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. The test results are the basis for the consult that is a case-by-case discussion of the horse and the disease process. Pathogenes Testing Options
S. neurona SAG 1, 5, 6
Anti-myelin protein antibodies
Borrelia screening-Lyme disease
Serum Chemistry (only with submission of other testing)
Tests for S. neurona antibodies : SAG ELISA
S. neurona SAG 1
S. neurona SAG 1 strains are virulent, and the most common serotype found 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 possible to find an infinite number of S. neurona strains.
This began a divergent view by scientists of how to test for S. neurona 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 affects 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
In 2009 a "new", very infectious S. neurona was discovered, it killed a lot of sea otters. In a surprising discovery, a marker (SAG 6) on this virulent S. neurona was identified as 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.
Testing Horses With Signs of EPM
In 2015 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. Until the significance of disease causing serotypes are known, it is important to us to differentiate the infections by reactivity to S. neurona 1, 5, and 6. When you ask us for an "EPM" test, meaning the horse has signs of EPM (syndrome) and are there S. neurona antibodies, we test for all three serotypes in the sample for one low price.
Test for S. fayeri
Neuromuscular disease in horses has been associated with S. fayeri muscle cysts. Our test detects antibodies to the toxin released from S. fayeri into the serum of horses with clinical signs of EPM. About 30% of horses in the United States harbor S. fayeri cysts.
You can test your horse for S. fayeri infection. ELISA Submission Form
The Sarcocystis 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 within hours after ingestion of feed material contaminated by opossum feces (S. neurona) or dog feces (S. fayeri). Once in the intestinal cells, sporozoites divide, becoming first generation merozoites. The merozoites undergo several replications.
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. Armadillo's are the right intermediate hosts for S. neurona. 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 are infectious to the definitive host, a dog or an opossum. Once ingested 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 definitive host. 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.
It was believed that S. fayeri sarcocysts were of no consequence to the horse. Generally, sarcocysts are not associated with inflammation. It is not the reaction cysts that causes inflammation, the reaction is to dying host cells. Inflammation is a natural process designed to recruit cells to kill parasites and then remove the debris. Natural killer cells 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 the genetics of the horse combine to limit the S. fayeri infection. Immune deficiency, stress, debilitation or genetics can allow significant infection and disease.
The interplay between cells and the molecules that control their actions, cytokines, are important. Horses that have dying cysts in their muscles make antitoxin. Antitoxins are antibodies against the S. fayeri toxin. The toxin antibodies in serum can be measured. Not all cysts breakdown to release toxins. In horses with clinical signs, the toxin stimulates cytokines, and cytokines increase inflammation that results in disease.
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.
The S. fayeri serum test measures the levels of antitoxin in the horses serum. A horse with S. fayeri toxicosis can have another infection caused by S. neurona because the infections aren't mutually exclusive. Duel infections are detected using 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)
Myelin protein and neurofilaments
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 neuromuscular disease in people.
Horses with chronic relapsing EPM (that are treated time and time again), have MPP or MP2 antibodies. Sometimes there is axon damage, neurofilaments can be detected in the serum, NfL. Neurofilaments can be present in demyelinating and non-demyelinating polyneuropathies.
It is important because horses with autoimmune disease will not respond to antiprotozoal drugs! The horse's signs will wax and wane, nerves are demyelinated due to inflammation and then repair by re-myelinating the nerve. The damaged nerve sheath eventually scars by calcification. When axons are damaged and proper treatment is initiated the NfL value will decrease quickly. This is a biomarker to determine if the treatment is useful. Horses with chronic disease should be tested because testing can change the treatment decisions. ELISA Submission Form
Test for inflammation: C-reactive protein
The most stable serum molecule for testing the inflammatory pathway associated with Sarcocystis is an acute phase protein, CRP. A normal 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. This cytokine is not pathognomonic (unique) for protozoal diseases. Elevated serum CRP is related to infection. CRP is a useful 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.
Vitamin E deficiency may result in equine motor neuron disease (EMD) and can is treated with supplementation. Horses that don't need supplementation are those on green grass or high quality green hay. Testing the serum levels will identify horses that are deficient and when to discontinue therapy. Samples for vitamin E testing should be collected in purple top tubes (EDTA tube). Remove the plasma and keep it cool and protected from light. Deliver to the Texas A&M diagnostic laboratory for testing with a cool pack. It is best to deliver to the laboratory within 24 hours. To read our discussion about vitamin E and your horse use this link. You may also find this blog useful.