| Salmonella enteritidis(SE)often colonizes in the ovary and other tissues of the adult poultry reproductive tract,causing direct contamination and vertical transmission of eggs that are difficult to eradicate and prevent.It not only affects the egg production,but also causes egg pollution as well as environmental pollution and harms human health.The immune function of the ovaries and oviduct is necessary to protect these tissues from infection and to produce hygienic eggs,but the complex immune regulation mechanism is not yet clear in the course of SE infection of the reproductive tract.In this study,high-yielding Shao ducks were used as research objects.Through the establishment of artificial infection models,the roles and molecular mechanisms of duck PERP in SE infection were investigated through RNA-seq transcriptome sequencing and yeast two-hybrid system.The aim of the study was to fully reveal the mechanism of PERP in regulating the apoptosis of duck granulosa cells(dGC)infected with SE.At the same time,it can also provide a certain reference for reducing SE infection and improving the safety of poultry eggs.The main findings are as follows:1.Dynamic distribution and partial immunoassay detection in the reproductive tract of artificially SE-infected ducksThe tissue distribution of SE was detected by specific PCR and immunohistochemistry,and the fecal discharge was systematically monitored 13 weeks after infection.The results showed that most of the SE-infected ducks were in carrier-stage but the mortality rate was not high.However,with long-term persistence of fecal shedding of SE after sexual maturity,the bacteria spread to the reproductive tract and often colonized in the ovarian stroma,small follicles,oviductal isthmus and vagina.In this study,the serum levels of immunoglobulin(IgA,IgG,IgM),T lymphocyte subsets(CD3+,CD4+,CD8+)and reproductive hormones were detected by ELISA.The results showed that serum levels of IgA,IgG and IgM of the susceptible group were significantly higher than those of the resistant group(P<0.05),While the T lymphocyte subsets(CD3+,CD4+,CD8+)showed the opposite trend.The level of reproductive hormone(PROG,E2,LH,FSH)of the susceptible group were significantly lower than that of the resistant group.In this study,RT-qPCR was used to detect the immune response gene expression profile of bacteria colonized tissues after 13 weeks infection.The results showed that after SE infection,the expression of Toll-like receptors(TLR2,TLR4-5,TLR15,TLR21),NOD-like receptors(NOD1,NLRX1,NLRP12),avian β-defensins(AvβD4-5,AvβD7,AvPD12),Cytokines(IL-6,IL-1β,IFN-γ)and MyD88 were significantly up-regulated in ovarian stroma,small follicles,oviductal isthmus and vaginal tissue(P<0.05).However,the expression of TLR3,TLR7,NLRC3 and TNF-a were significantly down-regulated in the above tissues(P<0.05).The above results indicate that the duck produces a strong immune response after SE infection in vivo.In addition,this study also examined the effect of SE infection on egg production performance and egg quality.The results showed that after SE infection,production was postponed.The egg production rate of SE susceptible ducks was only 67.14%at 24 weeks,which was significantly lower than that of resistant group(92.86%)and control group(98.57%).At the same time,it was also found that the index of eggshell weight,egg shell thickness,eggshell strength,protein height and egg yolk color decreased significantly after SE infection(P<0.05).2.Screening of key candidate genes for SE infection duck based on RNA-seqIn order to reveal the role of key candidate genes in duck SE infection,RNA-Seq high-throughput sequencing technology was conducted to analyze differentially expressed genes in ovarian tissues(small follicles and ovarian stroma)infected with SE based on the above established duck model of oral infection.The results showed that 915 and 1203 differential genes were screened out from small follicles and ovarian stroma tissue,respectively.Among them,23 genes such as PERP,EGFR,PAFAH1B2,DOK6 and RXFP2 are common differentially expressed genes of both group.KEGG enrichment analysis showed that differentially expressed genes are mainly enriched in p53 signaling pathway which involved in process of apoptosis.RT-qPCR was used to verify some of the differential genes,and their expression trends were consistent with the transcriptome sequencing results.The results confirmed the reliability of the RNA-Seq high-throughput sequencing and laid the foundation for further investigation of the pathogenic mechanism of SE.This study screened the key genes related to SE infection in duck ovary.3.Study on the role of PERP in SE-infected duck granulosa cellsIn order to explore the molecular mechanism of PERP in SE-infected ducks,the key candidate gene PERP in SE-infected duck was selected as the research object.We used knockdown or overexpressing PERP in duck granulosa cells to verify its effect on the SE infection process.The expressions of p53,MDM2,PERP,caspace-3 and Bcl-2 were detected by qPCR and WB,respectively.The results showed that the expression of p53,MDM2,PERP,caspace-3 and Bcl-2 were upregulated in SE-infected duck granulosa cells when the PERP were overexpressed(P<0.01),while the trend was opposite when we knocked down the PERP expression of duck granulosa cells.CCK8 and FACS were also used to detect the proliferation and apoptosis of PERP-overexpression and PERP-knockdown duck granulosa cells,respectively.The results showed that when overexpressed PERP were promoted the apoptosis of dGC(P<0.01),while knocked down the expression of PERP were enhanced the proliferation of dGC.Furthermore,the adhesion and invasion of SE of PERP-overexpression and PERP-knockdown duck granulosa cells were detected by colony forming unit.The results showed that the SE adhesion and invasion of dGC with overexpression of PERP was significantly increased(P<0.01).Knocking down the PERP reduced the amount of SE adhesion and invasion of dGC.These results fully confirmed that PERP mediated the p53 signaling pathway involved in duck granulosa cells infected by SE.4.Study on the interaction between duck PERP protein and SE SipC proteinIn order to reveal the involvement of duck PERP protein in the pathogenesis of SE in vitro,this study used Gateway technology to use SE MY1 SipC protein(effector protein secreted into host cells during SE infection)as a "decoy”protein,through GAL4 yeast two-hybrid system,Twelve proteins that may interact with SipC protein were screened from duck granulosa cell cDNA library,among them,the positive clones expressing PERP had a high frequency.The sipC gene deletion strain was constructed by constructing the,Red homologous recombination system,and the interaction between SipC and PERP was further verified by one-by-one yeast revertive hybridization and GST-pull down test.In vitro experiments have shown that overexpression of PERP can promote adhesion of SE to duck granulosa cells.This experiment laid the foundation for further revealing the molecular mechanism of SE infection.In summary,not only the immune response of duck ovary stroma,small follicles,oviduct isthmus and vagina,but also egg production performance and egg quality are affected after SE infection.In the process of SE infection,the key effect factor PERP mediated the p53 signaling pathway and interacted with SipC protein to promote SE invasion. |
