Theses and Dissertations

Permanent URI for this collectionhttps://hdl.handle.net/10217/100385

Browse

Now showing 1 - 20 of 106

Embargo
Anterior cruciate ligament injury as a driver of post-traumatic osteoarthritis: mechanisms and outcomes elucidated from preclinical models
(Colorado State University. Libraries, 2025) Van Zeeland, Emily, author; Sikes, Katie J., advisor; Easley, Jeremiah T., advisor; Santangelo, Kelly S., committee member; Palmer, Ross H., committee member
Anterior cruciate ligament (ACL) injuries are among the most prevalent orthopedic injuries in active populations and are a leading cause of knee post-traumatic osteoarthritis (PTOA), a debilitating joint condition. PTOA remains a challenging clinical condition, particularly due to its multifactorial pathogenesis and lack of disease-modifying therapies. Emerging research indicates that biological sex significantly influences both the susceptibility to ACL injuries and the progression of PTOA, yet the underlying mechanisms remain poorly understood. Additionally, limitations in current surgical approaches to ACL reconstruction continue to impede long-term joint preservation. The overarching goal of this dissertation was to investigate both biological and surgical determinants of PTOA with the intent of informing improved therapeutic strategies for individuals following ACL rupture. This work is structured around three aims: 1) investigating the role of sex in PTOA progression, 2) probing estrogen signaling as a modulator of PTOA development, and 3) evaluating novel surgical reconstruction strategies in a large animal model. In Aim 1, we used a non-surgical, mechanical mouse model of ACL rupture to explore sex-specific differences in the development of PTOA following joint trauma. Male and female C57BL/6J mice were subjected to unilateral ACL rupture and were monitored over time using functional and structural assessments to comprehensively characterize PTOA progression. These included weekly mobility analyses such as voluntary mobility, static weight-bearing, and gait analyses to track symptomatic development. In parallel, joint degeneration was evaluated longitudinally through radiographic imaging and at terminal timepoints via histological scoring. ACL tissues from injured and control limbs were collected at multiple early timepoints for untargeted, label-free proteomic profiling, enabling characterization of dynamic molecular responses to injury. Our findings revealed that male mice demonstrated greater impairment in mobility, including reduced overall activity and altered hindlimb stance, in comparison to females. Histological analysis further confirmed more severe joint pathology in males. Interestingly, radiographic measures showed limited sex-based differences, suggesting that early structural joint changes may be more sensitively captured by histology and behavior than by imaging alone. Proteomic analysis revealed a markedly different injury response between sexes. Female mice exhibited an early, robust molecular response peaking at three days post-injury, with upregulation of pathways associated with inflammation, coagulation, and estrogen signaling. Males showed a more attenuated and prolonged proteomic response, suggesting sex-specific differences in the temporal dynamics of molecular signaling post-injury. Collectively, these findings identify significant sex-based variation in both the symptomatic and molecular trajectory of PTOA following ACL rupture, reinforcing the need for sex-specific approaches to therapeutic development and clinical management. Aim 2 built upon these findings by exploring the role of estrogen signaling in modulating PTOA progression, with a focus on its contributions in the male mouse joint. Given the absence of early estrogenic signaling in male mice observed in Aim 1, we hypothesized that localized manipulation of estrogen pathways could influence PTOA disease trajectory. Using our established non-surgical ACL rupture model, we administered a single intra-articular (IA) injection of either 17β-estradiol to enhance estrogen signaling or Fulvestrant, an estrogen receptor antagonist, to suppress it. Mice were evaluated over a 35-day period for symptomatic and structural indicators of PTOA development, including voluntary mobility, gait, weight-bearing, and histopathologic scoring of the injured joint. 