Ticks are vectors of diverse pathogens, with transmission risk elevated in regions of close human-livestock-wildlife contact. In rural South Africa, non-malarial acute febrile illness (AFI) is common, yet the microbial diversity of ticks and human patients remains underexplored. We applied microbiome sequencing (16S rDNA for bacteria; 18S rDNA for eukaryotes) to four sample groups from Bushbuckridge, Mpumalanga, South Africa: Rhipicephalus sanguineus s.l. (n16S = 175, n18S = 30), Amblyomma hebraeum (n16S = 74, n18S = 21), human AFI patients (n16S = 168, n18S = 14), and apparently healthy humans (n16S = 67, n18S = 3). Bacterial alpha diversity did not differ significantly between groups (Shannon p = 0.81, Inverse-Simpson p = 0.36), whereas eukaryotic diversity was lower in human patients (Shannon p = 0.00026, Inverse-Simpson p = 0.00415). Beta diversity exhibited clear differences between groups, with ticks showing greater variability (bacteria p = 0.001; eukaryotes p = 0.001). Co-occurrence networks revealed structured clusters reflecting organised community interactions. Pathogen DNA detected included Plasmodium falciparum in an AFI patient, and sequences similar to several spotted fever group Rickettsia species, including the first report of Rickettsia barbariae in ticks from South Africa. This result requires confirmation by multi-gene sequence analysis. Novel Rickettsiales with uncertain pathogenicity were detected across sample groups. Opportunistic pathogens were detected including Acanthamoeba, Aspergillus and Malassezia. These findings provide baseline evidence on microbial diversity in a rural community in South Africa, underscoring the need for integrated surveillance to address AFI burden within a One Health framework.

Neglected, but economically significant, tropical diseases may be prevalent in domestic cats of Namibia. A multi-centre prevalence field study was conducted across Namibia to assess the distribution of vector-borne pathogens and retroviruses from domestic cats. Samples of blood and serum from 280 cats in 15 towns across eight regions (22-51 cats per region) were analysed. Screening for Ehrlichia, Anaplasma, Babesia, and Hepatozoon species, as well as feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV), was conducted using light microscopy, point-of-care serology, and quantitative real-time polymerase chain reaction (qPCR) assays. Haematology and serum biochemistry analyses were also performed. Several pathogens were identified in Namibian cats using these diagnostic tools. Comparatively, pathogen detection rates varied by modality, namely light microscopy (5 %), serology (42 %), and qPCR (27 %). More specifically, microscopy revealed an overall prevalence (3 %) for inclusions resembling large Babesia parasites, serology indicated a seroprevalence for FIV antibodies (4 %) and FeLV antigen (40 %), and an overall prevalence determined by qPCR for E. canis (2 %) and H. canis (26 %). The investigation also demonstrated the associations between tick presence, pathogen infection, and disease manifestations in Namibian cats. A particular significant positive association was found between H. canis infection and FeLV antigen seroprevalence (P = 0.005). Overall, the study highlighted the difference of various diagnostic tools for detecting pathogen prevalence in cats. Appropriate diagnostic testing - informed by known associations with disease manifestation - should be key in guiding responsible treatment strategies and evaluating potential zoonotic risks linked to domestic cats of Namibia.

