Master of Science / Department of Clinical Sciences / James K. Roush / The objective of this study was to evaluate the effects of intraarticular dextrose prolotherapy on osteoarthritis of the elbow or stifle in dogs. This was a randomized, double-blind, placebo controlled prospective trial of intraarticular dextrose prolotherapy given at 0 and 6 weeks for relief of osteoarthritis. Dogs with unilateral lameness were evaluated by orthopedic exam, visual lameness score, Canine Brief Pain Inventory (CBPI), goniometry, and by kinetic gait analysis at 0, 6 and 12 weeks. Joint radiographs were scored at 0 and 12 weeks. Ten client-owned dogs with naturally occurring osteoarthritis of the elbow or stifle were enrolled. Initial visual lameness, age, body weight, duration of lameness, and CBPI scores did not differ between groups. Change in CBPI PS score in the prolotherapy group from week 6-12 was significantly less improved than placebo with no other significant differences in CBPI Pain Severity (PS) or Pain Interference (PI) scores between groups. There were no significant differences for range of motion or radiographic scores between groups at any time. Kinetic forces improved in prolotherapy dogs, but were not significantly different between treatment groups at any time. There were no significant benefits of intraarticular dextrose prolotherapy for treatment of osteoarthritis of the elbow and stifle in dogs in this study. Larger enrollments and more stringent inclusion criteria should be considered in future evaluations of prolotherapy.

Master of Science / Department of Anatomy and Physiology / Jishu Shi / The development of reverse genetics systems for the manipulation of viral and bacterial genomes has provided platforms for identifying virulence genes, studying pathogenesis and developing vaccines. Replication-competent vaccines (e.g., modified live virus (MLV) vaccines and replicating viral/bacterial vectors) are considered the most efficacious approach for vaccine development. We constructed replication-competent candidate vaccines for two viral diseases in pigs via reverse genetics. The first vaccine we designed is to protect against highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). HP-PRRSV can cause high mortality in pigs of all ages. Vaccines to protect pigs from HP-PRRSV are not commercially available in the US. According to previous studies, the non-structural protein (NSP) coding region of HP-PRRSV is closely related to the high mortality rate and the structural protein (SP) coding region contributes to the induction of broadly protective neutralizing antibodies. We created a chimeric PRRSV, of which the SP coding region was derived from HP-PRRSV and NSP coding region was derived from a low-pathogenic strain. This chimeric PRRSV caused similar CPE in cells as parental viruses, but had slower growth kinetics. We hypothesize that this chimeric virus will have a low pathogenicity and could serve as a candidate vaccine that can provide protection against HP-PRRV. The second vaccine vector is a modified Listeria innocua (L.inn), a non-pathogenic strain of Listeria. Genetically related Listeria monocytogenes (L.m) is a well-known intracellular pathogen that encodes specialized virulent determinants facilitating its intracellular growth and spread. Our goal is to make L.inn a vaccine vector that can deliver classical swine fever (CSF) viral antigen into intracellular environments by complementation of L.inn with selected L.m virulence genes necessary for intracellular survival and induction of a robust immune response. In this study, we constructed a shuttle vector pHT-E2 that can express CSFV antigen E2 in L.inn. We cloned the plcA-prfA operon of L.m virulence gene cluster (vgc) into pHT-E2, which enhanced the expression of E2 in L.inn. In future studies, we plan to clone additional L.m virulence genes into the shuttle vector to increase immunogenicity of this recombinant L.inn and test its ability to protect pigs from CSFV.

Master of Science / Department of Clinical Sciences / Elizabeth Davis / Differences in pharmacokinetics and drug disposition exist between young and adult animals which become especially important for drugs with a narrow therapeutic index. While the pharmacokinetics and plasma concentrations of intravenous lidocaine have been studied in adult horses, determination of the disposition in foals is necessary before appropriate clinical use can be determined. This study examined the disposition of intravenous lidocaine in healthy (phase I) and hospitalized (phase II) foals. Phase I consisted of 6 healthy 4-10 week old foals administered a 6-hour intravenous lidocaine infusion. Phase II consisted of 8 hospitalized foals (2-136 days old) administered intravenous lidocaine. A bolus (1.3 mg/kg) of lidocaine was administered intravenously to all foals followed by a 50 µg/kg/min infusion. Plasma lidocaine and monoethylglycinexylidide (MEGX) concentrations were determined. In phase I, plasma lidocaine concentrations remained below the suggested adult target range of 1-2 µg/mL with MEGX concentrations approximately half that of the parent drug. Total body clearance of lidocaine was 72.2 ± 7.8 mL/min/kg, elimination half-life (t₁/₂) was 26.3 ± 3.7 min, peak concentration (C[subscript]m[subscript]a[subscript]x) was 0.79 ± 0.07 µg/mL, and the volume of distribution (V[subscript]d) was 1.8 ± 0.4 L/kg. The C[subscript]m[subscript]a[subscript]x for MEGX was 0.36 ± 0.11 µg/mL, t₁/₂ was 60 ± 6 min and time to peak concentration (T[subscript]m[subscript]a[subscript]x) was 279.6 ± 90.3 min. In phase II, the severely compromised foals that were eventually euthanized had the largest fluctuations in plasma lidocaine and MEGX concentrations; foals that were discharged from the hospital had plasma concentrations below the target adult range similar to foals in phase I. In conclusion, despite low plasma lidocaine concentrations, the clinical benefits observed in foals may be due to the presence of metabolites. Further research in a larger population of unhealthy foals is required before comprehensive dosing recommendations can be made.

