Current Research

Grayson-Jockey Club Research Foundation’s board of directors has announced a slate of 19 research projects which the Foundation will fund for a total of $1,003,580 in 2014. The list includes eleven new projects and five which are in their second year, as well as three Storm Cat Career Development Awards. The allotment brings the Foundation’s total impact since 1983 to $20.9 million to fund 310 projects at 41 universities.


WEIGHT BEARING; PERFUSION AND BIOENERGETICS IN LAMINITIS
Andrew van Eps, University of Queensland -First Year (2 Year Grant)

Equine laminitis continues to be a devastating complication to multiple equine diseases. Laminitis is reported to affect 2–7% of horses annually. Due to frequency and severity of the disease, it has been listed as the number one priority for equine research by the members of the American Association of Equine Practitioners (AAEP), and as a priority for the Grayson-Jockey Club Research Foundation, USDA, etc.

From research performed in the past decade it has emerged that there are three distinct major forms of laminitis. One of them is supporting-limb laminitis. Although it has never been proven, supporting limb laminitis is suspected to occur as a result of reduced blood supply to the connection between hoof and bone (the lamellar tissue). It is proposed that horses rely upon regular loading and unloading of the foot in order to move blood (containing nutrients and oxygen) through it. Our research groups (Queensland, U of Penn, and Ohio State), supported by the Grayson-Jockey Club Research Foundation, now have strong preliminary data that supports this theory.

This next step will utilize a protocol that combines real–time monitoring of lamellar tissue blood flow and energy balance with a suitable and humane model of preferential weight bearing on one limb. We seek to confirm the cause of supporting limb laminitis and to test potential therapeutic interventions. It is anticipated that the results of this study will directly guide the design of devices and/or protocols that can be used in the clinical setting to prevent supporting limb laminitis.

An effective preventative strategy would be a significant step forward for the welfare of horses and for the horse industry.


EVENTS AFFECTING LAMINAR ADHESION IN EQUINE SEPSIS
James Belknap, The Ohio State University -First Year (2 Year Grant)

Sepsis–related laminitis (SRL) is a common type of laminitis which occurs in animals experiencing far large breadth of diseases such as infectious diarrhea (i.e. Salmonella enterocolitis), pneumonia, intestinal compromise from colic (i.e. colon torsion), grain overload, and retained placenta/acute uterine infection post foaling. Although we have discovered some of the central laminar events occurring in SRL (including the same initial inflammation as occurs in organ injury and failure in human sepsis patients), we still have not established the events or signaling that result in laminar failure.

After many decades of frustration with failures of multiple therapies, we now have one therapy, continuous digital hypothermia (CDH), which has proven effective in the laboratory and clinical setting in many horses. We will use advanced biochemistry techniques, combined with two cutting edge research tools including kinome arrays and metabolomics analysis. The results will provide targets for pharmaceutical therapies to either replace or augment CDH, and will also for the first time determine effects of CDH in the normal digit (important to know for any commonly used therapy).

Advancing laminitis therapy will allow more at- risk animals to avoid the crippling consequences of SRL and return to their previous level of function in the equine industry as was witnessed with Paynter.


R. EQUI PNEUMONIA: CAN A NOVEL VACCINE PROTECT FOALS?
Noah Cohen, Texas A&M University -One Year

Pneumonia caused by Rhodococcus equi remains an important cause of disease and death in foals at equine breeding farms worldwide. Effective prevention for this disease is lacking. In recent years, the Foundation has funded several projects on this disease because of its importance. Currently, there is not an approved vaccine available in North America to protect against R. equi pneumonia. To date, the only vaccination strategy that has been demonstrated to be effective for protecting against experimental challenge with R. equi has been oral administration of modified live strains of R. equi to foals. However, this approach has limitations because of concerns that these live organisms might revert to virulence and cause disease in some foals.

We have developed a novel method for bacteria so they are absolutely unable to reproduce but remain immunologically intact by using electron–beam technology. Moreover, we have demonstrated that e–beam–irradiated isolates of R. equi administered orally can stimulate R. equi–specific immune responses in very young foals. Thus, the primary aim of this study is to determine whether administration of e–beam–irradiated R. equi administered orally to foals during early life can protect against experimental infection of the lungs with live R. equi. As a secondary aim, we will examine selected indicators of immune responses to the vaccine.

To summarize, we propose to evaluate the ability of this vaccine to protect foals against infection with R. equi as an essential next step in our efforts to develop a vaccine against R. equi. and subsequently other infections such as Strep equi, the causative agent of strangles.


