Investing in Discoveries

Your Gifts at Work

Each year, OVC Pet Trust invests nearly $500,000 into projects and equipment to benefit companion animal health and well-being. Funded projects aim to improve the diagnoses and treatments of a wide variety of health conditions in pets. From using stem cells to understand canine epilepsy; examining cartilage cells to develop better treatments for joint injuries; and investigating predictors of how well dogs with bone cancer will do after treatment; the questions our researchers hope to answer are ones that may have benefits for pets and humans alike. 

Your support helps us invest in discovery, and train student veterinarians and specialists who have gone on to careers in private practice and at universities across Canada and around the world.

 

Spring 2016 OVC Pet Trust Funded Projects
 

 

The effect of the concentration of blood thinners in the blood on platelet function tests

Various diseases can cause excessive blood clotting in dogs, resulting in thromboembolism (formation of clots and stroke-like events), which is often fatal. In dogs, the blood thinners aspirin and clopidogrel are used in an effort to decrease the risk. Those drugs blocks platelets, which are cells responsible for the first stage of clotting. Effectiveness of these drugs may vary, therefore monitoring platelet function is recommended to ensure the drugs are working. In a previously funded Pet Trust study, research results were successful in showing that three platelet function tests could detect aspirin and clopidogrel effects in dogs and were therefore potentially useful to monitor platelet function in dogs treated with those drugs. However, dogs’ individual results varied considerably and many of the tests did not detect changes in every dog. It is not known if this represents individual difference in absorption of drugs from the intestinal tract, individual differences in platelet response to those drugs, or if a drug carryover effect was present. To address these questions and help in the interpretation of the results, individual dogs’ drug levels will be indirectly assessed by measuring drug metabolites using high-performance liquid chromatography. If drug levels correlate with platelet function test results, the tests may have utility in adjusting drug doses.    

 

Understanding the link between inflammation and blood clotting in dogs with clinical disease

Canine immune mediated haemolytic anaemia (IMHA) has a high fatality rate, with mortality rates ranging up to 70% in some studies despite aggressive immunosuppressive therapy and supportive care. Autoimmune haemolytic anaemia (AIHA) is a disease that affects humans, and is similar to canine IMHA.

The immune system abnormalities of AIHA are becoming well defined, and this increased information is leading to novel treatment strategies. In autoimmune diseases in people, there are excess numbers of certain immune cells and their cellular products (cytokines). Cytokines are produced normally by cells in the body, and are especially important in modulating the normal immune system. Imbalances in the cytokines can lead to immune-mediated / autoimmune diseases. New treatment strategies are used to target these abnormalities in the immune system. For example, antibodies have been developed to attack the excessive immune cells or cytokines that are characteristic in such diseases.

Similar advances have not yet been made in defining the mechanisms responsible for creating IMHA in dogs. This project aims to describe the cytokine levels in dogs with IMHA in the acute treatment period. Cytokine levels will be compared to those of healthy dogs. By describing the cytokine abnormalities in dogs with IMHA, potential treatment targets could be uncovered. This will help lead to more effective treatment strategies and better survival of these patients.

Antibiotic resistance in bacteria from dogs

Antibiotic resistance is an increasing problem in bacteria from animals, in particular resistance to a very important group of antibiotics called extended-spectrum cephalosporins (ESC). Fecal bacteria are the main cause of urinary tract infections (UTIs) in dogs and the majority of the bacteria causing these infections belong to the Enterobacteriaceae family. Unfortunately, we don’t have recent data on the prevalence of antibiotic resistance in these bacteria and on the frequency of carriage of ESC-resistant Enterobacteriaceae in dogs in Ontario. This project will help to fill this gap.

Fecal samples from healthy dogs will be collected for this project at a variety of locations in Ontario and investigated for the presence of resistant Enterobacteriaceae in two different ways. First Escherichia coli (the main species among intestinal Enterobacteriaceae and leading cause of canine UTIs) will be isolated directly from these feces and tested for resistance to antibiotics commonly used to treat UTIs. Second, a more sensitive detection method based on enrichment cultures will also be applied to these fecal samples to detect ESC-resistant bacteria. Overall, this project will provide an estimate of antibiotic resistance frequencies in the fecal reservoir of bacteria which is the source of UTIs in dogs in Ontario and thus help to orient and adjust treatment options in the face of increasing resistance frequencies. The project will also allow us to assess the frequency of dogs carrying Enterobacteriaceae resistant to the ESCs in Ontario. Finally, by comparing ESC-resistant and susceptible Enterobacteriaceae in dogs, the project will help orient prudent antibiotic use guidelines to avoid co-selection of ESC-resistant bacteria when using other antibiotics to which they are also resistant.

