Pathogenesis and characterization of inflammatory airway disease in Ontario Thoroughbred and Standardbred racehorses
Janet Beeler-Marfisi, Assistant Professor, Pathobiology, BA, DVM, DVSc, Dipl. ACVP

Inflammatory Airway Disease is a type of lung inflammation in horses that is similar to mild asthma in people. It is common in racehorses, especially 2 and 3 year olds that are stabled, but the asthma is not caused by an infection and the horse's temperature remains normal. The asthma causes the horse to have a persistent cough, which is made worse by exercise. We think it is partly caused by breathing in barn dust from hay, bedding and the floor (during sweeping). In part, barn dust is made up of very fine mould spores (even from good looking hay), and little pieces of dead bacteria (from manure). Both of these things cause the horse’s lungs to react and that reaction causes the asthma. Another thing we think causes horse asthma is air pollution. It is true that in Ontario air pollution causes asthma in people, but we haven’t studied whether it does the same in horses. It’s important to study this disease because it is costly to treat and means missed training and racing days, so the earnings potential of the horse is decreased. In this project, we will work with Thoroughbred and Standardbred trainers and veterinarians at Woodbine and Mohawk racetracks. When a trainer has a horse that they think is coughing bad enough to call their veterinarian, the vet will do a physical examination on the horse and will use their scope to look in the horse’s windpipe and down into the lungs. The vet will then perform a ‘lung wash’ on the horse. This is a safe procedure where they put a small amount of sterile saline into the lung and suck it right back out. It doesn’t fix the problem, but it allows us to look at the cells and mucus in the lungs and tells us what the horse has wrong with it (asthma, lung infection, or bleeding). Based on what we find we will be able to work out approximately how many racehorses have asthma, and we will see whether air pollution, the mould spores and pieces of bacteria cause asthma. If we can get scientific evidence of these things we will be able to help racehorses to be healthier and improve their earnings potential to the benefit of all involved.

Ciliary function in horses with inflammatory lung disease
Dorothee Bienzle, Professor, Pathobiology, DVM Guelph, PhD McMaster, Diplomate ACVP

Cilia are small hair-like extensions on cells that line the nose, trachea and larger airways. The function of cilia is to move a layer of mucus produced by cells in the lower airways toward the mouth for swallowing, and therefore to protect the lung from inhaled particles. Thus, cells with cilia that constantly beat to move a layer of mucus “outward” line the entire upper airway. Inhaled bacteria, molds and particles “stick” to the mucus layer, and are then moved by beating ciliary toward the mouth. People and animals born with cilia that are defective in the proteins necessary for ciliary movement (“primary ciliary dyskinesia”) have recurrent pneumonia throughout their life as well as abnormalities in other organs. In conditions such as asthma and heaves, recurrent inflammation of the airways results in injury to cells lining the airways, including ciliated epithelial cells. It is thought that in these conditions cilia do not regenerate properly, leading to a vicious cycle of poor clearance of particles by shortened or poorly beating cilia and recurring inflammation. Viral infections of the nose and lungs have also very recently been recognized to affect the function of cilia. We will collect epithelial cells from healthy horses’ noses with a simple short medical brush, put the cells into culture and measure how well they move particles mimicking bacteria (fluorescent beads). We will compare the results from nasal samples to tracheal samples obtained with a bronchoscope and a longer medical brush. If cilia in samples from the nose function similar to those from the trachea, routine samples for assessments can be readily acquired. Once the functional assays are established we will collect samples from young and older horses with mild and severe lung inflammation (“inflammatory airway disease”, IAD, and “heaves”), respectively, to measure how cilia function in these conditions and to assess their appearance. If abnormal cilia function is a consistent feature of respiratory diseases of horses, inhaled medication to change the composition of mucus and/or to increase the production of mucus may be helpful.

