Comparison of different direct diagnostic methods to identify Babesia bovis and Babesia bigemina in animals vaccinated with live attenuated parasites

Blood smear examination, flow cytometry, duplex Polymerase Chain Reaction (PCR), and duplex nested PCR (nPCR) were evaluated for detection of Babesia bigemina and Babesia bovis infections in cattle vaccinated with live attenuated strains. Two groups of four cattle were immunized with either B. bigemina (Bi) or B. bovis (Bo). On day 23 post inoculation (PI), Bi cattle were vaccinated with B. bovis (BiBo) and Bo cattle were vaccinated with B. bigemina (BoBi). Babesia bigemina was first detected by blood smear examination 7.5+/-3.5 days PI in the Bi group and 32.2+/-1.7 days PI in the BoBi group. The first occurrence of B. bovis in blood smears was 8.0 days PI in the Bo group and 36.0+/-2.6 days PI in the BiBo group. Flow cytometry detected parasitized erythrocytes on day 1.7+/-1.5 and 2.2+/-1.5 PI in the Bi and Bo groups, respectively, but did not discriminate between the two Babesia spp. Duplex PCR detected B. bigemina on day 4.0+/-0.8 and 26.0+/-0.8 PI in the Bi and BoBi groups, respectively, and B. bovis on day 4.0 and 25.3+/-0.5 PI in the Bo and BiBo groups, respectively. The duplex nPCR detected B. bigemina on 3.0+/-0.8 and 25.0+/-0.0 days PI in the Bi and BoBi groups, respectively, and 4.7+/-1.7 and 27.7+/-6.2 days PI in the Bo and BiBo groups, respectively. Duplex nPCR outperformed the other tests in terms of specificity and sensitivity, indicating that it is the most useful method for identifying Babesia spp. in cattle following vaccination.