Background/Question/Methods Worldwide, including in Africa, rickettsioses are recognized as emerging or re-emerging infections. In southern Africa, several Rickettsia species are associated with disease in humans, including Ri. africae (causative agent of African tick bite fever) and Ri. conorii (causative agent of Mediterranean Spotted Fever). The aim of this study was to sample and screen a diversity of questing tick species for the presence of rickettsial bacteria in Eswatini and explore patterns in the distributions of ticks and rickettsial species. Questing ticks were collected from the vegetation at 26 savanna sites across conservation area, protected ranches, government ranches, and communal rangelands. Adult, nymph and larval ticks were tested for rickettsial DNA using a combination of real-time PCR assays, conventional PCR assays, and direct sequencing of multiple loci. Results/Conclusions We detected rickettsial DNA in three out of seven tick species tested (Amblyomma hebraeum, Haemaphysalis elliptica, and Rhipicephalus simus). Using concatenated sequences from three gene loci, rickettsial samples grouped into four distinct clades that included Ri. africae, Ri. conorii, Rickettsia massiliae, and Rickettisa barbariae. Notably, we found an average prevalence of 55% for Ri. africae in A. hebreaum, 4.2% for Ri. conorii in H. elliptica, and 4.4% for Ri. massiliaein Rh. simus. Amblyomma hebreaum were collected at 19 sites, of which we detected Ri. africae in 13, including sites in all land-use categories. Haemaphysalis elliptica were collected at 18 sites, of which we detected Ri. conorii in one communal rangeland and one government ranch. Rhipicephalus simus were collected at 18 sites, of which we detected Ri. massiliae at six sites within a single conservation area and in one government ranch. These results suggest the widespread presence of A. hebreaum ticks capable of spreading Ri. africae in Eswatini savannas while the presence of H. elliptica with Ri. conorii and R. simus with Ri. massiliae may be more concentrated to communal areas and conservation areas, respectively. These trends could be a result of stronger reservoir host or environmental constraints on the distribution of Ri. conorii and Ri. massiliae within the distribution of the vector as compared to Ri. africae. Wider surveillance of tick-borne diseases should be continued, as rickettsioses pose an ongoing threat to humans in the area.
