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Seroepidemiology of Sarcocystis neurona, Toxoplasma gondii and Neospora spp. among horses in the south of the state of Minas Gerais, Brazil

Seroepidemiology of Sarcocystis neurona, Toxoplasma gondii and Neospora spp. among horses in the... The present study used the indirect fluorescent antibody test (IFAT) to determine the seroprevalence of Sarcocystis neurona, Toxoplasma gondii and Neospora spp., and evaluated the variables associated with these infections among 506 apparently healthy horses, reared in the south of the state of Minas Gerais, Brazil. This study was conducted between April 2012 and October 2013. Among the horses, the true prevalence of S. neurona was 26% (95% CI: 22.0-30.4%), T. gondii 19.9% (95% CI: 15.5-24.8%) and Neospora spp. 23.9% (95% CI: 19.9-28.1%); and among the farms, 88.3% (95% CI: 74.4-91.6%), 71.6% (95% CI: 41-92.8%) and 85% (95% CI: 70.7-96.1%), respectively. Regarding mixed infection, 17 horses (3.4%) were seropositive for both S. neurona and T. gondii, 16 (3.2%) for T. gondii and Neospora spp. and 14 (2.8%) for S. neurona and Neospora spp. The associations between seropositivity and variables relating to the structure of the farm, management and health were analyzed using the logistic regression analysis, through the generalized estimating equations (GEE). The results suggest that the south of Minas Gerais is an enzootic area for S. neurona, T. gondii and Neospora spp. among horses, with prevalence of asymptomatic subclinical or chronic infections. Keywords: Apicomplexa, Sarcocystidae, indirect immunofluorescence, risk factors. Resumo Este estudo determinou, pela reação de imunofluorescência indireta (RIFI), a soroprevalência de Sarcocystis neurona, Toxoplasma gondii e Neospora spp., e avaliou as variáveis associadas com a infecção, em 506 equinos, aparentemente sadios, criados no Sul de Minas, Brasil. O estudo foi realizado no período de abril de 2012 a outubro de 2013. Entre equinos, a prevalência verdadeira para S. neurona foi de 26% (IC 95%= 22,0-30,4%); para T. gondii de 19,9% (IC 95%= 15,5-24,8%); e para Neospora spp. de 23,9% (IC 95%= 19,9-28,1%); e entre propriedades, 88,3% (IC 95%= 74,4-91,6%), 71,6% (IC 95% = 41-92,8%), e 85% (IC 95%= 70,7-96,1%), respectivamente. Em relação à infecção mista, 17 (3,4%) equinos foram soropositivos para S. neurona e T. gondii, 16 (3,2%) para T. gondii e Neospora spp., e 14 (2,8%) para S. neurona e Neospora spp. A associação entre soropositividade e variáveis relacionadas à estrutura da propriedade, manejo e sanidade, foi analisada, utilizando-se a análise de regressão logística estimada por Generalized Estimating Equations (GEE). Os resultados sugerem que o Sul de Minas é área enzoótica para S. neurona, T. gondii e Neospora spp. em equinos, com predomínio de infecções subclínicas ou crônicas, assintomáticas. Palavras-chave: Apicomplexa, Sarcocystidae, imunofluorescência indireta, fatores de risco. *Corresponding author: Antônio Marcos Guimarães. Departamento de Medicina Veterinária – DMV, Universidade Federal de Lavras – UFLA, CP 3037, CEP 37200-000, Lavras, MG, Brasil. e-mail: amg@dmv.ufla.br www.cbpv.org.br/rbpv Seroepidemiology of Sarcoystis neurona 143 v. 25, n. 2, abr.-jun. 2016 These three protozoa of the phylum Apicomplexa, family Introduction Sarcocystidae, are transmitted through consumption of water and food contaminated by sporocysts, in the case of S. neurona, and Brazil possesses one of the largest horse herds in the world, with sporulated oocysts, in the cases of T. gondii and Neospora spp., approximately 5.3 million head. Horse-rearing is an important which are released by the definitive hosts: respectively, possums, source of income and employment. In the southern region of the felids and canids (LANGONI et al., 2007; DUBEY & SU, 2009; state of Minas Gerais, horse rearing occupies an important space DUBEY et al., 2015). The infection is usually asymptomatic among in the local economic, touristic, historic and cultural scene, since horses, but may sporadically present nervous symptoms in the this was the birthplace of the Mangalarga Marchador horse breed, which is internationally admired (IBGE, 2013). cases of S. neurona, T. gondii and N. hughesi, and reproductive Equine protozoal myeloencephalitis, caused by Sarcocystis disorders, when involving T. gondii and N. caninum (LOCATELLI- neurona, is a disease characterized by a neurological condition DITTRICH et al., 2006; GENNARI et al., 2015). in which horses participates as an accidental host. In Brazil, the Because of the importance of horse-rearing in the south of the seroprevalence of S. neurona among horses has been estimated in state of Minas Gerais, determining the prevalences of S. neurona, 37.7% (Western blot) (LINS et al., 2012) and 33.7% (ELISA) T. gondii and Neospora spp., along with the factors associated with (PIVOTO et al., 2014) in Rio Grande do Sul, and 8.75% (IFAT) these infections, is fundamental for implementing effective control in Rio de Janeiro (STELMANN, 2014). Worldwide seroprevalence measures, so as to reduce the exposure of the animals and prevent varies between 0% and 89.2% (DUBEY et al., 2015). the economic losses caused by these parasites. Moreover, this type Toxoplasmosis, the disease caused by Toxoplasma gondii, is of investigation increases the knowledge on the distribution of a zoonosis of global interest due to its impact on public health. these agents among the horse population nationally. It can be transmitted through consumption of raw or undercooked Thus, due to the scarcity of studies in Brazil and lack of meat, including horse meat (DUBEY & SU, 2009). The levels of information regarding equine infection in the southern part of antibodies for T. gondii among horses in Brazil has been estimated the state of Minas Gerais, the present study had the objectives of in 12.8% (IFAT) in Minas Gerais (NAVES et al., 2005), 2.5% determining the prevalence of S. neurona, T. gondii and Neospora (IFAT) in Mato Grosso do Sul (LASKOSKI et al., 2015) and 28,4% spp., and identifying possible factors associated with seropositivity (IFAT) in states of Alagoas, Paraíba, Pernambuco, Rio Grande do among Mangalarga Marchador horses. Norte and Minas Gerais (GENNARI et al., 2015). Worldwide, seroprevalence varies between 0% and 72.2% (ALSHAHERY & Materials and Methods MANSOUR, 2012; MATSUO et al., 2014). Neosporosis is a parasitic disease caused by the protozoon Neospora Study area and animals caninum, which can infect several mammals, including horses, and by N. hughesi, which infects horses and is an uncommon etiological Blood samples were gathered from 506 Mangalarga Marchador agent for equine protozoal myeloencephalitis (GOODSWEN et al., horses that had been reared on 53 farms that were distributed among 2013). In Brazil, the prevalence of anti-Neospora spp. antibodies 27 municipalities in the microregions of Andrelândia, Lavras, São among horses has been estimated in 57.6% (IFAT) in São Paulo João Del Rey, São Lourenço and Varginha, in the southern portion (STELMANN  et  al., 2011), 25.7% (IFAT) in Santa Catarina of the state of Minas Gerais (Figure 1). The study was conducted (ABREU  et  al., 2014), and 1.87% (IFAT) in Rio de Janeiro (STELMANN, 2014) and, abroad, from 0% to 35% (DUBEY et al., between April 2012 and October 2013 after approval had been 1999; PATITUCCI et al., 2004). obtained from the Ethics Commission for Animal Use (COEA) Figure 1. Map of the state of Minas Gerais, Brazil, highlighting the location of the municipalities of the southern region that were involved in the study. 144 Ribeiro, M.J.M. et al. Braz. J. Vet. Parasitol. of the Federal University of Lavras (UFLA), and was registered temperature, they were stored in a freezer at –20 °C until the at this institution under number 023/11. time of use. The  IFAT for T. gondii was performed using the The farms were chosen randomly based on guidance from technique described by Langoni et al. (2007) and the antigens the Brazilian Association of Mangalarga Marchador Horse were the tachyzoites of the parasite. After successive inoculations, Breeders (Associação Brasileira de Criadores de Cavalos Mangalarga the tachyzoites were recovered through rinsing the peritoneal Marchador, ABCCMM). In this sampling, the proportions between cavity of mice with a saline solution. Ten microliters of a solution the mesoregions and the sizes of farms and their structures were containing tachyzoites was placed in each of the 12 slide wells maintained in order to attain homogeneous representativeness. for IFAT (about 10 tachyzoites / field at 40x magnification in At least ten horses from each farm were required for the prevalence an optical microscope). After the slides had been dried at room calculation on S. neurona, T. gondii and Neospora spp. In addition, temperature, they were stored in a freezer at –20 °C until the pregnant mares and foals of less than six months of age were not time of the serological tests. In the present