| The vertebrate immune system plays an essential role in resisting external antigenic stimuli and pathogenic microorganism invasion.Commonly,the immune system can eliminate pathogens or inhibits their growth by inhibiting their reproductive ability.Throughout long-term evolution,fish have formed a complete immune system that includes innate and adaptive immunity,which collaboratively ensures effective protection of the body.Fish gills are critical respiratory organs and one of the main breakthroughs in safeguarding against aquatic pathogen invasion.The gill mucosa constitutes the first line and plays an important role in the prevention of pathogenic infection invasion.This study explored the immune response function of gill mucosa in pathogenic infections(such as viruses and bacteria),holding great significance for the disease prevention and control of fish.Fish infectious hematopoietic necrosis virus(IHNV,Novirhabdovirus,Rhabdoviridae)is one of the most destructive pathogens in salmon trout.It primarily infects the juvenile stage of the fish,resulting in the extremely high mortality rate and causing serious damage to salmon trout farming.Therefore,in this study,we first constructed an IHNV model of rainbow trout immersion infection and analyzed the histopathological changes and viral load changes in the gill mucosa after viral infection.It was found that IHNV can significantly infect the gill mucosal tissue of rainbow trout.The infection period is mainly within two weeks with the highest load 4 days after infection(DPI).Severe tissue damage and inflammatory response appear in the gills with an increase in the goblet cells of the primary lamella(PL)and secondary lamellar(SL).Further analysis by transcriptomic sequencing demonstrated that the virus significantly induced a strong innate and adaptive immune response in the gill mucosa of rainbow trout.Interestingly,we noticed that the role of gill mucosa is not limited to the antiviral immune response but also includes a significant antibacterial immune response,indicating that the gills of fish developed a secondary bacterial infection during viral infection.Following IHNV infection,there was a disturbance in the equilibrium of commensal microorganisms in rainbow trout,as evidenced by 16 S r RNA sequencing analysis.The gill mucosa exhibited a significant decrease in both the richness and abundance of bacterial species.Moreover,even the microorganisms that survived the infection could not regain their original homeostatic state.Specifically,there was a substantial increase in the Proteobacteria phylum.Overall,our findings indicate that IHNV infection can significantly impact mucosal immunity and microbial community,leading to changes in pathological and bacterial structure.The columnar disease caused by Flavobacterium columnare(FC)leads to severe damage to the skin,gills,and other mucosal tissues of fish.We also found a significant increase in the abundance of FC in the gill mucosa of rainbow trout after IHNV infection.Subsequently,we proceeded to analyze microbial homeostasis changes and immune responses during bacterial infection with the gill mucosa of rainbow trout.After exposing fish to FC,we detected substantial bacterial proliferations in the gill tissue,as well as significant tissue damage and inflammation.Our analysis of the organism’s 16 S r RNA sequencing further revealed that the fish’s gill mucosa surface microorganisms were disrupted by the infection.This disruption could significantly impact the normal functions of the gill mucosa.As a result,when a virus infects a fish,the bacteria present on the surface of the gill mucosa may exploit the weakened state to reproduce,giving rise to a secondary infection which leads to another bacterial disease.Teleost fish were the first vertebrates with a well-developed adaptive immune system,comprising three immunoglobulin types(Ig M,Ig D and Ig T/Ig Z).In order to further explore the immune response of fish gill mucosa in the process of FC infection,we comprehensively analyzed the innate and adaptive immune-related gene expression,gill mucosal immunoglobulin and B cell-mediated humoral immune response of rainbow trout from the molecular,cellular and protein levels based on the rainbow trout mucosal bath infection model.Previous studies have shown that Ig T in fish gill mucosal tissue exerts important mucosal immune function during parasitic sensing,similar to Ig A in mammals.However,the function of immunoglobulins in the gill mucosal tissue during bacterial infection remains unclear.The results of this study revealed that rainbow trout’s gill structure experienced severe hyperplasia 12 hours post-infection(HPI)with FC.Additionally,the m RNA expression levels of NOS2 and cathelicidin-1 were significantly regulated throughout the infection process,while the expression of Ig T,Ig D,and Ig M heavy chain genes significantly increased 28 DPI.The findings showed the infectioninduced innate and adaptive immune responses in the trout’s gill mucosa.Furthermore,immunofluorescence and Western blot analyses demonstrated a significant increase in the content of Ig T and Ig T+ B cells in the gill mucosa,and a large quantity of Ig T in the gill mucus could specifically bind to FC.Hence,the results suggest that fish Ig T and Ig T+ B cells serve an essential immune function in the process of gill mucosa anti-bacterial infection.This study succeeded in inducing gill infections in juvenile rainbow trout by using the bath technique for both the IHNV virus and the FC bacteria,which had a significant impact on the trout’s gills.The infections triggered both innate and adaptive immune responses,with the former being responsible for the initial response.The study found that both types of pathogens significantly affected the trout’s gill microbiota.The critical period for observing changes in morphology,immune response,transcription profiles,and microbiota was the first week of exposure(4 DPI).Furthermore,the study on the trout’s gill transcriptome after infection revealed an increase in genes associated with virus and bacteria immunity.The study also focused on the Flavobacteriales family of bacteria known for causing gill diseases in cold water fish,which will be further explored in chapters three and four. |
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