- Feb 1, 2006
Although progress has been made studying equine protozoal myeloencephalitis (EPM) in the last 10 years, some aspects of the disease remain elusive. One researcher, half-jokingly, notes that EPM is considered the most diagnosed neurologic disorder in horses, and the most misdiagnosed neurologic disorder in horses! It's no surprise that researchers are seeking means to accurately identify horses that have EPM.
Along those lines, not knowing why some horses exposed to the protozoan parasite Sarcocystis neurona develop full-blown infection (resulting in brain and spinal cord inflammation) while other exposed horses remain impervious is another mystery engaging researchers. Mix in an interest to improve treatments, and you have the nutshell version of what's going on with the latest in EPM research.
Here are the details.
Know Thy Enemy
"Veterinarians have been hampered in dealing with Sarcocystis neurona by lack of understanding of its species identity, makeup, host range, and the mechanisms it uses to cause disease in horses," reports Linda S. Mansfield, VMD, MSc, PhD, professor of microbiology in the department of microbiology and molecular genetics at Michigan State University. "The most basic information needed is how to determine which Sarcocystis isolates are S. neurona and which are not in a rapid test."
To deal with those questions, Mansfield and her team assembled a large collection of Sarcocystis and other related cyst-forming coccidians, learned how to grow them in culture, and developed a "species identity" or "genetic fingerprint" for S. neurona isolates. "We applied this method to characterizing the available Sarcocystis isolates that have been assessed by preliminary methods as 'neurona-like,' " she says. "Our hypothesis is that S. neurona undergoes recombination during the sexual phase of parasite development, leading to isolates with varied genetic traits causing increased virulence." From that information, she hopes to:
1) Develop reproducible, sensitive methods for classification of clinical isolates of S. neurona based on molecular markers.
2) Explain the nature and extent of genetic variation of Sarcocystis species isolated from horses and opossums.
3) Improve the systematic classification of these organisms according to their evolutionary relationships.
"Once the method is established," Mansfield explains, "it can be used as a model to identify Sarcocystis we encounter in
animals to the appropriate species, to improve diagnostic methods for EPM, to track true sources of S. neurona for horses, to predict whether other closely related Sarcocystis will cause disease in horses, and to test whether S. neurona undergoes genetic recombination to produce new strains with pathogenic (disease-causing) potential. The term 'species' is similar to a breed and means 'a group of organisms that have a unique set of characteristics'--like body shape and behavior--that distinguishes them from other organisms and whose members can interbreed. Currently, we have finished analyzing a panel of S. neurona isolates." This means a number of isolates has been examined to determine similarities and differences.
Researchers at The Ohio State University have also cultured S. neurona out of an experimental horse, says William Saville, DVM, PhD, Dipl. ACVIM, extension epidemiologist/large animal internist in the department of veterinary preventive medicine. "The culture didn't come from nervous tissue, but came out of three different organs." Further details are expected to be released this year.
Know Thy Enemy, Genetically
Researchers at the University of Kentucky's Gluck Equine Research Center are focusing on the molecules that comprise S. neurona. Explains molecular parasitologist Daniel K. Howe, PhD, assistant professor in the department of veterinary science, "We are sequencing S. neurona--somewhat comparable to the human genome project, but on a much smaller scale."
To date, the lab has completed about 15,000 partial gene-sequencing reactions, allowing researchers to identify particular parasite genes. "We've identified a number of major surface proteins of the parasite, and we're currently using those in serological assays to examine the equine immune response during Sarcocystis infection," Howe says. This information could be instrumental in understanding how the parasite infects animals, how it evades the immune system of an infected horse, and whether the immune response differs in horses that develop the disease versus horses that don't. "Ultimately, we hope to be able to exploit that information to develop a better diagnostic test or a protective vaccine," he says.
S. Neurona in the Horse
Researchers working with experimentally induced and naturally occurring cases of EPM to determine the pathophysiology of S. neurona and protective immunity to the disease include Sharon Witonsky, DVM, PhD, Dipl. ACVIM, assistant professor in the department of large animal clinical sciences and colleagues at the Virginia-Maryland Regional College of Veterinary Medicine (VMRCVM), plus Frank M. Andrews, DVM, MS, Dipl. ACVIM, professor and section chief of large animal medicine at the University of Tennessee College of Veterinary Medicine.
