PCR-based detection and differentiation of piroplasms and malaria parasites from goats in Thailand

Abstract

A novel multiplex PCR (mPCR) was developed to identify and differentiate malaria parasite (Plasmodium caprae), and two piroplasms (Babesia bigemina and Theileria luwenshuni) in Thai goats. Three pairs of primer were designed to be species-specific, targeting cytochrome c oxidase subunit I (COI) gene in mitochondria. Plasmids containing each parasite’s DNA was serially diluted to evaluate the sensitivity of singleplex and mPCR assays. The specificity of the primer sets was confirmed by testing for amplification from DNA of each pathogen and other related hemoparasites. A total of 100 goat blood samples from five provinces of Thailand was used to validate the reliability and application of the assay. The detection limit of the mPCR in this study was 108 parasite copies and the diagnostic specificity in detection of P. caprae and T. luwenshuni was found to be high (94.9%-100%). The high level of diagnostic sensitivity (100%) in the detection of T. luwenshuni was also recorded. However, the diagnostic sensitivity of mPCR was found to be low (50%) in the detection of P. caprae from field-collected blood samples. These results demonstrated the successful establishment of the mPCR presented by its same level of sensitivity to each single PCR counterparts and its ability to simultaneous detection all three parasites in one reaction. This study also describes the first report of Theileria luwenshuni and Babesia bigemina in goats of Thailand.