Molecular detection and characterisation of Theileria parva and other Theileria species in cattle-associated ticks from southern Mozambique.

Tick-borne protozoa of the genus Theileria, particularly T. parva, represent a major challenge to cattle production in sub-Saharan Africa. Recent outbreaks clinically ascribable to East Coast fever (ECF) in Maputo Province, southern Mozambique, highlight the need to investigate the occurence of Theileria parva and other Theileria species in local tick populations. This study aimed to investigate the presence and molecular diversity of Theileria spp. in ixodid ticks collected from cattle in the buffer zone of Maputo National Park. Between May 2023 and February 2024, 881 adult ticks were collected from six farms located in three villages. Four species were identified: Rhipicephalus microplus, Rhipicephalus evertsi, Amblyomma hebraeum and Rhipicephalus appendiculatus. A total of 326 ticks were individually subjected to molecular analysis. Screening was performed using a real-time PCR targeting the LSU rRNA gene of Babesia and Theileria genera. Positive samples with a Ct ≤34 were further tested by a specific nested PCR targeting the T. parva p104 gene and by a nested PCR targeting the 18S rRNA gene, followed by Sanger sequencing and phylogenetic analysis. Overall, 62.6% (204/326) of ticks were positive at real-time PCR and further analysed. The p104 assay identified T. parva in 11 ticks and confirmed by sequencing. Most T. parva isolates clustered closely in phylogenetic analysis, indicating low genetic diversity among the analysed isolates, while one divergent isolate was detected in A. hebraeum. The 18S rRNA nested PCR yielded a positive result confirmed by sequencing in 1 specimen that was identified as T. velifera. These findings confirm the circulation of T. parva in tick populations from the study area, suggesting the need for continued clinical, serological and molecular surveillance in southern Mozambique.