 

Development of a novel minimally invasive lung lavage technique in cats
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Comparison of bronchoalveolar lavage suction techniques to sample feline lower airways

Cats are commonly taken to a veterinarian for respiratory diseases. A diagnostic test that may be recommended is a bronchoalveolar lavage (BAL), where a small volume of sterile fluid is instilled into the lungs via the windpipe, and then suctioned back out. The fluid is then examined for the presence of cells and bacteria. Reasons for performing a BAL include coughing and changes on chest x-ray images that indicate lung disease.

Our team recently developed a new BAL technique (F-BAL) that uses fluoroscopy (video x-ray) to direct location of sampling with a soft catheter. Compared to the standard technique, patients have their airway protected during the procedure and the use of an expensive bronchoscope is not required. F-BAL was found to be safe, effective and accurate for sampling the caudal lung lobes in 12 healthy cats in a Pet Trust funded Winter 2015 study. The next stage in development of this technique is to evaluate its use in patients with lung disease and correlate the BAL findings with imaging findings. 

X-rays are known to be less sensitive than CT for detecting subtle lung lesions and many patients with clinical respiratory disease have normal chest x-rays on presentation. This was also evident in the above-mentioned study where CT identified lung disease in cats that did not have any evidence of respiratory disease based on history, physical examination and chest x-rays. Until recently, CT in cats was performed only under general anesthesia limiting its use. With faster CT scanners it is now feasible to perform these scans with mild or no sedation, reducing the cost and patient risk. Given these factors CT is considered the gold standard in lung imaging. However, there is still no study correlating the CT findings with clinical diagnoses.

We anticipate that findings from this study will identify the utility of CT-scan for patients with respiratory disease and also develop a practical and safe technique for performing BAL to successfully sample small or subtle lung lesions in small veterinary patients.

 

Investigation of a novel biodegradable gel for delivery of antibiotics locally in surgical sites

Treatment of surgical site infections is typically accomplished by administration of antibiotics. Local conditions such as biofilm production (a 'slime' produced by bacteria) and bacterial adherence to metallic implants can lead to underestimation by the clinician of the amount of antibiotic needed for clinical resolution of infection. Unfortunately, an increasing number of these implant-associated infections are resistant to first line antibiotics and are causing clinicians to turn to antibiotics with less associated resistance. Dispersin B (DspB), an enzyme, has been recently shown to prevent bacterial biofilm formation. Use of DspB, combined with other agents, may provide a novel way of preventing surgical site infections locally. Some commercial preparations are available for use but they are difficult to obtain and have limited antibiotic choice. At OVC, formulations are being compounded. However, little is known about the homogeneity of the drug concentration and the stability of this product once compounded. There is also little known about the rate the drug is released and the length of time the drug is released for. These factors are important to know when deciding if local delivery of antibiotics will be effective for treatment or prevention of infection. If this product is found to be stable and releases the drug in a suitable concentration and time period, it may be useful for administration of antibiotics for prevention of infection at the time of surgery by delivering antibiotics and/or enzymes preventing biofilm formation locally around metallic orthopaedic implants. This could be accomplished by reducing the bacterial numbers, preventing bacteria laden biofilm formation and decreasing implant associated surgical site infections. 

 

Examining canine cartilage cells to develop better treatments for joint cartilage injuries in dogs

Regenerative medicine holds enormous promise to improve the welfare of dogs. This project focuses on using cells derived from cartilage (chondrocytes) to develop better treatments for joint cartilage injuries. Past Pet Trust funded work in the Koch lab has been successful in isolating stem cells from bone marrow and fat of the dog. Stem cell cultures are now banked in the lab from these tissues. These cells show promise to treat inflammatory conditions in the dog. However, our studies have shown that these stem cells do not easily become cartilage-producing chondrocytes. For the purpose of joint cartilage repair we plan to explore chondrocytes as a starting cell for tissue regeneration therapies. Historically, chondrocyte use have been hampered by their low natural numbers within cartilage that required the cells to be expanded in a laboratory prior to clinical use. Culture expansion unfortunately alters the cells so by the time there are sufficient cells for clinical use the cells are no longer normal chondrocytes. Recent advances in the area of molecular and genetic techniques can likely overcome these historical limitations. We have assembled a team of outstanding clinicians and basic scientists to apply these cutting-edge technologies towards novel cartilage repair therapies in the dog. The overall objectives of this application are to 1) isolate chondrocytes from the articular cartilage of dogs, 2) temporarily immortalize these cells to enable prolonged cell replication, and 3) compare these cells’ ability to differentiate into cartilage-producing cells to that of untreated cultured chondrocytes. The purpose is to generate chondrocytes that can be cryopreserved for long-term storage and provide an unlimited source of cells for effective cartilage repair therapies in dogs.

