The EPM society has organized a Special Interest Group (SIG) gathering at the ACVIM meeting for many years, as it did in 2018. The objectives of the EPM society are: to organize individuals with interests in EPM research to contribute to a greater understanding of disease; encourage cooperative research; promote awareness of current research and new developments; and evaluate guidelines for the diagnosis, treatment and prevention of EPM.
In addition to the SIG, the EPM society has conducted organized focus meetings at Rood and Riddle in Lexington, Kentucky, in October 2014, and Lake Tahoe, Nevada, in October 2017. At those meetings, a small group of dedicated clinicians, researchers and members from industry came together to discuss recent developments in the field and identify a path to advance our knowledge of this devastating disease.
Many of the recent advances were presented at last fall’s Focus meeting (which was reported on EquiManagement.com as “EPM Society Workshop,” so the agenda for this year’s EPM SIG was a bit different. Steve Reed, DVM, DACVIM, began the meeting with a short eulogy/tribute for Bill Saville, DVM, who passed away this year (to read a tribute to Saville, visit vet. osu.edu and search for “William Saville”).
Reed then introduced the new EPM society officers: President: Dr. Sharon Witonsky, Virginia Tech; Vice President: Dr. Amy Johnson, University of Pennsylvania; Treasurer: Dr. Jennifer Morrow, Equine Diagnostic Solutions. And he introduced the Executive Board members: Dr. Nicola Pusterla, UC Davis; Dr. Dan Howe, Gluck Equine Research Center; and Antoinette Marsh, Ohio State University.
Witonsky lauded Reed by saying, “Steve is an incredibly well-known and well-respected person in the field of veterinary medicine with a particular passion for neurology and EPM. He not only oversaw the organization of the annual EPM SIGs, but he and Dr. Jennifer Morrow and Jenny Evans put an incredible amount of effort into both planning and executing the first EPM focus meeting at Rood and Riddle in 2014. He was also instrumental helping Dr. Pusterla in organizing and securing donors for the Tahoe meeting. Steve will continue on the board as the past president.”
Mouse Model Research
The focus of this year’s SIG was a review of research and an interactive discussion between a panel of EPM experts in immunology and the audience.
In order to provide some background information, Witonsky reviewed the predicted protective immune response to Sarcocystis neurona, as well as potential other pathways when infected horses or experimental models (i.e., mice) are infected. With the initial exposure to a pathogen or antigen, there is initial non-specific recognition by the antigen presenting cells (APC) (i.e., macrophages, dendritic cells) of particular common molecules (i.e., LPS). This activates the antigen presenting cells.
Depending on which common molecules stimulate the APCs, and depending on which other cytokines are present in the environment, the antigen presenting cells then activate T-cells toward a particular response. Different types of T-cell responses produce different cytokines (i.e., IFN-gamma, IL-4, IL-10, IL-17), which are then responsible for helping control the infection (i.e., CD4 Th1 IFN-gamma), allergen (i.e., CD4 Th2 IL-4) and auto-immune response (i.e., CD4 T-regulatory, IL-10).
If the proper immune response is stimulated, the infection or allergen can be controlled. If not, this can lead to ongoing infection, spread of disease and potential death.
The predicted protective immune response to Sarcocystis neurona is thought to be macrophage 1, dendritic cell 1, CD4 T-helper 1 IFN-gamma and CD8 IFN-g (M1 DC1, CD4 Th1 CD8 IFN-g) response. There are some data that support this theory from mouse models. The advantages of using a mouse model are that you can control both the genetics and environment of the mouse study.
Those effects cause considerable variation in equine studies. It is much easier to get the number of mice needed for studies and it is often much less expensive. In addition, many of the reagents are readily available to assess many details of the immune response, whereas equine-specific or reagents that work to measure equine-specific responses have been very limited.
Another advantage of the mouse studies is that researchers can use mutant strains (i.e., those strains that have an altered genetic component, such as the IFN-gamma knock out mice). These strains can then be used to assess the role of a specific molecule in disease.
IFN-gamma is one of the most critical cytokines in the cell mediated protective immune response. Lindsay and Dubey performed some of the first infection studies in IFN-gamma knockout (GKO) mice. They found that the mice couldn’t live more than about 20 days. They also identified where S. neurona was present in the mice. This told investigators that IFN-gamma is critical for protection.
With this as a starting place, investigators performed other KO studies and determined that nude and CD8KO mice get disease, supporting a role for T-cells—and specifically CD8 cells—in protection. B-cell deficient mice don’t get disease, suggesting both that B-cells and the humoral immune response (antibodies) are not critical for protection.
Finally, studies in SCID mice, which lack both B- and T-cells but have a strong innate system, demonstrated that the infected BALB/c strain mice don’t get disease until they are depleted again with IFNgamma.
A different strain of mice, SCID mice on C57BL6 background, do get disease when infected with S. neurona. IFNgamma depletion is not needed. This suggests that there could be role for a genetic predisposition to disease.
