Hyperimmune plasma transfusion for the prevention of R. equi infections in foals
Dr. Luis G. Arroyo | Associate Professor | Lic. Med. Vet., DVSc, PhD, DACVIM

Rhodococcus equi causes severe pneumonia in foals worldwide as well as other forms of extrapulmonary infections that are also severe. Foals acquire the infection shortly after being born which makes prevention of this disease difficult. Administration of R. equi-specific hyperimmune plasma (HIP) intravenously is the only form of prophylaxis available, as a vaccine does not currently exist. While it does not prevent infection, HIP administration has been shown to decrease severity of pneumonia in newborn foals experimentally challenged, although the protective mechanism of HIP remains poorly understood. The aim of this study is to determine the clinical usefulness of HIP administration under field conditions in Canada, as this has not been previously evaluated.

The association of Equine Parvovirus-Hepatitis (EqPV-H) with Theiler’s disease and the prevalence of EqPV-H DNA and antibodies in horses in Ontario.
Luis G. Arroyo | Associate Professor | Lic. Med. Vet., DVSc, PhD, DACVIM

Theiler’s disease (TD) (aka: equine serum hepatitis) is an acute or subacute hepatitis of horses, and is one of the most commonly recognized causes of acute hepatic failure in adult horses. The disease is sudden in onset and rapidly progresses with death occurring within 24-48 hours. Up to 80% of affected horses show signs of hepatic encephalopathy (head pressing, aimless walking, yawning). Horses that develop hepatic encephalopathy have a grave prognosis. The majority of cases occur 4-10 weeks after the administration of an equine biologic (“biologic-associated TD”) with the most common biologic being tetanus antitoxin.

The administration of equine serum or plasma has also been implicated. In some cases, it has been observed there are horses affected that have not been treated with any equine biologic (“non-biologic associated TD”), and this led to the suggestion that an infectious and possibly contagious agent may be involved. A virus similar to the human hepatitis viruses was considered to be the most likely cause. Since 2011, three novel flaviviruses have been reported to be associated with equine hepatic disease. These include non-primate hepacivirus (NPHV), also known as equine hepacivirus (EqHV), equine pegivirus (EPgV) and Theiler’s disease-associated virus (TDAV). Of these viruses only NPHV has been shown to be hepatotropic in the horse. In 2018, a novel equine parvovirus, designated equine parvovirus-hepatitis (EqPV-H) was identified in the serum and liver of a horse that died of Theiler’s disease 65 days after treatment with equine origin tetanus antitoxin (TAT).

This virus was also found in the administered TAT and it has been suggested that EqPV-H can cause Theiler’s disease in horses. EqPV-H had been detected in horses suffering from liver disease in Ontario, therefore, the objective of this study is to determine the prevalence of the virus in a subset of horses in Ontario.

Dynamics of vitamin D metabolites in healthy and hospitalized horses with enterocolitis
Luis G. Arroyo | Associate Professor | Lic. Med. Vet., DVSc, PhD, DACVIM

Equine enterocolitis (diarrhea, colitis) is a major cause of morbidity and mortality worldwide. Consequences of equine colitis include loss of hormonal control, metabolic/ electrolyte / fluid derangements, and organ failure. Vitamin D has a multitude of functions, including regulation of calcium and phosphorus, promotes bone health, controls the immune system, and reduces inflammation. Low vitamin D levels have been linked to mortality in sick people. There is no information on how the blood levels of vitamin D change in sick and healthy adult horses and whether these changes are associated with other hormones, inflammatory factors, and mortality in hospitalized horses with colitis. The goal of this study is to determine whether changes in vitamin D during hospitalization of horses with colitis are associated with laboratory abnormalities, disease severity and outcome.

The relationship between Exercise Induced Pulmonary Hemorrhage and exposure to ambient air pollution in Thoroughbred and Standardbred racehorses
Janet Beeler-Marfisi. Assistant Professor | BA, DVM, DVSc, Dipl. ACVP

Veterinarians and trainers at Ontario at Woodbine and Woodbine-Mohawk Park are suspicious that outdoor air pollution and colder weather are causing horses to bleed; we want to find out whether this is true or not. Exercise induced pulmonary hemorrhage (EIPH) is a very common problem in Thoroughbred (TB) and Standardbred (SB) world-wide. The condition is important to Ontario racehorses as affected TB and SB are suspected to have shortened racing careers. In rare cases, horses can suffer sudden death from massive bleeding.

