Current Research

Grayson-Jockey Club Research Foundation’s board of directors has announced a slate of 18 research projects which the Foundation will fund for a total of $1,239,083 in 2018. The list includes eleven new projects and seven continuing proposals, as well as Two Research Career Development Awards.
The allotment brings the Foundation’s total impact since 1983 to over $26.3 million to fund 358 projects
at 43 universities.


HOST-DIRECTED CONTROL OF R. EQUI FOAL PNEUMONIA II
Angela Bordin, Texas A & M University – First Year (2 Year Grant)

One Sentence Summary: We propose to use an inhaled product applied directly into the lungs to increase immune responses to protect foals against Rhodococcus equi, a bacterium that causes severe pneumonia in foals.

Pneumonia caused by a bacterium called Rhodococcus equi (R. equi) is an important cause of illness and death in foals. Because no vaccine is available and because many foals are unable to fight infection on their own, control of R. equi pneumonia is based on treatment of foals with a specific class of antimicrobials (named macrolides). With extensive, repetitive use of macrolides, these bacteria become resistant to this group of antimicrobials. The lack of alternative antimicrobials that would efficiently kill R. equi, and the low likelihood of novel antimicrobial development indicate that new approaches to control are needed. The specter of antimicrobial resistance is a major problem for human and veterinary medicine.

We propose to use a self–cure strategy (named host–directed, where the foal is the host) to reduce the occurrence and mortality of R. equi pneumonia, and to decrease the chances of development of antimicrobial resistance. Foals appear to be infected with R. equi during the first weeks after birth, a period which precedes complete development of adaptive immunity (the type of immune response made of specialized cells, such as antibody–producing cells). Foals are born with a functional innate immune system, comprised of cells which respond to all invading organisms. Stimulation of innate immunity, therefore, is a logical approach for protecting foals. Some molecules located on the surface of innate immune cells, called Toll–like receptors or TLRs, are important mediators of innate immunity. They act primarily through secretion of substances called cytokines. PUL–042 is a product that combines two substances that can stimulate innate immunity via TLRs directly that can be inhaled into the lungs to prevent bacterial pneumonia, and we plan to investigate its use as a method of host–directed control of R. equi pneumonia. We propose to use this product via nebulization, which is the administration of mist inhaled into the lungs using a nebulizer (such as those used for asthma patients). The objectives of our proposal are to better understand the how the infection of R. equi occurs in lung cells of both horses and foals (Year 1), and to evaluate if nebulized PUL–042 is safe and if it increases the capacity of equine lung cells to kill R. equi (Year 2).

Our long–term goal is to demonstrate that PUL–042 nebulization of foals could be used at equine breeding farms to prevent pneumonia caused by R. equi. Once optimized, this approach could replace antimicrobial prophylaxis (a strategy in which antimicrobials are used to prevent the disease by treating the foals that are not sick). Furthermore, it might serve as a supplementary treatment to antimicrobials therapy in foals with R. equi pneumonia, which could potentially shorten treatment time and improve outcomes. The current standard for prevention of R. equi pneumonia at farms is transfusion of R. equi hyperimmune plasma. This approach has many disadvantages, such as being expensive, labor–intensive, and causing undesired side–effects in foals. Nebulization of PUL–042 could replace plasma transfusion. Last, by stimulating innate immune responses of the newborn foal, a reduction of other bacterial respiratory infections (e.g., the bacterium that causes strangles) might occur, as well as improved responses of foals to vaccines. Results of this project will have tremendous impact for the equine industry, specifically at horse– breeding farms where R. equi pneumonia can occur in 20–40% of the foal crop.


THOROUGHBRED SALES RADIOLOGY-ULTRASONOGRAPHY STUDY
C. Wayne McIlwraith, Colorado State University – First Year (2 Year Grant)

One Sentence Summary: This study will improve the industry’s understanding of the significance of sesamoiditis, ultrasonographic suspensory branch changes, and stifle lucencies in sales yearlings and two-year-olds.

Veterinary interpretation of certain findings on pre-sale radiographs often varies and this is a continued source of controversy within the Thoroughbred industry. We currently have insufficient information relating to sesamoiditis and stifle lucencies for veterinarians to give objective advice on the true significance of these lesions, and the risk of associated problems as the horse enters training. Present uncertainty over the meaning of these findings can negatively impact the sale of young horses and causes frustration amongst breeders, consignors, buyers and veterinarians. Veterinary advice that deems these findings an increased risk for resale makes pinhookers reluctant to bid on affected horses, therefore leaving the sellers to count solely on the interest of buyers looking to retain horses to race themselves. This drastically weakens the marketplace and negatively impacts the whole industry. In order to turn this around and enable the repository system to function as it was intended to strengthen the industry; our research group believes that a large-scale scientific investigation into the significance of sesamoiditis and stifle lucencies in sales Thoroughbreds is needed.

Suspensory branch ultrasonography is becoming more frequent in sales yearlings, as sellers and buyers sensitized to the issue of sesamoiditis seek to gather more information on which to base their decisions. Ultrasonography is being performed on yearlings with radiographic signs of possible sesamoiditis, as well as on valuable yearlings that have a radiographic sesamoid appearance considered to be within normal limits. As pre-sale suspensory branch ultrasonography is becoming commonplace, it is critical that evidence-based research be performed to provide objective data to explain the long-term significance of varying degrees of ultrasonographic findings.

This research will investigate both sesamoiditis and medial femoral condyle lucencies in sales horses, on an unprecedented scale. Researchers will gather radiograph and ultrasound information on a large number of sales yearlings and two-year-olds and then follow these horses into their racing careers. Researchers will analyze which, if any, changes seen on sales radiographs and ultrasounds are predictors of clinical injury or compromised racing ability, compared to other horses without these radiograph findings. Consignor permission for research inclusion was granted for 74% of yearlings at the 2016 Keeneland September Sale, showing an overwhelming desire for information to sort out these issues. Researchers will evaluate the radiograph information on these 2975 yearlings, combined with the ultrasound information on 704 of these yearlings. Permission for inclusion in the two-year-old phase of the study was granted by 45 two-year-old consignors, for 78% of eligible horses. Therefore, the yearling information will be paired with two-year-old sales radiographs on 473 of these same horses, and two-year-old sales ultrasounds on 415 of these horses, for those which presented at the five major 2017 two-year-old in training sales. Horses will then be followed into their two and three-year-old racing seasons.

The ultimate goal of this research is to provide the industry with a better understanding of sesamoiditis and stifle lucencies in Thoroughbreds. With the knowledge gained from large-scale scientific research, the information on pre-sale radiographs should allow the seller and prospective buyers to work with veterinarians to make informed decisions on the trade and future racing management of sale horses. Individual horses will benefit through appropriate management tailored to any significant abnormalities that have an increased risk of injury during training. The industry as a whole will benefit through increased knowledge and confidence in decision making surrounding bloodstock purchases and management. This will boost industry-wide confidence in the sales repository system and in the veterinarian’s role in the trade, management and care of the Thoroughbred horse.


UNDERLYING CAUSE OF RECURRENT EXERTIONAL RHABDOMYOLYSIS
Stephanie Valberg, Michigan State University –One Year Grant

One Sentence Summary: Stress-induced modification to the skeletal muscle calcium release channel forms the basis for tying up in thoroughbreds and pinpoints a target for development of effective new treatments.

The goal of our research is to establish preventative treatments for tying-up, also known as recurrent exertional rhabdomyolysis (RER) in Thoroughbred racehorses. Tying up affects 5-10 percent of racehorses and is responsible for substantial morbidity, veterinary expense, and days lost in training. We now have the expertise, samples and collaborations in place to determine the underlying basis for the form of tying up called RER which impacts one third of trainers at the track on any given day (1).

