Emergence of avian origin and equine origin canine influenza viruses (CIVs) in Asia and the United States brings important concerns. Humans are in closer and more frequent contact with dogs than other common hosts of influenza. Thus, CIV is a potential threat to human health. However, little is known about the determinants of CIV host tropism or the transmissibility of CIVs to humans. An amino acid change (W222L) was implicated in modifying hemagglutinin receptor binding by CIV. This was tested using reverse genetics, glycan microarray and virus histochemistry. Glycan microarray demonstrated that avian-origin CIV (H3N2-222W) bind predominantly to alpha-2, 3 linked glycans. Virus histochemistry indicated that rH3N2-222L had higher binding affinity with epithelial cilia of canine tracheal tissue and weaker binding with avian tracheal tissue. Ferret infection demonstrated that the avian-origin H3N2 CIV could cause infection and limited to rhinitis, suggesting that CIV could infect humans.

Background: Chronic enteropathies (CE) predispose dogs to thromboembolic disease, but the underlying mechanisms are poorly understood. Humans with CE have decreased activity of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), a von Willebrand factor (vWF) cleaving enzyme, and increased circulating vWF. The primary aim of this study is to assess plasma ADAMTS13 activity, vWF antigen (vWF:Ag) concentration, and vWF collagen binding activity (vWF:CBA) in dogs with CE. Hypotheses: Dogs with CE have reduced plasma ADAMTS13 activity, increased vWF:Ag, and increased vWF:CBA compared to healthy control dogs. Animals: Twenty privately-owned dogs with CE and 40 healthy dogs were recruited from a specialty hospital population. Methods: Prospective observational study. Dogs were confirmed to have CE using histopathology. ADAMTS13 activity was measured using a commercially available ELISA kit (DiapharmaⓇ) in 20 dogs with CE and 40 healthy control dogs. Plasma vWF:Ag was assessed in 20 dogs with CE and 20 healthy control dogs. Plasma vWF:CBA was assessed in 19 dogs with CE and 20 healthy control dogs. For statistical analysis, an unpaired t-test was used for normally distributed data and Wilcoxon rank sum was used for non-Gaussian distribution. Significance was set at P < 0.05. Results: Plasma ADAMTS13 activity and vWF:Ag were not significantly different compared to healthy dogs (P = 0.07, P = 0.16, respectively). Plasma vWF:CBA was significantly decreased compared to healthy dogs (P = 0.007). Conclusions and clinical relevance: Plasma ADAMTS13 activity was not significantly different in dogs with CE compared to healthy dogs and is unlikely to be the primary mechanism for hypercoagulability associated with CE. Forty-three percent of CE dogs with hypoalbuminemia had ADAMTS13 activity below reference interval. Further studies are warranted to evaluate ADAMTS13 activity in a subset of dogs with CE, including those with protein losing enteropathy and thromboembolism. / Master of Science / Background: Dogs with chronic gastrointestinal disease, or chronic enteropathies (CE), often have abnormal blood clotting which predisposes to thrombosis. The mechanisms leading to this abnormal blood clotting are poorly understood. Humans with CE also suffer from abnormal blood clotting and thrombosis. Decreased activity of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), a von Willebrand factor (vWF) cleaving enzyme, and increased circulating vWF have been reported as contributors to this abnormal blood clotting in people. Von Willebrand factor is a protein that allows platelets to stick together and adhere to damaged tissue to facilitate blood clot formation. The concentration of vWF can be measured using vWF antigen (vWF:Ag) and the activity of vWF can be measured using vWF collagen binding activity (vWF:CBA). Hypothesis: Dogs with CE have reduced plasma ADAMTS13 activity, increased vWF:Ag, and increased vWF:CBA compared to healthy control dogs. Animals: Twenty privately-owned dogs with CE and 40 healthy dogs were recruited from a hospital population. Methods: Prospective observational study. Plasma ADAMTS13 activity, vWF:Ag and vWF:CBA were assessed in dogs with CE and compared to healthy control dogs. Results: There was no difference in plasma ADAMTS13 activity and vWF:Ag between the two groups. The mean vWF:CBA was significantly lower in CE dogs compared to healthy dogs. Conclusions and clinical relevance: Reduced ADAMTS13 activity is unlikely to be the primary mechanism for abnormal blood clotting in dogs with CE.

