| Controlling and avoiding Salmonella(SE) infection in chicken breeding and industry is a universal attention. Toll like receptor 4(TLR4) and its signaling pathways have been proved playing a critical role not only in signal transduction but also in the activation of immune responses. In this study, single nucleotide polymorphisms (SNPs) were screened in the coding region of TLR4 gene based on sequences of several local and commercial chicken breeds. Frequencies of different SNPs vary among different breeds. Then SPF chickens were inoculated with SE and grouping based on different haplotypes. Immune response induced by S. infection, concentrations of total IgG and bacterial burden, mRNA expression of key genes involved in TLR4 signaling pathway were investigated in infected young chickens. Therefore, acquiring functional genetic markers relative to SE resistance and understanding more of molecular mechanism underlying the immune response of SE infection were expected.[Trial 1] Genetic Polymorphism Study of TLR4 Gene. The DNA direct sequencing approach was applied to assess the SNPs at all exons, partial introns and 5'region of TLR4 gene(4373 bp) in 12 chicken breeds (Beijing You Chicken, White earlobes Yellow Chicken, Liyang Chicken, Henan Game Chicken, Taihe Chicken, Big Bone Chicken, Langshan Chicken, Xianju Chicken and Chahua Chicken, from native China; Recessive White, Comb and White Leghorn, from foreign country). A total of 36 SNPs were identified, of which 19 SNPs were in the coding region (10 missense mutations and 12 SNPs in the exon 3) and 21 SNPs were new SNPs. The 13 SNPs in the exon 3 were genotyped in all chicken breeds (688). Results showed that the frequencies of various haplotypes differ significantly among various breeds and suggested that it might relate to gene function.[Trial 2] Comparison Study of Immune Response Differences Induced by SE among Chicken Groups With Various Haplotypes. Based on loci 903 and 1832, SPF chickens were genotyped of 1 day old and divide them into three groups (TT/GG, GG/AA and GT/GA), named A, E and F with each 35 individuals. Each chicken was intramuscular injected with 0.5ml of the bacterial suspension containing 108 CFU SE at 21 days old. The serological parameters and mRNA expression of key genes involved in TLR4 signaling pathway in caecum were determined at day 1, 2, 3, 4 and 5 post infection. Results showed that (1) SE repressed the growth of all chickens tested; The serum concentrations of IgG, IL-6 and IL-1 increased at 96h post infection; The serum concentrations of IgG, IL-6 and IL-1 of group A were significant higher than that of group E and F at the first three day post infection (P<0.05), separately. (2) Genes expression studies showed that the infected chickens had the highest expression of TLR4, MyD88 and NF-κB on day 1DPI; The mRNA expression of gene TLR4,TRAF6 and NF-κB of group A were significant higher than that of group E and F at the first three day post infection (P<0.05), separately, which is consistent with the results of the examination of serum concentrations of IgG, IL-6 and IL-1. In conclusion, this study suggested that two mutations at loci 903 and loci 1832 of TLR4 gene may affecting the recognition between protein TLR4 and the SE, then affecting the signal transduction through gene expression of TLR4,TRAF6 and NF-κB, consequently, changing the immune response against S infection. And also, this study proved that chickens with haplotype TT/GG have superior immune response against S infection. |
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