| Salmonella enteritidis (SE) is gram-negative facultative bacteria, and they cause one of the most common foodborne diseases. The adult animals or poultry infected with Salmonella enteritidis usually remain symptomless and unnoticed, and they cause lots of economic losses in poultry production in the world. Take a long view, breeding a new strain by genetics methods is the best choice to improve the chicken’s resistance to Salmonellosis caused by Salmonella enteritidis, and it also is the urgent need for the sustainable development of poultry industry.Geneticists and immunologists believed the theory "theory of genes determined animal resistance" for a long time, which meant that the pathogenesis of some diseases was related to the genetic background of the animals themselves. Host genetic background also influenced the Salmonella enteritidis infection things in chicken production, and exploring related genes was the essential means for studying the resistance of chicken to Salmonella enteritidis. It suggested that making an intensive study of genetic background of native chicken breeds is the key for solving the problem of Salmonella enteritidis infection in poultry production.Toll-like receptors (TLRs) are innate immune pattern recognition receptors, which can recognize and bind one or more pathogen-associated molecular patterns (PAMP), and deliver the information of antigen recognition from extracellular to intracellular and induce inflammatory response, and they were important candidate genes for studying poultry resistance to Salmonella enteritidis infection. Chicken TLR15(ChTLR15) gene is found to be the specific toll-like receptor in chickens in recent years. Current research suggested that chTLR15had a close relationship with poultry resistance to Salmonella enteritidis infection, and the relationship between expression level of chTLR15in heterophilic granulocytes and the ability of resisting the infection of pathogenic microorganisms have become the focus of recent research. Exploring the expression regulation of related genes is the necessary way for studying poultry resistance to Salmonella enteritidis infection.This study is focus on chTLR15considering genetic differences of resistance to Salmonella enteritidis infection among Chinese native chicken breeds. In this study, we analyzed the expression of chTLR15by real-time quantitative PCR in mRNA and DNA level and analysised chTLR15gene polymorphism to investigate the relationship between gene chTLR15and native chicken breeds’resistance to Salmonella enteritidis, which will provide scientific basis and technological supporting for native chicken breeding work in the future. In this study, these devided into the following three parts:1Comparison of SE Natural Infection Rate in Qingyuan Partridge and Baier Chicken and Correlation between Polymorphism of ChTLR15and SE InfectionIn order to test the Salmonella natural infection rates of two generations by agglutination test, selected Qingyuan Partridge and Baier chicken as the object of this study. In the first generation, the S.pullorum infection rates of Qingyuan Partridge and Baier chicken were41.41%and58.82%respectively, and SE infection rates were68.69%and76.47%respectively. In the second generation were48.83%and66.3%respectively. Results indicated that the results of natural infection rates were relatively stable. Qingyuan Partridge had weaker susceptibility of SE than Baier chicken. Three SNPs in the only exon of ChTLR15gene were scanned in Qingyuan Partridge and Baier chicken breeds by PCR-SSCP method, which were named G168A, C726T and A1166G respectively. G168A and C726T were silent mutations, and Lys was induced to change to Arg by A1166G. The genotypes distribution of G168A between the two breeds had no significant difference (P>0.05), and that of C726T and A1166G both were significant difference (P<0.05, P<0.01respectively). G168A of Baier breed was low-degree polymorphic (PIC<0.25), and in Qingyuan breed it was moderate-degree polymorphic. C726T in the two breeds were both moderate-degree point (0.5> PIC>0.25). A1166G in Baier breed was high-degree polymorphic (PIC>0.5), and in Qingyuan breed was low. Analysis of the correlation between ChTLR15SNPs genotype distribution and SE infection demonstrated that the genotype distribution of Baier in the C726T SNP between infection-positive group and negative group had significant difference (χ2=8.84, P<0.05), the number of BB genotype in negative group was significantly more than that of positive group, negative group had18%higher B allele frequency than the positive group, suggesting that T