Isolation And Identification Of Pathogenic Bacteria From Wild Silver Carp And Evaluation Of Inactivated Vaccine In Chagan Lake

Aeromonas caviae(A.caviae)is an important conditional pathogen,which can infect A variety of animals,such as human,animal and fish,and pose A threat to public health and safety.Especially to the aquaculture industry caused great harm,has attracted the attention of many experts and scholars at home and abroad.Child to explore early 2020 jilin Chagan Lake branch left bay,the reasons of silver carps widespread death phenomenon occurring in experiment by the onset of silver carps in the purification of a plant with a growth advantage strain,named AC-CY,then AC-CY for preliminary identification of bacteria morphology and physiological and biochemical test,and by using 16 s r DNA and housekeeping gene sequence analysis to identify the molecular level and the phylogenetic tree analysis,after the AC-CY strain animal regression test,carrying virulence genes,determination of growth curve,median lethal dose and drug susceptibility test.To provide further theoretical guidance for the identification of A.caviae.As the fishery big country,my country has caused the unreasonable application of antibiotics and chemical drugs,resulting in a huge hazard of antibiotics and chemical drugs,and the method of preventing water-raw animal disease is especially important,vaccination Various high mortality infectious diseases can be effectively prevented.Since the inactivated vaccine is to remove the toxicity of the pathogens by chemical or physical methods,the preparation process is simple,the immunogenicity is strong,high safety,and convenient storage and transportation is favored by the majority of animal breeding households.Therefore,this study evaluates the preparation of AC-CY to inactivate vaccine and immunization effects for the health and sustainable development of fisheries and aquaculture industry as the former theoretical support.The results showed that the colony morphology of AC-CY was semitransparent round colony,smooth surface and neat edge.The bacteria was Gram-negative bacilli by microscopic examination.Animal regression test showed that the pathogen was pathogenic,and the symptoms were similar to those on site.The results of physiological and biochemical tests were similar to those of Aeromonas,but the species could not be further identified,so they were identified by molecular identification.The 16S r DNA results showed that AC-CY was clustered with A.caviae,and the similarity was 99%.Subsequently,it was further confirmed by housekeeping gene identification that AC-CY belonged to A.caviae.The PCR results of AC-CY virulence genes showed that the isolated strain AC-CY carried seven virulence genes.The median lethal dose of AC-CY to zebrafish and silver carp was 2.51×10~2 CFU/tail and2×10~4 CFU/tail,respectively.Moreover,the results of drug sensitivity test showed that AC-CY strain was highly sensitive to Penicillin,Phenoxacillin,Ampicillin,Carboxpicillin,Cefalexin,Minocycline,Ceftazidime,Clindamycin,Medivycin,Vancomycin,Cefradine,Cefazoline which belonged to multi-drug resistant strains and proved that it was difficult to treat in production.After the preparation of AC-CY inactivated vaccine and evaluation of immune efficacy,inactivated group and PBS group respectively before immunization(0d),after the first immunization(7d),after the second immunization(14d),21d,after the third immunization(28d)and challenge test(35d)time to collect tissues and blood,using ELISA method to determine serum LYS,AKP,C4,C3 and Ig M antibodies;Leukocyte phagocytosis test;Immune protection rate test;Real-time fluorescent quantitative PCR detection.After the silver carp was immunized with AC-CY inactivated vaccine,the serum LYS,AKP,C4,C3 and Ig M contents in the inactivated group were increased to varying degrees compared with those in the PBS group.The expression levels of IL-10,IL-1β,IFN-γand TNF-αwere increased by real-time fluorescent quantitative PCR.the phagocytic ability of leukocytes was enhanced;The immune protection rate was 77%.The results showed that AC-CY inactivated vaccine could induce a strong immune response in the body.