Babesiosis, caused by Babesia rossi, is a common cause of morbidity and mortality of dogs in South Africa. Canine babesiosis can be classified either as uncomplicated or complicated based on the degree of anaemia and the severity of the presenting clinical signs.1,2 In uncomplicated babesiosis, the clinical signs are mostly attributable to the degree of the anaemia, whereas in complicated babesiosis the disease process is characterised by additional organ involvement.3,4 One of the most common haematological hallmarks of canine babesiosis, caused by B. rossi, is thrombocytopenia, which is not associated with clinical haemorrhage despite very low platelet counts that would normally cause inability to maintain normal primary haemostatic function.5 The aim of this study was to describe the thromboelastographic findings in uncomplicated canine babesiosis and compare them with those of normal, healthy control dogs. We hypothesised that these dogs would have a normal to hypercoagulable haemostatic capacity, despite the severe thrombocytopenia, and that this could be detected with thromboelastography (TEG), which has previously been shown to correlate well with clinical signs of haemorrhage in dogs.6 This was a prospective, cross sectional, observational study that included 20 client-owned dogs, diagnosed with uncomplicated canine babesiosis at the Onderstepoort Veterinary Academic Hospital (OVAH). Infection with B. rossi was confirmed by polymerase chain reaction (PCR) and reverse line blot (RLB) hybridisation assay. Blood samples were collected at the time of diagnosis. A group of 10 healthy control dogs were included for comparison. Antithrombin activity (AT) was measured using an automated spectrophotometric analyser (Cobas Integra 400, Roche, South Africa). D-dimer was measured using an immunometric flow-through principle (D-dimer Single test, Nycocard Reader II, Medinor A/S). Prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen assays were performed on the ST art® 4 analyser (Diagnostica Stago, Roche, South Africa). TEG analysis was performed using the TEG® 5000 Thromboelastograph® Haemostasis System (Haemoscope, Pro-Gen Diagnostics (Pty) Ltd, South Africa). A complete blood count was performed on the ADVIA 2120 (Siemens, South Africa). The results of the babesiosis and control groups were compared using the Mann-Whitney U test or the Students t-test based on normality. The normality assumption for distribution of the variables in the data was evaluated using the Shapiro-Wilk test. The statistical significance was set at p<0.01. The mean haematocrit (Ht) and median platelet count was significantly lower in the babesiosis group than the controls (0.29 vs. 0.50 L/L; p<0.01 and 22.0 vs. 374.5 x 109/l; p<0.01, respectively). There was no significant difference in any of the TEG parameters between the babesiosis group and the controls. The medians for the various TEG parameters in the babesiosis group versus the controls were; R: 5.5 vs. 4.4 min (p=0.05); K: 2.5 vs. 2.0 min (p=0.08); angle: 58.3 vs. 61.1 degrees (p=0.35); MA: 47.0 vs. 57.0 mm (p=0.02); G: 4.9 vs. 6.7 dyn/cm2 (p=0.02); LY30: 0.00 vs. 0.6% (p=0.20); and LY60: 0.00 vs. 3.0% (p=0.014). The median fibrinogen concentration was significantly higher in the babesiosis group than in the control group; 5.8 g/L (5.0 – 7.0) vs. 2.9 g/L (2.5 – 3.3); (p<0.01). The mean AT activity was significantly lower in the babesiosis group than in the control group; 102.6 mg/dl (89.9 – 112.8) vs. 127.8 mg/dl (110.6 – 134.8); (p<0.01). The median D-dimer concentration was not significantly different in the babesiosis group compared to the control group; 0.3 mg/L (0.1 – 0.4) vs. 0.1 mg/L (0.1 – 0.2); (p=0.016). Median PT was not significantly different in the babesiosis group compared to the control group; 6.5 sec (6.4 – 7.2) vs. 6.8 sec (6.6 – 7.5); (p=0.14). Median aPTT was significantly prolonged in the babesiosis group compared to the control group; 13.6 sec (12.4 – 14.5) vs. 11.5 sec (10.7 – 12.2); (p<0.01). Despite the severe thrombocytopenia, dogs suffering from uncomplicated babesiosis did not have clinical signs of haemorrhage. The thromboelastograms of the babesiosis group were normal to hypercoagulable and thus correlated well with the clinical phenotype. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Companion Animal Clinical Studies / unrestricted

