Current Research

Grayson-Jockey Club Research Foundation’s board of directors has announced a slate of 17 research projects which the Foundation will fund for a total of $986,863 in 2015. The list includes ten new projects and seven which are in their second year, as well as Two Research Career Development Awards. The allotment brings the Foundation’s total impact since 1983 to over $22 million to fund 322 projects at 41 universities.

Angela Bordin, Texas A&M University– First Year (2 Year Grant)

One Sentence Summary: A guinea pig model of R. equi pneumonia will help to better understand the disease in foals, and evaluate novel approaches for controlling and preventing R. equi pneumonia.

Pneumonia caused by Rhodococcus equi is an important cause of disease and death in foals. Important advances in R. equi research could be achieved using a small animal model of pneumonia, including the evaluation of novel approaches for controlling and preventing R. equi pneumonia before their test in horses. Currently, there is no model of pneumonia caused by R. equi: infection of mice does not cause pneumonia as recognized in foals with natural or experimental infections. As a result, instillation of bacteria into the airways of foals to cause lung infections is used as an experimental model of natural disease. Infection of foals is problematic for several reasons. First, it is labor–intensive and cost–restrictive, and, frequently, involves a team of trained personnel to monitor and administer treatments to infected foals several times a day. Second, it demands adequate infra–structure to keep foals and their respective dams in a confined space to allow physical examination and administration of treatments, while also providing access to paddocks. Third, the use of foals is restricted to the spring foaling season in the northern hemisphere, limiting the number of experiments that can be conducted in one year. Fourth, laboratory reagents to study the immune system of horses are quite limited relative to guinea pigs. Fifth, and perhaps most importantly, there are major welfare considerations of making foals severely ill. Thus, the objective of this study is to develop a guinea pig model of R. equi pneumonia. We intend to determine whether aerosolized R. equi can produce in guinea pigs the same form of pneumonia observed in naturally infected foals; and also to identify a dose of R. equi that consistently produces pulmonary lesions in guinea pigs that are proportional to lesions in foals naturally infected with R. equi. Guinea pigs will be infected by aerosol at various doses of R. equi and monitored daily to evaluate disease. They will be euthanized at 5 weeks post–infection or when clinical signs of pneumonia are detected, whichever comes first. This model will be useful for evaluating and optimizing novel strategies for therapeutics and vaccines, and for studying several aspects of R. equi pneumonia without the drawbacks of using foals, and will be a benefit to scientists studying R. equi on a world–wide basis.

The use of a guinea pig model of Rhodococcus equi would greatly benefit the equine industry. The impact of R. equi pneumonia on the equine industry is large. In the United States, respiratory disease is the third most common cause of disease in foals less than 6 months of age. Foals with R. equi pneumonia can either show clinical signs of disease, or have the lesions without clinical signs (known as subclinical disease), but the most common form of the disease is a chronic pneumonia with an insidious onset that hinder early detection. Often, the lung lesions are well–established and extensive damage has occurred by the time a diagnosis is made. Consequently, a high proportion of foals with R. equi pneumonia die, and foals that do survive the disease are less likely than age–matched cohorts to race.

Treatment and prevention of R. equi pneumonia are difficult: there is no vaccine commercially available, treatment with antimicrobials is lengthy, costly, has potential adverse effects, and is not uniformly successful. Also, there is great need to identify alternative antimicrobials because of evidence of increased resistance. Evaluation of antimicrobials in animals is necessary because drugs that are effective in the laboratory are not always effective in patients. Also, no efficacious vaccine against R. equi pneumonia exists. The equine industry would greatly benefit with the development of both alternative antimicrobials and vaccines that could protect foals against R. equi pneumonia. Thus, there is need to develop a small animal model of R. equi pneumonia, which would allow for evaluation of research questions of clinical importance (such as how the disease develops, and evaluation of new vaccines and antimicrobials) in guinea pigs before they are evaluated in horses or foals. A guinea pig model of R. equi pneumonia would allow for more rapid advances in the field resulting from experiments completed faster than if they were performed in the foal model.

Laurie Goodrich, Colorado State University – First Year (2 Year Grant)

One Sentence Summary: The completion of this project will answer the important question of whether allogeneic mesenchymal stem cells derived from bone marrow (BMDMSCs) are a viable alternative to autologous BMDMSCs in the horse. (Autologous means cells from the horse’s own bone marrow; allogeneic means from another, healthy horse.)

Regenerative therapies, specifically mesenchymal stem cells that are derived from a horse’s bone marrow (BMDMSCs) are a sought after and highly utilized treatment for diseases of tendons, ligaments and cartilage in racehorse and sport horse practices. When these cells are harvested from a horse’s own marrow cavity (autologous or self), they are grown and expanded over a period of two to four weeks and subsequently injected into the injured tendon, ligament or joint. The time required for this process can make this treatment inconvenient for owners and trainers as they must wait a few weeks before returning to get the therapy. Furthermore, the process of individual cell expansion is expensive. An alternative source may be cells from another, healthy horse’s (allogeneic or non–self) bone marrow that has already undergone expansion and is ready for injection. A lag time in treatment to injury has been found to sometimes be detrimental; therefore, an “off the shelf” treatment would potentially enhance the healing potential of these readily available cells in addition to making the treatment more convenient and less expensive to clients. BMDMSCs have been studied intensively in other species and have been established to have a local immunosuppressive effect so that the host’s immune system does not recognize them as foreign. This area has not been closely examined in the horse. If no differences exist between autologous and allogeneic stem cells then BMDMSCs from young, healthy horses may be a viable alternative source to autologous cells. Allogeneic (non–self) BMDMSCs would be highly available and less expensive for clients.

