Pet Trust Funded Projects – Fall 2010

Dr. Alexa Bersenas
$19,812

Post-operative evaluation of abdominal fluid in dogs with closed-suction drains

Dogs need surgery of the abdomen for many different reasons, such as obstructions from eating a “foreign body” like a ball or bone, cancer of the stomach or intestine, or having twisted a part of the intestine. After surgery, infections of the abdomen (septic peritonitis) can occur, especially if the intestines were leaking before surgery. This complication, a bacterial infection in the abdomen, is life-threatening; successful outcome requires repeat abdominal surgery. Survival in dogs and cats, despite surgical intervention ranges from 52-80%. Timely diagnosis of abdominal infection and surgical intervention is paramount for improved survival. Infection is currently diagnosed based on a combination of the patient’s clinical signs and laboratory findings. However, confirming the diagnosis can be difficult.

Tests that are used to aid in the diagnosis of infection include 1) evaluation of the abdominal fluid under the microscope to look for evidence of bacteria, 2) simultaneous comparison of glucose and lactate from the abdomen and the blood of the patient, and 3) bacterial culture and sensitivity of the abdominal fluid. Problems exist with each of these tests.

Microscopic examination of abdominal fluid can be difficult to interpret following surgery because surgical manipulation will cause inflammation and while this is expected, it can hide underlying infection. Currently, there are no reported normal values for abdominal drain fluid in dogs following surgery. A complimentary test is the comparison of glucose and lactate from the abdomen and blood. In two studies, these ratios have been shown to diagnose infection, but these studies have only looked at the values before surgery, not following surgery. Finally, abdominal fluid culture and sensitivity is considered the gold standard of diagnosing infection, but results take a minimum of 24-48 hours. This delay prevents the use of culture to provide tools for immediate patient management. As such, a better method to quickly and confidently diagnose post surgical infection is needed.

Dr. Shauna Blois
$5,225

Assessment of hypercoagulability in naturally occurring canine pituitary dependent hyperadrenocorticism

Canine pituitary-dependent hyperadrenocorticism (PDH) is a common endocrine condition in older dogs and can be associated with inappropriate blood clot formation leading to serious or fatal complications. Currently, the mechanism as to why PDH causes an increase in blood clot formation is unknown. In the Winter 2009 Pet Trust competition, our research group was granted funds to investigate this mechanism. Thus far, we have recruited 13 (of the planned 20) dogs with naturally occurring PDH. Thromboelastography with Platelet Mapping (TEG-PM) is being used to assess the entire blood clotting pathway in these patients. Many of the PDH patients enrolled in our study have increased blood clotting activity detected by TEG-PM. Early in the course of our research, it was noticed that many dogs with PDH are losing protein in the urine. The goal of the next phase of our project is to measure urinary protein loss as well as circulating antithrombin levels in our patients. Antithrombin is a protein that circulates in the body, serving as a natural blood clot inhibitor. Loss of antithrombin in the urine may be a mechanism that predisposes dogs with PDH to clotting problems. The overall goal of the study is to better understand why inappropriate blood clot formation occurs in PDH patients. It is hoped that this understanding will allow for future investigation into prevention of this potentially fatal complication.

Dr. Robert Cruz
$35,500

Evaluation of a MRI hepatobiliary contrast agent in dogs with splenic hemangiosarcoma for detection of metastasis

Liver nodules are commonly noted on routine abdominal ultrasound and differentiating between benign nodular hyperplasia and malignant liver nodules frequently not possible. The ability to accurately and efficiently (i.e. not wait for a biopsy) determine if liver nodules are malignant is particularly important in dogs with splenic hemangiosarcoma as the presence of metastatic disease may impact the owner’s decision to proceed with treatment. Our group has recently shown that a new liver specific MRI contrast agent (gaoxetic acid/Primovist) is safe and efficacious in dogs. This study will evaluate the ability of this contrast agent to differentiate benign hyperplastic liver nodules from metastasis in dogs presenting with cavitated splenic masses.