study, N. caninum included in the sampling. The reasons were that pregnant mares tachyzoites were used as the antigen in the IFAT, and their antibodies are more sensitive to manipulation, while foals younger than six presented cross-reactions with N. hughesi (GONDIM et al., 2009). months may present colostral antibodies (passive immunity), The IFAT for Neospora spp. was performed in accordance with which would interfere with the serological diagnosis. The blood the technique described by Paré et al. (1995), using glass slides samples were obtained by means of jugular venipuncture and were containing tachyzoites of N. caninum as the antigen (Laboratório collected in Vacutainer tubes. All the animals appeared to be healthy. Imunodot, Jaboticabal, Brazil). After centrifugation at 500 g for 10 minutes, the serum samples An equine anti-IgG conjugate (SIGMA, Saint Louis, MO, USA) from 506 horses were stored in individually identified cryotubes was used for performing the IFAT, at a dilution of 1:64, along with and frozen at –20 °C until the serological tests were performed. positive (serum samples of mares seropositive to S. neurona, T. gondii The definition of the number of horses (n) needed to estimate and Neospora spp.) and negative (serum samples of newborn foal the prevalence was based on the formula of the Pan-American before colostrum ingestion and serum samples of periparturient Zoonosis Center (CEPANZO, 1979), which is appropriate mare, both seronegative to respective protozoa) controls for the for calculating samples for research on chronic diseases: respective protozoa. The serum samples were considered positive if 2 2 n = [p x (100-p) x Z ]/(d x p/100) . Considering an estimated they presented complete fluorescence of the S. neurona merozoites prevalence (p) of 50%, a 95% confidence interval (z) of 1.96 and at a dilution of 1:80 (DUBEY et al., 2015), and of the T. gondii an acceptable error margin (d) of 10%, the minimum number and Neospora spp. tachyzoites, respectively, at dilutions of 1:64 (n) of horses was 384. (ABREU et al., 2014) and 1:50 (LOCATELLI-DITTRICH et al., 2006). Both the IFAT and the preparation of slides containing S. neurona and T. gondii antigens were performed at the Parasite Information-gathering on the farms Disease Laboratory of the Federal University of Lavras (UFLA). A questionnaire-based interview was carried out with either the owners or the person responsible for each farm. The questions Statistical analyses related to herd health, the breeder, management and the structure of the farm. Moreover, questions asking about any history of The EpiData software was used to tabulate the data relating to reproductive problems and the neurological condition of the herds the variables studied. These data were later ranked and analyzed were also devised. This information served as independent variables using the statistical software SPSS 20.0 statistical software (IBM, for subsequent analyses that evaluated the associations between 2011). In calculating true prevalence (TP) for the anti-S. neurona seropositivity to the protozoa and factors relating to sociocultural antibodies, values of 94% and 88.9% were used for sensitivity (SE) information, herd health and management practices observed and specificity (SP), respectively, as prescribed by Duarte et al. on the farms. In total, 305 variables were analyzed, which were (2003). In the case of TP for T. gondii, the SE and SP values were, obtained by means of the questionnaire. respectively, 90.9% and 100% (LU et al., 2013). In turn, TP for Neospora spp. presented values of 91.0% and 99.0%, respectively, for SE and SP, in accordance with Björkman & Uggla (1999). Serological tests Subsequently, the SE and SP between the herds were used to calculate the TP at herd level (NOORDHUIZEN et al., 2001), by The indirect fluorescent antibody test (IFAT) was the method means of the epidemiological calculators of the Epitools software chosen for investigating anti-S. neurona, T. gondii and Neospora spp. IgG antibodies. This is considered to be the gold-standard test for (SERGEANT, 2011). The variables relating to the structural, serologically diagnosing the latter two protozoa (LANGONI et al., sanitary and management characteristics of the farms, along with 2007; DUBEY & SCHARES, 2011). the inherent characteristics of the animals, were firstly subjected to In investigating anti-S. neurona antibodies, the IFAT was the chi-square test or Exact Test of Fisher (less than five observations performed as described by Johnson et al. (2013). The antigens were in any category) using the SPSS 20.0 software (IBM, 2011), so as to demonstrate their association with the seropositivity of the S. neurona merozoites, produced in cell cultures. Ten microliters of S. neurona merozoite solution was placed in each one of the 12 slide studied agents. This association, when statistically significant, wells for IFAT (about 10 merozoites / field at 40x magnification was quantified by the odds ratio and its confidence interval at in an optical microscope). After slides had been dried at room 95% (CI. 95%), which estimates the risk relationship between Seroepidemiology of Sarcoystis neurona 145 v. 25, n. 2, abr.-jun. 2016 independents variables (risk or protective factors) and the dependent among which 21 (44.7%) manufactured the concentrate on-site. variables (serology results). Variables that presented p values < 0.05 Regarding sanitary management, 50 farmers (94.3%) vaccinated were considered significant, meaning that they were statistically their animals, and 31 (58.5%) had veterinary assistance. Most of associated with seropositivity to S. neurona, T. gondii and Neospora the farms used artificial insemination techniques (62.3%). Horse- spp. Subsequently, the variables that presented p values < 0.20 in rearing was the main source of income on 13 farms (24.5%), and the chi-square test or Exact Test of Fisher were subjected to the 33 (62.3%) had been conducting this activity for over 20 years. logistic regression analysis, through the generalized estimating equations (GEE) (CORBELLINI et al., 2006; BRUHN et al., Seroprevalence and variables associated with infection 2013). Variables that presented p values < 0.05 in the GEE were considered to be significantly associated with seropositivity for Sarcocystis neurona. Out of the 506 horses, 117 (23.0%) S. neurona, T. gondii and Neospora spp. presented anti-S. neurona antibodies, with true prevalence (TP) The QIC (Quasilikelihood under the Independence model of 26.0% (95% CI: 22.0-30.4%). Out of the 53 properties Criterion) and the related QICC statistics, proposed by Pan analyzed, 44 had at least one seropositive horse, with TP of 88.3% (2001) as GEE logistic regression goodness of fit measure, was (95% CI: 74.4-91.6%). As shown in Table 1, the properties that used to compare the GEE models, considering that when QICC included forest reserves or that bordered a reserve presented a high approximates QIC the GEE model is correctly specified and that risk (p < 0.05) of having horses seropositive for S. neurona. Farms the model with the smaller QIC and QICC statistics is preferred. that manufactured their own feed also had greater risk (p < 0.05) of having infected horses. Toxoplasma gondii. In the case of T. gondii, 107 (21.1%) Results horses were seropositive, representing a TP of 19.9% (95% CI: 15.5-24.8%). On 45 farms, at least one horse was seropositive, Farm profile indicating a TP of 71.6% (95% CI: 41.0-92.8%). Farms that had more than 100 horses presented lower risk (p < 0.05) of having Among the 506 animals analyzed, 343 were female (67.8%) infected animals. Farms that vaccinated their horses against tetanus and 163 were male (32.2%). There were 61 foals (12.1%) and and influenza, and those that had an area for treating, cleaning 445 adults (87.9%). Among the 53 farms, horse-rearing was the and caring for the animals, presented lower risk (p < 0.05) of main activity on only 21 of them (39.6%). Regarding size, 27 farms infection. Horses over the age of five years were significantly (51.0%) covered more than 100 ha, with a mean area of 199.1 ha. (p < 0.05) more liable to infection by T. gondii (Table 2). There was Forty-eight farms (90.6%) either bordered or included a forest or a significant association (p < 0.05) between the use of embryo woodland reserve. Regarding the objective of the horse-rearing, transference and seropositivity, both through the Exact Test of 40 farmers (75.3%) reared horses for commercial purposes, and Fisher or chi-square test and through the multivariate analysis 34 (64.1%) for commercial purposes and reproduction. Forty-eight using the generalized estimating equations (GEE). In this latter farms (90.6%) reared both horses and cattle, concomitantly, among which 22 (45.8%) combined the two species in the same test, horses on farms that did not use embryo transference had twice as much risk of infection (95% CI: 1.086-3.003) as farms pasture. On 47 farms (88.7%), there were other