EPM has been around a long time. In 1999, I began to develop tools to understand the disease and develop diagnostic tests. Many tools were missing. The story that unfolded is told in our publications. The key to EPM was understanding the parasite, Sarcocystis neurona, and how it infects horses. How horses react to infection is as important as knowing what caused the disease.
Peer Reviewed Publications
Laboratory culture came first
The ability to create tools to study S. neurona and EPM was success in culturing it in the laboratory. In 1999 I learned what cells allowed the organism to grow, which cells held parasites in limbo, and chemicals that forced mature parasites to leave cells. I continued to build on the techniques described in this paper. Infecting primary equine cells was a first step in developing the infection model. The work with calcium ionophore and how these drugs affect S. neurona was another cornerstone of this fascinating work, important in drug development.
Molecular tools to identify infection
Another step in disease diagnosis and treatment is identifying a marker that can be evaluated for changes before and after infection. It is also useful to evaluate the marker after treatment. In this paper we describe identifying the major surface antigen of S. neurona from the most common serotype that infects horses. We cloned the gene for the major antigen and then expressed it as a recombinant protein to use in specific assays. We were first to identify, clone the genes, express the protein, and harness it for use as a bioassay so we named it SAG 1, for surface antigen 1.
The ELISA test detects SAG 1
The SAG 1 antigen is uniquely found on the surface of the most common Sarcocystis neurona that infects horses. The horse makes antibodies against SAG 1 that are found in the serum, and sometimes in the cerebrospinal fluid, of infected horses. A step in developing tools for diagnosis and treatment of disease is developing an assay and proving that it is directly related to the disease.
Early Signs of EPM
An initial step in developing tools to diagnose and treat disease started with identifying the disease clinically. Early detection of infection with Sarcocystis neurona, a cause of EPM, will give the best outcome. In this paper we describe the early signs of S. neurona encephalitis after experimental infection in six horses. Blinded examiners determined scores for ataxia, dysmetria, paresis, and spasticity as part of a gait assessment score. Other signs of infection are usually noticed by owners before classical signs are apparent. What surprised us was all horses followed a similar chronological presentation but not exactly the same timeline.
Building on previous works
Drug development starts in the laboratory with a lead molecule or two. Before one can move forward on drug development, the basics of safety, pharmacokinetics, toxicity, and target of engagement must be accomplished, this is called pre-clinical development. An assay for disease detection is required to ensure the correct animals are enrolled, requiring assays that can detect disease. When these pieces are in place, manufacture begins, initially in the laboratory and finally in a cGMP facility. These were the building blocks of our proposed treatment.
The hidden clue
The most perplexing issue about EPM, when I started my work in 1999, was why the organism was so difficult to find in EPM horses in brain or spinal tissue on post-mortem exam. Comments in the margins of my notebooks often questioned why inflammation and no organism. Finally, in 2015 we realized that a rare disease, polyneuritis equi, was related to S. neurona. The well studied myelin protein 2 was the key. We demonstrated that 78% of horses with Sarcocystis neurona infections also had antibodies against myelin protein 2.
A Disease Model for EPM
The missing piece of understanding any disease is the ability to create the disease and prove the disease was created, (Koch's Postulates). In this paper we explain how Dr. Ellison at Pathogenes was the first to fulfill Koch's Postulates using S. neurona to create clinical EPM and isolate the organism from the central nervous system of the horse.
Immune responses to rSAG1
After we knew that the immunodominant surface protein was SAG1 and antibodies were found in the serum and cerebrospinal fluid of EPM horses, it was necessary to show that recombinant protein induced a measurable response when given to horses. In this paper we were able to validate the SAG1 ELISA to detect antibodies in horses and show that an immune response to rSAG1 protected horses against disease by using our infection model.
What about dogs?
Sarcocystis neurona infects opossums, armadillos and rarely other species. The horse is an unusual host for S. neurona and that is why it doesn't form cysts in the muscles , but may induce inflammation that is related to polyneuritis. S. neurona was found in a dog with encephalitis. We worked with a small animal referral clinic to determine the serotypes of S. neurona that were present in dog serum. It was important to serotype the strains in this study.
A re-discovery
The outbreak of mad cow disease in England sent researchers looking for alternative tissues. Horse spinal tissues fit the bill! The research led to the discovery of a neuritogenic peptide on myelin protein 2, MPP. This region of the protein is involved with cellular immunity, inflammation, and disease. Read about the assay we developed to detect antibodies to MPP and our thoughts on the pathway in horses.
Equine muscular sarcocystosis
Horses are natural hosts for Sarcocystis fayeri. This infection can cause disease, but it is unusual. Some astute researchers in Japan published great tools that allowed us to detect disease-associated S. fayeri toxin in the serum of horses. EMS is another piece of the EPM puzzle.
EMS again
There are complications developing assays for S. fayeri. One would have to find a specific, non-cross reactive antigen to design an assay. Even if this was accomplished, only 6% of S. fayeri infected horses have disease-causing toxins, so infecting enough horses would be expensive. And only recently are clinicians thinking that S. fayeri contributes significantly to equine neuromuscular disease. Serendipitously, we discovered S. fayeri in horses with cyst-producing toxins that allowed us to develop our assay.
EPM mimics in horses
As we discovered, re-discovered, and uncovered clues to equine neurodegenerative diseases, we proposed in 2017 that relapses attributed to equine protozoal myeloencephalitis do not have parasites in the central nervous system and thus are not EPM. Read about our proof in this paper.
A Novel Retro-Inverso Pentapeptide for Treating Neuroinflammation
FC-12738, a retro-inverso pentapeptide developed by Neurodegenerative Disease Research, Inc., is currently under investigation for treating neuroinflammation associated with amyotrophic lateral sclerosis (ALS).
This study aimed to evaluate the pharmacokinetic properties of FC-12738, including absorption, distribution, metabolism, excretion, and drug-drug interactions.
Assessment of levamisole HCl and thymosin α1 in two mouse models of amyotrophic lateral sclerosis
Amyotrohpic lateral sclerosis (ALS) is a progressive neurodegenerative disease that causes generalized
muscle weakness and atrophy. Neuropathologically, ALS is defined by severe loss of upper and lower motor neurons with a robust neuroinflammatory response.