The effect of thyroxine supplementation on measures of performance in racehorses
Kritchevsky, J

Co-investigators: Laurent L. Couetil, Veterinary Clinical Sciences, Purdue University

For the pari-mutuel racing industry to maintain its integrity, it is important that horses compete without receiving substances that alter their performance. Investigative personnel in Ontario report seeing open containers of thyroxine in many barns visited during out-of-competition testing. Finding extremely elevated blood concentrations of thyroxine has been documented on numerous occasions on post-race blood testing of some horses from Ontario racetracks. Despite this, the effect of excess thyroid hormone on equine performance has never been reported. We are proposing an experiment designed to determine whether giving high doses of thyroid hormone to racehorses alters their performance in any way. We will do this by giving thyroid supplement at 2 and 5 times the recommended dosage to fit Standardbreds, and then evaluating how they perform during standardized exercise testing. Horses will also perform the standardized exercise testing with no thyroid supplement as a control. The exercise will be performed on a high speed treadmill that allows horses to reach exercise intensity that is similar to that reached during races.

In order to determine how the horses’ blood thyroid concentrations vary in response to strenuous exercise, we will measure blood thyroid hormone concentrations before, during, and then shortly after strenuous exercise in control and supplemented horses.

High blood thyroid concentrations have been associated with the abnormal heart rhythm atrial fibrillation in humans. The horses in this study will wear an ECG monitor during exercise so that their cardiac function can be monitored continuously and any abnormal activity can be documented.

Development of Osteochondral Constructs Using Equine Umbilical Cord-Derived Mesenchymal Stromal Cells for Treating Joint Cartilage Defects
Thomas G. Koch, Associate Professor, DVM Copenhagen, PhD Guelph

Co-investigators: Rita A. Kandel, M.D., Department of Pathology, Mount Sinai Hospital, Toronto, ON, Mark Hurtig, Clinical Studies, OVC, Judith Koenig, Clinical Studies, OVC, Ms. Sarah Lepage, PhD Candidate, Biomed Sci, OVC

Orthopedic injuries, constituted mainly of trauma to joint cartilage, are the most common cause of lost training days or premature retirement in the equine athlete. As cartilage tissue has a low intrinsic capacity to heal, repeated injury will eventually result in post-traumatic osteoarthritis in the joint. Though current regenerative therapies for the treatment of focal cartilage defects are showing promising preliminary results, they are wrought with potential complications from secondary surgical sites to isolate patient-specific cells or tissue. Therefore, we propose to investigate the potential of a novel cell type in the generation of osteochondral-like plugs without requiring a secondary surgical site on the patient.

We have successfully and reproducibly isolated mesenchymal stromal cells from umbilical cord blood (CB-MSC) and demonstrated their ability to generate cartilage in vitro. We found that by subsequently growing the engineered cartilage on a bone substitute, we could generate an osteochondral construct that could serve as an implant. In 2006, Dr. Rita Kandel’s group in Toronto generated similar constructs and implanted them into sheep with induced cartilage injuries; the implants successfully contributed to cartilage repair at 9 months post-surgery. We believe that with further optimization, we can generate an osteochondral-like implant using CB-MSC with superior properties for in vivo repair of focal cartilage defects. The biomechanical properties of cartilage generated in vitro are historically poor, making the graft susceptible to damage upon transplantation. We propose to mechanically mature the cartilage prior to implantation in order to better withstand the large loading forces within the equine joint upon transplantation.

