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sidewinder3 (2)Horses with neuromuscular disease that are unresponsive to the usual treatments can be frustrating for veterinarians and horse owners. This post encourages you to get in touch with us if you have a horse with "EPM" and is not responding to treatment. ELISA Submission Form

 

We are following 38 horses that have clinical signs consistent with polyneuritis equi or cauda equina neuritis, an often overlooked syndrome associated with EPM. Our goal is to develop a treatment protocol for veterinarians dealing with these cases. Sometimes these horses are weak on one side, they have hemiparesis, and that prevents them from tracking normally.  The weak rear end falls to the weak side.  A commonly used descriptive term is "sidewinder". Ellison 2015 MPP MP2 Assay

There are other clinical signs of this disease.  Dragging limbs, tripping, or even drooling and dropping feed (cranial nerve dysfunction) are observed. The tongue can be atonic.  Another sign in these horses can be dripping urine or an inability to empty the bladder.  The most common comment in the history is multiple EPM treatments. We developed tests that can lead to a diagnosis of polyneuritis equi. Pathogenes Testing Options

Historically, the distinction between polyneuritis equi and cauda equina neuritis was the presence of cranial nerve involvement.  Discerning the disease by cranial nerve signs may not be accurate because horses with cauda equina may also have cranial nerve deficits.  In terminal cauda equina, horses are unable to urinate and often colic.  We don’t distinguish the two diseases by cranial nerve function because the owners and veterinarians involved with the horses we track often report difficulties with chewing, droopy lips, or facial muscle atrophy. These signs are often responsive to specific treatment.

Two serum tests, MPP and MP2,  measure anti-myelin protein antibodies.  Horses with polyneuritis equi often have circulating antibodies that are reactive against myelin protein and/or a reactive site on myelin.  The presence of antibodies are evidence that an autoimmune reaction is involved in the disease process.  A positive test can assist in diagnosis and potentially life-saving treatment.  2015 EPM and MPP

Eventually we think we will be able to stage to degree of damage using these tests. The C-reactive protein assay (CRP) may be the most useful test for case monitoring after a diagnosis is made.  The CRP assay is useful because response to treatment precedes a reduction in the pathologic inflammatory cycle. It may be that polyneuritis is the early stages of disease that ends in cauda equina syndrome.  That is to say, chronic idiopathic polyneuritis left untreated will eventually develop into clinical cauda equina.

If we understand the disease process correctly, it may be possible to prevent the progression of disease.  We are following the clinical course of 38 horses, their therapy, and the outcome of treatment.  We are assembling the data for a statistical analysis of the diagnostic and treatment options.  This data-based approach will be very useful to veterinarians trying to manage these horses.  The long game is devising a standard treatment protocol for afflicted horses to keep them healthy,  but we are not there yet.

Recently Dr. H. wrote a quick email asking if it was concerning that the CRP (C-reactive protein) level remained elevated in his patient, a 7 year old mare with a 3-year history of relapsing disease.  The good news is that she is currently clinically great while receiving treatment.  He wanted to know what is next.  What can be expected?  Unfortunately for Dr. H.,  we don’t have all the answers.  We sent back all the variables, the possibilities, and the literature he could review to make his clinical decisions.

Dr. Laura Benedetti is busy contacting veterinarians, asking for gait scores to update our records and requesting blood samples on the horses we’ve identified that could have this condition.  She answers emails about polyneuritis equi.  What is missing from the process is a forum for owners to discuss their horses with other owners similarly diagnosed horses.  While there are numerous forums and posts about individual horses with suspected EPM, these sites aren’t that useful for those facing a polyneuritis equi diagnosis for their horse.

We were pleased with the support offered to Taylor Lin (Blaze was posted on January 22 at Pathogenes Inc. Facebook), who initially felt alone before finding others who had been there before and kindly posted comments.

Horses with polyneuritis equi and cauda equina should receive a potential diagnosis and an individualized treatment protocol based on data that includes clinical examination by a veterinarian and laboratory testing.  Sub-clinical disease is an important aspect of chronic idiopathic polyneuritis. Dr. H’s mare  looks great and is performing well.  However, serum testing indicates she still has disease that needs to be treated.  Often the CRP can be used determine an endpoint to treatment.

