Maintenance of Trypanosoma cruzi, T. evansi and Leishmania spp. by domestic dogs and wild mammals in a rural settlement in Brazil-Bolivian border

Domestic dogs are considered reservoirs hosts for several vector-borne parasites. This study aimed to evaluate the role of domestic dogs as hosts for Trypanosoma cruzi, Trypanosoma evansi and Leishmania spp. in single and co-infections in the Urucum settlement, near the Brazil-Bolivian border. Additionally, we evaluated the involvement of wild mammals’ in the maintenance of these parasites in the study area. Blood samples of dogs (n = 62) and six species of wild mammals (n = 36) were collected in July and August of 2015. The infections were assessed using parasitological, serological and molecular tests. Clinical examination of dogs was performed and their feeding habits were noted. Overall, 87% (54/62) of sampled dogs were positive for at least one trypanosomatid species, in single (n = 9) and co-infections (n = 45). We found that 76% of dogs were positive for T. cruzi, four of them displayed high parasitemias demonstrated by hemoculture, including one strain types TcI, two TcIII and one TcIII/TcV. Around 73% (45/62) of dogs were positive to T. evansi, three with high parasitemias as seen by positive microhematocrit centrifuge technique. Of dogs sampled, 50% (31/62) were positive for Leishmania spp. by PCR or serology. We found a positive influence of (i) T. evansi on mucous pallor, (ii) co-infection by T. cruzi and Leishmania with onychogryphosis, and (iii) all parasites to skin lesions of sampled dogs. Finally, feeding on wild mammals had a positive influence in the Leishmania spp. infection in dogs. We found that 28% (5/18) coati Nasua nasua was co-infected for all three trypanosamatids, demonstrating that it might play a key role in maintenance of these parasites. Our results showed the importance of Urucum region as a hotspot for T. cruzi, T. evansi and Leishmania spp. and demonstrated that dogs can be considered as incidental hosts.

2008; Rabinowitz et al., 2009;Wang et al., 2012). Predation and agonistic encounters between dogs and wild mammals have been reported as a source of spill-over and spill back for parasitic infection in rural areas (Daszak et al., 2000;Thompson et al., 2009). In rural settlements, dogs are used to guard the properties or for companionship in the field. Dogs are also frequently used for hunting, where exposure to pathogens could result from contact or consumption of wild animals.
Dogs are important domestic reservoirs for Trypanosoma cruzi and Leishmania spp., constituting a matter of public health concern worldwide (Gürtler et al., 1993;Maia-Elkhoury et al., 2008;Esch and Petersen, 2013;Gürtler and Cardinal, 2015). The Brazil state of Mato Grosso do Sul is enzootic for T. cruzi, Leishmania spp. and Trypanosoma evansi which are the etiological agents of Chagas disease, leishmaniasis and animal trypanosomiasis, respectively (Mello et al., 1988;Silva et al., 1995;Herrera et al., 2011;Santos et al., 2015). Furthermore, dogs have been suggested to provide a link between domestic and wild T. cruzi transmission cycles (Roque and Jansen, 2008), and have useful as sentinels for T. cruzi in enzootic areas (Roque and Jansen, 2008;Ramírez et al., 2013;Xavier et al., 2014). Nomenclature of T. cruzi is complex, and currently seven discrete typing units (DTUs) are recognized TcI-TcVI and TcBat (Zingales et al., 2012;Barros et al., 2017). Dogs are considered the main domestic reservoir for leishmaniasis, due to their close contact with humans and presence of high numbers of parasites in the skin (Reis et al., 2010;Nunes et al., 2016;Sevá et al., 2016).
Trypanosomiasis caused by T. evansi has been reported in many species of wild mammals and domestic mammals at the Pantanal region (Nunes et al., 1993;Herrera et al., 2004). Like horses, dogs are particularly susceptible to infection by T. evansi (Hoare, 1972;Franke et al., 1994;Herrera et al., 2004). Trypanosomiasis is characterized mainly by acute, progressive and severe anemia in dogs and horses (Silva et al., 1995;Aquino et al., 2002).
These three species of trypanosomatids are multi-host parasites, capable of parasitizing a large number of domestic and wild mammalian species (Herrera et al., 2011;Jansen et al., 2015). Although T. cruzi, T. evansi and Leishmania spp. have been reported in the Mato Grosso do Sul state, their prevalence and maintenance hosts in the rural settlements of Corumbá city is unknown. This study aimed to evaluate the role of domestic dogs and wild mammals as hosts for T. cruzi, T. evansi and Leishmania spp., in the maintenance of these parasites in the Urucum rural settlement, near the Brazil-Bolivian border. Additionally we aimed to evaluate the influence of single or co-infections on clinical aspects of dogs.

