Screening Of Immune Adjuvants For Attenuated Vaccine Of Aeromonas Veronii HisJ Gene Deletion

The pollution of water resources and the increase of breeding density lead to Aeromonas vickerii infection more easily in the breeding population.We should no longer rely on the use of chemicals and antibiotics to solve the current problem of disease.The use of microecological preparations,immune stimulants,vaccination and other measures for comprehensive disease management is more advocated in the new ecological agriculture model.Therefore,vaccination,as a green ecological control means,has become the mainstream technology of global aquatic animal disease prevention and control,and has a good prospect in the development of the world aquaculture industry.At present,most commercial aquatic vaccines are still inactivated vaccines,but inactivated vaccines have the disadvantages of short duration of immunization,large dose,multiple doses of vaccination and serious adverse reactions.Compared with inactivated vaccine,live attenuated vaccine has the advantages of simple vaccination,low manufacturing cost,rapid delivery,low immune dose,fewer shots and prolonged immunity.However,sometimes the immune stimulation ability of single attenuated vaccine is not strong enough,or it is not enough to stimulate the ideal immune response under certain conditions(such as individual immune insufficiency),so the immune effect of attenuated vaccine can be improved by adding adjuvants.The TH0426 hisJ gene deletion strain of Aeromonas veronii was constructed in the laboratory,and its pathogenicity was significantly weakened,which could be used as a candidate strain for attenuated vaccine.Therefore,in thisstudy,the hisJ gene deletion strain was prepared as attenuated vaccine,and four adjuvants including proprotic extract,aluminum hydroxide gel,β-glucan and fungus polysaccharide were selected to be used together with attenuated vaccine,respectively,to select adjuvants that could improve the protective effect of attenuated vaccine.Before preparing the attenuated vaccine,the four adjuvants were tested to determine whether they would affect the growth of hisJ gene deletion strains.After it was determined that the adjuvant had no bacteriostatic effect,four groups of adjuvant and attenuated vaccine were used together,and they were set as four adjuvant experimental groups: aluminum hydroxide gel adjuvant group,propolis extract adjuvant group,β-glucan adjuvant group and fungus polysaccharide adjuvant group.At the same time,unadjuvant attenuated vaccine group and blank control group were set up.Experimental crucian carp were immunized by intraperitoneal injection on day 1 and day 14 after two weeks of observation,and a certain number of crucian carp were randomly selected for blood collection and tissue collection on day 0d,day 7,day 14,day 21,day 28 and day 35.Part of the blood was added with anticoagulant to prepare anticoagulant and used for leukocyte phagocytosis activity test.The remaining blood was prepared into serum and used for serum index detection;The results showed that the non serum specific antibody(Ig M)was higher in all adjuvant groups,while the serum activities of acid phosphatase(ACP),alkaline phosphatase(AKP),superoxide dismutase(SOD),lysozyme(LZM)and catalase(CAT)were increased.The serum indexes of adjuvant groups were significantly higher than those of attenuated vaccine groups.The transcriptional levels of IL-1β,TNF-α,IL-10 and IFN-γ genes in liver,spleen,kidney and gill tissues were detected by fluorescence quantitative PCR technique.It was confirmed that the attenuated vaccine with immune adjuvant can promote the inflammatory response of the body,and the combination of adjuvant and attenuated vaccine can enhance the immune response of crucian carp.The results of leukocyte phagocytosis activity test and immune protection rate further verified the ability of each adjuvant group to enhance immunity of crucian carp.The survival rate of propolis extract adjuvant group was the highest(77.14%),followed by aluminum hydroxide gel adjuvant,fungus polysaccharide adjuvant and β-glucan adjuvant,and the survival rate of attenuated vaccine group was the lowest(65.71%).The results showed that the overall immune effect of crucian carp inoculated with attenuated vaccine supplemented with propolis extract adjuvant was the best.The immunological effect of aluminum hydroxide gel adjuvant attenuated vaccine was the second.The immune effect of fungus polysaccharide adjuvant and β-glucan adjuvant was slightly lower than that of the first two adjuvants,and they were not equal,but the immune effect was better than that of attenuated vaccine without adjuvant.Thisstudy comprehensively evaluated the immune-enhancing effects of four adjuvants on hisJ gene deletion attenuated vaccines,which will help lay the foundation for future vaccine adjuvant research.At the same time,it will further promote the vaccine research to prevent Aeromonas veronii infection,and also provide a new theoretical basis for the subsequent development of aquatic vaccines.