The main hypothesis of this grant is that there are no differences between autologous and allogeneic BMDMSCs in how these cells stimulate the immune system. We will first test this hypothesis by comparing the immunity of these cells in cell culture (in vitro) and attempt to determine the mechanisms by which these cells locally suppress the immune response (Specific Aim 1). In our second Specific Aim (Specific Aim 2) we will compare the immune response to intra–articular injection of each horse when autologous or allogeneic BMDMSCs are administered once and with repeated injection. The completion of this project will answer the important question of whether allogeneic BMDMSCs are a viable alternative to autologous BMDMSCs in the horse. If so, allogeneic cells could be more readily available and less expensive, allowing BMDMSC therapy to be more highly utilized and affordable to owners and trainers.

Regenerative medicine, especially mesenchymal stem cell therapy is a frequently utilized treatment in racehorse and sport horse practice. The lag time of individual cell culture expansion makes this therapy inconvenient and expensive. If allogeneic (non–self) BMDMSCs could be utilized instead of autologous (self) BMDMSCs without these cells being recognized as foreign, this regenerative therapy could be made available as a less expensive “off the shelf” treatment making BMDMSCs available to clients who would have previously foregone the therapy due to expense. Research studies are mounting with evidence of the benefits this therapy provides. When the questions in this proposal are answered, we will have an improved understanding of how these cells avoid immune recognition and potentially have a more available and affordable source of stem cell therapy.

Ramiro Toribio, The Ohio State University – First Year (2 Year Grant)

One Sentence Summary: This study will elucidate the importance of stress hormones as well as hormones that affect neurological function in the development and progression of diseases of newborn foals.

Sepsis (blood poisoning) is a condition where bacteria can multiply in the blood and set up widespread infections throughout the body of newborn foals often leading to death. The clinical signs and consequences of sepsis include severe stress, organ failure, abnormal hormonal and metabolic functions (e.g. low blood glucose), electrolyte disturbances (low blood sodium), dehydration, low blood pressure, impaired organ maturation and function, and death.

The hypothalamus–pituitary–adrenal gland axis (HPAA) is a complex system that regulates how foals respond to stressful events. Dysfunction of the HPAA results in abnormal concentrations of stress hormones, including steroids from the adrenal gland. These steroids are essential to regulate glucose, electrolytes, blood pressure, as well as organ maturation (lungs, brain, intestine). Some of these steroids (neurosteroids) protect nerve cells against injury and inflammation. We are proposing that in sick newborn foals the equilibrium between these steroid hormones is broken, and depending on its severity it can have deleterious effects on metabolic activities, immune protection, organ function, and likelihood for survival. For example, we suggest that critically ill and premature foals will have increased levels of progesterone (a sex hormone) and low levels of cortisol (a stress hormone).

We will investigate hormones from the brain, pituitary gland, and adrenal gland. We will determine their association with the clinical signs and blood abnormalities of sick foals, as well as whether their levels are linked to clinical improvement or mortality.

We have established collaborations with multiple institutions to investigate the importance of these hormones in the development and progression of diseases affecting newborn foals. We have the expertise to address these questions in a timely manner. The information generated will be relevant to the equine industry as it could lead to the development of novel treatments for foals.

Sepsis is a pathological condition where bacteria can multiply in the blood and tissues, releasing toxins throughout the body of newborn foals. Sepsis is the number one cause of mortality in newborn foals. Many foals that survive sepsis are left with disabilities that impair their future performance. Stress hormones including steroids from the adrenal gland that are essential to control glucose, electrolytes, and blood pressure are often altered. Other steroids are important for organ maturation / function (lungs, brain, intestine). Recent investigations indicate that other steroids (aka neurosteroids) for which minimal information exists in foals protect nerve cells against injury.

We are proposing that in sick newborn foals the equilibrium between these steroid hormones is broken and it can impair their ability to survive.

The goal of this project is to investigate the importance of these hormones in newborn foals with different levels of disease. Hormones will be measured during hospitalization, and their level/balance assessed. The association between hormones, blood abnormalities, and likelihood mortality will be determined.

This study has clinical relevance to the equine industry as a better understanding of equine sepsis will improve treatment. We will generate novel information that will benefit the well–being of horses. Results will be valuable in the implementation of novel therapies to restore the hormonal balance. For example, we may consider using progesterone (a mare hormone) to accelerate brain maturation in dummy foals. In other instances we may want to block the production of other hormones. This project will also be positive for the training of students and veterinarians with interest in equine research. We are confident that we can accomplish the goals of this project in the proposed time–frame.

Tanja Hess, Colorado State University – One Year

One Sentence Summary: In an effort to improve the diagnosis, management and treatment of the many horses and ponies who suffer from metabolic disorders related to insulin dysregulation, we will investigate the ability of novel, small, gene-regulating molecules called microRNAs to be used as convenient and accurate biomarkers for equine insulin resistance.

MicroRNAs are small molecules that reduce the ability of genes to express themselves, which, in turn, can limit or alter the function of cells or body tissues. Emerging research in humans and laboratory animals has shown that microRNAs play important roles in regulating genes associated with type II diabetes and metabolic syndrome, and that the levels of specific microRNAs circulating in the bloodstream can serve as potential biomarkers for the diagnosis and prognosis of these diseases. In this study, we apply this new idea to equine insulin resistance, a prevalent pathophysiologic disorder among horses. We hypothesize that horses and who are insulin resistant will have a different circulating microRNA profile than those who are healthy, and will test this hypothesis in two separate herds. Once we identify specific microRNAs which are differentially expressed between insulin resistant and healthy animals, we will then identify candidate genes involved in insulin resistance which may be regulated by microRNA in the horse. As a result, this project has the potential not only to generate new, minimally–invasive biomarkers for the diagnosis, treatment and possible prediction of equine insulin resistance, but also to generate new knowledge about the physiological processes and molecular mechanisms which become dysregulated in horses suffering from insulin resistance, yielding critical clues for the future development of therapeutic interventions.