Eight dogs were enrolled in a randomized, cross-over study that used optical aggregometry and a platelet function analyzer to evaluate platelet function before and after the administration of 5 aspirin dosages: 0.5 mg/kg q24h, 1 mg/kg q24h, 2 mg/kg q24h, 4 mg/kg q24h and 10 mg/kg q12h. Urine 11-dehydro-thromboxane-B2 (11-dTXB2) and 6-keto-prostaglandin-F1alpha (6-keto-PGF1alpha), were measured. Compared to pre-treatment, there were significant decreases in maximum aggregometry amplitude and increases in PFA-100 closure times for all doses except 0.5 mg/kg q24h. There was no difference in amplitude or closure time between the 2 mg/kg, 4 mg/kg, and 10 mg/kg q12h dosages. At 2 mg/kg q24h, 100 percent (aggregometry) of dogs were aspirin responders. There was a significant decrease in urinary 11-dTXB2- and 6-keto-PGF1alpha-to-creatinine ratios with aspirin administration. An aspirin dosage of 2 mg/kg q24h consistently inhibits platelet function in healthy dogs without decreasing prostacyclin synthesis significantly more than lower aspirin dosages.

Respiratory disease represents an important component of small animal emergency medicine. The morbidity and mortality of respiratory disease and inflammation, although poorly defined, is considered to be significant. Much of the therapy used in the stabilization and management of respiratory disease in veterinary patients has been taken from human medicine, including inhalation therapy. Heparin has been shown to have substantial anticoagulant, anti-inflammatory, and antiibrotic effects within the lungs when administered via inhalation in human patients. To date, no studies have evaluated the use of nebulized heparin in dogs. This study is the first to attempt to generate pharmacokinetic data regarding nebulized unfractionated heparin in the dog.

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We investigated the origins, neural modulation, ionic mechanisms, and electrical coupling properties of the pacemaker systems in the canine ileum by simultaneously recording the intracellular electrical activity and accompanying mechanical activity in cross-sectioned slabs of the muscularis extema or in the isolated circular muscle. In the whole thickness preparation, intracellular recordings were taken from the circular muscle near the myenteric plexus (MyP), deep muscular plexus (DMP), and intermediate areas between the MyP and DMP and in the isolated circular muscle preparation from similar areas except near the myenteric plexus. One type of slow wave, sigmoidal or triangular in shape, was recorded from impalements near the DMP region in the whole-thickness preparation. Another type observed from the MyP region oscillated at nearly the same frequency (9-10 cycles/min) and was characterized by a fast upstroke and a square shape. A mixture of these two patterns was recorded in intermediate areas (the outer circular muscle or OCM) while triangular slow waves were present near the the submucosal plexus (SMP) inner circular muscle. Neither type of slow waves was affected by atropine, guanethidine, propranolol, and phentolamine (all 1 μM). Under these conditions of inhibition of NANC (non-adrenergic, non-cholinergic) nerves, electrical field stimulation (EFS) produced a fast, monophasic inhibitory junction potential (IJP) followed by a triggered slow wave (TSW) which could be premature or delayed and whose amplitude was maximum near the MyP region and decayed progressively in the other areas (minimum in SMP region). The K+ channel blocker, apamin at 10⁻⁶ M, did not affect resting membrane potentials or spontaneous slow waves but inhibited the amplitude of the IJP up to 70% and slightly but significantly enhanced (30%) the amplitude of the TSW. Long duration, single pulses (50-100 msec square waves, 10-20 V) elicited TSWs without IJPs. Both the slow waves and TSWs were associated with contractions of circular muscle which were significantly enhanced by apamin but not by blockers of adrenergic and cholinergic nerves. When the IJPs recorded near the MyP or DMP were abolished by tetrodotoxin (TTX, 1 μM) or by the NO synthase (NOS) inhibitor, N^ω nitro L-arginine (L-NNA, 50 