Simultaneous detection of piroplasma infections in field Haemaphysalis qinghaiensis ticks by reverse line blotting.

Piroplasmosis is caused by tick-borne haemoprotozoan parasites in the genera of Theileria and Babesia, in which numbers of agents are highly pathogenic for cattle, sheep and goats. We developed a reverse line blot (RLB) assay for detection and differentiation of four different parasites, in which 18S ribosomal RNA gene sequence was amplified with a set of universal primers specific for all members in the genera of Theileria and Babesia; and the probes were designed on the basis of hypervariable region 4 (V4 region) of 18S rRNA gene. Three Theileria and one Babesia can be detected simultaneously on this system and it was sensitive to detect a parasitemia level between 10(-5) and 10(-8)%. A total of 149 Haemaphysalis qinghaiensis ticks collected from Lintan County of Gannan Tibetan Autonomous Region was tested by RLB. Among these, 136 tick samples were also tested by a nested PCR assay developed previously. After comparison of these results, it showed that more T. luwenshuni was detected in RLB assay, while more T. uilenbergi was detected in H. qinghaiensis ticks by nested PCR. The RLB has shown capability for simultaneous detection of four species of piroplasm in H. qinghaiensis ticks, indicating its usefulness for epidemiological studies of piroplasmosis.