Current Research

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The board of directors of Grayson-Jockey Club Research Foundation has authorized expenditure of $1,648,434 to fund 13 new projects at 12 universities, 12 continuing projects, and two career development awards worth $20,000 each. This marks the seventh straight year that more than $1 million has been approved. The 2021 slate of research brings Grayson-Jockey Club Research Foundation’s totals since 1983 to more than $30.6 million to underwrite 397 projects at 45 universities.


  • Click here for a printable list of our 2021 Projects


  • PASSIVE IMMUNIZATION OF FOALS WITH RNA-AB AGAINST R EQUI
    Jeroen Pollet, Baylor College of Medicine – One Year Grant

    One Sentence Summary: By inhalation therapy, we intend to deliver the genetic code for a protective antibody against Rhodococcus equi into the lung cells of newborn foals, to rapidly protect them against infection.

    The objective of this study is to make a unique vaccine to instantly protect newborn foals against a microbial infection caused by Rhodococcus equi (R. equi). This vaccine comprises a lab-made messenger RNA molecule (mRNA), which is a blueprint that can be used to instruct cells to produce a specific protein. We have previously shown that proteins, antibodies, binding microbial surface molecule, named poly-N-acetyl glucosamine (PNAG), can protect foals against R. equi. These protective antibodies can be acquired by the foals by either drinking milk from a vaccinated mare or by a blood transfusion. However, each of these approaches has limitations and vaccination of newborn foals cannot provide protection when foals are most susceptible to infection (it takes weeks before the foal can be vaccinated and has sufficient antibodies to be protected). Therefore, we would like to use the mRNA platform to produce mRNA that can instruct cells to produce protective PNAG binding antibodies. The mRNA therapy would provide near instant immunity because the body can translate the mRNA into proteins in just a few hours. In any other traditional way, reaching and maintaining the number of antibodies would be extremely difficult. We intend to administer the mRNA therapy by using a nebulizer, to let the foals inhale the RNA. This way, we hope to make the procedure less invasive and have mRNA delivered to lung cells where the antibodies are most needed for protection against R. equi. The proposed project would be the first study developing an mRNA therapy for horses. It would also be the first application of mRNA technology for the production of mAbs against microbial infections. Targeting PNAG would be highly beneficial for newborn foals, as PNAG is also present on the surface of other microbes. For that reason, the mRNA-encoded vaccine may be a great tool to protect foals not only from R. equi, but possibly also form other infections. Therefore, all scientific progress will be valuable as the potential for the platform is enormous, both in human and veterinary medicine.

    Pneumonia caused by Rhodococcus equi (R. equi) is one of the most important causes of disease and death in newborn foals. A vaccine against a microbial surface molecule, poly-N-acetyl glucosamine (PNAG), can induce highly protective proteins, antibodies that protect foals against R. equi. This vaccine is administered to pregnant mares such that immunity is transferred passively via the mare’s milk to their foals. Maternal vaccination is necessary because foals cannot mount protective immune responses to vaccination shortly after birth when they are most susceptible to R. equi infection. Unfortunately, relying on antibody transfer leaves many foals vulnerable when the transfer of antibodies is inadequate or absent. Thus, there is a great need for novel approaches for safely delivering anti-PNAG antibodies to foals.

    Importance to the Equine Industry: We propose to develop and evaluate a new vaccine to instantly protect newborn foals against a microbial infection caused by R. equi. This vaccine comprises lab-made messenger RNA molecules (mRNA), these are a blueprint which can be used to instruct cells to produce protective PNAG binding antibodies. We hope to establish almost instant in vivo production of significant numbers of neutralizing antibodies against R. equi in newborn foals. In contrast to DNA, mRNA is a non-infectious, non-integrating platform, constituting no potential risk of infection or mutations. Additionally, mRNA is broken down by normal cellular processes. Several different mRNA therapeutics and vaccines have now been tested in clinical studies in humans and they have been shown to be safe and well-tolerated.


    HYPERTHERMIA AND ACIDOSIS IN EXERTIONAL MUSCLE DAMAGE
    Michael Davis, Oklahoma State University – One Year Grant

    One Sentence Summary One Sentence Summary: This project will identify an underlying cause of exercise-associated muscle fatigue and soreness and allow trainers to more precisely condition horses with fewer training days lost to muscle soreness.

    Exercise-induced muscle damage is common in equine athletes. It is typically recognized soon after a bout of training or competitive exercise as varying degrees of muscle soreness and damage. In severe cases, the amount of muscle damage can create secondary life-threatening conditions such as shock and kidney failure. The condition has been described since the time of the Roman Empire, and in the last 2 decades progress has been made in identifying genetic defects in specific horse breeds that predispose horses to developing severe forms of the condition (Recurrent Exertional Rhabdomyolysis or RER), but non-genetic risk factors can still predispose ALL athletic horses to develop exercise-induced muscle damage. One important factor may be heat. Working muscle can get very hot – over 110oF – and studies have shown that when muscle gets that hot, the processes used by the muscle to convert nutrients and oxygen into the energy that powers muscle contractions become inefficient, creating a vicious cycle of producing more and more heat just to maintain the same level of exercise performance. Similarly, a build-up of acidic by-products can also impair muscle efficiency, and the fact that the heat and acid build-up frequently occur together highlights their potential role in not only fatigue, but the muscle damage that commonly occurs in fatigued equine athletes. However, past studies have been limited by: i) existing technology that failed to reproduce completely the conditions that actually exist during exercise, and ii) analytical techniques that were not exact or specific enough to identify exactly which part of the energy metabolism pathway was failing during exercise. These limitations can be overcome by the use of new technology called high-resolution respirometry, and preliminary studies in our laboratory using this technology have tentatively identified the specific elements in the muscle that fail in the face of excess heat and excess acid. Furthermore, our data suggest that this inefficiency can become progressively more severe, ultimately resulting in muscle damage. If this process occurs in enough muscle cells, outward signs of muscle soreness can occur. Our proposed studies are designed to confirm that this process occurs during high-intensity exercise and is responsible for non-genetic muscle disease. The studies will use new technology that allows the careful examination of muscle cells to determine the impact of high temperature and acid on muscle energy production, as well as help identify how these changes lead to muscle damage. Using these studies, we will not only provide answers to why horses can “tie-up” even when they do not possess a genetic defect, but also provide the scientific tools to facilitate understanding of why horses with genetic defects are more prone to tying-up.

    Importance to the Equine Industry: Successful conditioning of a horse for athletic activities requires the careful application of stress – too little stress and there is little or no improvement in exercise capacity, but too much stress results in damage to the horse. Like other tissues that must adapt positively to improve athletic performance, muscle requires this careful balance, and tying-up is believed to be the result of too much metabolic stress when compared to what the muscles can accept or withstand. When a horse ties-up, the trainer must provide for proper recovery, which leads to additional costs and lost time in training. Conversely, it is likely that optimal training intensity for muscle conditioning may not be consistently delivered in an effort to avoid triggering an episode of tying-up. This necessarily cautious approach causes training to take longer and to cost more than it would otherwise if it was known precisely what causes tying up. This information would enable the trainer to have greater knowledge and confidence regarding the amount of stress that can be placed safely on a horse in training. The proposed studies will provide information that will lead to greater understanding of the causes for tying-up, leading to a greater level of confidence in the process of conditioning horses for athletic performance. This in turn should eventually reduce the episodes of exercise- or training-induced damage while increasing the economic and physiologic efficiency with which horses are conditioned.


    DEVELOPING AN IMPROVED SEROLOGICAL TEST FOR STRANGLES
    Noah Cohen, Texas A&M University – One Year Grant

    One Sentence Summary: We propose to develop a more accurate blood test to identify horses infected with the bacterium that causes strangles to improve control and prevention of strangles.

    The disease known as strangles remains the most important infectious disease of horses in the United States (US) and other countries. Strangles is an ancient disease that has persisted for centuries because the bacterium that causes this disease (viz., Streptococcus equi subspecies equi [SEE]) can survive long-term in carrier horses that do not show clinical signs. Control and prevention of strangles is greatly hampered by the limited accuracy of testing to accurately identify infected horses, particularly carrier horses that do not show clinical signs. These in apparent carriers are difficult to detect using either microbiological culture or molecular testing using polymerase chain-reaction (PCR) because they can test negative by these methods for long periods of time. An accurate test to detect antibodies in blood indicative of strangles would be useful to detect carrier horses (and horses that have overt signs of strangles). However, available blood tests for detecting antibodies for SEE proteins lack diagnostic accuracy. We therefore propose to use an innovative protein microarray technology and blood samples from horses with and without strangles to identify proteins that can be used to make an improved blood (serum, the liquid portion of blood) test for strangles. If successful, this new test will help protect horses from this prevalent and impactful disease by enabling the detection of horses that are infected with SEE, including those not showing signs of strangles. Furthermore, it is our belief and hope that proteins identified using the protein microarray technology will identify novel targets for a more effective strangles vaccine.

    Importance to the Equine Industry: Equine industry stakeholders tell us that strangles is a high priority problem, and this is consistent with our clinical experience. This project is expected to create an improved blood-sample-based diagnostic test to help protect equine industries from this prevalent and impactful disease. Furthermore, it is our belief and hope that proteins identified using the protein microarray technology will identify novel targets for a more effective strangles vaccine.


    MITIGATION OF EQUINE RECURRENT UVEITIS THROUGH SOCS
    Joseph Larkin, University of Florida – Two Year

    One Sentence Summary: We seek to design a topical eye drop, using a natural protein, which helps to prevent pain and blindness associated with equine recurrent uveitis.

    Equine recurrent uveitis (ERU), or moon blindness, is a serious disease in horses that has no cure. ERU currently affects 10-25% of the horse population. ERU is caused by a misdirected immune system. Usually the immune system does an excellent job in destroying infectious agents and cancers. In ERU, the immune system attacks components of the eye. Although ERU symptoms vary between horses, it has two main varieties. In the first variety (relapsing-remitting), horses have reoccurring periods of severe eye pain and swelling followed by periods minimal disease (remission). Each time the horse experiences a relapse, the immune system figures out better ways to attack the eye, resulting in more severe disease. In the second variety (insidious), the immune system never turns off, promoting steady eye damage. Although treatments are available, ERU remains one of the leading causes of blindness in horses. This is because the available treatments do not work for all horses or stop working as the disease progresses. Another complication is that the current treatments have significant side-effects, making them unsuitable for prolonged treatment of this life-long disease. There is a very real need for safer, more effective treatments for ERU.

    Since ERU is caused by a misdirected immune response, our goal is to turn down the immune system enough to stop eye damage but allow it to maintain function against pathogens and cancers. Our team has developed a medicine that is based on a molecule, naturally present in horses and people, that turns down the immune system. Suppressor of Cytokine Signaling-1 (SOCS1) turns off the communication between immune cells causing the immune response, thereby telling them to stop. We have packaged a small portion of the SOCS1 molecule, SOCS1-KIR, and tested its ability to turn off misdirected immune responses in mice. Our results have been very exciting. We successfully reduced immune responses in mouse diseases that resemble multiple sclerosis and lupus (other diseases caused by misdirected immune systems). A very exciting moment came when we made an eye-drop containing SOCS1-KIR to treat a mouse disease that resembles ERU. Simple eye-drops were safe and very effective in treating the mouse model of ERU. Our preliminary horse experiments seem to indicate that the drug will work in horses as well. This grant application proposes to test the safety of this treatment in horses and see if it has the potential to work in horses, as it has in the mouse models of human and horse diseases. We believe that a simple eye-drop of SOCS1-KIR daily will safely reduce the steady damage mediated by the insidious disease and extend the period between relapse and remission. As such, SOCS1-KIR eye-drops will serve as a save, NOVEL treatment for an incurable disease that remains a major cause of blindness in horses.