The most common theory for why EIPH happens is that the tiny blood vessels in the lung rupture secondary to repeated high-intensity work. Veterinarians who regularly assess racehorses endoscopically at Woodbine and Woodbine-Mohawk Park in Ontario, Canada are concerned that their proximity, respectively, to Lester B. Pearson Airport and a major highway system is exposing racehorses to high amounts of air pollution, which might contribute to development of EIPH. No one has studied whether outdoor (ambient) air pollution contributes to the development of bleeding in the lungs, but previous research did find a relationship with cooler temperature.

Another study compared air pollution to race finish position, and while that study did not look at EIPH, the researcher did find that TB racing under hazardous ozone conditions had slower race times. Another research group conducted an experiment where horses exercising on a treadmill were exposed to ozone, and they found that this was associated with lung inflammation. These things have not been studied in SB racehorses.

The Ontario government has air quality monitoring stations and the federal government has weather stations located close to Woodbine and Woodbine-Mohawk Park. These stations collect air pollution data including nitrogen dioxide, ozone and fine particulate matter less than 2.5 microns in diameter – these are all components of smog, or air pollution. The weather station collects temperature data, and both the provincial and federal government offer free access to their databases via the internet. We think that increased smog, with or without a decrease in temperature is associated with an increased risk of EIPH in Ontario racehorses. We also think that increased smog, with or without a decrease in temperature is associated with a worse race finish position.

Do mast cells play a role in persistent breeding-induced endometritis in the mare?
Tracey Chenier. Associate Professor | DVM, DVSc, Diplomate ACT

Over 18% of Ontario’s 212,000 horses are designated as breeding stock, and reproductive health is critical to the economic success of the industry. Reproductive efficiency in horses is generally low, for a variety of reasons, including infertility caused by uterine disease such as endometritis. Endometritis is the third most common medical condition encountered overall in equine veterinary practice and approximately 60% of mares affected by endometritis are barren. One type of endometritis, persistent breeding induced endometritis, or PBIE, occurs in mares that have an abnormal inflammatory response after breeding. Up to 43% of broodmares suffer from this condition, depending upon the study and the definition used. Given the large number of horses in the breeding sector in Ontario, endometritis and more specifically PBIE, causes a significant economic impact to the breeding industry.

One type of inflammatory cell, the mast cell, may play a role in PBIE. While mast cells are generally thought of as inducing allergic reactions, they are also involved in many chronic inflammatory conditions and can induce fibrosis. Both of these are occur in PBIE-affected mares. This study will determine the number of mast cells in the endometrium of PBIE- affected and unaffected mares, and evaluate whether cell numbers change in response to artificial insemination. A better understanding of the abnormal inflammation that occurs in PBIE-affected mares will lead to new and improved treatments for this important condition that causes infertility.

The Safety and Efficacy of Intra-Articular Therapies
Mark Hurtig. Professor | DVM, MVSc, Diplomate ACVS

We intend to compare the efficacy of mesechymal stromal cells (MSCs) - previously known as mesenchymal stem cells - versus steroid injections in the equine fetlock for treatment of joint injury and osteoarthritis. This project also addresses the safety of steroid injections in joints which continue to be widely debated in the racing and sporting industry. We know from human studies that injection of MSCs result in better and longer results in the human knee than steroids. Also, preliminary studies in equine stifle and fetlock lameness seem to indicate the MSC therapies improve the recovery of the horses.

Our own work has shown that we can identify the best MSCs from the specific donors and use these cells in joints to reduce inflammation. In addition, the co-investigators in this proposal have shown that MSCs can be thawed, handled and injected without damaging the cells, and that a single injection of similar MSCs can reduce or stop the progress of osteoarthritis in the knee of sheep. Since chip fractures in the fetlock joint are relatively common source of lameness and contribute to cartilage damage, we will create a small chip fracture in the fetlock joint of research horses and follow the progress of physical and metabolic cartilage injury in horses that receive MSCs or steroids. In order make this model more aligned with real-life management of these injuries we propose to exercise the horses in the form of galloping and jumping during the evaluation of the treatments.

