2014-2015 Projects

Molecular epidemiology of organisms potentially causing diarrhea in foals: A longitudinal study to identify pathogenic genotypes with emphasis on clostridial organisms.
Dr. Henry Staempfli, Department of Clinical Studies

Collaborators: Dr Angelika Schoster University of Zurich; Dr Scott Weese OVC; Miranda Abrahams; Mohammad Jalali

Most foals experience an episode of diarrhea before 6 months of age. In this study, we collected fecal samples biweekly from normal foals, foals with diarrhea, and their dams (604 samples total) over their first four months of life. Samples were cultured for Clostridium difficile, a common bacterial pathogen isolated from foals with diarrhea. Samples that were cultured positive for C. difficile were also tested for the presence of toxins, which are thought to be required for the bacteria to cause diarrhea. Additionally ribotype strains were determined for all culture positive samples to determine genetic relationship to previously identified strains.

Mares, healthy foals, and diarrheic foals all shed C. difficile at different time points throughout the study. Foals under one month of age were significantly more likely to shed C. difficile than older foals, but foals with diarrhea did not shed more C. difficile than foals without diarrhea. All of the culture positive samples contained toxins, and 21 different ribotype strains were identified. Four of these strains have been previously identified in human C. difficile infections.

These results are significant because they highlight the continued importance of multiple pathogens in causing foal diarrhea; the fact that shedding of C. difficile was not significantly correlated with diarrhea in these foals suggests that a multitude of other pathogens may have been responsible for the diarrhea observed in these foals. Younger foals were more likely shed C. difficile, which is consistent with previous reports. Additionally, the identification of strains which cause disease in humans amongst our study population raises concerns for biosecurity and public health on equine breeding farms. Further analyses in the laboratory are currently under way to identify additional organisms present potentially causing diarrhea in foals.

Ex-vivo Pulmonary Arterial Perfusion System to model biomechanical and hemodynamic phenomena in equine pulmonary arteries
Dr Luis Arroyo, Department of Clinical Studies

Co-investigator: Bruce Guest

The behavior of catheters within the pulmonary artery was investigated using equine en bloc heart and lung preparations. An equine ex vivo heart and lung perfusion system (EVHLPS) was built and utilized to develop a blind technique for catheter placement into the distal main stem of the pulmonary artery utilizing balloon tipped catheters. A 7Fr, 200cm pancreatic sphincteroplasty catheter with a 16mm balloon was found to be an effective device to facilitate placement of a pressure measurement catheter within the pulmonary artery at the location where EIPH occurs in the horse.

The EVHLPS replicated basic aspects of equine pulmonary artery hemodynamics and mechanics. The navigation technique developed in the EVHLPS is currently being used to collect hemodynamic and mechanical data from the pulmonary arteries of live horses. Increased knowledge of the properties of the equine pulmonary may shed light on the causes of EIPH in horses. The EVHLPS will allow development of this knowledge without the use of live animal testing

Characterization of the respiratory microbiome of healthy horses
Dr Scott Weese, Department of Pathobiology

Co-investigator: Dr Gaelle Hirsch, DVSc grad student and advisor Dr. Henri Staempfli.

With advancement in testing methods, it has become abundantly clear that the body's microbiota (microbial populations) and microbiome (genetic composition of those populations) are incredibly large, diverse and critical for health. While focus has mainly been in the intestinal tract, other body sites also harbour important bacterial microbiomes.

The upper respiratory tract microbiome is a potential reservoir of various pathogens (e.g. Streptococcus equi) that can cause disease in the host or be spread to other horses. Its composition may play a key role in determination of whether an animal becomes infected or a carrier. Further, while the lower airways are often considered to be sterile or only transiently contaminated, evidence from humans indicates the presence of a diverse microbiome that plays a critical role in both development and prevention of disease. Understanding the respiratory microbiome is crucial for understanding the airway's normal protective mechanisms, how bacterial populations change in response to disease, how microbiome changes may cause disease and how microbiomes can be altered to prevent and treat disease.

This study will use advanced molecular and bioinformatics methods to characterize the equine respiratory microbiome, describe and compare the bacterial compositions of different locations in the respiratory tract, and determine the variability between different sites and between horses that are in active training versus those that are on pasture. With a better understanding of the respiratory bacterial population, studies investigating the role of this population in various diseases, as well as studies regarding treatment and prevention of disease, will be possible.

