Grayson-Jockey Club Research Foundation’s board of directors has announced a slate of 19 research projects which the Foundation will fund for a total of $1,478,542 in 2017. The list includes eleven new projects and eight 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 $24.8 million to fund 346 projects
at 43 universities.
From research performed in the past decade it has emerged that there are three distinct major forms of laminitis: laminitis associated with severe systemic disease (sepsis associated); laminitis associated with insulin dysregulation (endocrinopathic laminitis) and laminitis associated with excessive weight bearing on a foot (supporting limb laminitis). Although each form potentially represents a unique disease pathway in terms of the causal mechanisms, all still share the same outcome: weakening of the connections between hoof and bone (the lamellar tissue), irreversible changes to the morphology and mechanics of the foot, and characteristic, debilitating foot pain. By far the most common cause of laminitis is an underlying endocrine disturbance (either equine metabolic syndrome [EMS] or pituitary pars intermedia dysfunction [PPID, “Cushing’s disease”]) that results in insulin dysregulation. An exaggerated insulin response to ingested carbohydrates, often from pasture, initiates a cascade of events in prone horses leading to varying degrees of lamellar injury, lameness and resultant structural change. Repetition of this process occurs over time: the typical chronic endocrinopathic laminitis case suffers recurrent acute bouts of exacerbation, with each episode contributing further to loss of lamellar tissue organization and subsequent gross structural and mechanical derangements of the feet. Despite recent advances in our understanding of the role of insulin in endocrinopathic laminitis, there are still gaps in our knowledge. Furthermore, although we have developed management strategies aimed at controlling the underlying systemic causes of endocrinopathic laminitis, we do not have an effective therapy for horses suffering from an acute bout of endocrinopathic laminitis (whether it be an initial episode or exacerbation in a chronic case).
Our research groups, supported by the Grayson- Jockey Club Research Foundation, have developed continuous digital hypothermia (CDH) as an effective preventative and treatment for another form of the disease - sepsis-associated laminitis - and it has been widely adopted as a method of prophylaxis in severely ill horses in the clinical setting. Our previous studies also demonstrated a dramatic therapeutic effect in an experimental sepsis-related laminitis model even after the onset of lameness: CDH effectively halted progression and prevented lamellar structural failure. There is, however, currently no data on whether CDH is effective against endocrinopathic laminitis, limiting the clinical use of the technique in the most common clinical cases. High blood insulin has been purported to cause laminitis through dysregulation of processes including blood perfusion, metabolism, and cell signaling within the lamellar tissue. There is recent evidence that the growth factor receptor IGF-1R is activated by excess insulin, potentially triggering a cell division response and disruption of normal lamellar cell adhesions. We have strong preliminary data indicating that growth factor signaling is activated within the lamellar tissue in models of both sepsis-related and endocrinopathic laminitis. In addition, our preliminary data shows that digital hypothermia inhibits this growth factor signaling in a sepsis-related laminitis model. We also have preliminary evidence demonstrating the effects of hypothermia in slowing the metabolism of lamellar tissue, a potential protective effect in negative energy balance states due to reduced blood perfusion or excessive metabolic activation of lamellar tissue due to growth factor dysregulation. We hypothesize that CDH will ameliorate laminitis and prevent lamellar structural failure in endocrinopathic (hyperinsulinemic) laminitis. Using are liable model of endocrinopathic laminitis (the euglycemic, hyperinsulinemic clamp [EHC]) and combining our established cutting edge analysis techniques, we will evaluate a range of metabolic and molecular markers of lamellar energy metabolism, perfusion and signaling events in the EHC model for the first time in order to better elucidate the pathophysiology of hyperinsulinemic laminitis. We will also evaluate the effects of CDH on these processes, and test the principle of CDH as a treatment for endocrinopathic laminitis. The presence of a clinically relevant protective effect of CDH in hyperinsulinemic laminitis will be determined by carefully evaluating lamellar structural changes with light and electron microscopy. We anticipate that the proposed study will address the fundamental question of whether CDH is effective and therefore indicated in the management of endocrinopathic laminitis.
Equine herpes virus type 1 (EHV-1) is a virus that infects horses throughout the world, causing lung disease (pneumonia) and, even worse, abortion and a devastating neurologic syndrome called equine herpes myeloencephalopathy (EHM). Horses with EHM frequently suffer from permanent damage to the spinal cord, resulting in weakness, staggering gait and even paralysis. Severely affected horses often have to be humanely put down and those with less severe symptoms never attain their performance potential. Thus, this virus is a big problem for horse owners and trainers alike, not only because of treatment costs and the potential permanent effects on performance, but also because of the strict quarantine procedures and cancellation of events and races required to prevent spread of the virus. In this year alone, there have been 21 reported outbreaks of EHV-1 in various states across the US, which caused cancellation of races, quarantine of stables, and the unfortunate death of some horses. We have no predictive tests to identify which EHV-1 exposed horses are at highest risk for abortion and EHM. Even worse, we have no effective drugs to prevent abortion and EHM from occurring in EHV-1-infected horses. We now know that horses with abortion and EHM suffer from blood clots (thrombi) that occur in blood vessels providing vital nutrients and oxygen to the placenta and spinal cord. When these blood vessels are blocked by clots, the tissue dies because of lack of oxygen and this can result in abortion and EHM. We do not know why some horses with EHV-1 suffer from clots, but recent studies have shown that their blood becomes prone to clotting or hyper-clottable. In our previously published studies, we found that EHV-1 interacts with blood platelets, which then helps to make the blood clot. Importantly, our latest results show that we can stop the virus from causing the blood to become hyper-clottable if we add specific anticoagulants or “blood thinners” to horse blood that has been “treated” with virus in test tubes in the laboratory. In these studies, we tested two blood thinners (unfractionated heparin and low molecular weight heparin) that are currently available to veterinarians for use in clinical practice. We plan to test these two drugs in Aim 1, to determine whether blood samples taken from horses treated with these drugs become resistant to the hyper-clotting effects of EHV-1. With these results, veterinarians can finally develop a treatment strategy to help prevent abortion and paralysis in EHV-1-infected horses. Unfortunately, both heparin drugs must be given by injection into the neck and this can cause painful swelling. In the laboratory, we also tested a newly approved blood thinner for people that is taken by mouth. This drug, called apixaban, has not yet been tested in horses. In Aim 2, we plan to give this new oral blood thinner to healthy horses by mouth and determine whether the drug is absorbed into the bloodstream and if it can prevent EHV-1 from making the blood hyper-clottable. With these results, veterinarians may have access to a new blood-thinning drug to treat horses at risk for abnormal blood clotting due to EHV-1 and other common disorders such as severe colic.