    Equine recurrent uveitis (ERU) is a disease whereby owners watch their horses develop a painful debilitating disease which eventually results in blindness. Horses that become blind are a danger to themselves as they can no longer navigate through a field. In addition, the horse is negatively impacted because he/she can no longer perform the farming or industry duties they have grown accustomed to. The owners are then faced with the challenge of caring for a blind horse or choosing the very difficult decision to euthanize a companion animal. ERU currently affects 10-25% of the horse population. Although treatments are available, they do not work for all horses. In addition, severe negative side effects prevent the daily use of these drugs to reduce the eye damaging components of this chronic disease. As such, there remains a critical unmet need for safer, more effective novel therapeutic strategies to treat recurrent uveitis.

    Importance to the Equine Industry: We propose to build upon previous studies in small animal models, and preliminary horse studies, that demonstrate that this novel treatment may work in horses. Our strategy to provide daily eye drops to regulate ERU, while allowing the horse to maintain their necessary immune responses against pathogens would be a breakthrough if successful. Horses would maintain their sight and lifestyle, while owners would maintain their livelihoods and companion animals for years to come.


    ENVIRONMENTAL ORIGINS OF EQUINE ANTIMICROBIAL RESISTANCE
    Brandy Burgess, University of Georgia – Two Year

    One Sentence Summary: This study will elucidate how antimicrobial resistance and virulence determinants are shared among horses and hospital environment, as well as the role antimicrobial exposure plays at this interface..

    Antimicrobial resistant (AMR) pathogens are a critical threat to the health of horses and the personnel who care for them; this is perhaps most evident in the veterinary hospital setting, where AMR or multidrug-resistant (MDR) healthcare-associated infections (HAIs) can devastate both patient health and hospital budgets. A comprehensive understanding of the factors that allow AMR pathogens to emerge in veterinary hospitals is necessary to develop a targeted, efficient approach to preventing these infections. It is understood that the use of antimicrobial drugs (AMDs) gives a competitive advantage to the microorganisms that harbor resistance genes, allowing them to persist in bacterial populations. However, this fails to explain why exposure to the hospital environment alone also increases patients’ risk of harboring AMR bacteria. Our previous work has demonstrated that AMR among Escherichia coli isolated from horses is not only associated with AMD use, but also with hospitalization. A similar phenomenon has been demonstrated among cattle transitioning to a commercial feedlot; AMR genes detected in fecal samples were similar between AMD-treated and untreated cattle, suggesting that transition to and maintenance in the feedlot environment were more impactful on resistance genes in the fecal microbiome than was immediate exposure to AMDs. These studies demonstrate that the environment, which can serve as a reservoir for AMR genes, may play a much larger role in the emergence and maintenance of AMR than does the immediate exposure to antimicrobials. Therefore, if we are going to develop preventive policies and practices that target the emergence and maintenance of AMR in the veterinary hospital environment, then we must expand our perceptions of this intricate ecosystem. Developing an in-depth insight into the dynamics of AMR within microbial populations requires the application of next-generation sequencing technology, which demonstrates a more complete picture of the microbial ecology of an environment compared to traditionally utilized culture-based methods.

    As such, our long-term goal is to characterize the influence of AMD use and prescribing practices in veterinary hospitals on the emergence of AMR-pathogens that detrimentally impact animal and human health. To that end, this study aims to use metagenomics and bioinformatics tools to determine the capacity of the veterinary hospital environment to serve as a reservoir for virulence factors, resistance genes, and the emergence of pathogenic organisms. Our central hypothesis is that the long-term accumulation of resistance genes and, secondarily, virulence factors, in the environment has a greater impact on the fecal microbiome and its resistance genes than does recent AMD exposure. The objectives of this proposal are to 1) to characterize the fecal microbiome and its AMR genes for hospitalized horses with AMD exposure, hospitalized horses without AMD exposure, and healthy horses that have not been hospitalized nor had AMD exposure, 2) characterize the microbiome and its AMR genes for the veterinary hospital environment, and 3) evaluate the relationship of the microbiome and AMR genes between environmental and fecal samples using previously established bioinformatics tools.

    An observational study will be conducted at the University of Georgia to evaluate the fecal microbiome and AMR genes among hospitalized adult horses with AMD exposure (n=20), hospitalized horses without AMD exposure (n=20), healthy horses from the previous groups’ farms that have not been hospitalized nor had AMD exposure (n=40). Fecal samples will be collected from all horses, and environmental samples will be collected from the housing area of hospitalized horses. DNA will be extracted from all fecal and environmental samples and submitted for genetic sequencing. Data will be analyzed for differences in microbial composition and resistance genes between sample groups and microbial diversity between sample types.

    This study will characterize the relationship between AMD exposure, the equine fecal microbiome, and the veterinary hospital microbiome. Specifically, it will demonstrate that the long-term accumulation of resistance genes in the environment has a greater impact on resistance genes in the fecal microbiome than does have recent AMD exposure.

    Importance to the Equine Industry: Enacting effective hospital infection control and responsible antimicrobial use policies requires scientifically sound, evidence-based decision making. However, there remains a crucial gap in our understanding of how AMR pathogens emerge within veterinary hospitals – the role of the hospital environment in the development and dissemination of these pathogens. By implementing technology that allows for in-depth evaluation and comparison of microbial communities, this study will elucidate how antimicrobial resistance and virulence determinants are shared between equine patients and their surrounding hospital environment, as well as the role that AMD exposure plays at this interface. An improved understanding of these dynamics will ultimately allow veterinary hospitals and equine clinicians to better tailor their antimicrobial prescribing and infection control practices to curb the development of antimicrobial resistant HAIs. Furthermore, this innovative and fundamentally groundbreaking investigation will serve as a platform for continued investigations into the ecological dynamics of AMD use and its impact on human and animal health and the environment.


    TREATMENT OF JOINT INJURY WITH MESENCHYMAL STROMAL CELLS
    Thomas Koch, University of Guelph – Two Year Grant

    One Sentence Summary: Evaluation of equine umbilical cord blood-derived mesenchymal stromal cells to treat joint injuries in horses.

    Horses are multi-use livestock that contribute more than $122 billion to the U.S. economy. Injuries to joints are among the most common causes of lost training days or premature retirement in equine athletes. Large cartilage defects GT 4cm2 are a clinical challenge to treat. Left untreated, chondral or osteochondral defects can lead to osteoarthritis and further joint deterioration. As in humans, cartilage treatment strategies in the equine model are mainly palliative. Since the intrinsic repair capacity of cartilage is very limited, particularly in the case of large, full-thickness defects, surgical intervention is necessary to restore total joint function. Further, it is necessary to view the problem of damage to cartilage as one affecting the whole joint as an organ. Case reports of treatments such as microfracture and osteochondral graft transfers have shown some success but are limited in their capacity for full hyaline cartilage remodeling. These invasive techniques may eventually be replaced by cell-based therapies and tissue engineering approaches for cartilage regeneration.

    Chondrocyte transplantation techniques such as autologous chondrocyte implantation (ACI), matrix-induced ACI (MACI) and spheroids of human autologous matrix-associated chondrocytes have been shown to be effective but are impractical for most equine surgical practices due to the complexity of the multi-step procedures.

    Joint cartilage chips, particulate cartilage or morselized cartilage, as they are also sometimes referred to, have the advantage of being a one-step procedure compared to the above two-step procedures. In humans, cartilage chips have shown significant clinical promise, regardless of whether the cartilage was sourced from juvenile human cadavers, unrelated adult cadavers, or the patient themselves. Autologous cartilage chips have also been shown to induce robust cartilage healing in the horse. However, current cartilage chip methods are hampered by a limited supply of donor material, risk of disease transmission, suboptimal graft tissue, and donor site morbidity. Donor age, co-morbidities, and harvest site may also negatively influence the biological potency of present cartilage and cell grafting methods. We propose to solve these issues by using cartilage generated in the laboratory from equine umbilical cord blood mesenchymal stromal cells (eCB-MSC).

    MSCs have emerged as a desirable alternative to chondrocytes for the treatment of cartilage injuries. After isolation, MSCs can be expanded in culture to achieve the desired number for transplantation, and MSCs are capable of differentiating into cartilage in vitro. MSCs are also known to have the ability to regulate immune cells adding to their value as a cellular therapeutic agent. MSCs derived from the bone marrow or adipose tissues of the horse are disadvantaged by invasive tissue collection, extended culture time for autologous use, and reduced biological repair potential if from older patients. We were the first in the world to report the isolation of eCB-MSCs and have published extensively on their remarkable chondrogenic potency. Independent labs have confirmed the unique chondrogenic properties of eCB-MSCs. Importantly, neocartilage generated from eCB-MSC has been reported to have superior compressive properties comparable to that of natural equine cartilage. Significant work has been done in our lab to establish a robust platform for the chemical induction of eCB-MSC chondrogenesis. We are now able to consistently produce large amounts of neocartilage tissue from eCB-MSCs suitable for clinical implantation.

    We hypothesize that eCB-MSC-derived cartilage chips will provide enhanced anabolic effect on the joint compared to microfracture when treating full-thickness induced cartilage defects. Our objective is to compare and contrast the efficacy of allogeneic eCB-MSC-derived cartilage chips with microfracture repair in treating induced cartilage defects and assess their effect on the overall health of the surrounding joint when combined with follow-up eCB-MSC injections.

    Importance to the Equine Industry: These studies will assess a novel approach to managing joint disease secondary to focal cartilage repair using eCB-MSC-derived cartilage chips and undifferentiated MSCs. Successful completion of this project will pave the way for a second follow-up project grant with the following aim: In vivo assessment of MSC-derived cartilage-chips and undifferentiated MSCs for horses with natural spontaneous cartilage defects. If our long-term goals are realized, this new therapy would allow horses across disciplines to continue performing at a high level and likely delay the onset of post-traumatic osteoarthritis that is frequently associated with focal cartilage defects.


    OPTIMIZING BONE GROWTH TO REDUCE EQUINE FRACTURE
    Mariana Kersh, University of Illinois Urbana-Champaign – Two Year Grant

    One Sentence Summary: Reduction in distal limb fractures through exercise in young horses would have a significant positive impact on horse welfare and the economics and public perception of the horse industry.

    Musculoskeletal injuries, especially fractures to the lower leg, are a major reason for racehorse injury and are expensive to fix, but may be minimized using exercise to encourage bone adaptation. These fractures may be reduced by introducing an exercise intervention strategy in young horses to strengthen their bones.

    This project will identify how exercise-related biomechanical changes in young foals can lead to optimal bone adaptation and decreased fracture risk. By using a combination of imaging, mechanical testing, and computer modeling, the project provides a molecules-to-bone scale understanding of bone modeling and remodeling to improve bone strength.

    The objective of the current proposal is to (1) evaluate two exercise interventions that use different speeds (12 foals) and (2) identify markers (biomechanical and biological) of bone activity that can be used to predict adaptation to exercise. We hypothesize that a constant speed exercise regimen will produce similar bone adaptation compared to an increasing speed exercise regimen. Musculoskeletal simulations will be used to predict muscle and joint reaction loads on the bones, to be used in predictions of bone strength. Two exercise protocols will be implemented in a separate set of foals from ages 8-16 weeks. Intervention 1 will consist of ten minutes of daily exercise at a constant speed (measured via accelerometers). Intervention 2 will consist of a gradual increase in speed over the 8 weeks. We hypothesize that the greatest bone response to exercise for mid-shaft cross-sectional properties (total area, cortical area, etc.) will occur before the epiphyseal growth plate fuses. Additionally, we hypothesize that cross-sectional properties that resist bending loads in the cannon and compressive loads in the long pastern should increase with positive allometry (changes in proportion due to growth). The results of this study will provide a thorough rationale for how muscle and joint forces load bone during movement, and provide evidence of the efficacy of exercise intervention during growth, which may be translated to other species

    Importance to the Equine Industry: Horse breeders need an easy-to-follow exercise intervention strategy that has proven effective, if it is to be adopted and used. The fetlock is the location of the most distal limb fractures in racehorses. Characterization of normal growth and development of the cannon and long pastern bones is the first step in understanding the effects of early age exercise on bone health. Priming the bones during growth may cause them to strengthen in areas of common fracture. Using finite-element models will allow us to understand bone growth and development without the need to sacrifice foals. Validation of an exercise intervention that is easy to implement would increase the likelihood of its adoption by racehorse breeders.


