In addition to a neurologic examination and other tests used to rule in or out other neurologic disease, exposure to EPM may
be confirmed by diagnostic tests to detect the presence of IgG antibodies to the parasite. For deceased horses, EPM can be
confirmed if classic lesions of the brain and spinal cord are seen during postmortem examination.
A few diagnostic tests are available—the older Western blot test, the indirect fluorescent antibody test (IFAT) and a quantitative
ELISA based on S. neurona surface antigens (SnSAGs) SAG1 ELISA as well as the newer SAG 2,3,4 ELISA.
The initial diagnostic test was the Western blot, a complex, semiquantitative method first used in the early 1990s. The test
describes the presence of specific immunoreactive bands but relies on subjective interpretation for results, often with variation
from laboratory to laboratory. It is easy to inadvertently contaminate cerebrospinal fluid (CSF) samples with blood during
the sampling procedure, which can result in false positive results.
"A negative Western blot test from a blood sample is a reliable indicator that a horse does not have EPM," says Pusterela.
If the test result is positive, though, then a second Western blot test is performed on a sample of CSF to confirm intrathecal
antibody production, which is suggestive of CNS infection.
The IFAT is based on quantitative fluorescence of the entire organism under the microscope, SarcoFluor (specific for S. neurona) and NeoFluor (specific for N. hughesi). It's the only test that offers separate assays for the two organisms. However, the incidence of EPM caused by N. hughesi is thought to be low. IFAT was the first established quantitative antibody testing modality available that calculates the
theoretical probability of the disease depending on the titer of the immunologically affected animal. As with Western blot
test, this test method also is subjective in its interpretation.
ELISA tests based on the S. neurona SnSAGs are nonsubjective and quantitative, generating titers. However, there are significant differences in diagnostic accuracy
depending on the specific SAG used in the assay.
"Using the original surface antigen, SnSAG1, about 30 percent of the isolates in nature did not have that surface antigen,
causing false negative results," says Reed. "Because the SnSAG 2,3,4 ELISA uses three specific antigens expressed in all S. neurona isolates to date, there's an inherent enhancement of diagnostic power."
"While individual serum and CSF titers can be determined, it's the ratio of serum to CSF titers that's very predictive of
an EPM diagnosis," says Morrow. "There's a normal flow of serum antibodies back and forth across the slightly permeable blood-brain
barrier with a normal proportionality. As the intrathecal antibody production increases, the titer ratio decreases. Ratios
of more than 100 strongly correlate with EPM. Based on the results from over 100 necropsies, a ratio of more than 100 has
a sensitivity of 80 percent and a specificity of 97 percent."
Reed calls CSF sampling helpful, and at Rood & Riddle Equine Hospital they've switched to using the surface antigens SAG 2,3,4
titer ratio test for S. neurona. "In a validation study set, we now have more than 400 paired serum and CSF samples from field cases from several institutions
around the country, and more than 100 of them have gone to postmortem. So we have the gold standard of diagnosis on about
Pusterela notes that the IFAT and SnSAG 2,3,4 surface antigen diagnostics are both good quantitative tests, though there's
no published comparative study looking at both. "The UC Davis test [IFAT] is quantitative and has been evaluated using confirmed
EPM cases," says Pusterela. "Further, the titer appears to be directly related to the likelihood of disease in a neurologic