Genetic diversity in Babesia bovis from southern Africa and estimation of B. bovis infection levels in cattle using an optimised quantitative PCR assay.

Babesia bovis is a causal agent of bovine babesiosis, a disease which leads to mortality and morbidity and impacts the cattle industry worldwide. We amplified, cloned and sequenced the B. bovis merozoite surface antigen-2b (msa-2b) gene (∼940 bp) and the near full-length 18S rRNA gene (∼1600 bp) from cattle samples from South Africa and Mozambique to determine sequence variation between B. bovis parasites in the region. A TaqMan quantitative real-time PCR (qPCR) assay (18S rRNA gene) was optimised for the detection of B. bovis and estimation of parasitaemia in field samples from cattle from southern Africa. Phylogenetic analysis grouped the Msa-2b sequences in six clades and these were 59.7 to 99.6% identical to reference sequences. Sequence variation amongst B. bovis 18S rRNA sequences was found at 2 to 36 positions, and the sequences were 97 to 99% identical to published sequences. Mismatches between the B. bovis 18S rRNA sequences and a previously published qPCR forward primer (BoF) were observed; therefore, we developed a new forward primer (BoF2), and optimised the qPCR assay. Six 10-fold dilution series of B. bovis infected erythrocytes (2 × 108 to 2 × 103 infected red blood cells [iRBC]/ml) were analysed in triplicate in each of six separate qPCR runs, to determine the efficiency of the assay. The qPCR assay amplified the B. bovis 18S rRNA gene with 92.0 to 94.9% efficiency. The detection limit of the qPCR assay was approximately 6 iRBCs/µl. The performance of the optimised assay to diagnose B. bovis in field samples was assessed by testing DNA from 222 field samples of cattle from South Africa and Mozambique using three methods: the optimised qPCR assay, the reverse line blot (RLB) hybridisation assay, and the previously published qPCR assay. The detection rate of B. bovis using the optimised qPCR assay (31.1%, 69/222) was significantly higher (p<0.001) than both that using RLB (20.7%, 46/222) and the previously published qPCR assay (5.4%; 12/222). The B. bovis parasitaemia in samples from infected cattle ranged from 6 iRBCs/µl to 101,852 iRBCs/µl of blood. Our study revealed marked sequence variation between B. bovis parasites from southern Africa. The optimised qPCR assay will be useful in epidemiological studies and clinical diagnosis of B. bovis in southern Africa, and can be used to determine parasitaemia and potential carrier status in cattle populations, which is essential in the control of babesiosis.

Distribution and prevalence of ticks and tick-borne pathogens of wild animals in South Africa: A systematic review.

Ticks are significant ectoparasites of animals and humans. Published data indicate that most vectors that transmit livestock and human pathogens in sub-Saharan Africa, are native to the region and originate from wild animals. Currently, there is a paucity of information on the role of wild animals on the epidemiology of zoonotic tick-borne pathogens in South Africa. This systematic review focuses on the distribution of ticks and prevalence of tick-borne pathogens in different wild animals in South Africa to identify potential reservoir hosts and possible hotspots for emergence of novel tick-borne pathogens. Following several screening processes, 38 peer-reviewed studies published from 1970 to 2021, were deemed eligible. The studies reported on ticks collected from 63 host species of 21 host families, mostly Canidae, Felidae, Bovidae and Muridae. A total of 49 tick species of nine genera, i.e. Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes, Margaropus, Nuttalliella, Rhipicentor and Rhipicephalus, were reported. Nine tick species, i.e. Amblyomma marmoreum, Am. hebraeum, Haemaphysalis elliptica, Hyalomma truncatum, I. rubicundus, Rh. appendiculatus, Rh. (B.) decoloratus, Rh. evertsi evertsi and Rh. simus were the most commonly reported. Pathogens of the genera Anaplasma, Babesia, Hepatozoo n and Theileria were identified in the wild animals. This review provides more insight on the ecology of ticks and tick-borne pathogens of wild animals in South Africa and gives useful information for predicting their future spread. It also demonstrates that wild animals habour a diverse range of tick species. This level of diversity entails a similarly high potential for emergence of novel tick-borne pathogens. The review further indicates that wild animals in South Africa are sentinels of tick-borne protozoans of veterinary importance and some bacterial pathogens as most ticks they habour are known vectors of pathogens of domestic animals and humans. However, studies on potential tick-borne zoonoses are under-represented and should be included in future epidemiological surveys, especially in the light of climate change and other anthropogenic threats which might result in the emergence of novel tick-borne pathogens.

