Molecular detection of zoonotic pathogens in fleas collected from dogs and cats in Portugal

BACKGROUND: Fleas are common ectoparasites of companion animals and key vectors of pathogens of veterinary and zoonotic relevance. Although flea infestations are highly prevalent in dogs and cats in Portugal, nationwide data on flea-borne pathogens are limited. This study aimed to estimate the prevalence and characterize the molecular diversity of Bartonella spp., Rickettsia spp., and Dipylidium caninum in fleas collected from dogs and cats in mainland Portugal, and to identify associated epidemiological factors. METHODS: Fleas collected from dogs and cats across mainland Portugal were screened using conventional polymerase chain reaction (PCR) assays targeting the 16S-23S ribosomal RNA (rRNA) intergenic spacer region of Bartonella spp., the ompB gene of Rickettsia spp., and the 28S rRNA gene region of D. caninum. Positive amplicons were sequenced and analyzed phylogenetically. Associations between pathogen detection and epidemiological variables, including host species, host lifestyle, flea species, and geographic region, were evaluated using multivariable logistic regression models. RESULTS: A total of 1261 fleas were analyzed, with 24.7% positive for at least one pathogen. Bartonella spp. DNA was detected in 8.3% of fleas and was significantly more frequent in fleas from cats, particularly stray/sheltered animals, with regional differences also observed. Identified species included B. clarridgeiae, B. henselae, and B. rochalimae. Rickettsia spp. DNA was detected in 16.2% of fleas, predominantly in Ctenocephalides felis, with flea species, host species, and geographic region identified as independent predictors of positivity. Most sequences corresponded to R. felis, while R. asembonensis and Candidatus R. senegalensis, the latter reported for the first time in Europe, were also identified. Dipylidium caninum DNA was detected in 1.8% of fleas, all C. felis, with both canine- and feline-associated genotypes circulating in fleas from dogs and cats. Co-detection of pathogen DNA occurred in 2.1% of fleas, mostly Bartonella spp./Rickettsia spp. CONCLUSIONS: Fleas infesting companion animals in Portugal frequently harbor pathogens of veterinary and zoonotic relevance, with C. felis playing a central role in pathogen circulation at the human-animal interface. These findings reinforce the need for sustained surveillance and effective ectoparasite control within a One Health framework to reduce pathogen circulation and mitigate risks to both animal and public health.