Detection of Clostridium botulinum neurotoxins A and B in milk by ELISA and immuno-PCR at higher sensitivity than mouse bio-assay

An outbreak of botulism was suspected at an Austrian dairy farm in June 2010. Six Simmental cows, out of a herd of 29, were affected and showed the typical signs. The affected cows included either animals suffering from sudden recumbency and reduced tongue tone or others which developed paresis, which gradually led to recumbency. Most of the affected animals died. Two cases were submitted to the Clinic for Ruminants at the University of Veterinary Medicine Vienna in order to clarify the cause of illness. The animals had reportedly been fed recently with silage possibly polluted by discarded cat carcasses spread onto the pasture used for the silage in question. The two referred cases both showed recumbency and swallowing difficulties. Both cows had to be euthanized. One of the two cows was in the late stage of pregnancy and a healthy calf could be delivered by Caesarean section. The outbreak of botulism was diagnosed clinically as well as by confirmation of Clostridium botulinum neurotoxins C and D by mouse bioassay. To the authors’ knowledge, this is the first report in which a live calf has been delivered out of a cow suffering from acute botulism.  

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Strategies to Design Inhibitors of Clostridium Botulinum Neurotoxins

Infectious Disorders - Drug Targets ◽

10.2174/187152607780090667 ◽

2007 ◽

Vol 7 (1) ◽

pp. 47-57 ◽

Cited By ~ 26

Author(s):

S. Cai ◽

B. Singh

Keyword(s):

Clostridium Botulinum ◽

Botulinum Neurotoxins

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An Atypical Outbreak of Food-Borne Botulism Due to Clostridium botulinum Types B and E from Ham

Journal of Clinical Microbiology ◽

10.1128/jcm.02942-14 ◽

2014 ◽

Vol 53 (2) ◽

pp. 722-726 ◽

Cited By ~ 23

Author(s):

Christelle Mazuet ◽

Jean Sautereau ◽

Christine Legeay ◽

Christiane Bouchier ◽

Philippe Bouvet ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Clostridium Botulinum ◽

Gene Sequencing ◽

Whole Genome ◽

Content Type ◽

Food Borne ◽

Botulinum Neurotoxins

An outbreak of human botulism was due to consumption of ham containing botulinum neurotoxins B and E. AClostridium botulinumtype E strain isolated from ham was assigned to a new subtype (E12) based onbont/Egene sequencing and belongs to a new multilocus sequence subtype, as analyzed by whole-genome sequencing.

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Immunoprecipitation of Native Botulinum Neurotoxin Complexes from Clostridium botulinum Subtype A Strains

Applied and Environmental Microbiology ◽

10.1128/aem.02817-14 ◽

2014 ◽

Vol 81 (2) ◽

pp. 481-491 ◽

Cited By ~ 8

Author(s):

Guangyun Lin ◽

William H. Tepp ◽

Marite Bradshaw ◽

Chase M. Fredrick ◽

Eric A. Johnson

Keyword(s):

Gastrointestinal Tract ◽

Clostridium Botulinum ◽

Botulinum Neurotoxin ◽

Protein Complexes ◽

Host Strain ◽

Mouse Serum ◽

Content Type ◽

Strain Nctc ◽

Botulinum Neurotoxins ◽

Subtype A

ABSTRACTBotulinum neurotoxins (BoNTs) naturally exist as components of protein complexes containing nontoxic proteins. The nontoxic proteins impart stability of BoNTs in the gastrointestinal tract and during purification and handling. The two primary neurotoxin complexes (TCs) are (i) TC1, consisting of BoNT, nontoxin-nonhemagglutinin (NTNH), and hemagglutinins (HAs), and (ii) TC2, consisting of BoNT and NTNH (and possibly OrfX proteins). In this study, BoNT/A subtypes A1, A2, A3, and A5 were examined for the compositions of their TCs in culture extracts using immunoprecipitation (IP). IP analyses showed that BoNT/A1 and BoNT/A5 form TC1s, while BoNT/A2 and BoNT/A3 form TC2s. AClostridium botulinumhost strain expressing recombinant BoNT/A4 (normally present as a TC2) from an extrachromosomal plasmid formed a TC1 with complexing proteins from the host strain, indicating that the HAs and NTNH encoded on the chromosome associated with the plasmid-encoded BoNT/A4. Strain NCTC 2916 (A1/silent B1), which carries both anhasilentbont/bcluster and anorfX bont/a1cluster, was also examined. IP analysis revealed that NCTC 2916 formed only a TC2 containing BoNT/A1 and its associated NTNH. No association between BoNT/A1 and the nontoxic proteins from the silentbont/bcluster was detected, although the HAs were expressed as determined by Western blotting analysis. Additionally, NTNH and HAs from the silentbont/bcluster did not form a complex in NCTC 2916. The stabilities of the two types of TC differed at various pHs and with addition of KCl and NaCl. TC1 complexes were more stable than TC2 complexes. Mouse serum stabilized TC2, while TC1 was unaffected.

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Recombinant Holotoxoid Vaccine against Botulism

Infection and Immunity ◽

10.1128/iai.00843-07 ◽

2007 ◽

Vol 76 (1) ◽

pp. 437-442 ◽

Cited By ~ 42

Author(s):

Christina L. Pier ◽

William H. Tepp ◽

Marite Bradshaw ◽

Eric A. Johnson ◽

Joseph T. Barbieri ◽

...

Keyword(s):

Immune Response ◽

Heavy Chain ◽

Cortical Neurons ◽

Clostridium Botulinum ◽

Vaccine Candidate ◽

Primary Cortical Neurons ◽

Current Vaccine ◽

Botulinum Neurotoxins ◽

Double Amino Acid ◽

Limited Supply

ABSTRACT The botulinum neurotoxins (BoNT) are the most toxic proteins for humans and designated “Category A Select Agents.” The current vaccine against botulism is in limited supply, and there is a need to develop new vaccine strategies. A recombinant BoNT/A toxoid was produced in Clostridium botulinum that contained a double amino acid substitution, R363A Y365F (termed BoNT/ARYM). BoNT/ARYM was noncatalytic for SNAP25 and nontoxic for mice. Immunization with BoNT/ARYM protected mice from challenge at levels that were similar to chemically inactivated BoNT/A toxoid. BoNT/ARYM elicited an immune response against the light-chain and heavy-chain components of the toxin. Neutralizing anti-BoNT/ARYM sera blocked BoNT toxicity in primary cortical neurons and blocked ganglioside binding by the heavy chain. BoNT/ARYM represents a viable vaccine candidate for a holotoxoid against botulism.

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