Semi-nested PCR detection of Babesia orientalis in its natural hosts Rhipicephalus haemaphysaloides and buffalo

Babesiosis has recently been recognized as an emerging infectious disease of buffalo in China. In order to investigate the epidemiology and enzootic potential of this parasite in Hubei province, we sought to develop a semi-nested PCR to detect Babesia orientalis in buffalo and the potential tick vector-Rhipicephalus haemaphysaloides by amplifying a specific 257bp fragment of B. orientalis 18S rRNA gene. The practical limit of detection showed that it had high sensitivity and an approximate parasitemia of 0.00000012% was detected by the PCR system. The blood samples of 121 asymptomatic buffaloes collected from four babesia endemic counties and that of 71 asymptomatic buffaloes collected from three babesia free counties in Hubei province of China were examined for the presence of B. orientalis using both Wright-Giemsa stained blood smear and semi-nested PCR. Microscopic examination revealed that 5/121 animals were positive, whereas 24/121 animals were positive by the semi-nested PCR assay. Of 378 ticks (R. haemaphysaloides) collected from buffaloes and examined by the semi-nested PCR, 35 were positive. The results showed that the semi-nested PCR was a useful method to investigate the epidemiology of buffalo babesiosis (B. orientalis), which is widely distributed in Hubei province, China.