Serosurvey for Leishmania spp . , Toxoplasma gondii , Trypanosoma cruzi and Neospora caninum in neighborhood dogs in Curitiba-Paraná , Brazil Inquérito sorológico para Leishmania spp . , Toxoplasma gondii , Trypanosoma cruzi e Neospora caninum em cães comunitários em Curitiba-Paraná , Brasil

Neighborhood dogs may act as reservoirs for several zoonotic protozoan infections, particularly in urban areas, thus constituting a potential public health threat. Accordingly, the aim of the present study was to evaluate the exposure of neighborhood dogs to four protozoan pathogens in public areas with high levels of human movement in Curitiba, southern Brazil. Blood samples from 26 neighborhood dogs were screened by means of the indirect immunofluorescent antibody test (IFAT) for Leishmania spp., Toxoplasma gondii, Trypanosoma cruzi and Neospora caninum, and a questionnaire was answered by the respective keeper. A total of 8/26 dogs (30.7%) seroreactive to T. gondii, 3/26 (11.5%) to N. caninum and 2/26 (7.7%) to both were identified. All the samples were seronegative for T. cruzi and Leishmania spp. Pathogen seroreactivity was not associated with the daily human movements or other epidemiological variables investigated (p > 0.05). In conclusion, the low seroprevalence for T. gondii and N. caninum indicated low environmental and food risk for animal infection and the seronegativity for Leishmania spp. and T. cruzi may reflect the absence of these pathogens in urban areas of Curitiba. Moreover, neighborhood dogs may be used as environmental sentinels for the presence of protozoan pathogens and their vectors.

