| 摘要: |
| 从江苏连云港某养殖场养殖死亡的泥鳅肝脏、血液及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泥鳅具有很强的致病性。采用表观分类学及分子生物学方法, 对分离菌进行了形态特征、理化特性、胞外酶活性及溶血活性等生物学性状检验; 用PCR 方法同时扩增其16S rRNA和gyrB 基因, 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, 通过基因序列分析, 比较了两种基因在相似细菌的检测和鉴定能力; 基于16S rRNA 和gyrB 基因的系统发育学分析表明分离菌(LD081008B-1)所扩增的16S rRNA 和gyrB 基因序列均与GenBank 数据库中霍乱弧菌具有较高的相似性, 且gyrB基因用于细菌种间鉴定更具优越性; 16S rRNA 基因序列长度为1446bp(GenBank 登录号: GQ205447), gyrB 基因序列长度为1207bp (GenBank 登录号: GQ205452); 根据分离菌的表型特征及分子特征, 判定病原菌为弧菌属(Vibrio Pacini 1854)的霍乱弧菌(Vibrio cholerae)。胞外酶活性及溶血活性检测表明分离菌均具有蛋白酶、卵磷脂酶、淀粉酶、明胶酶及DNA 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。分离菌的耐药谱测定结果显示, 除对供试49 种抗菌药物中的杆菌肽耐药外, 对其它48 种药物敏感或高度敏感。
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| 关键词: 泥鳅, 霍乱弧菌, 生物学特性, 16S rRNA 基因, gyrB 基因 |
| DOI：10.11693/hyhz200906004004 |
| 分类号: |
| 基金项目:农业部淡水鱼类遗传育种和养殖生物学重点开放实验室开放基金资助，BZ2009-03 号；连云港市科技攻关项目资助，CN0826 号。 |
附件 |
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| PHENOTYPIC AND MOLECULAR IDENTIFICATION OF PATHOGENIC VIBRIO CHOLERAE ISOLATED FROM MISGURNUS ANGUILLICAUDATUS |
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BING Xu-Wen1, YAN Bin-Lun2, ZHANG Xiao-Jun2, QIN Lei2, BI Ke-Ran2
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1.Key Laboratory of Genetic Breeding and Aquaculture Biology of Freshwater Fishes, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Science;2.College of Ocean, Key Laboratory of Oceanic Biotechnology of Jiangsu, Huaihai Institute of Technology
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| Abstract: |
| A serious mortality of cultured loach Misgurnus anguillicaudatus occurred in some farms in Donghai County, Jiangsu Province in October 2008. Syndrome included bleeding in head, opercula, lower jaw, fins, and fin bases, swollen muscle, anus, liver, and spleen, and empty intestines. Tissues were sampled from liver, bleed, and ascitic fluid of diseased fish, from which the pathogen were isolated, and the biological characteristics were examined, including characteristics in morphology, physiology, and biochemistry, as well as the activity of extracellulase and hemolysin. The 16S rRNA and gyrB were amplified by PCR, with which sequences deposited in databases were compared, and molecular phylogenetic trees were constructed. The result indicates high homogeneity between the isolates and Vibrio cholerae from GenBank database, showing that gyrB gene analysis is a more useful tool. The sequenced 16S rRNA gene of strain LD081008B-1 (GenBank accession No.GQ205447) is 1446bp in length, the sequenced gyrB gene of strain LD081008B-1 (GenBank accession No.GQ205452) is 1207bp in length. The isolates were identified as V. cholerae (Pacini 1854) in terms of phenotepic and molecular characteristics. Detection of the activity of extracellulase and hemolysin shows that the isolates could produced proteinase, diastase, lecithinase, gelatinase, and DNase, and exhibited β-haemolysis on agar plates containing 7% defibrinated rabbit blood. Drug resistance of isolates to 49 antimicrobial agents was detected. The results show that isolates were sensitive or highly sensitive to all agents, except for bacitracin.
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| Key words: Misgurnus anguillicaudatus L., Vibrio cholerae, Biological characteristics, 16S rRNA gene, gyrB gene |
