Antibodies against Apicomplexa protozoa and absence sarcocysts in heart tissues from horses in southern Brazil

Anticorpos contra protozoários do filo Apicomplexa e ausência de sarcocistos no miocárdio de equinos no sul do Brasil

Luiza Pires Portella Gustavo Cauduro Cadore Luis Antonio Sangioni Luiz Fernando Vilani Pellegrini Rafael Fighera Fernanda Ramos Fernanda Silveira Flores Vogel About the authors

Abstract

Sarcocystis spp., Neospora spp., and Toxoplasma gondii are Apicomplexa protozoa that can infect horses. This study aimed to investigate the occurrence of antibodies against Sarcocystis spp., Neospora spp., and T. gondii in horses slaughtered in southern Brazil. The presence of histological lesions, tissue cysts, and Sarcocystis spp. DNA in the hearts of these horses was also investigated. A total of 197 paired serum and heart samples were evaluated by serology and direct microscopic examination; 50 of these samples were subjected to histopathological and PCR analyses. Antibodies against at least one of the protozoa were detected in 146 (74.1%) of the serum samples. The frequencies of positive serology were: 36% (71/197) against Sarcocystis spp., 39.1% (77/197) against Neospora spp., and 47.2% (93/197) against T. gondii. No cysts, Sarcocystis spp. DNA, or histopathological lesions were observed in myocardial tissue samples. The frequencies of antibody seropositivity against Sarcocystis spp., Neospora spp., and T. gondii showed that horses are frequently infected by these parasites in southern Brazil. The absence of sarcocysts in horse tissues is compatible with their role as aberrant/accidental hosts in the life cycle of Sarcocystis spp..

Keywords:
Accidental hosts; epidemiology; Sarcocystis spp.; Neospora spp.; Toxoplasma gondii

Resumo

Sarcocystis spp., Neospora spp. e Toxoplasma gondii são protozoários que pertencem ao filo Apicomplexa e que podem afetar equinos. O objetivo deste estudo foi investigar a ocorrência de anticorpos contra Sarcocystis spp., Neospora spp. e T. gondii. A presença de lesões histológicas, cistos teciduais e DNA de Sarcocystis spp. no miocárdio de equinos abatidos no sul do Brasil também foi investigado. Um total de 197 amostras de soro juntamente com as respectivas amostras de coração, foram avaliadas por sorologia e exame microscópico direto. Destas amostras, 50 foram selecionadas e submetidas a análise histopatológica e PCR. Anticorpos contra pelo menos um dos protozoários foi detectado em 146 (74,1%) das amostras de soro. As frequências de sorologia positiva foram: 36% (71/197) para Sarcocystis spp., 39,1% (77/197) para Neospora spp. e 47,2% (93/197) para T. gondii. Não foram encontradas lesões histopatológicas, cistos e DNA de Sarcocystis spp. nas amostras de miocárdio dos equinos. As frequências de soropositividade para Sarcocystis spp., Neospora spp. e T. gondii mostra que os equinos podem ser frequentemente infectados por estes parasitas no sul do Brasil. A ausência de sarcocistose no coração dos equinos é compatível com seu papel como hospedeiro errático/acidental no ciclo de vida deste protozoário.

Palavras-chave:
Hospedeiro acidental; epidemiologia; Sarcocystis spp.; Neospora spp.; Toxoplasma gondii