For centuries, racehorses have been selectively bred for speeds that require rapid-fire muscle contraction and relaxation. Muscle contraction involves release of calcium (Ca2+) from storage sites within the muscle cell to allow Ca2+ to interact with contractile proteins and trigger the muscle to shorten (contract). Relaxation of muscle after a contraction occurs by pumping Ca2+ back into the intracellular storage sites. Releasing more Ca2+ can increase the force of a muscle contraction and thus speed. Excitement and adrenaline will increase the release of Ca2+ within the muscle cell for a short term boost in speed. Beyond a threshold, however, too much Ca2+ can cause a prolonged contraction and muscle damage producing typical symptoms of tying up, a stiff painful gait. Selection for speed in Thoroughbreds has likely favored horses capable of generating slightly higher intracellular Ca2+ release from storage sites. Introduction of a nervous temperament in Thoroughbreds may also enhance this effect. We believe that in horses with RER, subjected to the chronic stress of training, an adrenaline-induced modification occurs in the Ca2+ release channel in skeletal muscle. This modification allows excessive amounts of Ca2+ to be released into the muscle during exercise, especially exercise in excited horses, tipping the balance of Ca2+ toward an episode of tying up.

Support for our theory comes from studies of heart muscle which contains a similar channel for Ca2+ release as skeletal muscle. Studies of heart failure have found that chronic stress changes the Ca2+ release channels such that they become persistently leaky. In the heart, this persistent leak causes arrhythmias and poor heart muscle function. We believe that the Ca2+ release in RER horses is particularly susceptible to this chronic modification by stress and this sets horses up for tying up when kept in a high stress environment.

In this grant we will first determine the DNA sequence of the Ca2+ release channel gene because we have found significant inaccuracies in the published sequence that will impact all future genetic studies. We will then examine the protein structure of the Ca2+ release channel. We will determine if the type of modification of this channel that occurs in human heart failure exists in Thoroughbred fillies susceptible to RER and determine if this modification is absent in fillies that don’t develop RER when they are in a high stress training environment. We will restrict our investigations to fillies because RER occurs most commonly in fillies and because we have evidence that Ca2+ regulation varies between fillies and colts. Finally we will look at global expression of all genes in skeletal muscle between Thoroughbred fillies in race training susceptible to RER and Thoroughbred fillies in race training not susceptible to RER.

The first two aims will focus specifically on the Ca2+ release channel and the third aim will broadly examine all genes in muscle for causal or compensatory modifications that occur in horses susceptible to tying up. By defining the specific genes and protein modifications that occur with RER we can use drug screening methodologies to develop targeted treatments for the disease. Considerable progress has been made toward these aims through preemptive sample collection and method validation. We have the expertise, samples and collaborations in place to determine the underlying basis for a disorder that affects 10% of racehorses and impacts one third of trainers at the track on any given day.

Tying-up or recurrent exertional rhabdomyolysis (RER) is an extremely frustrating, painful and common muscle disorder affecting 5 – 10% of Thoroughbred racehorses. Owners often lament that it frequently affects their young fillies showing the most promise at the beginning of their training. The term "tying-up" is used because of the stiff gait, muscle cramping, pain and reluctance to move that may develop following mild to moderate exercise. Countless dollars are spent on ineffective remedies to prevent this syndrome. Up to 75% of trainers have at least one horse with tying up and recurrence is so frequent in 17% of the affected horses that they do not race again that season (1, 7). The recurrent nature of RER, the long lay-up time between episodes of tying-up, the expense of training a horse with susceptibility to RER have led to a variety of legal and illicit strategies to prevent the disease. Presently treatment with dantrolene, which decreases Ca2+ leak through the Ca2+ release channel, is one of the few means to prevent RER (5). Dantrolene, however, is expensive, has to be given 60 -90 min before each ride and requires substantial withdrawal times. The scarcity of much needed information regarding the underlying basis for RER has limited the ability to develop therapeutic agents that specifically target the mechanisms that cause the disease. We believe that our experience, state-of-the-art methods and strong collaborations with equine practitioners at racing centers have positioned us to determine the underlying basis for RER and identify targets for therapeutic development.


FIROCOXIB PROPERTIES IN EQUINE PREGNANCY & PLACENTITIS II
Margo Macpherson, University of Florida –One Year Grant

One Sentence Summary: This project should provide a fundamental step toward determining if specific drugs (including firocoxibs) are performing expected functions, such as resolving inflammation, in pregnancies threatened by placentitis.

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

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

In 2015, the Grayson-Jockey Club supported work in our laboratories to examine characteristics of firocoxib when administered to mares with experimentally-induced placentitis. Specifically, we tested firocoxib in our model of ascending placentitis to 1. Characterize anti-inflammatory effects of firocoxib in mares with placentitis, 2. Determine if firocoxib combined with trimethoprim sulfamethoxazole and altrenogest would improve foal survival from mares with placentitis. We are in the process of analyzing a large and complex set of data from these studies. Notably, substances that initiate inflammation (cytokines) were lower in allantoic fluid of infected mares administered firocoxib as compared to infected, untreated mares. When mares with placentitis were administered firocoxib, trimethoprim sulfamethoxazole and altrenogest, they carried pregnancies longer and delivered more live foals than untreated, infected mares. While these data are very exciting, further validating the physiological basis for treatment success is a critical next step. Our goal for this application is to analyze samples, obtained from the 2017 mares having experimentally-induced placentitis, to determine if the drug combination of firocoxib, trimethoprim sulfamethoxazole and altrenogest minimized the disease-induced inflammatory response that initiated preterm delivery of foals. These data will be compared to samples from infected, untreated mares. Access to samples from these treatment groups offers us a unique opportunity to provide a rational basis for placentitis treatment choices. Information from these samples is expected to substantiate the promising results of improved foal survival from mares after administration of firocoxib, TMS and altrenogest.

Foals that are delivered prior to the last week of a normal gestation experience high rates of death. Foal death, in turn, exerts a tremendous financial burden on the owner and breeder, both directly, and as reflected in poor produce record for the mare. The single most important cause of abortion and premature delivery in horses is bacterial infection of the placenta (placentitis) which leads to uncontrolled inflammation and premature delivery of a foal. Identification of drugs that are effective for treating mares with placentitis is paramount to improving foal survival. A fundamental step toward making optimal therapeutic choices is to determine if specific drugs are performing expected functions, such as resolving inflammation, in mares with placentitis. Information from our recent study showed a high survival rate in foals delivered from mares treated with firocoxib, TMS and altrenogest. Information from the proposed study will help the equine community make educated choices when treating mares with placentitis. Given the high incidence of this condition in equine pregnancy, and the significant financial burden a lost pregnancy imparts, we feel that this work is paramount to the solvency of the equine industry.


EFFECTS OF LOW-DUST FORAGE ON LUNG HEALTH OF ATHLETIC HORSES
Laurent Couetil, Purdue University– First Year (2 Year Grant)

One Sentence Summary: This proposal seeks to provide a non-pharmaceutical solution to the widespread problem of equine asthma by evaluating the benefits of low-dust forage for horses engaged in athletic activity.

Horses, like people, commonly suffer from asthma. In fact, it is the second most common cause of disease in athletic horses including young Thoroughbred racehorses. While the condition causes decreased performance, it is difficult to detect. Affected horses typically train well and may only cough occasionally. The best way to detect equine asthma is by visualization of excess mucus by endoscopy of the airways after exercise or by identification of airway inflammation by lung wash. Asthmatic horses can be treated with anti-inflammatory drugs (corticosteroids); however, treatment may not be effective, and the risk of residue resulting in a positive drug test is always a concern.