animals besides that did use this technique (p = 0.023). horses and cattle, with the presence of dogs on 45 farms (84.9%). Neospora spp. The TP for Neospora spp. was 23.9% (95% Interaction between the different species of animals occurred in 39 (73.6%) herds, to a greater or lesser degree. Regarding the farming CI: 19.9-28.1%), with 105 seropositive horses. On the farms, the system, 36 farmers (67.9%) maintained their animals exclusively TP was 85% (95% CI: 70.7-96.1%), with at least one positive in picket-fenced enclosures. Considering food management, animal on 41 farms. Farms in the southern part of the state of 47 farms (88.7%) used grass and also offered feed to the animals, Minas Gerais that offered hay to their horses presented lower risk Table 1. Epidemiological variables associated with antibodies for S. neurona (IFAT 1:80), in the univariate analysis (P < 0.05), among horses reared in the southern part of the state of Minas Gerais, Brazil. Sarcocystis neurona Odds Factors Positive Negative 95% CI P* ratio (OR) n % n % Feed manufactured on the farm (n = 47**) 0.026 No 13 65.00 7 35.00 1 Yes 25 92.59 2 7.41 6.731 1.219-37.155 Farm includes or borders a forest reserve (n = 53) 0.030 No 2 33.33 3 66.67 1 Yes 42 87.50 6 12.50 10.500 1.445-76.289 IFAT: indirect fluorescent antibody test; CI: confidence interval. *Exact Test of Fisher (P < 0.05). **Six farms did not use feed. There was no significant association (P > 0.05) between seropositivity and the following variables: sex; age; keeping horses in stables; and purpose of horse-rearing. 146 Ribeiro, M.J.M. et al. Braz. J. Vet. Parasitol. (p < 0.05) of having animals seropositive for Neospora spp. Farms Mixed infection on which the horses were in contact with other animals, or in which horses over the age of five years were present, presented Mixed infection by S. neurona and T. gondii occurred in 3.4% greater risk (p < 0.05) of infection (Table  3). Horses on farms (17/506) of the horses, while 3.2% (16/506) were positive for both on which cattle and cats were also present showed greater risk T. gondii and Neospora spp. Horses infected by S. neurona and (p < 0.05) of being infected by Neospora spp., from the multiple Neospora spp. represented 2.8% (14/506) of the sample. Infection logistic regression through the generalized estimating equations involving the three agents occurred among only 1.4% (7/506) of (GEE) (Table 4). animals. In 11.3% (6/53) of the farms there was positivity for all Table 2. Epidemiological variables associated with antibodies for T. gondii (IFAT 1:64), in the univariate analysis (P < 0.05), among horses reared in the southern part of the state of Minas Gerais, Brazil. Toxoplasma gondii Odds Factors Positive Negative 95% CI P* ratio (OR) n % n % Total number of horses on the farm (n = 53) 0.033 >100 animals 6 60.00 4 40.00 1 <100 animals 39 90.69 4 9.31 6.493 1.272-33.333 Vaccine against tetanus (n = 53) 0.038 Yes 3 50.00 3 50.00 1 No 42 89.36 5 10.64 8.403 1.321-52.631 Vaccine against influenza (n = 53) 0.014 Yes 12 66.66 6 33.34 1 No 33 94.28 2 5.72 8.264 1.459-47.619 Use of embryo transfer (n = 53) 0.017 Yes 17 70.83 7 29.17 1 No 28 96.55 1 3.45 11.494 1.303-99.999 Use of a treatment, cleaning and care area for the animals (n = 53) 0.050 Yes 20 74.07 7 25.93 1 No 25 96.15 1 3.85 8.771 0.993-76.923 Age of the animals (n= 485**) 0.011 < 5 years 36 16.29 185 83.71 1 > 5 years 68 25.76 196 74.24 1.783 1.135-2.799 IFAT: indirect fluorescent antibody test; CI: confidence interval. *Exact Test of Fisher and chi-square test (P < 0.05). **The ages of 21 horses were not informed. There was no significant association (P > 0.05) between seropositivity and the following variables: presence of cats; rearing purpose and system; sex; and rearing of horses and cattle together. Table 3. Epidemiological variables associated with antibodies for Neospora spp. (IFAT 1:50), in the univariate analysis (P < 0.05), among horses reared in the southern part of the state of Minas Gerais, Brazil. Neospora spp. Odds Factors Positive Negative 95% CI P* ratio (OR) N % n % Supply of hay (n= 53) 0.045 Yes 13 61.90 8 38.10 1 No 28 87.50 4 12.50 4.310 1.096-16.949 Contact between horses and other animals (n= 53) 0.008 No 7 50.00 7 50.00 1 Yes 34 87.18 5 12.82 6.800 1.666-27.761 Age of the animals (n= 485**) 0.034 < 5 years 37 16.74 184 83.26 1 > 5 years 65 24.62 199 75.38 1.624 1.035-2.549 IFAT: indirect fluorescent antibody test; CI: confidence interval. *Exact Test of Fisher and chi-square test (P < 0.05). **The ages of 21 horses were not informed. There was no significant association (P > 0.05) between seropositivity and the following variables: presence of dogs on the property; sex of the animals; and occurrence of either reproductive or neurological disorders in the equine herds. Seroepidemiology of Sarcoystis neurona 147 v. 25, n. 2, abr.-jun. 2016 Table 4. Epidemiological variables associated with antibodies for Neospora spp. (IFAT 1:50), in the multiple logistic regression analysis using the generalized estimate equations, among horses reared in the southern part of the state of Minas Gerais, Brazil. Neospora spp. Adjusted Odds Factors Positive Negative CI 95% (OR) P Ratio (OR) n % n % Presence of cattle (n= 53) 0.007 No 2 40.00 3 60.00 1 Yes 39 81.25 9 18.75 5.280 1.585-17.598 Presence of cats (n= 53) 0.045 No 21 67.74 10 32.26 1 Yes 15 93.75 1 6.25 1.881 1.015-3.485 IFAT: indirect fluorescent antibody test; CI: confidence interval. QIC (Quasilikelihood under the Independence model Criterion): 448.2; QICC: 446.4 (The QIC and QICC was used as GEE logistic regression goodness of fit measure, considering that when QICC approximates QIC the GEE model is correctly specified and that the model with the smaller QIC and QICC statistics was preferred). three agents, and all farms presented horses infected by at least The possum ( Didelphis albiventris) is a broadly distributed animal one protozoon. However, 47.2% (239/506) of the horses were in South America for which primary and secondary woodlands are seronegative for all three parasites. its natural habitat (ORR, 1986). Given its high synanthropy, this species is a potential disseminator of pathogens between domestic animals and man (MULLER et al., 2005). This would explain the Discussion association between the presence of forest reserves either on the farms or bordering the farms and seropositivity for S. neurona, Farm profile which has also been reported in other studies (MORLEY et al., 2008; DUBEY et al., 2015). The greater risk of infected animals Most of the farms presented interaction between different found on farms that manufactured their own feed suggests that animals, mostly between horses and cattle and/or dogs, which the storage of the raw material used in the formulation was were the two predominant species on these farms. Moreover, the inadequate, thus allowing access by wild animals (possums) to herds were mostly located in pastures and near woodlands, thus the food (MORLEY et al., 2008). allowing contact with wild fauna and, consequently, favoring Toxoplasma gondii. The prevalence of anti- T. gondii antibodies exposure of the horses to the parasites studied in this investigation among the horses (19.9%) was within the range observed in serological (ABREU et al., 2014; DUBEY et al., 2015). Most of the horses studies carried in Brazil, 31.5% (IFAT) in states of Mato Grosso, were reared for commercial purposes, thus allowing a large flow Mato Grosso do Sul, Paraná and São Paulo (VIDOTTO et al., of animals, which favors dispersion of the agents (MOURA et al., 1997), and 2.1% (IFAT) in Rio de Janeiro (STELMANN, 2014), 2013). The majority of the farms showed good sanitary practices, and same 17.7% (IFAT) in Paraná (FINGER et al., 2013), and such as vaccinating the animals, sanitizing the installations, storing abroad, 2.6% (IFAT) in South Korea (GUPTA  et  al., 2002) food adequately and having veterinarian assistance. This demonstrated and 40.5% (IFAT) in Egypt (GHAZY et al, 2007). The high the owners’ concern for caring for the herd’s health, which could prevalence among farms (71.6%) indicated that there was wide have influenced the seroprevalence rates of the agents analyzed dispersion of the parasite in the region. Thus, we can infer that and the absence of clinical symptomatology among the animals. positive farms (those with at least one seropositive equine) had potential risk factors for infection with T. gondii. Considering the intense movement of horses between farms, as most farms Seroprevalence and variables associated with infection had animals that participated in commercial events, which may Sarcocystis neurona. The prevalence of S. neurona among have predisposed the animals towards infection by T. gondii, as horses (26.0%) was within the range observed in previous studies suggested by Kouam et al. (2010). conducted in Brazil, 37.7% (Western blot) in Rio Grande do In the present study, the lower risk of seropositivity for T. gondii Sul (LINS  et  al., 2012) and 