Some additional information supporting the CD4 and CD8 response is that both immunocompetent mice and GKO mice developed memory CD4 and CD8 responses. This information demonstrates the information and benefits that can be gained from using mouse strains.
However, all of the studies are experimental infection, with specific strains, doses and routes of infection. All of the information might also be applicable to horses, but the data are still being generated. In addition, the ability to generate has been limited due to the limited availability of equine specific reagents, having horses that meet case definitions and resources/funding for the studies.
The majority of information generated from equine studies has been with naturally occurring cases of EPM. Investigators have identified in some studies that EPM-affected horses have decreased antigen specific proliferation responses compared to control horses and seropositive horses (Tornquist, et al., 2001).
Another study identified that there was a decrease in the ability of cells to proliferate in response to SAG1. They also saw a decrease in IFN-gamma expression and an increase in IL-4 expression in EPM-affected horses vs. controls (Spencer, et al., 2004, 2005).
Some studies (Yang, et al., 2006; Witonsky et al., 2008) saw decreased proliferation response to PMA/I in naturally and experimentally infected horses.
EPM-affected horses also had increased CD4 and neutrophils compared to controls. There was a trend for decreased antigen-specific responses in experimentally infected.
Finally, there was additional data indicating that not only might T-cell responses be affected, but that the APCs might not be functioning, either.
An experimentally infected EPM model showed increased ability of CD4 cells stimulated with PMA/I to proliferate (Day 2) but decreased ability to proliferate to PMA/I at Day 28. There also was increased ability of CD8 cells to proliferate in response to PMA/I when stimulated in vitro at Day 2, but decreased ability of CD8 cells to respond to merozoites but not to PMA/I (Days 28, 56, 70) (Lewis et al., 2014).
Collectively, these data support that some EPM-affected horses have altered immune responses, but there are critical gaps in current knowledge about protection to S. neurona, as well as what happens with the immune response for horses that develop disease.
The question was also raised as to whether there is a difference in the immune response in horses that develop recurrent disease.
EPM and Other Diseases
At the 2014 EPM focus meeting, the question about the role of S. neurona in other disease was raised. At the time, Tom Divers brought up a study in which he was involved that was finding S. neurona in about 50% of the horses being necropsied that weren’t treated. In addition, the study found that treatment decreased the percentage of cases in which S. neurona was found.
New or Latent Infections?
With S. neurona, researchers and veterinarians don’t know when the horses are exposed or how long it takes from exposure to develop disease. They also don’t know whether the horses have latent infection, which means that when they have some other health event (i.e., shipping, pregnancy, colic, surgery, musculoskeletal disease), the latent infection can become activated to cause disease.
The high seroprevalence to S. neurona and the low incidence of disease suggests that most horses are capable of mounting the predicted immune response (i.e., M1, DC1, CD4 Th1, CD8 IFNgamma).
For investigators studying this disease, this can be a challenging situation. For the most part, the horses after exposure are trying to make the protective response. When they take samples, researchers have no idea when the horses were exposed or where they are in the schematic of the immune response.
If a veterinarian or researcher could take a sample, such as blood, to accurately measure the immune response to infection, that would be ideal. However, researchers don’t know if what is happening in the peripheral immune response is also happening in the brain and spinal cord (central nervous system) of the horse. They know that serum and CSF titers can be quite different, and based on other infectious disease models, they know the peripheral response isn’t consistent with the response in the CNS.
Therefore, for some of these studies to gain the required information, researchers need to have necropsies and samples from the CNS to most accurately identify samples and look at the difference in immune response in horses that develop disease.
David Horhov, PhD, and Witonsky led the audience in an interactive discussion about additional information regarding the following questions/topics. From those questions and answers the actionable items/outcomes were:
1. Witonsky and several collaborators will work on a project to identify the immune phenotype of EPM-affected horses and the role of S. neurona. More cases are needed. If you are interested in participating, please contact her at [email protected].
2. The group discussed the need for a potential biobank to store samples. As part of this, it will be critical to decide on case definitions, as well as what additional information is wanted (i.e., geographic location, clinical and treatment response, outcome) from all submitted cases.
3. Witonsky and others are working with Dr. Carrie Finno to identify whether EPM-affected horses have a genetic predisposition to EPM. More cases are needed. If you are interested in collaborating, please contact Witonsky.
4. There is the potential to provide a retrospective case study about N. hughesi cases.
5. Everything being done regarding methodology for S. neurona can be applied to N. hughesi. If samples are being biobanked, N. hughesi samples should also be saved.
6. Morrow and Evans will be developing a website.
Sharon Witonsky, DVM, PhD, DACVIM, is an Associate Professor of Equine Field Service (PMM) in the Department of Large Animal Clinical Sciences at the Virginia-Maryland College of Veterinary Medicine.