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Recently JAVMA published a letter to the editor and we’d like to give the author kudos!  He was disappointed that after years of using a particular product, a clinical trial revealed the drug was not effective.  He placed his faith on recommendations from trusted and respected colleagues, board certified specialists and continuing education speakers. They didn’t base their opinions on clinical data and that led to years of using a drug for pain in dogs that was just shown to have a complete lack of effect.  We all recognize the importance of new scientific evidence, lack of funding for many projects, and the burden on the people that provide treatment recommendations to know and understand the supporting data- or lack thereof-before dissemination of their opinion.  We concur with the statement in response to the letter that  many clinicians, if they looked at the information used in the daily treatment of patients, would likely be shocked to find out just how little clinical data are truly available to support current recommendations, or how many opinions are not based on experience or understanding-just a conflict of opinion.

We have taken a different path with our work.   Rather than patenting the intellectual property (IP) and  then licensing the technology, on completing research we decided to make our work available to those who needed it, and open the discussion.  Has it cost us in many ways?  Yes.  But then, I grew up in the scientific community at a different time.  Once upon a time, that would be the 1970’s, we shared data and discovery.  I was a lab rat back then, spending many hours with the fluorescent microscope-looking at how Chinese hamster ovary cells responded to ATP stimulation.  OK, I get it.  But the experience was good.  We shared samples and “stuff” that moved knowledge forward.  I used that experience to look at Leptospira and Moon Blindness, another immune mediated condition, for my Masters thesis.  Then off to veterinary school.  While I was practicing veterinary medicine in the field over the next 20 years, things changed.  There was the biochemical revolution that spawned the field of molecular biology, and PCR, and proteomics.  Suddenly things like PCR, had monumental value.  Everything was DNA and  genes.  Heck, whole organisms are patented-Neospora comes to mind, (quick update, the patent office won’t do that anymore).  To use the IP, one has to have a license and pay royalties.  The revolution was in the 80”s to the 90’s, the patent for PCR was filed on June 17, 1987.

When I returned in 1999 to work on my PhD, in molecular biology of course, everything went through the University’s Office of Licensing and Technology (OLT).  Universities recognized the value of IP.  No more sharing.  No more shared discovery.  Heck, recently I wanted to use a video to help veterinarians get CSF taps using a standing procedure.  I was told there would be a largish up front fee followed by a royalty due for each use…one fee for every veterinarian that viewed the tape. It was explained to me the video is like a Beatle’s song-and they wanted to hold my hand.  I made my own video and freely gave it out hoping to get veterinarians to collect CSF and support our Orogin trial. Oh, we were criticized, one veterinarian wanted the video removed and not shared because all those horse owners would be getting CSF taps on their own horses.

We persevered, partially due to the words of one of my PhD professors, Ellis Greiner, “Science is self-correcting”.  If I continued to do work, collaborate with those with true curiosity and an open mind, we would eventually see the day when our ideas would receive acknowledgement and open discussion.  Because that is what science is, open discussion of ideas. To that end we publish our work, in peer reviewed journals, and submit our papers and abstracts for presentation at scientific meetings.  We collected and shared samples, (DNA, RNA, organisms), and imparted ideas, traveled to NIH and Washington and beyond; we teleconferenced, (with Germany, the Netherlands, Japan, and even Kentucky), with those who  were open to the burden of review.  There was plenty of criticism from those who didn’t take the time to understand, review and hold an open discussion.  We carefully persevered, leaving breadcrumbs.  Sure, we patented some IP.  And made some IP  public so that  others can’t patent it.  The IP is available to everyone.

I think the long awaited day is dawning.  The discussion at the upcoming EPM special information session will have a panel discussion on the evidence of the role of S. neurona in infection and disease.  Is it a disease of low parasite burden-host mediated pro-inflammatory response or is disease a reflection of high parasite burden resulting in direct injury to the CNS? they ask.  We are presenting two papers, the most important is our recent data on polyneuritis equi.  Our work is important because our evidence supports, and may identify, one inflammatory pathway for disease.  The story we tell is one of relapsing disease and hope of treatment.  Our story identifies a protagonist molecule and the hero horse heading off with all four legs moving in proper order.  We look forward to SIG participants that will critically review what we have done.  We welcome suggestions or guidance on our errant interpretations. Perhaps some will return to their laboratories and repeat our work. Or collaborate without IP on the mind.  They may claim no funding-we are used to that because we are self-funded.  No big grants, although we’d gladly accept one.*  We are optimistic that this may be the moment.  We’ll let you know Dr. Greiner, and if our time has not come, we will continue to drop those breadcrumbs.

