Serological study of American Tegumentary Leishmaniasis in working horses and contact dogs in the city of Bandeirantes, Parana Estudo sorológico da Leishmaniose Tegumentar Americana em equídeos de trabalho e de cães contactantes na cidade de Bandeirantes, Paraná Estudio serológico de la Leishmaniasis Tegumentaria Americana en équidos de trabajo y perros de contacto en la ciudad de Bandeirantes, Paraná

The American Tegumentary Leishmaniasis (ATL) occurs in several parts of the world, in Brazil and also in the State of Parana, it is a disease of great importance in public health, since it affects animals and humans, with a zoonotic character. Leishmaniasis is found in two forms, tegumentary and visceral, and has sandflies as vectors and wild animals and domestic dogs as reservoirs. ATL is considered an endemic disease in the city of Bandeirantes, with Research, Society and Development, v. 10, n. 11, e03101119073, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19073 2 human cases occurring in several urban and rural locations, mainly in areas close to forest remnants, peri-urban and peripheral areas, where vulnerable populations live in general, and with precarious basic sanitation conditions. In these regions also live the participants of the wagon driver project, who own several working horses. The project provides guidance on management, good practices, disease prevention, and animal welfare. Since the role of horses as reservoirs and in the chain of transmission of ATL is not well known, the aim of this study was to carry out a serological study of ATL in horses and in the contact dogs of the participants of the " wagon driver" project, at UENPCLM, to evaluate seropositivity in these species. Blood samples were collected from 20 horses and 12 contact dogs. A serological study, using the ELISAi test for ATL diagnosis, was carried out on the horses and dogs in relation to this study population, to assess seropositivity. In the serological survey, eight horses out of 20 animals (40%) and four dogs out of 12 animals (33.33%) were seropositive, a result considered significant, indicating the presence of the disease and the risk of transmission where they live. The occurrence of human cases and the presence of ATL vectors have already been identified in several locations in the city. Due to the lack of knowledge about the disease and the difficulty in controlling the vectors, health education is necessary in order to provide the population with orientation on how to prevent the disease, as well as entomological surveillance and new serological studies in areas at risk for ATL transmission. The results indicate that there may be importance in the role of horses as reservoirs and in the transmission of Tegumentary Leishmaniasis, which still needs to be clarified.

human cases occurring in several urban and rural locations, mainly in areas close to forest remnants, peri-urban and peripheral areas, where vulnerable populations live in general, and with precarious basic sanitation conditions. In these regions also live the participants of the wagon driver project, who own several working horses. The project provides guidance on management, good practices, disease prevention, and animal welfare. Since the role of horses as reservoirs and in the chain of transmission of ATL is not well known, the aim of this study was to carry out a serological study of ATL in horses and in the contact dogs of the participants of the " wagon driver" project, at UENP-CLM, to evaluate seropositivity in these species. Blood samples were collected from 20 horses and 12 contact dogs. A serological study, using the ELISAi test for ATL diagnosis, was carried out on the horses and dogs in relation to this study population, to assess seropositivity. In the serological survey, eight horses out of 20 animals (40%) and four dogs out of 12 animals (33.33%) were seropositive, a result considered significant, indicating the presence of the disease and the risk of transmission where they live. The occurrence of human cases and the presence of ATL vectors have already been identified in several locations in the city. Due to the lack of knowledge about the disease and the difficulty in controlling the vectors, health education is necessary in order to provide the population with orientation on how to prevent the disease, as well as entomological surveillance and new serological studies in areas at risk for ATL transmission. The results indicate that there may be importance in the role of horses as reservoirs and in the transmission of Tegumentary Leishmaniasis, which still needs to be clarified. Keywords: Phlebotominae; Public Health; Zoonosis.