Dr. Alice Defarges
$6,997

Development of new techniques for bone marrow biopsy in cats: further studies

Bone marrow biopsy is needed in cats with blood disorders when a diagnosis cannot be made based on examination of the blood. Bone marrow core biopsy uses a special needle to remove a small core of bone marrow. It is a difficult procedure in cats because the biopsy needles are designed for human use and are too large for easy use in cats (the smallest is 13gauge). We previously investigated using a smaller (15gauge) needle designed for emergency fluid administration into the bone marrow, and which we modified for biopsy. A study in dogs (Pet Trust Spring 2009) showed that biopsy with this 15gauge needle attached to a high-speed drill was easier to perform than manual biopsy with a 13 gauge needle, and that it provided good quality samples. The previous study in cats (Pet Trust Spring 2010) compared 15gauge biopsies from the front leg to 13gauge biopsies from the pelvis, but the high-speed drill was not used as the depth of needle insertion was too difficult to control. Biopsy quality of both varied from poor to excellent, but the 15gauge biopsies were easier to obtain and less traumatic. The difference between obtaining a good or poor biopsy was a matter of difference of needle angle of several degrees, which is hard to judge. We wish to now evaluate 13 and 15 gauge biopsies from a standard biopsy site in the back leg. The needle angle for this site and the use of a new low-speed drill designed for bone marrow biopsy should improve biopsy results.

Dr. Alice Defarges
$13,367

Standardization of broncheoalveolar lavage suction techniques to optimize diagnostic yield of lower airway samples

Bronchoscopy is a technique used to visualize the lower airways in animals and humans. With bronchoscopy, the airways can be “washed” (bronchoalveolar lavage, BAL) to obtain samples for diagnostic analysis. Indications for bronchoscopy and BAL include chronic cough, unexplained changes on lung radiographs, presence of tracheal or bronchial foreign objects and lung masses. Side effects of bronchoscopy are usually minor and transient in nature. The basic technique for BAL consists of saline injection into the bronchi with re-aspiration. The methods for re-aspiration include manual suction using a syringe and active suction with a pump. Negative pressures used in human medicine range from 25 to 100 mm Hg. If high negative pressure is applied, fluid retrieval may be limited to airway collapse and cell preservation may be poor. Hence, we believe that a standardized protocol for BAL is required for consistent results and to allow for comparisons across clinicians, conditions and institutions.

Dr. Luis Gaitero
$4,500

Activity and silencing of DNA repair enzyme 06-methylguanine-DNA methyltransferase (MGMT) in canine intracranial oligodendrogliomas and mengingiomas

Brain tumours are as frequent in dogs as in humans. Meningiomas and gliomas are the most common primary brain tumours in dogs. Treatment guidelines for specific types of brain tumors are lacking in veterinary medicine and the management of dogs affected is usually not satisfactory. Often a combination of surgery, chemotherapy, and radiation therapy is used. The use of chemotherapy for brain tumours in veterinary medicine is usually anecdotal even though in human medicine some patients show a good response. Nowadays, many studies in human medicine are focus in the assessment and determination of factors that can predict which tumours benefit from the different modalities of therapy. One of those predictive factors is the methylation status of a tumour gene O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein. Methylation inactivates this gene decreasing the production of MGMT in tumour cells. Loss of function in MGMT is related to an increase in survival in human patients with intracranial gliomas treated with some chemotherapeutic drugs. Patients with strong methylation states significantly benefited from temozolomide treatment. The objective of this study is to determine the methylation status of the MGMT gene in samples of two of the most common canine intracranial tumours, oligodendrogliomas and meningiomas in order to estimate the ability of the tumour to response to a treatment. Based on the results of this study, we intend to proceed into a future study to correlate the MGMT methylation status to the overall patient survival and response to chemotherapeutic agents.