MicroRNAs as Equine Joint Health Biomarkers
Thomas G. Koch, Associate Professor, DVM Copenhagen, PhD Guelph

Co-investigators: Jonathan LaMarre, Biomedical Sciences, Judith Koenig, Clinical Studies

Injuries involving joint cartilage such as osteoarthritis (OA) are some of the most common causes of lameness and pain in horses. Sophisticated means of monitoring joint health status are needed to allow early detection and intervention as well as monitoring the effect of interventions. MicroRNAs are a class of short non-coding RNAs that participate in various biological processes including cartilage development and homeostasis. MicroRNAs have been measured at the tissue level, in synovial fluid and serum, and may reflect some aspects of the health status of the animal. MicroRNAs may therefore be useful biomarkers of joint health that could be evaluated through frequent blood and or joint fluid sampling and analysis. In the present proposal, operating funds are sought to determine robust methods for detecting microRNAs in synovial fluid and blood plasma from horses and to determine cut-off values for selected microRNAs in healthy horses and horses with joint inflammation.

Characterization of the epidemiology and clinical impact of Clostridium difficile infection in hospitalized foals
Scott Weese, Professor, Pathobiology, DVM, DVSc Guelph; Dipl ACVIM

Co-investigators: Nathan Slovis DVM, Dipl. ACVIM and Anne Kullmann DVM, Hagyard Equine Medical Institute (HEMI), Diego Gomez-Nieto DVM DipACVIM, Dept of Pathobiology

Neonatal foal diarrhea is a common problem in foals, and Clostridium difficile is an important cause. In Central Kentucky, there has been an increased recognition of C. difficile (CDI) infection in the foals on farms and at an equine hospital, with the latter characterized initially by a subclinical infection that develops clinical disease usually after 48 hours of hospitalization. Disease that is different from classical enterocolitis is consistent with recognition in humans that CDI is not always accompanied by overt diarrhea. Preliminary data have indicated 7 different ribotypes from nine C. difficile clinically affected foals, something that is consistent with multiple community sources rather than a hospital focus.

This prospective case control study will therefore evaluate the prevalence and epidemiology of Clostridium difficile colonization in hospitalized foals versus farm-matched controls, evaluate the impact of C. difficile colonization on development of disease during hospitalization, characterize C. difficile recovered from foals, evaluate diagnostic tests for CDI and evaluate the transmission of C. difficile between mares and foals. It will involve collection of fecal samples from foals presented to two equine hospitals (Kentucky, Ontario) for reasons other than diarrhea, plus their mares, age and farm-matched controls and foals with disease potentially attributable to CDI. This study will provide important information about the nature of this important pathogen, the risks associated with C. difficile shedding at hospital admission, transmission of C. difficile between mares and foals and risk factors for disease, with a goal of better understanding the disease to better diagnose, treat and prevent infections.

A prospective study on the use of fecal oral transfaunation as adjunct treatment in horses with acute colitis and diarrhea
Dr. Henry Staempfli, Department of Clinical Studies

Co-investigators: Scott Weese, Pathobiology

Acute equine enterocolitis concerns the equine industry because of high mortality and economic costs of treatments. The underlying cause for the diarrhea is rarely known and routine treatments are mainly supportive, with intravenous fluid treatment and if needed antibiotics and anti-inflammatories. Recently in human medicine there has been growing scientific evidence that oral or rectal transfaunation with fecal cocktails from healthy donor individuals are beneficial to treat and prevent recurrent C difficile infections. Central to acute diarrhea is disruption of a normal healthy bacterial flora maintaining health and function of the large colon, which in horses is crucial for providing energy in form of fatty acids. The main goal of this research is to use this technology by transfaunating fresh feces from a healthy horse via nasogastric intubation to the horse with acute diarrhea. The transfaunated feces should accelerate the healing of the colon and the reestablishment of a normal healthy functioning ceco-colonic flora. The research will be using a prospective objective study to compare horses not receiving the transfaunation with horses receiving transfaunation in order to find out benefit of such a cheap adjunct treatment. We will use complex DNA sequencing methods to establish evidence that indeed the transfaunated feces will accelerate recovery from diarrhea, will change the damaged colonic flora and will cause less devastating side effects often observed in such cases. This will be a practical study with client cases of diarrhea randomly assigned to transfaunation.

