Equine Protozoal Myeloencephalitis (EPM): Diagnosis
Equine protozoal myeloencephalitis (EPM) continues to be an elusive disease to prevent, diagnose, and treat.
What is EPM?
Equine protozoal myeloencephalitis describes an infection of the central nervous system with an intracellular (they cannot reproduce outside their host cell) protozoan parasite known as Sarcocystis neurona.
To complete its life cycle, this organism requires a definitive host (the opossum), which feeds on the muscles of a dead intermediate host (such as a raccoon, skunk, cat, or armadillo). S. neurona is contained in the muscles in the form of sarcocysts, which once ingested by the opossum will mature and pass in their infective stage (sporocysts) in opossum feces. Horse feed, hay, or pasture that is contaminated by opossum feces is then unintentionally consumed by a horse; the ingested sporocysts penetrate and infect a horse's leukocytes (white blood cells, predominantly monocytes) and endothelial cells that line the blood vessels. It is speculated that, like a "Trojan horse," the parasites cross the horse's blood-brain barrier by hiding in the leukocytes, then they are released within the central nervous system (CNS) tissues, where they inflict their damage.
A typical presentation of EPM is that of neurologic signs, specifically asymmetric atrophy (muscle wasting) and ataxia (incoordination), with the atrophy occurring in focal muscle areas. A horse may appear to have neurologic signs in one side of his body, such as one hind limb that drags, or an eyelid or lip that droops on one side. In more subtle cases, it is sometimes difficult to determine if a horse is lame or is exhibiting neurologic signs such as ataxia. Any number of neurological signs can be present with EPM. Those signs can mimic other neurological diseases or musculoskeletal diseases, particularly of the pelvic, sacroiliac, or lumbosacral regions, or conditions due to cervical vertebrae stenosis (narrowing of the vertebral canal) or instability, also known as wobbler's syndrome. A horse affected by EPM might demonstrate signs consistent with brain disease, cranial nerve damage, peripheral nerve problems, obscure lameness, behavioral changes, or any combination of these.
A physical exam helps identify the type and extent of a horse's incoordination and neurologic signs, and it helps localize the lesions to specific areas of the central nervous system. While a thorough clinical and neurologic exam corroborates the presence of a neurologic problem, without testing it is often difficult to know for certain whether EPM is the only cause of disease, or whether other problems are occurring concurrently (such as Wobbler's Syndrome). S. neurona can affect any portion of the central nervous system and can, therefore, mimic other diseases, making generalizations during diagnosis impractical.
Testing for EPM
One of the major areas of controversy surrounding EPM is testing. Testing requires identification of markers of infection, such as antibodies specific for S. neurona. Such antibodies might be found in the blood of a horse that has been exposed, but not necessarily infected (no overt disease resulted from exposure). Antibodies found in the cerebrospinal fluid (CSF) cast a higher suspicion that the neurologic signs exhibited by a horse might be linked to infection and subsequent parasitic damage of spinal cord tissues.Testing for the antibody IgG (immunoglobulin G, the protein with antibody activity that's found in highest concentration in serum) has never been promoted as a "screening" test for EPM, but rather this test is intended to be used as an adjunct to clinical assessment when diagnosing a horse showing neurologic signs.
PCR (polymerase chain reaction) testing identifies minute particles of DNA specific to S. neurona when protozoa are present in the spinal fluid. The parasite might not remain following its damage to the central nervous system, so a negative PCR test does not entirely eliminate S. neurona from the list of suspicion.
A big problem in tracking the presence of antibodies in the CSF is that any blood contamination in a CSF sample can yield a positive result even if a horse has only been exposed (there could be antibodies in the blood). The PCR test on CSF is very specific for the presence of S. neurona parasites, although it has poor clinical sensitivity due to the transient appearance of parasite DNA in this media. This results in many false negative results.
Western Blot or Immunoblot Assay
The Western blot (WB) or immunoblot assay is generally considered the best test currently available. This is the test that we recommended for most of the horses we evaluate for EPM. The test was initially developed at the University of Kentucky in 1991 and is now run by IDEXX. A Western blot test gives a reading of "positive" or "negative" as to the presence of antibodies in the serum or CSF fluid, but it does not quantify the extent. Nor does it determine if a horse is only showing antibodies from a previous exposure and is not actively infected with EPM (this can lead to false positives, or saying a horse is actively infected when it is not in danger of developing the disease).
Despite these limitations, this test remains the gold standard to which all other tests are compared. Many claims have been made about the development of other assays with greater sensitivity and/or specificity, but these assays have not performed as well as predicted in actual practice.
A positive result (on serum or CSF) by the standard Western blot supports a diagnosis of EPM in the presence of accompanying clinical signs. A negative standard Western blot result (serum or CSF) essentially rules it out, except for acute onset cases or in immune-compromised horses for which there might not be a detectable IgG response. While the Western blot test format is not intended to be a quantitative one and doesn't provide a number, it gives a semiquantitative assessment of antibody level, so samples collected at different times can be directly compared to each other, with observable changes in levels.