Dr. Robert Jacobs
$14,000

Immunoflurescent and confocal analysis of granule polarization in canine histiocytic diseases

Histiocytes are cells of the immune-system essential for fighting infectious agents. Some breeds of dogs are prone to develop malignant cancer of histiocycytes as well as severe, inflammation of histiocytes collectively referred to as canine histiocytic diseases (CHD). Exceedingly high prevalence of CHD in only few breeds implies a genetic causation. This research aims to discover the mechanism of CHD disease development by comparison to familial hemophgocytic histiocytosis (FHLH), a fatal, genetic disease of histiocytes that occur in young children. Defective genes in children with FHLH code for proteins that are involved in the killing machinery of immune-system. When intact the killing apparatus of the immune-system eradicates malignant cells at early stages of cancer development, eliminates viral infection and terminates inflammatory processes at the end of their favorable course. As tragically demonstrated in children with FHLH, lack of effective killing leads to development of perpetuating, fatal inflammation and high susceptibility to cancer. Given resemblance in disease characteristics and genetic pre-disposition, we suspect that a similar mechanism underlies CHD. Thus, the objective of this project is to assess the killing apparatus in dogs with histiocytic diseases. Proving identical mechanism of disease will promote reciprocal research that benefits both children and dogs and can leads towards effective therapeutic approaches. Proving an identifiable cause for these diseases will promote development of a diagnostic test for early detection of affected/carrier dogs and assist in eradication of these devastating diseases.

Dr. Jonathon Lamarre
$16,000

The role of IL-II Corneal Cell Migration, Proliferation and Survival

Eye diseases are very common problems in dogs that are painful to the patient and distressing to clients. Many eye diseases are challenging to manage and may result in chronic inflammation, visual impairment, vision loss or, in severe cases, loss of the eye. In general veterinary practice, diseases involving the cornea are the most frequently encountered serious eye problems. In many corneal diseases the expression of regulatory proteins that control inflammation, blood vessel growth, cell proliferation, migration and apoptosis are often abnormal. Collectively, these regulatory proteins are known as cytokines. As in other tissues, the balance between cytokines that promote inflammation and those that suppress it is one key to normal function and recovery from disease. After evaluating the relative expression of cytokines in the canine eye, our previous Pet Trust-funded research has focused on one particular cytokine, interleukin-11 (IL-11). Our studies to date have demonstrated that this cytokine is both present in the canine cornea and inducible by relevant stimuli. In several other tissues, IL-11 is generally considered to suppress potentially-damaging inflammatory responses and is actually used as a drug. In this capacity IL-11 modulates key cellular processes, including migration, proliferation and the promotion of cell survival (anti-apoptosis). These cellular processes are all integral to corneal health and to the cornea’s ability to respond to injury. This proposal builds on our previous identification of IL-11 expression in the cornea and examines the consequences of IL-11 expression on corneal cells. We expect that this will pave the way for novel treatments for canine corneal disease.

Dr. Tony Ogg
$12,360

Effect of Axillary Incubation and Sedation on Activated Clotting Time in Cats

Activated clotting time (ACT) is a test that measures the time it takes for a fresh blood sample to clot. It is a useful, practical and inexpensive test for detecting abnormal blood clotting and inflammation in dogs. Our impression is that it is also useful for these purposes in cats. However, the investigation of this is hampered by the lack of a reference range using the new ACT system. ACT used to be performed using a special blood tube that is no longer available. A new tube is now available (MAX-ACT, Helena Laboratories). The tube itself is relatively inexpensive, but warming the tube and measuring the clot uses an instrument (Aktalyte) which is cost-prohibitive to most practices. A recent study at OVC validated underarm incubation and visual clot detection as an alternative to using the Aktalyke for dogs. The purpose of this study is to validate this method in cats. ACT will be measured in blood samples using both methods and the results compared. ACT measurements will be repeated after sedation, as some cats require sedation for blood sampling and the effect of sedation on ACT is not known.

