Effects Of Hemolysis Factors Deletion On Pathogenicity And Immune Response Of Lateolabrax Maculatus Infected With Vibrio Anguillarum

Lateolabrax maculatus is an important marine fish in China.In recent years,due to the increase of breeding density and the increasingly serious water pollution,the resistance of L.maculatus to pathogen infection has been reduced,and the fishery losses caused by disease problems have become more and more serious.Vibrio anguillarum is a Gram-negative pathogenic bacterium widely distributed in seawater.It is one of the important sources of bacterial diseases in mariculture and can cause serious infections in fish.It is very important to understand the infection mechanism of V.anguillarum and the immune mechanism of L.maculatus for the prevention of diseases in L.maculatus culture,but so far there are few reports on this aspect.In this study,the strain with hemolysis-related genes deletion,V.anguillarum△vah1-vah4-rtx A previously constructed in our laboratory,was used to compare the infection ability and pathogenicity of the wild strain in L.maculatus.The pathological changes of L.maculatus and the expression of immune-related genes were compared between wild and this mutant strain as well.The results showed that:(1)Pathogenicity detection of V.anguillarum in infected L.maculatus:By injecting the wild strain and the deletion strain△vah1-vah4-rtx A of V.anguillarum into the young L.maculatus,it can be seen that the median lethal dose after injection of the wild strain was 2.1028×10~5 CFU/ml,and the median lethal dose after injection of the deletion strain△vah1-vah4-rtx A was 1.8372×10~6 CFU/ml.By counting the number of V.anguillarum wild strains and deletion strains in different tissues of L.maculatus,V.anguillarum showed a gradual increase in different parts of L.maculatus,especially in the intestine and gill.At 24 h after challenge,a large number of V.anguillarum was first observed in the intestine of L.maculatus,and at 48 h,a significant increase in the number of colonization was observed in the gill.At 48 h,the number of intestinal colonization was 2.13×10~5 CFU/g,and the number of gill colonization was 1.16×10~5CFU/g.The number of V.anguillarum in the intestine was about 1.84 times that of the gill,and 15～90 times that of the liver,spleen and kidney.By analyzing the pathological tissue sections of the wild strain and the deletion strain infected by the L.maculatus,it was found that the symptoms of the L.maculatus infected with the deletion strain were significantly lower than those of the wild strain infected with the wild strain,such as hepatocyte necrosis,spleen congestion,intestinal tissue vacuolization,and gill filament lesions.(2)The immune response of V.anguillarum infected L.maculatus:The results of GO enrichment analysis of differential genes showed that the enriched GO terms were related to immune response,such as biological regulation function category(GO:0065007)and protein binding category(GO:0005515).The immune-related pathways in the KEGG enrichment included hematopoietic cell lineage(ko04640),allograft rejection(ko05330),cell adhesion molecules(ko04514),Ig A production in the intestinal immune network(ko04672),antigen processing and presentation(ko04612),etc.Thirty-seven immune-related genes were screened from the significant difference genes,and immune-related antimicrobial peptide genes such as DUOX2 and hamp,BCR signaling pathway genes such as IGHV3-21 and cd22 were screened.In addition,some immune-related genes were also screened in antigen processing and presentation,chemokines,cytokines,cytokine receptors,etc.Further analysis of the important genes HLA-DRA,cd22 and the antimicrobial peptide hepcidin encoding gene hamp in the Hematopoietic cell lineage pathway showed that the homology of HLA-DRA,cd22 and hamp in fish was generally higher than 70%.RT-PCR expression analysis showed that the expression levels of six tissues such as liver and spleen were up-regulated after infection with V.anguillarum.After the deletion of hemolysis-related genes vah1,vah4and rtx A,the relative expression levels of immune-related genes HLA-DRA,cd22 and hamp were lower than those of wild strain infection.After deletion of hemolysis-related genes vah1,vah4,and rtx A,the pathogenicity of V.anguillarum infecting L.maculatus decreased significantly,and the immune response of L.maculatus decreased.The results of this study are helpful to understand the preliminary mechanism of V.anguillarum infection in L.maculatus,and it is expected to prepare immunopotentiators against V.anguillarum based on the△vah1-vah4-rtxA attenuated strain.