Immunofluorescent Antibody Test
An immunofluorescent antibody test (IFAT) identifies surface antibodies in a blood sample. This means the test is specific for antibodies against an entire protozoon rather than portions or protein components of the organism. The IFAT relies on antigens (substances foreign to the body that evoke an immune response) at the surface of the parasite to detect antibodies, which are shared, in part, with S. fayeri, another common equine parasite. Intracellular protein antigens are not exposed for detection. In theory, surface antigens should be available throughout active infection as parasites are continually released from dying cells and infecting new ones. The IFAT reads out a quantifying number, or titer, that expresses the concentration of antibodies circulating in the horse's blood. In theory, a high concentration of antibodies in the blood indicates the horse is, or has been recently, infected. Comparative titers taken a few weeks apart indicate if a horse's antibody level is rising, and, if so, this corroborates an active infection that could be linked with clinical signs of neurologic disease.
Proponents of the IFAT suggest it might be used as a screening test to determine if it is necessary to pursue a spinal tap for CSF testing on those horses with borderline titers.
Parasites cultured in the laboratory are fixed into small wells in microscope slides. Antibodies, commercially produced in the laboratory against the horse's natural antibodies and linked with fluorescent material, are added to these wells, followed by the blood serum of the horse being tested for EPM. If antibodies against EPM parasites are present in the blood of the horse being tested, they will adhere around the cells of the parasites in the slides. In a chain reaction, the anti-horse antibodies with the fluorescent material will stick to the horse's natural antibodies adhered around the parasite, making it glow. This is considered to be a positive reaction. If there are no natural antibodies against the EPM parasites in the blood of the horse being tested, then this 'chain reaction' will not occur and the parasite will not glow, reflecting a negative result.
The IFAT is considered an alternative diagnostic tool and offers some advantages over the most common test used for EPM, the Western blot. The main advantage is that you can quantify the amount of antibodies in serum and interpret the test differently based on the antibody concentrations. A high antibody concentration would be more indicative of infection compared to a low antibody concentration, even though both could be above the threshold concentration for a positive result on the Western blot.
In general, rising titers can be a useful diagnostic tool for many diseases. However, it's important to remember that horses may be exposed to S. neurona repeatedly without causing disease. An S. neurona titer may or may not be related to the current clinical picture. Therefore, we generally try to determine if the horse is positive or negative (i.e. has it been exposed or not). The overwhelming majority of negative horses (on serum or CSF) don't have EPM. Positive serum samples with a wide range of antibody concentration are extremely common with or without clinical disease.
An enzyme-linked immunosorbent assay (ELISA) is a test for detecting antibodies to surface proteins of the protozoa, and it is currently only available through Antech Diagnostics. The results are given as a quantitative titer as with the IFAT. Antibodies it detects are very specific, and some strains will not be detected, hence, leading to a negative titer in spite of a horse having an active infection.
The ELISA test relies on recombinant SAG1 (a specific protein) antigen to detect antibodies. However, it has been demonstrated that many S. neurona isolates do not produce SAG1. Horses infected with those strains will test falsely negative. Antibodies produced against S. fayeri, another common equine parasite, cross react with SAG1.
Gene Expression Identification
Different diseases express themselves in a DNA signature. Using this technology on a horse's blood sample should allow positive identification of a diseased horse based on six gene markers that have a high sensitivity and specificity for EPM.
When infected with a particular agent, a characteristic set or combination of genes is expressed or suppressed as compared to normal background values. The genes that are either expressed or suppressed are compared to a standard for horses known to be infected with S. neurona, and if the "pattern" of upregulated and downregulated genes matches, then the diagnosis is confirmed. Essentially, it is looking for a specific pattern of gene expression in the test horse and comparing it to a known positive case.
Experimentation found the test to be greater than 90% sensitive and specific for S. neurona infection during the first month of infection. At the present time, this test is not yet ready for field testing, which is required before it can be made commercially available.
Vaccine Interference with Testing
A horse that has been vaccinated against EPM (using the original Fort Dodge Animal Health product) will have developed antibodies to the protozoa and the component proteins of the protozoa, even though the horse hasn't actually been infected with EPM. This poses a problem for screening or testing since a blood or CSF test result will come up positive regardless of whether or not a vaccinated horse is truly neurologic due to EPM. This vaccine is no longer available, although new vaccine research is in progress.Vaccinated horses develop an IgG response in serum and CSF that can not be distinguished by Western blot from natural infection.
None of the tests are capable of validating the presence of clinical disease alone, despite claims to the contrary. A positive PCR test probably comes the closest, but parasite DNA is rarely present in sufficient quantities to detect. The rest of the assays detect the presence of antibody--some with more specificity and/or greater sensitivity than others. The best of these is nothing more than an adjunct to a good clinical history and a thorough neurologic exam.