μM), the occurrence of the TSW in response to EFS was advanced in time and increased in amplitude. The effects of L-NNA were reversed by L-but not D-arginine (both 1 mM). L-arginine significantly prolonged the durations of IJPs from the MyP and DMP regions. In contrast, the N-type Ca²⁺ channel blocker ω-conotoxin GVIA (ω-CTX, 1-3 x10⁻⁷M) abolished the IJP but delayed the induction of the TSW. Subsequent addition of either TTX or L-NNA advanced the onset of the TSW. The TSWs elicited by 50-100 msec single pulses were resistant to TTX, ω-CTX, or L-NNA. All treatments which abolished the IJP significantly increased contractions of circular muscle associated with spontaneous slow waves and TSWs. In the isolated circular muscle preparation (with the DMP intact) triangular slow waves were recorded near the DMP or close to the MyP border. The frequency and amplitude of the slow waves recorded near the DMP were significantly smaller than those recorded in similar areas in the full thickness preparation. EFS of this preparation evoked IJPs of 18-20 mV in amplitude. The IJPs were biphasic, lasted 5s and showed a fast and a slow component. No TSW occurred after the fast component of the IJPs; slow repolarization was observed instead. Long duration single pulses did not induce TSWs. In this preparation, the NOS inhibitor, N^ω nitro L-arginine methyl ester (L-NAME, 3x10⁻⁴ M), abolished the IJPs and regularized the slow waves, but TSWs could not be evoked. Superfusion of inhibitory neuromediators had different effects on pacemaking activity. SIN-1, a donor of NO, hyperpolarized the membrane, significantly increased slow wave frequency but reduced its amplitude, and abolished contractions. VIP (less effective) and PACAP (more effective) reduced slow wave frequency and amplitude without changing resting membrane potentials. P ACAP, but not VIP, increased circular muscle tone at 10⁻⁶ M. Nifedipine (10⁻⁷ and 3 X 10⁻⁷ M), an L-type calcium channel blocker, did not modify the shape of slow waves in any area of the full thickness preparation. It also did not reduce the amplitude of the IJP or TSW. Ni²⁺ at 200 μM, a Ca²⁺ channel blocker, inhibited slow wave frequency and amplitude and contractions. In Ca²⁺ -free Krebs (0.1 mM EGTA) for 10-15 min, the amplitude and frequency of the slow waves were gradually reduced. The TSW in response to 100 msec single pulses was still recorded near the MyP but never near the DMP region. The inhibitory effect of Ca²⁺ -free solution on slow wave amplitude was more rapid in onset near the DMP region. The intracellular Ca²⁺ store pump inhibitor, cyclopiazonic acid (10-30 μM), enhanced slow wave frequency and contractions. This differential sensitivity to removal of Ca²⁺ may be related to the morphological and functional observations which suggested that different electrical coupling properties between the pacemaker networks existed. The MyP pacemakers were less electrically well-coupled by visible gap junctions (low resistive cell-to-cell contacts) to outer circular muscle and hence showed greater susceptibility to 1 mM octanol (a gap junction blocker). The DMP pacemakers made numerous gap junction contacts to circular muscle, and slow waves paced from this region were less susceptible to 1 mM octanol. We conclude that 1) the pacemaker system of the canine ileum consists of two types of pacemakers that correspond to the presence of two networks of pacemaker cells found in the MyP and the DMP. The MyP network appeared to dominate pacemaking activity. 2) The slow waves and the TSW originated independently of neural activity but were delayed by IJPs. The MyP and the DMP provide two independent inhibitory neural inputs, where NO is released to mediate IJPs and relaxation and influence the delay in the occurrence of the TSW. 3) The TSW originates exclusively from the MyP region from which it spreads passively to other areas. It can reset the timing of slow waves in both pacemaker networks. 4) Ca²⁺ entry through non L-or N-type Ca²⁺ channels initiates slow waves. Intracellular Ca²⁺ stores modulate slow waves. 5) Different nature of electrical coupling of the MyP and DMP pacemakers to circular muscle may explain the differential sensitivity of slow waves to Ca²⁺ removal and gap junction blockade. / Thesis / Master of Engineering (ME)

