Current Research
The board of directors of Grayson-Jockey Club Research Foundation announced that it has authorized expenditure of $2,455,164 to fund 11 new projects and thirteen continuing projects at 14 universities as well as three career development awards. The 2024 slate of research brings Grayson’s totals since 1940 has provided more than $42.3 million to underwrite more than 437 projects at 48 universities.
Click here for a printable list of our 2024 Projects
Stem Cell Neotissue Implants for Equine Tendon Healing
EQUINE TENDINOPATHY: ADVANCED IMAGING AND TENEX EFFICACY
Brad Nelson, Colorado State University –Two Year Grant
One Sentence Summary: This study will enhance our knowledge of equine tendon repair by comprehensive clinical imaging, while simultaneously assessing a novel tendon therapy, improving veterinary and industry understanding.
Tendon and ligament injuries are a significant cause of musculoskeletal disease in equine athletes, which results in an inability to perform their athletic activities and often leads to early retirement. Once injured, tendons heal poorly, and repair tissue frequently consists of scar tissue. This lack of healing response predisposes horses to re-injure, even after several months of controlled rehabilitation. There are many treatments aimed at improving the tendon healing response, but none do this well enough to prevent re-injury. Percutaneous Ultrasonic Needle Tenotomy (PUNT) with a Tenex device has shown promise in humans with tendon injury by improving the healing process, though it is unknown if it is useful in treating horses. In this study, we propose using a common equine model of the superficial digital flexor tendon (SDFT), a commonly injured tendon in equine athletes, with a modification that more closely represents clinical disease scenarios. This new model will be used to determine whether PUNT has any benefit in horses. Our research team has expertise in advanced ultrasound and MRI techniques and in bench-top tendon analyses. We will use this knowledge to not only improve our understanding of tendon injury and repair, but these methods will also help us critically evaluate if PUNT is an effective treatment in horses. Completing this study will benefit equine veterinarians, owners, trainers and the industry by improving our understanding of tendon disease and has the potential to offer new imaging strategies that can be incorporated into equine clinical practice.
Importance to the Equine Industry: Upon completion of this study, we will be able to provide the equine industry with a more complete understanding of equine tendon healing and the potential benefit of a new tendon therapy for horses. Equine tendon and ligament injuries are frequent, and the long duration of healing needed to return to work and high re-injury rates are a serious problem. This study will provide us with highly detailed imaging data that can be used to better understand the healing response to tendon injury in horses and will inform clinicians how best to intervene to improve healing. These imaging methods have the capability of being incorporated into clinical practice. Furthermore, if this new tendon therapy results in improved healing, it will have the potential to improve the quality of tendon repair tissue and return horses to athleticism earlier with a lower chance of re-injury. Collectively, this work will improve our knowledge of tendon disease in the horse, which we can use to better inform the equine industry and drive future advancements in tendon injury and therapies.
DIET NONSTRUCTURAL CARBOHYDRATES ALTER OVARIAN FUNCTIONS
Elaine Carnevale, Colorado State University – One Year Grant
One Sentence Summary: This study will review how feeding feeding nonstructural carbohydrates (grains and sugars) to older mares can alter cell function and metabolism in ovarian follicles, impairing ovulation, oocyte and embryo viability, and fertility.
Diet can affect health and productivity of the mare, including reproductive success. The equine diet is often augmented with high energy feeds, such as nonstructural carbohydrates (NSCs) which include grains and sugars. However, the effects of diet components on cell function in horses have not been well studied; this is especially notable for the specialized and interdependent cells within the ovarian follicle. Human consumption of carbohydrates and sugars promotes reduced reproductive success and ovulatory failure; however, the effects of NSCs on reproductive cell metabolism are poorly understood in humans and have not been studied in horses - although this could be an important consideration for managing and optimizing fertility and health in broodmares and their offspring. In the proposed study, we will investigate if feeding NSCs to older mares alters the ability of cells within the ovarian preovulatory follicle to uptake and properly use energy substrates, which are essential for function and survival of the oocyte (egg) and early embryo. In addition, changes in concentrations or cell response to insulin and glucose could disrupt normal intrafollicular cell communications that coordinate oocyte maturation and ovulation, potentially affecting ovulation and fertility.
To study our hypothesis, older light-horse mares will be divided into two groups: (1) CON, with mares maintained on grass/alfalfa hay and (2) NSC, with mares fed hay and 4-6 pounds daily of NSCs (corn, oats, barley, and dry molasses). When mares have an estrous follicle GT 35 mm in diameter, a GnRH analog will be administered to induce follicle/oocyte maturation. We will collect contents of these follicles about 20 hours after induction or approximately 18 hours before the mare would naturally ovulate. Therefore, we will be studying the follicle that the mare has “selected” to ovulate during an estrus cycle. First, the metabolites, or the molecules in cells that participate in metabolism and cell function, will be assessed for the follicular cells. In addition, cells will be cultured with labelled (13C) glucose with or without insulin, allowing us to track the uptake and metabolism of glucose. Glucose functions in coordinating oocyte maturation in the follicle. In addition, follicle cells surrounding the oocyte need to take up glucose, convert it to pyruvate, and directly transfer pyruvate to the oocyte, providing an essential energy substrate for the oocyte and for early embryo development. Failure for these processes to occur within the follicle could leave the oocyte without enough energy for optimal function and/or to sustain early embryo development. Consequently, the likelihood that the embryo is less viable and/or dies early in development is increased. Second, we will study proteins within the follicle that participate in timing and coordination of ovulation, to allow release of the oocyte from the ovarian follicle at a stage of maturation that optimizes fertilization and further development. These processes are essential to establish a viable pregnancy. We anticipate that feeding NSCs will impact follicle cell responsiveness to insulin, glucose uptake, metabolic pathways, stored energy substrates, and ovulation signaling. Consequently, our results will provide valuable information for the management of the mare prior to breeding, which could affect reproductive efficiency, fertility, and potential to produce a healthy foal.
Importance to the Equine Industry: Producing a Thoroughbred racehorse is an investment in time and money. Each stage in the process, from breeding the mare to racing, is carefully managed for success. However, a step that is often overlooked is “feeding the oocyte”. Prior to ovulation, the oocyte prepares for maturation and fertilization by storing energy substrates and other cell components, which will be essential for oocyte function and for early embryo development. We are only starting to learn about the effects of a mare’s diet on follicle function, oocyte quality, and the offspring; this is especially true in the older mare who has a propensity for metabolic problems. Fertility in other species is affected by maternal diet; notably, carbohydrate and sugar consumption in women can impair ovulation (release of the oocyte from the follicle) and fertility. Similar research has not been performed in mares. Even more notable, the preconception diet has been shown to affect long-term health of the offspring of mares and women. In short, how we “feed” the oocyte can have a substantive effect on fertility, long-term health of the offspring and, potentially, athletic ability. Nonstructural carbohydrates (NSCs), including grains and sugar, are often fed to mares, although their effect on the equine preovulatory follicle and fertility is unknown. Potentially within the ovarian follicle, NSCs can affect insulin/glucose dynamics and/or inhibit energy acquisition and storage in the follicular cells and oocyte; factors which will negatively impact ovulation and quality of the oocyte and early embryo. These alterations can result in no pregnancy or early embryo death, problems recognized in older mares. Our research will determine if feeding NSCs affects functions of the ovarian follicle, which is pertinent to management of all mares, including mares performing or recently retired from racing. Our research can promote visibility and future investigations into an area of study essential for equine reproduction.
SHINING A LIGHT ON THE BLACK BOX OF EARLY PREGNANCY LOSS
Amanda de Mestre, Cornell University –Two Year Grant
One Sentence Summary: This study will determine how common genetic disorders of the early equine embryo occur as well as identify how they present to veterinarians and key factors that increase their risk.
Whilst progress has been made over the last two decades in getting more mares pregnant, owners and veterinarians still face a significant challenge in preventing pregnancy loss, particularly in the first two months after conception. This lack of progress is significantly hampered by our lack of understanding of the reason a pregnancy fails making treatment and management approaches close to impossible. Previous research has focused on the possible role of the mare’s uterus in providing a suitable environment for the developing embryo, but in contrast, a significant gap exists in understanding all other causes of early pregnancy loss (EPL) which account for around 60-80% of cases. Our lab has been investigating novel reasons why a pregnancy may fail and have approached this by working alongside vets, to pioneer new methods to obtain clinical cases of EPL and determine if their genetic composition is compatible with life.
Veterinarians submit tissue from clinical cases of EPL to our laboratory for assessment. In our first analysis of these failed conceptuses, we found that 20% of clinical cases were associated with a genetic disorder termed aneuploidy, that is, the loss or gain of a whole chromosome compared with the normal number. This condition is very well described in the human literature as a common cause of miscarriage in women but it was the first description of this disorder in cases of pregnancy loss in the mare. An abnormal number of chromosomes results in significant disruption to the genome usually incompatible with continuation of the pregnancy in any mammalian species. It represents the most common single cause of EPL described to date in intensively managed mares finally providing us with a new target to interrogate. We know the genome can be modified from its normal state in many other ways. Exactly if and how these other genetic variations in the genome lead to pregnancy loss in the mare remains unknown. Furthermore, we know very little about the clinical features of genetically abnormal pregnancies, key to vets being able to identify which pregnancies should be maintained, which are better off being terminated and the mare rebred.
This project will investigate two types of genetic disorders termed triploidy and aneuploidy. Instead of a single whole chromosome being duplicated or lost, as is the case of aneuploidy, in triploidy an individual has an extra copy of all the chromosomes resulting in three full sets as opposed to the usual two sets. The project will characterize how these embryos ended up with an abnormal number of chromosomes. We predict this will be very common (30-50% of cases) and it primarily due to a combination of (i) genetic errors in the egg and (ii) an error of the egg that allows two sperm to fertilize the egg, as opposed to the usual one sperm. Studies in women have shown that the source of the genetic defect impacts multiple clinical features of the pregnancy, which is incredibly useful for early detection and clinical management by the veterinarian. Therefore, will also ask the questions: What risks factors (exposures) increase the chance of these genetic disorders occurring? How do these pregnancies present clinically so that they can detected and managed appropriately by the veterinarian? Ultimately this will empower veterinarians with new knowledge that we predict will lead to new diagnostic tests, treatment approaches and ultimately reduce the occurrence of this common but frustrating condition.
