Molecular characterization of Theileria species and Theileria parva genetic diversity associated with bovine theileriosis outbreaks in Zimbabwe

Several Theileria species infect cattle and wildlife in eastern and southern Africa. Among them, Theileria (T.) parva is the most economically important, causing severe disease and substantial livestock losses in Zimbabwe. Recent shifts in spatio-temporal patterns of theileriosis outbreaks highlight the need for updated information on circulating Theileria species and strains to better understand the infection process and to guide control strategies. Between November 2020 and May 2021, 147 whole blood, 145 dried blood spots, 87 fecal, and 22 organ samples were collected from 158 cattle in Bindura and Makoni districts. Samples were analyzed using pan-Theileria 18S rRNA and T. parva-specific PCRs, followed by Sanger sequencing. Overall, Theileria DNA was detected in 83.3% of the cases. Species distribution was as follows: T. parva 63.1%, T. velifera 23.9%, T. taurotragi (2.3%), T. mutans (2.3%), with mixed infections (3.1%) detected mainly in organ samples. Partial sequences of the sporozoite surface antigen p67 and schizont antigen Tp2 were amplified from 44 and 23 T. parva-positive samples, respectively. Phylogenetic analyses showed clustering with the Kenyan Muguga isolate, a component of the widely used T. parva vaccine cocktail, rather than with Zimbabwe's Boleni strain, suggesting potential divergence from the local vaccine strain. In addition, dried blood spots (n = 145) and feces (n = 87) were screened by 18S rRNA PCR for Theileria. PCR detection from DBS showed 59.3% relative sensitivity, 64.0% relative specificity, and 60.1% overall relative accuracy compared to WB, while fecal PCR showed 12.7% relative sensitivity, 85.7% relative specificity, and 18.6% relative overall accuracy. Despite reduced sensitivity, non-invasive fecal sampling, an innovative method, remains useful for population-level surveillance in wildlife or areas where blood collection is impractical. Although not the primary focus of this study, WB were also screened for other tick-borne pathogens (E. ruminantium, A. marginale, B. bovis, and B. bigemina) to broaden the Theileria results. This study provides updated insight into Theileria species diversity and T. parva strain composition in outbreak-prone districts of Zimbabwe. The findings underscore the importance of integrating molecular surveillance into routine monitoring programs and considering circulating strain diversity when evaluating and adopting effective vaccination strategies.