In both Aims, we will not directly infect horses with EHV-1, but will mimic viral infection by combining blood from horses with EHV-1in test tubes.
Aim 1: To test heparin-based blood-thinning drugs for their ability to prevent EHV-1 from making blood hyper-clottable. These drugs are already in use by veterinarians to help stop abnormal blood clot formation in horses with other diseases, but have never been tested for their ability to stop EHV-1 from making blood hyper-clottable. Here we will inject healthy horses with standard doses of unfractionated and low molecular weight heparin and then collect blood from the treated horses to combine with EHV-1 in test tubes in the laboratory. We will then determine whether the treated horses’ blood is resistant to EHV-1-induced blood clot formation.
Aim 2: Here we will test the new oral blood-thinning drug, apixaban, by giving it to healthy horses by mouth and also injecting it into the blood (as a comparison for oral absorption). As for the heparin-treated horses in Aim 1, we will determine whether apixaban treatment prevents EHV-induced blood clot formation. By collecting blood from these horses at various times after giving the drug, we will also measure how much drug gets into the circulation and how long it stays there. This is crucial information required to develop a drug dosage schedule for treating horses with apixaban.
Joint infections can lead to devastating consequences to a horse’s soundness, athletic career or even their lives. Infections mainly occur after a trauma like a wound, but may also be a complication of joint injections or surgery that involves a joint. Bacterial infections in other locations can usually be eliminated with a course of antibiotics. However, when bacteria enter a joint, they interact with joint fluid, and group together in a protein-containing clump that allows them to evade antibiotics. When bacteria live and proliferate within the joint they cause inflammation that can lead to arthritis. Finding new drugs that can fight bacteria, even when clumped, and decrease inflammation will lead to a better prognosis for the horse’s life and career. We recently found that platelet-rich plasma (PRP), a therapy commonly used to help recovery from athletic injuries, is useful in fighting off bacterial infections. The main component of PRP is the platelet, a cell critical for blood clotting. An important additional function of platelets is as an immune cell that produces proteins that help the immune system recognize, fight and clear infection from bacteria and viruses. We hypothesize that the use of the antibacterial portion of PRP in combination with antibiotics will increase the effectiveness of those antibiotics in joint infection and allow successful eradication of the infection.
Late term pregnancy loss or the delivery of non-viable foals produces a substantial emotional and economic burden on horse owners and breeders. One of the most common causes of late term pregnancy loss in horses is caused by bacterial infection of the placenta, costing the Thoroughbred Industry as much as $45 million in 2015. Currently there are no screening tests available which are able to identify mares with placentitis early enough and reliably enough to alter the course of disease. In our laboratory, we aim to develop a screening assay that is affordable, sensitive, and can specifically identify placentitis. Using nuclear magnetic resonance imaging technology to study the metabolome (the set of all metabolites present in a tissue or fluid) of equine allantoic fluid, we have so far identified eight metabolites that vary between normal and infected mares. The objective of this proposal is to confirm these metabolite differences in a larger group of mares and identify differences in vaginal secretion and blood of those same mares. We will compare the onset of alterations in metabolites after experimental infection to changes occurring in conventional tests, such as ultrasound measurement of the uterus and placenta, hormonal testing and measurement of blood inflammatory markers, such as lactate and serum amyloid A, with the goal of detecting specific changes which occur earlier than conventional tests. If successful, plasma or swabs of the vaginal area could each serve as easily-obtained samples for veterinary screening and diagnosis of placentitis in the future.
Kisspeptins are small proteins that are integral to reproductive function and it is our overall objective to define the effect of kisspeptins on equine reproductive function and fertility. In other species, kisspeptins have been shown to integrate processes such as aging, stress, nutritional deprivation, and obesity in puberty and fertility. In women, kisspeptin levels are detectable in blood only during pregnancy, and rise almost 1,000 fold during the first trimester of pregnancy. Abnormally low kisspeptin levels have been associated with compromised pregnancies and pregnancy loss, and are being used by clinicians to identify “high risk” human pregnancies. While great advances are being made in the human field, there has been no work on this area in the horse. One reason for the absence of research activity has been that the scientific community lacked a way to accurately measure kisspeptins from a sample of horse blood. Our laboratory has since developed such an assay and we can demonstrate that kisspeptin is elevated in pregnant mares as compared to mares that are not pregnant. With the development of this crucial assay and the proposed series of experiments, we will be able to obtain insight as to how kisspeptins are involved in equine pregnancy and if they can serve as an indicator for fetal viability and as a biomarker for pregnancy compromise in the horse.
Osteoarthritis (OA), or simply arthritis, is the leading cause of lameness in horses. It is a debilitating disease characterized by progressive inflammation and loss of the normal joint surface, resulting in pain, difficulty getting around, and ultimately euthanasia. Arthritis can be very challenging to treat over the long term. Commonly used therapies often work for short periods of time or only provide partial relief of symptoms. There is a critical need for treatments that specifically target the cause of the problem and cannot only prevent disease from getting worse but even reverse the disease process and help the joint heal with a healthy covering of cartilage over the bone.