Exposure to dust in the stall, particularly from hay, is likely an important contributor to the onset of equine asthma. Feeding horses steamed hay or grass silage (haylage) is an effective way to decrease dust levels; however, it is unknown if it would be sufficient to control airway inflammation in asthmatic racehorses. The body has natural ways to resolve inflammation by using compounds present in the diet, such as omega-3 fatty acids, to decrease inflammation and heal tissue. Fresh grass and haylage have high levels of omega-3 fatty acids compared to dry hay. Racehorses and Sport Horses typically are fed dry hay and do not have access to fresh grass. Consequently, in addition to lowering dust levels, feeding haylage to racehorses may provide additional benefits compared to steamed hay due to higher omega-3 content.

The goal of the study is to compare the effect of feeding dry hay, steamed hay, or haylage on dust level, airway inflammation and blood omega-3 levels in Thoroughbreds actively racing in Indiana. We hope to find that horses fed haylage or steamed hay will be exposed to lower levels of dust, resulting in decreased airway inflammation compared to horses fed dry hay. In addition, we believe that haylage will result in the most significant improvement, and this will be associated with higher blood levels of omega-3 fatty acids.

Horse asthma is the second most common cause of disease in young Thoroughbred racehorses after bucked shins. Affected racehorses are commonly treated with anti-inflammatory drugs and antibiotics, despite only weak evidence to support their use. Drug elimination times are poorly documented, thereby presenting a risk of being detected during drug testing. Therefore, there is considerable interest in resolving lung inflammation without drugs. The purpose of the study is to compare the effect of dry hay, steamed hay and haylage on lung health of racing Thoroughbreds. If the study demonstrates that low-dust forages such as haylage and steamed hay help reduce asthma severity in racehorses, it would potentially be a major benefit to horses’ health and welfare and to the equine industry.


INVESTIGATING METABOLIC STRESS AND VIRAL HEPATITIS
Sabine Mann, Cornell University– First Year (2 Year Grant)

One Sentence Summary: We will study metabolic pathways and hepatic viral infection to find a relationship with maladaptation to training syndrome/high GGT to help improve the health and performance of race horses.

Maladaption to training syndrome is characterized by the increase in a specific enzyme, y-Glutamyl-Transferase (GGT), in the blood of racing Thoroughbreds. It is currently unknown what causes the abnormally high enzyme and if it is the cause of the poor performance of affected horses. We seek to shed light to the possible causes and will test different potential factors that may help us explain this syndrome in this study so that we can help prevent or treat the affected horses.

The first factor could be a metabolic problem that causes horses to have high GGT activity and makes them less able to respond to the challenge of a race. This may be due to the fact that strenuous exercise can lead to detrimental accumulation of the byproducts of burning fuel, so called free radicals, or oxidative stress. We will follow horses over time, beginning during the lower-intensity early training season, so that we can identify changes early and before abnormal enzyme levels develop. This way we will be able to show if changes in metabolic pathways develop before GGT activity increases.

The second factor is a possible infection with a recently discovered virus that targets the liver and causes an inflammation of the organ, called hepatitis. Such an infection could explain why GGT increases as it has a high concentration in the liver. We could show previously that approximately 18 % of horses racing in California had high GGT concentrations. Of those horses, 3-5% were infected with one of the two equine viruses, hepacivirus and parvovirus, respectively. Although the number of horses that were found to be infected is low, the risk to have high GGT concentrations was higher in infected horses, and thus it could be a contributing factor to high GGT activity. Therefore, we think it is important to determine if liver infection with viruses could be causing the maladaptation to training syndrome.

Both questions will be answered by a study of a group of healthy Thoroughbred horses that will be blood samples over time. This will allow us to detect early on when the GGT concentration is increasing and study the changes in metabolism or viral infection that happen concurrently or before GGT increases. To do this, we will quantify over 250 different metabolites in the serum samples of healthy and high GGT horses (15 animals each), and compare the findings to find metabolic clues to understand the syndrome. We will also subject these horses to a training race to see if some of the metabolic markers that we are looking for may only be changing for a short period of time after the animals are challenged. In addition, we will detect and quantify virus particles in the blood of these horses to determine if recent virus infection is more likely in horses that develop high GGT concentrations. Our goal is to find the reason for the increase in GGT activity so that we can help identify possible strategies to prevent and treat horses that develop this syndrome.

Maladaptation to training syndrome affects the performance of equine athletes. The syndrome is defined as an imbalance between training and recovery that is associated with unexplained signs of reduced performance. The “poor performance horse” is thought to be associated with abnormalities in the results of enzyme activities in the blood of affected. No cause has been established definitively to date.

Trainers and race track veterinarians report that a persistent elevation in GGT (above 50 U/l) is associated with a decline in performance. No cause has been found although our preliminary studies suggest it may be caused by either oxidative stress or recently discovered hepatitis viral infection. Regardless of the cause, affected horses generally require several weeks of rest before the GGT returns to normal causing a decrease in starts. Additionally, the economic impact of a perceived reduction in performance in horses that may continue racing might also have severe economic consequences for the owners and perpetuate the ill health in affected horses. In many horses the onset of the disorder is in late spring or early summer which might result in loss of the remaining season and in some cases the syndrome has prevented high profile horses from running in televised stake races and disappointment to the racing public. Given the relationship of high GGT concentrations as a marker or cause of poor performance, and the striking lack of studies investigating causal or associative pathways, we believe a strategic approach is needed that will allow us to create the necessary evidence to address this problem. We aim to address this gap in knowledge and if successful, will be able to identify specific management or treatment strategies.


NOVEL ANALGESIC COMBINATION IN HORSES
Alonso Guedes, University of Minnesota –One Year Grant

One Sentence Summary: We are developing a medication strategy for joint inflammation/pain that can provide pain relief as well as help protect the joint cartilage from damage caused by chemical mediators of inflammation.

A pain management strategy that also has disease-modifying effects is an unmet medical need. Using a well-established and transient model of joint pain in horses, our previous Grayson-funded grant defined the optimum doses and pain relief with inhibition of two proteins, cyclooxygenases (COX; with phenylbutazone) and soluble epoxide hydrolase (sEH; with t-TUCB), involved in two distinct cascades of fatty acid metabolism. The objective of the current application is to logically build on this knowledge by exploring whether this novel pain control strategy also offers desirable disease modifying effects. Guided by strong preliminary data, we now propose to study the effects of inhibiting these two proteins on chemical markers of inflammation and tissue destruction in the whole joint (Sp. Aim 1), and in the responses of specific joint cells called chondrocytes (the main joint cell type which forms the joint cartilage) to chemical mediators of inflammation (Sp. Aim 2). For Sp. Aim1, we will use joint fluid samples collected in our prior Grayson-funded grant mentioned above in which 6 adult healthy horses received a small amount of sterile bacterial toxin (LPS) in a joint and were treated with t-TUCB, phenylbutazone, and a combination of 3 different doses of t-TUCB with phenylbutazone. We wish to see if the addition of the sEH inhibitor helped reduce inflammation and protected the joint cartilage against the harmful effects of these chemicals. Joint fluid concentrations of 3 common pro-inflammatory chemical mediators (IL-1beta, TNF-alpha and IL-6) and 2 markers of joint cartilage degradation/synthesis (C2C, CP II) will be examined using standard techniques. The goal is to better understand the effects of the combined protein inhibition and determine whether or not it translates into less inflammation with protection of the cartilage matrix. For Sp. Aim 2, we propose to compare the effects of inhibiting the two proteins on chondrocyte responses to chemical mediators of inflammation. The goal of these studies is to confirm/refute whether blocking these two enzymes will protect the chondrocytes from damage induced by the inflammatory chemicals. Immortalized human-derived chondrocytes, which have been used extensively to study chondrocyte biology, will be used. Cultured chondrocytes will be incubated for 24 hours with distinct inflammatory chemicals (IL-1beta, TNF-alpha, LPS or tunicamycin) to induce cell stress and death, and treated with the COX-inhibitor phenylbutazone, the sEH inhibitor t-TUCB or with combinations of these as indicated. The treatment with best results in the immortalized human chondrocytes will be repeated using equine chondrocytes to add credibility of the treatment effect in equine cells. The culture media will be harvested for measurement of chemical mediators released by the cells. The cells will be harvested and used to assess markers of cell stress and death. Preliminarily, sEH inhibition significantly prevented tunicamycin-induced chondrocyte death. Collectively, these studies are developing a therapeutic framework for joint pain that is both symptom- and disease-modifying.