Master of Science / Department of Clinical Sciences / Warren L. Beard / Objectives: To quantify recovery of colored microspheres from cadaver tarsocrural joints via arthroscopic or needle lavage, and to compare recovery for 1-5L of lavage fluid. Study design: Randomized experimental trial. Methods: 8 adult Quarter Horse cadavers had 1.5 million 15μm, colored microspheres injected into each tarsocrural joint. Each joint was randomly assigned to receive lavage with an arthroscope and egress cannula (group A) or three (1 ingress, 2 egress) 14 gauge needles (group N) with 5L 0.9% NaCl. The egress fluid from each liter of lavage was collected separately, and the number of microspheres present in each recovered liter was determined via spectrophotometry. Results: A significant interaction (p<0.01) was present between treatment group and liter. The number of microspheres recovered in the first liter of lavage fluid was significantly higher in the needle group than in the arthroscope group (p<0.01). For both groups the number of microspheres recovered in the first liter of lavage fluid represented a majority of the total microspheres collected, and was significantly different from the subsequent liters collected (p<0.01). The number of microspheres recovered did not differ between liters 2, 3, 4, and 5, within or between treatment groups. Conclusions: In this model, tarsocrural lavage with three 14-gauge needles was more effective at removing colored microspheres from the joint than arthroscopic lavage, suggesting the number or placement of portals present may be more important than portal size and flow rate. No difference in microsphere recovery was seen with lavage volumes greater than 1L.

Master of Science in Biomedical Sciences / Department of Diagnostic Medicine/Pathobiology / Natalia Cernicchiaro / David G. Renter / Cattle shed Shiga toxin-producing Escherichia coli (STEC) in their feces. Therefore, cattle pose a risk to contaminate produce, water, and beef products intended for human consumption. The United States Department of Agriculture Food Safety and Inspection Service consider seven STEC serogroups (O26, O45, O103, O111, O121, O145, and O157) as adulterants in raw, non-intact beef products. Contrary to O157, the frequency and distribution of non-O157 serogroups and virulence genes have not been well-established in cattle. Therefore, the objectives of my thesis research were: 1) to appraise and synthesize data from peer-reviewed literature on non-O157 serogroup and virulence gene prevalence, and 2) to determine the prevalence of seven STEC in feedlot cattle feces across seasons. A systematic review and meta-analysis of published literature were conducted to gather, summarize, and interpret the existent data regarding non-O157 serogroup and virulence gene prevalence in cattle. Random-effects meta-analyses were used to obtain pooled non-O157 fecal prevalence estimates for continents worldwide and meta-regression analyses were conducted to evaluate effects of specific factors on between-study heterogeneity. Results indicated that non-O157 serogroup and virulence gene fecal prevalence significantly differed (P < 0.05) by geographic region, with North America yielding the highest pooled prevalence estimate worldwide. While previous research has demonstrated a strong seasonal shedding pattern of STEC O157, data regarding the seasonality of non-O157 STEC shedding in cattle is very limited. A repeated cross-sectional study was conducted to obtain serogroup and virulence gene prevalence data for the seven STEC in pre-harvest cattle feces, in summer and winter. We found that non-O157 serogroups were recovered in fecal samples collected in both seasons but virulence genes, thus STEC, were rarely detected in summer and undetected in winter. In conclusion, non-O157 STEC are present in cattle feces at very low frequencies, but STEC O103 and O157 significantly differed (P < 0.05) between seasons. Overall, the research described in this thesis greatly contributes to the limited body of data regarding non-O157 serogroup and virulence gene distribution in cattle and provides a better understanding of two major risk factors, season and geographic distribution, associated with STEC fecal shedding in cattle.

