A quantitative real-time PCR (qPCR) assay based on the cox III gene was evaluated for the simultaneous detection and
discrimination of Theileria species in buffalo and cattle blood samples from South Africa and Mozambique using melting
curve analysis. The results obtained were compared to those of the reverse line blot (RLB) hybridization assay for the
simultaneous detection and differentiation of Theileria spp. in mixed infections, and to the 18S rRNA qPCR assay results for
the specific detection of Theileria parva. Theileria parva, Theileria sp. (buffalo), Theileria taurotragi, Theileria buffeli and
Theileria mutans were detected by the cox III assay. Theileria velifera was not detected from any of the samples analysed.
Seventeen percent of the samples had non-species specific melting peaks and 4.5% of the samples were negative or below
the detection limit of the assay. The cox III assay identified more T. parva and Theileria sp. (buffalo) positive samples than the
RLB assay, and also detected more T. parva infections than the 18S assay. However, only a small number of samples were
positive for the benign Theileria spp. To our knowledge T. taurotragi has never been identified from the African buffalo, its
identification in some samples by the qPCR assay was unexpected. Because of these discrepancies in the results, cox III
qPCR products were cloned and sequenced. Sequence analysis indicated extensive inter- and intra-species variations in the
probe target regions of the cox III gene sequences of the benign Theileria spp. and therefore explains their low detection.
The cox III assay is specific for the detection of T. parva infections in cattle and buffalo. Sequence data generated from this
study can be used for the development of a more inclusive assay for detection and differentiation of all variants of the
mildly pathogenic and benign Theileria spp. of buffalo and cattle.