Functional Study On IgM Of Common Carp(Cyprinus Carpio) Infected With Aeromonas Hydrophila

Common carp(Cyprinus carpio)is among the major freshwater economic species in China,exceeding one-tenth of the nation’s freshwater fish breeding production.Common carp farming has provided a source of high-quality protein and jobs around the world,but it has also faced major problems.Aeromonas hydrophila is a very common bacteria in aquatic environments worldwide.This opportunistic zoonotic bacterium can cause hemorrhagic septicemia,abdominal dropsy,and skin ulceration,and is considered one of the most devastating pathogens of Cyprinoid fish.The continued use of antibiotics as antimicrobial agents in aquaculture can lead to the development of pathogen resistance,which not only disrupts the ecology and microbial homeostasis of the fish gut but also leads to the accumulation of excess antibiotics in the fish,which can be harmful to humans.To achieve sustainable development of aquaculture,it is necessary not only to optimize the agricultural environment rationally but also to study the interactions between host and pathogenic microorganisms.Immunoglobulin(Ig)M is an important immune effector that protects organisms from a wide variety of pathogens.Therefore,this paper studied the immune response of common carp IgM after bacterial infection by intraperitoneal injection of A.hydrophila.The following are the details and findings of this study:1.A polyclonal antibody against common carp IgM was successfully prepared.In this study,the complete c DNA sequence of common carp IgM heavy chain was obtained from NCBI and successfully cloned the CH2-CH4 domains into the p ET-32a prokaryotic expression plasmid.The purified recombinant IgM was used as an immunogen to prepare polyclonal antibodies against common carp IgM.It was verified by western blot,immunofluorescence staining,and mass spectrometry that the antibody prepared in this study had high specificity.2.To explore the immune response of common carp after bacterial infection,this study used A.hydrophila to infect common carp via intraperitoneal injection.In the infected group,typical symptoms of A.hydrophila appeared,such as ascites,anal redness and swelling,body congesting,and enteritis.Using hematoxylin-eosin(H&E)staining,the results showed severe pathological changes in the intestine after A.hydrophila infection,including a breakdown of intestinal epithelial tissue as well as a decrease in the intestinal villus aspect ratio.The aspect ratio of the intestinal villi decreased significantly at 0.5 days post-infection and began to recover gradually at 7days post-infection,with the fastest recovery occurring in the hindgut.Common carp develop strong innate and adaptive immune responses after A.hydrophila infection.Quantitative real-time PCR(q PCR)analysis showed that A.hydrophila infection induced the expression of antimicrobial peptide genes,pro-inflammatory factors,and Igs in the gut,spleen,and head and kidney tissues of common carp.3.Common carp IgM~+B cells and IgM undergo an adaptive immune response following A.hydrophila infection.By immunofluorescence analysis,the results showed a significant increase in the number of IgM~+B cells in the foregut,midgut,hindgut and head and kidney tissues of common carp after A.hydrophila infection.By western blot analysis,we found that bacteria-specific IgM titers were significantly increased in the gut mucus and serum of infected fish compared with control fish,suggesting that IgM plays an important role in the defense against bacterial invasion.Reinfected fish exhibit resistance to A.hydrophila.By q PCR,we detected significantly lower expression of the enterotoxin gene of A.hydrophila in the gut,spleen,and head and kidney tissues of common carp when infected fish were re-injected with the bacteria compared to the first infected fish.In addition,we found lighter intestinal histopathological changes and a higher survival rate in the reinfected group compared to the first infected fish.In conclusion,a polyclonal antibody with high specificity against the IgM heavy chain of common carp was prepared in this study.A.hydrophila can invade the gut and induce a strong immune response in the gut mucosa,and IgM and IgM~+B cells play an important role in resisting bacterial invasion.Therefore,the results in this paper provide crucial insights into the role of IgM in bacterial enteritis of teleost fish.