ANHIDROSIS IN FOALS TREATED WITH MACROLIDES
Robert MacKay, University of Florida -First Year (2 Year Grant)

Whereas most foals affected by R equi pneumonia used to die with this disease, they now can be treated successfully with erythromycin or similar drugs (e.g., clarithromycin, azithromycin). Treatment typically takes several weeks and recovering foals often are turned out for part of the day. Two important side–effects detract somewhat from the effectiveness of erythromycin: diarrhea and overheating. Diarrhea is typically mild but the apparent inability of treated foals to regulate body heat effectively can sometimes be fatal.

We presume that these deaths are caused by heatstroke. Our theory is that the overheating problem is similar to anhidrosis (nonsweating), in that the ability to sweat is impaired. We have good evidence for this in the case of erythromycin, based upon studies done in preparation for this proposal. This makes the foal unable to efficiently lose body heat, and on hot days, puts it at risk for heatstroke. We have developed a way to measure precisely the ability of horses to sweat. We plan to use this simple test to show that impaired sweating is the cause of overheating not only in foals given erythromycin but also in some foals treated with azithromycin, clarithromycin, and even the exciting new antibiotic, gamithromycin. Some additional studies will be dedicated to trying to sort out how this class of drugs affects sweating.

If we are right, we hope that these results will serve as a wake-up call for all concerned that foals on erythromycin–like drugs including azithromycin, clarithromycin, and gamithromycin are at risk for heatstroke during and even after treatment and they therefore need to be protected by implementation of common–sense heat control measures.


CHARACTERIZATION OF INTRA–ARTICULAR ISOFLUPREDONE
Heather Knych, University of California- Davis -First Year (2 Year Grant)

Corticosteroids are potent anti-inflammatory agents and as such are commonly used to prevent and treat performance related injuries in equine athletes. Due to their ability to affect performance and potential to allow a horse to compete when it otherwise should not, corticosteroid use is regulated in performance horses. Over the last 2 years, a concerted effort has been made to establish standardized regulatory thresholds and withdrawal times for this class of drugs. To date, recommendations have been made for methylprednisolone acetate, triamcinolone acetonide and betamethasone acetate. The extended withdrawal time for methylprednisolone in particular, has led some veterinarians to seek out alternative corticosteroids, such as isoflupredone acetate; however, there is limited data about the pharmacology of this drug, or on its anti-inflammatory effects in joints.

Data gathered in this study will provide valuable information for the duration of effect of this drug in the horse by measuring up-regulated genes and inflammatory proteins, in both normal and inflamed joints. If, as we have theorized, this drug has a prolonged effect relative to detection times, results from this study may support less frequent administration of intra-articular corticosteroids, which may ultimately prove beneficial to the health of the joint.


OPTIMIZATION OF RACETRACK SURFACE PROPERTIES
Susan Stover, University of California- Davis -First Year (2 Year Grant)

Evidence indicates that race surfaces affect the likelihood for injuries in racehorses. Race surface design has the potential to significantly reduce injuries because race surfaces affect all racehorses training and racing on a surface. Therefore, race surface design is an attractive factor to manage to reduce racehorse injuries.

Mild and severe injuries to the fetlock are very common. Catastrophic injuries to the fetlock are the greatest cause of death in racehorses, causing about 50% of injury related deaths. Fetlock injuries are due to circumstances that cause the fetlock to extend beyond its normal range of motion (hyperextension). We hypothesize that fetlock hyperextension, and thus related injuries, can be prevented by developing race surfaces that change the way the limb interacts with the surface. Our objective is to determine the characteristics that a race surface should have to prevent fetlock injuries.

It is not economically practical to build the number of race surfaces needed to determine the ideal surface for injury prevention. Thus, a computer modeling and simulation approach is being used because it is a powerful and economical tool for investigation of race surface characteristics on fetlock motion, and thus risk for injury. This approach can be used to assess the effect of surfaces presently installed, as well as those surfaces that do not presently exist. Racehorse fetlock motion will be predicted for a large number of surfaces with different characteristics. Because the predictions are only as good as the computer model, the model will be evaluated for accuracy (validated) by comparing predictions with actual race surface behavior and racehorse fetlock motion that we previously measured simultaneously at two racetracks, one with a dirt surface and one with a synthetic surface. After validation, the model will be used to determine race surface characteristics that prevent fetlock hyperextension. These race surface characteristics can be used as a standard guide.


CONTRAST ENHANCED CT FOR DETECTION OF CARTILAGE INJURY
Chris Kawcak, Colorado State University -First Year (2 Year Grant)

Osteoarthritis is the most common musculoskeletal problem in horses and leads to lameness, decreased athleticism, and early retirement. Proteins in articular cartilage (glycosaminoglycans, GAGs) are important for articular cartilage structure in that they provide a smooth gliding surface for normal joint mobility. GAG loss is one of the early signs of osteoarthritis. When that occurs, the articular cartilage becomes weaker and is more prone to tearing due to continued joint inflammation. Once articular cartilage becomes damaged it has limited ability to repair itself and therefore, the disease will progress and worsen over time. Because of this, early detection of articular cartilage injury is critical to successful long–term outcomes.

