The board of directors of Grayson-Jockey Club Research Foundation announced that it has authorized expenditure of $1,856,507 to fund 14 new projects and nine continuing projects at 13 universities as well as two career development awards. The 2023 slate of research brings Grayson’s totals since 1940 has provided more than $40 million to underwrite more than 426 projects at 45 universities.
Osteoarthritis is one of the most common disorders treated in horses, and is estimated to affect up to 80% of horses over the age of 15 years. Despite this high prevalence, treatment options remain limited and understanding of the mechanisms behind osteoarthritis progression are not fully understood. Cells of the innate immune system are increasingly thought to play a role in early stages of degenerative joint disease. The purpose of this study is to determine how the genetic code of cells, particularly cells of the innate immune system, changes during early osteoarthritis progression and how those values correlate to levels of inflammatory biomarkers, called cytokines, in joint fluid. This work will integrate new technologies including single cell RNA sequencing and multiplex biomarker analysis towards this goal. This study will highlight the role of immune infiltration contributing to disease biology in OA progression towards the overall objective of developing treatments tailored to disease stage.
Importance to the Equine Industry: This project will help clinicians to better understand how osteoarthritis changes the immune system and the genetic code of the cells in the joint when osteoarthritis is induced traumatically and then progresses. This in turn will help researchers develop appropriate targeted therapies depending on the stage of osteoarthritis (early, mid stage, late), ultimately helping clinicians to better treat horses with joint disease. This will impact the equine industry by improving understanding of changes occurring over time in osteoarthritis progression which will allow for development of targeted therapies tailored to disease stage for one of the most common disorders treated in horses.
Osteoarthritis (OA) results in chronic, progressive lameness and is a significant cause of morbidity and retirement in horses (Bertuglia 2016; McIlwraith 2012). Joint disease can occur spontaneously or secondary to joint trauma, and even with appropriate care, OA will develop in up to 50% of fractures involving the joint in human patients and following similar fractures in horses (Dirschl 2004; McIlwraith 1988; Niebauer 1988). OA involves several tissues within the joint, including articular cartilage, synovial membrane, and surrounding joint capsule and ligament tissues. Cartilage degeneration are the most recognizable lesion which occurs in OA, and these become irreversible rapidly during OA development.
Steroids and HA supplementation have been the mainstays of joint therapy in horses for more than two decades, in addition to oral or intravenous anti-inflammatories such as Bute, Banamine and Equioxx. Orthobiologics have become more available for the treatment of OA but require processing of minutes to weeks depending on the product, and the final product and clinical effects are inconsistent. HA formulations available to the veterinary market can improve synovial fluid consistency and lubrication, but these formulations only result in detectable HA in the joint for ~24 hours (Jackson 2006; Palmieri 2017). Lubricin is a highly sugarcoated molecule, and these sugars are responsible for its ability to provide effective cartilage lubrication (Jay 2001). Lubricin has the potential to treat both biological and physical aspects of joint disease, showing promise in rodent and mini-pig models of joint trauma (Jay 2012; Teeple 2011; Waller 2017). In addition, because lubricin can physically bind to the surface of articular cartilage, its sustained residence time of 28-42 days following a single intra-articular injection suggest that it may have a sustained effect (see Preliminary Studies). However, progress with lubricin therapy has been hampered by significant challenges with the largescale production and purification of the molecule. The field has been slow to translate lubricin therapy to large animals or veterinary clinical trials due to technical challenges associated with producing sufficient quantities of recombinant lubricin for a large animal trial and also due to competing demands for testing recombinant lubricin in human clinical trials. Notably, we have recently developed a novel strategy for production and purification of full-length, highly sugar-coated recombinant lubricin in sufficient quantities required for equine studies.
The long-term objective of this proposal is to translate lubricin therapy to equine clinical patients. We hypothesize that equine specific recombinant lubricin (rEqLub) can improve joint inflammation and arthritis by means of its anti-inflammatory and lubricating properties. In Aim 1, the efficacy of equine-specific synthetic lubricin to ameliorate the development of osteoarthritis in a surgical disease model. In Aim 2, we will study the signaling pathways that are affected by rEqLub in healthy equine fetlock joints, which will help identify mechanisms by which lubricin can protect joint health. We expect these studies to provide a baseline for the establishment of efficacy of lubricin joint therapy in the horse, which will springboard evaluation of lubricin therapy to future clinical trials in equine patients.
Importance to the Equine Industry: Lameness is the leading cause of early retirement across all horse breeds and disciplines. OA is responsible for more than 60% of all equine lameness (Caron 2003), and it is the second leading cause of premature racehorse retirement (Welsh 2013). Most currently available joint medications temporarily relieve symptoms but do not prevent progression of arthritis. Corticosteroids, with or without the addition of hyaluronic acid, remains the most common intra-articular therapy in both horses and humans (Fusco 2021; Velloso Alvarez 2020). With newer regulations restricting medication and corticosteroid use in racehorses and sport horses, equine therapeutics are limited, and an ideal intra-articular medication does not exist. Evidence in other species suggests that lubricin has immunomodulatory, chondroprotective, and lubrication effects, making lubricin a promising future therapeutic for OA (Alquraini 2015; Iqbal 2016; Waller 2013).
Production of sufficient quantities of lubricin for clinical studies in large animals, such as the horse, has not been feasible in the past due to challenges associated with making this complex, highly sugar-coated protein. However, we have developed a novel strategy for large-scale production of lubricin in amounts sufficient for joint injections in horses. This overcomes a significant hurdle and enables us to address a critical gap in understanding the biological functions of lubricin. In the proposed studies, we will assess equine-specific lubricin’s (rEqLub) ability to mitigate OA severity in an equine surgical disease model and identify cellular pathways affected by rEqLub to identify additional functions of lubricin in a healthy joint. We expect these studies to provide data about the ability of lubricin to treat joint disease in the horse. This study provides a path toward a potential real, clinical treatment that does more than mask the problem.
Stifle injuries are common career ending injuries for equine athletes. In particular, injuries to the meniscus result in a relatively poor prognosis for return to prior level of performance. A single, small, retrospective study concluded that compared to historical controls, mesenchymal stem cells (MSCs) resulted in an improvement in the ability to return to work compared to surgery alone. Further, a recent survey of large animal surgeons and sports medicine specialists, indicated MSCs were the most commonly used biologic therapy following surgery for meniscal injury. However, recent reports are controversial, as a new retrospective study of meniscal injuries showed no effect of biologic therapies, including MSCs, on outcome. There is a clear gap in knowledge as to whether intra-articular stem cell administration improves outcomes for meniscal injury. Part of the challenge of answering this question has been the lack of an experimental model of meniscal disease in the horse. Therefore, we will conduct a randomized, controlled, blinded prospective study evaluating the use of intra-articular mesenchymal stem cells for treatment of meniscal injury using a novel, experimental model of meniscal disease. This study will achieve two important objectives. Firstly, it will establish this experimental model of meniscal disease which can be used now and in the future for evaluating the effectiveness of intraarticular treatments for meniscal disease. Secondly, the study will evaluate the effectiveness of intra-articular MSCs for meniscal disease using multiple outcome measures including lameness, joint fluid characteristics, diagnostic imaging outcomes, joint evaluation following euthanasia and cellular tissue (histologic) outcomes. The study will have an immediate impact on the treatment of meniscal disease in the horse by determining the efficacy of intra-articular MSC use, and a longer-lasting impact by providing an experimental model for future investigation of therapeutic interventions.
Importance to the Equine Industry: Meniscal injury remains difficult to treat in the horse, despite surgical intervention, with little known about the efficacy of intraarticular treatments following surgery. With only approximately 40% of horses returning to their previous level of performance following meniscal injury, the industry is in dire need of effective therapies. Retrospective studies on intra-articular mesenchymal stem cells for the treatment of meniscal disease have been conflicting; some studies claim a significant improvement and other studies find no benefit to biologic therapies. The question of the efficacy of mesenchymal stem cells needs to be answered with a randomized, blinded, controlled, experimental study of meniscal injury. To answer this important treatment question, this study will utilize a novel equine model of meniscal injury and evaluate the effectiveness of intra-articular mesenchymal stem cells for equine meniscal injury. Importantly, the study employs a plethora of outcome parameters including lameness, diagnostic imaging outcomes, joint fluid characteristics and cellular tissue (histologic) characteristics. The combination of these outcome parameters will provide a comprehensive look at the effect of mesenchymal stem cells in meniscal disease. The study will have an immediate effect on how equine veterinarians treat meniscal disease, improving the use of owner resources and establishing a treatment protocol backed by scientific evidence to enhance outcomes in our equine athletes.
Tendon and ligament injuries are a leading cause of lost training days and early retirement among equine athletes. In adults, local cells rarely go to the injury to assist with healing. This explains, in part, the slow and incomplete healing that frequently occurs. Recent research shows that implanted stem cells direct tendon tissue stem cells to go to the injury and produce healthy tissue instead of scar tissue. Currently, there are limited ways to apply stem cells to tendon and ligament injuries or to keep them at the injury site during the healing process. We propose to overcome some of the hurdles to cell therapies for tendon injuries by turning stem cells into tendon cells that produce normal tendon tissue around them. Specifically, we developed a unique system that stimulates stem cells to become tendon cells and produce tissue after 21 days in highly customized fluid flowing through chambers that contain the cells on collagen templates. The proposed studies will confirm generation of tendon cells in new tissue that can be stored and shipped frozen so that it can be used promptly whenever and wherever necessary. The ability of the tissue to promote tendon healing will also be confirmed in equine tendon injuries. The ultimate goal of this work is to create a reproducible mechanism to generate tendon tissue implants that can be clinically applied to facilitate healing and restore normal limb function in equine athletes and companions.