Introduction

Sarcocystis neurona, Neospora spp., and Toxoplasma gondii are Apicomplexa protozoan parasites that can cause encephalomyelitis in horses. Equine protozoal myeloencephalitis (EPM) is a severe and debilitating neurological disease that is most often caused by S. neurona (DUBEY et al., 2001aDubey JP, Lindsay DS, Saville WJA, Reed SM, Granstrom DE, Speer CA. A review of and equine protozoal myeloencephalitis (EPM). Sarcocystis neuronaVet Parasitol 2001a; 95(2-4): 89-131. PMid:11223193. http://dx.doi.org/10.1016/S0304-4017(00)00384-8.
http://dx.doi.org/10.1016/S0304-4017(00)...
) and less frequently by Neospora hughesi (REED et al., 2016Reed SM, Furr M, Howe DK, Johnson AL, MacKay RJ, Morrow JK, et al. Equine Protozoal Myeloencephalitis: an updated consensus statement with a focus on parasite biology, diagnosis, treatment, and prevention. J Vet Intern Med 2016; 30(2): 491-502. PMid:26857902. http://dx.doi.org/10.1111/jvim.13834.
http://dx.doi.org/10.1111/jvim.13834...
). Commonly, a diagnosis of EPM is based on neurological signs and serology indicating prior exposure to Sarcocystis spp. and/or Neospora spp. (RENIER et al., 2016Renier AC, Morrow JK, Graves AJ, Finno CJ, Howe DK, Owens SD, et al. Diagnosis of equine protozoal myeloencephalitis using indirect fluorescent antibody testing and enzyme-linked immunosorbent assay titer ratios for Sarcocystis neurona and Neospora hughesi.J Equine Vet Sci 2016; 36: 49-51. http://dx.doi.org/10.1016/j.jevs.2015.10.010.
http://dx.doi.org/10.1016/j.jevs.2015.10...
). Clinical signs depend on the distribution of the parasite in the central nervous system (DUBEY et al., 2001aDubey JP, Lindsay DS, Saville WJA, Reed SM, Granstrom DE, Speer CA. A review of and equine protozoal myeloencephalitis (EPM). Sarcocystis neuronaVet Parasitol 2001a; 95(2-4): 89-131. PMid:11223193. http://dx.doi.org/10.1016/S0304-4017(00)00384-8.
http://dx.doi.org/10.1016/S0304-4017(00)...
). Equines become infected with S. neurona by ingesting sporocysts excreted in the feces of opossums (Didelphis spp.), which are definitive hosts for the pathogen (FENGER et al., 1995Fenger CK, Granstrom DE, Langemeier JL, Stamper S, Donahue JM, Patterson JS, et al. Identification of opossums (Didelphis virginianaSarcocystis neurona.) as the putative definitive host of J Parasitol 1995; 81(6): 916-919. PMid:8544064. http://dx.doi.org/10.2307/3284040.
http://dx.doi.org/10.2307/3284040...
; DUBEY et al., 2001aDubey JP, Lindsay DS, Saville WJA, Reed SM, Granstrom DE, Speer CA. A review of and equine protozoal myeloencephalitis (EPM). Sarcocystis neuronaVet Parasitol 2001a; 95(2-4): 89-131. PMid:11223193. http://dx.doi.org/10.1016/S0304-4017(00)00384-8.
http://dx.doi.org/10.1016/S0304-4017(00)...
). Horses are generally considered accidental hosts of this protozoan (DUBEY et al., 2006Dubey JP, Chapman JL, Rosenthal BM, Mense M, Schueler RL. Clinical Sarcocystis neurona, Sarcocystis canis, Toxoplasma gondii and infections in dogs. Neospora caninumVet Parasitol 2006; 137(1-2): 36-49. PMid:16458431. http://dx.doi.org/10.1016/j.vetpar.2005.12.017.
http://dx.doi.org/10.1016/j.vetpar.2005....
). However, Mullaney et al. (2005)Mullaney T, Murphy AJ, Kiupel M, Bell JA, Rossano MG, Mansfield LS. Evidence to support horses as natural intermediate hosts for Sarcocystis neurona.Vet Parasitol 2005; 133(1): 27-36. PMid:15970386. http://dx.doi.org/10.1016/j.vetpar.2005.05.016.
http://dx.doi.org/10.1016/j.vetpar.2005....
suggested that horses can act as natural intermediate hosts of S. neurona, since cysts have been found within their muscle tissues.