The team is uniquely placed to investigate genetic causes of EPL in the mare with a large tissue bank and clear ways to expand these in 2024/2025 achieved through established and strong partnerships with multiple collaborating veterinarians and a track record in reproduction, genomics, epidemiology and Thoroughbred research. An established collaboration with an international leader in equine genomics is also in place for consultation on genomic methodologies.
Importance to the Equine Industry: The ultimate goal of owners of breeding mares is to produce a viable and healthy foal each year she is bred. One of the greatest impediments to achieving this goal is the loss of her pregnancy in the first two months following conception, referred to here as early pregnancy loss (EPL). This condition is incredibly frustrating for the veterinarian and owner alike as it results in increased veterinary interventions and associated welfare implications for the mare, much higher costs for owners, and can be emotionally distressing for owners striving to produce that elusive foal. It can also mean a mare fails to produce a foal that season as a result of poor fertility related to endometrial cup formation, or indirectly due to drifting of the conception date so late in the season that it is no longer viable to breed her in the subsequent year. The first question owners often ask when the veterinarian finds a failed pregnancy is: why did it fail? Unfortunately, we know so little about the underlying causes of EPL that the answer is usually ‘I don’t know’. Scientific studies have shown that in around 60-80% of clinical cases of EPL the underlying cause is not identified using currently available knowledge. In the absence of specific diagnoses of the cause of EPL, stud farm veterinarians have very few options available to them to manage EPL. Generic treatments such as progesterone supplementation is sometimes used in a hope of preventing EPL, although low progesterone is usually not the reason that the pregnancy was lost and therefore in most cases it is ineffective. Further, pregnancies with genetic errors incompatible with life that are treated with progesterone are unnecessarily prolonged, and as a result, the mare may miss the opportunity to be rebred that season. Essential to overcoming these challenges is identification of new explanations as to why EPL occurs informing novel diagnostic tests, therapeutics and management approaches can be implemented in the future.
KETODOXAPRAM AS A TREATMENT FOR ATRIAL FIBRILLATION
Annelies Decloedt, Ghent University –Two Year Grant
One Sentence Summary: This study is to determine if Ketodoxapram is a promising novel treatment method for horses with atrial fibrillation.
Cardiac arrhythmias are frequently diagnosed in horses and can have serious consequences such as collapse or sudden death. Atrial fibrillation is the most common clinically important cardiac arrhythmia in equine athletes. Atrial fibrillation is found in about 0.3% of horses in the general equine population, and in up to 5% of racehorses. Although the mechanisms and consequences of atrial fibrillation are increasingly better understood, drug treatment options remain limited. Even in human medicine, the currently available medication is often ineffective and, in addition, there is a high risk of side effects and relapse after treatment. At the Faculty of Veterinary Medicine in Ghent (Belgium), horses are routinely treated with an intracardiac electrical shock under general anesthesia, with a success rate of about 95%. The electrical shock results in cardioversion of atrial fibrillation to normal sinus rhythm. This treatment can world-wide only be carried out in a few highly specialized centers. To avoid this technical procedure, pharmacological treatment can be performed using drugs. Traditionally, horses with atrial fibrillation are treated with the drug quinidine sulphate, but this medication is increasingly difficult to obtain in many countries: the product is disappearing from the market because of the frequent side effects in humans and animals. Common side effects that occur after the administration of quinidine sulphate in horses include nasal swelling, depression, high heart rates, colic, diarrhea, weakness, laminitis, collapse and even sudden death. The success rate of quinidine sulphate treatment is about 60-80%, which is also considerably lower compared to the electrical cardioversion treatment. Other available drugs are even less effective, with a maximal success rate of about 50%, and also cause different side effects.
Studies in human medicine are currently evaluating drugs which target specific ion channels in the heart, the TASK-1 potassium channels. Drugs which inhibit these TASK-1 channels, such as ketodoxapram, could be a novel treatment option for atrial fibrillation. The TASK-1 channels are mainly expressed at the level of the atria, which minimizes the occurrence of side effects when a TASK-1 inhibitor such as ketodoxapram is administered. Specific blocking of the TASK-1 channels slows down the atrial fibrillation rate and allows cardioversion to normal sinus rhythm. Preliminary results of ongoing research by our group demonstrated that the TASK-1 potassium channel is also present in horses. Moreover, the equine and human TASK-1 channel are very similar. Therefore, intravenous treatment by ketodoxapram seems like an ideal candidate for pharmacological treatment of atrial fibrillation, especially short duration atrial fibrillation after exercise or anesthesia.
We will test ketodoxapram treatment in horses in which atrial fibrillation is induced by a pacemaker. This way we can evaluate the effect of ketodoxapram for treatment of very recently developed atrial fibrillation as well as for atrial fibrillation during two months. The first aim of this study is to demonstrate that ketodoxapram can slow down the atrial fibrillation and treat experimentally induced atrial fibrillation in the horse. Using an implanted pacemaker, we will induce atrial fibrillation during 48 hours. By acquiring minimally invasive atrial biopsies using a forceps introduced through the jugular vein in the standing horse, we will evaluate whether this results in a higher expression of the TASK-1 channels. Next, we will evaluate whether administration of ketodoxapram will result in cardioversion to normal sinus rhythm. We will compare the ketodoxapram treatment to a placebo treatment. In addition, we will monitor different clinical parameters such as heart rate and respiration before and after administration of ketodoxapram (compared to placebo), and evaluate the ketodoxapram levels in the blood. The second aim of this study is to use ketodoxapram to treat horses with chronic experimentally induced atrial fibrillation (2 months). We hypothesize that ketodoxapram administration will slow down the atrial fibrillation, and will convert the horses to normal sinus rhythm. Our ultimate goal is to demonstrate that ketodoxapram is a safe and effective treatment option for horses with atrial fibrillation. Ketodoxapram may become the method of choice for pharmacological treatment of horses with atrial fibrillation.
Importance to the Equine Industry: A novel treatment option for atrial fibrillation is important for the equine industry, especially the sports horse industry of all disciplines (racing, jumping, eventing, dressage,...). Atrial fibrillation often results in decreased sports performance and potentially collapse or even sudden cardiac death. Cardiac disease is the third most common cause for poor performance, and atrial fibrillation is seen as the most common pathological arrhythmia causing performance loss in sport horses. Horses with atrial fibrillation that need to perform at high levels, have abnormally elevated heart rates during exercise or show additional arrhythmias should undergo cardioversion. In addition, prompt treatment after the initiation of atrial fibrillation is important to prevent atrial remodeling. Atrial remodeling stabilizes the arrhythmia and makes it even more difficult to treat. At the Faculty of Veterinary Medicine in Ghent, horses are routinely treated with transvenous electrical cardioversion, with a success rate of about 95%. However, this treatment can only be performed in highly specialized centers and is not available in every country. The other option is medical treatment. Traditionally, quinidine sulphate is used in horses with atrial fibrillation. However, the success rate is lower (60-80% depending on the type of horse treated) and this medication is increasingly difficult to obtain in many countries: indeed, the product is disappearing from the market because of frequent side effects in humans and animals. Other available products give conversion in 50% of cases at most. Moreover, serious side effects are described for each of these products. Therefore, a novel safe and effective treatment for atrial fibrillation is urgently needed. The aim of this study is to evaluate a new drug, ketodoxapram, for the treatment of atrial fibrillation. In the future, ketodoxapram may become the treatment of choice for horses with acute or chronic atrial fibrillation.
ADIPONECTIN AS A CLINICAL BIOMARKER FOR LAMINITIS RISK
Melody A de Laat, Queensland University of Technology –Two Year Grant
One Sentence Summary: The aim of this project is to improve early detection of the painful foot disease laminitis, this project will evaluate the value of a promising biomarker for the condition called adiponectin.
Our horses are eating better quality pastures and feeds than ever before, so they are becoming overweight and unhealthy. Just like in humans, diseases of metabolism are increasingly apparent in horses. In particular, metabolic conditions where insulin secretion is increased are increasingly recognized. Researchers do not know exactly what causes the insulin concentrations to reach and remain at these high levels in some horses. High insulin levels are a major problem in horses because they cause the painful hoof disease called laminitis. Laminitis results in lameness, and in about 30% of cases the horse needs to be euthanized.
This project aims to improve our ability to determine the risk of laminitis in an individual animal. To do this we will examine a hormone that is found to be at lower concentrations in horses that are at risk of developing laminitis. We currently do not know much about this hormone in horses, but this project will extensively examine the hormone. We will determine the normal levels and fluctuations of the hormone after eating, and how it changes in animals that have metabolic disease and are prone to laminitis. We will determine whether the hormone concentration differs between horse and pony breeds, and what concentration of the hormone can be used to predict the relative risk of laminitis.
In addition to inappropriate diet, metabolic disease in horses is also likely to be due to underlying genetic variations. Due to the complexity of metabolic disease in horses, it is likely that many genes are involved. However, these genetic factors have not yet been identified. As part of this project, we will examine the sequence of the gene that codes for adiponectin, and determine whether the sequence varies between healthy horses, and those with metabolic disease. If a difference in the genetic sequence between animals is identified, we will determine whether or not this genetic change contributes to the disease. If it does, it could become part of a genetic test that helps to identify animals at risk of laminitis in the future. Overall, this project will improve our ability to predict and prevent laminitis, lessening the impact of this important disease.
Importance to the Equine Industry:
Laminitis is the second most common reason for equine veterinary consultations (after colic), and most cases of laminitis are due to high insulin concentrations (and this is the type of laminitis being investigated here). The disease is intensely painful, and due to a lack of effective drug treatments for laminitis many sufferers do not survive. Losses associated with laminitis are increasing, because diets rich in sugars and starch are more commonly fed, even when the horses are not in work. This excess food contributes to high insulin concentrations, the root cause of disease. Thus, laminitis represents a significant economic, social and welfare burden to the horse industry and horse-owning communities worldwide.
By improving our ability to detect which individuals laminitis is more likely to occur in, this project will enable better management of horses at risk of laminitis. This will ultimately reduce the number of horse suffering from the disease through improved ability to predict its onset and earlier recognition of risk so that preventative measures can be introduced. A reduction in the number of cases of laminitis will improve horse welfare, reduce the costs associated with laminitis treatment and increase the productivity of horse industries, such as racing and sporting associations and breeding facilities.
NOVEL PROTEIN TARGETS FOR R. EQUI SEROLOGY AND VACCINES
Noah Cohen, Texas A&M University – One Year Grant
One Sentence Summary: This study will use a novel method for the first systematic evaluation of a large number of proteins to identify targets for developing a simple blood test and vaccines for rhodococcal foal pneumonia.
