| Summary: | <i>Bartonella</i> spp. comprises emergent and re-emergent fastidious Gram-negative bacteria with worldwide distribution. Cats are the main reservoir hosts for <i>Bartonella henselae</i> and dogs represent opportunistic hosts for the bacteria. Even though ticks may also play a role in transmission, their competence as vectors for <i>Bartonella</i> spp. has not been totally understood. Considering only a few studies had a focus on screening <i>Bartonella</i> in animals, humans and ectoparasites in Portugal, this study aimed to address the molecular occurrence of <i>Bartonella</i> sp. in 123 stray cats, 25 stray dogs, 30 humans from Lisbon and 236 questing ticks within the country. Using a qPCR targeting the <i>nuoG</i> gene, it was possible to detect <i>Bartonella</i> sp. DNA on 20.32% of cat samples (25/123). From these positive samples, 13 sequences were characterized as <i>B. henselae</i>, 11 as <i>B. clarridgeiae</i> and 1 presented co-infection with both species. The absolute quantification of <i>nuoG</i><i>Bartonella</i> DNA in sampled cats ranged from 2.78 × 10 to 1.03 × 10<sup>5</sup> copies/µL. The sampled dogs, humans and ticks were negative. These results showed that <i>B. henselae</i> and <i>B. clarridgeiae</i> are circulating in stray cats from Lisbon. Additional and more extended studies should be conducted to determine the impact of such infections on humans, particularly those in constant and direct contact with cats.
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