T. gondii and Neospora spp. are biologically similar protozoa (DUBEY et al., 2009Dubey JP, Jenkins MC, Kwok OCH, Zink RL, Michalski ML, Ulrich V, et al. Seroprevalence of Neospora caninum and Toxoplasma gondii antibodies in white-tailed deer () from Iowa and Minnesota using four serologic tests. Odocoileus virginianusVet Parasitol 2009; 161(3-4): 330-334. PMid:19285809. http://dx.doi.org/10.1016/j.vetpar.2009.01.002.
http://dx.doi.org/10.1016/j.vetpar.2009....
). Neosporosis is a widely recognized reproductive disease in cattle caused by Neospora spp.; this agent can also cause EPM (HAMIR et al., 1998Hamir AN, Tornquisyt SJ, Gerros TC, Topper MJ, Dubey JP. -associated equine protozoal myeloencephalitis. Neospora caninumVet Parasitol 1998; 79(4): 269-274. PMid:9831950. http://dx.doi.org/10.1016/S0304-4017(98)00178-2.
http://dx.doi.org/10.1016/S0304-4017(98)...
). Horses can be infected by both N. caninum and N. hughesi; however, EPM seems to be caused specifically by N. hughesi (REED et al., 2016Reed SM, Furr M, Howe DK, Johnson AL, MacKay RJ, Morrow JK, et al. Equine Protozoal Myeloencephalitis: an updated consensus statement with a focus on parasite biology, diagnosis, treatment, and prevention. J Vet Intern Med 2016; 30(2): 491-502. PMid:26857902. http://dx.doi.org/10.1111/jvim.13834.
http://dx.doi.org/10.1111/jvim.13834...
). Toxoplasmosis is a zoonotic disease caused by T. gondii and the infection with this protozoan in horses is usually subclinical (AL-KHALIDI & DUBEY, 1979Al-Khalidi NW, Dubey JP. Prevalence of Toxoplasma gondii infection in horses. J Parasitol 1979; 65(2): 331-334. PMid:448624. http://dx.doi.org/10.2307/3280181.
http://dx.doi.org/10.2307/3280181...
). However, clinical signs of infection, including hyperirritability, motor incoordination, ocular disorders, and abortions, have been reported (DUBEY & PORTERFIELD, 1986Dubey JP, Porterfield ML. -like sporozoa in an aborted equine fetus. ToxoplasmaJ Am Vet Med Assoc 1986; 188(11): 1312-1313. PMid:3087925.). Therefore, considering the importance of Apicomplexa protozoa as etiologies of neurological diseases in horses, the objectives of this study were to investigate (a) the presence of antibodies against Sarcocystis spp., Neospora spp., and T. gondii in horses slaughtered in Rio Grande do Sul, Brazil, and (b) the presence of histological lesions, tissue cysts, and Sarcocystis spp. DNA in the hearts of these horses.

Materials and Methods

Animals and samples

Heart tissues and serum samples of 197 horses were collected from a slaughterhouse located in São Gabriel, Rio Grande do Sul, Brazil. The samples were collected between September and November 2014. The animals were clinically healthy horses, ranging from 6 months to 18 years of age and originating from two states of southern Brazil: Rio Grande do Sul and Paraná. Collected blood samples were centrifuged at 250 × g for 10 minutes to obtain serum and then stored at −20 °C until testing. Harvested heart tissue samples (50 g) were stored individually in plastic bags and kept refrigerated until processing.

Direct examination and histopathology

Each heart tissue sample was directly examined. Approximately 25 g of tissue from each sample was macerated and homogenized with phosphate-buffered saline (PBS; 0.1 M phosphate, 0.33 M NaCl, pH 7.2) and filtered with gauze. This homogenate was examined for sarcocysts using an inverted microscope at 400×. For the histopathological examination, 50 of the 197 heart tissue samples were randomly selected, sectioned transversely, and fixed in 10% formaldehyde. Samples were steam autoclaved after 7 days of formaldehyde fixation, and then processed for histology and stained with hematoxylin and eosin. The resulting material was analyzed by light microscopy.

