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Occasionally horses that have been treated for EPM will relapse days or weeks later. Recurrence of signs of EPM can be due to S. neurona, S. fayeri, or polyneuritis equi. This blog introduces the experiment to investigate disease conditions in relapsing horses.

Clinical disease is associated with Sarcocystis fayeri and Sarcocystis neurona infections in horses. Both of these infections will produce inflammation that can produce a disease called polyneuritis equi.  Equine protozoal myeloencephalitis is a  tough disease to understand and the following question was posed by the EPM society as an area of high interest: a) can S. neurona establish a persistent but unapparent infection in the horse? We are investigating the seroprevalence of antibodies to S. neurona, S. fayeri, and polyneuritis equi in these relapsing horses in an effort to answer that question.

The Trojan horse model taught us that any horse can get disease, at least those we challenged showed signs. But some horses were more susceptible than others. We observed that organisms could more easily enter the cells of some horses.  Does that mean that genetics plays a part? We’re not sure yet.  The model taught us that antibodies horsegroupwere produced by day 17 of the infection, clinical signs could be as early as day 10, and in every case the signs were sequential.  That means each horse showed the same signs in a very predictable way.

In field cases of suspected EPM we start by measuring antibodies. In most of these cases, the disease is not acute, but has progressed to a chronic state.  A statistically significant finding in acute cases was that antibodies against S. neurona increased as the infection progressed, yet the clinical signs varied with the horse.  That means some horses which had experience with the organism would have more pronounced signs than those which were infected for the first time.  We learned about the immune response and protection against S. neurona by using our challenge model.  The immune responses were similar in the experimentally challenged horses and horses that were naturally infected.

What we couldn’t do with the model was create a chronic condition that mimicked what we see in field cases. Field cases offer various disease manifestations however we don’t know when the horse was infected or the immune background (experience) of the animal.  Sorting this out takes many observations over a long period.  Observations and measurements require objective tests, run many times, with the data evaluated statistically.

Our long term objective was to gather data from a lot of horses, over a year, and produce meaningful analysis.  We analyzed over 17,000 blood samples and saw a pattern in a hundred or so horses.  These horses were chronic and their signs reoccurred after treatment.  It could take a few weeks. or happen after years of being healthy.  Some horses responded to treatments we suggested but they would eventually show signs again.

After manikins-puzzle-four-orange-cartoon-characters-colored-pieces-white-background-34676756developing tools and identifying horses, we designed an experiment that could answer  the question posed by the EPM society.  The experiment is finishing up after 1 year.  Enrollment was staggered and new horses are enrolling, some horses are still completing 3, 6, 9 and 12 month evaluations.  The exciting, novel, and interesting findings are that we were able to define 3 disease conditions associated with EPM.  The data from the experiment is available from the International Journal of Applied Research in Veterinary Medicine, Vol 15, No 1, 2017.  The paper will show up on their web page. The next 3 blogs will define the three diseases and explain what it means to the horse and potential treatment.

For auld lang syne, my dear,
for auld lang syne,
we'll take a cup of kindness yet,
for auld lang syne.

As another year closes, Pathogenes gives a big THANK YOU to those who contributed to our research studies.  Each consult adds information to our system and renews our hope that we will find a cure for EPM.  In 2017, we are concentrating on FDA-approved studies which move our treatments closer to full licensing as well as publications that explain our work. We fondly remember those animals along the way that brought us here, like Lily a paint mare that was one our first cases in 2013.

We two have run about the slopes,
and picked the daisies fine;
But we've wandered many a weary foot,
since auld lang syne.

An autoimmune test was (developed in 2014) and  incorporated into our EPM panel in 2015.  The S. fayeri assay was added in 2015.  Results from these assays were published, hoping our information will make EPM a lot easier to understand.  Our work taught us how to better prevent disease. The road to prevention starts with a correct diagnosis.

Importantly, horses with a diagnosis of EPM may suffer from S. neurona or S. fayeri sarcocystosis.  Or they may have an autoimmune disease.  Autoimmune disease is present when the horse’s immune system attacks myelin protein, the covering found on nerve tissues.  Detecting antibodies against the horses own nervous tissues indicates the horse has polyneuritis.  We suggest that autoimmune polyneuritis may start with a protozoal infection which stimulates chronic inflammation in some animals.  Clinically, once the cause of the disease is identified, the horse can begin the recovery process. Antiprotozoal drugs aren’t the answer to autoimmune polyneuritis.

