Theileria sergenti and T. buffeli: Polymerase Chain Reaction-Based Marker System for Differentiating the Parasite Species from Infected Cattle Blood and Infected Tick Salivary Gland

Abstract

Benign Theileria species in cattle, Theileria sergenti and T. buffeli, are morphologically indistinguishable. The polymerase chain reaction (PCR) was used to amplify the genes encoding the 33- and 34-kDa major piroplasm antigens (p33/34) of T. sergenti and T. buffeli from cattle blood infected with these parasites and tick salivary gland infected with T. sergenti. Following amplification, the p33 gene from T. sergenti and the p34 gene from T. buffeli were clearly differentiated using the restriction enzyme sites that were not shared between them. The oligonucleotide primer set, designed from the p33/34 genes, was specific for these Theileria species, since no amplification was detected with DNA from Babesia ovata, B. bovis, Anaplasma marginale, A. centrale, Eperythrozoon wenyoni, bovine white blood cells, and uninfected tick salivary glands. One tenth vol of the template prepared from either 25 μl of blood with 0.5% parasitemia or individual tick salivary glands with six infected acini allowed sufficient amplification for differentiation of the two parasite species by restriction enzyme digestion. In addition, this system could be used to demonstrate the simultaneous, experimentally induced infection of cattle with T. sergenti and T. buffeli. The PCR-based marker system therefore provides a means to differentiate T. sergenti from T. buffeli in infected cattle blood and infected tick salivary glands. This system may also be useful for the characterization of other benign Theileria species in cattle.