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“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.

 

Sarcocystis neurona in host cells 100x

Results from experimentally infecting horses with Sarcocystis neurona supports the notion that equine protozoal myeloencephalitis is a syndrome caused by cell damage due immunological responses to protozoal infections. The disease has two phases. In the first phase, parasites turn off the horse immune responses (Witonsky 2008) and that allows the parasite to spread from the gut to other organs (Elitsur 2007). The second phase occurs when antibodies are produced to the protozoal infection and that is when clinical signs are apparent. Not all horses succumb to damaging immune responses, they don’t show signs of EPM, despite the presence of antibodies produced during infections.

Critically, this view of the pathogenesis of disease prevents a “diagnostic test for EPM” using tests to detect antibodies to protozoan parasites. Horse infections can be detected by antibody tests, however diagnosing EPM requires the develop biomarkers detecting pathologic cell processes associated with protozoal infections. We recognize that protozoal infections and EPM are not the same thing. This discussion touches three issues that are the forefront of our research: the pathogenesis of sarcocystis infections, diagnosis of EPM (a syndrome) must include inflammatory markers, and inflammation associated with protozoal infections in horses is detected by C reactive protein.

There are several pathogenic protozoa that are implicated in EPM, most commonly S. neurona and rarely Neospora hughesi, and perhaps the most successful parasite on the planet, Toxoplasma gondii. Diagnostic antibody tests have to accommodate detection of all these contenders. We assert the terminology should be “idiopathic” until a specific etiology is determined. Each of these parasites may, or may not, enter the CNS. However clinical signs are found in conjunction with inflammatory cells in neural tissues of infected horses and inflammation + parasites define the disease syndrome EPM. Inflammation as the cause of clinical signs is not a new idea, nor a hypothesis held by exclusively by us. Researchers in Germany (Olias) identified the same scenarios in pigeon sarcocystosis (proposed as a model for equine protozoal myeloencephalitis) and recently Do Carmo (2015) reports immunological responses and markers of cell responses in equine toxoplasmosis that include C reactive protein, CRP.

We take issue with the notion that parasites invade the central nervous system (CNS) to cause EPM—and we reject that documentation of these interlopers by CSF antibody is necessary for diagnosis and treatment of the clinical signs of EPM in a horse. We challenge the dogma that states “parasites that enter and remain in the CNS to cause disease”. This position assumes that disease and parasites go hand-in-hand ignoring the inflammatory part of the EPM syndrome. No doubt parasites are related to EPM. Occupation in the CNS tissue is not necessary. A marker for pathologic cell involvement is needed to address the disease EPM.

Consider this: parasites aren’t recovered from very many animals with terminal disease attributed to EPM. Animal experiments support inflammation, and not parasites, cause signs of EPM. Histological evidence of inflammation is used to make the diagnosis in the majority of cases because parasites aren’t found by any detection method. And most profoundly-- animals with long-term disease are treated with the right protocol. Inflammatory lesions in the CNS are treatable.  Reconciling these facts put more weight on inflammation in the pathogenesis of EPM. Logically, more emphasis should be placed on diagnostic tests that include inflammatory markers of cell damage over antibody detection. A panel of tests may be necessary. Releasing the grip on old dogma is necessary to design new tests to diagnose and treat EPM.

The terminology used in various publications confounds understanding EPM. Parasites in the CNS of a horse, supported by antibodies in the cerebrospinal fluid (CSF), would be more appropriately called sarcocystosis. By definition EPM requires clinical signs and those signs are related to inflammation present often in the absence of protozoa. Sarcocystosis requires the presence of parasites. Unfortunately, when the “presumptive diagnosis” of EPM was used (that included samples from horses with EPM and not sarcocystosis) to “validate EPM tests”, understanding EPM took an errant path.

Tests that detect antibody to a specific organism (ie sarcocystosis) are available. Simply calling parasite infections diagnosed by antibodies as sarcocystosis, followed by serotype, would allow researchers the ability to re-frame their view of the pathogenesis of disease in the EPM syndrome. A small caveat is that detecting active protozoa would be desirable. We have a pretty good idea how long antibodies linger in a horse that eliminated parasites. Likewise post-treatment success is measured by a reduction in serum antibodies if the animal isn't chronically exposed to parasites. There is substantial evidence that CSF antibodies found in challenged horses is transient despite continued progressive signs of EPM. That data has profound implications on the value of CSF antibodies for determining EPM.

Wendte (2010) pointed out that highly conserved parasite proteins are similar (he was discussing SAG’s 2, 3, and 4) to S. falcatula and the implications for the lack of specificity of diagnostic tests based on PCR and antibodies. The mutual exclusiveness of SnSAG1, 5, and 6 present an interesting unexplained phenomenon that requires more research. If we knew the function of SnSAG1, 5, and 6 proteins, we may more fully understand the pathogenesis of EPM. We propose these proteins function in host inflammatory pathways leading to pathology associated with sarcocystosis in turn, that leads to EPM. Parasites in the CNS aren’t required to cause signs in this scenario. Concentrating on antigens unique to pathogenic strains would allow researchers the ability to re-frame their view on the pathogenesis of disease in the EPM syndrome.

A recent paper (Do Carmo, 2015) gives us hope that others out there understand parasitic protozoal infections as we do. Toxoplasma affects many warm-blooded animals, horses included. Toxo uses horses as intermediate hosts and these infections are generally asymptomatic. Fulminant toxoplasmosis is often associated with immunosuppression. Sometimes signs are mild. Signs can indicate involvement of the central nervous system that include ataxia or sometimes excessive irritability. In South America antibodies to Toxoplasma occur in 32% of the animals. Do Carmo and co-workers suggest a hypothesis that equine immune responses against T. gondii are lasting, variable, and a contributing factor for the disease pathogenesis and cellular lesions. These researchers investigated the levels of several immunological variables and markers of cell damage in Toxoplasma-seropositive horses. They found higher levels of immunoglobulins, pro-inflammatory cytokines, and CRP when compared to seronegative horses. They found a correlation between high antibody levels and inflammatory mediators. They conclude that as a consequence of chronicity of disease, cellular lesions may lead to tissue damage with the appearance of clinical disease.

Our starting list for immunological variables for S. neurona included TNFα, IFNγ, IL1, IL4, and IL6 (Spenser 2004). We also investigated serum amyloid A (Schwab 2010) and CRP. After years of testing serum from experimental and suspect clinical cases, we have settled on the most useful serological markers for S. neurona that are: antibodies against SAG 1, 5, 6 and CRP. We currently investigate responses (down regulating the IL6 receptor) to levamisole HCl and the direct effects levamisole HCl has on Sarcocystis neurona and Toxoplasma.

The diagnosis and treatment of EPM is a changing field, effecting the predicted outcome for so many horses. The EPM dogma will have to change simply by correcting the terminology used in infections and disease. Inflammation will occupy a position that is front and center to the discussion.