Abstract

Sarcocystis neurona and Neospora hughesi are coccidian protozoa that can cause neurological illness in horses in America. In this study we report seroprevalence of Neospora spp. andS. neurona in sera of 333 donkeys from the northeastern region of Brazil. Antibodies to Neospora spp. were detected in 2% (7 donkeys) of 333 sera tested by the indirect fluorescent antibody test (IFAT) with a cut-off dilution of 1:40. Antibodies to S. neurona were found in 3% (10 donkeys) of the samples tested by IFAT (cut-off ≥50) and 21% (69 donkeys) by the direct agglutination test (SAT ≥50). The SAT and IFAT results for S. neurona showed a poor concordance (value of Kappa=0.051). This is the first report ofNeospora spp. antibodies in Brazilian donkeys and the first detection of antibodies against S. neurona in this animal species.

Keywords:
Neospora hughesi; Neospora caninum; Sarcocystis neurona; donkey; Brazil

Resumo

Sarcocystis neurona e Neospora hughesi são protozoários coccídios que infectam equídeos e são responsáveis por doenças neurológicas nessas espécies. Neste estudo, a soroprevalência de infecção porS. neurona e Neospora spp. foi determinada em amostras de 333 soros de jumentos da Região Nordeste do Brasil. Anticorpos contra Neospora spp. foram detectados em 2% (7 jumentos) dos 333 animais examinados pela reação de imunofluorescência indireta (RIFI), com ponto de corte de 40. Anticorpos contra S. neurona foram detectados em 3% (10 jumentos) das amostras pela RIFI (ponto de corte de 50) e em 21% (69 jumentos) pela técnica de aglutinação direta (SAT - ponto de corte de 50). SAT e RIFI, para diagnóstico de S. neurona, apresentaram uma baixa concordância (Kappa = 0,051). Essa é a primeira observação de anticorpos anti-N. caninum em jumentos brasileiros e a primeira detecção de anticorpos contra S. neurona nessa espécie.

Palavras-chave:
Neospora hughesi; Neospora caninum; Sarcocystis neurona; jumentos; Brasil

Introduction

Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses that is caused primarily by Sarcocystis neurona and less frequently byNeospora hughesi (DUBEY et al., 2001Dubey JP, Lindsay DS, Kerber CE, Kasai N, Pena HFJ, Gennari SM, et al. First isolation of from the South American opossum, , from Brazil. Sarcocystis neuronaDidelphis albiventrisVet Parasitol 2001; 95(2-4): 295-304. http://dx.doi.org/10.1016/S0304-4017(00)00395-2. PMid:11223209.
http://dx.doi.org/10.1016/S0304-4017(00)... , 2015Dubey JP, Howe DK, Furr M, Saville WJ, Marsh AE, Reed SM, et al. An update on infection in animals and equine protozoa myeloencephalitis (EPM). Sarcocystis neuronaVet Parasitol 2015; 209(1-2): 1-42. http://dx.doi.org/10.1016/j.vetpar.2015.01.026. PMid:25737052.
http://dx.doi.org/10.1016/j.vetpar.2015.... ). N. hughesi infection has also been reported to cause abortion in horses (PUSTERLA et al., 2014Pusterla N, Mackie S, Packham A, Conrad PA. Serological investigation of transplacental infection with and in broodmares. Neospora hughesiSarcocystis neuronaVet J 2014; 202(3): 649-650. http://dx.doi.org/10.1016/j.tvjl.2014.09.015. PMid:25438732.
http://dx.doi.org/10.1016/j.tvjl.2014.09... ).

