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"When Rare Becomes Common”, an editorial in the April 2017 issue of the Pharmaceutical Executive, points out that seven years is the average time it takes an individual to get a correct diagnosis for a rare disease in the United States.  In a twisted story parents that were unwilling to accept their doctor’s colic diagnosis saved their four-month-old from liver failure due to a rare hereditary disorder.  The disease is discoverable by newborn blood screening and treated with drugs.  The piece defines rare disease in the US as one that occurs in less than 200,000 Americans at a given time.  In Europe, a disease is characterized as rare when it affects fewer than one in 2,000.

Common diseases have phenotypes or subtypes.  Drugs may only work in a sup-population of those diagnosed with a specific gene.  Unfortunately people that take statins who have a certain genetic profile are at increased risk  to die from muscular wasting.

You are unique, defined by your genetic profile. The human genome is mapped.  But you are defined by a combination, and sometimes a variant, of those genes.  It’s important because cures come by addressing the specific gene’s mechanism.  Each of us have variant genes. Some drugs work for all of us, one drug may work better for your genetic makeup, and possibly a drug should not be used because of a variant gene you possess.

“All the individualized variants are rare, but then the rare becomes the norm.”

The International Rare Diseases Research Consortium (human) was launched in 2011 to facilitate cooperation and collaboration on a global scale among those active in rare disease research and maximize the output of rare disease R&D efforts around the world. What drives this group are policies and guidelines for data sharing and standards, diagnostics, biomarkers, biobanks, models, publication, intellectual property, and communication.  There are three scientific committees that guide the work: diagnostics, interdisciplinary, and therapies—and three constituent committees: funders, companies, and patient advocacy.  This model resulted in 200 new therapies four years earlier than expected.

Rare Disease Day occurs on February 28, participants host events that help create more awareness of and support for research collaborations that bring hope to patients.

Equine protozoal myeloencephalitis is a rare and unsolved problem worthy of EPM Awareness Day or maybe Protozoa Awareness Day.  Sarcocystosis is a common disease and is perhaps linked to the EPM syndrome.   We look forward to participating in the upcoming 2nd EPM workshop.  Organism biology, diagnosis, treatment and prevention are topics that will be discussed by clinicians, researchers, and industry companies, each with diverse interests in the field of EPM.  The meeting is in October, we will present our data on S. fayeri sarcocystosis and three inflammatory diseases that look like EPM.

Nicolas Faacci and Cheryl Heuton created a show called Num3ers in which a mathematician uses equations to help solve various crimes.  The strategy can apply to  equine protozoal myeloencephalitis.  The show teaches obvious and not so apparent factors should be considered.

EPM can be quite complicated to diagnose and treat if you ascribe to the opinion that the parasites (that cause disease) are in the horses central nervous system and an antibody test will identify these diseased horses. We have evidence that this “obvious” factor isn’t always true.

We assert that EPM is a syndrome that involves infection and inflammation.   Chronic inflammation may set the course for additional disease processes (see Intern J App Res Vet Med 2015, p164-170 and p175-181).  Protozoal infections may cause chronic inflammation.

Infectious processes use similar (innate) inflammatory pathways.  It’s not surprising several diseases confound EPM diagnosis when clinical signs include neuromuscular disease. The past 30 years were dedicated to reconciling antibody and EPM (due to S. neurona).  Often overlooked, S. fayeri, (the other Sarcocystis that infects horses in the United States) can be associated with neuromuscular disease, and certainly is an unaccounted variable in studies that defined ataxic horses with neuroinflammation as EPM horses.

The association between Sarcocystis and neuromuscular disease can be elucidated using algorithms.  The the database used by an algorithm should be large enough to get statistical analysis, attributable, and accurate.  No only does the analysis identify groups of similar animals, where and when animals are at risk, and possibly reasons for treatment outcome, the analyzed data can reveal new relationships. Our algorithms also apply to single cases and are the basis for many of our recommendations.

The EPM database contains more than 18,000 horses.  Attributable is defined as a medical record signed by a veterinarian.  All data is reviewed by quality assurance at each step: testing, recording, and analysis to ensure the data represents the veterinarians observations. The accuracy of the EPM data rests with the veterinary exam.