Insulin resistance is a growing problem among horse populations, and a contributor to such costly and debilitating conditions as obesity and pasture–associated laminitis. While a variety of clinical and field tests are available for the detection of insulin dysregulation, they may be labor intensive, require multiple blood samples, or be imprecise in their ability to assess insulin resistance in individual animals. Identification of a minimally–invasive, single–sample biomarker that allows practitioners to detect and monitor insulin resistance has the potential to advance animal well–being and reduce associated diagnostic and treatment costs to the horse owner. Circulating microRNAs, small molecules involved in gene regulation, have recently been recognized as effective biomarkers to screen for, and even predict, metabolic disease in humans and other species. If this novel technology can be applied to the equine population, it may provide practitioners and horse owners with a new and more convenient tool in the fight against equine metabolic syndrome, and yield important new knowledge about how cellular and physiological processes are regulated in insulin–resistant vs. healthy horses.

Ryan Ferris, Colorado State University – One Year

One Sentence Summary: Understanding the role of bacterial biofilm in conferring protection to the host immune system and antibiotics is crucial to development of therapeutic protocols for treatment of endometritis in mares. (Biolfilm: A matrix which provides a ‘protective” blanket’ for the wall of the uterus.)

Mares with fertility issues may have a chronic bacterial infection in their uterus that is resistant to traditional antibiotic therapy. Bacteria involved in chronic uterine infections may be capable of forming biofilms, a sugar–like matrix consisting of biological polymers that coats bacterial colonies adherent to the wall of the uterus in a ‘protective blanket’. The biofilm matrix is able to provide protection from the natural uterine immune system by altering the movement and function of white blood cells, and preventing antibodies from binding to bacteria. In addition, biofilms confer resistance to antibiotic therapy by reducing the amount of drug that reaches the bacteria and altering metabolism and growth of the bacteria so that antibiotics are not as effective. Ultimately, biofilms produce a population of bacteria that are protected and resistant to antimicrobial agents that are able to propagate and further disperse to other infection sites. Bacteria isolated from the equine uterus are capable of producing biofilms in the laboratory. However, it is unknown if these same bacteria produce a biofilm during natural clinical cases of endometritis. The goals of this proposal are to 1) determine if bacteria are capable of producing a biofilm using luminescent labeled bacteria in a model of infectious endometritis, and 2) evaluate the mare’s immune response to bacteria living within the protection of a biofilm.

Horses with chronic, resistant or repeated bacterial infections are often suspected of harboring biofilm, a carbohydrate–based matrix that coats colonies of bacteria. Biofilms are thought to protect the bacteria from the host immune system and from antimicrobial treatment. Equine–origin bacterial strains are capable of producing a biofilm that is highly resistant to degradation in the laboratory. The purpose of this project is to determine if bacteria isolated from the equine uterus produce a biofilm in the equine uterus and how the host responds to this infection.

The first breakthrough in this area was when it was determined that bacteria in the equine uterus may be associated with a biofilm. The goals of this proposal are to use luciferase producing bacteria to determine the location of bacteria in the uterus for guided biopsy sampling. This will allow the determination of the spatial location and identification of key bacterial sites of colonization and subsequent biofilm formation to determine if the bacteria are on the surface of the endometrium, deep in the glands, or penetrating into the submucosa. Additionally these studies will determine if the bacteria are producing a biofilm as part of the infection, and evaluate the resulting inflammatory response to the bacteria in a biofilm. It is anticipated that this study will provide a better understanding of the role biofilms play in cases of equine infectious endometritis. Future studies, based on results of this pilot study, will focus on evaluating various treatments and how the mare’s immune system is able to help eliminate these infections. Ultimately, these studies will lead to improved therapeutic strategies for management of mares with uterine infections caused by biofilm–producing bacteria.

Thomas Chambers, University of Kentucky – First Year (2 Year Grant)

One Sentence Summary: This project explores the mechanism of equine herpesvirus-1 blockage of an immune defense pathway and its relationship to equine herpesviral myeloencephalopathy, a serious condition affecting horses.

Equine herpesvirus–1 (EHV–1) is a one of the leading causes of respiratory disease in horses. It is an especially worrisome disease because it can produce symptoms including neurological signs known as equine herpesviral myeloencephalopathy (EHM) that could lead to the death of the affected horse. There are vaccines for EHV–1 but control of the disease has been very difficult because, like most herpesviruses, EHV–1 appears to have many ways of avoiding host immune responses. This project explores one way of avoiding the immune response that has not been explored elsewhere.

We have evidence that one of the ways EHV–1 avoids immune responses is by blocking the Type–1 interferon (IFN) response, also known as the IFN–alpha/beta response. The IFN–alpha/beta response is important as a non–specific first line of defense against many virus infections, by making cells more resistant to virus infection. Our preliminary studies show that EHV–1 causes an initial stimulation of IFN–alpha/beta but then blocks its further activity once the virus replication has gone into its late phase. These studies were done using an EHV–1 strain that causes EHM, and we do not know whether the results are applicable to all EHV–1 strains. We propose that one or more factors made by the virus are responsible for blocking the IFN–alpha/beta response, and here we want to find out how that block happens. We also propose that this block happens with the EHV–1 strains that cause EHM but not with the EHV–1 viruses that don’t cause EHM.

Specific aims are:

1. Determine which steps of the IFN–alpha/beta response pathway (sensitization, induction, or amplification) are blocked by EHV–1

2. Determine the relationship between the ability of an EHV–1 strain to cause EHM and its ability to block the IFN–alpha/beta response.

With this knowledge we can potentially design interventions to bolster innate immune responses against EHV–1 and improve control of this disease. This research could eventually lead to treatments for horses infected with EHV–1 that target the viral factors which cause the block in IFN–alpha/beta, with the goal of reducing the severity of disease and the likelihood of EHM.

