Development Of A Rapid,Immunochromatographic Strip Test For Bovine Theileriosis Based On RMPSP

Bovine theileriosis is one of hemoprotozoan disease which is caused by the infection of bovine erythrocyte and reticuloendothelial cells by Theileria genus,presenting some clinical symptoms like fever,hypohemia,icterus and lymph node enlargement,even death.Theileria annulata and Theileria pava are the most serious pathoginici pathogens among Theileria species.The disease caused by Theileria pava named as“East Coast fever”,which is distributed mainly in Africa but not present in China.In China,Three species of theilera have been reported which including T.annulata,T.sergenti and T.sinensis.The previous work has found the major piroplasm surface proteins could react with Theileria positive sera,which indicated that MPSP is a good candidate antigen to detect the three kinds of bovine Theileria spp.In the present study,we expressed and purified MPSP protein,then anti-MPSP polyclone antibodies were produced in rabbits.Finally,a rapid diagnostic strip for Theileria was established.The main contents are as follows:The MPSP gene has been cloned and ligated into vector pET28?a?,selected single clones were used for enzyme digestion and Ploymerase Chain Reaction?PCR?identification.The positive recombinant plasmid MPSP-pET28a was transformed into the Escherichia coli competent cells and induced to express the protein.The reactogenicity of the protein was analyzed by using Western-Blot.The results indicated that the MPSP protein could reacted with positive serum of T.annulata,T.sergenti,T.sinensis but not with B.bovis,B.bigemina,B.major and negative serum of bovines,which suggested that the MPSP have a good specificity and is a good candidate antigen for detection.A large amount of protein was purified and concentrated,with the final concentration was 1.6 mg/mL.Three healthy rabbit were prepared for producing anti-MPSP polyclone antibodies.Each rabbit was immunized for three times?1 d,14 d and 28 d?,100?g of protein was applied for each immunization.Ten days after the last immunization,rabbits were executed for isolation of the sera.The titers of serum determined by enzyme-linked immunosorbent assay were 1?25600,1?12800,1?12800,respectively.Purification of the antibodies from serum was using by protein A method,and the heavy chain and light chain of the immunoglobulin could be seen clearly through SDS-PAGE analysis.A rapid colloidal gold-based immunochromatographic assay?GICA?for bovine theileriosis was established using MPSP and anti-MPSP polyclone antibodies for detection of T.annulata,T.sergenti and T.sinensis.The nitrocellulose membrane of immunochromatographic strip is incubated with rMPSP antigen on the test line and anti-MPSP serum on the control line,respectively.MPSP antigen marked with colloidal gold particles was sprayed in glass fiber paper and used as Colloidal gold pad in the detection system.The sample mat,colloidal gold pad,nitrocellulose membrane and water absorbent pad are assembled on PVC board and then were cutted into 2.5 mm in width to make the final product of colloidal gold-based immunochromatographic strips.The established GICA method strip has a good specificity,easy to operate,and time saving?within 20 min?.Furthermore,the evaluation of the performance of the establied GICA strips shown that the strip could be able to detect antibodies from the experimentally infected animals with T.sergenti and T.sinensis from the week 3^th to the week 15^th,indicating that the strip is suitable for the detection of both parasites in early and late infections.The samples of yaks sera?n=80?collected from Gannan were detected by both rMPSP-ELISA and GICA based on rMPSP.The results compared between two method show that the positive rates are 65%and 72.5%,respectively.The agreement rates of strongly positive and negative are both 100%.However,the agreement rate of weakly positive is 83.3%.The result indicated that GICA has a good diagnostic effects to cattle suffering an acute infection with Theileria.GICA have many advantages,such as easy to perform in the field,does not require expensive instruments and cheaply et,which made it a good testing tool although the sensitivity of GICA is lower than ELISA.In conclusion,this study has developed a rapid,immunochromatographic strip test for Bovine theileriosis based on rMPSP,which provides a new tool to detection of bovine theileriosis.