Congenital and developmental limb deformities in canines are rare and can occur as a genetic disorder or be caused by extrinsic factors. Without surgery to correct the deformity, conservative management can be implemented to manage exercise and restrict high-intensity activity of the canine. However, any alteration to the normal gait and locomotive biomechanics of a canine can have significant long-term effects on the musculoskeletal health and quality of life of the canine. To improve quality of life and provide an alternative and more cost-effective approach to surgery, a custom prosthetic was designed and developed for a canine born with a congenital right forelimb deformity. Since canine prosthetics that are currently on the market are limited and expensive, the goal of this thesis was to create a durable and inexpensive prosthetic to stabilize the gait of a canine. A 1-year-old German Shepherd was the single subject of this research project. The major results indicated that the custom-designed, 3D printed prosthetic parts, which included the foot and the body of the prosthetic, were strong enough to withstand the high-impact forces and stresses experienced during the gait of a canine. The results also indicated that the prosthetic was comfortable and did not cause any pain or discomfort to the canine, as well as the prosthetic leg and foot being the correct length to stabilize the gait of the canine and redistribute the body weight of the tripod canine to that of a tetrapod canine. This study also developed and outlined a feasible fabrication process that could be repeated and used to produce other custom prosthetics for canines with rare congenital or development limb deformities as an alternative to surgery. In a future study, fatigue testing, tensile testing, and impact testing should be performed to determine the failure points. Fatigue testing is a critical factor in determining failure of a part.

<p> Treatment of canine colonic tissue with some O-alkyl and O-benzyl hydroxylamines potentiated the response of the tissue to histamine in different experimental environments.</p> <p> Sixteen O-substituted hydroxylamine compounds were synthesized using a modification of the Gabriel synthesis. These compounds were tested for their ability to potentiate histamine in canine colonic tissue through diamine oxidase inhibition.</p> <p> Three procedures were used to determine their activity: (1) secondary rise -hydroxylamine derivatives were added to epithelial tissue preparations in Ussing chambers after an initial dose of histamine. Active compounds caused a secondary increase in the short circuit current across the tissue, (2) dose-response profiles were constructed for several hydroxylamine compounds to determine whether they caused a significant shift to the left of the normal histamine curve (potentiated response), and (3) diamine oxidase enzyme assays were performed to examine the ability of the hydroxylamine derivatives to inhibit partially purified diamine oxidase. This aided in determining if inactive compounds could not potentiate histamine due to an inability to access the enzyme in the epithelial preparation.</p> <p> The structure-activity relation observed indicates that: (1) active compounds are oxygen and not nitrogen substituted hydroxylamines, (2) branched compounds are less active than their straight chain analogues, (3) greater steric bulk of the alkyl substituent can decrease the activity of the compound, (4) the presence of a carbon-carbon double (allyl) or triple (4-pentynyl) bond does not affect the activity of the compound, (5) longer straight chain O-alkyl hydroxylamines are less active than shorter chain derivatives, (6) steric bulk of the benzyl compounds is not likely to be the cause of its inability to inhibit diamine oxidase since the cinnamyl derivative is active, and (7) meta- and para-oxygen substituents (-OH, -OCH3) on O-benzyl hydroxylamines increase their diamine oxidase inhibiting properties.</p> / Thesis / Master of Science (MSc)

