Development and evaluation of two nested PCR assays for the detection of Babesia bovis from cattle blood.

We developed and evaluated two nested polymerase chain reaction (nPCR) assays for the diagnosis of Babesia bovis infection in cattle based on two membrane protein genes from B. bovis, BBOV_IV005650 (BV5650) and BBO_IV008970 (BV8970). The specificities and sensitivities of the tests were compared with B. bovis Rhoptery associated protein 1 gene (RAP-1) nPCR. The specificity of the tests was 100% for B. bovis DNA. The sensitivities of nPCR to B. bovis from the in vitro cultured parasites were as low as 10(-8)%, 10(-6)%, and 10(-7)% parasitemia for BV5650, BV8970, and RAP-1 nPCR, respectively. The nPCR detected as little as 1 fg genomic DNA per test for BV5650 and 100fg per test for both BV8970 and RAP-1 genes. For field applications, the sensitivity was evaluated to a total of 165 field samples from Ghana, Mongolia, Brazil and Japan. The nPCR assay of BV5650 was the most sensitive for the detection of B. bovis from the field samples. The BV5650 nPCR assay provides a good diagnostic tool for laboratory diagnostic assessment of B. bovis infection in cattle worldwide.