养殖大菱鲆病原菌迟缓爱德华氏菌的分离鉴定及快速检测技术的建立
摘要
大菱鲆(Scophthalmus maximus L)是一个经济价值很高的养殖品种,它生长迅速并且味道十分鲜美,从而深受人们的喜爱。自从20世纪90年代从欧洲引入中国后,大菱鲆的养殖得到了很快的发展。然而,随着养殖环境的不断恶化,使得大菱鲆的细菌性疾病不断发生。爱德华氏菌病(edwardsiellosis)就是经常发生的一种。
爱德华氏菌病是由迟缓爱德华氏菌(Edwardsiella tarda)引起的。迟缓爱德华氏菌,也被记为迟钝爱德华氏菌或缓慢爱德华氏菌,属肠杆菌科,是我国水产养殖中极具危害性的病原菌。在鱼类中,迟缓爱德华氏菌有着广谱的宿主范围,能够感染多种淡水和海水鱼类,包括牙鲆、鳗鲡、真鲷、虹鳟、鲻鱼和罗非鱼等,其感染造成鱼类出血性败血症以及内脏组织的坏死等病症,最终导致患鱼死亡,统称为爱德华氏菌病。另外,迟缓爱德华氏菌还可感染鸟类、哺乳动物及爬行动物等;也有造成人类胃和肠道感染的报道。
2006年夏季和秋季,山东省胶南和昌邑一带的许多海水养殖场养殖大菱鲆发生暴发性传染病,肉眼能观察到的症状为眼睛肿胀、有炎症、有出血点、有腹水和粘性液体。经分离得到一株致病菌,命名为LTB-4。病原菌的主要特征是:革兰氏染色阴性,短棒状,大小(0.5～0.9)×(1.0～2.0)μm,无鞭毛,吲哚、硫化氢、赖氨酸脱羧酶、鸟氨酸脱羧酶和葡萄糖阳性,VP反应阴性,不能利用丙二酸、蔗糖、甘露醇、山梨醇、鼠李糖和蜜二糖。腹腔注射大菱鲆的半数致死量为3×103 cfu/mL。16Sr DNA序列比对和构建进化树表明LTB-4与迟缓爱德华氏菌相似度最高。结合其他的鉴定手段包括形态观察、生理生化指标,判定这株菌为鞭毛缺失型迟缓爱德华氏菌。这是国内首次报道鞭毛缺失型迟缓爱德华氏菌。使用gyrB基因的通用引物扩增了LTB-4和ET2034(迟缓爱德华氏菌标准菌株)的部分片段并测序,通过比对设计迟缓爱德华氏菌gyrB基因的特异性引物。PCR结果表明可以在3h内结束,除了迟缓爱德华氏菌LTB-4和ET2034能扩增出特异性片段,其他对照菌株都为阴性;PCR的检测阈值是4.0×104 cfu/mL。用迟缓爱德华氏菌WY28作为抗原,免疫新西兰兔,获得效价为1:2048的多克隆抗体;以此多克隆抗体作为一抗,山羊抗兔IgG-HRP作为酶标二抗,建立迟缓爱德华氏菌的间接ELISA快速检测法。采用棋盘滴定法确定抗原与一抗的最佳工作浓度分别为106 cfu/mL和1:10000;酶标二抗的最适工作浓度为1:1000。将该方法标准化后进行检测实验,交叉实验结果表明,此多克隆抗体与肠杆菌科的其它菌株交叉反应结果为阴性。研究结果表明,本研究建立的间接ELISA技术在6 h内即可检测出浓度为105 cfu/mL的迟缓爱德华氏菌,且与所检测的其它菌株均无交叉反应。
本论文在国内首次报道了大菱鲆病原菌,鞭毛缺失型迟缓爱德华氏菌,并建立了两种迟缓爱德华氏菌的快速检测方法,为大菱鲆爱德华氏菌病的研究和防治提供了基础和保障。
Turbot (Scophthalmus maximus L) is an economically important marine fish species and is valued for its rapid growth and good taste. Since introduced into China from Europe in the 1990s, the commercial culture develops very rapidly. However, with the rapid development of the turbot culture in China, several diseases occurred and it suggestes that they resulted from high-intensive cultivation and improper management since 2001. Edwardsiellosis is very common now, there were several reports between 2006 and 2007.
