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 $1,160,556 in 2016. The list includes eleven new projects and eight which are in their second year, as well as Two Research Career Development Awards.
The allotment brings the Foundation’s total impact since 1983 to over $23.3 million to fund 335 projects
at 42 universities.


THYRO-HYOID MUSCLE TRAINING TO TREAT DDSP
Normand Ducharme, Cornell University – First Year (2 Year Grant)

One Sentence Summary: (DDSP is Dorsal Displacement of the Soft Palate.) A better knowledge of DDSP mechanism will give the basis for new treatment options and prophylactic training methods to prevent or reduce the occurrence of DDSP in young horses starting training.

Decrease in racing performance can result from multiple causes but airway obstruction is a prevalent cause in horses. Intermittent dorsal displacement of the soft palate (DDSP) causes an upper–airway obstruction that is associated with an abnormal sound (i.e. gurgling), and it has been recognized as a common cause of poor performance in racehorses with a reported prevalence of 10–20%..Management of DDSP remains controversial because of the lack of a clear cause of this disorder, resulting in several treatment strategies each directed toward one of the proposed causes.

In this study we are targeting the neuromuscular causes of DDSP associated with deficit of the TH muscles. The current standard treatment (laryngeal tie–forward) was developed to treat this change in laryngeal position. Success rates approaching 80+% have been reported for this treatment strategy, with minimal detrimental effects to the horse and return to race after surgery ranging 64–83%. As described by Cheetham et al (2008), before diagnosis and surgical treatment DDSP horses face a significant decrease in their racing performance and consequent earning; many horses were diagnosed while in training and resulted not able to start their racing carrier due to this disease. Moreover, despite surgery, up to 34% of DDSP horses did not return to racing.

Preliminary findings suggest a potential rationale for a new management approach of horses with DDSP, by increasing the muscle strength and resistance to fatigue through specific training protocols and/or functional electrical stimulation (FES) of the TH muscles. A better knowledge on DDSP causes would give the basis for new treatment options in affected horses, but also improve the chance of early diagnosis and prophylactic training methods to prevent or reduce the occurrence of DDSP in young horses starting training.


A NOVEL VACCINE AGAINST EQUINE STRANGLES
Noah Cohen,Texas A & M University – First Year (2 Year Grant)

One Sentence Summary: We have a new concept for a vaccine to protect horses against the disease known as Strangles and have good preliminary data suggesting this vaccine will be safe and effective.

Infectious diseases are leading causes of disease and death for all horses on a worldwide basis. Streptococcus equi subspecies equi (SE) is a bacterium that causes a highly contagious disease commonly known as Strangles. Strangles remains the most frequently diagnosed infectious disease of horses. Currently available vaccines against Strangles have limitations in safety (i.e., can cause disease or complications) and efficacy (i.e., they don’t always protect horses well). Consequently, great need exists for novel approaches to protect against SE, the bacterium that causes Strangles. Polysaccharides are chains of sugar molecules that may be found on the surface of bacteria (and other cells). Bacterial–specific surface polysaccharides have been demonstrated to be highly effective as vaccines against multiple bacterial pathogens, and the laboratory of Gerald Pier (co–Principal Investigator) at Harvard Medical School has discovered a conserved one, termed poly–N–acetyl glucosamine (PNAG), that can be targeted to generate protective immunity. Naturally–occurring PNAG does not stimulate the immune system to produce antibodies that can protect against infection. By chemically altering the PNAG molecule, however, a form of PNAG can be produced that elicits antibodies against PNAG and can provide protection against the many bacteria that have PNAG on their surfaces. Our laboratories (Cohen and Pier) have generated compelling preliminary data regarding the efficacy of an anti–PNAG vaccine. This project will extend our exciting preliminary data by evaluating the safety, immunogenicity, and efficacy of a PNAG to protect horses against Strangles. We expect to demonstrate that the PNAG vaccine does not cause serious adverse effects, induces detectable antibodies in serum and nasal wash fluid that recognize PNAG on the surface of SE, and that these antibodies in serum and nasal wash fluid will significantly increase killing of SE by immune cells of horses. We further expect to demonstrate that the PNAG vaccine protects horses against experimental infection with SE. A safe and effective vaccine against SE would be of great benefit to the health and welfare of horses and to the equine industry.


FITNESS AND PERSISTENCE AND OF DRUG RESISTANT R. EQUI
Steeve Giguère, University of Georgia– First Year (2 Year Grant)

One Sentence Summary: We will determine if drug-resistant Rhodococcus equi can persist in the environment and if resistant strains are more likely to cause disease than susceptible strains.

The standard treatment for pneumonia in foals caused by the bacterium Rhodococcus equi has been combination therapy using one of the macrolide antibiotics (erythromycin, clarithromycin, or azithromycin) with the antibiotic rifampin. Widespread resistance to these drugs is a major emerging problem facing the horse-breeding industry, with resistant isolates being cultured 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 macrolide and rifampin resistance in R. equi. The logical long-term approach to prevent expansion and spread of resistant R. equi would be to decrease widespread use of macrolides and rifampin in foals. However, strategies aimed at decreasing the use of these drugs will only be effective if carrying the genes that cause resistance alter the ability of R. equi to grow and survive (termed fitness), thereby conferring a competitive disadvantage to reproduce in the absence of antimicrobial selection pressure. Given that R. equi survives in the environment and has the ability to replicate in soil under suitable environmental conditions, the proportion of resistant R. equi would not be expected to decrease over time if its ability to survive is preserved or if virulence (the ability to cause disease) is enhanced. Therefore, it is of paramount importance to understand the effects of carrying macrolide or rifampin resistance mechanisms on overall fitness and virulence of R. equi. The overall objective of the proposed research is to determine the effects of carrying macrolide or rifampin resistance mechanisms on the fitness of R. equi and its ability to cause disease.

Our preliminary data indicate that resistant isolates of R. equi have decreased fitness compared to susceptible isolates as assessed by in vitro growth. In addition, resistant isolates have impaired ability to survive in soil. All the bacterial strains required are already available and 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. After completion of this work, we will have determined if the determinant conferring macrolide resistance or mutations conferring rifampin resistance affect: 1) in vitro growth of R. equi; 2) the ability of R. equi to survive in soil at various temperatures; and, 3) the ability of R. equi to cause disease, both in the presence or absence of antimicrobial selection pressure. In addition, the proposed work will characterize how long these resistance mechanisms persist in R. equi in the absence of antimicrobial selection pressure. 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. After completing these experiments, it will be possible to develop models to study eradication of macrolide-resistant R. equi. These models will be used to monitor emergence or disappearance of macrolide resistance in the environment of horse farms in response to various interventions and to devise strategies to control expansion of the problem of antibiotic resistance.


