Studies On The Changes Of Skin Mucosal Immunity And Symbiotic Microbiota In Rainbow Trout (Oncorhynchus Mykiss) Infected With IHNV

The skin is the largest mucosal immune tissue on the surface of teleost fish,serving as the first line of defense against the invasion of external pathogens.At the same time,there are many symbiotic microorganisms colonizing on the surface of fish skin.Because of the special living environment of fish,their skin is always threatened by various pathogenic microorganisms and harmful substances in the external water.There are complex interactions between the skin immune response of the host,external pathogens and symbiotic microorganisms,which affects the health of the body at all times.Due to the continuous expansion of aquaculture scale and the improvement of intensive level,the disease problem of rainbow trout(Oncorhynchus mykiss)has become increasingly prominent.Infectious hematopoietic necrosis is one of the serious infectious diseases,which is caused by infectious hematopoietic necrosis virus(IHNV).The mortality rate of rainbow trout after IHNV infection is very high,seriously hindering the development of rainbow trout farming industry.However,it is unclear whether virus infection can cause mucosal immune response of teleost skin,how virus infection affects the composition of microbiota on skin surface,and whether IgT and IgT~+B lymphocytes in skin tissue play an antiviral immune role.Therefore,this study used IHNV to infect rainbow trout and analyzed the pathological changes in skin tissue,expression changes of immune genes,and changes in the composition and structure of skin microbiota after IHNV infection.Finally,we detected the changes of B lymphocytes and mucosal immunoglobulins during adaptive immune response.The main results are as follows:1.IHNV successfully invaded rainbow trout and caused significant pathological changes in skin.After infected with IHNV,the diseased fish exhibit the following typical symptoms:darkening of the skin,exophthalmia and pale gills.The qPCR results showed that the viral load in the skin tissue was the highest 4-7 days post IHNV infection,and then gradually decreased.Pathological section staining at different time points revealed the histopathological changes of skin caused by IHNV invasion:the thickness of epidermis became thinner,the number of goblet cells decreased and epithelial cells fell off.These changes were most obvious around 7 dpi,and then gradually recovered.2.After IHNV infection,the skin first initiates an antiviral innate immune response,and then activates a strong antibacterial immune response.The expression of immune related genes in the skin and transcriptome sequencing results showed that the innate antiviral immunity was strongest 4-7 days after IHNV infection,while the anti-bacterial immune response was stronger about 14 days after IHNV infection.The sequencing results of the microbiota revealed that IHNV infection significantly destroyed the homeostasis of the microorganisms on the skin surface and caused significant increase in conditional pathogenic bacteria.Combined with transcriptome sequencing results,it is concluded that the invasion of IHNV destroys the homeostasis of microbiota on the skin surface,resulting in the increase of conditional pathogenic bacteria,thus activating the anti-bacterial immune response.3.IgT~+B lymphocytes and IgT play important roles in the adaptive immune response against IHNV invasion.Through immunofluorescence,flow cytometry and western blotting,it was found that the number of IgT~+B lymphocytes in the skin of survival fish immunized twice after IHNV infection was significantly increased,and the concentration of IHNV-specific IgT in skin mucus was also significantly increased.The results of this study indicate that IHNV infection can cause significant lesions in the skin tissue of rainbow trout,and after IHNV invasion,the skin tissue first initiates an antiviral immune response,which may subsequently lead to secondary bacterial infection due to disruption of mucosal microorganisms.During the adaptive immune response,IgT~+B lymphocytes and mucosal IgT may play important antiviral functions.In summary,the results of this study provide new insights into the interactions between fish skin mucosal immune response,invading viruses,and symbiotic microorganisms,and once again emphasize the important role of mucosal IgT in the skin mucosal immune response.