| dc.description.abstract | 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. | en |