NOVEL ANALGESIC COMBINATION IN HORSES
Alonso Guedes, Universityof Minnesota – First Year (2 Year Grant)

One Sentence Summary: We propose to study a novel, likely more efficacious and potentially safer approach than currently available options to manage pain in horses.

Pain is a debilitating factor in diseases such as laminitis (foot inflammation and pain) and osteoarthritis (joint inflammation and pain), and its effective control is one of the greatest challenges in equine clinical practice. We have preliminary in vitro data showing that t-TUCB is a potent inhibitor of equine sEH and in vivo data showing that 1 mg/kg t-TUCB, but not lower doses, produces significant pain relief using the joint pain model proposed here. Preliminary clinical studies in nine horses with laminitis and one horse with osteoarthritis suggest that dual COX/sEH inhibition with phenylbutazone/t-TUCB combination may result in additive/ synergistic responses, as previously found in pain models in rats. The significance of the proposed research is that it is expected to provide new knowledge on the modulation of different branches of fatty acid metabolism in horses that could be used for the design of clinical trials and for developing novel approaches for pain management in these animals. This has high potential to impact all horses by decreasing the negative effect that poorly controlled pain has on equine health and productivity, which in turn decreases the economic burden for the equine industry.


TRAINING AND SURFACES FOR INJURY PREVENTION
Susan Stover, University Of California-Davis – First Year (2 Year Grant)

One Sentence Summary: Risk for bone fracture in the fetlock joint due to training program and race surface properties will be determined using computer models that simulate bone damage and repair .

There are certainly better and worse training schedules and better and worse racetrack surfaces that are used to prevent, recover from, and resume training after, an injury or bone fracture. Our goal is to investigate and determine training programs and race surface properties that keep horses healthy and prevent bone fractures.

To prevent bone fractures, we must understand how they develop in our racehorses. We know that bone fractures rarely occur from a single bad step in athletes. Instead, fractures are the result of multiple factors that affect the accumulation of bone damage over racehorses’ careers. These factors are related to the distances that racehorses train and race, the speed of the horse during training and racing, and the stiffness of the race surface that training and racing occur on. In addition because damaged bone can heal given enough time, the training program--- the weekly timing of works and races---plays a large role in racehorses’ susceptibility to injury and bone fracture. Further, these factors interact with, and affect, each other over time.

In order to understand the impact of training schedules and race surface properties on racehorses’ susceptibility to injury over time, many things must be factored in--- the complex interaction of gait, speed, distance, and timing of exercise; alterations of limb loading due to race surface properties; and the time course of removal of damaged bone and the inherent time lag in the replacement by healthy new bone.. The complexity of the relationships require computer modeling approaches that take into account the time sensitive nature and interaction of the events to understand the resultant risk for injury.

We will use a computer model that simulates micro damage accumulation with training and racing, and repair of damaged bone with time. The model will be developed based on lifetime race and timed–work records of 60 racehorses that died during training or racing. The sesamoid bones at the back of the fetlock will be used to measure the actual bone damage and resulting repair from each of the same 60 racehorses to ensure or adjust the computer model to reflect what actually occurred in the racehorses. Subsequently (Part II), we will further calibrate and validate the model using lifetime race and timed–work records of 392 Thoroughbred racehorses; 98 case racehorses that died because of a proximal sesamoid bone fracture and 294 control Thoroughbred racehorses that did not die during their career. Lastly, we will use the validated model to determine combinations of training programs and race surfaces that prevent injury and combinations that put racehorses at high risk for injury.

The potential usefulness of the modeling approach is three–fold. Models can be used to evaluate existing training programs and race surface mechanical properties for risk for injury. Training programs and race surfaces that do not currently exist can be screened in a cost– and time–effective manner to design improved training programs and optimal race surface properties. Exercise and race venue histories of race entrants could be screened before races to help identify horses at high risk for catastrophic injury during the upcoming race.

Skeletal injuries take an insidious, but large and deleterious toll on equine and jockey welfare, and industry economics. Attrition due to musculoskeletal injuries is the largest cause of wastage in racing. Injuries are the cause of 83% and 71% of deaths in Thoroughbred and Quarter Horse racehorses, respectively Non–fatal injuries are the most common reason for exit of horses from training. Injuries resulting in an inability to train for more than 5 days occur 29 times more frequently than fatal injuries. It has been estimated that )950 million dollars in horse costs alone are lost in the United States each year due to the loss of 3% of horses from racing each month.

Fetlock injuries are the greatest cause of fatal musculoskeletal injuries in Thoroughbreds and 34–40% of deaths due to musculoskeletal injuries in Quarter Horses. Proximal sesamoid bone fractures are the most common cause of fetlock injury. Further, 36% of non–fatal injuries affected the proximal sesamoid bones (7%) or fetlock support structures (29%).

Catastrophic injuries of horses are the major cause of jockey falls and injuries, Jockeys are 162 times more likely to fall and 171 times more likely to be injured when riding a horse that died in a race than otherwise. Worker compensation claims were $6.5 million dollars over a 5 year period in California alone. When horses sustained a fatal fetlock injury during a race, jockeys fell 22% of the time, and were injured 13% of the time. Consequently, fetlock injuries take a large toll on equine and jockey welfare, and industry economics.


HOST-DIRECTED CONTROL OF R. EQUI FOAL PNEUMONIA
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.


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

One Sentence Summary: This proposal defines the critical next step to understand underlying mechanisms of disease by developing a database of tissue– specific gene expression and regulation in the healthy adult horse.

