Effect Of Immersion Temperature On Immune Response In Carassius Auratus Gibelio With Aeromonas Hydrophila Bacterin

Aeromonas hydrophila,as a common opportunistic pathogen,causes the huge economic losses to freshwater aquaculture in China.With the rapid growth of fish farming,the phenomena of blindly using antibiotics have become common.Studies have shown that the residues of chemical antibiotics have negative effects on fish,and pose a potential threat to food safety.So the development of vaccines has become a hot focus on fish immunology.However,the immune effect is influenced by vaccination strategies and the environmental factors.In this paper,Carassius auratus gibelio was used as the research object,and biotechnologies,such as immune toxicology,biochemical and molecular biology,were employed in this study.By controlling the temperature of dipping immersion,effects on immune response in carassius auratus gibelio with Aeromonas hydrophila bacterin were studied.In the present research,the inactivated Aeromonas hydrophila vaccine was diluted to 1×10~8 CFU/mL,and the Carassius auratus gibelio were immersed for 30min in the temperature of 20~oC,25~oC and 30~oC respectively.Sampling was performed at 3 h,12 h,48 h and 96 h post immersion,respectively.The enzymatic activity of lysozyme(LZM),super oxide dismutase(SOD),alkaline phosphatase(AKP),and acidic phosphatase(ACP)in the serum and mucus was determined by the enzyme reagent kits.Real-time quantitative PCR was carried out to analyze the expression of immune related genes:major histocompatibility complex I(MHC I),toll-like receptor 3(TLR3)and interleukin 11(IL–11)in the gill,intestines and spleen.Samples of serum and mucus were collected at 5d、7d、11d、18d and 25d after immersion respectively,and the titer of specific IgM antibody was detected by enzyme-linked immunosorbent assay.At 25~thh day after immersion,the artificial infection experiment was carried out with the Aeromonas hydrophila in a concentration of 3.74×10~6 CFU/mL,and the relative percent survival(RPS)was calculated.The main results were as follows:The activity of non-specific immune-related enzymes in the serum and mucus increased significantly after immersion.In mucus,the activity of LZM increased significantly with the increase of temperature in the short period after immersion,and the time reaching the peak was shorter.However,the enzyme activity gradually decreased and eventually stabilized over time.The activity of SOD,AKP and ACP showed a trend of decreasing first,and the enzyme activity reached a peak between 12h and 48h,having a positive relation with the increase of immersed temperature.In serum,there was no significant difference in LZM activity in all temperature immersion groups,the activity of enzyme significantly increased until 96h.The activity of SOD increased significantly at 3h in 30~oC treatment group,and the other two groups peaked around 12h,followed by a steady decline.This meant that the increase of immersion temperature would decrease the time for reaching the peak.The activity of the AKP and ACP increased sharply at 3h post the immersion,and then returned to the normal level,followed by gradually increasing over time.The expression levels of three genes changed in different tissues.The expression of gene in the intestine was significantly higher than the other two tissues.In 25~oC and30~oC groups,the expression of genes was significantly higher than that in 20~oC.The trend of gene expression in the gill was similar to the spleen,which was not significantly affected by immersion temperature.The expression of MHC I gene significantly up-regulated at 12h after immersion first,then down-regulated at 48h and followed by up-regulating again at 96h.The expression of TLR3 gene was up-regulated and followed by down–regulated,and the peak time in the gill and intestine was earlier than that in the spleen.The expression of IL-11 gene was decreased gradually over time,and the expression of the gene was significantly lower than that in the control group after 12h in the gill,and the gene expression in the intestine and spleen was significantly higher than that in the control group.In the group of 30~oC immersion,the titer of specific IgM antibody rised earlier and the antibody titer was higher.The immune effect was evaluated by artificial infection experiment with the Aeromonas hydrophila of 3.74 x 10~6 CFU/mL,and the results showed that the RPS in the three immersed temperatures(20~oC,25~oC and 30~oC)was38.9%,33.3%and 50%respectively.In this paper,the changes of the non-specific enzyme activity and the specific IgM antibody in the mucus and serum,and expression of immune related genes,in the Carassius auratus gibelio with the different treatments of immersed temperature,were studied,aiming to elucidate the effect of immersion temperature on Carassius auratus gibelio immune response.The results showed that immersion temperature could significantly affect the immune response,and immersion at 30~oC could significantly enhance the immune protective effect in fish.