| Edwardsiella tarda is one of the important pathogens in aquaculture, caused prevalent diseases in marine, freshwater aquaculture animals in the world, and caused serious economic losses to aquaculture industry. Currently, disinfectants, antibiotics and other chemicals are still main way to prevent and treat bacterial disease caused by Edwardsiella tarda, but there are many problems happen such as bacterial resistance, drug residues in aquaculture food.The dominant bacterial strain,named as TANX,was isolated from visceral tissues of dead Clarias fuscus whose symptoms suspected Edwardsiellosis. Through comprehensive analysis of the morphological characteristics in the culture dishes, Gram staining, SS medium for identification and specificity of PCR detection of Edwardsiella tarda and 16 S r DNA gene sequencing, the results confirmed that the strain TANX was Edwardsiella tarda.We detected the six kinds of Edwardsiella tarda common virulence genes.We can find that TANX bacteria containing only muk F and cit C two kinds of virulence genes from PCR detection. The challenge tests were carried out by artificially infecting healthy Tilapia Mossambica with the pure culture of the isolated strain.The test results showed that the pathogenicity of TANX was weak, and the mortality of experimental group is very low. Antibiotic sensitivity test showed that TANX strains were sensitive to cefuroxime, cefotaxime, ceftriaxone, cefepime, aztreonam, ofloxacin, levofloxacin and other drugs, and resistant to penicillin G, ampicillin, tetracycline, polymyxin B and other drugs.In order to explore the immune efficacy of Live-attenuated vaccine(LAV) for Oreochromis niloticus. In this study, we constructed Live-attenuated vaccine and Formalin killed cell. The use of intraperitoneal injection and oral immune Oreochromis niloticus, respectively.Collected serum, head kidney, spleen regularly.The parameters evaluated included the specific Ig M antibody titer, acid phosphatase(ACP), alkaline phosphatase(AKP) and superoxide dismutase(SOD) three kinds of enzymatic activity and the genes expression of IL-1,TNF,IFN,Caspase3 in the spleens and head kidney, and the immunologic potency(relative percent survival,RPS) of LAVand FKC.The immune the specific Ig M level were significantly increased, and LAV group and FKC group antibody levels were significantly higher than control group(P <0.01),their antibody levels were not significantly different statistically. But immunity for 21 days and 28 days immunity,LAV group antibody levels were significantly higher than FKC groups(P <0.05). Enzyme activity results confirmed that LAV groups and FKC groups there were no significant difference acid phosphatase vaccine group and Alkaline Phosphatase.But immune immunity 21 and 28 days, the SOD of LAV vaccine groups were significant higher than FKC vaccine groups(P <0.05).Real-time Fluorescence quantitative PCR results show LAV injection group the relative expression of IL-1 in head kidney was highly significant than control group(P <0.01). But immunization 28 days, LAV group IL-1, IFN, TNF, Caspase3 expression levels in head kidney and Caspase3 expression levels in spleen were higher than in FKC groups, were significant highly than control group(P<0.01). The relative protective survival of LAV by intraperitoneal injection and oral administration were 90.5%?63.6%, higher than FKC's 76.1%?59%. LAV group and FKC group were significant higher than control group(P <0.05). Based on the above, LAV in oral immune way can cause stronger immune response than in intraperitoneal injection way. Live-attenuated vaccine are good candidate vaccine in preventing edwardsiella tarda disease. |
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