PCR amplification and sequence of the p33 piroplasm surface antigen gene of a Theileria species isolated from cattle in west Java, Indonesia.

Abstract

Oriental theileriosis, a parasitic disease of cattle caused by protozoa of the Theileria orientalis/sergenti/buffeli group, has been reported in Indonesia but its causal agent had not yet been characterized. This study was carried out to isolate and characterize the parasite through comparison of its p33 piroplasm surface antigen gene sequence, with known p32 sequences of T. sergenti and T. buffeli isolates. A Theileria spp. isolate was collected from an Ongole cow in Jonggol, West-Java, and transferred into a splenectomized calf for antigen production. Piroplasms were extracted from erythrocytes by ammonium chloride-lysis, separated from unlysed leukocytes and parasitic DNA was phenol-extracted. Polymerase chain reaction (PCR) was carried out on genomic DNA with a pair of 20 bp primers showing consensus for the p32-35 nucleotide sequence of 7 known T. orientalis/sergenti/buffeli isolates. An 875 bp fragment was amplified, and further sequenced on both strands by the dye-labeled terminators method. It showed an 88% homology with the p33 nucleotide sequence of the Japanese T. sergenti Ikeda stock and a lesser homology with 6 other sequences of Australian T. buffeli or Japanese T. sergenti stocks. It was shown to share the presence of the Pst 1 and the absence of the HindIII restriction sites of the T. sergenti Ikeda stock and of one Australian T. buffeli stock, respectively. In conclusion, the affiliation to and the relative position of this Indonesian isolate within the T. orientalis/sergenti/buffeli group has been elucidated.