    NEW GENERATION EQUINE INFLUENZA BIVALENT VLP VACCINE
    Thomas Chambers, University of Kentucky – Two Year Grant

    One Sentence Summary: We propose to create a novel, safe and effective vaccine for equine influenza based on the 21st-century technology of noninfectious virus-like particles produced in plants.

    For viral diseases of horses, most existing vaccines are based on technologies originally developed in the 1960s. These conventional vaccines are mainly killed virus, whole or chemically broken into subunits. The main advantage of these over more modern alternatives such as modified live virus vaccines is the safety of administering an inactive (killed) virus. But equine influenza vaccines (unlike, e.g. rabies) need to be updated from time to time as the viruses in worldwide circulation continue to mutate, and updating the conventional vaccines has historically taken unexpectedly long periods of time, up to 5 years. The conventional vaccines have another drawback which is that, based on blood samples, it is very difficult to tell the difference between a horse that has been infected with influenza and a horse that has been vaccinated (in scientific jargon, the vaccines are not DIVAcompliant), and this failure complicates epidemiological investigations of disease outbreaks. In humans regarding SARScoronavirus, it is feared that the same drawback would complicate the analysis of outbreaks and for that reason the human coronavirus vaccines, when they finally appear, will NOT be conventional killed virus vaccines even though that is the traditional path to follow.

    We propose to use 21st century technologies to create a 21st-century equine influenza vaccine. Our co-investigator at CSIR, South Africa, has already successfully used this approach to create an avian influenza virus vaccine that works in chickens. We will modify plants to express the major equine influenza protein, hemagglutinin, in their leaves in the form of ‘virus-like particles’ or VLPs. VLPs are not infectious. Those leaves will be processed into a form that the VLPs can be injected into the horse’s neck. We predict (1) that the horse’s immune system will ‘see’ and respond to the VLP hemagglutinin with protective responses—in our proposal we will test this. We also predict (2) that this vaccine is fully as safe as the conventional vaccines, since the plant material is non-infectious—in our proposal we will test this also. Engineering the plant in this manner has become routine in agricultural research, so this vaccine will be easy to update. The vaccine will be DIVA-compliant as it contains the influenza hemagglutinin alone, whereas in nature the hemagglutinin never occurs without another protein, neuraminidase, that would be the basis for spotting infections. If our predictions are shown to be accurate, the outcome will be an equine influenza vaccine that meets the demands of the 21st century. An additional benefit is that the same approach can be adapted to many other equine vaccines, not just influenza.

    Importance to the Equine Industry: Equine influenza remains a threat to horses in most parts of the world, particularly including performance horses that must travel for competition or breeding purposes and are repeatedly exposed to contagious diseases. Vaccines for equine influenza have a long but checkered history: these are generally more effective at reducing the severity of disease once it is contracted, rather than preventing it, and a half-century of vaccination has not stopped outbreaks from occurring up to the present day: indeed the winter of 2018-19 was worse than most in this regard. The vaccines most widely used are based on half-century-old technologies and have the limitations of those technologies. This project will bring 21st-century technologies to bear and overcome the conventional limitations. If successful it will serve as a model for development of a new generation of equine vaccines across a spectrum of diseases.


    EXPANDING KNOWLEDGE OF THE MICRO-BIOME IN MARES & FOALS
    University of Kentucky, Gluck Equine Research Center – One Year Grant

    One Sentence Summary: This project will look to provide sampling and analysis including PCR, genomic and microbiology of the micro-biome in mares and foals.

    Foal diarrheas appeared earlier in the spring of 2021 and seems more aggressive than in years past. The mortality has been very low but high morbidity levels at several farms here and in Ohio and Illinois. Funds will be used for sampling and analysis including PCR, genomic and microbiology. Concern over the fact that it seems to be antibiotic resistant and a clear diagnosis of the causative organism has yet to emerge. There is concern that some farms are treating prophylactically which only leads to resistance and that a better understanding of the micro-biome of the mares and foals is needed. It is perceived and rightly so, that sampling should occur on farms from conception to foaling, not just in the window of crisis.


    INJURY PREDICTION FROM STRIDE DERIVED RACING LOAD
    Chris Whitton, University of Melbourne – Two Year Grant

    One Sentence Summary: By studying patterns in bone fatigue accrual over time in racehorses, we will better, and earlier, identify horses at risk of limb injury, facilitating timely evidence based preventative strategies.

    In a 1-year Grayson project, we have shown that speed and stride length decrease several races prior to injury, indicating that monitoring speed and stride characteristics during racing has potential for predicting musculoskeletal injury (MSI). However, changes in stride indicate a significant degree of injury has already occurred and not all horses that injure show this stride pattern. This project aims to advance our prediction of MSI in order to better detect horses at risk of MSI and to do so much earlier (2) increasing the specificity of our models in predicting injury by taking into account outcomes such as retirement and enforced rest.

    Limb injuries in racehorses are not a ‘yes or no’ outcome. Rather they develop gradually over time. When any material, including bone, is subjected to repeated high loads it slowly weakens and eventually fails or fractures through the process of material fatigue. This is a well understood process in the physical sciences. The higher the load the fewer the number of times the material can sustain the load before failure or fracture. We know this is happening in racehorses because a high proportion of horses with catastrophic fractures have evidence of pre-existing pathology at postmortem, showing that the injury came on gradually. In the galloping horse the bone is loaded with each stride and the magnitude of the load is proportional to the speed the horse is galloping at. Previous work by our research group has improved our understanding of the fatigue process in equine bone and this knowledge can now be applied to horses when racing. With the recent development in technology that has enabled individual measurements of stride parameters for each racehorse, quantification of the fatigue history of a horse’s skeleton can be achieved.

    In assessing the difference in quantification of bone fatigue between injured and non-injured horses, it is important to account for events not categorized as injury but may still be indicators of injury such as retirement and enforced rests. These events are termed “competing risks”, and not accounting for these events would otherwise assume a horse that retired or had an enforced rest to be a healthy horse. This would result in the healthy population of horses having a subpopulation of horses that had unreported lameness or minor injuries which would result in a much different result than if these competing risks were identified and dealt with.

    This proposed project will utilize a large-scale database containing five years of continuously recorded race day speed, and stride measurements. The data was collated for all races in Tasmania, Australia for the 2011 to 2016 racing seasons by StrideMASTER, a company that utilizes a GPS tracking and precision sensors that are worn in the saddle of every racehorse that starts in a race in Tasmania. Research by our group funded by the Grayson Jockey Club found that this type of data shows great promise for real time monitoring of injury risk as the rate of injury increases with detected decreases in speed and stride length during races over time. By including our understanding of how loading with each stride causes injury we will build on our previous work to greatly enhance our ability to predict injury from stride and speed characteristics and to do so earlier.

    Our aim is therefore to enhance the ability to predict MSI from stride and speed data collected during racing by including the effect of bone fatigue. Rather than only predicting the endpoint to MSI as per previous epidemiological studies, this research will allow us to map the development of MSI over time thus enabling much earlier detection of horses at risk of injury. The findings of this research could be used to inform the development of real-time injury risk tracking for individual horses.

    Lameness in Thoroughbred racehorses due to musculoskeletal injury (MSI) is the most common reason for poor performance and early exit from the industry, and accounts for upwards of 80% of fatalities. Horse breakdown is also the leading cause of serious jockey injuries. These factors result in substantial costs to the industry and affect the public’s confidence in the sport risking its social license to operate.

    Therefore, the ability to identify factors that are predictive of an impending injury is critical. Though various risk factors for MSI have been studied and identified, these have not proven useful for early detection of injury in individual horses. Much effort is currently directed towards advanced imaging for the early detection of bone injury and although such methods show promise, they are not upscaleable for constant monitoring of whole populations of horses. The methods developed in the current study will allow much better targeting of advanced imaging by identifying horses at risk of MSI.

    Importance to the Equine Industry: This proposed project will build on our advancing knowledge of bone fatigue in vitro with the utilization of a large-scale database of continuously recorded stride data to identify modifiable factors that contribute to bone fatigue as well as to determine if assessing bone fatigue over time can be used to predict, and therefore prevent injuries in racehorses.

    Our ultimate aim is to efficiently and cost effectively constantly monitor horses when racing in order to detect horses at risk of injury. Doing so would allow early interventions to modify workloads. Reducing racehorse injury rates will aid in improving public perception towards the racing industry, reduce economic costs, as well as improve both equine and jockey welfare.


    PREDICTING EXERCISING ARRHYTHMIAS WITH RESTING EGGS
    Molly McCue, University of Minnesota –Two Year Grant

    One Sentence Summary: We will use at rest ECGs to identify horses with irregular heart rhythms at exercise that can cause sudden cardiac death (SCD), allowing for increased monitoring and improved understanding of SCD.

    Irregular heart rhythms are an important cause of sudden death (SCD) in horses. Most horses that develop irregular rhythms cannot be detected using our standard diagnostic tools as electrocardiograms (ECGs) at rest appear visually normal and no structural abnormalities can be identified on cardiac ultrasound. Human athletes that develop irregular rhythms that are known to cause SCD can be identified at rest using computational ECG analysis, even when the ECGs appear normal on initial visual inspection. These individuals can then be regularly monitored during their athletic careers, allowing for retirement from high intensity exercise before the development of a potentially fatal rhythm. A simple to perform test to identify horses at increased risk of developing irregular rhythms that can cause SCD would allow for increased monitoring of these horses to reduce their risk of SCD.

    Racehorses commonly develop irregular rhythms at exercise. Many of these rhythms resolve without incident, however, some cause SCD. Detecting irregular rhythms at exercise requires an ECG to be performed during exercise which is laborious to obtain and frequently contains significant artefacts making their interpretation a challenge. Therefore, exercising ECGs are rarely used outside of research and full cardiac workup settings. In contrast, resting ECGs are easy to obtain and interpret. We have demonstrated that it is possible to identify horses with the most common cardiac arrhythmia (atrial fibrillation) using resting ECGs from affected horses when they do not have an irregular rhythm. In this proposal we will determine if it is possible to use resting ECGs in a normal rhythm to identify horses that will develop other important arrhythmias. We will develop standardized computational protocols to analyze these ECGs. These computational protocols convert the ECG wave into computer signals that can be used to identify subtle changes in the ECG that cannot be identified using visual inspection alone.

    We hypothesize that computational analysis of resting ECGs can be used to identify horses that will develop irregular heart rhythms at exercise. We are already funded to perform ECGs before, during, and after maximal exercise in 500 Standardbred and 500 Thoroughbred racehorses. Our aims will use two different types of computational analysis (complexity and restitution analysis) and a combination of the two methods to determine if it is possible to identify horses that develop exercise-associated irregular rhythms using at rest ECGs.