Importance to the Equine Industry: The results of this proposal will establish a way to create healthy tendon cells and tissue from stem cells and prove that the cells remain in a tendon injury to accelerate healing. Tendon and ligament injuries account for a large majority of musculoskeletal injuries that are responsible for up to 72% of lost training days and 14% of retirements by equine athletes. Among injuries, superficial digital flexor tendon and suspensory ligament injuries are the most common, comprising up to 46% of all limb injuries. Tendons and ligaments have poor healing capacity, and poor or abnormal tissue repair contributes to a reinjury rate as high as 67% within 2 years. Direct injection of stem cells into tendon injuries to augment natural healing has mixed results, and the cells may not always stay at the site of tissue damage. We propose to overcome the current limitations of stem cell tendon therapies in adult horses with tendon tissue produced by adipose tissue stem cells grown in conditions designed to create tendon cells. Confirmation that the implanted cells remain at the treatment site and do not change may help overcome some of the current regulatory restrictions on stem cell use. Additionally, confirmation that the new tissue is not damaged by freezing will make it possible for the treatment to be available to all horses. Overall, the work in this proposal will significantly advance regenerative therapies for tendon injury in a host of equine companions and athletes plagued by an all-too-common musculoskeletal problem.
An alternative approach to the use of intra-uterine antibiotics is the use of gallium as a disruptor of bacterial iron-dependent processes. Iron is essential for almost all pathogens and is required for enzymatic processes, such as DNA synthesis, electron transport and oxidative stress defense. Gallium has a nearly identical ionic radius as iron, and some bacterial uptake systems are unable to distinguish between the two elements. Gallium disrupts iron-dependent processes since it cannot be reduced under physiological conditions. Thus, gallium incorporates into iron-containing proteins disrupting their function resulting in bacterial death by essentially starving bacteria. Previous studies have found gallium compounds exhibit antibacterial activity against several pathogens, including Pseudomonas aeruginosa, Francisella tulerensis, Acinetobacter baumannii, several mycobacterial species and Klebsiella pneumoniae.
With increased public awareness of antimicrobial resistance there is a need to develop new, safe, and efficacious antimicrobial strategies. We propose to investigate the use of gallium as an intrauterine therapy in a three-part progressive process.
• The initial phase of this project is to confirm the effectiveness of gallium as an antimicrobial agent against Escherichia coli, Streptococcus equi subspecies zooepidemicus, Pseudomonas aeruginosa and Klebsiella pneumoniea, respectively.
• A second study to determine serum and endometrial gallium concentrations after various intra-uterine infusion doses and estimate the amount of active gallium to treat infections
• Then a safety study using the dose determined to be effective in the second experiment to determine if there is a negative impact of gallium on uterine health.
Importance to the Equine Industry: Growing public awareness of antimicrobial resistances underscores the need to develop alternative therapies for the treatment of uterine infections. Although antimicrobial resistance is often considered to be concerning in equine reproduction, there are few studies evaluating changes in antibiotic resistance in bacteria isolates from mares’ uteri. One research study evaluated uterine bacterial isolates in 8,296 cases between the years of 2003 and 2008 and found a significant change in resistance to chosen antibiotics over those years. This highlights the importance not only of sensitivity profiles for isolated bacterial pathogens prior to treatment, but also the distinct need to develop alternative antimicrobial treatments to preserve the effectiveness of current antibiotics.
Pneumonia caused by the bacterium Rhodococcus equi (R. equi) remains a major problem for the equine industry. Great need exists to develop an effective vaccine against R. equi because of the importance of R. equi pneumonia to the equine industry (please see Importance to the Equine Industry below). Our long-term goal is to develop a vaccine using messenger RNA (mRNA) that can be administered intramuscularly (IM) neonatal foals to protect them against pneumonia caused by R. equi. The vaccine will target a protein that is necessary for R. equi to cause disease in foals known as the virulence-associated protein A (VapA). The rationale for targeting VapA is that levels of antibodies against this protein in foals are correlated with protection against R. equi pneumonia. This project will address 2 essential steps to achieve this goal. First, we will demonstrate that the VapA mRNA we have designed can elicit immune responses when administered intramuscularly (IM) to foals at ages 1 and 21 days.
Specifically, we will compare anti-VapA antibody activity levels in serum and bronchoalveolar lavage fluid (i.e., lung washings) by an enzyme linked immunosorbent assay (ELISA) and cell-mediated immune responses specific for VapA of immune cells circulating in blood by a method known as ELISpot in immunized (n=12) and control (n=12) foals. Second, we will examine the efficacy of the vaccine to protect these same 12 vaccinated and 12 unvaccinated foals against experimental infection. At age 31 days, foals all foals will be infected intrabronchially with virulent R. equi and then monitored through age 9 weeks for signs of pneumonia. All foals that develop pneumonia will be treated with antimicrobials and any other supportive care needed. Results of this study will provide evidence of the safety and efficacy of immunizing foals with mRNA to protect them against rhodococcal pneumonia.
Results also will have important implications for immunizing foals against other infectious disease.
Importance to the Equine Industry: The horse industry is an agriculturally and economically important venture in the U.S., generating over $215 billion in total production in 2016. Producing healthy foals is necessary to maintain and improve the nation’s horse population, and the health of foals is important to the horse industry from a welfare perspective. Infectious diseases are leading causes of disease and death in foals, and pneumonia is a principal infectious disease of foals. Respiratory disease was the most common cause of disease and death in foals in Texas, and ranked 3rd as a cause of morbidity and 2nd as a cause of mortality in U.S. foals 1 to 6 months of age. R. equi is considered the most common cause of severe pneumonia. This disease is important to the equine industry for the following reasons. The disease is endemic at many horse-breeding farms with cumulative incidence often around 20% to 40% of the foal population. At endemic farms, the costs resulting from veterinary care, long-term therapy, and mortality of some foals are very high. In addition to significant immediate costs, R. equi pneumonia can have a long-term detrimental effect on the equine industry because it has been reported that North American foals that recover from the disease are less likely to race as adults and that performance was diminished by this disease for some Australian Thoroughbreds.
Despite the revolutionary technological advancements leading to unparalleled progress in the genomics of equine traits, diseases and disorders in the past decade, the genomics of equine reproduction has been given relatively less attention. Currently, only a few stallion reproductive disorders have been associated with candidate genes or candidate genomic regions, although without actual knowledge of the underlying molecular mechanisms. Among these, an autosomal gene FKBP6 in horse chromosome 13 (ECA13) is of particular interest because a certain genotype of this gene is significantly and invariably associated with Thoroughbred stallion subfertility. This association was initially discovered in 2012 by studying seven Thoroughbred stallions with a confirmed reproductive disorder - impaired acrosome reaction (IAR). Acrosome is a single secretory granule present in the head of mammalian sperm and secretion of acrosome, known as acrosome reaction (AR), is necessary for natural fertilization. Impaired acrosome reaction results in subfertility or infertility. More recently, a large-scale follow-up study compared breeding records of 150 Thoroughbred stallions with their FKBP6 genotype and confirmed the association of FKBP6 with stallion subfertility, regardless whether data for acrosome reaction were available or not. It was shown that the frequency of this ‘stallion subfertility’ genotype in global horse populations and Thoroughbreds separately is 4%.
The study also reported about the development of a reliable and easy-to-use genetic test to identify stallions with the ‘subfertility’ genotype. The main limitation of this and the earlier study is that despite of significant and consistent association between FKBP6 and stallion subfertility, there is still no knowledge WHY? Firstly, because the associated genotype does not affect the structure or function of the FKBP6 gene and secondly, there is no evidence that FKBP6 has functions in acrosome reaction. Furthermore, the FKBP6 ‘subfertility’ genotype has also been found in Hanoverian and Friesian stallions, but the genotype is associated with subfertility only in Thoroughbred stallions and not in other breeds. This suggests that the FKBP6 ‘subfertility’ genotype may not be the actual cause but is rather tagging in the nearby genomic regions a gene or genes with structural or functional genetic variants unique to Thoroughbreds. In addition, identification of the first genetic marker for IAR and stallion subfertility is of outstanding scientiﬁc interest because the Thoroughbred is so far the only horse breed where IAR has been conﬁrmed as a cause of stallion subfertility and because molecular causes for IAR have not been disclosed in any species, including humans.
The goal of the proposed research is to explore the genomic region around the FKBP6 gene in a cohort of 21 subfertile Thoroughbreds, including those with confirmed IAR, and compare it with the sequence landscape of this region in over 700 horses representing general Thoroughbred population and many other breeds. The aim is to pinpoint unique or rare sequence variants in the subfertile Thoroughbred cohort that could be likely candidates for IAR and stallion subfertility. This work will be done using multi-platform whole genome and testis transcriptome analysis. We anticipate that the findings will improve our understanding about the genetic regulation of acrosome functions in the stallion and mammals in general.
