| Piglet diarrhea is one of the common intestinal diseases in pig industry.As the disease progresses,immune system function of piglets would be decreased,growth would be slow,feed utilization would be decrease and mortality would be increased.Based on statistics,half of the deaths were caused by diarrhea each year,leading to huge economic losses and endangering the development of the pig industry.According to the cause of diarrhea,there are physiological,nutritional,stress,autogenic,and disease diarrhea.Disease diarrhea include viral,parasitic and bacterial diarrhea.Among them,Clostridium perfringens type C(C.perfringens)is one of the major factors causing diarrhea in piglets.It is extremely high that an incidence rate(60-80%)and mortality(80~100%)in 7 days piglets.Many studies on investigation,diagnosis,prevention and treatment of molecular epidemiology have conducted.However,it is still unclear that the structure and function of protein in diarrhea,especially the immune regulatory mechanism and signaling pathway activation caused by C.perfringens.In this study,30 piglets were selected as subjects.Five heads were randomly selected as control groups(NC).The remaining 25 pigs were challenged by orally bouillon culture-medium inoculated with C.perfringens type C to induce andestablish a model of diarrhea.The diarrhea score and mental status were recorded.The top five with and last bottom five piglets with based on total diarrhea scores were considered as susceptible group(IS)and resistan group(IR).qRT-PCR,Western Blot,and ELISA were used to study the innate immune responses of C.perfringens associated with diarrhea after infection in piglets,and to evaluate the molecular mechanism of diarrhea in the intestinal response of piglets.To solve the scientific problem of C.perfringens-resistance breeding for the binary pigs(Large White × Landrace),the reference was provided.The main findings are as follows:1.qRT-PCR detection found that some key genes were quantified in TLRs signaling pathwayincluding TLR2,TLR4,TLR9,MyD88,NF-?B,TRAF6,MAPK1,TBK1,TNF-?,IFN-?,TL-6.Compared with NC group and IR group,the expression of IL-8 was up-regulated.TBK1 gene was significantly up-regulated in IS group(P<0.01).2.Western blot analysis of TLR4,HMGB1,NF-?B protein concentration,the results showed thatin the jejunum and ileumTLR4,HMGB1,NF-?B in IS group were higher than those in NC group and IS group.In IS and IR group.The content of TLR4 was the highest,followed by NF-?B,and however the HMGB1 was the lowest.The results were consistent with the results of protein relative expression.3.The levels of interleukin(IL-1?,IL-6,IL-8,IL-10,IL-12)and interferon(IFN-?)in jejunum and ileum were measured by ELISA.The results showed that the content of factors increased,except for the IL-10 levels in the IS and IR groups were not significantly different from those in the NC group.The data in the other groups were significant.The levels of IgA,IgG and IgM in blood changed at different time points.CONCLUSION: The C.perfringens induced piglet diarrhea model can activate TLRs signaling pathway in the small intestine,increase the expression of related genes in the TLRs signaling pathway,and then regulate the release of downstream inflammatory factors,thereby supporting intestinal mucosal immunity.This experimental study found that compared with jejunum tissue,the expression of related genes in ileal was higher and the cytokine content was higher.And the main target organ of C.perfringens is the ileum segment. |
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