title: Improved detection methods by genetic and immunological techniques for botulinum C/D and D/C mosaic neurotoxins.
abstract: 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 LD(50), 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.
authors.affiliation: Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai-Kita, Izumisano, Osaka 598-8531, Japan.
authors.forename: Keiji
authors.initials: K
authors.lastname: Nakamura
authors.affiliation: Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai-Kita, Izumisano, Osaka 598-8531, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kohda
authors.affiliation: Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai-Kita, Izumisano, Osaka 598-8531, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Seto
authors.affiliation: Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai-Kita, Izumisano, Osaka 598-8531, Japan.
authors.forename: Masafumi
authors.initials: M
authors.lastname: Mukamoto
authors.affiliation: Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai-Kita, Izumisano, Osaka 598-8531, Japan. Electronic address: kozaki@center.osakafu-u.ac.jp.
authors.forename: Shunji
authors.initials: S
authors.lastname: Kozaki
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D000918:Antibody Specificity; D001715:Bird Diseases / Q000382:microbiology*; D001905:Botulinum Toxins / Q000032:analysis / Q000145:classification* / Q000276:immunology / Q000378:metabolism; D001906:Botulism / Q000175:diagnosis / Q000662:veterinary*; D002417:Cattle; D002418:Cattle Diseases / Q000382:microbiology*; D002845:Chromatography / Q000379:methods; D003014:Clostridium botulinum / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007158:Immunologic Techniques; D008297:Male; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; C096325:botulinum toxin type D; D001905:Botulinum Toxins; C096324:botulinum toxin type C