Effects Of Edwardsiella Live Attenuated Vaccine On The Turbot Gut Microbiome Based On 16S RRNA Amplicon Sequencing

Edwardsiella piscicida causes edwardsiellosis in a variety of fish species and has led to significant economic losses in aquaculture worldwide.Therefore,effective and secure control measures are urgently needed to control Edwardsiella infections.Live attenuated vaccine(LAV)is one of the most effective means to control bacterial infections in fish.The current live attenuated vaccines against E.piscicida are based on the deletion of the genes encoding type Ⅲ secretion system(T3SS),which is the core virulence system of E.piscicida.In this study,E.piscicida 10Δ exhibited more sensitivity to reactive oxygen species and an impaired ability of biofilm formation.The colonization of E.piscicida 10Δ in the host liver and intestine increased first and then declined due to the clearance of the host immune response.The intraperitoneal vaccination by E.piscicida 10Δ significantly upregulated the expression of genes encoding immune-related proteins(INF-γ,IL-12,IL-10,MHC Ⅰ,MHC Ⅱ,CD8,IL-1β,IgM)in the turbot spleen and enhanced the serum antibody titers of IgM.Microorganisms naturally exist in the form of communities.Different microorganisms compete or collaborate with each other,eventually forming a complex interaction network.Here,we analyzed the diversity of microbial community based on 16S rRNA amplicon sequencing.The 16S V4(515-806)segment was chosen to build the library by a two-step PCR method.DADA2 was used to denoise the raw data and generate amplicon sequence variants.QIIME2 was employed to create feature tables and tree files,and finally the trained classifier SILVA was used to obtain a species annotation table.This method can effectively determine the species and the relative abundance of microorganisms in the specific community.A complex and dynamic microbial community is attached to the mucosal surface of the fish intestine,which plays an important role in fish metabolism,immunity,and intestinal development.16S amplicon sequencing revealed that the vaccination of E.piscicida 10Δ slightly reduced the relative abundance of intestinal microbiota in turbot.The vaccination of E.piscicida 10Δ increased the relative abundance of Vibrio and decreased the relative abundance of Pseudoalteromonas,Oceanospirillum and Oleibacter compared to the healthy turbot gut microbial community.Meanwhile,E.piscicida 10Δ conferred the intestinal microbiota metabolic capacity associated with fructose and mannose metabolism,galactose metabolism,starch and sucrose metabolism,while impaired their metabolic capacity related to fatty acid degradation and valine,leucine,and isoleucine degradation.At the generic level,inoculation with E.piscicida 10Δ had a smaller effect on the level of species composition of the host major community and caused less change in the alpha diversity of the intestinal flora than that caused by infection with the wild type of E.piscicida,and these new features allow a more comprehensive evaluation of the safety of this strain as a potential live attenuated vaccine.In this study,we studied the physiological and biochemical phenotypes and stimulated host immune response characteristics of the candidate live attenuated vaccine E.piscicida 10Δ,established the method and standard for the identification of metagenomic identification of fish intestinal microorganisms,and further investigated the changes of host intestinal microflora caused by vaccine immunization,providing a new perspective and strategy for the evaluation and development of fish live attenuated vaccines.