Study area
The study was carried out in the Urucum rural settlement, located near to the Brazil-Bolivian border, approximately 18 km from the urban area of Corumbá, Mato Grosso do Sul state (Fig. 1). The total area is 1979 hectares, which is divided in 84 small properties. Approximately 87 families live in Urucum rural settlement. The main economic activities are cassava and vegetables growing, dairy farming and breeding of chickens, pigs and cattle. A census survey carried out by the Zoonoses Control Center (ZCC) estimated domestic dogs' population in approximately 300 animals (ZCC, Unpublished results).

Blood sample of dogs
Sampling was conducted in July and August of 2015. Approximately 4 ml of venous blood was collected from each animal using sterile techniques: 2 ml were stored in a tube with Ethylenediamine Tetra acetic Acid (EDTA) for parasitological and molecular testing, and the other 2 ml were stored in tubes without EDTA for serological testing.
2.3. Capture and blood sampling of wild mammals From July to August 2015 thirty tomahawk traps were deployed (90 × 45 x 50 Equipos Fauna ® ) in the region. Traps were placed on both the ground and in trees of forested areas. Traps set on the ground were baited with bacon, while traps set on the trees were baited with bananas. Traps were checked daily during ten days at two surveys, totaling a sample effort of 300 Trap-night. Animals were immobilization using a combination of Tiletamine and Zolazepam (Zoletil 50 ® ), with doses varying according to the species and weight of captured animal. Using sterile techniques approximately 4 ml of venous blood was collected from each animal, which were divided into EDTA and serum tubes.

Diagnostic of trypanosomatids
Infections by T. cruzi, T. evansi and Leishmania spp. were assessed in domestic dogs and wild mammals using parasitological, molecular and serological tests. Parasitological testing for T. cruzi and Leishmania spp. was carried out using hemoculture (HC) and inoculating 300 μl of blood in Novy McNeal Nicole (NNN) medium with Liver Infusion Tryptose (LIT) overlay and NNN with Schneider biphasic medium, in duplicate. Hemoculture tubes were incubated at 27°C during 30 days and monitored for parasite development once a week. When epimastigotes were present, they were subjected to DNA extraction using the previously described phenol-chloroform method (Vallejo et al., 1999), and deposited at the "Coleção de Trypanosoma de Mamíferos Silvestres, Domésticos e Vetores, Fiocruz -COLTRYP" (Oswaldo Cruz Foundation, Rio de Janeiro -RJ/Brazil). To determine the DTU of T. cruzi samples, all NNN + Lit positives were subjected to multiplex PCR amplification of the non-transcribed spacer of the mini-exon gene (SL-IR) according to Fernandes et al. (2001) and Westenberger et al. (2005). Each reaction included negative and positive control samples from T. cruzi strains representing the six DTUs. For T. evansi we used Microhematocrit Centrifuge Technique (MHCT) to determine if an animal was parasitemic (Woo, 1970).
For molecular testing genomic DNA was extracted from 200 μl of total blood using the QIAamp Blood DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Detection of Trypanosoma sp. infection in host blood was performed by nested Polymerase Chain Reaction (nPCR) that targeted a variable region of the trypanosome 18S rRNA gene, with external primers TRY927F and TRY927R, and internal primers SSU561Fand SSU561R, according to Smith et al. (2008). Positive samples were further subjected to D71 and D72 primers which amplify a conserved sequence of the large subunit of the ribosomal DNA gene (24Sα rDNA), to test for T. cruzi (Souto and Zingales, 1993). To test for T. evansi, positive samples were also run on TBR1 and TBR2 primers which amplify sequence of mini-chromosome satellite DNA for T. evansi (Masiga et al., 1992). Finally, samples were run using Leishmania specific primers A and B targeting kDNA of Leishmania sp. (Schubach et al., 1998). Each reaction included negative and positive control samples from Trypanosoma sp., T. cruzi, T. evansi and Leishmania spp. PCR products were visualized in 2% agarose gel after ethidium bromide staining under ultraviolet light.
For the detection of anti-T. evansi IgG antibodies we used an IFAT assay on dogs and C. thous only (Aquino et al., 2010). Currently there are no conjugated fluoresceine antibodies against IgG for N. nasua, S. cay, D. azarea, E. sexcinctus and D. novemcinctus. The cut-off value for the IFAT was 1∶40. The cut-off value for the ELISA was defined as the mean optical absorbance of the negative controls +20%. To each reaction plate, 2 positive and 2 negative control sera were run.
In the present study we considered high parasitemias (patent infection) the positivity in the parasitological tests HC and MHCT. An animal was considered infected when a positive result was found on any of the diagnostic tests used: parasitological, serology and molecular.

Physical examination of dogs
Clinical signs suggestive of trypanosomiasis (weight loss, lymphadenomegaly, onychogryphosis, mucous pallor, skin lesions and ocular signs) were evaluated by a veterinarian and noted for each dog. In addition, owners were asked about feeding habits and contact with wild mammals.