Horses become infected with S. neurona after ingesting sporocysts shed by opossums, Didelphis spp. In North America, D. virginiana is the definitive host for S. neurona, and in South America the opossum D. albiventris is a proven definitive host (DUBEY et al., 2001Dubey JP, Lindsay DS, Kerber CE, Kasai N, Pena HFJ, Gennari SM, et al. First isolation of from the South American opossum, , from Brazil. Sarcocystis neuronaDidelphis albiventrisVet Parasitol 2001; 95(2-4): 295-304. http://dx.doi.org/10.1016/S0304-4017(00)00395-2. PMid:11223209.
http://dx.doi.org/10.1016/S0304-4017(00)... ). The definitive host for N. huguesi is unknown. Serologically antibodies againstN. hughesi will cross-react with N. caninumantigen and antibodies to N. caninum will react to N. hughesi antigen (GONDIM et al., 2009Gondim LFP, Lindsay DS, McAllister MM. Canine and bovine Neospora caninum control sera examined for cross-reactivity using Neospora caninum and indirect fluorescent antibody tests. Neospora hughesiJ Parasitol 2009; 95(1): 86-88. http://dx.doi.org/10.1645/GE-1710.1. PMid:18613752.
http://dx.doi.org/10.1645/GE-1710.1... ). Sera reactive to N. caninum antigen in the present study were considered positive for antibodies to Neosporaspp.

In Brazil, antibodies to Neospora spp. and S. neurona have been reported in horses (see reviews DUBEY & SCHARES, 2011Dubey JP, Schares G. Neosporosis in animals – the last five years. Vet Parasitol 2011; 180(1-2): 90-108. http://dx.doi.org/10.1016/j.vetpar.2011.05.031. PMid:21704458.
http://dx.doi.org/10.1016/j.vetpar.2011.... ; DUBEY et al., 2015Dubey JP, Howe DK, Furr M, Saville WJ, Marsh AE, Reed SM, et al. An update on infection in animals and equine protozoa myeloencephalitis (EPM). Sarcocystis neuronaVet Parasitol 2015; 209(1-2): 1-42. http://dx.doi.org/10.1016/j.vetpar.2015.01.026. PMid:25737052.
http://dx.doi.org/10.1016/j.vetpar.2015.... ) but there is no report for these infections in donkeys.

Information regarding Neospora spp. and S. neuronaexposure in donkeys (Equus asinus) from other countries is scarce (MACHACOVÁ et al., 2013Machacová T, Bártová E, Di Loria A, Sedlák K, Guccione J, Fulgione D, et al. Seroprevalence and risk factors of spp. in donkeys from Southern Italy. NeosporaVet Parasitol 2013; 198(1-2): 201-204. http://dx.doi.org/10.1016/j.vetpar.2013.08.013. PMid:24050943.
http://dx.doi.org/10.1016/j.vetpar.2013.... ; BLANCO et al., 2014Blanco RD, Patarroyo JH, Vargas MI, Cardona JA, Araújo LS, Gomez VE. Ocorrência de anticorpos anti-Neospora spp. em jumentos () no estado de Sucre – Colômbia. Equus asinusArq Bras Med Vet Zootec 2014; 66(2): 450-454. http://dx.doi.org/10.1590/1678-41627002.
http://dx.doi.org/10.1590/1678-41627002... ). Donkeys are used traditionally for working roles, however in some parts of the world they are increasingly being used for milk production.

Here we report seroprevalence of Neospora spp. and S. neurona in donkeys from Brazil.

Materials and Methods

Serum samples were obtained from 333 donkeys (Equus asinus) from rural properties, located in the northeastern region of Brazil (Table 1). We used a convenience sampling technique and the animals included in the sample were those available at the time of the survey. All animals were from both genders and different ages, and were mainly bred for working roles. All procedures were conducted in accordance with the Animal Protocols approved by the Ethic Committee of the Faculty of Veterinary Medicine, University of São Paulo, Brazil.