DNA extraction and polymerase chain reaction (PCR)

Total DNA was extracted from approximately 50 mg of each heart tissue sample using a commercial kit (Wizard Genomic DNA Purification Kit, Promega, Madison, WI, USA) according to the manufacturer's instructions, with modifications in the lysis step, as outlined by Moré et al. (2011)Moré G, Abrahamovich P, Jurado S, Bacigalupe D, Marin JC, Rambeaud M, et al. Prevalence of spp. in Argentinean cattle. SarcocystisVet Parasitol 2011; 177(1-2): 162-165. PMid:21168276. http://dx.doi.org/10.1016/j.vetpar.2010.11.036.
http://dx.doi.org/10.1016/j.vetpar.2010....
.

Total DNA was subjected to PCR, using primers specific for the 18S rDNA region of Sarcocystis spp. (forward: 5′-CGCAAATTACCCAATCCTGA-3′ and reverse: 5′-ATTTCTCATAAGGTGCAGGAG-3′). The PCR reaction was performed in a total volume of 25 μL, containing 5 µL of buffer, 100 ng of each primer; 10 mM deoxynucleotide triphosphate (dNTPs); 1 U of DNA polymerase GoTaq® (Hot Start Polymerase, Promega, Madison, WI, USA), and 100 ng of total DNA used as a template. Amplification was performed with an initial denaturation for 4 min at 95 °C; followed by 40 cycles of denaturation for 40 sec at 94 °C, annealing for 30 sec at 59 °C, and extension for 60 sec at 72 °C; with a final extension for 6 min at 72°C. The PCR products were visualized by UV illumination after electrophoresis on a 1% agarose gel stained with GelRed® (Biotium Inc., CA, USA).

Antibody survey

Anti-Sarcocystis spp., anti-Neospora spp., and anti-T. gondii immunoglobulin G (IgG) antibodies were detected by indirect fluorescence antibody test (IFAT) on separate slides. Antigens used to detect antibodies were merozoites of S. neurona (SN-37R strain) cultivated in CV-1 cells, tachyzoites of N. caninum (NC-1 strain), and tachyzoites of T. gondii (RH strain), all maintained in Vero cells. The cell were cultured in RPMI 1640 culture medium (Invitrogen, Brazil), supplemented with 10% fetal bovine serum (Nutricell, Brazil) at 37 °C under 5% CO2.

Serum samples were diluted 1:50 in PBS, and equine sera that were known to be positive or negative for the tested protozoa were used as controls. Commercial fluorescein-labeled anti-equine IgG (Goat Anti-Equine IgG FITC®, 160A, Southern Biotech, Birmingham, USA) was used as the secondary antibody. Slides were observed at 400× magnification using a fluorescent microscope (Leica CTR 4000/EBQ 100, Leica Microsystems, Germany). Samples with titers ≥ 50 were considered positive.

Results

Serologic results for the 197 horses evaluated by IFAT showed that 146 (74.1%) were positive for antigens of at least one of the protozoa. The frequency of detection of antibodies against each of the protozoa were the following: 36% (71/197) anti-Sarcocystis spp., 39.1% (77/197) anti-Neospora spp., and 47.2% (93/197) anti-T. gondii. Mixed infection by Sarcocystis spp. and Neospora spp. occurred in 5.6% (11/197) of the horses, while 11.7% (23/197) were positive for Sarcocystis spp. and T. gondii, and equines infected by Neospora spp. and T. gondii was 12.7% (25/197). In 19 (9.6%) serum samples, antibodies were detected against all three protozoa. The antibody seropositivity of horses for Sarcocystis spp., Neospora spp., or T. gondii alone were 9.1% (18/197), 12.2% (24/197), and 13.2% (26/197), respectively.

