Analysis Of Genetic Diversity Of Theileria Annulata And The Effects Of Its Infection On TLRs And NLRs Signaling Pathways In Cattle

Tropical theileriosis,caused by Theileria annulata,is one of the fatal tick-borne diseases.T.annulata was one of the most pathogenic protozoa infective to cattle,causing seriously threat especially in tropical,subtropical and temperate endemic areas.During T.annulata infection,the innate immunity will be activated and help host defend against parasitic infection or eliminate the invaded parasite.In innate immune system,TLRs,NLRs and inflammasomes play pivotal roles during protozoa infection process.However,the role of innate immune mechanisms was remaining unclear during T.annulata infection.In the present study,we identified the key molecules of TLRs and NLRs signaling pathways in host immune responses against T.annulata Kashi strain infection by RNA-seq,PCR Array and ELISA methods.Firstly,LF-RPA method was established to provide technical support for the detection of T.annulata in vivo.The LF-RPA assay was evaluated via testing small number of samples,which demonstrated that LF-RPA could be used as an ideal diagnostic tool for detecting T.annulata in endemic regions with limited to fewer and local resources.Secondly,by analyzing the T.annulata population genetic structure from Gansu,Hainan,Inner Mongolia,Liaoning,Tianjin and Xinjiang populations,it was found that the genetic characteristics of Xinjiang population was distinct from others.The genetic diversity and MOI analysis revealed that Xinjiang population showed more diversity and multiple genotypes than other populations.DAPC analysis was proved to be the existence of population substructure in Xinjiang.Moreover,the Xinjiang population showed a high genetic differentiation,which indicated limited genetic exchange of T.annulata with other populations in China.Thirdly,the transcription expression profile of PBMC stimulated by T.annulata sporozoites was analyzed through RNA-seq technique.In TLRs signaling pathway,the downstream chemokine CXCL9 and CXCL10 were up-regulated.In NLRs signaling pathway,up-regulation of NOD2,ASC and CCL2 were observed.These data suggested that both signaling pathways play an immunomodulatory role at 72 h post infection.Furthermore,ELISA results showed that the concentration of IL-1?,IL-6 and TNF? in supernatant was significantly decreased at primary infection,however,the concentrations of proinflammatory cytokines was increased at 72 h p.i..These data suggested that the innate immune response of PBMC was not evident in the early stage of T.annulata infection,which may indicate that the pathogen could suppress the innate immune response,and consequently establish a parasitism in host.Lastly,the Custom RT2 Profiler PCR Array were used to explore the mRNA levels of 42 TLRs and NLRs signaling pathways relevant genes.In comparison with pre-infection,the transcriptional level of 42 relevant genes were obviously changed at 72 h,96 h,120 h and 168 h p.i.,12 of which(including TLR1,TLR6,TLR10,MyD88,NLRP1,IRF3,TBK1,CD40,CD86,IFNA1,IFNB1 and TNF?)showed significantly differences(P < 0.05).ELISA results demonstrated that the concentration of IL-6,IL-1? and TNF? in serum were significantly increased at the late stage(168 h p.i.),suggesting that Th1-type immune response was dominant role in this process.In a word,these findings not only contribute to a better understandings of the immune mechanisms during the T.annulata infection,and but also provide theoretical basis for an optimal therapeutic approach to control theileriosis.