The board of directors of Grayson-Jockey Club Research Foundation has authorized expenditure of $1,661,180 to fund 15 new projects and 10 continuing projects at 16 universities, and three career development awards worth $20,000 each. This marks the 8th straight year that more than $1 million has been approved. The 2022 slate of research brings Grayson-Jockey Club Research Foundation’s totals since 1983 to more than $32.1 million to underwrite 412 projects at 45 universities.
Multidrug resistant (MDR)-R. equi has emerged in the environment of horse-breeding farms in Kentucky due to overuse of antibiotics to prevent disease caused by R. equi in foals. Because MDR-R. equi leads to a higher probability of death in affected foals, it is necessary to mitigate antimicrobial resistance in R. equi to promote animal health and welfare, and to protect efficacy of antibiotics in the long term to treat diseases in animals and people. The effect of measures to decrease antimicrobial use on the dynamics of (MDR)-R. equi in the environment of horse-breeding farms is still unknown. In this proposal, we will: 1) determine temporal trends of prevalence of MDR-R. equi in the environment of horse-breeding farms over a 5-year period; 2) identify temporality of risk factors associated with persistence of resistance; 3) determine temporal trends in selective pressure in the environment; and 4) determine associations between antibiotic use in foals (from questionnaires), selective pressure in the environment (by measuring concentration of antimicrobials in soil), and frequency of MDR-R. equi in the environment (by testing soil samples). We will also perform an evolutionary analysis to compare genetic variability of R. equi isolates between 2017 and 2021 samples to investigate genetic diversification over a period of 5 years. This analysis will allow identification of genetic elements acquired or mutated that allow bacteria to adapt and evolve to maintain antimicrobial resistance genes that are potentially costly for bacteria fitness in soil. By having an overall understanding of IF and HOW MDR-R. equi persists in the environment, we will be able to recommend strategies to decrease the risk of foals getting contaminated with MDR-R. equi from the environment.
Importance to the Equine Industry: Rhodococcus equi is a great threat to equine health and welfare, and the leading cause of disease and death in foals in many states in the USA. Nonetheless, highly effective ways to prevent this disease in foals are lacking. Thoracic ultrasonography for early identification of lung lesions followed by antimicrobial treatment of subclinically affected foals to prevent the development of severe R. equi disease has led to the emergence of multidrug resistant (MDR)-R. equi in both horses and foals and their environment. In 2017, we found MDR-R. equi in the soil of 76 of 100 horse farms in Kentucky. Foals infected with MDR-R. equi are 7-fold more likely to die than foals infected with susceptible isolates. Therefore, there is a great need to mitigate antimicrobial resistance in R. equi. It is necessary that we understand the effect of measures to reduce antimicrobial use in horse-breeding farms on the dynamics of MDR-R. equi populations to better predict the risk of infection of foals with MDR-R. equi and to develop strategies to control its spread among foals and farms. Widespread macrolide and rifampin resistance in R. equi isolates has become a major emerging problem facing the horse–breeding industry and might adversely impact human health. The work proposed in this application represents the first important steps in understanding the evolution of MDR-R. equi at horse farms in response to changes in antimicrobial pressure over time. Given the importance of rhodococcal disease for the equine industry, our ability to curtail antimicrobial resistance in R. equi is of urgent importance.
Persistent breeding-induced endometritis (PBIE) in mares can cause early pregnancy loss, negatively impacting reproductive efficiency and economic returns. Following mating, a transient inflammation in the uterus (endometritis) may occur for 24 hours as an inflammatory reaction to clear dead sperm and other contaminants from the mare’s uterus. The subset of mares that are unable to resolve this inflammation are considered susceptible to PBIE, which may be the result of variation from the normal immune response to breeding. Mares that suffer from PBIE often endure repeated cycles of ineffective treatments. While many studies have evaluated PBIE in live mares (in vivo), few studies have utilized in vitro conditions (growing uterine cells in a dish in an incubator), which have largely been limited to a 2-dimensional approach and may not accurately capture the mare’s normal immune response. Three-dimensional (3D) cell cultures (organoids) are a novel method for creating a physiologically relevant representation of equine endometrium (inner lining of cells in the uterus). Created from endometrial biopsies taken during routine clinical procedures, the organoid model system provides an in vitro system to study reproductive health in mares. As we explore which aspects of the immune system are causing PBIE in mares, novel biological agents such as natural stem cell secretions (extracellular vesicles; EVs) to treat endometritis will require testing. A validated, in vitro 3D model of equine endometritis would eliminate the current dependence on mares for endometritis-related research until clinical-phase testing of evidence-based therapeutic options are developed. Endometritis is not a new problem, but it is one that requires a novel approach, such as use of an organoid model system, to understand the complex nature of the disease and resolve it without extensive use of research mares.
Importance to the Equine Industry: Up to 20% of Thoroughbred mares are affected by persistent breeding-induced endometritis (PBIE), which reduces pregnancy rates leading to significant economic cost and compromised mare welfare. A validated, in vitro 3D model of equine endometritis would eliminate the current dependence on research mares for endometritis-related research until clinical-phase testing of evidence based therapeutic options are developed, which will improve mare welfare. Endometritis is not a new problem, but it is one that requires a novel approach, such as use of an organoid model system, to understand the complex nature of the disease and resolve it without extensive use of research mares.
Palmar osteochondral disease (POD) is a well-recognized cause of poor performance and chronic lameness in Thoroughbred racehorses. Racehorses subject their fetlock joints, especially those in the forelimbs, to extremely high loads while racing and training. The repetitive stresses experienced in this region result in fatigue of the bone underlying the joint surface (subchondral bone). The body lays down more bone in an attempt to strengthen these areas, as well as heal areas of microscopic damage, but the bone that is placed is of inferior quality. In the early stages of this process, the cartilage along the back of the condyles is still viable; however, as the disease progresses, focal areas of bone become devitalized, and eventually, the overlying joint surface collapses. Historically, it wasn’t until reaching this later stage that POD was typically diagnosed by veterinarians in the field. Once this stage has been reached, the only treatment options currently available are palliative and fail to cure the disease. Unfortunately, the progression of this disease is difficult to predict and varies between individual horses. Not only do we lack an effective treatment strategy for POD, but we also lack a way to study the progression of disease. The development of an experimental model of POD that would allow researchers to study the progression in a controlled, prospective manner would open the doors for future studies on variables that may affect lesion severity and facilitate the development of an optimized treatment plan.
A technique that produces changes in the subchondral bone without affecting the articular cartilage is critical for modeling the early stages of POD. A minimally-invasive model has recently been developed and validated at CSU for the creation of bone marrow lesions (BMLs) in both sheep and rats, without damage to the overlying articular cartilage. BMLs have been identified as a frequent finding on magnetic resonance imaging (MRI) in horses with varying degrees of POD and have also been associated with the occurrence of joint surface collapse in humans. The goal of this proposed study is to evaluate the ability of this model to create BMLs in horses. This will be combined with a controlled exercise program previously shown to induce subchondral bone changes in young, untrained horses, with the goal of creating lesions similar to those seen in the early stages of POD. The investigators believe that this technique will consistently induce changes similar to those seen in the early stages of POD and in humans with flattening of the joint surface, thus allowing us to use said model to potentially improve our understanding of disease progression and develop suitable treatment strategies.
