| Toll-like receptors (TLRs) are a family of type I transmembrane receptors classified as pattern-recognition receptors (PRRs), with which hosts recognize pathogen-associated molecular patterns (PAMPs). This recognition leads to the activation of the subsequent signaling pathway, which results in the development of host immune responses. TLR5is important in the host defense against bacterial pathogens, which recognizes bacterial flagellin; TLR15appears to be unique to poultry and is molecularly distinct from all other known TLRs and has no mammalian counterpart, that recognizes some component of Salmonella. Salmonella contamination of poultry products is a global threat to public health. In recent years, Salmonella enterica serovar Enteritidis (SE) has been identified as one of the most common causes of food poisoning in both China and other countries. An improved understanding of the host innate immune response to SE would provide more options for reducing pathogen contamination of poultry products. China has a wealth of chicken genetic resources with more than80different indigenous chicken breeds. These resources could provide valuable breeding materials for the poultry industry in China and even for the rest of the world. However, there are no descriptions, as yet, of the innate immune response to SE among Chinese chicken lines. This study was aimed to clone and compare sequences of TLR5and TLR15genes of Chinese different chicken lines, and investigate the differences in the ability of distinct genetic lines of chicken to defend against SE infection from the perspective of innate immunity, so as to accumulate some theoretical basis for the further study of chicken TLRs function as well as the resistance of avian to Salmonella infection.1. Cloning and sequence analysis of TLR5and TLR15genes of Chinese different chicken lines and goose TLR5geneSpecific primers were designed based on the sequences of chicken TLR5and TLR15genes published in GenBank, and the targeted genes were amplified by PCR from chicken genomic DNAs, and then the comparisons for the sequences were performed. Meanwhile, goose TLR5gene was successfully cloned using rapid amplification of cDNA ends (RACE) and compared in homology with the sequences of TLR5genes of other species. Prediction of protein domains of chicken TLR5, TLR15and goose TLR5genes by the SMART program revealed that they were all type I transmembrane glycoproteins and had the typical TLR structure that consists of extracellular leucine-rich repeats (LRRs), a transmembrane domain and an intracellular Toll/interleukin-1(TIR) domain. The open reading frame (ORF) of goose TLR5was2583bp in length encoding860amino acids, which consisted of a single exon; the amino acid sequence of goose TLR5shared50.3%,49.9%and82.7%-82.8%homology with human, mouse and chicken TLR5, respectively. The TLR5or TLR15gene of different chicken lines shared high homology that were relatively conserved; TLR5gene of different chicken lines shared more than99.8%homology in nucleotides and99.4%-99.9%homology in amino acids; TLR15gene of different chicken lines shared more than99.7%homology in nucleotides and99.4%-99.9%homology in amino acids. There were9varied nucleotide sites in the ORF of TLR5gene among different chicken lines, of which4sites were non-synonymous substitutions mainly occurred in Wugu chicken and/or Qingke chicken; the4sites were all located in extracellular domain of which1site was located in LRR (E341K) and1site in LRRCT (A619E). There were16varied nucleotide sites in the ORF of TLR15gene among different chicken lines, of which7sites were non-synonymous substitutions;4sites of the7sites were located in extracellular domain (E158K, A309E, L332F and K.389R), but not in LRR; the other3sites were located in intracellular domain, of which1site was located in TIR (F710L) that occurred in Anka chicken. These variant sites might affect the recognition of ligands by TLRs, that influence the immune responses to the invaded pathogens. 2. Innate immune responses of Chinese different chicken lines against infection with Salmonella EnteritidisWhole blood samples were collected from the chickens of four different Chinese chicken lines (Qingjiaoma, Sanhuang, Wugu, and Xueshanma) and their peripheral blood mononuclear cells (PBMCs) were isolated, which were in vitro exposured to Salmonella Enteritidis (SE) strain50041, and the mRNA expression levels of relative TLRs (TLR4, TLR5and TLR15) and cytokines (IL-1β, IL-6, TGF-β4, CXCLi2and CCLi2) were determined at2and4h post-infection using quantitative real-time PCR (qRT-PCR). It was showed that after exposured to SE, Wugu and Xueshanma lines expressed higher levels of TLR4and TLR15mRNA than the two others, while TLR5was expressed less in the Sanhuang line than in others; Wugu and Xueshanma lines produced more IL-1β, IL-6, CXCLi2and CCLi2mRNA than Qingjiaoma and Sanhuang lines; the Qingjiaoma line showed significantly higher expression level of TGF-β4mRNA than the Xueshanma line at4h post-infection (p<0.05).It was initially seen by the above experimental results of in vitro infection that Wugu and Xueshanma lines had higher resistance to SE. In view of this, the mRNA expression levels of relative TLRs and cytokines in spleens and cecal tonsils of three chicken lines (Sanhuang, Wugu, and Xueshanma) after oral infection with SE were then evaluated using qRT-PCR. After SE infection, three TLRs (TLR4, TLR5and TLR15) mRNA were expressed much higher in spleens of the Xueshanma line and cecal tonsils of the Wugu line than others. Wugu and/or Xueshanma lines produced higher expression levels of IL-1β, IL-6and CCLi2mRNA in spleens and cecal tonsils after infection, and the expression of these cytokines in cecal tonsils of the Sanhuang line were downregulated compared to the uninfected chickens. In addition, infection with SE also increased CXCLi2mRNA expression in spleens of the Xueshanma line and cecal tonsils of the Wugu line, which suggested that Wugu and/or Xueshanma lines had certain advantages in the inflammatory cytokine response to SE infection. It was demonstrated by the experiments of in vitro and in vivo infection that Wugu and/or Xueshanma lines be more responsive to SE infection in the aspect of innate immunity. |
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