Comparison of umbilical cord tissue and cord blood stem cells
Thomas G. Koch, Associate Professor, DVM Copenhagen, PhD Guelph

Equine mesenchymal stromal cells are increasingly explored as novel therapeutic agents in equine medicine for a variety of conditions. The field is increasingly focused on using these cells in an allogeneic manner due to ease of application and reduced cost of cell production. Cells from neonatal tissues have higher proliferative potential than similar cells derived from adult tissues. The use of cells with high proliferative potential is essential in order to leverage the potential advantages of using allogeneic cells. However, extended culture expansion may result in cellular drift with regards to phenotype, function and cytogenetic stability (normal chromosome numbers and sizes). The Koch lab has extensively studied equine umbilical cord blood-derived mesenchymal stromal cells and found the cells to have higher proliferative potential than similar cells from bone marrow and adipose tissue. One limitation of cord blood cells is the collection of the umbilical cord blood and the relatively few progenitor cells present in the blood. Advanced foaling management is required at the farm level and trained personnel have to be present during foaling to obtain the blood. Extensive laboratory cell expansion is needed due to the few progenitor cells present, which is associated with increased laboratory selection pressure on the cells. Mesenchymal stromal cells have been isolated from the connective tissue of equine umbilical cords. This cell source is easier to obtain and higher number of putative progenitor cells has been reported. However, it is unclear if mesenchymal stromal cells from cord tissue and cord blood are equivalent with regards to phenotype, proliferation, function and cytogenetic stability. The goal of this project is to compare donor-matched mesenchymal stromal cells from cord tissue and cord blood with regards to the above-mentioned parameters. The influence of oxygen tension on these cell parameters will also be evaluated for both cell sources.

Nutritional and feeding behaviour effects of steaming or soaking hay for horses
Katrina Merkies, Associate Professor

Dried hay is the most common feedstuff for horses in North America. Horse owners supply hay in a variety of formats for a variety of reasons. Two such methods in which dried hay is altered before being offered to horses is soaking and, to a lesser extent, steaming. When grass hay is soaked or steamed the resulting wet or steamed hay has less carbohydrates. Soaking, but not steaming, also results in a loss of protein and micronutrients. A lower carbohydrate-content hay can help to reduce blood glucose and this is appealing for horses with laminitis, insulin resistance (Cushings), Equine Polysaccharide Myopathy, and those who are overweight. The extent of the effects of soaking and steaming on timothy/alfalfa hay, the most commonly available hay in Ontario, is unknown as the majority of research has been conducted in the United Kingdom where grass hays are more common. Therefore, under commonly-used soaking and steaming practices, this research seeks to understand how soaking and steaming affect the nutrient content of timothy/alfalfa hay and additionally whether soaking or steaming alters the preference horses have for dried, soaked or steamed hay.

Comparing loading between the forelimbs of racing Thoroughbred to investigate possible causes of laterality in catastrophic injury
Jeffrey J. Thomason, Professor, BA, MSc, PhD Toronto

The U.S. Jockey Club’s Equine Injury Database (EID) has information on more than 1.6 million racing starts by Thoroughbreds in North America. A recent survey of the approximately 3000 catastrophic injuries (CI) appearing in the database confirmed that the frequency of CI is related to the surface type, being greater on dirt, intermediate on turf and least on synthetics. In addition the study found that the left fore and right hind limbs were significantly more prone to CI on dirt tracks than the opposite limbs, but this effect was not evident on synthetic or turf tracks. The researchers speculated that this laterality of injury might be due to uneven forces acing on the left and right limbs in turns combined with relative inconsistency of support during the stance on dirt. These findings are significant for Ontario’s TB racing industry because races are on turf or synthetics while most training is on dirt. The aims of this study are to assess whether laterality of biomechanical loading exists on dirt and synthetic surfaces, and whether epidemiological data identify that as an issue in Ontario. We will tackle this project in 4 ways: (a) We’ll collaborate with the researcher currently studying the Ontario Death Registry, to look for evidence of injury laterality. The emphasis will be on the forefeet, which sustain higher rates of injury. (b) We will take another look at data we have from previous experiment on forces acting on the feet of Thoroughbreds galloping on the straight and turns of different tracks, and will look for evidence of laterality in loading at all stages of the stance for both front feet. We have data from dirt, turf and synthetic surfaces, using the same horse but on different days. (c) We will record new biomechanical data from the front feet of horses galloping on a training dirt track and a racing synthetic track within a few minutes, to find whether and how much laterality of loading exists on each surface. (d) We combine the analyses from all of the data from parts a-c to assess whether laterality is a problem in Ontario, and to categorize which parts of the complex mechanical loading acting on the horse’ hoof during galloping might be at the root of the problem. The results should identify whether turning or the surface induces the higher risk for injury, from a biomechanical perspective.