The investigators plan to induce lesions in the medial metacarpal condyles of both front fetlock joints in 3 horses using the BML technique developed in sheep. Lesion progression and horse response will be monitored over a 6-month period postoperatively using digital radiography, MRI, and computed tomography (CT). A radiologist with expertise in equine imaging will evaluate these images to help characterize each lesion. During this time, horses will be exercised on an equine treadmill to simulate active race training. Horses will be humanely euthanized at the end of the study period in order to examine the joint surface and characterize lesions on a microscopic level. The investigators anticipate that the response of each horse and progression of disease will be similar to that in horses with POD, with diagnostic imaging being suitable to monitor disease progress and help identify specific time points during which therapeutic intervention may be targeted. A successful experimental model of POD will not only help us develop improved treatment strategies for POD, but it will also help us to fill the gaps in our current knowledge of this prevalent and potentially career-altering disease process. Lastly, research in humans with advanced osteoarthritis has demonstrated the importance of treating the SCB in addition to the joint surface. Since the horse is considered one of the best models for studying orthopedic disease in humans, the results of this work and future studies could also help further develop techniques for the treatment of joint disease in humans
Importance to the Equine Industry: Palmar osteochondral disease (POD) is common in Thoroughbred racehorses and leads to significant osteoarthritis and pain in the fetlock joints. Osteoarthritis causes up to 60% of clinical lameness cases in equine athletes and accounts for the greatest single economic loss to the horse industry in the US with a loss of approximately $1 billion per year. Not only does POD limit a racehorse’s potential, but it can also result in chronic pain and increase their susceptibility to fracture.
Our goal is to develop a model of POD in horses that will allow us to better study the progression of disease and develop suitable treatment strategies. If successful, future studies using this model may also allow us to identify thresholds in the intensity or duration of training and racing that lead to the formation of POD. This information would better enable trainers to optimize a horse’s career while reducing the risk for injury. The success of this technique would also give us a way to test the effects of novel treatment strategies at various stages of disease so that we can help preserve the well-being and athletic potential of our patients.
Equine rotavirus A (ERVA) is one of the most common causes of diarrhea in foals within their first 3 months of age. This virus infects and damages the intestinal lining, impeding appropriate absorption of food and water, leading to diarrhea and severe dehydration that requires rapid implementation of medical treatment in sick foals. These sick foals are also very contagious and shed up to 109 virus particles per gram of feces, contaminating the environment. In addition, rotavirus particles are highly resistant in the environment, therefore sustaining transmission to healthy but susceptible foals through the contaminated environment. ERVA is responsible for anywhere between 20% and 77% of diarrhea cases in very young foals, outbreaks occur every foaling season, and can cause important losses to breeding enterprises. Surveillance efforts around the world have identified that there are two main genotypes of ERVA that cause diarrhea in foals, namely G3 and G14. Control and prevention strategies rely on: 1) enhancing maternal immunity to the foals via vaccination of pregnant mares and 2) biosecurity measures (including hygiene) to interrupt transmission. There are only 3 vaccines available around the world (one of which is commercially available in the US), all of which are solely based on one of two prototype ERVA G3 strains isolated in the 1980’s. All of these vaccines are inactivated, i.e., based on a killed virus preparation that is poorly immunogenic, which means that it does not elicit a strong immune response by the horse and multiple doses are required to reach adequate immunity. Mares are vaccinated during pregnancy with the hope to induce good maternal immunity. Even after multiple doses, the efficacy of this vaccine is limited as these are poorly immunogenic, induce G3-specific antibodies with limited cross-reactivity with other circulating strains of ERVA, depend on colostrum quality and intake, among other factors related to the mare, foal and environment that influence response to the vaccine. An additional limitation on the development of rotavirus vaccines is related to the difficulties in isolating and propagating viral strains for use in vaccines. In light of this, there is a critical need to develop more effective, modern next generation vaccines that induce strong and effective maternal immunity and using novel platforms that do not depend on rotavirus propagation in the laboratory and that can be updated easily based on circulating strains. We hypothesize that a viral-vectored vaccine will elicit better protection to ERVA compared to the current vaccine, and could be rapidly adapted to incorporate emerging rotaviruses from the field, including the recently described group B rotavirus. This proposal focuses on developing a viral vectored vaccine that can address existing issues related to equine rotavirus vaccination using a vector that has proven efficacious in horses against the deadly African horse sickness virus. Specifically, we will use a modified and attenuated vaccinia virus (Ankara) which will be modified in such a way that will express a highly immunogenic protein of the surface of ERVA specific to G3 and G14 strains known as VP7. Antibodies generated by the immune system against this VP7 protein have shown to effectively block rotavirus and confer protection. This attenuated vaccinia virus is non-pathogenic to horses, does not cause disease or is transmitted to other horses. We plan to evaluate the vaccine in mares and in a neonatal murine model to determine immunogenicity, safety and efficacy as a pre-clinical model and proof-of-concept. We expect this novel vaccine to significantly improve the efficacy of current vaccines.
Importance to the Equine Industry: Diarrhea in foals due to equine rotavirus continues to be a significant health issue causing outbreaks of disease in farms during every foaling season. Twenty to 77% of diarrhea cases in foals within their first three months of age are associated with equine rotavirus, while other causes of diarrhea such as those caused by bacteria and Cryptosporidium parvum are of lower impact. The impact of the disease is variable but most frequently associated with high numbers of sick foals (high morbidity), which require intensive medical management. These sick foals are highly contagious and, therefore, control of the disease also requires strict biosecurity protocols to try to minimize the contamination of the environment and continued spread. While vaccination of pregnant mares is a common method of control and prevention, the current available vaccine has limited and controversial efficacy and, therefore, the level of protection is far from optimal, undermining its wide use. No changes to this vaccine formulation have been made since its licensure in order to improve its efficacy and keep up with more current circulating strains of this virus in the horse farms. Therefore, this proposal brings a novel vaccine platform that can significantly enhance current control and prevention efforts to minimize the impact of this disease in young foals.
Superficial digital flexor tendon (SDFT) injuries are common debilitating injuries in Thoroughbred racehorses. Persistent inflammation during tendon healing and the poor intrinsic healing capacity of tendon ultimately results in poor quality of tendon repair. This in turn leads to decreased elastic strength of ‘healed’ tendon tissue and increases re-injury rates in horses. Circulating macrophages are key immune cells that become localized within tissues throughout the healing period and produce inflammatory and regulatory factors that control inflammation, pain and healing responses with the tissue. Tenocytes function to maintain tendon structure by synthesizing and secreting tendon matrix proteins. Recent findings show that inflammatory processes operating during tendon healing affect tenocyte activities such that their matrix synthesis capacity is altered and consequently, contributes to tendon degeneration. In this research, we will evaluate the effects of macrophage polarization and macrophage-derived inflammation on tenocyte activities in a tendon matrix culture model.
