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Abstract. Antigens of cultured Sarcocystis neurona merozoites were examined using immunoblot analysis. Blotted proteins were probed with S. cruzi, S. muris, and S. neurona antisera produced in rabbits, S. fayeri (pre-and post-infection) and S. neurona (pre-and post-inoculation) sera produced in horses, immune sera from 7 histologically confirmed cases of equine protozoa1 myeloencephalitis (EPM), and pre-suckle serum from a newborn foal. Eight proteins, 70, 24, 23.5, 22.5, 13, 11, 10.5, and 10 Kd, were detected only by S. neurona antiserum and/or immune serum from EPM-affected horses. Equine sera were titered by the indirect immunofluorescent antibody (IFA) method using air-dried, cultured S. neurona merozoites. Anti-Sarcocystis IFA titers were found in horses with or without EPM. Serum titers did not correspond to the number of specific bands recognized on immunoblots.Equine protozoa1 myeloencephalitis (EPM) is an often debilitating central nervous system (CNS) disease of the horse.9 It has been reported in horses native to North, 5 South, 1 and Central America. 6 The etiologic agent of EPM was recently cultured from naturally occurring cases in the US 2,3 and Panama 6 and named Sarcocystis neurona.Serological diagnosis of EPM caused by S. neurona has been complicated by the fact that species within the genus Sarcocystis share antigens. 4 For this reason, cross-reactions are observed when antigen from one species is recognized by serum antibody developed polyacrylamide gel electrophoresis (SDS-PAGE) in 0.75 mm, vertical, 5-20% or 10-20% linear gradient polyacrylamide resolving gels and 4% stacking gels using a discontinuous buffer method 8 and silver stained. 7 High-and low-range molecular weight markers were included in separate lanes. Immunoblots were prepared using 1 x 10 7 solubilized merozoites separated in 1 -well SDS-PAGE gels. Separated proteins were electrophoretically transferred to 0.45 µm nitrocellulose in Towbin buffer (0.192 M glycine, 0.025 M tris pH 8.3, 20% v/v methanol) using 1.0 A constant current for 1 hr. Prestained molecular weight markers were used in a separate lane.c Blotting efficiency was demonstrated by India ink staining of the blot and Coomassie blue staining of the against another species. The objective of this study was transferred gel.7 Blots were blocked with 5% non-fat dry milk, to identify S. neurona-specific proteins for antemortem 0.1% nonionic emulsifier, d and 0.1% sodium azide in 0.01 diagnostic use that would not cross-react with antibody M PBS, pH 7.2 for 1 hr and subdivided into lanes using a against other species. molded plexiglass press. e Each of the following sera were diluted in blocking buffer and incubated for 2 hr in individual Materials and methods lanes: S. cruzi (bradyzoites), S. muris (bradyzoites), and S.Sarcocystis neurona merozoites were harvested from boneurona (merozoites) antisera prepared in rabbits 7 normal vine monocyte (M617) cell cultures and purified on an isosrabbit serum, S. faveri (pre-and post-infection) and S. neumotic colloidal silica a st...
Abstract. A 5-year (1985-1989) retrospective immunohistochemical study was conducted using an avidinbiotin complex (ABC) immunoperoxidase method to demonstrate Sarcocystis neurona in histologically suspect cases of equine protozoa1 myeloencephalitis (EPM). Primary antibodies against S. neurona and S. cruzi were utilized for the ABC technique. The findings were compared with those from cases in which the organisms were detected by examination of hematoxylin and eosin (HE)-stained neuronal sections. HE-stained sections detected the presence of the organisms in 20% of the suspect cases; whereas the ABC technique confirmed the presence of S. neurona in 51% and 67% of the cases by S. neurona and S. cruzi antibodies, respectively. A review of clinical case histories showed that 21/47 (45%) of the EPM horses with parasites in the tissue sections had prior treatment with antiprotozoal drugs and/or steroids. Using the test results of S. neurona and S. cruzi as a standard reference, HE test sensitivity based on examination of up to 30 neuronal sections per case was only 25%, and test specificity was 91%. Equine protozoal myeloencephalitis (EPM) is a progressive disease of horses affecting the central nervous system (CNS).1,2,4,6 The causative agent is a protozoan, Sarcocystis neurona.5 An antemortem diagnostic test for the disease is not yet available. Postmortem diagnosis is based on the presence of characteristic histopathologic lesions and on the identification of the etiological agent.2,9 However, the detection of the protozoan by histopathology is laborious and often unrewarding 1,9 In a recent retrospective investigation of horses necropsied at the Laboratory of Large Animal Pathology, New Bolton Center (NBC), Kennett Square, Pennsylvania, over 20% of the cases had neurologic disease.7 Of these neurologic cases, 34% were considered to have an infectious cause (protozoal, viral, fungal, or bacterial). Within this group, EPM was the most frequent etiologic diagnosis based on the presence of characteristic microscopic lesions.2,4,9 However, the etiologic agent, S. neurona, 5 was identified in only 14 of the 70 positive cases of EPM. 7In the present study, we report of the immunohistochemical findings on these 70 histologically suspect cases of EPM. Materials and methodsClinical case histories and necropsy reports of 70 cases with lesions histopathologically compatible with EPM that were diagnosed between 1985 and 1989 at the NBC were examined. These records showed that an average of approximately 30 neuronal hematoxylin and eosin (HE)-stained sections were examined per case. Two histologic sections with the presence of S. neurona (determined from the necropsy report) or, in cases where the organisms were not observed, 2 sections with the most severe and extensive morphologic lesions from each of the suspect EPM cases were selected for immunohistochemistry. Consecutive tissue sections from the selected paraffin blocks (2 per horse) were cut at 5 µm and stained by an avidin-biotin complex (ABC) immunoperoxidase method ...
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