The following analysis, perhaps worthy of a Num3ers show, incriminates S. fayeri as a factor in equine neuromuscular disease.

We intended to demonstrate that C-reactive protein, CRP, an acute phase inflammatory biomarker, is elevated in a horse with S. neurona infection.  Intuitively, horses with EPM would have an elevated CRP.  Horses that are effectively treated for EPM would show a decrease in CRP.

We use the definition of EPM that says horses have antibody against S. neurona and a gait score (GAS) that is abnormal.  A GAS of 0 is normal and any GAS value >0 is abnormal.  Most scales are 0-5, a horse with GAS 1 has minimal signs and a horse with a score GAS 5 is  recumbent. For brevity, the analysis uses a GAS 0 and GAS +, the data is not distorted by clumping positive horses together.

The GAS as a parameter for EPM is a fundamental premise identifying a diseased horse.  An issue with using a GAS is that some horses with neurological disease not manifested as a gait deficits won’t fit the analysis.  One paper (that assessed the utility of antibody tests for EPM) described most cases in their population with predominant neurologic signs dysphagia, changes in vocalization, vestibular dysfunction, facial paralysis, and changes in mentation, therefore they were assigned “brainstem” lesions (see J Intern Med 2010 p 1184).  Case selection is a confounding factor in between-study analysis, it is difficult to compare studies that primarily use gait as a marker for EPM with studies that don’t.  Also, this is evidence that at least one group of researchers recognize that S. neurona infections don’t always cause gait anomalies.  It is unlikely they considered S. fayeri in their study.

The studies we conduct are federally mandated to use assessment of EPM by gait only.  Serum CRP concentrations (ug/ml) in horses with normal gaits and abnormal gaits due to neuromuscular disease are shown in the charts below.  Our numbers show that different cut-off values are important in patient evaluation.


Horses with gait deficits (due to non-infectious causes) may be confused with a horse with EPM. A diagnosis of EPM  requires gait deficits and antibodies against S. neurona.  Sarcocystis neurona seronegative horses with weakness, ataxia, and neuromuscular disease are called idiopathic encephalomyelitis, IE, in this analysis. It is apparent that any test that included other Sarcocystis (S. fayeri) would confound the data, therefore we use species specific ELISA tests to define S. neurona antibodies. The analysis of species specific serum biomarkers for S. neurona and serum CRP concentration from untreated horses (GAS+) with attributable data (EPM, n=832; IE, n=914 ) was investigated.

There is little difference between the groups although it is noteworthy that slightly more horses with IE have a normal CRP concentration.  Careful analysis, review of the literature, and common sense led us to examine several factors that could explain these data.

The next algorithm compared normal (serum CRP concentration <17ug/ml) or abnormal (serum CRP>16 ug/ml) with (clinical) and without (normal) clinical signs of neuromuscular disease by serum antibody status against S. neurona, and S. fayeri.  The serum was obtained from untreated horses with attributable data.

Normal horses with an elevated CRP were more likely to have S. fayeri antibodies than S. neurona antibodies.  These data indicate that sub-clinical S. fayeri infection can cause inflammation that is detected by CRP.

Research developments newly reported for Sarcocystis neurona may impact horse owners their veterinarians.  A novel genotype XIII was reported by Barbosa et al in the International Journal for Parasitology (2015).  This novel genotype is a sea mammal-virulent SAG 1 strain supporting SAG 1 and 5 antigen types dominate animal disease. This strain is vertically transmitted, from the mother to the fetus indicating S. neurona is more like than unlike other pathogenic protozoa.  Our pending publications, reviewed in our last two blogs, report new tests for horses with recurrent or residual signs of EPM that seek to clarify the role of inflammation in suspect-EPM horses.  The bottom line is that the key to maintaining a healthy horse is management through testing and examinations and understanding the pathogenesis of disease.

