2011-2012 Projects

Etiologic agents and their interaction with macrophages in heaves
Dr. Dorothee Bienzle, Department of Pathobiology

Inflammatory airway disease (IAD) and recurrent airway obstruction (RAO, or heaves) affect horses of different ages. Both conditions lead to inability to function as performance or pleasure horse, and result in loss of revenue. The cause of both conditions is thought to be chronic exposure to dusty environments, and the disease may be worsened by concurrent viral infections.

The role of other infectious agents in IAD and RAO is unknown. Exposure to dusty environments is inevitable for horses stabled indoors, and results in inhalation of fungal spores and bacterial components. Spores from molds that reach the lower airway of horses are taken up by macrophage cells, which starts inflammation. Previously, we showed that horses with RAO had exacerbation of disease if they inhaled a combination of dust and molds. We therefore know that experimentally, inhalation of molds can cause lung inflammation, but whether molds or bacterial components are present in the lungs of naturally affected or healthy horses is unknown. Further, the effect of mold spores and bacteria on macrophages is not understood.

Therefore, the goals of this study are 1) to collect lung washes form horses with naturally occurring RAO, and from horses with no lung disease; 2) to identify the molds by culture and PCR assays; 3) to measure a key bacterial component' and 4) to culture lung wash fluid with macrophages to determine whether these interactions initiate inflammatory responses.

Results from these studies will define whether presence of fungal spores in the lung is associated with RAO, and whether fungal spores can initiate inflammation.

Learn more about: respiratory research.

Evaluation of techniques to improve stem cell homing in the horse
Dr. Judith Koenig, Department of Clinical Studies

Tendon injuries are common in the equine athlete leading to initial loss of training days and significant economic losses. Upon return to strenuous exercise the repaired tendon often breaks down again due to the inferior quality of the repaired tendon. At this time the horse is often retired from athletic performance. Stem cells in combination with soluble biologics injected into a tendon lesion seem to improve the quality of the repaired tendon.

Currently, stem cell therapy does not return the horse to training and racing faster than conventional treatments, but appears to reduce the number of horses sustaining re-injury. The cause of this enhanced healing is unknown. It could be due to the stem cells themselves or be due to growth factors contained in the solution the cells are suspended in prior to injection. Some growth factors, like transforming growth factor ?, appear to stimulate stem cells to transform into different tissues. In addition, extracorporeal shock wave treatment has shown to accelerate differentiation of stem cells into different tissue types.

In an experimentally bowed tendon in horses, shock wave treatment increased the concentration of transforming growth factor ? and healed the tendon faster, which may be caused by an increased influx of stem cells. Similarly, when a bone defect was created in the thighbone of rats, and shock wave was applied, significantly more labelled stem cells settled into the defect ('homing') and exhibited faster healing compared to untreated bone defects.

Very little is known about homing of stem cells in the horse, and if it is possible to use shock wave treatment to attract stem cells to a tendon lesion (similar to other species); therefore, this study is designed to evaluate how equine stem respond to shock wave in vitro and if they grow faster into the different cell types then untreated stem cells. If successful in vitro, we will evaluate the use of shockwave to accelerate the differentiation of stem cells into tendon tissue in horses with experimental tendonitis in vivo.

Metagenomic characterization of the equine intestinal microflora
Dr. J Scott Weese, Pathobiology

The intestinal tract contains one of the most dense, dynamic and complex bacterial populations of any environment on the planet, containing up to 1000 different bacterial species at concentrations of 1 -100 trillion bacteria per gram. This microflora plays a critical role in health and disease, helping prevent disease caused by numerous potentially harmful bacteria that are regularly ingested and playing a critical role in digestion. Increasingly, effects on other areas such as immune function, inflammation and weight gain are being identified.

Disruption of the intestinal microflora most notably occurs with colitis (severe diarrhea), but may also result in some types of colic, laminitis, obesity, inflammatory bowel disease, metabolic diseases and allergic disorders. However, despite the clear importance of the intestinal microflora, our understanding of what constitutes 'normal' and 'abnormal' is woefully inadequate. It is estimated that a large percentage of bacteria in the equine intestinal tract are species that have not been identified, and many may not be culturable using standard culture methods. Studies relying on culture provide only a superficial assessment of the microflora by investigating a small subset of known, culturable organisms.

Only recently has technology become available to comprehensively assess the intestinal microflora, including assessment of unknown and uncluturable bacteria. This study will use next generation gene sequencing to provide the most comprehensive study of the equine gastrointestinal microflora, and to allow us to more confidently determine the 'normal' microflora and determine whether this differs between different types of horses.

An investigation of alternative methods of dehydration and cryoprotectant addition for equine embryos
Dr. Tracy Chenier, Department of Population Medicine

Embryo transfer in horses continues to be under-utilized as it is inefficient and expensive.

Current limitations mean that most embryos are transferred fresh upon flushing, and large recipient herds must be maintained to meet this need. Freezing horse embryos has met with limited success. Pregnancy rates with day 7-8 embryos are at or near 0% in most studies. Embryos tend to explode on thaw, evidence that current freezing methods do not remove enough water, and lethal ice formation results. Recent studies show that protective compounds such as glycerol which are present in freezing solutions do not cross into the horse embryo in high enough levels to prevent ice from forming.

This Study will evaluate alternative methods of both water removal and delivery of protective compounds inside the equine embryo. If successful, these methods will solve both the problem of inadequate water loss and low penetration of protective chemicals. Both of these factors are thought to be significant reasons for low pregnancy rates seen with frozen-thawed horse embryos.

