致病性哈氏弧菌生物学及分子特征

Biological and molecular characteristics of pathogenic Vibrio harveyi

从2尾病死的观赏用长鳍真鲨中分离到大量优势生长的细菌,致病性试验表明该菌对牙鲆及大菱鲆均有较强的致病性。在对分离菌进行了形态特征、理化特性、胞外酶及溶血素活性等生物学性状检验的同时,使用API-ID32E对分离菌做了进一步鉴定;同时,测定了该菌的16S rRNA和gyrB基因序列,分析了与相关细菌16S rRNA和gyrB基因序列的同源性,构建了系统发生树,比较了2种基因在相似细菌的检测和鉴别能力,结果gyrB基因用于细菌种间鉴定更具优越性。根据分离菌的表型特征及分子特征,判定分离菌为弧菌属(VibrioPacini 1854)的哈氏弧菌(Vibrio harveyiJohnson and Shunk 1936)。胞外酶及溶血活性检测表明,分离菌均能产生淀粉酶、蛋白酶、脂肪酶、DNA酶和卵磷脂酶,在含7%家兔脱纤血液营养琼脂培养基上呈β型溶血。以哈氏弧菌溶血素基因的保守区段设计的特异性引物,能够扩增出特异性片段。

Virtually pure dense cultures were isolated from diseased Oceanic whitetip shark, Carcharhinus longi manus L. The isolates had a strong pathogenicity to flounder and turbot by the challenge test. The biological characteristics of the isolates were examined with morphological, physiological and biochemical parameters, extracellulase activity and hemolytic activity, and API-ID32E system were applied in the bacterial classification. In addition, the 16S rRNA and gyrB gene were partially sequenced and compared with the sequences deposited in databases, molecular phylogenetic trees were constructed based on two genes,indicating that gyrB gene analysis may be a more useful tool for interspecies identification. The isolates were identified Vibrio harveyi （Johnson and Shunk 19a6） based on their phenotepic and molecular characteristics. The isolates could produce diastase,proteinase, Lipase,DNase, leeithinase, and exhibited β- haemolysis on agar plates containing 7% defibrinated rabbit blood. The conserved region of hemolysin gene of Vibrio harveyi could be amplified by PCR using specific primer.