Abstract:
Theileria parva is the causative agent of Corridor disease in cattle in South Africa. The African
buffalo (Syncerus caffer) is the reservoir host, and, as these animals are important for eco-tourism in
South Africa, it is compulsory to test and certify them disease free prior to translocation. A T.
parva-specific real-time polymerase chain reaction (PCR) test based on the small subunit ribosomal
RNA (18S rRNA) gene is one of the tests used for the diagnosis of the parasite in buffalo and cattle
in South Africa. However, because of the high similarity between the 18S rRNA gene sequences of
T. parva and Theileria sp. (buffalo), the latter is also amplified by the real-time PCR primers,
although it is not detected by the T. parva-specific hybridization probes. Preliminary sequencing
studies have revealed a small number of sequence differences within the 18S rRNA gene in both
species but the extent of this sequence variation is unknown. The aim of the current study was to
sequence the 18S rRNA genes of T. parva and Theileria sp. (buffalo), and to determine whether all
identified genotypes can be correctly detected by the real-time PCR assay. The reverse line blot
(RLB) hybridization assay was used to identify T. parva and Theileria sp. (buffalo) positive
samples from buffalo blood samples originating from the Kruger National Park, Hluhluwe-iMfolozi
Park, the Greater Limpopo Transfrontier Park, and a private game ranch in the Hoedspruit area.
Theileria parva and Theileria sp. (buffalo) were identified in 42 % and 28 %, respectively, of 252
samples, mainly as mixed infections. The full-length 18S rRNA gene of selected samples was
amplified, cloned and sequenced. From a total of 20 sequences obtained, 10 grouped with
previously published T. parva sequences from GenBank while 10 sequences grouped with a
previously published Theileria sp. (buffalo) sequence. All these formed a monophyletic group with
known pathogenic Theileria species. Our phylogenetic analyses confirm the distinction between
Theileria sp. (buffalo) and T. parva and indicate the existence of a single group of T. parva and two
Theileria sp. (buffalo) 18S rRNA gene variants in the African buffalo. Despite the observed
variation in the full-length parasite 18S rRNA gene sequences, the area in the V4 hypervariable region where the RLB and real-time PCR hybridization probes were developed was relatively
conserved. The T. parva specific real-time PCR assay was able to successfully detect all T. parva
variants and, although amplicons were obtained from Theileria sp. (buffalo) DNA, none of the
Theileria sp. (buffalo) 18S rRNA sequence variants were detected by the T. parva-specific
hybridization probes.
