Master of Science / Department of Diagnostic Medicine/Pathobiology / Shafiqul I. Chowdhury / Bovine herpesvirus type 1 (BHV-1) is an important pathogen of cattle that can cause severe respiratory tract infection known as infectious bovine rhinotracheitis (IBR), abortion in pregnant cows, and is an important component of the Bovine Respiratory Disease Complex (BRDC, “Shipping fever”). The ability of BHV-1 to transport anterogradely from neuron cell bodies in trigeminal ganglia to axon termini in the nasal and ocular epithelia of infected cattle complicates the control of the disease in both vaccinated and infected cattle populations. In calves and rabbits, Us9 deleted viruses have defective anterograde neuronal spread from cell bodies in the trigeminal ganglia to nerve termini in the nose and eye but retrograde spread remains unaffected. To characterize the neuronal spread of BHV-1, we developed primary neuronal cultures using the dorsal root ganglia (DRG) of rabbit embryos. We successfully used microfluidic chamber devices to isolate DRG in the somal compartment and allowed for efficient growth of axons into the axonal compartment. This enabled us to study axon mediated neuronal spread of infection as well as viral transport in axons. Thus, rabbit DRG neuronal culture was susceptible to BHV-1 mutant and wild-type infection, and the method allowed visualization of viral spread in chamber cultures using live cell imaging and fluorescent microscopy. Lastly, using the microfluidic chamber compartmentalized neuron culture system we showed that Us9 acidic domain-deleted and Us9 null mutant BHV-1 viruses had defective anterograde neuronal transport relative to BHV-1 wild type and/or Us9 rescued viruses.

Master of Science / Department of Clinical Sciences / Elizabeth Davis / The first aim of the current study was to investigate the pharmacokinetics of oral meloxicam tablets and the gastrointestinal and renal effects after a 14-day treatment period. Meloxicam was orally administered to six adult horses once daily at a dosage of 0.6 mg/kg for 14 consecutive days. Blood was collected prior to each administration and at 20 and 40 min, and 1, 2, 4, 8, 12, and 24 hours after administration on days 1, 7, and 14 for the determination of meloxicam plasma concentrations by mass spectrometry. In addition, trough samples were taken on days 3 and 10. Complete blood count, serum biochemical analysis, urinalysis, and gastroscopy were performed at baseline and conclusion of the investigation. Complete blood count, serum chemistry, and urinalysis results were unchanged through the study period. Gastroscopy scores were not significantly increased. The Cmax was 1.82 ± 0.80 µg/mL at Tmax 3.48 ± 3.30 hr on day 1, 2.07 ± 0.94 µg/mL at Tmax 1.24 ± 1.24 hr on day 7, and 1.81 ± 0.76 µg/mL at 1.93 ± 1.30 h on day 14 (p = 0.30). The mean half-life was 4.99 ± 1.11 h. The second aim of the study was to compare the analgesic effects and gastrointestinal and renal adverse effects of oral meloxicam tablets (0.6 mg/kg) to oral phenylbutazone tablets (4.4 mg/kg) orally once daily for 4 days in induced and naturally occurring lameness in adult horses. The study was performed on 4 healthy but lame adult horses. Complete blood count, serum biochemistry, urinalysis, and gastroscopy were performed prior to entrance to the study. Lameness was exacerbated in two horses using lipopolysaccharide (LPS; E. coli O55:B5) injected into the right metacarpophalangeal joint. The remaining two horses had Grade 3 or Grade 4 lameness due to naturally occurring laminitis. Meloxicam or phenylbutazone was administered to two horses each in a blinded, randomized manner once daily for four days. Lameness was evaluated using a pressure mat system and contact pressure, force, and stride length were evaluated at baseline and twice daily. Complete blood count, serum chemistry, and urinalysis were unremarkable for all four horses except one horse with an increased GGT. This horse experienced hepatic rupture secondary to amyloidosis the final day of the study. Gastric ulcer scores did not change during the study period. Phenylbutazone administration resulted in a greater response (force and contact area) in the right front and left hind limbs compared to meloxicam administration. There were not enough data points to evaluate the other two limbs. A third aim of the study was two-fold and first evaluated the effects of ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) with LPS on cyclooxygenase (COX) messenger RNA (mRNA) expression. The second portion documented the effects of LPS-induced joint inflammation and treatment with non-steroidal anti-inflammatory drugs on the mRNA and protein expression of COX-2 in PBMCs. The results indicate that LPS upregulates COX-2 gene expression in PBMCs. Additionally, injection of LPS into the metacarpophalangeal joint increases both COX-2 mRNA and protein expression in PBMCs at 24 hours after injection. The relative expression of COX-2 after treatment with meloxicam or phenylbutazone indicates a stronger inhibition with phenylbutazone; however, further study with additional horses is needed. Pharmacokinetic analysis of the oral tablet formulation of meloxicam indicates the pharmacokinetics are similar to the oral suspension formulation. Meloxicam appears to be inferior to phenylbutazone in its analgesic properties for induced lameness and naturally occurring laminitis, however the small sample size used in the study makes interpretation difficult.

