Characterization And Application Of TuIP And Tu88 Protein From Theileria Uilenbergi

Theileriosis of small ruminants caused by Theileria species infection is a protozoan disease, whichis transmitted by ixodid Heamaphysalis ticks. T. uilenbergi and T. luwenshuni are the main pathogens ofthis disease in China. In previous study, two antigenic gene Tu IP and Tu88 were identified byimmunoscreening T. uilenbergi merozoite c DNA library and both recombinant Tu88(r Tu88) andrecombinant Tu IP(r Tu IP) expressed in bacteria reacted strongly with the positive sera of T. uilenbergi.The aim of this study is characterizing of the functions of r Tu88 and r Tu IP and developing two rapidmethods for the diagnosis of theileriosis.The subcellular localization of both Tu88 and Tu IP proteins were stained by immunofluorescenceassay(IFA) and examined by laser scanning confocal microscope. The result demonstrated that bothrabbit anti-r Tu88 serum and rabbit anti-r Tu IP serum could specifically recognize the native proteins,indicating that both Tu88 gene and Tu IP gene are expressed in the cytoplasm of the T. uilenbergimerozoites. These findings suggest that Tu88 and Tu IP are potential candidates for the development of asero-diagnostic tool.The recombinantly expressed T. uilenbergi immunodominant protein(r Tu IP) was used to develop arapid colloidal gold-based immunochromatographic strip test for the detection of T. uilenbergi or/and T.luwenshuni infection in the field. The nitrocellulose membrane is incubated with r Tu IP antigen on thetest(T) line and anti-r Tu IP serum on the control(C) line, respectively. The r Tu IP antigen conjugated tocolloidal gold particles was used as the detection system for visualization on the line. Then the samplepad, conjugate pad, nitrocellulose membrane and absorbent pad were assembled on a backing plate inthe appropriate order. The strip test could be performed within 15 min, which did not require any specialequipment or skills. Furthermore, the strip test was conducted with samples collected on 0, 14, 19, 52,85 day post infection. The result revealed the strip test was able to detect antibodies in the sera ofanimals experimentally infected with T. uilenbergi from the day 14 th to the day 85 th, indicating the test issuitable for the detection of both early and late infections.Species-specific primers were designed based on Tu IP gene. Then a real-time quantitative PCRassay was developed for T. uilenbergi infection. The real-time PCR could detect 100 copies of Tu IPgene and no cross-reactivity was found in genomic DNA of T. luwenshuni, Babesia sp. and Anaplasmaovis, indicating satisfactory sensitivity and specificity. The coefficient of variation(CV) of intra-assayranged from 0.93 % to 3.25 % while the coefficient of variation(CV) of inter-assay ranged from 1.55 %to 4.32 %, demonstrating satisfactory stability and repeatability.In conclusion, a rapid gold immunochromatographic strip test and real-time quantitative PCR wereestablished based on Tu IP gene for the detection of theileriosis, which can be used for the diagnosis andepidemiological investigation of theileriosis.