It is difficult to figure out what is going on in horses which “relapse” with supposed “EPM”.  That is because horses can have one or a combination of  three syndromes that we have identified as associated with S. neurona.  The three syndromes are: S. neurona sarcocystosis, S. fayeri sarcocystosis, or autoimmune polyneuritis.  Each of these diseases which looks like EPM has a different treatment protocol. Some “EPM” tests can’t distinguish S. neurona from S. fayeri. Our unique approach does just that.

The BIG question we wanted to answer was “Can we prevent EPM?”  The answer is suggested in data from a complicated study that started a year ago.  The study was non-blinded (everyone got the medication) and uncontrolled (no placebo was used).  Treatment was given to horses with known relapsing/remitting EPM.  To enter the study, the horses had to be successfully treated, they had a normal neurological gait score.  It was important that there was a history of at least one EPM relapse that followed successful EPM treatment.

Horses were categorized as EPM (antibody against S. neurona), SF (S fayeri antitoxin present in the serum), or MPP (antibody against myelin protein or the against the neuritogenic peptide contained on the myelin protein). 2016 S fayeri Ellison 2015 MPP MP2 Assay

It was interesting to see that there were more S. fayeri-infected horses than S. neurona-infected horses.  There were more autoimmune horses than expected; in fact the autoimmune group comprised the largest number of cases!  The smallest group were horses in the EPM category, meaning there were antibodies against S. neurona present but not S. fayeri or antimyelin protein antibodies.  It was obvious that horses with relapsing/remitting signs of EPM that have antimyelin protein antibodies needed an alternate diagnosis.

We interpreted a “treatment failure” as the horse getting signs consistent with EPM and the prophylaxis was not working.  These horses were removed from the study and received an alternate treatment.  Horses with a diagnosis of autoimmune polyneuritis received treatment according to protocols that have worked for the majority of cases.  Horses with evidence of autoimmune polyneuritis failed in the first 3 months of treatment.  Interestingly, the horses with a diagnosis of Sarcocystis infections did not fail.

We discovered some more interesting and surprising results.  For example, we have evidence that horses with recurring EPM are re-exposed continually from the environment. We found that treating horses in the SF category eliminated the S. fayeri toxin that may be responsible for neuromuscular disease.  And, it was possible to detect sub-clinical disease.  Right now, we are working on a lab value to predict when sub-clinical disease turns into apparent disease.

We discussed this study with veterinarians at the 2016 AAEP meeting in Orlando, Florida, a gathering of veterinarians interested in EPM treatment and prophylaxis. Again, thank you to those who took the time to stop by and discuss your cases! The EPM prophylaxis paper is available , however we will share our view of the data and how it affects treatment decisions concerning your case now.  Just give us a call. We need the consult form completed.  Find it on our web site: www.pathogenes.com.

Overall, in 2016 we learned more about chronic disease, how to prevent EPM and the incidences of autoimmune polyneuritis.  This year, we will add more information to the pathogenesis of disease and work out a method to stage polyneuritis in the diseased animal.  We will continue to  transfer our information to the EPM community and promote positive discussion among practitioners. And so we’ll take a cup o’ kindness yet, for auld lang syne.

 

Some horses with equine protozoal myeloencephalitis (EPM) resolve their issues with treatment, these cases are simple.  Some horses continue to have gait problems that span years, they “relapse” year after year.  These are complex cases.  One common strategy is to treat the EPM-suspect horse and see what happens.  If the horse fails on treatment or “relapses” some suggest that the relapses “must reflect either new infections or reactivation of latent  infections” (MacKay 2008).

The comments about treating relapses states: “there is no obvious rationale for using a different drug to treat recrudescent EPM than was used to treat the primary presentation.  In my (MacKay) experience, regardless of the drug used, the response to treatment of each successive relapse is incrementally less complete.”  This approach leads veterinarians to extend the course of therapy, use higher doses of the same therapy, combine antiprotozoal, switch from one approved drug to another, institute “maintenance” antiprotozoal drug therapy…

Complex neurologic cases may be difficult to treat, not due to a lack of treatment effectiveness, but rather a lack of understanding of the pathogenesis of disease and the the drugs that should be used for the disease process.  Treatment response is a part of elucidating the disease in horses.Testing is critical to understanding what the disease process is in the horse.

