| Summary: | Bovine anaplasmosis, caused by <i>Anaplasma marginale</i>, is one of the most important tick-borne diseases of cattle. <i>Anaplasma marginale</i> 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 (<i>n</i> = 10) in two habitats in the study area over 12 months. A duplex real-time PCR assay targeting the <i>msp1β</i> gene of <i>A. marginale</i> and the <i>groEL</i> gene of <i>A. centrale</i> confirmed the presence of <i>A. marginale</i> 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 <i>A. marginale msp1α</i> 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 <i>A. marginale</i> 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 <i>A. marginale</i> 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.
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