Background: Histotripsy is a non-thermal, non-invasive high-intensity focused ultrasound (HIFU) ablative technique that causes mechanical fragmentation of tissue, resulting in liquefied acellular debris with histologically clear demarcated boundaries between treated and non-treated tissues. The acellular debris may include tumor antigens with preserved immunogenicity and the potential to generate systemic immune response against tumor cells. Soft tissue sarcomas (STS) are a common form of cancer in dogs with biological behavior similar to STS in humans. Long-term tumor control requires complete removal with extensive surgical resection, which in many cases is not feasible. As a result, there is need for alternative therapies. Objectives: The primary objective of this study was to demonstrate safety and feasibility of histotripsy in a small animal model of spontaneous STS. The secondary objective was to characterize the impact of histotripsy on the immunologic response. Materials and methods: Pet dogs diagnosed with spontaneous STS were recruited. CT scan of the chest, abdomen, and the tumor was performed for staging and treatment planning. Pretreatment biopsies were obtained. Safety was monitored with physical examinations, owner reports, and CBC/serum biochemistry. Partial tumor ablation was performed using a 500 kHz prototype histotripsy system. A spherical treatment zone of up to 3 cm diameter in each tumor was treated with histotripsy according to the patient-specific treatment plan using 1-2 cycle pulses applied at a pulse repetition frequency (PRF) of 500 Hz. Anatomical ablation zones were evaluated with contrast CT at 1- and 4-days post-treatment, with tumor resection at 4-6 days post-treatment. Tumor microenvironment (TME) gene expression was evaluated with the Nanostring Canine IO panel, and the systemic immune response was evaluated using multiplex serum cytokine levels. Results: Ten dogs were recruited and treated. Tumor histologies included 3 grade III STS, 4 grade II STS, 2 grade I STS, and 1 malignant mesenchymoma. Six dogs were alive, three dogs were euthanized due to disease progression, and one dog was lost to follow up. Histotripsy-related complications were generally self-limiting, with only one patient having increased cutaneous injury score from 1 to 2 (scale 1-5) post-treatment, likely due to prefocal cavitation at the skin. No significant adverse events impacting patient outcome were noted in any of the patients. Visible histotripsy cavitation bubble clouds were seen on real-time ultrasound imaging in nine of ten treatments. Post-treatment histopathology indicated sharply defined regions of ablation that were clearly identifiable grossly and histologically in all samples. Treatment zones were characterized by loss of cell viability, hyalinization, and acute hemorrhage. Post-treatment contrast-enhanced CT images revealed clear, demarcated regions of histotripsy ablated tissue in seven of ten patients. Differential gene expression analysis identified 79 genes with at least 2-fold change following treatment. Genes associated with inflammation, immune cell migration, and immune cell interactions were the highest upregulated. Amongst the gene set analyses, the myeloid compartment gene sets obtained the highest significance score. There were no statistically significant differences between pre- and post-treatment cytokine concentrations for any of the analytes. Conclusions: Histotripsy can achieve safe and effective tumor ablation in dogs diagnosed with STS. Histotripsy induced pro-inflammatory changes within the tumor microenvironment. Histotripsy as an immunotherapeutic treatment option needs to be further investigated. Histotripsy has a potential to be a precise, non-invasive treatment for canine STS. / Master of Science / Histotripsy is a non-thermal high-intensity focused ultrasound (HIFU) ablative technique that uses controlled acoustic cavitation to cause mechanical fragmentation of tissue. To date, there are no reports investigating histotripsy for the treatment of soft tissue sarcoma (STS). This study aimed to investigate the in vivo feasibility of ablating STS with histotripsy and to characterize the impact of partial histotripsy ablation on the acute immunologic response in canine patients with spontaneous STS. CT of the chest, abdomen, and the tumor was performed for staging and treatment-planning. Pretreatment biopsies were obtained. Safety was monitored with physical examinations, through owner reports, and CBC/serum biochemistry. A custom 500 kHz histotripsy system was used to treat ten dogs with naturally occurring STS. Anatomical ablation zones were evaluated with contrast CT at 1- and 4-days post-treatment, with tumor resection at 4-days post-treatment. Safety was determined by monitoring vital signs during treatment and post-treatment physical examinations, routine lab work, and owners' reports. Ablation was characterized using radiologic and histopathologic analyses. Systemic immunological impact was evaluated by measuring changes in cytokine concentrations, and tumor microenvironment changes were evaluated by characterizing changes in infiltration with tumor-associated macrophages (TAMs) and tumor-infiltrating lymphocytes (TILs) using multiplex immunohistochemistry and differential gene expression. Results showed histotripsy ablation can achieve safe and effective tumor ablation in all ten dogs. Immunological results showed histotripsy induced pro-inflammatory changes in the tumor microenvironment. Histotripsy as an immunotherapeutic treatment option needs to be further investigated. Overall, this study demonstrates histotripsy's potential as a precise, non-invasive treatment for STS.

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