Importance to the Equine Industry: Fertility and reproductive health of Thoroughbred breeding stallions, particularly the most popular ones from elite high-performance pedigrees, are important cornerstones of the Thoroughbred industry. It is therefore important to improve and expand the methods for breeding soundness evaluation, particularly for cases of unexplained subfertility where a stallion has poor breeding records (low pregnancy rates) despite normal physical characteristics and good semen quality. The proposed study aims to dissect the underlying genomics of one such conditions – subfertility due to impaired acrosome reaction which typically manifests as unexplained subfertility because acrosome reaction test is not part of routine breeding evaluation. The IAR condition has been found only in Thoroughbred stallions and is significantly associated with a certain genotype in the FKBP6 gene. Carriers of this genotype are invariably subfertile. However, we do not know why? The findings of the proposed study are expected to throw light on that so that the use of the FKBP6 genetic test for stallion evaluation will be supported by a molecular explanation. These types of diagnostic measures are of particular importance for breeds such as the Thoroughbred, where artiﬁcial insemination or any other forms of assisted reproduction are not allowed. In the long run, genetic monitoring of breeding stallions for IAR may help to eradicate the condition from the population.
Fetlock injuries are a common cause of lameness and poor performance in sport horses, including jumper, eventer and dressage horses. These injuries can involve ligaments and bones. X-rays and ultrasound remain the most commonly used tools for assessment of the fetlock; however these modalities may not recognize early bone lesions. Magnetic Resonance Imaging (MRI) has become the reference technique for joint assessment and several studies have demonstrated the value of MRI in the sport horse fetlock, especially to identify injuries to the articular bones. The importance of articular bone injuries in early development of osteoarthritis is increasingly recognized. Early identification of these lesions is key to prevention of progression of joint disease. Positron Emission Tomography (PET) is an imaging technique, involving the injection of a small amount of a radioactive dye that has become increasingly available for imaging the equine limb. Using the bone specific dye 18F-Sodium Fluoride (18F-NaF), PET provides 3-dimensional assessment of bones, detecting changes occurring at the molecular level, prior to the development of structural defects. PET has recently been shown highly helpful in the racehorse fetlock, not only for assessment of lameness, but also for monitoring of rehabilitation and screening of the general population. There is currently limited data available regarding PET imaging of the sport horse fetlock.
This proposal suggests performing both 18F-NaF PET and MRI of both fetlocks in a sport horse population with lameness localized to the fetlock. This would be a collaborative multicenter study including four sites: the University of California Davis Veterinary Medical Center (UCD VMC), Rood and Riddle Equine Hospital (RREH), the University of Florida equine hospital at the World Equestrian Center (UFL WEC) and Ocala Equine Hospital (OEH). Each of these centers is equipped with a PET scanner (MILEPET, LONGMILE Veterinary Imaging) designed to image the horse limb using standing sedation. Scanning two fetlocks in a standing horse is typically achieved within 10 to 15 minutes. Two of the centers (UCD VMC and UFL WEC) are equipped with low-field MRI scanner designed to scan standing horses (Hallmarq Veterinary Imaging) and the other two (RREH and OEH) use high-field MRI on anesthetized horses. MRI acquisition is typically 30 to 45 minutes per joint. Sixteen horses would be recruited at each site, for a total of 64 horses in the study. The aims of the study would be to describe the 18F-NaF PET findings in a sport horse population and compare with MRI. A total of 35 regions of interest including specific articular bone sites and areas of attachment of ligaments will be assessed independently both on PET and MRI by five independent experienced observers. Correlation between PET findings and both low-field and high-field MRI will be assessed. An additional aim of the study would be to assess an Artificial Intelligence (AI) software specifically designed to assess equine fetlock PET. This program will provide automated measurements of bone turn-over. These measurements will be correlated with the subjective grades from the observers. Based on the knowledge from racehorse studies and early clinical use of 18F-NaF PET in sport horses, we expect to demonstrate a good agreement between 18F-NaF PET findings and MRI abnormalities. It is also likely that 18F-NaF PET would identify early changes in articular bone and at the attachment of ligaments prior to the identification of MRI changes. The AI data would likely confirm and refine the assessment from the expert observers and ultimately contribute to simplify and speed up the assessment of PET scans.
Importance to the Equine Industry: Fetlock injuries are responsible for poor performance and early retirement in sport horses. 18F-NaF PET is a diagnostic technique that was proven successful in fetlock injury diagnosis, rehabilitation and prevention in racehorses. It is very likely that the sport horses would also benefit from this technique. Current knowledge suggests than 18F-NaF PET will compare favorably with MRI for articular bone lesion diagnosis. 18F-NaF PET has the potential to perform better than MRI for early identification of bone changes, both in joints and at attachment of ligaments. The ability of PET to perform measurements, potentially optimized by the development and validation of Artificial Intelligence programs, could offer a very helpful tool for monitoring of healing and rehabilitation of clinical cases and assessment of different treatment options in research studies. 18F-NaF imaging of fetlocks is faster and more economical than fetlock MRI imaging. For these reasons, 18F-NaF PET has a lot of potentials to become a broadly used diagnostic, monitoring and screening tool for assessment of fetlock injuries in sport horses. 18F-NaF PET is likely to improve the outcome for horses with fetlock injuries at a lower cost than current techniques.
In horses, neurological disease often results in a career-ending diagnosis. The major causes of spinal cord disease in the horse include cervical vertebral compressive myelopathy (CVCM; Wobbler syndrome) and equine neuroaxonal dystrophy / equine degenerative myeloencephalopathy (eNAD/EDM), The clinical signs observed with CVCM and eNAD/EDM are often highly similar, and it is challenging to arrive at a definitive diagnosis while the horse is alive. Unfortunately, these diagnoses are often made after the horse has been euthanized. Currently, there are not magnetic resonance imaging (MRI) machines large enough to accommodate the size of a horse’s neck. Therefore, the current way to diagnosis CVCM is with x-rays and computed tomography (CT), with or without the use of contrast (i.e. myelogram). With eNAD/EDM, there is no definitive diagnostic test yet available. Additionally, in Quarter horses (QH) and in Warmbloods (WB), the clinical presentation of eNAD/EDM differs, with QHs displaying an abnormally quiet mentation and incoordination at a young age, while WB often present between 5-15 years of age with a sudden onset of behavioral changes and incoordination. A vitamin E deficiency has been associated with eNAD/EDM in the QHs but not yet in the WB. Thus, there may be subcategories of eNAD/EDM. Since the definitive diagnosis in many cases of spinal cord disease in the horse can only be achieved after the horse has been euthanized, these career or life-ending decisions without a definitive diagnosis creates a stressful and emotional burden for owners, trainers, and veterinarians involved with affected horses.
Through a novel approach, where we were able to screen 367 proteins involved in neurologic disease in humans, we have defined specific proteins in the blood (serum) and cerebrospinal fluid (CSF) from confirmed cases of CVCM and eNAD/EDM in QHs and WBs. Based on these results, we now propose to validate these findings, using bench-side antibody tests, in both our initial set of samples and an additional validation group of confirmed cases of equine spinal cord disease. We hypothesize that concentrations of five specific proteins; R spondin-1 (RSPO1), neurofilament light chain (NEFL), dickkopf WNT signaling inhibitor 1 (DKK1), WAP, Follistatin/Kazal, Immunoglobulin, Kunitz And Netrin Domain Containing 1 (WFIKKN1) and calretinin (CALB2), will define the serum and CSF proteome from horses with CVCM and eNAD/EDM, thereby distinguishing between diseases. Specifically, we expect concentrations of CSF NEFL, CSF DKK1, CSF CALB2 and serum WFIKKN1 to be higher in CVCM versus eNAD/EDM versus healthy horses, while CSF RSPO1 will be highest in healthy horses and lowest in CVCM horses. Additionally, we hypothesize that in the spinal fluid, RSPO1 concentrations will be higher in eNAD/EDM QHs versus WBs.
Importance to the Equine Industry: The inability to sensitively and specifically diagnose equine spinal cord disease is a significant financial burden for horse owners, trainers and insurance underwriters. We anticipate that this study will provide highly specific and sensitive tests in blood and spinal fluid that, when combined with the results of standard diagnostic testing, can be used to diagnose horses with spinal cord disease due to CVCM or eNAD/EDM.
The healthy uterus was previously considered a sterile environment. Now, it is recognized that the uterus is colonize by a high number of bacteria of different species (i.e., uterine microbiome) that is necessary to prevent infection, modulate inflammation, and establish pregnancy. Endometritis is an inflammation of the lining of the uterus that occurs with the contact of bacteria and sperm present in the semen. Persistent breeding-induced endometritis (PBIE) is the most common reproductive problem encountered in the equine breeding industry and the third most frequent clinical problem affecting horses. Mares are classified as resistant and susceptible to PBIE based on their ability to clear the post-breeding endometritis. Our preliminary assessment indicates that mares susceptible to PBIE have a lower number of bacteria and species of bacteria than resistant mares. Therefore, the uterine microbiome of mares that are susceptible to PBIE seems to be distinct enough that it may be helpful to estimate susceptibility to PBIE, which would allow for the application of on-target therapy. Antibiotics are essential in veterinary and human medicine for the treatment of bacterial infections. Resistance has been demonstrated in all classes of antibiotics. The antibiotic resistome is the collection of genes contributing directly and indirectly to resistance. Post-mating uterine infusion with antibiotics is commonly administered to prevent endometritis. A recent study reported that 50% of Thoroughbred mares received post-mating antibiotics and up to 100% of mares in some farms. The extensive use of post-mating antibiotics likely contributes to the emergence of resistance. Therefore, there is a critical need to better understand the effects of antibiotics on the resistome and develop advanced diagnostic techniques for the detection of antibiotic resistance in clinical practice. The current method for testing antibiotic susceptibility is the disc diffusion test, and the turnaround time for results is up to 5 days. The use of molecular diagnostic techniques such as PCR could significantly reduce the time required to determine antibiotic susceptibility and the onset of treatment.