    In aim 1 we will use ECG strips without any artefacts that show a normal heart rhythm and rate. The pipeline will involve establishing: 1) the best complexity analysis algorithm; 2) the best method of converting the ECG trace to a computational signal; and 3) how to use these methods to differentiate between horses with and without irregular heart rhythms at exercise. In aim 2 we will use ECG strips without any artefacts that show a normal heart rhythm and rate. We will filter the ECGs to generate a computational pattern, then 1) develop a pipeline to perform restitution analysis; and 2) use restitution analysis to differentiate between horses with and without arrhythmias. Finally, in aim 3 we will use a combination of approaches to determine if the method from aim 1 or aim 2 or a combination of the methods from aim 1 and aim 2 are most accurate for differentiating between horses with and without cardiac arrhythmias.

    We will be able to identify horses that develop irregular heart rhythms at exercise using resting ECGs, without the need for a more complicated exercise ECG. Once identified, these horses can then be monitored more frequently to identify any cardiac changes that could further increase their risk of SCD. The knowledge gained during the follow-up on these horses will provide key information about why certain horses with irregular heart rhythms go on to die suddenly. This information could then be used to make recommendations for monitoring horses at high risk for SCD, which is key to reducing SCD in racehorses.

    Sudden cardiac death (SCD) is a major concern for the equine industry. The collapse and death of around 500 racehorses each year in front of crowds of people and potentially on national or international television has a devastating impact on the image of the racing industry. Not to mention, the damaging impact on the surrounding horses, jockey, trainer, groom, and owners. At this time, very little is known about the underlying causes of SCD in horses making it virtually impossible to reduce the rate of SCD. Reducing the rate of SCD in racehorses is of critical importance for the future success of racing in the United States, and across the world.

    Importance to the Equine Industry: Identifying horses at increased risk of developing SCD is a key first step to determining why certain horses die on the track, which is critically important for reducing the rate of SCD in racehorses. We have already demonstrated that it is possible to identify horses with the most common irregular heart rhythm (paroxysmal atrial fibrillation) using computational analysis of resting ECGs at a normal rhythm. In this proposal, we will determine if it is possible to identify horses that develop other irregular heart rhythms at exercise that cause SCD, using computational analysis of at rest normal rhythm ECGs. If our hypothesis is true and we are able to identify horses using this technique, we will demonstrate that horses that develop irregular heart rhythms at exercise have underlying electrical abnormalities in their hearts that predispose them to developing these irregular rhythms. This will be the first evidence for underlying cardiac abnormalities in horses with exercise-associated irregular rhythms and will allow for easy identification of horses at increased risk of developing these irregular rhythms, and therefore SCD. This will allow for increased monitoring of these horses to identify additional risk factors for why certain horses go on to develop SCD, ultimately allowing for a reduction in the rate of SCD.


    UNDERSTANDING AND PREVENTING SUPPORTING LIMB LAMINITIS
    Andrew Van Eps, University of Pennsylvania –Two Year Grant

    One Sentence Summary: We aim to make supporting limb laminitis preventable through analysis of archived model tissues, a multi-center limb motion study of horses at risk, and development of a prototype therapeutic device.

    Our knowledge of the processes that lead to laminitis in horses remains incomplete and as a consequence, successful prevention and therapy for this crippling disease remain elusive. Whilst much research has focused on laminitis associated with excess insulin and sepsis, comparatively little has focused on supporting limb laminitis. Supporting limb laminitis occurs in the opposite limb in horses with painful limb injuries, and is thought to be a consequence of increased limb load. Through our previous Grayson Jockey Club Research Foundation funded studies, we have developed a non-painful model to study the effects of increased limb load. We have combined this with cutting edge techniques including microdialysis and molecular analyses and have identified 3 key factors important to the development of supporting limb laminitis: 1) lamellar microvascular perfusion is dependent on cyclic loading/ unloading of the limb, 2) persistent increases in limb load specifically interfere with blood perfusion within the lamellar tissue of the foot, resulting in ischemia, and 3) not only increased limb load but simply a lack of normal walking/weight shifting can cause damage specifically to the cells in the foot (parabasal keratinocytes) which are furthest from the blood supply and most vulnerable to nutrient deprivation. Our studies of tissue from natural cases of supporting limb laminitis corroborate these experimental findings: laminitis lesions are often present in multiple limbs (not just the supporting limb) and the parabasal keratinocytes are the focus of mechanical failure.

    Based on this, our overall working hypothesis is that a combination of increased load/insufficient load cycling can inhibit blood supply sufficiently to cause tissue death in the supporting limb; whereas reduced load cycling alone (insufficient movement/weight shifting), can also interfere with perfusion sufficiently to trigger certain cell stress and death processes in these vulnerable cells, contributing to multiple limb laminitis in these cases. In this proposal, we plan to take a multifaceted approach to further understanding and developing means to prevent supporting laminitis. We hypothesize that horses hospitalized due to painful limb conditions have reduced limb load cycling activity (offloading frequency) that can be identified using a simple, inexpensive accelerometer-based sensor system that we have developed and validated for this purpose. We aim to deploy this system across multiple hospitals to gather data on patients with painful limb injuries. Analysis of this data will allow us to develop a mathematical model that can predict imminent laminitis risk in these cases, and ultimately will lead to development of this system as a clinical monitoring tool. An ideal preventative therapy for supporting limb laminitis would improve blood flow within the foot without transferring load back onto the primarily injured limb. After previous failed attempts to accomplish this, we have tested a range of interventions in a cadaver limb model and we now have pilot data showing that cyclic frog and sole pressure can enhance blood perfusion, even in limbs under constantly increased (45% bodyweight equivalent) load. Using a prototype pneumatic device, hoof wall load is cyclically transferred to the frog and sole in the standing horse, relieving load on the hoof wall without transferring it to the opposite limb. We hypothesize that lamellar perfusion can be enhanced even in limbs under increased load using these dynamic manipulations of frog and sole pressure and we aim to test this in the live horse using our validated tissue microdialysis system.

    Understanding the processes that cause cell stress and death specifically in the parabasal keratinocytes in supporting limb laminitis will present opportunities for treatments even in the face of reduced blood flow. We hypothesize that the parabasal cell dysfunction and death in supporting limb laminitis is mediated by signaling pathways triggered by low nutrient availability and we aim to characterize these processes in archived tissues from our preferential weight bearing model, paving the way for the development of evidence-based therapeutic strategies to prevent and limit progression of laminitis in these cases.

    Importance to the Equine Industry: 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. The development of effective strategies to monitor for and prevent supporting limb laminitis would be a significant step forward for the welfare of horses and for the horse industry.


    DIAGNOSIS OF INCIPIENT CONDYLAR STRESS FRACTURE
    Peter Muir, University of Wisconsin–Madison – Two Year Grant

    One Sentence Summary: This study will save the lives of racehorses by establishing screening using fetlock CT for diagnosis of horses with a high risk of imminent serious injury for personalized clinical care.

    Injuries to the Thoroughbred racehorse that lead to euthanasia are termed catastrophic. In 2019 the incidence of catastrophic injury was 1.53 fatalities per 1,000 starts in the USA, representing a loss of hundreds of horses. Condylar stress fracture represents ~25% of catastrophic injury. Race performance after surgical treatment of Thoroughbreds with condylar fracture is often disappointing. There is, therefore, a critical need to comprehensively improve preventative screening of Thoroughbred racehorses for the presence of concerning bone injuries that increase the risk of catastrophic injury from stress fracture. Our long-range goal is to reduce the incidence of catastrophic injury in Thoroughbreds by improving clinical screening using routine computed tomography (CT) imaging in the sedated standing horse to check concerning bone injury in the fetlock, particularly injuries that precede condylar stress fracture. The objective of this application is to develop a standing CT screening approach for evaluation of racing Thoroughbreds with concerning fetlock bone injuries that are associated with high risk of condylar stress fracture and potential catastrophic injury. The rationale for this work is that use of routine standing CT imaging to detect concerning bone injuries in the fetlock that cannot be identified by radiography will improve management of racehorses and rapidly lead to substantial reductions in serious injury through improved screening. To accomplish our objective, we will perform mechanical testing of cannon bone specimens after CT imaging. We will relate the dimensions of concerning bone injuries that would not be detectable by radiography to the propagation of a condylar stress fracture, which is a serious injury clinically. We will also use these data to build a computer (finite element) model of the fetlock joint to undertake more detailed analysis of the relationship between fetlock joint loading associated with galloping and propagation of condylar stress fracture from the initial small fatigue injury to bone adjacent to the joint surface of the fetlock. This work will be undertaken using limbs collected from Thoroughbred racehorses that have euthanatized at the racetrack because of catastrophic injury. Under loading that models racing, mechanical testing of bones with concerning bone injuries will be performed. Local features of the site of bone injury will be determined from CT images. This information will then be used to build the 3D fetlock computer model and tune and validate the model to identify horses with imminent risk of serious injury. As a prelude to this project, we have designed and built a state-of-the-art CT scanner for the standing horse that has enabled routine fetlock CT scanning in a clinical setting. The proposed research is innovative, because it capitalizes on routine availability of standing CT imaging. With regard to outcomes, the work is expected to exploit the substantial clinical value in routine fetlock CT imaging. Ultimately, the outcomes of this project will save many horses from serious fetlock injury and death. This is a major advance, as the initial focal fetlock bone injury that is a prelude to condylar stress fracture cannot be reliably identified on radiographs of the fetlock making it impossible for equine veterinarians up to now to effectively screen racing Thoroughbreds thoroughly for concerning bone lesions that represent high risk of injury. In the future, the advances that will arise from this research will help establish an injury prevention program for racing Thoroughbreds that is centered around CT screening. We are particularly well positioned to pursue this research because of the expertise of our multidisciplinary team regarding injury prevention in Thoroughbreds.

    Importance to the Equine Industry: This work will save the lives of many racehorses. Completion of this research will enhance knowledge of the relationship between specific features of the focal bone injury in the fetlock and imminent risk of condylar stress fracture, a serious injury that is often catastrophic. Since standing computed tomography (CT) imaging in horses is now routine, this knowledge is directly translatable into clinical practice because racehorses with concerning bone lesions in the fetlock that are not evident on radiographs can easily be identified and managed appropriately. Our approach and initial tuning and validation of the computer model will enable preemptive longitudinal monitoring of horses in training to further validate research findings and implement improved personalized clinical care for racehorses.


    SECOND YEAR GRANTS

    IMPROVING FUNGAL DIAGNOSIS IN HORSES
    Soon Hon Cheong, Cornell University – Second Year (2 Year Grant)

    One Sentence Summary: Develop a diagnostic test that can rapidly detect, identify, and determine the antifungal susceptibility profile of clinical equine samples to improve treatment outcomes of fungal infection in horses.

    There is a potentially long lag-time for veterinarians to obtain results from fungal culture and antifungal susceptibility testing. This delay forces veterinarians to guess which treatment to start with while waiting for results and even discourages veterinarians from submitting the sample at all. The unintended consequences of making the wrong antifungal treatment include: worsening of the disease, selection of resistant fungal agents, the high cost of treatment, and the erosion of trust between the owner and the veterinarian. The longer the delay in getting the results, the more likely that unintended consequences will occur. There is also very little information that is available to help guide the treatment selection for the veterinarian. This proposal seeks to explore two primary areas: the first is to use new molecular tools for rapid diagnosis and identification of fungal infections; and the second is to improve our knowledge of antifungal resistance in fungal isolates from clinical horse samples. Our long-term goals are to develop a rapid diagnostic test for fungal infection in horses and improve antifungal susceptibility screening to help guide clinician treatment selection for fungal infections in horses.

