Master of Science / Diagnostic Medicine and Pathobiology / Carol R. Wyatt / Currently, porcine reproductive and respiratory syndrome virus (PRRSV) is the most economically significant disease affecting the swine industry. PRRSV is known for its restricted cell tropism, primarily infecting porcine alveolar macrophages (PAM) via receptor-mediated endocytosis. PRRSV infects only a portion of the PAM population both in vivo and in vitro, which suggests that not every macrophage is PRRSV-permissive. Three surface proteins that can act as receptors for PRRSV have been identified on PAM, however, little else is known about the regulation of macrophage tropism. Factors determining cellular permissibility or resistance to PRRSV infection remain largely uncharacterized, although a recent study from our laboratory demonstrated that 1) permissiveness to PRRSV infection increased with time in culture, 2) macrophages from infected pigs could be superinfected, and 3) addition of actinomycin D, which inhibits mRNA synthesis, blocked infection. These data suggest that a PRRSV-permissive subpopulation of cells derives from a non-permissive precursor population and depends on new mRNA synthesis. The current studies were designed to examine the effects of PRRSV on both infected and uninfected PAM cells in vitro, specifically focusing on the expression of MHC I, MHC II, CD14, CD163 and CD172a surface proteins. The results show upregulation of MHC II, CD14, CD163 and CD172a expression in PRRSV-infected cells and a downregulation on the uninfected cells within the PRRSV-inoculated cultures. The role of apoptosis in the PRRSV-inoculated cultures was investigated, with results showing similar, low levels of apoptosis in control and infected PAM. PAM cytokine responses to PRRSV and LPS were also examined and, although they were uniquely different relative to control PAM, no trends were detected in the responses of PAM infected with PRRSV compared to uninfected and classically stimulated PAM. These data confirm that there are at least two subsets of macrophages within the alveolar population and suggests that the subsets are differentially affected by PRRS virus. We also demonstrated that MHC I becomes undetectable on PAM as a result of the freezing process, and that PRRSV-permissiveness is greater in the cell population after freezing.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Raymond R. R. Rowland / Infectious cDNA clones of PRRSV make it possible to construct marker viruses for the study of virus replication and pathogenesis. The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the single largest protein produced during virus replication. The cDNA of the pCMV-129 infectious PRRSV clone was modified by creating unique Mlu I and SgrA I restrictions sites at nucleotide (nt) positions 3,219 and 3,614, respectively: both located within the C-terminal region of nsp2. cDNAs coding for oligo- and polypeptide tags, including FLAG, enhanced green fluorescent protein (EGFP) and firefly luciferase were inserted into the newly created restriction sites. The results showed that only the EGFP-containing genomes were properly expressed and produced virus. EGFP fluorescence, but not EGFP immunoreactivity, was lost during passage of recombinant EGFP viruses in culture. Sequencing of a fluorescence-negative EGFP virus showed that the EGFP remained intact, except for the appearance of mutations that may affect chromophore formation. The results show that nsp2 can be a site for the expression of foreign proteins. Removal of the region between Mlu I and SgrA I sites resulted in a virus that contained a 131 amino acid deletion. The deleted region was replaced with EGFP or an eight amino acid influenza hemagglutanin (HA) tag. Recombinant viruses were used to infect pigs. Gross and micro-histopathology showed reduced pathogenesis when compared to the parent wild-type virus. The 131 amino acid peptide, when expressed as a recombinant protein and coated onto enzyme linked immunosorbent assay (ELISA) plates, was recognized by sera from pigs infected with wild-type virus, but not the deletion mutants. The results from this study show that nsp2 is a potential target for the development of marker vaccines that can differentiate infected from vaccinated animals (DIVA) and for virus attenuation.