Improved Detection and Sequencing of <i>Rickettsia</i> spp. DNA in South African Wildlife

BACKGROUND: Rickettsia spp. are obligate intracellular bacteria of the spotted fever group (SFG), typhus group (TG) and transitional group, with diverse vectors and significant zoonotic potential. While Rickettsia africae is well documented in South African ticks, the role of wildlife hosts in pathogen maintenance remains unclear. METHODS: We re-screened 73 spleen samples opportunistically collected from 11 wild mammal species that previously tested negative for Rickettsia by reverse line blot hybridization (RLB). The samples were collected from Kruger National Park, Timbavati Private Nature Reserve, Mountain Zebra National Park and Lapalala Wilderness Reserve. In addition, liver, lymph node and blood samples from two impala were included to preliminarily assess multi-organ dissemination. To increase detection sensitivity, we employed a nested PCR targeting the conserved gltA gene, in contrast to the 23S-5S intergenic spacer (IGS) targeted by RLB. RESULTS: The gltA region was successfully amplified by PCR from 32/73 animals (44%; confidence intervals [CI]: 32%-56%). Sequencing of 27 amplicons revealed SFG Rickettsia (n = 22), TG Rickettsia (n = 3) closely related to R. prowazekii and transitional group (n = 1) Rickettsia felis-like sequences. One zebra (Equus quagga) sequence was phylogenetically unresolved, and one sample showed a mixed SFG-TG infection. Rickettsial DNA detections included new host records in giraffe (Giraffa camelopardalis), hippopotamus (Hippopotamus amphibius), impala (Aepyceros melampus), warthog (Phacocoerus africanus), white rhinoceros (Ceratotherium simum) and zebra. Detection across multiple tissues in impala supports systemic dissemination, consistent with experimental evidence of rickettsial persistence in vertebrate tissues. CONCLUSIONS: These findings broaden the known host range and illustrate the complex dynamics of pathogen persistence, host diversity and co-infection, although interpretation is constrained by the limited and uneven sample sizes, which preclude robust population-level inference. Integrating high-sensitivity molecular diagnostics with ecological and vector surveillance within a One Health framework will be critical to clarify wildlife reservoir status and mitigate zoonotic risk at the wildlife-livestock-human interface.