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检测、脉冲场凝胶电泳、Southern blot杂交等方法，对中国食源性肉毒中毒6株菌株、中国微山湖地区土壤5株菌株和意大利婴儿肉毒中毒2株菌株进行毒素基因分析。试验菌株按照病原来源分为3个聚类。这种类型符合阿拉伯糖和菊糖的发酵模式。(2)测定了11株中国菌株和1株意大利菌株毒素基因的核苷酸序列。11个中国菌株的毒素基因核苷酸序列相同。中国菌株与意大利菌株氨基酸同源性为95.0%，中国菌株与E型肉毒杆菌氨基酸同源性为96.9%。来自意大利菌株（BL5262）和来自中国食源性肉毒杆菌菌株（KZ1885…更多）的丁酸梭菌神经毒素具有抗原性多样性。ELISA和BL5262神经毒素单克隆抗体免疫印迹分析表明，KZ1885神经毒素轻链与E型神经毒素具有相同的抗原性，而重链具有部分不同的抗原性。对BL5262神经毒素重链有特异性反应的单克隆抗体对KZ1885神经毒素几乎没有识别。这些观察结果也被核苷酸测序证实。此外，KZ1885与E型神经毒素之间的氨基酸序列差异似乎集中在重链的羧基末端。KZ1885神经毒素的特异性毒性比E型神经毒素低约15倍。结论(1)产神经毒素丁酸梭菌很可能是无性分布在各地的。(2)与C. butyricum BL5262神经毒素相比，C. butyricum KZ1885神经毒素在结构和抗原性上与C. botulinum E型神经毒素非常相似。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