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Gary A. Anderson / Foot-and-mouth disease (FMD) is a highly infectious viral disease of cattle, pigs, sheep, goats, buffalo, and artiodactyl wildlife species. Foot-and-mouth disease virus is endemic and periodic devastating epidemics have occurred and caused heavy economic losses in Iraq for a long time. The first official cases of FMD were recorded in 1937, while the first record of a specific FMD serotype in Iraq was serotype A in 1952. Other serotypes have been reported since then; serotypes O, SAT-1 and Asia1 were recorded in 1957, 1962, and 1975, respectively. Veterinary Services in Iraq has been severely weakened over the past two decades, and its infrastructure has been devastated as a consequence of previous political conflicts, wars and international sanctions. The breakdown of Veterinary Services led to the disruption of disease control strategies, collapse of disease surveillance and monitoring, and weakening of response systems. The destruction of the Al-Dora FMD laboratories for diagnosis and vaccine production by the United Nation in 1996, and the restrictions placed on the importation of vaccines have strongly affected the FMD control program. A severe epidemic of FMD occurred in Iraq in 1998, affecting 2.5 million ruminants and causing heavy losses in newly born animals. It is estimated to have killed about 550,000 animals. The outbreak was due to the serotype O1 Middle East strain which has affected large and small ruminants. In 2009, Iraq was severely affected by new serotype A (subtype A Iran 05). The major efforts of Veterinary Services in Iraq have been directed towards control of FMD by vaccination strategies. Two types of vaccine have been used, trivalent vaccine (O, A 22, and Asia 1) for cattle and buffalo and monovalent vaccine (O Manisa) for sheep and goats. Vaccination has been implemented once yearly on a voluntary basis. Sometimes other limited control measures have accompanied vaccination, which include quarantine, movement control, focused vaccination, disinfection, and public awareness programs. The FMD control program in Iraq has been confronted by many challenges: deficits in FMD surveillance and emergency preparedness, limited diagnostic capabilities, difficulties in restricting animal movement, and lack and irregular supply of appropriate vaccines.

Master of Science / Department of Clinical Sciences / David E. Anderson / Lameness in cattle is a common cause of pain however there are no approved cattle analgesic drugs. Flunixin meglumine, the only non-steroidal anti-inflammatory drug approved for use in adult dairy cattle, is labeled for pyrexia associated with bovine respiratory disease, endotoxemia, acute mastitis and associated inflammation. There is currently a lack of objective data regarding the analgesic efficacy of flunixin meglumine in cattle. The objectives of this study were to characterize an amphotericin B-induced lameness model and to ascertain the analgesic effects of flunixin meglumine using multimodal assessment. We hypothesized that flunixin meglumine would provide analgesia as evidenced by increased activity levels as well as increased exerted force and contact area on the affected limb in flunixin treated steers. Amphotericin B-induced synovitis arthritis was induced in the distal interphalangeal joint of 10 dairy steers. The cattle were randomly allocated between a treatment and a control group. The treatment steers received flunixin meglumine at the time of arthritis induction and at 12 hours post-induction. Accelerometric, gait, pressure mat, vital parameter and plasma cortisol data were gathered in the pre and post-induction phases. The data were analyzed using linear mixed models with treatment and time designated as fixed effects. Induction of amphotericin B arthritis produced a moderate, transient lameness. Control steers were more than twice as likely to be lame as flunixin meglumine treated steers using visual lameness assessment (92.2% ± 8.1 versus 40.7% ± 2.5) (P<0.03). Flunixin meglumine treated steers placed significantly greater force and contact area on the affected foot. Control steers also placed significantly greater force, impulse and contact area on the paired claw as compared to control steers. Flunixin treated steers spent considerably less time in recumbency than their control counterparts, particularly in the immediate post-induction time period. This is one of the first studies to document the character of an amphotericin B-induced synovitis arthritis model in cattle as well as to document analgesic efficacy of a nonsteroidal anti-inflammatory drug in an induced lameness model. Flunixin meglumine was efficacious in providing analgesia in an amphotericin B-induced lameness model in dairy steers.

