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Biomarkers

Here is a picture of a muscle cyst in a horse.  It is caused by a parasitic protozoa and is commonly found on post mortem exam.  You may be surprised to know that we can detect these cysts without killing the horse!

Sarcocysts in horse muscle are the final destination of infections caused by Sarcocystis fayeri.  This is a very common pathogenic protozoal infection called EMS (equine muscular sarcocystosis).  More than 80% of the horses in the United States are infected.  Muscle cysts aren’t generally  associated with clinical signs although disease is recognized in thin, debilitated, or stressed horses.  These infections are sometimes associated with muscle loss and weakness in a performance horse.  Rarely, they are associated with death. `The clinician  differentiates EMS from other neurologic diseases because it affects muscles, is slowly progressive, and doesn’t respond to common treatments. When infected muscles are ingested S. fayeri cysts release an enterotoxin that can make dogs and humans sick.  Consuming raw horse meat is the cause with different outcomes, both dogs and people can get enteritis from the toxin.  When the cysts are consumed by dogs the parasites can finish the life cycle and the dog  infects more horses. People get food poisoning, or enteritis, but the parasite can’t complete the life cycle.

Until recently the only definitive diagnosis for EMS was post-mortem examination.  The cysts are identified by histopathology using muscles from the tongue, esophagus, or skeletal muscles.  To recognize disease a cyst has to be present on that particular slide so it can sometimes be missed.  More often the cysts that are observed on slides are unrelated to the cause of death. Recently, Japanese scientists did an exhaustive evaluation of a case of food poisoning following raw horse meat ingestion by picnic goers.  They elucidated  the molecular signature of S. fayeri toxin. We were able to use that signature to identify S. fayeri infections in living horses by using an assay for anti-toxin. An advantage to the assay is that it can also differentiate sarcocystosis caused by S. neurona or S. fayeri. S. neurona infection in horses doesn’t terminate in the production of sarcocysts so the molecular signature of the toxin and surface antigens 1, 5, and 6 are uniquely different.  S. fayeri does have shared molecular markers with S. neurona, these are the surface antigens called 2, 3 and 4. The toxin is common to other cyst forming pathogenic protozoa but fortunately the only one of significance to the horse is S. fayeri.

We compared the histologic diagnosis of EMS in 32 horses to serum bioassay and found the results were very similar.  The histopathologist used 3 slides (one each from a different tissue in a horse) and we used a serum sample to bioassay for anti-toxin.  The diagnosis was correct in 78% of the horses by histopathology and 74% of the horses with the serum analysis.  The obvious advantage is that the bioassay is in the living horse.  There are several reasons that the analysis differs between the tests.  The toxin is present before cysts are formed so the serum test will detect infected horses before cysts could be seen on histopathology.  The animal will have cysts in the muscle about 77 days after the initial infection so the first phase of the infection isn’t detected on post mortem exam.  It is possible that some cysts don’t produce the toxin.  When the toxin isn’t produced differences in pathogenicity between strains may exist. Cysts that don’t produce the toxin won’t be detected by the serum test.  This isn’t a limitation on the serum test because the toxin is responsible for the clinical signs.

Another test that is useful for neurodegenerative disorders is the anti-myelin protein antibody test.  These tests were developed over thirty years ago specifically using horse samples and tissues. The horse tissues were used in myasthenia gravis (MG) research, a neurodegenerative disease in people, anti-myelin protein antibody is a hallmark of MG. It was prudent for researchers to substitute horse tissue instead of bovine tissue when Mad Cow disease was identified in Europe, this was great for equine researchers!  The molecular signature for myelin protein was identified and diseased horses were soon recognized as positive reactors when they had a disease called polyneuritis equi.

The anti-myelin protein assay  improved when myelin protein was mapped for epitopes.  Epitopes are reactive sites on proteins and and are the signal that reagents in tests detect. Another improvement in disease detection came along with the molecular biology revolution. Scientists realized that myelin needed to be folded properly.  When proteins are denatured they are like string, epitopes sit side by side.  Visualize a string, the two ends are far apart. However, when the protein (or a string)  is properly folded, conformational epitopes are recognized in the assay.  If you fold your string in half the two ends can form a unique epitope where the ends touch.

Another advance in assay development was determining that an inflammatory cytokine receptor was one of those epitopes found on the end of the folded myelin.  That was a useful discovery to figure out how the disease worked.  When the mechanism of disease in known, that is the pathogenesis of disease, a treatment can be designed. ,Fortunately the inflammation recruited by the cytokine doesn’t destroy the myelin producing cells it is active on the myelin proteins.  That means when the inflammatory disease is properly treated, and treatment is instituted early enough,  the nerves are repaired.  If the disease is untreated the nerves will be protected from inflammation by scar tissue.  Scar tissue won’t allow the proper function of the nerve. The assay can detect antibody against the myelin (diseased nerves) before there is irreparable damage. It is necessary to know what kind of inflammatory pathways are triggered, there are different types of inflammatory pathways that require different drugs.

Bioassays are useful when they detect markers that are characterized and understood.  They can be life-saving.