8.3% (IFAT) in Rio de Janeiro that was found on farms with more than 100 horses can be partly (STELMANN, 2014), and worldwide, same 26.1% (Western explained by the fact that these larger farms were technologically blot) in Argentina (MORÉ et al., 2014) and 27.6% (IFAT) in driven and their owners were better informed. On the other hand, EUA (PUSTERLA  et  al., 2014). The high prevalence among the precarious manner of storing the food offered to the horses herds (88.3%) can be explained by the fact that most horses were (DUBEY & SU, 2009), as well as environmental conditions favorable reared in picket-fenced enclosures, thus facilitating their contact towards development and survival of parasites (KOUAM et al., with wild fauna, which predisposed the animals towards infection 2010), helps to explain the greater risk of contamination of horses by S. neurona (MORLEY et al., 2008). In addition, the presence on small farms. of woodlands near the pastures in the absolute majority of the Although there was no direct relationship with the form of farms (90.57%), probably increased the risk of contact between transmission of T. gondii, the lower risk of seropositivity observed horses and wild animals, such as possums (DUBEY et al., 2015). in farms that had their animals vaccinated against tetanus and 148 Ribeiro, M.J.M. et al. Braz. J. Vet. Parasitol. influenza, or that used a specific area to care for the animals, allows indicates that on the farms where hay is supplied as food, there the inference that the owners are concerned about the health of is greater confinement of the animals in stalls and lower contact their animals. Thus, use of hygienic care and sanitary measures, with pastures. Thus, there is a lower risk of contact between the including those that are protection factors against other agents horses in stables and the definitive hosts (ABREU et al., 2014). and not directly against T. gondii, indirectly contribute towards Greater positivity of Neospora spp. among horses above the age reducing the risk of exposure or preventing immunosuppressant of five years suggests that horizontal transmission predominates, coinfections, which would be able to increase the risk of infection considering that only 16.7% (37/221) of the animals below the though this parasite (TASSI, 2007). age of five years were infected. This lower efficiency of congenital Greater seropositivity among horses over the age of five years transmission among horses, in comparison with cattle, can be can be explained by the fact that older animals have had more partly explained by placentation differences (PITEL et al., 2003). chances of becoming exposed to T. gondii over the course of their In the south of the state of Minas Gerais, horse farms with lives and, consequently, of becoming infected and producing the presence of cattle presented a significant association with antibodies against this protozoon. infection by Neospora spp. These results corroborate the findings The farms in the south of the state of Minas Gerais that used of Moura et al. (2013) and Abreu et al. (2014), thus suggesting embryo transfer techniques presented lower risk of horses becoming that cattle are able to maintain the agent on the farms, since dogs infected by T. gondii. Embryo transfer is a modern form of animal commonly have access to bovine meat and their placental remains. reproduction technology and is not directly related to transmission The significant association between seropositivity for the of T. gondii. However, we can infer that these properties were agent and the presence of cats in the farms suggests that there is more technologically-oriented, with use of good hygiene-sanitary greater presence of rodents on these farms. These animals can act practices capable of controlling various illnesses on the farm, and as intermediate hosts for Neospora spp. and are able to maintain that either directly or indirectly, these practices contributed towards reducing the risk of horse infections by T. gondii. Thus, these farms the parasite’s cycle on the farm and infect dogs, which due to were better prepared to implement preventive measures against their carnivorous habit, hunt these rodents. A similar result was T. gondii, such as safe storage of food and hygiene of installations observed by Stelmann (2014), who correlated equine neosporosis (DUBEY & SU, 2009). Thus, these farms avoided environmental with access of cats to food. Although there are studies that have conditions that would favor development and survival of parasite correlated infection by Neospora spp. with the presence of dogs oocysts, such as warm and humid locations (LANGONI et al., and contact between them and horses (MOURA et al., 2013), 2007; KOUAM et al., 2010). this was not shown on these farms in the southern part of the Although the presence of cats on the farm is a risk factor state of Minas Gerais. This result may suggest that the dogs were (DUBEY & SU, 2009), this situation was not observed in the probably not infected, or were not in the habit of feeding off present study, as also reported by Stelmann (2014). The presence placental remains and tissues of the animals. of seronegative cats on the farms and/or intense traffic of animals In the present study there was no significant association in the region could explain the absence of this correlation between sex and seropositivity to Neospora spp., which was also (LANGONI et al., 2007). There was no significant association observed previously by Moura et al. (2013). Similarly, animals between sex, rearing purpose or rearing system (stable or pasture) with a history of either reproductive or neurological disorders and seropositivity, and thus our results were in agreement with did not present any significant correlation with the infection. those of Stelmann (2014), although in disagreement with those In the case of reproductive disorders, there was also no association of Kouam et al. (2010). with positivity in the study by Moura  et  al. (2013). However, Neospora spp. The seroprevalence of Neospora spp. (23.9%) correlations between reproductive alterations and infection by among the horses was within the range observed in other the parasite were reported by Locatelli-Dittrich et al. (2006) and studies (IFAT) carried out in Brazil, 57.6% (IFAT) in São Abreu et al. (2014). Paulo (STELMANN  et  al., 2011) and 4.1% (IFAT) in Santa Catarina (MOURA  et  al., 2013), and same 25.7% (IFAT) in Santa Catarina (ABREU et al., 2014), and abroad, 8.9% (IFAT) Conclusions in Israel (KLIGLER  et  al., 2007) and 28.0% (IFAT) in Italy (CIARAMELLA  et  al., 2004), and same 24,0% (ELISA) in The results from the present study allow it to be stated that Czech Republic (BÁRTOVÁ et al., 2010). The wide distribution the southern part of the state of Minas Gerais is an enzootic of the infection among the farms (85.0%) was probably due to area for S. neurona, T. gondii and Neospora spp., with a wide the environmental conditions of the southern part of the state of distribution of these parasites among Mangalarga Marchador horse Minas Gerais, typical of Brazilian tropical regions, which favor farms. However, asymptomatic subclinical or chronic infection sporulation of Neospora spp. oocysts (MOURA et al., 2013). predominates in the region. Horizontal transmission seems to be The association between seropositivity for Neospora spp. and the main form of infection for these parasites, and different factors “no supply of hay” suggests that farms that are able to provide are associated with seropositivity among horses. More research is hay for their horses are more technologically-oriented and their needed in order to establish precisely which factors increase the owners are better informed. Thus, these owners take greater care risk of transmitting these agents, as well as to determine which regarding storage and supply of food, thereby minimizing the risk of animal infection. Moreover, hay is a source of roughage, which species of Neospora (N. caninum or N. hughesi) infects horses. Seroepidemiology of Sarcoystis neurona 149 v. 25, n. 2, abr.-jun. 2016 190-195. http://dx.doi.org/10.1590/S0074-02762009000200011. Acknowledgements PMid:19430643. To the Research Support Foundation of the State of Minas Dubey JP, Venturini MC, Venturini L, Mckinney J, Pecoraro M. Prevalence of antibodies to Sarcocystis neurona, Toxoplasma gondii, and Neospora Gerais (Fundação de Amparo à Pesquisa do Estado de Minas Gerais, caninum in horses from Argentina. Vet Parasitol 1999; 86(1): 59-62. 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Seroepidemiology of Sarcocystis neurona, Toxoplasma gondii and Neospora spp. among horses in the south of the state of Minas Gerais, Brazil

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Abstract