Pneumonia caused by the bacterium Rhodococcus equi (R. equi) remains a major problem for the equine industry (please see section below). Despite the importance of rhodococcal pneumonia, neither a simple diagnostic test nor a vaccine has been developed for this disease. The absence of a simple diagnostic test contributes to over-use of antibiotics to treat foals that results in selection for strains of R. equi resistant to those antibiotics. Thus, we propose to print 250 R. equi proteins onto a small slide and to then apply samples of serum (the liquid part of blood) from foals at breeding farms that have foals with rhodococcal pneumonia (i.e., foals naturally exposed to R. equi) or foals that have been infected experimentally with R. equi by infusing it into their lungs via the trachea. Our overarching hypothesis is that serum antibodies recognizing specific rhodococcal proteins can differentiate foals that develop pneumonia from those that do not. Identifying such proteins would serve 2 great needs for control and prevention of R. equi pneumonia. First, it would allow for developing a simple blood test to detect foals with R. equi pneumonia. Second, it would identify proteins that could be used as targets for an R. equi vaccine. For both goals, we will use serum from 2 groups of foals: 1) foals naturally exposed at Thoroughbred breeding farms in New York and 2) Quarter Horse foals experimentally infected in Texas; data from the 2 groups will be analyzed separately.
For developing a blood test for R. equi pneumonia, we will compare results of microarrays probed with serum from foals with rhodococcal pneumonia with those from foals in the same environments that do not develop pneumonia. We expect to identify proteins to which pneumonic foals develop higher antibody levels than foals that remain free of pneumonia. We will test for antibodies of 2 types: immunoglobulin G (IgG, the most abundant type of antibody in serum) or immunoglobulin M (IgM, a type of antibody that rises early after infection; IgM is the basis for the West Nile virus blood test, for example). The proteins identified will be candidates for a blood test in diagnostic platforms that are commonly used in diagnostic laboratories and possibly even in stall side tests. We will consider proteins both individually and in combination to identify the most accurate diagnostic test.
To identify proteins that will be targets for vaccines, we will probe microarrays with serum collected from foals before the onset of disease (for naturally exposed foals) or before intrabronchial infection (for experimentally infected foals), and compare the results between foals that subsequently developed pneumonia with those that do not develop pneumonia. We expect to identify proteins for which antibody levels (either IgG or IgM) are significantly higher in foals that do not develop pneumonia. These proteins correlated with resistance to natural or experimental infection will be appropriate targets for vaccines to protect foals against experimental infection. Conversely, we might find proteins for which antibody levels are higher in foals that develop pneumonia (markers of susceptibility).
Previous studies have examined serological reactivity only to a small number of specific proteins or preparation of the whole bacterium. To our knowledge, this study will be the first systematic evaluation of a wide array of rhodococcal proteins in foals for discovery of proteins that can be used for development of diagnostic tests and vaccines. Thus, findings from this study will yield important results even if either one of our 2 goals (protein targets for diagnostic tests or vaccines) is not achieved.
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 was 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. It occurs recurrently at many horse-breeding farms often with around 20% to 40% of the foal crop affected. At farms affected by rhodococcal pneumonia, the costs can be very high for veterinary care, long-term treatment, and death of some foals. 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.
Developing a non-invasive diagnostic test will improve control of this important disease and permit more prudent and accurate use of antimicrobials to limit the problem of antimicrobial resistance in R. equi. Identifying novel targets for vaccines could lead to the breakthrough needed to develop a vaccine for this devastating disease.
ROLE OF C1Q IN RHODOCOCCAL INFECTION
Angela Bordin, Texas A&M University – One Year Grant
One Sentence Summary: We will study the effects of complement in preventing Rhodococcus equi infection, a cause of severe pneumonia in foals, for future design of vaccines and more efficacious hyperimmune plasma products.
The complement system, also known as complement cascade, consists of a number of small molecules that circulate in the blood and enhance (complement) the ability of antibodies and cells that “eat” pathogens (phagocytes) to clear microbes from the body. This system can be activated when specific “triggers” initiate an amplifying cascade that results in killing microbes; one trigger is he "tagging" of microbes to antibodies, which are large Y-shaped proteins that stick to the microbe surface. When a molecule of the complement complex, called C1q, encounters this complex formed by antibody and microbe the activation of the classical complement pathway initiates. There are many consequences to this activation, and all include activation of mechanisms that will result in killing the "tagged" pathogen. It has been shown in human beings and other animals that the lack of C1q can impair antibody responses, but studies evaluating the role of C1q in foals are lacking, particularly in regards to Rhodococcus equi infection. R. equi is a microbe (bacterium) that causes the most common and severe form of pneumonia in foals less than 6 months of age, and protecting foals from this disease is important to the horse industry because it is a major cause of disease and death in foals. Furthermore, evidence exists that young foals, which are more susceptible to R. equi pneumonia than older foals, have a lower “activity” of C1q, the actual level (concentration) of C1q in foals remains unknown. Our recent work indicates that giving neonatal foals live R. equi via nasogastric tube (called enteral administration, using a tube similar to those used in colic cases) protects them against R. equi. Because this is not a strategy that can be used in farms to protect foals, we need to better understand the protective mechanisms behind this method. We found that C1q is one of the important mechanisms that is induced by this strategy, and further understanding this pathway is important to design targets for vaccines and other products that will stimulate the immune system (called immunomodulators). Additionally, this knowledge could be used by plasma manufacturers to improve protocols for vaccinating donor horses and increase the efficacy of R. equi hyperimmune plasma, used in many breeding farms to protect foals. Therefore, the focus of our proposal is to further characterize the impact of C1q in response to Rhodococcus equi infection.
Our long-term goal is to develop clinically useful preventive and therapeutic strategies for respiratory infections caused by R. equi (and potentially other pathogens). Our objectives with this application are to demonstrate that the concentration of C1q increases with age and enteral administration of R. equi at birth (Aim 1), and to demonstrate that serum C1q concentration increases killing of R. equi by macrophages (specialized cells that phagocyte, or eat, pathogens; Aim 2). Completing these aims will generate knowledge about serum concentration and the concentration needed for optimal killing of R. equi by macrophages, both important for understanding C1q responses that are essential for an effective antibody response. These can potentially be targeted to develop novel, safe, and effective methods for preventing R. equi pneumonia in foals, such as vaccines or hyperimmune plasma with increased efficacy. It could potentially also be used by veterinarians to assess C1q levels to identify foals with higher risk of developing pneumonia, and prompt exploring alternatives to transfusion of hyperimmune plasma (e.g., mRNA therapies and or immunomodulators) for increasing C1q. concentrations in newborn foals. Improving R. equi control and prevention methods 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). 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 rhodococcal pneumonia on the equine industry is large. In the US, respiratory disease is the 3rd most common cause of disease in foals less than 6 months of age, and it is the 2nd cause of mortality in foals. Respiratory diseases are the #1 reason for administration of antibiotics in farms. Treatment is difficult, and in many cases, extensive lung damage has occurred by the time a diagnosis is made, extending the treatment time, sometimes leading to a negative outcome. Therefore, Rhodococcus equi pneumonia is an important cause of disease and death in foals, with a significant economic impact to the equine industry. 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 rhodococcal pneumonia were less likely to race as adults. 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 as well as less side effects, such as diarrhea. Our proposal will focus on the complement cascade, which is a part of the immune system that enhances the ability of antibodies to clear microbes such as R. equi, among other functions. In particular, we will focus on C1q: 1) the concentration of C1q in serum of foals; and, 2) the effects of different levels of C1q in killing R. equi. If we find that higher concentrations of C1q increases killing of R. equi, it will be important for hyperimmune plasma manufacturers to assess and maximize C1q in plasma and for veterinarians to assess C1q activity to identify at-risk foals. It also would prompt exploring alternatives to transfusion (e.g., mRNA therapies and or immunomodulators) for increasing complement concentrations in newborn foals.
INSULIN DYSREGULATION: PLACENTAL CHANGES AND FOAL HEALTH
Elaine Norton, University of Arizona –Two Year Grant
One Sentence Summary: This study investigates the role of equine metabolic syndrome on placental function and foal health and metabolic outcomes in order to develop effective treatment and management regimens.
Equine metabolic syndrome (EMS), characterized by insulin dysregulation, is the leading cause of laminitis, affects all horse breeds and is one of the most common diseases facing horse owners. However, we do not understand the impact of EMS in pregnant mares on her foal’s heath or metabolism. EMS is common in broodmares due to their diets, lack of exercise, and the practice of maintaining broodmares at a higher body condition to ensure that the foal is receiving adequate nutrition to grow. Humans suffering from diabetes, a similar condition to EMS, experience long bouts of high blood glucose and insulin levels, which have been shown to significantly change how the placenta functions. These changes increase the risk of the mother going into early labor, having difficulty giving birth, stillbirths, or the baby requiring hospitalization. Therefore, there is a gap in knowledge of the effects of a mare having EMS on placental function and foal health outcomes which prevents owners knowing how to manage their pregnant mares to have a healthy, athletic foal.
In this study, we will investigate the role that EMS has on placental function and foal health and metabolic outcomes in order to develop effective treatment and management strategies. We hypothesize that EMS leads to an increased risk of mare placental dysfunction, which is associated with health and metabolic outcomes in their offspring.
In Aim 1 we will assess the placenta at the microscopic level for changes in cellular structure and damage in mares with and without EMS. We hypothesize that placentas from EMS mares will have changes similar to what has been found in pregnant women with diabetes.
In Aim 2 we will assess changes in how genes are expressed (turned on or turned off) in the placenta of mares with and without EMS. We hypothesize that alterations in placental gene expression in pathways related to inflammation, metabolism and growth will be associated with a mare’s EMS status.
In Aim 3 we will compare events at birthing, neonatal health and metabolic outcomes between foals within their first year of life born from mares with and without EMS. We hypothesize that foals born to mares with EMS will have a higher incidence of adverse events in the first few days of life and changes in growth and insulin resistance in their first year. This is supported by evidence in horses that maternal obesity, a risk factor for EMS, leads to an increased incidence of poor-quality colostrum, fetal insulin resistance, and chronic inflammation.
