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

Keiji Nakamura, Tomoko Kohda, Yoshiyuki Seto, Masafumi Mukamoto, Shunji Kozaki

Research output: Contribution to journalArticle

15 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)881-890
Number of pages10
JournalVeterinary Microbiology
Volume162
Issue number2-4
DOIs
Publication statusPublished - Mar 23 2013
Externally publishedYes

Fingerprint

botulinum toxin
Immunologic Techniques
Genetic Techniques
Dilatation and Curettage
neurotoxins
Neurotoxins
immunologic techniques
botulism
Botulism
cattle
methodology
Clostridium botulinum D
serotypes
genes
Clostridium botulinum type D
Clostridium botulinum C
Clostridium botulinum type C
Immunochromatography
immunoaffinity chromatography
Genes

All Science Journal Classification (ASJC) codes

  • Microbiology
  • veterinary(all)

Cite this

Improved detection methods by genetic and immunological techniques for botulinum C/D and D/C mosaic neurotoxins. / Nakamura, Keiji; Kohda, Tomoko; Seto, Yoshiyuki; Mukamoto, Masafumi; Kozaki, Shunji.

In: Veterinary Microbiology, Vol. 162, No. 2-4, 23.03.2013, p. 881-890.

Research output: Contribution to journalArticle

Nakamura, Keiji ; Kohda, Tomoko ; Seto, Yoshiyuki ; Mukamoto, Masafumi ; Kozaki, Shunji. / Improved detection methods by genetic and immunological techniques for botulinum C/D and D/C mosaic neurotoxins. In: Veterinary Microbiology. 2013 ; Vol. 162, No. 2-4. pp. 881-890.
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