The present study represents the most comprehensive investigation on FVBD performed in Portugal, in terms of the number of tested cats and extension of the covered geographical area, and reveals a considerable prevalence of infection in domestic cats from the north and centre regions of Portugal. Anaplasma/Ehrlichia spp., B. canis, B. vogeli, H. felis, L. infantum and Rickettsia spp. were detected among the assayed feline population.
Several ehrlichial and rickettsial infections are shared by man and companion animals . In the present study, Anaplasma/Ehrlichia spp. and Rickettsia spp. DNA were detected in 0.6% and 1.3% of the cats, respectively. In cats from southern Portugal, seroprevalences (by immunofluorescence antibody tests) were 13.5% for Anaplasma phagocytophilum and 18.9% for Rickettsia conorii, and 26.3% for R. conorii/Rickettsia felis. Furthermore, Breu et al.  also reported feline infection by Ehrlichia canis in Portugal. A national serological study on canine vector-borne diseases in Portugal detected a significantly higher seroprevalence of antibodies to Anaplasma spp. and E. canis in dogs from southern Portugal, when compared to dogs from the northern and central regions .
The present study represents the first report on the prevalence of Babesia spp. in cats from Portugal. A higher prevalence of Babesia spp. was found in Portuguese cats (9.4%), in comparison with that detected in cats from Barcelona, Spain (0/100) by Tabar et al.. Interestingly, it can also be presumed that the most prevalent piroplasm in the Portuguese feline population is B. vogeli, instead of B. canis, which was the piroplasm most frequently detected in dogs with babesiosis from the north of Portugal . So far, feline infection with B. vogeli has only been described in cats from Trinidad, Trinidad and Tobago , and Bangkok, Thailand . To the best of our knowledge, this is the first time that infection with B. vogeli has been detected in cats from Europe.
Feline co-infections with other erythrocytic pathogens such as Mycoplasma spp., Cytauxzoon felis or other species of Babesia may be possible [24, 25]. In the present study, only one cat was found co-infected with B. canis and B. vogeli out of the 29 cats infected with Babesia spp. Infection with B. canis and/or the Babesia microti-like piroplasm (syn. Theileria annae) was previously described in three cats from Portugal , but no information is available on the geographical origin of those cats. The B. microti-like piroplasm has also recently been found in dogs from northern Portugal affected by babesiosis . In the present study, other species of the genera Babesia and Theileria were not assessed. Nevertheless, as the entire results positive to the genus Babesia had an assigned species (i.e. B. vogeli, B. canis or both), although not impossible, a co-infection with the B. microti-like piroplasm seems unlikely.
Age has been described as a predisposing factor for feline infection with Babesia spp., with younger cats (less than 3 years old) more predisposed to infection in endemic areas [24, 28], and older cats more susceptible to the disease following relocation to an endemic area or in conjunction with concurrent disease, immunosuppression or severe trauma . In the present study, juvenile cats (≤ 1.5 year) had a significantly higher prevalence of B. vogeli infection in comparison with geriatric cats (≥ 7 years), probably because of the less mature immune status of young cats.
Infection with Hepatozoon spp. is frequently reported in dogs  but not in cats. Furthermore, the Hepatozoon species that infect cats have not been definitely characterized [30, 31]. Some authors have suggested that H. canis is the agent responsible for feline infection, but a new, yet unnamed, species of Hepatozoon genetically distinct from H. canis was recently detected in cats from southern and northeastern Spain [30, 32]. Infection with H. felis was firstly described by Tabar et al. in cats from Barcelona .
This is also the first report of feline infection with H. felis in Portugal. Molecularly confirmed infection with H. canis in cats from southern Portugal  and in a dog from northern Portugal  had already been described. The detected 15.6% prevalence of infection with H. felis in the present study is similar to that of Hepatozoon spp. (16%) described by Ortuño et al. in stray cats from Barcelona (p = 0.960), but higher than the 0.6% of Hepatozoon spp. found in Spanish domestic cats from a non-identified geographical background  and the 4.0% of H. felis in domestic cats from Barcelona  (p < 0.001 and p = 0.002, respectively). Moreover, the present study sustains the fact that Hepatozoon infection is widespread in the feline population of the Iberian Peninsula. Baneth et al., in a study from Israel, detected that most infected cats were young domestic short-haired males and that there was an over-representation of cats with retroviral disease. In the present study, no statistically significant association was found between infection with H. felis and independent variables including clinical status and FIV/FeLV infection (Table 1).
Leishmaniasis is an endemic zoonosis prevalent in the Mediterranean basin [34, 35]. The increase in the number of infections and disease cases reported in recent years, together with the results described in different prevalence studies, suggest that cats can act as a secondary reservoir host instead of an accidental one in areas where Leishmania spp. are endemic [36–38]. Several surveys of Leishmania spp. infection in cats have been performed in different countries by different techniques, with prevalences ranging between 0% and 68% [9, 16, 18, 39–43]. In the present study, detected prevalence (0.3%) might have been different if the qPCR was carried out with another tissue sample, such as bone marrow, spleen or liver. Results are lower than the 2.8% seroprevalence (by the direct agglutination test and an enzyme-linked immunosorbent assay) found in cats from northern Portugal  and also lower than the 20.3% obtained in blood samples from cats of Greater Lisbon (southern-central Portugal) by PCR  (p = 0.01 and p < 0.001, respectively). For the appropriate agents under assessment in the present study, comparative differences in the prevalence values can be related to different detection techniques (serology versus molecular analysis) as well as to a different geographical origin of cats (north and centre versus south). The latter may determine differences in climatic conditions, arthropod vector survival and agent transmission rate.
Associations between housing conditions and prevalence of infection were not found among cats for any one of the agents. Conversely, in a comparable study on canine vector-borne diseases, also in Portugal, a significantly higher sero-positivity to at least one agent (i.e. Dirofilaria immitis, E. canis, Borrelia burgdorferi sensu lato, Anaplasma spp. and/or L. infantum) has been found in clinically suspect dogs with an outdoor or mixed lifestyle .