Unfortunately, routine diagnostic methods (radiographs , ultrasound, MRI, CT) all have shortcomings in evaluating articular cartilage, although MRI is currently the preferred imaging method. However, when contrast agents are injected into the joint prior to CT imaging (CCECT), they have the potential to provide far more information about the health of cartilage. In preliminary experiments we have demonstrated that the amount of CCECT contrast in the cartilage is strongly related to the amount of GAG content within articular cartilage in normal joints. Our hypothesis is that CCECT is a safe and useful method to improve early diagnosis of joint disease. We will test this hypothesis by determining GAG concentrations in normal and diseased articular cartilage and comparing those amounts to CCECT attenuation. We will also test the safety of the contracts agent by evaluating articular cartilage samples for toxicity and lastly, compare the CCECT imaging modality to MRI. It is anticipated that in these experiments we will be able to demonstrate that decreasing GAG content is measurable using contrast CT studies allowing for the early detection of cartilage injury using readily available equipment.

The completion of this project will allow for the critical evaluation of CCECT as a method for the detection of early osteoarthritis in horses and will allow for applications of its use in clinical patients. If this method proves to be effective, it can be a useful tool for monitoring future treatment protocols in horses as well as future cartilage research as it offers a way of ante-mortem in depth evaluation of cartilage health.


PLATELET INHIBITORS POTENTIAL ANTITHROMBOTICS FOR EHV1
Tracy Stokol, Cornell University -One Year

Equine herpesvirus type 1 (EHV1) is a prevalent respiratory pathogen that is increasingly recognized as a cause of neonatal deaths, abortion, and neurological disease in performance horses. While viremia is common to mild and severe forms of EHV1, major gaps exist in our current understanding of how EHV1 targets the reproductive tract and spinal cord, and how to render infected horses resistant to this attack. Our preliminary data reveal that platelets act as a "Trojan Horse" to enable EHV1 tissue targeting and that inhibiting platelet activation represents a novel approach for EHM prevention.

Under experimental conditions, we have shown that equine platelets exposed to EHV1 undergo a procoagulant activation response and then bind to and transfer EHV1 infection to equine endothelial cells. Our proposal builds on these observations to test the hypothesis that experimental infection with EHV1 induces an in vivo procoagulant platelet phenotype, and that administration of phosphodiesterase inhibitors will decrease EHV1 induced platelet activation.. Results from EHV1 infected and sham infected horses will be compared and we will relate changes in platelet and coagulation parameters to recorded clinical signs (e.g. fever), platelet counts and level of viremia.

We expect to find evidence of platelet and coagulation activation during the acute phase of EHV1 infection and that horses demonstrating more profound activation response will also have more severe fever.

We will also compare the inhibitory effects of 4 antiplatelet drugs on EHV1 induced platelet activation. We expect that, the phosphodiesterase inhibitors will have the most inhibitory action on the ability of EHV1 to induce platelet procoagulant activity. EHV1 infection results in microvascular thrombosis that contributes to the clinical syndromes of abortion and EHM. For instance, horses that have severe neurological symptoms, such as paralysis or paresis, frequently have to be euthanized. Horses that do survive may not be able to race or may have decreased future racing performance due to the persistence of neurologic signs. By preventing or minimizing thrombosis, we can potentially decrease or eliminate these two severe clinical manifestations of EHV1 infection.

Successful completion of this study will show that, for the first time, platelets are activated in vivo in horses with EHV1 infection and that we can potentially inhibit platelets from participating in thrombosis, using available phosphodiesterase inhibitors. These affordable drugs, which are currently used in horses to treat airway disease, endotoxemia, laminitis and navicular disease, could readily be administered to exposed or known infected horses, including pregnant mares, to reduce or prevent the devastating sequelae of EHV1 infection. By inhibiting platelet function, we have our first real chance of preventing thrombosis and the clinical syndromes of abortion and EHM in horses.


ASSESSMENT OF A RHODOCOCCUS EQUI VACCINE IN FOALS
Jose Vazquez–Boland, University of Edinburgh -One Year

The pathogenic bacterium Rhodococcus equi produces a severe lung disease in foals that causes significant losses to the equine industry. Research efforts have failed so far in developing an effective vaccine against this disease. Understanding the intimate mechanisms used by pathogens to cause infection can lead to the identification of microbial “Achilles’ heels” that could be targeted by novel vaccines and diagnostics. With this in mind, we recently deciphered the composition of the R. equi genome (the entire repertoire of genes that make up this bacterium) and used sophisticated analytical techniques and molecular experimentation to identify suitable targets on a rational basis. This preliminary research was undertaken by an international consortium co–funded by Grayson -Jockey Club Research Foundation.

