Characterization of the D/C mosaic neurotoxin produced by Clostridium botulinum associated with bovine botulism in Japan

Clostridium botulinum types C and D are related to avian and mammalian botulism. Bovine botulism occurred at various farms from 2004 to 2007 in Japan. Since culture supernatants of isolates from cases of bovine botulism were neutralized completely and partially with type D and C antitoxins, respectively, we attempted to confirm the nucleotide sequences of the neurotoxin gene in isolates. The neurotoxin gene comprised two-thirds of the type D neurotoxin gene and one-third of the type C neurotoxin gene, indicating that the neurotoxin of bovine isolates is a mosaic of type D and C neurotoxins, D/C mosaic neurotoxin. We prepared four sets of primers to differentiate the genes of the mosaic and authentic forms with PCR. The results showed that all bovine botulism-related isolates possess the gene for the D/C mosaic form. Pulsed-field gel electrophoresis analysis demonstrated that isolates from bovine botulism which had occurred between 2004 and 2007 were genetically homologous, except for the isolate from one area. We further examined the biological and antigenic properties of the D/C mosaic neurotoxin, which was found to exhibit the highest lethal activity in mice compared with other types of neurotoxins. In the D/C mosaic neurotoxin, three epitopes recognized by monoclonal antibodies that specifically react to and neutralize the toxin were located in the carboxyl-terminal domain of the heavy chain. These results indicate that D/C mosaic neurotoxin is a pathogenic agent causing bovine botulism and has unique characteristics different from other type C and D neurotoxins.