A new targeted therapy that is starting to gain attention for a variety of diseases is the injection of bone marrow mononuclear cells (BMNC). BMNC are readily collected by a simple and quick (2 hour) procedure in the laboratory. A sample of bone marrow is collected from the girth area of the horse and processed in the lab to collect the cells. These cells are made up primarily of the immature cells that eventually become blood cells. In their immature state these cells have been used successfully to treat tissues with limited ability to heal (regenerate) and therefore are traditionally associated with difficult to cure diseases (for example in the nervous system, pancreas, and joints). Their regenerative potential ease of collection for immediate injection, make BMNC a promising alternative to existing treatments. Although the mature forms of these blood cells are known to be the actual cause of inflammation and tissue breakdown, some forms of these blood cells can also play a fundamental role in promoting tissue healing. Understanding how a group of cells could have seemingly opposite effects is the focus of our proposal. Macrophages, one of the cell types in BMNC, can either change into to inflammatory (M1) or non–inflammatory (M2) cells in response to their environment. In this proposal we outline how we plan to investigate this relationship between the joint environment (normal and inflamed) and BMNC in cell culture (Aim 1) and following injection into equine joints (normal and inflamed; Aim 2). The knowledge gained from these studies will lead to clinical trials using BMNC to treat inflamed joints in horses. Our long term goal is to use this information to develop BMNC as a treatment strategy for use in horses (and possibly people, dogs and cats) to provide lasting relief to joint pain and inflammation.
The equine injury database (EID) that was established by The Jockey Club in 2008, has enabled us to estimate the frequency of fatal equine injury during racing from the majority of racetracks in North America. These figures have been calculated for different types of racehorses (e.g. males vs. females; two-year olds vs. older horses) and different racing conditions (e.g. dirt vs. turf vs. synthetic surfaces; different race distances). Additionally in the last 3-years we have worked with the EID data to identify many risk factors for fatal injury and fracture in horses racing in North America. Our aim is to be able to predict, with some certainty, which horses are at significantly higher risk of fatal injury. To date the models we have produced have not been of sufficient predictive ability to enable firm recommendations to be produced. One of the reasons for this is the broad, non-specific nature of the outcomes used so far. [We had to use broad definitions in initial analyses so that we had sufficient statistical power to enable us to identify risk factors]. We are now in a position, with 8-years of data being available (from early next year), to refine our outcome definitions to specific reasons for fatal injury or euthanasia that refer to fracture of individual bones or indeed sudden death. The study being proposed as part of further analyses of these data will help move toward much greater certainty as to the factors that are most strongly associated with different causes of fatal injury and also help to identify how predictable fatal injury may be for individual horses taking part in different types of race. This will enable potential intervention before the fatal injury occurs.
The output from this work will be a set of risk profiles for particular types of horse in different types of race. This will help racehorse trainers, track managers, racing secretaries.
The goal of this research is to improve measures for controlling disease caused by equine herpesvirus types 1 (EHV-1). This project will generate crucial knowledge about how the horse’s immune system responds to EHV-1 vaccination and infection, identifying key components of the virus that stimulate immunity. The focus of this project is on immune responses made by the type of white blood cells known as cytotoxic T lymphocytes (CTL), which kill virus infected cells. CTL are considered to be important contributors to effective immunity against herpesviruses. However, measuring CTL responses is difficult, and the individual proteins of EHV-1 that initiate CTL responses are not well defined. This lack of technology and knowledge makes evaluation of natural infections or the effectiveness of vaccines in the horse very difficult and in some cases impossible.
We have assembled an international team of veterinary scientists with expertise in equine immunology and virology to address this problem. Our laboratories have developed sophisticated assays and techniques that enable us to assess CTL responses toEHV-1 in ways that have not previously been possible in the horse. In addition, Cornell University maintains a herd of specially bred Thoroughbred horses that are available for this research. The key immune system genes required for CTL responses (Major Histocompatibility Complex [MHC] genes) have been thoroughly characterized in these horses, which include the donor horse for the equine genome sequence (the Thoroughbred mare Twilight). These horses are a unique resource in equine research that allow us to conduct experiments on CTL responses that cannot be undertaken at any other location worldwide with the degree of precision proposed in this application. The MHC genes of the Thoroughbred horses to be studied in this project are commonly found in Thoroughbreds throughout the world and in many other horse breeds. Thus this research will be of general relevance to all horses.
The current proposal has three goals: 1) To determine if the current modified live virus (MLV) vaccine against EHV-1 induces protective cytotoxic T lymphocyte (CTL) responses. Surprisingly, although CTL responses are believed to be a critical component of a protective anti-EHV-1 response induced by vaccination, there is little published evidence on the ability of the MLV vaccine to induce CTL responses in vaccinatedhorses.2) To identify the individual proteins of EHV-1 that are most important for inducing protective CTL responses, and3) To identify the immune system molecules of the horse which present the EHV-1 antigens to CTL.
Achieving goals 2 and 3 will advance our understanding of how horse cytotoxic T lymphocytes provide immune protection against EHV-1.
This state-of-the-art research in equine immunology and virology will provide important new information and assays that can be used to evaluate current vaccines against equine herpesvirus type 1 and to design new and more effective vaccines in the future.