Our work with the genome led us to find that R. equi produces specific appendages termed “pili” that mediate attachment to host cells. We also found that these pili are essential for lung colonization and confer full protection when used as a vaccine in mice. This project aims to test the protective efficacy of this novel R. equi pili vaccine candidate in foals. R. equi infection is contracted during the first days of life and the immune system of young foals is incapable of stopping the pathogen once it is established deep in the lung tissue, resulting in life–threatening purulent pneumonia. We believe that protection against this pathogen can be achieved by targeting the pili with a vaccine that will prevent infection when still at its initial “colonization” stages in the airways before the bacteria become established in the lung. If the trial that will be conducted in this project is successful, the R. equi pili will form the basis of a commercial vaccine to control rhodococcal infection in stud farms.


ETIOLOGY OF IAD AND PERFORMANCE IN RACEHORSES
Laurent Couetil, Purdue University -First Year (2 Year Grant)

Inflammatory airway disease (IAD) is the second most common cause of poor performance in athletic horses. Inflammation and accumulation of mucus in the airways interferes with the movement of oxygen from the lung into the blood stream, limiting the performance of horses during strenuous exercise. The type of inflammation in the lung varies with the age of the horse. Younger horses most often exhibit airway inflammation characteristic of an allergic reaction with eosinophils and mast cells. In mature horses, airway inflammation usually is marked by neutrophils, which is considered a non-allergic response.

The triggering factors for IAD are not known but studies have implicated exposure to barn dust. On the other hand, studies have also supported a role for bacterial infection of the airways. The levels of airborne irritants horses are exposed to in the barn environment can be measured, and airway secretions can be sampled in order to classify the type of airway inflammation present in an individual horse. Combining these techniques will allow examination of the relationship between barn exposure and airway inflammation. In the past, investigations of the role of bacteria in IAD have relied upon time-consuming and relatively insensitive culture of mucus. Currently, DNA sequencing is a highly sensitive method that can be used to identify all the bacteria present in mucus, thereby characterizing the entire airway microbe population, or so-called “microbiome.” In addition to bacteria, viruses may cause airway inflammation. Again, DNA sequencing has improved the ability to detect viruses. This study is designed to explore the possibility that the various types of airway inflammation differ in their impact upon racing performance in horses and are due to different causes. Studying these differences is important to develop targeted prevention and treatment strategies.


PHARMACOKINETIC/PHARMACODYNAMIC EVALUATION OF OMEPRAZOLE
Paul Mills, University of Queensland -One Year

The horse’s stomach is a single chamber with two different linings. The top half of the stomach is lined by a squamous mucosa, akin to the lining of our esophagus. It is well recognized that ulcers in this area are the result of increased acid exposure to an area unaccustomed to acid exposure. The bottom half of the stomach is lined by a glandular mucosa, akin to the lining of our stomach. It is thought that ulcers in this area more closely resemble gastric ulceration in humans. The architecture of the lining of the bottom half of the stomach is significantly more complex than that of the top half and the cause of ulcers in this area is believed to be related more to a failure of normal defense mechanism than increased acid exposure per se. Omeprazole at a dose of 4 mg/kg once daily is well documented for the treatment of ulceration in the squamous mucosa with success rates of 70 – 80% consistently reported.

More recently a clinical trial reported success rates of 89% and 94% for 1 and 2 mg/kg once daily, respectively, with a number of factors potentially contributing to the efficacy of the lower doses observed. Of these factors it is possible that the administration immediately prior to work favored the low doses as exercise is the period in which the most damage to the squamous mucosa occurs. Likewise, administration following fasting may have been beneficial since fasting will improve omeprazole absorption. However, in a study comparing pre– vs. post–exercise administration of an alkaline–buffered omeprazole formulation (similar to GastroGard®), no clear benefit of these two factors was observed, which suggested that the formulation of omeprazole was responsible for the improved efficacy at lower doses. However, while direct comparison of the formulations has not been reported, preliminary data from an ongoing study suggests, at least indirectly, that absorption of the two formulations is similar. If equivalence could be demonstrated then the cumulative effect of the above discussion is that lower doses of omeprazole could be used in the treatment of squamous ulceration. This, combined with the imminent expiration of the GastroGard® patent and entry of generic formulations onto the market, would greatly increase affordability of the drug for the treatment of horses worldwide.

With the above in mind the aims of the study are: (1) to compare absorption of a range of commercially available omeprazole formulations; (2) to investigate the magnitude and duration of acid suppression achieved following administration of two doses of omeprazole under two different feeding conditions; (3) to develop a pharmacokinetic/pharmacodynamic model, where the concentration of drug in a blood sample is used for the prediction of an individual’s therapeutic response, for omeprazole in the horse; and (4) combining the findings from 1 – 3, develop a revised set of treatment recommendations for the treatment of gastric ulcers in the horse.