Our long-term goal is to understand the role of the uterine microbiome and resistome in uterine health to enhance diagnostic techniques. Studying the effects of post-mating antibiotics under controlled conditions will allow us to develop evidence-based guidelines regarding this procedure in mares. Our central hypothesis is that post-mating antibiotics affect uterine microbiome composition and resistome and that PCR can be used as a rapid and accurate test to determine antibiotic susceptibility.
Sixteen mares previously classified as susceptible (n=8) or resistant (n=8) to PBIE in the preliminary studies will be subjected to a five-day course of antibiotics starting 6 hours post-mating. A fertile stallion will be used to live cover all mares. The estrous cycle 1 of each mare will start as the control with no antibiotic infusion. Then treatment cycles 2 to 5 of each mare will be randomized in a crossover design to receive uterine infusions of gentamicin, ceftiofur sodium, procaine G penicillin, and ticarcillin-clavulanate. These drugs were chosen as they are the most frequently administered post-mating antibiotics. A washout cycle will be carried out after each treatment cycle and before the next. Uterine samples will be collected the day before, three days, and five days after mating for microbiome and resistome analyses, endometrial culture, and cytology. This sampling will emulate a 3- and 5-daycourse of antibiotics. The microbiome composition will be evaluated with advanced molecular techniques based on the sequencing of particular regions (16S) of the bacterial genome. The number of bacteria and species will be evaluated before and after antibiotic treatment, between groups (resistant vs. susceptible to PBIE), and between control and antibiotic treatment.
Positive bacterial cultures will be identified by their morphology and microbiological techniques. Bacterial isolates will be assessed for antibiotic susceptibility testing with the disc diffusion method and PCR for the four antibiotics used in the study. The antibiotic resistance genes expression levels will be investigated with PCR (3 to 4 genes per antibiotic) and compared with disc diffusion’s results.
The team of multi-disciplinary investigators is uniquely positioned to complete the project, as it comprises experts in uterine health, microbiome, bioinformatics, and molecular techniques applied to antibiotic resistance. We foresee that the results of this study will have an immediate positive impact on the horse industry and serve as the foundation for practical assessment of the microbiome composition in commercial Thoroughbred broodmares and diagnostic of antibiotic resistance in horses.
Importance to the Equine Industry: Endometritis is an inflammation of the lining of the uterus. It is the most common cause of subfertility in the horse. It is associated with a reduction in fertility, pregnancy loss, increased costs, and risks of bacterial infection. Post-mating antibiotics are commonly administered in horse breeds like the Thoroughbred to prevent endometritis, but its impact is unknown. It is recognized that a “core” uterine microbiome (i.e., bacteria that reside in the healthy uterus) is necessary to prevent infection, modulate inflammation, and establish pregnancy. The proposed research project focuses on understanding the impact of post-mating antibiotics in the mare uterus to enhance our knowledge of this disease and develop fast and accurate diagnostic techniques. Our central hypothesis is that post-mating antibiotics affect the bacteria that populate the uterus and their antibiotic resistance and that PCR, a widely used molecular test, can be used as a rapid and accurate test to determine antibiotic susceptibility. Our experimental design will closely simulate the Thoroughbred industry while evaluating the presence of antibiotic resistance. Antibiotic susceptibility testing is the standard method, and the turnaround time for results is 3-5 days. Treatment of bacterial diseases is often initiated and sometimes treated completed empirically due to the long turnaround time. Thus, it might be possible that screening antibiotic resistance with PCR in an isolate will speed up the choice of antibiotic, and the standard method could confirm the early rapid molecular diagnosis. In humans, this approach has proven to have high sensitivity and specificity. The methods proposed here can be extrapolated into other areas of equine practice and validated in commercial diagnostic laboratories, improve precision, and reduce the time from susceptibility testing to antibiotic treatment in different bacterial diseases of horses, which could be lifesaving in a clinically ill patient.
Emergence of novel equine rotavirus B (ERVB) that caused extensive foal diarrhea outbreaks during the 2021 and 2022 foaling seasons has been viewed as a significant concern to the equine industry due to its effects on foal health and subsequent economic impact. Currently, there are no effective vaccines for control of this highly contagious disease. This proposal will support our efforts to develop a ERVB nanoparticle vaccine candidate that can elicit durable and high titer neutralizing antibodies in mares, and as a result, foals born to vaccinated mares will be protected against ERVB infection through the maternal colostral antibodies. Once an effective and safe vaccine has been identified from this proposal, we will actively seek a collaboration with a biotech company to produce the vaccine for commercial purchase and distribution, which will likely be through other funds. The Gluck Center is currently exploring relationships that could be helpful in developing and brining a commercial ERVB vaccine to market.
Importance to the Equine Industry: A short-term deliverable to the equine industry is a vaccine candidate that can be used to protect foals against ERVB infection in future foaling seasons. Our long-term goal is to provide equine industry a trivalent rotavirus vaccine containing equine rotavirus A (G3 and G14 strains) as well as ERVB, that can protect foals against infections of both equine rotavirus A and B.
An effective EPM vaccine is desperately needed to prevent both the immediate and long-term effects of this debilitating disease on affected horses. Therefore, our long-term goal is to generate an effective vaccine against Sarcocystis neurona, the most common cause of EPM. Our theory is that we can use a software program called immune epitope database (IEDB) to screen the known S. neurona genetic sequences for potential protective peptides/epitopes. These peptides are small segments of protein that have the potential to stimulate a protective immune response against S. neurona, which is what we need with an efficacious vaccine. We can use this IEDB software program to generate a list of these proteins/peptides from the S. neurona sequence/genome, but then we have to test the peptides for their ability to provide protection. Before we can see if the peptides can actually protect a horse from EPM, we have to “test” the peptides in a much less expensive cell-based assay to see if they stimulate some of the same molecules/cytokines, such as IFN-γ, necessary for protection. We know that for protection against S. neurona, we need a certain type of immune response, a cell mediated immune response. Based on other studies, we know this consists of CD4 and CD8 cells producing IFN-γ. Therefore, we will use neurologically normal horses that have been exposed to S. neurona, but have not developed disease, as the horses that have made a protective immune response. We will then test all the different peptides for their ability to stimulate this in vitro protective CD4 and CD8 IFN-γ response. The peptides which stimulate the best CD4 and CD8 IFN-γ responses are the most likely ones to provide protection. Screening and ranking these peptides based on their ability to stimulate IFN-γ is the goal of the study. Those peptides which stimulate in vitro and in vivo protection are called protective epitopes. With this knowledge, the goals of this study will be complete. The next step would be to test these peptides/potential protective epitopes in a mouse model to see if they protect mice from developing EPM. Those protective epitopes would then be screened in horses for their ability to protect against EPM.
Importance to the Equine Industry: Equine protozoal myeloencephalitis (EPM) is a devastating disease caused by S. neurona, and for which there are no commercially efficacious vaccines available. EPM is one of the most significant and devastating neurologic diseases in the United States, with an increasing recognition of its presence and severity in South America as well. In 2001 the National Animal Health Monitoring System (NAHMS) reported annual costs of $55-110 million annually to the industry. No updated costs are available; however, costs continue to skyrocket. Owners suffer a financial and emotional burden due to time lost from performance, incomplete recovery and relapse of disease. Additionally, diagnostics, treatment and rehabilitation often total many thousands of dollars. Furthermore, with the current state of diagnosis and treatments, many horses do not recover completely and must be rehomed for another career. Moreover, at least 10% of these horses develop recurrent signs and must be retired at best, and sometimes euthanized due to their poor prognosis. Our theory is that we can use a software program called immune epitope database (IEDB) to screen the known S. neurona genetic sequences for potential protective peptides/epitopes. These peptides are small segments of protein that have the potential to stimulate a protective immune response against S. neurona, which is what we need with an efficacious vaccine. We can use this IEDB software program to generate a list of these proteins/peptides from the S. neurona sequence/ genome, but then we have to test the peptides for their ability to provide protection. The peptides which stimulate the best CD4 and CD8 IFN-γ responses are the most likely ones to provide protection. Those peptides which stimulate in vitro and in vivo protection are called protective epitopes. With this knowledge, the goals of this study will be complete. We would then test the peptides in a mouse model to see which ones provide protection.
Equine herpesvirus-1 (EHV-1) infection results in sporadic but devastating outbreaks of neurological disease in the equine population caused by a myeloencephalopathy with a poorly understood pathogenesis. The impact of EHV-1 myeloencephalopathy (EHM) on equine health and the industry is highlighted by a series of major outbreaks in North America and Europe over the past decade, including the two largest outbreaks in 2011 and 2021 in North America and Valencia, Spain. Despite the importance of EHV-1 in horses, effective prevention remains elusive and there is currently no vaccine available to prevent EHM. This is partially a consequence of the absence of a reliable experimental equine model of EHM and partially due to a lack of innovation in vaccine technology in the equine vaccine market in the past 40 years. In this proposal, five investigators with complementary expertise in equine herpesvirus virology, vaccinology and pathogenesis/animal models have come together to close a major gap in vaccine development against EHV infection. The goal of this application is to exploit innovations in RNA vaccine technology that have come about during the COVID-19 pandemic and are currently the first line of defense. We propose to refine this mRNA vaccine technology for immunization of horses. Based on our extensive preliminary data, we will test vaccines containing the parts of EHV-1 that are important for inducing protective immunity in horses. We are ideally placed to accomplish this task because we have previously developed a unique model for experimentally inducing EHM in horses that now allows us to test potential vaccine candidates . Moreover, given the high degree of similarity among equine herpesviruses genetically related to EHV-1, namely EHV-3, EHV-4, EHV-8 and EHV-9, our vaccine candidates, if proven successful, are likely to also offer protection to other equine herpesviruses. Additionally, they could readily be extended as a platform to induce protective immunity against an array of EHVs or other equine viruses.