No current treatment regimen for joint pain can improve symptoms and the disease process itself, and many of the symptomatic treatments can cause other problems with chronic administration. Our laboratory is working towards narrowing this gap as can be seen in our previously funded Grayson grant as well as in the current research proposal. Preliminary in vitro studies using chondrocytes suggest that sEH inhibition also protect chondrocytes, the main cell type that forms the joint cartilage, from the effects of chemical mediators of inflammation. Taken together, results of the previous Grayson grant and the in vitro data generated in our preliminary studies strongly suggest that sEH inhibition can both enhance analgesia when combined with phenylbutazone as well as be protective to the joint cartilage. The significance of the proposed research is that it is expected to provide a new treatment framework for joint pain in horses, one that is not only symptomatic but, most importantly, can protect the joint cartilage and thus be disease modifying. This has high potential to impact all horses by decreasing the negative effects of joint pain on equine health and productivity, which in turn decreases the economic burden for the equine industry


AMPK AGONISTS AND INSULIN DYSREGULATION IN HORSES
Teresa Burns, The Ohio State University – First Year (2 Year Grant)

One Sentence Summary: This project directly impacts the treatment of equine metabolic syndrome by assessing the efficacy of two drugs, metformin and acetylsalicylic acid, in the treatment of equine insulin dysregulation.

Equine metabolic syndrome (EMS) and EMS-associated laminitis (EMSAL; founder) are important and increasingly prevalent conditions in horses and ponies; EMSAL is the most common cause of founder in equine populations in developed nations, and it imposes a significant financial and animal welfare burden on the equine industry worldwide. Abnormalities in regulation of insulin and blood glucose (somewhat similar to that seen in pre-diabetic humans) is a hallmark of EMS and is directly involved in the risk of founder in affected animals; treatment strategies for EMS patients primarily revolve around reducing abnormalities in insulin and glucose responses to food in attempt to minimize this risk in affected horses. Currently, strategies to minimize the amount of sugar and starch in the diet (which cause increased insulin levels) and maximize regular aerobic exercise are central to this goal. However, even though many medications are available for improving insulin/glucose dynamics in humans, very few have been shown to be effective in horses; given that many affected horses are unable to participate in aerobic exercise due to the pain and debilitation of founder, treatment with drugs whose effects mimic those of aerobic exercise would be useful for managing these difficult cases. An enzyme termed 5’-Adenosine-monophosphate-activated protein kinase (AMPK) is important in humans and animals because it regulates metabolism in virtually all cells of the body and is important for sensing energy deprivation in the body; medications that activate the AMPK enzyme are a mainstay of medical treatment of metabolic syndrome and type II diabetes mellitus in human medicine. These medications promote normal blood sugar levels, burn fat, and improve insulin function in treated humans; therefore, since horses with EMS have similar metabolic problems as people with pre-diabetes, AMPK activation would also be an attractive treatment strategy for them. However, the ability of these human medications to activate the AMPK enzyme in horses is unknown. Metformin hydrochloride, a drug that activates AMPK that can be given orally, improves glucose and insulin levels in humans and is prescribed widely for this reason. Although metformin is given to horses clinically already, it has not been widely studied in this species. It is not absorbed from the GI tract in horses as well as in humans, and some investigators have suggested that it acts locally within the intestine to block absorption of sugars in feed (minimizing effects on blood sugar following meals). Recently, aspirin has also been shown to activate AMPK in people, and in contrast to metformin, aspirin is well-absorbed from the intestines in horses when given orally and has been given to horses for many years to treat inflammatory conditions. Further, aspirin and metformin have been shown act synergistically in improving insulin and glucose levels in humans; this would also be incredibly attractive for treatment of horses with EMS but hasn’t been investigated to date. Therefore, we plan to induce temporary insulin and glucose abnormalities (i.e. induce insulin dysregulation similar to pre-diabetes in human) in 14 adult horses using dexamethasone (a steroid medication used frequently to treat inflammatory and allergic diseases in horses, such as arthritis and heaves); following a 7-day induction period, 7 horses will be treated with metformin, and 7 horses will be treated with aspirin for 6 days. After this period, 7 horses will be treated with metformin and aspirin combination therapy for an additional 7 days. Blood samples, liver biopsy, and skeletal muscle biopsy samples will be collected at 1) baseline, 2) following induction of insulin and glucose abnormalities (i.e. insulin dysregulation similar to what occurs in equine metabolic syndrome), 3) following metformin or aspirin therapy, and 4) following combination therapy; these samples will be used to measure lipid and hormone concentrations and levels of tissue AMPK activation in each horse. Two tests (oral glucose test and combined insulin and glucose test) will also be performed at these same time points to evaluate the effects of the drug treatments on blood levels of insulin and glucose (and the insulin/glucose response to sugar challenge). Finally, the concentration of metformin will be measured in blood and liver samples collected from the MET treatment group to see if metformin is absorbed from the GI tract. We anticipate that the results of this project will provide valuable information about the ability of metformin and aspirin to activate the AMPK enzyme and improve abnormalities in insulin and glucose levels present during insulin dysregulation in horses and evaluate whether either or both of these medications might be suitable treatments for horses with EMS and EMSAL. Finally, we expect to clarify the likely predominant site of action of metformin (gastrointestinal tract mucosa vs. liver) in horses.

Laminitis is a crippling disease of horses and ponies that remains a major cause of morbidity and mortality worldwide. Laminitis has been identified as the most important priority for equine research by the American Association of Equine Practitioners due to both the high incidence of the disease (annual incidence of 2–7% of horses in recent studies), the severe and painful nature of the disease (horses often humanely euthanized or retired from competitive/useful activity secondary to chronic, intractable lameness), and the lack of effective therapies for treating it. In one of the largest studies of the incidence of lameness in the U.S. in recent history, a USDA National Animal Health Monitoring System study published in 2000 including data from approximately 3000 horse farms in 28 states stated that 13% of these farms reported a case of laminitis in a one year period. Of the different forms of laminitis, so-called “endocrinopathic” laminitis is by far the most common, accounting for almost 90% of laminitis cases presented to the equine general practitioner in a recent study. This form of laminitis affects adult horses of any age and breed, but, in the Thoroughbred industry, it is most common in brood mares and stallions where it is a major cause of loss of usage as well as premature death. Equine metabolic syndrome, an endocrine disease in the horse where excess nutrition and obesity lead to insulin disturbances, is the most common cause of endocrinopathic laminitis. We hypothesize that two drugs, metformin and acetylsalicylic acid (aspirin) will improve insulin and glucose dynamics in the horse with insulin dysregulation. This work may result in the establishment of an effective drug protocol for addressing insulin dysregulation-the primary component of endocrine disease in the horse that leads to laminitis.


HOST FACTORS INVOLVED IN EHM PATHOGENESIS AND LATENCY
Gisela Soboll Hussey, Michigan State University – First Year (2 Year Grant)

One Sentence Summary: The development of tools to protect horses from EHV-1 infection, compare the immune responses in old and young horses to identify the mechanisms causing clinical EHM.

