大菱鲆内脏结节病病原的分离与鉴定  

Isolation and identification of the pathogen causing granulomas in internal organs of turbot Scophthalmus maximus

作　　者：罗璋[1] 张振国[1] 郝爽[1] 白晓慧[1] 贾文平[1] 高谦 冯守明[1,3] LUO Zhang;ZHANG Zhenguo;HAO Shuang;BAI Xiaohui;JIA Wenping;GAO Qian;FENG Shouming(Tianjin Fishery Research Institute,Tianjin 300221,China;College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;Tianjin Aquatic Animal Disease Prevention and Control Center,Tianjin 300221,China)

机构地区：[1]天津市水产研究所,天津300221 [2]上海海洋大学水产与生命学院,上海201306 [3]天津市水生动物疫病预防控制中心,天津300221

出　　处：《中国水产科学》2019年第3期559-568,共10页Journal of Fishery Sciences of China

基　　金：天津市自然科学基金项目(16JCYBJC30000);天津市水产产业技术体系创新团队项目(ITTFR2017008);天津市农业科技成果转化与推广项目(201501030);天津市科技计划项目(18ZXBFNC00030)

摘　　要：2017年8月,天津市滨海新区某养殖场大菱鲆(Scophthalmus maximus)发生病害,累计死亡率约为25%,患病鱼体表无明显症状。通过肉眼和病理组织切片观察发现,患病鱼肾脏、脾脏、肝脏和肠道存在大量圆形结节,肾脏、肝脏和脾脏组织病变明显,肾小管坏死,肝细胞脂肪变性,脾脏中存在大量的坏死细胞。此外,在脾脏和肾脏组织中发现大量的抗酸杆菌。利用传统病原菌分离方法,从具有典型症状的濒死大菱鲆肾脏组织分离到优势菌株myco-10,利用该菌株注射攻毒能导致健康大菱鲆66.7%的死亡率,且表现出与自然发病鱼相同的症状。采用16S rDNA、Hsp65、ropB基因序列分析,构建系统发育树并结合细菌形态特征、生理生化测试对菌株myco-10进行鉴定,结果显示,菌株myco-10的16SrDNA、Hsp65、ropB基因序列与分枝杆菌属细菌(Mycobacteriumspp.)相似度最高,且在系统发育树中与海分枝杆菌(Mycobacteriummarinum)和溃疡分枝杆菌(Mycobacteriumulcerans)聚为一簇,生理生化反应与海分枝杆菌一致。综合生理生化特征和基因序列分析结果,将菌株myco-10鉴定为海分枝杆菌。这是中国首例分枝杆菌引起大菱鲆病害的报道,可为大菱鲆内脏结节病的防治提供参考资料。Turbot (Scophthalmus maximus) is an economically important marine fish species cultured in China. However, disease is a major problem in the turbot aquaculture fishery, causing high mortality. In August 2017, a disease outbreak among turbot in a marine farm in Tianjin led to a cumulative mortality rate of 25%. The main symptom of the disease was abundant white granulomas in the internal organs of the turbot. To define the causa- tive pathogens, bacteria were isolated from the spleen and kidneys of diseased fish using conventional methods. An artificial infection experiment was conducted to determine the pathogenicity of the isolated strain. The 16S rDNA, Hsp65, and ropB gene sequences, as well as morphological and biochemical characteristics of the strain, were examined. In addition, histopathological analysis was performed on the diseased turbot. Abundant white granulomas were observed in the spleen, kidneys, livers, and intestinal tracts of diseased fish. The hepatocytes in the livers of the diseased fish were damaged, and several irregular vacuolations were observed. Some of the spleen cells were enlarged and necrotic, and epithelial cells from the renal tubule showed vacuolar degeneration and ne- crosis. Furthermore, in the center of the pregranulomas, as well as kidney and spleen macrophages contained sev- eral phagocytized red, rod-shaped acid-fast bacteria. Strain myco-10 was isolated from spleens of diseased fish. Healthy turbot were subjected to bacterial challenge tests by intraperitoneal injection of 3×108 cfu/fish of the iso- lated myco-10. The accumulative mortality rate was 66.7% within 30 days post-infection. Diseased turbot dis- played symptoms similar to those observed in naturally infected individuals. Strain myco-10 showed positive pigmentation production after exposure to visible light, Tween-80 hydrolysis, urease, catalase at 68℃, and aryl- sulfatase, and negative results in terms of nitrate reductase and growth on 5% NaCl. Phylogenetic trees were con- structed by matching 16S rDNA, Hsp65,

关 键 词：大菱鲆 结节 海分枝杆菌 鉴定 

分 类 号：S941[农业科学—水产养殖]