Development of 3D printed models of the sacroiliac, hip, and cervical articular facet joints for ultrasound guided joint injections training of equine practitioners
Alex zur Linden, Assistant Professor, DVM, DACVR

Three-dimensional models are used to teach various skills and techniques without needing cadavers or practice on live patients. Ultrasound guided injections of the neck, hip, and sacroiliac joints are challenging to perform, but very useful for diagnosing and treating arthritis in sports and racehorses. Joint injections are used for diagnosing lameness by injecting local anesthetics, or treating arthritis by injecting steroids or other similar substances. These specific injections are often only performed by radiologists or those with advanced training, and access to ultrasound machines. With the increased use of ultrasound by equine veterinarians for the diagnosis of tendon injuries in racehorses, this imaging modality can also be used for diagnostic and therapeutic joint injections. One goal of this project is determine the proper 3D printed material to mimic equine bones via ultrasound examination. The next step is to scan various body parts of a cadaver (neck, sacroiliac joints, and hips) with computed tomography (CT) to create highly accurate digital versions of these joints. These body parts will then be 3D printed in high resolution and embedded in clear ballistics gel to mimic tissue, while allowing the user to directly see the needle and underlying bones. The models will then be assessed for validity by radiologists, equine surgeons, and residents at the Ontario Veterinary College. Once validated, these models will be used to teach interested equine veterinarians in Ontario how to perform these techniques via seminars.

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 has 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, it 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.

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. The 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.

Proteins involved in failure of early pregnancy
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).

Sustained release drug formulations for equine joint conditions: Formulation and forensic considerations
Mark Hurtig, Professor, DVM, MVSc, Diplomate ACVS

In past studies we have studied long acting drugs that were created by a drug company for the human market to control inflammation in joints for up to 90 days. Using the sheep knee as a model we showed that these drug and polymer combinations take up residency in the joint lining where they are slowly broken down to release the drug and control pain and inflammation. The duration and drug levels in joints can be changed by altering the polymer-drug binding, so release times of a few days to several months are possible. Through a collaboration with polymer chemist Dr. Elizabeth Gillies (Western University, Ontario) we will create non-steroidal anti-inflammatory drug (NSAIDs) formulations that will last from a few days to several months after injection into joints. The advantage of this delivery system is that no drug leaks from the joint into the rest of the horse's body to cause the all too common complications of colic and gut ulcers. Dr. Gillies' role will be to create the polymer carriers from safe, food grade chemicals. These are widely used in human sustained release drugs for the eye conditions, cancer therapies etc. Dr. Gillies will modify NSAID drugs so they can be trapped in the polymer carrier system. Laboratory experiments will be used to estimate the rate of drug release into joint fluid over 21 days. With this information we will conduct trials of sustained release NSAID formulations by injecting them into the fetlocks of horses at the University of Guelph Arkell Research Station. By injecting the drug and then challenging the horse joint with an inflammation-causing molecule (lipopolysaccharide) at intervals of up to 90 days, we will be able to determine the duration of the drugs' anti-inflammatory effect. Many horses have low grade inflammation in joints that is difficult to address, and requires repeated, costly treatments. Such medication regimes are expensive and not without risk, particularly when steroids are used since they may promote cartilage loss over time. An additional consideration is the potential for doping with similar drug formulations. By measuring drug levels in plasma, urine and joint fluid we will be able to estimate when drugs would be detected in doping tests. We will share all our data with racing and clean sport/doping jurisdictions and provide samples of our drug formulations for their own use. This project will provide the basis for commercialization of a sustained release formulation that would improve the safety of NSAID use in the horse, and potentially reduce the incidence and rate of osteoarthritis progression in horses.

