| Aeromonas spp. is one of the main pathogenic bacteria of aquatic animals. Itnot only leads to serious sapraemia of many aquatic animals, but also infectshuman and animals by food chain causing diarrhea and food poisoning toendanger peoples' health. Anhui is an important freshwater-breeding province inChina and the breeding output increases continuously year after year.Unfortunately, the bacteriosis of aquatic animals, especially Aeromonas disease(morbidity 60%, lethal rate 25%)breaks out more frequently than before, butthe research on Aeromonas is not relatively enough. Large quantities of aquaticanimals die because of not identifying pathogeny quickly and correctly andtaking effective prevention and cure measures in time. It has badly restricted thehealthy aquicultural development of Anhui province. In recent years, 15 strainsof bacteria have been isolated from aquatic animals suffering from sapraemia inour laboratory. In order to study the molecular epidemiology characters ofAeromonas Anhui strains comprehensively, ascertain their location inphylogenetic tree, which will provide scientific evidences for inspection,prevention and control of aquatic animals' Aeromonas infection, accumulate thedata of molecular biology characteristics of Aeromonas spp. and lay a foundationfor developing Aeromonas DNA vaccine as well. Numerical identification ofbacteria was used to define phenospecies of Aeromonas Anhui strains, multiplexPCR assay with rapid diagnosis of Aeromonas spp. and detection of its virulencegene had been established, the main virulence genotypes of Aeromonas Anhuistrains were determined and three virulence genes were cloned and analyzed inthis study. Firstly, 15 strains bacteria were identified by a whole set of numericalidentification mediums of gram-negative bacteria made in China. At the sametime, the pathogenicity of those bacteria was determined by animal experiments.The results show that all of the 15 strains bacteria isolated in our laboratory areAeromonas spp.. Among them, 6 strains (BA1,BA3,BA17,HA6,HA7 and CA2 )are A.hydrophila, 8 strains (BA11,BA16,JA23,JA24,CA1,CA3,GA1 andHSA1) are A.sobria, 1 strain(BA18) is A.caviae. Except CA2 and HSA1, other 13strains bacteria are pathogenic to experimental animals and there are somevirulence differences in both different phenotypes and strains,the lethal rate ofBA1,BA3,BA11,BA17,BA18,CA3,GA1,HA7,JA23 and JA24 strains toexperimental animals are 100%, while HA6,CA1 and BA16 strains are 66.7%,50% and 30% respectively. Secondly, multiplex PCR assay for fast diagnosis and molecularepidemiology survey of Aeromonas disease. we designed three pairs of specialprimers(adhesin gene, hemolysin gene and enterotoxin gene)basing on theirhigher conservative regions and reactive conditions were optimized by thecombination of primers' concentrations, magnesium ion concentration, thevolume of Taq DNA polymerase, anneal temperature, extension time and cycletimes. The built multiplex PCR assay is specific and sensitive. Thirdly, virulence genes of 15 Aeromonas Anhui strains were detected bymultiplex PCR and the virulence genotypes were defined. In the experiment, weextracted genome DNA of the 15 strains Aeromonas spp. as template respectivelyto amplify alt, hly and aha1 gene. The results show that 13 strains out of 15Aeromonas Anhui strains carry one or more virulence genes, 13 strainspathogenic Aeromonas distribute three kinds of virulence genotype, of which 2strains are alt+ahal-hly-(15.38%), 1 strain is alt+ahal+hly- (7.69%) and 10 strainsare alt+ahal+hly+ (76.92%). The results demonstrate that alt gene exists widely indifferent phenospecies Aeromonas spp. alt+ahal+hly+ virulence genotype is thedominant prevailing genotype in Anhui province. Finally, the virulence genes of 6 representative Aeromonas Anhui strainswere cloned and analyzed their ORF sequence. Three pairs of primers, whichwere special for the total ORF sequence of aha1, hly and alt gene respectively,were designed and synthesized. aha1, hly and alt gene were amplified from thegenome of 6 stains... |
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