The Analysis Of Genetic Diversity Of Vibrio Harveyi And Its Virulence-Associated Genes

Vibrio harveyi is known to be on a major pathogenic vibrio in mariculture animal.Disease outbreaks attributed to V.harveyi has been reported in variety of aquatic animalfarm production. In recent years， mass mortalities of many cultured kinds of mariculturedfishes，such as P. vannamei、Pseudosciaena crocea and Lutjanus sanguineus have occurredfrequently infected by V.harveyi. However for knowing the disease cause by V.harveyitime is short and its epidemiological studies is not deeply enough, especially in domesticlack of V.harveyi serum type molecular type analysis research. In this study, we isolated425strains from the diseased mariculture fish and marine water from the Guangdong,Guangxi and Hainan province, and identificed them have101strains V.harveyi by PCRmolecular identification biochemistry identification system. Numbers have been given toV.harveyi we have. A serum type and molecular classification（RAPD classification andBOX points type）were used to investigate the variability among101V. harveyi strains andanalysis of virulence-associated genes. The most results on are as follow:1. With Lutjanus sanguineus to the pathogenic experiment, we found that in101strains, strong virulence strains have20strains:1201,1203,1205,1206,1208,1213,1223,1236,1240,1243,1248,1251,1257,1260,1263,1277,1278,1279,1284,1285;weak virulence strains have12strains:1202,1204,1209,1215,1216,1219,1226,1227,1231,1258,1261,1264.2. According to the serum type, the results show that101strains of experiments ofV.harveyi can be divided into two kinds of serum type, one is the1261strains ofrepresentatives with eight before serum （O1208, O1261, O1250, O1203, O1223, O1277, O1279,O1205）are reaction, and the other is to1247strains of represented only with serum O1263，O1211reaction. Another1229,1246,1255,1260,1268,1269,1271,1272,1273,1275,1278,1281,1284,1287,1288,1290,1293,1296,1299and preparation for not of10in the serumof any kind of reaction, so not the division, therefore, it is necessary to use any othermethods serum division type.3. According to molecular classification: the results show that a total of10RAPDprimers were designed for their specificity in detecting V. harveyi, and only the primerPM2was highly reproducible and found suitable to use in RAPD-PCR. The geneticdiversity among V. harveyi isolates assessed by RAPD-PCR by PM2primer yielded15 different RAPD patterns which clustered the isolates into18groups at65%similarity level.Similarly, BOX-PCR clustered the20patterns into15groups at40%similarity. However,with RAPD-PCR and BOX-PCR,101V. harveyi could be divided into18groups at50%similarity. We could obtain the excellent results of molecular genetic diversity by both ofRAPD-PCR and BOX-PCR.4. Six virulence genes: toxs, zot, tcpA, tlh, tdh and trh have been choosen Accordingto the design of the gene GenBank primers, through the PCR, to detect101strains ofVibrio harveyi of distribution. The results showed that: the toxs gene whose detection ratewhich was in the101strains is17.8%but in the32strains toxic strain, whose detectionrate is25%; the zot gene’s detection rate is6.9%, its detection rate in the toxic strain is12.5%; the tlh gene’s detection rate is14.9%, its detection rate in the toxic strain is3.1%;the tdh gene’s detection rate is7.9%, its detection rate in the toxic strain is15.6%; but theresults of the tcpA and trh gene have not been detected. The toxs and zot virulence genesdetection rates of strains are far higher than those without strain, this shows that toxs, zotvirulence genes are relevant to the pathogenicity of V.harveyi. Virulence-associated genesdetection could reveal molecular character of various strains and offer significance formolecular epidemiological studies and effective prevention and control of V.harveyi.