Occurrence of Neospora caninum and Toxoplasma gondii antibodies in dogs from rural properties surrounding a biological reserve, Espirito Santo, Brazil

The aim of this study was to determine the presence of antibodies to Neospora caninum and Toxoplasma gondii in serum samples of 187 dogs from 30 rural properties surrounding Córrego do Veado Biological Reserve, Espírito Santo State, Brazil. The Reserve is one of the last lowland Atlantic Forest remnants of the region, surrounded by agriculture farms and cattle pastures. The presence of IgG antibodies was determined by the Indirect Fluorescent Antibody Test for T. gondii (cut-off 1:16) and N. caninum (cut-off 1:50). Positive samples were diluted 2-fold until the last positive dilution. Antibodies to T. gondii were found in 77 (47.05%) dogs and antibodies to N. caninum in 22 dogs (11.76%) and one only dog was positive for both infections. No association between T. gondii and N. caninum infection and sex was observed (p>0.05). Control measures to prevent those infections in dogs that living surrounding the reserve and that had contact with wild animals are important to avoid the introduction of N. caninum in wild animals. This was the first study of frequency of occurrence of T. gondii and N. caninum in dogs from Espírito Santo, Brazil.

In Brazil T. gondii is a common parasite, and the infection in dogs ranges from 2.6% to 90% (revised by DUBEY et al., 2012;FOURNIER et al., 2014;LOPES et al., 2011LOPES et al., , 2015RAIMUNDO et al., 2015). Also common, N. caninum is found to infect dogs in rates that go from 2.6% to 67.6% (revised by DUBEY, 2013).
In the State of Espírito Santo, information related to these parasites in dogs is not available. In this State, in the municipality of Pinheiros, is located the Córrego do Veado Biological Reserve, a protected area (2.357 ha) and one of the last lowland Atlantic Forest remnants of the region. Agriculture and cattle farms surround all the Reserve. Companion animals, mostly dogs, are often present and, because they are close to the forest, they can easily get in contact with wild animals. The objective of this study was to determine the frequency of occurrence of antibodies to T. gondii and N. caninum in dogs living in rural environment close to a forest matrix, in the state of Espírito Santo, where no data is available.
The present study was conducted in 30 rural properties surrounding the Córrego do Veado Biological Reserve (Figure 1), municipality of Pinheiros, State of Espírito Santo, southeastern region of Brazil (40°08'48"S, 18°20'33"W). Serum samples were collected from all 187 dogs (70 females and 117 males) that were present in the farms on the day of the visit from January 2011 to November 2012. The blood was collected from a jugular or cephalic vein and serum were kept at -20 o C until analyses. The presence of IgG antibodies against T. gondii and N. caninum was determined by means of an Indirect Fluorescent Antibody Test (IFAT) with a cut-off of 1:16 for T. gondii (CAÑÓN-FRANCO et al., 2004) and 1:50 for N. caninum (SOUZA et al., 2002). All positive samples were diluted serially 2-fold until the last positive dilution. Positives and negatives samples were added in each slide.
The Pearson's chi-square test (χ 2 ) was applied to evaluate whether the presence of antibodies against both parasites is associated with gender of dogs.
The handling of the dogs was previously authorized by the owners and the study was approved by the Ethics Committee for Animal Use of the Faculty of Veterinary Medicine, University of São Paulo, Brazil.
Antibodies to T. gondii were found in 77 (47.05%) of 187 dogs from 16 of 30 farms with titers of 32 in six dogs, 64 in 13 dogs, 128 in 18 dogs, 256 in 14 dogs, 512 in 22 dogs and 1.024 in four dogs. Also, 22 dogs (11.76%) from three of the 30 farms tested positive for N. caninum antibodies, with titers of 100 in two dogs, 200 in five dogs, 800 in seven dogs, 12,800 in six dogs and 25,600 in two dogs. Sampling sites are shown in Figure 1.
The results demonstrate that T. gondii and N. caninum are distributed along the farms surrounding the Córrego do Veado Reserve, in Espírito Santo State, Brazil. The farms from where the dog`s samples were collected, originally, were all Atlantic Rainforest territory, that is the biome with the highest degree of degradation and is currently restricted to remnants (ICMBio, 2000;ACOSTA et al., 2014). In the Reserve natural fauna and flora are preserved, and dozens of species of animals and plants that are under threat of extinguishment are present (ICMBio, 2000;CHIARELLO, 2000).
As show in Figure 1, around 50% of the farms surrounding the Reserve presented positive dogs to one or two studied coccidian. The farm dogs and cats, definitive hosts for N. caninum and T. gondii, respectively, easily can go to the Reserve territory and leave fecal oocysts in the area. Studies showed that both avian and mammals' species can clinically react diversely to T. gondii infection and for some species the parasite could be fatal (DUBEY, 2010). With N. caninum much less information is available in wild animals in the world and in Brazil (revised by ALMERIA, 2013).
In relation to T. gondii, an important zoonotic agent, the dogs are only intermediate hosts and, in this study, they represent sentinels of the environmental contamination. Due the proximity of the farms with the protected areas, special care should be introduced to reduce the wildlife infection possibilities. The occurrence of 47.05%, found in the rural dogs from Espírito Santo, is in agreement with results found in other parts of Brazil (revised by DUBEY et al., 2012;LOPES et al., 2011).
N. caninum infections are higher in rural dogs, probably related to availability of dead cattle carcasses and abortion products (DUBEY, 2013) in those areas. In the present study the occurrence of 11.76% is in the range of the results found in Brazilian dogs from rural areas (reviewed by DUBEY, 2013). This is the first survey of frequency of occurrence of antibodies to T. gondii and N. caninum in dogs in the State of Espírito Santo, Brazil.