EVALUATION OF A RAPID TEST FOR SALMONELLA
Dr. Paul Morley, Colorado State University -Second Year

Salmonella enterica is the most commonly reported cause of outbreaks of hospital–acquired infections in veterinary teaching hospitals and the most common cause of closure of equine hospitals. Congregating horses from multiple sources is associated with increased risks for spread of contagious disease such as Salmonella. Control of Salmonella is further complicated by the fact that both horses and environment can appear clean and still be contaminated with Salmonella.

Developing methods for testing that can be performed at veterinary clinics and performing objective comparisons of Salmonella detection methodologies were recently identified as critical needs for infection control in equine populations by an international panel of infection control experts. Rapid and reliable testing methods for S. enterica
in environmental and fecal samples are considered essential for facilitating effective infection control in horse populations. Current detection methods typically require 48–96 hours and 24–72 hours from submission to reporting, respectively, in addition to time needed for transportation of samples to the laboratory.

Recently, commercial tests have been developed for use in food safety microbiology which may provide a practical and useful alternative to traditional culture and polymerase chain reaction (PCR) methods when screening for Salmonella. These lateral flow immunoassays could be economically employed in point–of–care testing providing a 24 hour test result for fecal samples and 48 hour test results for environmental samples with reasonable sensitivity. This will allow more extensive yet less expensive use of screening to control Salmonella in veterinary settings. The Central Hypothesis for this work is: Commercially available test strips used in the food production industries can be adapted for use as a rapid, point–of– care test to detect Salmonella in horse feces or environmental samples with sensitivity that is equivalent to optimized culture or PCR testing. To address this central hypothesis we will identify an optimal culture technique for fecal cultures and use an established culture protocol for environmental samples. We will also test these samples with a validated commercial PCR test kit, and with the Reveal® 2.0 rapid test strip. We will use these test results to estimate the sensitivity and specificity of these tests using state–of–the art, best–practice epidemiological methods.


DEVELOPMENT OF ALTERNATIVE MODELS TO STUDY EHM
Dr. Gisela Soboll Hussey, Michigan State University -Second Year

Equine Herpesvirus-1 (EHV-1) infection has resulted in devastating outbreaks of neurological disease 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 research community’s emphasis on EHM, a significant knowledge gap still limits our ability to prevent this devasting disease, and this is the focus of this proposal. Reasons for this knowledge gap include the fact that the naturally occurring disease remains relatively rare, and EHM can only be experimentally reproduced in aged and immunosenescent horses which are unrepresentative of the naturally affected population.

The long-term goal of our research is to control the occurrence of EHM. To accomplish this goal, which is the objective of this proposal, is the development of equine models in which to study EHM pathogenesis and to determine the efficacy of preventive measures. The rationale of this proposal is based on powerful preliminary data showing that endothelial cell infection and pathology of the eye is a common event and that EHV-1 infection of the eye mirrors the disease seen in the CNS during EHM.

We predict that we will find evidence of similar infection of the CNS and identify markers of sub-clinical disease in CSF. Using cutting-edge mass spectrometry techniques, we have already identified a number of exciting candidate markers. There is good evidence in a number of studies in humans and other species, that infection and injury of the spinal cord or brain are commonly associated with specific biomarkers, and that these markers can be detected using spectrometry. The use of both the ocular model and biomarkers for studying EHM is a highly innovative approach that, if successful, will answer critical questions about the pathogenesis of EHM and provide practical and ethical models for studying vaccines and therapeutics for protection from the devastating outcomes of EHM.


ACOUSTOELASTOGRAPHY TO MONITOR INJURED EQUINE TENDON
Dr. Sabrina Brounts, University of Wisconsin–Madison -Second Year

The superficial digital flexor tendon (SDFT) is a commonly injured soft tissue structure in performance horses. Injury of this structure is site–specific and commonly affects the mid–portion of the SDFT. Prognosis for return to full athletic function is guarded due to the fact that often horses are placed back into work to soon and re–injury of the tendon occurs. Therefore, it is important to be able to assess quantitatively the mechanical properties of tendons under load before the horse returns to full athletic use. At the University of Wisconsin a novel technique called “Acoustoelastography (AEG) was developed, which interrelates the ultrasound wave propagation to the local tissue biomechanical stiffness. Pathological diseased tendon has less stiffness than normal healthy tendon. AEG can assess this in real–time without knowing/recording tissue loads or performing extensive numerical analysis on images. The goals of this project are: To determine whether Acoustoelastography (AEG) can measure mechanical properties and strains non–invasively in abnormal equine tendons and secondly evaluate the healing of the equine superficial digital flexor tendon (SDFT) during recovery from tendon injury. In this study, we will evaluate tendon stiffness in the SDFT that had an injury and therefore pathologic changes. This stiffness will be compared to the normal stiffness of a SDFT. We will then demonstrate secondly that the stiffness of a tendon changes during its healing process. Horses with injury to their SDFT will be followed for 6–12 months to show that stiffness increases as the tendon heals. We expect that this study will show that a simple non–invasive evaluation of tendon function is possible and is able to detect, monitor and help in the rehabilitation of tendon injury.


