Molecular Mechanism Of Vibrio Alginolyticus Infection In Litopenaeus Vannamei And Study On Bacteriophage Control

In order to understand the pathogenic mechanism of Vibrio alginolyticus infecting in Litopenaeus vannamei,and to detect the practical application effect of bacteriophage.Vibrio alginolyticus(1×10~6CFU/m L)was used to challenge Litopenaeus vannamei.At different time points(0,3,6,12,24,48h),samples from three groups that include control group(CK)of injection of PBS buffer,bacteriophage group(BP)of shrimp after immersion using bacteriophage product(LUXON)after injection with Vibrio alginolyticus and Vibrio group(VA)injected only with Vibrio alginolyticus.The effects of Vibrio alginolyticus on tissue structure,antioxidation,expression of immune-related genes and intestinal microbiome of Litopenaeus vannamei were analyzed.The specific results are as follows:At 3h compared with CK group,the arrangement of gill tissue in VA group and BP group was loose,the tissue structure was damaged and deformed by an optical microscope.With the extension of infection time,the epithelial cells were deformed or even broken,and vacuoles appeared in the gill filament cells.The hepatopancreas of VA group and BP group showed obvious epithelial cell exfoliation,lumen deformation and a large number of nuclei scattered in the interstitial space of hepatic tubules at 3h.The intestinal mucosal epithelial cells of VA group and BP group showed obvious damage at 6h and large area exfoliation at12h.It was proved that Vibrio alginolyticus could quickly break the defense system of healthy shrimp in the early stage of infection,and the tissue morphology of hepatopancreas and intestinal tract recovered after 48h,which indicated that the tissue structure of Litopenaeus vannamei could recover after being damaged to a certain extent.In this study,the content of MDA,T-AOC,ALT,AST and gene expression of pro PO gene and IMD gene in gill,hepatopancreas and hemocytes of Litopenaeus vannamei were detected.The results showed that MDA,ALT and AST in VA group increased significantly at 3h,and oxidative stress was the most serious.The activity of ALT in BP group was significantly lower than that in VA group at 3h and then increased significantly at 24h.In addition,the change trend of AST activity was similar to that of ALT.The expression of Lvpro PO in hepatopancreas was significantly up-regulated in VA group at 3h.Between 3h and 48h,the expression of Lvpro PO in BP group was significantly lower than that in VA group(P<0.05).The expression of Lvpro PO and Lv IMD in BP group was significantly higher than that in VA group at 12h,and then increased significantly at 48h(P<0.05).It shows that infection with Vibrio alginolyticus,the antioxidant capacity of shrimp enhanced with the activation of humoral immunity of innate immune system,which enhanced the resistance.And the use of bacteriophage is helpful to the antioxidant stress of shrimp,but the effect is not long-lasting.The protective effect on gill tissue is the most obvious.In order to study the changes of intestinal microbiome of Litopenaeus vannamei infected with Vibrio alginolyticus,shrimps was divided into three groups:blank group(NC),PBS control group(CK)and Vibrio alginolyticus infection group(VA).The intestinal samples were collected at 12h and 48h.The 16S r DNA gene V3-V4 region of bacteria was sequenced by Illumina Mi Seq platform.Compared with NC group,the content of Proteobacteria increased significantly(P<0.05),while the content of Actinobacteria and Tenericutes decreased significantly after infection with Vibrio alginolyticus.The abundance of Shewanella in intestinal microbiome of Litopenaeus vannamei increased significantly at12h(P<0.05),but the abundance of Candidatus＿Bacilloplasma decreased significantly at48h after infection(P<0.05).The intestinal microbiome of healthy shrimp maintained a dynamic balance.After infection with Vibrio alginolyticus,the multiplicity of intestinal microbiome decreased significantly at 12h,which could be caused by the proliferation of Candidatus＿Bacilloplasma and Shewanella.However,the specific mechanism needs to further study.The results show that the stability of the dynamic balance of microbiome in the intestine helps Litopenaeus vannamei resist pathogen colonization.