Studies On Isolation, Identification&Genetic Diversity Of Aeromonas From Conventional Freshwater Fish Ponds In Jiangsu Province

There were182strains isolated by the selective medium RS from the conventional freshwater fish ponds including normal case and disease case. By oxidase, catalase, and O/F experiment,152suspected Aeromonas strains were got which were oxidase and catalase positive, and fermented.16S rRNA sequence of all strains could be got by16S rRNA universal primers, then NCBI-BLAST software was used to do homology search in the GenBank database. The results showed that there were89Aeromonas strains, of which63strains could be identified to species (63/89,70.8%). Other26strains could be identificated to species by the gyrB gene. The results showed that Aeromonas veronii was widely distributed in all Aeromonas not only from normal water but also from diseased fish, and had a largest number(environmental strains:31/37,83.8%; pathogenic strains:39/52,75%). However Aeromonas hydrophila was in the opposite side of A. veronii (environmental strains:1/37,2.7%; pathogenic strains:10/52,19.2%). Besides, there were A.salmonicida, A.media and A.jandaei isolated from them.Virulence Genes such as aer, hly and alt were detected in89Aeromonas strains. There were20strains with aer genes positive (22.5%),34strains with hly genes positive(38.2%),34strains with alt genes positive(38.2%). None of the three virulence genes could be found in9of37envrionmental strains, and varying amounts of the three virulence genes can be found in other28strains. None of the three virulence genes coould be found in19of52envrionmental strains, and varying amounts of the three virulence genes can be found in other33strains. It had no significant rules of virulence genotype combination of33pathogenic strains. As A. veronii was widely distributed in the conventional freshwater fish ponds, the16S rRNA sequences and housekeeping genes sequences of these A. veronii strains were used to investigate there phylogenetic relationships. From the NJ-phylogenetic trees based on16S rRNA sequences, it could be found that the A. veronii strains isolated from no-diseased and diseased ponds could be divided into two genetic groups. The first group had30A. veronii strains, and the second group had14A. veronii strains. All strains isolated from no-diseased and diseased ponds were cross-distributed. The A. veronii strains isolated from diseased ponds in different regions could be divided into three genetic groups. The first group had20A. veronii strains which included11strains isolated from Changzhou,8from Wuxi, and1from Yancheng; the second group had11A. veronii strains which included6strains isolated from Changzhou,4from Wuxi, and1from Yancheng; the third group had7A. veronii strains which included4strains isolated from Changzhou,2from Wuxi, and1from Yancheng. All A. veronii strains in the three groups were cluster together in the same region.From the NJ-phylogenetic trees based on the combined stretch of16S rRNA-gyrB-dnaJ sequences, it could be found that the A. veronii strains isolated from no-diseased and diseased ponds could be divided into four genetic groups. The first group had18A. veronii strains which included10strains from no-diseased pond and8from diseased ponds; the second group had15A. veronii strains which included11strains from no-diseased pond and4from diseased ponds; the third group had6A. veronii strains which included3strains from no-diseased pond and3from diseased ponds; the forth group had9A. veronii strains which included4strains from no-diseased pond and5from diseased ponds. The A. veronii strains isolated from diseased ponds in different regions could be divided into two genetic groups. The first group had22A. veronii strains which included12strains isolated from Changzhou and10from Wuxi; the second group had16A. veronii strains which included9strains isolated from Changzhou,4from Wuxi, and3from Yancheng. All A. veronii strains in the two groups were cluster together in the same region.