Allogeneic Equine Umbilical Cord Blood Mesenchymal Stem Cells for Treatment of Exercise Induced Pulmonary Hemorrhage
Thomas G. Koch, Associate Professor, DVM Copenhagen, PhD Guelph

Co-investigators: Laurent Viel, Department of Clinical Studies

Exercised induced pulmonary hemorrhage (EIPH) is a common condition in race horses and a significant equine welfare issue. The etiology of EIPH is poorly understood but pulmonary inflammation is a downstream consequence of frequent bleeding. Mesenchymal stem cells (MSC) have non-progenitor functions through secretion of various proteins. Through such secreted factors MSC have demonstrated immune modulatory properties in vitro and in vivo. In our lab we have demonstrated that equine umbilical cord blood-derived (CB) MSC are more immune-modulatory than the more commonly used MSCs derived in vitro from adipose tissue or bone marrow aspirates.

We have also shown that CB-MSC suppress lymphocyte proliferation in vitro equally immediately after thawing or after one week of culture expansion post-thawing. These CB-MSC have low to no expression of MHC-I and II, suggesting they can be applied in an allogeneic manner. In vivo studies in the horse may lend support to allogeneic application of CB-MSC. Attenuation of pulmonary inflammation from bleeding following intravenous MSC injection has been shown in vivo in humans and non-equine species. The hypothesis of this project is that allogeneic equine CB-MSC will attenuate EIPH associated pulmonary inflammation. Funds are sought to perform a feasibility study to test this hypothesis. Use of allogeneic CB-MSC holds the promise of developing a novel off-the-shelf cell-based product for this important equine disorder.

Seroprevelence of Potomac Horse Fever in Ontario
Luis G. Arroyo, Associate Professor, Lic. Med. Vet., DVSc, PhD, DACVIM

Potomac Horse Fever (PHF) is a seasonal infectious disease caused by the bacterium Neorickettsia risticii. PHF occurs in Ontario mainly during the summer months (July to September). The clinical disease is usually sporadic with most of the cases reported from eastern and south-western Ontario. There severity of the clinical signs varies between horses, but in general characteristic clinical signs include depression, decreased appetite, fever, diarrhea and colic. A high percentage of cases (up to 40%) of horses suffering from PHF may develop laminitis, which is the most serious and life-threatening complication.

Recent studies conducted on the life cycle of N. risticii have shown that fresh water snails and aquatic insects, such as caddisflies, mayflies, and dragonflies, are common carriers of the bacterium. Ingestion of contaminated hay, grain, pasture or drinking water with dead insects may result in clinical disease. Currently, there is an increasing concern among horse owners in Ontario because of the lack of information about the epidemiology and prevalence of the disease, natural reservoir of the bacterium, and biosecurity measures to prevent and control the disease. Preliminary data at the Ontario Veterinary College support clinical, serological and molecular evidence of the presence of the disease in different parts of the province. However, up to date there are only 2 published cases of PHF from horses in Ontario.

The objectives of this preliminary study are to investigate the seroprevalence of PHF in horses from Ontario, and b) to isolate the endemic strain (s) of N. risticii from horses with clinical disease in Ontario for molecular analysis.

Characterization of the equine hindgut microflora and metabolites using an in-vitro model
Luis G. Arroyo, Associate Professor, Lic. Med. Vet., DVSc, PhD, DACVIM

Co-investigators: Dr. Emma Allen-Vercoe, Department of Molecular and Cellular Biology, University of Guelph

The equine gastrointestinal tract is a large tubular structure that houses complex microbial ecosystems within multiple compartments along its length. The hindgut compartment (cecum and colon) comprises the larger portion of the tract and it is here where complex sugars are fermented by the resident microbes, providing between 60 - 70% of the daily energy requirements of the horse.

