Master of Science / Department of Diagnostic Medicine and Pathobiology / Raymond R. R. Rowland / Three economically important swine diseases: Porcine Reproductive and Respiratory Syndrome (PRRS), Porcine Circovirus Associated Disease (PCVAD) and Swine influenza cost the US swine industry more than a billion dollars each year. This study developed a fluorescent microsphere immunoassay (FMIA) to simultaneously detect antibodies to the causative pathogens: PRRSV, porcine circovirus (PCV2) and swine influenza virus (SIV). The results showed that the multiplex assay possessed the predicted specificities. In the case of PRRSV NA, the assay displayed higher sensitivity when compared to a commercially available ELISA. The assay was employed to measure both IgG and IgM responses. The FMIA was found to possess several advantages over standard ELISA which include reduced sample volume, time and cost and provides a new tool for veterinary diagnostics. The FMIA was applied for swine disease surveillance in Hawaiian and Texan feral swine populations. The antibodies against PCV2 showed the highest prevalence among these three pathogens in both Hawaii and Texas. Hence we consider PCV2 as the most prevalent pathogen in Hawaiian and Texan feral pigs and this pathogen poses the greatest threat to commercial pigs. SIV seroprevelance increased from 2007 to 2010 in Hawaii State, suggesting an increasing risk for commercial pigs. Moreover, yearly surveillance in Texas State shows growth in seropositive response to all pathogens, particularly PCV2. The development of FMIA for detection of antibodies to multiple swine pathogens in serum samples offers an important alternative for swine disease surveillance in commercial and feral herds.

Doctor of Philosophy / Department of Diagnostic Medicine and Pathobiology / Brad J. White and Robert L. Larson / Bovine respiratory disease (BRD) is the most costly disease of cattle in US feedyards and diagnosis based on clinical signs of illness is challenging. Over the course of five independent studies we evaluated the precision of multiple observers assigning clinical illness scores (CIS) to calves with induced Mycoplasma bovis pneumonia. We also evaluated the accuracy of CIS in relation to lung lesions at necropsy. Agreement among observers over all five studies was slight ({kappa]= 0.16; 95% confidence interval, 0.10 to 0.24) and ranged from 0.10 to 0.21 for individual trials. The accuracy of CIS varied based on the pulmonary consolidation score chosen to represent a truly ill animal. Inflammation associated with BRD can lead to significant pulmonary damage and reduced lung function. Treatment for BRD frequently involves antimicrobial administration and occasionally non-steroidal anti-inflammatory drugs. We evaluated how calves experimentally challenged with Mannheimia haemolytica respond to treatment with flunixin meglumine, alone or in combination with the antimicrobial florfenicol. Individual calf response to bacterial pneumonia was highly variable in this study. None of the changes in serum biomarkers, CBC or chemistry parameters provided reliable indicators of the pulmonary inflammation associated with the mild severity of bronchopneumonia in our study. Metaphylaxis is frequently administered to manage the risk of BRD within cohorts of cattle. We evaluated the impact of metaphylactic antimicrobial administration 10 days prior to experimental Mannheimia haemolytica inoculation to mitigate pulmonary lesions. We found that calves receiving tildipirosin had less lung damage and fewer clinical signs of illness compared to calves treated with tulathromycin or saline. Finally, the ability to predict those animals that would not finish the production cycle normally would provide benefits in effectively managing cattle. We evaluated the ability of classification algorithms to accurately predict an individual calf’s outcome based on data available at first identification of and treatment for BRD. We found accuracy of classifiers was dependent on the data recorded by the feedyard and there are sub-groups of calves within feedyard populations where classifiers were highly accurate. These data suggest the importance of pairing the proper classifier with the data available.

Doctor of Philosophy / Department of Anatomy and Physiology / Jishu Shi / Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important swine diseases worldwide that leads to severe reproductive failure in sows and high mortality in young pigs. Vaccination is currently the most effective way to control this disease. The protection ability provided by vaccines however is limited due to the large diversity of field PRRSV strains. In chapter 2, we compared immune responses induced by vaccination and/or PRRSV infection by using IngelVac® Modified Live PRRSV vaccine (MLV), its parental strain VR-2332, and the heterologous KS-06 strain. Our results showed that MLV provide complete protection to homologous virus and partial protection to heterologous challenge. The protection was associated with the levels of PRRSV neutralizing antibodies at the time of challenge. Besides developing new vaccines to combat PRRSV, adjuvants have been applied to PRRSV MLV vaccines to induce vaccination-mediated cross-protection against genetically dissimilar PRRSV strains. In chapter 3, we demonstrated that a commercial MontanideTM Gel01ST adjuvant provides enhanced protection to homologous PRRSV infection by regulating the production of PRRSV-specific antibodies. In chapter 4, we tested a novel peptide nanofiber hydrogel acting as a potent adjuvant for PRRSV MLV vaccines. We found that the hydrogel adjuvant enhanced vaccine efficacy by developing of higher titers of neutralizing antibodies and stronger IFN-γ cellular immune responses. Chinese highly pathogenic PRRSV (HP-PRRSV) variants were isolated in 2006 and they belong to genotype 2 of PRRSV. Compared with classic PRRSV, HP-PRRSV is characterized by robust proliferation ability and high morbidity/mortality with all ages of pigs. In chapter 5, we compared the difference of immune responses elicited by HV-PRRSV, a Chinese HP-PRRSV, and a US virulent strain of PRRSV NADC-20. Traditional PRRSV MLV vaccines developed in US offer no protection to HP-PRRSV. Vaccines specific to HP-PRRSV strains available in China provide protection to HP-PRRSV. In chapter 6, we demonstrated that pigs challenged with US NADC-20 strain were protected by vaccination with Chinese MLV HP-PRRSV vaccines. The availability of Chinese HP-PRRSV vaccines in North America may act to increase the preparedness of possible transmission of HP-PRRSV to North America.