No sarcocysts were found in the 197 cardiac muscle tissue by direct examination. Of the 50 cardiac muscle samples subjected to histopathological examination and PCR, 25 (50%) were from animals seropositive for Sarcocystis spp. antibodies. No Sarcocystis spp. DNA was amplified by PCR of the 50 samples tested. Furthermore, no morphological alterations, lesions, or tissue cysts consistent with Sarcocystis spp. infection were observed in the histopathological analysis.

Discussion

Serological analysis indicated a frequency of 36% (71/197) seropositivity to Sarcocystis spp. in clinically healthy horses slaughtered in southern Brazil. This result was similar to the 35.6% seropositivity found in 101 thoroughbreds in Brazil (DUBEY et al., 1999aDubey JP, Kerber CE, Granstrom DE. Serologic prevalence of Sarcocystis neurona, Toxoplasma gondii and Neospora caninum in horses in Brazil. J Am Vet Med Assoc 1999a; 215(7): 970-972. PMid:10511862.). In addition, Hoane et al. (2006)Hoane JS, Gennari SM, Dubey JP, Ribeiro MG, Borges AS, Yai LEO, et al. Prevalence of Sarcocystis neurona and Neospora spp. infection in horses from Brazil based on presence of serum antibodies to parasite surface antigen. Vet Parasitol 2006; 136(2): 155-159. PMid:16310955. http://dx.doi.org/10.1016/j.vetpar.2005.10.023.
http://dx.doi.org/10.1016/j.vetpar.2005....
detected seropositivity to S. neurona in 69.6% of horses tested in Brazil. These results indicate consistent, widespread distribution of this parasite in Brazil. This is probably a result of the free access of definitive hosts to horse pastures and feed, resulting in broad environmental contamination by sporocysts and oocysts from this protozoan. Didelphis albiventris is a broadly distributed opossum that was confirmed to be definitive host of S. neurona in South America (DUBEY et al., 2001bDubey JP, Lindsay DS, Kerber CE, Kasai N, Pena HPJ, Gennari SM, et al. First isolation of Sarcocystis neurona from the South American opossum, Didelphis albiventris, from Brazil. Vet Parasitol 2001b; 95(2-4): 295-304. PMid:11223209. http://dx.doi.org/10.1016/S0304-4017(00)00395-2.
http://dx.doi.org/10.1016/S0304-4017(00)...
) and due to its synanthropy, this species is a potential disseminator of pathogens between domestic animals and man (MULLER et al., 2005Muller G, Brum JGW, Langoni PQ, Michels GH, Sinkoc AL, Ruas JL, et al. Didelphis albiventris Lund, 1841, parasitado por Neumann, 1899, e . Ixodes loricatusAmblyomma aureolatum (Pallas, 1772) (Acari: Ixodidae) no Rio Grande do SulArq Inst Biol 2005; 72(3): 319-324.).

