| Sebastes schlegelii is an ideal fish species for aquaculture,resource enhancement and recreational fishing in the northern coastal areas of China.In recent years,with the expansion of S.schlegelii culture scale and the limitation of disease prevention and control technology,culture diseases occur from time to time,and there are more and more types of diseases,among which bacterial diseases are the most common.Vibrio harveyi is a bacterium widely distributed in the ocean,and vibriosis caused by V.harveyi has been considered as one of the major diseases in aquaculture,which seriously restricts the healthy and sustainable development of the aquaculture industry.In this study,a dominant strain of P5W was isolated and purified from the foci of diseased S.schlegelii,which was identified as V.harveyi by 16S rDNA gene sequence analysis,and its physicochemical characteristics,extracellular product activity,histopathology and drug sensitivity were investigated to analyze the pathogenicity of P5W,explore the pathogenesis of P5W,and clarify the drug sensitivity of P5W.Further,based on RNA-sequencing technology,we analyzed the transcriptome differences of P5W-infected redfish,and clarified the main signaling pathways and immune genes in response to V.harveyi infection in redfish,and gained insight into the immune response mechanism of V.harveyi to Vibrio-like pathogens.The results of the study provide data for the prevention and control of V.harveyi disease.1.Isolation and identification of V.harveyi P5W as the causative agent of the S.schlegeliiIn this study,a distinctive and dominant strain of P5W was isolated and purified from the lesions of diseased S.schlegelii.The colonies were grayish white with smooth and moist surface on 2216E medium and yellow with regular and smooth edges on TCBS medium.Based on morphological observation,physiological and biochemical characteristics and 16S rDNA gene sequence analysis,it was identified as V.harveyi.Artificial infection experiments determined the pathogenicity of P5W to S.schlegelii with an LD50 of 5.7×106 CFU/mL.2.Growth characterization,histopathological observation,extracellular product activity analysis and drug susceptibility testing of V.harveyi P5WIn this study,the effects of different temperature,pH,salinity and other physicochemical factors on the growth of P5W were analyzed,and the results showed that the suitable growth conditions for P5W were 15～35℃,pH5～10,Na Cl content1～8.Histopathological observations were made on the liver and spleen of healthy and diseased redfish.The results showed that the hepatocytes of the diseased fish were obviously vacuolated,the hepatocytes were atrophied and the intracellular material was darkened,the spleen had discrete cells and iron-containing heme deposits,some epithelial cells of the renal tubules were necrotic,the glomeruli were atrophied and the muscle fiber structure was broken and necrotic.The results showed that the extracellular products of P5W had protease,lipase,lecithin,amylase,urease and gelatinase activities,and were hemolytic to sheep blood;the sensitivity of P5W to different antibacterial drugs was examined,and the results showed that P5W was particularly sensitive to ceftriaxone and tetracycline,and moderately sensitive or insensitive to erythromycin,cefoperazone and gentamicin.3.Transcriptome analysis of S.schlegelii blood before and after stimulation by V.harveyiThe blank control group（B-0h）,12h post-infection experimental group（B-12h-E）and control group（B-12h-C）,48h post-infection experimental group（B-48h-E）and control group（B-48h-C）were selected as the experimental time points,and 15 samples of S.schlegelii blood were sequenced by high-throughput sequencing.A total of1,019,795,876 raw reads were obtained,and 889,746,122 clean reads were obtained after quality control,and 104,976 unigene sequences were obtained by splicing,with an average length of 1,080.28 and N50 of 1,478;the spliced genes were annotated by NR,GO,COG,KEGG and Swiss-Prot databases.Functional annotation was performed,and36,822 genes were successfully annotated.In the differential gene expression analysis,8,391 differentially expressed genes were screened between the 12h control and 12h experimental blood samples,and 2,805 differentially expressed genes were screened between the 48h experimental and 48h control blood samples.The screened differential genes were analyzed by GO enrichment and KEGG enrichment,and a large number of immune-related terms such as bacterial defense,immune activation,immune regulation,and pathways such as NOD-like signaling pathway,necroptosis,and T cell receptor signaling pathway were obtained.Further analysis of immune genes enriched into the above pathways resulted in the screening of immune-related differentially expressed genes such as CCL4,CCNB1,and FADD.4.Transcriptome analysis of S.schlegelii spleen before and after stimulation by V.harveyiThe blank control group（S-0h）,12h post-infection experimental group（S-12h-E）and control group（S-12h-C）,48h post-infection experimental group（S-48h-E）and control group（S-48h-C）were selected as the experimental time points,and 15 samples of S.schlegelii spleen were sequenced by high-throughput sequencing.A total of992,503,854 raw reads were obtained,and 872,919,720 clean reads were obtained after quality control,accounting for 87.95%of the raw reads.In the differential gene expression analysis,4,571 differentially expressed genes were screened between spleen samples of 12h control and 12h experimental groups,and 5,378 differentially expressed genes were screened between blood samples of 48h experimental and 48h control groups.The screened differential genes were analyzed by GO enrichment and KEGG enrichment,and a large number of immune-related terms such as regulation of immune cell apoptosis,immunoglobulin,regulation of immune cell activity,and pathways such as necroptosis,antigen processing and delivery were obtained.Further analysis of immune genes enriched into the above pathways resulted in the screening of MHC1,CD22,IL4I1 and other immune-related differentially expressed genes. |
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