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 industry is highlighted by a series of major outbreaks in North America over the past decade, including the largest outbreak ever in 2011. Despite the importance of EHM, effective prevention remains elusive. This inability to protect horses from EHV-1 and neurological disease is thought to be due to the fact EHV-1 modulates host defense mechanisms and hides in the blood cells and nerve cells after initial infection where it coexists with the horse for life. Interestingly, for EHV-1, infection can be controlled by host immunity in younger individuals and EHM occurs in LT 10% of infected horses. In contrast, the incidence of EHM dramatically increases in horses GT 20 years to ~ 70%. Specific elements of the horses’ immunity are probably responsible for the increased incidence of clinical EHM in younger horses and those are likely highlighted in aged horses. We propose to use this phenomenon to our advantage and compare the immune responses in old and young horses to identify the mechanisms that lead to the virus’s “waking up” and causing neurological disease in horses. This will allow us to (1) understand how the virus stays hidden and what leads to it “waking up”, which is critical as avoiding the virus from hiding in the first place or alternatively keeping it from waking up, is the cornerstone herpesviral control long term, (2) identify factors that contribute to EHM; and (3) elucidating target points for therapeutic and preventative intervention.

EHV-1 and its devastating sequel EHM continues to cause significant disease in the affected animal as well as causing extensive economic losses through closure of race tracts and sales barns, delays in training schedules and death of valuable animals. The substantial impact of EHV-1 on equine health is highlighted by a series of major outbreaks in North America over the past decade, including the largest outbreak ever in 2011 which infected over 90 horses in 10 states, resulting in at least 13 fatalities and enormous financial costs to the equine industry. Despite years of research, major outbreaks of EHV-1 remain a worldwide problem demonstrating the limitations of current vaccination strategies in the horse. The major current limitation in our preventative efforts are that EHV-1 modulates host defense mechanisms and hides in the blood cells and nerve cells after initial infection where it coexists with its host for life. During the aging process of the horse the balance between the horse and the hidden virus shifts to where the virus starts multiplying again and the overzealous immune response to the virus causes damage in multiple parts of the body, including the spinal cord and leads to neurological disease. We propose to use this phenomenon to our advantage and compare the immune responses in old and young horses to identify the mechanisms that lead to the virus’s “waking up” and causing neurological disease in horses. Understanding how the virus hides and what causes it to “wake up” will lead to the development of therapeutics to avoid the virus from hiding in the first place or alternatively keeping it from waking up. Further, the identification of factors that contribute to EHM will elucidate target points for therapeutic and preventative intervention. Finally, some of the tools used to study differences between young and old horses might also provide new diagnostic tools that could be used in outbreak conditions to identify horses at risk for developing EHV-1.


UNRAVELING COMPLEX TRAITS BY DEFINING GENOME FUNCTION 2
Carrie Finno, University of California- Davis – First Year (2 Year Grant)

One Sentence Summary: An Atlas of gene regulation in the horse.

The sequencing of the horse genome in 2009 resulted in significant advancements in the field of equine genetics. Currently, there are 41 equine traits, including genetic diseases and coat colors, for which a genetic test is available. Of these, 31 were discovered since the horse genome was made publically available. The next step forward in the study of complex equine diseases is to define gene expression (which genes are making proteins in which tissues and how much protein is being made) and gene regulation (which genes are turned “on” or “off” in each tissue). Similar to other species, the horse has approximately 21,000 genes in its genome, which make up only a very small portion of the horses’ entire 2.7 billion bases of DNA. While it is known that changes in coding DNA (i.e., DNA that creates protein) may lead to disease, what is not known is the consequence of changes in non-coding DNA (i.e. DNA between coding regions). The vast majority of the equine genome consists of this non-coding DNA, whose function remains a mystery. It is apparent, however, that non-coding regions of DNA can bind proteins that will turn a gene “on” in one tissue, lung for example, while that same gene is “off” in another tissue, such as liver. The future of equine disease discovery lies in unraveling the location and function of these important DNA sites and determining, on a tissue-specific basis, how these noncoding regions of the genome regulate gene expression. In human research investigating complex genetic disease, as many as 93% of studies attempting to link a disease with a region in the genome identify an associated region within non-coding DNA. Similarly, many complex genetic diseases in the horse map to non-coding regions of the genome. The study proposed here parallels advances in human genomics and puts equine research on the cutting edge of disease discovery. This study will provide the foundation for not only equine genetic research but also research into the function and dysfunction of many specific equine disorders from laminitis to typing-up to heaves, among many others.

For the first time, we have the available tools and expertise to unravel these DNA and protein interactions that regulate gene expression. Our goal is to provide an open-access resource, a genomic roadmap outlining the location and function of non-coding “on/off switches,” for all researchers.

With support from the Grayson Jockey Club Foundation in 2016, a tissue-specific equine biobank and database of gene expression and regulation is being built. This proposal seeks to expand upon this initial progress to determine the location of additional regulatory elements. This annotation will extend to include the location of additional modifiers of gene expression using a technique called CTCF-sequencing, and discover important regulatory regions with a technique called ATAC-sequencing. While the initial analyses are being performed on 8 tissues, we have archived GT 80 tissues from each horse that is available for future studies. In fact, this biobank has already been leveraged through an international collaboration, whereby 24 individuals representing 16 research institutions in 10 countries provided funding, totaling $44,000, for the tissue(s) most relevant to their own research for RNA-sequencing. This has maximized the initial investment from Grayson Jockey Club. All data is publically available to the entire research community. We propose that development of this roadmap is as important to the equine industry today as the sequencing of the equine genome was in 2009.

Genetic studies have been performed for many important complex diseases, including fracture risk and tying-up in Thoroughbred racehorses, recurrent airway obstruction (“heaves”), osteochondrosis dissecans (OCD), navicular disease, and recurrent uveitis (“moon blindness”), among many others. Despite the results of these studies, which have successfully identified locations in the genome that may be associated with a specific disease, functional genetic mutations have not been discovered in any genes within these regions to date. For example, a region on chromosome 13 was associated with heaves in Warmbloods, but no mutations were identified in neighboring protein-coding genes. These genetic associations may indicate that a non-coding region of the genome is contributing to development of clinical disease. Additionally, many other active areas of equine research, including laminitis, immune response to infection, reproductive health, tendon injuries, and inflammatory conditions use gene expression analyses to identify biomarkers of disease. A publically available tissue-specific reference for all DNA-protein interactions in the horse would provide evidence that these non-coding regions play an important role in gene expression. In human medicine, genetic tools are used to analyze an individual’s DNA sequence, gene expression and DNA-protein interactions, thereby focusing on disease prevention and early diagnostics. Our long-term goal is to move towards this type of personalized medicine in the horse. This would allow veterinarians to screen horses and optimize treatment recommendations for the prevention of developmental disorders as well as provide specific targeted therapies for a variety of diseases based on gene expression changes in a particular individual. In addition to unraveling disease mechanisms, the DNA-protein interactions identified in this proposal would uncover new targets for biomarkers, prevention and treatment of disease.


DEVELOPMENT OF LIMITED VIEW 3D IMAGING
Chris Kawcak, Colorado State University – First Year (2 Year Grant)

One Sentence Summary: The goal of this proposal is to develop a point-of-care, 3-dimensional imaging technique that can be used to better characterize and prevent injuries in racehorses.

Racehorse injuries continue to have a negative effect on the racing and sport horse industries, especially with the widespread public display and social discussions around high-profile catastrophic injury events. The Grayson Jockey Club Research Foundation and other funding bodies have invested millions of dollars in solving this problem, and have helped to improve the understanding of such injuries. However, a key missing item in solving this problem is the development of an easy-to-use imaging device that can detect the subtle lesions that lead to injury. A device that can be used to better characterize injuries and as a surveillance technique to identify “at-risk” equine athletes is needed. The goal of this proposal is to develop a 3-dimensional imaging technique that can be used in the field for diagnosis and characterization of limb injuries in horses. It is the hope that development of such a device, with the goal of being inexpensive, easy-to-use and able to detect subtle changes within the legs of horses will lead to prevention of injury.