Importance to the Equine Industry: The EHV-1 “G” strain (SNP at ORF 30, G2254) has been associated with more severe outbreaks and a greater occurrence of EHM than the EHV-1 “A” strain (A2254 genotype). The enhanced replication capacity with the “G” strain appears to be associated with an enhanced frequency of more severe disease; however, both strains cause EHM. Several factors appear to be contributing to an uptick in EHV-1, particularly in Europe and the United States, with equine venues being closed or quarantined as a consequence. We propose an mRNA vaccine delivered in an extended release, biodegradable polyanhydride vaccine depot. Compared with traditional vaccines, we anticipate that this approach will be more effective and require fewer inoculations.
Ongoing EHV-1 outbreaks, including cases of neurologic disease caused by EHV-1 and called ‘equine herpesvirus myeloencephalopathy’ (EHM), emphasize the fact that currently available EHV-1 vaccines are not fully protective. These disease outbreaks are occurring despite widely used vaccination and many available EHV vaccines on the market. The fact that currently available inactivated or attenuated vaccines have not been sufficient to completely protect from EHV-1 outbreaks has caused the demand for novel EHV vaccine formulations that can prevent horses from infection and severe disease, especially neurologic disease. Here, we propose to design and evaluate a novel DNA vaccine formulation against EHV-1/EHM for horses. We are combining our expertise in equine immunology and EHV-1 research (Dr. Wagner) and virology and vaccine design (Dr. Diel) to first perform a comprehensive analysis of the best DNA vaccine candidate and then test the optimized vaccine candidate for its protective effects in horses. The DNA vaccine platform that we are testing in this project has already been used successfully for induction of immunity and protection from infectious disease in other species. The project will result in an established new EHV vaccine candidate and provide a new approach to effectively protect horses from EHV-1 infection and disease. Overall, the project is relevant to the prevention of disease induced by EHV-1 by increasing immunity and protection in the equine population and reducing severe neurological disease outbreaks in the US.
Importance to the Equine Industry: Equine herpesvirus type-1 (EHV-1) has a significant impact on animal health and the equine economy worldwide. Every year, several neurologic outbreaks are reported throughout the US (http://www.equinediseasecc.org/alerts/outbreaks). In an outbreak scenario, EHM is first confirmed by an EHV-1 positive diagnostic test in the affected horse(s). The positive EHV-1 test result causes immediate regulatory actions with stringent quarantine and biosecurity of all horses at the facility where the EHM affected horse is kept to prevent further spread of the contagious disease. Quarantine is in place for 21-28 days after the last suspected infection. Currently, all potentially exposed horses at a quarantined location are kept there for the same time for general precaution and preventing the possible spread of EHV-1 to other horses. During this time all movements of horses to and from the quarantine facility stop. Depending on the location of the first affected horse, quarantine can apply to race, show and auction premises, or secondary locations if a potentially exposed horse already moved to another site, and can thus stop the movement of involved horses for weeks. Consequently, EHV-1 quarantine represents a high economic burden for the horse industry through loss of severely affected horses, medical treatment charges of diseased horses, quarantine management efforts, and lost training, racing and/or competition time for all horses involved in the quarantine scenario. EHV-1/EHM outbreaks and quarantine have not only a serious impact on equine health, they also restrict horse transport, racing and competitions, and economically affect horse owners, equine businesses, and the equine industry.
Multidrug resistant (MDR)-R. equi has emerged in the environment of horse-breeding farms in Kentucky due to overuse of antibiotics to prevent disease caused by R. equi in foals. Because MDR-R. equi leads to a higher probability of death in affected foals, it is necessary to mitigate antimicrobial resistance in R. equi to promote animal health and welfare, and to protect efficacy of antibiotics in the long term to treat diseases in animals and people. The effect of measures to decrease antimicrobial use on the dynamics of (MDR)-R. equi in the environment of horse-breeding farms is still unknown. In this proposal, we will: 1) determine temporal trends of prevalence of MDR-R. equi in the environment of horse-breeding farms over a 5-year period; 2) identify temporality of risk factors associated with persistence of resistance; 3) determine temporal trends in selective pressure in the environment; and 4) determine associations between antibiotic use in foals (from questionnaires), selective pressure in the environment (by measuring concentration of antimicrobials in soil), and frequency of MDR-R. equi in the environment (by testing soil samples). We will also perform an evolutionary analysis to compare genetic variability of R. equi isolates between 2017 and 2021 samples to investigate genetic diversification over a period of 5 years. This analysis will allow identification of genetic elements acquired or mutated that allow bacteria to adapt and evolve to maintain antimicrobial resistance genes that are potentially costly for bacteria fitness in soil. By having an overall understanding of IF and HOW MDR-R. equi persists in the environment, we will be able to recommend strategies to decrease the risk of foals getting contaminated with MDR-R. equi from the environment.
Importance to the Equine Industry: Rhodococcus equi is a great threat to equine health and welfare, and the leading cause of disease and death in foals in many states in the USA. Nonetheless, highly effective ways to prevent this disease in foals are lacking. Thoracic ultrasonography for early identification of lung lesions followed by antimicrobial treatment of subclinically affected foals to prevent the development of severe R. equi disease has led to the emergence of multidrug resistant (MDR)-R. equi in both horses and foals and their environment. In 2017, we found MDR-R. equi in the soil of 76 of 100 horse farms in Kentucky. Foals infected with MDR-R. equi are 7-fold more likely to die than foals infected with susceptible isolates. Therefore, there is a great need to mitigate antimicrobial resistance in R. equi. It is necessary that we understand the effect of measures to reduce antimicrobial use in horse-breeding farms on the dynamics of MDR-R. equi populations to better predict the risk of infection of foals with MDR-R. equi and to develop strategies to control its spread among foals and farms. Widespread macrolide and rifampin resistance in R. equi isolates has become a major emerging problem facing the horse–breeding industry and might adversely impact human health. The work proposed in this application represents the first important steps in understanding the evolution of MDR-R. equi at horse farms in response to changes in antimicrobial pressure over time. Given the importance of rhodococcal disease for the equine industry, our ability to curtail antimicrobial resistance in R. equi is of urgent importance. Persistent breeding-induced endometritis (PBIE) in mares can cause early pregnancy loss, negatively impacting reproductive efficiency and economic returns. Following mating, a transient inflammation in the uterus (endometritis) may occur for 24 hours as an inflammatory reaction to clear dead sperm and other contaminants from the mare’s uterus. The subset of mares that are unable to resolve this inflammation are considered susceptible to PBIE, which may be the result of variation from the normal immune response to breeding. Mares that suffer from PBIE often endure repeated cycles of ineffective treatments. While many studies have evaluated PBIE in live mares (in vivo), few studies have utilized in vitro conditions (growing uterine cells in a dish in an incubator), which have largely been limited to a 2-dimensional approach and may not accurately capture the mare’s normal immune response. Three-dimensional (3D) cell cultures (organoids) are a novel method for creating a physiologically relevant representation of equine endometrium (inner lining of cells in the uterus). Created from endometrial biopsies taken during routine clinical procedures, the organoid model system provides an in vitro system to study reproductive health in mares. As we explore which aspects of the immune system are causing PBIE in mares, novel biological agents such as natural stem cell secretions (extracellular vesicles; EVs) to treat endometritis will require testing. A validated, in vitro 3D model of equine endometritis would eliminate the current dependence on mares for endometritis-related research until clinical-phase testing of evidence-based therapeutic options are developed. Endometritis is not a new problem, but it is one that requires a novel approach, such as use of an organoid model system, to understand the complex nature of the disease and resolve it without extensive use of research mares.
Superficial digital flexor tendon (SDFT) injuries are common debilitating injuries in Thoroughbred racehorses. Persistent inflammation during tendon healing and the poor intrinsic healing capacity of tendon ultimately results in poor quality of tendon repair. This in turn leads to decreased elastic strength of ‘healed’ tendon tissue and increases re-injury rates in horses. Circulating macrophages are key immune cells that become localized within tissues throughout the healing period and produce inflammatory and regulatory factors that control inflammation, pain and healing responses with the tissue. Tenocytes function to maintain tendon structure by synthesizing and secreting tendon matrix proteins. Recent findings show that inflammatory processes operating during tendon healing affect tenocyte activities such that their matrix synthesis capacity is altered and consequently, contributes to tendon degeneration. In this research, we will evaluate the effects of macrophage polarization and macrophage-derived inflammation on tenocyte activities in a tendon matrix culture model.