Occurrence and diversity of avian haemosporidia in Afrotropical landbirds.

Avian haemosporidian infections are widespread and can result in the decline of wild bird populations or in some cases contribute to extinction of species. We determined the prevalence and genetic diversity of avian haemosporidia in 93 samples from 22 landbird species from South Africa (N = 76) and West Africa (N = 17), of which six are intra-African migrants and one is a Palearctic migrant. The samples were analysed for the presence of avian haemosporidian DNA using real-time quantitative PCR (qPCR) and nested PCR assays targeting specific mitochondrial genes of these parasites. The cytochrome b (cytb) gene was sequenced for all samples that tested positive and phylogenetic analysis was conducted in order to determine the relationship of the new sequences with previously published sequences from the MalAvi database. The overall prevalence of avian haemosporidiosis was 68.82% (95% CI: 56.4%-78.87%) and 82.80% (95% CI: 65.68%-86.11%) as determined by qPCR and nested PCR respectively. Eighteen (19.36%; 95% CI; 10.78%-29.97%) samples had mixed infections. Infection prevalence of all haemosporidian spp. were significantly higher (p < 0.05) in samples from West Africa. Forty-six mitochondrial sequences obtained from 14 avian species grouped into three distinct clusters of Haemoproteus (36), Leucocytozoon (8) and Plasmodium (2). These represent eight published and nine new cytb lineages. The most common lineage was Haemoproteus sp. (VIMWE1) which was identified in two bird species from West Africa and seven bird species from South Africa. This study adds to our knowledge of host-parasite relationships of avian haemosporidia of Afrotropical birds.

Evidence confirming the phylogenetic position of Anaplasma centrale (ex Theiler 1911) Ristic and Kreier 1984.

In 1911, Sir Arnold Theiler isolated and described a parasite that was very similar to Anaplasma marginale but which was more centrally located within the erythrocytes of the host cells, and was much less pathogenic than A. marginale. He named the parasite A. marginale variety centrale. The name Anaplasma centrale, referring to the same organism, was published in Validation List No. 15 in 1984, but the publication was based on an erroneous assumption that Theiler had indicated that it was a separate species. Many authors have subsequently accepted this organism as a separate species, but evidence to indicate that it is a distinct species has never been presented. The near full-length 16S rRNA gene sequence, and the deduced amino acid sequences for groEL and msp4 from several isolates of A. marginale and A. centrale from around South Africa were compared with those of the A. marginale type strain, St Maries, and the A. centrale Israel strain and other reference sequences. Phylogenetic analyses of these sequences demonstrated that A. centrale consistently forms a separate clade from A. marginale, supported by high bootstrap values (≥90 %), revealing that there is divergence between these two organisms. In addition, we discuss distinctive characteristics which have been published recently, such as differences in Msp1a/Msp1aS gene structure, as well as genome architecture that provide further evidence to suggest that A. centrale is, in fact, a separate species. Our results, therefore, provide evidence to support the existing nomenclature, and confirm that A. centrale (ex Theiler 1911) Ristic and Kreier 1984 is, indeed, a distinct species.

Detection and Characterisation of Anaplasma marginale and A. centrale in South Africa.

Bovine anaplasmosis is endemic in South Africa and it has a negative economic impact on cattle farming. An improved understanding of Anaplasma marginale and Anaplasma marginale variety centrale (A. centrale) transmission, together with improved tools for pathogen detection and characterisation, are required to inform best management practices. Direct detection methods currently in use for A. marginale and A. centrale in South Africa are light microscopic examination of tissue and organ smears, conventional, nested, and quantitative real-time polymerase chain reaction (qPCR) assays, and a reverse line blot hybridisation assay. Of these, qPCR is the most sensitive for detection of A. marginale and A. centrale in South Africa. Serological assays also feature in routine diagnostics, but cross-reactions prevent accurate species identification. Recently, genetic characterisation has confirmed that A. marginale and A. centrale are separate species. Diversity studies targeting Msp1a repeats for A. marginale and Msp1aS repeats for A. centrale have revealed high genetic variation and point to correspondingly high levels of variation in A. marginale outer membrane proteins (OMPs), which have been shown to be potential vaccine candidates in North American studies. Information on these OMPs is lacking for South African A. marginale strains and should be considered in future recombinant vaccine development studies, ultimately informing the development of regional or global vaccines.

Molecular detection and phylogenetic analysis of Anaplasma marginale and Anaplasma centrale amongst transhumant cattle in north-eastern Uganda.

