Expression Of TLRs MRNA In Chiken Infected With E.tenella

Eimeria. tenella is the most virulent strains of coccidium. E.tenella has been widely studied in pathogens, life history and epidemiology, but only a little information on immune mechanism, especially on innate immune response, which restrited the research of coccidium vaccine. Until the theory of pattern recognition receptor (PRR) was raised, that widened the recognition of coccidium innate immune. Now researchers had studied deeply and compresively in type, ligand, biological function of TLRs family in mammals, which offered a new thinking on development of coccidium vaccine. Moreover, the studies on avian TLRs had appearsed to be increasing, however mainly focused on the change of TLRs mRNA expression with bacteria, virus infected, only have rarely reports in parasite infected with poultry.This test explored innate immune response of TLRs resisting E.t enella infected with chicken by molecular biology, cell biology and biology information, main researches as followed:(1) Detected the expression of TLRs mRNA of chiken cecum in6-21different-stage days;(2)15days chiken was inoculated two different-dose E.t enella, then detected TLRs mRNA of chicken cecum after1-7days;(3)Used the E.tenella second merozoite to stimulate chicken peripheral blood monocyte-macrophage and detected TLRs mRNA of cell; meanwhile collected supernate of cell as to detect content of IL-6、IL-12、IL-10and IFN-γ.1.The expression of TLRs mRNA of chicken cecum in6-12day stage.Respectively colleted chiken cecums on6、9、12、1518days,SYBR Green I fluorescence quantitative PCR to detect TLRs transcriptional expression of chicken cecum. The results showed:ChTLRs were all expressed in6-21different-day chicken cecum, the expression of ChTLRs mRNA in6day were relative high;except TLR3,the expression of ChTLRs mRNA in9day compared in6、12day were significant decreased(P<0.05);the expression of ChTLR mRNA in12days were all higher then other days,besided TLR3,moreover the expression of ChTLR2、ChTLR4、 ChTLR5、ChTLR7、ChTLR15mRNA were elavated(P<0.05); there was no evident change of ChTLR3mRNA expression in6、9、12days,but in15、18、21days the expression of TLRs mRNA of chicken cecum were significant lower than in6、12days(P<0.01),and the expression of ChTLR1、ChTLR3、ChTLR4、ChTLR7、ChTLR15mRNA compared9day were also decreased. The result indicated:besided TLR3,the change of TLRs mRNA expression was similar, the changeof TLR3mRNA expression in6、9、12days were no evidence.2. The effect of two different-dose E.tenella on TLRs mRNA expression in chicken cecumTo study the effect of E.tenella infective dose on immune respone of chicken cecum, this test repectively used two different infective dose5,000and150,000E.tenella oocyst every single to inoculated15days chicken,moreover eatablished no-infected group. Fluorescence quantitative PCR to detect the expressional level of TLRs mRNA of chicken cecum after infected1-7days.result discovered the expression of TLR2、TLR4、TLR5、TLR7、TLR15and TLR21mRNA of the chick inoculated5,000oocysts were increased in the4-5th day(P<0.05),and the expression were significant increased in lth day of TLR4、TLR5mRNA,the fold-value were4.7、5.3times compared to control group;but the expression of TLR5、TLR21mRNA were supression in7th day,were0.32、0.31times compared to control group.The expression of TLR3、TLR4、TLR5、TLR7、TLR15and TLR21mRNA of the chick inoculated150,000oocysts were decreased in2th、4th and5th day(P<0.05), while the expression of TLR7、TLR15mRNA in6th were elevated, the expression of TLR5、 TLR21mRNA in7th day were also decreade,the value were respectivly0.24、0.34times compared to control group.The expression TLR2、TLR3、TLR4、TLR5、TLR7、TLR15、TLR21mRNA were elevated of chicke infected with5,000oocysts in4-5th,but the expression of TLRs mRNA of chicke infected with150,000oocysts in2th、4th and5th day were decreased,the result menifested strong destructibility in organizition and cell with large-dose infective coccidium has caused supression of innate immune in chiken.Moreover,we could found out multiple ChTLRs participated in the immune response resisting coccudium,mainly were ChTLR3、ChTLR5、ChTLR7、ChTLR15and ChTLR21.3. The change of TLRs mRNA expression of chicken mononuclear-macrophage stimulated.by E. tenella second generation merozoite This test applied live and heat-killed E.tenella second generation merozoit to sitmulate chicken monocyte-macrophage, after disposed3h detecteed the change of TLRs mRNA expression of cell, meanwhile set up PBS as control group; then collected supernate of cell,ELISA detected the content of IL-6、IL-12、IL-10and IFN-γ.Result showed,compared to control group,the expression of ChTLR1、ChTLR2、ChTLR4、ChTLR5and ChTLR21mRNA were significant increased (P<0.01)after E.tenella second generation merozoit stimulated chicken mono-macrophage,the valule respectively were17.22、64.2、5.9、88.4and226.7times.Moerover we found the expression of ChTLR1、ChTLR5and ChTLR21mRNA in live E.tenella merozoit compared to heat-killed were respectivly rised2.9、3.0and9.1times.But the expression of ChTLR3、ChTLR7、ChTLR15mRNA in live E.tenella and heat-killed merozoit compared to control group were slightly reduced.The result of ELISA revealed after live and heat-killed E.tenella merozoit stilmulated3h,the content of IL-6in mono-macrophage cellar supernate respectively were42.15、28.7pg/ml,compared with the content of control group(6.87pg/ml) elevated(P<0.05), the content of IFN-γ in the supernate of live E.tenella merozoit stimulated mono-macrophage was elevated compared to conterol group,and the concentration were separately208.2pg/ml、163.5pg/ml;the content of IL-10in cellar supernate of live E.tenella merozoit was reduced compared contol group and heat-killed group(P<0.05).Results indicated:ChTLR1、ChTLR2、ChTLR4、ChTLR5、ChTLR21involved in the immune response of E.tenella merozoit stimulated mono-macrophage,deduced ChTLR1、ChTLR5、ChTLR21may recognizated the antigen-protein of E.tenella merozoit and could elevated the content of IFN-γ,the elevation of IL-6in cellar supernate may be relate to the increased expression of ChTLR1、ChTLR2、 ChTLR4.