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A Skipping Stones Day

benchThe exceptional Lakeside Trail, which shadows the perimeter of Lake Tahoe in Tahoe City and provides splendid views of the water and surrounding mountains, is dotted with clusters of memorial benches, each with a plaque commemorating a life, often the life of someone who once enjoyed that very bench.  In addition to merely citing the years of birth and death, most of the plaques include a lighthearted comment relative to the person memorialized.  My favorite is a simple wooden bench with a plaque which advises the dearly departed Isabel Brown that “It’s a skipping stones kind of day.”

You remember skipping stones.  Locating the flattest, most aerodynamic rock you could find and sidearming it out over the water in such a way that it bounces off the surface as many times as possible.  A cleverly launched stone can travel forever, a mediocre effort will sink, if you’ll excuse the expression, like a stone.  I likened the stones to the 23 papers researchers would be presenting the next day at the 2nd EPM Society Workshop at the nearby Granlibakken Resort.  Some surely would skip across the surface, to Isabel’s delight, others would suffer a fate less kind.

The 2nd EPM Society Workshop was held October 25-27th at the Granlibakken Resort, Tahoe City, CA.Twenty-three abstracts, divided into 6 sections, were presented in fifteen minute bursts.  After skimming the surface of the obviously intensive research, there was discussion among the 43 scientists in attendance. Some ideas were accepted, some tolerated, and some sunk to the bottom without much discussion.  The clinical diagnosis of EPM in the horse remains a major concern among the group. It was encouraging to have Dr. Patricia Conrad (UC Davis) pressing presenters to challenge the dogma that surrounds EPM and accept out-of-the-box ideas that should be discussed and tested, egos aside.

Five abstracts touched on the Biology of S neurona and Neospora  (the other organism that causes EPM).  Alice O’Byrne presented data based on the molecular association of S. neurona found in marine mammals and opossums in western Washington, hypothesizing that sporocysts shed from opossums contaminate the marine environment via overland runoff, resulting in marine mammal infections. Not a new idea, discussions focused on sporocyst-contaminated water and the relationship to equine infections, practical information for field veterinarians and owners.

Antoinette E. Marsh presented novel data demonstrating a procedure to take two geographically and antigenically distinct S neurona isolates from animals with clinical EPM, manipulate them in vitro producing morphologically similar small sarcocysts that are distinguishable from larger sarcocysts produced by a raccoon isolate.  The  message was the importance of immunohistochemistry staining for detecting the very small sarcocysts that can be overlooked due to size and lack of tissue inflammation. The practical application of the research will be re-examining 20 years worth of histological samples looking for S neurona  tissue cysts proving the horse is an intermediate host for this parasite.  It is a big deal, to us it is relevant to selecting for drug resistance with over use of anti-protozoals.

A sinker presentation was the Proportional Morbidity Rate (Incidence) of EPM in North America. Of note was that practitioners are treating EPM in the field and fewer cases are being referred to veterinary teaching hospitals.  We find the discord between field veterinary diagnosis and the ivory tower is the CSF collection issue.  Academicians are all-in for CSF as a diagnostic while field veterinarians are uncomfortable with the procedure.  Proficiency for field CSF collection is lacking, perhaps because it is not part of the veterinary teaching school curriculum, mostly due to liability associated with teaching the procedure.

The Seroprevalence of S neurona and Neospora hughesi in healthy equines in the US demonstrated that only 18% of healthy horses were seronegative to both parasites using IFAT.  The presenter, Kaitlin James (UC Davis) noted that horses from the south and older Warmblood breeds were associated with seropositivity to S neurona while there was no difference in seroprevalence of N hughesi across the country. This should spur lively discussion of predictive values of tests for N hughesi by field veterinarians in upcoming regional veterinary meetings.

Monica Aleman’s presentation S fayeri infection associated with neuromuscular disease in horses examined equine muscular sarcocystosis (EMS) from a different angle than our approach. She showed videos of horses with myositis that were lethargic, apparently painful, stiff, and reluctant to move.  Aleman’s group did not establish causality but did report a possible association with S fayeri .  They concluded that assuming Sarcocystis spp is an incidental finding in every case might be inaccurate. Discussions on EMS centered on the pathologists’ handling of post mortem findings because tissue sarcocysts are universally marked as incidental.  Our abstract, S fayeri associated anti-toxin in serum from horses with neuromuscular disease,  reported an association of the S fayeri  toxin (determined by the presence of serum antitoxin) with inflammation.  There was some anecdotal discussion on treatment of EMS, though these anecdotes did not rise to Conrad’s Bar Of Acceptability.  Effectiveness studies are relatively easy to conduct.  Industry funding for S fayeri projects are sinkers. Field evidence can be obtained and papers written but noone is holding his breath for a licensed treatment.  It’s wiser to hold out for good data-driven studies before accepting a few anecdotes as evidence.