Our long term goal is to elucidate the reason(s) exercise-induced pulmonary hemorrhage (EIPH) occurs during high speed equine exercise. The fundamental cause is that the smallest blood vessels in the lung (capillaries) break when horses gallop fast. The question we seek to answer is, "Why does this happen?" Until now the emphasis in this matter have been on the blood pressure responses of the pulmonary artery to this type of exercise. This artery takes the blood from the right side of the heart to the lungs. It has gradually been accepted that the development of EIPH is not compatible with this approach, and that breaking of lung capillaries is more mechanistically compatible with increased pressures in the left atrium (LA) which is the heart chamber into which the blood flows after leaving the lungs. Identifying the basic cause of EIPH must be a high priority if better treatments for the condition are to be found. Because human athletes with too much blood in their left atria (this is called volume overload) can also experience mild EIPH and because athletic horses can naturally increase their blood volume by up to 50% when they compete at the gallop, we will investigate whether this huge increase in circulating blood volumes is more than the LA can handle and so causes 3- to 10-fold increases in the LA pressure from resting levels. Horses'''''''' LA pressures when galloping hard are much higher than those recorded in trained human athletes exercising at comparable intensities. We aim to test the theory that the occurrence of EIPH and its severity in horses is directly related to increases in LA pressures during exercise; i.e., we believe that the greater the LA pressure, the more marked the EIPH. We will do this by measuring the LA pressure in 2 ways -- 1. Measuring the pressure in smaller pulmonary arteries (known as wedge pressure), and 2. Measuring the pressure in the left heart chamber (the left ventricle) into which the LA empties. Both measurements are recognized as valid in direct measures of LA pressure. We will further assess the link between LA pressure and EIPH by temporarily lowering this pressure during exercise by reducing the circulating blood volume and also by administering 0.5 mg/kg furosemide IV 4 hours before exercise. Furosemide (Lasix) is the only scientifically recognized drug that can significantly reduce the severity of EIPH but how it works is not clear. We will use 12 horses in this study and each will complete a number of strenuous exercise tests on the treadmill with normal and reduced blood volumes and also following Lasix administration. The wedge pressure and the left ventricular filling pressure will be measured throughout all these exercise tests. Following completion of every test each horse will be evaluated for the presence of EIPH and its severity by examining the trachea with and endoscope and also by infusing a small volume of sterile fluid in to the lower airways and then collecting that fluid and counting the number of red blood cells in it. (This is a very safe procedure that is widely practiced on animals and people.)The results of the post-exercise assessment for degree of EIPH will be related to the LA blood pressures determined from the wedge pressure and LV pressure measurements. We expect to find that lower LA pressures are equated with reductions in the severity of EIPH and that, similarly, if LA pressure is increased the EIPH is worse. With this relationship clearly established, we and others will be able to turn their attention to identifying treatments that can effectively lower the LA pressure response to exercise without interfering with the ability of the horse to perform. Then, and only then, will the veterinary profession be in a position to truly tackle the challenge of preventing EIPH, and this will be because the mechanisms responsible for EIPH will finally be well understood.
Our long-term goal is to improve survival in horses with large colon volvulus or twisted gut. Large colon volvulus occurs when the bowel twists 360 degrees or more on its axis, cutting off the blood supply to the intestine. Despite surgical treatment only about 60% of horses live because the intestine is damaged so quickly by the twist. Ethyl pyruvate is an antioxidant that improves tissue healing and survival in research models of disease where the blood supply to tissues is blocked. We studied ethyl pyruvate in research horses to show that ethyl pyruvate was safe. We also studied ethyl pyruvate in research horses treated with toxins to show that horses got less sick and recovered faster. The next logical step is to study ethyl pyruvate in horses with naturally occurring disease, like colic. Because ethyl pyruvate improves tissue healing after the blood supply is blocked, we want to see if ethyl pyruvate will lessen colon injury and improve survival in horses with large colon volvulus. We will perform a clinical trial using client owned horses admitted to the hospital for treatment of large colon volvulus at 5 different horse hospitals including Michigan, The Ohio, and North Carolina State University Veterinary Medical Centers, University of Pennsylvania-New Bolton Center, and Rood and Riddle Equine Hospital. After surgery and with owners'' consent, half the horses will receive ethyl pyruvate and half will receive saline. We will perform blood tests, monitor clinical signs, postoperative problems and survival to determine if ethyl pyruvate is effective. The results of our proposed work may offer a new treatment for horses with large colon volvulus that reduces colon damage and improves horse survival.
Osteoarthritis is a very common cause of lameness in horses. Traditionally thought of as a disease of articular cartilage, it has been shown that the soft tissue lining of the joint, the synovial membrane, is an important contributor to the protracted inflammation that accompanies the disease. The cells of the synovial membrane produce a wide variety of substances which contribute to the progression or resolution of the characteristic imbalanced and destructive environment in a joint with incipient or established osteoarthritis. Among these substances are products of fatty acids that reside in the cell membranes of the synovial tissue called oxylipids. These metabolic products of cell membrane metabolism can enhance or diminish inflammation, depending on the specific blend of oxylipids released. It has been shown that for a number of diseases, the makeup of the membrane of cells involved in an inflammatory process materially influences the overall profile of oxylipids in the tissue environment.
It is increasingly evident that the specific composition of dietary fatty acids can lead to changes in the makeup of cell membranes, thereby favorably or unfavorably influencing the array of oxylipids produced by the cells. Specifically, omega-3 fatty acids, such as alpha linolenic acid, can be preferentially incorporated into cell membranes leading to an oxylipid profile that favors resolution of inflammation, compared to the omega-6 fatty acids, such as arachidonic acid, that are generally associated with inflammation. Thus, appropriate modification the omega-6: omega-3 ratio in tissues can result in considerable health benefits. Based on the relatively sparse literature on the topic and our preliminary experiments, we hypothesize that synovial cells exposed to omega-3 fatty acids will preferentially incorporate them into their cell membranes. Subsequently, when an inflammatory stimulus is encountered, the overall biological effects of the oxylipids generated will favor an anti-inflammatory or resolving state, thereby diminishing the duration and severity of inflammation that is a hallmark of osteoarthritis.
We will test this hypothesis by exposing synovial cells in culture to a number of omega-3 and omega-6 fatty acids. Following this exposure, we will use established methods to quantify the relative amounts of a number of key fatty acids in their membranes. Once doses that result in optimal incorporation are established, we will repeat these incubations and then stimulate the cells with a molecule that is known to be central in establishing the chronic inflammatory events in osteoarthritis. Subsequently, we will identify and quantify the oxylipids produced by the cells and assess if the overall mix of oxylipids is favoring inflammation or its resolution. In addition, we will quantify the expression of genes of known non-lipid pro-and anti-inflammatory molecules to see if an omega-3 supplemented oxylipid environment influences their synthesis. We anticipate that omega-3 incorporation will have favorable effects in this model. These studies are an important first step in establishing science-based nutritional recommendations that can be used to optimize the omega-6: omega-3 ratio in the joint tissues of horses. The long-term goal of this research is to reduce the need for conventional symptomatic therapies for osteoarthritis in horses by generating information that will be useful in establishing ideal levels of omega-3 fatty acids in the ration that would serve as a means to treat and, perhaps more importantly, prevent or significantly delay the onset of the disease in the equine athlete.
