| In recent years,there are many reports about Aeromonas infecting aquatic animals,which has caused serious economic losses to aquaculture industry.Previously,most aquatic animal diseases caused by Aeromonas were prevented or treated with antibiotics or chemical drugs,which not only caused serious pollution to the water environment,but also increased the resistance of farmed animals after repeated use.In contrast,bacterial inactivated vaccine is not only simple and convenient to use,high safety,but also green and pollution-free,low production cost,very suitable for large-scale promotion.Although our aquatic vaccine started relatively late,but with the unremitting efforts of scientific research workers,great achievements have been made.Although the protection rate is high,the protection object is single and can only prevent the disease caused by a single pathogen.However,most of the diseases in aquatic animals are caused by a combination of multiple pathogens.In this study,the Aeromonas Vickeri strains and Aeromonas guinea-pig strains isolated and preserved in the laboratory were inactivated with formalin.Two vaccines were prepared,and the concentration of the vaccine was set into three different gradients of low concentration,medium concentration and high concentration.Crucian carp was immunized by intraperitoneal injection,and then the immune protection rate was determined by challenge test.Firstly,the inactivation temperature and formalin concentration were explored.The optimal inactivation temperature was 28℃,the final formalin concentration was 0.2%,and the inactivation time was 12 h.After safety was determined,the prepared inactivated vaccines were adjusted with PBS to three different concentrations:2.0×10~9CFU/m L,4.0×10~8CFU/m L,and 8.0×10~7CFU/m L.The vaccine was divided into three groups according to different concentrations,and a blank control group was set up.A number of crucian carp were randomly selected before immunization(0 d),7 d,14 d(the second immunization),21 d,28 d and 35 d after anesthesia for tail vein blood collection.The collected blood was placed in a refrigerator at 4℃overnight,and the upper serum was absorbed after centrifugation.The contents of SOD,CAT,Ig M,AKP,LZM,ACP,complement C3 and complement C4 in serum were determined by ELIAS kit.Liver,spleen,kidney and intestinal tissues were collected for real-time fluorescence quantitative PCR detection.Challenge test was conducted on the 35th day to determine the immune protection rate of the vaccine.Results showed that high levels of Ig M antibodies were produced in serum in all vaccine groups after the first immunization and continued to rise after the second booster immunization.Meanwhile,the contents of SOD,CAT,AKP,LZM,ACP,C3 and C4 in serum also increased significantly after the first immunization,and continued to rise after the second immunization,reaching the maximum at 21 days.There was no significant difference between PBS group and vaccine group during the whole immune cycle(P<0.05).Real-time quantitative PCR results showed that the expression levels of IL-10,IL-1β,IFN-γand TNF-αin all organs were increased after the first immunization,and continued to increase after the second immunization,reaching the maximum on day 21,and decreased on day 28.The results of immune protection rate showed that the protection rate of Aeromonas Vickerii was the highest in the medium-concentration vaccine group,reaching 75%,and the protection rate of Aeromonas Vickerii was the same in the low-concentration vaccine group and the high-concentration vaccine group,reaching 70%.The protection rate of guinea pig Aeromonas was the highest in the medium concentration group(80%),and the lowest in the high concentration group(70%).In this study,the effects of different vaccine concentrations on the immune protection rate of crucian carp were investigated in order to provide a theoretical basis for the preparation of the double vaccine. |