Master of Science / Department of Clinical Sciences / Warren L. Beard / The flexion test is routinely used in lameness and pre-purchase examinations. There is no accepted standard for duration of flexion or evidence that interpretation of results would differ with different durations of flexion. We hypothesized there would be no difference in interpretation of full hindlimb flexion for 5 or 60 seconds. Video recordings of lameness examinations of 34 client owned horses were performed that included: baseline lameness, upper hindlimb flexion for 60 seconds, and flexion of the same leg for 5 seconds. Videos were edited to blind reviewers to the hypothesis being tested. The baseline lameness video from each horse was paired with each flexion to make 2 pairs of videos for each case. Twenty video pairs were repeated to assess intra-observer repeatability. Fifteen experienced clinicians reviewed the videos and graded the response to flexion as either positive or negative. Potential associations between the duration of flexion and the likelihood of a positive flexion test were evaluated using generalized linear mixed models. A kappa value was calculated to assess the degree of intra-observer agreement on the repeated videos. Full hindlimb flexion of 60 seconds was more likely to be called positive than flexion of 5 seconds (p<0.0001), with the likelihood of the same interpretation 74% of the time. The first flexion performed was more likely to be called positive than subsequent flexions (p=0.029). Intra-assessor agreement averaged 75% with κ=0.49. Full hindlimb flexion of a horse for 5 seconds did not yield the same result as 60 seconds.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Daniel U. Thomson / A total of 284,300 animals from 10 commercial feedyards in Nebraska and Colorado were used to evaluate the effectiveness of a siderophore-receptor/porin protein-based vaccine under commercial feedlot conditions. Individual feedlots were assigned to 1 of 2 treatments: 1) all incoming cattle injected with 2 ml of SRP E. coli O157:H7 vaccine subcutaneously at arrival and at time of re-implant (VAC) or 2) all incoming cattle were not vaccinated, and were used as negative controls (CON). Twenty freshly voided fecal samples were taken from 5 pen floors of market ready cattle at each feedyard once a month during May, June, July, and August of 2010. Pre-harvest blood samples were collected on 3 occasions throughout the summer (June, July, and August). For each sampling month, 1 lot of 5 animals representing each feedyard was sampled. Fecal and blood samples were shipped to Epitopix, LLC for subsequent microbiology and anti-SRP antibody testing. Samples were coded such that laboratory personnel were blinded to the location and treatment of samples. Cattle receiving VAC treatment had reduced prevalence of E.coli O157:H7 in their feces relative to the E. coli O157:H7 prevalence in the feces of CON cattle (12.83% vs. 20.25% for VAC and CON, respectively; P = 0.07). Anti-SRP antibody titer was higher in the serum from VAC cattle relative to the SRP titer levels in serum obtained from CON cattle (0.622 and 0.075 for VAC and CON, respectively; P < 0.001). These data suggest that vaccination of feedlot cattle with SRP upon arrival at the feedlot and again 70-100 days pre-harvest reduces shedding of E. coli O157:H7.

