Molecular Characterization of Theileria orientalis Piroplasm Protein Encoded by an Open Reading Frame (To ORF2) in a Genomic Fragment

Abstract

ABSTRACT. In the present study, a novel antigenic protein expressed in the piroplasm stage of Theileria orientalis was characterized. A 4,707 bp genomic fragment amplified by PCR contained two open reading frames (ORFs). The deduced amino acid sequence of the first ORF showed significantly high similarlity to the ubiquitin carboxy terminal hydrolases/proteases while the second ORF (To ORF2) showed homology to several surface antigens of plasmodia. To ORF2 was expressed to determine whether the protein product is expressed by the parasite. In western blot analysis, bovine antiserum from a T. orientalis-infected calf recognized the recombinant protein containing a C-terminal part of the ORF expressed by baculovirus system. Western blot analysis with the anti-To ORF2 mouse serum recognized a 48 kDa protein in T. orientalis piroplasm lysates. Indirect immunofluorescence antibody test by confocal scanning laser microscopic analysis showed that antisera against the recombinant protein recognized T. orientalis piroplasm in the infected erythrocyte. The results from this study indicate that To ORF2 protein is expressed at the piroplasm stage and is immunogenic. This novel antigenic To ORF2 protein could be exploited for vaccine development against bovine piroplasmosis. KEY WORDS: Apicomplexa, parasite vaccine, piroplasm, Theileria orientalis.J. Vet. Med. Sci. 66(8): 957-963, 2004 Theileria parasites are tick-transmitted, intracellular protozoa belonging to the phylum Apicomplexa, which cause disease in wild and domestic ruminants. Two Theileria species, namely T. parva and T. annulata, are highly pathogenic, causing lymphoproliferative diseases in cattle. They cause significant economic losses to animal husbandry in tropical and subtropical countries [18,22,28,35]. On the other hand, T. orientalis cause bovine piroplasmosis, a disease characterized by anemia in infected cattle [17]. The disease occasionally cause death especially in severe cases [34] and lead to great economic loss in cattle production mainly in Japan and Korea [1,17].Recent attempts to develop subunit vaccines against Theileria parasites have focused on the sporozoite surface antigens (p67 of T. parva and SPAG-1 of T. annulata). These antigens were reported to give partial protection when different delivery systems and adjutants were tried [21,26]. However, little is known on stage specific expression of T. orientalis proteins. Only two piroplasm proteins, the major piroplasm surface protein (MPSP) [12,15] and P23 [29] have been reported although the erythrocytic stage is responsible for the pathology associated with theileriosis caused by T. orientalis. For other stages such as sporozoite or schizont, no specific genes have been identified so far. It was reported that vaccination with synthetic MPSP peptides reduced parasitemia [24], but effective and safe vaccine have not yet been developed for T. orientalis.In efforts to develop vaccines against T. orientalis, we have searched for genes specifically expressed in the sporozoite, schizont or piropl...

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