Due to limited documentation on vector-borne pathogens of companion animals in Namibia, a country-wide, multi-site field study was conducted to estimate the prevalence of these pathogens in domestic dogs. Samples of whole blood and serum from 375 dogs in 15 towns across eight regions were analysed. Vector-borne pathogens were screened by light microscopic examination of blood smears, point-of-care serology, and quantitative real-time polymerase chain reaction (qPCR). Haematology and serum biochemistry analyses were also performed. Collectively, the SNAP® 4Dx® Plus Test provided 64% seropositive results, comprising Ehrlichia species (59%), Anaplasma species (45%), Dirofilaria immitis (2%), and Borrelia burgdorferi (< 1%). Altogether, prevalence as determined by probe-based qPCR assays was 54%, comprising Ehrlichia canis (27%), Hepatozoon canis (25%), Anaplasma species (13%), and Babesia vogeli (8%). Light microscopy yielded the least number of positives, indicating a collective positive result of only 11% in screening for Ehrlichia, Anaplasma, Hepatozoon, Babesia, and microfilaria species. On the whole, Kunene and Otjozondjupa regions showed the highest pathogen prevalence (75%), and the lowest was from Erongo region (38%), on qPCR testing. Significant associations between tick presence and infection by E. canis (P = 0.001), Anaplasma species (P = 0.006), and B. vogeli (P = 0.008) were demonstrated. Likewise, relevant associations between haemoparasite infection and variables of patient signalment, history, and various disease manifestations were shown. Finally, significant associations were found between pathogen infection and numerous clinical pathology abnormalities of the erythron, leukon, and thrombon, including thrombocytopenia (P = 0.022). Diagnostic modalities should be used contextually to test for canine pathogens, with due consideration of the limitations. Appropriate diagnostic testing such as qPCR, guided by relevant known associations with disease manifestation, should guide responsible treatment strategies and identify potential zoonotic risks in pets.

Tick-borne bacterial pathogens from animals play a significant role in the (re)emergence of human diseases. Rhipicephalus sanguineus sensu lato, a globally prevalent tick, predominantly parasitises domestic dogs but can also feed on humans. We characterised temporal changes in the bacterial microbiome of the midgut and salivary gland tissues of R. sanguineus s.l. ticks and analysed their potential as reservoirs for pathogenic bacteria. A 16 S microbiome and amplicon sequence variant (ASV) approach was used to study the bacteria present in the tissues of R. sanguineus s.l. ticks collected from dogs in Hluvukani, a village in a rural community in Bushbuckridge, Mpumalanga, South Africa, in 2016, 2017 and 2019. Post processing, we obtained 43,161 total sequence reads which were clustered into ASVs by sample year. The final ASVs dataset consisted of seven genera: Coxiella , Anaplasma , Escherichia/Shigella , Ehrlichia , Borrelia , Rickettsia and Wolbachia . No differences in the microbiome profiles of the MG and SG tissues were noted. Coxiella endosymbionts dominated the microbiome in all years. Anaplasma was first detected in 2017, and an increase in Anaplasma levels was detected in 2019, when compared to 2017. All other genera were present at low levels. With the exclusion of Wolbachia , the other detected genera could have pathogenic potential, highlighting the role that R. sanguineus s.l. might play as a reservoir of pathogens.

The Bushbuckridge-East community in Mpumalanga Province, South Africa is bordered by nature reserves, including the Manyeleti Game Reserve. Murid rodents are prevalent in both Manyeleti and communal rangelands adjoining the community households. Although rodents are reservoir hosts for a broad range of viral, bacterial and parasitic pathogens, the rodent microbial diversity and transmission of zoonotic agents to humans in the community is understudied. In this study we investigated bacterial diversity in wild and commensal rodents sampled from different habitats. The 16S rRNA gene was amplified from DNA extracted from the blood of 24 wild Mastomys and one Steatomys sp. and subjected to PacBio circular consensus sequencing. As Bartonella species were dominant in the blood microbiome, gltA gene characterization was performed to delineate species. Rodents sampled from peri-urban and communal rangelands had higher proportions of Bartonella spp. [Hlalakahle (77.7%), Gottenburg (47.8%), Tlhavekisa (83.8%)] compared to those from the protected habitat (43.8%). Ehrlichia spp., Anaplasma spp., and Coxiella burnetii were detected at <1% of the sequence reads. Conventional PCR and sequencing validated the detection of Bartonella spp. with the first confirmation of Bartonella mastomydis infection in Mastomys in South Africa. Additionally, 317 mites, 90 fleas, 10 ticks and eight lice were collected from the rodents, providing evidence of possible vectors of the organisms detected. The detection of zoonotic agents in rodents in Bushbuckridge-East community, together with prior serological confirmation of Bartonella and Coxiella in non-malarial acute febrile patients from this community, highlights the possible risks that commensal rodents pose to human health.