Decrease in racing performance can result from multiple causes but airway obstruction is a prevalent cause in horses. Intermittent dorsal displacement of the soft palate (DDSP) causes an upper–airway obstruction that is associated with an abnormal sound (i.e. gurgling), and it has been recognized as a common cause of poor performance in racehorses with a reported prevalence of 10–20%..Management of DDSP remains controversial because of the lack of a clear cause of this disorder, resulting in several treatment strategies each directed toward one of the proposed causes.
In this study we are targeting the neuromuscular causes of DDSP associated with deficit of the TH muscles. The current standard treatment (laryngeal tie–forward) was developed to treat this change in laryngeal position. Success rates approaching 80+% have been reported for this treatment strategy, with minimal detrimental effects to the horse and return to race after surgery ranging 64–83%. As described by Cheetham et al (2008), before diagnosis and surgical treatment DDSP horses face a significant decrease in their racing performance and consequent earning; many horses were diagnosed while in training and resulted not able to start their racing carrier due to this disease. Moreover, despite surgery, up to 34% of DDSP horses did not return to racing.
Preliminary findings suggest a potential rationale for a new management approach of horses with DDSP, by increasing the muscle strength and resistance to fatigue through specific training protocols and/or functional electrical stimulation (FES) of the TH muscles. A better knowledge on DDSP causes would give the basis for new treatment options in affected horses, but also improve the chance of early diagnosis and prophylactic training methods to prevent or reduce the occurrence of DDSP in young horses starting training.
Infectious diseases are leading causes of disease and death for all horses on a worldwide basis. Streptococcus equi subspecies equi (SE) is a bacterium that causes a highly contagious disease commonly known as Strangles. Strangles remains the most frequently diagnosed infectious disease of horses. Currently available vaccines against Strangles have limitations in safety (i.e., can cause disease or complications) and efficacy (i.e., they don’t always protect horses well). Consequently, great need exists for novel approaches to protect against SE, the bacterium that causes Strangles. Polysaccharides are chains of sugar molecules that may be found on the surface of bacteria (and other cells). Bacterial–specific surface polysaccharides have been demonstrated to be highly effective as vaccines against multiple bacterial pathogens, and the laboratory of Gerald Pier (co–Principal Investigator) at Harvard Medical School has discovered a conserved one, termed poly–N–acetyl glucosamine (PNAG), that can be targeted to generate protective immunity. Naturally–occurring PNAG does not stimulate the immune system to produce antibodies that can protect against infection. By chemically altering the PNAG molecule, however, a form of PNAG can be produced that elicits antibodies against PNAG and can provide protection against the many bacteria that have PNAG on their surfaces. Our laboratories (Cohen and Pier) have generated compelling preliminary data regarding the efficacy of an anti–PNAG vaccine. This project will extend our exciting preliminary data by evaluating the safety, immunogenicity, and efficacy of a PNAG to protect horses against Strangles. We expect to demonstrate that the PNAG vaccine does not cause serious adverse effects, induces detectable antibodies in serum and nasal wash fluid that recognize PNAG on the surface of SE, and that these antibodies in serum and nasal wash fluid will significantly increase killing of SE by immune cells of horses. We further expect to demonstrate that the PNAG vaccine protects horses against experimental infection with SE. A safe and effective vaccine against SE would be of great benefit to the health and welfare of horses and to the equine industry.
The standard treatment for pneumonia in foals caused by the bacterium Rhodococcus equi has been combination therapy using one of the macrolide antibiotics (erythromycin, clarithromycin, or azithromycin) with the antibiotic rifampin. Widespread resistance to these drugs is a major emerging problem facing the horse-breeding industry, with resistant isolates being cultured from up to 40% of affected foals at some farms. Foals infected with such resistant isolates are more likely to die than foals infected with susceptible isolates. We have recently identified the genetic mechanisms of macrolide and rifampin resistance in R. equi. The logical long-term approach to prevent expansion and spread of resistant R. equi would be to decrease widespread use of macrolides and rifampin in foals. However, strategies aimed at decreasing the use of these drugs will only be effective if carrying the genes that cause resistance alter the ability of R. equi to grow and survive (termed fitness), thereby conferring a competitive disadvantage to reproduce in the absence of antimicrobial selection pressure. Given that R. equi survives in the environment and has the ability to replicate in soil under suitable environmental conditions, the proportion of resistant R. equi would not be expected to decrease over time if its ability to survive is preserved or if virulence (the ability to cause disease) is enhanced. Therefore, it is of paramount importance to understand the effects of carrying macrolide or rifampin resistance mechanisms on overall fitness and virulence of R. equi. The overall objective of the proposed research is to determine the effects of carrying macrolide or rifampin resistance mechanisms on the fitness of R. equi and its ability to cause disease.
Our preliminary data indicate that resistant isolates of R. equi have decreased fitness compared to susceptible isolates as assessed by in vitro growth. In addition, resistant isolates have impaired ability to survive in soil. All the bacterial strains required are already available and all the techniques and methods proposed in this application have already been optimized and are applied routinely in our laboratories. We are, therefore, in an ideal position to successfully complete the research proposed in this application. After completion of this work, we will have determined if the determinant conferring macrolide resistance or mutations conferring rifampin resistance affect: 1) in vitro growth of R. equi; 2) the ability of R. equi to survive in soil at various temperatures; and, 3) the ability of R. equi to cause disease, both in the presence or absence of antimicrobial selection pressure. In addition, the proposed work will characterize how long these resistance mechanisms persist in R. equi in the absence of antimicrobial selection pressure. These are the first important steps in understanding the ecology and epidemiology of macrolide-resistance on horse farms and in developing strategies to prevent antimicrobial resistance. After completing these experiments, it will be possible to develop models to study eradication of macrolide-resistant R. equi. These models will be used to monitor emergence or disappearance of macrolide resistance in the environment of horse farms in response to various interventions and to devise strategies to control expansion of the problem of antibiotic resistance.