The indirect fluorescent antibody test (IFAT) was used to detected antibodies againstNeospora spp. and was conducted according to the method described by Dubey et al. (1988)Dubey JP, Hattel AL, Lindsay DS, Topper MJ. Neonatal infection in dogs: isolation of the causative agent and experimental transmission. Neospora caninumJ Am Vet Med Assoc 1988; 193(10): 1259-1263. PMid:3144521. using tachyzoites of NC-1 N. caninum isolate as antigen.Sarcocystis neurona merozoites of the SN3 isolate (GRANSTROM et al., 1992Granstrom DE, Alvarez O Jr, Dubey JP, Comer PF, Williams NM. Equine protozoal myelitis in Panamanian horses and isolation of Sarcocystis neurona.J Parasitol 1992; 78(5): 909-912. http://dx.doi.org/10.2307/3283328. PMid:1403437.
http://dx.doi.org/10.2307/3283328... ) were used as the antigen in S. neurona IFAT and in the direct agglutination test (SAT) for S. neurona. For IFAT, tachyzoites or merozoites were collected from cultures, washed in PBS (NaCl 0.142M; KCl 0.003M; Na₂HPO₄ 0.008M; NaH₂PO₄ 0.001M, pH 7.4), counted with an hemocytometer to a concentration of 2x10⁷/mL, distributed in 12-well slides, air dried, fixed with methanol and stored at –20°C until used. Sera were distributed on the wells, incubated at room temperature in wet chambers for 30 min, and then the slides were soaked in PBS three times for 5 min; after the slides were air dried, the conjugate was applied, and the slides were incubated and processed as described above. Fluorescein-labeled affinity-purified antibodies against horse IgG were used as conjugate in both IFATs. For S. neurona IFAT, the cut-off value was 1:40 and for N. caninum, 1:50. Every positive serum was retested using a twofold serial dilution. Positive and negative S. neurona and N. caninum horse samples were added in each slide. The SAT was performed according Lindsay & Dubey (2001)Lindsay DS, Dubey JP. Direct agglutination test for the detection of antibodies to in experimentally infected animals. Sarcocystis neuronaVet Parasitol 2001; 95(2-4): 179-186. http://dx.doi.org/10.1016/S0304-4017(00)00389-7. PMid:11223198.
http://dx.doi.org/10.1016/S0304-4017(00)... with a cut-off value of 1:50. Positive and negative control sera were used to validate the results of each SAT.

The proportions of positive samples from the Brazilian states were compared forNeospora spp. and S. neurona using the Fisher’s exact test (ZAR, 2010Zar JH. Biostatistical analysis. Upper Saddle River: Prentice Hall; 2010.) with a P value <0.05 being significant.

Results and Discussion

The prevalence of antibodies to Neospora spp. in donkeys was 2% (95% CI: 0.8%-4.3%) and only donkeys from the states of Alagoas and Pernambuco were positive (Table 1). Occurrence of S. neurona was 21% (95% CI: 17%-26%) by SAT (titers between 50 and 200) and 3% (95% CI: 1.5%-5.5%) by IFAT (titers between 40 and 160) and positive donkeys were found in all five analyzed States by SAT, but not in the state of Paraíba by IFAT (Table 1). Five of the 10 IFAT positive donkeys were also SAT positive. The SAT and IFAT results for S. neurona showed a poor agreement (value of Kappa=0.051).

In Italy, occurrence of antibodies to Neospora spp. in donkeys was 11.8% using a competitive-ELISA (MACHACOVÁ et al., 2013Machacová T, Bártová E, Di Loria A, Sedlák K, Guccione J, Fulgione D, et al. Seroprevalence and risk factors of spp. in donkeys from Southern Italy. NeosporaVet Parasitol 2013; 198(1-2): 201-204. http://dx.doi.org/10.1016/j.vetpar.2013.08.013. PMid:24050943.
http://dx.doi.org/10.1016/j.vetpar.2013.... ) and in Colombia, 11 of the 56 examined donkeys presented antibodies against Neospora spp. measured by Dot-ELISA (BLANCO et al., 2014Blanco RD, Patarroyo JH, Vargas MI, Cardona JA, Araújo LS, Gomez VE. Ocorrência de anticorpos anti-Neospora spp. em jumentos () no estado de Sucre – Colômbia. Equus asinusArq Bras Med Vet Zootec 2014; 66(2): 450-454. http://dx.doi.org/10.1590/1678-41627002.
http://dx.doi.org/10.1590/1678-41627002... ). Due the different methodology used in the studies, comparisons between these studies and the present study are difficult to make.