Master of Science / Department of Clinical Sciences / Elizabeth G. Davis / Objective: To determine pharmacokinetic and pharmacodynamic properties of oral dexamethasone solution and powder compared to intravenous dexamethasone solution in healthy horses. Animals: 6 horses, 13-27 years if age, 385-630 kg Procedures: In a randomized, cross-over block design six healthy adult horses each received the following treatments 1) dexamethasone solution IV 0.05 mg/kg, 2) dexamethasone solution orally (PO) 0.05 mg/kg, and 3) dexamethasone powder PO 0.05 mg/kg all in the fed and fasted state. Each horse acted as an untreated control as secretion of cortisol was monitored for normal circadian rhythm. Quantification of plasma dexamethasone concentration and serum cortisol activity was determined by LC/MS and chemiluminescent enzyme immunoassay, respectively. Results: Each horse exhibited a circadian rhythm in cortisol secretion; however there was variation present between each horse. Mean cortisol concentrations at 6:00 AM and 8:00 AM were significantly higher than concentrations at 8:00 PM and 10:00PM. Cortisol concentrations were significantly less than base-line starting 1 hour post-administration of dexamethasone through 72 hours for the fasted treatment groups, and 2 hours through 48 hours for the fed groups. Pharmacokinetic modeling resulted in a two compartment model for the IV administration with elimination from the central compartment, and a one compartment model for orally administered dexamethasone. Oral, fasted, compounded powder achieved a significantly higher maximum concentration (Cmax) than both fasted and fed oral dexamethasone solutions. The AUC0inf for the orally administered compounded powder was significantly different when comparing fasted versus fed treatment groups. Bioavailability ranged between 33% and 70% among treatment groups, but due to the high variability there was not a significant difference. Conclusions and Clinical Relevance: Hospitalization of the horses did not have an effect on their circadian rhythm of cortisol secretion. Oral and intravenous administration of dexamethasone resulted in adrenal suppression with cortisol concentrations returning to base-line 48-72 hours post-administration. Although bioavailability was variable cortisol suppression was similar among all treatment groups. The variability in oral absorption will need to be taken in to account for oral dosing of dexamethasone.

Master of Science / Department of Clinical Sciences / Mike D. Apley / Lameness is a common, costly, and painful affliction in cattle at all production levels. There are currently no compounds specifically approved for analgesia in cattle in the United States. We hypothesized that intra-articular amphotericin B produces a controlled, transient synovitis-arthritis in cattle and that this model would allow characterization of the analgesic effects of intravenous sodium salicylate. This study examined the efficacy of sodium salicylate for providing analgesia in an amphotericin B-induced bovine synovitis/arthritis model utilizing ten male Holstein calves, 4-6 months old, and weighing approximately 250 kg. The study used a repeated measures partial cross-over design with 2 phases consisting of 3 treatment periods within each phase. Calves were blocked by weight and randomly assigned to sodium salicylate (50mg/kg intravenously) or placebo group for phase 1. In period 1, lameness induction was simulated with a needle-prick of the coronary band, followed by drug or placebo administration. At predetermined timepoints, serial blood samples for cortisol and salicylate concentrations, electrodermal activity measurements, heart rates, and pressure mat data were collected. Visual lameness scores were recorded by a blinded observer. In period 2, lameness was induced with injection of amphotericin B into the distal interphalangeal joint followed by drug or placebo administration with sample collection as previously described. In period 3, drug or placebo was administered to the respective calves with sample collection. After a 10-day washout, Phase 2 was conducted with treatments crossed over between groups. Cortisol and salicylate samples were analyzed by competitive chemiluminescent immunoassay and fluorescence polarization immunoassay, respectively. The pharmacokinetic data were analyzed using compartmental analysis. Mean intravenous salicylate apparent volume of distribution (V[subscript]d) was 0.2 ± 0.005 L/kg, total body clearance (CL[subscript]B) was 4.3 ± 0.2 mL/min*kg, and elimination half life (T[subscript]1/2 el) was 36.9 ± 1.2 minutes. The repeated measures data were analyzed based on a univariate split-plot approach with a random effects-mixed model. Differences in stance phase duration and serum cortisol concentration values were seen between both periods and treatment group*periods; differences in heart rate, contact surface area, and contact pressure values were seen between periods, suggesting that our lameness model was effective. No differences were seen between treatment groups. When analyzed by visual lameness score, differences were seen in heart rate, contact surface area, contact pressure, and cortisol concentrations. Area under the time-effect curves, determined using the trapezoidal rule, had results similar to the repeated measures data, except for a difference in period for electrodermal activity. This amphoterecin B-induced synovitis/arthritis model is a useful tool for studying changes associated with lameness in cattle. Sodium salicylate was not effective in providing analgesia following lameness.