Objectives: To investigate the feasibility of real-time three-dimensional (3D) echocardiographic analysis of right ventricular (RV) function in healthy dogs and to compare conventional and 3D echocardiographic (3DE) indices of RV function in dogs with various stages of myxomatous mitral valve disease (MMVD), classified per the guidelines of the American College of Veterinary Internal Medicine, to those from healthy dogs. Animals: Twenty-two healthy dogs and 56 dogs with MMVD Methods: All dogs underwent conventional and 3D echocardiographic examinations. Measurements of 3DE RV function indices including RV end-diastolic volume (EDV), RV end-systolic volume (ESV), RV stroke volume (SV), and RV ejection fraction (EF) were recorded. Measurements of conventional indices of RV function were also obtained. RV EDV, ESV, and SV were indexed to bodyweight (BW) and analyzed using commercially available software. Results: Three-dimensional RV datasets could be acquired and analyzed in all dogs. Intra- and inter-observer coefficents of variation were > 20% for all 3D RV indices. Right ventricular EDV and ESV were decreased and RV EF was increased in dogs with advanced MMVD when compared to controls. Several conventional echocardiographic indices of RV function also differed between the control group and various MMVD groups. Conclusions: Real-time 3DE RV assessment is feasible in normal dogs with acceptable intra- and inter-observer variability. Several 3DE indices of RV systolic function differ between dogs with advanced MMVD when compared to normal dogs. Further investigation is required to determine if these differences have clinical implications. / Master of Science

Background: Although lymphoma is one of the most common malignancies in dogs, remission rates and survival times remain stagnant. Treatment with a multi-agent chemotherapy protocol induces remission for less than one year and the majority of patients relapse. Fewer than 25% of dogs live longer than two years with the currently available treatments. Targeted immunotherapy using checkpoint molecule blockade of PD-1 and PD-L1 shows promise for various types of human cancer, including relapsed/refractory lymphoma; however, little is known regarding the role of these checkpoint molecules in canine lymphoma. Objectives: To determine the patterns of expression of mRNAs encoding PD-1 and its ligands PD-L1 and PD-L2 in lymphoma and reactive lymphoid hyperplasia controls. Methods: Retrospective: formalin-fixed paraffin-embedded (FFPE) tissue from dogs with untreated lymphoma (n=10) and reactive lymphoid hyperplasia (n=10). Prospective: fine-needle aspirates (FNAs) from dogs with untreated lymphoma (n=10) and reactive lymphoid hyperplasia (n=10). Total RNA was extracted, and expression of PD-1, PD-L1, and PD-L2 was measured using qRT-PCR analysis of random-primed cDNA. Checkpoint molecule expression levels were determined using the 2^∆∆CT method. Lymphoma immunophenotype was assessed using immunohistochemical analysis of CD3 and CD79a (FFPE) and review of patient medical records (FNA). Data analysis included Wilcoxon ranksum tests, Dunn's procedure of multiple comparisons, Kruskal-Wallis tests, and regression within an ANOVA. Significance at P < 0.05. Results: PD-1, PD-L1, and PD-L2 expression (normalized internally to 18S rRNA) was lower in lymphoma compared to reactive lymphoid hyperplasia (FFPE); the difference was significant for PD-1 and PD-L2. PD-1 and PD-L2 expression was lower in lymphoma compared to reactive lymphoid hyperplasia (FNA); the difference was significant for PD-1. PD-1, PD-L1, and PD-L2 expression was lower in B cell lymphoma compared to reactive lymphoid hyperplasia (FFPE); this difference was significant for PD-1 and PD-L2. PD-1 and PD-L2 expression was lower in B cell lymphoma compared to reactive lymphoid hyperplasia (FNA); the difference was significant for PD-1. The higher relative abundance of PD-L1 vs PD-1 and PD-L2 vs PD-1 was significantly different between lymphoma and reactive lymphoid hyperplasia (FFPE and FNA). Conclusions: In this study, checkpoint molecule expression was not upregulated in canine lymphoma relative to canine reactive lymphoid hyperplasia, suggesting a limited application