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Molecular detection of Bartonella spp. in humans, domestic animal and ectoparasites samplied in Lisbon, Portugal
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Bartonella spp. compreende pequenas bactérias fastidiosas com distribuição mundial. São consideradas agentes patogénicos zoonóticos negligenciados, cuja transmissão ao ser humano ocorre através do contato com mamíferos infetados e artrópodes que se alimentam de sangue. Os gatos são o principal reservatório e hospedeiro de pelo menos três Bartonella spp.: B. henselae e B. clarridgeiae, ambos agentes causadores da doença da arranhadura do gato, e B. koehlerae, agente de endocardite em humanos; adicionalmente, os cães representam um importante reservatório, especialmente para B. vinsonii subsp. berkhoffii. Embora as pulgas desempenhem um papel importante na transmissão das Bartonella spp. felina, outros potenciais vetores artrópodes foram identificados para a presença de DNA de Bartonella sp. Desta forma, as carraças representam um grupo importante para a identificação molecular de Bartonella sp.. Considerando que existem poucos estudos que abordam a ocorrência de Bartonella em Portugal, o presente trabalho teve como foco a identificação e caracterização molecular de Bartonella spp. em gatos e cães vadios, humanos e em carraças recolhidas no país referido. As recolhas de carraças foram realizadas usando o método da bandeira de arrasto de 2012-2018 em 19 pontos geográficos organizados em 8 distritos do continente. Para este estudo, foram doados 400 microlitros de sangue total em EDTA de cães e gatos não domiciliados que participavam do programa de esterilização promovido pela Casa dos Animais de Lisboa (CAL) e 1000 μl de sangue de indivíduos que estiveram em contato com gatos, recolhido após consentimento prévio. O DNA de gatos, cães e humanos foi extraído e a integridade do DNA confirmada por PCR convencional direcionado para os genes endógenos GAPDH e 18SRNA para mamíferos e carraças, respetivamente. A deteção e quantificação molecular foram realizadas por qPCR visando um fragmento do gene nuoG. As amostras positivas foram caracterizadas por PCR convencional para os genes de Bartonella spp. gltA e ribC. Das 123 amostras de sangue de gato, 25 (20.32%) foram positivas (quantificação) para o gene referido; destas amostras positivas, 13 foram caracterizadas como Bartonella henselae, 11 como B. clarridgeiae e 1 apresentaram co-infecção de ambas as espécies. A quantificação absoluta do DNA nuoG da Bartonella sp. em gatos amostrados variou de 2,78 × 10 a 1,03 × 105 cópias / μL. Pelo contrário, das 25 amostras de sangue de cães, 30 amostras de sangue humano e 236 amostras de carraças adultas (154 Ixodes spp. e 82 Rhipicephalus sanguineus sensu lato), todas foram negativas para o gene nuoG.
Os resultados obtidos sugerem que as espécies B. henselae e B. clarridgeaiae circulam em gatos vadios mas nenhum cão, humano ou carraça apresentaram resultados positivos para o agente patogénico. Apesar de nossos resultados não sugerirem os cães e humanos como reservatórios e hospedeiros de Bartonella spp., respectivamente, e as carraças como vetores competentes, mais estudos usando novas abordagens precisam de ser realizados para clarificar o seu papel na transmissão de Bartonella spp.
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.
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.
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Ciências biomédicas Microbiologia médica Biologia molecular Bartonella Carraças Gatos Cães Humanos Portugal