Sarcocystis neurona possesses one of six major surface antigen genes, SAG’s 1-6, on its outer surface.  The horse makes antibodies to these SAG’s and the antibodies are detected in the serum by ELISA testing.  Minor differences within the SAG genes allows classification into genotypes, or antigen types.  For example a SAG 1 S. neurona may be antigen type II or XIII.  The horse can only distinguish between SAG’s 1, 5, or 6 (serotypes) not antigen types.  The SAG’s 2, 3, and 4 are genetically variable between serotypes, are present in all Sarcocystis, and allow molecular biologist to examine differences between SAG genes.  Geneticists look at allelic variation within the SAG genes and that allows them to sub-classify S. neurona into genotypes or antigen types.

We developed three SAG specific ELISA tests based on recombinant SAG 1, 5, and 6, the strains that infect horses .  The specificity of these tests allows us to distinguish between serotypes by the antibodies made in response to infection. The majority of all disease caused by S. neurona in animals is due to SAG 1 and SAG 5 serotypes.  There may be virulence differences between the S. neurona SAG 1: antigen type II or XIII (discussed in Barbosa’s paper).  What is clinically relevant in the sick horse is recognizing the  serotype.  Measuring specific antibodies allows the veterinarian to identify resistant infections, determine the response to treatment, and distinguish relapse versus re-infection.

Our newest work identifies horses that have chronic inflammation.  Inflammatory responses cause the clinical signs often associated with EPM.  Some horses won’t respond to antiprotozoal agents because the protozoa are gone.  A frustrating clinical presentation is identifiable with our new serum testing, MPP and IL6 ELISA’s.  Our approach to managing these horses has not changed, we still measure SAG antibodies pre- and post-treatment.  We assess the horses by gait score before and after treatment.  We monitor the CRP serum concentration.  What has changed is that we can identify horses that will relapse and give the veterinarian an explanation why and a management program.

It is well known that equine serum samples show variation in reactivity to different surface antigens of S. neurona.  The most useful clinical point: it is not the level of antibody (titer) present in a horse’s serum that is important, but noting that the levels rise with duration of infection.  Another general rule is that the first experience with infection (naïve horse) will induce antibody production. The levels are minimal and short lived (8 weeks or so).  A horse experienced with infections will get and maintain a higher antibody level up to 5 months in some animals.  Management of EPM cases requires multiple serum analysis.  A single point test can’t decipher a new infection or a relapse. Multiple tests can suggest it the animal has naive infection or chronic exposure.  The horse with chronic exposure is more likely to experience abnormal immune responses that may look like EPM but really suffer from chronic polyneuritis.  It is important to distinguish these infections because the clinical management differs.

There is a report for a new trivalent SAG chimera ELISA test for efficient detection of antibodies against S. neurona .  This is an ELISA test that seeks to reduce the time, materials, and cost associated with running multiple ELISA’s using SAG 2, 4/3.  The diagnostic protocol involves using the the SAG ELISA to determine a consensus serum-to-CSF ratio, ratios less than 100 suggest that antibodies against S. neurona are being produced in the CNS and therefore parasites are suspected in the CNS.  Diagnosis of EPM based on CSF results is still confounded by normal passive transfer of antibodies across the blood-brain barrier.  The changes to detection of SAG 2, 4/3 antibodies by the third generation test don’t identify the issues concerning non-specific testing, it can’t discern serotype, doesn’t indicate a treatment failure due to strain resistance, or point the clinician in the direction of inflammation when parasites aren’t there. It remains to be seen if the reduction in costs for time and materials will transfer to the client.

The most exciting new information is in the Barbosa paper.  They report vertical transmission in S. neurona in a sea lion, a harbor porpoise, five harbor seals, and a pygmy sperm whale. We suspected and reported S. neurona in the lung tissue of a fetus from a mare experimentally infected with S. neurona in 2004. We suggest that there is a unique window of opportunity for fetal infection, before the fetus gains cellular immunity.  The observations of Barbosa and sea mammal infections may change the opinion that S. neurona is not vertically transmitted in horses (Dubey).

The possibility that mares can transmit infections to the fetus may stimulate management changes on farms with a high incidence of EPM.  It would be a very rare condition and the veterinarian is the best source to analyze risks on a case-by-case basis.

