Development of a triplex polymerase chain reaction assay for the detection and identification of the bovine hemoparasites, Anaplasma marginale, Babesia bovis, and Babesia bigemina.

Anaplasma marginale, Babesia bovis, and Babesia bigemina are hemoparasites that can co-infect ruminant livestock and cause symptoms that result in substantial economic loss and even death. These parasites are carried by ticks, which bite the host and transfer the hemoparasite into the blood. The standard approach to identify hemoparasites is Giemsa-stained thin blood smears observed under a microscope, but it is time-consuming and can be difficult to identify co-infections. Thus, we developed a novel triplex PCR to simultaneously detect B. bovis and B. bigemina (based on 18S ribosomal DNA [18S rDNA]) and A. marginale (based on the major surface protein 4 [MSP4] gene). The specific primers produced an amplicon of 168 base pairs (bp) for B. bigemina, 267 bp for B. bovis, and 478 bp A. marginale. The triplex PCR required a minimum of 0.1 pg of DNA to identify B. bovis, B. bigemina, or mixed infection, and 1 pg of DNA to identify A. marginale. There was no cross-amplification with Theileria spp. and Trypanosoma evansi DNAs. The triplex PCR could successfully amplify the three DNA targets simultaneously in cattle blood samples (n = 114) from Thailand. The triplex PCR detected more positive samples (27/114; 23.68%) than microscopic examination (5/114; 4.39%) for all three hemoparasites. Our study offers an alternative, specific, sensitive, and accurate identification and detection tool for co-infection of B. bovis, B. bigemina, and A. marginale in bovine blood. The developed triplex PCR assay could be useful for routine diagnostics in veterinary laboratories and for epidemiological monitoring of hemoparasites.