This project will have a great impact on the horse industry as this is the first time that placental function will be assessed in EMS affected mares at both the cellular and genetic level. This will allow us to link foal health outcomes with placental dysfunction. This will also allow us to identify changes that can be screened during a mare’s pregnancy to help guide timely treatment and management strategies The impact on the horse industry will be notable as EMS affects all breeds, and nearly a quarter of broodmares. If EMS is a risk factor for placental dysfunction, resulting in failure of passive transfer or neonatal disease, early management strategies in mares could prevent foal loss and save owners $5000-15,000 in hospitalization costs and emotional distress. Our strong team with expertise in neonatal, metabolic and reproductive health is well suited to assess the complexities afforded by maternal/ placental/fetal interactions in order to best identify treatment and management strategies needed to improve foal health outcomes.
Importance to the Equine Industry: The industry impact is notable as equine metabolic syndrome affects all breeds of horses and is the leading cause of laminitis, costing the equine industry over $15 million dollars annually. This number is continuing to rise as the reported prevalence of EMS has reached up to 25% in high-risk populations, with our preliminary data confirming that broodmares are amongst the high-risk groups. Impacts on foal health due to placental dysfunction, such as failure of passive transfer, sepsis or dummy foals can have a mortality of up to 25% and cost owners $5000-15,000. Identification of EMS as a significant risk factor for the above has the potential to improve foal health and thus reduce the financial and emotional cost to owners.
POLYACRYLAMIDE FOR JOINT THERAPY–CRITICAL THINGS UNKNOWN
Bruno C. Menarim, University of Kentucky –Two Year Grant
One Sentence Summary: Polyacrylamide for joint therapy–critical things unknown
Osteoarthritis (OA) is the leading cause of lameness in horses. OA is a debilitating disease characterized by chronic inflammation and a progressive loss of the normal joint surface, resulting in pain, lameness or other compromised mobility, which often gets so serious that the horse is euthanized. OA can be very challenging to treat over the long term. Commonly used therapies often work for short periods of time or only provide partial relief of symptoms. There is a critical need for treatments that can not only prevent disease from getting worse but offer lasting effects.
A new treatment called polyacrylamide hydrogel (PAAG) has been investigated as a treatment for OA showing lasting improvements (up to 24 months). Even though clinical improvements from PAAG in the treatment of OA are very positive, how PAAG works to provide joint pain relief is uncertain and debated. Resolving joint inflammation is a primary goal of joint therapies. However, research into how PAAG affects joint inflammation has not been reported. Further, concerns have been raised among equestrian authorities that PAAG could affect pain sensitivity, preventing detection of imminent more serious injuries. Indeed, for Thoroughbred racehorses, a national regulatory rule states, “Intra-articular injections of polyacrylamide hydrogels are prohibited within 180 days prior to Post Time.” A previous study in 3 goats suggested that PAAG-treated joints had normal nerve counts and shape. As such, significant work is still required to characterize how PAAG injection modulates the inflammatory response in joints, and confirm, on a larger scale, that PAAG does not impair proper function of pain-sensing nerves. Therefore, we propose the following studies as high priority questions in addressing concerns from equine authorities and the safe use of this promising joint therapy.
Study 1: Characterize the changes in the inflammatory response of OA joints after treating with PAAG. This will be accomplished by studying mediators of inflammation and pain recognition in the joint fluid and internal lining.
Study 2: Characterize how pre-treatment of joints with PAAG affects the inflammatory response and recognition of pain long term after inducing joint inflammation experimentally.
These are very important question directly related to the safety of intraarticular PAAG therapy in Thoroughbred racehorses and other equine athletes. In addition to studying mediators of inflammation and pain recognition in the joint fluid and internal lining as describe for Study 1, we will use load-sensing boots on both front limbs with experimentally inflamed joints (previously treated with PAAG or inert vehicle), to assess pain recognition.
Our long-term goal is to assess the safety of using polyacrylamide hydrogel (PAAG) to treat equine osteoarthritis (OA). Knowledge generated by this study will provide tools for informed decision making regarding the effects of PAAG and its appropriate use.
Importance to the Equine Industry: Importance to Industry: The equine industry is a huge economic driver in many states in the US and globally. Significant sums of money are invested annually in breeding and training the most outstanding equine athletes. Intensive training and competition pressures frequently lead to joint inflammation and degeneration (arthritis) that prevent horses from reaching their potential or necessitate retirement. Estimated annual financial losses of between 600 million and 1 billion dollars occur due to treatment and early retirement of equine athletes in the US. In addition to the significant economic cost of equine joint disease, there is the very important emotional impact that cannot be quantified and affects most horse owners in their lifetime. Regardless of the cause of the joint disease, the ultimate result for horses and their owners can be devastating.
Despite many advances over the years in the prevention, early detection, and treatment of equine joint disease, a critical need still remains to identify more effective ways to control inflammation in the joint and hold the advancement of arthritis in horses, enabling them to have an extended and good quality of life. The use of polyacrylamide hydrogel (PAAG) to treat OA has shown significant results with remarkably long-lasting effects. Data collected in this study will provide essential information of the effect of this promising treatment, to allow veterinarians, owners and authorities to make an informed decision about the safety of using PAAG to treat arthritis. Should we confirm PAAG provides improvements by resolving inflammation but not masking pain, PAAG treatment could improve the life quality and athletic longevity of thousands of horses.
NOVEL METHOD FOR DIAGNOSIS OF NOCARDIOFORM PLACENTITIS
Shavahn Loux, Louisiana State University–Two Year Grant
One Sentence Summary: A sensitive and specific diagnostic test for Nocardioform Placentitis.
Placentitis is the most common cause of late-term abortion in the mare, costing the equine industry millions of dollars in lost revenue every year. Despite the devastation caused by this disease, accurate diagnostic tests for placentitis are not currently available. The tests which are available cannot separate affected mares from non-affected mares, leading missed diagnoses, late-term abortions and sick foals. In our previous work, we have developed a diagnostic tool for Nocardioform placentitis which is accurate and sensitive. In this proposal, we are promoting the expansion of this work to include a prospective study to better understand when disease forms and how it progresses, as well as the strengths and limitations of this diagnostic test.
Additionally, we will work to understand why these bacteria are capable of causing disease and how they communicate and interact with the mare during disease. Initial work will be focused on characterizing the proteins and lipids which the bacteria produce in isolated culture as well as when they are infecting the placenta. Our early work shows that these bacteria will modify the mare’s fatty acids to create lipoglycans which are integrated into the bacterial cell wall. As similar lipoglycans cause an immune response as well as drive the progression of disease, identifying these molecules will help create a better understanding of the mechanisms of disease. In the process, we will uncover which specific molecules cause a response of the hosts’ immune system as well as work to improve our diagnostic test while evaluating its strengths and limitations. In total, this work should increase understanding of the proteins and lipids which drive disease and illicit an immune response by the mare. Additionally, we should have a better understanding of when disease starts and how it progresses which should help us improve the focus of future research into Nocardioform placentitis.
Importance to the Equine Industry:
Nocardioform placentitis is a devastating reproductive disease affecting equine populations worldwide. It is characterized by focal sites of infection which are frequently covered by a thick brown exudate. If Nocardioform placentitis isn’t diagnosed in a timely manner, mares will frequently abort their foals or give birth to small, runty foals. Despite its profound impact, diagnosing Nocardioform placentitis remains challenging, often relying on clinical signs such as premature udder development which are nonspecific and may only become apparent late in disease. As such, many farms treat their high-risk mares with antibiotics on a monthly basis whether there are signs of disease or not. Through the development and refinement of a sensitive and specific diagnostic test, we are able to minimize late-term abortion and stunted neonates while working to minimize this unnecessary antibiotic use. This will not only improve the health of pregnancies but will also help prevent the increasing problem of antibiotic resistant strains of bacteria. Moreover, through a more comprehensive understanding of how the disease originates and progresses, researchers working in this area will have better tools available to focus their research in ways to produce better results and better improve the health of the horse.
RISK ASSESSMENT FOR PROXIMAL SESAMOID BONE FRACTURE
Peter Muir, University of Wisconsin–Madison –Two Year Grant
One Sentence Summary: This study will save the lives of racehorses by establishing screening using fetlock standing computed tomography for diagnosis of horses with a high risk of serious injury for personalized care.
Injuries to the Thoroughbred racehorse that lead to euthanasia are termed catastrophic. In 2022 the incidence of catastrophic injury was 1.25 fatalities per 1,000 starts in the USA, representing a loss of hundreds of horses. Stress fracture of the proximal sesamoid bone (PSB) is a common cause of fatal injury. Repair of PSB fractures, particularly biaxial midbody fractures, or fetlock salvage is associated with a poor prognosis. 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 PSB stress fracture. Our long-range goal is to reduce the incidence of catastrophic injury in Thoroughbreds by improving clinical screening using routine standing computed tomography (sCT) imaging in the sedated horse to check for concerning bone injury in the fetlock, particularly PSB injury that precedes stress fracture. The objective of this application is to develop a sCT screening approach for evaluation of racing Thoroughbreds with concerning fetlock bone injuries using computerized analysis of sCT images to identify horses with high imminent risk of PSB stress fracture and potential catastrophic injury. Our hypothesis is that our computerized virtual mechanical testing approach will be able to identify horses with high risk of PSB stress fracture. Our hypothesis is based on the substantial progress that we have made implementing a similar computer modeling approach for predicting risk of condylar stress fracture.
The rationale for this work is that implementation of sCT screening with computer model analysis of sCT scans in populations of racehorses will lead to substantial reductions in fatal injuries associated with fetlock stress fractures by accurately identifying horses with imminent risk of PSB fracture. An advantage of our research approach is that other information, such as exercise history and information about the training surface are not needed for risk prediction. Risk for individual horses is predicted directly from imaging information. To accomplish our objective, we will perform mechanical testing of PSB specimens after sCT imaging. We will relate the structural changes in the PSB, features that would not be detectable by radiography, to the propagation of a PSB stress fracture, which is a serious injury clinically. We will use these data to build a computer (finite element) model of the PSB to undertake detailed analysis of the mechanical properties of the PSB so that bones with reduced mechanical properties can be easily identified. 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 PSBs will be performed. Relevant features in the PSB will be determined from sCT images. This information will then be used to build the 3D PSB computer model and optimize and validate the mechanical predictions from the computer model to identify horses with imminent risk of serious injury. As a prelude to this project, we have designed and built a helical sCT scanning system that enables routine fetlock CT imaging. The proposed research is innovative because it capitalizes on routine availability of sCT imaging. Regarding outcomes, the work is expected to contribute in meaningful ways to 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 incipient changes in the PSB that increase risk of 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. Advances from this research will improve the effectiveness of injury prevention programs for racing Thoroughbreds across the world as sCT imaging becomes more widely adopted. We are well positioned to pursue this research because of the expertise of our team regarding injury prevention.
