Sarcocystis neurona is the primary causative agent of equine protozoal myeloencephalitis (EPM), a common neurologic disease of horses in the Americas. We have developed a set of enzyme-linked immunosorbent assays (ELISAs) based on the four major surface antigens of S. neurona (SnSAGs) to analyze the equine antibody response to S. neurona. The SnSAG ELISAs were optimized and standardized with a sample set of 36 equine sera that had been characterized by Western blotting against total S. neurona parasite antigen, the current gold standard for S. neurona serology. The recombinant SnSAG2 (rSnSAG2) ELISA showed the highest sensitivity and specificity at 95.5% and 92.9%, respectively. In contrast, only 68.2% sensitivity and 71.4% specificity were achieved with the rSnSAG1 ELISA, indicating that this antigen may not be a reliable serological marker for analyzing antibodies against S. neurona in horses. Importantly, the ELISA antigens did not show cross-reactivity with antisera to Sarcocystis fayeri or Neospora hughesi, two other equine parasites. The accuracy and reliability exhibited by the SnSAG ELISAs suggest that these assays will be valuable tools for examining the equine immune response against S. neurona infection, which may help in understanding the pathobiology of this accidental parasite-host interaction. Moreover, with modification and further investigation, the SnSAG ELISAs have potential for use as immunodiagnostic tests to aid in the identification of horses affected by EPM.Sarcocystis neurona is a coccidian parasite that can infect horses and occasionally cause the neurologic disease equine protozoal myeloencephalitis (EPM) (6, 9). Horses become infected with S. neurona by ingesting sporocyst-contaminated food and water sources (8, 15). Ultimately, S. neurona can invade the central nervous system of the infected horse, causing focal or multifocal inflammation and EPM. S. neurona infection in horses is assessed by the detection of antibodies against the parasite in either the serum or cerebrospinal fluid (CSF); however, not all horses that seroconvert to S. neurona will develop EPM (9, 27). The seroprevalence of S. neurona infection in horses in the United States ranges between 0 and 89.2%, depending upon geographic locale (1-3, 10, 34, 37, 39, 40). In contrast, the incidence of clinical EPM has been estimated at Ͻ1% (28). It is not well understood what factors are responsible for the dichotomy between inapparent infection and clinical disease, but this ambiguity creates a major hindrance to EPM diagnosis and disease control.Current technologies for detecting S. neurona antibodies in equine serum and CSF samples include Western blotting (17), a modified version of Western blotting (35), an S. neurona direct-agglutination test (SAT) (25), and an indirect fluorescent-antibody test (5). Each of these current serodiagnostic assays utilizes complete S. neurona merozoite preparations as the antigen source, which has several drawbacks. Specifically, propagation of parasite cultures is relatively time-consuming ...