Neighborhood dogs, defined as semi-restricted or free roaming animals with semi-dependence on one or more individuals for food and shelter (WHO & WSPA, 1990), may act as reservoirs or sentinels for several zoonotic and protozoan infections, particularly in urban areas, thus constituting a potential threat to animal and public health (CABEZÓN et al., 2010;CASTILLO-NEYRA et al., 2015;GÜRTLER & CARDINAL, 2015;MEIRELES et al., 2004;ROBERTSON et al., 2000;SALB et al., 2008).Although protozoan infection has previously been assessed in different canine populations (AZZAG et al., 2015;COLLANTES-FERNÁNDEZ et al., 2008;LANGONI et al., 2013;TENNEY et al., 2014), the health status of neighborhood dogs remains to be fully established.
Accordingly, the aim of the present study was to evaluate the exposure of neighborhood dogs to four protozoan pathogens in public areas with highly levels of human movement in the city of Curitiba (bus stations and public parks), the eighth biggest city in Brazil.
This study was approved by the Ethics Committee for Animal Experimentation and Animal Welfare of the Federal University of Paraná, state of Paraná, Brazil (protocol number 027/2015).
Bus stations and public parks were included in this study because they are public areas with high and daily human concentration and movement, besides to their good conditions for neighborhood dogs housing.Dog inclusion criteria was applied according to the WHO definition (WHO & WSPA, 1990) and dogs only included if their correspondent keeper were identified.All 22 bus stations of the city of Curitiba were visited in attempt to identified neighborhood dogs and their respective keeper.In 26 public parks, the neighborhood dogs and their corresponding keeper were registered by population demand by means of city Hall Curitiba phone.
Whole blood samples (5 mL) were collected by means of venipuncture of the jugular vein using sterile vacuum tubes containing serum separator gel (BD Vacutainer, Franklin Lakes, NJ, USA) and were stored at room temperature (25 °C) until visible clot retraction.The samples were centrifuged at 1500 g for 5 minutes, serum separated and stored at -20 °C until serological analysis.The serum samples were tested for specific IgG antibodies against Leishmania spp., Toxoplasma gondii, Trypanosoma cruzi and Neospora caninum by means of the indirect immunofluorescent antibody test (IFAT), as previously described (CAMARGO, 1974).Both, positive and negative, control sera were provided by the Núcleo de Pesquisa em Zoonoses (NUPEZO), Universidade Estadual Paulista (UNESP), Botucatu -SP for all the four diseases.Samples were considered reactive when antibody titers ≥ 40 for Leishmania spp., ≥ 16 for T. gondii, ≥ 20 for T. cruzi and ≥ 25 for N. caninum were found.The titers were determined to the largest dilution at which fluorescence was viewed around the protozoa (endpoint titers).Fisher's exact test was used to determine differences relating to whether individual factors were associated with seroreactivity through IFAT.Moreover, the Spearman correlation coefficient was calculated to verify the correlation between daily human movement and seroreactivity.Results were considered to be statistically significant when p < 0.05.Data were stored and analyzed using commercially available software (Epi Info, version 7.1.5.2,CDC, Atlanta, USA).
In total, 3/26 neighborhood dogs (11.5%; 95% CI: 2.35-29.16%)were seroreactive to N. caninum, with antibody titers ranging from 25 to 200; 8/26 (30.7%; 95% CI: 14.33-51.79%)were seroreactive to T. gondii, with antibodies titers ranging from 16 to 64; and only 2/26 (7.7%) were seroreactive to both (Figure 1).All the dogs were seronegative to Leishmania spp.and T. cruzi.No significant associations were found between gender, diet, raw meat intake or hunting practices and seroreactivity to N. caninum and/or T. gondii.The seroprevalence results for N. caninum and T. gondii among the neighborhood dogs and the respective variables are presented in Table 1 and Table 2. Likewise, no correlation was found between daily high human movement and seroreactivity to N. caninum (Spearman correlation coefficient = -0.174;p = 0.631) and/or T. gondii (Spearman correlation coefficient = 0.302; p = 0.397) (Figure 1).However, the neighborhood dogs appear at bus stations where there are a higher population density, and also that the dogs seronegative for all surveyed pathogens represent the most these locations (Figure 2).Surprisingly, to the best of our knowledge, this study was the first serosurvey on this dog population worldwide.Despite the outdoor and free-roaming habits of neighborhood dogs, which may increase their exposure to protozoan infections (AZEVEDO et al., 2005;COLLANTES-FERNÁNDEZ et al., 2008;MOURA et al., 2009;NAZIR et al., 2014), they presented in this study a low seroprevalence for T. gondii and N. caninum compared with literature results (MEIRELES et al., 2004;SEABRA et al., 2015;VALADAS et al., 2010), which may indicate that there was a situation of low environmental oocyst contamination (MEIRELES et al., 2004).Since dogs play a secondary role in the toxoplasmosis cycle, neighborhood dogs may be considered to be true environmental sentinels for T. gondii, particularly when sharing a contaminated urban environment and contaminated food and water (MEIRELES et al., 2004).In contrast, as the definitive hosts of N. caninum, dogs may play an important role in spreading this disease (NAZIR et al., 2014).
As expected, all the neighborhood dogs tested negative for Leishmania spp.and T. cruzi in the present study.In fact, the city of Curitiba has been considered to be a non-endemic area for both of these diseases and, thus, these results may reflect the historical absence of vectors and pathogens (BRASIL, 2014a, b).However, canine visceral leishmaniasis was previously detected in 1/364 stray dogs (0.0027%) from a city situated close to Curitiba (FREHSE et al., 2010), and triatomine (T.cruzi vector) infestation was detected in 7/9 cities (77.8%) in the northern part of the state of Paraná (FALAVIGNA-GUILHERME et al., 2004).Moreover, the first autochthonous case of human visceral leishmaniasis in the state of Paraná was registered in 2015 in city of Foz do Iguaçu, and the first death due to this disease was reported in January 2016 (AEN, 2015).Since infection of dogs by Leishmania spp.may precede human cases (OLIVEIRA et al., 2001), periodic serosurveys among neighborhood dogs may be used as a means of active surveillance in non-endemic areas such as the city of Curitiba.

Figure 1 .
Figure 1.Map of Brazil showing the location of the state of Paraná and city of Curitiba.Enlarged map shows the spatial distribution of neighborhood dogs according to blood test results, in relation to Leishmania spp., Toxoplasma gondii, Trypanosoma cruzi and Neospora caninum, and daily human movements in bus stations (BS) and public parks (PP) included in the study, city of Curitiba, state of Paraná, southern Brazil, 2016.

Figure 2 .
Figure 2. Map of Brazil showing the location of the state of Paraná and city of Curitiba.Enlarged map shows the spatial distribution of neighborhood dogs according to blood test results in relation to population density of city of Curitiba, state of Paraná, southern Brazil, 2016.

Table 1 .
Seroprevalence for Neospora caninum and Toxoplasma gondii among neighborhood dogs according to IFAT, in relation to each variable studied, city ofCuritiba, state of Paraná, southern Brazil, 2016.

Table 2 .
Seroprevalence for Toxoplasma gondii and Neospora caninum among neighborhood dogs according to IFAT, in relation to each gender and housing location of neighborhood dogs, city ofCuritiba, state of Paraná, southern Brazil, 2016.