Margo Macpherson, University of Florida – First Year (2 Year Grant)

One Sentence Summary: The potent anti–inflammatory properties of firocoxib have the potential to significantly inhibit inflammation, and subsequent preterm delivery of foals, from mares with placentitis.

Nearly one–third of all premature births or stillborn deliveries in mares are caused by bacteria moving through the cervix and into the placenta. After infection, the placental tissues become inflamed and cause uterine contraction. Premature contractions cause birth of the foal. Foals born prematurely have a poor chance at survival, so delaying premature delivery is essential to saving a foal’s life. We believe that the key to preventing premature birth of foals from mares with placentitis is by treating mares with drugs to eliminate bacteria, stop inflammation and quiet the uterus.

Our group at the University of Florida treated mares with placentitis using trimethoprim sulfa tablets (antibiotic), pentoxifylline (anti– inflammatory) and Regumate™ (progestin to induce uterine quiescence). Treated mares delivered more live foals (10/12; 83%) when compared to untreated mares (five of five foals dead). Unfortunately, the same treatment protocol used in a clinical setting does not result in this high percentage of live born foals. Theories as to why this treatment protocol fails in some cases include: 1. resistance of bacteria to trimethoprim sulfa, 2. poor ability to diagnose placentitis which causes treatment to be delayed and, 3. uncontrolled inflammation. Research in women has demonstrated that inflammation, with or without bacterial infection, resulted in early delivery of babies. Survival of these babies was poor. Therefore, research efforts have been devoted to early identification and treatment of inflammation in pregnancy. Placentitis in mares closely mimics conditions causing preterm labor in women. It makes perfect sense that our efforts toward improving foal survival from mares with placentitis would involve investigation of drugs that attack inflammation (anti–inflammatory agents).

Our objectives in this work are to examine characteristics of firocoxib, a potent anti–inflammatory drug formulated for use in horses, after it is administered to mares with placentitis. First, we will determine if firocoxib can effectively reduce factors that cause inflammation in placentitis. Second, we will treat mares with placentitis using firocoxib, trimethoprim sulfa and Regumate™ to see if pregnancy is maintained and foal survival improved. We will compare this treatment to a commonly used placentitis treatment of pentoxifylline, trimethoprim sulfa and Regumate™. Our preliminary studies have shown us that firocoxib is found in the fluids of pregnancy (allantoic and amniotic), colostrum and plasma from foals of treated mares. Since firocoxib can penetrate the placenta effectively, we expect that firocoxib will reduce inflammation that occurs after placental infection. Further, we expect that infected mares treated with firocoxib, trimethoprim sulfa and altrenogest will carry pregnancies longer and deliver more live foals than mares administered a more traditional treatment. These objectives fulfill the over–arching goal of our research program which is to improve foal survival after placentitis.

Foals that are delivered prior to the last week of a normal gestation experience high rates of death. Foal death, in turn, exerts a tremendous financial burden on the owner and breeder, both directly, and as reflected in poor produce record for the mare. The single most important cause of abortion and premature delivery in horses is bacterial infection of the placenta (placentitis) which leads to uncontrolled inflammation and premature delivery of a foal. Identification of drugs that are effective for treating mares with placentitis is paramount to improving foal survival. A fundamental step toward making educated therapeutic choices is to determine if specific drugs are performing expected functions, such as resolving inflammation, in mares with placentitis. It is also important to determine if drugs are effective in stalling preterm labor so that the foal has more time to mature, and ideally, survive the infection. Given the high incidence of this condition in equine pregnancy, and the significant financial burden a lost pregnancy imparts, we feel that this work is paramount to the solvency of the equine industry.

Anthony Blikslager, North Carolina State University – First Year (2 Year Grant)

One Sentence Summary: This project will provide an evidence-based approach to the optimal treatment of horses with small intestinal strangulating obstruction in order to reduce endotoxemia and increase survival.

Horses with severe colic require surgery to correct intestinal obstruction, and it is this population of horses that requires the most intensive care to save their lives. Survival rates have continued to increase as equine anesthesia and surgery has progressively improved. This has shifted the emphasis of colic treatment to intensive medical care that is required before and particularly after surgery. There are a number of treatments that have become ‘the standard of care’ for horses with severe colic, including intravenous fluids and anti-inflammatory medications, such as flunixin meglumine (Banamine®). However, just as there have been advances in anesthesia and surgery, there have also been advances in medical therapy. One such advance has been the development of drugs called COX-2 inhibitors, which have an action similar to flunixin, but target inflammation much more specifically. Recent scientific discoveries have shown that the COX enzymes are responsible for beneficial functions, such as digestive and kidney health via COX-1, and detrimental functions, such as pain and inflammation via COX-2. Flunixin inhibits both COX enzymes, so it has benefits to horses with colic (pain relief and reduced inflammation) as well as side effects (stomach ulcers, kidney damage). We have recently discovered a new side effect of flunixin – it slows down repair of the gut because it inhibits COX-1. However, since 2010, an intravenous COX-2 inhibitor has been released on the market specifically for horses called firocoxib (Equioxx®). We have shown in research trials comparing flunixin and firocoxib that while both provided excellent levels of pain relief, flunixin reduced the rate at which the bowel could recover from colic-induced injury, whereas firocoxib allowed the bowel to recover more rapidly. We believe this may be a very important consideration when treating horses with surgical colic. To properly test this, we are proposing a multi-institutional clinical trial in which owners of colic patients going to surgery at NC State University, Michigan State University, or the University of Pennsylvania will have the option of enrolling their horse in a clinical trial in which flunixin or firocoxib will be given. The drug being used will be hidden from the veterinarians (they will know one of two anti-inflammatory drugs are being given). We will also give the drugs to horses in random order. This is all part of running a high level clinical trial, to make sure the results are as meaningful as possible. Clients will be given a small financial incentive to enroll their horses in this trial, and all other aspects of medical and surgical treatment will be kept the same. We will determine if one drug is advantageous over the other by measuring a comprehensive series of clinical measures (e.g., level of shock) as well as blood levels of bacterial toxins and markers of septic shock. The outcome will be a far greater understanding of the effects of both flunixin and firocoxib in horses with surgical colic, which we believe will help clinicians enhance their level of medical care in these critically ill horses.