Explains Witonsky, "We are interested in determining the mechanisms by which ingested S. neurona passes through the intestine, penetrates the blood/brain barrier, and eventually causes disease, as well as assessing changes in immune function associated with disease. In addition, our goal is to evaluate current diagnostic tests (i.e., SAG-1 ELISA, agglutination assay), and possibly develop additional diagnostic or therapeutic monitoring assays."
Researchers at Pathogenes, Inc., in Fairfield, Fla., are exploring the immune system's response to EPM infection, protective immune responses, novel treatment protocols, and the mechanism of early signs produced prior to ataxia (incoordination).
"We are currently working on specific methods to stimulate the immune system (with a recombinant vaccine) and assisting other groups in developing novel diagnostic tests using our model," states Siobhan P. Ellison, DVM, PhD, CEO of Pathogenes. "We feel accurate diagnosis prior to classic signs of disease, a stall-side screening assay for the horseman, and effective treatment and prevention will assist the owners of horses with or at risk to develop EPM."
Cats Aren't Where It's At
Because cats were found to be intermediate hosts for S. neurona, MSU researchers conducted a study to determine the prevalence of antibodies in the serum against S. neurona in a population of domestic cats previously tested for antibodies against Toxoplasma gondii. "Toxoplasma causes neurological disease in cats, so we reasoned that these cats may have a higher likelihood of having S. neurona because they are likely to be outdoor cats with high exposure to wild animals," Mansfield says. "Sarcocystis neurona must cycle between opossums and another host like the cat to keep the parasite going in the wild. Usually, opossums pass it along to raccoons, skunks, and, in the southern U.S., armadillos."
After cat samples were tested for antibodies against S. neurona by two different tests, researchers concluded that domestic cats don't play a substantial role as intermediate hosts in the natural life cycle of S. neurona.
Mansfield says, "Cats have the potential to harbor this organism and are able to pass it on to opossums if the opossum were to ingest cat tissue with the parasite. However, based on testing of a large number of cats throughout the U.S., it appears that they are unlikely to be exposed to opossum feces carrying the parasite and, thus, are negative. Certainly even an infected cat would pose no risk to horses. Only the opossum can give the parasite to a horse in its contaminated feces."
Researchers at VMRCVM and Equine Biodiagnostics recently demonstrated that vaccination with the EPM vaccine could, at least transiently, produce a positive Western blot test result for antibodies to S. neurona in cerebrospinal fluid. Also, one can't distinguish by Western blot between a horse that is seropositive due to natural exposure versus vaccination. Explains Witonsky, "It is possible that vaccination of a horse with the EPM vaccine has the potential to interfere with diagnostic testing if that same horse develops neurologic signs (soon after vaccination)."
Traditionally, the most accurate diagnosis is based on clinical neurologic signs and a positive Western blot test (WBT) for S. neurona antibodies in the serum and cerebrospinal fluid (CSF) of horses. But two new tests were recently developed to produce approximate estimates of the amount of anti-S. neurona antibody present in equine serum samples. The amount of antibody might indicate the level of infection--the higher the titer, the more organisms. "Both assays rely on whole parasites to detect the presence of anti-S. neurona antibodies," says Andrews.
In the S. neurona direct agglutination test (SAT), serum samples are mixed with killed S. neurona merozoites (merozoites are cells that arise from the asexual division of the protozoa). "A positive SAT result depends on the ability of anti-Sarcocystis antibodies present in the serum sample to cross-link merozoites, forming a distinct pattern on the bottom of a test tube," Andrews notes. "The developers claim approximately 90% agreement with the standard WBT; however, these results have not been published for peer review."
