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. (C) 2012 Elsevier B.V. All rights reserved.