17β-estradiol delivery led to measurable improvements in multiple outcome parameters, including enhanced mobility and smaller enthesophyte formation compared to saline controls. These findings suggest that estrogen may exert protective effects on PTOA development. However, interpretation of the therapeutic potential of 17β-estradiol was complicated by the biological activity of the vehicle control, β-cyclodextrin, which independently influenced mobility outcomes, osteophyte maturity, and estrogen receptor beta (ERβ) expression. Fulvestrant treatment produced an opposing phenotype characterized by decreased voluntary mobility, increased osteophyte maturity, and altered estrogen receptor dynamics. Notably, Fulvestrant led to a transient increase in estrogen receptor alpha (ERα)) expression in ACL remnants at 3 days post-injury, potentially suggesting a compensatory feedback loop following initial receptor antagonism. These receptor-specific findings, combined with the distinct treatment effects on joint morphology and function, underscore the complex role of estrogen signaling in PTOA. Collectively, these data highlight both the promise and the challenge of developing hormone-based therapies for PTOA and emphasize the need for refined delivery methods and better understanding of sex-specific receptor profiles. In Aim 3, we transitioned to a preclinical ovine model to investigate novel surgical strategies aimed at improving ACL graft healing and minimizing downstream joint degeneration. One reason for the inability of the ACL to spontaneously heal is the presence of plasmin in synovial fluid, which prevents a fibrin-platelet clot from forming between the torn ends of the ACL. Thus, we sought to determine if delivering vascular supply to ACL remnants via a graft could improve overall healing. In this pilot study, we evaluated two innovative reconstruction techniques designed to preserve native blood supply to the graft: one utilizing the lateral digital extensor tendon (LaDET) and the other employing the infrapatellar fat pad (IFP). Post-operative outcomes were evaluated through static weight-bearing, MRI, arthroscopy, macroscopic joint scoring, and histology over six months. The LaDET graft showed successful graft incorporation, histologic continuity with native ACL remnants, minimal synovial inflammation, and restored limb function. In contrast, the IFP graft demonstrated poor structural integration, persistent inflammation, and inferior functional recovery. These results suggest that vascularized tendon grafts, such as the LaDET, may provide a biologically informed surgical strategy for enhancing graft healing in ACL reconstruction. Overall, this dissertation advances our understanding of how biological sex, hormonal signaling, and surgical technique influence joint outcomes following ACL injury. The findings emphasize that PTOA development is driven by a combination of molecular and mechanical factors, and that effective treatment must address both. Sex-based differences in disease progression and the complex role of estrogen signaling suggest that personalized, biology-informed approaches may be potential therapeutic avenues to prevent or slow joint degeneration. Additionally, the exploration of vascularized grafts presents a promising direction for improving ACL reconstruction outcomes. Together, this work provides insight for future research aimed at integrating biological sex, molecular pathways, and surgical strategies to develop more effective, personalized interventions that improve outcomes and reduce the long-term impact of ACL injury.
Embargo
Investigation of treatment with molnupiravir and immune stimulants for use in the management of feline infectious peritonitis and associated comorbidities
(Colorado State University. Libraries, 2025) Cerna, Petra, author; Lappin, Michael R., advisor; Dow, Steven, advisor; Santangelo, Kelly, committee member; Dean, Gregg, committee member
Feline coronavirus (FCoV) belongs to the order Nidovirales; family Coronaviridae; subfamily Coronavirinae; genus Alphacoronavirus; species Alphacoronavirus 1. Feline coronavirus exists in two biotypes; feline enteric coronavirus (FECV), which mainly replicates in the enteric epithelial cells, and feline infectious peritonitis virus (FIPV), which results in lethal infection with efficient systemic replication in monocytes or macrophages. Efficient FCoV replication in activated monocytes and macrophages is a key event in feline infectious peritonitis (FIP) pathogenesis. Fortunately, only a small proportion of FCoV-infected cats go on to develop FIP which typically manifests as a vasculopathy resulting in ('effusive') form (up to 80% of FIP cases have effusions), or granuloma formation resulting in ('non-effusive') mass lesions, or a combination of the two. The effusive form of disease generally is believed to develop in cats with poor cell-mediated immune responses, and the non-effusive form develops in cats with partial cell-mediated immunity. Recently, new antiviral drugs like the nucleoside analog