The effect of a newly designed probiotic on prevention of diarrhea and fecal pathogen shedding of foals during the first two months of life
Dr. Henry Staempfli, Department of Clinical Studies

Collaborators: Dr Angelika Schoster University of Zurich; Dr Scott Weese OVC; Miranda Abrahams; Mohammad Jalali

Acute diarrhea in foals is an important disease complex causing large economic losses to the equine industry. Up to 80% of foals have an episode of diarrhea in their first 6 months of life. Causes are often multifactorial; however, infectious agents, particularly Clostridium difficile and C: perfringens may play an important role. Preventative measures are currently limited to standard infection control practices and novel approaches are needed. Probiotics have received increasing interest in human and veterinary medicine to prevent and treat enteric disease. Probiotics exert their beneficial effect through various pathways, including production of antimicrobial compounds targeting intestinal pathogens, as well as general immune stimulation and colonization resistance.

Over the counter probiotics often do not contain the promised ingredients and peer reviewed clinical studies proofing the claimed effects are not available. Few studies in horses have been performed to evaluate the efficacy of home-made probiotics but promising results have been reported. Recently, several commercial bacterial strains (Lactobacillus rhamnosus LHR 19 and SP1, L. plantarum LPAL and BG112 and Bifidobacterium animalis lactis) produced for the human food industry have been shown to inhibit growth of C. difficile and C. perfringens in vitro. These strains were also able to grow in the presence of acid and bile, making them suitable for further evaluation as animal probiotics.

These strains should be evaluated in a placebo controlled clinical trial for their ability to decrease the incidence of foal diarrhea and reduce fecal pathogen shedding, particularly of C. difficile, C. perfringens.

A Immuno-histochemical staining of adrenergic and cholinergic fibers in the equine sympathetic and parasympathetic nerves of the lower and upper airways in 6 horses
Dr Laurent Viel, Clinical Studies and Aitor Gallastegui

The current understanding of the role of innervation in upper and lower equine airway disease in horses is limited even though there is a significant impact of respiratory disease upon equine athletic performance. Attempt to correct surgically recurrent laryngeal neuropathy (roarer) in horses has some short-term benefit but has also a high relapse rate.

Recently Vanschandevij et al. (2011) demonstrated the benefit of using electro stimulation of some nerve branches of the recurrent nerve with some very promising results. Nevertheless one of the difficulties of those studies in the horse has been convincing identification of the nerve branches as to whether the branches are totally sympathetic and parasympathetic autonomic innervation. A recent anatomical study in our lab intended to accurately describe the cervical and thoracic autonomic neural networks that supply the airways in horses. The autonomic network innervating the airways was dissected in 10 horses. In this group of horses studied, it was identified that sympathetic and parasympathetic nerves intermingled mainly at the thoracic region.

In addition, the autonomic nervous supply was provided to the lower airways principally by branches from the vagus nerve (a parasympathetic nerve). The latter finding questioned the anatomic presence of sympathetic nerve branches needed to maintain bronchodilation of the airways. However the precise fibre type causing bronchodilation or constriction within the respiratory tract of horses has not been determined. We hypothesize that adrenergic fibers frequently reach the airways within the vagus nerve branches, and that some cholinergic fibers also travel within sympathetic nerves.

The proposed study will selectively identify these fibers at different levels of the autonomic neural network that supply the airways in horses. Describing the physical pathways of these fibers in horses will improve the understanding on equine airway physiology, and establish a basic knowledge necessary for the development of new diagnostic and therapeutic tools in the field of the equine neuro stimulation.

Calibration and assessment of capsule-endoscopy technology as a diagnostic tool for imaging the small intestine of the horse
Dr. Jeff Thomason, Department of Biomedical Sciences

Co-investigators: Dr. Judith Koenig, Dr. Heather Chalmers, Diane Gibbard, Grad Student, Dr. Laura Frost, Dr. Ken Armstrong, Warren Armstrong, Technical Support at Halton Equine Veterinary Services.

Horses are at a high risk of developing gastrointestinal tract (GIT) diseases such as ulcers and colic that are often undiagnosed given the limitations of available diagnostic tools (i.e. gastroscopy and rectal examinations). The horse has a very large GIT and it is a physical challenge to reach and view sections of the GIT without the use surgery.

Capsule endoscopy (CE) is a new diagnostic tool that has been successfully used in human medicine to help diagnose gastrointestinal disease by collecting images of the GIT as the capsule is traveling through it. The first two objectives of this study are to develop modifications to the human CE device and a procedure for tracking it, in order to image the small intestine of the horse.

After 4 trials on each of 2 live horses, we found that the size of the animal prevented continuous imaging of the small intestine. The images that were obtained were of very good quality (showing small lesions in the gut wall and even showing a few small pin worms in one.

