The Pathogenicity And Differential Expression Of Immune-Related Genes From Intestine Of Cynoglossus Semilaevis Induced By The Infection Of Shewanella Algae

In this study,a Gram-negative bacterium was isolated from diseased Cynoglossus semilaevis and tentatively named strain CSG-15.It was preliminary identified as Shewanella algae based on morphological,physiological and biochemical characteristics.The sequenced16 S r RNA gene of CSG-15 strain(Genbank accession no.KX455851)was 1509 bp.And it exhibited 99.86%,99.86% and 99.80% of identities compared with those of Shewanella algae strains ATCC51192(NR117771),YJ06114(EF542799),and MAS2737(GQ372875),respectively.Phylogenetic analysis grouped CSG-15 strain in the Shewanella algae cluster,and showed the closest relation to Shewanella algae ATCC51192 and YJ06114.Infection experiment showed that the median lethal dosage(LD50)for CSG-15 was calculated as 1.0×106 CFU/g fish weight.The pathological observation on the intestine showed that CSG-15 strain caused noticeable histological lesions,such as inflammatory cell infiltrating and villi shedding.Antimicrobial susceptibility test showed that it was highly sensitive to most of aminoglycosides,?-lactams,tetracyclines,quinolones,macrolides aminocyclitol,sulphonamides,chloramphenicols antibiotic.Growing characteristics showed that CSG-15 could grow under the condition of temperature 26-34°C,p H 5-9 and 20-50 ppt,and had a better adaptability to environment.The growth curve of strain CSG-15 under the condition of30°C,p H 7.0 and 30 ppt showed four distinct phases of bacterial growth.To the best of our knowledge,this is the first report of Shewanella algae linked to diseased C.semilaevis.The change of the structure of intestinal flora from Cynoglossus semilaevis Günther artificial infected with Shewanella algae was studied by 16 s r DNA high-throughput sequencing method.The gut microbes were collected at 0 h,24 h,48 h,72 h,and 96 h after infection.There are total 4545 OTUs from the intestinal microbes of 5 groups of half smooth tongue sole.There are 902 OTUs in common between the group CSH and CSY.The number of OTU for the samples at 0 h was the least,and it increased after infection with the pathogen and reached the peak for the sample at 72 h.And the number decreased obviously for the sample at 96 h,similar with that at 48 h.Result of species richness and diversity of the intestinal flora showed that numbers and species of intestinal flora increased with the time from 0 to 72 h after infection and decreased after 72 h.Results on beta diversity study showed that the groups of CSY(96 h)and CSH(24 h)clustered together,simalilsr with the groups ofCSB(0 h)and CSS(48 h).The groups of CST,CSB,and CSS also clustered together.Gut microbial diversity of the samples between at 24 h and 96 h,or at 0h and 48 h was similar.Compared with the other groups,Microbial diversity of the samples at 72 h was the most similar with those at 0 h and 48 h.In this study,the changes of intestinal dominant bacteria and pathogenic bacteria after infection with Shewanella algae were analysed.This can provide the clues for illuminating the pathogenesis,and also attribute to the process of the prevention and control for the aquatic animal disease.Teleost fish gut mucosa-associated lymphoid tissue played important roles in the immune response.We established an infection model of Cynoglossus semilaevis infected with Shewanella algae.And the gut samples were RNA-sequenced and the DEGs were analyzed.Total 23356 genes were indentified and the number was 19604,19534,20267,19568,19773,19530,19736,19799 and 19179,and account for the percent of 83.94%,83.64%,86.77%,83.78%,84.66%,83.62%,84.5%,84.77%and 82.12% for the sample of STH-1,STH-2,STH-3,STZ-1,STZ-2,STZ-1,STR-1,STR-2 and STR-3,respectively.GO and KEGG pathways analysis showed the screened immune DEGs were involved in complement system,Chemokines,phagocytosis,natural killer cell mediated cytotoxicity,Rap,GF-beta,Jak-STAT,NF-k B signal pathways.As a result,26 DEGs accociated with immune functions were indentified,including AMBP,FGB,CFD,C9,FGG,CCK and so on.There were 19 genes were downregulated,including AMBP,FGB,CFD,C9,FGG,VIT,COL6A1,MDK,CALML6,TGFBI,GSN,TAGLN,MDK,PCK1,MAMDC2,VAT1,ABCA12,FHL1 and ID4.And 7 genes were upregulated,including CCK,PTX3,HSP90B1,SOCS3,TMIGD1,TP53I3 and SOCS3.Then,the gene SOCS-3,CCK,COL6A1,TGFBI,FHL1 and HSP90B1 were validated by the real time quantitative PCR,and the results were consistent with RNA-seq.This is the first study on the RNA-sequencing and the DEGs of gut from Cynoglossus semilaevis infected with Shewanella algae and could attribute to understanding the gut mucosal immune response to pathogens.