Master of Science / Department of Clinical Sciences / Matt Miesner / Minimal research is available pertaining to the suspected acute cardiac injury of cattle experiencing the early signs of respiratory disease. One of the limitations of cardiac disease diagnosis in cattle is the lack of accurate measurement of cardiac disease. Blood biomoarkers such as cardiac troponin I could be used. Cardiac troponin I is a serum biomarker for cardiac injury in humans and many veterinary species. The goal of this thesis is to evaluate biophysical changes including cardiac injury that holstien calves experience during acute pneumonia. The first objective of this research was to evaluate a point of care cardiac troponin I assay for use in the bovine. Purified bovine cardiac troponin I was used to validate the accuracy of the assay over a wide range of concentrations in an in vitro experiment. This point of care assay was capable of accurately identifying bovine cardiac troponin I. An in vivo experiment was conducted to evaluate concentrations of cardiac troponin I in healthy Holstein calves. Concentrations evaluated with the point of care assay were similar to concentrations of a previously validated immunoassay. The second objective was to evaluate biophysical changes including serum biochemistry, complete blood count, cardiac troponin I, and high resolution digital thermography in Holstein calves during the first fourteen days after induction of pneumonia. Fibrinogen concentration increased with lung disease severity. The changes observed were considered clinically significant. Cardiac troponin I increased as pneumonia progressed during the study in this population.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Sanjeev K. Narayanan / Antibiotics are substances produced by bacteria or fungi that are inhibitory to other bacteria and fungi. Antimicrobial compounds include substances that are naturally produced, chemically modified or completely synthetic (chemically designed or synthesized). The chemical modification of naturally produced antibiotic generally results in increase stability, solubility, increased spectrum of activity, or efficacy. Antimicrobial compounds are used in animals to treat and control infectious diseases, and also for growth promotion. Bacteria may gain resistance to antibacterial agents via a variety of mechanisms. There is growing evidence that antimicrobial resistance has significant public health consequences. Rationale use of antimicrobial drugs using appropriate medication at the proper dosage and for duration is one of the important means to reduce selective pressure that helps reduce life of resistant organism. It is also vital to reduce the spread of multi drug resistant organisms in the environment especially in health care facilities. Bacteria evolve rapidly not only by mutation, but also by horizontal gene transfer through the transformation, transduction, and conjugation. Conjugation involves a close contact between two bacteria and transfer of the plasmid that carry many genetic elements. The pathogenic bacteria have the ability to sense as well as respond to the stress in the recipient. The epinephrine and norepinephrine play a key role in stress situations in animals. A previous study showed that norepinephrine (NE), a catecholamine at physiological concentrations promoted the conjugation efficiencies of a conjugative plasmid from a clinical strain of Salmonella typhimurium to an E. coli recipient in vitro. The objective of this study was to determine the effect of norepinephrine on conjugation of two E. coli strains. Both filter mating and liquid mating assays were used. The results revealed that there was no significance difference between the presence and the absence of norepinephrine on conjugative transfer of RP4 plasmid between E. coli strains (FS1290 and C600N) either in filter mating or liquid mating. Further studies are needed to determine whether higher concentration of (more than 20 mM) has any effects on conjugation in E. coli.

Master of Science / Department of Clinical Sciences / Hans Coetzee / Dehorning is a painful husbandry procedure that is commonly performed in dairy calves. Parenteral meloxicam combined with local anesthesia mitigates the physiological and behavioral effects of dehorning in calves. The purpose of this study was to determine the influence of timing of oral meloxicam administration on physiological responses in calves after dehorning. Thirty Holstein bull calves 8- 10 weeks of age (28-70 kg) were randomly assigned to one of three treatment groups: placebo-treated control group (CONT) (n=10), calves receiving meloxicam administered orally (1 mg/kg) in powdered milk replacer 12 h prior to cautery dehorning (MEL-PRE) (n=10) and calves receiving meloxicam administered as an oral bolus (1 mg/kg) at the time of dehorning (MEL-POST) (n=10). Following cautery dehorning, blood samples were collected to measure cortisol, substance P (SP), haptoglobin, ex-vivo prostaglandin E2 (PgE2) production after lipopolysaccharide stimulation and meloxicam concentrations. Maximum ocular temperature (MOT) and mechanical nociceptive threshold (MNT) was also assessed. Data were analyzed using non-compartmental pharmacokinetic analysis and repeated measures ANOVA models. Mean peak meloxicam concentrations were 3.61 ± 0 .21 μg/mL and 3.27 ± 0.14 μg/mL with average elimination half-lives of 38.62 ± 5.87 h and 35.81 ± 6.26 h in the MEL-PRE and MEL-POST groups respectively. Serum cortisol concentrations were lower in meloxicam-treated calves compared with control calves at 4 h post-dehorning (P=0.0004). SP concentrations were significantly higher in control calves compared with meloxicam-treated calves at 120 h after dehorning (P=0.038). PgE2 concentrations were lower in meloxicam-treated calves compared with control calves (P=0.001). MNT was higher in control calves at 1 h after dehorning (P=0.02) but meloxicam-treated calves tended to have a higher MNT at 6 h after dehorning (P=0.07). There was no effect of timing of meloxicam administration on plasma cortisol concentrations (P= 0.69), SP concentrations (P=0.86), haptoglobin concentrations (P=0.86), MOT (P=0.90), or MNT (P=0.99). However, PgE2 concentrations in MEL-PRE calves were similar to CONT calves after 12h post-dehorning, while MEL-POST calves had lower PgE2 concentrations for 3 d post dehorning. These findings suggest that meloxicam reduced cortisol, SP and PgE2 after dehorning but only PgE2 production was significantly affected by the timing of meloxicam administration.