 

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*Disclaimer: We are a for profit company.  We do not accept tax-exempt donations. Nor do we accept generously offered dowry’s from grateful horse-owning newly weds-although we gratefully appreciated the gesture.

IMG_0329What does it take to consult with a veterinarian about a neurologic horse?  Is a consult only an opinion?  And how does the consultant form an opinion?

We adhere first to the Veterinarians Oath https://www.avma.org/KB/Policies/Pages/veterinarians-oath.aspx and the veterinarians client-patient relationship https://www.avma.org/KB/Resources/Reference/Pages/VCPR.aspx. These are part of our assessment of a case and documented on our submission form.  Because, in the consulting/research business if it isn’t recorded and validated then it didn’t happen!

An opinion is based on education and experience.  Our opinion continually evolves by our research and the research published by others.  And then very carefully analyzing what we can apply for each case. Our evaluations are a lengthy process resulting in information that is relayed back to veterinarians, one-on-one.

Initially, our opinion is based on evaluation of a blood or cerebrospinal fluid sample. There is a lengthy process of evaluation for each sample we receive.  We review and validate the data (and data entry) that we get from the veterinarian, a minimum of 5 reviews , at least one documented by the QA  officer, and this is before the samples are tested.  Each sample is tested against known values and the results of testing are documented. The results are first captured by computer,  these data are evaluated, recorded in the consultation record, and then interpreted. The validation process for each result involves another five reviews starting with the equipment measuring the reactions to the last step, result interpretation. The interpretation draws information from the case history that was recorded on the submission form by the veterinarian and a review of the prior data.  Did the gait score change? Was the horse treated and with what? Is there other information that is useful?

At this point, we have a basis for the consult.  The consulting report is sent to the veterinarian.  If comments are needed-- something is obvious to us but may not be to the busy veterinarian, we send an email.  We use the data that was entered for each parameter into many algorithms that are programed into our computer.  These are the algorithms that spit out reports for field studies, FDA forms, or new research.  In house, we tag some samples for more testing or more analysis.  When the veterinarian calls we quickly call up the complete consultation record on the animal and discuss our view of the case and listen to what questions they may have.  This may result in more testing, sending peer reviewed literature, or sending summary tables of data under review by our statistician.  We may have more discussion after the information is reviewed by the attending veterinarian.

Another review of the data for each horse is made by Dr. Benedetti. She selects cases that are more complex and would benefit from more testing, or at least a more detailed discussion with the clinician.  At each point notes are made to the file if there are any significant values for the data that we track.  One horse may change our algorithm, that’s how new ideas happen.

We find interesting things. For example, unexpected results prompt new queries of our data base.  We build in more parameters into our new query as appropriate. That is the power of a data base.  Here is a recent case, a 20 year old untreated horse presented acutely with an abnormal gait.  The veterinarian requested testing for auto-reactive antibodies against myelin and a CRP. The anti-myelin protein antibodies were present but the CRP was 0 micrograms per ml of serum.  That’s unexpected!  Generally we expect untreated horses with MP2/MPP antibodies to have elevated CRP levels.

Dr. Benedetti queried 19,161 records and found 487 records that were similarly tested for a minimum of MP2/MPP.  Of those, 403 had detected MP2/MPP antibodies and an elevated CRP while 26 had a CRP of 0.  Our result is unexpected, only 5% of samples we tested are similar! Other parameters that we captured from submission forms are age and treatment history, the start of our understanding of this case. The group of samples with this result (antimyelin antibody positive and tested for CRP, CRP=0) come from horses that are teenagers, and 12 of them had no history treatment.   We can do more evaluations for region of the country,  time of year, or how the samples were shipped. We may take the 12 horse data set and design a questionnaire that would go to many veterinarians, targeted to a specific case to get more data for our query.  The case may be one they saw years ago, we would be looking for outcome, or a recent submission that would spur additional testing. Based on the results of the investigation we evolve our algorithms.  We expect veterinarians to call us and ask and discuss the case.  More discussion will most likely lead us to the correct interpretation or lead us to another investigation.