    One of the most commonly used molecular methods to quickly diagnose the presence of an infectious agent is the use of polymerase chain reaction (PCR) which detects and amplifies specific DNA sequences. Most rapid PCR tests are designed to be specific to one type of infectious agent, for example for equine herpesvirus-type1, to reduce mistakes. However, there are many different types of fungal agents that can cause disease in horses making it difficult to design a rapid PCR test for all of the common fungal organisms that infect horses. We propose to use a PCR strategy devised to study bacterial communities which detects and amplifies two common genes that are present in all fungal organisms yet have unique signatures that can be used to identify the species of fungal agents. This test has not yet been validated for use with fresh samples and not for horse samples. One limitation is the sensitivity of this test which makes it prone to over-diagnosis if the samples are from a ‘dirty’ site that is contaminated with environmental fungal organisms. We, therefore, propose to first test this assay on the supposedly clean uterine samples of horses that are collected using sterile technique and compare the results to the conventional fungal culture methods. We believe that the development of this test will be an invaluable diagnostic tool to improve the treatment of uterine fungal infection in horses.

    Resistance to antifungal drugs is an important consideration for treatment selection. Many fungal agents form a biofilm which is a complex mixture of secretions by the fungal organisms and often includes a change in the structure of the fungal organisms themselves. This layer of biofilm protects the fungal organisms from both the immune system and some antifungal drugs. The materials that make up biofilm are quite variable between different types of fungal organisms and therefore the protection against antifungal drugs also varies between different species of fungal organisms. The effect of biofilm on antifungal drug resistance is poorly studied in general and has not been examined in fungal isolates from horses. Current tests to determine antifungal drug susceptibility are done on cultures that are on agar or in liquid culture medium and the fungal organisms are not in biofilm. There are assays that test fungal organisms in biofilm for resistance to antifungal drugs but these have not been done on fungal organisms relevant to horses. Therefore we propose to perform antifungal susceptibility profile of fungal organisms from horses that are from different body sites and compare the antifungal susceptibility profile in biofilm with the conventional antifungal susceptibility test. We will save the fungal organisms that are resistant to antifungal drugs and determine if they have genes known to provide antifungal resistance. The hope is that these genetic markers can be used to develop rapid PCR diagnostic tests in the future. We expect biofilm from different fungal species will have different antifungal drug susceptibility and this information will be beneficial to guide treatment decisions for fungal infection in horses.

    This proposal seeks to validate a rapid diagnostic test for fungal infections in horses starting with uterine samples and determine if biofilm affects antifungal susceptibility resistance and how this may differ in different species of fungal agents. Ultimately, we hope to improve the health of horses and provide references for veterinarians to improve the treatment of fungal infection in horses.

    Importance to the Equine Industry: Fungal infections in horses can cause major health concerns and are difficult and expensive to treat. Treatment for fungal infections such as uterine fungal infection requires persistence and ideally information on the antifungal susceptibility but the lag-time from submission until a final result may take as long as three weeks and is a major impediment for diagnosis and treatment of this disease. Treatment of fungal infections should ideally be based on fungal culture and antifungal susceptibility profile. However, a primary limitation of the current diagnostic tests for fungal infection is the slow turn-around time taking as much as three weeks before final results are reported. Therefore, veterinarians have to resort to treating horses without a definitive diagnosis and without antifungal susceptibility profile. The proportion of fungal organisms that are resistant to antifungal drugs are increasing over time for some types of antifungal drugs. Treating horses with ineffective antifungal drugs are not only expensive but the lingering infection can cause longer-term health consequences. Therefore, a rapid diagnostic test would greatly enhance the willingness of veterinarians and owners to pursue diagnostics and minimize disease duration. The second goal is to provide a reference for veterinarians on the antifungal susceptibility profiles of common fungal species affecting horses.


    BISPHOSPHONATES AND FATAL MUSCULOSKELETAL INJURY
    Heidi Reesink, Cornell University – Second Year (2 Year Grant)

    One Sentence Summary: Determining the prevalence of bisphosphonate use in racehorses and whether bisphosphonates are associated with fatal musculoskeletal injury is essential to equine welfare and the future of racing.

    Horseracing is under increased scrutiny, especially due to recent clusters of fatal musculoskeletal injury (FMI) events at prominent racetracks. Aside from the unfortunate loss of equine life and risk to human jockeys, racehorse fatalities significantly impact public perception and lead to substantial economic losses for the equine industry. Fractures of the proximal sesamoid bones are the most common cause of racehorse fatality in New York, California, Kentucky, Florida and Hong Kong(1–6). While the epidemiology of FMI is complex and multi-factorial, there is widespread concern that recent clusters of fatalities may be due, in part, to the approval of the bisphosphonates clodronate (Osphos®) and tiludronate (Tildren®) in 2014. Bisphosphonates are known to interfere with the normal remodeling process of replacing old bone with new bone and result in increased microfractures in human bone.

    There is concern that bisphosphonates might impair the normal adaptive remodeling and repair processes in equine bone during race training. However, whether or not bisphosphonates are associated with equine FMI is an open question, and one that the racing industry needs to answer.

    Whereas bisphosphonates can only be detected in serum and urine for a relatively short period of time (~30 days), these drugs significantly accumulate in bone, resulting in a long-term reservoir for continuous release of bisphosphonate for months to years, even after the medication is discontinued(8). Bone concentrations of tiludronate have been detected in horses for up to 1 year post-treatment(9), and the half-life of clodronate in rodent bone is estimated to be months to years. Although harvesting bone biopsies is not a realistic approach for screening for bisphosphonates in live racehorses, it is the ideal tissue for estimating how commonly bisphosphonates are administered to the racing population.

    In Aim 1 of this proposal, a case control study, especially suited to the rapid acquisition of large data sets for investigating “rare” events such as FMI, will be employed to estimate how common bisphosphonate administration is in the young, exercising Thoroughbred racing population in New York using a sample of 82 animals that have already been acquired, plus an additional 100 animals that will be collected over the next year. Here, we propose to leverage Cornell University’s expertise in nuclear magnetic resonance (NMR) spectroscopy and SUNY-Morrisville/New York’s Equine Drug Testing and Research Program’s expertise in ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) small molecule detection methods to identify bisphosphonates in equine bone tissues. This expertise, coupled with our access to a cohort of 66 TB racehorse cadaver bone samples (expected to be 82 by 4/1/20) that have been extensively characterized through advanced imaging approaches, including computed tomography (CT) and bone density and bone quality imaging methods, will enable us to address important questions about the effects of bisphosphonate administration on bone remodeling. In Aim 1.3, we will evaluate the effects of bisphosphonates on bone remodeling in the young racing Thoroughbred population, with ~50% of our cohort including 2- and 3-year old horses with complete training and racing exercise histories.

    In Aim 2, we will apply similar techniques to determine how a single dose of the bisphosphonates clodronate and tiludronate results in detection of bisphosphonates in blood, urine, bone, hair and hoof tissues. While LC-MS/MS approaches can detect lower concentrations of bisphosphonates than NMR approaches, NMR is faster, less expensive, requires less sample preparation and may have value as a screening method. Furthermore, this Aim 2 dovetails with ongoing work in the New York Equine Drug Testing and Research Program laboratory directed towards improved method development for low-level detection of bisphosphonates in blood.

    Optimization of techniques for measuring bisphosphonates will advance the current state-of-art in racing medication drug testing efforts. Our ultimate goal is to help racehorse veterinarians, owners and trainers make informed decisions about fracture risk while improving equine welfare, mitigating negative publicity, and enhancing our understanding of the impact of bisphosphonates on bone remodeling and fracture risk.

    Importance to the Equine Industry: Fatal musculoskeletal injury (FMI) is of utmost concern to the horseracing industry, as public outcry about racehorse deaths is threatening the future of the sport and the livelihood of trainers, jockeys, veterinarians and other stakeholders. Speculation abounds about whether bisphosphonate administration in young racehorses could be a contributing factor to recent spates of fatal musculoskeletal injury (FMI) at prominent racetracks; however, there is a paucity of evidence to support or refute these claims.

    No data is available about how common bisphosphonate administration is in the young Thoroughbred population, with estimates spanning from LT 1 to GT 80% of horses. We are literally racing blind—often with owners, trainers, and racing jurisdictions unaware of whether their horses have been treated with bisphosphonates; if they have been treated, how frequently or how many doses they’ve received; and whether administration of bisphosphonates places horses at risk of injury due to altered bone remodeling, analgesic properties, or a combination of both.

    This proposal leverages advances in NMR and high-sensitivity mass spectrometry techniques for extracting and quantifying bisphosphonates in bone tissue, as well as in plasma and urine. In addition to estimating the prevalence of bisphosphonate administration in NY Thoroughbred racehorses, this study will allow us to determine whether bisphosphonates are associated with catastrophic proximal sesamoid bone fracture—the leading cause of FMI in U.S. racehorses—and to determine whether bisphosphonates are associated with altered bone remodeling in the fracture-prone distal MC3 condyle and proximal sesamoid bones. Our ultimate goal is to help racehorse veterinarians, owners and trainers make informed decisions about fracture risk while improving equine welfare, mitigating negative publicity, and enhancing our understanding of the impact of bisphosphonates on bone remodeling and fracture risk.


    Enhancing The Efficacy Of Mesenchymal Stem Cells For Tendon Healing
    Lauren Schnabel, North Carolina State University – Second Year (2 Year Grant)

    One Sentence Summary: This proposal examines the tendon inflammatory environment following acute injury and the effect of such an environment on mesenchymal stem cells (MSCs), with the goal of improving MSC treatment efficacy.

    Digital flexor tendon injuries occur frequently in athletic horses and are particularly debilitating and costly due to both their extensive healing time and their high re-injury rate (50% to 70%). One of the most promising treatments for tendon injury over the past decade has been the onset of mesenchymal stem cell (MSC) therapy. MSC therapy has been shown in experimental models to improve the quality of tendon repair in horses and other species and has also been shown clinically to reduce re-injury rates of naturally occurring superficial digital flexor tendon (SDFT) injuries in horses. Despite these positive advances, the precise mechanisms associated with MSC-induced tendon repair have yet to be elucidated.

    New evidence suggests that MSCs improve healing through signaling with the tendon injury environment, enhancing many important processes, including recruitment of blood vessels and nearby cells that are important for tendon healing. A key question relating to MSC therapy is the timing of treatment, as there is little doubt that the post-injury inflammatory environment within the tendon is critical to MSC-initiated healing. Although the traditional paradigm is to wait until the acute inflammation has subsided in the tendon before initiating MSC treatment, we propose the opposite, that the peak inflammatory environment is in fact the most beneficial for MSC transplantation to facilitate improved tendon healing, as this environment will cause the MSCs to produce pro-healing factors. The overall goal of this proposal is to characterize the inflammatory environment in acute tendon injury and assess how the inflammatory environment affects the factors produced by MSC. We hypothesize that expression of inflammatory cytokines changes over time following acute tendon injury and that the inflammatory cytokines present following acute tendon injury will actually enhance the ability of MSCs to heal tendons.

    Importance to the Equine Industry: Digital flexor tendon injuries occur frequently in athletic horses and are particularly debilitating and costly due to both their extensive healing time and their high re-injury rate (50% to 70%). Rehabilitation from a typical tendon injury takes at least 6 months and often up to 1 year for full return to work. A re-injury during or after this rehabilitation period is devastating to any horse’s career, but is especially devastating to that of a racehorse with a limited age and time frame for peak performance.

