Untargeted Metabolomic Analysis Of Bovine Lymphocytes Transformed With Theileria Annulata And Molecular Diagnosis By PCR Of Theileria Annulata

Theileria annulata is a Theileria blood parasite belonging to the subphylum Apicomplexa,class Theileriidae,which can be transmitted to bovines by hard ticks as a vector.Theileria annulata can infect the red blood cells,lymphocytes and lymphocytes in catde,causing bovine tropical theilenosis.The disease is widely distributed all over the world,causing serious losses to the local livestock economy and seriously restricting the development of animal husbandry in various countries.In addition,the metabolic pattern of Theileria annulata transforming host cells is similar to that of tumor cells,so non-targeted metabolomics studies were carried out on transformed cells of Theileria annulata,and metabolites that changed during the transformation process were screened,which provided ideas for the future development of new anti-Theileria annulata drugs.Meanwhile,it was found that TashHN,a specific secretory protein of Theileria annulata,was more active in the process of host cell transformation and had high research value.Therefore,two PCR molecular diagnostic methods were established for the TashHN gene of Theileria annulata,which could provide technical support for future research on the mechanism by which Theileria annulata transformed host cells,as well as a new method for clinical diagnosis of bovine theileriosis in the future.The main research results are as follows:1.Untargeted Metabolomics technology was used to detect and analyze the transformed cells of Theileria annulata before and after the bupavaquone treatment,and 68 metabolites with significant differences were screened,including 48 up-regulated metabolites and 20 down-regulated metabolites.Patlivvay enrichment analysis revealed that 13 metabolic pathways,including glutamine and glutamate metabolism,alanine,aspartate and glutamate metabolism and pentose phosphate pathway,were significantly enriched,with the most significant differences in alanine,aspartate and glutamate metabolism and phenylalanine metabolism.It is hypothesized from the experimental results that glutamine may be required as a major source of energy during the process by which Theileria annulata transformed host cells,and the results provide ideas for the progressive development of new anti-Theileria annulata drugs.2.The fluorescent quantitative PCR method established by the TashHN gene of Theileria annulata was more sensitive than me common PCR method,and improved the accuracy of Theileria annulata.Fluorescence quantitative PCR was performed on 135 samples of yellow cattle blood DNA from the Guyuan area of Ningxia,and the positive rate was 5.9%(8/135),and the positive rate of the common PCR method was 3.7(5/135).The samples tested positive by the common PCR method were also tested positive by this fluorescence quantitative PCR method,indicating that the method is reliable.The fluorescent quantitative PCR method established in this experiment is suitable for the detection of a large number of clinical samples as well as epidemiological investigations and can be applied to the clinical diagnosis and epidemic monitoring of bovine theileriosis,providing technical support for the prevention and control of the disease.3.A total of 135 blood samples from the Guyuan area of Ningxia were clinically tested by the common PCR method based on the TashHN gene of Theileiia annulata,and genetic evolutionary analysis was performed.It was found that the overall infection rate of Theileria annulata in Guyuan area was 3.7%.which was a low infection rate,and most of its genotypes belonged to the subbranches of T005,T029 and T038 in the Asian branch of Theileria annulata.In summary,this study identified differential metabolites and metabolic pathways that may be involved in cell transformation through untargeted Metabolomics analysis of transformed cells of Theileria annulata.At the same time,a reliable fluorescent quantitative PCR molecular diagnostic method was established based on the TashHN gene,and TashHN molecular genetic evolution analysis was performed on positive samples,which provided data and technical support for the prevention and treatment of bovine theileriosis in this area in the future.