Specifically, Limited View 3D technology will be developed for the equine distal limb, which will be achieved by taking a minimal number of radiographs to acquire a 3-dimensional image of the leg. This type of imaging technique should be achievable in the field, and better placed as a screening tool compared to existing imaging technology. This would allow the racing and sport horse industries to be better positioned to deal with the negative image that catastrophic injury causes.

The investigators propose to first use computer simulation techniques to design the device. The co-investigators at the University of Chicago have successfully done this in the human field, and along with the clinical expertise from the investigators at CSU, a device can be constructed that can be used the veterinarian in the field. Computed tomographic (CAT scan) images of the fetlock joint from normal Thoroughbred racehorses will be used as the basic model for computational modeling. Once the computer simulation is finalized the model will be validated by imaging the fetlock joints in the laboratory and comparing the results to the model. Any computer configurations found not to be valid will be replaced with options that are sound and clinically possible. This allows for real-time development of the Limited View 3D technique.

Once the imaging configuration is determined and validated, then fetlock joints with known problems will be imaged using the Limited View 3D technique and the results compared to traditional computed tomographic images. A board-certified radiologist with expertise in equine imaging will blindly evaluate images from both Limited View 3D and computed tomography to determine the value of Limited View 3D imaging. Once optimized, the results of this study will be used to develop the software and hardware configurations needed to acquire 3-dimensional images at the point of care. At this stage, a combination of funding strategies will be used to bring the technology to the equine industry, first through the development of a prototype that will be validated and clinically optimized at CSU, and then through investment strategies and collaborations with industry partners. Investigators at CSU currently possess the relationships, regulatory insight and fundraising capacity to bring such a device to the equine market. Clinicians at CSU also possess the infrastructure and experience to provide continuing education focused around such a device.

Injury in the athletic horse continues to be a major concern to the equine industry. Not only is it the most common cause of euthanasia, early retirement and lost days of training in the racing industry, but the negative press and public perception that surrounds catastrophic injury events inflicts a negative image about the industry. The negative press and perceptions around these events will likely continue to escalate with the increased reliance of instantaneous, public communications on social media. Many sources have contributed to funding research that has investigated that causes of such injuries, and we know now that these injuries are the end stage of a chronic process. A better understanding of the changes that occur in the legs of horses prior to injury is now better understood because of this investment by the equine community. However, there is a clear gap in our ability to acquire the necessary clinical information essential to identify horses that are at risk of injury. An easy to use, low-cost 3-dimensional imaging device that can be used by the veterinarian in the field is needed within the equine industry. Development of such a device will not only allow for improved characterization of lower limb injuries in racehorses, but will help to screen horses prior to racing to better prevent injury.

The investigators propose to use established computational modeling techniques to develop such a device. Imaging techniques that display 3-dimensional information about the health of the horse’s legs can be developed with a minimal number of x-ray projections. Preliminary data by the research team has shown that nearly all of the information obtained by a computed tomographic can be obtained with the proposed technique (Limited View 3 Dimensional imaging). The unique benefit of this technique is that it can be performed with a limited number of radiographic views, making it practical to use in the field by veterinarians. Results of this study will lay the groundwork for software and hardware design of this device.


EPIDEMIOLOGY OF DRUG-RESISTANT R. EQUI AT HORSE FARMS
Steeve Giguere, University of Georgia – First Year (2 Year Grant)

One Sentence Summary: We will determine if isolates of Rhodococcus equi highly resistant to antibiotics are widespread at horse breeding farms in Kentucky.

The standard treatment for pneumonia caused by the bacterium Rhodococcus equi in foals has been combination therapy using one of the macrolide antibiotics (erythromycin, clarithromycin, or azithromycin) with rifampin. In the absence of an effective vaccine, control of R. equi infections at many endemic farms relies on early detection of disease using thoracic ultrasound and initiation of treatment with a macrolide in combination with rifampin prior to development of clinical signs. This practice has considerably increased the number of foals being treated with antimicrobial agents. Recently, we have documented that mass antimicrobial treatment of all the foals with lung lesions detected by ultrasound has selected for antimicrobial resistance over time, with isolates of R. equi resistant to all macrolides and rifampin being cultured from more than 10 % of affected foals in Kentucky and from up to 40% of affected foals at some farms. Foals infected with such resistant isolates are more likely to die than foals infected with susceptible isolates. We have recently identified the genetic mechanisms of leading to antimicrobial resistance in R. equi. Our preliminary data indicate that a highly resistant strain of R. equi has been transmitted from farm to farm in Kentucky and has spread to other states as well but the true frequency of resistant isolates at horse-breeding farms and the factors associated with their presence are unknown. All the techniques and methods proposed in this application have already been optimized and are applied routinely in our laboratories. We are, therefore, in an ideal position to successfully complete the research proposed in this application. Our long-range goal is to eradicate or limit the spread of resistant isolates of R. equi. However, achievement of our long-range goal will be possible only if we have a good understanding of the epidemiology and ecology of macrolide and rifampin resistance in isolates of R. equi at horse-breeding farms. After completion of this work, we will have determined: 1- how widespread are isolates of R. equi resistant to macrolides, rifampin, or both at horse breeding farm in Kentucky; 2- whether the proportion of isolates of R. equi resistant to macrolides, rifampin or both varies by sample type (soil vs air), location (stall vs paddock) and month; 3- whether the rate of detection of isolates of R. equi resistant to macrolides, rifampin or both in the environment of farms that rely on early detection of disease using thoracic ultrasound is different to that of farms that do not use ultrasound for early detection; and 4- whether resistant isolates of R. equi currently detected at farms in Kentucky still represent dissemination of the same strain or if resistance is spreading to other isolates. These are the first important steps in understanding the ecology and epidemiology of macrolide-resistance on horse farms and in developing strategies to prevent antimicrobial resistance.

Pneumonia is the leading cause of disease and death in foals in Texas and ranks 3rd as a cause of morbidity and 2nd (after a combined category of trauma, injury, and wounds) as a cause of mortality in the United States. Given the importance of infectious diseases for the equine industry, our ability to curtail antimicrobial resistance is of pressing importance. Although many microorganisms cause respiratory disease in foals, R. equi is considered the most common cause of severe pneumonia, and is important to the equine industry for the following reasons. The disease is extremely common at many horse-breeding farms with sometimes 20% to 40% of the foal crop being affected. At those 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 has a long-term detrimental effect on the equine industry because foals that recover from the disease are less likely to race as adults. For decades, the standard treatment for R. equi foal pneumonia has been combination therapy using one of the macrolide antibiotics (erythromycin, clarithromycin, or azithromycin) with the antibiotic rifampin; effective alternatives to this combination are lacking. These antimicrobial agents are classified as critically important for human medicine by the World Health Organization. Widespread resistance to these 2 classes of drugs in R. equi isolates has become a major problem facing the horse-breeding industry and might adversely impact human health. The work proposed in this application represents the first important steps in understanding the ecology and epidemiology of antimicrobial resistance on horse farms and in developing strategies to prevent antimicrobial resistance.


ENDOCRINOPATHIC LAMINITIS: PATHOPHYSIOLOGY AND TREATMENT
James Belknap, Ohio State University – Second Year (2 Year Grant)

One Sentence Summary: This study will determine if continuous digital hypothermia is effective and therefore indicated in the management of endocrinopathic laminitis, the most common form of the disease.

