Isolation, Identification And Molecular Typing Of Aeromonas Hydrophila From Jiangsu And Characterizations Of Lowly Virulent Strains

Aeromonias hydrophila widely distributes in the water environment. It is an important broad-host-range pathogen. Especially it is an important aquatic pathogenic bacterium, and can cause the huge economic loss to aquaticulture industry. Therefore, it is important to investigate the epidemic strains of A. hydrophila and their pathogenicity.In the study we collected the169samples from the water environment and fish in Jiangsu district. Then we isolated and identified41strains among the samples, which could account for24.3%. It indicated that A. hydrophila was widespread in the environment. We selected the5main virulence genes, aer, act, alt, eprCAI and ahp to design the pairs of primers and detected them in41isolates by PCR. The results showed that aer and ahp had the higher detection rates,80.5%and78.0%, respectively; the rates of act and alt were65.9%and61.0%; the eprCAI had the lowest rates with39.0%. The strains carrying the5virulent genes accounted for17.1%, and those which had none of the5virulent genes accounted for4.88%. It suggested that the aer and ahp genes were widespread in the A. hydrophila isolates and one of the most important virulent genes, however, eprCAI was lowerly detected than aer and ahp. Moreover, most of the strains could detect the above virulent genes. From the above we concluded that the virulent genes were widespread in A. hydrophila isolates and the strains had the potential pathogenicity.We typed84A. hydrophila isolates using ERIC-PCR and RAPD. The results of agarose gel electrophoresis were figured by the Quantity One software, and then clustered by SPSS. The results showed that the84strains of A. hydrophila obtained the well-polymorphism fingerprints using the two methods. Four to16bands between100bp and5800bp were amplified by ERIC-PCR, and the84strains of A. hydrophila could be divided into4main groups and17subdivisions. There was a tendency that the strains from same time or geographic area could be clustered into one group. One to12bands between200bp and4500bp were amplified by RAPD, and the84strains of A. hydrophila could be divided into4main groups and12subdivisions and3main groups and19subdivisions using two different primers. Both the two methods showed the fine typing capability. But ERIC-PCR had more reproducibility and stability than RAPD. And ERIC-PCR had more powerful typing capability and had the merit that could amplify template without knowing its sequences. Therefore ERIC-PCR was more suitable to type A hydrophila strains.On the basis of the detection of the above5virulent genes, we investigated the characterizations of3strains of A. hydrophila carrying little virulent genes, NJ-4, NJ13and CS-13. The results showed that the3strains had no hemolytic activity and the capability of dissolving protein. Both NJ-4and CS-13had no cytotoxicity, but NJ13had. NJ-4had no adhesion ability to the Hep-2cells, but NJ13and CS-13had. The supernatant of NJ-4, NJ13and CS-13could not cause the zebra fish to death. The bacteria had pathogenicity for zebra fish, and their LD50were2.6×107CFU/mL,7×106CFU/mL and1.22×107CFU/mL, respectively. The outer membrane protein (OMP) profiles of NJ-4and CS-13were similar to that of BSK-10, but not to that of NJ13. Considering the virulence genes, NJ-4had none of the five genes, NJ13only had the ahp gene, and CS-13only had the aer gene. Combined with the results of characterizations, we concluded that there exists the relationship between the virulent genes and pathogenicity, and NJ-4was the representative of the lowly virulent strains.