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Lyme disease (infection with Borrelia bugdoferi) can confound EPM research because the diseases may have similar presentations. Clinically, there is no clear distinction that indicates a horse with Lyme disease from a horse with EPM.  Researchers are making an effort to describe the typical Lyme case.  Signs consistent with Lyme disease include ataxia, peripheral neuropathies, cranial nerve inflammation, muscle wasting, skin sensitivity, stiff neck, and possibly uveitis.  Changes may be observed in CSF fluid prompting collection of fluid by a spinal tap to look for organisms or antibodies against B bugdorferi.

There is no good diagnostic test to define the actively infected horse with neuroborelliosis (organisms in the central nervous system).  Detecting the organism in the central nervous system isn’t easy and requires molecular analysis and reasonable certainty that there is antibody production in the CSF, not antibody contamination from the periphery.  Similar to a supportive diagnosis of EPM, some clinicians use the serum/CSF ratio—generally a referral facility.  A test-positive interpretation may necessitate detection of antibodies against several Borrelia antigens as well as paying close attention to dilution factors used in the laboratory.

 

CDC Map of Lyme Disease in the US

Where the horse lives is an important consideration when including Lyme as the cause of neurologic disease because Borreliosis is a regional disease.  Check out the CDC’s map, shown above. Serum testing for Borrelia antibodies by ELISA is helpful to support a diagnosis, most Borrelia infected horses have serum antibodies.  This is another disease in which ruling out other causes of the signs is key.  And a negative serum test supports another cause of neurologic dysfunction and maybe helpful to rule in other diseases on the differential diagnosis list.

Several treatment protocols for Lyme are published, however, the ability to eliminate the organism by antibiotic therapy (in some infections) may not be possible.  Chronic disease is suspected in horses just like the syndrome in people.  Lyme disease does cause inflammation increasing the expectation that inflammatory markers like C-reactive protein will be elevated. Likewise, treating the inflammation can alleviate some signs of the disease and is useful.

Because horses can have antibodies against S. neurona, horses with and without EPM can have Lyme.  Lyme and EPM are a serious combination and can be lethal.  In our research it is important to include horses that have disease exclusively due to EPM.  It helps our final FDA submission that we are conducting laboratory studies because we know the horses don’t have Lyme disease.  But we’d like to avoid the  issues that would complicate our analysis of field cases.  That is why we ask you if Lyme disease is a consideration, you’ll find it on our submission form.  Being aware of Lyme, the regional nature of the disease, and ruling it in or out in a horse with ataxia is important to us.

“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.