Pain is a debilitating factor in diseases such as laminitis (foot inflammation and pain) and osteoarthritis (joint inflammation and pain), and its effective control is one of the greatest challenges in equine clinical practice. We have preliminary in vitro data showing that t-TUCB is a potent inhibitor of equine sEH and in vivo data showing that 1 mg/kg t-TUCB, but not lower doses, produces significant pain relief using the joint pain model proposed here. Preliminary clinical studies in nine horses with laminitis and one horse with osteoarthritis suggest that dual COX/sEH inhibition with phenylbutazone/t-TUCB combination may result in additive/ synergistic responses, as previously found in pain models in rats. The significance of the proposed research is that it is expected to provide new knowledge on the modulation of different branches of fatty acid metabolism in horses that could be used for the design of clinical trials and for developing novel approaches for pain management in these animals. This has high potential to impact all horses by decreasing the negative effect that poorly controlled pain has on equine health and productivity, which in turn decreases the economic burden for the equine industry.
There are certainly better and worse training schedules and better and worse racetrack surfaces that are used to prevent, recover from, and resume training after, an injury or bone fracture. Our goal is to investigate and determine training programs and race surface properties that keep horses healthy and prevent bone fractures.
To prevent bone fractures, we must understand how they develop in our racehorses. We know that bone fractures rarely occur from a single bad step in athletes. Instead, fractures are the result of multiple factors that affect the accumulation of bone damage over racehorses’ careers. These factors are related to the distances that racehorses train and race, the speed of the horse during training and racing, and the stiffness of the race surface that training and racing occur on. In addition because damaged bone can heal given enough time, the training program--- the weekly timing of works and races---plays a large role in racehorses’ susceptibility to injury and bone fracture. Further, these factors interact with, and affect, each other over time.
In order to understand the impact of training schedules and race surface properties on racehorses’ susceptibility to injury over time, many things must be factored in--- the complex interaction of gait, speed, distance, and timing of exercise; alterations of limb loading due to race surface properties; and the time course of removal of damaged bone and the inherent time lag in the replacement by healthy new bone.. The complexity of the relationships require computer modeling approaches that take into account the time sensitive nature and interaction of the events to understand the resultant risk for injury.
We will use a computer model that simulates micro damage accumulation with training and racing, and repair of damaged bone with time. The model will be developed based on lifetime race and timed–work records of 60 racehorses that died during training or racing. The sesamoid bones at the back of the fetlock will be used to measure the actual bone damage and resulting repair from each of the same 60 racehorses to ensure or adjust the computer model to reflect what actually occurred in the racehorses. Subsequently (Part II), we will further calibrate and validate the model using lifetime race and timed–work records of 392 Thoroughbred racehorses; 98 case racehorses that died because of a proximal sesamoid bone fracture and 294 control Thoroughbred racehorses that did not die during their career. Lastly, we will use the validated model to determine combinations of training programs and race surfaces that prevent injury and combinations that put racehorses at high risk for injury.
The potential usefulness of the modeling approach is three–fold. Models can be used to evaluate existing training programs and race surface mechanical properties for risk for injury. Training programs and race surfaces that do not currently exist can be screened in a cost– and time–effective manner to design improved training programs and optimal race surface properties. Exercise and race venue histories of race entrants could be screened before races to help identify horses at high risk for catastrophic injury during the upcoming race.
Skeletal injuries take an insidious, but large and deleterious toll on equine and jockey welfare, and industry economics. Attrition due to musculoskeletal injuries is the largest cause of wastage in racing. Injuries are the cause of 83% and 71% of deaths in Thoroughbred and Quarter Horse racehorses, respectively Non–fatal injuries are the most common reason for exit of horses from training. Injuries resulting in an inability to train for more than 5 days occur 29 times more frequently than fatal injuries. It has been estimated that )950 million dollars in horse costs alone are lost in the United States each year due to the loss of 3% of horses from racing each month.
Fetlock injuries are the greatest cause of fatal musculoskeletal injuries in Thoroughbreds and 34–40% of deaths due to musculoskeletal injuries in Quarter Horses. Proximal sesamoid bone fractures are the most common cause of fetlock injury. Further, 36% of non–fatal injuries affected the proximal sesamoid bones (7%) or fetlock support structures (29%).
Catastrophic injuries of horses are the major cause of jockey falls and injuries, Jockeys are 162 times more likely to fall and 171 times more likely to be injured when riding a horse that died in a race than otherwise. Worker compensation claims were $6.5 million dollars over a 5 year period in California alone. When horses sustained a fatal fetlock injury during a race, jockeys fell 22% of the time, and were injured 13% of the time. Consequently, fetlock injuries take a large toll on equine and jockey welfare, and industry economics.
Pneumonia caused by a bacterium called Rhodococcus equi (R. equi) is an important cause of illness and death in foals. Because no vaccine is available and because many foals are unable to fight infection on their own, control of R. equi pneumonia is based on treatment of foals with a specific class of antimicrobials (named macrolides). With extensive, repetitive use of macrolides, these bacteria become resistant to this group of antimicrobials. The lack of alternative antimicrobials that would efficiently kill R. equi, and the low likelihood of novel antimicrobial development indicate that new approaches to control are needed. The specter of antimicrobial resistance is a major problem for human and veterinary medicine.