The present study used the indirect fluorescent antibody test (IFAT) to determine the seroprevalence of Sarcocystis neurona, Toxoplasma gondii and Neospora spp., and evaluated the variables associated with these infections among 506 apparently healthy horses, reared in the south of the state of Minas Gerais, Brazil. This study was conducted between April 2012 and October 2013. Among the horses, the true prevalence of S. neurona was 26% (95% CI: 22.0-30.4%), T. gondii 19.9% (95% CI: 15.5-24.8%) and Neospora spp. 23.9% (95% CI: 19.9-28.1%); and among the farms, 88.3% (95% CI: 74.4-91.6%), 71.6% (95% CI: 41-92.8%) and 85% (95% CI: 70.7-96.1%), respectively. Regarding mixed infection, 17 horses (3.4%) were seropositive for both S. neurona and T. gondii, 16 (3.2%) for T. gondii and Neospora spp. and 14 (2.8%) for S. neurona and Neospora spp. The associations between seropositivity and variables relating to the structure of the farm, management and health were analyzed using the logistic regression analysis, through the generalized estimating equations (GEE). The results suggest that the south of Minas Gerais is an enzootic area for S. neurona, T. gondii and Neospora spp. among horses, with prevalence of asymptomatic subclinical or chronic infections. Keywords: Apicomplexa, Sarcocystidae, indirect immunofluorescence, risk factors. Resumo Este estudo determinou, pela reação de imunofluorescência indireta (RIFI), a soroprevalência de Sarcocystis neurona, Toxoplasma gondii e Neospora spp., e avaliou as variáveis associadas com a infecção, em 506 equinos, aparentemente sadios, criados no Sul de Minas, Brasil. O estudo foi realizado no período de abril de 2012 a outubro de 2013. Entre equinos, a prevalência verdadeira para S. neurona foi de 26% (IC 95%= 22,0-30,4%); para T. gondii de 19,9% (IC 95%= 15,5-24,8%); e para Neospora spp. de 23,9% (IC 95%= 19,9-28,1%); e entre propriedades, 88,3% (IC 95%= 74,4-91,6%), 71,6% (IC 95% = 41-92,8%), e 85% (IC 95%= 70,7-96,1%), respectivamente. Em relação à infecção mista, 17 (3,4%) equinos foram soropositivos para S. neurona e T. gondii, 16 (3,2%) para T. gondii e Neospora spp., e 14 (2,8%) para S. neurona e Neospora spp. A associação entre soropositividade e variáveis relacionadas à estrutura da propriedade, manejo e sanidade, foi analisada, utilizando-se a análise de regressão logística estimada por Generalized Estimating Equations (GEE). Os resultados sugerem que o Sul de Minas é área enzoótica para S. neurona, T. gondii e Neospora spp. em equinos, com predomínio de infecções subclínicas ou crônicas, assintomáticas. Palavras-chave: Apicomplexa, Sarcocystidae, imunofluorescência indireta, fatores de risco. *Corresponding author: Antônio Marcos Guimarães. Departamento de Medicina Veterinária – DMV, Universidade Federal de Lavras – UFLA, CP 3037, CEP 37200-000, Lavras, MG, Brasil. e-mail: amg@dmv.ufla.br www.cbpv.org.br/rbpv Seroepidemiology of Sarcoystis neurona 143 v. 25, n. 2, abr.-jun. 2016 These three protozoa of the phylum Apicomplexa, family Introduction Sarcocystidae, are transmitted through consumption of water and food contaminated by sporocysts, in the case of S. neurona, and Brazil possesses one of the largest horse herds in the world, with sporulated oocysts, in the cases of T. gondii and Neospora spp., approximately 5.3 million head. Horse-rearing is an important which are released by the definitive hosts: respectively, possums, source of income and employment. In the southern region of the felids and canids (LANGONI et al., 2007; DUBEY & SU, 2009; state of Minas Gerais, horse rearing occupies an important space DUBEY et al., 2015). The infection is usually asymptomatic among in the local economic, touristic, historic and cultural scene, since horses, but may sporadically present nervous symptoms in the this was the birthplace of the Mangalarga Marchador horse breed, which is internationally admired (IBGE, 2013). cases of S. neurona, T. gondii and N. hughesi, and reproductive Equine protozoal myeloencephalitis, caused by Sarcocystis disorders, when involving T. gondii and N. caninum (LOCATELLI- neurona, is a disease characterized by a neurological condition DITTRICH et al., 2006; GENNARI et al., 2015). in which horses participates as an accidental host. In Brazil, the Because of the importance of horse-rearing in the south of the seroprevalence of S. neurona among horses has been estimated in state of Minas Gerais, determining the prevalences of S. neurona, 37.7% (Western blot) (LINS et al., 2012) and 33.7% (ELISA) T. gondii and Neospora spp., along with the factors associated with (PIVOTO et al., 2014) in Rio Grande do Sul, and 8.75% (IFAT) these infections, is fundamental for implementing effective control in Rio de Janeiro (STELMANN, 2014). Worldwide seroprevalence measures, so as to reduce the exposure of the animals and prevent varies between 0% and 89.2% (DUBEY et al., 2015). the economic losses caused by these parasites. Moreover, this type Toxoplasmosis, the disease caused by Toxoplasma gondii, is of investigation increases the knowledge on the distribution of a zoonosis of global interest due to its impact on public health. these agents among the horse population nationally. It can be transmitted through consumption of raw or undercooked Thus, due to the scarcity of studies in Brazil and lack of meat, including horse meat (DUBEY & SU, 2009). The levels of information regarding equine infection in the southern part of antibodies for T. gondii among horses in Brazil has been estimated the state of Minas Gerais, the present study had the objectives of in 12.8% (IFAT) in Minas Gerais (NAVES et al., 2005), 2.5% determining the prevalence of S. neurona, T. gondii and Neospora (IFAT) in Mato Grosso do Sul (LASKOSKI et al., 2015) and 28,4% spp., and identifying possible factors associated with seropositivity (IFAT) in states of Alagoas, Paraíba, Pernambuco, Rio Grande do among Mangalarga Marchador horses. Norte and Minas Gerais (GENNARI et al., 2015). Worldwide, seroprevalence varies between 0% and 72.2% (ALSHAHERY & Materials and Methods MANSOUR, 2012; MATSUO et al., 2014). Neosporosis is a parasitic disease caused by the protozoon Neospora Study area and animals caninum, which can infect several mammals, including horses, and by N. hughesi, which infects horses and is an uncommon etiological Blood samples were gathered from 506 Mangalarga Marchador agent for equine protozoal myeloencephalitis (GOODSWEN et al., horses that had been reared on 53 farms that were distributed among 2013). In Brazil, the prevalence of anti-Neospora spp. antibodies 27 municipalities in the microregions of Andrelândia, Lavras, São among horses has been estimated in 57.6% (IFAT) in São Paulo João Del Rey, São Lourenço and Varginha, in the southern portion (STELMANN  et  al., 2011), 25.7% (IFAT) in Santa Catarina of the state of Minas Gerais (Figure 1). The study was conducted (ABREU  et  al., 2014), and 1.87% (IFAT) in Rio de Janeiro (STELMANN, 2014) and, abroad, from 0% to 35% (DUBEY et al., between April 2012 and October 2013 after approval had been 1999; PATITUCCI et al., 2004). obtained from the Ethics Commission for Animal Use (COEA) Figure 1. Map of the state of Minas Gerais, Brazil, highlighting the location of the municipalities of the southern region that were involved in the study. 144 Ribeiro, M.J.M. et al. Braz. J. Vet. Parasitol. of the Federal University of Lavras (UFLA), and was registered temperature, they were stored in a freezer at –20 °C until the at this institution under number 023/11. time of use. The  IFAT for T. gondii was performed using the The farms were chosen randomly based on guidance from technique described by Langoni et al. (2007) and the antigens the Brazilian Association of Mangalarga Marchador Horse were the tachyzoites of the parasite. After successive inoculations, Breeders (Associação Brasileira de Criadores de Cavalos Mangalarga the tachyzoites were recovered through rinsing the peritoneal Marchador, ABCCMM). In this sampling, the proportions between cavity of mice with a saline solution. Ten microliters of a solution the mesoregions and the sizes of farms and their structures were containing tachyzoites was placed in each of the 12 slide wells maintained in order to attain homogeneous representativeness. for IFAT (about 10 tachyzoites / field at 40x magnification in At least ten horses from each farm were required for the prevalence an optical microscope). After the slides had been dried at room calculation on S. neurona, T. gondii and Neospora spp. In addition, temperature, they were stored in a freezer at –20 °C until the pregnant mares and foals of less than six months of age were not time of the serological tests. In the present study, N. caninum included in the sampling. The reasons were that pregnant mares tachyzoites were used as the antigen in the IFAT, and their antibodies are more sensitive to manipulation, while foals younger than six presented cross-reactions with N. hughesi (GONDIM et al., 2009). months may present colostral antibodies (passive immunity), The IFAT for Neospora spp. was performed in accordance with which would interfere with the serological diagnosis. The blood the technique described by Paré et al. (1995), using glass slides