Zoology and Entomology
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Browsing Zoology and Entomology by Subject "African buffalo"
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Item Open Access Assessment and improvement of molecular diagnosis of Theileria parva of African buffalo (Syncerus caffer) in Southern Africa(University of the Free State, 2015-10-30) Pienaar, Ronel; Thekisoe, Oriel M. M.; Man, Ben J.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.Item Open Access Comparison of real-time polymerase chain reaction with the conventional PCR assay for the diagnosis of Theileria parva in South Africa(University of the Free State, 2015-08-04) Papli, Natasha Ektha; Latif, A. A.; Mbati, P.Theileria parva (T. parva) is transmitted from carrier buffalo to cattle causing Corridor disease in cattle. The 989/990 conventional Polymerase Chain Reaction (PCR) assay used for the detection of T. parva is labour-intensive and has the potential for contamination due to the need for post-amplification handling. Real-time PCR offers a way of addressing these limitations. This thesis describes the development of a TaqMan assay for the detection of T. parva and a comparison between this real-time assay with the real-time Hybridization probe assay and the conventional PCR assay for the diagnosis of T. parva. Theileria general forward and reverse primers and a T. parva TaqMan probe specific for the recognition of a conservative region of the T. parva 18S rRNA gene was designed. The TaqMan PCR assay could detect T. parva DNA at a 2x10-5% parasitaemia with a 93% certainty. The primer pairs and probe only cross-reacted with Theileria sp. (buffalo) and no amplification with other Theileria species, bacteria or related haemoparasites was observed. Theileria sp. (buffalo) is genetically closely related to T. parva. However, its biology and disease relations are not known. The TaqMan probe assay detected 87% of all positive samples for evidence of the diagnostic sensitivity and 100% of all negative samples tested negative for the diagnostic specificity assay. These results were compared with those obtained from 989/990 conventional PCR and BioPAD Hybridization probe PCR which targeted the same gene. The Hybridization probe PCR appeared to be more sensitive than the TaqMan probe PCR or conventional PCR assay. With the specificity test, the Hybridization probe PCR proved to be more specific than the other two assays. All three tests gave similar results for the diagnostic specificity. The TaqMan probe assay with its high sensitivity, wide range of detection ability and simplicity is particularly useful in the detection of T. parva. However, further studies are required to improve the specificity of the TaqMan PCR assay in order to eliminate the detection of Theileria sp. (buffalo).