We propose to use a self–cure strategy (named host–directed, where the foal is the host) to reduce the occurrence and mortality of R. equi pneumonia, and to decrease the chances of development of antimicrobial resistance. Foals appear to be infected with R. equi during the first weeks after birth, a period which precedes complete development of adaptive immunity (the type of immune response made of specialized cells, such as antibody–producing cells). Foals are born with a functional innate immune system, comprised of cells which respond to all invading organisms. Stimulation of innate immunity, therefore, is a logical approach for protecting foals. Some molecules located on the surface of innate immune cells, called Toll–like receptors or TLRs, are important mediators of innate immunity. They act primarily through secretion of substances called cytokines. PUL–042 is a product that combines two substances that can stimulate innate immunity via TLRs directly that can be inhaled into the lungs to prevent bacterial pneumonia, and we plan to investigate its use as a method of host–directed control of R. equi pneumonia. We propose to use this product via nebulization, which is the administration of mist inhaled into the lungs using a nebulizer (such as those used for asthma patients). The objectives of our proposal are to better understand the how the infection of R. equi occurs in lung cells of both horses and foals (Year 1), and to evaluate if nebulized PUL–042 is safe and if it increases the capacity of equine lung cells to kill R. equi (Year 2).
Our long–term goal is to demonstrate that PUL–042 nebulization of foals could be used at equine breeding farms to prevent pneumonia caused by R. equi. Once optimized, this approach could replace antimicrobial prophylaxis (a strategy in which antimicrobials are used to prevent the disease by treating the foals that are not sick). Furthermore, it might serve as a supplementary treatment to antimicrobials therapy in foals with R. equi pneumonia, which could potentially shorten treatment time and improve outcomes. The current standard for prevention of R. equi pneumonia at farms is transfusion of R. equi hyperimmune plasma. This approach has many disadvantages, such as being expensive, labor–intensive, and causing undesired side–effects in foals. Nebulization of PUL–042 could replace plasma transfusion. Last, by stimulating innate immune responses of the newborn foal, a reduction of other bacterial respiratory infections (e.g., the bacterium that causes strangles) might occur, as well as improved responses of foals to vaccines. Results of this project will have tremendous impact for the equine industry, specifically at horse– breeding farms where R. equi pneumonia can occur in 20–40% of the foal crop.
The sequencing of the horse genome in 2009 resulted in significant advancements in the field of equine genetics. Currently, there are 32 equine traits, including genetic diseases and coat colors, for which a genetic test is available. Of these, 22 were discovered since the horse genome was made publically available. The next step forward in the study of complex equine diseases is to define gene expression (which genes are making proteins in which tissues and how much protein is being made) and gene regulation (which genes are turned “on” or “off” in each tissue). Similar to other species, the horse has approximately 21,000 genes in its genome, which make up only a very small portion of the horses’ entire 2.7 billion bases of DNA. While it is known that changes in coding DNA (i.e., DNA that creates protein) may lead to disease, what is not known is the consequence of changes in non-coding DNA (i.e. DNA between coding regions). The vast majority of the equine genome consists of this non-coding DNA, whose function remains a mystery. It is apparent, however, that non-coding regions of DNA can bind proteins that will turn a gene “on” in one tissue, lung for example, while that same gene is “off” in another tissue, such as liver. The future of equine disease discovery lies in unraveling the location and function of these important DNA sites and determining, on a tissue-specific basis, how these noncoding regions of the genome regulate gene expression. In human research investigating complex genetic disease, as many as 93% of studies attempting to link a disease with a region in the genome identify an associated region within non-coding DNA. Similarly, many complex genetic diseases in the horse map to non-coding regions of the genome. The study proposed here parallels advances in human genomics and puts equine research on the cutting edge of disease discovery. This study will provide the foundation for not only equine genetic research but also research into the function and dysfunction of many specific equine disorders from laminitis to typing-up to heaves, among many others.
For the first time, we have the available tools and expertise to unravel these DNA and protein interactions that regulate gene expression. Our goal is to provide an open-access resource, a genomic roadmap outlining the location and function of non-coding “on/off switches,” for all researchers. Development of this roadmap is as important to the equine industry today as the sequencing of the equine genome was in 2009. In Years 1-2, we will characterize tissue-specific gene expression across 8 tissues (skeletal muscle, laminae, liver, ovary or testis, cerebral cortex, lung, spleen and heart) in one male and one female horse using RNAsequencing and identify tissue-specific “on/off switches” using ChIP-sequencing. This data will be linked to the underlying DNA sequence. In Years 3-4, we will extend our studies to include the location of additional “off switches” using CTCF-sequencing, as well as the discovery of important regulatory regions using a technique called DNAse-I hypersensitivity assays. While the initial analyses will be performed on 8 tissues, we will archive )50 tissues from each horse that will be available for future studies. Data will be publically accessible in existing genome browsers. Given the importance of this work, funding to cover sample collections has been secured through USDA NRSP-8, National Animal Genome Research Program equine coordinator’s funds ($15,000).
Genetic studies have been performed for many important complex diseases, including fracture risk and tying-up in Thoroughbred racehorses, recurrent airway obstruction (“heaves”), osteochondrosis dissecans (OCD), navicular disease, and recurrent uveitis (“moon blindness”), among many others. Despite the results of these studies, which have successfully identified locations in the genome that may be associated with a specific disease, functional genetic mutations have not been discovered in any genes within these regions to date. For example, a region on chromosome 13 was associated with heaves in Warmbloods, but no mutations were identified in neighboring protein-coding genes. These genetic associations may indicate that a non-coding region of the genome is contributing to development of clinical disease. Additionally, many other active areas of equine research, including laminitis, immune response to infection, reproductive health, tendon injuries, and inflammatory conditions use gene expression analyses to identify biomarkers of disease. A publically available tissue-specific reference for all DNA-protein interactions in the horse would provide evidence that these non-coding regions play an important role in gene expression. In human medicine, genetic tools are used to analyze an individual’s DNA sequence, gene expression and DNA-protein interactions, thereby focusing on disease prevention and early diagnostics. Our long-term goal is to move towards this type of personalized medicine in the horse. This would allow veterinarians to screen horses and optimize treatment recommendations for the prevention of developmental disorders as well as provide specific targeted therapies for a variety of diseases based on gene expression changes in a particular individual. In addition to unraveling disease mechanisms, the DNA-protein interactions identified in this proposal would uncover new targets for biomarkers, prevention and treatment of disease.
