Published August, 2012

Dan Howe, PhD, a molecular parasitologist at the University of Kentucky Gluck Equine Research Center, has developed a new assay to aid in diagnosing equine protozoal myeloencephalitis (EPM). This enzyme-linked immunosorbent assay, or ELISA, measures antibodies to the surface antigens (SAGs) SnSAG2, SnSAG3, and SnSAG4 ofSarcocystis neurona,the parasite that most commonly causes EPM.

These specific SAGs are proteins on the outer surface of the parasite that trigger the horse's immune system during an S. neurona infection. By measuring a colorimetric change relative to the amount of antibodies in a horse's blood sample, the assay provides quantitative data expressed as a titer (a measurement of antibody levels in a blood sample). Most horses have varying levels of serum antibodies against S. neurona due to a high natural exposure rate (seroprevalence) to the parasite.

"Although many horses have been exposed to S. neurona and have antibodies against the parasite, this doesn't necessarily correlate with an ongoing active infection, and relatively few horses ever develop EPM disease," Howe said.

Because of this high seroprevalence rate but low disease incidence, simply detecting antibodies in a horse's serum provides modest diagnostic information, said Howe. Instead, cerebrospinal fluid (CSF) obtained via spinal tap is the best method for diagnosing EPM.

"The new assay incorporates three unique antigens of S. neurona to measure the antibody response to parasite infection in serum or CSF," he said.

The Western blot, developed at the Gluck Center in the early 1990s, was the first commercially available test to detect a pattern of S. neurona antibodies. Subsequent tests employed different formats for antibody detection, each with their own limitations of use and interpretation. Recent studies indicate that detection of antibodies against SAGs 2, 3, and 4 provide a reliable and accurate indication of EPM disease when both serum and CSF are tested.

A field study on nearly 400 neurologic horses was conducted in collaboration with Rood & Riddle Equine Hospital, Lexington, Ky.; University of Pennsylvania New Bolton Center, Kennett Square, Pa.; University of Florida, Gainesville, Fla.; and the Marion duPont Scott Equine Medical Center, Leesburg, Va. The researchers' goals were to evaluate this ELISA test's clinical usefulness and diagnostic parameters and to reassess the value of obtaining CSF for accurate EPM diagnosis. Neurologic status was determined at case presentation, horses were grouped by diagnosis, and paired serum and CSF from each horse were tested with the ELISA. While serum ELISA titers did not correlate well with an EPM diagnosis, higher CSF titers were more often associated with disease. However, researchers obtained the most diagnostically predictive ELISA result when the CSF titer was compared to the serum titer and expressed as a ratio, Howe said.

"Horses suspected to have EPM will therefore benefit most by having CSF tested in conjunction with the serum," he said.

According to Howe, the serum to CSF comparison provides more accurate information and strongly supports whether there is active infection in the central nervous system. The information from the field study also reaffirmed that performing spinal taps to obtain CSF is an important diagnostic tool, he said.

The assay has been licensed exclusively to Equine Diagnostic Solutions LLC, a private reference laboratory in Lexington, co-owned by two University of Kentucky graduates.

Shaila Sigsgaard is a contributing writer for the Bluegrass Equine Digest.