The results for the comparisons between the proportions of positive animals from the Brazilian states are shown in Table 1. ForS. neurona statistical analyzes were done using both the SAT and IFAT results. For the SAT results, significant differences (P <0.05) were observed for S. neurona prevalence between the samples from Alagoas and the samples from Pernambuco, Piauí and Rio Grande do Norte, and between the samples from Pernambuco and Piauí. No differences were found between the prevalence value and location for the IFAT results for S. neurona and also when antibodies to Neospora spp. were analyzed.

This is the first report of Neospora spp. antibodies in Brazilian donkeys and the first detection of antibodies against S. neurona in this animal species.

This is the first study related to S. neurona occurrence in donkeys and the prevalence of 21% (95% CI: 17%-26%) by SAT found was lower than the prevalence of 36.0% to 69.6% found in Brazilian horses (DUBEY & SCHARES, 2011Dubey JP, Schares G. Neosporosis in animals – the last five years. Vet Parasitol 2011; 180(1-2): 90-108. http://dx.doi.org/10.1016/j.vetpar.2011.05.031. PMid:21704458.
http://dx.doi.org/10.1016/j.vetpar.2011.... ; PIVOTO et al., 2014Pivoto FL, Macêdo AG Jr, Silva MV, Ferreira FB, Silva DAO, Pompermayer E, et al. Serological status of mares in parturition and the levels of antibodies (IgG) against protozoan Family Sarcocystidae from their pre colostral foals. Vet Parasitol 2014; 199(1-2): 107-111. http://dx.doi.org/10.1016/j.vetpar.2013.10.001. PMid:24183649.
http://dx.doi.org/10.1016/j.vetpar.2013.... ). In this study there was a poor agreement between IFAT and SAT. There is no comprehensive study of the sensitivity and specificity of the SAT in equids (DUBEY et al., 2001Dubey JP, Lindsay DS, Kerber CE, Kasai N, Pena HFJ, Gennari SM, et al. First isolation of from the South American opossum, , from Brazil. Sarcocystis neuronaDidelphis albiventrisVet Parasitol 2001; 95(2-4): 295-304. http://dx.doi.org/10.1016/S0304-4017(00)00395-2. PMid:11223209.
http://dx.doi.org/10.1016/S0304-4017(00)... , 2015Dubey JP, Howe DK, Furr M, Saville WJ, Marsh AE, Reed SM, et al. An update on infection in animals and equine protozoa myeloencephalitis (EPM). Sarcocystis neuronaVet Parasitol 2015; 209(1-2): 1-42. http://dx.doi.org/10.1016/j.vetpar.2015.01.026. PMid:25737052.
http://dx.doi.org/10.1016/j.vetpar.2015.... ). Immunoblot is considered the golden test for seroprevalence studies of S. neurona in horses, but it is expensive and laborious. DUARTE et al. (2003)Duarte PC, Daft BM, Conrad PA, Packham AE, Gardner IA. Comparison of a serum indirect fluorescent antibody test with two Western blot tests for the diagnosis of equine protozoal myeloencephalitis. J Vet Diagn Invest 2003; 15(1): 8-13. http://dx.doi.org/10.1177/104063870301500103. PMid:12580288.
http://dx.doi.org/10.1177/10406387030150... showed that IFAT could be an alternative to immunoblot forS. neurona antibody detection in horses, with good specificity and sensitivity. Cross-reactivity of both tests with other protozoa from donkeys is not known.

Acknowledgements

The present study was supported, in part, by funds from Conselho Nacional de Pesquisa (CNPq), Brazil. SMG and MA were supported by a fellowship from CNPq.

References

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