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A giant ship’s engine failed. The ship’s owners tried one expert after another, but none of them could figure but how to fix the engine.

Then they brought in an old man who had been fixing ships since he was  young. He carried a large bag of tools with him, and when he arrived, he immediately went to work. He inspected the engine very carefully, top to bottom.

Two of the ship’s owners were there, watching this man, hoping he would know what to do. After looking things over, the old man reached into his bag and pulled out a small hammer. He gently tapped something. Instantly, the engine lurched into life. He carefully put his hammer away. The engine was fixed!

A week later, the owners received a bill from the old man for ten thousand dollars.

“What?!” the owners exclaimed. “He hardly did anything!”

So they wrote the old man a note saying, “Please send us an itemized bill.”

The man sent a bill that read:

Tapping with a hammer………………….. $ 2.00

Knowing where to tap…………………….. $ 9,998.00

Effort is important, but knowing where to make an effort makes all the difference! Just ask someone who lost a horse to EPM.  If the wrong disease is diagnosed and then ineffectively treated, the horse is lost.  Just what goes into the effort to give clinicians the tools they need to evaluate the horse?  Veterinarians spend four years in college, they undergo a vigorous selection process that is followed by four years of post-graduate education.  Some were fortunate enough and had a support system, some are still paying off those college loans.  Armed with his academic qualifications, the veterinarian accrues knowledge one field case at a time.  That is the art of practice.  Practicing veterinary medicine isn’t learned from a book.  Each animal is different and how it presents clinically and expectations on treatment effectiveness are a skill learned by putting in many  years of work.

What does it take to root out diagnostics that give us a picture of what to fix?  In our case, it took BS, MS, and two doctorate degrees  (DVM, PhD), a total of 13 years.  After that, seventeen years were spent in total immersion in the study of one parasite, Sarcocystis neurona.  The outcome was an understanding of how the parasite lives and infects cells, what cells are infected, how to infect horses, and how to interpret the response of each cell in the body to infection.  That was coupled with 30 years of field experience in equine medicine.   Finally, putting that knowledge into a story that made sense to veterinarians by translating science to a successful outcome.

An experienced clinician has to understand our bench science and layer that onto his or her education and experience.   Field veterinarians spend hours digesting the work we have done by reading our published work, and that isn’t easy.  It took me 17 years of total dedication, 24/7, to work out a theory, test the model, and present the knowledge to field practitioners, giving them tests and a interpretation of test results as a starting point for them.  No one can take a laboratory number and use that to cure animals.  Most lay people don’t have the background to understand the work.  We make it available and are happy to answer questions, but a few graduate level courses in immunology, parasitology, and molecular biology (biochemistry) are needed.

In addition to our milestones, we have a team of experts which also dedicated years to gaining background knowledge and experience in their fields. I learned to take a parasite from an animal, clone and express genes, and produce recombinant proteins to use in diagnostic tests, but there is more. Experts were needed at each step.

The cost of producing a licensed pharmaceutical for a horse is between five and ten million dollars.  Obtaining FDA protocols takes 3 people many hours to perfect, studies cost two hundred and fifty thousand to five hundred thousand dollars to conduct.  Manufacturing a tablet to use in studies cost hundreds of thousands of dollars (validating assays, testing the ingredients, testing the final product).  Each step under the watchful eye of the respective expert.

Is this important to you?  It should be. For example, we developed a drug to treat parasite mediated inflammation.  Little did anyone know that tossing the drug into water for as few as four hours or mixing it into a paste changes the chemical into a pro-inflammatory molecule.  It took us 4 years conducting many experiments in consult with a veterinary school to prove the effect that water has on levamisole.  What are the effects of levamisole on the parasite?  Our studies are almost ready to share. These are aspects of the experiments we conduct.  Right now,  we are possibly the only group investigating how to effectively treat and prevent relapses in horses with autoimmune polyneuritis.  We present and publish our work, sharing it with those who are interested.  We provide continuing education to veterinarians.  The protocol for treatment of autoimmune polyneuritis won’t be worked out for another year or two but we are training veterinarians on the protocol we are finding effective today.

Most pharmaceutical companies keep their information in-house, preventing  competition and perhaps hindering progress.  Negative data is rarely published.  Companies take the time to secure patents and license a product before the big rollout.  That prevents competition from compounders.  Compounders can provide a valuable service, unfortunately that has evolved into a business paradigm that is harmful and outside the intent of the services they can legally provide.  Compounders use loopholes to provide meds that are similar to licensed products, thereby avoiding the development costs and expenses for all the science behind the work.  They just change the flavor.  They don’t validate the amount of active drug in the formulation or understand the ramifications of their product.  Compounders are privy to information for which they invested neither time nor money in the science. When you shop for a drug to save a few dollars, you are making a choice in the future of drug development.

