Bhoora, Raksha VasantraiMbaba, Tshenolo VincentiaTroskie, MilanaAckermann, RebeccaCollins, Nicola E.2025-10-212025-10-212025-05Bhoora, R.V., Mbaba, T., Troskie, M., Ackermann, R.E. & Collins, N.E. 2025, 'Quantitative detection of Theileria haneyi in South African horses', Ticks and Tick-borne Diseases, vol. 16, no. 3, art. 102487, pp. 1-10, doi : 10.1016/j.ttbdis.2025.102487.1877-959X10.1016/j.ttbdis.2025.102487http://hdl.handle.net/2263/104783DATA AVAILABILITY : Data will be made available on request.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. HIGHLIGHTS • Theileria haneyi ema-11 sequences form two clades. • A TaqMan quantitative real-time PCR (qPCR) assay was developed to detect T. haneyi. • The Thema-11 and T. equi ema-1 qPCR assays detect and differentiate between T. haneyi and T. equi infections. • Mixed T. haneyi and T. equi infections are common in South Africa. • Theileria haneyi is always detected in samples that test positive for genotype C.en© 2025 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).Theileria haneyiEquine piroplasmosisQuantitative real-time PCR (qPCR)South Africa (SA)Polymerase chain reaction (PCR)Article