Anaplasmosis, babesiosis, and heartwater are the three most important tick-borne diseases of cattle in South Africa and result in a large number of mortalities. Endemic stability contributes to disease control, but little is known about the conditions required for maintenance of endemic stability. Through the on-going Health and Demographic Surveillance System in Livestock in the study area of the Mnisi One Health Platform, Mpumalanga, a great deal of information is being collected about cattle in the area, with the eventual aim of developing mathematical models to describe and predict infections. More than 15000 cattle have been identified for tick burden assessment, serological analyses and parasite identification. However, little is known about the time-course of infection of cattle with various tick-borne haemoparasites. Therefore, this study aimed to investigate the time-course of infection in new-born calves (n=10) and the presence of haemoparasites in adult ticks over a one year period using reverse line blot (RLB) hybridization and quantitative polymerase chain reaction assays. Blood samples and adult ticks were collected monthly from new-born calves in two areas of the Mnisi communal area: five located in a peri-urban area and five at the wildlife/livestock interface. A total of 119 blood samples and 805 adult ticks were collected. The RLB results confirm the exposure of most new-born calves in the Mnisi communal area to non-pathogenic and pathogenic tick-borne haemoparasites in the genera Anaplasma, Babesia, Ehrlichia and Theileria in their first year of life. A total of 805 adult ticks were identified to species level using identification keys and molecular methods. Only two tick species, Amblyomma hebraeum and Rhipicephalus microplus, were found on the calves during the year. Non-pathogenic and pathogenic haemoparasites in the genera Anaplasma, Babesia, Ehrlichia and Theileria were detected in pooled DNA extracted from ticks that had digested their blood meal. Pathogen-specific qPCR results indicated that some of the pathogens could not be detected in the calves until six to seven months of age and A. marginale was not detected at all in three calves at the wildlife/livestock interface. These calves were either infected at levels below the detection limit of our assays, or they were not infected at all. If the latter, it is possible that exposure to related non-pathogenic haemoparasites might help to establish and maintain endemic stability. Factors such as cattle density and dipping methods within different areas in the Mnisi communal area may play a role in the number of infected tick vectors in an area, and thus in the time-course of infection in new-born calves. It is clear that detailed information for cattle in different localities in the Mnisi communal area will be required in order to build accurate mathematical models to describe and predict infections.