Diagnosis of EPM is difficult and it is common knowledge that there are no “EPM” tests.   EPM is a syndrome that involves protozoal infection and signs that result from Sarcocystis-associated  infection.  An antibody test that detects protozoa won’t diagnose the inflammatory component of disease.  The presence or absence of antibodies can rule in or rule out the involvement of Sarcocystis. The inflammatory processes can linger long after the horses immune system resolved the active infection.  Inflammation makes EPM complicated to detect and treat.  The inflammatory process is not detected by circulating or CSF antibodies.

We are piecing together puzzling aspects of neuromuscular disease in horses and can help you understand some of those difficult cases.  Important aspects to consider are the presence/absence of Sarcocystis neurona antibodies, the serotype of the S. neurona infection, the host’s inflammatory response to protozoal infections, the presence of Sarcocystis fayeri toxins in horses, autoimmune antibodies, and the pharmacodynamics of treatment on both the horse and the parasites.

Published data clearly showed the clinical difference between the S. neurona challenged immune-competent horse and the S. neurona challenged immune-deficient horse.  Immune-competent horses cleared parasites from the blood and suffered clinical signs of disease.  The immune-deficient horses were unable to clear the parasitemia and surprisingly, did not show any signs of disease (Sellon 2004)! One take home message was that immune cells were involved in parasite clearance as well and clinical signs.  Interestingly, the immuno-competent horses did not have parasites that could be recovered indicating the parasites were eliminated.  Parasites were recovered from the immune-deficient horses. Understanding disease involves animal models, these horse experiments show the immune system impacts the results of infection. We have a disease model that makes a normal horse act like an immune-deficient horse and that helps us understand the disease processes and the effects of some treatments.

Another telling experiment showed that anti-protozoal drugs can delay the production antibodies--but not prevent disease (Furr).  The message is that antibodies are not a measure of disease, but they are a method to associate disease to a particular organism.  Horses on “maintenance” doses of antiprotozoal drugs may test negative for antibodies yet have treatable disease.  One reason that antibodies are decreased (or delayed) is that some anti-protozoal drugs alter the protozoa’s expression of antigens that are used as markers in antibody tests. Using antiprotozoals to prevent antibody production is not a rational approach to preventing “EPM”.

A recent topic is the contribution of Sarcocystis fayeri to neuromuscular disease in horses.  Previously S. fayeri was considered a common and benign muscle infection in horses.  Researchers at UC Davis and Pathogenes believe that Fayeri may not be so benign. The UC Davis group associated a slight increase in muscle cysts in horses with neuromuscular disease (over the number of cysts found in clinically normal horses) while we associated neuromuscular disease with inflammation and S. fayeri antitoxin.

We have determined that autoimmune antibodies, not detected by S. neurona antibody assays, are present in some “relapse” horses.  These horses are not effectively treated with antiprotozoal drugs.  They respond to judicious use of steroids.  It is this disease process that showed us the obvious rationale for using a different drug to treat recrudescent EPM than was used to treat the primary presentation.  Of course, testing and defining the presence of autoimmune antibodies is as critical as the proper treatment.

No doubt these additional conditions found in the “EPM” horse are rare.  In our experience these diseases account for 50% of the horses that are diagnosed with the rare condition EPM.  We assayed sera from 7601 horses with neuromuscular disease and found only 3745 were “EPM” suspect.  The other 3856 were clinically ill due to undetermined causes that included autoimmune disease and S. fayeri toxins.

In the past two years we have identified 195 horses with autoimmune disease that have not, and would not, respond to any antiprotozoal treatment protocol.  We also identified 121 horses presumably suffering from S. fayeri toxins.  These horses become worse with some antiprotozoal treatment protocols.

It is important to understand the components of equine neuromuscular disease and focus less on branding these cases as EPM.  When EPM is the correct diagnosis understanding the inflammatory component of the disease process is important because pathological inflammation goes hand-in-hand with infection.

Call us with questions, we are happy to share our approach with you.

 

Nicolas Faacci and Cheryl Heuton created a show called Num3ers in which a mathematician uses equations to help solve various crimes.  The strategy can apply to  equine protozoal myeloencephalitis.  The show teaches obvious and not so apparent factors should be considered.

EPM can be quite complicated to diagnose and treat if you ascribe to the opinion that the parasites (that cause disease) are in the horses central nervous system and an antibody test will identify these diseased horses. We have evidence that this “obvious” factor isn’t always true.