Master of Science / Department of Clinical Sciences / Daniel U. Thomson / Acute Interstitial Pneumonia (AIP) is a costly issue that affects feedlot cattle, especially during hot and dry summers. Research has yet to elucidate the exact etiology of AIP; therefore this study was conducted to determine possible factors that contribute to AIP in feedlot cattle. During the summer of 2011 in a 55,000 head feedyard in southwest Kansas, animals exhibiting clinical signs of AIP were selected for ante-mortem examination and data collection. The animal population within the feedlot consisted of 75% heifers and 25% steers. Approximately 50% of the animal population was black hided animals. Ante-mortem data consisted of rumen gas cap measurement for NH3 and H2S, rumen pH, serum chemistry, rectal temperature, and body weight. Post-mortem cases with and without ante-mortem evaluations were also selected for an additional examination. Postmortem examination included similar data as ante-mortem examination with addition of visual and histological examination of lung tissue. There were 31 ante-mortem cases of clinical AIP with the following observations (mean ± SD): rectal temperature 105.3 ± 0.7 °F, weight 1098 ± 123 lbs., H2S 136 ± 133.3 ppm, and rumen pH 6.4 ± 0.5. Twenty-five healthy cohorts were selected from identical pens to serve as controls. Observations from control animals were: rectal temperature 103.7 ± 1.1 °F, weight 1113 ± 133.3lbs, H2S 269.8 ± 311.6 ppm and rumen pH 6.2 ± 0.6. A total of 61 post-mortem cases with a preliminary diagnosis of AIP were analyzed and displayed the following values: H2S 1279.7 ± 1569 ppm, and rumen pH 6.3 ± 0.36. Fifty-three of these postmortem cases had diffuse, focal and /or patchy AIP confirmed by histology.

Master of Science / Department of Clinical Sciences / Daniel U. Thomson / Cross-bred horned steers and heifers (n = 40; BW = 311.8 ± 4.7 kgs.) were used to determine the effect of dehorning methods on pain, cattle behavior and wound healing. Cattle were blocked by weight and randomly assigned to 1 of 4 treatments: 1) control (CON); 2) banded using high tension elastic rubber (BAND); 3) mechanically removed (MECH); or 4) tipped (TIP). Vocalization and behavior were recorded during the dehorning process. Wound healing scores, attitude, gait and posture, appetite, and lying were recorded daily. Data were analyzed using the Wilcoxon Rank Sum Test of SAS (Cary, NC). Vocalization scores were highest for MECH, and the BAND cattle vocalized more than TIP and CON (P < 0.05). There were treatment effects for gait and posture (P = 0.03), appetite (P = 0.01) and lying scores (P < 0.01), BAND cattle had higher scores (P < 0.10) in these parameters compared to MECH, TIP and CON cattle. There was treatment by time interactions for attitude (P < 0.01), horn bud (P < 0.01) and bleeding (P < 0.01). BAND cattle had increased attitude scores in weeks 1, 3 and 4 (P < 0.10), increased hornbud scores in weeks 3 and 4 (P < 0.05) and increased bleeding scores in week 3 (P < 0.05). These data indicate that MECH is a painful procedure for cattle at the time of the procedure. Banding to remove horns from cattle is not recommended based on the data and observations from this study.

