中国对虾糠虾幼体病原哈氏弧菌的鉴定及毒力基因检测

2011年春季对江苏连云港某对虾育苗场中国对虾Fenneropenaeus chinensis病死糠虾幼体分离到优势生长菌,对分离菌进行致病性、形态与生理生化特征及16S rRNA和gyrB基因同源性与系统发育分析。结果显示,引起糠虾幼体大量死亡的病原为哈氏弧菌Vibrio harveyi,菌株kx1对中国对虾仔虾和日本对虾仔虾的半数致死量LD50分别为2.0×106CFU/ml和7.0×105CFU/ml。为进一步明确分离菌株毒力基因的携带情况,进行了分离鉴定的4株病原菌对群体效应调节基因(luxR)、毒力调控基因(toxR)、溶血素基因(vhhA和vhhB)、金属蛋白酶基因(vhpA和vhpB)、毒力相关基因(toxS)、鞭毛结构基因(flaA)及锌金属蛋白酶基因(pap6)共9种毒力基因的检测,结果表明,4株病原菌均可检测到luxR、toxR、vhhA、vhhB和pap6毒力基因,扩增片段大小分别为679、390、1324、216和355 bp,其他4种毒力基因未检测到。分离鉴定的4株病原哈氏弧菌携带相同的毒力基因,这些毒力基因可作为检测致病性哈氏弧菌的生物学标记。

Identification and virulence genes detection of pathogenic Vibrio harveyi isolated from mysis of Fenneropenaeus chinensis L.

Vibrio harveyi is widely distributed in the marine estuarine environments and has been widely recognized as a primary pathogen of many commercially cultured invertebrate species over the world. Breeding season of spring, 2011, the outbreak of mass mortality of mysis of Fenneropenaeus chinensis L. occurred in a hatchery in Lianyungang, Jiangsu Province. In this study, we isolated and identified the pathogenic bacteria, analyzed the pathogenicity of isolated strains, and detected the presence of virulence genes of isolated strains. Dominant bacteria isolated from the diseased mysis were identified based on morphological characteristics, physiological and biochemical characteristics, sequences similarity and phylogenicity analysis of the 16S rRNA and gyrB genes. The phylogenetic trees were constructed using the neighbor-joining method, and pathogenicity of the tested strain was analyzed by bath challenge experiment on postlarvae F. chinensis and P. japonicus, and virulence genes of isolated strains were detected by a specific PCR assay. The characteristics of identified strains （kx1-kx4） consist with V. harveyi, and the 16S rRNA and gyrB genes of the tested strains exhibited high similarity with V. harveyi. Two strains （kxl and kx2） were clustered with V. harveyi, which was supported by a high bootstrap value. The tested strain （kxl） was lethal to the healthy postlarvae F. chinensis and P. japonicus with the LDso of 2. 0 × 10^6 CFU/ml and 7.0 × 10^5 CFU/ml, respectively. The genes （luxR, toxR, vhhA, vhhB and pap6） existed in these four strains with gene fragments were 679 bp, 390 bp, 1324 bp, 216 bp and 355 bp, respectively. These results suggested that the diseased mysis of F. chinensis were infected by V. harveyi, and the isolated four strains have the same virulence genes, which can be used as the biomarkers to detect pathogenic V. harveyi.