Importance to the Equine Industry: This work will ultimately save the lives of many racehorses. Completion of this project will enhance knowledge of the relationship between specific structural changes in the proximal sesamoid bone and imminent risk of stress fracture propagation, a serious injury that is often catastrophic. Since true standing computed tomography imaging in horses is now routine, this knowledge is directly translatable into clinical practice because racehorses with concerning bone lesions that are not evident on radiographs can easily be identified and managed appropriately without disruption to training and racing schedules. Our virtual mechanical testing approach using computer modeling enables wide preemptive longitudinal monitoring of horses in training to implement improved personalized care for racehorses and reduced catastrophic injury.
SECOND YEAR GRANTS
Transcriptomic Response To Osteoarthritis
Lynn Pezzanite, Colorado State University –Second Year
One Sentence Summary: This study will highlight the role that cells of the immune system play to contributing to disease progression of osteoarthritis toward the goal of developing treatments for each stage of disease.
Osteoarthritis is one of the most common disorders treated in horses, and is estimated to affect up to 80% of horses over the age of 15 years. Despite this high prevalence, treatment options remain limited and understanding of the mechanisms behind osteoarthritis progression are not fully understood. Cells of the innate immune system are increasingly thought to play a role in early stages of degenerative joint disease. The purpose of this study is to determine how the genetic code of cells, particularly cells of the innate immune system, changes during early osteoarthritis progression and how those values correlate to levels of inflammatory biomarkers, called cytokines, in joint fluid. This work will integrate new technologies including single cell RNA sequencing and multiplex biomarker analysis towards this goal. This study will highlight the role of immune infiltration contributing to disease biology in OA progression towards the overall objective of developing treatments tailored to disease stage.
Importance to the Equine Industry: This project will help clinicians to better understand how osteoarthritis changes the immune system and the genetic code of the cells in the joint when osteoarthritis is induced traumatically and then progresses. This in turn will help researchers develop appropriate targeted therapies depending on the stage of osteoarthritis (early, mid stage, late), ultimately helping clinicians to better treat horses with joint disease. This will impact the equine industry by improving understanding of changes occurring over time in osteoarthritis progression which will allow for development of targeted therapies tailored to disease stage for one of the most common disorders treated in horses.
Efficacy of Recombinant Equine Lubricin for Osteoarthritis
Heidi Reesink, Cornell University –Second Year
One Sentence Summary: This study will assess efficacy of recombinant equine lubricin (rEqLub) in mitigating equine joint disease and identify gene and protein pathways affected by rEqLub in equine joints.
Osteoarthritis (OA) results in chronic, progressive lameness and is a significant cause of morbidity and retirement in horses (Bertuglia 2016; McIlwraith 2012). Joint disease can occur spontaneously or secondary to joint trauma, and even with appropriate care, OA will develop in up to 50% of fractures involving the joint in human patients and following similar fractures in horses (Dirschl 2004; McIlwraith 1988; Niebauer 1988). OA involves several tissues within the joint, including articular cartilage, synovial membrane, and surrounding joint capsule and ligament tissues. Cartilage degeneration are the most recognizable lesion which occurs in OA, and these become irreversible rapidly during OA development.
Steroids and HA supplementation have been the mainstays of joint therapy in horses for more than two decades, in addition to oral or intravenous anti-inflammatories such as Bute, Banamine and Equioxx. Orthobiologics have become more available for the treatment of OA but require processing of minutes to weeks depending on the product, and the final product and clinical effects are inconsistent. HA formulations available to the veterinary market can improve synovial fluid consistency and lubrication, but these formulations only result in detectable HA in the joint for ~24 hours (Jackson 2006; Palmieri 2017). Lubricin is a highly sugarcoated molecule, and these sugars are responsible for its ability to provide effective cartilage lubrication (Jay 2001). Lubricin has the potential to treat both biological and physical aspects of joint disease, showing promise in rodent and mini-pig models of joint trauma (Jay 2012; Teeple 2011; Waller 2017). In addition, because lubricin can physically bind to the surface of articular cartilage, its sustained residence time of 28-42 days following a single intra-articular injection suggest that it may have a sustained effect (see Preliminary Studies). However, progress with lubricin therapy has been hampered by significant challenges with the largescale production and purification of the molecule. The field has been slow to translate lubricin therapy to large animals or veterinary clinical trials due to technical challenges associated with producing sufficient quantities of recombinant lubricin for a large animal trial and also due to competing demands for testing recombinant lubricin in human clinical trials. Notably, we have recently developed a novel strategy for production and purification of full-length, highly sugar-coated recombinant lubricin in sufficient quantities required for equine studies.
The long-term objective of this proposal is to translate lubricin therapy to equine clinical patients. We hypothesize that equine specific recombinant lubricin (rEqLub) can improve joint inflammation and arthritis by means of its anti-inflammatory and lubricating properties. In Aim 1, the efficacy of equine-specific synthetic lubricin to ameliorate the development of osteoarthritis in a surgical disease model. In Aim 2, we will study the signaling pathways that are affected by rEqLub in healthy equine fetlock joints, which will help identify mechanisms by which lubricin can protect joint health. We expect these studies to provide a baseline for the establishment of efficacy of lubricin joint therapy in the horse, which will springboard evaluation of lubricin therapy to future clinical trials in equine patients.
Importance to the Equine Industry: Lameness is the leading cause of early retirement across all horse breeds and disciplines. OA is responsible for more than 60% of all equine lameness (Caron 2003), and it is the second leading cause of premature racehorse retirement (Welsh 2013). Most currently available joint medications temporarily relieve symptoms but do not prevent progression of arthritis. Corticosteroids, with or without the addition of hyaluronic acid, remains the most common intra-articular therapy in both horses and humans (Fusco 2021; Velloso Alvarez 2020). With newer regulations restricting medication and corticosteroid use in racehorses and sport horses, equine therapeutics are limited, and an ideal intra-articular medication does not exist. Evidence in other species suggests that lubricin has immunomodulatory, chondroprotective, and lubrication effects, making lubricin a promising future therapeutic for OA (Alquraini 2015; Iqbal 2016; Waller 2013).
Production of sufficient quantities of lubricin for clinical studies in large animals, such as the horse, has not been feasible in the past due to challenges associated with making this complex, highly sugar-coated protein. However, we have developed a novel strategy for large-scale production of lubricin in amounts sufficient for joint injections in horses. This overcomes a significant hurdle and enables us to address a critical gap in understanding the biological functions of lubricin. In the proposed studies, we will assess equine-specific lubricin’s (rEqLub) ability to mitigate OA severity in an equine surgical disease model and identify cellular pathways affected by rEqLub to identify additional functions of lubricin in a healthy joint. We expect these studies to provide data about the ability of lubricin to treat joint disease in the horse. This study provides a path toward a potential real, clinical treatment that does more than mask the problem.
Treatment Of Meniscal Injury With Mesenchymal Stem Cells
Aimee Colbath, Cornell University –Second Year
One Sentence Summary: This study will determine whether intra-articular mesenchymal stem cells lead to improved meniscal healing, providing an immediate impact on how veterinarians treat equine meniscal disease.
Stifle injuries are common career ending injuries for equine athletes. In particular, injuries to the meniscus result in a relatively poor prognosis for return to prior level of performance. A single, small, retrospective study concluded that compared to historical controls, mesenchymal stem cells (MSCs) resulted in an improvement in the ability to return to work compared to surgery alone. Further, a recent survey of large animal surgeons and sports medicine specialists, indicated MSCs were the most commonly used biologic therapy following surgery for meniscal injury. However, recent reports are controversial, as a new retrospective study of meniscal injuries showed no effect of biologic therapies, including MSCs, on outcome. There is a clear gap in knowledge as to whether intra-articular stem cell administration improves outcomes for meniscal injury. Part of the challenge of answering this question has been the lack of an experimental model of meniscal disease in the horse. Therefore, we will conduct a randomized, controlled, blinded prospective study evaluating the use of intra-articular mesenchymal stem cells for treatment of meniscal injury using a novel, experimental model of meniscal disease. This study will achieve two important objectives. Firstly, it will establish this experimental model of meniscal disease which can be used now and in the future for evaluating the effectiveness of intraarticular treatments for meniscal disease. Secondly, the study will evaluate the effectiveness of intra-articular MSCs for meniscal disease using multiple outcome measures including lameness, joint fluid characteristics, diagnostic imaging outcomes, joint evaluation following euthanasia and cellular tissue (histologic) outcomes. The study will have an immediate impact on the treatment of meniscal disease in the horse by determining the efficacy of intra-articular MSC use, and a longer-lasting impact by providing an experimental model for future investigation of therapeutic interventions.
Importance to the Equine Industry: Meniscal injury remains difficult to treat in the horse, despite surgical intervention, with little known about the efficacy of intraarticular treatments following surgery. With only approximately 40% of horses returning to their previous level of performance following meniscal injury, the industry is in dire need of effective therapies. Retrospective studies on intra-articular mesenchymal stem cells for the treatment of meniscal disease have been conflicting; some studies claim a significant improvement and other studies find no benefit to biologic therapies. The question of the efficacy of mesenchymal stem cells needs to be answered with a randomized, blinded, controlled, experimental study of meniscal injury. To answer this important treatment question, this study will utilize a novel equine model of meniscal injury and evaluate the effectiveness of intra-articular mesenchymal stem cells for equine meniscal injury. Importantly, the study employs a plethora of outcome parameters including lameness, diagnostic imaging outcomes, joint fluid characteristics and cellular tissue (histologic) characteristics. The combination of these outcome parameters will provide a comprehensive look at the effect of mesenchymal stem cells in meniscal disease. The study will have an immediate effect on how equine veterinarians treat meniscal disease, improving the use of owner resources and establishing a treatment protocol backed by scientific evidence to enhance outcomes in our equine athletes.
Stem Cell Neotissue Implants for Equine Tendon Healing
Mandi J. Lopez, Louisiana State University –Second Year
One Sentence Summary: This study will determine if viable neotissue implants generated from stem cells will augment current therapies to treat debilitating tendon injuries in equine athletes and companions.