I had hoped to stir highly relevant discussions on the biology of S neurona and S fayeri reflecting on evidence published by Edith Box in 1984 for S falcatula that sporocysts are derived from gut infections (not from bradyzoites released from degrading sarcocysts)  and the longevity of sarcocysts seen in horsesOur work would tie in Dr. Marsh’s observations and perhaps lead to enlightenment on recurrence of clinical signs due to inflammation of EPM. Unfortunately, my EMS antitoxin paper preceded the presentation for the Development and Validation of Pathogenes Assays. Discussion on our EMS presentation centered on polyneuritis equi assays (little positive experience in the group and anecdotal experience with one in-house assay developed against P2) and co-morbidity of sarcocystosis in horses. Lost was my point that EPM is over diagnosed and confused with highly treatable diseases. A positive outcome was a possible collaboration with Dr. Aleman on our mutual interest in using our polyneuritis equi assay using her post-mortem samples.

Dr. Sharon Witonsky presented an overview of host genetics and immunology and Identifying the immune phenotype in EPM horses.  Based on the data their group gathered thus far using histopathology, horses that were acutely affected appeared more likely to have acute inflammation vs. horses with more recurrent signs.  Horses with recurrent signs had degenerative changes with some evidence of previous inflammation. The ongoing study may determine the frequency at which S neurona is present in the CNS lesions of EPM infected horses. The frequency of the presence of thecal antibody producing antigens in the CNS of diseased horses would clarify the validity of using “histopathological lesions consistent with S neurona” in the absence of parasites as Gold Standard material to diagnose disease.  The abstract used a definition of a horse with EPM that was based on clinical signs consistent with EPM and are positive for S neurona antibodies in the CSF as positive (diseased) horses.

The abstracts presented in the Co-morbidity Between Apicomplexan Protozoa section presented ideas that polyparasitism is linked to increased disease severity (as in marine mammals).  The results of a study presented by Sarah Schale did not indicate a substantial role for polyparasitism in EPM in the eastern US. And Kaitlyn James suggested in her presentation that serologic testing of CSF and isolation of T gondii  in EPM suspect cases should be considered in the relationship between T gondii and EPM.

By far the most presented and discussed topics were those on laboratory diagnostics, highlighting the lack of a good diagnostic test for horses with EPM despite the CSF debate. Dr. Stephen Reed examined SAA as a biomarker for EPM diagnosis.  In his study, a total of 101 samples were included. 49 were “EPM positive” samples (7 confirmed by necropsy, 42 were diagnosed by serum:CSF SnSAG 2, 4/3 titer ratio of <100). He reported that SAA concentration is not of significance when used to identify and diagnose EPM in a single time point sample. Accurate antemortem diagnosis of EPM is challenging and it is accepted that evidence of intrathecal antibody production (e.g.SnSAG2, 4/3 serum:CSF titer ration <100) is diagnostic, however this criteria does not meet the bar for a Gold Standard EPM diagnosis. As a last minute head-to-head comparison, we were delighted to receive and test (using SAG 1, 5, 6 Elisa) 10 clinical samples provided by Steve Reed.  We identified all 5 horses that were given a diagnosis of EPM correctly and identified one animal as a “false positive” giving us a sensitivity of 83% and a specificity of 100% based on these samples. Only one sample was a Gold Standard positive and will wait for any future PCR data on these cases.

Amy Johnson presented data examining C-reactive protein (CRP) and serum amyloid A (SAA) in the diagnosis of EPM and other equine nervous system diseases.  They used a reference range of <0.1-10 mg/L for CRP and found no consistent relationships between SnSAG 2, 4/3 antibody levels and serum CRP or SAA.  Nor did they find a relationship between the two acute phase proteins in cases of EPM (diagnosed by serum:CSF antibody against SnSAG 2, 4/3  at <25). Not discussed but noteworthy is that we successfully use SAG 1, 5, 6 ELISA results with CRP in our case analysis.

