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Molecular detection of Babesia bigemina involves a nested PCR protocol and reverse line blot hybridization (RLBH) assay based on the 18S gene. In this study, we report the development of molecular tools for improving B. bigemina detection in bovine blood-a one-step PCR assay based on the amplification of rap-1a paralogous and a new RLBH Babesia spp. 18S probe. The one-step PCR assay is highly specific, with an estimated analytical sensitivity corresponding to 0.00002% parasitemia. The RLBH assay, with a new B. bigemina probe, allows the detection of all tested B. bigemina isolates showing no cross-hybridization with B. bovis 18S gene. By developing this highly specific and sensitive one-step PCR and upgrading the RLBH assay for B. bigemina, we have improved molecular assays which, together with serologic methods, provide valuable tools for epidemiologic studies of bovine babesiosis.

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