Using stem cells to understand canine epilepsy

Canine epilepsy is an important and common problem in both general and specialty veterinary practice. Increasing evidence in human medicine suggests that idiopathic epilepsy is highly associated with behavioural abnormalities, impaired learning and memory. These issues have recently been described in epileptic dogs and exacerbate the primary problems associated with the underlying disease itself. The growth of new abnormal neurons after seizures has been recently implicated in progressive increases in seizure frequency and the development of these concurrent problems. Newly formed neurons display atypical shapes and abnormal connections with the normal neuronal circuits. Moreover, preventing the development of these neurons in mouse models improves memory and reduces seizure frequency in the long term. This highlights the important potential roles played by neural stem cells in epileptic dogs. Experimental models show that neural stem cell proliferation and differentiation are mediated by the activation of glutamate and y-aminobutyric acid (GABA) receptors respectively. Our project aims to identify the cellular factors that regulate the expression of these receptors in neurons that develop from cultured canine stem cells isolated and characterized under several previous Pet Trust projects. In particular, we will focus on small RNAs that can be used as both markers for the disease and targets for eventual therapy. With a better understanding of the neural stem cell role in canine epilepsy and the mechanisms behind epilepsy-induced neurogenesis in the dog, we aim to improve the diagnosis and management of this serious disease.

Managing canine heart failure

Several large breed dogs including Doberman Pinschers, Great Danes, and Newfoundlands demonstrate high susceptibility to a form of heart disease called dilated cardiomyopathy (DCM). For example, up to 60% of Doberman Pinschers will develop DCM. Although the basis for most cases of canine DCM is genetic, the specific mechanisms which cause the heart to fail are unknown. Significant progress has been made in treating dogs with DCM, but this disease remains a fatal condition. Early diagnosis of DCM requires specialized equipment to screen dogs with echocardiography and ambulatory electrocardiograms. The development of a simple and widely accessible assay for use in veterinary clinics would allow for early treatment that prolongs the quality and quantity of the patient’s life. MicroRNAs (miRNAs) are short non-coding RNAs that bind to and prevent translation of target mRNA into protein. In failing hearts altered miRNA expression contributes to disease by causing pathological changes in protein expression. The release of miRNA from damaged heart muscle cells into the blood of patients with heart disease creates a signature that could be used as a diagnostic and prognostic tool. The objective of this project is to identify circulating miRNA profiles in the serum of dogs with and without DCM to create a diagnostic and prognostic miRNA profile. We aim to collect serum samples from non-failing and DCM dogs that are routinely seen at the Ontario Veterinary College. Our ultimate goal is to create a new tool for early and accurate diagnosis of canine DCM.

 

Predictors of disease outcome in dog bone and mammary cancers by digital microscopy

Currently, there are no reliable predictors of how well dogs with bone or mammary cancers will fare after treatment, other than tumour grade. Grade is assessed by standard microscopy on a biopsy specimen and is used to determine how aggressively (badly) a tumour is likely to behave. Our recently findings show that grading of bone cancer in dogs does not predict how long they live after standard treatment. For mammary tumours, grade is also not very informative for many cases. This leaves owners and veterinarians in the difficult position of deciding the best course of action for dogs with these common cancers. We have constructed tissue microarrays (TMAs) that contain small tissue cores of cancers from hundreds of patients. This technique allows for simultaneous analysis of the expression of multiple proteins by standard techniques on glass slides. Using these TMAs we have identified candidate biomarkers (proteins) that correlate with post-treatment survival in dogs with these two common cancers. However, the amount of time required to manually evaluate and score the hundreds of cores reduces the number of markers that we can evaluate. To solve this problem, we are now digitally scanning microscope slides and then applying digital image analysis to help evaluate the images. In addition to accelerating and broadening our analysis, computerized image analysis is unbiased and highly reproducible. These techniques have become standard in human cancer biomarker discovery and are routinely used to predict the behaviour of breast cancer. Our ultimate goal is to establish the most clinically relevant and powerful predictive biomarkers for application in routine biopsy analysis of bone and mammary cancers in dogs.