Identification, Detection And Construction Of Green Fluoredcent Protein Maeker Strains Of Pathogenic Aeromonas Hydrophila Isolated From Megalobrama Amblycephala

Megalobrama amblycephala, commonly known as the Wuchang fish. The development of intensive farming techniques and the increase of breeding density, leading to the deterioration of the ecological environment and a variety of diseases.In2011, an outbreak hemorrhagic occurred among cultured Megalobrama amblycephala with high mortality in Changzhou, the diseased fish could be seen that different levels of bleeding symptoms appeared in its chest, pelvic fin base, caudal fin base, jaw, orbital margin, gill cover edge, etc; autopsy revealed within the gut without food, liver was enlargement and bleeding. A dominant bacteria WJ-8was isolated from the liver of sick Megalobrama amblycephala. The challenge test results of using WJ-8to infect Megalobrama amblycephala displayed that the prevalence of symptoms and spontaneous onset symptoms were the same. It proved that WJ-8was pathogenic to Megalobrama amblycephala. The phenotypic characteristics of isolated strain was examined, including morphological characteristics, physiological, and biochemical characteristics, and the construction of phylogenetic tree, the strain was Aeromonas hydrophila. In order to choose the appropriate antibiotic the drug sensitive was tested. The testing results by paper extension methods for susceptibility test showed that florfenicol, Mepiquat, Rifamycin, streptomycin, kanamycin and other21kinds of drugs had the most inhibitive effects on the strain. At the same time, it showed resistance to penicillin, ampicillin, carbenicillin, oxacillin, and other12kinds of drugs. The histopathology research conducted with tissue technology, hematoxylin and eosin staining. The pathological studies were carried out about the liver, kidney and spleen tissues of both diseased Megalobrama amblycephala and health Megalobrama amblycephala. The results showed that some karyopyknosis or swelling, and some cells damaged, the kidney and spleen tissues had haemosiderin.A. hydrophila is an important aquatic animal pathogen. With primers designed according to the gene sequences of alt and aerA published in GenBank, duplex PCR was applied to amplify the genes of cytotoxic enterotoxin (alt) and Aerolysin gene (aerA) of A. hydrophila. The reaction conditions of the duplex PCR were optimized. Products of alt gene and aerA gene of A. hydrophila strains with duplex PCR amplification was of two Electrophoresis strips. The amplified fragment all and aerA were200and280bp. Furthermore, Aeromonas sobria, Aeromonas salmonicida, Aeromonas caviae, Vibrio fluxialis were used to determine the specificity of that rapid detection, the results were negative. The results of sensitivity of dulplex PCR showed that the two primers could detect A. hydrophila at a level of as few as3×104CFU/mL, and detect purified chromosomal DNA at a level of as few as49fg/uL. The virulent strain and attenuated strain have the alt and aerA gene, but the avirulent strains didn’t have. The results demonstrated that a new duplex PCR method for simultaneously detecting alt and aerA genes were successfully established. It is a practical method for pathogenic A hydrophila detection.10antibacterial fishery drugs had be chosen to detect the antibacterial effect, the results showed that the best drug is chlortetracycline hydrochloride, its minimum inhibitory concentration (MIC) and minimum bactericidal concentration were2μg/mL and4μg/mL; followed by florfenicol, enrofloxacin and streptomycin sulfate, the three kinds of drugs’MIC and MBC were all less than10μg/mL.In this study, the recombinant plasmid pEGFPuv-Kar was constructed by plasmid pET-28a (+) and plasmid pEGFPuv, and then transformed into the strain WJ-8. The transformants had bright green performance under fluorescent microscope. The results of stability test showed that after WJ-8GFP was passaged for12generation times, the stability rate of the recombinant plasmid was up to100%and reduced to5%after21generations. The artificial infection experiment results showed that WJ-8GFPhave strong pathogenicity. Aeromonas hydrophila marked with green fluorescent protein gene (WJ-8GFP) was used to inject Megalobrama amblycephala by intraperitoneal injection. Then the tissues of injected Megalobrama amblycephala were observed in6h,12h,24h,36h,48h after intraperitoneal injection to find the strain WJ-8GFP and the strain WJ-8GFP were be caculated. The results showed that the spleen and kidney organizations were the main infection organ, in which the kidney was the the most serious attacked one; the infected peak of the organs was in12h.