Edwardsiella tarda is the causative agent of edwardsiellosis in many commercially important freshwater and marine fish such as channel catfish, eels, mullet, chinook salmon, flounder, carp, tilapia and striped bass. It causes septicemia with extensive skin lesions, affecting internal organs and leads to extensive losses in both freshwater and marine aquaculture. It reported in humans as the cause of gastroenteritis and generalized infections mainly among individuals with impaired immune systems.
During the spring and summer of 2006, an epizootic occurred among cultured turbot (Scophthalmus maximus L.) in several fish farms in Qingdao, China. Eye tumefaction, inflammation, haemorrhages, ascites and the presence of a purulent fluid were the main macroscopic lesions observed. A Gram-negative, rod shaped bacterium (designated as LTB-4) was isolated from the infected fish. Isolate LTB-4 was gram-negative rod-shaped bacteria, occurring singly or in pairs with round-ends, (0.5～0.9)μm×(1.0～2.0)μm in size. Biochemically the isolate was positive for indole, H2S production, lysine decarboxylase, ornithine decarboxylase and D-glucose utilization, meanwhile negative for V.P. (voges proskauer) test, and malonate, sucrose, D-mannitol, D-sorbitol, L-rhamnose, and melibiose utilization. The LD50 was established as 3×103 cfu/g by intraperitoneal (i.p.) injection. Isolate LTB-4 is identified as E. tarda by morphology, physiological and biochemical tests and 16S rDNA sequence analysis. Unlike those commonly described E. tarda strains reported in China, no flagellum was observed. Partial gyrB genes was amplified from E. tarda using the universal primers of gyrB genes and sequenced. The PCR primers for the gyrB gene specific to E. tarda were designed. It revealed positive amplification of the gyrB fragment in E. tarda, whereas other bacterial species were negative; with the detection limit of 4.0×104 cfu/mL. Moreover, the technique enabled the recognition of E. tarda from diseased fish. An indirect Enzyme Linked Immunosorbent Assay (ELISA) for the rapid diagnosis of E. tarda, the pathogen of edwardsiellosis, is developed. The best working concentration of antigen and antiserum were 106 cfu/mL and 1∶10000 separately, determined by using checkerboard titration. The working concentration of HRP-labeled goat anti-bovine IgG was 1∶1000. The sensitivity of the serum was tested, and the lowest E. tarda suspension was 105 cfu/mL. Cross-reactions of antisera with other bacteria were detected, all results were negative.
爱德华氏菌病是由迟缓爱德华氏菌(Edwardsiella tarda)引起的。迟缓爱德华氏菌,也被记为迟钝爱德华氏菌或缓慢爱德华氏菌,属肠杆菌科,是我国水产养殖中极具危害性的病原菌。在鱼类中,迟缓爱德华氏菌有着广谱的宿主范围,能够感染多种淡水和海水鱼类,包括牙鲆、鳗鲡、真鲷、虹鳟、鲻鱼和罗非鱼等,其感染造成鱼类出血性败血症以及内脏组织的坏死等病症,最终导致患鱼死亡,统称为爱德华氏菌病。另外,迟缓爱德华氏菌还可感染鸟类、哺乳动物及爬行动物等;也有造成人类胃和肠道感染的报道。
2006年夏季和秋季,山东省胶南和昌邑一带的许多海水养殖场养殖大菱鲆发生暴发性传染病,肉眼能观察到的症状为眼睛肿胀、有炎症、有出血点、有腹水和粘性液体。经分离得到一株致病菌,命名为LTB-4。病原菌的主要特征是:革兰氏染色阴性,短棒状,大小(0.5～0.9)×(1.0～2.0)μm,无鞭毛,吲哚、硫化氢、赖氨酸脱羧酶、鸟氨酸脱羧酶和葡萄糖阳性,VP反应阴性,不能利用丙二酸、蔗糖、甘露醇、山梨醇、鼠李糖和蜜二糖。腹腔注射大菱鲆的半数致死量为3×103 cfu/mL。16Sr DNA序列比对和构建进化树表明LTB-4与迟缓爱德华氏菌相似度最高。结合其他的鉴定手段包括形态观察、生理生化指标,判定这株菌为鞭毛缺失型迟缓爱德华氏菌。这是国内首次报道鞭毛缺失型迟缓爱德华氏菌。使用gyrB基因的通用引物扩增了LTB-4和ET2034(迟缓爱德华氏菌标准菌株)的部分片段并测序,通过比对设计迟缓爱德华氏菌gyrB基因的特异性引物。PCR结果表明可以在3h内结束,除了迟缓爱德华氏菌LTB-4和ET2034能扩增出特异性片段,其他对照菌株都为阴性;PCR的检测阈值是4.0×104 cfu/mL。用迟缓爱德华氏菌WY28作为抗原,免疫新西兰兔,获得效价为1:2048的多克隆抗体;以此多克隆抗体作为一抗,山羊抗兔IgG-HRP作为酶标二抗,建立迟缓爱德华氏菌的间接ELISA快速检测法。采用棋盘滴定法确定抗原与一抗的最佳工作浓度分别为106 cfu/mL和1:10000;酶标二抗的最适工作浓度为1:1000。将该方法标准化后进行检测实验,交叉实验结果表明,此多克隆抗体与肠杆菌科的其它菌株交叉反应结果为阴性。研究结果表明,本研究建立的间接ELISA技术在6 h内即可检测出浓度为105 cfu/mL的迟缓爱德华氏菌,且与所检测的其它菌株均无交叉反应。