We assert that EPM is a syndrome that involves infection and inflammation.   Chronic inflammation may set the course for additional disease processes (see Intern J App Res Vet Med 2015, p164-170 and p175-181).  Protozoal infections may cause chronic inflammation.

Infectious processes use similar (innate) inflammatory pathways.  It’s not surprising several diseases confound EPM diagnosis when clinical signs include neuromuscular disease. The past 30 years were dedicated to reconciling antibody and EPM (due to S. neurona).  Often overlooked, S. fayeri, (the other Sarcocystis that infects horses in the United States) can be associated with neuromuscular disease, and certainly is an unaccounted variable in studies that defined ataxic horses with neuroinflammation as EPM horses.

The association between Sarcocystis and neuromuscular disease can be elucidated using algorithms.  The the database used by an algorithm should be large enough to get statistical analysis, attributable, and accurate.  No only does the analysis identify groups of similar animals, where and when animals are at risk, and possibly reasons for treatment outcome, the analyzed data can reveal new relationships. Our algorithms also apply to single cases and are the basis for many of our recommendations.

The EPM database contains more than 18,000 horses.  Attributable is defined as a medical record signed by a veterinarian.  All data is reviewed by quality assurance at each step: testing, recording, and analysis to ensure the data represents the veterinarians observations. The accuracy of the EPM data rests with the veterinary exam.

The following analysis, perhaps worthy of a Num3ers show, incriminates S. fayeri as a factor in equine neuromuscular disease.

We intended to demonstrate that C-reactive protein, CRP, an acute phase inflammatory biomarker, is elevated in a horse with S. neurona infection.  Intuitively, horses with EPM would have an elevated CRP.  Horses that are effectively treated for EPM would show a decrease in CRP.

We use the definition of EPM that says horses have antibody against S. neurona and a gait score (GAS) that is abnormal.  A GAS of 0 is normal and any GAS value >0 is abnormal.  Most scales are 0-5, a horse with GAS 1 has minimal signs and a horse with a score GAS 5 is  recumbent. For brevity, the analysis uses a GAS 0 and GAS +, the data is not distorted by clumping positive horses together.

The GAS as a parameter for EPM is a fundamental premise identifying a diseased horse.  An issue with using a GAS is that some horses with neurological disease not manifested as a gait deficits won’t fit the analysis.  One paper (that assessed the utility of antibody tests for EPM) described most cases in their population with predominant neurologic signs dysphagia, changes in vocalization, vestibular dysfunction, facial paralysis, and changes in mentation, therefore they were assigned “brainstem” lesions (see J Intern Med 2010 p 1184).  Case selection is a confounding factor in between-study analysis, it is difficult to compare studies that primarily use gait as a marker for EPM with studies that don’t.  Also, this is evidence that at least one group of researchers recognize that S. neurona infections don’t always cause gait anomalies.  It is unlikely they considered S. fayeri in their study.

The studies we conduct are federally mandated to use assessment of EPM by gait only.  Serum CRP concentrations (ug/ml) in horses with normal gaits and abnormal gaits due to neuromuscular disease are shown in the charts below.  Our numbers show that different cut-off values are important in patient evaluation.

 

Horses with gait deficits (due to non-infectious causes) may be confused with a horse with EPM. A diagnosis of EPM  requires gait deficits and antibodies against S. neurona.  Sarcocystis neurona seronegative horses with weakness, ataxia, and neuromuscular disease are called idiopathic encephalomyelitis, IE, in this analysis. It is apparent that any test that included other Sarcocystis (S. fayeri) would confound the data, therefore we use species specific ELISA tests to define S. neurona antibodies. The analysis of species specific serum biomarkers for S. neurona and serum CRP concentration from untreated horses (GAS+) with attributable data (EPM, n=832; IE, n=914 ) was investigated.

There is little difference between the groups although it is noteworthy that slightly more horses with IE have a normal CRP concentration.  Careful analysis, review of the literature, and common sense led us to examine several factors that could explain these data.

The next algorithm compared normal (serum CRP concentration <17ug/ml) or abnormal (serum CRP>16 ug/ml) with (clinical) and without (normal) clinical signs of neuromuscular disease by serum antibody status against S. neurona, and S. fayeri.  The serum was obtained from untreated horses with attributable data.

Normal horses with an elevated CRP were more likely to have S. fayeri antibodies than S. neurona antibodies.  These data indicate that sub-clinical S. fayeri infection can cause inflammation that is detected by CRP.