An antibody seropositivity of 39.1% (77/197) against Neospora spp. is relatively high when compared with another study performed in southern Brazil, in which 15.4% seropositivity was found in draft horses (SANGIONI et al., 2011Sangioni LA, Botton SA, Cargnelutti JF, Cadore GC, Cezar AS, Weiblen R, et al. Pesquisa de anticorpos anti- spp. e anti-herpersvírus equino em cavalos de tração no município de Santa Maria, RS, Brasil. NeosporaCienc Rural 2011; 41(2): 321-323. http://dx.doi.org/10.1590/S0103-84782011000200023.
http://dx.doi.org/10.1590/S0103-84782011...
) and 23.9% detected in horses from state of Minas Gerais (RIBEIRO et al., 2016Ribeiro MJM, Rosa MHF, Bruhn FRP, Garcia AM, Rocha CMBM, Guimarães AM. Seroepidemiology of Sarcocystis neurona, Toxoplasma gondii and spp. among horses in the south of the state of Minas Gerais, Brazil. NeosporaBraz J Vet Parasitol 2016; 25(2): 142-150. PMid:27334814. http://dx.doi.org/10.1590/S1984-29612016029.
http://dx.doi.org/10.1590/S1984-29612016...
). Moreover, Hoane et al. (2006)Hoane JS, Gennari SM, Dubey JP, Ribeiro MG, Borges AS, Yai LEO, et al. Prevalence of Sarcocystis neurona and Neospora spp. infection in horses from Brazil based on presence of serum antibodies to parasite surface antigen. Vet Parasitol 2006; 136(2): 155-159. PMid:16310955. http://dx.doi.org/10.1016/j.vetpar.2005.10.023.
http://dx.doi.org/10.1016/j.vetpar.2005....
detected a very low prevalence (2.5%) of antibodies against Neospora spp. in Brazilian horses. Neosporosis in horses has been described in several countries with different infection rates, which may be a result of environmental conditions, sampling methods, or the type of tests used for diagnosis (DUBEY et al., 1999aDubey JP, Kerber CE, Granstrom DE. Serologic prevalence of Sarcocystis neurona, Toxoplasma gondii and Neospora caninum in horses in Brazil. J Am Vet Med Assoc 1999a; 215(7): 970-972. PMid:10511862., bDubey JP, Venturini MC, Venturini L, McKinney J, Pecoraro M. Prevalence of antibodies to Sarcocystis neurona, Toxoplasma gondii and in horses from Argentina. Neospora caninumVet Parasitol 1999b; 86(1): 59-62. PMid:10489203. http://dx.doi.org/10.1016/S0304-4017(99)00127-2.
http://dx.doi.org/10.1016/S0304-4017(99)...
). Regardless of the variation, these results indicate that Neospora spp. infection is significant in the horses tested. Environmental contamination by oocysts excreted by dogs is a potential route for infection of horses by N. caninum (GONDIM et al., 2004Gondim LF, McAllister MM, Pitt WC, Zemlicka DE. Coyotes (Canis latrans) are definitive hosts of Neospora caninum.Int J Parasitol 2004; 34(2): 159-161. PMid:15037103. http://dx.doi.org/10.1016/j.ijpara.2004.01.001.
http://dx.doi.org/10.1016/j.ijpara.2004....
); however, the transplacental route also allows Neospora spp. to be maintained in horse populations (DUBEY & PORTERFIELD, 1990Dubey JP, Porterfield ML. (Apicomplexa) in an aborted equine fetus. Neospora caninumJ Parasitol 1990; 76(5): 732-734. PMid:2213418. http://dx.doi.org/10.2307/3282991.
http://dx.doi.org/10.2307/3282991...
). Based in these results, the neosporosis should be included in the differential diagnosis as a potential etiologic agent of EPM in southern Brazil.