MOLECULAR DETERMINANTS OF EHV-1 FUSION AND SPREAD
Dr. Arthur Frampton, University of North Carolina Wilmington -Second Year

Equine herpesvirus 1 (EHV-1) is the cause of significant illness in horses worldwide. The virus belongs to a family
of herpesviruses called the alphaherpesviridae, which includes the human viruses, herpes simplex virus 1 and 2 HSV-1, HSV-2). While EHV-1 has many features that are similar to the human herpesviruses, such as structure and genetic makeup, there are also important differences between the equine and human viruses. These differences include the mechanism of transmission or spread of the viruses and the clinical symptoms that arise after infection. Horses become infected with EHV-1 after coming into contact with airborne droplets containing virus particles or through contact with infected surroundings such as contaminated bedding. Once infected, horses exhibit clinical signs including respiratory distress, fever, lack of appetite, and other signs associated with a “common cold”. While most horses recover from this infection with no long-lasting negative effects, some horses will experience serious complications resulting from EHV-1 infection. Two major complications that may arise include neurological problems including the disease equine herpesvirus myeloencephalopathy (EHM) and abortion in pregnant mares. These secondary conditions occur after the virus spreads from the upper respiratory tract to very sensitive sites such as the central nervous system (CNS) and endometrium (uterine cavity). Recently, there has been a significant increase in the number of neurologic cases caused by EHV-1 across the U.S. prompting the USDA to declare EHM a potential emerging disease. In the late Spring/early Summer of 2011, there were 90 cases of EHM reported in 10 states and 13 horses succumbed to the disease [1]. Due to the increase in EHM cases it is critical that the underlying molecular mechanisms by which EHV-1 causes EHM be investigated so that appropriate therapeutic interventions can be developed and delivered. Although research is ongoing to identify why some horses develop these severe conditions while others recover without incident, much more information needs to be obtained in this area.

Previous work by other groups identified a mutation within the viral DNA polymerase enzyme that is correlated with EHM disease [2, 3, 10]. While this mutation does contribute to neurologic disease, other viral and host factors must also contribute to EHM as viruses that do not contain this mutation have been isolated from EHM horses and infection with some EHV-1 strains that do harbor this mutation do not lead to neurologic disease. For these reasons, studies aimed at identifying additional neurovirulence factors are warranted.

Studies outlined in this proposal will be performed to investigate the interaction of neurologic and non-neurologic strains of Equine herpesvirus 1 (EHV-1) with cells in order to identify specific viral and cellular factors that contribute to efficient fusion and cell to cell spread of EHV-1. Preliminary data obtained from our lab showed that the MHC-I receptor is used by a non-neurologic EHV-1 strain (RacL11) to enter and spread from one cell to the next. Results obtained from the experiments outlined in Aim #1 will reveal whether MHC-I is similarly used by neurologic EHV-1 strains for entry and spread and will also aim to identify additional cellular factors that contribute to disease outcome. In addition to identifying the key cellular factors, the essential set of viral glycoproteins that are needed to mediate fusion with cellular membranes will be determined in Aim #2. Once these key fusogenic glycoproteins are identified, we will determine whether specific mutations in any of the glycoproteins isolated from neurologic strains are responsible for their ability to increase fusion as this property is positively correlated with virulence. Finally, in Aim #3 the contribution of accessory glycoproteins including glycoprotein I and E to cell to cell spread will be evaluated.


SERUM BIOMARKERS FOR EQUINE LAMINITIS
Dr. Hannah Galantino–Homer, University of Pennsylvania -Second Year

The goal of this study is to improve the understanding and early diagnosis of equine laminitis. Equine laminitis is a disease that affects the suspension of the bone of the foot, and hence the weight of the horse, to the hoof capsule via the interdigitating lamellae of the hoof and underlying connective tissue. Laminitis causes failure of digital suspension resulting in excruciating pain and often resulting in humane destruction of the horse. Our previous studies, using archived samples from two experimental models of laminitis, detected molecular changes that are helping us understand the steps preceding full-blown laminitis. In human diseases, molecular changes in the affected tissue are often reflected in molecular changes in the blood. These “disease markers” can then be used to diagnose early stages of a disease, when therapies are the most effective. Our preliminary studies have determined that one of our potential markers is elevated in blood samples from an experimental model of laminitis and from horses with laminitis associated with a non-weight-bearing orthopedic injury (supporting limb laminitis). The goal of the proposed study is to identify at least three serum markers that would be useful for diagnosing the early, inapparent or “developmental” phase of laminitis and to detect the resulting tissue damage and the body’s immune response to that damage.

