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: Keiji Nakamura, Tomoko Kohda, Yoshiyuki Seto, Masafumi Mukamoto, Shunji Kozaki
• Journal: Veterinary microbiology (Vet Microbiol) Vol. 162 Issue 2-4 Pg. 881-890 (Mar 23 2013) ISSN: 1873-2542 [Electronic] Netherlands
• PMID: 23206412 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2012 Elsevier B.V. All rights reserved.

Chemical References

• Antibodies, Monoclonal
• botulinum toxin type D
• Botulinum Toxins
• botulinum toxin type C

Topics

• Animals
• Antibodies, Monoclonal (chemistry, immunology)
• Antibody Specificity
• Bird Diseases (microbiology)
• Botulinum Toxins (analysis, classification, immunology, metabolism)
• Botulism (diagnosis, veterinary)
• Cattle
• Cattle Diseases (microbiology)
• Chromatography (methods)
• Clostridium botulinum (classification, genetics, isolation & purification, metabolism)
• Immunologic Techniques
• Male
• Mice
• Polymerase Chain Reaction (methods)