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Tick-borne rickettsioses are considered among the oldest known vector-borne zoonotic diseases. Among the rickettsiae, Rickettsia africae is the most reported and important in Africa, as it is the aetiological agent of African tick bite fever (ATBF). Studies describing the prevalence of R. africae in southern Africa are fragmented, as they are limited to small geographical areas and focused on Amblyomma hebraeum and Amblyomma variegatum as vectors. Amblyomma spp. ticks were collected in Angola, Mozambique, South Africa, Zambia and Zimbabwe during the sampling period from March 2020 to September 2022. Rickettsia africae was detected using the ompA gene, while characterisation was conducted using omp, ompA, ompB and gltA genes. In total, 7734 Amblyomma spp. ticks were collected and were morphologically and molecularly identified as Amblyomma eburneum, A. hebraeum, Amblyomma pomposum and A. variegatum. Low levels of variability were observed in the phylogenetic analysis of the R. africae concatenated genes. The prevalence of R. africae ranged from 11.7% in South Africa to 35.7% in Zambia. This is one of the largest studies on R. africae prevalence in southern Africa and highlights the need for the inclusion of ATBF as a differential diagnosis when inhabitants and travellers present with flu-like symptoms in the documented countries. [ABSTRACT FROM AUTHOR]

• Afrotropical A tylotus horse flies are often misidentified morphologically. • COI struggles to distinguish between aftrotropical A tylotus species. • Horse flies transmit disease and must be correctly identified. The Afrotropical fly genus, Atylotus has previously shown little differentiation into species groups using the barcode gene COI. This study analysed all available Atylotus COI sequences from GenBank and BOLD to determine if COI is suitable for delimiting species of this genus. Morphological assessments of the different Afrotropical species were done to determine if these species have been accurately identified in recent publications. The results show that COI does not separate the species of this genus into species clades and these species are often misidentified in the literature. This is of concern as species of this genus are known vectors of pathogens and misidentifications have serious implications for management practices. Additional genes need to be used in future molecular studies to differentiate species. [ABSTRACT FROM AUTHOR]