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blue day before oroquinInfection with Sarcocystis is called sarcocystosis. Equine sarcocystosis, or equine protozoal myeloencephalitis (EPM), is an infection by protozoa that results in inflammation of the nervous tissues.  These parasites can sometimes be found in the central nervous system (CNS) although inflammation can cause signs without the parasite entering the CNS.  Note that EPM is a syndrome, it is caused by infection and inflammation. Infection causes a variety of signs that include muscular disease and neurological dysfunction. Sarcocystosis can be chronic in horses, those that undergo treatment frequently relapse. Relapse is most likely due to re-exposure of the protozoa in the feed or environment.

There are two protozoa that infect horses, Sarcocystis neurona and Sarcocystis fayeri. The horse is a natural host for S fayeri and may be less inflammatory that S neurona.  The horse is not a natural host for S neurona, the infection is called aberrant. Both of these organisms can cause clinical signs that look like EPM. There are tests to detect infections and exposure to these organisms. 2016 S fayeri

Protozoal infection stimulates immunity in the horse.  Innate (cellular) responses that trigger specific defensive pathways are activated quickly. These pathways remain active as long as the infectious stimulus is present.  Sometimes, these pathways become chronic by creating a “feedback” loop.  The innate response stimulates the production of proinflammatory  molecules, these molecules feed a signal to initiate the inflammatory reaction again, hence the results of  starting inflammation can be a chronic cycle.  These pathways are not specific.  Any infection can stimulate the initial response. Normally, when the infection is treated effectively the protective responses turn off.  Chronic inflammation is a dysfunction of the immune system.

We test for the presence of proinflammatory molecules found in the blood stream.  This is useful and an adjunct to other tests that are specific.  In our laboratory we quantitate C-reactive protein (CRP) to identify the extent of the pro-inflammatory pathway.  Values are measured from 0-99.  An absolute value isn’t as valuable as evaluating a trend.  A value that is above 16 is abnormal.  Values that are above 39 are statistically significant and suggest the clinical signs are due to inflammation related to specific conditions that we can also detect.

Another type of immune response to protozoal infection is acquired immunity.  Acquired immune responses result in specific antibodies and memory cells that target the infection.  When the horse gets re-infected, memory cells are primed and ready to act faster that in the initial insult.  Re-infected horses are “experienced” with the infecting organism, whereas a horse with no experience is called “naïve”. Antibodies against S. neurona are used to link a causal agent with clinical signs seen in horses with EPM.  Similarly, S. fayeri  produces antibodies in horses.  We can use different tests to distinguish between S. neurona and S. fayeri  infections.

Opossum_03-HangingBranchThe horse acquires Sarcocystis neurona by eating hay or feed that is contaminated with neurona-infected opossum feces. Opossums are the definitive host.  Definitive hosts exclusively produce the infective stage of Sarcocystis.  The infectious “eggs” (sporocysts) hold sporozoites that are short-lived in the horses’ intestine. There are three serotypes of S. neurona that can cause sarcocystosis in horses.  Often, the opossum is infected with multiple serotypes!  Dogs are definitive hosts for S. fayeri . Horses are infected by eating hay or feed that is contaminated with fayeri-infected dog feces.  It was long thought that S. fayeri   infections in horses were benign, unless a horse was debilitated from starvation or another disease.  Up to one third of horses in the United States are infected with S. fayeri.

The horse is an unnatural host for S. neurona.  After the initial infection, the parasite goes through its life cycles: sporozoitesbovine produce 1st generation merozoites and this stage kills host cells. First generation merozoites mature and produce 2nd generation merozoites.  These second generation merozoites must enter muscle cells and turn into cysts.  The horse is an unfriendly host to neurona merozoites and S. neurona is unable to make cysts. The horse is very friendly to S. fayeri and the second generation fayeri merozoites encyst in muscles. The cysts eventually mature and die and this is when they release toxins that result in inflammatory reactions.