Although it is accepted that M1 macrophages promote inflammation and M2 macrophages are beneficial to tissue healing, the precise mechanisms associated with these said effects are not known. Moreover, recent studies have also reported controversy pertaining to these effects. Therefore, in this proposal we aim to characterize M1 and M2 macrophage-derived inflammatory factors and assess their impact on SDF tenocyte activities (gene expression profiles and matrix synthesis). We hypothesize that M1 macrophages augment inflammation and negatively affect tenocyte activity, whereas M2 macrophages promote tenocyte matrix synthesis. The second objective of this research is to evaluate the immunomodulation potential of extracellular vesicles/exosomes isolated from bone marrow stem cells, a novel orthobiologic that is cell-free, mitigates immunogenic concerns and offer off-the shelf potential to enhance tendon healing. We will utilize state-of-the-art techniques to evaluate SDF tenocyte responses to macrophages and exosomes. On completion of this research, we will identify biological and immunological factors that can be subsequently targeted to improve the quality of tendon repair. Understanding the macrophage-derived factors that control tenocyte activity is necessary for our overall goal of identifying/ developing therapies that enhance tendon healing.
Importance to the Equine Industry: SDFT injuries are one of the most common reasons for retirement in Thoroughbred racehorses. Despite requiring prolonged periods for healing, the ‘healed’ tendons are fibrotic and results in high incidence of re-injuries. Growing evidence suggests that immunomodulation is an effective strategy to improve the quality of tendon repair. Macrophages are key immune cells that modulate inflammation, pain during healing and therefore, this research will characterize macrophage-derived inflammation, and identify biological/cellular mechanisms affecting tenocyte activity and, subsequently contribute to tendon degeneration during healing. This research is will also help evaluate the beneficial effects of a novel off-the-shelf biologics/ regenerative therapy for tendon healing as several recent studies have demonstrated that immunomodulation is key mechanism to enhance tendon healing. Collectively, this work will impact develop therapies that can improve tendon injury outcomes in horses.
The novel concept of “trained immunity” has been demonstrated to be extremely useful against various microbes in many different species, including people. The bone marrow is the soft tissue inside bones that makes blood-forming cells (blood stem cells or precursors) that generate white blood cells. In this project, we are interested specifically in the white blood cell neutrophil (after they are released from bone marrow), which is an “innate cell” that kills microbes inside other cells. When molecules from microbes attach to the surface of these precursors (to molecules called “receptors”, which stimulates the cell), they generate neutrophils that will travel to the circulation with an increased capacity of killing bacteria compared to those generated by precursors that were not stimulated (i.e., did not attach to microbes). There are a few examples of microbes and substances that stimulate these cells, for example, the attenuated tuberculosis vaccine bacillus Calmette-Guérin (BCG) or beta-glucan (a molecule of the cell wall of fungi). More recently, BCG has been used against infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has demonstrated the broad impact of trained immunity. This concept, however, has not been studied in horses. Newborn foals have an immature immune system, such that many are susceptible to Rhodococcus equi infection, an important cause of disease and death in foals. There is no vaccine commercially available, and the only strategy that has repeatedly protected foals against pneumonia by R. equi is the use of live bacteria given to foals using a foal stomach tube (same as used in colic cases). We believe this protection is attributable to stimulation of white blood cell precursors, specifically precursors of monocytes and neutrophils, which will “turn on or off” genes (called epigenetic modifications) that are important for the immune response. This method, however, is not feasible to be used in farms, therefore, we must learn what is inducing protection to be able to mimic these effects in a vaccine.
Our long-term goal is to develop a strategy that will be used in farms to prevent respiratory infections caused by R. equi (and potentially other infections, such as strangles) based on induction of trained immunity in foals. Our overall objective is to demonstrate that giving live R. equi via a stomach tube (called enterally) to newborn foals induces trained immunity that protects them against pneumonia caused by R. equi, while giving them fluids (saline) enterally does not. We plan to specifically: 1) demonstrate that giving foals live enterally in the first days after birth will induce trained immunity which will result in protection against pneumonia when foals receive R. equi in their lungs (Aim 1); and 2) demonstrate that neutrophils from foals that receive live R. equi enterally have improved capacity of killing R. equi in laboratory experiments (we give foals live bacteria enterally but collect blood to separate neutrophils and only expose them to bacteria in the laboratory; Aim 2).
In Aim 1, we expect to confirm several studies in which administration of live R. equi enterally results in protection against R. equi challenge in the lungs, and that this protection is related with changes in neutrophils. We do not intend to use this as a vaccine because giving live R. equi to foals in farms is not feasible, but these results are important because: 1) they are ESSENTIAL to better understand why these live bacteria in their stomach helps protect them against pneumonia later in life; and, 2) they will provide useful information about epigenetic changes (which genes are turned “on” or “off”) in innate immune cells (monocytes and neutrophils) following enteral administration of live R. equi, which has not been previously documented. In Aim 2, we expect to show that enteral administration of live R. equi generates blood neutrophils with improved ability (trained) to kill bacteria in the laboratory, while saline does not.
Our long-term goal is to develop strategies for prevention and control that could be used at equine breeding farms to prevent foals from developing pneumonia caused by R. equi. We believe that the key to designing these strategies that protect foals lies in better understanding an approach that is known to protect foals. This approach could potentially replace the use of antibiotics to prevent the disease (a strategy used by some farms to reduce the number of cases of R. equi pneumonia). We also believe that by stimulating the newborn foals’ innate immune responses, a decline of other infections (e.g., strangles) might occur, as well as better responses to vaccines. This will have a remarkable impact in the equine industry, specifically at horse-breeding farms in which the number of cases of the disease can exceed 20-40% of the foal crop.
Importance to the Equine Industry: The impact of R. equi pneumonia on the equine industry is large. In the United States, respiratory illness is the 3rd most common cause of disease in young foals, and the 2nd cause of death in foals. R. equi pneumonia is a disease difficult to detect and treat, and in many cases, extensive lung damage has occurred by the time a diagnosis is made, increasing the length of treatment, which sometimes is in vain. Consequently, a large number of foals R. equi succumb to the disease. Moreover, the economic impact of R. equi pneumonia to the industry is significant. There are 7.2 million horses in the US, with a direct contribution of over $50 billion and indirect of $122 billion to the economy. Racing has the highest direct contribution ($15.6 billion), and it has been shown that foals that recovered from R. equi pneumonia were less likely to race as adults, directly impacting the racing industry. Therefore, preventing R. equi pneumonia in foals would greatly benefit the equine industry: 1) less foals would develop the disease, directly benefiting horse-farms; 2) less foals receiving antibiotics, and, thus, less resistance to antibiotics would occur and less side effects, such as diarrhea; 3) improving the innate immunity of foals will have a broader impact in their health, such as reduction of other bacterial infections (e.g., strangles or equine distemper) because these cells are not “microbe-specific”, and better responses to vaccines; 4) the better understanding of the mechanisms of how enteral R. equi protects foals against pneumonia by limiting its survival and multiplication inside equine lung cells would direct benefit R. equi researchers. To date, a clear understanding of the changes associated with the disease and specifically what protect some foals against R. equi are unknown, and by studying a strategy known to prevent pneumonia in foals we will have useful information about the disease for vaccine development.
Rationale: Antimicrobial resistance (AMR) is clearly one of the most important public health issues facing societies globally. A majority of resulting scrutiny has focused on antimicrobial drug (AMD) use in food producing animals, but use of AMDs in horses has received increased attention. It is an accepted principle that all antimicrobial drug uses can promote resistance, and many propose restriction on AMD uses (especially in animals) without distinguishing the potential that some AMD uses may be safer than others, in relation to promotion of AMR. As veterinarians and caretakers of the wellbeing of horses, it is critical to maintain access and judicious use of AMDs in horses to treat bacterial infections, but it is also an incumbent public health responsibility that we select AMD use practices that are minimally impactful on AMR. Unfortunately, data from studies that objectively compare specific use practices in horses are relatively rare, and are critically need to promote evidence-based decision-making regarding this issue. Studies have traditionally evaluated AMR through characterization of individual cultured isolates. While this previous adds to our growing understanding of AMR in horses, there is a clear need to investigate the impacts of AMD use in horses using study designs and methods that, a) compare common antimicrobial use regimens in a single study to improve ability to make direct comparisons among treatments; b) yield results that are less likely to impacted by biases which can interfere with understanding the true impact of these exposures; c) examine impacts of AMD exposures in a larger context that characterizes all antimicrobial resistance found in entire microbial communities using sensitive, state-of-the-art methods; and d) evaluate how impacts of AMD exposures may be mirrored or may vary among different ecological niches in a horse.
Study Objective: Our overall long-term goal is to provide empirical evidence regarding the impacts of common use practices for antimicrobial drugs (AMDs) regarding promotion of AMR. This will allow us to identify treatment regimens that are beneficial to treatment of infections in horses and also have lower risks for promotion of antimicrobial resistance (AMR). The central hypothesis for this work is that common AMD treatment regimens in horses are not equivalent in their likelihood of promoting AMR, and specific hypotheses will investigate the impact of 4 common AMD treatment regimens on the entire communities of AMR genes and populations of bacteria from 3 different anatomical/ecological niches.
Study Design: Forty healthy, adult mares will be randomly assigned to 1 of 5 Study Groups. Groups 1-4 will be treated for 4 days with 4 of the most common AMD treatment regimens used in horses (ceftiofur crystalline free acid given IM; sodium penicillin G and gentamicin given IV; oxytetracycline given IV, and trimethoprim-sulfamethoxazole given orally), and Group 5 will be untreated controls. Horses will be examined daily by veterinarians, and samples will be collected on Days 0 and 1 prior to initial treatments, and after treatments are completed on Days 5 and 6, as well as 21 and 22. DNA extracted from samples on Days 0-1 will be pooled for genomic analysis, as will samples from Days 5-6, and also Days 21-22; this pooling of samples will decrease the potential for individual sample variability to affect results and thereby increase study power. Samples on all days will include feces obtained per rectum, and swabs of the deep nasal cavity and the vagina. After extraction and purification, DNA recovered from samples will be processed for 2 types of genomic sequencing that will, a) characterize all types of AMR genes, and b) characterize all members of the microbiome (i.e., entire microbial communities). We will then compare the compositions of resistomes and microbiomes among treatment and control groups across time points, and also among different microbial niches (feces, deep nasal cavity, vagina).