Colic is the leading cause of death in horses behind old age according to studies performed by the United States Department of Agriculture. The main reason for death in horses with colic is absorption of bacterial toxins from the gut into the bloodstream. This causes shock, which is often difficult to manage. Treatment has greatly improved over the last 20-30-years, but periodically requires re-evaluation to make sure veterinarians are treating with the best possible mediations. One very important drug used to combat colic is the non-steroidal anti-inflammatory drug (NSAID) flunixin (Banamine®) because it is a painkiller as well as reversing some of the shock-causing effects of bacterial toxins. However, it has side effects, including one that we have recently discovered in which flunixin slows down intestinal healing. This paradoxically increases absorption of bacterial toxins. Interestingly, a new NSAID on the market for horses, firocoxib (Equioxx®), provides more targeted treatment of pain and inflammation while limiting side effects, including delayed intestinal repair. To find out the clinical importance of this potentially critical difference in medications, we want to perform a multi-university clinical trial in which flunixin and firocoxib will be compared in horses going to surgery for colic. We will set it up so that use of firocoxib and flunixin will be given in a random order, and without the veterinarian knowing which drug they are using. In this way, we can remove bias and gather clinical information as well as important indicators of the level of shock that will facilitate decision-making on colic treatment in the future.

Anne Wooldridge, Auburn University – First Year (2 Year Grant)

One Sentence Summary: Injectable hydrogel microsphere scaffolds containing endothelial progenitor cells are a potential novel therapy to decrease healing time in distal limb wounds in the horse.

Although the field of stem cell therapy in the horse has made great strides, the fate of injected stem cells (i.e. do the cells stay at the site of injury, stay alive, and cause repair or do they improve healing through other means) is still unknown. Biomaterial scaffolds are engineered materials that provide a framework to ensure that stem cells remain at the site they are administered and optimize the function of the cells. Endothelial progenitor cells (EPCs) are cells that form new blood vessels or repair the lining (endothelium) of existing blood vessels. These cells come from stem cells in the bone marrow, and they circulate in the bloodstream, so obtaining EPCs for analysis and culture only requires a simple blood draw from the horse. The ability of EPCs to form new blood vessels and repair damaged blood vessels makes them very attractive for treatment of conditions where blood flow is reduced. Wounds on the horse’s cannon bones frequently take long periods of time to heal and tend to form proud flesh, partially due to abnormal or reduced blood flow. Our first aim is to culture the equine EPCs within injectable, hydrogel microspheres and test the ability of the cells to form blood vessels from those spheres. We will also optimize our ability to detect the equine EPCs after they have been injected into the horse so that we can determine if they are contributing to new blood vessel formation. The second aim will evaluate healing in a distal limb wound model in the horse. Using 6 adult horses, 2 full thickness wounds (each the size of a quarter) will be created on the front and rear cannon bones (8 wounds per horse) and treated with EPCs alone, EPCs encapsulated in microspheres, microspheres alone, or sham injection. Healing and blood vessel formation will be analyzed and compared between treatments. We expect that the EPCs in microspheres will cause increased blood vessel formation and decreased healing time compared to all other treatments. This proposal is part of an ongoing collaborative effort between the College of Veterinary Medicine and the College of Engineering at Auburn University. Using our interdisciplinary expertise, our goal is to develop new regenerative therapies for horses that leverage the use of progenitor cells combined with engineered biomaterials as scaffolding.

Delayed healing and proud flesh formation involving wounds of the horse’s lower limbs is one of the most common problems facing veterinarians and horse owners, and new effective treatments are needed. Our research group is a combination of equine veterinary specialists and biomedical engineers that are working on developing new therapies for wounds or other diseases that are caused by or associated with poor blood flow. Endothelial progenitor cells (EPCs) are cells that form new blood vessels or repair the lining (endothelium) of existing blood vessels. Combining stem or progenitor cells with engineered biomaterials allows the veterinarian to deliver stem cells directly to the area of interest and keep them in that location for a longer period of time. The cells are more likely to have the ability to heal tissues and form blood vessels if they are not immediately carried away. In addition to a potential new treatment for distal limb wounds, the cell/biomaterial combination therapy could have implications for many different diseases and many different cell types.

Andrew van Eps, University of Queensland – First Year (2 Year Grant)

One Sentence Summary: This study has potential to show the efficacy of a novel pneumatic boot to prevent lamellar changes leading to supporting limb laminitis (SLL) and may provide an effective clinical preventive for SLL.

Research in the past decade has more clearly defined that there are three different types of laminitis: sepsis–associated laminitis (e.g. laminitis in the horse with gastrointestinal disease, pneumonia, or retained placenta), endocrinopathic laminitis (e.g. laminitis in the overweight horse [also called metabolic syndrome] and in the older horse with Cushings disease) and supporting limb laminitis (SLL, the horse with a severe lameness [e.g. fracture or joint infection] on one limb which develops laminitis in the opposite/ supporting limb). Although established experimental models in the past decade have more clearly defined lamellar (synonymous with laminar) events in laminitis associated with sepsis and endocrinopathies, the lack of an appropriate model limited advancement of our understanding of SLL.