Although it is accepted that M1 macrophages promote inflammation and M2 macrophages are beneficial to tissue healing, the precise mechanisms associated with these said effects are not known. Moreover, recent studies have also reported controversy pertaining to these effects. Therefore, in this proposal we aim to characterize M1 and M2 macrophage-derived inflammatory factors and assess their impact on SDF tenocyte activities (gene expression profiles and matrix synthesis). We hypothesize that M1 macrophages augment inflammation and negatively affect tenocyte activity, whereas M2 macrophages promote tenocyte matrix synthesis. The second objective of this research is to evaluate the immunomodulation potential of extracellular vesicles/exosomes isolated from bone marrow stem cells, a novel orthobiologic that is cell-free, mitigates immunogenic concerns and offer off-the shelf potential to enhance tendon healing. We will utilize state-of-the-art techniques to evaluate SDF tenocyte responses to macrophages and exosomes. On completion of this research, we will identify biological and immunological factors that can be subsequently targeted to improve the quality of tendon repair. Understanding the macrophage-derived factors that control tenocyte activity is necessary for our overall goal of identifying/ developing therapies that enhance tendon healing.
Importance to the Equine Industry: SDFT injuries are one of the most common reasons for retirement in Thoroughbred racehorses. Despite requiring prolonged periods for healing, the ‘healed’ tendons are fibrotic and results in high incidence of re-injuries. Growing evidence suggests that immunomodulation is an effective strategy to improve the quality of tendon repair. Macrophages are key immune cells that modulate inflammation, pain during healing and therefore, this research will characterize macrophage-derived inflammation, and identify biological/cellular mechanisms affecting tenocyte activity and, subsequently contribute to tendon degeneration during healing. This research is will also help evaluate the beneficial effects of a novel off-the-shelf biologics/ regenerative therapy for tendon healing as several recent studies have demonstrated that immunomodulation is key mechanism to enhance tendon healing. Collectively, this work will impact develop therapies that can improve tendon injury outcomes in horses.
Mycophenolate mofetil (MMF) is an effective immunosuppressant is used in human medicine to prevent solid organ transplant rejection. Significance: MMF, is rapidly converted in the body to mycophenolic acid (MPA), and is used to suppress the immune system to inhibit the body’s immune cells from attacking its organ systems (immune-mediated disease). Horses develop immune mediated diseases. Management of equine immune-mediated disease is difficult due to the limited number of drugs, lack of response, medication costs and adverse effects. Evaluation of a safe, effective and inexpensive immunosuppressant drug for management of equine immune-mediated disease is a critical unmet need.
Our overall working hypothesis is that establishing the parameters of absorption, distribution, metabolism and excretion (pharmacokinetics, PK) of orally dosed MMF in healthy horses will provide dosing guidelines for clinical trials that evaluate its efficacy for treatment of equine immune-mediated diseases. First hypothesis: Adult healthy horses that have been fed and subsequently administrated MMF via a stomach tube will have a decrease in maximum concentration of MPA. Aim 1: Determine the influence of feeding the combination of grain and hay on the PK profile of MMF following a single dose administered via the stomach route in healthy adult horses. In humans, food-MMF interactions decrease the maximum plasma concentrations of mycophenolic acid (MPA), the active metabolite of MMF, by 40%; however, the extent of drug absorption is unchanged.
Our objective is to comprehensively characterize the PK of MMF in healthy horses in both fed and fasted states. Six horses of one breed and sex will receive a 10 mg/kg dose of a crushed aqueous slurry of generic MMF tablets via the stomach route after a 12 hour fast and 1 hour after consuming grain and hay. Blood samples will be collected time 0 (predose) then at 12 time points thereafter. PK profiles of MMF and MPA will be quantified using a validated high performance assay that we have previously developed. Determination of the PK parameters of oral MMF in fed and fasted states is required to determine the drug availability in the horse tissues and thus possible dose adjustments needed for horses receiving the drug in a fed state.
Second hypothesis: MMF given at an oral dose of 10 mg/kg every 12 hours for 7 days will not result in drug accumulation, will be safe and well tolerated. Aim 2: Investigate the PK properties, safety and tolerability of multiple repeated oral doses of MMF in healthy adult horses. Our rationale is that MMF must be given at least daily or up to every 8 hours to manage autoimmune and immune-mediated diseases in dogs and humans. As such, PK, safety and tolerability of the drug must be evaluated after subsequent multiple daily doses for an extended period of time to determine feasibility of clinical use in the equine patient. Our objective is to define the PK values, effects on blood counts, urine parameters, kidney, protein, and liver values and physiologic parameters in horses receiving MMF every 12 hours over a sequential 7-day period.
To test this hypothesis, six apparently healthy adult Standardbred mares will be administered a dose of 10 mg/kg crushed MMF generic tablets via an oral dosing syringe as a corn syrup slurry (as is common practice in equine practice) for a period of seven consecutive days. A full PK sampling screen using pre-dose (time 0) and 12 time points will be performed on Day 1 and Day 7 as will complete blood counts, biochemical profiles and urinalysis. Blood samples will be taken in the morning immediately before the next am dose of MMF on Days 2-6. Physiologic parameters including respiratory rate, heart rate, abdominal sounds, temperature, oral gum color and response as well as visuals will be recorded every 8 hours for 7 days. The results from these studies will provide the foundation for clinical trial MMF dosing regimens to investigate treatment of equine immune-mediated disease.
Importance to the Equine Industry: Addressing horse health is one of the most important and challenging issues for the equine industry. Immune-mediated diseases can either be primary (or autoimmune) in which antibodies or activated cells start to attack normal body constituents or secondary (immune-mediated) in which the antigen is foreign to the body and may include drugs, bacteria, viruses that then stimulate a reaction that results in host tissue damage. Immune-mediated disease can affect every organ system in the horse. Examples of these diseases include but are not limited to pemphigus foliaceus, respiratory disease, hemolytic anemia, recurrent uveitis and muscle disorders. For most immune-mediated diseases the prognosis is poor due to refractory disease and the life-ending adverse reactions from chronic steroid administration thus resulting in significant economic loss. New immunosuppressive medications that have a safer toxicity profile need to be investigated for treatment of horses with immune-mediated disease.
Pharmacology studies of new drugs need to be performed in apparently healthy horses to lay the foundation for the development of evidence-based therapeutic regimens for affected horses. This proposal will evaluate the drug metabolism and distribution of an immunosuppressive medication, mycophenolate mofetil. Mycophenolate mofetil has been used successfully in dogs, cats and humans to control many different immune-mediated diseases.
