Quantitative detection of Theileria haneyi in South African horses

Abstract

Theileria haneyi is an apicomplexan parasite that is closely related to Theileria equi, a known causative agent of equine piroplasmosis. Theileria equi genotypes A, B, C, and D have been reported to occur in South African equids. Preliminary studies in South Africa indicated an association between T. equi genotype C and T. haneyi infections. The molecular distinction between these parasites is reliant on a nested PCR assay, which has been reported to be unreliable. A recently reported indirect ELISA based on the equi merozoite antigen (ThEMA-11) of T. haneyi can detect geographically diverse T. haneyi strains. Based on the exclusivity of the ema-11 gene to T. haneyi, we developed a TaqMan minor groove binder (MGB™) quantitative real-time PCR (qPCR) assay to amplify and detect the ema-11 gene. Published Thema-11 gene sequences were used to design primers for the amplification of the ema-11 gene from South African samples. Thema-11 amplicons were cloned and sequenced. An alignment of the South African ema-11 gene sequences with published 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 shown to be rapid, specific, and sensitive in detecting T. haneyi infections. The diagnostic utility of the Thema-11-specific qPCR assay was evaluated together with a T. equi ema-1-specific qPCR assay. Theileria haneyi was detected in 75% of the South African field samples screened, while the occurrence of T. equi based on the quantitative amplification of the ema-1 gene was much higher (100%). These results suggest that used in combination, the Thema-11-specific qPCR assay, and the T. equi ema-1-specific qPCR assay could detect and differentiate between T. haneyi and T. equi infections.