If we find it appropriate, we may design a survey.  For horse owners we may make the survey a quiz. Our consultation program is designed to analyze a unique case against a large data base giving veterinarians the wisdom of the crowd.

1Recently we quizzed horse owners about their interest in equine protozoal myeloencephalitis. Eighty-three percent of horse owners look to their veterinarian first for information about EPM! Ninety-seven percent knew that EPM was caused by a protozoa and 92% knew the opossum transmits the disease to horses. Eighty-five percent thought the infection was treatable and 10% felt the disease was very difficult to treat.  An accurate diagnosis only rated with 33% of owners.  There are a couple of areas veterinarians have some work to do, for example 13% of owners think that vitamin E will treat EPM and 7% think vitamin C is involved.  The majority of owners recognize the importance of inflammation in disease.

Veterinarians learned the basics about sarcocystosis in school.  And as they developed the art of practicing veterinary medicine they found new information provided by drug companies that paid for the, research.  The resulting  papers described aspects of infections.  Most papers concentrated on antibody detected following infections.  The disease syndrome (infection plus inflammation) wasn’t explored because horses didn’t develop EPM from the induced infections.  Information was dispersed at continuing education meetings and peer reviewed reports.  Controlled studies using horses are useful however, these studies are limited by expense and the interests of those funding the investigations.

Published field case reports provide an overview of disease pathologies interpreted through the lens of academia because these reports describe particularly difficult cases referred to a university. Typically, horses suspected of EPM are seen by veterinarians and these cases are not referred.  A veterinarian may form opinions referenced on just a handful of cases.

We offer consulting to veterinarians.  Our reference comes from statistical data from thousands of cases of suspected sarcocystosis seen by veterinarians across the country.  Our statistician evaluates the data we provide and renders an opinion.  Some things are obvious.  Some things we think are true, but he tells us our data is biased by the samples we get.  It is unlikely that veterinarians send us samples on horses without suspect EPM.  We are able to condense the information and tailor it to each case.  And just in case a vet is too busy, if a case starts to drop through the cracks we send an email with suggestions.  As we discover new areas of interest we will test samples and we provide that information back to the veterinarian, just in case it will make a difference.  The power in our observations are the data base.  We make the single field case relevant to statistical data from the crowd.

Pretty soon we will resend a survey out to veterinarians.  It is designed to gather data that we will forward to our statistician.  Our goal is to define some unusual presentations of neurological disease and determine the prevalence of those diseases.  When we are ready, we’ll give you the heads up so you can fill out the survey for us.  And we will also ask horse owners to prod their veterinarians to complete the survey.  Numbers are important because the decision to produce new treatments is based on need.  Common disease are interesting to large pharmaceutical companies.  Rare diseases rely on grass roots companies.  Micro companies work on rare diseases because there is a need and there is a wealth of information to be exposed. We have inquiring minds.  And when something is obvious to us we begin to let you know.  And here is where we start, a survey. Thank-you in advance.

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.

The EPM Society is meeting this month in California.  Pathogenes is presenting 3 papers, one is of particular interest to the Society and that is Development and validation of assays offered by Pathogenes, these are tests used in our consulting services.  The nuts and bolts of our tests are published, including test format, dilution factors, assay development, samples used for validation and testing, and results interpretation.  A big part of testing is the sensitivity and specificity of the test.  We have enough experimental and field data to allow veterinarians to use a predictive value for EPM.  The predictive value depends on the amount of disease in the population.  For example, disease is more prevalent in Texas, the predictive value of a positive test is more meaningful in Texas compared to Idaho, we found no EPM in Idaho.   Yet, some details interest the group, such as strains of organisms and the validation protocols.

