Recognization Of Chicken Toll Like Receptors On Eimeria Tenella And Signaling Pathway

Avian coccidiosis is recognised as the most significant parasitic disease with high incidence rate and difficulty in prevention in the poultry industry. The annual economic loss of this disease in the world was estimated more than7billion US$. While in China, the costs just in anticoccidial drugs was2-3billion yuan each year, which took up1/3of expense in chicken disease prevention. Presently, the main means for prevention and therapy of chicken coccidiosis is to treat the infected chickens with anti-coccidiosis drug. However, the development of poultry industry is restricted because of the biological characteristic of the coccidian and poultry production, the problem of food safety, drug resistant and chemical medicines residue. In recent years, the application of new vaccines to prevent and cure the chicken coccidiosis is imminent. Till now the commercial anticoccidial vaccines used in the world were live vaccines. However, the use of live vaccines is limited somewhat by the pathogenicity of parasites used. While there has not been any major breakthrough yet in research of genetic engineering vaccine, the possible reason may be connected with the unclear of immunologic mechanism, especially innate immunity in host with coccidian.Janeway raised the theory of pathogen-associated molecular patterns (PAMPs) recognition by receptors of inate immune cells in the1990s. Toll-like receptors (TLRs) were identified as a group of pattern recognition receptors (PRRs), play important roles in the innate immunity and adaptive immunity by recognizing PAMPs. In this study, We analyzed the expression of chicken toll-like receptors (ChTLRs) in different tissues of Luo-man chicken. Then, detected the mRNA expression of these genes in target organ and immune organ of chicken infected with Eimeria tenella, and in innate immune cells stimulated with sporozoites of E. tenella, to screen the ChTLRs which were involved in E. tenella recognization. Finally, we studied the function of ChTLRs in E. tenella recognization and signal pathway. The major results were given below:1. Development of real-time PCR for the detection of chicken ChTLRs, singal adaptors and cytokines mRNA expressionIn the experiment, we designed and synthesized the primers, and established real-time quantitative PCR to detect mRNA transcription of ChTLRs (ChTLRl,2,3,4,5,7,15and21), singal adaptors (MyD88、TRIF、TIRAP、TRAF6and NF-κB) and cytokines (IFN-γ、IL-6、IL-10and IL-12). The results showed that the amplification efficiency (E values) of eighteen genes (8ChTLRs,5signal adaptors,4cytocines and1reference genes) were95.5%-108.3%, the correlation coefficients (R2values) were greater than0.989. The assays were very sensitive and had a detection limit of10-13g/mL DNA. The established real-time quantitative PCR methods were highly specific, the melting curve showed a single peak for every genes. It was highly reproducible and had a coefficient of variation less than2.03percent. The results indicated that the methods could be used for quantification of ChTLRs, singal adaptors and cytokines mRNA expression in chicken.2. Expression profile of ChTLRs、MyD88、TRIF and TIRAP mRNA in different tissues of chickenIn order to determine the expression of ChTLRs, MyD88, TRIF and TIRAP in taret organs of chicken, we detected mRNA expression of these genes in heart, liver, spleen, lung, kidney, thymus, bursa, duodenum, jejunum, cecum, muscle and skin of10days Luo-man chicken. The results demonstrated that there were extensive expression of ChTLRs, MyD88, TRIF and TIRAP mRNA in immune organs and intestinal tissues. Among them, ChTLRl, ChTLR15, MyD88and TIRAP were expressed with the highest level in bursa, while ChTLR2, ChTLR7and ChTLR21were expressed at most in spleen, the maximum expression of ChTLR3were found in kidney, but ChTLR4and TRIF had the highest levels of expression in duodenum. Compare with other ChTLRs, the expression difference of ChTLR4mRNA was relatively low. The expression of ChTLR5in liver and skin, ChTLR15in lung and kidney were extremely low. While we didn’t detect any expression of ChTLR2mRNA in lung and skin, and ChTLR3mRNA in lung. There were now obvious difference in MyD88, TRIF and TIRAP mRNA expression in detected tissues.3. Dynamic change of ChTLRs、MyD88and TRIF mRNA expression in spleen of chicken infected with E. tenella in vivoIn this experiment,15days chicken were infected with50000E. tenella