Improved detection methods by genetic and immunological techniques for botulinum C/D and D/C mosaic neurotoxins

Clostridium botulinum type C and D strains produce serotype-specific or mosaic botulinum neurotoxin (BoNT). Botulinum C/D and D/C mosaic neurotoxins (BoNT/CD and /DC) are related to avian and bovine botulism, respectively. The two mosaic BoNTs cannot be differentiated from authentic type C and D BoNTs by the conventional serotyping method. In this study, we attempted to establish novel methods for the specific detection of BoNT/CD or/DC. Comparison with nontoxic component genes in type C and D strains revealed that the nucleotide sequence of the ha70 gene is well conserved among either serotype-specific or mosaic BoNT-producing strains. A multiplex PCR method with primers for the light chain of boNT, ntnh, and ha70 gene detection was developed for typing of the boNT gene in type C and D strains. Upon applying this method, twenty-seven type C and D strains, including authentic strains and the isolates from avian and bovine botulism, were successfully divided into type C, C/D mosaic, type D, and D/C mosaic BoNT-producing strains. We then prepared an immunochromatography kit with specific monoclonal antibody showing high binding affinity to each mosaic BoNT. BoNT/CD and /DC in the culture supernatant were detected with limits of detection of 2.5 and 10 LD50, respectively. Furthermore, we confirmed the applicability of the kit for BoNT/DC using crude culture supernatant from a specimen from a bovine suspected of having botulism. These results indicate that the genetic and immunological detection methods are useful for the diagnosis of avian and bovine botulism.