Introduction
The leishmaniases are considered anthropozoonoses and constitute a serious public health problem and present tegumentary (LT) and visceral (VL) forms of occurrence, with variations in the clinical presentation and ecological diversity.
According to estimates by the World Health Organization (WHO), more than a billion people live in areas at risk of infection.
It has a worldwide distribution, occurring in 92 countries on four continents (Americas, Africa, Asia, and Europe) with an annual record of 700 thousand to 1.3 million new cases. It is considered to be one of the six most important infectious diseases in the world, due to the large coefficient of occurrence and the capacity to produce deformities (WHO 2020;Brasil 2017;Brasil 2021).
In the Americas, leishmaniases occur in 18 countries and the most common clinical form is cutaneous leishmaniasis ATL is associated with environmental factors and knowledge of its reservoirs is of great importance to establish risk factors for transmission and infection of the disease. In areas without deforestation peridomestic animals such as dogs, horses and rodents may act as reservoirs, therefore, it is important to conduct studies to investigate the role of these animals in the prevalence of leishmaniasis (Truppel et al. 2014).
In endemic areas for leishmaniasis it is frequent the presence of infected dogs and phlebotomine sandflies in the peridomicile, and considering that horses are long-lived animals and are in endemic areas, they have great chances of becoming infected by several organisms, including the Leishmania spp, and for being one of the food sources of the sandflies, they can contribute to the maintenance of the peridomiciliary cycle of ATL (Silveira et al. 1996;Evers et al. 2017).
ATL is considered endemic in the northern region of Parana, as well as in the city of Bandeirantes, where phlebotomine sand fly vectors have already been identified in households in several locations in the city, including peri-urban and rural areas. The proximity of domestic animal shelters, the accumulation of organic matter, and soil moisture due to domestic water disposal are risk factors that may favor the presence of vectors and the transmission of the disease (Cruz et al. 2013;Massafera et al. 2005).
Research, Society andDevelopment, v. 10, n. 11, e03101119073, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i11.19073 Tegumentary Leishmaniasis can affect animals and humans, and dogs and horses can serve as reservoirs. Equine leishmaniasis is still a neglected tropical disease and its importance in the transmission cycle of human leishmaniasis is still unclear. Leishmaniasis should be included in the differential diagnosis of equine skin diseases that do not respond to antibiotic and antifungal treatments (Vieira et al. 2020).
This study aimed to perform a serological study to evaluate the seropositivity for ATL in horses and contact dogs of owners participating in the wagon driver project at the Northern Parana State University (UENP) Luiz Meneghel campus (CLM), in the city of Bandeirantes, Parana State.

Methodology
The study was conducted in the municipality of Bandeirantes, located in Northern Parana and southern region of The horses that participated in the study belonged to six different owners participating in the wagon driver project (UENP-PR), which provides equine evaluation and clinical care, as well as guidance on management and prevention of endemic diseases. They came from the neighborhoods of Habitar Brasil (periruban), Vila São Pedro (periruban) and Bairro rural Yara, places with remaining forest areas, animal breeding and the presence of organic matter near the houses.
Blood samples were collected from 20 horses used for work or leisure. The animals belonged to owners living in the city of Bandeirantes, who participated in the wagon driver project of the Universidade Estadual do Norte do Paraná. The owners and animals lived in periurban and rural areas near the city, where there were forest remnants, with poor sanitation, constituting a vulnerable population, with low socioeconomic and cultural power.
The collections occurred during the period from January to July 2019 and a 30x8 needle and 5 milliliter (mL) syringe were used as materials, and the access was made in the jugular vein for blood collection, which after removal was immediately deposited in a dry tube and stored in a thermal box with ice packs until the moment of centrifugation, which was performed 2 hours after collection in the parasitology laboratory of the UENP. After centrifugation, the serum was deposited in eppendorfs and frozen in a freezer at -20 degrees Celsius (Cº) and sent to the State University of Londrina (UEL) in an isothermal box with ice packs to perform the serology for ATL.
Enzyme Linked ImmunonoSorbent Assay (ELISA) was performed as described by Szargiki et al. (2009) andFerreira et al. (2018) with adaptations, using L. amazonensis antigen, serum and peroxidase-conjugated protein A conjugate (Sigma Aldrich, USA). Samples were tested in duplicate and the test cut-off point was obtained using the mean absorbance of the negative serums with the addition of three standard deviations. To estimate the corrected cut-off point, the Roc curve from the MedCalc Statistical Software program was used as described by Schoonjans, Zalata, Depuydt and Comhaire (1995).
A general physical examination of the horses was performed to assess their general condition, the examination included measuring heart rate, respiratory rate, rectal temperature, capillary perfusion time in oral mucosa, evaluation of oral mucosal staining, and size and consistency of palpable lymph nodes. The results were compared with the parameters of normality found in the literature of Muir and Hubbell (2009). Research, Society and Development, v. 10, n. 11, e03101119073, 2021(CC BY 4.0) | ISSN 2525 5 The body condition score of the animals was also identified by visual analysis and palpation of the areas where fat deposition occurs, where number nine is considered an extremely obese animal and number one is a very thin animal, using the methodology described by Bender et al. (2014).
Blood samples were also collected from 12 dogs that had direct contact with these horses and lived in the same place and belonged to the same owners. Blood was collected with a 25x7 needle and a 5 mL syringe, using the jugular or cephalic vein as access. After the collection, the blood was deposited in a dry tube and refrigerated until centrifugation, which was performed in the parasitology laboratory at UENP, using the same methodology described above.
For the serology of the dogs, the Enzyme Linked ImmunonoSorbent Assay (ELISA) immunoassay technique was also used as described by Benitez et al. (2018) with adaptations, using the antigen L. amazonenses, tests performed in duplicate with positive and negative control on each blade and using the statistical analysis of the ROC curve with MedCal Statistical Software program, as described by Metz (1978).