of PD-1 and PD-L1 blockade in canine lymphoma. The ligand:receptor relative abundance imbalances reflect the lower PD-1 expression relative to PD-L1 and PD-L2 in lymphoma. Although these results do not suggest that checkpoint inhibitors would be useful for treatment, they give insight into the mechanisms of unchecked lymphocyte proliferation in canine lymphoma. / Master of Science / Lymphoma, a cancer of the white blood cells in the body, is one of the most common malignancies in dogs. Although treatment with a multi-agent chemotherapy protocol results in high remission rates, the remission duration is usually less than one year, with the majority of patients relapsing. In an effort to improve remission rates and survival times, scientists have been working to develop therapeutic interventions that target specific points in the development and replication cycle of a cancer cell. One such strategy, targeting checkpoint molecules programed death (PD)-1 and PD-L1, has shown promise for several different types of human cancers, including lymphoma. PD-1 is a receptor on T cells, which together with its ligands, PD-L1 and PD-L2, decreases lymphocyte function when activated. This is a protective mechanism, acting to inhibit sustained harmful inflammation in a normal healthy dog. Some cancers have taken advantage of this pathway, increasing expression of PD-L1 or L-L2 in order to evade detection by the immune system. To date, little is known regarding the role and expression of these immune checkpoint molecules in dogs with lymphoma. We sought to evaluate if PD-1, PD-L1 and PD-L2 expression is significantly increased in canine lymphoma compared to reactive lymphoid hyperplasia controls. Tissue samples were collected from two sources. Cytology samples of lymphoma and reactive lymphoid hyperplasia were collected by fine needle aspiration from clinical patients. Formalin fixed paraffin embedded tissue samples of lymphoma and reactive lymphoid hyperplasia were collected from the archived tissue bank. Using a molecular analysis technique called quantitative reverse transcription PCR (qRT-PCR) we measured the amount of messenger RNA (mRNA) encoding PD-1 and its ligands PD-L1 and PD-L2 in lymphoma and in reactive lymphoid hyperplasia controls. In our results we did not observe an upregulation in the expression of checkpoint molecules in canine lymphoma relative to canine reactive lymphoid hyperplasia. This suggests there may be a limited therapeutic application for PD-1 and PD-L1/PD-L2 blockade in canine lymphoma. Although these results do not suggest that checkpoint inhibitors would be useful for treatment, they give insight into the mechanisms of unchecked lymphocyte proliferation in canine lymphoma.

The object of this study was to compare growth factor and cytokine profiles in canine autologous conditioned serum (ACS) to canine plasma. Blood collected from 16 medium to large breed dogs was used to produce ACS (Orthokine® vet irap 10 syringes) and citrated plasma (control). Canine-specific ELISA assays were run per manufacturers’ instructions for interleukin (IL)-10, IL-4, tumor necrosis factor (TNF)-α, insulin-like growth factor (IGF)-1, fibroblast growth factor (FGF)-2, transforming growth factor (TGF)-β1, IL-1β, and interleukin-1 receptor antagonist (IL-1ra). Serum, in addition to plasma and ACS, was collected from an additional 6 dogs for TNF-α, IL-1β, and IL-1ra analysis (total of 22 dogs). Data were analyzed for differences in cytokine concentrations between ACS, plasma, and serum using the Wilcoxon signed-rank test with significance set at P<.05.There was a large variability in growth factor and cytokine concentrations between individual dogs in both plasma and ACS. There were no significant differences in IL-10, TNF-α, IGF-1, FGF-2, and TGF-β1 concentrations between ACS, plasma, or serum. ACS concentrations of IL-1β (median, range; 46.3 pg/mL, 0-828.8) and IL-4 (0.0 pg/mL, 0-244.1) were significantly increased compared to plasma (36.6 pg/mL, 0-657.1 and 0.0 pg/mL, 0-0, respectively). IL-1ra concentrations in ACS (median, range; 3458.9 pg/mL, 1,243.1-12,089.0) were significantly higher than plasma (692.3 pg/mL, 422.5- 1,475.6), as was the IL-1ra:IL-1β ratio (39.9 and 7.2, respectively). / Master of Science