The goal of the proposed studies is to ultimately improve the health and welfare of the horse affected by immune-mediated disease by evaluating a novel treatment that has the potential to rapidly induce disease control and remission without undesirable multisystemic and life-threatening side effects as is prevalent with the use of corticosteriods, ultimately providing a higher quality of life while reducing morbidly and mortality.
We propose to use CD40-targeted vaccine delivery of three proteins that we consider to have a high-probability of being protective immune targets of SEE: 1) a conserved polypeptide of the M protein, 2) EndoSe; and, 3) IdeE. These three proteins will be attached to an antibody that specifically recognizes the CD40 receptor on the cell surface of the antigen-presenting “sentinel” cells (APCs) of the immune system. This strategy not only achieves targeted delivery of a vaccine “payload” into the heart of the immune system, the anti-CD40 antibody also flips the master switch that critically controls the positive outcome of the immune response. Our previous research of CD40-targeted delivery in chickens has demonstrated that a single vaccination established immunity much sooner than classical formulations, that more antibodies were produced, that they bound more strongly to the target pathogen, and that immunity was long-lasting. In one study, we reported 100% protection against infectious bursal disease virus (IBDV) after a single subcutaneous vaccination, and in another study we established 60% protection against a lethal challenge with highly pathogenic avian influenza (HPAI) after one shot, and 100% protection after a booster injection was given.
For this strategy to work in horses, we need to develop an antibody that strongly binds to and activates the CD40 receptor of the horse (equine CD40), since the chicken CD40 receptor is different from its equine counterpart. After the antibody has been designed, produced and tested for biological activity (the capacity to stimulate the APCs in the horse), we will use it to assess its capacity to enhance and amplify the immune response against a cocktail of three new protective bacterial proteins of SEE. A follow-up study in year 2 will then assess the efficacy of this novel vaccine to protect horses from strangles.
Importance to the Equine Industry: Strangles remains the most frequently diagnosed infectious disease of horses. It is a highly contagious disease caused by infection with Streptococcus equi subsp. equi, (SEE) which results in fever, profuse nasal discharge, swollen lymph nodes, and subsequent abscess formation in the nodes draining the upper respiratory tract. Morbidity is generally very high, and death may occur when the upper airways get blocked by swollen lymph nodes. Therefore, this disease remains of the utmost importance to the equine industry. The fact is that natural infection produces long-lasting immunity, that the bacterium has limited genetic diversity, and that it doesn’t survive long in the environment make it theoretically possible to control strangles with a vaccine. Nevertheless, no entirely safe and effective strangles vaccines are available for use in North America. On the one hand, live modified vaccine strains sometimes produce unacceptable residual morbidity; moreover, intranasal inoculation tends to lead to strong avoidance behavior by horses. On the other hand, dead vaccines such as killed bacteria or bacterial components are much safer but have been shown to lack the capacity to induce a sufficiently strong, protective immune response. This problem can be solved by combining the vaccine with a safe and effective immune-enhancing formulation (known as “adjuvant”) that kick starts the immune system so as to provoke a vigorous response against synthetic bacterial (SEE) compounds - that by themselves are harmless - but that will induce an immune response that will protect the host from being infected with SEE, leading to long lasting resistance against strangles.
The novel concept of “trained immunity” has been demonstrated to be extremely useful against various microbes in many different species, including people. The bone marrow is the soft tissue inside bones that makes blood-forming cells (blood stem cells or precursors) that generate white blood cells. In this project, we are interested specifically in the white blood cell neutrophil (after they are released from bone marrow), which is an “innate cell” that kills microbes inside other cells. When molecules from microbes attach to the surface of these precursors (to molecules called “receptors”, which stimulates the cell), they generate neutrophils that will travel to the circulation with an increased capacity of killing bacteria compared to those generated by precursors that were not stimulated (i.e., did not attach to microbes). There are a few examples of microbes and substances that stimulate these cells, for example, the attenuated tuberculosis vaccine bacillus Calmette-Guérin (BCG) or beta-glucan (a molecule of the cell wall of fungi). More recently, BCG has been used against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has demonstrated the broad impact of trained immunity. This concept, however, has not been studied in horses. Newborn foals have an immature immune system, such that many are susceptible to Rhodococcus equi infection, an important cause of disease and death in foals. There is no vaccine commercially available, and the only strategy that has repeatedly protected foals against pneumonia by R. equi is the use of live bacteria given to foals using a foal stomach tube (same as used in colic cases). We believe this protection is attributable to stimulation of white blood cell precursors, specifically precursors of monocytes and neutrophils, which will “turn on or off” genes (called epigenetic modifications) that are important for the immune response. This method, however, is not feasible to be used in farms, therefore, we must learn what is inducing protection to be able to mimic these effects in a vaccine.
Our long-term goal is to develop a strategy that will be used in farms to prevent respiratory infections caused by R. equi (and potentially other infections, such as strangles) based on induction of trained immunity in foals. Our overall objective is to demonstrate that giving live R. equi via a stomach tube (called enterally) to newborn foals induces trained immunity that protects them against pneumonia caused by R. equi, while giving them fluids (saline) enterally does not. We plan to specifically: 1) demonstrate that giving foals live enterally in the first days after birth will induce trained immunity which will result in protection against pneumonia when foals receive R. equi in their lungs (Aim 1); and 2) demonstrate that neutrophils from foals that receive live R. equi enterally have improved capacity of killing R. equi in laboratory experiments (we give foals live bacteria enterally but collect blood to separate neutrophils and only expose them to bacteria in the laboratory; Aim 2).
In Aim 1, we expect to confirm several studies in which administration of live R. equi enterally results in protection against R. equi challenge in the lungs, and that this protection is related with changes in neutrophils. We do not intend to use this as a vaccine because giving live R. equi to foals in farms is not feasible, but these results are important because: 1) they are ESSENTIAL to better understand why these live bacteria in their stomach helps protect them against pneumonia later in life; and, 2) they will provide useful information about epigenetic changes (which genes are turned “on” or “off”) in innate immune cells (monocytes and neutrophils) following enteral administration of live R. equi, which has not been previously documented. In Aim 2, we expect to show that enteral administration of live R. equi generates blood neutrophils with improved ability (trained) to kill bacteria in the laboratory, while saline does not.
Our long-term goal is to develop strategies for prevention and control that could be used at equine breeding farms to prevent foals from developing pneumonia caused by R. equi. We believe that the key to designing these strategies that protect foals lies in better understanding an approach that is known to protect foals. This approach could potentially replace the use of antibiotics to prevent the disease (a strategy used by some farms to reduce the number of cases of R. equi pneumonia). We also believe that by stimulating the newborn foals’ innate immune responses, a decline of other infections (e.g., strangles) might occur, as well as better responses to vaccines. This will have a remarkable impact in the equine industry, specifically at horse-breeding farms in which the number of cases of the disease can exceed 20-40% of the foal crop.