The horse responds to both Sarcocystis infections by innate immunity.  Elevated CRP levels are seen in both diseases. In fact, the horse can look normal and have an elevated CRP.  This is called sub-clinical inflammatory disease. Sporozoites of Sarcocystis don’t produce enough identifying molecules to simulate acquired immunity.  The acquired immune response to S. neurona is elicited by the merozoitesSometimes, as disease progresses or with some treatments, these organisms can hide the molecules that stimulate antibodies. However, if first generation merozoites are being produced from sporozoites, antibodies will be stimulated to the “new” infection, even if the infection doesn’t progress from the gut into the blood stream of the horse.

The foregoing is a description of the pathogenesis of sarcocystosis in horses and can be used to understand the results of testing.  The disease process, from infection to immunity, was used to form our three disease model of EPM which are: 1) S. neurona sarcocystosis, 2) S. fayeri sarcocystosis, and 3) post- treatment EPM inflammatory syndrome (PTEDS).  Pathologic PTEDS progresses into the treatable autoimmune polyneuritis or a condition known as polyneuritis equi.

An elevated CRP concentration indicates inflammation.  If there are no apparent clinical signs when the sample was taken, the horse has “sub-clinical” disease.  An elevated CRP value, found along with antibodies to a specific organism (S. neurona, S. fayeri, or Borrelia, Lyme)…indicates there is active disease.  We associate elevated CRP values in horses without Sarcocystis antibodies with pathologic, chronic inflammation that results in autoimmune polyneuritis.

Research developments newly reported for Sarcocystis neurona may impact horse owners their veterinarians.  A novel genotype XIII was reported by Barbosa et al in the International Journal for Parasitology (2015).  This novel genotype is a sea mammal-virulent SAG 1 strain supporting SAG 1 and 5 antigen types dominate animal disease. This strain is vertically transmitted, from the mother to the fetus indicating S. neurona is more like than unlike other pathogenic protozoa.  Our pending publications, reviewed in our last two blogs, report new tests for horses with recurrent or residual signs of EPM that seek to clarify the role of inflammation in suspect-EPM horses.  The bottom line is that the key to maintaining a healthy horse is management through testing and examinations and understanding the pathogenesis of disease.

Sarcocystis neurona possesses one of six major surface antigen genes, SAG’s 1-6, on its outer surface.  The horse makes antibodies to these SAG’s and the antibodies are detected in the serum by ELISA testing.  Minor differences within the SAG genes allows classification into genotypes, or antigen types.  For example a SAG 1 S. neurona may be antigen type II or XIII.  The horse can only distinguish between SAG’s 1, 5, or 6 (serotypes) not antigen types.  The SAG’s 2, 3, and 4 are genetically variable between serotypes, are present in all Sarcocystis, and allow molecular biologist to examine differences between SAG genes.  Geneticists look at allelic variation within the SAG genes and that allows them to sub-classify S. neurona into genotypes or antigen types.

We developed three SAG specific ELISA tests based on recombinant SAG 1, 5, and 6, the strains that infect horses .  The specificity of these tests allows us to distinguish between serotypes by the antibodies made in response to infection. The majority of all disease caused by S. neurona in animals is due to SAG 1 and SAG 5 serotypes.  There may be virulence differences between the S. neurona SAG 1: antigen type II or XIII (discussed in Barbosa’s paper).  What is clinically relevant in the sick horse is recognizing the  serotype.  Measuring specific antibodies allows the veterinarian to identify resistant infections, determine the response to treatment, and distinguish relapse versus re-infection.

Our newest work identifies horses that have chronic inflammation.  Inflammatory responses cause the clinical signs often associated with EPM.  Some horses won’t respond to antiprotozoal agents because the protozoa are gone.  A frustrating clinical presentation is identifiable with our new serum testing, MPP and IL6 ELISA’s.  Our approach to managing these horses has not changed, we still measure SAG antibodies pre- and post-treatment.  We assess the horses by gait score before and after treatment.  We monitor the CRP serum concentration.  What has changed is that we can identify horses that will relapse and give the veterinarian an explanation why and a management program.

