Assessment and improvement of molecular diagnosis of Theileria parva of African buffalo (Syncerus caffer) in Southern Africa

Abstract

English: Buffalo-adapted Theileria parva causes Corridor disease in cattle. Strict control measures therefore apply to the movement of buffalo in South Africa and include mandatory testing of buffalo for the presence of T. parva. The official test is a real-time hybridization PCR assay that amplifies the V4 hypervariable region of the 18S rRNA gene of T. parva, T. sp. (buffalo) and T. sp. (bougasvlei). The effect that mixed T. parva and T. sp. (buffalo)-like infections have on accurate T. parva diagnosis was investigated. In-vitro mixed infection simulations indicated PCR signal suppression at 100 to 1000-fold T. sp. (buffalo) excess at low T. parva parasitaemia. Suppression of PCR signal was found in field buffalo with mixed infections. The T. parva-positive status of these cases was confirmed by selective suppression of T. sp. (buffalo) amplification using a locked nucleic acid clamp and independent assays based on the p67, p104 and Tpr genes. Conventional and SYBR® Green touch-down PCR methods were developed for each protein coding gene and buffalo from the endemic Kruger National Park were screened. The protein gene assays compared well with the negative and T. parva positive samples diagnosed on the current real-time assay however, they did detect additional positive samples diagnosed as negative on the real-time hybridization. These samples were all T. sp. (buffalo) positive. This confirmed the suppressive effect on PCR signal due to template competition in the current real-time PCR assay. Some positive samples were not detected by the protein genes, possibly due to sequence variation in the primer regions. These independent markers proved useful as supplementary assays in the accurate diagnosis of T. parva infections where mixed infections occur in the buffalo host. The development of the Hybrid II assay, a real-time hybridization PCR method, which compared well with the official hybridization assay in terms of specificity and sensitivity revolutionized the diagnosis of the disease for the main reason that it is not influenced by mixed infections of T. sp. (buffalo)-like parasites and is as such a significant improvement on the current hybridization assay. While the incidence of mixed infections in the Corridor disease endemic region of South Africa is significant, little information is available on the specific distribution and prevalence of T. sp. (buffalo) and T. sp. (bougasvlei). Specific real-time PCR assays were developed and a total of 1211 samples known to harbor these parasites were screened. Both parasites are widely distributed in southern Africa and the incidence of mixed infections with T. parva within the endemic region is similar (~25-50%). However, a significant discrepancy exists with regard to mixed infections of T. sp. (buffalo) and T. sp. (bougasvlei) (~10%). Evidence for speciation between T. sp. (buffalo) and T. sp. (bougasvlei) is supported by phylogenetic analysis of the COI gene, and their designation as different species. This suggests mutual exclusion of parasites and the possibility of hybrid sterility in cases of mixed infections.