Thirteen steps to where we are
Equine protozoal myeloencephalitis (EPM) was the most commonly diagnosed neurological disease in horses in North America in 1997. In 1999, there was a lot of missing information about the disease and Sarcocystis neurona. The first successful isolation of the protozoa from the spinal cord of a horse with EPM was in 1991 (Dubey), and an antemortem diagnostic test, the Granstrom Western Blot, was developed in 1993 using the Dubey isolate.
The lack of specificity of the Western Blot test led clinicians to uncover subclinical S. neurona infections, they were prevalent because more than half the horses in the US had intestinal infections and were seropositive on tests. Scientists quickly recognized variable antibody reactivity on the WB, and when a new antigenically different organism was recovered from a horse in 2001, the diagnostic waters were muddied.
The PhD project I selected in 1999 was to create tools needed to study S. neurona. Since then we have developed 13 tools, each tool is a step to understanding the disease EPM.
The organism had to be cultured in the laboratory and then a marker to identify antibodies from infected animals could lead to a useful diagnostic test. A diagnostic test not only has to differentiate organisms in culture but must also distinguish between diseased and non-diseased individuals. After molecularly characterizing the most likely-to-be-important gene from a disease causing S. neurona, the next step was creating a disease model. The gene was the prominent 29kDa SAG 1 gene of S. neurona. Creating a horse model of EPM had stymied researchers since the initial isolation of the organism in 1991.
Using unique culturing techniques (Step 1: Ellison 2001) and information from the characterization of SAG1 and expressing the recombinant protein, rSAG1 (Step 2: Ellison 2001), the first successful experimental model of EPM was developed (Step 3: Ellison 2004). The unique culture techniques used horses’ cells to be infected in vitro and returned back to the donor, creating an environment that transported the organism to the central nervous system, just as it is accomplished in nature. The term Trojan Horse Model was appropriate for the techniques used. The Trojan Horse model led to validation of the SnSAG1 ELISA (Step 4: Ellison 2003) as a diagnostic test and tied the clinical signs to antibody detection.
The Trojan Horse model was used by pharmaceutical companies to test the efficacy of their anti-protozoal drugs. A unique opportunity was the ability to detect early signs of EPM (Step 5: Ellison 2003) that were observed in all infected horses. Another use of the Trojan Horse model was examining immune responses to the recombinant protein, rSAG1, and determining that the antibody responses were protective against challenge, rSAG1 was a useful vaccine.
By 2012 the Trojan Horse model was used test effectiveness of drugs, and drugs that would kill the protozoa (Step 6: Ellison 2012) and modulate the immune system to prevent adverse immune reactions (Step 7: Ellison 2012). Other species were implicated in infections with S. neurona, they included dogs (Step 8: Ellison 2012) and cats. Modulating the inflammatory response to S. neurona was important, it had been a conundrum since 1991 that horses would have signs of EPM, but isolation of the organism eluded researchers. Inflammation and no parasites needed an explanation.
We found some hidden clues in the rare disease, polyneuritis equi, and inflammation associated with equine myelin pro
tein 2 (Step 9: Ellison 2015). A re-discovery led us to a neuritogenic peptide (Step 10: Ellison 2015) and the association with S. neurona. We were not the first to think of this, others tried and failed. Our success depended on using recombinant proteins and not a myelin protein homogenate. When others abandoned this path, they didn’t return and try and improve the assay. We knew that success of an ELISA depended on recombinant protein over antigens used in Western Blots and applied our knowledge to the neural tissues. If you hear someone tried and failed, don’t forget to ask about the source of the antigen!
By now we had important tools to ask some questions. How do horses with equine muscular sarcocystis, EMS, compare with horses with EPM? Horses are commonly infected with S. fayeri cysts and they are seen in histopathology of muscles from horses, almost 80% of horses have cysts. Generally, horses with EMS do not show any clinical signs, but we suspected some might. We developed an ELISA to detect a toxin from S. fayeri (Step 11: Ellison 2016) that was apparent in horses with neuromuscular disease. The horses infected with a toxin-secreting S. fayeri can show neurological disease.
Another conundrum in the equine community is that S. neurona causes relapses, not because the drug is ineffective, but because there are undetected, microscopic, single cell cysts that re-infect a horse. The prevailing opinion is EPM horses relapse, and relapses are a parameter for pre-mortem diagnosis of EPM. We questioned that hypothesis. Were chronic relapsing EPM cases really inappropriately treated EMS or polyneuritis equi horses? We answered the question in a long term field study (Step 12: Ellison 2017). An issue was the S. fayeri assay. We needed enough post-mortem cases with S. fayeri infections to make an association between our anti-S. fayeri toxin ELISA and histopathology. That opportunity presented itself to us when we conducted a safety study in horse (Step 13: Ellison 2019).