K Molecular mechanisms of prostaglandin-induced embryonic loss in mares
Dr Keith Betteridge, Department of Biomedical Science

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

We study how conceptus attachment to the equine uterus proceeds or fails between Days 12-21 of gestation while the conceptus is still enclosed in a capsule. We collect conceptuses and endometrium of defined ages transcervically and analyse them for important changes in proteins, cytokines and steroids. We are most interested in changes that help to explain the differences between successful and unsuccessful early pregnancy during a period of frequent early embryonic loss in mares. Normal pregnancies are compared with pregnancies that fail after mares are injected with cloprostenol, an analogue of prostaglandin that induces luteolysis. Our most recent findings confirm that prostaglandin treatment at Day 18 has a direct and immediate harmful effect on the conceptus.

In the proposed studies, we will compare the temporal and spatial changes in the production of estrone sulfate and other potential signalling factors produced by the trophoblast of healthy and failing conceptuses. In addition, changes such as expression of attachment and nutrient transfer factors indicative of a successful endometrial response will be further characterized. These assays will employ immunohistochemical and proteomic analyses and a recently available horse gene expression microarray to compare changes in samples collected via our established protocols.

K Molecular mechanisms of prostaglandin-induced embryonic loss in maresCandidate markers for success or failure will be confirmed by quantitative RT-PCR and protein identification using mass spectrometry or immunoassays. 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.

Electro arthrography for non-invasive on-farm assessment of fetlock joint cartilage health
Dr Mark Hurtig, Department of Clinical Studies

Co-investigators: Dr. Adele Changoor, Dr. Mohamed Hoba, MSc candidate, Dr. Karen Gordon, Dr. Don Trout, Dr. Lance Bassage.

Cartilage has unique biomechanical properties resulting from interactions among extracellular matrix components, consisting of hydrated proteoglycan trapped in a collagen network, and interstitial fluid. During compression, cartilage produces electrical signals, known as streaming potentials, due to negatively charged functional groups on proteoglycan1. Streaming potentials reflect cartilage composition and structure and are more sensitive to cartilage load bearing properties than purely biomechanical measurements2-4. A new method called electroarthrography (EAG) measures streaming potentials non-invasively5 and has the potential to become a clinical tool that may contribute to the diagnosis and treatment of degenerative joint diseases.

This study aims to develop a diagnostic EAG method suitable for on-farm assessment of fetlock cartilage. Specific objectives include correlating EAG with direct macroscopic, biomechanical, histological and biochemical measurements of cartilage properties, and comparing EAG to current clinical assessments of joint health.

We hypothesize that (1) EAG signals are strongly correlated to cartilage composition and load bearing properties, and (2) EAG can consistently distinguish early cartilage degradation from more advanced erosion.

Testing hypothesis (1) will involve experiments in fetlock explants where EAG obtained during simulated joint loading is compared to direct assessments of normal, enzymatically degraded, and osteoarthritic cartilage.

Testing hypothesis (2) will require clinical application of EAG in normal horses and those exhibiting clinical signs of joint disease.

An EAG-based diagnostic would provide veterinarians with a sensitive tool for monitoring joint health. Detecting cartilage degeneration at treatable stages may prevent horses from being lost to joint disease and may lower costs associated with training these animals.

Effect of allogeneic umbilical cord blood mesenchymal stromal cells on induced synovitis in horses
Dr Judith Koenig, Department of Clinical Studies

Co-investigator: Thomas Koch, DVM, PhD, Dept. of Biomedical Sciences

Currently the focus of systemic and intra-articular therapies involves slowing the progression of osteoarthritis since reversal of established osteoarthritis has yet to be demonstrated.

In equine joints with naturally occurring disease intraarticular administration of both, adipose derived and bone marrow derived, MSC were reported to improve lameness in horses unresponsive to conventional treatment. Recently, a comparable physiologic response to the intra-articular injection of autologous and allogeneic stem cells was reported in the horse. This suggests there may be potential for developing frozen allogeneic stem cell products that could be available for treatment of the equine athlete at the time of diagnosis of injury. Therefore, our hypothesis is that allogeneic umbilical cord blood mesenchymal stromal cells have an anti-inflammatory effect in induced synovitis in horses.

The first part of the study will evaluate the in vitro anti-inflammatory potential of allogeneic umbilical cord blood mesenchymal cells. For the in vivo study, inflammation will be temporarily induced with endotoxin in the midcarpal joint of 6 horses, and allogeneic umbilical cord blood mesenchymal cells will be injected and with repeat synovial fluid samples the effect of the MSC on various joint parameters evaluated.