There is little molecular data from Anaplasma marginale and Anaplasma centrale isolates from cattle in Uganda. Between November 2013 and January 2014, blood was collected from 240 cattle in 20 randomly-selected herds in two districts of the Karamoja Region in north-eastern Uganda. A duplex quantitative real-time polymerase chain reaction (qPCR) assay was used to detect and determine the prevalence of A. marginale (targeting the msp1ß gene) and A. centrale (targeting the groEL gene). The qPCR assay revealed that most cattle (82.9%; 95% confidence interval [CI] 78.2-87.7%) were positive for A. marginale DNA, while fewer cattle (12.1%; 95% CI 7.9-16.2%) were positive for A. centrale DNA. A mixed effects logistic regression model showed that the age of cattle was significantly associated with A. centrale infection, while the prevalence of A. marginale varied significantly according to locality. The near full-length 16S ribosomal RNA (16S rRNA) gene and the heat shock protein gene, groEL, for both Anaplasma species were amplified from a selection of samples. The amplicons were cloned and the resulting recombinants sequenced. We found three novel A. marginale 16S rRNA variants, seven A. marginale groEL gene sequence variants and two A. centrale groEL gene sequence variants. Phylogenetic trees were inferred from sequence alignments of the 16S rRNA gene and GroEL amino acid sequences determined here and published sequences using maximum likelihood, Bayesian inference and parsimony methods Phylogenetic analyses classified the 16S rRNA gene and GroEL amino acid sequences into one clade for A. marginale and a separate clade for A. centrale. This study reveals a high prevalence and sequence variability of A. marginale and A. centrale, and is the first report on the phylogenetic characterisation of A. marginale and A. centrale from cattle in Uganda using molecular markers. Sequence variation can be attributed to mobile pastoralism, communal grazing and grazing with wildlife. These data support future epidemiological investigations for bovine anaplasmosis in Uganda.

Co-infections with multiple genotypes of Anaplasma marginale in cattle indicate pathogen diversity.

BACKGROUND: Only a few studies have examined the presence of Anaplasma marginale and Anaplasma centrale in South Africa, and no studies have comprehensively examined these species across the whole country. To undertake this country-wide study we adapted a duplex quantitative real-time PCR (qPCR) assay for use in South Africa but found that one of the genes on which the assay was based was variable. Therefore, we sequenced a variety of field samples and tested the assay on the variants detected. We used the assay to screen 517 cattle samples sourced from all nine provinces of South Africa, and subsequently examined A. marginale positive samples for msp1α genotype to gauge strain diversity. RESULTS: Although the A. marginale msp1ß gene is variable, the qPCR functions at an acceptable efficiency. The A. centrale groEL gene was not variable within the qPCR assay region. Of the cattle samples screened using the assay, 57% and 17% were found to be positive for A. marginale and A. centrale, respectively. Approximately 15% of the cattle were co-infected. Msp1α genotyping revealed 36 novel repeat sequences. Together with data from previous studies, we analysed the Msp1a repeats from South Africa where a total of 99 repeats have been described that can be attributed to 190 msp1α genotypes. While 22% of these repeats are also found in other countries, only two South African genotypes are also found in other countries; otherwise, the genotypes are unique to South Africa. CONCLUSIONS: Anaplasma marginale was prevalent in the Western Cape, KwaZulu-Natal and Mpumalanga and absent in the Northern Cape. Anaplasma centrale was prevalent in the Western Cape and KwaZulu-Natal and absent in the Northern Cape and Eastern Cape. None of the cattle in the study were known to be vaccinated with A. centrale, so finding positive cattle indicates that this organism appears to be naturally circulating in cattle. A diverse population of A. marginale strains are found in South Africa, with some msp1α genotypes widely distributed across the country, and others appearing only once in one province. This diversity should be taken into account in future vaccine development studies.

Comparison of three nucleic acid-based tests for detecting Anaplasma marginale and Anaplasma centrale in cattle.