Based on our earlier studies, we will investigate blood levels of specific proteins using samples from horses without laminitis, horses in the early stage of the disease prior to lameness, and horses with overt laminitis. The studies will involve methods for the identification and quantification of proteins in tissue and blood samples and for the detection of specific classes of antibodies involved in the immune response. Positive results from this study will then be used to develop diagnostic tests to detect incipient laminitis in horses that are believed to be at risk of developing the disease, such as horses with a prior history of laminitis, severe orthopedic injuries, colic, obesity, or horses that have ingested excess grain, and to assess lamellar tissue damage. New technology that allows the “multiplexing” of assays for several markers into a single assay will be used to generate an assay for lamellar tissue damage.


STORM CAT CAREER DEVELOPMENT AWARD

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. In each of the last two years, the Foundation board had authorized two Storm Cat Awards in addition to the award provided by Mrs. Hamilton. Three candidates have been selected to receive the Annual Storm Cat Career Development Award. They are:

Bradley B. Nelson, Colorado State University
Contrast CT for Cartilage Injury in Impact OA Model

Osteoarthritis (OA) is a well-recognized disease in horses and humans. The impact of this disease incurs significant costs causing persistent lameness and decreased athletic performance. Joint injury leads to progressive inflammation and damage to articular cartilage with an early mark being the loss of glycosaminoglycan (GAG) content. The disease is ultimately progressive, since cartilage healing is limited. Therefore, early detection of OA is critical to successful therapy. Currently, MRI is the gold standard imaging method to evaluate cartilage in horses and humans, but presents difficulties that can lead to false positives.

A more sensitive method to evaluate cartilage with decreased anesthetic time is warranted in horses. With the advent of multi-slice computed tomography (CT), costs and scan length have decreased (minutes) while having improved spatial resolution compared to MRI.

Contrast agents can be injected into the joint to help outline articular structures for X-ray based imaging including contrast enhanced computed tomography (CECT). Preliminary experiments in our group have demonstrated a strong correlation between equine articular glycosaminoglycan (GAG) content and CT attenuation in normal cartilage explants. We hypothesize that decreased equine articular cartilage GAG content will be detectible and correlative with CT attenuation using a CA4+ contrast agent in an impact model of osteoarthritis. To test this hypothesis, we aim to determine correlations between articular cartilage GAG content and CT attenuation (CECT) with cationic contrast agent (CA4+) in horses where degenerative GAG concentrations in articular cartilage are created by an impact injury.


Heidi Reesink, Cornell University
Lubricin as a biotherapeutic for equine joint disease

The Research Problem. Traumatic injury to joints such as the knee and fetlock are common in racehorses, often chipping off bone fragments, but also precipitating more widespread cartilage injury that culminates in osteoarthritis (OA). Joint injury and OA rank second only to tendon injury as the leading cause of racehorse retirement. A significant unmet need in research is the evolution of better intraarticular medications that have a more regenerative effect that might bolster both cartilage restoration and quell the reaction in the joint that follows impact injury. There are no treatments that markedly alter the progression of OA, and most therapies have focused on symptom-modifying treatments, including analgesics and physical therapy, and a variety of joint injections. The aims of the proposed research are to examine a new molecule called lubricin, that may provide better and more long-term amelioration of the symptoms of arthritis.

We hypothesize that lubricin may enhance functional repair throughout the joint, and in-so-doing, reduce pain and the progression of disease. We contend that current joint medications don’t mimic resident biology in a joint, and provide a short term resolution for a chronic problem. Lubricin has been identified as the primary boundary lubricant in synovial fluid, which prevents cartilage roughening and degradation under high-load, low-motion conditions by decreasing friction at the articular cartilage gliding surfaces. In addition, lubricin is proposed to protect against the development of OA by decreasing joint lining cell adhesion to the articular cartilage surfaces, inhibiting joint lining overgrowth and scar tissue formation, and stimulating synthetic action from the joint lining to increase other joint fluid constituents. These features make lubricin an ideal joint medication when administered by injection. Despite this obvious potential, little is really known about the changes in lubricin levels or molecular organization with joint disease (fracture or arthritis), the effect of lubricin on tissues that make up the joint, on ways to enhance its impact as a high-load lubricant, and on dosing or longevity of the molecule after injection.

