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• Of 109 dogs tested in a South African settlement, 31% were seropositive to Babesia rossi. • These seropositive dogs had no history of severe babesiosis. • Clinical signs varied amongst other dogs in the settlement which were PCR-positive to B. rossi. • Tick control and treatment for babesiosis are not routine in the settlement. • Acquired immunity through repeated exposure to ticks might explain these observations. Babesia rossi is an important, tick-borne intraerythrocytic protozoan parasite; however, its natural history and epidemiology is poorly understood. Babesia rossi is the most virulent Babesia sp. in domestic dogs and is generally considered to cause severe babesiosis, which is fatal if left untreated. However, subclinical infections and mild disease from B. rossi have been reported, although the clinical progression of these cases was not reported. Therefore, to better understand B. rossi under field conditions, we evaluated its clinical progression and seroprevalence in an owned, free-roaming dog population in Zenzele, South Africa, where the parasite is endemic and prevention is not routine. The entire dog population in Zenzele was monitored intensively at the individual level from March 2008 until April 2014, primarily for a longitudinal study on rabies control. Subsequent evaluation of B. rossi comprised analyses of clinical and laboratory data collected from the Zenzele dog population during the 6 year study period. A substantial proportion (31% (n = 34)) of 109 dogs (randomly selected from every available dog in February/March 2010 older than ~6–8 weeks (n = 246)) tested by Indirect Fluorescent Antibody Test had seroconverted strongly to B. rossi. All 34 dogs were generally consistently healthy adults, determined from regular clinical examinations between March 2008 and April 2014. Blood smear examinations at multiple time points between July 2009 and February 2011 were also undertaken for almost all of these (34) seropositive dogs and all those tested were consistently negative for Babesia spp. Subclinical infections and mild disease were also the main findings for a separate group of 18 dogs positive for Babesia spp. on blood smear examination and confirmed to be infected with B. rossi by Polymerase Chain Reaction – Reverse Line Blot. Almost all of these dogs were positive at only one time point from repeat blood smear examinations between July 2009 and February 2011. We suggest that these observations are consistent with immunity acquired from repeated, low-level exposure to the parasite, generating transient subclinical infections or mild disease. Should this be the case, the use of tick control, particularly in adult dogs in free-roaming populations in B. rossi endemic regions, should be carefully considered. [ABSTRACT FROM AUTHOR]