The investigators of this proposal have recently been funded by GJCRF for two SLL studies using very different models and experimental techniques; however, results from both studies indicate that lamellar hypoxia (meaning decreased tissue oxygen levels, most likely from decreased lamellar blood flow) is the driving force behind lamellar failure in SLL. Because the two laboratories use different approaches (AVE/DWR using a novel microdialysis catheter placed in the lamellar tissue to obtain lamellar interstitial fluid [fluid in the space between the cells which reflects the activities of the cells] for assessment of energy metabolites and blood flow[ by injecting urea into the interstitial space and measuring how long it takes to clear] and JKB using molecular and biochemistry/histologic imaging techniques in lamellar tissue) to detect lamellar cellular dysregulation/ hypoxia, we can now combine these approaches (and our recent results) to fully assess the efficacy of a therapeutic intervention as a prophylactic treatment for the horse at risk of SLL. The results of the van Eps/Richardson study strongly indicate that it is not the excessive weight bearing but is the lack of movement of the supporting limb which results in lamellar hypoxia; they also demonstrated that pharmacologic intervention most commonly used by veterinarians in attempt to increase lamellar blood flow (acepromazine) not only did not improve lamellar metabolism but actually worsened the parameters assessed. Thus, the culmination of these studies indicates that the only effective treatment may be a dynamic shoe which provides the same motion to the digit as walking while allowing the animal to maintain weight on the supporting limb.

Using a custom “V shoe” designed by the Belknap laboratory as a non–painful cause of decreased weight bearing on one limb (and excessive weight bearing on the opposite limb), we are now well situated to test a novel, practical pneumatic shoe device (which was produced by the SoftRide company at our request and to our specifications) on the supporting limb. In the proposed study, we will first perform short term animal protocols (( 6 hours) using a custom stocks present in the van Eps laboratory in order to: 1) determine the ability of different combinations/ cycles of heel and toe elevations to improve lamellar oxygenation/blood flow (assessing lamellar interstitial fluid obtained from the microdialysis catheters inserted in the lamellae in the front fee), and 2) determine the effect of these movements of the supporting limb digit by the pneumatic shoe on the weight bearing on the opposite limb (due to the fact that the opposite/injured limb may only be able to sustain a moderate degree of weight bearing in the clinical case). Once we have established the best combination and frequency of heel/toe elevations which do not cause a dramatic increase in weight bearing on the opposite limb, we will then perform a longer term study in which we will assess (using the microdialysis technique in the van Eps laboratory and biochemistry techniques on tissue samples in the Belknap lab) the efficacy of the pneumatic shoe using the chosen heel/toe elevation protocol (from the first experiment) in horses with the V shoe on the opposite limb for 96 hours.

We expect to determine the efficacy of the use of a pneumatic device to maintain digital movement of the supporting limb in ameliorating the lamellar cellular dysregulation/ hypoxia that occurs with excessive weight bearing on one limb; we feel it is likely that we will establish a protocol using the pneumatic shoe which will be effective in protecting the lamellae from decreased blood flow/hypoxia. In conclusion, the results from the proposed study will not only further our understanding of SLL, but have a great potential of resulting in the rapid establishment of a commercially viable prophylactic treatment for SLL.

Laminitis been voted the number one priority for equine research by the American Association of Equine Practitioners due to both the high incidence of the disease (annual incidence of 2–7% of horses in recent studies), the severe nature of the disease (high incidence of humane destruction or chronic lameness due to crippling nature) and the lack of effective therapies for treating the disease. In one of the largest studies of the incidence of lameness in the U.S. in recent history, a USDA National Animal Health Monitoring Study published in 2000 of approximately 3000 horse farms in 28 states stated that 13% of these farms reported a case of laminitis in a one year period. Supporting limb laminitis occurs in all breeds, and is particularly devastating due to the much higher mortality rate (50%) compared to the other types of laminitis (e.g. endocrinopathic laminitis). Supporting limb laminitis is perhaps the most familiar form of the disease to the racing industry and general public, being the condition that led to the demise of Kentucky Derby winner Barbaro, in 2007 and more recently of Kentucky Derby contender Intense Holiday in 2014. This highlights the fact that, despite great advances in the treatment of even the most catastrophic limb fractures and infections in adult horses, supporting limb laminitis remains the major cause of treatment failure and euthanasia for humane reasons in these cases. Because the horse at risk of supporting limb laminitis is easily detectable in most cases, an effective preventative treatment strategy would be a significant step forward for the welfare of horses and for the horse industry.

Andrew van Eps, University of Queensland – Second Year

Equine laminitis continues to be a devastating complication to multiple equine diseases. Laminitis is reported to affect 2–7% of horses annually. Due to frequency and severity of the disease, it has been listed as the number one priority for equine research by the members of the American Association of Equine Practitioners (AAEP), and as a priority for the Grayson-Jockey Club Research Foundation, USDA, etc.

From research performed in the past decade it has emerged that there are three distinct major forms of laminitis. One of them is supporting-limb laminitis. Although it has never been proven, supporting limb laminitis is suspected to occur as a result of reduced blood supply to the connection between hoof and bone (the lamellar tissue). It is proposed that horses rely upon regular loading and unloading of the foot in order to move blood (containing nutrients and oxygen) through it. Our research groups (Queensland, U of Penn, and Ohio State), supported by the Grayson-Jockey Club Research Foundation, now have strong preliminary data that supports this theory.

This next step will utilize a protocol that combines real–time monitoring of lamellar tissue blood flow and energy balance with a suitable and humane model of preferential weight bearing on one limb. We seek to confirm the cause of supporting limb laminitis and to test potential therapeutic interventions. It is anticipated that the results of this study will directly guide the design of devices and/or protocols that can be used in the clinical setting to prevent supporting limb laminitis.

An effective preventative strategy would be a significant step forward for the welfare of horses and for the horse industry.