The sequencing of the horse genome in 2009 resulted in significant advancements in the field of equine genetics. Currently, there are 32 equine traits, including genetic diseases and coat colors, for which a genetic test is available. Of these, 22 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. Development of this roadmap is as important to the equine industry today as the sequencing of the equine genome was in 2009. In Years 1-2, we will characterize tissue-specific gene expression across 8 tissues (skeletal muscle, laminae, liver, ovary or testis, cerebral cortex, lung, spleen and heart) in one male and one female horse using RNAsequencing and identify tissue-specific “on/off switches” using ChIP-sequencing. This data will be linked to the underlying DNA sequence. In Years 3-4, we will extend our studies to include the location of additional “off switches” using CTCF-sequencing, as well as the discovery of important regulatory regions using a technique called DNAse-I hypersensitivity assays. While the initial analyses will be performed on 8 tissues, we will archive )50 tissues from each horse that will be available for future studies. Data will be publically accessible in existing genome browsers. Given the importance of this work, funding to cover sample collections has been secured through USDA NRSP-8, National Animal Genome Research Program equine coordinator’s funds ($15,000).

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.


EHV-1 AND LATENCY
Lutz Goehring, Ludwig Maximilians University – First Year (2 Year Grant)

One Sentence Summary: We will know about EHV–1 latency locations; about prevalence in horse populations, and if different latency stages exist. Finding ’stages’ will allow us to speculate on interventional strategies.

Latency is a hallmark mechanism of all herpesviruses and is best described as a dormant, reclusive state of an infection. Equid Herpesvirus–1 (EHV–1) is the cause of respiratory disease in horses, but also leads to devastating outbreaks of myeloencephalopathy and late term abortions. A horse that is suffering from an acute infection with EHV–1 will simultaneously develop a latent infection, specifically in the trigeminal ganglion or in lymph nodes of the respiratory tract. While the horse overcomes the acute infection within 2 – 3 weeks, the silent or latent stage of infection is permanent. Latency is often compared to hibernation in mammals which illustrates that specific signals, yet unidentified, are existing that can reactivate virus, resulting in a return to the respiratory tract and in a spurious replication and shedding of virus into the environment and into other horses. This scenario happened at the National Cutting Horse Association’s Western National Championships in Ogden, Utah in 2011, and resulted in a multi–state outbreak. Many horses were euthanized, barns were quarantined and financial losses were significant. Yet, we don’t have a good understanding of what causes viral reactivation or are there strategies on how to intervene other than we realized that an outbreak can start with reactivation of virus in a single animal. EHV–1 latency research is difficult as latent–infected tissue is more likely inaccessible, and tissues collected during post mortem exam may or may not contain latent virus, and if so, there will be no information on the time between infection and the moment of sample analysis. Here we got access to a unique tissue sample collection from horses that were purpose–infected with EHV–1 and euthanized 7 weeks after infection. A time interval of 7 weeks should allow latency to develop. Testing for latency has so far focused on detection of viral DNA. As latent virus does not fully replicates but we assume it produces a stop signal to stop replication, we believe this stop signal, an RNA transcript, can be quantified in analogy to procedure in other herpesviruses. In human HSV–1 latency the quantity of stop signals correlates with depth of latency. The sample collection from the purpose–infected horses will serve as a positive control, and we will determine the amount of this product (a.k.a. stop signal) in relation to viral genome copies in a cell and to cellular transcriptional activity. Besides the results we will also screen other neural tissues and lymph nodes for latency to identify alternative sites in horses. However, the stop signal quantity in these tissues should be high in many samples, as latency has been just recently established, while in samples collected from random tissue specimen from abattoir horses we expect the stop signal amount to be very diverse and depending on the interval between infection and collecting the sample. This will be the first attempt to show different stages of latency based on the quantity of specific latency marker mRNA and it will show the importance of this marker in staging latency. This will be the first step towards identification of more and other host or virus–derived factors that control latency at the cellular level, and results will initiate more research on how to intervene or how to rebalance between deep hibernation and reactivation. In addition, prevalence data of latency in the general horse population will be answered, and if latency is detected at one tissue site, what are the chances that other sites are latent–infected, too, or in other words whether there is random or ranked latency in horses with naturally occurring latency.

The EHV–1 outbreak at the National Cutting Horse Association’s Western National Championships in Ogden, Utah in 2011, which resulted in a multi–state outbreak and many horses were euthanized due to serious neurological disease, barns were quarantined and travel restrictions were in place for a number of weeks, provides a dramatic example of the disease capacity of this virus. Important to understand is the fact that an outbreak typically starts with a single horse where dormant virus becomes re-activated, is returning to the respiratory tract and is being shed into the environment and into other horses. Infections with EHV–1 can occur early in life and immunity is short–lived, which implies that re–infection with same or similar virus can occur. Already with the first infection the dormant or latent stage is induced. Hence, we will not be able to prevent latency establishment in the horse population; however, with this research proposal we will study virus activity during this dormant or hibernating stage, which will hopefully allow us to find ways to counterbalance awakening activities of this virus when in latency. Vaccination, for example, has been the strategy of choice to prevent flare–ups of shingles in the elderly. Shingles is the result of reactivated chickenpox virus (Varicella Zoster virus, also a herpesvirus); however, we first have to find sensitive ways to identify virus in latency, and we have to study its activity level in a latent–infected cell. The aim of this project is to identify markers of viral activity during latency followed by finding strategies that possibly intervene with viral behavior during latency.


PET IMAGING OF THE EQUINE DISTAL LIMB
Mathieu Spriet, University Of California-Davis – One Year

One Sentence Summary: Position Emission Tomography (PET) imaging is a diagnostic tool, newly available to the horse, that will allow detection of lesions not identified with other techniques.

This project proposes to evaluate an imaging technique newly available for the horse. Due to configuration of the machine, Positron Emission Tomography (PET) was not available for use in the horse, but recent technological innovations have led to the development of portable scanners that can be used to image the horse limb. Our team was the first one to acquire images on 3 horses with such a system. We propose to image 6 horses from a research herd to optimize the technique and then conduct a clinical trial, with 6 horses in the first year and 16 horses in the second year.

The advantage of this imaging modality is that instead of providing information primarily about the size and shape of the body part imaged, PET brings information regarding the activity that occurs at the molecular level. It is well known that many lesions are preceded by changes at the molecular level that could not be assessed without invasive tests. PET opens the door to improving early detection of injuries leading to lameness, but also helps better understand the causes of injury and provide valuable information in the assessment of the healing process. PET will also be helpful in determining the efficacy of treatment and help refine rehabilitation programs.


