Development of a novel multiplex PCR assay for the detection and differentiation of <i>Plasmodium caprae</i> from <i>Theileria luwenshuni</i> and <i>Babesia</i> spp. in goats

Intraerythrocytic parasites are traditionally identified by the microscopic examination of Giemsa-stained blood smears. However, this method does not always allow for the identification of individual species in goat's RBCs. Moreover, its unreliability in detecting low levels of parasitemia makes it unsuitable for epidemiological investigations and leaves goat farms vulnerable to potential outbreaks. In the present study, a novel multiplex PCR (mPCR) targeting the cytochrome c oxidase subunit I (COI) gene was developed to detect and subsequently differentiate Plasmodium caprae, Theileria luwenshuni, and Babesia spp. The specificity of each primer set was assessed both in silica and with a panel of DNA samples from the hosts themselves and other goat hemoparasites. Amplicons generated from each pair of primers were 664, 555, and 320-bp for P. caprae, Babesia spp., and T. luwenshuni, respectively. These products were further confirmed by sequencing. Our novel mPCR reactions successfully demonstrated the accurate and simultaneous amplification of the three parasites' DNA samples. The current mPCR method showed no cross-amplification with unintended targets. The detection limit of the mPCR in this study was 10(8) parasites' DNA copies per reaction. The current mPCR was able to detect the minimum parasitemia of approximately 0.001%, 0.000005%, 0.00001% for P. caprae, Babesia spp. and T. luwenshuni, respectively. The diagnostic specificity in the detection of P. caprae and T. luwenshuni ranged from 94.9 to 100 %. The mPCR was further applied to a collection of field blood samples from five provinces in Thailand to validate its reliability and applicability. The results demonstrated the successful detection of P. caprae, Babesia spp. and T. luwenshuni in goat samples with the same sensitivity levels as conventional PCR methods. This study also confirmed the presence of T. luwenshuni and Babesia spp. in Thai goats. The current mPCR method offers an alternative for the diagnosis of P. caprae, T. luwenshuni, and Babesia spp., either single or under co-infection conditions, and for large-scale surveillance.