samples were obtained by means of jugular venipuncture and were containing tachyzoites of N. caninum as the antigen (Laboratório collected in Vacutainer tubes. All the animals appeared to be healthy. Imunodot, Jaboticabal, Brazil). After centrifugation at 500 g for 10 minutes, the serum samples An equine anti-IgG conjugate (SIGMA, Saint Louis, MO, USA) from 506 horses were stored in individually identified cryotubes was used for performing the IFAT, at a dilution of 1:64, along with and frozen at –20 °C until the serological tests were performed. positive (serum samples of mares seropositive to S. neurona, T. gondii The definition of the number of horses (n) needed to estimate and Neospora spp.) and negative (serum samples of newborn foal the prevalence was based on the formula of the Pan-American before colostrum ingestion and serum samples of periparturient Zoonosis Center (CEPANZO, 1979), which is appropriate mare, both seronegative to respective protozoa) controls for the for calculating samples for research on chronic diseases: respective protozoa. The serum samples were considered positive if 2 2 n = [p x (100-p) x Z ]/(d x p/100) . Considering an estimated they presented complete fluorescence of the S. neurona merozoites prevalence (p) of 50%, a 95% confidence interval (z) of 1.96 and at a dilution of 1:80 (DUBEY et al., 2015), and of the T. gondii an acceptable error margin (d) of 10%, the minimum number and Neospora spp. tachyzoites, respectively, at dilutions of 1:64 (n) of horses was 384. (ABREU et al., 2014) and 1:50 (LOCATELLI-DITTRICH et al., 2006). Both the IFAT and the preparation of slides containing S. neurona and T. gondii antigens were performed at the Parasite Information-gathering on the farms Disease Laboratory of the Federal University of Lavras (UFLA). A questionnaire-based interview was carried out with either the owners or the person responsible for each farm. The questions Statistical analyses related to herd health, the breeder, management and the structure of the farm. Moreover, questions asking about any history of The EpiData software was used to tabulate the data relating to reproductive problems and the neurological condition of the herds the variables studied. These data were later ranked and analyzed were also devised. This information served as independent variables using the statistical software SPSS 20.0 statistical software (IBM, for subsequent analyses that evaluated the associations between 2011). In calculating true prevalence (TP) for the anti-S. neurona seropositivity to the protozoa and factors relating to sociocultural antibodies, values of 94% and 88.9% were used for sensitivity (SE) information, herd health and management practices observed and specificity (SP), respectively, as prescribed by Duarte et al. on the farms. In total, 305 variables were analyzed, which were (2003). In the case of TP for T. gondii, the SE and SP values were, obtained by means of the questionnaire. respectively, 90.9% and 100% (LU et al., 2013). In turn, TP for Neospora spp. presented values of 91.0% and 99.0%, respectively, for SE and SP, in accordance with Björkman & Uggla (1999). Serological tests Subsequently, the SE and SP between the herds were used to calculate the TP at herd level (NOORDHUIZEN et al., 2001), by The indirect fluorescent antibody test (IFAT) was the method means of the epidemiological calculators of the Epitools software chosen for investigating anti-S. neurona, T. gondii and Neospora spp. IgG antibodies. This is considered to be the gold-standard test for (SERGEANT, 2011). The variables relating to the structural, serologically diagnosing the latter two protozoa (LANGONI et al., sanitary and management characteristics of the farms, along with 2007; DUBEY & SCHARES, 2011). the inherent characteristics of the animals, were firstly subjected to In investigating anti-S. neurona antibodies, the IFAT was the chi-square test or Exact Test of Fisher (less than five observations performed as described by Johnson et al. (2013). The antigens were in any category) using the SPSS 20.0 software (IBM, 2011), so as to demonstrate their association with the seropositivity of the S. neurona merozoites, produced in cell cultures. Ten microliters of S. neurona merozoite solution was placed in each one of the 12 slide studied agents. This association, when statistically significant, wells for IFAT (about 10 merozoites / field at 40x magnification was quantified by the odds ratio and its confidence interval at in an optical microscope). After slides had been dried at room 95% (CI. 95%), which estimates the risk relationship between Seroepidemiology of Sarcoystis neurona 145 v. 25, n. 2, abr.-jun. 2016 independents variables (risk or protective factors) and the dependent among which 21 (44.7%) manufactured the concentrate on-site. variables (serology results). Variables that presented p values < 0.05 Regarding sanitary management, 50 farmers (94.3%) vaccinated were considered significant, meaning that they were statistically their animals, and 31 (58.5%) had veterinary assistance. Most of associated with seropositivity to S. neurona, T. gondii and Neospora the farms used artificial insemination techniques (62.3%). Horse- spp. Subsequently, the variables that presented p values < 0.20 in rearing was the main source of income on 13 farms (24.5%), and the chi-square test or Exact Test of Fisher were subjected to the 33 (62.3%) had been conducting this activity for over 20 years. logistic regression analysis, through the generalized estimating equations (GEE) (CORBELLINI et al., 2006; BRUHN et al., Seroprevalence and variables associated with infection 2013). Variables that presented p values < 0.05 in the GEE were considered to be significantly associated with seropositivity for Sarcocystis neurona. Out of the 506 horses, 117 (23.0%) S. neurona, T. gondii and Neospora spp. presented anti-S. neurona antibodies, with true prevalence (TP) The QIC (Quasilikelihood under the Independence model of 26.0% (95% CI: 22.0-30.4%). Out of the 53 properties Criterion) and the related QICC statistics, proposed by Pan analyzed, 44 had at least one seropositive horse, with TP of 88.3% (2001) as GEE logistic regression goodness of fit measure, was (95% CI: 74.4-91.6%). As shown in Table 1, the properties that used to compare the GEE models, considering that when QICC included forest reserves or that bordered a reserve presented a high approximates QIC the GEE model is correctly specified and that risk (p < 0.05) of having horses seropositive for S. neurona. Farms the model with the smaller QIC and QICC statistics is preferred. that manufactured their own feed also had greater risk (p < 0.05) of having infected horses. Toxoplasma gondii. In the case of T. gondii, 107 (21.1%) Results horses were seropositive, representing a TP of 19.9% (95% CI: 15.5-24.8%). On 45 farms, at least one horse was seropositive, Farm profile indicating a TP of 71.6% (95% CI: 41.0-92.8%). Farms that had more than 100 horses presented lower risk (p < 0.05) of having Among the 506 animals analyzed, 343 were female (67.8%) infected animals. Farms that vaccinated their horses against tetanus and 163 were male (32.2%). There were 61 foals (12.1%) and and influenza, and those that had an area for treating, cleaning 445 adults (87.9%). Among the 53 farms, horse-rearing was the and caring for the animals, presented lower risk (p < 0.05) of main activity on only 21 of them (39.6%). Regarding size, 27 farms infection. Horses over the age of five years were significantly (51.0%) covered more than 100 ha, with a mean area of 199.1 ha. (p < 0.05) more liable to infection by T. gondii (Table 2). There was Forty-eight farms (90.6%) either bordered or included a forest or a significant association (p < 0.05) between the use of embryo woodland reserve. Regarding the objective of the horse-rearing, transference and seropositivity, both through the Exact Test of 40 farmers (75.3%) reared horses for commercial purposes, and Fisher or chi-square test and through the multivariate analysis 34 (64.1%) for commercial purposes and reproduction. Forty-eight using the generalized estimating equations (GEE). In this latter farms (90.6%) reared both horses and cattle, concomitantly, among which 22 (45.8%) combined the two species in the same test, horses on farms that did not use embryo transference had twice as much risk of infection (95% CI: 1.086-3.003) as farms pasture. On 47 farms (88.7%), there were other animals besides that did use this technique (p = 0.023). horses and cattle, with the presence of dogs on 45 farms (84.9%). Neospora spp. The TP for Neospora spp. was 23.9% (95% Interaction between the different species of animals occurred in 39 (73.6%) herds, to a greater or lesser degree. Regarding the farming CI: 19.9-28.1%), with 105 seropositive horses. On the farms, the system, 36 farmers (67.9%) maintained their animals exclusively TP was 85% (95% CI: 70.7-96.1%), with at least one positive in picket-fenced enclosures. Considering food management, animal on 41 farms. Farms in the southern part of the state of 47 farms (88.7%) used grass and also offered feed to the animals, Minas Gerais that offered hay to their horses presented lower risk Table 1. Epidemiological variables associated with antibodies for S. neurona (IFAT 1:80), in the univariate analysis (P < 0.05), among horses reared in the southern part of the state of Minas Gerais, Brazil. Sarcocystis neurona Odds Factors Positive Negative 95% CI P* ratio (OR) n % n % Feed manufactured on the farm (n = 47**) 0.026 No 13 65.00 7 35.00 1 Yes 25 92.59 2 7.41 6.731 1.219-37.155 Farm includes or borders a forest reserve (n = 53) 0.030 No 2 33.33 3 66.67 1 Yes 42 87.50 6 12.50 10.500 1.445-76.289 IFAT: indirect fluorescent antibody test; CI: confidence interval. *Exact Test of Fisher (P < 0.05). **Six farms did not use feed. There was no significant association (P > 0.05) between seropositivity and the following variables: sex; age; keeping horses in stables; and purpose of horse-rearing. 