2010-2011 Projects

Evaluation of anatomic, inter-horse and geographic variation in the equine gastrointestinal microflora.
Dr. J Scott Weese, Pathobiology, Co-investigators: Angelika Schoster, DVSc student, Dr. Luis Arroyo, Clinical Studies

The bacterial population of the intestinal tract plays a key role in health, nutrition and disease in horses. Disruption of this population can cause diarrhea while enhancement (i.e. through administration of probiotics) could represent an effective way to prevent or treat disease.

Our ability to diagnose, prevent and treat disease is severely hampered by our superficial understanding of this complex environment. Bacterial culture has typically been used to assess intestinal bacterial populations however it is widely accepted that we only know the identity of a minority of bacterial species and cannot culture a large percentage of those.

Additionally, we have relied on fecal samples but we do not know whether these are a good indicator of what is happening in the areas where disease is actually occurring. Poor understanding of the important protective components (Lactobacillus spp) of the intestinal tract has hampered development of effective probiotics. There are clear and rather remarkable deficiencies in our basic understanding of the intestinal bacterial population in horses, something that compromises various research efforts.

This study will compare the bacteria of different parts of the intestinal tract, and in the same locations between horses. It will investigate variations over the course of a year to determine whether seasonal variation needs to be considered. It will characterize the presence or absence of selected bacterial groups between horses, within horses and over time. This study will provide critical information for interpretation of older studies, design of future studies, identification of new causes of disease and development of effective probiotics.

Arrhythmia and Sudden Death in Thoroughbred Racehorses
Dr. Peter W. Physick-Sheard, Population Medicine, Co-investigator: Kim. J. McGurrin, Clinical Studies

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 cause of death, when the term 'sudden cardiac death' (SCD) is applied. It is assumed, whatever underlying problems may be present, that the primary cause is serious disturbance in heart rhythm, but evidence is lacking because affected animals are not wearing an ECG at the time of death.

In an attempt to better understand the causes, we recently performed a study in Standardbred racehorses in which heart rhythm was followed from harnessing until the end of the race during normal competition. We identified disturbances in rhythm immediately after the race that would be entirely capable of causing a fatal outcome, but there were no fatalities, and we saw clear indications of strategies that might reduce the risk of a fatal outcome. SCD associated with racing occurs more commonly in the Thoroughbred than in the Standardbred, and there is every possibility that similar events are taking place.

To resolve this question we plan to monitor heart rate and rhythm during normal competition in Thoroughbred horses, from saddling to unsaddling and beyond, to see if similar disturbances are taking place. The objective would be to characterise the range of usual rhythm variations and provide guidance as we develop strategies to minimise risk.

The objective would be to characterise the range of usual rhythm variations and provide guidance as we develop strategies to minimise risk.

Does Type A Clostridium perfringens cause acute typhlocolitis in horses?
Dr. John F. Prescott, Pathobiology Co-investigator: Luis Arroyo, Clinical Studies

Because of their large colon and caecum, horses are particularly susceptible to acute, severe and sometimes fatal diarrheal illness. Considerable progress has been made in the last decade in understanding what used to be called 'colitis X'. In particular, a bacterium called Clostridium difficile, that has recently become very prominent as a serious problem in Canadian hospitals in older human patients treated with antibiotics, has also been shown to be a common cause of fatal colitis in horses. However, perhaps 60% of cases of fatal colitis in horses have no known cause.

We think that another clostridial bacterium, Clostridium perfringens, may be involved in fatal colitis in both adult horses and in foals, and want to find out whether this is correct. We are especially interested in strains of C. perfringens that are called type A and that have the potential to produce a newly described toxin, called Beta2 toxin, and a another toxin associated with food poisoning in people, called enterotoxin.

Our study will examine faeces from foals or adult horses with acute diarrhea (typhlocolitis) for known causes of acute colitis (Salmonella, C. dfficile) as well as for C. perfringens. We will identify these agents by culture and by the presence of toxins (C. difficile toxin, C. perfringens enterotoxin), as well as for Beta2 toxin using an ELISA we are developing for this purpose in other animal species. We will also count the number of C. perfringens per gram of faces and also examine them for the genes encoding enterotoxin and Beta2 toxin. Finally, we will sequence the genome of an isolate from fatal colitis in a foal, and try to identify any novel toxin that could affect horses. These results will be compared to faeces from age matched but healthy foals and adult horses. In this way we hope to show the association of C. perfringens with acute colitis in horses.

CD117 positive equine cord blood-derived cells – a fountain of cells suitable for equine cell-based therapies such as cartilage repair?
Dr. Dorothee Bienzle, Department of Pathobiology, Co-investigators: Thomas Koch, Department of Clinical Studies; Dean H. Betts, Department of Physiology & Pharmacology, UWO

The novel isolation of mesenchymal stem cells from equine cord blood reported by Koch and colleagues in 2007 from the Ontario Veterinary College has initiated a whole new area of equine veterinary research which is now being pursued at multiple institutions worldwide.

The long-term goal of this proposal is identification of cells suitable for novel cell-based therapies in the horse.

The objective of the proposal is to isolate cells from equine cord blood, which are superior for improving repair of focal cartilage injuries in the horse. Equine cord blood contains cells with potential to produce cartilage in a laboratory setting.

However, differences in cartilage potential were noted between different cell cultures. There is, therefore, a need to establish cell lines with a consistent and reproducible cartilage potential before these cells can be introduced into mainstream equine clinical practice.

The hypothesis of this proposal is that cord blood contains few, but very potent cells, which can be identified on a molecular level, and possess high proliferative and chondrogenic potential. The availability of well-characterized cell lines is the first prerequisite for pursuing commercialization of cell-based therapies.