Pathogenes has many partners in our quest to bring a safe equine therapy to market.  We have scientific partners, financial partners, veterinary partners, horse owners, and the government.  The government is everyone's partner in business.   In return for income tax the FED provides infrastructure and many other services that fly under the radar.

Our partnership with the FED’s will allow us to bring two drugs to the equine community.  In return for their services we provide a percent of fees for the life of the commercialized product. As part of the drug licensing process the center for veterinary medicine provides milestone goals and a path to complete those goals.  Our path is directed by guidance information documents, GFI’s.  As we accomplish each goal, we take the next step.

Our first milestone was developing a treatment and providing information to FDA that equine protozoal myeloencephalitis (EPM) is an important, rare disease.  Unprofitable for big pharmaceutical companies to develop the treatment of EPM falls under the (orphan) drug act, or MUMS.  Pathogenes commitment is to provide a safe and effective therapy that all can afford and its designation under MUMS makes that possible.  The MUMS program provides help with submissions, fee waivers, and guidance so we can accomplish our goals. the FDA has a big say in the studies that we run to meet milestones that lead to commercialization.

Starting the next field trial with NeuroQuel is a milestone. Before we could move forward with our therapies we had to produce a drug that had rigorous scrutiny in the manufacturing process. Unlike compounding (one- drug one-animal, no quality control for the drug substance activity/stability in the product) an FDA licensed drug must be carefully evaluated--that means nearly a million dollars is associated with  production.  A drug manufactured in a facility compliant with cGMP’s is licensed because it has achieved the rigors of ingredient and production testing.

A next step is the NeuroQuel PLACEBO CONTROLLED FIELD TRIAL.  There seems to be misinformation about placebo controlled trials so here are some facts to clear the air:

  • You must enroll to be considered for a placebo controlled trial.  You must sign a paper informing you of the nature of the study and by enrolling you agree that you understand the conditions of the study.
  • Enrollment documents explain the statistical chances that you will receive a placebo or the active drug.  If you haven’t signed an enrollment document, you are not in a placebo controlled trial!
  • Enrollment requires that information is given back to the study monitor!  If you have not completed in-trial and end of trial forms you are not in a placebo controlled study.
  • A horse enrolled in the study must satisfy some entrance criteria.  Entrance criteria can include age, weight, and recent treatment history.  A huge entrance factor in our placebo controlled study is diagnosis of EPM.

The up-side of these controlled studies is the quality of the information that is given to FDA.  The down side may be the number of forms that have to be signed!  We spent the last couple of years training veterinarians about EPM.  We gave our trained veterinarians continuing education for their help and attention.  Most discovered some new things about their EPM case, veterinarians didn’t learn everything they needed to understand EPM in veterinary school!  Most importantly, we found diseases that mimic EPM.  We spent a few years developing tests to identify EPM-mimicking disease so that our study isn’t compromised.  The recognition of these EPM-mimicking diseases did lead to publications that educated those veterinarians that are current with EPM topics.  Effective treatment of some of these horses (that didn’t respond to commercialized EPM therapies because they didn’t have EPM) became possible.

We are contacting our trained veterinarians that treat horses with EPM so they may enroll horses that have failed on commercial EPM treatments.  NeuroQuel is for the treatment of horses with residual or relapsing signs due to EPM caused by S. neurona.  Horses eligible for enrollment in the placebo controlled NeuroQuel study include a diagnosis of EPM with a cerebrospinal fluid analysis (CSF test) followed by treatment with the label dose of a licensed antiprotozoal therapy (no compounded products or off label drugs) within 90 days of enrollment.  Other conditions include age and the degree of deficit.  If you have a horse that was diagnosed with EPM by CSF tap, has not completely responded to Marquis, Protozil, or ReBalance using the dose on the label and has residual signs you may have a case that will qualify for our study.

For full information on the enrollment of horses into the placebo controlled field study please check out the downloadable documents at https://pathogenes.com/w/clinical-trial/.  Or use the contact information at www.pathogenes.com and we can help with the enrollment process.  Enrollment in these studies will help bring our treatment to market and bring new information to the equine community.

If you have a horse that does not qualify, please give us a call. It is important to document the reason the animal couldn’t participate.

For more information about placebos and the “placebo effect” please consider http://www.wired.com/2009/08/ff-placebo-effect/