Dr. Tony Ogg
$6,224

Prevalence of antinuclear and anti-red cell antibodies in normal cats

Antinuclear antibodies (ANA) and anti-red cell antibodies (detected with a Coombs test) are antibodies directed against a patient’s own DNA and red blood cells, respectively. They are non-specific markers of autoimmune diseases (diseases where a patient’s own immune system attacks the patient), as well as specific markers of systemic lupus erythematosus (“lupus”) and immune-mediated hemolytic anemia (anemia caused by antibody destruction of red cells), respectively. We are performing a long-term clinical study of cats with chronic anemia not due to infections with feline leukemia virus (FeLV), feline immunodeficiency virus (FIV), Mycoplasma haemofelis and M. haemominutum bacteria, or chronic kidney disease. We have treated 42 cats. Forty percent of the cases tested have had positive ANA tests, and 40% (not all the same cats) have had a positive Coombs tests. This suggests that at least in some of these cats the anemia is due to autoimmunity, implying they should be treated with immunosuppressive drugs. However, it is not known how many normal healthy cats have a positive ANA or Coombs test. It is known that in most species some normal animals have positive tests.

Dr. Glen Pyle
$14,970

A molecular and cellular investigation of dilated cardiomyopathy in Doberman Pinschers

Up to 60% of Doberman Pinschers die of an inheritable form of heart failure called dilated cardiomyopathy (DCM)1-3. Although the age of onset can vary, once the condition manifests clinically as overt congestive heart failure (CHF), dogs experience a rapid and devastating decline: average survival time is 14 days17. Despite the high prevalence of DCM in the Doberman population, little is known about the underlying mechanisms of this disease4.

Work in other hereditary forms of DCM has found that mutations in genes encoding for the group of contractile proteins collectively known as “myofilaments” are the most common cause of heart failure8,10,24. Interestingly, the most effective treatment for DCM in Doberman Pinschers is a group of drugs called calcium sensitizers that alter myofilament function17-20. Together these data have led us to hypothesize that abnormal cardiac myofilament function is the underlying cause of DCM in Doberman Pinschers. To test this hypothesis we are working in collaboration with the clinical cardiology service at the Veterinary Teaching Hospital at the Ontario Veterinary College to measure myofilament function in normal and diseased heart tissue. Moreover, we will examine the composition and modification of myofilament proteins to determine if changes in myofilament proteins occur with DCM. Using calcium sensitizers that target unique regions of the myofilaments will allow us to map any dysfunction to a specific region of the myofilament complex, permitting us to identify a subset of candidate genes that may be altered in this genetically transmitted form of heart failure.

Our results will determine if calcium sensitizers are so effective because they correct a functional defect, or simply mask dysfunction. The use of calcium sensitizers that target different regions of the myofilaments will also allow us to compare the effectiveness of compounds in this drug class, and evaluate whether changing current therapeutic protocols might improve survival and quality of life for our DCM patients. Finally, our assessment of cardiac myofilament composition and function will test the hypothesis that this subcellular structure is a causative player in canine DCM. Overall our work will simultaneously test the rationale and effectiveness of currently used therapies, while searching for the cause of this naturally occurring and devastating disease.

Dr. Ameet Singh
$4,000

Post-hospital discharge procedure-specific site infection surveillance in small animal patients

Surgical site infections (SSI) are an inherent risk of any surgical procedure. They are associated with substantial morbidity and lead to increased cost of treatment as a result of prolonged hospital stay in human patients. The causes of SSI are multi-factorial and likely originate from the bacteria normally present on the patients skin and from that of the operating room (OR) staff. Numerous SSI surveillance programs have been completed in human surgical patients and these have contributed to the reduction in SSI rates. Despite the clear benefit of proper SSI surveillance and the changes that have been made to surgical practices in humans based on surveillance data, only a few prospective SSI surveillance programs have been performed in veterinary surgical patients. These studies have identified several risk factors for SSI including number of people in the OR, duration of surgery, time from shaving the surgical site to surgery, infection of the surgical site and the use of propofol (anesthetic drug). Despite this useful information, these studies have had significant limitations such as sample size, reliance on medical record data to identify infections, failure to use standard (Centers for Disease Control and Prevention, CDC) definitions, failure to clearly differentiate infection from inflammation and failure to provide adequate follow-up time to ensure that all infections were identified. Long-term, prospective, active post-hospital discharge SSI surveillance programs in veterinary medicine are lacking. The limitations in available SSI data limit interventions that can be implemented to reduce SSIs. They also hamper proper design of studies investigating SSIs since adequate baseline SSI rate information is needed for proper study design.

