Establishment And Application Of Dot-ELISA Method For Detection Of Theileria Annulata

Tropical theileriosis is a tick-borne protozoan disease caused by Theileria annulata(T.annulata)infecting the lymphocytes and red blood cells of susceptible cattle,causing fever,salivation,superficial lymphnode enlargement,acute anaemia,jaundice and other symptoms.The disease is mostly acute and has a high morbidity and mortality rate,causing severe economic losses to the cattle industry.In this paper,the prevalence of the disease was analysed by PCR on whole blood 275 from cattle collected in the Turpan and Altay regions of Xinjiang.The Ta SP gene of T.annulata in Xinjiang was cloned and the characteristics of its coding protein were analyzed.The p GEX-4T-1-Ta SP plasmid was constructed,and the p GEX-4T-1-Ta SP plasmid was expressed by Escherichia coli(E.coli).The Dot-ELISA method was established by screening condition using the r Ta SP protein as an antigen.The method was used to detect the disease in the farming area and to screen for latent infections,so that the timely detection,treatment and elimination of latent infections could be carried out to promote the healthy development of the local cattle industry.(1)To understand the status of T.annulata infection in Turpan and Altay regions.In this study,275 randomly collected bovine whole blood samples from two regions were tested for T.annulata using the Tams1 gene as the target gene and the prevalence of T.annulata infection in the examined regions was statistically analysed.The ML algorithm MEGA software was used to calculate and analyse the interspecific genetic distances of T.annulata using the T92+G+I model.The results showed that the total infection rate of T.annulata in two area was 26.9%(74/275),and the infection rate in Turpan area was28.8%(59/205),which was slightly higher than 21.4%(15/70)in Altay area.Significance analysis showed that P > 0.1 showed no difference in infection rate between the two regions.The phylogenetic tree was constructed,and the two regions were not in the same evolutionary branch.Therefore,this study provides data for the prevention and control of the disease in these two regions.(2)In order to analyze the Ta SP gene and its biological characteristics of T.annulata and obtain the recombinant protein.Reference to the Ta SP gene sequence(FJ895154)of T.annulata in Gen Bank,the Primer software was used to design the primers.The Ta SP gene was cloned by PCR using positive DNA samples of T.annulata,and the homology,physicochemical properties,antigenic epitopes of B cell and transmembrane regions were analyzed;The recombinant expression plasmid was constructed by selecting the truncated region of the Ta SP gene,and the r Ta SP protein was expressed by E.coli and verified by SDSPAGE and Western blot.The results showed that the Ta SP gene of T.annulata was successfully cloned,and the homology of the gene sequence with the NCBI uploaded sequence of Xinjiang strain(FJ895154)was found by NCBI-blast comparison.The phylogenetic tree was constructed and the gene sequence showed close affinity to the Sudanese region(EU032566)and the Inner Mongolia sequence(DQ073055).The predictive analysis showed that the protein was an acid and hydrophilic amino with strong antigenicity.The recombinant expression plasmid was constructed as p GEX-4T-1-Ta SP.The r Ta SP protein was expressed in E.coli and specifically reacted with T.annulata positive serum as verified by Western blot.Therefore,the r Ta SP protein of T.annulata in Xinjiang was obtained.(3)In order to establish a Dot-ELISA method of T.annulata.A nitrocellulose membrane was used as a solid phase carrier for the antigen.The purified r Ta SP protein as a detectable antigen.The optimal concentration of antigen,the concentration of the blocking solution,the optimal dilution ratio of serum,the secondary antibody and the time of action were selected step by step.The method was used for serological investigations in the Turpan and Bayinbruck regions.The results showed that the optimal dilution of the antigen was 150 μg/m L,the 2%BSA was the optimal blocking solution and the blocking time was 1.5 h.The optimal dilution ratio and action time for the serum and enzyme secondary antibody were 1∶100 1.5 h,1∶8 000 1 h,respectively,and the action time for DAB chromogenic solution was 6 min.The conformance results showed that the overall compliance rate of the Dot-ELISA and PCR methods(88.25%)was higher than that of the indirect ELISA(80.0%)and its positive rate was higher than that of the indirect ELISA method suggesting that the Dot-ELISA developed in this study is more sensitive than the indirect ELISA method.