Molecular mechanisms of prostaglandin-induced embryonic loss in mares
Dr. Keith J. Betteridge, Biomedical Sciences, Co-investigators: M. Anthony Hayes and Brandon Lillie, Pathobiology; Luis Arroyo, Clinical Studies; James I. Raeside, Biomedical Sciences

We study how the horse embryo attaches to the endometrium (lining of the uterus) during the critical third week of pregnancy. At this stage the embryo is still enclosed in a 'capsule' and is known as a conceptus. We collect conceptuses of defined ages and small pieces of the uterine lining (endometrium) through the cervix from normal pregnancies, and also from pregnancies that have been 'compromised' by injection of the mare with a hormone to induce pregnancy failure. We then characterize important changes in proteins, steroid hormones and other molecules that change in the conceptus and uterus to identify those that best explain success and failure of pregnancy.

We have already found several molecules that help us understand how the embryo exchanges signals with the mare and becomes attached. Recent availability of the horse genome and various new discovery tools such as gene expression arrays mean that we can now examine these events in a more comprehensive manner. Having developed methods for sampling tissues and fluids in both the conceptus and its uterine environment, we can now analyze them for the presence and absence of our target molecules in normal and failing pregnancies and assess the significance of differences that are recognized.

The work will help explain conceptus interaction with the endometrium that is essential to pregnancy maintenance and which, when disrupted, results in pregnancy failure. This will be key to the development of diagnostic tests of reproductive health and, possibly, to new treatments for infertility.

Intravascular Assessment of the Pulmonary Artery Haemodynamics: A pilot study.
Dr. R John Runciman, School of Engineering; Luis Arroyo, Department Clinical Studies, Co-investigators: Laurent Viel, Department of Clinical Studies; Alexander Valverde, Department of Clinical Studies

Approximately 80% of racehorses can be expected to develop Exercise-Induced Pulmonary Haemorrhage (EIPH) during their career. The management and treatment of EIPH have a substantial economic impact to the equine industry, with the cost of treating EIPH estimated to exceed $100 million annually in the United States alone.

There is currently a poor understanding regarding the cause of EIPH and potential mechanisms include capillary stress failure, pulmonary fibrosis, and small airway disease. Currently, the most accepted cause for EIPH is exercise-induced pulmonary hypertension, resulting in pulmonary capillary rupture.

Recently we observed the presence of calcification and fibrosis of the pulmonary artery wall in a large proportion of racehorses. Such lesions are known to interfere with elasticity that the vessel needs in order to accommodate the cardiovascular pumping action of the heart during strenuous exercise. Indeed, arterial calcification is the most important cause of vascular stiffness in humans and is considered a predictive factor for cardiovascular mortality, coronary morbidity and fatal stroke.

Similarly, we suspect that the compliance of the pulmonary arterial wall of horses with artery calcification is affected. This impaired compliance will directly affect the capacity of the vessel to deal with large fluctuations in blood pressure, which may in turn cause the downstream microvasculature damage seen with EIPH.

We hypothesize that the pulmonary artery compliance of horses with calcified arteries is impaired. This study will establish a protocol on how to catheterize the pulmonary artery and measure the relative stiffness of the pulmonary arterial tree.

Histopathological pulmonary lesions in actively racing horses submitted for Post-Mortem examination in the Ontario Death Registry; an investigation into Inflammatory Airway Disease prevalence.
Dr. Laurent Viel, Clinical Studies, Co-investigators: Dr. Federica ter Woort, Dr. Luis Arroyo – Department of Clinical Studies

Inflammatory Airway Disease (IAD) in horses results from an inflammatory response of the lungs to an unknown stimuli, although it is commonly referred to as an allergic airway disorder.

Many studies document the exaggerated response to inhaled particulate, including dust and mold spores from feed and bedding as a major component of IAD. IAD has been recognised as a different condition from heaves or Recurrent Airway Obstruction, since it primarily affects young athletic horses in training and racing. At the present time, it is speculated in both the scientific literature and lay press that IAD may contribute to as much as 50-60% of coughing horses and subsequently poor athletic performance.

The Ontario Racing Commission Death Registry program examines between 110-125 horses per year which have been euthanized based upon humane grounds or which have died suddenly.

Hence, this study proposes to examine a total of 150-200 horses for the prevalence of airway pathology and to describe semi-quantitatively the observed pathology lesions. The study results should provide a relatively accurate indication of the disease prevalence as well as the severity of the lesions associated with IAD. This knowledge may contribute to future recommendations and regulatory changes for treatment options for airway disease in racing horses.

Is blood doping detectable with hemoglobin assays?
Dr. Dorothee Bienzle, Department of Pathobiology

Erythropoietin (EPO) is a small glycoprotein hormone produced by specialized cells in the kidney. The hormone stimulates production of red blood cells (RBC) in the bone marrow, which in turn increases the number of RBC and the amount of hemoglobin in blood. A higher concentration of hemoglobin correlates to greater ability to transport oxygen, and therefore enhanced performance in human, equine and other athletes.

Synthetic human EPO (rEPO) is available in various formulations, and if given to horses increases hemoglobin production. All forms of rEPO are difficult and expensive to detect since only small amounts are administered and they remain in circulation only very briefly. An alternative to direct measurement of rEPO is to evaluate the effect on hemoglobin concentration. This approach has been successfully instituted in many human sports jurisdictions, and typically comprises multiple measurements of hemoglobin in human athletes before, during and after competition.

Through this proposal funds are sought to establish 'normal' hemoglobin concentrations in horses before and after race performance, with the intent to use this information to establish limits for permissible racing.