    One of the most promising treatments for tendon injury over the past decade has been the onset of mesenchymal stem cell (MSC) therapy. MSCs have been shown to both improve the quality of tendon healing and reduce the tendon re-injury rate in horses, yet the mechanism of action is unknown as is the optimal time to treat with MSCs. While the current strategy has been to wait until acute inflammation has subsided in the tendon before initiating MSC treatment, we propose that an injured tendon should be treated with MSC during the peak of inflammation based on our preliminary data. In this proposal, we seek to characterize the tendon cytokine inflammatory environment and then test if the main proteins present in that environment can be used to stimulate MSCs to secrete factors that result in improved tendon healing compared to unstimulated MSCs. Knowledge gained from these studies will guide future in vivo studies as well as the clinical use of MSCs in horses for the treatment of tendon injuries in order to maximize the benefit of MSCs and significantly reduce tendon re-injury rates.


    AMPK AGONIST COMBINATION THERAPY AND ID IN HORSES
    Teresa Burns, The Ohio State University – Second Year (2 Year Grant)

    One Sentence Summary: By completing this work, we hope to characterize a combination therapy to improve equine insulin resistance that is administered orally and well tolerated.

    Our research team has recently shown that two drugs, metformin and aspirin, act synergistically (greater effect than either drug alone) to improve insulin resistance (IR) when administered as a combination to adult light-breed horses. Resveratrol (RES), another drug that acts like metformin and aspirin, would be another attractive therapeutic option for equine IR, both due to its ability to influence multiple pathways that affect IR (in addition to those affected by metformin and aspirin). Our team has shown that there are pathways that are associated with laminitis that could be inhibited by RES, which would be a highly desirable therapeutic strategy (especially given that RES is safe, palatable, and effective). It also may be effective when given in combination with other drugs like metformin and aspirin, but this hasn't been evaluated in horses to date. Given our laboratory’s interest in establishing practical, effective, and safe medical therapies for equine IR and laminitis, we would like to build on our recent work by evaluating RES and combinations in a model of dexamethasone-induced IR. If RES further enhances the ability of the MET/ASP combination to improve IR, this will provide support for a novel combination medical approach; if RES given alone is as effective as MET/ASP, then a simplified approach (that is also palatable and well-tolerated) may be feasible. The effects of this drug combination in equine ID have not been previously investigated.

    In this proposal, we hypothesize that RES therapy will improve systemic IR in light-breed horses and will act synergistically with MET and ASP in improving IR when co-administered with these drugs. Further, we hypothesize that treatment with the AMPK agonists proposed in this study will inhibit multiple pathways that might confer ability to prevent and treat endocrinopathic laminitis. Finally, we will investigate how the drug combinations in this study alter equine IR by evaluating parameters measured by the frequently sampled insulin modified IV glucose tolerance test. In the proposed study, we will induce IR with dexamethasone in 35 horses and assess the effects of RES, MET/ASP, RES/ASP, RES/MET/ASP, and placebo (CON) treatment by 1)assessing the effect of RES on systemic IR and on tissue signal pathway activation in light-breed horses, 2) comparing the relative abilities of co-administration of various AMPK agonist combinations (RES,MET/ASP, RES/ASP, RES/MET/ASP) to mitigate IR, and 3) identifying which facets of IR(systemic IR, insulin secretion from the pancreas) are affected by these medications.

    Importance to the Equine Industry: Equine metabolic syndrome-associated laminitis (EMSAL) is the most common form of laminitis in horses, ponies, and donkeys in industrialized nations. Similar to human metabolic syndrome, the condition is now known to affect not only obese animals, but also horses maintained even in ideal body condition for their occupation or breed. Nutritional and exercise management are central to treatment of this condition, but both can be difficult in the face of severe IR and the severe pain of laminitis, especially in genetically predisposed breeds (like ponies, Arabians, and Tennessee Walking Horses, for example). Thus, in addition to dietary and exercise management, it is critical to establish medications that improve insulin and glucose dynamics in animals at risk for or already suffering from EMSAL; drugs that both improve systemic IR and protect the digital lamellae are particularly attractive prospects. Many of the therapeutics available for treating IR activate important metabolic signaling pathways, like an enzyme called AMPK that is present in virtually all cells. This enzyme has been referred to as ''exercise in a bottle'', and drugs that activate it might be particularly useful for foundered horses that can’t work due to foot pain. Few drugs that activate this enzyme have been critically evaluated in horses, particularly when administered in combinations.


    SDFT ADAPTATION IN THOROUGHBRED RACEHORSES
    Sushmitha Durgam, The Ohio State University – Second Year (2 Year Grant)

    One Sentence Summary: The impact of training and racing on (mal)adaptations in superficial digital flexor tendon hierarchical structure will be evaluated to delineate the pathophysiology of this common injury in racehorses.

    Superficial digital flexor tendon (SDFT) injury is a common cause of wastage in Thoroughbred racehorses. At high speeds, this tendon functions close to its maximal capacity and therefore prone to strain injury. The tensile strength of SDFTs is primarily due to sliding motion that occurs between collagen fiber bundles, called fascicles and is facilitated by the interfascicular matrix present between the fascicles. The interfascicular matrix is rich in elastin and imparts tendon its elasticity.

    The SDFT samples analyzed in this research will be obtained from 2-, 3- and 4-yo Thoroughbred racehorses necropsied through California Animal Health and Food Safety Laboratory System (CAHFS) for this research. We will determine if the specialized hierarchical structure (fascicle size, interfascicular matrix thickness and interfascicular elastin content) and function (mechanical strength) undergoes (mal)adaptation in response to race training.

    The existing studies in this regard is minimal and uses outdated methodologies focused strictly on longitudinal fiber pattern. This research will evaluate the effects of training/racing on the specialized structure of SDFT which is primarily responsible for tendon elasticity. We will use Second Harmonic Generation (SHG) microscopy which is the current research industry standard for assessing collagen-rich biological tissues and combine with elastin immuno-staining.

    These findings will improve our understanding of mechanisms involved in SDFT remodeling and injury development, since most if not all are cumulative in nature. These outcomes will lay a strong foundation for ongoing research.

    Importance to the Equine Industry: There is growing evidence that superficial digital flexor tendon (SDFT) injuries are due to chronic over-strain pathology, that is, a consequence of cumulative changes/degeneration within tendon structure rather than a single traumatic event. This research will generate a data base documenting the adaptive (and, potentially, maladaptive) responses of Thoroughbred flexor tendons to athletic training. This in turn will contribute to our understanding of pathophysiology of SDFT injuries in Thoroughbreds, a major problem in the racing industry and a source of considerable frustration with clinicians, trainers and owners, given the high recurrence rate of these injuries. We will specifically focus on evaluating the effects of athletic training/racing on SDFT hierarchical structure and function.


    DYNAMICS OF VITAMIN D IN HOSPITAL FOALS
    Ramiro Toribio, The Ohio State University – Second Year (2 Year Grant)

    One Sentence Summary: Critically ill foals often have low blood levels of vitamin D; our goal is to investigate if their levels over time are associated with the severity of their disease and mortality.

    Sepsis is a condition where bacteria multiply in blood and set up widespread infections throughout the body, often leading to death. Consequences of sepsis include loss of hormonal control, metabolic/ electrolyte/fluid derangements, and organ failure. Vitamin D has a multitude of functions. It is important for the intestinal absorption and kidney reabsorption of calcium and phosphorus, it is essential to maintain healthy bones, controls the immune system, reduces inflammation, it has antimicrobial properties, it helps with the integrity of the skin, and participates in energy metabolism. For vitamin D to go the different organs it requires a transporting protein (DBP).

    We have documented that critically ill foals often have low levels of vitamin D that are associated to the severity of their illness and mortality. In critically ill people, low vitamin D levels have been linked to outcome. Information on vitamin D in sick foals is minimal. There is also a lack of information on the association between vitamin D with other hormones, calcium and phosphorus, inflammatory factors, and severity of illness.

    Our goal is to measure the blood levels of different forms of vitamin D over time in hospitalized and healthy foals, to determine their association with other hormones and proteins, as well as with severity of disease and mortality. This research will be relevant in understanding diseases of newborn foals, which could have clinical and therapeutic implications.

    Importance to the Equine Industry: This project will investigate a recently documented problem in critically ill foals (vitamin D deficiency) for which information is minimal, its implications to foal health are unknown, but preliminary data indicates an association with disease severity and mortality. Elucidating the importance of vitamin D in the pathogenesis of equine neonatal illnesses will have diagnostic, prognostic, and therapeutic implications.

    During sepsis, various homeostatic systems try to maintain the foal viable. It is then reasonable to assume that by better understanding endocrine and metabolic processes in critically ill foals, our ability to reduce mortality will improve. Cost of treating a critically ill foal in the US varies from $200 to $1500/day, and the prognosis of survival ranges from 35-60%. Thus, sepsis has a negative economic impact on the equine industry.

    We have made substantial contributions in understanding hormonal regulation in critically ill foals and horses. Our research have shifted some paradigms on the pathophysiology and therapeutic approaches for a number of conditions in hospitalized foals. This project will be a continuation of our work on equine neonatal endocrinology, calcium dysregulation, and mechanisms leading to systemic inflammation, organ dysfunction, and mortality. We will take advantage of our clinical and research expertise, as well as collaborations with other academic institutions and the private sector.

    Information from this study will lead to future research (interventional, mechanistic) on disorders of the critically ill equine neonate. It will contribute to the training of students/graduate students interested in equine clinical and translational research (relevant to the profession and the equine industry). It will enhance collaborations between academia and the private sector. Therefore, this research is in line with the mission of the GJCRF regarding impactful investigations.


    ASTHMA, PERFORMANCE AND OMEGA-3S IN RACING THOROUGHBREDS
    Laurent Couetil, Purdue University – Second Year (2 Year Grant)

    One Sentence Summary: The main purpose of the study is to investigate the variability of asthma severity in horses racing across the US, its effect on performance and determine if omega-3 PUFA supplementation is beneficial.

    Equine asthma is an important cause of poor racing performance. A study of Thoroughbreds racing in Indiana demonstrated that 80% of examined horses had mild asthma. It is not known if asthma is equally common among horses racing elsewhere in the US or if the type of inflammation varies based upon climate or geographic location.

    The effect of asthma upon racing performance varies with the type of inflammation present. The airways of asthmatic horses may have an increase in one or any combination of inflammatory cells: mast cells, neutrophils, or, rarely, eosinophils. In Indiana Thoroughbreds, mast cell inflammation has roughly twice the negative effect on racing performance that neutrophil inflammation does. The degree of mast cell inflammation varies with fungal exposure, while the degree of neutrophil inflammation is related to overall dust exposure. Thus, it is likely that different environmental triggers lead to different types of inflammation and is likely to vary with different climates and geographic regions.

    Because reducing exposure to organic dust and its fungal components is important to prevent and treat equine asthma, low-dust forages such as steamed hay, pelleted hay, and haylage have been investigated. Haylage appears to offer additional benefit beyond that of reduced dust exposure alone, presumably due to a more beneficial nutritional profile higher in omega-3 polyunsaturated fatty acids (omega-3 PUFA). Omega-3 PUFA work to resolve inflammatory processes and offer a potential natural way to treat and prevent asthma in the equine athlete. However, haylage is not readily available in most US locations and can carry a risk of botulism. Rather than changing a horse’s forage, adding a dietary supplement high in omega-3 PUFA would be an attractive alternative.

    Therefore, this study has 3 main goals: 1. Investigate the variability of asthma severity and type in horses racing in differing climate conditions and geographic regions across the US, 2. Examine the effect of asthma upon racing performance, and 3. Determine if omega-3 PUFA supplementation can reduce the asthmatic inflammatory response.

    Importance to the Equine Industry: Equine asthma, formerly referred to as inflammatory airway disease, is extremely common in racehorses, with up to 80% of horses racing in Indiana affected. It is unknown whether the frequency of asthma diagnosis in actively racing horses or the type of inflammation diagnosed varies across the US.