Dr. Ameet Singh
$7,100

Hepatocyte expression of endothelial and inducible nitric oxide synthase in dogs with congenital portosystemic shunt

Congenital portosystemic shunt (CPSS) is a common vascular anomaly in dogs where an embryological vessel persists at birth leading to an inappropriate communication between the portal and systemic circulation that bypasses the liver. The portal system carries blood from the intestines and other abdominal organs to the liver and contains nutrients, growth factors and intestinal-derived toxins all produced from the digestion of food. The inability to clear portal blood of intestinal derived toxins can lead to alterations in central nervous system function termed hepatic encephalopathy. Redirecting blood flow into the liver by surgical attenuation of the CPSS will promote hepatic growth, decrease hepatic encephalopathy and prolong lifespan of affected animals. A proportion of animals respond poorly after surgical intervention and little is known about predicting a positive clinical outcome.

Nitric oxide (NO) is an inorganic gas that is involved in numerous physiological processes in the mammalian body as a biological messenger molecule. NO release leads to vascular smooth muscle relaxation and subsequent dilation of blood vessels and NO scavenging by hemoglobin results in blood vessel constriction. Nitric oxide is essential for the regulation of blood flow through the liver and alterations in NO levels may have deleterious consequences during pathological processes. Recent work, has shown that serum levels of NO metabolites are decreased in children with CPSS suggesting that liver derived NOS is correlated with abnormal blood flow within the liver. Furthermore, NOS may influence outcome after surgical intervention for CPSS. In this proposal we wish to determine the levels of NOS in the livers of dogs diagnosed with CPSS.

Dr. Scott Weese
$15,060

Identification of Encephalitozoon cuniculi in pet rabbits

Encephalitozoon cuniculi is a microsporidial organism, a strange class of microorganisms that have been considered protozoa but which are now classified as fungi. E. cuniculi is an important and often overlooked cause of kidney and neurological disease in pet rabbits, and anecdotal evidence indicates it is probably a leading cause of infectious disease in pet rabbits. One problem with identifying and understanding E. cuniculi infection is the limitations in available diagnostic tests. Serological (blood) testing is often used but this simply shows that a rabbit has been exposed to the organism at some point, not that it is actively infected. The organism can be isolated from urine in some infected rabbits, but current tests are suboptimal, potentially because of inadequate sensitivity. This study will evaluate different culture and molecular assays for detection of E. cuniculi and provide needed information about potential diagnostic tests to ultimately allow for better diagnosis, earlier treatment and better disease surveillance. This study is part of a broader research program evaluating clinical, epidemiological and microbiological aspects of E. cuniculi in rabbits.

Dr. Scott Weese
$18,840

Metagenomic characterization of canine intestinal microflora and investigation of diarrhea outbreak

The intestinal tract is one of the most, if not the most, complex microbial environments on Earth. Each gram of intestinal contents contains trillions of bacteria, along with diverse populations of fungi, protozoa and viruses, but this population is poorly understood in both health and disease. Despite the commonness of diarrhea in dogs, identification of the cause is often difficult, in part because of limitations in available tests and the understanding of the intestinal microbial population. Typically, the focus on intestinal bacteria and viruses has been on a very narrow range of known pathogens. It is likely that the canine intestinal tract houses a diverse viral microflora, along with the highly complex bacterial microflora, and that various unknown microorganisms could be the cause of diarrhea. Recently, new technologies have allowed for revolutionary breakthroughs in characterization of the intestinal tract. These facilitate ‘metagenomic’ analysis; broad genomic study of microbial diversity of complex populations. Metagenomic studies can groundbreaking information about the bacterial and viral composition of the intestinal microflora and may be able to identify novel pathogens, such as could be responsible for diarrhea in dogs. Using a metagenomic approach, this study will provide the most comprehensive investigation of the intestinal bacterial and viral microflora in healthy dogs, and try to identify the cause of a large outbreak of diarrhea.