From research performed in the past decade it has emerged that there are three distinct major forms of laminitis: laminitis associated with severe systemic disease (sepsis associated); laminitis associated with insulin dysregulation (endocrinopathic laminitis) and laminitis associated with excessive weight bearing on a foot (supporting limb laminitis). Although each form potentially represents a unique disease pathway in terms of the causal mechanisms, all still share the same outcome: weakening of the connections between hoof and bone (the lamellar tissue), irreversible changes to the morphology and mechanics of the foot, and characteristic, debilitating foot pain. By far the most common cause of laminitis is an underlying endocrine disturbance (either equine metabolic syndrome [EMS] or pituitary pars intermedia dysfunction [PPID, “Cushing’s disease”]) that results in insulin dysregulation. An exaggerated insulin response to ingested carbohydrates, often from pasture, initiates a cascade of events in prone horses leading to varying degrees of lamellar injury, lameness and resultant structural change. Repetition of this process occurs over time: the typical chronic endocrinopathic laminitis case suffers recurrent acute bouts of exacerbation, with each episode contributing further to loss of lamellar tissue organization and subsequent gross structural and mechanical derangements of the feet. Despite recent advances in our understanding of the role of insulin in endocrinopathic laminitis, there are still gaps in our knowledge. Furthermore, although we have developed management strategies aimed at controlling the underlying systemic causes of endocrinopathic laminitis, we do not have an effective therapy for horses suffering from an acute bout of endocrinopathic laminitis (whether it be an initial episode or exacerbation in a chronic case).

Our research groups, supported by the Grayson- Jockey Club Research Foundation, have developed continuous digital hypothermia (CDH) as an effective preventative and treatment for another form of the disease - sepsis-associated laminitis - and it has been widely adopted as a method of prophylaxis in severely ill horses in the clinical setting. Our previous studies also demonstrated a dramatic therapeutic effect in an experimental sepsis-related laminitis model even after the onset of lameness: CDH effectively halted progression and prevented lamellar structural failure. There is, however, currently no data on whether CDH is effective against endocrinopathic laminitis, limiting the clinical use of the technique in the most common clinical cases. High blood insulin has been purported to cause laminitis through dysregulation of processes including blood perfusion, metabolism, and cell signaling within the lamellar tissue. There is recent evidence that the growth factor receptor IGF-1R is activated by excess insulin, potentially triggering a cell division response and disruption of normal lamellar cell adhesions. We have strong preliminary data indicating that growth factor signaling is activated within the lamellar tissue in models of both sepsis-related and endocrinopathic laminitis. In addition, our preliminary data shows that digital hypothermia inhibits this growth factor signaling in a sepsis-related laminitis model. We also have preliminary evidence demonstrating the effects of hypothermia in slowing the metabolism of lamellar tissue, a potential protective effect in negative energy balance states due to reduced blood perfusion or excessive metabolic activation of lamellar tissue due to growth factor dysregulation. We hypothesize that CDH will ameliorate laminitis and prevent lamellar structural failure in endocrinopathic (hyperinsulinemic) laminitis. Using are liable model of endocrinopathic laminitis (the euglycemic, hyperinsulinemic clamp [EHC]) and combining our established cutting edge analysis techniques, we will evaluate a range of metabolic and molecular markers of lamellar energy metabolism, perfusion and signaling events in the EHC model for the first time in order to better elucidate the pathophysiology of hyperinsulinemic laminitis. We will also evaluate the effects of CDH on these processes, and test the principle of CDH as a treatment for endocrinopathic laminitis. The presence of a clinically relevant protective effect of CDH in hyperinsulinemic laminitis will be determined by carefully evaluating lamellar structural changes with light and electron microscopy. We anticipate that the proposed study will address the fundamental question of whether CDH is effective and therefore indicated in the management of endocrinopathic laminitis.


PLATELET LYSATE THERAPY IN INFECTIOUS ARTHRITIS
Lauren Schnabel, North Carolina State University – Second Year (2 Year Grant)

One Sentence Summary: This proposal examines the antibacterial properties of platelets to treat joint infections in horses more effectively than conventional therapies, with the goal of reducing morbidity and mortality.

Joint infections can lead to devastating consequences to a horse’s soundness, athletic career or even their lives. Infections mainly occur after a trauma like a wound, but may also be a complication of joint injections or surgery that involves a joint. Bacterial infections in other locations can usually be eliminated with a course of antibiotics. However, when bacteria enter a joint, they interact with joint fluid, and group together in a protein-containing clump that allows them to evade antibiotics. When bacteria live and proliferate within the joint they cause inflammation that can lead to arthritis. Finding new drugs that can fight bacteria, even when clumped, and decrease inflammation will lead to a better prognosis for the horse’s life and career. We recently found that platelet-rich plasma (PRP), a therapy commonly used to help recovery from athletic injuries, is useful in fighting off bacterial infections. The main component of PRP is the platelet, a cell critical for blood clotting. An important additional function of platelets is as an immune cell that produces proteins that help the immune system recognize, fight and clear infection from bacteria and viruses. We hypothesize that the use of the antibacterial portion of PRP in combination with antibiotics will increase the effectiveness of those antibiotics in joint infection and allow successful eradication of the infection.


EVALUATION OF KISSPEPTIN THE PREGNANT MARE
Christianne Magee, Colorado State University – Second Year (2 Year Grant)

One Sentence Summary: This proposal will allow us to gain insight as to how kisspeptins are involved in equine pregnancy and if they can serve as a biomarker for pregnancy compromise.

Kisspeptins are small proteins that are integral to reproductive function and it is our overall objective to define the effect of kisspeptins on equine reproductive function and fertility. In other species, kisspeptins have been shown to integrate processes such as aging, stress, nutritional deprivation, and obesity in puberty and fertility. In women, kisspeptin levels are detectable in blood only during pregnancy, and rise almost 1,000 fold during the first trimester of pregnancy. Abnormally low kisspeptin levels have been associated with compromised pregnancies and pregnancy loss, and are being used by clinicians to identify “high risk” human pregnancies. While great advances are being made in the human field, there has been no work on this area in the horse. One reason for the absence of research activity has been that the scientific community lacked a way to accurately measure kisspeptins from a sample of horse blood. Our laboratory has since developed such an assay and we can demonstrate that kisspeptin is elevated in pregnant mares as compared to mares that are not pregnant. With the development of this crucial assay and the proposed series of experiments, we will be able to obtain insight as to how kisspeptins are involved in equine pregnancy and if they can serve as an indicator for fetal viability and as a biomarker for pregnancy compromise in the horse.


BONE MARROW MONONUCLEAR CELLS FOR EQUINE JOINT THERAPY
Linda Dahlgren, Virginia Maryland CVM – Second Year (2 Year Grant)

One Sentence Summary: The results from this study will pave the way to investigate a new cell therapy from equine bone marrow as a targeted regenerative therapy for horses suffering from arthritis.

Osteoarthritis (OA), or simply arthritis, is the leading cause of lameness in horses. It is a debilitating disease characterized by progressive inflammation and loss of the normal joint surface, resulting in pain, difficulty getting around, and ultimately euthanasia. Arthritis can be very challenging to treat over the long term. Commonly used therapies often work for short periods of time or only provide partial relief of symptoms. There is a critical need for treatments that specifically target the cause of the problem and cannot only prevent disease from getting worse but even reverse the disease process and help the joint heal with a healthy covering of cartilage over the bone.