We welcome you to post your questions and observations on Pathogenes Inc. Facebook page.  The information will be useful for others facing this disease in their horse.  Pictures and short videos are also welcome.  Individual treatment options are discussed privately with veterinarians because these need to be tailored to the specific cases.

Testing the horse with suspect EPM should yield values that assist in treatment decisions.  The IFAT shown in the photo is a positive result.  That doesn’t necessarily mean this horse should be treated with anti-protozoal medications!  Our study that defined relapses attributed to EPM categorized three diseases that have three different treatments. The diseases warrant different treatment because horses may have a one-time exposure or  be chronically exposed. Post-disease conditions that leave the animal with signs due to inflammation are also treated differently.

Sarcocystosis can be caused by S. neurona or S. fayeri.  Some horses are chronically exposed to Sarcocystis in the environment and continued exposure will result in new disease.  These cases are often interpreted as relapses. Disease in horses can be managed and prevented if the correct process is recognized.  The reagents we use to measure S. neurona allow us to determine if a horse has active disease, relapsing disease due to chronic exposure, or signs due to another condition.

Similarly, disease due to S. fayeri can be resolved and prevented if there is suspected environmental exposure.  About thirty percent of horses have S. fayeri and it is usually harmless, horses don’t show disease.  However, some horses do have disease or sub-clinical disease associated with S. fayeri.  We measure the toxin associated with disease-causing fayeri cyst and associate disease with the CRP test.  The CRP allows us to give significance to the presence of S. fayeri  toxin and determine if the infection is due to re-exposure.

The most important criteria in evaluating a horse before, during, and after treatment is the gait score or the presence of signs that indicate disease.  Lab values are important and give insight into the disease process.  The trend. obtained from repeating lab testing, has the most significance in long term case management.  Combined, signs and test results are a valuable method to manage horses.

Quick reference for sarcocystosis testing in a horse that improved with treatment:

S. neurona antibody declined after treatment, CRP trending to normal: indicates that the horse does not have repeat exposure to S. neurona in the environment.  MONITORING THE HORSE FOR CLINICAL SIGNS IS SUFFICIENT.

S. neurona antibody declined after treatment, CRP elevated: indicates that the horse does not have repeat exposure to S. neurona in the environment and there is another disease process. FURTHER TESTING TO DEFINE THE DISEASE PROCESS IS NEEDED.  TESTING FOR S. FAYERI TOXIN, LYME, OR AUTOIMMUNE DISEASE IS USEFUL. DEWORMING TO REMOVE ENCYSTED SMALL STRONGYLES IS USEFUL IN A FEW CASES.

S. neurona antibody increased within 1 month after treatment, CRP trending to normal: indicates that the horse may not have repeat exposure in the environment and responded to the elimination of parasites. A RISE IN TITER IS NOT UNUSUAL, ANTIBODIES WILL REMAIN FOR 5-8 MONTHS. RETESTING THE S. NEURONA SAG’S AT 6-9 MONTHS IS USEFUL.

S. neurona antibody increased after treatment, CRP elevated: indicates that the horse does have repeat exposure in the environment or there is another disease process. FURTHER TESTING TO DEFINE THE DISEASE PROCESS IS NEEDED.  TESTING FOR S. FAYERI TOXIN, LYME, OR AUTOIMMUNE DISEASE IS USEFUL. DEWORMING TO REMOVE ENCYSTED SMALL STRONGYLES IS USEFUL IN A FEW CASES.

S. fayeri antitoxin declined after treatment, CRP trending to normal: indicates that the horse does not have repeat exposure in the environment.   MONITORING THE HORSE FOR CLINICAL SIGNS IS SUFFICIENT.  If the horses was treated for 6 months for S. fayeri the horse can discontinue treatment.

S. fayeri antibody declined after treatment, CRP elevated: indicates that the horse does not have repeat exposure to S. fayeri in the environment and there is another disease process. FURTHER TESTING TO DEFINE THE DISEASE PROCESS IS NEEDED.  TESTING FOR S. FAYERI TOXIN, LYME, OR AUTOIMMUNE DISEASE IS USEFUL. DEWORMING TO REMOVE ENCYSTED SMALL STRONGYLES IS USEFUL IN A FEW CASES.

In our testing/treatment protocol we will not see an increase in S. fayeri antibody after treatment to indicate acute disease was resolved. The anti-toxin is a response to cysts that produce toxin. The CRP is the indicator of disease.