An older methodology to detect antibodies, the indirect fluorescent antibody test (IFAT) relies on fluorescence to detect anti-S.neurona antibodies. Says Andrews, "Initial results published using this technique compared favorably with the standard EPM test. However, the number of horses tested was limited. Since S. neurona and S. fayeri share many proteins, it seems unlikely that this assay will perform as well as the standard WBT under the rigors of routine clinical testing (30% of U.S. horses are thought to be exposed to S. fayeri)."
Researchers continue to analyze the WBT test. One study of WBT sensitivity and specificity found that test sensitivity was about the same as reported previously (87% vs. 89%); high sensitivity indicates few false negative results, reports Andrews. "In this study, a negative test result was 98% effective in ruling out disease, which is in agreement with earlier estimates (92%)."
On the other hand, test specificity (the percentage of horses without EPM that were correctly identified by the test) in the study was lower than previously reported among neurologic horses, indicating a higher number of false positive CSF test results. In other words, some horses were identified by the test as having EPM, but did not at necropsy.
Andrews concludes, "The presence of clinical neurologic signs and the WBT are still recommended for the diagnosis of EPM in horses and certainly indicate exposure to the parasite."
At Ohio State, researchers examined the immunofluorescent antibody (IFAT) test and Western blot test to see if either could differentiate between S. fayeri and S. neurona infection. Saville reports the IFAT test was less effective at distinguishing between the two infections, but the Western blot distinguished between them.
Pathogenes researchers have been evaluating the SAG-1 ELISA diagnostic assay. Explains Ellison, "The SAG-1 ELISA detects antibodies against S. neurona in the serum or CSF. The test is based on recombinant protein attached to a plate that is then used as antigen (target) to capture specific antibodies found in serum from horses with EPM. The horse antibodies are then detected using specific reagents."
The important properties of the SAG-1 ELISA are:
- The recombinant protein target is the immunodominant protein on the surface of S. neurona (unique to S. neurona).
- By using this antigen, veterinarians can determine a specific titer to S. neurona.
Says Ellison, "The titer is useful to look at samples over time and determine if the antibody titer is increasing or decreasing. An increasing titer (a two-fold increase in titer) would be seen in acute disease, and a decreasing titer would be detected at the end of successful treatment."
Additionally, the use of a specific titer allows calculation of a specific antibody index in the CSF. Both the total level of antibodies in the CSF and the specific amount attributed to S. neurona antibodies can be determined from the titer obtained by the SAG-1 ELISA. Says Ellison, "We have determined what levels of specific antibodies are relevant to clinical signs of disease using an experimental model developed at Pathogenes."
"One important aspect of knowing when a horse first contracted EPM was the ability to detect visible signs (such as asymmetrical eyelid drooping or muscle wasting) present before a horse shows lameness or ataxia, which is what is usually associated with EPM," Ellison continues. "We feel early recognition of infection will allow the horse owner to treat the horse prior to spinal cord damage that permanently affects the horse."
Ohio State researchers are working on a new diagnostic test for a major drug testing company. Additional details are not available at this time.
Michigan State researchers investigated the effectiveness of pyrantel tartrate, the active ingredient in the daily dewormer Strongid C, against S. neurona. "We tested the hypothesis that pyrantel tartrate can kill S. neurona merozoites growing in equine skin cell cultures," says Mansfield.
S. neurona merozoites exposed to higher concentrations of pyrantel tartrate did not produce abnormalities suggestive of EPM infection in equine dermal cells. "This demonstrates that pyrantel tartrate has activity against S. neurona merozoites in vitro and suggests that it may have activity against the infective stage of the parasite found in the horse's gut," Mansfield observes. "Subsequently, further testing was conducted to determine whether pyrantel tartrate at the standard marketed dose could be used to prevent EPM in horses. These studies will be published in the near future."
Horses are still becoming infected with S. neurona and still developing EPM, and owners are still dealing with horses that don't recover fully from the disease. While much has been learned in the past 10 years, and much research is ongoing, there still is an air of mystery about this condition that might take years to unravel.
About the Author
Marcia King is an award-winning freelance writer based in Ohio who specializes in equine, canine, and feline veterinary topics. She's schooled in hunt seat, dressage, and Western pleasure.
POLL: University Equine Hospitals