Master of Science / Department of Clinical Sciences / Thomas Schermerhorn / The use of continuous blood glucose monitors (CBGMs) has recently come into favor in human medicine for the control and monitoring of the diabetic patient. It allows for a higher degree of accuracy of the true glucose curve throughout a 72-hour period. With this information, physicians are better equipped to treat and manage diabetic patients. Recently, this modality has been verified for use in veterinary patients including cats and dogs. This is an excellent source of information, especially in the management of difficult to regulate veterinary patients. This device has potential for use in various applications, particularly for the monitoring of patients with various diseases under general anesthesia. In order to ensure accurate results do occur when an animal is under general anesthesia, the continuous blood glucose monitor was evaluated on apparently healthy patients under anesthesia for routine procedures such as ovariohysterectomies and orchiectomies. In this manner, the monitor was tested on anesthetized patients that had the potential to experience hypothermia, hypotension, and other anesthesia-associated complications that can be typical of patients that could benefit from the CGMS.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Thu Annelise Nguyen / Gap junctional intercellular channels allow the cells to communicate with each other. A breach in gap junctional intercellular communication (GJIC) affects cell growth and proliferation. In addition, many neoplastic cells exhibit a decrease in GJIC. Many factors that decrease GJIC have been shown to potentiate cancer formation. 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), an environmental pollutant, is a carcinogen; however, its mechanism of carcinogenicity is unclear. Therefore, we examined the effect of TCDD on GJIC in MCF-7, a human breast cell line and normal mammary epithelial cells (HMEC). TCDD showed a decrease in GJIC in MCF-7 cells caused by increased phosphorylation of gap junctional protein, Cx43. PKCα-mediated phosphorylation of Cx43 was confirmed by inhibitor studies using calphostin C. Interestingly, TCDD affected GJIC in HMEC through a novel pathway involving redistribution of Cx43 to the perinuclear membrane. Our studies suggest that TCDD causes decrease in GJIC which could potentially lead to cancer. This also indicates that if GJIC is restored it could decrease cell growth and proliferation. Therefore, we investigated the role of substituted quinolines (PQ1), shown to bind with gap junctional proteins by computational docking. The results showed that indeed PQ1 significantly increases GJIC and exerts anti-tumor effect in human breast cancer cells compared to control without treatment or HMEC. We found an increase in GJIC, growth attenutation and increased apoptosis in T47D human breast cancer cell line. Our studies suggest that PQ1 is a novel gap junctional activator causing a decrease in tumor growth. Since PQ1 alone is effective in decreasing tumor growth in breast tumors, we proposed to test its efficacy with the current drug of choice for breast cancer, tamoxifen. The combinational treatment of tamoxifen and PQ1 showed a significant decrease in cell viability, increase in BAX (Bcl2-associated X), and, increase in caspase 3 activation compared to individual treatments. Hence, combinational treatment of PQ1 and tamoxifen can potentiate decrease in tumor growth. In conclusion, downregulation of gap junctions can potentiate tumor growth while restoration of GJIC can induce apoptosis and decrease tumor growth.

Master of Veterinary Biomedical Science / Department of Diagnostic Medicine/Pathobiology / Michael W. Dryden / Patricia A. Payne / Giardiasis is a common intestinal protozal parasitic infection of the pet dog and cat population. Veterinarians often have difficulty correctly diagnosing this parasite. Studies were conducted to compare the zinc sulfate double centrifuge fecal flotation to the SNAP (registered trademark) Giardia fecal ELISA test manufactured by IDEXX laboratories Inc. in purpose bred beagles and shelter and commercial kennel dogs. In these evaluations the zinc sulfate double centrifuge fecal flotation and fecal ELISA test performed similarly. Both tests performed better in the shelter and commercial kennel dog population than the chronically infected purpose bred beagles. There was an increase in number of positive animals identified when 3 consecutive daily samples were evaluated as compared to any one individual day for either test method. Post treatment evaluation of the diagnostic tests was performed in 23 laboratory beagles. Each beagle was treated for 3 consecutive days with Drontal plus and then bathed on the last day of treatment and fecal samples were collected from the treated dogs every other day starting one day post treatment for 21 days. It was found that all beagles were negative on zinc sulfate double centrifugation fecal flotation, fecal ELISA and IFA within 24 hours of treatment and nineteen (82.6%) of the beagles did not re-shed cysts during the 21 day post-treatment evaluation period. Four beagles returned to shedding cysts (Flotation or IFA positive) between days 17 and 21. These findings suggest that a positive test within a week of treatment is likely the result of inappropriate treatment. After the prepatent period, positive results may occur due to a return to shedding, reinfection or inappropriate treatment. Chronically infected laboratory beagles may not be a good model for acute Giardia infections as these dogs are rarely clinically ill and detection is more difficult.