Arrythmia and sudden death in Thoroughbred racehorses
Dr. Peter W. Physick-Sheard, Department of Population Medicine

Sudden death in a racehorse is distressing for everyone involved in racing. It raises animal welfare, economic, and safety concerns, and represents horrendous public relations for the industry. As for human athletes, neither circumstances surrounding the episode nor findings at post-mortem necessarily provide information on underlying cause. In these cases the term 'sudden cardiac death' (SCD) is applied. It is assumed the primary cause is serious disturbance in heart rhythm, but evidence is lacking because affected animals' ECG's are not being monitored at time of death.

In an attempt to better understand causes, a study was recently performed in Standardbred racehorses in which heart rhythm was followed from harnessing until the end of the race during normal competition. Disturbances in rhythm immediately after the race were identified that would be capable of causing a fatal outcome, but there were no deaths. Clear indications of strategies that might reduce risk of a fatal outcome were noted, however. SCD associated with racing occurs more commonly in the Thoroughbred than in the Standardbred, and it is possible arrhythmias are taking place here also. To resolve this question, heart rate and rhythm will be monitored during normal competition in Thoroughbred horses, from saddling to unsaddling and into early recovery.

The objectives will be to characterise the range of usual rate and rhythm variations, and to provide guidance as strategies to minimise risk are developed.

Assessing post-operative outcomes in horses treated for dorsal displacement of the soft palate
Dr. Heather Chalmers, Clinical Studies

Affecting between 10-20% of 2 -3 year-old racehorses, dorsal displacement of the soft palate (DDSP) is a condition resulting in upper airway obstruction, noise, and inability to perform at peak exercise1. Towards the end of a race, a palatial displacement can cause a horse to pull up suddenly and be unable to complete the race.

There are many treatments for DDSP, characterized as non-surgical (eg. tack modifications) and surgical. The two most common surgical treatments are 1) the laryngeal tie forward and 2) the myectomy (Llewellyn) procedure. 1 The choice of surgery depends on factors both intrinsic (age, breed, value) and extrinsic (availability, clinician preference) to the horse. Importantly, the efficacy of these surgeries has never been assessed in clinical patients and the relative outcomes have never been compared. This gap in knowledge is likely because traditionally DDSP is only accurately diagnosed by endoscopic exam during treadmill exercise, which limits its practicality.

Recently, two additional tools have become available to diagnose DDSP in horses – 1) ultrasound of the upper airway2, performed stall side and 2) over-ground wireless endoscopy, performed at the race track. We propose to assess horses following surgery for DDSP at the trackside using both ultrasound and the new dynamic respiratory endoscopy system.

The goal of this study is to evaluate the post-operative outcomes of horses that underwent surgery for DDSP, to establish if these treatments reduce the incidence of DDSP during exercise, and to compare the relative success of the two surgeries. We anticipate that this novel information will greatly improve our ability to make treatment and prognostic recommendations for affected horses.

An experimental model of duodenitis-proximal jejunitis in horses challenged with Clostridium difficile toxins. A preliminary study.
Dr. Luis Arroyo, Department of Clinical Studies

Duodenitis-proximal jejunitis (DPJ) is an acute disease in horses clinically characterized by signs of colic, depression, fluid accumulation in the small intestine and stomach, ileus, and endotoxaemia. The cause remains unknown; but Clostridia spp., Salmonella spp. and mycotoxins have been suggested. Although Salmonella spp. have been sporadically isolated from horses with clinical signs of DPJ, the histopathological lesions observed in the small intestine of such cases do not resemble those described for salmonellosis. Mycotoxins fail to produce clinical disease in experimentally challenged horses, although histopathological lesions appeared to be similar to those described in DPJ cases. A clostridial cause had been suspected because a large number of Gram-positive rods are observed on nasogastric reflux samples. Additionally, histopathological lesions in affected horses are similar to those produced by Clostridium spp toxins. However, specific clostridial species have not been consistently and repeatedly isolated from affected horses.

C. difficile was isolated from gastric contents of 10/10 horses suffering from DPJ at the OVC, and it was proposed that this enteropathogen may be an etiologic candidate. Although C. difficile is mainly recognized as a cause of colitis in horses and humans, it has also been associated with severe lesions in the small intestinal mucosa of foals naturally infected or experimentally challenged with C. difficile. It has also been isolated from the small intestine of horses and ponies affected with various small-intestinal disorders. It had been demonstrated that C. difficile toxins (A and B) can profoundly affect the motility of the small intestine.

We hypothesize that C. difficile toxins can disturb small intestinal motility in horses leading to ileus with subsequent development of clinical signs of DPJ.

The objective of this study is to experimentally reproduce clinical signs of DPJ by inoculating C. difficile toxins into the small intestine of horses.

Genome-based vaccination against Rhodococcus equi
Dr. John F. Prescott, Department of Pathobiology

The pathogenic bacterium Rhodococcus equi is recognized as a major cause of mortality in horses between 1 and 5 months of age and is endemic in many breeding farms. There is currently no vaccine to prevent the infection. We recently published the complete genome sequence of a foal clinical isolate of R. equi.

This proposal aims at using the genome information as framework for the rational development of a R. equi vaccine.

Two strategies will be used. In one we will use functional genomics to identify R. equi genes involved in infection. These genes will be removed from the R. equi genome. Mutant strains showing attenuation in a mouse model of lung infection will be tested as live vaccines for protective efficacy. The second 'reverse vaccinology' strategy involves the identification of immunodominant antigens using sera from naturally infected foals. The corresponding genes will be identified in the genome, their products expressed in vitro and purified, and tested for protective efficacy in vaccine trials in mice.

Funds for this translational research project are requested to support for two years the work of a Canadian PhD student at the University of Edinburgh, where R. equi post-genomic research is being coordinated. The student originates from Dr. Prescott's lab (MSc, University of Guelph, 2009) at the Ontario Veterinary College, and the exchange will strengthen the collaboration between two laboratories.