欧洲鳗鲡肝肾病病原菌的分离与鉴定

Isolation and Identification of Bacteriosis Pathogen from Anguilla anguilla with Edwardsiellasis

患肝肾病的养殖欧洲鳗鲡(Anguilla anguilla)出现肝、肾肿大,且肝脏具白色溃疡灶的症状。从肝脏中分离、纯化到优势细菌,定名AL60306NA1。人工感染试验证实:向欧洲鳗鲡腹腔内注射1.0×108cfu/mL菌株悬液,实验鱼死亡率达100.0%;注射1.0×107cfu/mL,死亡率为60.0%;形态学观察、API20E细菌鉴定试剂盒鉴定实验证明菌株AL60306NA1有动力、拥有革兰氏反应阴性、氧化酶阴性、H2O2酶阳性、兼性厌氧、发酵葡萄糖产酸产气、不发酵蔗糖和蜜二糖及L一阿拉伯糖、赖氨酸脱羧酶阳性、柠檬酸盐弱阳性、产生硫化氢和吲哚、MR阳性和典型的β-溶血等特征;16S rRNA特异性基因分析鉴定实验说明克隆的AL60306NA1 16S rRNA基因部份序列为1507bp、与迟缓爱德华氏菌(AB050829.1)的同源性达99.7%。综合上述结果,确定该菌株为养殖欧洲鳗鲡肝肾病的病原菌,并鉴定为迟缓爱德华氏菌(Edwardsiella tarda)野生型。用菌株AL60306NA1感染小白鼠实验测定该菌对小白鼠的LD50为7.1×105cfu/mL,同时自该菌株PCR扩增到长度约1000bp的溶血素基因特异性片段,结果表明菌株AL60306NA1有一定的致病力。

An outbreak of disease of the culture Anguilla anguilla showed the symptom of some white ulcerative parts in swelled liver and swelled kidney.A strain of bacterial pathogen was isolated from diseased Anguilla anguilla named as AL60306NA1.By intraperitoneal injection test,the isolated strain AL60306NA1 could conduce 100% mortality rate at 1.0？？08cfu/mL concentration,and the mortality rate was 60.0%at 1.0？？07 cfu/mL concentration.Strain AL60306NA1 revealed motility positive,gram negative,oxydase negative,catalase positive,facultative anaerobes,acid and gas production from glucose,mannitol negative,D-melibiose negative,L-arabinose negative,L-lysine positive,trisodium citrate weakly positive,sodium thiosulfate positive,L-tryptophane positive,MR positive.The strain was ？-hemolysis.The sequence of 16SrRNA of AL60306NA1 was 1507bp and the phylogenetic tree showed that AL60306NA1 shared homologies of 99.7% with Edwardsiella tarda（AB050829.1）.Based on the physiological results,API 20Ebiochemical items and 16SrRNA gene tests,strain AL60306NA1 was confirmed as the causative pathogen bacteria of Edwardsiellasis of Anguilla anguilla,and was identified as Edwardsiella tarda wild-type.The results of the toxicity test in mouse indicated that the LD50 of strain AL60306NA1 was 7.1？？05 cfu/mL.PCR assay showed that the specificity fragment of hemolysin Gene of the bacterium AL60306NA1 was 1 000bp,and the strain was fairly pathogenic.