Statistical analysis
Single and co-infections were expressed as frequency of occurrence (percentage of single and co-infection regarding all animals sampled). Due to the number of samples collected, statistical analysis was performed only on dogs.
Path Analysis was used to estimate the influence in the relationship between positive dogs and clinical signs, contact with wildlife and feeding on wildlife. The variables were considered to be statistically significant when values of p were ≤0.05.
To test possible associations among the three trypanosomatids evaluated, we used a Chi-squared test and statistical significance was measures at p ≤ 0.05. Data were analyzed using R 3.4.2 (R Development Core Team, 2015).

Discussion
This study demonstrated that T. cruzi, T. evansi and Leishmania spp. circulate in domestic dogs and wild mammals in Urucum settlement in the extreme west of Brazil. The high occurrence of co-infections in the majority of domestic dogs (73%) is noteworthy. Their role as maintenance hosts and sentinels of these three parasites should be carefully observed within Mato Grosso do Sul border region. Among the wild mammals sampled, N. nasua proved to be a key species, where overall 67% were positive with one or more species of parasite and 44% were co-infected. Cerdocyon thous was also co-infected by T. cruzi and Leishmania spp., demonstrating the importance of carnivores in the maintenance of these parasites in sylvatic cycles. Although the most common scenario is to find hosts infected by multiple parasites, the numerous variables involved may be related to different outcomes. Coinfections may alter the frequency or occurrence of infection, as well as the pathogenicity to the hosts, consequently impacting transmission dynamics (Budischak et al., 2012;Enriquez et al., 2016;Fountain-Jones et al., 2017).
Two thirds of dogs at Urucum settlement were positive for T. cruzi, half of dogs were positive by PCR with high parasitemias observed in four dogs: one TcI, two TcIII and one TcIII/TcV mix. Primarily, TcI and TcIII have been identified in wild mammals (Brenière et al., 2016;Barros et al., 2017), as observed in the Brazilian Pantanal and in the neighbouring Paraguayan Chaco (Herrera et al., 2008;Acosta et al., 2017). However, TcI and TcIII have also been also reported infecting humans, domestic dogs and synathropic wild mammals in domestic and peridomestic cycles in Bolivia, Paraguay, Colombia, Venezuela and the Amazon (Yeo et al., 2005;Barnabé et al., 2011;Brenière et al., 2016;Enriquez et al., 2016;Barros et al., 2017).
Our data suggests that TcV is more geographic widespread than previously thought. This DTU seems to be associated with human rather than dogs, and wild and synathropic mammals, mainly in Bolivia, Paraguay and Argentina (Barnabé et al., 2011;Monje-Rumi et al., 2015;Brenière et al., 2016). Nevertheless, this DTU has been reported also in dogs of Argentina, Bolivia and northern Brazil (Brenière et al., 2016;Enriquez et al., 2016). In wild mammals, TcV has been identified in six orders in seven countries of South America (Brenière et al., 2016). Importantly, the Gran Chaco region which borders the study location, has been proposed as the origin of TcV, where it is commonly found (Perez et al., 2013).
The different DTUs found infecting dogs in our study may be related to their predation of wild mammals and/or close contact with the T. cruzi enzootic environment. In the Gran Chaco, armadillos' species have been reported as the main synathropic sylvatic host of TcIII (Alvarado-Otegui et al., 2012;Acosta et al., 2017). Since armadillos were one of the most cited animals hunted by the dogs, these animals may be getting infected through the oral route. TcV has been found infecting small mammals such as Monodelphis sp. and Rattus rattus in Bolivia, and TcI was recorded parasitizing Didelphis albiventris and N. nasua in Gran Chaco and Pantanal region, respectively (Herrera et al., 2008;Alvarado-Otegui et al., 2012). Circulating T. cruzi in these wild mammals' could infect dogs when they hunt or feed on these wild mammals. However, the vector mediated transmission should not be ruled out in enzootic areas, and dogs in the Urucum settlement could be infected by this route, as well as the ingestion of infected triatomine bugs (Montenegro et al., 2002;Reinthinger et al., 2005). Because dogs develop a chronic form of Chagas disease with a low number of circulating parasites (Pineda et al., 1998;Araújo et al., 2002), the source of infection for triatomine vectors is likely low, and therefore the risk to humans unlikely. Co-infections with multiple DTUs in dogs, has been previously found; TcI/TcII was reported in dogs near Serra da Canastra National Park, southeast region of Brazil (Rocha et al., 2013). Dogs have also been documented as co-infected in Colombia by TcI/TcII and TcI/TcIV (Ramírez et al., 2013), which are characteristic genotypes of domestic and sylvatic transmission cycles.