Several nucleic acid-based assays have been developed for detecting Anaplasma marginale and Anaplasma centrale in vectors and hosts, making the choice of method to use in endemic areas difficult. We evaluated the ability of the reverse line blot (RLB) hybridisation assay, two nested polymerase chain reaction (nPCR) assays and a duplex real-time quantitative polymerase chain reaction (qPCR) assay to detect A. marginale and A. centrale infections in cattle (n = 66) in South Africa. The lowest detection limits for A. marginale plasmid DNA were 2500 copies by the RLB assay, 250 copies by the nPCR and qPCR assays and 2500, 250 and 25 copies of A. centrale plasmid DNA by the RLB, nPCR and qPCR assays respectively. The qPCR assay detected more A. marginale- and A. centrale-positive samples than the other assays, either as single or mixed infections. Although the results of the qPCR and nPCR tests were in agreement for the majority (38) of A. marginale-positive samples, 13 samples tested negative for A. marginale using nPCR but positive using qPCR. To explain this discrepancy, the target sequence region of the nPCR assay was evaluated by cloning and sequencing the msp1ß gene from selected field samples. The results indicated sequence variation in the internal forward primer (AM100) area amongst the South African A. marginale msp1ß sequences, resulting in false negatives. We propose the use of the duplex qPCR assay in future studies as it is more sensitive and offers the benefits of quantification and multiplex detection of both Anaplasma spp.

Characterization of Anaplasma marginale subsp. centrale Strains by Use of msp1aS Genotyping Reveals a Wildlife Reservoir.

Bovine anaplasmosis caused by the intraerythrocytic rickettsial pathogen Anaplasma marginale is endemic in South Africa. Anaplasma marginale subspecies centrale also infects cattle; however, it causes a milder form of anaplasmosis and is used as a live vaccine against A. marginale There has been less interest in the epidemiology of A. marginale subsp. centrale, and, as a result, there are few reports detecting natural infections of this organism. When detected in cattle, it is often assumed that it is due to vaccination, and in most cases, it is reported as coinfection with A. marginale without characterization of the strain. A total of 380 blood samples from wild ruminant species and cattle collected from biobanks, national parks, and other regions of South Africa were used in duplex real-time PCR assays to simultaneously detect A. marginale and A. marginale subsp. centrale. PCR results indicated high occurrence of A. marginale subsp. centrale infections, ranging from 25 to 100% in national parks. Samples positive for A. marginale subsp. centrale were further characterized using the msp1aS gene, a homolog of msp1α of A. marginale, which contains repeats at the 5' ends that are useful for genotyping strains. A total of 47 Msp1aS repeats were identified, which corresponded to 32 A. marginale subsp. centrale genotypes detected in cattle, buffalo, and wildebeest. RepeatAnalyzer was used to examine strain diversity. Our results demonstrate a diversity of A. marginale subsp. centrale strains from cattle and wildlife hosts from South Africa and indicate the utility of msp1aS as a genotypic marker for A. marginale subsp. centrale strain diversity.

Molecular investigation of tick-borne haemoparasite infections among transhumant zebu cattle in Karamoja Region, Uganda.

Tick-borne diseases (TBDs) are a major constraint to cattle production in pastoral areas in Africa. Although information on tick-borne infections is important to prioritise control approaches, it is limited for transhumant zebu cattle in Karamoja, Uganda. We conducted a study to determine the occurrence and level of tick-borne infections among cattle in Karamoja Region. A total of 240 cattle were selected for blood collection using systematic sampling in 20 randomly-selected herds in two districts. The hypervariable V4 region of the 18S rRNA gene for Theileria/Babesia and the V1 region of the 16S rRNA gene for Ehrlichia/Anaplasma were amplified and hybridised to genus- and species-specific oligonucleotide probes on a reverse line blot (RLB) membrane. A duplex quantitative real-time polymerase chain reaction (qPCR) assay based on msp1ß and groEL genes was used for the detection of Anaplasma marginale and A. centrale, while monoplex qPCR assays were used for the detection of Ehrlichia ruminantium (226bp fragment of the pCS20 region) and Theileria parva (18S rRNA gene). The RLB hybridisation assay demonstrated the presence of tick-borne haemoparasites in all but one sample (99.6%), mostly as mixed infections (97.5%). The most frequently detected species were Theileria mutans (88.3%, 95% confidence interval: 84.6-91.7%), A. marginale (73.8%: 68.3-78.8%), Theileria velifera (71.3%: 65.8-76.7%) and Anaplasma sp. Omatjenne (63.3%: 57.5-68.8%). Other virulent pathogens, namely Babesia bigemina (5.0%) and T. parva (2.9%), were also detected with RLB, but not E. ruminantium. The proportions of qPCR positive samples were 82.9% (A. marginale), 12.1% (A. centrale), 3.3% (T. parva), and 1.7% (E. ruminantium). The full-length 18S rRNA genes from 6 out of 47 samples that were positive on RLB for the Babesia genus-specific probe and not for any of the Babesia species-specific probes were amplified, cloned and sequenced. The sequences were used to construct phylogenetic trees. Variations (5 to 9 nucleotides) in the 18S rRNA gene sequences of B. bigemina were identified, when compared with B. bigemina sequences from other parts of the world. Three nucleotide differences in the B. bigemina probe region may explain the failure of the RLB hybridisation assay to detect B. bigemina in some samples. T. mutans and B. bigemina sequences grouped in separate clades from previously published sequences. In conclusion, this study demonstrated high and widespread occurrence, and sequence variation of tick-borne haemoparasites among cattle in the pastoral area of Karamoja, which is useful for diagnosis and control of TBDs.

