Characterization of the D/C mosaic neurotoxin produced by Clostridium botulinum associated with bovine botulism in Japan
Characterization of the D/C mosaic neurotoxin produced by Clostridium botulinum associated with bovine botulism in Japan
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DOI：
10.1016/j.vetmic.2009.07.023
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发表时间：
2010-01-06
影响因子：
3.3
通讯作者：
Kozaki, Shunji
中科院分区：
文献类型：
--
作者：
Nakamura, Keiji;Kohda, Tomoko;Kozaki, Shunji
C 型和 D 型肉毒梭菌与鸟类和哺乳动物肉毒杆菌中毒有关。 2004年至2007年，日本多个农场都发生了牛肉毒杆菌中毒事件。由于牛肉毒中毒病例的分离株的培养上清液分别被 D 型和 C 型抗毒素完全和部分中和，因此我们试图确认分离株中神经毒素基因的核苷酸序列。神经毒素基因由三分之二的D型神经毒素基因和三分之一的C型神经毒素基因组成，表明牛分离株的神经毒素是D型和C型神经毒素的嵌合体，即D/C嵌合型神经毒素。我们准备了四组引物，通过 PCR 来区分镶嵌型和真实型的基因。结果表明，所有与牛肉毒杆菌中毒相关的分离株均具有 D/C 嵌合体形式的基因。脉冲场凝胶电泳分析表明，2004年至2007年间发生的牛肉毒杆菌中毒分离株除了来自一个地区的分离株外，在遗传上是同源的。我们进一步检查了 D/C 嵌合神经毒素的生物学和抗原特性，发现与其他类型的神经毒素相比，它对小鼠表现出最高的致死活性。在 D/C 嵌合神经毒素中，单克隆抗体识别的三个表位位于重链的羧基末端结构域，这些表位与毒素发生特异性反应并中和毒素。这些结果表明D/C嵌合神经毒素是引起牛肉毒杆菌中毒的病原体，并且具有不同于其他C型和D型神经毒素的独特特征。 (C) 2009 Elsevier B.V. 保留所有权利。
Clostridium botulinum types C and D are related to avian and mammalian botulism. Bovine botulism occurred at various farms from 2004 to 2007 in Japan. Since culture supernatants of isolates from cases of bovine botulism were neutralized completely and partially with type D and C antitoxins, respectively, we attempted to confirm the nucleotide sequences of the neurotoxin gene in isolates. The neurotoxin gene comprised two-thirds of the type D neurotoxin gene and one-third of the type C neurotoxin gene, indicating that the neurotoxin of bovine isolates is a mosaic of type D and C neurotoxins, D/C mosaic neurotoxin. We prepared four sets of primers to differentiate the genes of the mosaic and authentic forms with PCR. The results showed that all bovine botulism-related isolates possess the gene for the D/C mosaic form. Pulsed-field gel electrophoresis analysis demonstrated that isolates from bovine botulism which had occurred between 2004 and 2007 were genetically homologous, except for the isolate from one area. We further examined the biological and antigenic properties of the D/C mosaic neurotoxin, which was found to exhibit the highest lethal activity in mice compared with other types of neurotoxins. In the D/C mosaic neurotoxin, three epitopes recognized by monoclonal antibodies that specifically react to and neutralize the toxin were located in the carboxyl-terminal domain of the heavy chain. These results indicate that D/C mosaic neurotoxin is a pathogenic agent causing bovine botulism and has unique characteristics different from other type C and D neurotoxins. (C) 2009 Elsevier B.V. All rights reserved.