Doctor of Philosophy / Department of Anatomy and Physiology / Frank Blecha / L Tonatiuh Melgarejo / Background - Cardiovascular disease (CVD) impacts the lives of millions, and ranks as the number one killer world-wide. Despite significant research efforts, CVD remains a major burden on the national health care system, and novel therapeutic modalities to effectively and curatively fight many debilitating diseases of the heart and vasculature are urgently needed. The role of inflammation in the development of CVD has been increasingly in focus through the past decade. Elucidating upon the plethora of innate immune mechanisms likely involved in CVD therefore becomes of immediate interest. Host defense peptides (HDPs) are central elements of innate immunity, encompassing molecules (including the defensin peptides) with wide-reaching biological effects, including immunomodulation and antimicrobial activity. Hypothesis & Specific Aims - The study's main hypothesis relies upon the basic concept that the heart possesses a local innate defense system, which actively aids in fighting off a variety of "danger signals", and that a disarray in this defense contributes to development of CVD. The heart expresses beta-defensin peptides (BDs), and we theorized that these HDPs act as a local defense system within the myocardium - or in other words as "guardians of heart health". The specific aims of the experimental studies were to 1) Evaluate expression of cardiac BDs in response to inflammatory mediators, and 2) Assess the functional properties (including antimicrobial activity and immunomodulation) of synthetic BD peptides in vitro. Design & Methods - To test our hypothesis, we studied myocardial beta-defensin expression (rBDs) in a rat model, comparing levels among two experimental and one control group. Animals were exposed to lipopolysaccharide (LPS) or high-fat diet (HFD) intake - representative of exposure to either an infectious (LPS) or non-infectious (HFD) inflammatory mediator. Serum samples were collected for measurement of cytokines, inflammatory and cardiac biomarkers and lipid-profiling. Beta-defensin levels were assessed using customized Superarray assays and qRT-PCR, and all amplicon sizes on the PCR products were subsequently confirmed using agarose gel electrophoresis. Serum levels were assessed on commercial ELISA kits. Functional assessment of select rBDs included computational modeling as well as in vitro antimicrobial and cell migration assays. Results & Conclusion - Exposure to high-fat diet feeding for a period of three weeks resulted in a multifold-increase in cardiac mRNA expression of select rBDs, while short-term LPS exposure resulted in a smaller, but statistically non-significant, elevation in the myocardial expression of rBDs. Synthetic analogues of two naturally occurring cardiac rBDs were evaluated for in vitro activity. The synthetic rBD11 peptide exhibited antimicrobial activity against Staph aureus, and both rBDs exhibited chemoattraction of rat leukocytes. Our data suggests that rBDs might play a central role in the intrinsic immune mechanisms of the cardiovasuclar system, and possibly act as protectors of heart health.

Master of Science / Department of Clinical Sciences / David S. Hodgson / Peste des petits ruminants (PPR) is an economically important and highly contagious disease of sheep and goats. It is characterized by enteritis, stomatitis, pneumonia, and discharge from the nose and eyes. This report contains a review of PPR and its epidemiology in Afghanistan and other PPR- endemic countries followed by recommendations for dealing disease in Afghanistan. Studies showed that PPR is still endemic in Afghanistan’s neighboring countries including Pakistan, Iran, Tajikistan, and China. From January of 2009 to January of 2010, 852 outbreaks of PPR were reported to the OIE from 24 different countries. However, this study focuses on Afghanistan and some neighboring countries (Iran, Tajikistan). Animal clinics and Veterinary Field Units (VFUs) reported 7,741 cases of PPR from 2008 to 2009 in different parts of Afghanistan. A study by the Food and Agriculture Organization (FAO) in 2009 showed that PPR is endemic in various parts of Afghanistan. Seroprevalence of PPR varied from 0% in Kapisa to 48% in Herat province of Afghanistan. The last chapter of this report includes recommendations and guidelines regarding prevention and eradication of PPR from Afghanistan. These recommendations could help improve animal health and the economy of Afghanistan in the future.

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.