Molecular diversity of tick-associated Rickettsia species in multiple provinces of Zambia

Ticks harbor diverse Rickettsia spp., including several spotted fever group rickettsiae of veterinary and public health importance. However, information on tick-associated Rickettsia spp. in Zambia remains geographically limited. In this retrospective molecular survey, we examined 1,587 individual ticks collected from vegetation and domestic animals in eight provinces of Zambia during 2016-2018. Ticks were identified morphologically and screened for Rickettsia spp. by PCR targeting the citrate synthase gene (gltA). Representative gltA positive samples were further characterized by sequencing of ompA, ompB, htrA, sca4, and the 16S rRNA gene. In total, 204 ticks (12.9%) were tested positive for gltA and detected in all eight provinces examined. Sequencing of gltA revealed 26 sequence variants, of which 25 clustered within the spotted fever group and one belonged to transitional group rickettsiae. Multilocus phylogenetic analysis identified sequence variants related to recognized or putative tick-borne rickettsiae, including R. africae, R. conorii, R. massiliae, R. aeschlimannii, Candidatus R. jingxinensis, Candidatus R. rhabdomydis, and R. hoogstraalii. These findings indicate that ticks in Zambia harbor genetically diverse Rickettsia lineages and provide a geographically broad molecular baseline for future surveillance, isolation attempts, and genomic characterization of tick-borne rickettsiae in the region.