Molecular detection of Bartonella spp. in humans, domestic animals and ectoparasites sampled in Lisbon, Portugal

Abstract

Bartonella spp. es una bacteria gramnegativa emergente de distribución mundial. Los gatos son los principales reservorios de Bartonella henselae y los perros son huéspedes oportunistas. Aunque las garrapatas también pueden desempeñar un papel en la transmisión, su competencia como vectores de Bartonella spp. no se conoce del todo. Teniendo en cuenta que sólo unos pocos estudios se han centrado en la detección de Bartonella en animales, humanos y ectoparásitos en Portugal, este estudio pretende abordar la presencia molecular de Bartonella sp. en 123 gatos callejeros, 25 perros callejeros, 30 humanos de Lisboa y 236 garrapatas buscadoras dentro del país. Utilizando una qPCR dirigida al gen nuoG, fue posible detectar ADN de Bartonella sp. en el 20,32% de las muestras de gatos (25/123). De estas muestras positivas, 13 secuencias se caracterizaron como B. henselae, 11 como B. clarridgeiae y 1 presentaba coinfección con ambas especies. La cuantificación absoluta de ADN del género Bartonella (nuoG) en los gatos muestreados osciló entre 2,78 × 10 y 1,03 × 105 copias/μL. Los perros, humanos y garrapatas muestreados fueron negativos. Estos resultados mostraron que B. henselae y B. clarridgeiae circulan en gatos callejeros de Lisboa.

Bartonella spp. comprises fastidious gram-negative bacteria with worldwide distribution. They are considered neglected zoonotic pathogens, whose transmission to humans occurs through contact with infected mammals and blood-sucking arthropod. Cats are the main reservoir host for at least three Bartonella spp.: B. henselae and B. clarridgeiae, both causative agents for cat scratch disease, and B. koehlerae agent for endocarditis in humans, in adittion, dogs represent an important reservoir, especially for B. vinsonii subsp. berkhoffii. Although fleas play a major role in transmission of feline Bartonella, other potential arthropod vectors have been identified to harbor Bartonella DNA. In this way, ticks represent an important group for Bartonella molecular identification. Considering that there are few studies addressing the occurrence of Bartonella in Portugal, the present study is focused on the molecular identification and characterization of Bartonella spp. circulating in stray cats and dogs, humans and in questing ticks collected in the referred country. Tick collections were performed using the drag-flag method from 2012-2018 in 19 geographical points organized in 8 districts of mainland Portugal. Four hundred microliters of EDTA whole blood from non-domiciled cats and dogs participating in the sterilization program promoted by Casa dos Animais de Lisboa (CAL) was donated for the purpose of this study and 1000 μl human blood was taken after prior consent from individuals that have been in contact with cats. DNA from cats, dogs and humans was extracted and DNA integrity was confirmed by a conventional PCR targeting the endogenous GAPDH and 18SRNA genes, for mammals and ticks, respectively. Molecular detection and quantification were carried out by qPCR targeting a fragment of nuoG. Positive samples were characterized by a conventional PCR for the gltA and ribC Bartonella spp. genes. From 123 cat blood samples, 25 (20.32%) were positive (quantification) for the referred Bartonella gene; from those positive samples, 13 were characterized as B. henselae, 11 as B. clarridgeiae and 1 presented a co-infection of both species. Furthermore, the absolute quantification of nuoG Bartonella DNA in sampled cats ranged from 2.78 × 10 to 1.03 × 10⁵ copies/μL. On the contrary, from 25 dog blood samples, 30 human blood samples, and 236 adult tick samples (154 Ixodes spp. and 82 Rhipicephalus sanguineus sensu lato), all were negative for the nuoG gene. These results suggest the species B. henselae and B. clarridgeaiae are circulating in stray cats but, in contrast, no dogs, humans or ticks were positive for the pathogen. Despite our results were not supportive regarding dogs and humans as Bartonella spp. reservoirs and hosts, respectively, and ticks as competent vectors, more studies using new approaches need to be performed in order to clarify their role in Bartonella spp. transmission.