It is impossible to know what we will discover next and how it may affect your horse or even human medicine.  We choose to try to do what we do by funding our own work and providing a direct pipeline to clinicians, via our consulting.  The next time you think you may be paying us or your veterinarian too high a price for goods or services, please consider the people, time and expenses which went into our ability to successfully manage your horses.  Without that background and the costs incurred in reaching this point, some of these animals would not be alive today. It takes a village.

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.

It’s been 20 years since the discovery that S. neurona was the causative agent of EPM. The disease has been described as “an enigma wrapped in a mystery”! The confusion? Despite widespread exposure to S. neurona in horses, as shown by a high seroprevalence against this parasite, the disease is uncommon. How did we contribute to solving the EPM mystery? We use specific chemicals to block certain reactions. Successfully targeting, and understanding the effects of targeted therapy, helps define the course of disease.

The existing paradigm is that clinical disease, caused by S. neurona in horses, is due to protozoal parasites that are randomly infecting the central nervous system (CNS) tissues. This view of infection is a barrier to understanding EPM. Our Trojan horse model indicates horses show a biphasic disease that is not random.

Acutely, ingested S. neurona sporocysts undergo division and the daughter cells (merozoites) enter the blood stream (parasitemia) from the gut. EPM afflicted horses respond by down regulating white blood cell reactions, including proliferation responses and production of the cytokine interferon gamma. The absence of IFN γ allows S. neurona to invade the brains of mice. The rapid clearance of the parasitemia and appearance of merozoites in organs, like the liver, induce protective antibodies by the horse. The protective antibody respond to major surface antigens of S. neurona and appear at the same time as encephalomyelitis. Central nervous system inflammation is recognized by cranial nerve signs and ataxia. The appearance of these signs begin the chronic disease phase.

The severity and onset of central nervous signs are dose dependent. However, histopathologic lesions that contain parasitic stages, found rarely in clinical cases, are not found in sporocyst challenge experiments.  Therefore parasites entering the brain and spinal cord are apparently independent of sporocyst challenge dose. The conflict between the number of parasites in CNS tissues that are not dose related and the direct association between immune response and clinical signs may suggest the mechanisms of acute and chronic disease phases are immune mediated.

It might not be a requirement of disease that parasites or inflammatory cells enter the CNS. The choroid plexus is important mediator between the periphery and the brain. This selective gateway permits bidirectional communication between the CNS and blood circulation by cytokine transfer. The immunopathological basis of the central nervous system lesions in EPM may be due, acutely, to decreased IFNγ (IFNγ is a protective response against S. neurona infection). A decrease in IFNγ may permit parasite entry. Chronic disease is characterized by an increase in destructive IL6.

The cytokine IL6 is proinflammatory in the CNS and can cause the clinical signs of EPM. It is possible to directly target white blood cells resulting in reducing the production of IL6. Drugs may exert effects on leukocytes stationed on the circulatory side of the choroid plexus by decreasing the amount of cytokine IL6 produced and thereby,  passage into the CNS.

The regulation of IL6 consists of a collection of proteins: the cytokine IL6, the cytokine receptor IL6R, and a signaling protein, called gp 130. The unique aspect of the cytokine IL6 is that it is a species specific protein, horse cytokine IL6 is unique to horses. The gp 130 is not unique to a species. Paradoxically, acute and chronic EPM can use the same IL6 pathway. All three proteins, IL6, IL6R, and gp 130, as well as antibody against these proteins, can stimulate the pathway to different outcomes!

We discovered that some S. neurona proteins mimic some of the cytokine pathway proteins—the SAG 1 and 5 are similar to the IL6 cytokine while the SAG 2, 3, 4 and 6 proteins are not. The SAG 1, 5, and 6 are mutually exclusive phenotypes of S. neurona that infect horses and cause clinical signs.  The SAG 1 and 5 phenotypes can invade horse brains. That makes the SAG 6 phenotype an exception to the rule that S. neurona invades CNS tissues.

When we recognized the SAG 6 phenotype, we realized that this is an example of an exceptional S. neurona. Unlike the other S. neurona's, this one has not been recognized in EPM. This phenotype allowed us to examine S. neurona infections and use an analysis matrix to explain disease on a molecular basis. Most animal disease caused by S. neurona (horses too) is caused by SAG 1 and 5 phenotypes (entering the CNS by a white blood cell in a mechanism that may include the species specific IL6 receptor). Inflammation, the cause of the signs of EPM, is likely caused by the hosts’ immune response against Sarcocystis. We know how to block this inflammatory reaction thereby reducing IL6 production. We speculate that it occurs at the choroid plexus gateway.

Our studies contribute to the understanding of EPM, solves specific problems associated with understanding disease, challenges the existing paradigm and ultimately changes clinical practice. The critical barrier to progress in the field of EPM isn’t understanding molecular regulation at the choroid plexus gateway, it just may be teaching an alternate view of how EPM works.