Importance to the Equine Industry: The impact of R. equi pneumonia on the equine industry is large. In the United States, respiratory illness is the 3rd most common cause of disease in young foals, and the 2nd cause of death in foals. R. equi pneumonia is a disease difficult to detect and treat, and in many cases, extensive lung damage has occurred by the time a diagnosis is made, increasing the length of treatment, which sometimes is in vain. Consequently, a large number of foals R. equi succumb to the disease. Moreover, the economic impact of R. equi pneumonia to the industry is significant. There are 7.2 million horses in the US, with a direct contribution of over $50 billion and indirect of $122 billion to the economy. Racing has the highest direct contribution ($15.6 billion), and it has been shown that foals that recovered from R. equi pneumonia were less likely to race as adults, directly impacting the racing industry. Therefore, preventing R. equi pneumonia in foals would greatly benefit the equine industry: 1) less foals would develop the disease, directly benefiting horse-farms; 2) less foals receiving antibiotics, and, thus, less resistance to antibiotics would occur and less side effects, such as diarrhea; 3) improving the innate immunity of foals will have a broader impact in their health, such as reduction of other bacterial infections (e.g., strangles or equine distemper) because these cells are not “microbe-specific”, and better responses to vaccines; 4) the better understanding of the mechanisms of how enteral R. equi protects foals against pneumonia by limiting its survival and multiplication inside equine lung cells would direct benefit R. equi researchers. To date, a clear understanding of the changes associated with the disease and specifically what protect some foals against R. equi are unknown, and by studying a strategy known to prevent pneumonia in foals we will have useful information about the disease for vaccine development.
Pneumonia caused by the bacterium Rhodococcus equi (R. equi) remains a major problem for the equine industry. Great need exists to develop an effective vaccine against R. equi because of the importance of R. equi pneumonia to the equine industry (please see Importance to the Equine Industry below). Our long-term goal is to develop a vaccine using messenger RNA (mRNA) that can be delivered by inhalation to neonatal foals to protect them against pneumonia caused by R. equi. The vaccine will target a protein that is necessary for R. equi to cause disease in foals known as the virulence-associated protein A (VapA). The rationale for nebulization is to stimulate immune responses in the lungs, which are the site of infection of inhaled R. equi that cause pneumonia. This project will address 2 essential first steps for developing an mRNA vaccine against R. equi for foals. First, we will compare the ability of 4 different constructs of mRNA to produce VapA after the mRNA is introduced to equine lower respiratory tract cells that are grown in culture, so that we can identify the best candidates for a vaccine to evaluate in foals. Second, we will evaluate the effectiveness of the mRNA vaccine candidates selected based on results of our cell-culture work to produce immune responses against VapA (and thus against R. equi) in foals after the mRNA is delivered to the foals by inhalation at ages 1 and 21 day. Specifically, we will compare antibody activity levels against VapA in serum and lung fluid and cellular immunity specific for VapA in peripheral blood samples of foals that were nebulized with mRNA vaccine candidates. We will compare the immune responses in the foals vaccinated by inhalation to those of foals either nebulized with saline solution (negative control) or immunized intramuscularly with mRNA encoding VapA (positive control); saline nebulization or intramuscular injection also will be performed at ages 1 and 21 days. We expect to find that foals can produce antibody responses to VapA following inhalation of mRNA encoding VapA, even in the face of antibodies from their dams that are transferred via colostrum that can interfere with other vaccines. Results of this study will provide evidence for ensuing studies to evaluate the efficacy of an mRNA vaccine construct to protect foals against intrabronchial infection with R. equi. To our knowledge, this would be the first mRNA vaccine delivered by inhalation in any species other than mice.
Importance to the Equine Industry: The horse industry remains an agriculturally and economically important venture in the U.S., generating over $215 billion in total production in 2016. Producing healthy foals is necessary to maintain and improve the nation’s horse population, and the health of foals is important to the horse industry from a welfare perspective. Infectious diseases are leading causes of disease and death in foals, and pneumonia is a principal infectious disease of foals. Respiratory disease was the most common cause of disease and death in foals in Texas, and ranked 3rd as a cause of morbidity and 2nd as a cause of mortality in U.S. foals 1 to 6 months of age. R. equi is considered the most common cause of severe pneumonia. This disease is important to the equine industry for the following reasons. The disease is endemic at many horse-breeding farms with cumulative incidence often around 20% to 40% of the foal population. At endemic farms, the costs resulting from veterinary care, long-term therapy, and mortality of some foals are very high. In addition to significant immediate costs, R. equi pneumonia can have a long-term detrimental effect on the equine industry because it has been reported that North American foals that recover from the disease are less likely to race as adults and that performance was diminished by this disease for some Australian Thoroughbreds.
Rationale: Antimicrobial resistance (AMR) is clearly one of the most important public health issues facing societies globally. A majority of resulting scrutiny has focused on antimicrobial drug (AMD) use in food producing animals, but use of AMDs in horses has received increased attention. It is an accepted principle that all antimicrobial drug uses can promote resistance, and many propose restriction on AMD uses (especially in animals) without distinguishing the potential that some AMD uses may be safer than others, in relation to promotion of AMR. As veterinarians and caretakers of the wellbeing of horses, it is critical to maintain access and judicious use of AMDs in horses to treat bacterial infections, but it is also an incumbent public health responsibility that we select AMD use practices that are minimally impactful on AMR. Unfortunately, data from studies that objectively compare specific use practices in horses are relatively rare, and are critically need to promote evidence-based decision-making regarding this issue. Studies have traditionally evaluated AMR through characterization of individual cultured isolates. While this previous adds to our growing understanding of AMR in horses, there is a clear need to investigate the impacts of AMD use in horses using study designs and methods that, a) compare common antimicrobial use regimens in a single study to improve ability to make direct comparisons among treatments; b) yield results that are less likely to impacted by biases which can interfere with understanding the true impact of these exposures; c) examine impacts of AMD exposures in a larger context that characterizes all antimicrobial resistance found in entire microbial communities using sensitive, state-of-the-art methods; and d) evaluate how impacts of AMD exposures may be mirrored or may vary among different ecological niches in a horse.
Study Objective: Our overall long-term goal is to provide empirical evidence regarding the impacts of common use practices for antimicrobial drugs (AMDs) regarding promotion of AMR. This will allow us to identify treatment regimens that are beneficial to treatment of infections in horses and also have lower risks for promotion of antimicrobial resistance (AMR). The central hypothesis for this work is that common AMD treatment regimens in horses are not equivalent in their likelihood of promoting AMR, and specific hypotheses will investigate the impact of 4 common AMD treatment regimens on the entire communities of AMR genes and populations of bacteria from 3 different anatomical/ecological niches.
