Toxicological, Genetic and Physiological analyses of neurotoxigenic Clostridium butyricum from food-borne botulism and environment origins

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1. Genetic and physiological analyses(1) Six strains derived from food-borne botulism cases in China, five strains from soil of Weishan lake area, China, and two strains from infant botulism cases in Italy were analyzed with random amplified polymorphic DNA assay, pulsed-field gel electrophoresis, and Southern blot hybridization for the toxin gene. Test strains were divided into three clusters in accordance with the origin. This typing agreed to fermentation patterns of arabinose and inulin.(2) Nucleotide sequences of the toxin gene of eleven Chinese strains and a Italian strain were determined. Nucleotide sequences of the toxin gene of eleven Chinese strains were identical. The amino acid identity between Chinese and Italian strains was 95.0% and that between Chinese strains and C. botulinum type E was 96.9%.2. Toxicological analysisThere is antigenical diversity between C. butyricum neurotoxins from an Italian strain (BL5262) and from a Chinese strain from food-borne botulism (KZ1885 … More ). ELISA and immunoblotting analyses with the monoclonal antibodies (mAbs) against C. botulinum type E neurotoxin and BL5262 neurotoxin indicated that the light chain of KZ1885 neurotoxin possesses the same antigenical characteristics as that of type E neurotoxin, while the heavy chain shares partially antigenically different portion. The mAbs reacting specifically to BL5262 neurotoxin heavy chain hardly recognized KZ1885 neurotoxin. These observations was also confirmed by nucleotide sequencing. Furthermore, the difference of amino acid sequences between KZ1885 and type E neurotoxins appeared to be concentrated at the carboxyl-terminal portion of the heavy chain. The specific toxicity of KZ1885 neurotoxin was about 15 times lower than that of type E neurotoxin.3. Conclusion(1) It is likely that neurotoxigenic C. butyricum are clonally distributed over each vast area.(2) C. butyricum KZ1885 neurotoxin is structurally, antigenically much similar to C. botulinum type E neurotoxin, in comparison with C. butyricum BL5262 neurotoxin. Less