It is well known that equine serum samples show variation in reactivity to different surface antigens of S. neurona.  The most useful clinical point: it is not the level of antibody (titer) present in a horse’s serum that is important, but noting that the levels rise with duration of infection.  Another general rule is that the first experience with infection (naïve horse) will induce antibody production. The levels are minimal and short lived (8 weeks or so).  A horse experienced with infections will get and maintain a higher antibody level up to 5 months in some animals.  Management of EPM cases requires multiple serum analysis.  A single point test can’t decipher a new infection or a relapse. Multiple tests can suggest it the animal has naive infection or chronic exposure.  The horse with chronic exposure is more likely to experience abnormal immune responses that may look like EPM but really suffer from chronic polyneuritis.  It is important to distinguish these infections because the clinical management differs.

There is a report for a new trivalent SAG chimera ELISA test for efficient detection of antibodies against S. neurona .  This is an ELISA test that seeks to reduce the time, materials, and cost associated with running multiple ELISA’s using SAG 2, 4/3.  The diagnostic protocol involves using the the SAG ELISA to determine a consensus serum-to-CSF ratio, ratios less than 100 suggest that antibodies against S. neurona are being produced in the CNS and therefore parasites are suspected in the CNS.  Diagnosis of EPM based on CSF results is still confounded by normal passive transfer of antibodies across the blood-brain barrier.  The changes to detection of SAG 2, 4/3 antibodies by the third generation test don’t identify the issues concerning non-specific testing, it can’t discern serotype, doesn’t indicate a treatment failure due to strain resistance, or point the clinician in the direction of inflammation when parasites aren’t there. It remains to be seen if the reduction in costs for time and materials will transfer to the client.

The most exciting new information is in the Barbosa paper.  They report vertical transmission in S. neurona in a sea lion, a harbor porpoise, five harbor seals, and a pygmy sperm whale. We suspected and reported S. neurona in the lung tissue of a fetus from a mare experimentally infected with S. neurona in 2004. We suggest that there is a unique window of opportunity for fetal infection, before the fetus gains cellular immunity.  The observations of Barbosa and sea mammal infections may change the opinion that S. neurona is not vertically transmitted in horses (Dubey).

The possibility that mares can transmit infections to the fetus may stimulate management changes on farms with a high incidence of EPM.  It would be a very rare condition and the veterinarian is the best source to analyze risks on a case-by-case basis.

Give us a call if you have questions or concerns about EPM .  We outline management protocols for horses as part of our consulting service.  We haven’t seen any new evidence that prods us to change our approach to the diagnosis of sarcocystosis or inflammatory mediated neuropathy.  We advise multiple exams, even in a recovered horse, once healthy let’s keep them that way!  We are committed to testing for SAG 1, 5, and 6 in independent ELISA tests, we won’t combine our three tests for convenience or price.  Confirming the presence of inflammation and distinguishing peripheral from central neuropathy are current goals.

We are committed to developing diagnostic tests and effective treatments for parasitic disease.

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.

Between the dark and the daylight,

When the sun is ascending to power,

Comes a pause in the day's occupation

That is known as the Scientist's Hour.

It is dawn in tiny Fairfield, Florida, but Pathogenes is not sleeping in. Before daybreak, we begin to answer the emails which slithered in overnight when nobody was looking. It's also time to send out the documents which had gone directly to spam and to watch videos of ataxic horses from every state but Hawaii and Alaska, not to mention a few from our friends up North. We make early phone calls to our team of advisors across the country, wishing we had been more alert in Geography so that we'd recall that it is two hours earlier in the Mountain Time Zone. We are repaid for this failing later in the day when other poor Geography students telephone us at Pathogenes from Sacramento at ten p. m.

Our drop box at the end of the gated driveway oftentimes offers up samples from local vets and these are carried back to the lab. FedEx runs an early delivery route before the regular noon call so the gates must be opened well before eight a. m. The team arrives beginning at nine, a courier is dispatched to the small Farifield Post Office forthwith and the delivery trucks begin rolling in, culminating with UPS, the final visitor of the day, at seven p.m.

