半滑舌鳎病原鳗利斯顿氏菌表型及分子特征研究

2008年12月,江苏赣榆某渔场养殖半滑舌鳎出现大量死亡,症状主要表现为:头部、鳃盖及鳍基出血,尾鳍腐烂,腹腔膨胀并积有大量腹水,部分病鱼肠管脱出肛外。从病鱼肝脏、腹水中分离出大量优势生长的细菌,人工感染试验证明其对半滑舌鳎有较强的致病性。对3株分离菌进行了形态特征、理化特性、胞外酶及溶血素活性等表型生物学性状检验;测定了代表菌株的16S rRNA和gyrB基因序列,分析了16S rRNA和gyrB两种基因序列的同源性,并构建了系统发生树,比较了两种基因对分离菌的鉴定能力;结果表明gyrB基因用于细菌种间鉴定更具优越性;基于16S rRNA和gyrB基因的系统发育学分析表明分离菌与鳗利斯顿氏菌具有高同源性,根据分离菌的表型及分子特征,判定分离菌为利斯顿氏菌属(Listonella MacDonell and Colwell 1986)的鳗利斯顿氏菌[Listonella anguillarum (Bergeman 1909) MacDonell and Colwell 1986]。胞外酶活性及溶血活性检测表明3株分离菌均具有淀粉酶、蛋白酶、卵磷脂酶等胞外酶活性,在含7%家兔脱纤血液营养琼脂培养基上呈β型溶血,PCR检测3株供试菌均可扩增出大小约493 bp的溶血素基因和大小约248bp的金属蛋白酶基因。分离菌的耐药谱测定结果显示,对供试49种抗菌药物中的青霉素G等13种药物耐药,对羧苄青霉素等6种药物存在敏感与耐药的株间差异。

Phenotypic and molecular characterization of pathogenic Listonella anguillarum isolated from half-smooth tongue sole Cynoglossus semilaevis

In December 2008,continuous high-level mortalities of culturedtongue soleoccurredin one farm of Ganyu County of Jiangsu Province,China,gross signs of disease exhibited haemorrhages at the head,opercula and base of fin,rot of dorsal fin,swelling of the abdomen filled with ascitic fluid,and exposure of intestine tube outside of anus.Virtually pure dense cultures were obtained from liver and ascitic fluid of diseased tongue sole,and strong pathogenicity of the isolates to tongue sole were determined by inoculation.The phenotypic characteristics of isolates (3 strains) were examined,including morphological characteristics,physiological and biochemical characteristics,and the activity of extracellulase and hemoly sin;the 16S rRNA and gyrB were amplified by PCR and compared with sequences deposited in databases,molecular phylogenetic trees were constructed.The results of phylogenetic analyses based on 16S rRNA and gyrB genes were compared,this confirmed that gyrB gene analysis may be a more useful too l.Phylogenetic analysis of 16S rRNA and gyrB genes indicated highhomogeneity between the isolates and Listonena anguill arum from GenBank database.The isolates were identified Listonella anguillarum [(Bergeman 1909) MacDonell and Colwell 1986] of Listonella (MacDonell and Colwell 1986) based on their phenotepic and molecular characteristics.Detection of the activity of extracellulase and hemolysin showed that the isolates could produced diastase,proteinase and lecithinase,exhibited β-haemolysis on containing 7% defibrinated rabbit blood agar plates,and hemolysin gene (493 bp) and metalloprotease gene (248 bp) of 3 isolates could be amplified.Drug resistance of isolates to 49 antimicrobial agents were detected,the results showed that isolates were resistant to 13 agents including penicillin G,and have difference in sensitivity and resistance to six antimicrobial agents among different strains.