Give us a call if you have questions or concerns about EPM .  We outline management protocols for horses as part of our consulting service.  We haven’t seen any new evidence that prods us to change our approach to the diagnosis of sarcocystosis or inflammatory mediated neuropathy.  We advise multiple exams, even in a recovered horse, once healthy let’s keep them that way!  We are committed to testing for SAG 1, 5, and 6 in independent ELISA tests, we won’t combine our three tests for convenience or price.  Confirming the presence of inflammation and distinguishing peripheral from central neuropathy are current goals.

We are committed to developing diagnostic tests and effective treatments for parasitic disease.


A horse is evaluated for muscle wasting, an abnormal gait, and paralysis/paresis of limbs and or cranial nerves and given a presumptive diagnosis of equine protozoal myeloencephalitis.  This horse may or may not be positive on tests that measure antibodies to S. neurona, most commonly it will test positive.  What can be confusing is that the horse can improve with treatment.  And sometimes the horse will improve without treatment.  Often horses with this clinical presentation relapse.

We may have found a link between these chronic-relapsing EPM-suspect horses and polyneuritis equi.  The etiology of polyneuritis equi is unknown but is generally considered an immune-mediated peripheral polyneuritis.  Serum antibodies against equine myelin proteins are found in horses with polyneuritis equi, the test was developed in England decades ago. The relapsing signs are related to demyelination followed by re-myelination of the nerves.  Eventually the auto-immune polyneuritis horses succumb to the disease because the inability to urinate or control bowel function are untreatable.

The link between EPM and immune-mediated clinical signs were discussed more than 14 years ago.  In 2002 researchers at Ohio State (Soflay, Reed, and others) considered cytokine mediated neuropathy in explaining their clinical observations in S. neurona challenged horses that did not have parasites in the CNS.  Inflammatory lesions in the CNS were the criteria for validation sample selection for “EPM” tests.  And that is the issue with “EPM” tests.  We stand our ground that antibody tests detect Sarcocysis antibodies and some antibodies are specific for S. neurona.  EPM is related to S. neurona antibodies but the inflammatory component of disease is not, and by definition a horse with EPM has clinical signs-and by all indications the clinical signs are due to inflammation.  And some of these horses may have polyneuritis equi.  And perhaps S. neurona is one etiology of polyneuritis equi.

What did we show in our more recent studies?  First, we determined that serum antibodies against a reactive site of equine myelin protein 2 that is linked to polyneuritis equi are found in horses with a diagnosis of EPM.  Possibly there is an IL6 reactive site on the disease producing region of equine myelin 2.  Drugs that decrease the synthesis of IL6 can reduce or eliminate clinical signs of disease due to inflammation.

Proteomics is the study of genes (sequences) and how they function.  The tests that we used in our proteomic studies are useful in clinical cases. The clinical cases we examined take us back to the CRP levels in horses. We found the higher the CRP value the more likely the horse has anti-equine myelin proteins.  Now we want to relate our observations to IL6 mediated polyneuropathy.  That would allow us to associate the IL6 pro-inflammatory pathway to disease in horses.  It may take a few more months of experiment to predict outcome of cases with our new tests.  What we can do now is more completely evaluate inflammation by a serum test and discuss our work and how it may affect your case.

“What's in a name?” wrote Shakespeare. “That which we call a rose, by any other name would smell as sweet.”

Koch’s designed postulates to establish an agents responsibility for disease.  The pathogen must be present in all disease, the pathogen must be isolated from the diseased host and grown in pure culture and these lab organisms must induce disease, to satisfy Koch.  His tests are the biological gold standard for demonstrating a causative relationship between a microbe and a disease. The monumental task that has prevented achieving these goals for Sarcocystis neurona is the two-host life cycle of Sarcocystis.

The definitive host, the opossum, serves as a host for many species of Sarcocystis, however the more discriminating intermediate host can be used to differentiate between Sarcocystis species.  Thus bioassay may be highly intermediate host dependent.  Other schemes used to identify the pathogenic agent are molecular: differentiation of organisms that are morphologically similar using specific genetic markers (genotype, antigen types) and antigen based assays that depend on an immune response to infection creating antibodies for assays.

