The present study is the most extensive study ever in Chile, including the most relevant CVBP in dogs. We found a widespread occurrence of CVBP in rural, free-ranging dogs of central Chile, with three quarters of the dogs positive to at least one pathogen. Previous studies in other parts of the world showed that rural dogs are frequently exposed to or infected by different vector-borne pathogens [59,60]. The outdoor activity of these dogs exposes them to a range of vectors, which is, together with the absence of antiparasitic prophylactic management, the most likely reasons for our findings. In this sense, higher rates of exposure or infection were found in rural dogs when compared with their urban counterparts [61–63]. In the Metropolitan Region of Chile, the prevalence of R. sanguineus and Ctenocephalides canis was indeed higher in rural than in urban dogs .
Anaplasma platys was the only Anaplasmataceae confirmed in this study. This picture is similar to that reported in other geographical areas where R. sanguineus is the only tick species infecting dogs (e.g. [65,66]). The absence of E. canis, a bacterium that was reported in northern Chile , is not surprising considering that only the tropical lineage of R. sanguineus is able to transmit E. canis , and that in central Chile (where our study was conducted), only the temperate lineage has been previously reported . Accordingly, the seroprevalence of 69% using and A. phagocytophilum-based IFI commercial kit registered in dogs from Santiago  probably corresponded to A. platys, due to the serologic cross-reaction among species within the family Anaplasmaceae . The observed prevalence in our study can be considered high when compared with other studies in rural dogs. It is close to the prevalences reported by Brown et al.  in free-roaming dogs associated with remote Aboriginal communities in Australia, but higher than other studies carried out in Brazil , Ivory Coast , Kenya  and Uganda . The higher probability of A. platys infections in young dogs in our study was already been recorded in a previous study in Africa , most likely due to a primary exposure of juvenile individuals to the pathogen [71,72] and might be related to the lower levels CD8 T lymphocytes found in young dogs , which has a role in clearance of rickettsial infections . In agreement with our results, other studies found that dogs infested with R. sanguineus were more likely to be infected with or exposed to Anaplasma spp. than uninfested dogs [62,75,76]. Overall, our results suggest that the risk of infection with A. platy is more associated with the tick abundance than just the presence of the tick.
The overall prevalence of hemoplasmas in in our study was similar to the prevalence observed in rural dogs in southern Chile , and in rural or free-ranging dogs worldwide, such as Australia  and Brazil . Rural environments were suggested to be a risk factor for hemoplasma infections when compared with urban environments [21,79], and, accordingly, in free-ranging compared with domestic pet dogs . We found similar prevalence for both hemoplasma species, coinciding with the findings by Soto et al. , although these authors did not detect any co-infection. The observed higher infection percentage in older dogs may be explained by an increased probability of exposure throughout life and/or by the characteristic long-term bacteremia of hemoplasma infection [81,82]. Lack of hemoplasma clearance was reported in infection follow-up studies [83,84].
To the best of our knowledge, this survey represents the first molecular detection of T. cruzi in dogs in the Metropolitan Region of Chile, although the presence of parasitized dogs in this region was known [23,85]. The only previous study in the country was conducted in two regions in the North of Chile . It is known that dogs are competent hosts of importance in the cycle of T. cruzi in endemic areas [87,88]. Despite of this, there have been few molecular studies in rural dogs in the Americas [89–91]. Rural dogs have been suggested as a bridge between the domestic and sylvatic transmission cycles , and this can be the case in our study area, where all of the studied dogs live outdoor and some of them accompany muleteers in areas where triatomines abound . Further studies should aim to characterize the genetic diversity of T. cruzi in the region.
The presence of Leishmania spp. is herein reported for the first time in Chile, though it is endemic in some neighboring areas of Argentina [29,92,93]. Phlebotomine sandflies are present in Chile (i.e. Lutzomyia isopsi and Lutzomyia sp.) , but none of these species has known vectorial capacity. If confirmed, our findings would markedly extend the distribution of Leishmania spp., though the species identity has not been ascertained. Due to the importance of this parasite for veterinary medicine and public health, the proper characterization of the cases should be further investigated.
A higher prevalence of microfilariae of A. reconditum was detected at Knott´s test in previous studies conducted in Chile [27,28] and this could be due to the number of animals tested or to the lower occurrence of specific vectors (i.e., fleas and lice) in the sampled animals. Ctenocephalides felis, in particular, is considered a well stablished intermediated host [95–97]. The globally distributed C. felis is also a common parasite of dogs in the Metropolitan Region of Chile [13,64] although they were found at low prevalence on the dogs included in this study and lice were not found at all (data not shown).
Absence or a very low number of Rickettsia and Bartonella PCR-positive dogs reported here has been previously observed elsewhere [98,99], though a high prevalence of the pathogen has been detected in ectoparasites vectors [17,100]. In absence of acute infections, low blood bacterial levels could be observed for these pathogens, being detect only by cPCR [101,102].
Pirosplasmid and Hepatozoon spp. DNA was not detected during this survey. Babesia vogeli was recently found in Chile , is surprising we had not found it despite been present the competent vector R. sanguineus. Absent of Hepatozoon spp. and B. gibsoni could be due to the geographical isolation of Chile from the rest of the continent. Nevertheless, the arrival of imported cases of dogs infected with H. canis, and/or B. gibsoni must be considered a risk for the Chilean canine population, also considering the widespread presence of R. sanguineus in the country and the circulation of dogs among countries.
A third of the studied dogs were co-infected with two or more pathogens. Co-infection is considered frequent in CVBD-endemic areas, especially in dogs living environments with high vector density and without antiparasitic treatment . Interestingly, although A. platys was the most prevalent agent in our study, was not the pathogen most commonly associated with co-infections in dogs, in contrast with previous studies in areas where R. sanguineus is prevalent  in areas. In our case, hemoplasma species were common in cases of coinfection, and multiple infections has indeed been considered as risk factor for hemoplasma infection [80,103].
Higher WBC and segmented neutrophil level were found in co-infected animals. No consistent leukogram abnormalities have been associated with canine hemoplasmosis or anaplasmosis [21,81,104]. However, increased leukogram values have been associated with T. cruzi infections . On the other hand, higher GGT values were found in co-infected animals. Anyway, almost all the GGT values were into the range of the reference values . It is possible that our findings may be explained by absence of acute stages of infection. Chronically infected dogs usually present low bacteremia or parasitemia . Thus, dogs with chronic or “hidden” infections used to be apparently healthy with absent or minor hematological abnormalities [8,72] . For example, most of the cases of canine haemoplasmosis used to be chronic subclinical infections and infected dogs seemed unable to clear the infection . Therefore, as suggested before, co-infection complicates the diagnosis based on clinical examination and hematological and biochemistry abnormalities alone .