Molecular detection and characterization of emerging pathogens of Rickettsia- felis and felis-like organisms from peri-domestic fleas in Uganda

Background: Flea-borne spotted fever is an emerging zoonosis caused by Rikecttsia felis , a Gram-negative obligate intracellular bacterium. The agent is believed to be cosmopolitan, following the distribution pattern of its host and reservoir, Ctenocephalides felis . However, the epidemiology and public health risk it poses remains poorly understood in sub-Saharan Africa, including Uganda. Yersinia pestis , is primarily transmitted by rodent fleas, Xenopsylla cheopis , but other fleas, particularly C. felis , have vectoral capacity. They are neglected in Ugandan entomological surveillance and public health practices, particularly outside endemic foci of bubonic plague. Methods: We collected 14,641 fleas from domestic animals, rodents and homestead environment; compared their diversity and abundance. Pooled into 714 flea pools by species, collection time, host, and host species, 172 pools were selected based on seasons and analyzed for Yersinia pestis Pla genes, while 62 pools were tested for Rickettsia species gltA , ompA , and 17kDA genes by qPCR and Sanger sequencing. Results: Five flea species were identified from the collections: Ctenocephalides canis, C. felis, Echidnophaga gallinacea, Pulex irritans, and Xenopsylla cheopis . Ctenocephalides was the predominant genus, accounting for 84.8% of fleas collected, mostly found on dogs and goats. Except for P. irritans (which was found in Gulu district) the other four flea species were found across all districts, year-round, with higher numbers collected in dry seasons compared to rainy seasons (c 2 =47.64, df =20, p <0.001). Rattus rattus constituted 74% of rodents captured from human dwellings and was the only rodent species with fleas, where X. cheopis was the predominant species and E. gallinacea found on only three rodents. All 172 pools of fleas tested negative for Yersinia pestis . Of the 62 pools tested for Rickettsia spp., 29 (46.8%) were positive. Twenty-five PCR amplicons were successfully sequenced for 17kDa and two for ompA genes. Based on 17kDa , two were identified as R. felis from C. canis and 23 were R. asembonensis from multiple flea species, including C. canis collected goats and C. felis from cats. Conclusion: Our survey identified a high pooled detection rate (~50%) of Rickettsia spp. in fleas tested, suggesting a potential risk of human exposure and infection. Rickettsia felis and R. asembonensis were the predominant flea-borne Rickettsia spp. identified, with this study also representing the first report of Rickettsia spp. in E. gallinacea in Uganda.