spp. are more prevalent in GS case samples than in control samples.The isolates with a positive result in the mouse bioassay were tested by PCR. Only six of them were positive by PCR, three type C and three type D. One type C and one type D isolate were lipase-negative and their result was not reproduced.The isolates tested by MB-ELISA were only 28, from those neutralized by type CD botulinum antitoxin mixture in the bioassay. Only three isolates had positive results, which were also positive by PCR. Two of them type C and one type D. MB-ELISA was specific, but less sensitive than other diagnostic means.The isolation and identification results showed that bovine botulism in Germany could be due to types D and C and avian botulism due to type C.The current study recommends the usage of Fastidious Anaerobic Agar medium for isolation of Clostridium species, including C. botulinum, C. perfringens and C. tetani. The inhibitory effect of other bacteria was confirmed by the finding that none of the four C. botulinum isolates in this study was obtained after enrichment in liquid medium; they were isolated either by direct plating, direct heat or alcohol treatment.