The last disease process that is related to sarcocystosis is inflammation.  Chronic neuromuscular inflammation is polyneuritis.  Pathologic inflammation can be related to new or old disease.  That will be the final topic in this series.  Stay tuned.

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.

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.

Sarcocystis fayeri produces a toxin that may cause weakness in horses

Sarcocystis spp. are prevalent in the muscles of horses in various regions of the United States and Europe, as many as 30% of the population are infected. There are four species that infect the muscles of horses, S. asinus, S. bertrami, S. equicanis, and S. fayeri. All of these species use canids as a definitive host and trans-placental infection can occur. The pathogenicity of the four species is variable.

Sarcocystosis can be mild or severe. Signs include fever, apathy, anorexia, myositis, muscle weakness, autoimmune disease, or hair loss. In chronic disease signs include weight loss, difficulty in chewing and swallowing, depression, ataxia, emaciation, and generalized weakness. Generally, the mild histological lesions of myositis don’t correlate with the profound muscular weakness seen in afflicted horses. The role of toxins associated with bradyzoites from sarcocysts is not defined, but may be associated with lost strength.

The pathway to chronic inflammation in horses is unknown. Evidently there is a prepatent period of greater than 79 days in naive horses because three S. neurona seronegative ponies that were fed S. fayeri oocysts remained clinically normal at day 79 when the experiment was terminated. Sarcocystis fayeri sarcocysts were found in striated muscles of all inoculated ponies. Perhaps degenerating cysts release toxin-producing bradyzoites. This pathway may explain profound muscle weakness in debilitated or chronically infected horses. The toxin presumably sensitizes CD4+ T cells. Upon re-exposure to S. fayeri in the gut inflammation may ensue. Inflammation may be measured by serum C-reactive protein (CRP) levels. Serum antibodies against S. fayeri toxin are detectable in horses.

The S. fayeri toxin, called sarcocystine, was first associated with sarcocysts in 1899. There is occasional mild interest in Sarcocystis toxins. In 1997 sarcocystine drew interest, there was an association between human sarcocystosis and multiple sclerosis. Sarcocysts, identified as S. gigantea and S. cruzi, produce a small, heat labile protein-toxin that causes increased respiration, depression, ascending paralysis, even death, in rats and rabbits. Last year severe food poisoning was reported in people eating raw horse meat.

Food poisoning was documented in up to 78% of people consuming contaminated raw horsemeat. Sarcocystis fayeri caused enteritis in people that ingested the muscle cysts. Sarcocysts in the meat were identified by morphology and completing the parasite lifecycle in dogs. The enterotoxin was isolated and characterized. Toxicity was confirmed in rabbits receiving 15 µg of S. fayeri sarcocystine. Molecular analysis indicated the toxin is 15kDa and has homology with virulence and cell-invasion factors of Toxoplasma gondii.

Toxoplasma-induced inflammatory bowel disease is studied in a pathogen-driven mouse model. Acute, lethal ileitis is induced in susceptible mice by single antigen of T. gondii. The susceptible mouse required antigen specific CD4+ effector cells and these cells could transfer susceptibility to resistant mice. The intestinal pathology induced by T. gondii required the induction of proinflammatory cytokines that included INF-γ and TNF-α. Neutralization of INF-γ or CD4+ T cells prevented necrosis of the ilea and prevented acute mortality. This model demonstrated that disease requires the production of chemokines that elicit a proinflammatory responses from CD4+ cells by transferring disease susceptibility to mice using these cells.

After S. fayeri infection, serum antibodies against S. neurona are detected by IFAT.* Sarcocystis fayeri infections may be misdiagnosed as being S. neurona infections using IFAT tests. Treatment for S. fayeri cysts in muscles may differ from treatments for S. neurona merozoites. It is important to determine the effects EPM treatments on S. fayeri cysts. Toxin-specific assays may associate degenerating S. fayeri sarcocysts to muscle weakness in some horses. We developed a serum ELISA to detect S. fayeri toxin-specific antibodies in equine serum. Unlike IFAT, tests the S. fayeri toxin antibodies do not cross-react on SAG 1, 5, or 6 ELISA tests.

*Saville, Dubey, Oglesbee, Sofaly, Marsh, Elitsur, Vianna, Lindsay, Reed 2004