Molecular detection and characterization of flea-associated bacteria: genetic diversity and co-occurrence patterns involving Bartonella, Rickettsia, and Wolbachia in Ctenocephalides felis felis from an Atlantic Forest region of Brazil.

Ctenocephalides felis felis is a widespread ectoparasite of dogs and cats and has been associated with transmission of several microorganisms of veterinary and public health relevance. This study investigated the molecular occurrence, genetic diversity, and co-occurrence patterns of Bartonella spp., Rickettsia spp., and Wolbachia spp. in fleas collected from dogs and cats in an Atlantic Forest area encompassing the Ilhéus and Itabuna microregions, Bahia State, northeastern Brazil. A total of 1,489 fleas were analyzed, including 1,301 collected from 81 dogs and 188 from 39 cats. PCR targeting the endogenous cox1 gene successfully amplified DNA from 95.77% of fleas collected from dogs and 97.34% of those collected from cats. Quantitative PCR (qPCR) targeting the 16S-23S rRNA internal transcribed spacer (ITS) revealed a higher molecular detection rate of Bartonella spp. in fleas from cats (34.43%) than in those from dogs (8.98%). Conversely, Rickettsia spp. was detected more frequently in fleas collected from dogs (24.8%) than from cats (3.28%). Wolbachia spp. was more frequently detected in female fleas from both dogs (52.8%) and cats (50.3%) than in male fleas (p < 0.05). Triple co-occurrence of the three microorganisms was detected in 0.08% (11/1246) of flea DNA samples positive the cox-1 endogenous gene and subjected to downstream molecular analyses for Bartonella spp., Rickettsia spp., and Wolbachia spp.. Phylogenetic analyses based on multiple molecular markers for Bartonella spp. (gltA, ftsZ, nuoG, and rpoB), Rickettsia spp. (gltA, htrA, ompA, and ompB), and Wolbachia spp. (gatB and wsp) confirmed the detection of Bartonella henselae, Bartonella clarridgeiae, Rickettsia felis, and Wolbachia spp. belonging to supergroups B and F. In fleas collected from dogs, the detection of Wolbachia spp. was associated with a higher predicted probability of Rickettsia spp. detection, whereas in fleas collected from cats it was associated with a lower predicted probability of Bartonella spp. detection. Overall, the findings revealed distinct host-associated molecular detection patterns of Bartonella spp., Rickettsia spp., and Wolbachia spp. in C. felis felis, demonstrating the circulation and genetic diversity of these bacterial agents in flea populations. The detection of zoonotic bacterial agents in fleas highlights their relevance for integrated veterinary and public health surveillance within a One Health framework.