Toxoplasmosis is a zoonosis of great interest due to its impact on public health and that can be transmitted through consumption of raw or undercooked meat (DUBEY & SU, 2009Dubey JP, Su C. Population biology of Toxoplasma gondii: what’s out and where did they come from. Mem Inst Oswaldo Cruz 2009; 104(2): 190-195. PMid:19430643. http://dx.doi.org/10.1590/S0074-02762009000200011.
http://dx.doi.org/10.1590/S0074-02762009...
). T. gondii infections in equines have been reported in Brazil, with variable antibody detection rates (DUBEY et al., 1999aDubey JP, Kerber CE, Granstrom DE. Serologic prevalence of Sarcocystis neurona, Toxoplasma gondii and Neospora caninum in horses in Brazil. J Am Vet Med Assoc 1999a; 215(7): 970-972. PMid:10511862.; RIBEIRO et al., 2016Ribeiro MJM, Rosa MHF, Bruhn FRP, Garcia AM, Rocha CMBM, Guimarães AM. Seroepidemiology of Sarcocystis neurona, Toxoplasma gondii and spp. among horses in the south of the state of Minas Gerais, Brazil. NeosporaBraz J Vet Parasitol 2016; 25(2): 142-150. PMid:27334814. http://dx.doi.org/10.1590/S1984-29612016029.
http://dx.doi.org/10.1590/S1984-29612016...
). In the present study, seropositivity to T. gondii was 47.2% (93/197), which is high when compared to similar studies (DUBEY et al., 1999aDubey JP, Kerber CE, Granstrom DE. Serologic prevalence of Sarcocystis neurona, Toxoplasma gondii and Neospora caninum in horses in Brazil. J Am Vet Med Assoc 1999a; 215(7): 970-972. PMid:10511862., bDubey JP, Venturini MC, Venturini L, McKinney J, Pecoraro M. Prevalence of antibodies to Sarcocystis neurona, Toxoplasma gondii and in horses from Argentina. Neospora caninumVet Parasitol 1999b; 86(1): 59-62. PMid:10489203. http://dx.doi.org/10.1016/S0304-4017(99)00127-2.
http://dx.doi.org/10.1016/S0304-4017(99)...
), but similar results were found in Egypt (GHAZY et al., 2007Ghazy AA, Shaapan RM, Abdel-Rahman EH. Comparative serological diagnosis of toxoplasmosis in horses using locally isolated Toxoplasma gondii.Vet Parasitol 2007; 145(1-2): 31-36. PMid:17174034. http://dx.doi.org/10.1016/j.vetpar.2006.11.010.
http://dx.doi.org/10.1016/j.vetpar.2006....
). In state of Minas Gerais, Brazil, Ribeiro et al. (2016)Ribeiro MJM, Rosa MHF, Bruhn FRP, Garcia AM, Rocha CMBM, Guimarães AM. Seroepidemiology of Sarcocystis neurona, Toxoplasma gondii and spp. among horses in the south of the state of Minas Gerais, Brazil. NeosporaBraz J Vet Parasitol 2016; 25(2): 142-150. PMid:27334814. http://dx.doi.org/10.1590/S1984-29612016029.
http://dx.doi.org/10.1590/S1984-29612016...
showed a wide dispersion of T. gondii among horse farms, revealing that in 71.6% of the assessed farms, at least one presented one seropositive equine. The great differences in seroprevalence may be associated with various factors, including the type of feed given and the source of the water provided to animals; the intrinsic resistance to infection of horses tested; the population density of domestic or wild cats in the environment; environmental conditions; and the type of diagnostic test used (KOUAM et al., 2010Kouam MK, Diakou A, Kanzoura V, Papadopoulos E, Gajadhar AA, Theodoropoulos G. A seroepidemiological study of exposure to Toxoplasma, Leishmania, Echinococcus and in equids in Greece and analysis of risk factors. TrichinellaVet Parasitol 2010; 170(1-2): 170-175. PMid:20197215. http://dx.doi.org/10.1016/j.vetpar.2010.02.004.
http://dx.doi.org/10.1016/j.vetpar.2010....
). The high frequencies of antibodies detected against each of the parasites tested might be related to the origin of the blood samples. Although only clinically normal horses were sampled, it is important to highlight that most (85%) of these animals were over 10 years of age, which increases the probability of horizontal infection.