Importance to the Equine Industry: Expected Results: Our prior research shows that using the state-of-the-art genomic methods for investigation of AMR in animal populations provides novel insights on the ecology of antimicrobial resistance when considering the totality of all AMR genes in context of the entirety of microbial populations. This holistic approach provides important novel perspective on the impacts of factors influencing the ecology of antimicrobial resistance. We anticipate that some of the AMD treatment regimens will have greater impact on the resistome, as well as the microbiome, and that these changes will vary among the ecological niches being investigated.
Potential Impacts for Animal Health: These results will address an important need for veterinarians and producers regarding antimicrobial drug use and antimicrobial resistance. Clearly, as stewards entrusted with the wellbeing of horses, we need to continue to use AMDs for treatment of important bacterial infections in horses. However, we must also address the critical need for prevention of AMR through improvement in use practices and stewardship of this critical resource for the future benefit of animals and public health. This proposed research has important practical relevance and provide immediate benefit to veterinarians and horse producers as the results will demonstrate the magnitude of changes to the resistome and microbiome that can be expected in response to common AMD treatments and over time; these findings can help to guide treatment protocols so as to have lesser impacts on the development of AMR in treated horses. This research also has important foundational relevance as it will provide novel information about ecology of the entire resistome and microbiome in three important microbial niches of the horse.
Infection of the placenta and subsequent placental disease (placentitis) is the most common problem of late gestation in mares [2,4,5]. Besides the effects on the mares’ well-being and the emotional impact on the owners, placentitis results in abortion or the birth of pre-mature or sick foals, leading to multimillion-dollar losses in the equine breeding industry [2,4,5,7,8]. Although placentitis is one of the costliest diseases in the equine breeding industry, the exact cause of the disease is still not fully understood, leading to inefficient preventive, diagnostic, and treatment protocols . Previously, we characterized the molecular changes in placental tissue at the final stage of this condition . However, there is no information about the exact mechanism of infection, the interaction of the placenta (host) and the causative bacteria (pathogens), and progression of the disease. It is currently assumed that only a few bacteria (mainly Streptococcus equi subspecies zooepidemicus (Strep. equi) in ascending placentitis and Amycolatopsis or Crossiella in nocardioform placentitis) cause placentitis [5,8]. In our preliminary data, we identified several bacteria present in large abundancies in placentitis cases. Surprisingly, Strep. equi, Amycolatopsis, and Crossiella were not always the most abundant pathogens. However, with the 16S rRNA sequencing approach that we used, we were not able to distinguish between contaminating, environmental bacteria and metabolically active bacteria involved in the pathogenesis of placentitis.
Therefore, in this proposal, we aim to identify the bacteria involved in placentitis using dual-RNA sequencing which identifies the gene expression of both host and pathogens simultaneously and identifies only those bacteria that are metabolically active. Within the last few years, with the advances of molecular technology and high-throughput data, studying the host-pathogen interaction has become feasible. A recently developed technique, known as dual RNA sequencing (dual RNA-seq), is designed to understand the interaction between the host and pathogen, identifying the gene expression patterns that result in disruption of normal host physiology, manifesting as infection and disease in the organism . This novel technique leads to identifying new virulence factors in the pathogen or pathways in the host cell that respond to the exposure to specific pathogens [27,28]. For example, this technique led to the identification of genes that play a key role during Salmonella typhimurium infection in humans, leading to identification of a targeted treatment .
We hypothesize that by using this technique, we can further understand the pathogenesis and the host-pathogen interaction in equine placentitis. Similar to human medicine, this information will provide therapeutic targets for placentitis. To test our ability to initiate this research and as proof of concept, we successfully utilized a publicly available dataset from equine Strep. equi -induced placentitis along with a recently generated RNA-seq dataset from equine nocardioform placentitis (conventional RNA-seq datasets) to investigate the gene expression in both the bacteria and the placenta. However, our results are limited due to the low sequencing depth of these datasets. Nevertheless, our analysis shows bacterial reads in RNA-sequenced samples, with an apparent ability to accurately identify and quantify this population, providing a novel method for detecting pathogenic bacteria during disease. Our goal for this proposal is to repeat this procedure in a larger sample set and perform more in-depth sequencing (dual RNA-seq; 150-200 million reads) to increase the resolution on the gene expression patterns in the bacteria and in the placental transcriptome, as well as to provide enough sequence to improve the accuracy of pathogen identification. We will test our hypothesis on previously collected samples from clinical cases of ascending and nocardioform placentitis and experimentally induced cases of ascending placentitis. In vitro experiments are also planned (1) to confirm the causative agents of placentitis, (2) to monitor the crosstalk between the placenta and pathogen(s), and (3) to characterize the molecular changes during the progression of ascending placentitis, using our novel equine placental organoid model. Our results will hold potential for the development of new diagnostic tools and therapies to forestall placentitis-induced preterm labor.
Importance to the Equine Industry: Placentitis is one of the most common problems of late gestation in mares [1–5]. Approximately 3-7% of equine pregnancies worldwide are affected by placentitis 6 and 10-30% of all abortions, premature deliveries and neonatal deaths are caused by placentitis 6, leading to multimillion-dollar losses in the equine breeding industry [1–5,7,8]. Furthermore, placentitis has an effect on the mares’ well-being and an emotional impact on the owners [1–5,7,8]. Currently the initial diagnosis of placentitis is based on clinical signs while the essential key to a successful treatment of placentitis is an early diagnosis [8,10]. Unfortunately, the clinical signs occur at the final stages of the disease, reducing the efficiency of the treatment. Several groups have attempted to identify biomarkers for earlier diagnosis but with limited success: either they are not sensitive or specific enough and/or are not (yet) useful in clinical practice [5,29,30]. Our study will ultimately lead to the identification of diagnostic biomarkers and the development of treatment and prevention strategies for equine ascending placentitis. This proposal will (1) identify the bacteria involved in placentitis in order to have targeted treatment; (2) identify gene expression patterns of the pathogens during ascending and nocardioform placentitis, in order to identify important genes for the pathogenesis and survival; (3) Identify gene expression patterns of the host during ascending and nocardioform placentitis in order to identify genes important in the host reaction to the pathogen; (4) Identify interacting signaling pathways between the pathogen and host during placentitis cases. Finally, our study will also focus on evaluating the specific and most relevant genes and pathways identified in this study through an in vitro system utilizing placental organoids and primary trophoblastic cells in order to better understand the molecular mechanisms occurring at the host-pathogen interface.
The most common site of fatal musculoskeletal injuries in racehorses is the fetlock joint. Within the fetlock joint, fracture of the proximal sesamoid bones is the most common fatal fracture. Fractures occur in characteristic configurations that indicate that specific limb loading circumstances cause the fractures. We have evidence that leads us to believe that movement of the long pastern bone (P1) toward the outside of the horse during extension of the fetlock joint during high speed exercise is the circumstance that leads to fetlock fractures. If this is true, hoof trimming, shoeing, and racetrack surface management could limit abnormal P1 motion and prevent fetlock fractures.
The fetlock joint is comprised of four bones: the cannon bone, the long pastern bone (P1), and the medial and lateral proximal sesamoid bones. The cannon bone articulates with both P1 and the proximal sesamoid bones. However, the proximal sesamoid bones do not articulate with P1; instead, their motion is coupled through soft tissue attachments within the fetlock joint. One set of soft tissue structures is the medial and lateral collateral sesamoidean ligaments. These two ligaments connect the medial and lateral proximal sesamoid bones to the top of P1. The three-dimensional rotations of the cannon bone relative to P1 are well described for all phases of equine locomotion. However, little is known about the three-dimensional movements of the proximal sesamoid bones.
The geometry of the fetlock joint largely confines its motion to flexion and extension (i.e., sagittal plane motion). However, small movements occur outside of flexion and extension. These motions include rotation about P1 and about the cannon bone (i.e., internal or external rotation) and movement of the bottom of P1 inside or outside of the limb. Recently, we determined that the PSBs experience motion other than flexion and extension when the fetlock joint is loaded at racing-speed levels and the hoof is flat. Importantly, these motions of the proximal sesamoid bones are highly consistent with the development of common fractures in racehorses. Shoeing, uneven footing, and other factors (e.g., turning a corner) affect the three-dimensional motions of P1 relative to the cannon bone. We do not know if these factors affect proximal sesamoid bone motion. Uneven footing has been caused during laboratory tests by wedges placed under the inside or outside of the hoof, but banked racetracks, unbalanced hooves, and/ or unharrowed racetracks likely have a similar affect. Since non-sagittal plane motions of P1 relative to the cannon bone are exaggerated by uneven footing, we believe that proximal sesamoid bone motions will also be exaggerated by uneven footing due to the soft tissue connections to P1 and the joint articulation with the cannon bone. Therefore, the primary goal of this proposal is to determine how uneven footing affects the motion of the proximal sesamoid bones relative to the cannon bone. The secondary goal is to determine if the ligaments connecting the PSBs to P1 are the mechanism for the motions. These research goals will improve our understanding of how proximal sesamoid bone fracture may be linked to hoof conformation, shoeing, and racetrack surface conditions.