VALIDATION OF STALL-SIDE STRANGLES DIAGNOSIS USING LAMP
Ashley Boyle, University of Pennsylvania – One Year

One Sentence Summary: We aim to validate a stall–side test that could be used for fast, sensitive, accurate, and cost efficient diagnosis of strangles (S. equi) carriers (a highly infectious equine respiratory disease). [LAMP means loop– mediated isothermal nucleic acid amplification.]

Strangles is a highly infectious upper respiratory disease of horses caused by the bacteria Streptococcus equi subsp equi (S. equi). When a horse has strangles, the animal can develop the following: enlarged swellings surrounding the throat that can block the horse’s airway, chronic abscesses throughout the body leading to weight loss and colic, severe allergic reactions, and chronic bacterial infections of the throat that are a source of infection for other horses (resulting in "carrier horses"). Once a herd is exposed, up to 100% of the horses can be affected by the disease depending on the effectiveness of control methods implemented. Current methods of disease detection can have low sensitivity and accuracy depending on the type of sample collected from the horse. These tests take a minimum of one to three days to process in a laboratory. Identification and use of low cost point of care “stall– side” diagnostics would greatly reduce the spread of this infectious disease by identifying strangles in an index or carrier horse.

Strangles carriers are the hardest horses to detect with the disease for they appear outwardly healthy. Our laboratory recently proved the best sample to use to detect carriers is a throat sample from the guttural pouch (the Eustachian tubes of the horse).

Current diagnostic technology is available to change the way we test for strangles with the ability to test for the bacteria that causes strangles without the need for the laboratory setting. The recent development of a molecular diagnostic tool known as loop– mediated isothermal nucleic acid amplification (LAMP) is available in a disposable, water–activated device that requires only the external instrumentation of a cellular telephone offering stall–side disease detection. The purpose of this study is to validate this cartridge for fast, sensitive, accurate, and cost efficient diagnosis of strangles carriers in the field.


IGGS(T) ANTIBODIES IDENTIFY FOALS AT RISK FOR R.EQU
David Horohov, University of Kentucky – One Year

One Sentence Summary: This project involves the validation of a new test for Rhodococcus equi infections in foals.

Rhodococcus equi is of worldwide importance as a cause of pneumonia and of a variety of extra pulmonary lesions (EPLs) in young foals. Control of R. equi pulmonary infections currently relies on early detection of disease using thoracic ultrasonography and initiation of treatment with antimicrobial agents prior to the development of clinical signs. Although this approach is widely used, it fails to differentiate those foals with progressive disease from those that will regress their lesions spontaneously. We have recently demonstrated that a specific antibody response to the bacterium had a strong positive correlation with rhodococcal pneumonia. Our hypothesis is that this early antibody response is a predictor of rhodococcal pneumonia and/or extra pulmonary lesions in young foals and can be used to identify those specific foals that will require antibiotic treatment. To test this hypothesis we propose collect sera and clinical data from foals on a rhodococcus–endemic farm, and test their antibody profile. We expect that foals diagnosed with R. equi infections will exhibit a different antibody profile from those that remain disease-free. Further, we expect that this difference will be detectable prior to the development of clinical signs and thus allow for the strategic treatment of only those foals at greatest risk for disease even before disease is seen.

Pneumonia caused by Rhodococcus equi remains a significant financial burden and welfare concern for the equine industry. Young foals are uniquely susceptible to R. equi infections. Currently there are no effective vaccines or other measures to prevent Rhodococcus infections in foals. Most foals are infected early in life, as evidenced by thoracic ultrasound screening. While this screening can identify foals infected with this bacterium, it does not identify those at risk for developing disease. Most foals will resolve their infection without significant consequences. However, since there is no way to identify these resistant foals at the time of screening, this can result in the antibiotic treatment of resistant foals. Not only is this expensive and a management issue, it also increases the risk for antibiotic–resistant strains of the bacterium to develop. The goal of this project is to test a new approach for identifying foals at risk for developing rhodococcal disease. If successful, this should reduce the number of foals being treated unnecessarily with antibiotics.


A GUINEA PIG MODEL OF RHODOCOCCUS EQUI PNEUMONIA
Angela Bordin, Texas A&M University–Second Year

One Sentence Summary: A guinea pig model of R. equi pneumonia will help to better understand the disease in foals, and evaluate novel approaches for controlling and preventing R. equi pneumonia.

Pneumonia caused by Rhodococcus equi is an important cause of disease and death in foals. Important advances in R. equi research could be achieved using a small animal model of pneumonia, including the evaluation of novel approaches for controlling and preventing R. equi pneumonia before their test in horses. Currently, there is no model of pneumonia caused by R. equi: infection of mice does not cause pneumonia as recognized in foals with natural or experimental infections. As a result, instillation of bacteria into the airways of foals to cause lung infections is used as an experimental model of natural disease. Infection of foals is problematic for several reasons. First, it is labor–intensive and cost–restrictive, and, frequently, involves a team of trained personnel to monitor and administer treatments to infected foals several times a day. Second, it demands adequate infra–structure to keep foals and their respective dams in a confined space to allow physical examination and administration of treatments, while also providing access to paddocks. Third, the use of foals is restricted to the spring foaling season in the northern hemisphere, limiting the number of experiments that can be conducted in one year. Fourth, laboratory reagents to study the immune system of horses are quite limited relative to guinea pigs. Fifth, and perhaps most importantly, there are major welfare considerations of making foals severely ill. Thus, the objective of this study is to develop a guinea pig model of R. equi pneumonia.
We intend to determine whether aerosolized R. equi can produce in guinea pigs the same form of pneumonia observed in naturally infected foals; and also to identify a dose of R. equi that consistently produces pulmonary lesions in guinea pigs that are proportional to lesions in foals naturally infected with R. equi. Guinea pigs will be infected by aerosol at various doses of R. equi and monitored daily to evaluate disease. They will be euthanized at 5 weeks post–infection or when clinical signs of pneumonia are detected, whichever comes first. This model will be useful for evaluating and optimizing novel strategies for therapeutics and vaccines, and for studying several aspects of R. equi pneumonia without the drawbacks of using foals, and will be a benefit to scientists studying R. equi on a world–wide basis.