Despite the importance of S. neurona as an etiologic agent of EPM, the epidemiology of this protozoan disease in horses is not completely known. There are three other Sarcocystis species that infect equines and form cysts in their muscle tissue, S. bertrami, S. equicanis, and S. fayeri (LEVINE, 1986Levine ND. The taxonomy of Sarcocystis (Protozoa, Apicomplexa) species. J Parasitol 1986; 72(3): 372-382. PMid:3091802. http://dx.doi.org/10.2307/3281676.
http://dx.doi.org/10.2307/3281676...
). Therefore, equine cardiac muscle was examined for sarcocysts to investigate whether these horses could serve as intermediate hosts in the parasite’s life cycle. For most authors, in spite of the clinical importance of EPM, equines are considered as accidental hosts of S. neurona, where the infection does not determine the tissue cysts formation (DUBEY et al., 2006Dubey JP, Chapman JL, Rosenthal BM, Mense M, Schueler RL. Clinical Sarcocystis neurona, Sarcocystis canis, Toxoplasma gondii and infections in dogs. Neospora caninumVet Parasitol 2006; 137(1-2): 36-49. PMid:16458431. http://dx.doi.org/10.1016/j.vetpar.2005.12.017.
http://dx.doi.org/10.1016/j.vetpar.2005....
). However, Mullaney et al. (2005)Mullaney T, Murphy AJ, Kiupel M, Bell JA, Rossano MG, Mansfield LS. Evidence to support horses as natural intermediate hosts for Sarcocystis neurona.Vet Parasitol 2005; 133(1): 27-36. PMid:15970386. http://dx.doi.org/10.1016/j.vetpar.2005.05.016.
http://dx.doi.org/10.1016/j.vetpar.2005....
found sarcocysts in the tongue muscle tissue of a horse with clinical signs of EPM, and they confirmed the presence of S. neurona DNA by restriction fragment length polymorphism and PCR. Thus, more studies are needed to clarify the epidemiological role of horses in the life cycle of this protozoan. In this study, no cysts or Sarcocystis spp. DNA were detected in the myocardium tissue samples evaluated, reinforcing the idea that infected horses act as accidental or final hosts of the parasite. S. neurona infection in Brazil is associated with cases of EPM (MASRI et al., 1992Masri MD, Alda JL, Dubey JP. -associated ataxia in horses in Brazil. Sarcocystis neuronaVet Parasitol 1992; 44(3-4): 311-314. PMid:1466140. http://dx.doi.org/10.1016/0304-4017(92)90128-V.
http://dx.doi.org/10.1016/0304-4017(92)9...
) and the presence of the definitive host D. albiventris (DUBEY et al., 2001bDubey JP, Lindsay DS, Kerber CE, Kasai N, Pena HPJ, Gennari SM, et al. First isolation of Sarcocystis neurona from the South American opossum, Didelphis albiventris, from Brazil. Vet Parasitol 2001b; 95(2-4): 295-304. PMid:11223209. http://dx.doi.org/10.1016/S0304-4017(00)00395-2.
http://dx.doi.org/10.1016/S0304-4017(00)...
). This opossum is frequently encountered in rural areas, and usually lives in forests. However, with an increase in deforestation, the opossums are in closer proximity to farms and urban areas, increasing the possibility of environmental contamination and potential for infection of horses (DUBEY et al., 2001aDubey JP, Lindsay DS, Saville WJA, Reed SM, Granstrom DE, Speer CA. A review of and equine protozoal myeloencephalitis (EPM). Sarcocystis neuronaVet Parasitol 2001a; 95(2-4): 89-131. PMid:11223193. http://dx.doi.org/10.1016/S0304-4017(00)00384-8.
http://dx.doi.org/10.1016/S0304-4017(00)...
).

Conclusion

The high frequencies of antibody seropositivity against Sarcocystis spp., Neospora spp., and T. gondii in clinically healthy horses showed that these animals are at risk of infection by Apicomplexa parasites in southern Brazil. The absence of sarcocysts in the heart tissues of anti-Sarcocystis spp. seropositive horses is compatible with their role as aberrant/accidental hosts in the life cycle of S. neurona.

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Publication Dates

  • Publication in this collection
    16 Mar 2017
  • Date of issue
    Jan-Mar 2017

History

  • Received
    13 July 2016
  • Accepted
    19 Sept 2016
Colégio Brasileiro de Parasitologia Veterinária FCAV/UNESP - Departamento de Patologia Veterinária, Via de acesso Prof. Paulo Donato Castellane s/n, Zona Rural, , 14884-900 Jaboticabal - SP, Brasil, Fone: (16) 3209-7100 RAMAL 7934 - Jaboticabal - SP - Brazil
E-mail: cbpv_rbpv.fcav@unesp.br