The broad objectives of this proposal are to examine the lubricin levels in joints with various stages of joint damage (from normal through reactive to obvious arthritis), to assess the organization of lubricin on the cartilage surface, and to determine the impact of lubricin on cartilage and joint lining tissues. This information will provide key information in the development of a lubricin treatment protocol. Studies so far suggest that lubricin protects articular cartilage from fibrillation by functioning as a boundary lubricant and by preventing joint lining cell overgrowth and protein deposition. We seek to understand the underlying mechanisms by which lubricin confers these properties in the joint.


Katarzyna Dembek, The Ohio State University
Adrenal Steroids and Neurosteroids in Sick Newborn Foals

Sepsis, defined as a systemic inflammatory response to pathogenic microorganisms, is the number one cause of foal mortality. The prognosis of survival in septic foals ranges from 35-60%.

In foals, endocrine maturation occurs in late gestation and continues into the early post-natal period. These adaptations are accompanied by changes in the Hypothalamus-Pituitary-Adrenal Axis (HPAA), energy metabolism, and the cardiovascular system. Premature birth, failure of transfer of passive immunity, and infections often result in foal sepsis and death. Critically ill foals are sent to intensive care units with hypotension, poor tissue perfusion, energy dysregulation, acid-base and electrolyte abnormalities, and organ failure. Survival under these circumstances is dictated by the ability of various homeostatic systems to overcome these derangements and control systemic inflammation. Unfortunately, a number of these foals remain refractory to current therapies and succumb to sepsis, likely the result of endocrine failure, which our group has shown to be associated with foal mortality.

In response to stress, the hypothalamus releases corticotropin-releasing hormone (CRH) and vasopressin (AVP) to stimulate the pituitary gland to secret adrenocorticotropic hormone (ACTH), which induces the adrenal cortex to release cortisol, aldosterone, and sex steroids. Except for cortisol, information on adrenocortical function in sick foals is limited. We, and others, have shown that in most critically ill foals, HPAA activation is characterized by increased AVP, ACTH and cortisol concentrations. However, the role of hypothalamic or pituitary failure in adrenal insufficiency (AI) in sick newborn foals remains unclear.

A major complication of sepsis in children is relative adrenal insufficiency (RAI) or critical illness- related corticosteroid insufficiency (CIRCI), in which the secretion of one or multiple adrenocortical steroids is impaired and contributes to mortality. In people, adrenal gland function is not evaluated only by measuring cortisol and the response to ACTH stimulation, but also other adrenal steroids including dehydroepiandrosterone (DHEA), aldosterone, corticosterone, and androstenedione.6 In equine practice, the evaluation of adrenal gland function is limited to cortisol concentrations, and ACTH stimulation tests are rarely performed. Drs. Aleman and Madigan have provided persuasive evidence that progestagens affect newborn foal behavior and may be involved in the pathogenesis of maladjustment syndrome.

HPAA dysfunction in septic foals occurs primarily at the adrenal gland level; however, based on work by our group, hyposecretion of hypothalamic (CRH, AVP) and pituitary factors (ACTH) likely contributes to this process. Endocrine factors that regulate hypothalamic function in other species include brain derived neurotrophic factor (BDNF), endocannabinoids, cytokines, LPS, NMDAR (N-methyl-D-aspartate receptor), and GABAAR (GABAA receptor).10 BDNF is a key modulatory factor for CRH and AVP secretion. Low BDNF levels were associated with mortality in critically ill human patients.11 In our pilot data we found a trend for low BDNF levels in 8 septic foals. Relevant to this proposal, adrenal steroids (DHEA, progesterone, pregnenolone) can be transformed into neurosteroids (allopregnanolone), which provide neuroprotection by inhibiting NMDAR, stimulating GABAAR, modulating BDNF, and promoting CRH and AVP secretion. We propose that BDNF is a key player in hypothalamic dysfunction in septic foals. We also suggest that inappropriate neurosteroid secretion will be linked to low BDNF levels in septic foals with HP dysfunction. Thus, the overall goal of this project is to elucidate the role of hypothalamic regulatory factors (e.g. BDNF) and their interaction with adrenal steroid precursors / neurosteroids in the development of RAI in critically ill foals.

We hypothesize that while in critically ill foals AI is mainly the result of adrenal failure (primary RAI), in a number of foals impaired secretion of steroid precursors/neurosteroids and BDNF leads to hypothalamic (tertiary RAI; ↓AVP, ↓CRH) and subsequently pituitary (secondary RAI; ↓ACTH) and adrenal gland dysfunction, which will be associated with multiple clinical derangements and a worse prognosis for survival.

This study will show that in addition to its effects on metabolic and immune functions (↓cortisol), RAI can also lead to electrolyte and fluid dysregulation, , delayed organ maturation, and neurological abnormalities. This information will have prognostic and therapeutic value. A comprehensive understanding on the HPAA response to sepsis will provide the opportunity for the development of novel therapies for equine neonatal sepsis. For example, the use of neurosteroids to modulate HPAA function may have potential use in the clinic.