In this study, half of dogs were infected with Leishmania spp. The region of Corumbá is an important focus for American Visceral   Leishmaniasis in Brazil since the 80's Correa Antonialli et al., 2007). While no dogs or wild mammals were positive by hemoculture, the high prevalence found through PCR and serology indicate that these animals may constitute a source of infection to sand flies. In fact, dogs play a central role in the epidemiology of leishmaniasis while they do not present high parasitemias in the blood, they are main reservoirs due to high parasitic load in the skin (Ciaramella et al., 1997;de Queiroz et al., 2011). The occurrence of Leishmania spp. in N. nasua, S. cay and C. thous reinforces the enzootic characteristic of this parasite in the study area. This is the first record of Leishmania infecting N. nasua and S. cay in the central-western region of Brazil, since C. thous was previously reported by Mello et al. (1988). Path Analysis supported that consumption of wild mammals was positively correlated with the infection in dogs by Leishmania spp. These findings support previous findings regarding oral infection as an important route of transmission of trypanosomatids (Herrera et al., 2011;Roque and Jansen, 2014). We found a high prevalence of T. evansi in dogs (73%) and N. nasua (50%), and our results support previous findings by Silva et al. (1995Silva et al. ( , 1996 and Herrera et al. (2004), where a high occurrence of infected dogs and N. nasua were found in the nearby Pantanal region. The high prevalence of T. evansi in the study area suggest that N. nasua is an important reservoir host for this parasite, as seen in the other sites in the Pantanal wetland (Nunes et al., 1993;Herrera et al., 2004). This high prevalence in N. nasua could affect the infection in dogs. In enzootic areas, dogs that live in close contact with wild reservoirs are often infected with T. evansi (Franke et al., 1994;Herrera et al., 2004).
In dogs, the main clinical signs of T. evansi infection is severe anemia, which was seen in our study by the association between T. evansi infection and mucous pallor. Nevertheless, as reported by other authors, intermittent fever, subcutaneous edema, blindness, lethargy, haemostatic changes, and a lack of coordination may also be observed in dogs infected by T. evansi (Singh et al., 1993;Brandão et al., 2002).
Although we did not find a significant association among co-infections in dogs, our data showed that the co-infection by Leishmania spp. and T. cruzi were related to onychogryphosis. Additionally, we found that infection with Leishmania spp., T. cruzi and T. evansi were related to skin lesions. A natural case of co-infection by the hemoparasites Leishmania chagasi and T. evansi in one dog was reported in the south western of Mato Grosso do Sul (Savani et al., 2005). However, to the best of our knowledge, this study is the first to registered co-infection by T. cruzi, T. evansi and Leishmania spp. in dogs. When managing the conservation of wild mammals, it is important to consider that co-infection by T. cruzi and T. evansi influence the health of free-living N. nasua (Alves et al., 2011;Olifiers et al., 2015).
We found discordant results between serological and molecular assays indicating differences in infection. The chronicity of T. cruzi and Leishmania spp. infection is exemplified in our study by several dogs and N. nasua that were serology positive and PCR negative. In the case of T. evansi, the low occurrence of cryptic infection in dogs is probably associated with fatal characteristic of this disease.
The occurrence of seronegative, but PCR positive dogs and N. nasua could be associated with the initial acute infection, characterized by the low level of serum immunoglobulins. We observed that half of the dogs and 17% of N. nasua displayed T. cruzi in the blood. Trypanosoma cruzi parasitemia is higher in the acute phase than in the chronic phase (Pineda et al., 1998;Araújo et al., 2002). Alternatively, the presence of T. cruzi in the blood could also be due to reinfection by the same or different DTUs (Machado et al., 2001). This might also be seen in T. evansi and Leishmania spp. infected animals. The high occurrence of PCR positive dogs and N. nasua suggests active transmission in the studied area.

Conclusions
In conclusion, our results reveal the importance of monitoring trypanosomatids in dogs in cross-border Brazil-Bolivia due to their susceptibility. We found that dogs in the Urucum settlement serve as incidental hosts in the active sylvatic cycle of T. cruzi, T. evansi and Leishmania spp. which was seen by the infected wild mammals. The high prevalence of these three parasites found in dogs demonstrates that they are important domestic reservoirs and potentially important sentinels to human infection in the studied area. Finally, Urucum settlement could be considered a hotspot to emergent neglected diseases caused by trypanosomatids due to the high occurrence of the three trypanosomatids investigated.

Ethical approval
All field procedures and laboratory studies were conducted in accordance with a license granted by the Biodiversity Information and Authorization System of the Chico Mendes Institute for Biodiversity Conservation (license number 47821-3). The present study was approved by the Ethics Committee for Animal Use of Dom Bosco Catholic University, Campo Grande, MS (license number 011/2015). All dog owners signed a consent form before dogs were sampled.

Declarations of interest
None.

Conflict of interest
The authors have declared that no competing interests exist.