Study Design: Forty healthy, adult mares will be randomly assigned to 1 of 5 Study Groups. Groups 1-4 will be treated for 4 days with 4 of the most common AMD treatment regimens used in horses (ceftiofur crystalline free acid given IM; sodium penicillin G and gentamicin given IV; oxytetracycline given IV, and trimethoprim-sulfamethoxazole given orally), and Group 5 will be untreated controls. Horses will be examined daily by veterinarians, and samples will be collected on Days 0 and 1 prior to initial treatments, and after treatments are completed on Days 5 and 6, as well as 21 and 22. DNA extracted from samples on Days 0-1 will be pooled for genomic analysis, as will samples from Days 5-6, and also Days 21-22; this pooling of samples will decrease the potential for individual sample variability to affect results and thereby increase study power. Samples on all days will include feces obtained per rectum, and swabs of the deep nasal cavity and the vagina. After extraction and purification, DNA recovered from samples will be processed for 2 types of genomic sequencing that will, a) characterize all types of AMR genes, and b) characterize all members of the microbiome (i.e., entire microbial communities). We will then compare the compositions of resistomes and microbiomes among treatment and control groups across time points, and also among different microbial niches (feces, deep nasal cavity, vagina).
Importance to the Equine Industry: Expected Results: Our prior research shows that using the state-of-the-art genomic methods for investigation of AMR in animal populations provides novel insights on the ecology of antimicrobial resistance when considering the totality of all AMR genes in context of the entirety of microbial populations. This holistic approach provides important novel perspective on the impacts of factors influencing the ecology of antimicrobial resistance. We anticipate that some of the AMD treatment regimens will have greater impact on the resistome, as well as the microbiome, and that these changes will vary among the ecological niches being investigated.
Potential Impacts for Animal Health: These results will address an important need for veterinarians and producers regarding antimicrobial drug use and antimicrobial resistance. Clearly, as stewards entrusted with the wellbeing of horses, we need to continue to use AMDs for treatment of important bacterial infections in horses. However, we must also address the critical need for prevention of AMR through improvement in use practices and stewardship of this critical resource for the future benefit of animals and public health. This proposed research has important practical relevance and provide immediate benefit to veterinarians and horse producers as the results will demonstrate the magnitude of changes to the resistome and microbiome that can be expected in response to common AMD treatments and over time; these findings can help to guide treatment protocols so as to have lesser impacts on the development of AMR in treated horses. This research also has important foundational relevance as it will provide novel information about ecology of the entire resistome and microbiome in three important microbial niches of the horse.
Infection of the placenta and subsequent placental disease (placentitis) is the most common problem of late gestation in mares [2,4,5]. Besides the effects on the mares’ well-being and the emotional impact on the owners, placentitis results in abortion or the birth of pre-mature or sick foals, leading to multimillion-dollar losses in the equine breeding industry [2,4,5,7,8]. Although placentitis is one of the costliest diseases in the equine breeding industry, the exact cause of the disease is still not fully understood, leading to inefficient preventive, diagnostic, and treatment protocols . Previously, we characterized the molecular changes in placental tissue at the final stage of this condition . However, there is no information about the exact mechanism of infection, the interaction of the placenta (host) and the causative bacteria (pathogens), and progression of the disease. It is currently assumed that only a few bacteria (mainly Streptococcus equi subspecies zooepidemicus (Strep. equi) in ascending placentitis and Amycolatopsis or Crossiella in nocardioform placentitis) cause placentitis [5,8]. In our preliminary data, we identified several bacteria present in large abundancies in placentitis cases. Surprisingly, Strep. equi, Amycolatopsis, and Crossiella were not always the most abundant pathogens. However, with the 16S rRNA sequencing approach that we used, we were not able to distinguish between contaminating, environmental bacteria and metabolically active bacteria involved in the pathogenesis of placentitis.
Therefore, in this proposal, we aim to identify the bacteria involved in placentitis using dual-RNA sequencing which identifies the gene expression of both host and pathogens simultaneously and identifies only those bacteria that are metabolically active. Within the last few years, with the advances of molecular technology and high-throughput data, studying the host-pathogen interaction has become feasible. A recently developed technique, known as dual RNA sequencing (dual RNA-seq), is designed to understand the interaction between the host and pathogen, identifying the gene expression patterns that result in disruption of normal host physiology, manifesting as infection and disease in the organism . This novel technique leads to identifying new virulence factors in the pathogen or pathways in the host cell that respond to the exposure to specific pathogens [27,28]. For example, this technique led to the identification of genes that play a key role during Salmonella typhimurium infection in humans, leading to identification of a targeted treatment .
We hypothesize that by using this technique, we can further understand the pathogenesis and the host-pathogen interaction in equine placentitis. Similar to human medicine, this information will provide therapeutic targets for placentitis. To test our ability to initiate this research and as proof of concept, we successfully utilized a publicly available dataset from equine Strep. equi -induced placentitis along with a recently generated RNA-seq dataset from equine nocardioform placentitis (conventional RNA-seq datasets) to investigate the gene expression in both the bacteria and the placenta. However, our results are limited due to the low sequencing depth of these datasets. Nevertheless, our analysis shows bacterial reads in RNA-sequenced samples, with an apparent ability to accurately identify and quantify this population, providing a novel method for detecting pathogenic bacteria during disease. Our goal for this proposal is to repeat this procedure in a larger sample set and perform more in-depth sequencing (dual RNA-seq; 150-200 million reads) to increase the resolution on the gene expression patterns in the bacteria and in the placental transcriptome, as well as to provide enough sequence to improve the accuracy of pathogen identification. We will test our hypothesis on previously collected samples from clinical cases of ascending and nocardioform placentitis and experimentally induced cases of ascending placentitis. In vitro experiments are also planned (1) to confirm the causative agents of placentitis, (2) to monitor the crosstalk between the placenta and pathogen(s), and (3) to characterize the molecular changes during the progression of ascending placentitis, using our novel equine placental organoid model. Our results will hold potential for the development of new diagnostic tools and therapies to forestall placentitis-induced preterm labor.
Importance to the Equine Industry: Placentitis is one of the most common problems of late gestation in mares [1–5]. Approximately 3-7% of equine pregnancies worldwide are affected by placentitis 6 and 10-30% of all abortions, premature deliveries and neonatal deaths are caused by placentitis 6, leading to multimillion-dollar losses in the equine breeding industry [1–5,7,8]. Furthermore, placentitis has an effect on the mares’ well-being and an emotional impact on the owners [1–5,7,8]. Currently the initial diagnosis of placentitis is based on clinical signs while the essential key to a successful treatment of placentitis is an early diagnosis [8,10]. Unfortunately, the clinical signs occur at the final stages of the disease, reducing the efficiency of the treatment. Several groups have attempted to identify biomarkers for earlier diagnosis but with limited success: either they are not sensitive or specific enough and/or are not (yet) useful in clinical practice [5,29,30]. Our study will ultimately lead to the identification of diagnostic biomarkers and the development of treatment and prevention strategies for equine ascending placentitis. This proposal will (1) identify the bacteria involved in placentitis in order to have targeted treatment; (2) identify gene expression patterns of the pathogens during ascending and nocardioform placentitis, in order to identify important genes for the pathogenesis and survival; (3) Identify gene expression patterns of the host during ascending and nocardioform placentitis in order to identify genes important in the host reaction to the pathogen; (4) Identify interacting signaling pathways between the pathogen and host during placentitis cases. Finally, our study will also focus on evaluating the specific and most relevant genes and pathways identified in this study through an in vitro system utilizing placental organoids and primary trophoblastic cells in order to better understand the molecular mechanisms occurring at the host-pathogen interface.
