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EPM 2011

EPM 2011

Many groups of scientists have researched Sarcocystis neurona and EPM. Each group has added new knowledge to form a big picture of infections and the relationship to disease. Until recently, the pieces didn’t seem to fit into one picture and that leaves different groups of scientists with polarizing ideas.

We are exploring the antigenicity, pathogenicity, and molecular traits of Sarcocystis neurona in horses. We also explore the horse’s reactions to infections beyond antigens. Our results give us new insights into past research. Hindsight, as you know, is 20-20.

About Hosts

Realizing and proving that the opossum was a definitive host for S. neurona was a big step forward in EPM research, attributed to Clara Fenger, University of Kentucky. Researchers could focus on sporocysts, which are infective to horses. Sporocysts used in challenge experiments were important in order to determine the intermediate host(s) and complete Koch’s postulates for EPM. For example, nude or immunodeficient mice (ID mice) are susceptible to S. neurona  sporocysts.

The lab strain, isolated from horse with clinical EPM, and the sporocysts from feral opossums, when compared, were 99.8% the same genetically. One important gene of S. neurona was merely 0.2% different than the same gene in S. falcatula. Once the genetic similarity was determined, intermediate host susceptibility took center stage. Surprisingly, the intermediate host specificity for S. falcatula includes budgies, not immunodeficient mice. The biological difference, the ability to infect one host and not another, is a founding principle in pathogenic protozoal identification. Intermediate host specificity highlighted important biological differences between the two closely related parasites. The idea that S. neurona was identical to S. falcatula was wrong. Twenty years later, the molecular differences between S. neurona and S. falcatula remain complicated, but distinct. Antigenic differences are complicated and confound our understanding of EPM.

 

 

Equine protozoal myeloencephalitis was recognized in horses in the 50’s and S. neurona was isolated in 1991. The cultured S. neurona enabled Clara Fenger, then a student at the University of Kentucky, and her associates to compare the in vitro material to sporocysts obtained from wildlife. Fenger’s group used molecular markers of Sarcocystidae to suggest a definitive host would be in the dog family. (It was later discovered that S. neurona does infect dogs–there are ongoing investigations to determine if S. canis could be S. neurona). These markers were used to identify S. neurona in feces and intestinal digest of wildlife specimens. These authors concluded that the opossum was involved in EPM. Aflutter with excitement over her new and important data, Dr. Fenger visited another research group and discussed her ideas. The secret was out and history reveals that it was highly suspicious  that S. neurona cycled between opossums and birds.

The race was on to experimentally induce EPM from opossum oocysts. Fenger and her co-workers used wild caught opossum oocysts to infect horses and induce clinical signs of EPM. The infections didn’t result in organisms in the CNS. They failed to complete Koch’s postulates because they failed to re-isolate S. neurona from the CNS of an experimentally infected horse. They described ataxia in the challenged horses and inflammatory lesions that did not include the organism. Likewise, S. falcatula failed to induce EPM. There were no antibodies detected (using what were the antigens du jour) in the S. falcatula experiments when the samples were tested by the Western Blot (EBI, KY). The field was open to those that could identify sporocysts as S. neurona, produce sufficient numbers for challenge experiments and isolate the organism from the CNS of a horse. This task remains unfulfilled to this day. What followed was gathering data about the intermediate host range of S. neurona and the realization that the opossum harbors more than S. rileyi, S. neurona, and S. falcatula as was thought in 1997.

Dr. Fenger and her group identified an outbreak of EPM in 12 of 21 horses on a farm. She found “EPM may develop as an epizootic. Fenger reported subtle clinical signs that were originally considered unimportant that ultimately progressed to obvious neurologic signs.” She co-patented the use of pyrimethamine and trimethoprim-sulfamethoxazole for the treatment of EPM. Also, she reported “adverse effects associated with EPM treatment (pyrimethamine and trimethoprim-sulfamethoxazole) included worsening of neurologic signs, anemia, abortion, and leukopenic and febrile episodes.”

From these papers we found the roots of some long held ideas. The body of work contributed by CK Fenger and her associates (1994-1997) identified the opossum as the definitive host of S. neurona, a cornerstone in this field. They were able to recognize that subtle signs are important in horses with EPM. They recognized that disease is not isolated to an individual horse when sufficient infectious material is present. It is expected that farms with one case of EPM will have others. Most important, in hind sight, is realizing there are CNS lesions associated with clinical signs but no parasites.

Despite recognizing and reporting the initial subtlety and ubiquity of EPM, there is a long standing belief by some that only a few horses are susceptible to infections, presumably due to some defect of their immune systems that allow them to succumb to disease. Sharon Witonsky, in conjunction with Pathogenes used our research model to show that the parasite itself can manipulate the equine immune system. The parasite uses strain specific down regulation measured by proliferation responses. Our research showed that any horse is susceptible to infection using pathogenic strains. Most horses have mild infections that can resolve. Our work also shows that the level of challenge for a horse is low, in the thousands, not millions of organisms, as used in most studies. We also observed that infected horses have a statistically significant rise in titer the longer the infection continues. The higher the titer, the longer the infection. It is beneficial for the diagnosis of EPM to show that a horse has a two to four fold rise in titer in conjunction with signs consistent with EPM.

Fenger’s group presented the proof that opossums are definitive hosts for S. neurona and they clearly believed that S. neurona and S. falcatula were synonymous. They conducted an experimental challenge in horses using oocysts derived from opossums fed sparrows, hosts of S. falcatula. We now know S. neurona and S. falcatula are not the same genetically or biologically. Foals in their study immuno-converted on immunoblots post challenge and demonstrated clinical signs consistent with EPM–this observation was the opposite of the University of Florida S. falcatula (Florida) infection challenge in which horses did not seroconvert, again based on Western blot (EBI, KY). In 2010 we determined that S. falcatula (Florida) displays a surface antigen that is genetically identical to a surface antigen of S. neurona.

Experimental infections of these horses with oocysts were, at best, a mixed oocyst population challenge because wild caught opossum oocysts (these oocysts could have S. neurona or S. falcatula) were added to the challenge dose derived from sparrows. There is no definitive evidence that S. neurona was present in this challenge study, they reported seroconversion on immunoblots, this in contrast to the UF S. falcatula study and indicates S. neurona may have been present . They did prove, by bird challenge (budgie), that S. falcatula comprised some, if not all, of the oocyst population that they used. They demonstrated inflammation and saw clinical signs of ataxia.

In hindsight, we know that some S. falcatula strains could be confused with S. neurona on immunoblots. These confounding S. falcatula strains may infect horses, induce antibodies, and share surface antigens that are almost identical to one phenotype of S. neurona. Fenger’s work illustrates that some S. falcatula strains induce ataxia in horses, but S. falcatula doesn’t cross into the central nervous system. An alternate view is that they introduced S. neurona from the wild caught opossums using a mixed oocyst challenge. A challenge with a small number of S. neurona oocysts could also support their results and based on the work of others is the most likely scenario.

Repeated exposure of horses in an environment contaminated with S. neurona is highly likely. Dr. Fenger’s work supports the idea that it would take very few oocysts to infect horses, ataxia would be seen clinically, and no organisms would be present in the central nervous system, although there would be lesions consistent with inflammation. It is apparent that treatment for organisms that do not enter the CNS would differ from those that do and that the pathology of the neural tissue inflammation is crucial to our understanding of EPM in the horse.

 

 

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