Microbial communities (microbiota) disturbances may affect the health of the host leading to potentially life threatening disorders in horses, such as colitis and laminitis. The composition and function of the equine hindgut microflora is currently a topic undergoing intense research and much knowledge had been generated. However, a better understanding of the microbial ecosystem, in particular how disturbances in microbiota homeostasis change its function and the consequences of these changes to host health, is needed. In order to understand the effects of gut microbiota disturbances, is imperative to know the status of the microbial ecosystem under healthy conditions. Culture of whole gut microbial communities in vitro, under physiologically relevant conditions, can be achieved by using continuous culture (chemostat) systems. The aim of this proposal is to develop an in vitro system that mimics the nutritional and environmental conditions of the hindgut of the horse, which (when seeded with GI samples obtained from horses) will allow analysis of the microbial population composition, abundance and distribution and their metabolic products.

Determination of practical physical measures of equine welfare
Katrina Merkies, Associate Professor

Co-investigators: Derek Haley, Pop Med, Penny Lawlis, OMAFRA, Gayle Ecker, Equine Guelph (consultation role), Cordy Dubois, PhD student – APS

Equine Guelph declared 2014 as the year of Equine Welfare, dedicated to promoting the welfare for all equines in all stages. “Welfare” is a buzz word attracting media attention and public scrutiny, and most equine organizations have stated that equine welfare in their sport is of paramount importance. The racing associations in Canada are no exception, boldly declaring that they are educated to racehorse welfare and encourage routine inspection to achieve the highest possible standards. The issue of who is conducting routine inspections, what their qualifications are, and what exactly they are inspecting is the focus of this research. In cooperation with the National Farm Animal Care Council (NFACC), Equine Canada, Equine Guelph and OMAFRA, an equine welfare assessment model is being developed to provide a standardized testing procedure for animal, resource, and management-based measures. Inherent in this, specific measures need to be selected, tested and validated for inclusion in such a model. For example, an animal-based measure might look at body condition score; a resource-based measure might determine feed trough height; and a management-based measure could record feeding intervals. These three measures together could provide insight to the quality of welfare a horse experiences in regards to good feeding practices. Once appropriate measures are selected and tested for reliability and repeatability, a standardized training program to certify equine welfare evaluators can be developed. This proposal is seeking funding for the portion of the larger study to select and validate these measures.

The global lung epithelial response to inhaled dust
Dorothee Bienzle, Professor, Pathobiology, DVM Guelph, PhD McMaster, Diplomate ACVP

Co-investigators: L. Viel, Clinical Studies

Horses that are stabled indoors are prone to develop Recurrent Airway Obstruction (RAO) or “heaves”. The disease is caused be an exaggerated response to inhaled mould spores and bacterial components in dusty air and poor quality hay. Affected horses have lung inflammation that improves when they are moved to non-dusty environments and treated with anti-inflammatory and broncho-dilatory drugs. Lung inflammation recurs when horses are re-exposed to poor quality air. RAO is slowly progressive, and affected horses cough and eventually become unable to race or to be ridden. From past work we have good understanding of the nature of the inflammatory response during active RAO. However, the lung epithelium is the first tissue to respond to inhaled antigen and to initiate inflammation, and little is known about the factors that cause release of inflammatory mediators from the bronchial epithelium and increase in mucus production. Recent advances in molecular techniques (RNA-Seq technology) allow sequencing of all expressed genes in tissues less than 1 gram. The resultant sequences are analyzed relative to the equine genome to determine how many copies of each gene are expressed, what the function of the gene is and whether related genes are expressed together. We will apply this technique to tiny biopsy samples collected by endoscopy from horses with and without RAO before and after exposure to dust, and then perform a global molecular analysis. We anticipate that this study will identify key differences between horses that develop RAO and those that do not develop RAO.

