Theileriosis, babesiosis, heartwater and anaplasmosis are considered to be amongst the most important tick-borne diseases of livestock in sub-Saharan Africa‟s tropical and subtropical regions resulting in extensive economic losses to farmers in endemic areas. It is well-known that the African buffalo (Syncerus caffer) is the natural reservoir host of various tick-borne haemoparasites of veterinary importance. In this study, the occurrence of tick-borne haemoprotozoan parasites (Theileria, Babesia, Anaplasma and Ehrlichia species) in buffalo from two geographically isolated national parks in South Africa (Kruger National Park and Hluhluwe-iMfolozi Park) was determined using the reverse line blot (RLB) hybridization assay. The RLB results revealed the presence of Theileria, Babesia and Anaplasma species either as single or as mixed infections. Although not detected with the RLB assay, 5% of the buffalo blood samples from the KNP tested positive for the presence of Ehrlichia ruminantium using the pCS20 real-time PCR assay. Previous studies on the occurrence of haemoparasites in the South African buffalo population have mainly focussed on the prevalence of Theileria species only. The finding on the presence of Anaplasma, Ehrlichia and Babesia species is therefore a novel contribution. This study has confirmed the findings of previous studies that buffalo is the natural reservoir host of both pathogenic and non-pathogenic Theileria species namely, T. parva, Theileria sp. (buffalo), T. mutans, T. velifera and T. buffe1i.In this study, the most frequently occurring Theileria species detected in the KNP were T. mutans (81%), Theileria sp. (sable) (61%), T. parva (40%), Theileria sp. (buffalo) (13%) and T. velifera (11%). Theileria buffeli was not detected in the KNP. In the Hluhluwe-iMfolozi Park, the most occurring Theileria species were T. mutans (55%), T. velifera (54%), T. parva (53%), Theileria sp. (sable) (53%), Theileria sp. (buffalo) (49%) and T. buffeli, (49%). Theileria sp. (sable) causes fatal clinical disease in roan and sable antelope in South Africa and we can only speculate whether the presence of Theileria sp. (sable) DNA in the buffalo population was a true and/or incidental finding. An interesting finding was the presence of Babesia occultans DNA in 50% of the buffalo from the Hluhluwe-iMfolozi Park. Babesia occultans is the causative agent of a benign form of cattle babesiosis in South Africa and, to date; this organism has not been identified in wildlife in South Africa. The significance of this finding warrents further investigation and confirmation using gene cloning, sequencing and phylogenetic analysis. Ehrlichia ruminantium has been reported to infect not only domesticated ruminants but also wild ruminants, however most wildlife species appear to carry the organism asymptomatically. In this study, we were not able to detect E. ruminantium DNA in any of the buffalo samples tested using the RLB hybridization assay. However, using the quantitative pCS20 real-time PCR assay we detected E. ruminantium DNA in 5% of the KNP samples. None of the Hluhluwe-iMfolozi Park samples tested positive for E. ruminantium using the real-time PCR assay. These results suggest that buffalo is not the natural reservoir host of E. ruminantium. However, a subclinical carrier state in buffalo has been experimentally shown to occur after tick transmission from carrier animals and further studies will have to be conducted to confirm whether this finding holds any potential risk to domestic animals. In Southern Africa, two Anaplasma species are known to infect cattle, A. marginale and A. centrale. Clinical bovine anaplasmosis is usually caused by A. marginale; whilst A. centrale generally results in mild disease. Because there is partial cross immunity between the two species, A. centrale is used as a live vaccine for cattle in Israel, South Africa, South America and Australia. Apart from cattle, Anaplasma marginale has been described in wild ruminants which can become persistently infected serving as reservoirs for infection of susceptible hosts; it has been recovered from 10 wild ruminants. Subclinical occurrence of A. marginale, either natural or after artificial infection has been confirmed in the African buffalo and various other wildlife species. In this study, the Anaplasma species detected from HluhluweiMfolozi Park buffalo samples were A. centrale (75%), A. marginale (42%) and Anaplasma (formerly Ehrlichia) sp. Omatjenne (28%). DNA of these species was also detected in buffalo from KNP; A. centrale (49%), A. marginale (24%) and Anaplasma (Ehrlichia) sp. Omatjenne (5%). The presence of A. marginale in the buffalo population suggests that buffalo may be a factor in the epidemiology and spread of bovine anaplasmosis because, as reservoir hosts of A. marginale, they could serve as a source of infective blood for mechanical spread by various routes and biological transmission by ticks. Factors such as climate, host abundance, tick host diversity, and topography have, however, all been shown to also impact on the epidemiology of A. marginale. Subsequently 64 samples were selected that either tested (i) positive for a specific Anaplasma spp. (A. centrale, A. marginale and/or Anaplasma (Ehrlichia) sp. Omatjenne) using the RLB assay, or (ii) in which the PCR products hybridized only with the Anaplasma/Ehrlichia genus-specific probes for molecular characterization by cloning and sequencing of the 16S rRNA gene. Aplification of the full-length and/or partial parasite 16S rRNA gene of any of the selected samples that previously tested positive for the presence of Anaplasma (Ehrlichia)sp. Omatjenne (using the RLB assay) or E. ruminantium (using the pCS20 real-time PCR assay) was unsuccessful. This was most probably due to low rickettsaemia. However, amplification of either the near full-length parasite 16S rRNA gene or a partial 16S rRNA gene from seven samples from the KNP and three from Hluhluwe-iMfolozi Park was successful. Results indicated that the obtained sequences of 12 of the 18 clones were highly similar to published A. centrale 16S rRNA gene sequences, four of the clones were highly similar to the published A. marginale sequences and the sequences of the remaining two clones were closely similar to Anaplasma (Ehrlichia) sp. strain Omatjenne. The observed sequence similarities were confirmed by phylogenetic analyses. An interesting finding was the presence of one full-length parasite 16S rDNA sequence that was 100% identical to that of the published A. centrale vaccine strain sequences. It is well known that A. centrale is widely used as live vaccine for the control of bovine anaplasmosis. The occurrence of A. centrale vaccine strain DNA in the South African buffalo population is therefore of great interest. It can only be speculated whether A. centrale has evolved in the African buffalo, and/or if buffalo act as natural reservoir hosts, or if is it merely being maintained in the buffalo population by in utero transmission. This also serves as the first report of Anaplasma (Ehrlichia) sp. Omatjenne DNA in the African buffalo which warrents further investigation. In conclusion, the findings suggest that buffalo is a natural reservoir of Anaplasma spp. infection and could play an important role in the epidemiology and spread of anaplasmosis and may represent a serious threat to the livestock industry.