| Some research have proved the ability of flagellin to induce the innate immunity in host, but rare research was about the role played by flagellin in innate immunity triggered by recombinant attenuated Salmonella strains. This study was to investigate the role played by flagellin in innate immune of chickens and mice induced by an attenuated Salmonella Typhimurium, providing evidences for choosing suitable attenuated Salmonella strain as vaccine vector.The specific primers were designed according to the conserved sequences of IL-6, IL-1β, IFN-γ, TGF-β4, β-actin mRNA. Total RNA was extracted from chicken macrophage cell line HD11stimulated by flagellin, and was transcribed reversely into cDNA. The qRT-PCR was established to evaluate the relative expression level of four cytokines’mRNA. In result, melting curve was single peak, and amplification curves of the same cDNA after three same reactions’amplification were superpositions. The results showed that qRT-PCR was a specific and repeatable method. On the stimulation of flagellin, inflammatory IL-6, IL-1β genes were up-regulated, while the expression level of IFN-y, anti-inflammatory TGF-β4genes were not changed in HD11. Then the heterophils from one-week chicks’blood were isolated and interacted with flagellin, attenuated Salmonella Typhimurium X4550(pYA3334-F) and the flagellin-deficient flhD mutant X4550△FlhD(pYA3334-F) respectively. After detecting several cytokines on different stimulation time with qRT-PCR, it was found that the relative expression of IL-6, IL-1β were up-regulated, while the expression level of TGF-β4was not changed obviously on stimulation of the flagellin. Heterophils can up-regulate the expression of IL-6, IL-1β after being stimulated by two Salmonella strains on2h,3h, and4h, while TGF-β4was up-regulated only on4h. IFN-y were not detected in heterophils of normal or experimental chickens’ heterophils. After interacting with X4550(pYA3334-F) for3h or4h, heterophils obviously improved the transcriptional expression of IL-6, IL-1β and TGF-β4than X4550△FlhD(pYA3334-F).chickens of One-week’s old were challenged with X4550(pYA3334-F) and X4550△FlhD(pYA3334-F) on the dose of5×109CFU. Salmonella Typhimurium from the cecum, liver and spleen of chicks infected by the two strains were isolated and counted on the LB solid medium containing30μg/ml nalidixic acid at different time. The qRT-PCR was applied to measure the differential expressions of cytokines and TLR5in heterophils, cecum and spleen on early stage of infection. The results showed that X4550△FlhD(pYA3334-F) had stronger ability to colonize than X4550(pYA3334-F) on the early stage of infection, but can’t stay as long as the later. Compared with X4550△FlhD(pYA3334-F), X4550(pYA3334-F) could cause intensive inflammatory reaction by inducing the expression of CXCLi2, IL-1β, TGF-β4and TLR5in heterophils in vivo on the third day, eventually controlling its colonizational number on the early stage of infection. X4550(pYA3334-F) could also fiercely induce the expression of CXCLi2, IL-1β, TGF-β4and TLR5in cecum and spleen on the early stage of infection.BALB/c mice of6-8weeks’old were challenged with X4550(pYA3334-F) and X4550△FlhD(pYA3334-F) on the dose of109CFU. Salmonella from PPs, MLN, liver and spleen of mice infected by the two strains were isolated and counted on the LB solid medium containing30μg/ml nalidixic acid at different time. The qRT-PCR was applied to compare the differential expressions of cytokines in small intestine and spleen on early stage of infection. The result suggested that X4550△FlhD(pYA3334-F) have stronger ability to clonize than X4550(pYA3334-F) on the early stage of infection. Compared with X4550△FlhD(pYA3334-F), X4550(pYA3334-F) can obviously induce the expression of MIP-2, MCP-1, IL-1β(P<0.05). In spleen, the transcriptional level of TNF-a in mice infected by X4550(pYA3334-F) was notably higher than X4550△FlhD(pYA3334-F), while it wasn’t changed in small intestine. The transcriptional level of IFN-y in small intestine on the first day after infected showed significant change among two experimental groups, but it wasn’t changed in spleen. IL-4showed low transcriptional level in small intestine and spleen of both experimental groups.It was suggested by the infection in vivo that compared to X4550△FlhD(pYA3334-F), X4550(pYA3334-F) could induce stronger cytokine responses, controlling the infection in chickens and mice on the early stage. The result indicated that the presence of flagellin could enhance the level of innate immunity triggered by the attenuated strain, which provide some theoretical basis for the research on the attenuated Salmonella Typhimurium as a vaccine vehicle. |
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