Each technique is has issues:  viability of pathogenic material for bioassay, mixed infections in the definitive host, misidentification of genetic markers between highly similar organisms, and antigenic cross-reactivity of antibodies.  Antigenic cross-reactivity can limit antibody dependent assays to identification of genera and not species. It is accepted that there are 12 antigen types with 35 genotypes (Wendte) or four groups (Howe) of S. neurona.  As far as the horse is concerned, there are three serotypes of S. neurona: 1, 5, and 6.

Thirteen years ago M. Butcher demonstrated there were differences between Sarcocystis isolates, specifically an S. neurona isolate from a horse and a suspiciously similar one from a cat.  Bioassay experiments are used to correct science.  For example, antigenic and molecular similarity between S. falcatula and a horse isolate of S. neurona  were so minute researchers believed them to be the same, until animal infection studies proved otherwise.

It doesn’t surprise us that using the raccoon-opossum derived S. neurona  organism may be a flawed model to satisfy Koch’s postulates for EPM.  The organism was never demonstrated in the CNS tissues of many experimentally infected horses, a critical misstep if the disease is  by parasite-mediated pathology.  These challenged horses showed clinical signs that were unrelated to parasites in the CNS.  Maybe these experiments validated an immune mediated pathogenesis of disease in EPM irrespective of strain of S. neurona.

Experimental material from raccoon-opossum-horse infections have served as a cornerstone to current dogma.  Especially confounding is when biomarkers are validated using material from these experiments that induce encephalitis that is not directly parasite mediated.  It was shown that the raccoon-opossum material was a mixed infection; does that mean there is a biological difference in the S. neurona’s transmitted from the raccoon to the opossum and parasites weren’t found in equine neural tissues due to strain?  Or did multiple strains all induce inflammation, the true disease?

A new paper by Dryburgh (2015) attempts to clarify the biological differences among isolates of S. neurona by bioassay in raccoons and opossums.  In a nut shell, the experiment tests an organism identified as S. neurona that represents all the serotypes that induce antibodies in horses, SAG 1, 5, and 6. They used organisms from a sea otter SAG 6, raccoon (the strain used in the equine infection studies) SAG 1, a horse strain SAG 1, and a cat isolate SAG 5.  The pathogens were isolated in culture and cultured material was used to challenge the raccoons.

All infected raccoons developed antibodies to S. neurona although differences in immune-reactivity was observed between strains.   Raccoons did not develop neurological disease.  It was determined that the SAG 6 (sea otter) and SAG 1 (raccoon) isolate infected raccoons and were infective for opossums while the SAG 5 (cat) strain infected the cat, but not the raccoon.  The SAG 1 horse strain did not infect the raccoon.  Infected raccoon tissues (sea otter and raccoon) did infect opossums and produced more material (sometimes very few sporocysts-opossum intestines had to be scraped to demonstrate the infection) for future infections.  This work SUGGESTS that antigenic differences and biological differences exist among the S. neurona isolates.

These experiments affirm our position that it is important to determine the S. neurona serotype that infects horses using SAG 1, 5, 6 and EPM is an inflammatory disease.  It is important to point out that strains of S. neurona that cause disease in raccoons were biologically different than the strain isolated from a horse with EPM. Strains that induce antibodies in horses aren’t necessarily going to produce CNS infections--affirming our belief that detecting antibodies in CSF fluid will not determine which horse has EPM.  Demonstrating that strains of S. neurona that infect raccoons don’t invade the CNS of horses (shown by multiple experiments) but produce clinical signs and inflammatory lesions in the CNS is evidence that inflammation is a large part of EPM.

                                      ROSETTES OF S. NEURONA IN CULTURE

When taken together what is important is determining when protozoa are a factor in a horse with clinical signs of EPM.  Those horses need an anti-protozoal treatment and immune modulation.  Horses with clinical signs attributed to EPM, that have no evidence of protozoa, need alternate treatment.  The key to successful treatment is a good clinical examination and multiple supportive diagnostic tests.