Comparative research-comparing and combining data quantitatively (whether the data is collected at the same site, by a group of researchers, or by independent researchers) is sometimes necessary in complicated situations.  Such is the case with equine protozoal myeloencephalitis (EPM) because the disease is difficult and expensive to study. Conditions that can complicate comparative EPM research are the methodological differences and the limitations in the experimental design of the published studies

The data obtained from EPM studies often includes unintended consequences. For example, the organisms that are used in infections induced by sporocysts don’t use the same organism! When the organism is passed through an intermediate host, a bias is introduced into the study. Intermediate host bias expressed by S. neurona is well documented. A classic example of host bias is used in EPM research to identify sporocysts.  S. neurona sporocysts collected from feral opossums feces will infect immunodeficient mice while the same sample will infect birds, an indication that the organism is S. falcatula. Logically, using two intermediate hosts will identify mixed infections, mixed infections are often the case with opossums.

Hosts don’t always have the same stringency, the ability to sift out one parasite’s species from another. However, increasing the passage of sporocysts through the less-discriminating host increases selection bias for that host. The host can also discriminate on a finer level, even selecting phenotypes of strains.

An example of phenotype selection in the EPM literature is evident from the Transport Model experiments. These experiments are a series of 3 published studies that use sporocysts from feral opossums that are passed through a raccoon. An independent study found that these sporocysts were two different organisms! When the sporocysts were introduced into a horse, S. neurona SAG 1 was isolated from the infection. The same sporocysts were cultured in the lab and a strain of S. neurona was isolated that didn’t contain SAG 1. The two S. neurona strains were called 37R-744 and 37R-138, respectively. There may be further evidence that the 37R-138 contains low levels of a SAG 1 strain and this confounding situation would not be unexpected.

Comparing serum antibodies by phenotype has been compromised by methodological differences and the limitations in the experimental design between labs running these studies. It isn’t often we get a chance to make a direct comparison of antibody tests in the same horses, but the opportunity has presented, and we review it here. A model was designed that transported merozoites. The Merozoite Transport model used strains of S. neurona identified as a SAG 1 and SAG 5 phenotypes for the infection.

There is a paper that may indicate that the SAG 5 strain (SN4) has some SAG 1 organisms--an issue that won’t affect our analysis, yet illustrates the complicated relationships in S. neurona infections.

This study challenged six horses with S. neurona. The “control, unchallenged” horse did have pre-challenge reactivity for SAG 5 for all IgG isotypes, but fell below what was considered positive on the serum test--that’s why the researchers decided to include the animal in the study. If we ignore the control unchallenged horse (it didn’t seroconvert based on the parameters set for a positive result) we can observe what phenotype antibodies are detected by the ELISA tests.  This is a head-to-head comparison of SAG 1, 5 ELISA and 2, 4/3 ELISA. The SAG 6 strain was not tested, nor used in the challenge.  The question posed by the study is “Which ELISA’s detect infection and are better diagnostically in this study?”

The animals were tested before the challenge and at 42 and 89 days after challenge. The author concluded that the horses had an active infection at day 42.  All the horses were negative for serum antibodies to both phenotypes of S. neurona before challenge using the test conditions and all the horses (100%) seroconverted by day 42 to SAG 1. Four of the five horses converted to SAG 2 by day 42, (80%). None of the horses had serum antibodies detected by the 4/3 ELISA! The serology for the SAG 5 phenotype was also 80%, four of the five horses seroconverted after challenge.

The author states “diagnostically the SnSAG2, SnSAG3, and SnSAG4 are usually more dependable markers for infection than SnSAG1 and SnSAG5”. We couldn’t disagree more. The data clearly indicates in this head-to-head analysis of phenotype antibodies detected after infection that SAG 1 ELISA is superior to detect a SAG 1 challenge. This study may indicate there is a virulence difference between SAG 1 and SAG 5 strains or a host bias that needs further investigation.

There are reasons that SnSAG2, SnSAG 3, and SnSAG 4, antigens, that are not specific to S. neurona, are not good markers for S. neurona infections in horses. The strongest of the arguments are that these proteins have variable expression during infections as demonstrated in at least one published paper. More importantly, common antigens can’t discern species of Sarcocystis that don’t result in EPM.

The debate over what pool of antigens is optimum for detecting EPM is put to rest. In October there is a meeting in Kentucky that will bring EPM researchers together to brainstorm the issues surrounding this terrible disease. In this head-to-head debate, we hope to tip the discussion toward redefining the disease as one of infection and inflammation. A simple step in redefining as EPM syndrome will shift the paradigm for those involved in studying and treating this disease to the factors that contribute to encephalomyelitis-inflammation.