146 Ribeiro, M.J.M. et al. Braz. J. Vet. Parasitol. (p < 0.05) of having animals seropositive for Neospora spp. Farms Mixed infection on which the horses were in contact with other animals, or in which horses over the age of five years were present, presented Mixed infection by S. neurona and T. gondii occurred in 3.4% greater risk (p < 0.05) of infection (Table  3). Horses on farms (17/506) of the horses, while 3.2% (16/506) were positive for both on which cattle and cats were also present showed greater risk T. gondii and Neospora spp. Horses infected by S. neurona and (p < 0.05) of being infected by Neospora spp., from the multiple Neospora spp. represented 2.8% (14/506) of the sample. Infection logistic regression through the generalized estimating equations involving the three agents occurred among only 1.4% (7/506) of (GEE) (Table 4). animals. In 11.3% (6/53) of the farms there was positivity for all Table 2. Epidemiological variables associated with antibodies for T. gondii (IFAT 1:64), in the univariate analysis (P < 0.05), among horses reared in the southern part of the state of Minas Gerais, Brazil. Toxoplasma gondii Odds Factors Positive Negative 95% CI P* ratio (OR) n % n % Total number of horses on the farm (n = 53) 0.033 >100 animals 6 60.00 4 40.00 1 <100 animals 39 90.69 4 9.31 6.493 1.272-33.333 Vaccine against tetanus (n = 53) 0.038 Yes 3 50.00 3 50.00 1 No 42 89.36 5 10.64 8.403 1.321-52.631 Vaccine against influenza (n = 53) 0.014 Yes 12 66.66 6 33.34 1 No 33 94.28 2 5.72 8.264 1.459-47.619 Use of embryo transfer (n = 53) 0.017 Yes 17 70.83 7 29.17 1 No 28 96.55 1 3.45 11.494 1.303-99.999 Use of a treatment, cleaning and care area for the animals (n = 53) 0.050 Yes 20 74.07 7 25.93 1 No 25 96.15 1 3.85 8.771 0.993-76.923 Age of the animals (n= 485**) 0.011 < 5 years 36 16.29 185 83.71 1 > 5 years 68 25.76 196 74.24 1.783 1.135-2.799 IFAT: indirect fluorescent antibody test; CI: confidence interval. *Exact Test of Fisher and chi-square test (P < 0.05). **The ages of 21 horses were not informed. There was no significant association (P > 0.05) between seropositivity and the following variables: presence of cats; rearing purpose and system; sex; and rearing of horses and cattle together. Table 3. Epidemiological variables associated with antibodies for Neospora spp. (IFAT 1:50), in the univariate analysis (P < 0.05), among horses reared in the southern part of the state of Minas Gerais, Brazil. Neospora spp. Odds Factors Positive Negative 95% CI P* ratio (OR) N % n % Supply of hay (n= 53) 0.045 Yes 13 61.90 8 38.10 1 No 28 87.50 4 12.50 4.310 1.096-16.949 Contact between horses and other animals (n= 53) 0.008 No 7 50.00 7 50.00 1 Yes 34 87.18 5 12.82 6.800 1.666-27.761 Age of the animals (n= 485**) 0.034 < 5 years 37 16.74 184 83.26 1 > 5 years 65 24.62 199 75.38 1.624 1.035-2.549 IFAT: indirect fluorescent antibody test; CI: confidence interval. *Exact Test of Fisher and chi-square test (P < 0.05). **The ages of 21 horses were not informed. There was no significant association (P > 0.05) between seropositivity and the following variables: presence of dogs on the property; sex of the animals; and occurrence of either reproductive or neurological disorders in the equine herds. Seroepidemiology of Sarcoystis neurona 147 v. 25, n. 2, abr.-jun. 2016 Table 4. Epidemiological variables associated with antibodies for Neospora spp. (IFAT 1:50), in the multiple logistic regression analysis using the generalized estimate equations, among horses reared in the southern part of the state of Minas Gerais, Brazil. Neospora spp. Adjusted Odds Factors Positive Negative CI 95% (OR) P Ratio (OR) n % n % Presence of cattle (n= 53) 0.007 No 2 40.00 3 60.00 1 Yes 39 81.25 9 18.75 5.280 1.585-17.598 Presence of cats (n= 53) 0.045 No 21 67.74 10 32.26 1 Yes 15 93.75 1 6.25 1.881 1.015-3.485 IFAT: indirect fluorescent antibody test; CI: confidence interval. QIC (Quasilikelihood under the Independence model Criterion): 448.2; QICC: 446.4 (The QIC and QICC was used as GEE logistic regression goodness of fit measure, considering that when QICC approximates QIC the GEE model is correctly specified and that the model with the smaller QIC and QICC statistics was preferred). three agents, and all farms presented horses infected by at least The possum ( Didelphis albiventris) is a broadly distributed animal one protozoon. However, 47.2% (239/506) of the horses were in South America for which primary and secondary woodlands are seronegative for all three parasites. its natural habitat (ORR, 1986). Given its high synanthropy, this species is a potential disseminator of pathogens between domestic animals and man (MULLER et al., 2005). This would explain the Discussion association between the presence of forest reserves either on the farms or bordering the farms and seropositivity for S. neurona, Farm profile which has also been reported in other studies (MORLEY et al., 2008; DUBEY et al., 2015). The greater risk of infected animals Most of the farms presented interaction between different found on farms that manufactured their own feed suggests that animals, mostly between horses and cattle and/or dogs, which the storage of the raw material used in the formulation was were the two predominant species on these farms. Moreover, the inadequate, thus allowing access by wild animals (possums) to herds were mostly located in pastures and near woodlands, thus the food (MORLEY et al., 2008). allowing contact with wild fauna and, consequently, favoring Toxoplasma gondii. The prevalence of anti- T. gondii antibodies exposure of the horses to the parasites studied in this investigation among the horses (19.9%) was within the range observed in serological (ABREU et al., 2014; DUBEY et al., 2015). Most of the horses studies carried in Brazil, 31.5% (IFAT) in states of Mato Grosso, were reared for commercial purposes, thus allowing a large flow Mato Grosso do Sul, Paraná and São Paulo (VIDOTTO et al., of animals, which favors dispersion of the agents (MOURA et al., 1997), and 2.1% (IFAT) in Rio de Janeiro (STELMANN, 2014), 2013). The majority of the farms showed good sanitary practices, and same 17.7% (IFAT) in Paraná (FINGER et al., 2013), and such as vaccinating the animals, sanitizing the installations, storing abroad, 2.6% (IFAT) in South Korea (GUPTA  et  al., 2002) food adequately and having veterinarian assistance. This demonstrated and 40.5% (IFAT) in Egypt (GHAZY et al, 2007). The high the owners’ concern for caring for the herd’s health, which could prevalence among farms (71.6%) indicated that there was wide have influenced the seroprevalence rates of the agents analyzed dispersion of the parasite in the region. Thus, we can infer that and the absence of clinical symptomatology among the animals. positive farms (those with at least one seropositive equine) had potential risk factors for infection with T. gondii. Considering the intense movement of horses between farms, as most farms Seroprevalence and variables associated with infection had animals that participated in commercial events, which may Sarcocystis neurona. The prevalence of S. neurona among have predisposed the animals towards infection by T. gondii, as horses (26.0%) was within the range observed in previous studies suggested by Kouam et al. (2010). conducted in Brazil, 37.7% (Western blot) in Rio Grande do In the present study, the lower risk of seropositivity for T. gondii Sul (LINS  et  al., 2012) and 8.3% (IFAT) in Rio de Janeiro that was found on farms with more than 100 horses can be partly (STELMANN, 2014), and worldwide, same 26.1% (Western explained by the fact that these larger farms were technologically blot) in Argentina (MORÉ et al., 2014) and 27.6% (IFAT) in driven and their owners were better informed. On the other hand, EUA (PUSTERLA  et  al., 2014). The high prevalence among the precarious manner of storing the food offered to the horses herds (88.3%) can be explained by the fact that most horses were (DUBEY & SU, 2009), as well as environmental conditions favorable reared in picket-fenced enclosures, thus facilitating their contact towards development and survival of parasites (KOUAM et al., with wild fauna, which predisposed the animals towards infection 2010), helps to explain the greater risk of contamination of horses by S. neurona (MORLEY et al., 2008). In addition, the presence on small farms. of woodlands near the pastures in the absolute majority of the Although there was no direct relationship with the form of farms (90.57%), probably increased the risk of contact between transmission of T. gondii, the lower risk of seropositivity observed horses and wild animals, such as possums (DUBEY et al., 2015). in farms that had their animals vaccinated against tetanus and 148 Ribeiro, M.J.M. et al. Braz. J. Vet. Parasitol. influenza, or that used a specific area to care for the animals, allows indicates that on the farms where hay is supplied as food, there the inference that the owners are concerned about the health of is greater confinement of the animals in stalls and lower contact their animals. Thus, use of hygienic care and sanitary measures, with pastures. Thus, there is a lower risk of contact between the including those that are protection factors against other agents horses in stables and the definitive hosts (ABREU et al., 2014). and not directly against T. gondii, indirectly contribute towards Greater positivity of Neospora spp. among horses above the age reducing the risk of exposure or preventing immunosuppressant of five years suggests that horizontal transmission predominates, coinfections, which would be able to increase the risk of infection considering that only 16.7% (37/221) of the animals below the though this parasite (TASSI, 2007). age of five years were infected. This lower efficiency of congenital Greater seropositivity among horses over the age of five years transmission among horses, in comparison with cattle, can be can be explained by the fact that older animals have had more partly explained by placentation differences (PITEL et al., 2003). chances of becoming exposed to T. gondii over the course of their In the south of the state of Minas Gerais, horse farms with lives and, consequently, of becoming infected and producing the presence of cattle presented a significant association with antibodies against this protozoon. infection by Neospora spp. These results corroborate the findings The farms in the south of the state of Minas Gerais that used of Moura et al. (2013) and Abreu et al. (2014), thus suggesting embryo transfer techniques presented lower risk of horses becoming that cattle are able to maintain the agent on the farms, since dogs infected by T. gondii. Embryo transfer is a modern form of animal commonly have access to bovine meat and their placental remains. reproduction technology and is not directly related to transmission The significant association between seropositivity for the of T. gondii. However, we can infer that these properties were agent and the presence of cats in the farms suggests that there is more technologically-oriented, with use of good hygiene-sanitary greater presence of rodents on these farms. These animals can act practices capable of controlling various illnesses on the farm, and as intermediate hosts for Neospora spp. and are able to maintain that either directly or indirectly, these practices contributed towards reducing the risk of horse infections by T. gondii. Thus, these farms the parasite’s cycle on the farm and infect dogs, which due to were better prepared to implement preventive measures against their carnivorous habit, hunt these rodents. A similar result was T. gondii, such as safe storage of food and hygiene of installations observed by Stelmann (2014), who correlated equine neosporosis (DUBEY & SU, 2009). Thus, these farms avoided environmental with access of cats to food. Although there are studies that have conditions that would favor development and survival of parasite correlated infection by Neospora spp. with the presence of dogs oocysts, such as warm and humid locations (LANGONI et al., and contact between them and horses (MOURA et al., 2013), 2007; KOUAM et al., 2010). this was not shown on these farms in the southern part of the Although the presence of cats on the farm is a risk factor state of Minas Gerais. This result may suggest that the dogs were (DUBEY & SU, 2009), this situation was not observed in the probably not infected, or were not in the habit of feeding off present study, as also reported by Stelmann (2014). The presence placental remains and tissues of the animals. of seronegative cats on the farms and/or intense traffic of animals In the present study there was no significant association in the region could explain the absence of this correlation between sex and seropositivity to Neospora spp., which was also (LANGONI et al., 2007). There was no significant association observed previously by Moura et al. (2013). Similarly, animals between sex, rearing purpose or rearing system (stable or pasture) with a history of either reproductive or neurological disorders and seropositivity, and thus our results were in agreement with did not present any significant correlation with the infection. those of Stelmann (2014), although in disagreement with those In the case of reproductive disorders, there was also no association of Kouam et al. (2010). with positivity in the study by Moura  et  al. (2013). However, Neospora spp. The seroprevalence of Neospora spp. (23.9%) correlations between reproductive alterations and infection by among the horses was within the range observed in other the parasite were reported by Locatelli-Dittrich et al. (2006) and studies (IFAT) carried out in Brazil, 57.6% (IFAT) in São Abreu et al. (2014). Paulo (STELMANN  et  al., 2011) and 4.1% (IFAT) in Santa Catarina (MOURA  et  al., 2013), and same 25.7% (IFAT) in Santa Catarina (ABREU et al., 2014), and abroad, 8.9% (IFAT) Conclusions in Israel (KLIGLER  et  al., 2007) and 28.0% (IFAT) in Italy (CIARAMELLA  et  al., 2004), and same 24,0% (ELISA) in The results from the present study allow it to be stated that Czech Republic (BÁRTOVÁ et al., 2010). The wide distribution the southern part of the state of Minas Gerais is an enzootic of the infection among the farms (85.0%) was probably due to area for S. neurona, T. gondii and Neospora spp., with a wide the environmental conditions of the southern part of the state of distribution of these parasites among Mangalarga Marchador horse Minas Gerais, typical of Brazilian tropical regions, which favor farms. However, asymptomatic subclinical or chronic infection sporulation of Neospora spp. oocysts (MOURA et al., 2013). predominates in the region. Horizontal transmission seems to be The association between seropositivity for Neospora spp. and the main form of infection for these parasites, and different factors “no supply of hay” suggests that farms that are able to provide are associated with seropositivity among horses. More research is hay for their horses are more technologically-oriented and their needed in order to establish precisely which factors increase the owners are better informed. Thus, these owners take greater care risk of transmitting these agents, as well as to determine which regarding storage and supply of food, thereby minimizing the risk of animal infection. Moreover, hay is a source of roughage, which species of Neospora (N. caninum or N. hughesi) infects horses. Seroepidemiology of Sarcoystis neurona 149 v. 25, n. 2, abr.-jun. 2016 190-195. http://dx.doi.org/10.1590/S0074-02762009000200011. Acknowledgements PMid:19430643. To the Research Support Foundation of the State of Minas Dubey JP, Venturini MC, Venturini L, Mckinney J, Pecoraro M. Prevalence of antibodies to Sarcocystis neurona, Toxoplasma gondii, and Neospora Gerais (Fundação de Amparo à Pesquisa do Estado de Minas Gerais, caninum in horses from Argentina. Vet Parasitol 1999; 86(1): 59-62. FAPEMIG), for financial support. To Dr. Luis Fernando Pita http://dx.doi.org/10.1016/S0304-4017(99)00127-2. PMid:10489203. Gondim (UFBA), for the S. neurona merozoites, and to Dr. Ricardo Wagner de Almeida Vitor (UFMG), for the T. gondii tachyzoites. Finger MA, Villalobos EMC, Lara MCCSH, Cunha EMS, Barros-Filho IR, Deconto I, et al. Detection of anti-Toxoplasma gondii antibodies in To Conselho Nacional de Desenvolvimento Científico e Tecnológico carthorses in the metropolitan region of Curitiba, Paraná, Brazil. Rev for research scholarship to C.M.B.M.R. Bras Parasitol Vet 2013; 22(1): 179-181. http://dx.doi.org/10.1590/ S1984-29612013005000001. PMid:23459849. References Gennari SM, Esmerini PO, Lopes MG, Soares HS, Vitaliano SN, Cabral AD,  et  al. Occurrence of antibodies to Toxoplasma gondii and Abreu RA, Weiss RR, Thomaz-Soccol V, Locatelli-Dittrich R, Laskoski its isolation and genotyping in donkeys, mules, and horses in Brazil. LM, Bertol MAF, et al. 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