Tendon and ligament injuries are a leading cause of lost training days and early retirement among equine athletes. In adults, local cells rarely go to the injury to assist with healing. This explains, in part, the slow and incomplete healing that frequently occurs. Recent research shows that implanted stem cells direct tendon tissue stem cells to go to the injury and produce healthy tissue instead of scar tissue. Currently, there are limited ways to apply stem cells to tendon and ligament injuries or to keep them at the injury site during the healing process. We propose to overcome some of the hurdles to cell therapies for tendon injuries by turning stem cells into tendon cells that produce normal tendon tissue around them. Specifically, we developed a unique system that stimulates stem cells to become tendon cells and produce tissue after 21 days in highly customized fluid flowing through chambers that contain the cells on collagen templates. The proposed studies will confirm generation of tendon cells in new tissue that can be stored and shipped frozen so that it can be used promptly whenever and wherever necessary. The ability of the tissue to promote tendon healing will also be confirmed in equine tendon injuries. The ultimate goal of this work is to create a reproducible mechanism to generate tendon tissue implants that can be clinically applied to facilitate healing and restore normal limb function in equine athletes and companions.
Importance to the Equine Industry: The results of this proposal will establish a way to create healthy tendon cells and tissue from stem cells and prove that the cells remain in a tendon injury to accelerate healing. Tendon and ligament injuries account for a large majority of musculoskeletal injuries that are responsible for up to 72% of lost training days and 14% of retirements by equine athletes. Among injuries, superficial digital flexor tendon and suspensory ligament injuries are the most common, comprising up to 46% of all limb injuries. Tendons and ligaments have poor healing capacity, and poor or abnormal tissue repair contributes to a reinjury rate as high as 67% within 2 years. Direct injection of stem cells into tendon injuries to augment natural healing has mixed results, and the cells may not always stay at the site of tissue damage. We propose to overcome the current limitations of stem cell tendon therapies in adult horses with tendon tissue produced by adipose tissue stem cells grown in conditions designed to create tendon cells. Confirmation that the implanted cells remain at the treatment site and do not change may help overcome some of the current regulatory restrictions on stem cell use. Additionally, confirmation that the new tissue is not damaged by freezing will make it possible for the treatment to be available to all horses. Overall, the work in this proposal will significantly advance regenerative therapies for tendon injury in a host of equine companions and athletes plagued by an all-too-common musculoskeletal problem.
Gallium Nitrate to Treat Bacterial Endometritis in Mares
Dale Kelley, Oklahoma State University –Second Year
One Sentence Summary: This study proposes to develop new, safe, and efficacious antimicrobial strategies to treat antimicrobial resistance.
An alternative approach to the use of intra-uterine antibiotics is the use of gallium as a disruptor of bacterial iron-dependent processes. Iron is essential for almost all pathogens and is required for enzymatic processes, such as DNA synthesis, electron transport and oxidative stress defense. Gallium has a nearly identical ionic radius as iron, and some bacterial uptake systems are unable to distinguish between the two elements. Gallium disrupts iron-dependent processes since it cannot be reduced under physiological conditions. Thus, gallium incorporates into iron-containing proteins disrupting their function resulting in bacterial death by essentially starving bacteria. Previous studies have found gallium compounds exhibit antibacterial activity against several pathogens, including Pseudomonas aeruginosa, Francisella tulerensis, Acinetobacter baumannii, several mycobacterial species and Klebsiella pneumoniae.
With increased public awareness of antimicrobial resistance there is a need to develop new, safe, and efficacious antimicrobial strategies. We propose to investigate the use of gallium as an intrauterine therapy in a three-part progressive process.
• The initial phase of this project is to confirm the effectiveness of gallium as an antimicrobial agent against Escherichia coli, Streptococcus equi subspecies zooepidemicus, Pseudomonas aeruginosa and Klebsiella pneumoniea, respectively.
• A second study to determine serum and endometrial gallium concentrations after various intra-uterine infusion doses and estimate the amount of active gallium to treat infections
• Then a safety study using the dose determined to be effective in the second experiment to determine if there is a negative impact of gallium on uterine health.
Importance to the Equine Industry: Growing public awareness of antimicrobial resistances underscores the need to develop alternative therapies for the treatment of uterine infections. Although antimicrobial resistance is often considered to be concerning in equine reproduction, there are few studies evaluating changes in antibiotic resistance in bacteria isolates from mares’ uteri. One research study evaluated uterine bacterial isolates in 8,296 cases between the years of 2003 and 2008 and found a significant change in resistance to chosen antibiotics over those years. This highlights the importance not only of sensitivity profiles for isolated bacterial pathogens prior to treatment, but also the distinct need to develop alternative antimicrobial treatments to preserve the effectiveness of current antibiotics.
A VapA mRNA Vaccine for R. equi Pneumonia
Noah Cohen, Texas A&M University –Second Year
One Sentence Summary: This grant evaluates an mRNA vaccine administered intramuscularly to foals to protect against pneumonia caused by the bacterium Rhodococcus equi, a major cause of disease and death in foals worldwide.
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 administered intramuscularly (IM) 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 targeting VapA is that levels of antibodies against this protein in foals are correlated with protection against R. equi pneumonia. This project will address 2 essential steps to achieve this goal. First, we will demonstrate that the VapA mRNA we have designed can elicit immune responses when administered intramuscularly (IM) to foals at ages 1 and 21 days.
Specifically, we will compare anti-VapA antibody activity levels in serum and bronchoalveolar lavage fluid (i.e., lung washings) by an enzyme linked immunosorbent assay (ELISA) and cell-mediated immune responses specific for VapA of immune cells circulating in blood by a method known as ELISpot in immunized (n=12) and control (n=12) foals. Second, we will examine the efficacy of the vaccine to protect these same 12 vaccinated and 12 unvaccinated foals against experimental infection. At age 31 days, foals all foals will be infected intrabronchially with virulent R. equi and then monitored through age 9 weeks for signs of pneumonia. All foals that develop pneumonia will be treated with antimicrobials and any other supportive care needed. Results of this study will provide evidence of the safety and efficacy of immunizing foals with mRNA to protect them against rhodococcal pneumonia.
Results also will have important implications for immunizing foals against other infectious disease.
Importance to the Equine Industry: The horse industry is 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.
Genomics of Thoroughbred Stallion Subfertility
Terje Raudsepp, Texas A&M University –Second Year
One Sentence Summary: The proposed project aims to identify candidate genes and regulatory variants underlying impaired acrosome reaction and subfertility in Thoroughbred stallions using multi-platform genomics.
Despite the revolutionary technological advancements leading to unparalleled progress in the genomics of equine traits, diseases and disorders in the past decade, the genomics of equine reproduction has been given relatively less attention. Currently, only a few stallion reproductive disorders have been associated with candidate genes or candidate genomic regions, although without actual knowledge of the underlying molecular mechanisms. Among these, an autosomal gene FKBP6 in horse chromosome 13 (ECA13) is of particular interest because a certain genotype of this gene is significantly and invariably associated with Thoroughbred stallion subfertility. This association was initially discovered in 2012 by studying seven Thoroughbred stallions with a confirmed reproductive disorder - impaired acrosome reaction (IAR). Acrosome is a single secretory granule present in the head of mammalian sperm and secretion of acrosome, known as acrosome reaction (AR), is necessary for natural fertilization. Impaired acrosome reaction results in subfertility or infertility. More recently, a large-scale follow-up study compared breeding records of 150 Thoroughbred stallions with their FKBP6 genotype and confirmed the association of FKBP6 with stallion subfertility, regardless whether data for acrosome reaction were available or not. It was shown that the frequency of this ‘stallion subfertility’ genotype in global horse populations and Thoroughbreds separately is 4%.
The study also reported about the development of a reliable and easy-to-use genetic test to identify stallions with the ‘subfertility’ genotype. The main limitation of this and the earlier study is that despite of significant and consistent association between FKBP6 and stallion subfertility, there is still no knowledge WHY? Firstly, because the associated genotype does not affect the structure or function of the FKBP6 gene and secondly, there is no evidence that FKBP6 has functions in acrosome reaction. Furthermore, the FKBP6 ‘subfertility’ genotype has also been found in Hanoverian and Friesian stallions, but the genotype is associated with subfertility only in Thoroughbred stallions and not in other breeds. This suggests that the FKBP6 ‘subfertility’ genotype may not be the actual cause but is rather tagging in the nearby genomic regions a gene or genes with structural or functional genetic variants unique to Thoroughbreds. In addition, identification of the first genetic marker for IAR and stallion subfertility is of outstanding scientific interest because the Thoroughbred is so far the only horse breed where IAR has been confirmed as a cause of stallion subfertility and because molecular causes for IAR have not been disclosed in any species, including humans.
The goal of the proposed research is to explore the genomic region around the FKBP6 gene in a cohort of 21 subfertile Thoroughbreds, including those with confirmed IAR, and compare it with the sequence landscape of this region in over 700 horses representing general Thoroughbred population and many other breeds. The aim is to pinpoint unique or rare sequence variants in the subfertile Thoroughbred cohort that could be likely candidates for IAR and stallion subfertility. This work will be done using multi-platform whole genome and testis transcriptome analysis. We anticipate that the findings will improve our understanding about the genetic regulation of acrosome functions in the stallion and mammals in general.
Importance to the Equine Industry: Fertility and reproductive health of Thoroughbred breeding stallions, particularly the most popular ones from elite high-performance pedigrees, are important cornerstones of the Thoroughbred industry. It is therefore important to improve and expand the methods for breeding soundness evaluation, particularly for cases of unexplained subfertility where a stallion has poor breeding records (low pregnancy rates) despite normal physical characteristics and good semen quality. The proposed study aims to dissect the underlying genomics of one such conditions – subfertility due to impaired acrosome reaction which typically manifests as unexplained subfertility because acrosome reaction test is not part of routine breeding evaluation. The IAR condition has been found only in Thoroughbred stallions and is significantly associated with a certain genotype in the FKBP6 gene. Carriers of this genotype are invariably subfertile. However, we do not know why? The findings of the proposed study are expected to throw light on that so that the use of the FKBP6 genetic test for stallion evaluation will be supported by a molecular explanation. These types of diagnostic measures are of particular importance for breeds such as the Thoroughbred, where artificial insemination or any other forms of assisted reproduction are not allowed. In the long run, genetic monitoring of breeding stallions for IAR may help to eradicate the condition from the population.