Equine protozoal myeloencephalitis is a disease that is characterized by multiple signs. Often the presence of gait deficits, such as lameness or ataxia, lead to a presumptive diagnosis of EPM by a veterinarian. Early signs of EPM are often characterized by behavior changes, loss of muscle mass, and weakness (failing to perform well can be a sign of weakness). A question perplexing EPM researchers is the source of infection. Are infections new or are they persistent, existing infections? A new infection can be seen after the horse has been effectively treated while a persistent infection means the parasite was never eliminated. The far reaching implications of the question concern the biology of the organism and how the horse fights the infection. The answer to the infection question will lead to more effective therapy and possibly lead to preventing disease.

New infections come from oocysts (sporocysts) that are ingested by the horse, picked up from the environment (hay, feed) or a resident opossum, depositing infectious material where horses graze or pick up spilled grain. One possible mechanism that would explain persistent infection is that the parasite remains in the animal in a dormant state. If the S. neurona the merozoite “hibernates” in a cyst stage then the horse would be considered a true intermediate host. The horse was proposed as a natural intermediate host for S. neurona by T. Mullaney in 2005.

Another possibility is that a drug kills only some stages of the parasites, upon removal of the drug at the end of treatment, parasites resume the infection. Levant Dirikolu reported “ the results of in vivo and in vitro studies of triazine agents (diclazuril, toltrazuril) with various apicomplexan parasites clearly indicate that removal of triazines after appropriate treatment time results in regrowth of parasites” in the Feb. 2013 issue of the Journal of the American Veterinary Medical Association. This maybe stage related drug resistance that can lead to persistent EPM. Ineffective treatment leaves the parasite infection intact.

If you follow our research, you already know that we believe that EPM is a syndrome. A syndrome is a collection of signs that are observed in, and are characteristic of, a single condition. The EPM syndrome consists of infection and inflammation. Treating the infection may be effective in eliminating the parasite but the protozoal induced inflammation (that is not responsive to phenylbutazone, flunixin, or firocoxib) can leave a horse with chronic, relapsing disease.

Our definitions are as follows:

EPM: EPM horses are defined as those horses that had clinical signs, including gait deficits consistent with a diagnosis of EPM (ataxia) and had a significant level of S. neurona antibodies in the serum. These horses had a drop in serum antibodies after treatment and responded to treatment. There were 55% of the animals that had "EPM".

IE: Horses with clinical signs, including gait deficits consistent with a diagnosis of EPM, but did not have S. neurona antibody (and did not show a rise in titer after treatment). About 20% of EPM suspect horses with no serum antibody will develop an increase in antibody after treatment. A pre and post (convalescent) titer is a valid method to implicate the cause of a disease. These symptomatic horses were classified as idiopathic encephalomyelitis (IE). Idiopathic is the term applied to conditions from an obscure or unknown cause—we use it to say all ataxic horses that do not have a definitive diagnosis. Horses that  had IE were 48% of this data set.

PTEDS: A significant issue is treating the protozoa but not effectively treating the inflammation, our soap box. Horses that fell into this category had clinical signs that included gait deficits consistent with a diagnosis of EPM, antibody levels dropped to undetectable in the serum after treatment, however the horse maintained some signs. The veterinarian saw some response to treatment but either the signs quickly returned or were never completely eliminated. These horses were classed as horses with Post Treatment EPM Disease Syndrome. Although these cases are related to S. neurona these are not new infections and are “residual” signs associated with EPM syndrome. Alternatively, Dirikolu had concluded “In unsuccessful cases, relapse can occur because of failure of the removal of some of these stages (some stages are inhibited and retain the ability to begin development again once the drug is removed”. Dirikolu’s definition of relapse falls under our definition of clinical EPM. The data set showed that 40% of horses had PTEDS.

New cases of EPM in a horse (that was effectively treated) were identified in our data set by evaluating a recurrence of disease (that included a rise in antibody titer from undetected post-treatment) after a prolonged period. Three percent of horses had new infections.

A simple answer to the question posed as “Are horses with EPM new cases or are they persistently infected?” is difficult! To get to the answer, EPM has to be re-defined as a syndrome and horses with IE have to be identified. Certainly PTEDS must be differentiated from IE that is not related to S. neurona. Long term evaluation of horses that experienced S. neurona infections by clinical examination and serum testing will clarify these issues. Developing a diagnostic test for horses with IE is important. But first, the veterinarian must recognize and consider IE as a condition. While intuitive, the component of inflammation must become the primary topic in EPM discussions. As Dirikolu and others have speculated, the horse’s intact immune response can likely remove S. neurona. Turning off the inflammatory component of EPM may be difficult for some animals.

Diagnostic testing and elucidating the pathogenesis of inflammation resulting in IE are the next big research steps necessary to solve the EPM enigma. We found a molecular link between viral, bacterial, and protozoal infectious organisms associated with IE and cytokine mediated inflammation.