Our interpretations of the results from samples sent to us are the culmination of more than forty years of lab and field experience and fourteen years dedicated to EPM. And now we have yet another addition to the team. We call him Hal 9000P and he is tasked with test result interpretations and reporting.


Hal 9000’s people skills are renowned-- earning a top 100 award in his previous career, see his resume on Wikipedia. Hal 9000-P is a Cracker Jack at numbers, especially the numbers -1, 0 and 1. True/false and “not null” are Hal’s forte…beyond that Hal 9000 doesn’t read or speak. Austin trained Hal 9000-P to use our algorithm.

Submission Forms The submission form accompanies serum samples and gives us all the data we need to help you with your case of EPM. Our interpretation, coupled with a veterinarian’s neurological exam and field experience, simplifies treating equine protozoal myeloencephalitis. Sometimes, we find no submission form in the box, just a blood sample. Careful sleuthing by the lab crew can often reveal the source from the return address sticker. Failing that, we wait. Eventually, someone will call for the results.

Gait Assessment Score The veterinary exams are critical to a meaningful interpretation of our test. A trigger for “no interpretation” is absence of information on the submission form. A horse name and reporting email won’t give us anything to interpret—it may even trigger an email from Hal 9000.

It is well known that most horses in the United States are exposed to S. neurona resulting in serum antibodies, but yet there is no disease. This conundrum plagued EPM diagnosis for 25 years. We rely heavily on the gait assessment score (GAS), our hands are tied if there is no GAS on the form. A behavior problem should be scored as a “1”, issues such as head shaking and Horner’s (syndrome) make sense to us but Hal 9000 doesn’t interpret comments. He can evaluate a “1” listed as “Normal-deficit”, just check the box. We also need the submission form signed in order to use it for our FDA endeavors. You will get an interpretation without a signed submission, but the data is lost to our research. Be sure and download our newest, easy to fill out form on the services tab.

Send pre- and post treatment samples The follow-up sample is also critical to our analysis. If the second submission isn’t available, we won’t know how drugs. We hear that the once Grade 4 ataxic horse is now happily galloping in the meadow and can’t be caught…to us that is a treatment failure. We need that second signed submission form and blood sample.

Treatment response Initially our logic is based on the horse’s treatment history. If the horse has not been treated with an anti-protozoal AND there are antibodies to S. neurona then we want how the animal did clinically after treatment. Send us a quick email and let us know the response! Alternately, if there are no antibodies to S. neurona we still want your veterinarian to understand the disease process, this will lead to treatment options.

No S. neurona antibodies, GAS >0 We base our recommendations on statistics. We have tested and evaluated thousands of horses. We realize that some horses have no antibody; there are several situations could exist. No antibodies to S. neurona can indicate early infection (less than 17 days) or the horse has been exposed to anti-protozoals. Less likely reasons for lack of specific antibodies are that the animal has no ability to respond due to a defective immune system. This is a long held myth that there is a genetic predisposition to develop EPM—we don’t believe that. There is another theory…there are many unrecognized to S. neurona strains, thus all Sarcocystis may cause EPM—this is the Great Divide between our testing (specific) and the others (non-specific tests).

And, here is the most logical reason: it isn’t EPM. Yes, the horse can have neuroinflammation but not have active infection. In these cases we believe that a past infection with S. neurona, treatment of protozoa and not inflammation, is the problem. Our statistics show that in 80% of the cases without specific antibodies there is no infection. These cases can be treated. The numbers also indicate that 20% of the time we would miss early infections. When there is an early infection a post treatment test can determine active infection. A fourfold rise in titer (antibodies in the serum) indicates that there was active infection. The treatment decision is based on the veterinarian’s experience and risk assessment.

If a veterinarian is risk-averse, we offer C-reactive protein (CRP) testing. If the inflammation is due to ongoing S. neurona, Lyme, enteritis, gastritis, colitis, respiratory disease, tumor or other cause of inflammation the CRP will be high.