Master of Science / Department of Anatomy & Physiology / Masaaki Tamura / Rat umbilical cord matrix stem cells (UCMSC) have been shown to exhibit a remarkable ability to control rat mammary adenocarcinoma (Mat B III) cell proliferation both in vivo and in vitro. To study the underlying mechanisms and genes involved in Mat B III growth attenuation, total RNA was extracted from the naïve rat UCMSC alone and those co-cultured with Mat B III in Transwell culture dishes. Gene expression profiles of naive rat UCMSC alone and those cocultured with Mat B III cells were investigated by microarray analysis using an Illumina RatRef- 12 Expression BeadChip. The comparison of gene expression profiles between untreated and cocultured rat UCMSC identified five up-regulated candidate genes (follistatin (FST), sulfatase1 (SULF-1), glucose phosphate isomerase (GPI), HtrA serine peptidase (HTRA1), and adipocyte differentiation-related protein (ADRP)) and two down-regulated candidate genes (transforming growth factor, beta-induced, 68kDa (TGFβI) and podoplanin (PDPN)) based upon the following screening criteria: 1) expression of the candidate genes should show at least a 1.5 fold change in rat UCMSC co-cultured with Mat B III cells; 2) candidate genes encode secretory proteins; and 3) they encode cell growth-related proteins. Following confirmation of gene expression by real time-PCR, ADRP, SULF-1 and GPI were selected for further analysis. Addition of specific neutralizing antibodies against these three gene products individually in co-cultures of 1:20 rat UCMSC:Mat B III cells significantly increased cell proliferation, implying that these gene products are produced under the co-cultured condition and functionally attenuate cell growth. Immunoprecipitation followed by Western blot analysis demonstrated that these proteins are indeed secreted into the culture medium. Individual over-expression of these three genes in rat UCMSC significantly enhanced UCMSC-dependent inhibition of cell proliferation in co-culture. These results suggest that ADRP, SULF-1 and GPI act as tumor suppressor genes, and these genes might be involved in rat UCMSC-dependent growth attenuation of rat mammary tumors.

Doctor of Philosophy / Department of Diagnostic Medicine and Pathobiology / Ronette Gehring / The absence of pain management for common husbandry procedures, such as dehorning and castration of cattle, is considered to be an important animal welfare consideration, but there are currently no drugs approved by the FDA for the purpose of providing pain relief in cattle. The FDA’s Center for Veterinary Medicine (CVM) recognizes the need for the availability of pain-relieving drugs and has encouraged research into the development of behavioral and physiologic measures which can reliably demonstrate the effectiveness in that species. The USDA has also recognized this need by providing grants for research into investigating pain models for cattle that can be used for the development of pain mitigation methods. The studies reported in this dissertation were funded by the USDA and the American Association of Bovine Practitioners. They add to the body of knowledge from which a pain model in cattle may eventually be validated for use in the drug approval process and also contribute to knowledge base for a candidate non-steroidal anti-inflammatory drug for convenient use in cattle. The study reported in Chapter 2 was conducted to support research of a candidate pain-assessment variable, substance P. This study provides future researchers with recommended sample handling procedures for obtaining reliable and repeatable results, which is important if substance P is to be validated as pain biomarker in cattle. The study in Chapter 3 investigated the use of several variables for use in a pain model. The results provided researchers, veterinarians and policy-makers with evidence to support the common practice of castrating and dehorning calves at the same time rather than as individual procedures separated by a healing interim. The study in Chapter 4 investigated the pharmacokinetics of oral meloxicam when administered to juvenile ruminant and pre-ruminant calves. This study added to the growing knowledge base of the pharmacokinetics of oral meloxicam in cattle and also provided practitioners with practical information concerning the administration of the drug in milk replacer. Chapter 5 investigated the use of oral meloxicam in a production setting and indicated that meloxicam administration prior to surgical castration may reduce the incidence of respiratory disease in the post-surgical period.

Master of Science / Department of Biomedical Sciences / Warren Beard / Reasons for performing study: Caecal acidosis is a central event in the metabolic cascade that occurs following grain overload. Buffering the caecal acidosis by enterally administered sodium bicarbonate may be beneficial to affected horses. Objectives: To determine the effect and duration of enterally administered sodium bicarbonate (NaHCO[subscript]3) on caecal pH in healthy horses. Study design: Prospective controlled study using normal horses with caecal cannulas Methods: 9 horses previously fitted with a caecal cannula. 6 horses received 1.0 g/kg bwt NaHCO[subscript]3 via nasogastric tube and 3 control horses were given 3 L of water via nasogastric tube. Clinical parameters, water consumption, venous blood gases, caecal pH, faecal pH and faecal water content were measured at 6 hour intervals over a 36 hour study period. Results: Horses that received enterally administered NaHCO[subscript]3 had a significantly increased caecal pH that lasted the duration of the study. Treated horses increased their water intake, developed metabolic alcalemia, significantly increased sodium concentrations and significantly decreased potassium concentrations. Conclusions and potential relevance: Enterally administered NaHCO[subscript]3 may be beneficial in buffering the caecal acidosis that occurs following an acute carbohydrate overload