Testing

If you have any questions about testing please give us a call us at 352-591-3221. VETERINARIAN ELISA Submission Form.  Please sign the form and assign the gait assessment score.

Testing for the cause of neuromuscular weakness and disease is important! There are three diseases caused by Sarcocystis that look alike when the horse undergoes a clinical examination.  If the underlying cause of the disease isn't treated appropriately, the disease becomes chronic and progressive.

Our testing protocol allows the veterinarian to select one test or several, using a serum (or CSF) sample. Our tests are disease specific, a combination of tests are useful to select the proper treatment.  We freeze and hold the sample for 6 months to keep your options open. Our trained staff is available to suggest the most appropriate test.  Pathogenes Testing Options

 

TESTING OPTIONS

  • S. neurona SAG 1, 5, 6

  • S. fayeri

  • Anti-myelin protein antibodies

  •  Borrelia screening-Lyme disease

  • C-reactive protein

Tests for S. neurona antibodies : SAG ELISA

S. neurona SAG 1

We have no doubt that SAG 1 infections are more virulent, and the most common among equine S. neurona infections. The majority of all disease caused by S. neurona is due to SAG 1 or SAG 5 serotypes. Some of these strains are sensitive to ponazuril and diclazuril, but more importantly, some strains are quite resistant.

The SAG 1 serotype may have a relationship with inflammatory molecules (cytokines) that produce clinical signs of disease. The SAG 1 strains may have evolved to stimulate inflammation, increasing the presence of leukocytes in the intestines ensure the ability to hitch a ride to spread in the horse. It is perhaps most important to address the inflammatory aspect of the EPM syndrome in the SAG 1-infected horses.

S. neurona SAG 5

The discovery of the first SAG 5 infection in a horse is noteworthy because research is directed by one’s idea of disease causing organisms. Depending on unique serotype tests meant that all the disease-causing serotypes must be represented in the tests. A false negative could result if an unrecognized serotype wasn’t included in the analysis. Finding two serotypes convinced some researchers that it was impossible to ensure all serotypes would be represented. If there are two, is it possible that there are infinite numbers of S. neurona strains?

This began a divergent view by scientists of how to test for antibodies. We like specificity.  The alternate view from ours was to include markers from non-disease- causing Sarcocystis and prevent false-positive results by dilution. They used "common" antigens as markers. These tests would show false-positive results unless the cross-reactive antibodies were removed by dilution; another strategy was to calculate a likelihood ratio of disease based on some horses presumably known to have EPM. As time went by, and tests evolved the EPM positive horses didn't have organisms isolated, nor were they found (by special testing) and some of these horses were treated.  Some treatments prevent antibody production while some treatments will increase these antibodies.  This will affect tests.

Experimental infections with at least one SAG 5 strain were mild and self-limiting. Inflammation is a hallmark of this type of infection. The molecular interactions between inflammatory molecules and SAG 5 are different from those of SAG 6.

S. neurona SAG 6

Early on we hooked our wagon to the “specific” path, concentrating on SAG 1, the most infectious strain found in horses.  Others followed the “common” path, they selected markers common to all Sarcocystis. In 2009 a "new", very infectious S. neurona was discovered, it killed a lot of sea otters.  In a surprising discovery, we identified a marker (SAG 6) on this virulent S. neurona was identical to a Sarcocystis found in birds, S. falcatula. We provided a bit of DNA to NIH researchers and they made the link.  Luckily, this is the exact S. falcatula strain used to prove the bird-infecting strain couldn't infect horses!  The way to differentiate the two organisms is by determining which animal they infect. We did this by antibody testing.  What we found is that horses, and cats that live on horse farms with EPM, have antibodies to SAG 6.

Horses have antibodies to SAG 6.  The bird-infecting strain can't infect horses.  The logical conclusion is that S. neurona SAG 6 strains infect horses and S. falcatula SAG 6 strains infect birds.  It wasn’t until 2015 that the population geneticists concluded it is very unlikely there will be any more serotypes in addition to SAG 1, 5, and 6.

S. neurona SAG 1, 5, and 6 are the serotypes that formed the basis for our tests since the discovery of the SAG 6 serotype.  We looked at over a thousand horses that had SAG 6 antibodies and found evidence of inflammation in more than half of them. Inflammation was associated with disease.

Until the significance of disease causing serotypes are known, it is important to us to differentiate the infections by reactivity to S. neurona serotypes.  When you ask us for an "EPM" test, meaning the horse is suspicious of having EPM syndrome and does S. neurona play a part in the disease, we test for all three serotypes in the sample for one low price.

No denying it, these are antibody tests and antibody tests are limited. But, antibody tests are what we have to work with. Antibody tests are limiting because the disease is inflammation.  Testing is quick, available, inexpensive, minimally invasive, and easily understood.

Test to detect disease due to S. fayeri (SF)

Sarcocystis fayeri

It was believed for a very long time that S. fayeri infections were benign in horses. Disease was only to be found in debilitated horses. In 2014, a Japanese paper related S. fayeri food poisoning in some horse-eating humans.  That stimulated us and workers at CA Davis to related neuromuscular disease in horses to S. fayeri cysts.  The California researchers looked for cysts in muscle tissues of horses. We took an alternate path and detected antibodies to the toxins released from S. fayeri in the serum of horses that were suspected of EPM. It was the toxins from the cysts that made the Japanese people sick. About 30% of horses in the US have S. fayeri cysts.