A new targeted therapy that is starting to gain attention for a variety of diseases is the injection of bone marrow mononuclear cells (BMNC). BMNC are readily collected by a simple and quick (2 hour) procedure in the laboratory. A sample of bone marrow is collected from the girth area of the horse and processed in the lab to collect the cells. These cells are made up primarily of the immature cells that eventually become blood cells. In their immature state these cells have been used successfully to treat tissues with limited ability to heal (regenerate) and therefore are traditionally associated with difficult to cure diseases (for example in the nervous system, pancreas, and joints). Their regenerative potential ease of collection for immediate injection, make BMNC a promising alternative to existing treatments. Although the mature forms of these blood cells are known to be the actual cause of inflammation and tissue breakdown, some forms of these blood cells can also play a fundamental role in promoting tissue healing. Understanding how a group of cells could have seemingly opposite effects is the focus of our proposal. Macrophages, one of the cell types in BMNC, can either change into to inflammatory (M1) or non–inflammatory (M2) cells in response to their environment. In this proposal we outline how we plan to investigate this relationship between the joint environment (normal and inflamed) and BMNC in cell culture (Aim 1) and following injection into equine joints (normal and inflamed; Aim 2). The knowledge gained from these studies will lead to clinical trials using BMNC to treat inflamed joints in horses. Our long term goal is to use this information to develop BMNC as a treatment strategy for use in horses (and possibly people, dogs and cats) to provide lasting relief to joint pain and inflammation.


PREDICTING THE RISK OF EQUINE FATAL INJURY DURING RACING
Tim Parkin, University of Glasgow – Second Year (2 Year Grant)

One Sentence Summary: We will use the Equine Injury Database (EID) to better predict and identify horses at greatest risk of fatal injury during racing and provide measures to further reduce the number of horses dying on North American racetracks.

The equine injury database (EID) that was established by The Jockey Club in 2008, has enabled us to estimate the frequency of fatal equine injury during racing from the majority of racetracks in North America. These figures have been calculated for different types of racehorses (e.g. males vs. females; two-year olds vs. older horses) and different racing conditions (e.g. dirt vs. turf vs. synthetic surfaces; different race distances). Additionally in the last 3-years we have worked with the EID data to identify many risk factors for fatal injury and fracture in horses racing in North America. Our aim is to be able to predict, with some certainty, which horses are at significantly higher risk of fatal injury. To date the models we have produced have not been of sufficient predictive ability to enable firm recommendations to be produced. One of the reasons for this is the broad, non-specific nature of the outcomes used so far. [We had to use broad definitions in initial analyses so that we had sufficient statistical power to enable us to identify risk factors]. We are now in a position, with 8-years of data being available (from early next year), to refine our outcome definitions to specific reasons for fatal injury or euthanasia that refer to fracture of individual bones or indeed sudden death. The study being proposed as part of further analyses of these data will help move toward much greater certainty as to the factors that are most strongly associated with different causes of fatal injury and also help to identify how predictable fatal injury may be for individual horses taking part in different types of race. This will enable potential intervention before the fatal injury occurs.

The output from this work will be a set of risk profiles for particular types of horse in different types of race. This will help racehorse trainers, track managers, racing secretaries.


CYTOTOXIC T-CELL IMMUNITY TO EQUINE HERPESVIRUS TYPE 1
Doug Antczak, Cornell University – Second Year (2 Year Grant)

One Sentence Summary: This research will develop critically needed knowledge about how the horse immune system responds to equine herpesvirus type1 vaccination and infection.

The goal of this research is to improve measures for controlling disease caused by equine herpesvirus types 1 (EHV-1). This project will generate crucial knowledge about how the horse’s immune system responds to EHV-1 vaccination and infection, identifying key components of the virus that stimulate immunity. The focus of this project is on immune responses made by the type of white blood cells known as cytotoxic T lymphocytes (CTL), which kill virus infected cells. CTL are considered to be important contributors to effective immunity against herpesviruses. However, measuring CTL responses is difficult, and the individual proteins of EHV-1 that initiate CTL responses are not well defined. This lack of technology and knowledge makes evaluation of natural infections or the effectiveness of vaccines in the horse very difficult and in some cases impossible.

We have assembled an international team of veterinary scientists with expertise in equine immunology and virology to address this problem. Our laboratories have developed sophisticated assays and techniques that enable us to assess CTL responses toEHV-1 in ways that have not previously been possible in the horse. In addition, Cornell University maintains a herd of specially bred Thoroughbred horses that are available for this research. The key immune system genes required for CTL responses (Major Histocompatibility Complex [MHC] genes) have been thoroughly characterized in these horses, which include the donor horse for the equine genome sequence (the Thoroughbred mare Twilight). These horses are a unique resource in equine research that allow us to conduct experiments on CTL responses that cannot be undertaken at any other location worldwide with the degree of precision proposed in this application. The MHC genes of the Thoroughbred horses to be studied in this project are commonly found in Thoroughbreds throughout the world and in many other horse breeds. Thus this research will be of general relevance to all horses.

The current proposal has three goals: 1) To determine if the current modified live virus (MLV) vaccine against EHV-1 induces protective cytotoxic T lymphocyte (CTL) responses. Surprisingly, although CTL responses are believed to be a critical component of a protective anti-EHV-1 response induced by vaccination, there is little published evidence on the ability of the MLV vaccine to induce CTL responses in vaccinatedhorses.2) To identify the individual proteins of EHV-1 that are most important for inducing protective CTL responses, and3) To identify the immune system molecules of the horse which present the EHV-1 antigens to CTL.

Achieving goals 2 and 3 will advance our understanding of how horse cytotoxic T lymphocytes provide immune protection against EHV-1.

This state-of-the-art research in equine immunology and virology will provide important new information and assays that can be used to evaluate current vaccines against equine herpesvirus type 1 and to design new and more effective vaccines in the future.


RESEARCH CAREER DEVELOPMENT AWARD

There are two Career Development Awards offered through the Foundation in 2018.

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

Stephanie Bond Stephanie Bond, University of Calgary
Airway Inflammation: On Pathogenesis and Performance

The selection process has resulted in the recommendation of Dr. Stephanie Bond as recipient of the Storm Cat Career Development Award for 2018. Statement by Dr. Renaud Leguillette, Calgary Chair in Equine Sports Medicine: “Dr. Bond has been awarded an Eyes High II DVM Graduate Recruitment Scholarship . . . This program seeks to award scholarships to candidates deemed to be highly accomplished and whose potential for research excellence is judged to be outstanding . . . The Storm Cat Award is intended to provide supplemental funds for graduate student development [and] would be entirely used as a salary supplement for Dr. Bond. This has been agreed by the Associate Dean of Graduate Education at University of Calgary Veterinary Medicine.”


The Elaine and Bertram Klein Development Award is a competitive program intended to promote development of promising investigators by providing a one year salary supplement of $15,000. This program is restricted to one award per year and is named in honor of Elaine and Bertram Klein. The grant is funded by the Klein Family Foundation.
The 2018 award winner is:

Jessica Gilbertie Jessica M. Gilbertie, North Carolina State University
Platelet Lysate Therapy In Infectious Arthritis

The selection process has resulted in the recommendation of Dr. Jessica Gilbertie for the The Elaine and Bertram Klein Development Award of 2018. As is often the case, Dr. Gilbertie’s studies already have touched different institutions. Although she is applying via North Carolina State University, the following recommendation comes from the renowned Dr. Dean Richardson of New Bolton Center, University of Pennsylvania:

“This letter confirms my support for Dr. Jessica Gilbertie submitting a revision of the grant proposal entitled ‘Platelet-rich Plasma Therapy in Infectious Arthritis’ with Dr. Lauren Schnabel at North Carolina State University’s College of Veterinary Medicine. Dr. Gilbertie was largely responsible for the preliminary data and study design with Dr. Thomas Shaer, who will be serving as co-investigator. Dr. Gilbertie will be continuing her research on the antimicrobial properties of platelets to support her PhD thesis in Dr. Schnabel’s laboratory.”