Theileria haneyi is an apicomplexan parasite closely related to Theileria equi, a known causative agent of equine piroplasmosis. The molecular distinction between these parasites relies on a nested polymerase chain reaction (PCR) assay, which has been reported to be unreliable. A recently reported indirect ELISA based on equi merozoite antigen 11 (Thema-11) of T. haneyi can detect geographically diverse T. haneyi strains. Since the ema11 gene is exclusive to T. haneyi, it was chosen as the target for developing a TaqMan minor groove binder (MGBTM) quantitative real-time PCR (qPCR). Published T. haneyi ema-11 gene sequences were used to design primers to amplify the ema-11 gene, and ema-11 amplicons from South African samples were cloned and sequenced. An alignment of the South African ema-11 gene sequences with published T. haneyi ema-11 gene sequences enabled the identification of a conserved region for the design of the qPCR assay. The T. haneyi ema-11 (Thema-11) qPCR assay was efficient, specific, and sensitive in detecting T. haneyi ema-11. The detection limit was determined to be 1.169 x 10-3 % parasitized erythrocytes. The performance of the Thema-11 qPCR assay was evaluated together with a T. equi ema-1-specific qPCR assay. Theileria haneyi was detected in 67.6 % of the South African field samples screened, while the occurrence of T. equi based on the quantitative amplification of the ema-1 gene was higher (91.8 %). Our results suggest that combined, the Thema-11 and T. equi ema-1 qPCR assays could detect and differentiate between T. haneyi and T. equi infections.

Background: Tick-borne bacterial pathogens from both domestic and wild animals play a significant role in the (re)emergence of human diseases. Primary tick endosymbionts have considerable influence on tick fitness and pathogen acquisition or transmission, while secondary endosymbionts are more likely to be pathogens. Rhipicephalus sanguineus is one of the most widespread tick species as they predominantly parasitise domestic dogs, though they have also been documented to feed on domestic animals and humans. This makes them ideal vectors of bacterial pathogens that can pose a significant threat to human health. Rhipicephalus sanguineus is host to a species-specific Coxiellaendosymbiont. Tick-borne pathogens and endosymbiotic bacteria can be studied through a targeted microbiome approach. Methods: We utilised a 16S rRNA microbiome and amplicon sequence variant (ASV) approach to study the bacterial groups present in the midgut and salivary gland tissues of R. sanguineus ticks collected from dogs in a rural community in Bushbuckridge, Mpumalanga, South Africa, from 2016 to 2019. Results: Post processing, we obtained 43,161 total sequence reads which were clustered into ASVs by sample year. After contaminants were removed there were ASVs belonging to seven genera: Coxiella, Anaplasma, Escherichia/Shigella, Ehrlichia, Borrelia, Rickettsia and Wolbachia. Coxiella endosymbionts dominated the microbiome. In 2017 Anaplasma was introduced to the microbiome and increased at the 2019 sampling. All other genera were present at low levels. Conclusions: Our study highlights the changes in the microbiome of the R. sanguineus ticks over time. We found high numbers of two pathogenic Anaplasma species, A. platys and A. centrale, which cause disease in dogs and cattle, respectively, although A. platys infections in humans have been documented. With the exclusion of Wolbachia, the other detected genera could have pathogenic potential. Given our findings of pathogenic bacterial species, our study highlights the role that R. sanguineusmight play as a reservoir of pathogens.

[This corrects the article DOI: 10.1016/j.crmicr.2023.100198.].

Bovine anaplasmosis, caused by Anaplasma marginale, is one of the most important tick-borne diseases of cattle. Anaplasma marginale is known to be present in the Mnisi community, Mpumalanga Province, with frequent cases of anaplasmosis reported. This study investigated the infection dynamics in calves (n = 10) in two habitats in the study area over 12 months. A duplex real-time PCR assay targeting the msp1β gene of A. marginale and the groEL gene of A. centrale confirmed the presence of A. marginale in five calves in a peri-urban area from the first month, but in only two calves at the wildlife–livestock interface and only after six months. These results were confirmed by 16S rRNA microbiome analysis. Over 50 A. marginale msp1α genotypes were detected in the calves along with five novel Msp1a repeats. Calves in the peri-urban area were more likely to be infected with A. marginale than calves in the wildlife–livestock interface. Cattle management, acaricide treatment, and cattle density could explain differences in infection prevalence in the two areas. Our results revealed that most calves were superinfected by distinct A. marginale strains within the study period, indicating continuous challenge with multiple strains that should lead to robust immunity in the calves and endemic stability in the area.