Discussion
Regarding the physical examination performed, all horses were within normal vital and clinical parameters and only four animals had a low body score, score between 2 and 3, after visualizing low fat in areas such as neck and tail base, according to the description of Muir andHubberl (2009) andBender et al. (2014).
The dogs belonged to three owners, and the other participating equine owners reported not owning any dogs. 50% (6/12) of the dogs were female and 50% (6/12) were male. About the serology, there was positivity in 33.3% of animals (4/12), but no animal had skin lesions.
In the ELISAi technique for LTA serology, three cut-off points were used, following the methodology of Schoonjans et al. (1995) for the calculation, where the results were 0.09125 as the first cut-off point, 0.018738 as the second cut-off point, and 0.147465 as the third cut-off point.
The results in horses showed seropositivity of 40% (8/20) at the third cut-off point (Table I), such animals belonged to 4 different owners and no animal had skin lesions.
In the results of the horses, the seropositivity for LT was higher than in the study by Ferreira et al. (2018), who found positivity of 25.4% for indirect ELISAi in the maranhense lowlands and described that the role of horses in the transmission of leishmaniasis is uncertain, but that seroconversion occurs in these animals. The results of seropositivity in this study regarding horses differ from those of Benvenga (2013) in São Paulo, who found much lower positivity of 2.5% of the horses analyzed, although this author used the indirect immunofluorescence technique.
This study also differed from Limeira et al (2019), which with a meta-analysis, showed a prevalence for combined ATL of 25%, but with great heterogeneity among the studies, related to differences in the methods used to diagnose the disease.
The seropositivity of the dogs was four dogs in a total of 12 animals (33.33%), which is higher than the results of Lonardoni et al. (2006) that found 19% of the analyzed animals positive. None of the positive dogs had clinical signs of the disease, and the seropositivity was higher than the results found by Madeira et al. (2000), in the state of Rio de Janeiro, where they found 11.94% of reactors. The presence near the peridomicile and work animals such as horses and companion animals such as dogs is attractive to sandflies, which may increase the risk of contracting ATL in transmission areas. Measures are needed to control the ATL vector population, especially in the household environment, in addition to educational measures for the population, observations also described by Cruz et al. (2013).
Studies such as those by Bresalier, Cassidy and Woody (2015) comparing the health of humans and animals on the transmission of zoonosis such as leishmaniases should be conducted in an integrated and simultaneous manner. Bandeirantes was one of the municipalities that stood out in the study by Melo, Rossoni and Teodoro (2017), due to the high number of cases of ATL in humans, among the 268 municipalities in the state of Paraná with recorded cases.
The present study showed in the serological results found that there are horses and dogs seropositive for tegumentary leishmaniasis in the region studied, indicating the presence of the disease in the areas sampled. Studies already carried out in the region, in which vectors were found and cases of the disease in humans already reported, reaffirm the endemicity of ATL in the city and indicate the need for vigilance and control of vectors and health education activities to make the population aware of the prevention of the disease should be carried out in the region, since there are many rural areas and remaining forest areas, areas with accumulation of organic matter that can be risk factors for the transmission of ATL in the region.

Conclusions
Both horses and dogs seropositive for American Tegumentary Leishmaniasis (ATL) were found in the region studied, there are other studies that show the presence of the vector and registered cases of the disease in the city, which reaffirms the endemic form of ATL in Bandeirantes and shows the need to prevent the disease and spread forms of prevention to the population.
There is a necessity to realize serological studies for ATL covering a larger number of horses and dogs in the transmission areas where human cases have been reported, for a better evaluation of the risk factors and to better understand the role of horses in the transmission of the disease, considering that there are still few studies on the subject.