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.