Study On The Effect And Mechanism Of Hypertonic Immersion Inactivated Aeromonas Hydrophila Vaccine In Different Salinities On The Immune Response Of Carassius Auratus Gibelio

Aeromonas hydrophila(Ah)is one of the most serious diseases in Carassius auratus gibelio rearing by causing bacterial septicemia.Immune prevention is an important strategy for the aquaculture development.In this study,inactivated Aeromonas hydrophila and Carassius auratus gibelio were used as experimental materials.The immune responses of gibel carp post direct immersion vaccination(DI)and hypertonic immersion vaccination with inactivated A.hydrophila were studied.We measured changes of non-specific immune-related enzyme activity,the proportion of membrane-surface immunoglobulin-positive(s Ig M~+)cells,specific antibody levels,intestinal microbial abundance and relative immune protection rates.The immune protective effect of the hypertonic immersion Aeromonas hydrophila inactivated vaccine on Carassius auratus gibelio was comprehensively evaluated from the antibodies,cells and gut microbes levels,the effect mechanism of hyperosmotic immersion immunity on fish immune response is explained from the level of immune-related enzyme activity,s Ig M~+cell level,specific antibody level,and intestinal microbial level.Provide technical support for improving vaccine immunization strategies and methods,improving the efficiency of vaccine use,and evaluating vaccine effects.It has accumulated data references for the selection of the optimal salinity of the Carassius auratus gibelio.In this experiment,a blank control group and three experimental groups were set up,and each group was repeated three times.The experimental group divided into the direct immersion group and the hypertonic immersion group.The direct immersion group was to immersion the Carassius auratus gibelio in the inactivated vaccine of Aeromonas hydrophila at a concentration of 1×10~8 cfu/m L(DI group)for 30 min.The hypertonic immersion group was to immersion the Carassius auratus gibelio in a NaCl solution at a concentration of 2.6%(group H1)and 5.2%(group H2)for 10minutes,and then transferred to 1×10~8 cfu/m L Aeromonas hydrophila vaccine to immersion for 30 minutes.The mainresults are as follows:(1)At 6,12,24,48 and 96 hours after immunization,the mucus and serum were collected.The activities of alkaline phosphatase(AKP),acid phosphatase(ACP),lysozyme(LZM)and superoxide dismutase(SOD)in serum and mucus were detected by enzyme activity assay kit.The results showed that after immersion immunization,the activities of the four enzymes in the DI group,the H1 group,and the H2 group were significantly higher than the control group,and the enzyme activities in the H1and H2 groups were higher than the DI group;The basic activity of ACP,AKP and SOD in mucus is lower than that in serum,while the basic activity of LZM is higher;It can be seen that immersion immunity can significantly increase the activity of non-specific immune-related enzymes in the serum and mucus of allogynogenetic crucian carp;immersion the inactivated vaccine after hypertonic immersion,the enzyme activity can be further increased.(2)On the 3rd,5th,7th,11th,18th,25th,and 32th days after immunization,take mucus and serum,and use enzyme-linked immunosorbent assay(ELISA)to measure the changes of Aeromonas hydrophila specific Ig M levels in mucus and serum;At 1,2,3,4,5,and 6 weeks,the peripheral blood and spleen leukocytes of Carassius auratus gibelio were collected,and the proportion of membrane surface immunoglobulin-positive(s Ig M~+)cells in leukocytes was detected by flow cytometry;After 32 days,the challege test was carried out to compare the relative percentage survival(RPS).The results showed that the proportion of s Ig M~+cells in peripheral blood and spleen of fish was significantly increased after immunization,and the proportion of s Ig M~+cells in H1 group was higher than that in the other two immersion groups;In the immersion treatment group,the peak time of the antibody level in the mucus was relatively earlier than the serum,but the antibody in the serum lasted longer.The peak value of the specific antibody level in the mucus of the H1group was higher than H2 group.The RPS was 0,48%,60%and 52%in control group,DI group,H1 group and H2 group.There was a close positive correlation between the proportion of s Ig M~+cells and the level of specific antibodies and the immune protection rate.(3)Take the midgut of the Carassius auratus gibelio on the 14th day after immunization and when death is about to occur after challenge.changes in intestinal microbial abundance of Carassius auratus gibelio were measured by Mi Seq sequencing.The results show that the hypertonic immersion Aeromonas hydrophila inactivated vaccine can significantly increase the abundance level of intestinl microbes of Carassius auratus gibelio;On the 14th day after immunization,the abundance of the genus Cetobacterium of the H1 group was higher than the H2 and DI groups.when death is about to occur after challenge,the abundance of Aeromonas in the intestinal tract of the H1 and H2 groups was lower than that of the DI group,and the H1 group was the lowest;The results of intestinal microbial abundance change showed that the dominant colonies of H1 group had stronger colonization ability and better immune effect.To sum up,this test explored the effects and mechanism of immune response of the Carassius auratus gibelio after hypertonic immersion Aeromonas hydrophila Vaccine of different salinities from the aspects of non-specific immune-related enzyme activity,s Ig M~+cell proportion,specific antibody levels,intestinal microorganism abundance and immune protection rate.The results showed that immersion immunity can significantly improve the immune response of Carassius auratus gibelio;Immersion the Aeromonas hydrophila vaccine after suitable hypertonic NaCl solution immersion treatment can further improve its immune effect.The results of this study show that the best effect is obtained by immersion with 2.6%NaCl solution.