Master of Science / Department of Clinical Sciences / Warren L. Beard / Pharmacokinetics of Intramuscular Morphine in the Horse Elizabeth Devine, DVM; Butch KuKanich, DVM, PhD, DACVCP; Warren Beard, DVM, MS, DACVS Objective - To determine the pharmacokinetics of morphine after intramuscular administration in a clinical population of horses Design – Prospective, clinical study Animals – Pilot study included 2 normal horses and the clinical study included 75 horses Procedures – Morphine was administered at 0.1mg/kg, IM and 2-3 blood samples were obtained from each horse at various times from 0-9 hours after administration. Plasma morphine concentrations were measured using liquid chromatography and mass spectrometry. Results – Data was analyzed using a naïve pooled pharmacokinetic model. The half-life for the elimination phase was approximately 1.5 hours, the volume of distribution (per bioavailability) was approximately 4.5 L/kg and the clearance (per bioavailability) was approximately 35 mL/kg/min. The peak plasma concentration was 21.6 ng/mL and occurred approximately 4 minutes after administration. Plasma concentrations of morphine were below the limit of quantification by 7 hours in 74 horses. Conclusions and Clinical Relevance – The relatively short half-life of morphine indicates the need for frequent dosing to maintain targeted plasma concentrations. Adverse effects were uncommon in this study and morphine was well tolerated at a dose of 0.1 mg/kg, IM. Morphine may be a useful adjunctive therapy in painful horses, but the variable plasma concentrations suggest the dose and dosing interval may need to be adjusted to the individual patient’s response.

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.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Ludek Zurek / Transfer of bacteria, including antibiotic resistant strains between companion animals and people is likely due to close physical contacts. However, surveillance programs on prevalence of antibiotic resistance are focused mainly on food animals and very little is known about the role of companion animals in the development and spread of antibiotic resistant bacteria. For this study, enterococci were chosen as model organism due to intrinsic and acquired antibiotic resistance and several virulence traits that make them the 3rd most important nosocomial pathogens. In addition, increased fecal shedding of antibiotic resistant bacteria from stressed animals has been reported from studies on food animals. To determine whether the gut microbiota of pet animals serves as a reservoir of clinically important enterococci, 360 enterococcal isolates from two groups: healthy group and pyoderma (stressed) group with 9 dogs in each were identified and screened for resistance to 10 antibiotics and 4 virulence traits. The transferability of resistance determinants and clonality of selected isolates were assessed by horizontal gene transfer assays and pulsed-field gel electrophoresis, respectively. In addition, overall diversity of bacteria as well as antibiotic and metal resistance genes in feces of healthy dogs was assessed by tag-encoded parallel pyrosequencing and microarray analysis, respectively. The most prevalent enterococcal species identified was E. faecalis: healthy group (70.5%); pyoderma group (44.0%). In the pyoderma group, antibiotic resistance and virulence traits (esp, gelE) were more frequent than in the healthy group; however, the overall prevalence of antibiotic resistant strains was low (< 37%) in both groups. The most prevalent resistance genes were tet(M)and tet(S). The antibiotic resistance traits were transferable in-vitro in E. faecalis (tetracycline, erythromycin, doxycycline) and E. faecium (tetracycline). Genotyping revealed less diverse E. faecalis community in pyoderma infected dogs. Pyrosequencing (~7,500 sequences per dog) revealed Firmicutes as the dominant phylum and most common genera included Turicibacter, Lactobacillus, Ruminococcus, Clostridium, and Fusobacterium. Two phyla Lentisphaerae (<1%) and Fibrobacteres (<1%) are reported for the first time from healthy dogs. Microarray data revealed the presence of several tetracycline, erythromycin, aminoglycoside, and copper resistance genes; however, most of these originated from one animal with history of chronic skin infection two year prior to our sampling. Higher prevalence of antimicrobial resistance in pyoderma infected dogs may be related to stress; however, this requires further investigation. In conclusion, based on our data, healthy and pyoderma infected dogs do not represent an important reservoir of clinically significant antibiotic resistant microbiota.

Master of Science / Department of Clinical Sciences / Gregory F. Grauer / Chronic kidney disease (CKD) is a common cause of morbidity and mortality in cats. The purpose of this study was to investigate the effects of Chinese rhubarb (Rheum officinale) supplementation on the progression of feline CKD. Cats with stable IRIS stage II or III CKD and without certain comorbidities were included in the study. Cats were randomly divided into 3 treatment groups and administered Chinese rhubarb extract (Group 1, Rubenal®, Vetoquinol, Forth Worth, TX; 75 mg tablet by mouth every 12 h), benazepril as a positive control (Group 2, 0.5 mg/kg by mouth every 24 h), or both (Group 3). Cats were fed a commercial renal specific diet and enteric phosphate binder as appropriate. Body weight, laboratory data, and blood pressure were recorded every 3 months. Variables between groups at enrollment and within groups over visits were compared with ANOVA and repeated measures ANOVA, respectively. A treatment by visit interaction term was included in all repeated measures models. Significance was set at p ≤ 0.05. Except for body weight there was no significant differences between treatment groups at enrollment. There was no significant change in body weight, hematocrit (Hct), UPC, serum creatinine, or systemic blood pressure over time as compared to baseline within any group. There was no significant difference between groups over time in regards to change in body weight, Hct, UPC, serum creatinine, or systemic blood pressure. The treatment by time interaction was non-significant in all models. Based on easily measured clinical parameters, this study failed to detect a significant difference in cats administered a Chinese rhubarb supplement, benazepril, or both.