Treated The best time to retest is 8 weeks following treatment. We want to see that the antibodies are down to the undetected range. However, it the GAS is > 0 after treatment, we suggest determining the CRP and further treatment.

Relapse A relapse of EPM defined by us as insufficient treatment of IL6 mediated inflammation. Bute and banamine don’t work to treat this inflammatory path. A relapse can be due to incomplete removal of S. neurona, re-infection with S. neurona, or a reaction to another cause of IL6 mediated inflammation. We are documenting other reasons for IL6 inflammation, we recognize vaccination can induced ataxia in 0.3% of disease reported on our submission forms.

It’s the end of the day…we’ve used our lifetime of acquired knowledge to help you with your horse. We encourage you to forward your questions to Hal 9000…he’s waiting…

“A lie told often enough becomes the truth”. --Vladimir Lenin

If you’re not talking about inflammation, then-- by exclusion-- you’re lying about equine protozoal myeloencephalitis.  Reviewing only the causative agents (protozoa), life-cycle of the parasite, antibody testing, and treatment perpetuates misunderstanding.  Old ideas (EPM is enzootic and effectively untreatable, most horses are doomed to relapse) repeated often enough become a self-fulfilling prophesy.

Instead of viewing EPM as a population of horses infected with S. neurona containing a sub-group of untreatable, relapsing horses, chew on this.  View the population of ataxic horses as the whole pie (data set).  Ataxia is ubiquitous in horses and ataxia has several etiologies.  Sometimes ataxia in horses is caused by S. neurona.  Our data associates ataxia and S. neurona  in 54% of ataxic horses.

The ideas are very similar, however the practical difference between the views is that we can treat the latter group successfully—we are all too familiar with the treatment success of the former group.  Treating inflammation appropriately is key.  Understanding and managing the two components of EPM, inflammation and infection, are important to long term success.  The successful management of a horse with EPM will not be achieved by treatment alone, management takes an understanding the disease process.  It takes some work and it isn’t hard.

We make the association based on a gait assessment score, GAS, and the presence of specific serum antibody to the predominant surface antigens of S. neurona-SAG 1, 5, or 6.  A GAS of >1 and an ELISA SAG 1, 5, or 6 titer >8 attributes the ataxia to S. neurona.  A response to treatment supports the diagnosis.

Horses that do not have antibodies to S. neurona, with a positive GAS, are considered the rest of the pie, 46%. Just for arguments sake, call this group of animals IE-- disease due to inflammatory encephalitis.

If licensed anti-protozoal drugs are used for treatment the treatment efficacy is (published) 58%.  The difference in treating inflammation along with an anti-protozoal in suspect cases of EPM is 35%.  Are we just treating IE horses with an effective immune modulator or is there reason to think S. neurona played a role in disease? That is an experimental question for scientists, horse owners and veterinarians want results.

When horses show signs after "EPM" treatment they are called “relapses”.  Relapses are attributed to ineffective killing of anti-protozoals or re-infection.  The relapse rate with conventional antiprotozoal drugs are reported as 25% (ReBalance) and 20% (Triazines)—all of these drugs decrease the detectable antibodies in serum leaving, on average, 17% of horses in the IE category—ataxic with no antibodies--these are post-treatment IE cases but are attributable to S. neurona.  The rate of relapse (inflammation post-treatment) is three times higher in horses in which the inflammation was not addressed at the time of protozoal killing. Seventy one percent of ataxic horses with a presumptive diagnosis of EPM (no alternate diagnosis) had a root cause of inflammation that was most likely due to S. neurona infections.  This should be the topic of conversation about EPM.

The math leaves us to believe that 22% of horses with IE are horses with inflammation with an undetermined cause.  In some cases, we detect the effectiveness treatment for IE by serum CRP.  The anticipated treatment failure rate of treating inflammation and ignoring the inciting infectious cause, including S. neurona, would be high.  Our data indicates that S. neurona is a significant cause of IE in horses and should be a top consideration in treatment options.  Our research indicates that inflammation is the larger issue.  When treatment fails other obvious causes of IE should be investigated.