Quantitative detection of Theileria haneyi in South African horses

Theileria haneyi is an apicomplexan parasite closely related to Theileria equi, a known causative agent of equine piroplasmosis. The molecular distinction between these parasites relies on a nested polymerase chain reaction (PCR) assay, which has been reported to be unreliable. A recently reported indirect ELISA based on equi merozoite antigen 11 (Thema-11) of T. haneyi can detect geographically diverse T. haneyi strains. Since the ema-11 gene is exclusive to T. haneyi, it was chosen as the target for developing a TaqMan minor groove binder (MGB™) quantitative real-time PCR (qPCR). Published T. haneyi ema-11 gene sequences were used to design primers to amplify the ema-11 gene, and ema-11 amplicons from South African samples were cloned and sequenced. An alignment of the South African ema-11 gene sequences with published T. haneyi ema-11 gene sequences enabled the identification of a conserved region for the design of the qPCR assay. The T. haneyi ema-11 (Thema-11) qPCR assay was efficient, specific, and sensitive in detecting T. haneyi ema-11. The detection limit was determined to be 1.169 × 10^-3 % parasitized erythrocytes. The performance of the Thema-11 qPCR assay was evaluated together with a T. equi ema-1-specific qPCR assay. Theileria haneyi was detected in 67.6 % of the South African field samples screened, while the occurrence of T. equi based on the quantitative amplification of the ema-1 gene was higher (91.8 %). Our results suggest that combined, the Thema-11 and T. equi ema-1 qPCR assays could detect and differentiate between T. haneyi and T. equi infections.