You can test your horse now, or read on to learn more about this type of infection.   ELISA Submission Form

It’s useful to review the biology of Sarcocystis that can infect the horse.
The parasite life cycle starts as infectious eggs (sporocysts, shown at right).  Bile in the horses gut stimulates the release of sporozoites (the comma shaped organisms at right) and they burrow into the intestinal cells.  This happens within hours after ingestion of feed material contaminated by opossum feces. Once in intestinal cells, sporozoites divide, becoming first generation merozoites.  The merozoites undergo several replications.

Sarcocyst in muscle tissue

The merozoite stage only becomes dormant, forming a sarcocyst (muscle cyst), if it is in the right host. Horses are the right intermediate host for S. fayeri. Cysts can form in 7 days from the time the sporozoites are released into the intestine. Even in a natural intermediate host, many parasites die and fail to form cysts.  The muscle cysts wait for the definitive host, a dog, to eat the infected horse muscles, and the cycle begins anew.

Occasionally sarcocysts may contain two different merozoite species! A cyst-forming species can harbor another species that isn’t able to encyst in a host by itself.  Once a sarcocyst forms, it is nearly dormant. Sarcocysts contain bradyzoites, they are very slow growing organisms, ready for ingestion by the dog. A cyst remains viable by slowly metabolizing nutrients. Dormant cysts are still dynamic structures, they grow and die. When they die they release toxins. Muscle cysts can breakdown and release some slow growing organisms that may be capable of making new cysts in adjacent muscle cells.

If the cyst is metabolizing at all, the bradyzoites would be susceptible to some anti-protozoal therapies, but none are licensed for this purpose, because it hasn't been shown that they are effective. However, the biology of the parasite may have given us a way to test drugs for this purpose. We are doing it with a Field Trial, the results will be published soon. You can read about the Field Trial and if your horse qualifies call Dr. Laura (see About Us) to enroll.

It was believed that the sarcocyst was of no consequence to the horse. Generally, sarcocysts don't induce inflammation. It is the reaction to dying cysts that causes inflammation, the reaction is to dying host cells. Inflammation is a natural process that recruits cells that kill parasites and then removes the debris. Natural killer cells, really, that is what they are called, are a type of cytotoxic lymphocyte that is critical to the innate immune system that can be deployed as soon as 3 days after infection. Immunity, natural killer cells and possibly genetics of the horse combine to limit the S. fayeri infection. Immune deficiency, stress, debilitation and genetics of the horse can allow significant infection and disease.

The interplay between cells and the molecules that control their actions, cytokines, are important.  The point is that horses that have dying cysts in their muscles make antitoxin, antitoxins are antibodies against the S. fayeri toxin.  We measure these toxin antibodies.  Not all cysts breakdown releasing toxins, but in horses with clinical signs, the toxin stimulates cytokines, and cytokines increase inflammation.

Inflammatory molecules cause clinical signs in horses. These pro-inflammatory molecules can travel to peripheral nerves or even into the central nervous system where the clinical signs become profound. The result is polyneuritis.

Our serum test measures the levels of antitoxin in the horse.  A horse with S. fayeri toxicosis can have another infection with S. neurona, the infections aren't mutually exclusive, we can detect that by running the SAG tests and the S. fayeri test. The treatment for these conditions will be different and that is an important consideration for testing.

Tests to detect autoimmune polyneuritis (MPP/MP2)

anti-myelin Protein Tests

A common theme so far is the role of inflammation in equine sarcocystosis.  Way back in 1981 some English researchers realized three important things:

  • disease due to polyneuritis (inflamed nerves) in horses (and rats) could be related to circulating anti-myelin protein (MP2) antibodies,
  • chronic disease led to remitting/relapsing disease due to demyelination of peripheral or central nervous system nerves,
  • and equine myelin has an IL6 receptor in a very reactive part of the myelin protein (MPP).

These researchers were studying MS, multiple sclerosis, in people. And as an aside, a little interest was generated looking at the number of people with MS that also were infected with a people strain of Sarcocystis. They looked for evidence of the organism and some  people even looked at toxins produced by sarcocystosis in people. There was a connection, but not enough  to ignite the fires of inquisition.

We were intrigued.  Do horses with EPM, or more importantly horses with chronic relapsing EPM (that are treated time and time again), have MPP or MP2 antibodies? The answer was yes.  And then we asked ourselves: what is the disease process? The disease is autoimmune, but was it possible that S. neurona or S. fayeri stimulate this autoimmune disease?  When is disease due to parasites, which serotypes, and when does disease become autoimmune? That is what we study now, the relationships, or pathogenesis of disease due to sarcocystosis, in horses.

It is important because horses with autoimmune disease will not respond to antiprotozoal drugs!   The horse's signs will wax and wane, nerves are demyelinating due to inflammation and then being repaired; the damaged nerve sheath eventually scars by calcification. This is a life-ending event. Horses with chronic disease should be tested because testing will change your treatment decisions.ELISA Submission Form

Test for inflammation: C-reactive protein

The most stable serum molecule for testing the inflammatory pathway is an acute phase protein, CRP.  The value should be less than 16 micrograms/ml in the serum of a healthy horse.  Elevated values are seen in both S. neurona and S. fayeri infections. Unfortunately, this cytokine is not specific for protozoal diseases although multiple studies have related CRP to infection.  We have done that with statistical significance in several of our studies.  This is our choice for monitoring a horse with autoimmune polyneuritis.  If the horse looks good clinically but the CRP remains high (evidence of sub-clinical disease) we suggest continuing with appropriate therapy.  We are monitoring over 60 cases of autoimmune polyneuritis and will discuss our approach to treatment.  We looked at another inflammatory marker, serum amyloid A, years ago.  We published our work, the bottom line is that it was not useful for managing the horse with EPM.