The use of a guinea pig model of Rhodococcus equi would greatly benefit the equine industry. The impact of R. equi pneumonia on the equine industry is large. In the United States, respiratory disease is the third most common cause of disease in foals less than 6 months of age. Foals with R. equi pneumonia can either show clinical signs of disease, or have the lesions without clinical signs (known as subclinical disease), but the most common form of the disease is a chronic pneumonia with an insidious onset that hinder early detection. Often, the lung lesions are well–established and extensive damage has occurred by the time a diagnosis is made. Consequently, a high proportion of foals with R. equi pneumonia die, and foals that do survive the disease are less likely than age–matched cohorts to race.

Treatment and prevention of R. equi pneumonia are difficult: there is no vaccine commercially available, treatment with antimicrobials is lengthy, costly, has potential adverse effects, and is not uniformly successful. Also, there is great need to identify alternative antimicrobials because of evidence of increased resistance. Evaluation of antimicrobials in animals is necessary because drugs that are effective in the laboratory are not always effective in patients. Also, no efficacious vaccine against R. equi pneumonia exists. The equine industry would greatly benefit with the development of both alternative antimicrobials and vaccines that could protect foals against R. equi pneumonia. Thus, there is need to develop a small animal model of R. equi pneumonia, which would allow for evaluation of research questions of clinical importance (such as how the disease develops, and evaluation of new vaccines and antimicrobials) in guinea pigs before they are evaluated in horses or foals. A guinea pig model of R. equi pneumonia would allow for more rapid advances in the field resulting from experiments completed faster than if they were performed in the foal model.


IMMUNE PROPERTIES OF AUTOLOGOUS AND ALLOGENEIC BMDMSCS
Laurie Goodrich, Colorado State University- Second Year

One Sentence Summary: The completion of this project will answer the important question of whether allogeneic mesenchymal stem cells derived from bone marrow (BMDMSCs) are a viable alternative to autologous BMDMSCs in the horse. (Autologous means cells from the horse’s own bone marrow; allogeneic means from another, healthy horse.)

Regenerative therapies, specifically mesenchymal stem cells that are derived from a horse’s bone marrow (BMDMSCs) are a sought after and highly utilized treatment for diseases of tendons, ligaments and cartilage in racehorse and sport horse practices. When these cells are harvested from a horse’s own marrow cavity (autologous or self), they are grown and expanded over a period of two to four weeks and subsequently injected into the injured tendon, ligament or joint. The time required for this process can make this treatment inconvenient for owners and trainers as they must wait a few weeks before returning to get the therapy. Furthermore, the process of individual cell expansion is expensive. An alternative source may be cells from another, healthy horse’s (allogeneic or non–self) bone marrow that has already undergone expansion and is ready for injection. A lag time in treatment to injury has been found to sometimes be detrimental; therefore, an “off the shelf” treatment would potentially enhance the healing potential of these readily available cells in addition to making the treatment more convenient and less expensive to clients. BMDMSCs have been studied intensively in other species and have been established to have a local immunosuppressive effect so that the host’s immune system does not recognize them as foreign. This area has not been closely examined in the horse. If no differences exist between autologous and allogeneic stem cells then BMDMSCs from young, healthy horses may be a viable alternative source to autologous cells. Allogeneic (non–self) BMDMSCs would be highly available and less expensive for clients.

The main hypothesis of this grant is that there are no differences between autologous and allogeneic BMDMSCs in how these cells stimulate the immune system. We will first test this hypothesis by comparing the immunity of these cells in cell culture (in vitro) and attempt to determine the mechanisms by which these cells locally suppress the immune response (Specific Aim 1). In our second Specific Aim (Specific Aim 2) we will compare the immune response to intra–articular injection of each horse when autologous or allogeneic BMDMSCs are administered once and with repeated injection. The completion of this project will answer the important question of whether allogeneic BMDMSCs are a viable alternative to autologous BMDMSCs in the horse. If so, allogeneic cells could be more readily available and less expensive, allowing BMDMSC therapy to be more highly utilized and affordable to owners and trainers.

Regenerative medicine, especially mesenchymal stem cell therapy is a frequently utilized treatment in racehorse and sport horse practice.
The lag time of individual cell culture expansion makes this therapy inconvenient and expensive. If allogeneic (non–self) BMDMSCs could be utilized instead of autologous (self) BMDMSCs without these cells being recognized as foreign, this regenerative therapy could be made available as a less expensive “off the shelf” treatment making BMDMSCs available to clients who would have previously foregone the therapy due to expense. Research studies are mounting with evidence of the benefits this therapy provides. When the questions in this proposal are answered, we will have an improved understanding of how these cells avoid immune recognition and potentially have a more available and affordable source of stem cell therapy.


STEROID / NEUROSTEROID DYNAMICS IN CRITICALLY ILL FOALS
Ramiro Toribio, The Ohio State University- Second Year

One Sentence Summary: This study will elucidate the importance of stress hormones as well as hormones that affect neurological function in the development and progression of diseases of newborn foals.

Sepsis (blood poisoning) is a condition where bacteria can multiply in the blood and set up widespread infections throughout the body of newborn foals often leading to death. The clinical signs and consequences of sepsis include severe stress, organ failure, abnormal hormonal and metabolic functions (e.g. low blood glucose), electrolyte disturbances (low blood sodium), dehydration, low blood pressure, impaired organ maturation and function, and death.

The hypothalamus–pituitary–adrenal gland axis (HPAA) is a complex system that regulates how foals respond to stressful events. Dysfunction of the HPAA results in abnormal concentrations of stress hormones, including steroids from the adrenal gland. These steroids are essential to regulate glucose, electrolytes, blood pressure, as well as organ maturation (lungs, brain, intestine). Some of these steroids (neurosteroids) protect nerve cells against injury and inflammation. We are proposing that in sick newborn foals the equilibrium between these steroid hormones is broken, and depending on its severity it can have deleterious effects on metabolic activities, immune protection, organ function, and likelihood for survival. For example, we suggest that critically ill and premature foals will have increased levels of progesterone (a sex hormone) and low levels of cortisol (a stress hormone).

We will investigate hormones from the brain, pituitary gland, and adrenal gland. We will determine their association with the clinical signs and blood abnormalities of sick foals, as well as whether their levels are linked to clinical improvement or mortality.

We have established collaborations with multiple institutions to investigate the importance of these hormones in the development and progression of diseases affecting newborn foals. We have the expertise to address these questions in a timely manner. The information generated will be relevant to the equine industry as it could lead to the development of novel treatments for foals.