The most common site of fatal musculoskeletal injuries in racehorses is the fetlock joint. Within the fetlock joint, fracture of the proximal sesamoid bones is the most common fatal fracture. Fractures occur in characteristic configurations that indicate that specific limb loading circumstances cause the fractures. We have evidence that leads us to believe that movement of the long pastern bone (P1) toward the outside of the horse during extension of the fetlock joint during high speed exercise is the circumstance that leads to fetlock fractures. If this is true, hoof trimming, shoeing, and racetrack surface management could limit abnormal P1 motion and prevent fetlock fractures.
The fetlock joint is comprised of four bones: the cannon bone, the long pastern bone (P1), and the medial and lateral proximal sesamoid bones. The cannon bone articulates with both P1 and the proximal sesamoid bones. However, the proximal sesamoid bones do not articulate with P1; instead, their motion is coupled through soft tissue attachments within the fetlock joint. One set of soft tissue structures is the medial and lateral collateral sesamoidean ligaments. These two ligaments connect the medial and lateral proximal sesamoid bones to the top of P1. The three-dimensional rotations of the cannon bone relative to P1 are well described for all phases of equine locomotion. However, little is known about the three-dimensional movements of the proximal sesamoid bones.
The geometry of the fetlock joint largely confines its motion to flexion and extension (i.e., sagittal plane motion). However, small movements occur outside of flexion and extension. These motions include rotation about P1 and about the cannon bone (i.e., internal or external rotation) and movement of the bottom of P1 inside or outside of the limb. Recently, we determined that the PSBs experience motion other than flexion and extension when the fetlock joint is loaded at racing-speed levels and the hoof is flat. Importantly, these motions of the proximal sesamoid bones are highly consistent with the development of common fractures in racehorses. Shoeing, uneven footing, and other factors (e.g., turning a corner) affect the three-dimensional motions of P1 relative to the cannon bone. We do not know if these factors affect proximal sesamoid bone motion. Uneven footing has been caused during laboratory tests by wedges placed under the inside or outside of the hoof, but banked racetracks, unbalanced hooves, and/ or unharrowed racetracks likely have a similar affect. Since non-sagittal plane motions of P1 relative to the cannon bone are exaggerated by uneven footing, we believe that proximal sesamoid bone motions will also be exaggerated by uneven footing due to the soft tissue connections to P1 and the joint articulation with the cannon bone. Therefore, the primary goal of this proposal is to determine how uneven footing affects the motion of the proximal sesamoid bones relative to the cannon bone. The secondary goal is to determine if the ligaments connecting the PSBs to P1 are the mechanism for the motions. These research goals will improve our understanding of how proximal sesamoid bone fracture may be linked to hoof conformation, shoeing, and racetrack surface conditions.
Importance to the Equine Industry: Fractures of bones in the fetlock are some of the most common fatal and non-fatal injuries that occur during racing. Proximal sesamoid bone fracture is one of the most common fatal fractures worldwide. It causes 44% of all fatal injuries in California Thoroughbred racehorses, 55% of fatal injuries in South African racehorses, and 37% of fatal injuries in Australian racehorses. Fracture of the lateral side of the cannon bone, at the end that articulates with the proximal sesamoid bones, is one of the most common non-fatal injuries and the second most common fatal injury. This cannon bone fractures accounts for 10-25% of racehorse deaths. Both proximal sesamoid bone and cannon bone condylar fractures have typical fracture configurations that indicate they result from specific limb loading circumstances. Motions of the P1 and of the proximal sesamoid bones relative to the cannon bone outside of fetlock flexion and extension are consistent with these fracture configurations that occur in Thoroughbred racehorses. Medial proximal sesamoid bone fracture is associated with development of a weak spot against the opposing cannon bone that is the likely consequence of a specific limb loading condition that promotes rotation of the medial proximal sesamoid bone into the cannon bone. We have observed this motion during fetlock extension at gallop loads in our laboratory. We believe this motion will be exacerbated by unbalanced hoof trimming, lateral wedge horseshoes, and uneven race surfaces (such as banked racetracks and unharrowed footing). Racetrack surfaces in the United States are banked in the straights for drainage and in the turns. If our hypothesis is supported, hoof trimming, shoeing, and racetrack design recommendations could be made to prevent fetlock injuries.
Rhodococcus equi causes severe pneumonia in foals, but rarely causes disease in adult horses. Many foals clear the infection without need for treatment, but others require long-term antibiotic treatment or even die. Unfortunately, we do not understand why foals in general, and some foals in particular, are so susceptible to R. equi infection. This project aims to increase our understanding of why some foals die from R. equi pneumonia, while others clear the infection without showing signs of illness, with the ultimate goal of more effectively treating and preventing this devastating equine infectious disease.
Increasing evidence suggests vitamin D and cortisol are vital in the immune response to bacteria like R. equi in other species. Vitamin D is important for producing specific proteins within immune cells that can kill bacteria. Cortisol is important in directing the immune response during infection. However, the role of these hormones in the development and severity of R. equi pneumonia in foals is not known.
This proposed study will investigate the relationship between blood levels of cortisol and vitamin D and the development and progression of R. equi pneumonia in foals on a large horse-breeding farm with a high rate of R. equi infection. Vitamin D and cortisol concentrations and associated immune proteins will be measured in blood samples taken at strategic time points from birth to weaning. We will look for associations between these measurements and disease development and severity. This is the first study to investigate these immune-regulatory hormones in R. equi pneumonia in foals. By conducting this study in foals with naturally-occurring R. equi exposure and infection rather than in experimental infection or in laboratory models, we will be able to define factors that influence disease development in this real-world setting.
Importance to the Equine Industry: Pneumonia in general, and R. equi pneumonia in particular, are leading causes of disease and death in foals. Costs associated with disease screening, veterinary care, antibiotic treatment, loss of foals, and impacts on future athletic performance are extreme. Additionally, the efficacy of frequently used antibiotics to treat R. equi may be decreasing as resistance to those drugs is increasing. The effects of steroid hormones such as vitamin D and cortisol on R. equi susceptibility have not been explored to date. A better understanding of how vitamin D and cortisol impact the foal's risk of R. equi infection after natural exposure will open up new avenues for both prevention and treatment of this common, costly, and important disease. This work directly supports the mission of the Grayson Jockey Club Research Foundation and addresses a target research area by working to reduce the impact of R. equi pneumonia on the equine industry.
