Study On The Isolation, Identification, Genotype And The Effect Of Environmental Factors Of Aeromonas Hydrophila

Aeromonas hydrophila can cause the bacterial septicemia disease inCarassius auratus gibelio, Megalobrama amblycephala Yih,Hypophthalmichthys molitrix, Cyprinus carpio Linnaeus, etc. It causeshuge economic losses to aquaculture industry. Accurate identification ofthe bacteria before it can be convenient for subsequent research and theprevention and control of disease. In regular bacteria identification, thegeneral method is the morphological identification of bacteria andsupplemented by biochemical tests. The bacteria as single-celled organismis polymorphism and variability. Some biochemical reactions also areunstable. Only reling on the morphology and biochemical reaction isdifficult to accurately identified. Therefore searching for other accurate andfast method of bacteria identification is of great significance. The16SrDNA sequence analysis of prokaryote has become the standard method ofbacteria species identification and classification.The experiment synthesizes morphological observation, molecularidentification, physiological determination, biochemical tests to construct phylogenetic tree and homologic analysis of bacteria isolated from differentregions and ecological niche. Physiological and biochemical tests showedthat bacteria can decompose the Mannose, Sucrose, Citrate, Malonate,Carbamide, Bile Esculin, etc. and can not ferment Melezitose, Melibiose,Phaseomannite. Glucose fermented by bacteria can product acid and gas.Genetic sequencing results display that the length of16S rRNA of strainWJ11-5is1450bp. The GenBank NO. is JN836334. The homology of theamplfied gene of strain is comparised through the NCBI Blast andphylogeny tree is constructed by the Neighbor-Joining method. The resutshowed that16S rRNA of strain WJ11-5in phylogenetic tree belong to thesame bacterium with Aeromonas hydrophila. The experiment finallyconfirmed the isolated bacteria is A.hydrophila.In the experiment, enterobacterial repetitive intergenic consensus(ERIC)-PCR was used to analyze the typing of the105strains ofAeromonas hydrophila collected from Freshwater aquaculture area inJiangsu, Henan, Fujian and so on. The genotyping technique was used toapply in the epidemiological investigation of the strains and to evaluate therelationship of the genotype, regional distribution and the ecological niches.Electrophoresis prints was analysed by using Quantity one4.6and usedUPGMA to make a tree plot to analyse hereditary character.28specialgenotypes were identified from the105isolates. Genotype XV was the dominant genotype, consisting of20strains, whereas genotype IV, XI,XXII, XXV, XXVIII, XXVII and XXVI contained only one strain. There isa tendency that the strains from the same geographic aera or ecologicalniches could be clustered into one group. In addition, A. hydrophila fromdifferent regions and ecological niche might get together for the samegenotype. The results suggest that there is likely some relevance betweenthe genotype and ecological niche among A. hydrophila strains.The experiment which choosed Megalobrama amblycephala Yih as thetested fish verified the toxicity intension of the isolated A.hydrophilatrough artificial infection test. In the environmental stress experiment, westudy the relationship between toxicity strengh of A.hydrophila withenvironmental factors. The orthogonal experimental has a4level factor(temperatures) and three2level factors(pH, nitrite, ammonia nitrogen).The factor which affects bacteria virulence significantly is analysed bysimulated natural aqucaculture environment. Constructing regressionequation y=164.713-6.399A+14.367B-11.914B×C. In the equation, y(h)denotes fish survival time. The scope of A(tenmperature) is from20to32℃. The scope of B (pH) is from6.5to8. The scope of C (ammonianitrogen) is from0.02to0.04mg/L.Results show that temperature and ammonia nitrogen with pH areextremely significant influence on the survival time of fish. The influence of PH stress is significant. The effect of nitrite stress is not significant.Theoretically, when the temperature is20℃, pH=8, ammonia nitrogenconcentration is0.02mg/L, the fish’s longest survival time in7days is149.76hours.The paper discuss the relationship between A.hydrophila isolated fromdifferent regional ecological niches. In the environmental stress experiment,we analysd the factors which affects bacteria virulence significantly by thestrain choosed through artificial infection test. Constructing regressionequation in order to forecast the incidence in aquafarm. Our study willprovide the theory basis for the research of bacterium epidemiology andwill have significative guidance to the prevention of bacterial septicemiadisease.