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

1.（1）采用随机扩增多态性DNA（RAPD）、脉冲场凝胶电泳（PFGE）和Southern印迹杂交（Southern blot）技术，对我国6株食源性肉毒杆菌中毒菌株、5株微山湖地区土壤肉毒杆菌中毒菌株和2株意大利婴儿肉毒杆菌中毒菌株的毒素基因进行了分析。根据来源将测试菌株分为三个簇。该分型与阿拉伯糖和菊粉的发酵模式一致。(2)测定了11株中国分离株和1株意大利分离株的毒素基因的核苷酸序列。11株中国分离株的毒素基因核苷酸序列完全相同。中国株与意大利株的氨基酸同源性为95.0%，中国株与C. E型肉毒杆菌阳性率为96.9%。毒理学分析表明，C.来自意大利菌株（BL 5262）和来自食源性肉毒中毒的中国菌株（KZ 1885）的丁酸菌神经毒素 ...更多信息 ).用抗C. E型肉毒神经毒素和BL 5262神经毒素的抗原性比较表明，KZ 1885神经毒素的轻链具有与E型肉毒神经毒素相同的抗原性，而重链具有部分抗原性不同的部分。与BL5262神经毒素重链特异性反应的单克隆抗体几乎不识别KZ1885神经毒素。这些观察结果也通过核苷酸测序得到证实。此外，KZ1885和E型神经毒素之间的氨基酸序列的差异似乎集中在重链的羧基端部分。KZ1885神经毒素的比毒力约为E型神经毒素的15倍.结论（1）神经源性C.丁酸菌属的植物在每个广阔的地区都有无性系分布。(2)C.丁酸菌KZ 1885神经毒素在结构、抗原性上与C. E型肉毒毒素与C.丁酸菌BL5262神经毒素。少

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