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.

Organisms in the genus Anaplasma are obligate intracellular alphaproteobacteria. Bovine anaplasmosis, predominantly caused by Anaplasma marginale, is the most prevalent tick-borne disease (TBD) of cattle worldwide. Other Anaplasma species are known to cause disease; these include A. ovis, A. platys in dogs, A. capra in goats and humans, and A. phagocytophilum in humans. The rapid advancement of next-generation sequencing technologies has led to the discovery of many novel sequences ascribed to the genus Anaplasma, with over 20 putative new species being proposed since the last formal organization of the genus. Most 16S rRNA gene surveys for Ana -plasma were conducted on cattle and to a lesser extent on rodents, dogs, and ticks. Little is known about the occurrence, diversity, or impact of Anaplasma species circulating in wildlife species. Therefore, we conducted a 16S rRNA gene survey with the goal of identifying Anaplasma species in a variety of wildlife species in the Kruger National Park and neighbouring game reserves, using an unbiased 16S rRNA gene microbiome approach. An Anaplasma/Ehrlichia-group specific quantitative real-time PCR (qPCR) assay revealed the presence of Anaplasma and/or Ehrlichia species in 70.0% (21/30) of African buffalo, 86.7% (26/30) of impala, 36.7% (11/30) of greater kudu, 3.2% (1/31) of African wild dog, 40.6% (13/32) of Burchell's zebra, 43.3% (13/30) of warthog, 22.6% (7/ 31) of spotted hyena, 40.0% (12/30) of leopard, 17.6% (6/34) of lion, 16.7% (5/30) of African elephant and 8.6% (3/35) of white rhinoceros samples. Microbiome sequencing data from the qPCR positive samples revealed four 16S rRNA sequences identical to previously published Anaplasma sequences, as well as nine novel Anaplasma 16S genotypes. Our results reveal a greater diversity of putative Anaplasma species circulating in wildlife than currently classified within the genus. Our findings highlight a potential expansion of the Anaplasma host range and the need for more genetic information from other important genes or genome sequencing of putative novel species for correct classification and further assessment of their occurrence in wildlife, livestock and companion animals.

In Africa, ticks continue to be a major hindrance to the improvement of the livestock industry due to tick-borne pathogens that include Anaplasma, Ehrlichia, Rickettsia and Coxiella species. A systemic review and meta-analysis were conducted here and highlighted the distribution and prevalence of these tick-borne pathogens in African ticks. Relevant publications were searched in five electronic databases and selected using inclusion/exclusion criteria, resulting in 138 and 78 papers included in the qualitative and quantitative analysis, respectively. Most of the studies focused on Rickettsia africae (38 studies), followed by Ehrlichia ruminantium (27 studies), Coxiella burnetii (20 studies) and Anaplasma marginale (17 studies). A meta-analysis of proportions was performed using the random-effects model. The highest prevalence was obtained for Rickettsia spp. (18.39%; 95% CI: 14.23–22.85%), R. africae (13.47%; 95% CI: 2.76–28.69%), R. conorii (11.28%; 95% CI: 1.77–25.89%), A. marginale (12.75%; 95% CI: 4.06–24.35%), E. ruminantium (6.37%; 95% CI: 3.97–9.16%) and E. canis (4.3%; 95% CI: 0.04–12.66%). The prevalence of C. burnetii was low (0%; 95% CI: 0–0.25%), with higher prevalence for Coxiella spp. (27.02%; 95% CI: 10.83–46.03%) and Coxiella-like endosymbionts (70.47%; 95% CI: 27–99.82%). The effect of the tick genera, tick species, country and other variables were identified and highlighted the epidemiology of Rhipicephalus ticks in the heartwater; affinity of each Rickettsia species for different tick genera; dominant distribution of A. marginale, R. africae and Coxiella-like endosymbionts in ticks and a low distribution of C. burnetii in African hard ticks.

