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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.

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.

“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.

Like Jimmy Buffett says: changes in latitudes, changes in attitudes, nothing remains quite the same.  And nothing is truer than for the SarcocystisSarcocystidae have been around a long time giving them plenty of time to hone their skills as very successful parasites. The parasites strategy is change.  Toxoplasma and Sarcocystis are closely related and use similar infection strategies, that by design, make them very selective for the hosts they infect. These parasites display different surface proteins (SAG’s), during different parts of the infection cycle, that keeps the parasite ahead of protective responses that may be mounted by the host. Stage related protein expression is thought to maintain host specificity and also allow the parasite to manipulate the host’s immune system.

Sarcocystis fayeri infects horses and, unless the horse is debilitated, little pathology results because the horse is a natural intermediate host. Horses have adapted to this species and not surprisingly, horses don’t produce measurable antibodies to the S. fayeri  parasites. Sometimes SAG proteins aren’t abundant enough for laboratory detection, but are important in the animal’s response to infection. Undetectable (laboratory) levels of a parasite protein trigger mechanisms that allow the host to tolerate the parasite.  It is surprising that parasitic protozoa prominently display some SAG’s that become the dominant target of antibody production. This recognition must give some advantage to the parasites.  The resulting serum antibodies are instrumental in controlling infections and that can also favor the ultimate survival of the parasite.

Examples of dominant Sarcocystis neurona SAG’s are SAG 1, 5, and 6.  These SAG’s are detected by day 14-17 of infection, the levels of antibodies rise as the duration of unresolved infection continues. An example of SAG’s that do not induce a measureable antibody response are SAG 3 and 4, as was shown in a horse experiment run at the University of Kentucky.  Variable expression was shown by SAG 2 in the Kentucky experiment, initially measured SAG 2 antibody resolved before the end of the experiment.

In some cases the parasite proteins initiate pathological host immune responses.  These harmful responses can be in full force before other SAG’s are recognized by the host.  This is by parasite design.  For example, in early infections of Toxoplasma gondii, the most successful pathogenic protozoa on the planet, responses to TgSAG1 induce local pathological inflammation.  Experimentally deleting this SAG from the surface of the Toxo also eliminates the pathological immune response.  Likewise, in Sarcocystis neurona, we believe SnSAG1 recruits these adaptive immune responsive cells and these cells may provide the parasite a ride into the central nervous system: inside a host cell.

A large conundrum in Toxoplasmosis is why the intestinal stages of the parasite don’t induce an immune response in the (intermediate) host.  The same proteins displayed on these infectious organisms will induce good immune responses when given as adjuvanted vaccines, proving that they can induce an immune response.  As the infection progresses the parasites display SAG’s that make them recognizable to the host-- eliciting their own demise.  Controlling an infection may benefit Toxo because limiting the infection can ensure the hosts survival.

The horse is an aberrant host for S. neurona, the parasite-horse relationship evolved to limit infections. Some of these adaptive proteins, in some horses, stimulate pathological inflammation.This is perhaps why inflammation persists long after parasite elimination. And a reason why parasites are rarely found in the CNS of experimentally infected horses.  Testing for parasite antibodies can distinguish between animals with inflammatory pathology and those that need an anti-parasitic agent.

It is suggested that the lack of TgSAG1 compromises the ability of the parasite to persist in the brain tissues of mice.  Perhaps the lack of TgSAG1 changes the surface structure of the parasites and hinders its ability to enter host cells. Or it is possible that eliminating the pathological inflammatory process somehow hinders infections in mice. Also, again using Toxoplasma as an example, dendritic cells that are present in the intestinal tissue can extend across the intestinal epithelium and this may be an important mechanism used by parasites to disseminate in the body.

It is our hypothesis that S. neurona also uses dendritic cells to modulate IL6 inflammation. Currently under investigation at Virginia Tech University is the ability of levamisole to regulate the dendritic cells  response in regulating equine leukocytes.  We can provide circumstantial evidence of mechanisms of action of a drug if we can selectively block the action.  Parasites up-regulate IL6 in a detrimental fashion while levamisole turns off the inflammatory component of the IL6 response.  Decreasing the IL6 response by levamisole has been shown in mice and the IL6 response was implicated in dogs with visceral leishmaniasis.  Leishmania is a pathogenic protozoa and stimulates inflammatory molecules to invade the CNS causing signs of disease.  The parasites don’t need to enter the CNS to cause disese, just the cytokine.  If we can turn off the cytokine responses we can alleviate clinical signs of disease.

Part of our work is  to establish the mechanism of action of the drugs used to fight sarcocystosis.  Minor modifications of drugs can make them more efficient or render them ineffective.  For example, the efficacy decoquinate is 15 fold greater, due to increased bioavailability, when smaller particles are used in the formulation.  These effects are demonstrated in PK (pharmakokinetic) studies. Other changes are seen with levamisole.  Levamisole phosphate is toxic while the hydrochloride molecule is not.  And levamisole HCl breaks down very quickly, in just days if it is diluted in water.  That is why levamisole solutions can’t be prepared ahead of time and stored, these solutions won’t work properly.  It doesn’t remain quite the same.


The idea that EPM is primarily an immune mediated disease, perhaps obvious, is not readily accepted.  Parasites were isolated from brain tissue in experimentally infected horses (Ellison.  Intern. J. App. Res. Vet Med. 2004. p 79-89).  In this experiment, immuno-affinity beads were used to trap parasites from large volumes of neurological tissues—a technique that was key to proving the success of this model.  A highly virulent strain of S. neurona was employed in these studies. The organism was passed through a horse (with re-isolation) to increase the organisms virulence for horses.  Passing the organism through other species (such as a raccoon) result in parasites that are less virulent for horses.

The evidence that parasites (S. neurona) are associated with brain lesions is lacking in the majority of clinically diseased animals as well as experimentally infected horses (Saville. Vet Parasitol. 2001. Feb; p 211-22). What was clearly demonstrated in the Saville model is that clinical signs are associated with inflammation—not protozoa. Therefore it is generally accepted that clinical signs (neuroinflammation) and the presence of S. neurona antibodies define a horse with EPM pre-mortem. Even post-mortem diagnosis of EPM does not depend on demonstration of the protozoa (Saville).

Treating a horse with antiprotozoal drugs for EPM can result in elimination of antibodies and, in some cases, incomplete resolution of clinical signs.  Treating specific neuroinflammation can result in resolution of disease in these horses.  There are several inflammatory molecules that play a role in the modulation of the early immune response  to parasitic protozoa.  Molecules and their receptors are active areas of research.

We found data from a recent paper showing levels of specific neuroinflammatory molecules (found in brain and blood) distinguished between treated animals that had cleared systemic parasites, but not those in the brain, interesting.  It will take years of research to apply these tests to horses with EPM, should researchers even take on the work.  The first step in investigating the inflammatory component of EPM is recognizing the importance of neuroinflammation in disease. However, these tests may be instrumental in accruing more evidence that the vast majority of horses with EPM don’t have active parasites in the CNS.  Certainly the outcome will change the approach to treating EPM.