Sepsis is a pathological condition where bacteria can multiply in the blood and tissues, releasing toxins throughout the body of newborn foals. Sepsis is the number one cause of mortality in newborn foals. Many foals that survive sepsis are left with disabilities that impair their future performance. Stress hormones including steroids from the adrenal gland that are essential to control glucose, electrolytes, and blood pressure are often altered. Other steroids are important for organ maturation / function (lungs, brain, intestine). Recent investigations indicate that other steroids (aka neurosteroids) for which minimal information exists in foals protect nerve cells against injury.

We are proposing that in sick newborn foals the equilibrium between these steroid hormones is broken and it can impair their ability to survive.

The goal of this project is to investigate the importance of these hormones in newborn foals with different levels of disease. Hormones will be measured during hospitalization, and their level/balance assessed. The association between hormones, blood abnormalities, and likelihood mortality will be determined.

This study has clinical relevance to the equine industry as a better understanding of equine sepsis will improve treatment. We will generate novel information that will benefit the well–being of horses. Results will be valuable in the implementation of novel therapies to restore the hormonal balance. For example, we may consider using progesterone (a mare hormone) to accelerate brain maturation in dummy foals. In other instances we may want to block the production of other hormones. This project will also be positive for the training of students and veterinarians with interest in equine research. We are confident that we can accomplish the goals of this project in the proposed time–frame.


INHIBITION OF TYPE–I INTERFERON RESPONSE BY EHV–1
Thomas Chambers, University of Kentucky –Second Year

One Sentence Summary: This project explores the mechanism of equine herpesvirus-1 blockage of an immune defense pathway and its relationship to equine herpesviral myeloencephalopathy, a serious condition affecting horses.

Equine herpesvirus–1 (EHV–1) is a one of the leading causes of respiratory disease in horses. It is an especially worrisome disease because it can produce symptoms including neurological signs known as equine herpesviral myeloencephalopathy (EHM) that could lead to the death of the affected horse. There are vaccines for EHV–1 but control of the disease has been very difficult because, like most herpesviruses, EHV–1 appears to have many ways of avoiding host immune responses. This project explores one way of avoiding the immune response that has not been explored elsewhere.

We have evidence that one of the ways EHV–1 avoids immune responses is by blocking the Type–1 interferon (IFN) response, also known as the IFN–alpha/beta response. The IFN–alpha/beta response is important as a non–specific first line of defense against many virus infections, by making cells more resistant to virus infection. Our preliminary studies show that EHV–1 causes an initial stimulation of IFN–alpha/beta but then blocks its further activity once the virus replication has gone into its late phase. These studies were done using an EHV–1 strain that causes EHM, and we do not know whether the results are applicable to all EHV–1 strains. We propose that one or more factors made by the virus are responsible for blocking the IFN–alpha/beta response, and here we want to find out how that block happens. We also propose that this block happens with the EHV–1 strains that cause EHM but not with the EHV–1 viruses that don’t cause EHM.

Specific aims are:

1. Determine which steps of the IFN–alpha/beta response pathway (sensitization, induction, or amplification) are blocked by EHV–1

2. Determine the relationship between the ability of an EHV–1 strain to cause EHM and its ability to block the IFN–alpha/beta response.

With this knowledge we can potentially design interventions to bolster innate immune responses against EHV–1 and improve control of this disease. This research could eventually lead to treatments for horses infected with EHV–1 that target the viral factors which cause the block in IFN–alpha/beta, with the goal of reducing the severity of disease and the likelihood of EHM.


FIROCOXIB PROPERTIES IN EQUINE PREGNANCY AND PLACENTITIS
Margo Macpherson, University of Florida –Second Year

One Sentence Summary: The potent anti–inflammatory properties of firocoxib have the potential to significantly inhibit inflammation, and subsequent preterm delivery of foals, from mares with 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).

Our objectives in this work are to examine characteristics of firocoxib, a potent anti–inflammatory drug formulated for use in horses, after it is administered to mares with placentitis. First, we will determine if firocoxib can effectively reduce factors that cause inflammation in placentitis. Second, we will treat mares with placentitis using firocoxib, trimethoprim sulfa and Regumate™ to see if pregnancy is maintained and foal survival improved. We will compare this treatment to a commonly used placentitis treatment of pentoxifylline, trimethoprim sulfa and Regumate™. Our preliminary studies have shown us that firocoxib is found in the fluids of pregnancy (allantoic and amniotic), colostrum and plasma from foals of treated mares. Since firocoxib can penetrate the placenta effectively, we expect that firocoxib will reduce inflammation that occurs after placental infection. Further, we expect that infected mares treated with firocoxib, trimethoprim sulfa and altrenogest will carry pregnancies longer and deliver more live foals than mares administered a more traditional treatment. These objectives fulfill the over–arching goal of our research program which is to improve foal survival after placentitis.

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 educated therapeutic choices is to determine if specific drugs are performing expected functions, such as resolving inflammation, in mares with placentitis. It is also important to determine if drugs are effective in stalling preterm labor so that the foal has more time to mature, and ideally, survive the infection. 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.


FLUNIXIN OR FIROCOXIB IN POSTOPERATIVE COLIC PATIENTS
Anthony Blikslager, North Carolina State University–Second Year

One Sentence Summary: This project will provide an evidence-based approach to the optimal treatment of horses with small intestinal strangulating obstruction in order to reduce endotoxemia and increase survival.

