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.

Study on some molecular characterization of Babesia orientalis.

The study on buffalo babesiosis indicated that its pathogen was different from other Babesia on many aspects such as morphology, transmission and pathogenicity. Therefore, it was named as a new species-Babesia orientalis. In order to prove the validity of this taxon, molecular taxonomic study on the pathogen was done in this experiment. The complete 18S rRNA gene sequence of B. orientalis was determined by PCR. It was sequenced and blasted. The results indicated that the classification of the parasite belonged to the genus Babesia. The 1700 bp complete sequence was compared with 15 other Babesia sp. available in GenBank. The data were analyzed and a phylogenetic tree was established. The results indicated that the hereditary distance of the parasite was close to that of Babesia sp. from South Africa and Babesia ovis, and the hereditary distance was far from Babesia bigemina and B. bovis.

Culture-derived Babesia orientalis exoantigens used as a vaccine against buffalo babesiosis.

Buffalo babesiosis represents a major problem for the livestock industry in China. The pathogen of this disease was isolated and identified as Babesia orientalis and subsequently propagated in vitro, using the microaerophilus stationary phase culture system. The aim of this study was to determine the efficacy of exoantigens derived from B. orientalis cultures to induce a protective immunity against challenge exposure to virulent organisms in laboratory and field. The results showed that exoantigens of B. orientalis could induce a high degree of protection against challenge with a virulent B. orientalis strain. The animals in the vaccinated group exhibited a slight decrease in haemoglobin levels and blood cell counts, whereas animals in the control group showed typical clinical symptoms and died between days 11 and 16 after challenge. In another approach, 82 buffaloes kept in B. orientalis-endemic areas were injected with exoantigens plus Freund's adjuvant in two doses at an interval of 2 weeks, one month before the endemic period. An additional 86 buffaloes served as unvaccinated controls. During the endemic period, the clinical signs of all buffaloes were monitored and the antibody response was investigated, using the latex agglutination test. During the endemic period, none of the vaccinated buffaloes showed clinical signs or died, whereas five control buffaloes showed clinical signs and two of them died. The mean antibody titre of vaccinated animals was higher than that of the control animals. These results showed that the culture-derived exoantigens of B. orientalis could be used as a vaccine to prevent buffalo babesiosis.