Screening Of Virulence Genes And Establishment Of Rapid Identification Method Of Vibrio Harveyi

Vibrio harveyi is Gram-negative, luminous bacterium and widly distributed in aquatic environment. It is one of the most important pathogenic agents for aquatic animals. It is important to establish a rapid, specific method of detecting V. harveyi. The virulence factors of V. harveyi that have been found were extracellular hemolysin, proteases (including metalloprotease and serine protease), phospholipases and lipases. Whether there are other virulence genes are not clear. In this study, five virulence-related genes to other Vibrio species were chosen, and their distribution in V. harveyi was detected.toxR(toxin regulator)is widly distributed in Vibrio species, which was discovered as a positive transcriptional regulator of many virulence genes in Vibrio species, and the homology is low in different Vibrio species. In this study, a pair of specific primers based on toxR gene were designed, and it was used to detect V. harveyi by PCR amplification from 56 Vibrio reference strains. All the 20 strains of V. harveyi(including two type strains of V. harveyi)could amply a 382 bp fragment. The detecting percent is 100%. The PCR amplification targeting the toxR gene of V. harveyi showed amplification of 382 bp DNA fragments in nine of the 21 isolates from the diseased fish, i.e. XVP5, TA-1, FA-1, FM-1, C-12D, R2-16K, CY-16D, CC-36D and Vang. Two isolates (XVP5 and FA-1) were selected for 16SrDNA amplification and sequencing. Then, alignment of these 16SrDNA were made with other Vibrio type strains which were published in the GenBank, and a phylogenetic tree were made by DNASTAR software. The results showed that both of them were V. harveyi. It took less than 5h from DNA extraction to get the result of PCR amplification. The detection limit of the PCR was 4.0×103 cells/ml. It was proved that this method was rapid, specific and sensitive.toxR plays an important role in regulating the expression of virulence gene of V. cholerae and V. parahaemolyticus. It also can stimulate the expression of hemolysin and regulate the expression of OMP(outer membrane proteins)of V. vulnificus. However, toxR regulation of OMP expression is not associated with virulence expression in V. anguillarum. Whether toxR was associated with the virulence expression in V. harveyi was not known. At the start of our study, only a partial sequence of toxR of V. harveyi was cloned. In this study, effort was conducted to clone the whole sequence of toxR gene for further study the function of this gene. The whole sequence of toxR was cloned by the method of LAPCR, For this, two pairs of PCR primers were designed, and two turns of PCR was performed. Part of unknow toxR sequences were cloned and sequenced successfully.toxS,tcpA, zot, flaB and flaC were important virulence genes distributed in V. cholerae, V. parahaemolyticus, V. alginolyticus and V. mimicus. In this study, the distributions of these five genes in V. harveyi were detected. The sequences of these five genes in different Vibrio species were obtained from GenBank and were aligned, respectively. PCR primers were designed from the conserved reginon, and were used to detect their distribution in V. harveyi by PCR amplification. The results showed that the specific toxS fragment were got in 8 strains (the results of 2 type strains of V. harveyi were negative) from 25 Vibrio type strains, and 4 strains from other isolates of V. harveyi. zot fragment were amplified from 2 out of 25 Vibrio type strains (the results of 2 type strains of V. harveyi were negative), and no amplification from any V. harveyi strains. tcpA were amplified from 4 Vibrio type strains, but none was V. harveyi. There were 11 strains amplified specific fragment of flaB from 25 Vibrio type strains (2 type strains of V. harveyi were positive), and the results of 28 V. harveyi strains were also positive in other Vibrio isolates. flaC gene was amplified from 13 out of 25 Vibrio type strains (the results of two V. harveyi type trains were negative), 12 were positive from other V. harveyi isolates. In conclusion, flaB, flaC and toxS were widly distributed in V. harveyi isolates, but tcpA and zot was not found in V. harveyi. Zebrafish were infected with different V. harveyi strains and the results showed that there was no obvious correlation between the pathogenicity of V. harveyi and the possession of the 5 virulence genes.