Horses with severe colic require surgery to correct intestinal obstruction, and it is this population of horses that requires the most intensive care to save their lives. Survival rates have continued to increase as equine anesthesia and surgery has progressively improved. This has shifted the emphasis of colic treatment to intensive medical care that is required before and particularly after surgery. There are a number of treatments that have become ‘the standard of care’ for horses with severe colic, including intravenous fluids and anti-inflammatory medications, such as flunixin meglumine (Banamine®). However, just as there have been advances in anesthesia and surgery, there have also been advances in medical therapy. One such advance has been the development of drugs called COX-2 inhibitors, which have an action similar to flunixin, but target inflammation much more specifically. Recent scientific discoveries have shown that the COX enzymes are responsible for beneficial functions, such as digestive and kidney health via COX-1, and detrimental functions, such as pain and inflammation via COX-2. Flunixin inhibits both COX enzymes, so it has benefits to horses with colic (pain relief and reduced inflammation) as well as side effects (stomach ulcers, kidney damage). We have recently discovered a new side effect of flunixin – it slows down repair of the gut because it inhibits COX-1. However, since 2010, an intravenous COX-2 inhibitor has been released on the market specifically for horses called firocoxib (Equioxx®). We have shown in research trials comparing flunixin and firocoxib that while both provided excellent levels of pain relief, flunixin reduced the rate at which the bowel could recover from colic-induced injury, whereas firocoxib allowed the bowel to recover more rapidly. We believe this may be a very important consideration when treating horses with surgical colic. To properly test this, we are proposing a multi-institutional clinical trial in which owners of colic patients going to surgery at NC State University, Michigan State University, or the University of Pennsylvania will have the option of enrolling their horse in a clinical trial in which flunixin or firocoxib will be given. The drug being used will be hidden from the veterinarians (they will know one of two anti-inflammatory drugs are being given). We will also give the drugs to horses in random order. This is all part of running a high level clinical trial, to make sure the results are as meaningful as possible. Clients will be given a small financial incentive to enroll their horses in this trial, and all other aspects of medical and surgical treatment will be kept the same. We will determine if one drug is advantageous over the other by measuring a comprehensive series of clinical measures (e.g., level of shock) as well as blood levels of bacterial toxins and markers of septic shock. The outcome will be a far greater understanding of the effects of both flunixin and firocoxib in horses with surgical colic, which we believe will help clinicians enhance their level of medical care in these critically ill horses.

Colic is the leading cause of death in horses behind old age according to studies performed by the United States Department of Agriculture. The main reason for death in horses with colic is absorption of bacterial toxins from the gut into the bloodstream. This causes shock, which is often difficult to manage. Treatment has greatly improved over the last 20-30-years, but periodically requires re-evaluation to make sure veterinarians are treating with the best possible mediations. One very important drug used to combat colic is the non-steroidal anti-inflammatory drug (NSAID) flunixin (Banamine®) because it is a painkiller as well as reversing some of the shock-causing effects of bacterial toxins. However, it has side effects, including one that we have recently discovered in which flunixin slows down intestinal healing. This paradoxically increases absorption of bacterial toxins. Interestingly, a new NSAID on the market for horses, firocoxib (Equioxx®), provides more targeted treatment of pain and inflammation while limiting side effects, including delayed intestinal repair. To find out the clinical importance of this potentially critical difference in medications, we want to perform a multi-university clinical trial in which flunixin and firocoxib will be compared in horses going to surgery for colic. We will set it up so that use of firocoxib and flunixin will be given in a random order, and without the veterinarian knowing which drug they are using. In this way, we can remove bias and gather clinical information as well as important indicators of the level of shock that will facilitate decision-making on colic treatment in the future.


MICROSPHERE ENCAPSULATED EPCS AND WOUND VASCULARIZATION
Anne Wooldridge, Auburn University–Second Year

One Sentence Summary: Injectable hydrogel microsphere scaffolds containing endothelial progenitor cells are a potential novel therapy to decrease healing time in distal limb wounds in the horse.

Although the field of stem cell therapy in the horse has made great strides, the fate of injected stem cells (i.e. do the cells stay at the site of injury, stay alive, and cause repair or do they improve healing through other means) is still unknown. Biomaterial scaffolds are engineered materials that provide a framework to ensure that stem cells remain at the site they are administered and optimize the function of the cells. Endothelial progenitor cells (EPCs) are cells that form new blood vessels or repair the lining (endothelium) of existing blood vessels. These cells come from stem cells in the bone marrow, and they circulate in the bloodstream, so obtaining EPCs for analysis and culture only requires a simple blood draw from the horse. The ability of EPCs to form new blood vessels and repair damaged blood vessels makes them very attractive for treatment of conditions where blood flow is reduced. Wounds on the horse’s cannon bones frequently take long periods of time to heal and tend to form proud flesh, partially due to abnormal or reduced blood flow. Our first aim is to culture the equine EPCs within injectable, hydrogel microspheres and test the ability of the cells to form blood vessels from those spheres. We will also optimize our ability to detect the equine EPCs after they have been injected into the horse so that we can determine if they are contributing to new blood vessel formation. The second aim will evaluate healing in a distal limb wound model in the horse. Using 6 adult horses, 2 full thickness wounds (each the size of a quarter) will be created on the front and rear cannon bones (8 wounds per horse) and treated with EPCs alone, EPCs encapsulated in microspheres, microspheres alone, or sham injection. Healing and blood vessel formation will be analyzed and compared between treatments. We expect that the EPCs in microspheres will cause increased blood vessel formation and decreased healing time compared to all other treatments. This proposal is part of an ongoing collaborative effort between the College of Veterinary Medicine and the College of Engineering at Auburn University. Using our interdisciplinary expertise, our goal is to develop new regenerative therapies for horses that leverage the use of progenitor cells combined with engineered biomaterials as scaffolding.

Delayed healing and proud flesh formation involving wounds of the horse’s lower limbs is one of the most common problems facing veterinarians and horse owners, and new effective treatments are needed. Our research group is a combination of equine veterinary specialists and biomedical engineers that are working on developing new therapies for wounds or other diseases that are caused by or associated with poor blood flow. Endothelial progenitor cells (EPCs) are cells that form new blood vessels or repair the lining (endothelium) of existing blood vessels. Combining stem or progenitor cells with engineered biomaterials allows the veterinarian to deliver stem cells directly to the area of interest and keep them in that location for a longer period of time. The cells are more likely to have the ability to heal tissues and form blood vessels if they are not immediately carried away. In addition to a potential new treatment for distal limb wounds, the cell/biomaterial combination therapy could have implications for many different diseases and many different cell types.


PREVENTION OF SUPPORTING LIMB LAMINITIS
Andrew van Eps, University of Queensland –Second Year

One Sentence Summary: This study has potential to show the efficacy of a novel pneumatic boot to prevent lamellar changes leading to supporting limb laminitis (SLL) and may provide an effective clinical preventive for SLL.

Research in the past decade has more clearly defined that there are three different types of laminitis: sepsis–associated laminitis (e.g. laminitis in the horse with gastrointestinal disease, pneumonia, or retained placenta), endocrinopathic laminitis (e.g. laminitis in the overweight horse [also called metabolic syndrome] and in the older horse with Cushings disease) and supporting limb laminitis (SLL, the horse with a severe lameness [e.g. fracture or joint infection] on one limb which develops laminitis in the opposite/ supporting limb). Although established experimental models in the past decade have more clearly defined lamellar (synonymous with laminar) events in laminitis associated with sepsis and endocrinopathies, the lack of an appropriate model limited advancement of our understanding of SLL.