Why the difference in opinion on antigens that are diagnostically useful and the relationship to CRP? A sidebar conversation with  keynote speaker Jeroen Saeij, presenting A comparative approach to the Apicomplexan protozoal organisms was productive. He understands the ramifications of using proteins or spliced peptides (SnSAG 2, 3/4) that rely on linear epitopes for antibody detection versus recombinant proteins (SAG 1, 5, 6) that are designed (expressed and folded) to use conformational epitopes. The importance of  conformational epitopes are well reported in T gondii work, Dr. Saeij’s foundation, as well as in early S neurona molecular work reported by Marsh, myself, and others. He touched on the importance of strain profiling in TG (SAG 1, 5, and 6 tests profile strains).  Examining serotype specific strains may determine that exposure to SAG 6 displaying organisms are immunogenic and protective against disease.

Saeij spoke of work published by Dr. Grigg’s group at NIH (S neurona found in sea otters).  The NIH group collaborated with us in discovering that SAG 6 from S falcatula and neurona were identical.  University of Florida researchers had long since shown that S falcatula (SAG 6 phenotype) didn’t cause equine disease reporting that no immune response was detected in these infected horses.  If the serology was correct, logic says the SAG 6 antibody we detect in horses is due to an un-isolated strain of SAG 6-expressing S neuronaWe collaborated with David Lindsay to show that cats on horse farms with EPM did have circulating antibody against SAG 1 and SAG 6 strains of S neurona, in addition to the SAG 5 strain used in his report.  That data may be public when he has some time.  Briefly touching on the suggestion by Philip Olias that pigeons (the intermediate hosts for S calchaci) could model EPM brought back memories of hosting his graduate student, Kristina Myer, for the summer.  We helped her develop skills to take back to the project in Germany.  Our conversations with Dr. Olias had supported our hypothesis that an immune mediated inflammatory reaction causes clinical signs in horses and pigeons. The immune mediated hypothesis was bolstered by his finding (unpublished) that immunosuppressants prevented pigeon encephalomyelitis while untreated controls succumb to disease. This finding may be public at the end of the German patent process.

Additional topics, such as host specificity of Sarcocystis and the production of a parasitopherous vacuole by S neurona, were welcome reminders of our unproven hypothesis.  Long hours as a PhD candidate were spent looking for a double membrane captured on electron micrographs, taken in early cell cultures, that would prove the existence of a transient and elusive parasitopherous vacuole. We suspect that host specificity is hidden in parasite mimicry of host cytokines.   It’s too soon to know if Dr. Saeij’s presence in the Society will have the impact we need to challenge the dogma.

Akinyi Nyaoke presented data from post mortem examinations on horses conducted over 26 years in California. The post-presentation discussions centered on the absence of pathognomonic lesions for the diagnosis of S neurona EPM and while histopathology may be diagnostic in cases in which merozoites and/or schizonts are seen microscopically, other ancillary tests including immunohistochemistry on many sections of the CNS are needed to confirm a diagnosis. Rodney Belgrave presented data comparing results for SAG 2, 4/3 ELISA and IFAT in which EPM positive samples were defined by an antibody value.  The abstract stimulated discussion because the data showed the ratio calculations for both assays required positive CSF samples; positive SAG CSF samples occasionally yielded negative ratios.  Not treating a horse with a positive CSF and a negative ratio caused more angst among clinicians in the room than the seasoned academicians. A comparison of specific antibody index and Goldmann-Witmer coefficient (C-value) to evaluate intrathecal immunoglobulin G production in EPM concluded that a C-value, previously defined at a 1.0 cut off value, was found to be improved using a 4.0 cut off value. A clinically useful nugget.

Pathogenes research was summarized in three abstracts in the co-morbitity, diagnostics, and treatment (prevention) sections.  Our biggest disappointment  was that the meeting leaned heavily toward S neurona and not the host response to infections.

Common among researchers were regrets about insufficient funding to carry on their work.  Most have champagne wishes and caviar dreams, but, alas, there are few Scrooge McDucks out there opening their money bins to aid in the battle against EPM.  Much of the subsequent research will, of necessity, be self-funded.

All in all, a worthwhile meeting.  Some stones skipped glibly over the surface, a few kept their heads above water and only a few fell to the bottom.  Isabel Brown, who I have it on good authority was a researcher herself, would have been proud.