Dr. Darren Wood
$19,740

Development and characterization of recombinant canine activated protein C for treatment of sepsis in dogs

Sepsis is common in dogs and often results in morality despite standard treatment. In humans, activated protein C (APC) is an effective treatment for severe sepsis. APC is derived from protein C (PC) in plasma and has an anti-coagulant, anti-inflammatory, pro-fibrinolytic, and cytoprotective effects. Reduced PC levels are associated with mortality in humans with sepsis. Currently, recombinant human activated protein C (rhAPC) is the only US FDA-approved therapy that increases survival in adult patients with high-risk, severe sepsis. Similar to humans, PC activity is significantly decreased in canine sepsis and such decreases predict mortality. Success in treating sepsis in humans with rhAPC provides a rationale for treating septic dogs with APC. However, rhAPC is highly antigenic in dogs, rendering it inappropriate for canine use, and knowledge of PC biology in dogs is limited. Therefore, canine-specific PC needs to be isolated or genetically engineered and better characterized before intervention with the PC pathway in septic canine patients is attempted. We propose a study that examines the anti-coagulant, anti-inflammatory, pro-fibrinolytic, and cytoprotective effects of PC purified from canine plasma. Canine PC is expected to have similar biological properties as its human counterpart. The results from this study will provide a basis for developing and characterizing recombinant canine protein (rCnAPC).

Dr. Tony Yu, Clinical Studies
$8,700

Measurement of pseudomonas aeruginosa antimicrobial sensitivity to topically versus systemically delivered concentrations of antibiotics for use in the treatment of otitis externa

Ear infections (otitis externa) are one of the most common presentations to a veterinary office. Despite advances in therapeutic options, ear infections are often refractory to treatment owing to the increasing presence of antibiotic resistant strains of bacteria. Among these microorganisms, Pseudomonas aeruginosa is reported to have a high level of resistance to our first, and now second-line antibiotics in veterinary medicine. Growing concern regarding treatment failure, extremely high client cost of third-line of antibiotics, and potential cross-resistance of these antibiotics in the human field has sparked the need for further evaluation of our diagnostic and therapeutic choices when dealing with bacterial infections in pet’s ears.

Currently, antimicrobial sensitivities are reported as the concentration of drugs systemically delivered to infected tissue. Topically applied medications, however, achieve concentrations well above 10 to 1000 times the concentration of systemically administered drugs at the site of infection. Hence, a better method of antimicrobial sensitivity evaluation is needed that would reflect activity of topical therapy against Pseudomonas infections in the ear. We would propose generating and verifying a Topical Otic Product Sensitivity (TOPS) panel that would asses Pseudomonas sensitivities to concentrations of antibiotics consistent with topically applied medications currently on the market for veterinarians.

Culture isolates from patients with “resistant” Pseudomonas infected ears will be obtained from local laboratories. A representative collection of 200 Pseudomonas aeruginosa isolates will then be tested using gradient concentrations of topical antimicrobials commonly used in veterinary medicine for the treatment of ear infections. The aim of this project would be to provide more appropriate antimicrobial therapy recommendations, help to minimize the need for systemic and third-line antibiotics, and, detect the true incidence of Pseudomonas “resistance” in our pet population.

$222,395 was distributed in new research funds.

$25,476 was awarded to Dr. Darren Wood et al for the purchase of a whole blood aggregometer for the Ontario Veterinary College Hemostasis Research Laboratory.