The investigators of this proposal have recently been funded by GJCRF for two SLL studies using very different models and experimental techniques; however, results from both studies indicate that lamellar hypoxia (meaning decreased tissue oxygen levels, most likely from decreased lamellar blood flow) is the driving force behind lamellar failure in SLL. Because the two laboratories use different approaches (AVE/DWR using a novel microdialysis catheter placed in the lamellar tissue to obtain lamellar interstitial fluid [fluid in the space between the cells which reflects the activities of the cells] for assessment of energy metabolites and blood flow[ by injecting urea into the interstitial space and measuring how long it takes to clear] and JKB using molecular and biochemistry/histologic imaging techniques in lamellar tissue) to detect lamellar cellular dysregulation/ hypoxia, we can now combine these approaches (and our recent results) to fully assess the efficacy of a therapeutic intervention as a prophylactic treatment for the horse at risk of SLL. The results of the van Eps/Richardson study strongly indicate that it is not the excessive weight bearing but is the lack of movement of the supporting limb which results in lamellar hypoxia; they also demonstrated that pharmacologic intervention most commonly used by veterinarians in attempt to increase lamellar blood flow (acepromazine) not only did not improve lamellar metabolism but actually worsened the parameters assessed. Thus, the culmination of these studies indicates that the only effective treatment may be a dynamic shoe which provides the same motion to the digit as walking while allowing the animal to maintain weight on the supporting limb.

Using a custom “V shoe” designed by the Belknap laboratory as a non–painful cause of decreased weight bearing on one limb (and excessive weight bearing on the opposite limb), we are now well situated to test a novel, practical pneumatic shoe device (which was produced by the SoftRide company at our request and to our specifications) on the supporting limb. In the proposed study, we will first perform short term animal protocols (( 6 hours) using a custom stocks present in the van Eps laboratory in order to: 1) determine the ability of different combinations/ cycles of heel and toe elevations to improve lamellar oxygenation/blood flow (assessing lamellar interstitial fluid obtained from the microdialysis catheters inserted in the lamellae in the front fee), and 2) determine the effect of these movements of the supporting limb digit by the pneumatic shoe on the weight bearing on the opposite limb (due to the fact that the opposite/injured limb may only be able to sustain a moderate degree of weight bearing in the clinical case). Once we have established the best combination and frequency of heel/toe elevations which do not cause a dramatic increase in weight bearing on the opposite limb, we will then perform a longer term study in which we will assess (using the microdialysis technique in the van Eps laboratory and biochemistry techniques on tissue samples in the Belknap lab) the efficacy of the pneumatic shoe using the chosen heel/toe elevation protocol (from the first experiment) in horses with the V shoe on the opposite limb for 96 hours.

We expect to determine the efficacy of the use of a pneumatic device to maintain digital movement of the supporting limb in ameliorating the lamellar cellular dysregulation/ hypoxia that occurs with excessive weight bearing on one limb; we feel it is likely that we will establish a protocol using the pneumatic shoe which will be effective in protecting the lamellae from decreased blood flow/hypoxia. In conclusion, the results from the proposed study will not only further our understanding of SLL, but have a great potential of resulting in the rapid establishment of a commercially viable prophylactic treatment for SLL.

Laminitis been voted the number one priority for equine research by the American Association of Equine Practitioners due to both the high incidence of the disease (annual incidence of 2–7% of horses in recent studies), the severe nature of the disease (high incidence of humane destruction or chronic lameness due to crippling nature) and the lack of effective therapies for treating the disease. In one of the largest studies of the incidence of lameness in the U.S. in recent history, a USDA National Animal Health Monitoring Study published in 2000 of approximately 3000 horse farms in 28 states stated that 13% of these farms reported a case of laminitis in a one year period. Supporting limb laminitis occurs in all breeds, and is particularly devastating due to the much higher mortality rate (50%) compared to the other types of laminitis (e.g. endocrinopathic laminitis). Supporting limb laminitis is perhaps the most familiar form of the disease to the racing industry and general public, being the condition that led to the demise of Kentucky Derby winner Barbaro, in 2007 and more recently of Kentucky Derby contender Intense Holiday in 2014. This highlights the fact that, despite great advances in the treatment of even the most catastrophic limb fractures and infections in adult horses, supporting limb laminitis remains the major cause of treatment failure and euthanasia for humane reasons in these cases. Because the horse at risk of supporting limb laminitis is easily detectable in most cases, an effective preventative treatment strategy would be a significant step forward for the welfare of horses and for the horse industry.


RESEARCH CAREER DEVELOPMENT AWARD

There are two Career Development Awards offered through the Foundation in 2016.

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:

Elaine Norton, University of Minnesota
Genetic Bases of Metabolic Syndrome and Laminitis Risk

Dr. Norton received her DVM from Colorado State and did a residency/masters program in large animal medicine at Auburn University. She is currently enrolled in the PhD program at the University of Minnesota, College of Veterinary Medicine. Her field of study is comparative and molecular biosciences. She is under the mentorship of Dr. Molly McCue and her research project for the period of the award is the identification of the underlying genetic risk factors in horses with Equine Metabolic Syndrome. She presented an excellent plan for her year of study and her support letters were filled with accolades for her abilities and dedication to equine research.


The Elaine 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 renowned horsewoman, Elaine Klein. The grant is funded by $15,000 donations by the Klein Family Foundation.
The 2016 award winner is:

Amanda L. Ziegler, North Carolina State University
Flunixin and Firocoxib in Post-Surgical Colic Patients

Dr. Zeigler is a graduate student at NCSU in the college of Veterinary Medicine’s Comparative Biomedical Sciences program. Her year of study was well defined and is being spent on a project funded by GJCRF in 2015 working under Dr. Anthony Blikslager. The project deals with improving drug selection for postoperative colic pain. She received excellent letters of support from faculty, mentors and the Dean of the college, Dr. Paul Lunn. This is a multi-institutional effort combining cases from N.C. State, Michigan State and New Bolton Center. It will help tremendously to know this information to improve the survival rate of surgical colic.