本论文在国内首次报道了大菱鲆病原菌,鞭毛缺失型迟缓爱德华氏菌,并建立了两种迟缓爱德华氏菌的快速检测方法,为大菱鲆爱德华氏菌病的研究和防治提供了基础和保障。
Turbot (Scophthalmus maximus L) is an economically important marine fish species and is valued for its rapid growth and good taste. Since introduced into China from Europe in the 1990s, the commercial culture develops very rapidly. However, with the rapid development of the turbot culture in China, several diseases occurred and it suggestes that they resulted from high-intensive cultivation and improper management since 2001. Edwardsiellosis is very common now, there were several reports between 2006 and 2007.
Edwardsiella tarda is the causative agent of edwardsiellosis in many commercially important freshwater and marine fish such as channel catfish, eels, mullet, chinook salmon, flounder, carp, tilapia and striped bass. It causes septicemia with extensive skin lesions, affecting internal organs and leads to extensive losses in both freshwater and marine aquaculture. It reported in humans as the cause of gastroenteritis and generalized infections mainly among individuals with impaired immune systems.
During the spring and summer of 2006, an epizootic occurred among cultured turbot (Scophthalmus maximus L.) in several fish farms in Qingdao, China. Eye tumefaction, inflammation, haemorrhages, ascites and the presence of a purulent fluid were the main macroscopic lesions observed. A Gram-negative, rod shaped bacterium (designated as LTB-4) was isolated from the infected fish. Isolate LTB-4 was gram-negative rod-shaped bacteria, occurring singly or in pairs with round-ends, (0.5～0.9)μm×(1.0～2.0)μm in size. Biochemically the isolate was positive for indole, H2S production, lysine decarboxylase, ornithine decarboxylase and D-glucose utilization, meanwhile negative for V.P. (voges proskauer) test, and malonate, sucrose, D-mannitol, D-sorbitol, L-rhamnose, and melibiose utilization. The LD50 was established as 3×103 cfu/g by intraperitoneal (i.p.) injection. Isolate LTB-4 is identified as E. tarda by morphology, physiological and biochemical tests and 16S rDNA sequence analysis. Unlike those commonly described E. tarda strains reported in China, no flagellum was observed. Partial gyrB genes was amplified from E. tarda using the universal primers of gyrB genes and sequenced. The PCR primers for the gyrB gene specific to E. tarda were designed. It revealed positive amplification of the gyrB fragment in E. tarda, whereas other bacterial species were negative; with the detection limit of 4.0×104 cfu/mL. Moreover, the technique enabled the recognition of E. tarda from diseased fish. An indirect Enzyme Linked Immunosorbent Assay (ELISA) for the rapid diagnosis of E. tarda, the pathogen of edwardsiellosis, is developed. The best working concentration of antigen and antiserum were 106 cfu/mL and 1∶10000 separately, determined by using checkerboard titration. The working concentration of HRP-labeled goat anti-bovine IgG was 1∶1000. The sensitivity of the serum was tested, and the lowest E. tarda suspension was 105 cfu/mL. Cross-reactions of antisera with other bacteria were detected, all results were negative.
引文
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