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

Abstract:

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 (kx1 and kx2) were clustered with V. harveyi, which was supported by a high bootstrap value. The tested strain (kx1) was lethal to the healthy postlarvae F. chinensis and P. japonicus with the LD₅₀ of 2.0×10⁶ CFU/ml and 7.0×10⁵ 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.