Phylogeny of Theileria buffeli genotypes identified in the South African buffalo (Syncerus caffer) population.

Theileria buffeli/orientalis is a group of benign and mildly pathogenic species of cattle and buffalo in various parts of the world. In a previous study, we identified T. buffeli in blood samples originating from the African buffalo (Syncerus caffer) in the Hluhluwe-iMfolozi Game Park (HIP) and the Addo Elephant Game Park (AEGP) in South Africa. The aim of this study was to characterise the 18S rRNA gene and complete internal transcribed spacer (ITS1-5.8S-ITS2) region of T. buffeli samples, and to establish the phylogenetic position of this species based on these loci. The 18S rRNA gene and the complete ITS region were amplified from DNA extracted from blood samples originating from buffalo in HIP and AEGP. The PCR products were cloned and the resulting recombinants sequenced. We identified novel T. buffeli-like 18S rRNA and ITS genotypes from buffalo in the AEGP, and novel Theileria sinensis-like 18S rRNA genotypes from buffalo in the HIP. Phylogenetic analyses indicated that the T. buffeli-like sequences were similar to T. buffeli sequences from cattle and buffalo in China and India, and the T. sinensis-like sequences were similar to T. sinensis 18S rRNA sequences of cattle and yak in China. There was extensive sequence variation between the novel T. buffeli genotypes of the African buffalo and previously described T. buffeli and T. sinensis genotypes. The presence of organisms with T. buffeli-like and T. sinensis-like genotypes in the African buffalo could be of significant importance, particularly to the cattle industry in South Africa as these animals might act as sources of infections to naïve cattle. This is the first report on the characterisation of the full-length 18S rRNA gene and ITS region of T. buffeli and T. sinensis genotypes in South Africa. Our study provides invaluable information towards the classification of this complex group of benign and mildly pathogenic species.

Evaluation of a real-time PCR test for the detection and discrimination of theileria species in the African buffalo (Syncerus caffer).

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.

Sequence variation identified in the 18S rRNA gene of Theileria mutans and Theileria velifera from the African buffalo (Syncerus caffer).

The African buffalo (Syncerus caffer) is a natural reservoir host for both pathogenic and non-pathogenic Theileria species. These often occur naturally as mixed infections in buffalo. Although the benign and mildly pathogenic forms do not have any significant economic importance, their presence could complicate the interpretation of diagnostic test results aimed at the specific diagnosis of the pathogenic Theileria parva in cattle and buffalo in South Africa. The 18S rRNA gene has been used as the target in a quantitative real-time PCR (qPCR) assay for the detection of T. parva infections. However, the extent of sequence variation within this gene in the non-pathogenic Theileria spp. of the Africa buffalo is not well known. The aim of this study was, therefore, to characterise the full-length 18S rRNA genes of Theileria mutans, Theileria sp. (strain MSD) and T. velifera and to determine the possible influence of any sequence variation on the specific detection of T. parva using the 18S rRNA qPCR. The reverse line blot (RLB) hybridization assay was used to select samples which either tested positive for several different Theileria spp., or which hybridised only with the Babesia/Theileria genus-specific probe and not with any of the Babesia or Theileria species-specific probes. The full-length 18S rRNA genes from 14 samples, originating from 13 buffalo and one bovine from different localities in South Africa, were amplified, cloned and the resulting recombinants sequenced. Variations in the 18S rRNA gene sequences were identified in T. mutans, Theileria sp. (strain MSD) and T. velifera, with the greatest diversity observed amongst the T. mutans variants. This variation possibly explained why the RLB hybridization assay failed to detect T. mutans and T. velifera in some of the analysed samples.

Identification of Theileria parva and Theileria sp. (buffalo) 18S rRNA gene sequence variants in the African Buffalo (Syncerus caffer) in southern Africa.

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. T. 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.