Studies On Variation Analysis Of TLR4Gene And The Association Of TLRs Gene With Chicken Response To Salmonella Infection

Salmonella which has a lot of serotypes and distributes in nature widely can infectdifferent day-old chickens. Salmonella disease is an acute systemic disease that results in ahigh mortality rate in young chicks but rarely causes such severe clinical disease in adult birds,though it can result in loss of weight, decreased laying, diarrhea, and lesions andabnormalities of the reproductive tract, which leads to enormous economic losses to thepoultry industry. Human eating meat, eggs, milk or other food which were contaminated bySalmonella will cause food poisoning, resulting in a serious public health crisis. Therefore,the key to solving this situation is breeding varieties of anti-Salmonella chicken and theestablishment of healthy breeding flocks.Toll-like (TLRs) is the bridge connecting natural immune and acquired immune, it plays avery important role in resisting to bacterial infection and triggering the body’s immuneresponse. The purpose of this study is to explore the genetic polymorphism of TLR4of thelocal chicken breeds, as the starting point of TLR4genetic differences of different kinds ofchicken, we carry out the experiment of Salmonella typhimurium inoculation to exploreanti-Salmonella difference between Jining Bairi chicken and Shouguang chicken, contributingto reveal the local chicken breeds disease characteristics, and lay the foundation for poultrydisease genetics and breeding.In this study, the DNA pooling direct sequencing approach was applied to screen the SNPsat all three exons of TLR4gene in5shandong local and2commercial chicken breeds (JiningBairi chicken, Laiwu Black chicken, Luhua chicken, Luxi game chicken, shouguang chicken,Hy-Line Brown and AA broiler). C77T and G247A were selected to genotype in all the trialchickens basing on direct sequencing approach. The results showed that17SNPs of TLR4gene were found in seven flocks; the C77T locus in five local chicken breeds only Luhuachicken deviated from Hardy-Weinberg equilibrium, polymorphic information content only inShouguang chicken was moderate and the remaining4local groups were low-grade; theG247A locus in five local chicken breeds were in Hardy-Weinberg equilibrium, polymorphicinformation content in Shouguang chicken were low polymorphism, the other4groups are moderate polymorphism. This study showed that TLR4gene had different degrees of singlenucleotide polymorphisms in the seven flocks, the polymorphism information of C77T andG247A loci in shouguang chiken were different from the other4local chichen breeds.In order to comparing the differences against Salmonella infection between ShouguangChicken and Jining Bairi Chicken,1-day-old Shouguang Chicken and Jining Bairi Chickenwhich were Salmonella negative were randomLy divided into two groups and inoculated with0.3mL PBS and Salmonella typhimurium respectively. For Shouguang Chicken serumantibody levels were detected in1,3,7and14days post inoculation. TLR4gene expressionwas measured in jejunum, ileum, cecum and duodenum, respectively. Correlation analysis forgenotyping and antibody levels was done to all the chickens. For Jining Bairi Chicken serumantibody levels and TLR4, TLR5, TLR15and TLR21gene expression in cecum and spleenwere detected in1,3and7days post inoculation. The morphological changes of duodenumwere measured in7days post inoculation.The results showed that in control gropups, the serum antibody levels of ShouguangChicken were significantly higher than Jining Bairi Chicken at1and3days after inoculation,while the antibody levels were significantly lower than Jining Bairi Chicken at7days postinoculation. TLR4gene expression in cecum of Shouguang Chicken was significantly lowerthan Jining Bairi Chicken at1and7days post inoculation, while the expression level was notsignificant at3days after inoculation. In challenged groups, the serum antibody levels of thetwo varieties of chicken and TLR4gene expression in cecum were not significant at1and3days after inoculation, but the serum antibody levels and TLR4gene expression in cecum ofJining Bairi chicken were significantly higher than that of the Shouguang chicken.The correlation analysis between Shouguang TLR4gene C77T and G247A locusgenotypes, different combinations of genotypes (CC/GG and CT/GG) and serum antibodylevels found that the difference of antibody levels of different genotypes and the combinationof genotypesthe was not significant at the same time; the antibody levels of differentgenotypes and genotype combinations change over time and significant difference. TLR4expression levels were highly significant in the intestinal tissues of Shouguang Chicken at7days post inoculation. TLR4gene expression levels were highest in duodenum and lowest incecum. The duodenal villus length of Jining Bairi chicken between the control group and thechallenged group were highly significant at3and7days post inoculation. The ratio of theduodenal villi length and crypt depth was extremely significant at7days post inoculation.TLR5gene expression levels of the cecal tissue was significantly higher than the controlgroup at1and3days after inoculation, TLR15gene expression levels were extremelysignificant at3and7days after inoculation, TLR21gene expression levels was significantlyhigher at7days after inoculation. TLR5and TLR15gene expression levels at the three timepoints had no significant difference, TLR21gene expression levels was higher than those inthe control group at1day post inoculation.Summarized above, the colonization of Salmonella in different intestinal parts isdifferent and damage to the intestinal morphology. TLR4of two breeds have the differentresponses to Salmonella and different TLRs play an important role in Salmonella infection.