The published literature on schizont-"transforming," or pathogenic theileriosis, in African wild artiodactyls is dated and based on limited information. Here the authors review the taxonomy, diagnosis, epidemiology, hematology, pathology, and aspects of control in various species. Molecular studies based on 18S and 16S rRNA gene sequences have shown that African wild artiodactyls are commonly infected with diverse Theileria spp., as well as nontheilerial hemoprotozoa and rickettsia-like bacteria, and coinfections with pathogenic and nonpathogenic Theileria species are often recorded. Although theileriosis is still confusingly referred to as cytauxzoonosis in many species, the validity of a separate Cytauxzoon genus in artiodactyls is debated. The epidemiology of theileriosis is complex; the likelihood of fatal disease depends on the interplay of parasite, vertebrate host, tick vector, and environmental factors. Roan calves (Hippotragus equinus) and stressed animals of all host species are more susceptible to fatal theileriosis. Even though regenerative anemia is common, peripheral blood piroplasm parasitemia does not correlate with disease severity. Other than anemia, common macroscopic lesions include icterus, hemorrhages (mucosal, serosal, and tissue), fluid effusions into body cavities, lung edema, and variably sized raised cream-colored foci of leukocyte infiltration in multiple organs. Histopathologic findings include vasocentric hyperproliferation and lysis of atypical leukocytes with associated intracellular schizonts, parenchymal necrosis, hemorrhage, thromboembolism, and edema. Immunophenotyping is required to establish the identity of the schizont-transformed leukocytes in wild ungulates. Throughout the review, we propose avenues for future research by comparing existing knowledge on selected aspects of theileriosis in domestic livestock with that in African wild artiodactyls.

DNA samples from 74 patients with non-malarial acute febrile illness (AFI), 282 rodents, 100 cattle, 56 dogs and 160 Rhipicephalus sanguineus ticks were screened for the presence of Anaplasma phagocytophilum DNA using a quantitative PCR (qPCR) assay targeting the msp2 gene. The test detected both A. phagocytophilum and Anaplasma sp. SA/ZAM dog DNA. Microbiome sequencing confirmed the presence of low levels of A. phagocytophilum DNA in the blood of rodents, dogs and cattle, while high levels of A. platys and Anaplasma sp. SA/ZAM dog were detected in dogs. Directed sequencing of the 16S rRNA and gltA genes in selected samples revealed the presence of A. phagocytophilum DNA in humans, dogs and rodents and highlighted its importance as a possible contributing cause of AFI in South Africa. A number of recently described Anaplasma species and A. platys were also detected in the study. Phylogenetic analyses grouped Anaplasma sp. SA/ZAM dog into a distinct clade, with sufficient divergence from other Anaplasma species to warrant classification as a separate species. Until appropriate type-material can be deposited and the species is formally described, we will refer to this novel organism as Anaplasma sp. SA dog.

Babesia bigemina is one of the aetiological agents of bovine babesiosis, which causes economic losses through mortality, loss of production and control costs. Effective means of detecting and quantifying B. bigemina in cattle populations is therefore important to inform control approaches. In order to examine the parasite genetic diversity in African countries, B. bigemina 18S rRNA genes from cattle from South Africa, Uganda and Angola were sequenced. The 25 distinct B. bigemina 18S rRNA gene sequences obtained in this study showed 99 to 100% identity with previously published sequences of strains from African and other continents. The sequences of the previously published B. bigemina 18S rRNA gene-specific quantitative PCR (qPCR) primers and probe, developed based on American and Asian strains, were conserved in the African B. bigemina sequences. The qPCR assay was evaluated using 10-fold and 2-fold serial dilutions of B. bigemina-infected erythrocytes to determine the efficiency and analytical sensitivity. The qPCR assay had an efficiency of 98.14 ± 1.71%, and the limit of detection was approximately 1.5 infected red blood cells (iRBCs) per microlitre (μl) of blood. The detection rate of B. bigemina from duplicates of field-collected blood samples from cattle from South Africa, Mozambique and Angola was 37% (30/81), 12% (6/49) and 50% (38/76), respectively. Reverse line blot hybridisation (RLB) results obtained from the same samples in previous studies, using a previously published B. bigemina-specific probe, detected the parasite DNA in only 1.5% (3/206) of the samples. A new B. bigemina-specific RLB oligonucleotide probe was designed in the hypervariable V4 region of the 18S rRNA gene. Screening of field blood samples from cattle showed that the new probe was specific, and its frequency of detection of B. bigemina was three times higher than the previously published probe. The qPCR assay and the newly developed B. bigemina-specific RLB probe provide good tools for epidemiological studies, which are essential in the control of bovine babesiosis.

