一株引起大弹涂鱼出血症的维氏气单胞菌的分离及鉴定  

Isolation and Identification of a Aeromonas veronii Strain Causing the Hemorrhagic Disease of Great Blue-spotted Mudskipper(Boleophthalmus pectinirostris)

作　　者：王维[1,2] 李长红 詹萍萍[2] 陈炯 WANG Wei;LI Chang-Hong;ZHAN Ping-Ping;CHEN Jiong(State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agroproducts,Ningbo University,Ningbo 315211,China;Laboratory of Biochemistry and Molecular Biology,School of Marine Science,Ningbo University,Ningbo 315832,China)

机构地区：[1]宁波大学农产品质量安全危害因子与风险防控国家重点实验室,宁波315211 [2]宁波大学海洋学院/生物化学与分子生物学实验室,宁波315832

出　　处：《农业生物技术学报》2022年第2期344-355,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31972821);浙江省自然科学基金(LY21C190003);宁波大学研究生科研创新基金(IF2021006)。

摘　　要：2020年8~9月,浙江慈溪市某大弹涂鱼(Boleophthalmus pectinirostris)养殖场发生爆发性病害,病鱼主要表现出典型的出血症症状,给养殖户造成严重的经济损失。为了鉴定该病害的病原,本研究通过解剖观察、细菌分离培养、电镜观察、生化鉴定、药敏实验、动物回归感染实验、16S rRNA及DNA旋回酶亚基B(DNA gyrase subunit B,gyrB)基因系统发育分析等方法对病原进行鉴定。结果从发病鱼体内分离得到一株优势菌,将其命名为AV20211212。通过革兰氏染色的光镜观察及扫描与透视电镜的超微结构观察发现,AV20211212为革兰氏阴性短杆菌,两端钝圆,可见中空极生单鞭毛。生理生化特征鉴定结果显示,AV20211212与维氏气单胞菌(Aeromonas veronii)的生理生化特征相似。16S rRNA核苷酸序列及gyrB编码氨基酸序列同源性分析显示,AV20211212与维氏气单胞菌的16S rRNA的同源性为93.6%~99.4%,gyrB同源性为99.5%~99.7%。16S rRNA核苷酸序列及gyrB编码氨基酸序列的系统发育分析结果均显示,AV20211212与维氏气单胞菌紧密成簇。推测AV20211212为维氏气单胞菌。药敏实验结果显示,AV20211212对青霉素、氨苄西林等7种抗生素高度耐药,对阿奇霉素中度敏感,而对诺氟沙星、丁胺卡那等12种抗菌药物敏感。对健康大弹涂鱼进行回归感染,结果显示症状与养殖场发病鱼体一致,半致死剂量为4.56×10^(3) CFU/g。本研究首次将维氏气单胞菌鉴定为大弹涂鱼的致病菌,研究结果为慈溪地区细菌性鱼类病害的有效预防及合理用药提供了理论依据及技术支持。From August to September 2020,an outbreak of hemorrhagic disease in the great blue-spotted mudskipper(Boleophthalmus pectinirostris)occurred in a farm in Cixi City,Zhejiang Province,causing serious economic losses to farmers.In order to identify the pathogen of the disease,methods including the anatomical observation,bacterial isolation and culture,electron microscope observation,biochemical test,drug sensitivity test,artificial infection test,and phylogenetic analysis of the 16S rRNA and DNA gyrase subunit B gene(gyrB)were used for identification of the pathogen.The results showed that one dominant strain,named AV20211212,was isolated from the diseased fish.Observation by light microscope with gram staining and ultrastructural observation by scanning and transmission electron microscope showed that the isolated strain AV20211212 was Gram-negative with blunt round ends and hollow polar monotrichous flagella.Results from physiological and biochemical characteristics identification showed that AV20211212 had the similar physiological and biochemical characteristics to Aeromonas veronii.Homology analysis demonstrated that the16S rRNA of the isolated strain shared 93.6%~99.4%homology with those of other A.veronii and the amino acid sequence of the gyrB from the isolated strain shared 99.5%~99.7%homology with those of other A.veronii.Phylogenetic analysis showed that the isolated strain was closely clustered with A.veronii.The isolated strain was highly resistant to 7 antibiotics such as penicillin and ampicillin,moderately sensitive to azithromycin,and sensitive to 12 antibiotics such as norfloxacin and amikacin.The mudskipper infected by the isolated strain AV20211212 showed the same symptoms with naturally diseased mudskipper on the farm,and its medium lethal dose(LD50)was 4.56×10^(3) CFU/g to mudskipper.In the present study,A.veronii was identified as the pathogen of mudskipper for the first time.All the results provided theoretical basis and technical support for the effective prevention and rational medicine us

关 键 词：大弹涂鱼 维氏气单胞菌 形态观察 生理生化特征 系统发育分析 

分 类 号：S941[农业科学—水产养殖] R378.3[农业科学—水产科学]