Enhancement of Immune-Modulatory Properties of eCB-MSCs
Thomas G. Koch. Associate Professor | DVM Copenhagen, PhD Guelph

Horses are multi-use livestock that contribute more than $19 billion annually to the Canadian economy. Joint inflammation due to osteoarthritis or injury is among the most common causes of lost training days or premature retirement in equine athletes. Stem cells hold the promise of novel therapeutic approaches to these difficult-to-treat problems. Mesenchymal stromal cells (MSCs) can be expanded in culture to achieve the desired number for treatment and are capable of modulating cells of the immune system even after long-term storage, which makes them desirable for treatment of immune and inflammatory disorders.

We expect that equine cord blood-derived (CB-) MSCs can be enhanced to have improved anti-inflammatory and immunomodulatory properties for future use as treatment of a variety of inflammatory or immunerelated disorders in horses. Our sole objective of this study is to determine possible in vitro enhancement of equine CB-MSCs using a novel proprietary culture engineering method to improve these immunomodulatory properties. The work will generate pivotal data to support the clinical evaluation of enhanced allogenic equine CBMSCs to treat joint inflammation in research horses. Upon proving the concept in research horses with induced inflammation, the enhanced equine CB-MSC would be tested in a randomized clinical trial for the treatment of joint inflammation in client-owned horses suffering from osteoarthritis.

Fully implemented, this therapy would provide a safe, efficacious, and technically simple treatment for selected inflammatory or immune-related disorders in horses.

Vitrified Equine MSC Cartilage for Cartilage Repair
Thomas G. Koch. Associate Professor | DVM Copenhagen, PhD Guelph

Horses are multi-use livestock that contribute more than $19 billion annually to the Canadian economy. Injuries to joints are among the most common causes of lost training days or premature retirement in equine athletes. Stem cells hold the promise of novel therapeutic approaches to these difficult-to-treat problems. Large cartilage defects >4cm2 are a challenge to treat, but so-called cartilage chips (1x1x1 mm) have shown significant clinical promise in human medicine, regardless of whether the cartilage was sourced from juvenile human cadavers, unrelated adult cadavers, or from the patient.

Preclinical work in the horse model has also shown promising results using cartilage harvested from the joints of horses. However, current cartilage chip methods are hampered by a limited supply of donor material, risk of disease transmission, suboptimal graft tissue, and donor site morbidity. Donor age, comorbidities, and harvest site may also negatively influence the biological potency of present cartilage and cell grafting methods.

We propose to solve these issues by using cartilage generated in the laboratory from equine cord blood mesenchymal stromal cells (eCB-MSC). We will vitrify (cryopreserve) the cartilage tissue for indefinite storage and availability as an off-the-shelf live-cell cartilage graft that can be implanted using standard surgical methods. We are seeking support to establish a robust vitrification protocol for eCB-MSC-derived neocartilage.

The work will generate pivotal data to support the clinical evaluation of cryopreserved allogenic eCBMSC cartilage chips to repair focal cartilage defects in research horses. Fully implemented, this therapy would provide a safe, efficacious, and technically simple treatment for horses as well as provide an opportunity for a Canadian biotechnology business to bank and distribute vitrified cartilage tissue in unlimited quantities to the world market.

Effect of Extracorporeal Shock Wave Therapy on Intracellular Stress and Immunomodulatory Function of Equine Umbilical Cord Blood Mesenchymal Stromal Cells
Judith Koenig. Associate Professor | Mag Med vet, Dr Med Vet, DVSc, DACVS, DECVS, DACVSMR

Electrohydraulic shock wave (ESWT) has been shown to increase proliferation and differentiation of equine umbilical cord blood derived mesenchymal cells (CB-MSCs). The mechanism of this effect is unknown. It could be the result of cell stress or alternatively, due to production of cytokines or other secreted substances. Further evaluation of this mechanism is needed to develop a combined shockwave-CB-MSC treatment to enhance tissue healing.

Equine umbilical cord blood derived MSCs will be treated in a special water bath with shockwave. For objective 1 we will determine the level of the cellular stress response in CBMSCs directly after shockwave treatment (1 hour), after one to a few population doublings (24 hours and 3 days), and after 1 passage (7 days) by measuring oxygen radicals and how many cells stay healthy or get damaged. For objective 2 we will determine whether shockwave treatment increases the secretion of inflammatory mediators over untreated CB-MSCs in vitro, which will shed insight on the mechanism behind the positive effects of ESWT and CB-MSC therapy for soft tissue injuries.

Results of this research will benefit all horses regardless of breed or sport. Better understanding the benefits of shockwave to enhance stem cell function will allow us to decide if shockwave should be used to pre-treat stem cells prior to injection into a tendon.