James Belknap, The Ohio State University - Second Year

Sepsis–related laminitis (SRL) is a common type of laminitis which occurs in animals experiencing far large breadth of diseases such as infectious diarrhea (i.e. Salmonella enterocolitis), pneumonia, intestinal compromise from colic (i.e. colon torsion), grain overload, and retained placenta/acute uterine infection post foaling. Although we have discovered some of the central laminar events occurring in SRL (including the same initial inflammation as occurs in organ injury and failure in human sepsis patients), we still have not established the events or signaling that result in laminar failure.

After many decades of frustration with failures of multiple therapies, we now have one therapy, continuous digital hypothermia (CDH), which has proven effective in the laboratory and clinical setting in many horses. We will use advanced biochemistry techniques, combined with two cutting edge research tools including kinome arrays and metabolomics analysis. The results will provide targets for pharmaceutical therapies to either replace or augment CDH, and will also for the first time determine effects of CDH in the normal digit (important to know for any commonly used therapy).

Advancing laminitis therapy will allow more at- risk animals to avoid the crippling consequences of SRL and return to their previous level of function in the equine industry as was witnessed with Paynter.

Robert MacKay, University of Florida - Second Year

Whereas most foals affected by R equi pneumonia used to die with this disease, they now can be treated successfully with erythromycin or similar drugs (e.g., clarithromycin, azithromycin). Treatment typically takes several weeks and recovering foals often are turned out for part of the day. Two important side–effects detract somewhat from the effectiveness of erythromycin: diarrhea and overheating. Diarrhea is typically mild but the apparent inability of treated foals to regulate body heat effectively can sometimes be fatal.

We presume that these deaths are caused by heatstroke. Our theory is that the overheating problem is similar to anhidrosis (nonsweating), in that the ability to sweat is impaired. We have good evidence for this in the case of erythromycin, based upon studies done in preparation for this proposal. This makes the foal unable to efficiently lose body heat, and on hot days, puts it at risk for heatstroke. We have developed a way to measure precisely the ability of horses to sweat. We plan to use this simple test to show that impaired sweating is the cause of overheating not only in foals given erythromycin but also in some foals treated with azithromycin, clarithromycin, and even the exciting new antibiotic, gamithromycin. Some additional studies will be dedicated to trying to sort out how this class of drugs affects sweating.

If we are right, we hope that these results will serve as a wake-up call for all concerned that foals on erythromycin–like drugs including azithromycin, clarithromycin, and gamithromycin are at risk for heatstroke during and even after treatment and they therefore need to be protected by implementation of common–sense heat control measures.

Heather Knych, University of California- Davis - Second Year

Corticosteroids are potent anti-inflammatory agents and as such are commonly used to prevent and treat performance related injuries in equine athletes. Due to their ability to affect performance and potential to allow a horse to compete when it otherwise should not, corticosteroid use is regulated in performance horses. Over the last 2 years, a concerted effort has been made to establish standardized regulatory thresholds and withdrawal times for this class of drugs. To date, recommendations have been made for methylprednisolone acetate, triamcinolone acetonide and betamethasone acetate. The extended withdrawal time for methylprednisolone in particular, has led some veterinarians to seek out alternative corticosteroids, such as isoflupredone acetate; however, there is limited data about the pharmacology of this drug, or on its anti-inflammatory effects in joints.

Data gathered in this study will provide valuable information for the duration of effect of this drug in the horse by measuring up-regulated genes and inflammatory proteins, in both normal and inflamed joints. If, as we have theorized, this drug has a prolonged effect relative to detection times, results from this study may support less frequent administration of intra-articular corticosteroids, which may ultimately prove beneficial to the health of the joint.

Susan Stover, University of California- Davis - Second Year

Evidence indicates that race surfaces affect the likelihood for injuries in racehorses. Race surface design has the potential to significantly reduce injuries because race surfaces affect all racehorses training and racing on a surface. Therefore, race surface design is an attractive factor to manage to reduce racehorse injuries.

Mild and severe injuries to the fetlock are very common. Catastrophic injuries to the fetlock are the greatest cause of death in racehorses, causing about 50% of injury related deaths. Fetlock injuries are due to circumstances that cause the fetlock to extend beyond its normal range of motion (hyperextension). We hypothesize that fetlock hyperextension, and thus related injuries, can be prevented by developing race surfaces that change the way the limb interacts with the surface. Our objective is to determine the characteristics that a race surface should have to prevent fetlock injuries.

It is not economically practical to build the number of race surfaces needed to determine the ideal surface for injury prevention. Thus, a computer modeling and simulation approach is being used because it is a powerful and economical tool for investigation of race surface characteristics on fetlock motion, and thus risk for injury. This approach can be used to assess the effect of surfaces presently installed, as well as those surfaces that do not presently exist. Racehorse fetlock motion will be predicted for a large number of surfaces with different characteristics. Because the predictions are only as good as the computer model, the model will be evaluated for accuracy (validated) by comparing predictions with actual race surface behavior and racehorse fetlock motion that we previously measured simultaneously at two racetracks, one with a dirt surface and one with a synthetic surface. After validation, the model will be used to determine race surface characteristics that prevent fetlock hyperextension. These race surface characteristics can be used as a standard guide.

Chris Kawcak, Colorado State University - Second Year

Osteoarthritis is the most common musculoskeletal problem in horses and leads to lameness, decreased athleticism, and early retirement. Proteins in articular cartilage (glycosaminoglycans, GAGs) are important for articular cartilage structure in that they provide a smooth gliding surface for normal joint mobility. GAG loss is one of the early signs of osteoarthritis. When that occurs, the articular cartilage becomes weaker and is more prone to tearing due to continued joint inflammation. Once articular cartilage becomes damaged it has limited ability to repair itself and therefore, the disease will progress and worsen over time. Because of this, early detection of articular cartilage injury is critical to successful long–term outcomes.

