Isolation and characterization of a gene encoding a potential vaccine and diagnostic antigen of Theileria lestoquardi
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Theileria are tick-transmitted, haemoprotozoan parasites infecting wild and domestic ungulates throughout many areas of the world. The most economically important parasite of sheep and goats in many parts of Asia is T. lestoquardi (syn. T. hirci) which causes the disease known as malignant ovine/caprine theileriosis. Its diagnosis is confused by the widespread presence of the benign ovine species T. ovis and this will persist until a recognised diagnostic test is developed. The object of this study was to express a T. lestoquardi major merozoite/piroplasm surface antigen (mMPSA), a candidate diagnostic antigen, as a recombinant protein in bacteria and characterise it using sheep infection sera. A fragment equivalent to 89.7 % of the protein coding region for the mMPSA was peR-amplified, cloned into the pQE-30 expression vector and transformed into Escherichia coli strain XLI-Blue. Following analysis by agarose gel electrophoresis, six recombinant colonies were selected and sequenced. A consensus mMPSA amino acid sequence was found to have a homology of > 95 % to other T. lestoquardi mMPSA sequences available in the Genfsank database. One clone was IPTG-induced to express the recombinant mMPSA as a HisTag protein. The HisTag-mMPSA was immunoblotted and probed with sequential sera from four sheep that had been infected with a T. lestoquardi sporozoite stabilate. A variable antibody response to mMPSA was observed between the four sheep; only one sheep showed a strong response. Antibody detection ELISAs using HisTag-mMPSA failed to work even after further purification of the antigen by dialysis. However, ELISA with recombinant mMPSA produced as a fusion to glutathione S-transferase (GST) generated results that matched those of the western blots. Polypeptides of T. lestoquardi infected lymphocytes were probed with the same sequential sera in an immunoblotting procedure and three antigens (- 70 kDa, 100 kDa and 150 kDa) were found to elicit immune responses in two or more sheep. The T. lestoquardi mMPSA may not be of value in a subunit vaccine or sero-diagnostic test because of the variability of the antibody response. However, the schizont antigens warrant further study to assess their true potential in sero-diagnostics.