    While asthma is very common in racehorses, signs of the disease are subtle and may not be recognized in all cases. When asthma is diagnosed, racehorses are commonly treated with corticosteroids, bronchodilators, antimicrobials, and nebulized substances, despite a lack of evidence to support their effectiveness. In addition, these treatments carry the risks of violation of medication statutes, adverse side effects, drug resistance, and negative public perception.

    Therefore, means of managing equine asthma with natural dietary products are of great interest to veterinarians, trainers, and racing boards alike. Reducing horses’ exposure to dust is often limited by real-world constraints: haylage is not universally available and carries the risk of botulism, steaming hay is labor-intensive, and feeding pelleted hay can be costly while increasing the risk of unwanted behaviors in stalled horses, such as cribbing. Dietary supplementation that corrects the relative deficiency of omega-3 PUFA in stabled horses fed dry hay may aid in the resolution of airway inflammation, reduce the use of potentially harmful drugs, and improve the welfare of not only racing horses, but equine athletes in general.


    EFFECT OF NEBULIZED LIDOCAINE IN TREATING EQUINE ASTHMA
    Melissa Mazan, Tufts University – Second Year (2 Year Grant)

    One Sentence Summary: We propose to evaluate the efficacy of inhaled lidocaine in equine asthma in reducing airway inflammation and hyper-responsiveness by promoting an anti-inflammatory lung environment.

    We propose to evaluate the efficacy of nebulized lidocaine in equine asthma in reducing airway inflammation and hyper-responsiveness by promoting an anti-inflammatory lung environment. Equine asthma is endemic in stabled horses of all ages, is a pervasive cause of poor performance, and is characterized by airway inflammation, ‘twitchy’ airways, and cough. Equine asthma results as a response to both classic allergens in the environment, such as the molds spores in hay, as well as a non-specific response to irritants and inflammagens in even the best of barns; it is unlikely that even good management will be successful in eliminating equine asthma where horses are stabled.

    The pharmaceutical approach to equine asthma remains reliant on corticosteroids and bronchodilators such as albuterol, both of which have undesirable systemic side effects. Corticosteroids can impede healing, suppress the immune system, and cause muscle wasting, in addition to worsening common metabolic diseases; bronchodilators often lose efficacy due to downregulation of receptors, and can cause cardiac abnormalities. While inhaled versions of these drugs are generally safer, they are often prohibitively expensive; moreover, pharmaceutical industries are phasing out the commonly used metered dose inhalers further impeding our ability to effectively treat this disease. Lidocaine, a drug which is commonly used as a local anesthetic, has been shown to have anti-inflammatory effects when used intravenously in horses with colic and people with post-surgical inflammation. Nebulized lidocaine has been demonstrated to ameliorate chronic cough and improve lung function in people with asthma, as well as improve lung function through modulating immune responses; it has also been shown to improve lung function in an allergen model of asthma in cats.

    We hypothesize that nebulized lidocaine will be a safe and effective anti-inflammatory treatment in horses with equine asthma. Our preliminary data show improvements in clinical signs, lung function, and airway inflammation in horses with moderate to severe equine asthma and all horses treated tolerated treatment well, but these early data do not compare lidocaine to placebo, and important questions about safety, effectiveness, and mechanism of drug action remain. The next steps to leveraging this novel and potentially safer and effective treatment are 1) to use unaffected horses from our research herd to determine the extent of systemic absorption and penetration to the lower airways, document if any adverse effects occur to the upper airway, and to ensure that normal horses do not experience lower airway inflammation, and 2) to use client-owned horses with equine asthma to compare treatment with nebulized lidocaine v. placebo to determine if lidocaine results in improved clinical signs and decreased cough, decreased BAL cytological inflammation, and decreased airway reactivity in equine asthma, and is accompanied by a change to an anti-inflammatory immunologic response in the lung. Lidocaine is inexpensive, has a wide safety margin when given by nebulization, and represents a real opportunity for a new approach to this devastating disease.

    In order to prove these hypotheses we will first treat horses from our research herd with a single dose of nebulized lidocaine and monitor blood, urine, and BAL for lidocaine levels as well as endoscopically monitoring upper airway function. We will then recruit horses with veterinarian-diagnosed mild-to-moderate asthma for initial enrollment. We will characterize horses through use of physical examination with clinical and exercise scoring, lung function, and analysis of lung fluid (bronchoalveolar lavage, BAL) to quantify airway inflammation. Horses will be randomly assigned to one of 3 groups (8/group) for twice daily nebulization for 7 treatments with either 1) 4% lidocaine solution (1 mg/kg); 2) 0.9% sterile saline at equal volume; or 3) mask placement only as a control. We will repeat testing as well as monitoring blood and urine levels of lidocaine in order to determine if lidocaine treatment successfully ameliorates signs of equine asthma and in order to assess systemic deposition. We will harvest lung fluid (BAL) both before and after treatment to determine if lidocaine induces an anti-inflammatory profile in the lung. It is of critical importance to develop effective, inexpensive, and safe methods to treat equine asthma and thereby relieve the equine industry of an important impediment to equine performance, and the combined strengths of our laboratories in clinical, physiological, and immunologic testing puts us in a strong position to do so.

    Importance to the Equine Industry: Equine asthma takes a severe toll on the equine industry by negatively affecting athletic performance and decreasing days spent competing as well as by incurring considerable veterinary costs to the owner. Equine asthma in its early stages, if left untreated, increases the risk that a horse will develop the crippling form of disease known as recurrent airway obstruction (RAO, or heaves). Diagnosis of equine asthma can be subtle, as small diminutions in respiratory function can have marked negative effects on high performance athletes. Currently available treatments, namely corticosteroids and beta-2 adrenergic bronchodilators, are little different to what the veterinary field was able to offer 20 years ago. Corticosteroids are remarkably effective in combatting inflammation and bronchospasm, but are accompanied by adverse side effects such as poor wound healing, muscle wasting and exacerbation of equine metabolic syndrome. Bronchodilators such as albuterol and clenbuterol effectively relieve bronchospasm, but can result in cardiac abnormalities and, when given repeatedly, will cease to work at all. Inhaled corticosteroids have fewer systemic effects, but they are prohibitively expensive, thus many owners cannot afford this treatment modality. Moreover, metered dose inhalers are being systematically phased out by pharmaceutical industries in favor of dry powders that are not yet suitable for use in horses. For all of these reasons, it is imperative that we identify as yet unexplored treatments that are safe, effective, and inexpensive: nebulized lidocaine, which already has demonstrated efficacy and safety in human asthmatics, presents an untapped resource in the treatment of airway inflammation and cough that characterizes equine asthma and prevents equine athletes from achieving their full potential.


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    Bisphosphonate Effects on Biomarkers
    Heather Knych, California-Davis – Second Year (2 Year Grant)

    One Sentence Summary: This study will allow for development of sensitive and alternate methods for detection of bisphosphonates.

    Bisphosphonates are anti-resorptive agents that have been used for several years in human medicine. Recently, two veterinary products, labeled for use in horses over 4 years of age, were approved by the Food and Drug Administration. Although not recommended for use in young horses, there are reports of bisphosphonate administration in young racehorses. There are currently no published reports describing the effects of bisphosphonates on the skeleton of growing horses, however, by virtue of their effects on bone, administration of bisphosphonates is theorized to have detrimental effects on normal bone development in young horses. Inhibition of bone resorption is also believed to interfere with normal bone healing, which is imperative to the repair of microcracks observed in the bones of racehorses. Due to concerns regarding the use of bisphosphonates in young animals in active training, where bone development and remodeling are especially important, some racing jurisdictions have adopted a policy prohibiting the use of this class of drugs in racehorses.

    Although analytical methods for the detection of bisphosphonate administration have been developed and are utilized by drug testing labs, they only remain in the blood for a short period of time, make regulation of their use in performance horses challenging. In the current grant proposal, we propose to use determine drug concentrations, assess proteins known to be altered by bisphosphonate administration in other species and genetic biomarkers to develop a sensitive method by which to detect administration of the bisphosphonate, clodronate.

    In the proposed study, 10 exercised two-year-old Thoroughbred horses will be studied. Six horses will receive clodronate according to the manufacturer’s instructions and 4 horses will receive an equivalent volume of saline. Blood, urine and hair samples will be collected prior to, during and post drug administration for drug concentration determination and analysis of genetic and protein biomarkers.


    NOVEL DELIVERY OF ANTIMICROBIALS INTO EQUINE JOINTS
    Simon Bailey, University of Melbourne – Second Year (2 Year Grant)

    One Sentence Summary: The development and testing of, a novel (gel) carrier formulation for the antibiotic Cefuroxime, injection into horses’ joints for application as a treatment of joint infections.

    Antibiotics are used in humans and animals for the treatment and prevention of infections. The development of resistance by bacteria against antibiotics is occurring worldwide and is a crisis for the future heath of animals and people; the One Health principle recognizes inextricable links of human and animal health. Antibiotic resistance is recognized as an urgent, global, One Health emergency by the Centers for Disease Control and Prevention (CDC). Humans are dying from infections with bacteria that are resistant to every known antibiotic. This crisis has developed, in part, from complacency in critical thinking of the most appropriate way antibiotics are used. The likelihood of new classes of antibiotics being discovered is low, and therefore we need to ensure the ongoing effectiveness of the antibiotics that are currently available.

    Due to this One Health emergency of antibiotic resistance, veterinary use of antibiotic necessitates a twofold approach. First veterinarian must reconsider the use of some ‘older’ antibiotics that have in recent decades been replaced with newer antibiotics. Second, methods of administering the antibiotics that both optimize the effectiveness in killing bacteria and decrease the risk of bacteria developing resistance to antibiotics need to be developed e.g. by adjusting the amount of drug given, timing of doses and route by which the antibiotic is administered to the animal.

    When selecting antibiotics for treatment of bacterial infections in horses, veterinarians are guided by a tiered scale of antibiotics in terms of their importance to human health (as dictated by the WHO). Currently there are few options from which veterinarians can choose in the lowest tiers of antibiotic importance. The number of antibiotics that are suitable to be instilled into the joints of horses are even fewer. If there is no available antibiotic to which the infection is sensitive in this lower class, veterinarians must use the more reserved drugs. This puts veterinarians in a challenging ethical position attempting to balance the horse’s treatment and recovery with the risk to human health.

    The second-generation (2ndG) cephalosporin antibiotics are nowadays no longer considered to be among the latest drug classes used to treat bacteria that are potentially resistant to other antibiotics, having been replaced with newer ‘generations’ of cephalosporin antibiotics. The World Health Organization (WHO) does not consider protection of the use of the 2ndG cephalosporin class of antibiotic as critically important for use in human medicine, so they are attractive for re-evaluation for veterinary use. Furthermore, these drugs may have a perfectly suitable antibiotic activity for combatting all the different species of bacteria that are commonly found in infections of horses, including infections in joints.

    Importance to the Equine Industry: Horses commonly sustain wounds due to accident or misadventure. If the wound involves a joint, then infection in the joint can be established. Infections in joints are very difficult to clear and can lead to the death of the horse if not able to be cleared.

    Despite being discovered approximately 45 years ago, there is little use of 2ndG cephalosporins in veterinary medicine; there is one licensed 2ndG cephalosporin preparation for use into the udder of cows. Theoretically, and supported by our preliminary data, Cefuroxime, a 2ndG cephalosporins has a good spectrum of activity for killing the bacteria which commonly cause infections in horses. More information is required on the sensitivity of these bacteria to 2ndG cephalosporin antibiotics.