Proteins involved in failure of early pregnancy
Dr Keith Betteridge, Department of Biomedical Science

Co-investigators: M. Anthony Hayes (Co-PI) and Brandon Lillie, Pathobiology; James I. Raeside, Biomedical Sciences

During the third week of pregnancy, the horse conceptus (embryo and associated membranes) moves around in the uterus, transiently protected by a thin capsule. At about Day 17, the capsule starts to degrade so the placenta can develop. At this critical stage of early pregnancy, the embryo appears to be more vulnerable. Some proteins normally involved in protecting the non-pregnant uterus from infection are potentially harmful to the unprotected embryo so their production is normally suppressed when the capsule is lost. However, those that persist in excess during uterine inflammation or spontaneous pregnancy failure have the potential to harm the embryo when the capsule becomes more permeable. Using various new technologies, we have identified many proteins produced by the equine endometrium, and several that are suppressed as pregnancy is established but increased in pregnancy failure of inflammation. Our prime objectives are to identify the proteins that are most likely to harm the embryo, and those that might be most useful in the assessment of uterine health in barren mares. To do this, we are comparing sequential changes in uterine proteins in cycling mares, mares with endometritis, mares in which pregnancy failure occurs spontaneously, and mares in which pregnancy failure is induced experimentally by injection of cloprostenol at Day 18 (when the conceptus is more vulnerable to early embryonic death). Funding is requested for the sample collection and laboratory analysis needed to further characterize several of these endometrial proteins that are most likely to be involved in early embryonic death.

Nitric Oxide’s role in Persistent Endometritis in Mares
Elizabeth Scholtz, Population Medicine

Co-investigators: Tracey Chenier, Population Medicine; Firdous Khan, Population Medicine.

Endometiritis, or uterine inflammation, causes huge economic losses to the horse breeding industry in Ontario and worldwide. It is in fact considered “the third overall most important clinical problem in equine practice after colic and respiratory tract disorders” (Troedsson, 1999). Persistent endometritis is thought to occur due to failure of the uterine muscles to contract, resulting in accumulation of debris in the uterus following breeding. Recent research shows that mares susceptible to this problem have increased levels of an enzyme (iNOS) that synthesizes nitric oxide (NO), a smooth muscle relaxant, and greater levels of NO in the uterus than resistant mares. Nitric oxide may be involved in the development of persistent endometritis. We hypothesize this is mediated through the relaxant effect of NO on uterine muscles. This study will test whether a) NO inhibits uterine contractions in the mare, b) the activity of NO synthesizing enzymes is greater in the uterus of susceptible mares than in resistant mares, and c) inhibiting iNOS activity will reduce NO production in the uterus of susceptible mares. In experiment 1, uterine tissue obtained from mares immediately after euthanasia/slaughter, will be used to test the effect of NO on uterine contractions. In experiment 2, production of NO, with or without the addition of an iNOS inhibitor, will be measured in uterine biopsy samples from susceptible and resistant mares. This study will provide valuable insight into the development of persistent endometritis and form a basis for the therapeutic use of iNOS inhibitors in controlling the problem.

ORC Death Registry: An in-Depth Analysis
Peter Physick Sheard, Professor Emeritus/Emerita (College), BVSc (Bristol), DipVetSurg, MSc (Guelph), FRCVS (UK)

The Ontario Racing Commission (ORC) Death Registry was inaugurated in 2003, and contains a record of all racehorses dying within 60 days of participation in racing in the Province of Ontario. Most recorded horses were subjected to post mortem examination. General overviews of Registry data have previously been presented and subsets have been extracted and published to support specific studies. In-depth analysis of accumulated data has not been performed and overall Registry integrity has not been assessed. Over the last two years we have compiled a secure, detailed database of all horses entering the Registry to the end of 2011. This database contains Registry data, plus detailed race line information. It adds expanded results from post-mortem and ancillary reports, including categorization of post-mortem findings not appearing in the list of final diagnoses, and expanded details on circumstances of death. Each record now contains almost 1000 variables. All entries have been error-checked. Data has also been gathered on races taking place in the province from 2003-2011 to allow outcomes such as gender, age, and (where applicable) gait-specific mortality risk to be assessed. These expansions allow much greater detail to be extracted from Registry data, aiding in group-specific selection of targeted preventive strategies. Analysis will provide information of great significance to Ontario racing, while direction will be provided to aid in further development of this most valuable Provincial resource.