Unfortunately, routine diagnostic methods (radiographs , ultrasound, MRI, CT) all have shortcomings in evaluating articular cartilage, although MRI is currently the preferred imaging method. However, when contrast agents are injected into the joint prior to CT imaging (CCECT), they have the potential to provide far more information about the health of cartilage. In preliminary experiments we have demonstrated that the amount of CCECT contrast in the cartilage is strongly related to the amount of GAG content within articular cartilage in normal joints. Our hypothesis is that CCECT is a safe and useful method to improve early diagnosis of joint disease. We will test this hypothesis by determining GAG concentrations in normal and diseased articular cartilage and comparing those amounts to CCECT attenuation. We will also test the safety of the contracts agent by evaluating articular cartilage samples for toxicity and lastly, compare the CCECT imaging modality to MRI. It is anticipated that in these experiments we will be able to demonstrate that decreasing GAG content is measurable using contrast CT studies allowing for the early detection of cartilage injury using readily available equipment.

The completion of this project will allow for the critical evaluation of CCECT as a method for the detection of early osteoarthritis in horses and will allow for applications of its use in clinical patients. If this method proves to be effective, it can be a useful tool for monitoring future treatment protocols in horses as well as future cartilage research as it offers a way of ante-mortem in depth evaluation of cartilage health.

Laurent Couteil, Purdue University - Second Year

Inflammatory airway disease (IAD) is the second most common cause of poor performance in athletic horses. Inflammation and accumulation of mucus in the airways interferes with the movement of oxygen from the lung into the blood stream, limiting the performance of horses during strenuous exercise. The type of inflammation in the lung varies with the age of the horse. Younger horses most often exhibit airway inflammation characteristic of an allergic reaction with eosinophils and mast cells. In mature horses, airway inflammation usually is marked by neutrophils, which is considered a non-allergic response.

The triggering factors for IAD are not known but studies have implicated exposure to barn dust. On the other hand, studies have also supported a role for bacterial infection of the airways. The levels of airborne irritants horses are exposed to in the barn environment can be measured and airway secretions can be sampled in order to classify the type of airway inflammation present in an individual horse. Combining these techniques will allow examination of the relationship between barn exposure and airway inflammation. In the past, investigations of the role of bacteria in IAD have relied upon time-consuming and relatively insensitive culture of mucus. Currently, DNA sequencing is a highly sensitive method that can be used to identify all the bacteria present in mucus, thereby characterizing the entire airway microbe population, or so-called “microbiome.” In addition to bacteria, viruses may cause airway inflammation. Again, DNA sequencing has improved the ability to detect viruses. This study is designed to explore the possibility that the various types of airway inflammation differ in their impact upon racing performance in horses and are due to different causes. Studying these differences is important to develop targeted prevention and treatment strategies.


There are two Career Development Awards offered through the Foundation this year.

The Storm Cat Career Development Award, inaugurated in 2006, is a $15,000 grant designed as an early boost to an individual considering a career in equine research. It has been underwritten annually by Mrs. Lucy Young Hamilton, a Grayson-Jockey Club Research Foundation board member whose family stood the retired champion stallion Storm Cat at Overbrook Farm.
This year the award winner is:

Aimee Colbath, Colorado State University
Immune Properties of Autogenous and Allogeneic BMDMSCs

The central hypothesis, based on preliminary data and earlier published reports in other species, is that there are no significant differences in immune profiles between autologous and allogeneic BMDMSCs in vitro and in vivo.

Specific aims include : 1)To compare the immunogenic and the mmunosuppressive properties of autologous to allogeneic bone marrow derived mesenchymal stem cells in vitro and identify a mechanistic explanation for their immunosuppressive properties. The hypothesis is that no significant differences exist between autologous and allogeneic BMDMSCs when compared in vitro and the mechanism of action of immunosuppression may be due to the production of PGE2, indoleamine 2,3-dioxygenase (IDO), TGFß, nitric oxide, or reactive oxygen species. 2) To compare the intra-articular response of autologous versus allogeneic bone marrow derived mesechymal stem cells in vivo and determine if responses change with repeated injections. Our hypothesis is that the joints of horses will not respond significantly differently when autologous or allogeneic BMDMSCs are injected once, or repeatedly.

The Elaine Klein Development Award is a new competitive program intended to promote development of promising investigators by providing a one year salary supplement of $15,000. This program is restricted to one award per year and is named in honor of renowned horsewoman, Elaine Klein. The grant is funded by $15,000 donations by the Klein Family Foundation.
The 2015 award winner is:

Sophie H. Bogers, Marion DuPont Scott Equine Medical Center
Stem Cells to Treat Osteoarthritis in Horses

The hypothesis is that three-dimensional spheroid culture of equine bone marrow-derived mesenchymal stem cells (eMSCs) will increase production of anti-inflammatory and immunomodulatory mediators and not pro-inflammatory mediators compared to monolayer culture. Futhermore, that spheroid eMSCs will have the following effects in two in vitro models of osteoarthritis: 1) Increase anti-inflammatory/immunomodulatory mediators 2) Increase gene expression of growth factors 3) Reduce the levels of pro-inflammatory mediators 4) Reduce matrix metalloproteinase enzyme (MMP) gene expression and 5) Reduce tissue inhibitor of MMP (TIMP) expression.

Specific aims include optimizing the anti-inflammatory properties of eMSCs to treat synovitis and osteoarthritis in horses. This study aims include determining whether: 1)Three-dimensional spheroid culture of eMSCs alters the expression of pro- and anti-inflammatory mediators, MMPs and growth factors compared with monolayer culture. 2) To determine if spheroid or monolayer eMSCs reduce the pro-inflammatory cascade in two in vitro models of synovitis: lipopolysaccharide (LPS)-stimulated synoviocytes and LPS-stimulated cartilage cultures.