    For 2ndG cephalosporin antibiotics to effectively kill bacteria, they must remain above a critical concentration of antibiotic. If they drop below this concentration, they are less effective and allow bacteria to multiply. The first bacteria to start re-growing are those that are least sensitive to the antibiotic: this is the cause of increasing antibiotic resistance. Because cephalosporins are cleared rapidly from joints of horses, to use the drug preparations currently available it would be necessary to injecting them into the joint multiple times a day. If a formulation was able keep the antibiotic in the joint for a prolonged time (for example three days), it would have benefits for the treatment effectiveness and for the welfare of the horse.

    An ideal formulation would be an antibiotic impregnated gel that when injected into joints of horses that keeps the 2ndG cephalosporin antibiotic above the critical concentration for effective bacterial killing for three days. Developing such a formulation is the purpose of this research project.


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    DIAGNOSTIC ASSAY FOR RECURRENT EXERTIONAL RHABDOMYOLYSIS
    Molly McCue, University of Minnesota – Second Year (2 Year Grant)

    One Sentence Summary: Our goal is to identify a comprehensive set of genetic markers that allow RER risk prediction before horses tie-up and preemptive management to decrease the frequency and severity of clinical disease.

    Exercise-induced tying-up (rhabdomyolysis) is a painful condition characterized by muscle cramping, rapid-onset muscle damage and possibly death. Approximately 10–25% of Thoroughbred (TB) and Standardbred (STDB) racehorses tie-up repeatedly with exercise, a syndrome collectively termed “recurrent exertional rhabdomyolysis” (RER). In addition to affecting the horse’s welfare, RER impacts the ability of affected horses to train and race, which has large economic consequences. Recent studies have conclusively shown that RER is a complex genetic disorder, with ~ 40 – 50% of RER risk in both breeds due to a horse’s genetic makeup. However, RER is not a simple genetic disease caused by a single mutation, as there are likely hundreds of alleles contributing to the overall genetic risk in both breeds.

    Our hypothesis is that an individual’s genetic risk for RER results from the combination of multiple alleles in genes that are responsible for skeletal muscle metabolism, excitability, contractility, structure, and repair. In prior work we have identified ~2,500 genes that may harbor alleles that increase a horse’s risk for developing RER. Further, we have identified an initial test set of genetic markers that can determine if a horse will become an RER case with up to 92% accuracy. Using this information coupled with whole genome sequencing we identified a far more comprehensive set of 27,000 genetic markers for further follow-up in RER cases and controls.

    In this proposal, we will use genotype data from all 27,000 markers in 2,304 horses to accomplish two important objectives:

    Aim 1. Refine and validate a genetic test that predicts an individual’s risk for RER. We have developed a genotyping assay that will allow us to quickly determine each horse’s genotype for each of the 27,000 alleles of potential interest. In this aim, we will use these genotypes and statistical modeling in 495 TBs and 476 STDBs to develop a genetic risk model that provides a probability that an individual horse will develop RER. After we have developed this initial model, we will confirm that it is accurate in an independent group of 674 TB and 659 STDB horses.

    Aim 2. Identify functional alleles underlying RER susceptibility. We will use the combined genotype data from all 1,169 TB and 1,135 STDB horses from objective 1 to prioritize alleles with moderate to large effects on RER risk for functional validation, and we will identify the genes and pathways harboring these alleles as targets for novel treatments and/or interventions to prevent RER.

    To date, there are no known published scientific studies describing the genetic mutations underlying RER, nor are there published, validated genetic tests for this important disease. The work outlined in this proposal will provide both a genetic test and critical information regarding RER pathophysiology.

    Importance to the Equine Industry: Exercise-induced tying-up causes a range of signs from undetectable muscle damage, to mild muscle soreness, to severe and extensive muscle cell damage, to life-threatening sequelae, such as inability to stand and kidney failure. It is now well established that genetics contributes to repeated episodes of tying-up in TB and STDB racehorses, a condition termed recurrent exertional rhabdomyolysis (RER) to distinguish it from sporadic cases of tying-up. RER is an important cause of illness, medical expenses, and loss of training and racing days. In fact, tying-up recurrence is so frequent in ~17% of affected TBs, that after a single bout of rhabdomyolysis, these horses do not race again that season1. Identifying individuals at risk for RER before the first episode of tying-up will allow owners and trainers to put management and training strategies in place to prevent clinical disease, loss of training and racing days.

    Upon completion of this work we will have identified and validated a set of genetic markers that can be used to estimate the probability that an individual will develop RER. This work will thereby create a valid diagnostic test that can be made available to owners, trainers and veterinarians. This RER risk model will allow us to identify individual horses before they tie-up, allowing preemptive management changes to decrease the frequency and severity of their disease. Genetic tests offer advantages over clinical diagnosis and muscle biopsy, including being more sensitive and specific and allowing veterinarians to eliminate a surgical biopsy procedure, which in turn decreases morbidity and lost training time. We will also have identified genes, pathways and putative functional alleles for RER susceptibility. Understanding the genes and pathways that lead to RER will allow for the development of treatment and management strategies that target the underlying molecular changes responsible for RER with the promise to lead to better outcomes.


    INHIBITING EHV-1 WITH ANTI-INFLAMMATORY DRUGS
    Arthur Frampton, University of North Carolina Wilmington – Second Year (2 Year Grant)

    One Sentence Summary: This study will use a tissue culture model system to test the ability of specific drugs to reduce the damaging hyper-inflammatory response that is observed in EHV-1 infected horses suffering from Equine Herpesvirus Myeloencephalopathy (EHM).

    Equine herpesvirus type 1 (EHV-1) is a major pathogen of horses and results in the loss of millions of dollars each year in the U. S. The virus is highly contagious and is readily spread from horse to horse via nasal secretions and contact with infected surroundings. Each year multiple EHV-1 outbreaks occur throughout the US and the number of horses that suffer serious consequences subsequent to infection is on the rise. Currently, while horses are routinely vaccinated for EHV-1, none of the vaccines have been shown to provide protection against the severe neurologic disease termed Equine Herpesvirus Myeloencephalopathy (EHM). Thus, in the absence of any effective vaccines, there is a desperate need for new treatment strategies for EHM that are both safe and effective. In our previous study, we showed that EHV-1 infection triggers the expression of many host immune response genes including the inflammatory chemokines CXCL9, CXCL10, CXCL11.

    While a normal inflammatory response is needed for clearance of the virus from the EHV-1 infected tissues, too much inflammation can lead to tissue pathology and, if it occurs in the central nervous system, this hyper-inflammation can lead to EHM. In this study, we will examine the ability of specific drugs to reduce the expression of CXCL9, CXCL10, and CXCL11. Four of the 5 drugs that we will test are non-steroidal anti-inflammatory drugs (NSAIDs) that are routinely prescribed for equines experiencing inflammation due to a variety of stimuli and the final drug (AG490) has been shown to strongly inhibit the expression of these 3 chemokines. In addition to reducing the inflammatory response, both the NSAIDs and AG490 have been shown in multiple studies to reduce herpesvirus and other virus infections. We hypothesize that one or more of the drugs will significantly reduce the expression of the inflammatory chemokines CXCL9, CXCL10, and CXCL11 as well as inhibit the ability of EHV-1 to productively infect cells. The new knowledge obtained in this study will reveal which drugs are best able to depress this damaging inflammatory response and decrease EHV-1 infection. One or more of these drugs can then be included as part of a treatment regimen against EHV-1, either prophylactically to limit the number of EHM cases, or post-EHV-1 exposure to reduce the severity of EHM disease.

    Importance to the Equine Industry: EHV–1 continues to be a major problem afflicting horses and the diseases associated with this virus continue to cause a significant drain on financial resources for those in the equine industry. Due to the inability of current EHV–1 vaccines to protect against the severe neurologic disease Equine Herpesvirus Myeloencephalopathy (EHM), coupled with the rise in the number of EHM cases in the U.S., there is a desperate need for new treatment options. In this study we will examine two different classes of drugs to determine which are most effective at reducing the expression of damaging host cell inflammatory mediators, as well as, reduce EHV-1 infection.

    We propose that an effective treatment option for EHV–1 infected horses will be intervention with drugs that inhibit the specific, damaging, host cell inflammatory proteins including the chemokines CXCL9, CXCL10, and CXCL11. Good disease surveillance programs and health monitoring coupled with early intervention with these anti-inflammatory drugs would be expected to reduce the hyper–inflammation observed in the central nervous system of EHM–afflicted horses post infection. If administered early after EHV–1 infection, these drugs could significantly lessen the probability that the infected horse will become paralyzed. In addition to treating horses after infection, these drugs may also be able to be administered prophylactically to reduce the chance that an EHV-1 infected horse will develop EHM.


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    RESEARCH CAREER DEVELOPMENT AWARDS

    There are two Career Development Award recipients in 2021.

    The Storm Cat Career Development Award, inaugurated in 2006, is a $20,000 grant in 2021 and is 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's winner is:


     Callum G. Donnelly Callum G. Donnelly, University of California-Davis
    Proteomic Investigation Of Equine Spinal Ataxia

    Dr. Donnelly has completed his residency program and is in a research training position under the mentorship of Dr. Carrie Finno. His project, Proteomic investigation of equine spinal ataxia, offers him an excellent opportunity to improve his research skills and put him at the forefront of precision medicine for the horse.

    The key training areas for this proposal will serve as fundamental career development opportunities, in addition to building the skills necessary for completion of the proposed study. For this project, the principal skill area will be proficiency in computational biology, with particular emphasis on the handling of large datasets. Additionally, this project has already realized the need for collaboration and will offer further opportunities in networking, scientific writing, communication and presentation skills. These are the major areas of focus for the proposed year of the project.

    The project is expected to identify novel protein biomarkers that differentiate normal horses from those with spinal ataxia, with high sensitivity and specificity. Further, we expect to demonstrate that the proteome will differ depending on the prevailing spinal ataxia etiology, thereby allowing for the discovery of biomarkers unique to each disease. The impact of this work will be profound, enabling the rapid screening of proteins that can readily be transitioned into diagnostic tests. Ultimately, it will propel equine neurologic disease diagnosis into the precision medicine age.

    Faculty supervisor: Dr. Carrie Finno, Associate Professor of Veterinary Genetics


    The Elaine and Bertram Klein Development Award is a competitive program intended to promote development of promising investigators by providing a one year salary supplement of $20,000 in 2021. This program is restricted to one award per year and is named in memory of a renowned horsewoman and her late husband, a Thoroughbred owner and breeder. The first grant was in 2015 and was funded for $15,000 with donations by the Klein family.


     Aileen Rowland  Aileen Rowland, Texas A & M University
    Efficacy Of Xenogen-Free MSCs For Osteoarthritis

    Dr. Rowland’s project will be under the mentorship of Dr. Ashlee Watts. Dr. Rowland presented a very detailed year in her research with a goal of obtaining a position as a clinical researcher in academia with the aim of advancing regenerative medicine for equine athletes and humans alike. Her training towards this goal is directed in two foundational areas: first, a solid understanding of the scientific process and research design, knowledge of molecular and cellular biology, and advancement of skills needed to be an effective researcher; second, to enhance her communication skills in regard to interactions with other veterinarians, medical professionals and scientists, as well as the public. Both of these are supported by her current position as a graduate student and member of the Comparative Orthopedics and Regenerative Medicine Laboratory at Texas A&M University.

    The objective of this study is to use a randomized clinical trial, for the treatment of naturally occurring osteoarthritis, to test the effectiveness of MSCs prepared with autologous xenogen-free culture media (XFREEMSCs) against industry-standard, FBS supplemented MSCs (FBS-MSCs) as the control treatment. The expected outcome of this clinical trial will have extreme value to the equine sports medicine community, and will also be directly applicable to human regenerative medicine.

    Faculty supervisor: Dr. Ashlee Watts, Associate Professor