allele may be beneficial gene against Salmonella enteritidis infection. Other SNPs didn’t have significant difference in the positive and negative groups, suggesting that the genotype distribution of G168A, C726T (in the Qingyuan Partridge) and A1166G had no significant correlation with Salmonella enteritidis infection.2Test of LD50in Qingyuan partridge and Baier chicken and the Influence of Body-Weight in Early Growing Stage after Salmonella Enteritidis InfectionIn order to evaluate the sensitivity of Qingyuan partridge and Baier chicken to SE, twice LD50tests were operated. There were gender groups in test two, but no in test one. Divided2-day-old non-SE-infected chickens into one control group and four infection groups, each infection group was inoculated with Salmonella Enteritidis at106、107、108、109dose by subcutaneous injection, and control group with PBS. Observed incidence of clinical change and death of each group, carried out bacteriological identification, and determined median lethal dose (LD50). LD50of Qingyuan partridge and Baier chicken were5.89×107and1.10×108in test one, and1.29×108cfu and1.5×108cfu in test two, and the LD50of Qingyuan and Baier males were3.18×108cfu and1.29×108cfu, and females were4.22×108cfu and1.79×108cfu in test two. The LD50results showed that there was no significant difference of sensitivity between Qingyuan partridge and Baier chicken. Test of isolation and identification of bacteria showed that Salmonella Enteritidis distributed in liver, spleen, lung, kidney and cecum after injection, and the isolate strain was identified to be same as inoculation strain, and organs of infection group showned typical clinical symptoms of Salmonella Enteritidis. Selected106cfu dose which lead to no death as the injection dose, recorded the body weight of control and infection group, and sampled spleen on the1,3and10day after infection (for experiment three). The body weight of Qingyuan partridge and Baier was significantly effected by the cross action of treatment and age after infection, in breed (F197=7.7, P=0.000; F342=16.1, P=0.000) and between breeds (F142=7.5, P=0.000; F439=16.8, P=0.000). Body weight of Baier control group was significantly lower than hatching weight (P=0.0257) after injection with PBS on the first day, which might be caused by catching stress, and body weight shown increasing after three days, significantly higher than hatching weight(P=0.000), while body weight of Qingyuan partridge treated with PBS shown increasing during the test period. These results indicated that Qingyuan partridge had stronger anti-stress ability than Baier breed. Body weight of Baier tested group was significantly lower than hatching weight (P=0.0047,0.0003), and exceed the hatching weight on the eighth day post infection, showing that body weight of Baier infection group was got seriously retardant; while body weight of Qingyuan partridge infection group was significantly lower than control group, but had no difference on the first day with hatching weight (P=0.1446), and on the third day had exceed hatching weight (P=0.0000). Infection group got both catching and Salmonella Enteritidis stimulation, under the circumstance of same inoculation dose and higher hatching weight of Baier, indicating that Baier was more sensitive to Salmonella Enteritidis than Qingyuan partridge.3Expression of ChTLR15mRNA in Spleen in the Early Growing Stage after Salmonella Enteritidis InfectionQingyuan partridge and Baier (two indigenous chicken breeds) were sleeted as the object of this study. Real-time PCR was carried out to compare the expression levels of ChTLR15genes between control and infection groups of two indigenous chicken breeds in spleen at day1,3and10post infection. The results indicated that, compared with control group, ChTLR15gene in spleens of106group of Baier were significant upregulated (P=0.033,0.001) at day1and3post infection, and Qingyuan partridge were also extremely significant upregulated (P=0.016,0.022,0.001) at3time points. There were no defferences between the expression of Qingyuan and Baier infection group, at three time points, compared with day1, the expression level of ChTLR15gene in spleens upregulated constinuously in the course of salmonella infection. At day1post infection, the level of ChTLR15of Qingyuan patridge breed was lower than that of Baier breed. The level between test group and control group of Qingyuan patridge breed and Baier breed did not differ significantly. In a word, ChTLR15gene took part in the immune response in the course of salmonella infection. |
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