GS-441524 and molnupiravir (MPV) has been used in successful treatment of FIP. However, more treatment options for cats with FIP, especially those that not responding or relapse, are needed and the goal of this work was to evaluate the use of nucleoside analogue molnupiravir and immune stimulants to determine the best treatment protocols for cats with FIP. We compared two different immune stimulants for antiviral activity in cats: a TLR 2/6-activating compound polyprenyl immunostimulant; (PI) and a liposome Toll-like receptor 3/9 agonist complexes (LTC) to determine relative abilities to stimulate immune responses in vitro and to study the effects of treatment on immune responses in healthy cats. In these studies, both LTC and PI protocols induced immune enhancing effects, suggesting a possible clinical use for the management of chronic infectious diseases like FIP. We concluded that activating the TLR 3 and 9 pathways (LTC) induced superior broad interferon production in vitro than the activation of the TLR 2 and 6 pathways (PI). We also determined the pharmacokinetics of molnupiravir in cats with naturally occurring FIP by measuring MPV and EIDD-1931 (β-D-N4-hydroxycytidine; NHC) serum levels in seven cats diagnosed with naturally occurring FIP treated with MPV. The mean NHC concentrations at all time points were at least 4 times the reported in vitro EC50 for feline coronavirus strains and twice daily dosing for seven days did not lead to accumulation of drug within the serum. Minimal accumulation of drug was seen in trough concentrations following twice-daily dosing for 7 days. These results supported the use of MPV in the treatment of FIP. We have conducted a prospective, open‐label longitudinal single‐center clinical trial with MPV in cats with FIP. A total of 78% cats survived to 6 months and 22% cats died or were euthanized. We also found that the median total bilirubin concentrations were significantly different (p= 0.0007) between the survivors vs non-survivors. Relapses occurred in 12% of the cats and all achieved remission during a second course of treatment. No adverse effects necessitated discontinuation of the treatment. This study showed effectiveness of using 12 week therapy with MPV in treatment of FIP. While most cats with FIP were euthanized in the past; more cats are now being treated with antiviral drugs. As a result of this, new disease processes that could be associated with FIP are being recognized; for example, immune-mediated hemolytic anemia (IMHA), myocarditis or other cardiac changes, and other comorbidities and the goal of this study was to evaluate the development of IMHA and myocarditis in cats with FIP and its impact on survival and prognosis of these cats. We have evaluated 45 cats with associative IMHA to FIP in a multicentric study. The median hematocrit for these cats was 18% and anemia was non-regenerative in 36/45 cats with concurrent thrombocytopenia was present in 18/45 cats. All 45 cats were treated with nucleoside analogs and 44 cats with glucocorticoids and at the time of last follow-up 33 cats had survived while 12 had died or were euthanized. Although FIP is likely an uncommon cause of associative IMHA, as more cats with FIP are treated with antiviral therapy, it is important to consider IMHA as a possible cause of anemia in cats with FIP. We documented the cardiac changes in cats diagnosed with FIP using echocardiography and cardiac troponin I (cTnI). Twenty cats diagnosed with FIP and tested negative for concurrent infections associated with myocarditis underwent an echocardiographic examination. The left ventricular posterior wall from right parasternal long axis and short axis was thickened in 55% (11/20) and 25% (5/20) of cats, respectively. The median cTnI at initial evaluation was 0.37 ng/ml (IQR 0.20-0.83) and recheck of cTnI performed at 12 weeks following antiviral therapy in 6 cats that initially had elevated cTnI showed normalized cTnI to <0.20 ng/ml. Cats with FIP can present with elevations in cTnI and ventricular wall thickening, which are suggestive of myocarditis and/or myocardial injury. The results of the studies described the use of antivirals and immune stimulants in FIP and outlined the possible comorbidities clinicians will be facing during the FIP treatment. Further investigation into FIP treatment with antivirals such as nucleoside analogues and protease inhibitors and immune stimulants are needed to increase the success rate in treatment of critically ill cats and prevent relapses in the future. Moreover, studies evaluating comorbidities in cats with FIP such as IMHA, myocarditis, sepsis and gastrointestinal disease are needed to better understand how to manage these cases.