Validation of Biomarkers for Equine Neurodegeneration
Carrie J. Finno, University of California-Davis –Second Year
One Sentence Summary: It is expected that this study will improve the diagnosis of spinal cord disease in horses.
In horses, neurological disease often results in a career-ending diagnosis. The major causes of spinal cord disease in the horse include cervical vertebral compressive myelopathy (CVCM; Wobbler syndrome) and equine neuroaxonal dystrophy / equine degenerative myeloencephalopathy (eNAD/EDM), The clinical signs observed with CVCM and eNAD/EDM are often highly similar, and it is challenging to arrive at a definitive diagnosis while the horse is alive. Unfortunately, these diagnoses are often made after the horse has been euthanized. Currently, there are not magnetic resonance imaging (MRI) machines large enough to accommodate the size of a horse’s neck. Therefore, the current way to diagnosis CVCM is with x-rays and computed tomography (CT), with or without the use of contrast (i.e. myelogram). With eNAD/EDM, there is no definitive diagnostic test yet available. Additionally, in Quarter horses (QH) and in Warmbloods (WB), the clinical presentation of eNAD/EDM differs, with QHs displaying an abnormally quiet mentation and incoordination at a young age, while WB often present between 5-15 years of age with a sudden onset of behavioral changes and incoordination. A vitamin E deficiency has been associated with eNAD/EDM in the QHs but not yet in the WB. Thus, there may be subcategories of eNAD/EDM. Since the definitive diagnosis in many cases of spinal cord disease in the horse can only be achieved after the horse has been euthanized, these career or life-ending decisions without a definitive diagnosis creates a stressful and emotional burden for owners, trainers, and veterinarians involved with affected horses.
Through a novel approach, where we were able to screen 367 proteins involved in neurologic disease in humans, we have defined specific proteins in the blood (serum) and cerebrospinal fluid (CSF) from confirmed cases of CVCM and eNAD/EDM in QHs and WBs. Based on these results, we now propose to validate these findings, using bench-side antibody tests, in both our initial set of samples and an additional validation group of confirmed cases of equine spinal cord disease. We hypothesize that concentrations of five specific proteins; R spondin-1 (RSPO1), neurofilament light chain (NEFL), dickkopf WNT signaling inhibitor 1 (DKK1), WAP, Follistatin/Kazal, Immunoglobulin, Kunitz And Netrin Domain Containing 1 (WFIKKN1) and calretinin (CALB2), will define the serum and CSF proteome from horses with CVCM and eNAD/EDM, thereby distinguishing between diseases. Specifically, we expect concentrations of CSF NEFL, CSF DKK1, CSF CALB2 and serum WFIKKN1 to be higher in CVCM versus eNAD/EDM versus healthy horses, while CSF RSPO1 will be highest in healthy horses and lowest in CVCM horses. Additionally, we hypothesize that in the spinal fluid, RSPO1 concentrations will be higher in eNAD/EDM QHs versus WBs.
Importance to the Equine Industry: The inability to sensitively and specifically diagnose equine spinal cord disease is a significant financial burden for horse owners, trainers and insurance underwriters. We anticipate that this study will provide highly specific and sensitive tests in blood and spinal fluid that, when combined with the results of standard diagnostic testing, can be used to diagnose horses with spinal cord disease due to CVCM or eNAD/EDM.
Antibiotic Effects On Uterine Microbiome And Resistome
Igor Canisso, University of Illinois –Second Year
One Sentence Summary: This is a study of uterine microbiome and resistome of mares resistant and susceptible to endometritis treated with post-mating antibiotics.
The healthy uterus was previously considered a sterile environment. Now, it is recognized that the uterus is colonize by a high number of bacteria of different species (i.e., uterine microbiome) that is necessary to prevent infection, modulate inflammation, and establish pregnancy. Endometritis is an inflammation of the lining of the uterus that occurs with the contact of bacteria and sperm present in the semen. Persistent breeding-induced endometritis (PBIE) is the most common reproductive problem encountered in the equine breeding industry and the third most frequent clinical problem affecting horses. Mares are classified as resistant and susceptible to PBIE based on their ability to clear the post-breeding endometritis. Our preliminary assessment indicates that mares susceptible to PBIE have a lower number of bacteria and species of bacteria than resistant mares. Therefore, the uterine microbiome of mares that are susceptible to PBIE seems to be distinct enough that it may be helpful to estimate susceptibility to PBIE, which would allow for the application of on-target therapy. Antibiotics are essential in veterinary and human medicine for the treatment of bacterial infections. Resistance has been demonstrated in all classes of antibiotics. The antibiotic resistome is the collection of genes contributing directly and indirectly to resistance. Post-mating uterine infusion with antibiotics is commonly administered to prevent endometritis. A recent study reported that 50% of Thoroughbred mares received post-mating antibiotics and up to 100% of mares in some farms. The extensive use of post-mating antibiotics likely contributes to the emergence of resistance. Therefore, there is a critical need to better understand the effects of antibiotics on the resistome and develop advanced diagnostic techniques for the detection of antibiotic resistance in clinical practice. The current method for testing antibiotic susceptibility is the disc diffusion test, and the turnaround time for results is up to 5 days. The use of molecular diagnostic techniques such as PCR could significantly reduce the time required to determine antibiotic susceptibility and the onset of treatment.
Our long-term goal is to understand the role of the uterine microbiome and resistome in uterine health to enhance diagnostic techniques. Studying the effects of post-mating antibiotics under controlled conditions will allow us to develop evidence-based guidelines regarding this procedure in mares. Our central hypothesis is that post-mating antibiotics affect uterine microbiome composition and resistome and that PCR can be used as a rapid and accurate test to determine antibiotic susceptibility.
Sixteen mares previously classified as susceptible (n=8) or resistant (n=8) to PBIE in the preliminary studies will be subjected to a five-day course of antibiotics starting 6 hours post-mating. A fertile stallion will be used to live cover all mares. The estrous cycle 1 of each mare will start as the control with no antibiotic infusion. Then treatment cycles 2 to 5 of each mare will be randomized in a crossover design to receive uterine infusions of gentamicin, ceftiofur sodium, procaine G penicillin, and ticarcillin-clavulanate. These drugs were chosen as they are the most frequently administered post-mating antibiotics. A washout cycle will be carried out after each treatment cycle and before the next. Uterine samples will be collected the day before, three days, and five days after mating for microbiome and resistome analyses, endometrial culture, and cytology. This sampling will emulate a 3- and 5-daycourse of antibiotics. The microbiome composition will be evaluated with advanced molecular techniques based on the sequencing of particular regions (16S) of the bacterial genome. The number of bacteria and species will be evaluated before and after antibiotic treatment, between groups (resistant vs. susceptible to PBIE), and between control and antibiotic treatment.
Positive bacterial cultures will be identified by their morphology and microbiological techniques. Bacterial isolates will be assessed for antibiotic susceptibility testing with the disc diffusion method and PCR for the four antibiotics used in the study. The antibiotic resistance genes expression levels will be investigated with PCR (3 to 4 genes per antibiotic) and compared with disc diffusion’s results.
The team of multi-disciplinary investigators is uniquely positioned to complete the project, as it comprises experts in uterine health, microbiome, bioinformatics, and molecular techniques applied to antibiotic resistance. We foresee that the results of this study will have an immediate positive impact on the horse industry and serve as the foundation for practical assessment of the microbiome composition in commercial Thoroughbred broodmares and diagnostic of antibiotic resistance in horses.
Importance to the Equine Industry: Endometritis is an inflammation of the lining of the uterus. It is the most common cause of subfertility in the horse. It is associated with a reduction in fertility, pregnancy loss, increased costs, and risks of bacterial infection. Post-mating antibiotics are commonly administered in horse breeds like the Thoroughbred to prevent endometritis, but its impact is unknown. It is recognized that a “core” uterine microbiome (i.e., bacteria that reside in the healthy uterus) is necessary to prevent infection, modulate inflammation, and establish pregnancy. The proposed research project focuses on understanding the impact of post-mating antibiotics in the mare uterus to enhance our knowledge of this disease and develop fast and accurate diagnostic techniques. Our central hypothesis is that post-mating antibiotics affect the bacteria that populate the uterus and their antibiotic resistance and that PCR, a widely used molecular test, can be used as a rapid and accurate test to determine antibiotic susceptibility. Our experimental design will closely simulate the Thoroughbred industry while evaluating the presence of antibiotic resistance. Antibiotic susceptibility testing is the standard method, and the turnaround time for results is 3-5 days. Treatment of bacterial diseases is often initiated and sometimes treated completed empirically due to the long turnaround time. Thus, it might be possible that screening antibiotic resistance with PCR in an isolate will speed up the choice of antibiotic, and the standard method could confirm the early rapid molecular diagnosis. In humans, this approach has proven to have high sensitivity and specificity. The methods proposed here can be extrapolated into other areas of equine practice and validated in commercial diagnostic laboratories, improve precision, and reduce the time from susceptibility testing to antibiotic treatment in different bacterial diseases of horses, which could be lifesaving in a clinically ill patient.
Nanoparticle Vaccines for Equine Rotavirus B
Feng Li, University of Kentucky –Second Year
One Sentence Summary: The vaccine candidate developed from this project will help the equine industry to control and prevent equine rotavirus B infection in future foaling seasons.
Emergence of novel equine rotavirus B (ERVB) that caused extensive foal diarrhea outbreaks during the 2021 and 2022 foaling seasons has been viewed as a significant concern to the equine industry due to its effects on foal health and subsequent economic impact. Currently, there are no effective vaccines for control of this highly contagious disease. This proposal will support our efforts to develop a ERVB nanoparticle vaccine candidate that can elicit durable and high titer neutralizing antibodies in mares, and as a result, foals born to vaccinated mares will be protected against ERVB infection through the maternal colostral antibodies. Once an effective and safe vaccine has been identified from this proposal, we will actively seek a collaboration with a biotech company to produce the vaccine for commercial purchase and distribution, which will likely be through other funds. The Gluck Center is currently exploring relationships that could be helpful in developing and brining a commercial ERVB vaccine to market.
Importance to the Equine Industry: A short-term deliverable to the equine industry is a vaccine candidate that can be used to protect foals against ERVB infection in future foaling seasons. Our long-term goal is to provide equine industry a trivalent rotavirus vaccine containing equine rotavirus A (G3 and G14 strains) as well as ERVB, that can protect foals against infections of both equine rotavirus A and B.