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.

A prospective study on the use of fecal oral transfaunation as adjunct treatment in horses with acute colitis and diarrhea
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.

Evaluation of the intestinal microbiota in horses with gas colic
Scott Weese, Professor, Pathobiology, DVM, DVSc Guelph; Dipl ACVIM

Colic is a leading cause of illness and death in horses, and root causes are poorly understood. One important factor in development of colic is thought to be excessive gas production or accumulation in the intestinal tract, which can result in colic itself or potentially lead to life-threatening displacement or torsion of the colon. The horse’s intestinal tract harbours a large, complex and diverse microbial population (the microbiota) that plays critical roles in health and disease. Gas production by intestinal bacteria is likely a critical factor in development of gas colic, but the role of the microbiota in colic has not been adequately evaluated. A recent study by the applicant identified a group of ‘indicator’ bacteria that were evaluated with risk of, or prevention of, large colon torsion in postpartum mares. This was the first study to identify changes in the microbiota that precede disease and suggests that certain changes might be able to both predict colic and allow for development of methods to modify the microbiota to prevent colic. This study will evaluate horses that are predisposed to gas colic and evaluate their fecal microbiota before, during and after colic episodes, as well as compare them to horses that are not predisposed to colic. This study will provide important new insight into this important equine disease and provide information needed for development of new preventive methods.

Evaluation of the seroprevalence of Borrelia burgdorferi and Anaplasma phagocytophilum in Ontario horses
Scott Weese, Professor, Pathobiology, DVM, DVSc Guelph; Dipl ACVIM

In recent years, there has been a dramatic change in the range of ticks and the diseases they carry in Ontario. Climate change is a likely driving factor and climate and tick range models indicate that this trend will continue, with steady increases in ticks, even in northern regions. Unfortunately, as ticks move into an area, tick-borne diseases accompany them. Lyme disease caused by Borrelia burgdorferi, a bacterium that has a life cycle involving small mammals (e.g. mice), large mammals (e.g. deer) and Ixodes scapularis (called the black legged or deer tick). Ticks become infected when feeding from an infected animal, then can pass the bacterium on when feeding on another animal, including horses. In recent years, Lyme disease has been increasingly identified in humans and dogs in Ontario, with anecdotal infection indicating infections in horses. Since Lyme disease can be quite variable, often vague in nature, but potentially serious, compromising the health and performance of affected horses, understanding of this disease in Ontario horses is needed. Another tick borne bacterium, Anasplasma phagocytophilum, is also of concern. It is also transmitted by Ixodes scapularis, and an infected horse was identified in eastern Ontario in 2015. It is unclear whether that was a rare infection, a more common problem that has gone undiagnosed or an indication of movement of this disease into Ontario. This study will evaluate the presence of antibodies against these two tick borne bacteria in horses from across Ontario. The presence of antibodies indicates the horse has been exposed sometime in the past. Identification of how common antibodies are and factors that are associated with the presence of antibodies (e.g. region of Ontario, access to woodlands, travel) will provide important information about these diseases. It will also investigate the relevance of finding antibodies against B. burgdorferi in healthy horses. It is unknown whether there is any clinical relevance of this; whether it denotes previous exposure that has been controlled, whether it indicates the potential that disease is developing or whether there is a risk of subsequent complications. This is important for understanding how to interpret any screening results and to help determine when testing is appropriate.