sporulated oocysts each. Quantitative real-time PCR (qRT-PCR) anlysis of ChTLRs, MyD88and TRIF mRNA expression in cecum and spleen of chicken infected with E. tenella for3,12,24and72hours, were carried out. The results demonstrated that the expression level of ChTLRs and TRIF mRNA in spleen and ceum were down-regulated, while MyD88mRNA were up-regulated after E. tenella infection for3hours, with no significant difference.12hours after inoculation, ChTLRs, MyD88and TRIF mRNA expression were increased, the expression level of ChTLR3、ChTLR15and MyD88mRNA were significant higher than that of uninfected chicken. ChTLR3mRNA expression in spleen and MyD88, TRIF mRNA expression in cecum were up-regulated24hours after inoculation,.72hours after infection only ChTLR7, MyD88, TRIF mRNA expression in spleen and MyD88mRNA expression in cecum were up-regulated, with no significant difference.4. ChTLRs、MyD88and TRIF mRNA expression in heterophiles and macrophages stimulated by E. tenella sporozoites in vitroThe aim of this experiment was to determine the ChTLRs, singal adaptors and inflammatory cytokines expression accociated with E. tenella sporozoites stimulation in heterophiles and macrophages isolated from chicken. The results showed that heterophils and macrophages stimulated with live sporozoites had significant higher expression of ChTLR4and ChTLR15mRNA than unstimulated cells. When stimulated for4hours, the expression of ChTLR15and IL-6mRNA in heterophiles were significant up-regulated. The heat-killed E. tenella sporozoites had ability of stimulated higher expression of ChTLRs, singal adaptors and cytokines mRNA in heterophiles and macrophages than live sporozoites. When compared with unstinulated heterophiles, the expression of ChTLR3, ChTLR4, ChTLR15, MyD88, IFN-y, IL-6, IL-10and IL-12mRNA in stimulated heterophiles for2hours were up-regulated significantly, and there were significant increase of ChTLR4, ChTLR15, IL-6, IFN-y and IL-10mRNA expression in stimulated heterophiles for4hours. Macrophages stimulated with heat-killed E. tenella sporozoites for2hours had the significant up-regulated of ChTLR4, ChTLR15、MyD88、IL-6、IL-12and IFN-y mRNA expression than unstimulated cells. The results suggested that ChTLR4and ChTLR15were involved in E. tenella recognization.5. Recognization of E. tenella by ChTLR4and ChTLR15and signal pathwayTo determine the role of ChTLR4and ChTLR15in response to E. tenella, we used RNAi-mediated gene silencing of ChTLR4and ChTLR15mRNA expression, then detected the expression of singal adaptors and cytokines mRNA in macrophages, and content of cytokines in supernatant of macrophage culture. The results demonstrated that siChTLR4#l-transfected macrophages had73.12%reduction in ChTLR4mRNA expression compared with negative control siRNA-transfected cells. Accompany with the ChTLR4interference, the expression of MyD88、TIRAP、TRAF6、NF-κB、IFN-y. IL-6、IL-10and IL-12mRNA, the content of IFN-γ、IL-6、IL-10and IL-12were decreased, with no significant difference. When siChTLR15#1mediates69.68%reduction of ChTLR15mRNA expression, the expression of MyD88. TIRAP、TRAF6、 NF-κB、IFN-γ、IL-6and IL-10mRNA were decreased, while IL-12mRNA expression was reduced significantly, the content of IL-12in supernatant was decreased significantly24hours after E. tenella sporozoites stimulation. In addition, interference of ChTLR4and ChTLR15mRNA expression in macrophages led to a concomitant significant reduction of MyD88、TRAF6、IFN-y and IL-12mRNA expression and content of IL-12in supernatant of macrophage stimulated by sporozoites for24hours. In order to determine the role of MyD88in singaling pathway of ChTLR4and ChTLR15recognizing E. tenella,69.75%reduction of MyD88mRNA expression were mediated by siMyD88#1compared with control siRNA. The interference of MyD88mRNA expression in macrophages led to a concomitant significant reduction of TIRAP、TRAF6、NF-κB、IFN-y and IL-12mRNA expression and content of IL-12in supernatant of macrophages stimulated by sporozoites for24hours.In summary, the results of this paper suggested that ChTLR4and ChTLR15were involved in E. tenella recognization. ChTLR15played a key role in E.tenella-induced innate immunity of chicken on IL-12producing. There maybe a cooperation of ChTLR4and ChTLR15on recognization of E. tenella for induction expression of IL-12and IFN-y in macrophages. After recognization of E. tenella, ChTLR4and ChTLR15mediated MyD88dependent pathway and activated the production of IL-12and IFN-y.