An Efficacious EPM Vaccine is on the Way
Sharon Witonsky, Virginia Maryland CVM –Second Year
One Sentence Summary: This study plans to identify potential MHC class I CD8 and MHC class II CD4 protective epitopes for an efficacious vaccine against EPM, due to Sarcocystis neurona.
An effective EPM vaccine is desperately needed to prevent both the immediate and long-term effects of this debilitating disease on affected horses. Therefore, our long-term goal is to generate an effective vaccine against Sarcocystis neurona, the most common cause of EPM. Our theory is that we can use a software program called immune epitope database (IEDB) to screen the known S. neurona genetic sequences for potential protective peptides/epitopes. These peptides are small segments of protein that have the potential to stimulate a protective immune response against S. neurona, which is what we need with an efficacious vaccine. We can use this IEDB software program to generate a list of these proteins/peptides from the S. neurona sequence/genome, but then we have to test the peptides for their ability to provide protection. Before we can see if the peptides can actually protect a horse from EPM, we have to “test” the peptides in a much less expensive cell-based assay to see if they stimulate some of the same molecules/cytokines, such as IFN-γ, necessary for protection. We know that for protection against S. neurona, we need a certain type of immune response, a cell mediated immune response. Based on other studies, we know this consists of CD4 and CD8 cells producing IFN-γ. Therefore, we will use neurologically normal horses that have been exposed to S. neurona, but have not developed disease, as the horses that have made a protective immune response. We will then test all the different peptides for their ability to stimulate this in vitro protective CD4 and CD8 IFN-γ response. The peptides which stimulate the best CD4 and CD8 IFN-γ responses are the most likely ones to provide protection. Screening and ranking these peptides based on their ability to stimulate IFN-γ is the goal of the study. Those peptides which stimulate in vitro and in vivo protection are called protective epitopes. With this knowledge, the goals of this study will be complete. The next step would be to test these peptides/potential protective epitopes in a mouse model to see if they protect mice from developing EPM. Those protective epitopes would then be screened in horses for their ability to protect against EPM.
Importance to the Equine Industry: Equine protozoal myeloencephalitis (EPM) is a devastating disease caused by S. neurona, and for which there are no commercially efficacious vaccines available. EPM is one of the most significant and devastating neurologic diseases in the United States, with an increasing recognition of its presence and severity in South America as well. In 2001 the National Animal Health Monitoring System (NAHMS) reported annual costs of $55-110 million annually to the industry. No updated costs are available; however, costs continue to skyrocket. Owners suffer a financial and emotional burden due to time lost from performance, incomplete recovery and relapse of disease. Additionally, diagnostics, treatment and rehabilitation often total many thousands of dollars. Furthermore, with the current state of diagnosis and treatments, many horses do not recover completely and must be rehomed for another career. Moreover, at least 10% of these horses develop recurrent signs and must be retired at best, and sometimes euthanized due to their poor prognosis. Our theory is that we can use a software program called immune epitope database (IEDB) to screen the known S. neurona genetic sequences for potential protective peptides/epitopes. These peptides are small segments of protein that have the potential to stimulate a protective immune response against S. neurona, which is what we need with an efficacious vaccine. We can use this IEDB software program to generate a list of these proteins/peptides from the S. neurona sequence/ genome, but then we have to test the peptides for their ability to provide protection. The peptides which stimulate the best CD4 and CD8 IFN-γ responses are the most likely ones to provide protection. Those peptides which stimulate in vitro and in vivo protection are called protective epitopes. With this knowledge, the goals of this study will be complete. We would then test the peptides in a mouse model to see which ones provide protection.
Use Of Nucleic Acid Vaccines To Protect From EHV-1/EHM
Gisela Soboll Hussey Michigan State University – Second Year of Three Year Grant
One Sentence Summary: This proposal is to develop a novel mRNA-based equine herpesvirus (EHV) vaccine that protects horses from EHV-1 myeloencephalopathy and will also likely cross-protect against other equine herpesviruses
Equine herpesvirus-1 (EHV-1) infection results in sporadic but devastating outbreaks of neurological disease in the equine population caused by a myeloencephalopathy with a poorly understood pathogenesis. The impact of EHV-1 myeloencephalopathy (EHM) on equine health and the industry is highlighted by a series of major outbreaks in North America and Europe over the past decade, including the two largest outbreaks in 2011 and 2021 in North America and Valencia, Spain. Despite the importance of EHV-1 in horses, effective prevention remains elusive and there is currently no vaccine available to prevent EHM. This is partially a consequence of the absence of a reliable experimental equine model of EHM and partially due to a lack of innovation in vaccine technology in the equine vaccine market in the past 40 years. In this proposal, five investigators with complementary expertise in equine herpesvirus virology, vaccinology and pathogenesis/animal models have come together to close a major gap in vaccine development against EHV infection. The goal of this application is to exploit innovations in RNA vaccine technology that have come about during the COVID-19 pandemic and are currently the first line of defense. We propose to refine this mRNA vaccine technology for immunization of horses. Based on our extensive preliminary data, we will test vaccines containing the parts of EHV-1 that are important for inducing protective immunity in horses. We are ideally placed to accomplish this task because we have previously developed a unique model for experimentally inducing EHM in horses that now allows us to test potential vaccine candidates . Moreover, given the high degree of similarity among equine herpesviruses genetically related to EHV-1, namely EHV-3, EHV-4, EHV-8 and EHV-9, our vaccine candidates, if proven successful, are likely to also offer protection to other equine herpesviruses. Additionally, they could readily be extended as a platform to induce protective immunity against an array of EHVs or other equine viruses.
Importance to the Equine Industry:
The EHV-1 “G” strain (SNP at ORF 30, G2254) has been associated with more severe outbreaks and a greater occurrence of EHM than the EHV-1 “A” strain (A2254 genotype). The enhanced replication capacity with the “G” strain appears to be associated with an enhanced frequency of more severe disease; however, both strains cause EHM. Several factors appear to be contributing to an uptick in EHV-1, particularly in Europe and the United States, with equine venues being closed or quarantined as a consequence. We propose an mRNA vaccine delivered in an extended release, biodegradable polyanhydride vaccine depot. Compared with traditional vaccines, we anticipate that this approach will be more effective and require fewer inoculations.
A Novel DNA Vaccine Platform To Control EHV-1 and EHV-4
Bettina Wagner Cornell University –Second Year
One Sentence Summary: The goal of this project is to develop an effective DNA vaccine for equine herpesvirus to protect horses against infection and disease and to prevent EHV outbreaks in the US.
Ongoing EHV-1 outbreaks, including cases of neurologic disease caused by EHV-1 and called ‘equine herpesvirus myeloencephalopathy’ (EHM), emphasize the fact that currently available EHV-1 vaccines are not fully protective. These disease outbreaks are occurring despite widely used vaccination and many available EHV vaccines on the market. The fact that currently available inactivated or attenuated vaccines have not been sufficient to completely protect from EHV-1 outbreaks has caused the demand for novel EHV vaccine formulations that can prevent horses from infection and severe disease, especially neurologic disease. Here, we propose to design and evaluate a novel DNA vaccine formulation against EHV-1/EHM for horses. We are combining our expertise in equine immunology and EHV-1 research (Dr. Wagner) and virology and vaccine design (Dr. Diel) to first perform a comprehensive analysis of the best DNA vaccine candidate and then test the optimized vaccine candidate for its protective effects in horses. The DNA vaccine platform that we are testing in this project has already been used successfully for induction of immunity and protection from infectious disease in other species. The project will result in an established new EHV vaccine candidate and provide a new approach to effectively protect horses from EHV-1 infection and disease. Overall, the project is relevant to the prevention of disease induced by EHV-1 by increasing immunity and protection in the equine population and reducing severe neurological disease outbreaks in the US.
Importance to the Equine Industry:
Equine herpesvirus type-1 (EHV-1) has a significant impact on animal health and the equine economy worldwide. Every year, several neurologic outbreaks are reported throughout the US (http://www.equinediseasecc.org/alerts/outbreaks). In an outbreak scenario, EHM is first confirmed by an EHV-1 positive diagnostic test in the affected horse(s). The positive EHV-1 test result causes immediate regulatory actions with stringent quarantine and biosecurity of all horses at the facility where the EHM affected horse is kept to prevent further spread of the contagious disease. Quarantine is in place for 21-28 days after the last suspected infection. Currently, all potentially exposed horses at a quarantined location are kept there for the same time for general precaution and preventing the possible spread of EHV-1 to other horses. During this time all movements of horses to and from the quarantine facility stop. Depending on the location of the first affected horse, quarantine can apply to race, show and auction premises, or secondary locations if a potentially exposed horse already moved to another site, and can thus stop the movement of involved horses for weeks. Consequently, EHV-1 quarantine represents a high economic burden for the horse industry through loss of severely affected horses, medical treatment charges of diseased horses, quarantine management efforts, and lost training, racing and/or competition time for all horses involved in the quarantine scenario. EHV-1/EHM outbreaks and quarantine have not only a serious impact on equine health, they also restrict horse transport, racing and competitions, and economically affect horse owners, equine businesses, and the equine industry.
RESEARCH CAREER DEVELOPMENT AWARDS
There are three Career Development Award recipients in 2024.
The Storm Cat Career Development Award, inaugurated in 2006, is a $20,000 grant in 2024 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.
Rebecca Bishop,
University of Illinois Urbana-Champaign
Multiomic Investigation of Peritoneal Fluid in Colic
Faculty supervisors Dr. Annette McCoy, Associate Professor, Equine Surgery and Dr. Pamela Wilkins, Professor, Veterinary Clinical 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 2024. 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.
Rebecca Legere,
Texas A & M University
Passive immunization against Rhodococcus equi using MRNA
Faculty supervisor Dr. Noah Cohen, Distinguished Professor and Associate Department Head
In early 2024, Nancy C. Kelly passed away after an extended illness. Nancy helped raise millions
of dollars for equine research, as the vice president of development for Grayson-Jockey Club Research Foundation. She was also the executive director of The Jockey Club Safety Net Foundation, organizing countless fund-raising events ranging from golf tournaments and ladies’ luncheons to formal gala dinners.
In her honor, Grayson is funding a special Career Development Award in 2024.
Lauren Hughes,
University of Minnesota
Genetics of Pituitary Pars Intermedia Dysfunction Risk
Faculty supervisor Dr. Molly McCue, Professor, Department of Veterinary Population Medicine (VPM)