The objective of this study is to investigate if an opioid pain medication, fentanyl, can be administered to horses via a patch placed on the skin. The drug is absorbed from the patch through the skin as opposed to being administered by injection. Fentanyl, administered in this way, has the potential to provide clinically relevant pain relief to horses without the need for expensive intravenous catheters and frequent injections. We aim to describe how well fentanyl is absorbed through the skin from the patch and the degree and duration of pain relief that it provides at different dosages in horses. Armed with the information obtained from this proposed study, we will then be able to make a recommendation for dose and frequency of fentanyl administration via a patch in horses. Ultimately, the ability of veterinarians to provide long-lasting opioids for pain relief in horses without the need for intravenous catheters or multiple, painful injections will be a game changer in pain management for horses.
Importance to the Equine Industry: Management of patient pain is a constant challenge for the large animal veterinarian. Opioids are commonly used for management of pain in the horses but as they must be given by injection in horses, they require maintenance of an intravenous catheter and/ or multiple injections in the muscle to provide constant pain relief. This is time consuming to the veterinarian, painful for the horse, and expensive for the owner. With completion of this project, we aim to show that fentanyl absorbed through the skin via patch application represents an effective option for pain management in equine patients and has the potential to change the face of pain management for horses around the world. As such, the results of this project will be of great importance to the equine industry.
Laminitis in horses is most commonly associated with either equine metabolic syndrome (EMS) or pituitary pars intermedia dysfunction (PPID;”Cushing’s Disease”). In both cases, high blood insulin, resulting from insulin dysregulation (ID) is what drives the development of laminitis. An exaggerated insulin response to dietary carbohydrates, often from pasture, can causes varying degrees of damage to the feet, resulting in laminitis. We now understand that high blood insulin is the key event that drives the development (and recurrence) of laminitis in these horses: high blood insulin concentrations directly damage the lamellae within the feet. Successful prevention and treatment of laminitis in these cases therefore depends on our ability to control the blood insulin responses in horses and ponies with ID. Since management strategies (including dietary regulation) are often insufficient alone to control blood insulin, there is a need for medications that specifically control the insulin response to feeding in order to prevent laminitis in these cases. Sirolimus (rapamycin) is a drug that is primarily for the treatment of cancer and prevention of organ rejection, but it also has anti-aging effects and has variable effects on insulin dynamics across multiple species. We have preliminary data demonstrating that sirolimus has potent effects on insulin production in horses: a single intravenous dose of sirolimus can suppress insulin production (in response to an oral sugar challenge) for at least 24 hours. Furthermore, our preliminary pilot data from an experimentally-induced ID model demonstrates that once-daily administration of oral sirolimus normalizes the insulin response to an oral glucose challenge, without causing apparent adverse effects during 7 days of sirolimus treatment. These potent effects of sirolimus on insulin production in the horse have great potential for the treatment of ID (and prevention of laminitis). We hypothesize that treatment with sirolimus will prevent high blood glucose in response to ingested carbohydrate in horses with both experimentally-induced and naturally-occurring ID and will be safe and well tolerated. Using an established experimental model of ID, we will test the effects of 2 different dose rates of oral sirolimus on insulin dynamics in response to oral sugar challenge. We will then test the effects of oral sirolimus treatment in horses with naturally-occurring ID using a randomized, placebo-controlled crossover study. Blood sirolimus concentrations as well as hematological parameters and serial clinical evaluations will be used to monitor for any adverse effects. We anticipate that this study will provide fundamental information on the efficacy and safety of sirolimus for the treatment of ID in horses. In line with our preliminary data, we expect that sirolimus therapy will rapidly and profoundly suppress insulin production in response to ingested sugar, without causing adverse effects. The results of this study have the potential to drive the development of novel treatments for insulin control and prevention of laminitis in the horse.
Importance to the Equine Industry: Laminitis is a crippling disease of horses and ponies that remains a major cause of morbidity and mortality worldwide. Laminitis has 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. Of the different forms of laminitis, so called “endocrinopathic” laminitis is by far the most common, accounting for almost 90% of laminitis cases presented to a first opinion practice in a recent study. This form of laminitis affects adult horses of any age and breed, but in the Thoroughbred industry it is most common in brood mares and stallions where it is a major cause of loss of usage as well as premature death. Although preventative strategies including early identification and dietary management are useful for reducing the incidence, horses still succumb to endocrinopathic laminitis and a rational, evidence based therapy is essential in managing the acute case. We hypothesize that sirolimus will be an effective and safe therapy for controlling insulin dysregulation and preventing endocrinopathic laminitis in horses.
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.
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.
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.
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.
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.
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.
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.
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.
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.
There are three Career Development Award recipients in 2022.
The Storm Cat Career Development Award, inaugurated in 2006, is a $20,000 grant in 2022 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 there are two award winners:
Dr. Bayless has completed his residency program and is in a research training position under the mentorship of Dr. Katie Sheats. Her overall career goal is to contribute to improve outcomes in equine patients through research, teaching, and clinical practice as a veterinary clinician scientist. Throughout her training as an equine internal medicine specialist at Colorado State University and PhD studies at North Carolina State University, she developed research expertise, clinical acumen, communication skills, and teaching experience. As she prepares to transition to an independent researcher in a faculty position, she looks forward to continuing to hone these essential professional attributes during the final year to receiving her PhD.
Faculty supervisor Dr. Mary Katherine Sheats, Assistant Professor of Equine Primary Care
Dr. Shaffer will receive her PhD in Mechanical and Aerospace Engineering from the University of California, Davis in December 2021. She will be employed full-time as a post-doctoral researcher in the J.D. Wheat Veterinary Orthopedic Research Laboratory. Her post-doctoral work consists of the following three projects; all are related to racehorse stress fractures. Her goal is to obtain a faculty position and continue research on equine biomechanics, specifically factors that affect risk for injury.
Faculty supervisor Dr. Susan Stover, Professor School of Veterinary Medicine
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 2022. 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.
This project is the most logical next step in this line of research created by Dr. Menarim, and an important stepping stone in the progress of his career as an independent investigator. Dr. Menarim's background as an experienced sports medicine and surgery clinician (over 10 years), combined with his contemporary research skills including recent training in "omic analyses" (RNA-seq and lipidome) at the Gluck Center, is allowing him to further advance the field of synovial immunology in equine health and disease. Dr. Menarim's accomplishments in this field, leading to several awards (5 in the last 3 years); include the development of an innovative in vitro model of synovial inflammation that yields data of highly significant clinical translation. This research has the potential to yield new and innovative therapeutic approaches for synovial inflammation and OA in equine patients.Faculty supervisor Dr. James MacLeod, Director, UK Ag Equine Programs