Importance to the Equine Industry: Fractures of bones in the fetlock are some of the most common fatal and non-fatal injuries that occur during racing. Proximal sesamoid bone fracture is one of the most common fatal fractures worldwide. It causes 44% of all fatal injuries in California Thoroughbred racehorses, 55% of fatal injuries in South African racehorses, and 37% of fatal injuries in Australian racehorses. Fracture of the lateral side of the cannon bone, at the end that articulates with the proximal sesamoid bones, is one of the most common non-fatal injuries and the second most common fatal injury. This cannon bone fractures accounts for 10-25% of racehorse deaths. Both proximal sesamoid bone and cannon bone condylar fractures have typical fracture configurations that indicate they result from specific limb loading circumstances. Motions of the P1 and of the proximal sesamoid bones relative to the cannon bone outside of fetlock flexion and extension are consistent with these fracture configurations that occur in Thoroughbred racehorses. Medial proximal sesamoid bone fracture is associated with development of a weak spot against the opposing cannon bone that is the likely consequence of a specific limb loading condition that promotes rotation of the medial proximal sesamoid bone into the cannon bone. We have observed this motion during fetlock extension at gallop loads in our laboratory. We believe this motion will be exacerbated by unbalanced hoof trimming, lateral wedge horseshoes, and uneven race surfaces (such as banked racetracks and unharrowed footing). Racetrack surfaces in the United States are banked in the straights for drainage and in the turns. If our hypothesis is supported, hoof trimming, shoeing, and racetrack design recommendations could be made to prevent fetlock injuries.
Rhodococcus equi causes severe pneumonia in foals, but rarely causes disease in adult horses. Many foals clear the infection without need for treatment, but others require long-term antibiotic treatment or even die. Unfortunately, we do not understand why foals in general, and some foals in particular, are so susceptible to R. equi infection. This project aims to increase our understanding of why some foals die from R. equi pneumonia, while others clear the infection without showing signs of illness, with the ultimate goal of more effectively treating and preventing this devastating equine infectious disease.
Increasing evidence suggests vitamin D and cortisol are vital in the immune response to bacteria like R. equi in other species. Vitamin D is important for producing specific proteins within immune cells that can kill bacteria. Cortisol is important in directing the immune response during infection. However, the role of these hormones in the development and severity of R. equi pneumonia in foals is not known.
This proposed study will investigate the relationship between blood levels of cortisol and vitamin D and the development and progression of R. equi pneumonia in foals on a large horse-breeding farm with a high rate of R. equi infection. Vitamin D and cortisol concentrations and associated immune proteins will be measured in blood samples taken at strategic time points from birth to weaning. We will look for associations between these measurements and disease development and severity. This is the first study to investigate these immune-regulatory hormones in R. equi pneumonia in foals. By conducting this study in foals with naturally-occurring R. equi exposure and infection rather than in experimental infection or in laboratory models, we will be able to define factors that influence disease development in this real-world setting.
Importance to the Equine Industry: Pneumonia in general, and R. equi pneumonia in particular, are leading causes of disease and death in foals. Costs associated with disease screening, veterinary care, antibiotic treatment, loss of foals, and impacts on future athletic performance are extreme. Additionally, the efficacy of frequently used antibiotics to treat R. equi may be decreasing as resistance to those drugs is increasing. The effects of steroid hormones such as vitamin D and cortisol on R. equi susceptibility have not been explored to date. A better understanding of how vitamin D and cortisol impact the foal's risk of R. equi infection after natural exposure will open up new avenues for both prevention and treatment of this common, costly, and important disease. This work directly supports the mission of the Grayson Jockey Club Research Foundation and addresses a target research area by working to reduce the impact of R. equi pneumonia on the equine industry.
The objective of this study is to investigate if an opioid pain medication, fentanyl, can be administered to horses via a patch placed on the skin. The drug is absorbed from the patch through the skin as opposed to being administered by injection. Fentanyl, administered in this way, has the potential to provide clinically relevant pain relief to horses without the need for expensive intravenous catheters and frequent injections. We aim to describe how well fentanyl is absorbed through the skin from the patch and the degree and duration of pain relief that it provides at different dosages in horses. Armed with the information obtained from this proposed study, we will then be able to make a recommendation for dose and frequency of fentanyl administration via a patch in horses. Ultimately, the ability of veterinarians to provide long-lasting opioids for pain relief in horses without the need for intravenous catheters or multiple, painful injections will be a game changer in pain management for horses.
Importance to the Equine Industry: Management of patient pain is a constant challenge for the large animal veterinarian. Opioids are commonly used for management of pain in the horses but as they must be given by injection in horses, they require maintenance of an intravenous catheter and/ or multiple injections in the muscle to provide constant pain relief. This is time consuming to the veterinarian, painful for the horse, and expensive for the owner. With completion of this project, we aim to show that fentanyl absorbed through the skin via patch application represents an effective option for pain management in equine patients and has the potential to change the face of pain management for horses around the world. As such, the results of this project will be of great importance to the equine industry.
Laminitis in horses is most commonly associated with either equine metabolic syndrome (EMS) or pituitary pars intermedia dysfunction (PPID;”Cushing’s Disease”). In both cases, high blood insulin, resulting from insulin dysregulation (ID) is what drives the development of laminitis. An exaggerated insulin response to dietary carbohydrates, often from pasture, can causes varying degrees of damage to the feet, resulting in laminitis. We now understand that high blood insulin is the key event that drives the development (and recurrence) of laminitis in these horses: high blood insulin concentrations directly damage the lamellae within the feet. Successful prevention and treatment of laminitis in these cases therefore depends on our ability to control the blood insulin responses in horses and ponies with ID.
Since management strategies (including dietary regulation) are often insufficient alone to control blood insulin, there is a need for medications that specifically control the insulin response to feeding in order to prevent laminitis in these cases. Sirolimus (rapamycin) is a drug that is primarily for the treatment of cancer and prevention of organ rejection, but it also has anti-aging effects and has variable effects on insulin dynamics across multiple species. We have preliminary data demonstrating that sirolimus has potent effects on insulin production in horses: a single intravenous dose of sirolimus can suppress insulin production (in response to an oral sugar challenge) for at least 24 hours. Furthermore, our preliminary pilot data from an experimentally-induced ID model demonstrates that once-daily administration of oral sirolimus normalizes the insulin response to an oral glucose challenge, without causing apparent adverse effects during 7 days of sirolimus treatment. These potent effects of sirolimus on insulin production in the horse have great potential for the treatment of ID (and prevention of laminitis).
We hypothesize that treatment with sirolimus will prevent high blood glucose in response to ingested carbohydrate in horses with both experimentally-induced and naturally-occurring ID and will be safe and well tolerated. Using an established experimental model of ID, we will test the effects of 2 different dose rates of oral sirolimus on insulin dynamics in response to oral sugar challenge. We will then test the effects of oral sirolimus treatment in horses with naturally-occurring ID using a randomized, placebo-controlled crossover study. Blood sirolimus concentrations as well as hematological parameters and serial clinical evaluations will be used to monitor for any adverse effects. We anticipate that this study will provide fundamental information on the efficacy and safety of sirolimus for the treatment of ID in horses. In line with our preliminary data, we expect that sirolimus therapy will rapidly and profoundly suppress insulin production in response to ingested sugar, without causing adverse effects. The results of this study have the potential to drive the development of novel treatments for insulin control and prevention of laminitis in the horse.
Importance to the Equine Industry: Laminitis is a crippling disease of horses and ponies that remains a major cause of morbidity and mortality worldwide. Laminitis has been voted the number one priority for equine research by the American Association of Equine Practitioners due to both the high incidence of the disease (annual incidence of 2–7% of horses in recent studies), the severe nature of the disease (high incidence of humane destruction or chronic lameness due to crippling nature) and the lack of effective therapies for treating the disease. In one of the largest studies of the incidence of lameness in the U.S. in recent history, a USDA National Animal Health Monitoring Study published in 2000 of approximately 3000 horse farms in 28 states stated that 13% of these farms reported a case of laminitis in a one year period. Of the different forms of laminitis, so called “endocrinopathic” laminitis is by far the most common, accounting for almost 90% of laminitis cases presented to a first opinion practice in a recent study. This form of laminitis affects adult horses of any age and breed, but in the Thoroughbred industry it is most common in brood mares and stallions where it is a major cause of loss of usage as well as premature death. Although preventative strategies including early identification and dietary management are useful for reducing the incidence, horses still succumb to endocrinopathic laminitis and a rational, evidence based therapy is essential in managing the acute case. We hypothesize that sirolimus will be an effective and safe therapy for controlling insulin dysregulation and preventing endocrinopathic laminitis in horses.
There are two Career Development Award recipients in 2023.
The Storm Cat Career Development Award, inaugurated in 2006, is a $20,000 grant in 2022 and is designed as an early boost to an individual considering a career in equine research. It has been underwritten annually by Mrs. Lucy Young Hamilton, a Grayson-Jockey Club Research Foundation board member whose family stood the retired champion stallion Storm Cat at Overbrook Farm. This year there are two award winners.
Hodgson Chair of Equine Studies
The Elaine and Bertram Klein Development Award is a competitive program intended to promote development of promising investigators by providing a one year salary supplement of $20,000 in 2022. This program is restricted to one award per year and is named in memory of a renowned horsewoman and her late husband, a Thoroughbred owner and breeder. The first grant was in 2015 and was funded for $15,000 with donations by the Klein family.