Latency is a hallmark mechanism of all herpesviruses and is best described as a dormant, reclusive state of an infection. Equid Herpesvirus–1 (EHV–1) is the cause of respiratory disease in horses, but also leads to devastating outbreaks of myeloencephalopathy and late term abortions. A horse that is suffering from an acute infection with EHV–1 will simultaneously develop a latent infection, specifically in the trigeminal ganglion or in lymph nodes of the respiratory tract. While the horse overcomes the acute infection within 2 – 3 weeks, the silent or latent stage of infection is permanent. Latency is often compared to hibernation in mammals which illustrates that specific signals, yet unidentified, are existing that can reactivate virus, resulting in a return to the respiratory tract and in a spurious replication and shedding of virus into the environment and into other horses. This scenario happened at the National Cutting Horse Association’s Western National Championships in Ogden, Utah in 2011, and resulted in a multi–state outbreak. Many horses were euthanized, barns were quarantined and financial losses were significant. Yet, we don’t have a good understanding of what causes viral reactivation or are there strategies on how to intervene other than we realized that an outbreak can start with reactivation of virus in a single animal. EHV–1 latency research is difficult as latent–infected tissue is more likely inaccessible, and tissues collected during post mortem exam may or may not contain latent virus, and if so, there will be no information on the time between infection and the moment of sample analysis. Here we got access to a unique tissue sample collection from horses that were purpose–infected with EHV–1 and euthanized 7 weeks after infection. A time interval of 7 weeks should allow latency to develop. Testing for latency has so far focused on detection of viral DNA. As latent virus does not fully replicates but we assume it produces a stop signal to stop replication, we believe this stop signal, an RNA transcript, can be quantified in analogy to procedure in other herpesviruses. In human HSV–1 latency the quantity of stop signals correlates with depth of latency. The sample collection from the purpose–infected horses will serve as a positive control, and we will determine the amount of this product (a.k.a. stop signal) in relation to viral genome copies in a cell and to cellular transcriptional activity. Besides the results we will also screen other neural tissues and lymph nodes for latency to identify alternative sites in horses. However, the stop signal quantity in these tissues should be high in many samples, as latency has been just recently established, while in samples collected from random tissue specimen from abattoir horses we expect the stop signal amount to be very diverse and depending on the interval between infection and collecting the sample. This will be the first attempt to show different stages of latency based on the quantity of specific latency marker mRNA and it will show the importance of this marker in staging latency. This will be the first step towards identification of more and other host or virus–derived factors that control latency at the cellular level, and results will initiate more research on how to intervene or how to rebalance between deep hibernation and reactivation. In addition, prevalence data of latency in the general horse population will be answered, and if latency is detected at one tissue site, what are the chances that other sites are latent–infected, too, or in other words whether there is random or ranked latency in horses with naturally occurring latency.
The EHV–1 outbreak at the National Cutting Horse Association’s Western National Championships in Ogden, Utah in 2011, which resulted in a multi–state outbreak and many horses were euthanized due to serious neurological disease, barns were quarantined and travel restrictions were in place for a number of weeks, provides a dramatic example of the disease capacity of this virus. Important to understand is the fact that an outbreak typically starts with a single horse where dormant virus becomes re-activated, is returning to the respiratory tract and is being shed into the environment and into other horses. Infections with EHV–1 can occur early in life and immunity is short–lived, which implies that re–infection with same or similar virus can occur. Already with the first infection the dormant or latent stage is induced. Hence, we will not be able to prevent latency establishment in the horse population; however, with this research proposal we will study virus activity during this dormant or hibernating stage, which will hopefully allow us to find ways to counterbalance awakening activities of this virus when in latency. Vaccination, for example, has been the strategy of choice to prevent flare–ups of shingles in the elderly. Shingles is the result of reactivated chickenpox virus (Varicella Zoster virus, also a herpesvirus); however, we first have to find sensitive ways to identify virus in latency, and we have to study its activity level in a latent–infected cell. The aim of this project is to identify markers of viral activity during latency followed by finding strategies that possibly intervene with viral behavior during latency.
There are two Career Development Awards offered through the Foundation in 2017.
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:
Dr. Loux is a postdoctoral fellow at the Gluck Equine Research Center at the University of Kentucky. Her project will be analyzing the microRNA (miRNA) population in mares throughout normal gestation, as well as during experimentally induced placentitis. RNA isolated from chorioallantois (6, 8, and 10 months of gestation) will be sent to the R. J. Carver Biotechnology Center at the University of Illinois (Urbana, IL) for next-generation sequencing to fully characterize the miRNA population at each given interval and to maximize the data generated by each sample. Significant changes will be verified by qPCR, and will encompass a greater number of samples. Professor and Albert Clay Endowed Chair, Dr. Barry A. Ball is her advisor on the project.
The Elaine Klein Development Award is a 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 2016 award winner is:
Dr. Jacob and Dr. Patty Weber of the Michigan State University College of Veterinary Medicine will be working in collaboration with Dr. Molly McCue of the University of Minnesota on the project entitled, “Biomarkers of Equine Metabolic Syndrome, Age and Diet”. Veterinarians have identified equine metabolic syndrome (EMS) as the most common cause of laminitis. A key component of EMS1 is insulin resistance characterized by hyperinsulinemia and/or abnormal glycemic/insulinemic responses to oral or intravenous glucose challenge. A causal role for insulin in laminitis development has been supported by studies of hyperinsulinemia in horses and ponies. Similar to type 2 diabetes mellitus in humans, EMS is a complex trait with risk of disease due to the interaction between innate (i.e. genetics, breed, age) and environmental (e.g. diet, exercise) factors. .The emerging clinical importance of EMS and insulin dysregulation justifies studies that advance understanding of underlying pathophysiology; knowledge that will lead to improved methods for identification of at-risk individuals before the onset of disease and identify new therapeutic targets.