Background for non-test developers: The USDA,  under animal plant health inspection service (APHIS) directs animal health and veterinary  biologics.  The Veterinary Biologics Program implements provisions assuring that effective diagnostics are available and appropriate standards are developed.  They also issue licenses and inspect products and facilities.  They issue memoranda that relate to licensing diagnostic tests.  These directives outline the steps to provide sensitivity and specificity data, ruggedness,  receiver operating characteristic curves (ROC) that test sensitivity versus a false positive rate. The ROC curve is useful visualizing the compromise between sensitivity and specificity for different cutoff values and for selecting a cutoff value.

Our mission is to develop patent protected technologies for licensing and we use USDA’s requirements to guide our work.  For example, developing assays.  A first step is understanding what you want to measure, the analyte (that can be an antigen-a protein related to a specific organism, an antibody, or a genetic sequence). The next steps are  deciding on a range of concentration, sample type, and the potential for cross-reactions.

A big hurdle is the Gold Standard selected for a diagnostic test.  You will read about “Gold Standards” and “EPM” testing making this topic worth understanding.

USDA’s context for a Gold Standard is to provide “an accepted means of determining the diagnostic status (positive versus negative) of a diseased animal”.  These same animals should be evaluated by the test and the Gold Standard samples are used to determine the accepted “true diagnosis”.  Using EPM as an example, USDA requires reference standards (serum and CSF for example) from 20 animals.  They accept samples from field cases.  USDA requires confirmation of disease: organisms in the brain of the animal-the accepted definition of EPM and, here is the kicker, enough of the samples (serum and CSF) to provide the reference standard over the life of the test.  Our discussions with USDA confirm this is not an achievable goal.

Gold standard samples from 20 natural cases of EPM are difficult to obtain. The horses must not be treated prior to sampling because treatment changes the diagnostic analyte. S neurona has three serotypes (each analyte is unique to each serotype) that must be detected by the test. Sixty horses with natural disease are needed for SAG 1, 5, and 6 testing.  Common analytes SAG 2, 3, and 4 are not specific for the disease EPM because these SAG’s are present in non-EPM causing protozoa.

A licensed EPM test is not happening.

A core issue with the disease caused by S neurona is the contribution of inflammation to the EPM syndrome.  The organisms can be eliminated leaving some horses with treatable clinical signs of disease.  Detecting analytes that identify S neurona in conjunction with inflammation is clinically useful.  Each veterinarian must weigh the “Gold Standard” that was used for the test and it’s usefulness in their treatment decisions.

USDA will license an antibody test that detects a specific antigen.  The Gold Standard for these tests can be obtained by vaccination. For example, we vaccinated 50 animals with recombinant SAG 1 to obtain enough reference material for submission to USDA.  In addition, USDA requires the “master seed”, a number of vials of the analyte used in test development.  The master seed is validated from serial passages of the recombinant organism, all tested and documented, so a 3rd generation seed (serial)  is proven to be unchanged by passage. The requirements to license our SAG 1, 5, 6 tests are 3 master seeds (with validated serials) and 20 vaccinated animals.  The test is “for the detection of antibody against serotype 1 (or 5 or 6) of S neurona in the serum or CSF of horses”. We do not diagnose the disease EPM. We report the level of antibody against the analyte SAG 1, 5, or 6 of S neurona. We also use a serum test to evaluate the inflammatory reaction to infections.  This panel of tests forms the basis of our consultation.

We are pursuing a license for the ante-mortem diagnosis of equine muscular sarcocystosis due to S. fayeri  in horses. The assay will detect disease (the presence of muscle cysts) so the USDA Gold Standard for disease applies.  Reference standards are produced from field cases of EMS.  EMS is also easy to produce experimentally, a source of material for reference standards.

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Another disease that warrants a licensed diagnostic test is polyneuritis equi, a demyelinating polyneuropathy that results in ataxia (classical) or cranial nerve deficits (atypical).  The USDA Gold Standard for polyneuritis equi is met with field cases.   We identify cases of polyneuritis equi by clinical signs and antibody against myelin protein.  The disease process, a response to an infectious organism, results in demyelination of nerves followed by remyelination of that nerve.  The diagnostic we are developing identifies the demyelinating process.