When a horse doesn’t survive the debilitating neurologic disease equine protozoal myeloencephalitis (EPM), the body might be sent to necropsy so pathologists can confirm the cause of disease, whether for the owner’s understanding, insurance purposes, or both. To confirm the diagnosis of EPM, pathologists must be able to see the causative protozoa, Sarcocystis neurona or Neospora hughesi, in tissue samples under the microscope or detect protozoal DNA with polymerase chain reaction (PCR) testing. But until recently it’s been unclear whether EPM treatment, disease duration, and tissue sample preservation methods interfere with results.

Rebecca Ruby, BVSc, MS, assistant professor at the University of Kentucky Veterinary Diagnostic Laboratory (UKVDL), in Lexington, recently sought to examine the effects of these variables, along with Jennifer Janes, DVM, PhD, Dipl. ACVP. Ruby presented their results at the Dorothy Russell Havemeyer Foundation’s EPM Advances in the Field Symposium, held in October 2022 in Asheville, North Carolina.

Ruby looked at tissue samples and PCR test results for S. neurona, the primary causative agent of EPM (and the one they typically test for in Central Kentucky), from 199 horses with neurologic disease tested from 2010 to 2021—the lab has a high equine caseload and keeps an extensive library of samples for research. Of those horses, 78 had detectable DNA with cycle threshold (CT) values from 26.56 to 40.08 (average of 34.30). A CT value indicates the point where a real-time PCR diagnostic result changes from negative to positive (think about the time it takes for the test line to appear, or not appear, on a COVID-19 PCR test, for instance).

Within this group 60 (75%) horses had experienced neurologic signs for a duration of one day to three years, with a mean of 60 days and a median of 10 days. Ruby reported that 31 (39%) of the horses were known to have received antiprotozoal treatment prior to death.

Pathologists tested 56 formalin-fixed paraffin embedded (FFPE) samples and 19 fresh nervous tissue samples, with average CT values of 34.78 and 32.83, respectively.

She described additional findings:

• In two cases pathologists detected neurona DNA in horses with noncompatible histopathologic lesions—in other words, they didn’t see spinal cord damage that would suggest the horses had EPM, but the animals tested positive for it regardless.
• In 121 horses they didn’t find neurona DNA. Pathologists observed nervous system inflammation in 40 (33%) cases, warranting a presumptive diagnosis of EPM. “It’s like our constellation of inflammatory changes considered consistent with EPM, so we’re not seeing the protozoa necessarily, we are not identifying the DNA,” she explained. “We’re just saying, ‘We don’t know things that do this in horses that are not EPM.’”
• In the remaining 66% of cases, they pinpointed something other than EPM or a noninflammatory neurologic condition (i.e., cervical vertebral stenotic myelopathy, aka Wobbler syndrome) as the primary diagnosis.

“Out of this whole group, it’s really important to recognize 66% of those horses had a diagnosis that was not EPM,” said Ruby. “So, these are horses that might have been on the necropsy floor due to a history of acute onset neurologic disease, resulting in testing for S. neurona immediately, and then they were diagnosed with something else.”

Ruby said this subset of suspected, but not confirmed, cases of EPM is the group of horses she and her colleagues hope to study further, looking for other protozoal organisms besides S. neurona that could be causing the neurologic signs.

In the meantime, as far as post-mortem testing for EPM, “From a financial perspective it’s probably smarter to run the test on targeted PCR than formalin-fixed, because this allows the pathologist to identify a site with active inflammation, which makes it more likely the protozoa will be in that section of the nervous system.”

In summary, Ruby explained that S. neurona is detectable in FFPE tissue in horses with acute and chronic neurologic disease, with and without antiprotozoal treatment, and pathologists rarely detected S. neurona DNA in horses with no histopathologic lesions that would support that diagnosis.