Bovine anaplasmosis is a globally economically important tick-borne disease caused by the obligate intraerythrocytic rickettsia, Anaplasma marginale. A live Anaplasma centrale blood-based vaccine is available, but it does not protect against all A. marginale field strains and may also transmit other blood-borne pathogens. Five potential outer membrane protein (OMP) vaccine candidates have been well-characterised in A. marginale strains from the USA, however, their levels of conservation in other countries must be ascertained in order to inform their use in a vaccine with regional or global efficacy. This study assessed the amino acid variation in vaccine candidate OMPs in South African strains of A. marginale, and also compared the immunogenic properties between South African and US strains. OMP genes Am779, Am854, omp7, omp8 and omp9 were amplified and sequenced from a set of genetically diverse South African samples with different msp1 alpha-genotypes. OMPs Am854 and Am779 were highly conserved, with 99-100 % amino acid identity, while Omp7, Omp8 and Omp9 had 79-100 % identity with US strains. As has been shown previously, Omp7-9 possess conserved Nand Ctermini, a central variable region, and a highly conserved CD4 T-cell epitope, FLLVDDA(I/V)V, in the N-terminal region. Western blot analysis of recombinant OMPs indicates strong antigenic conservation between South African and US strains of A. marginale, suggesting that they are good candidates for use in a novel global vaccine cocktail, although further work on the best formulation and delivery methods will be necessary.

Recently reported substantial genetic diversity within Theileria equi 18S rRNA gene sequences has led to the identification of five genotypes A, B, C, D, and E, complicating molecular and serological diagnosis. In addition, T. haneyi has lately been reported as a species closely related to the T. equi 18S rRNA genotype C (Knowles et al., 2018). Theileria spp. of this group have a monophyletic origin and are therefore referred to as Equus group to distinguish them from the remaining Theileria lineages (Jalovecka et al., 2019). In this study, we report on the development of genotype-specific quantitative real-time PCR assays capable of detecting and distinguishing between each parasite genotype. Alignment of complete 18S rRNA sequences available on GenBank allowed for the design of a single primer pair and five TaqMan minor groove binder (MGB™) probes specific for each genotype (A–E). The assays, evaluated as qPCR simplex and two qPCR multiplex formats (Multiplex EP–ABC and Multiplex EP–DE), were shown to be both efficient and specific in the detection of T. equi genotypes. The developed qPCR assays were used to study (i) the intra-specific diversity of parasite genotypes within horse and zebra, (ii) the inter-specific differences in parasite genotype diversity in horses as compared to zebra, and (iii) the geographic distribution of T. equi 18S rRNA genotypes in South Africa. In addition, (iv) the presence of T. haneyi in South Africa was evaluated. An assessment of 342 equine field samples comprising 149 field horses, 55 racehorses, and 138 wild zebra confirmed the previously reported presence of T. equi 18S rRNA genotypes A, B, C, and D, and absence of genotype E in South African equids. Theileria equi genotypes A, B, C, and D, were detected in zebra, whereas only genotypes A, C and D, could be identified in field horses, and only genotypes A and C in racehorses. Genotypes B and D were the dominant genotypes identified in zebra in South Africa, while horses were predominantly infected with T. equi genotypes A and C. The greater diversity of T. equi genotypes in zebra suggests that it is an ancestral host for this piroplasmid lineage. Importantly, evidence is presented that each identified T. equi genotype segregates independently in each of the three studied equid populations reinforcing the notion that they represent individual separate entities corresponding to species. Preliminary investigations of the relationship between T. equi genotype C infections and Theileria haneyi, suggest that in addition to the five currently known T. equi genotypes, South African equids are also infected with T. haneyi.