养殖梭鱼格氏乳球菌的分离鉴定及致病性研究  

Isolation,Identification and Pathogenicity Study of Lactococcus garvieae from Cultured Liza haematocheila

作　　者：洪宝华 马荣荣 袁娜 张克鑫 李锦锦 倪思圳 朱卫东 张鸿鹄 钱冬[1] HONG Bao-Hua;MA Rong-Rong;YUAN Na;ZHANG Ke-Xin;LI Jin-Jin;NI Si-Zhen;ZHU Wei-Dong;ZHANG Hong-Hu;QIAN Dong(College of Marine Sciences,Ningbo University,Ningbo 315823,China;Yuyao Fisheries Technology Extension Center,Yuyao 315400,China)

机构地区：[1]宁波大学海洋学院,宁波315823 [2]余姚市水产技术推广中心,余姚315400

出　　处：《农业生物技术学报》2020年第8期1458-1470,共13页Journal of Agricultural Biotechnology

基　　金：浙江省科技重点研发项目(2020C02020);浙江省教育厅建设项目(202000305)。

摘　　要：2018年夏浙江宁波某淡水游钓场梭鱼(Liza haematocheila)出现大规模持续发病,6~8月累计死亡率达60%以上.濒死鱼池边独游或水面打转,突眼、眼膜增厚或不透明,腹部膨胀;解剖可见腹水,肝、脾肿大和局部出血,腹内鳔和肾相邻处有花斑状假膜形成,隔层内充气.为进一步探究病因,用营养琼脂、脑心浸液琼脂培养基从3尾典型症状病鱼的脑、肝、脾和肾分离到圆形、湿润、灰白色,菌落直径1~4 mm的8株革兰阳性短链球菌(命名为Lh-1~Lh-8);用VITEK-2-Compact生理生化鉴定,与格氏乳球菌(Lactococcus garvieae)相似性达99%;用16S rDNA、gyrB(gyrase B)等基因进行分子鉴定,序列同源性比对显示与格氏乳球菌相似性分别达100%和93.5%,均表明为格氏乳球菌.取Lh-3腹腔注射健康梭鱼,感染2~5 d后可见自然发病症状,并重新分离到格氏乳球菌,LD50为5.86×10^4 CFU/尾.Lh-3在血琼脂平板呈典型α溶血,脱脂乳平板未出现透明圈.Lh-3腹腔注射和灌胃4周龄美国癌症研究所(Institute of Cancer Research,ICR)小鼠(Mus musculus),12 h后出现全身竖毛、眼部红肿、四肢颤栗等现象,1×10^9 CFU/只攻毒组24 h内死亡率达100%,感染小鼠可见肝、肺、腹腔内壁充血等病理变化.证实所分离的格氏乳球菌是本次梭鱼发病的病原体,对梭鱼有很强毒性,是养殖梭鱼的重要病原体;对小鼠有一定的致病力,是潜在的人鱼共患病原.本研究为梭鱼乃至水产养殖病害防控提供了一定的参考.In the summer of 2018,a large-scale persistent outbreak of Liza haematocheila occurred in a freshwater fishing farm in Ningbo,Zhejiang province,with the cumulative mortality rate more than 60%.The moribund fish swimming alone or spinning on the water surface with unresponsive,anorexic,swollen abdomen and exophthalmos,thickened opaque eyes.The dissected mullet showed ascites,enlarged liver,spleen,petechiae and local bleeding and other symptoms,reddish macular pseudomembrane was observed in the connection of the swim bladder and kidney in the abdomen with the inflated compartment.To study the pathogen and main pathogenesis of cultured L.haematocheila and other related species for further diseases scientific control,eight bacterial strains(named Lh-1~Lh-8)were isolated from the brain,liver,spleen and kidney of 3 diseased fish with typical symptoms by using nutrient agar(NA)and brain heart infusion agar(BHIA)medium.Round,smooth and greyish white colonies sized 1~4 mm were found on BHIA and confirmed as Gram-positive coccus with short chain.Lh-3,one of the isolates,was identified physiologically and biochemically as Lactococcus garvieae with 99%similarity by using VITEK-2-Compact.The 16S rDNA and gyrase B(gyrB)were used for molecular identification.Sequence homology alignment and construction of a phylogenetic tree showed that the similarity to Lactococcus garvieae reached 100%and 93.5%,respectively,and both showed Lactococcus garvieae.The symptoms similar to natural disease occurred in L.haematocheila 2 d after was injected intraperitoneally with Lh-3,with the LD50 of 5.86×10^4 CFU/ind.Lh-3 showed typical alpha hemolysis on blood agar plates,and no transparent circles appeared on skimmed milk plates.Institute of Cancer Research(ICR)mice(Mus musculus)were infected by intraperitoneal injection of Lh-3.The mice showed bristled,trembling and dispirited with reddish and swollen eyes after 12 h of challenging.Pathological changes such as congestion of liver,lung and abdominal wall were observed in infected mice,and the mo

关 键 词：梭鱼 格氏乳球菌 防治 致病性 

分 类 号：S943[农业科学—水产养殖]