Embargo
Mechanosensitivity of myoblast-derived extracellular vesicles: implications for skeletal muscle regenerative therapeutics
(Colorado State University. Libraries, 2025) Williams, Katherine B., author; Ehrhart, Nicole, advisor; Hamilton, Karyn L., advisor; LaRocca, Thomas J., committee member; Duval, Dawn, committee member
Skeletal muscle regenerative capacity and function declines with advancing age. This contributes to poor recovery following surgery or injury, and to age-related muscle loss and dysfunction (i.e. sarcopenia). Extracellular vesicles (EVs) are membrane-bound vesicles that regulate intercellular signaling through the delivery of complex cargo including RNAs, proteins, and lipids. Over the past decade, EVs have emerged as promising biologic therapeutics for musculoskeletal repair due to their ability to deliver bioactive cargo to recipient cells, recapitulating many of the benefits of mesenchymal stromal cells. However, strategies to enhance EV therapeutic potential and to optimize their cargo for regeneration remain limited. Mechanical forces are a potent regulator of skeletal muscle development and homeostasis. Key processes for effective muscle regeneration, including muscle stem (i.e. satellite cell) and progenitor cell (myoblast) quiescence, activation, proliferation, and differentiation, are all impacted by mechanical signaling. EV biogenesis and function are also affected by mechanical cues. Because EV composition and function are highly dependent on the physiology and environment of the parent cell, we hypothesized that applying mechanical strain to myoblasts will modulate their EV cargo and function to enhance myogenesis and muscle regeneration. Broadly, this dissertation investigates how mechanical strain alters the production, microRNA (miRNA) cargo, and regenerative capacity of EVs derived from myoblasts, with the overarching goal of optimizing EV-based therapies for aged muscle regeneration. Chapter 1 discusses the potential of EVs as an alternative to mesenchymal stromal cells for regenerative medicine, the impact of aging on EV biogenesis, and mechanical cues as a tunable factor to modulate EV cargo and function. Chapter 2 demonstrates that mechanical strain applied to C2C12 myoblasts increases EV production, enhances proliferation and differentiation of naïve myoblasts receiving EVs derived from strained myoblasts, and modulates EV miRNA cargo. In Chapter 3, we describe an optimized protocol to isolate and expand primary C57BL/6 murine myoblasts, characterize the EVs they produce, and compare the effects of EVs derived from bone marrow MSCs to EVs derived from primary myoblasts on myoblast proliferation. Chapter 4 investigates the impact of mechanical strain on primary myoblast EV miRNA cargo, as well as the impact of mechanically strained primary myoblast EVs on gene expression, proliferation, and differentiation in recipient myoblasts. Finally, Chapter 5 evaluates the effects of strained myoblast-derived EVs on in vivo muscle regeneration in an aged mouse model of muscle injury. Together, this research demonstrates that mechanical strain is a powerful tool to modulate myoblast EV miRNA cargo and myogenic function, possibly enhancing the therapeutic potential of myoblast-derived EVs to accelerate recovery of aged muscle function following injury. This work advances our understanding of EV biology in the context of optimizing biologic therapeutics for muscle regeneration and muscle aging.
Open Access
Polyclonal IgY supplementation at birth did not affect preweaned dairy calf health
(Colorado State University. Libraries, 2025) De La Cuadra Rojas, Luisa Fernanda, author; Raabis, Sarah, advisor; Cramer, Catie, committee member; Applegate, Tanya, committee member
Diarrhea in dairy calves has a significant impact on farm economics, primarily due to weight loss, increased labor for farm staff, and treatment costs. Antimicrobials are commonly used for the prevention and treatment of this disease, but their overuse contributes to antimicrobial resistance (AMR), making calves key reservoirs of resistance genes. Alternatives to the use of antimicrobials have been widely researched, and immunoglobulin Y (IgY) is one of them. This is an immunotherapy that utilizes IgY extracted from egg yolk to reduce pathogen colonization, with promising results in the treatment of calf diarrhea. IgY can be either monoclonal or polyclonal. Monoclonal IgY products target single pathogens, however, they may be less effective in neonatal calves exposed to multiple enteric pathogens simultaneously. Gastrointestinal infections can also predispose calves to respiratory illnesses, such as bovine respiratory disease (BRD), thereby compounding health and economic burdens, including increased use of antimicrobials. This study evaluated the effect of a commercial polyclonal IgY supplement on pre-weaned calf health. A randomized controlled trial was conducted between December 2023 and March 2024 on a commercial dairy farm. Sample size (n=80) was calculated to detect a 20% difference in days with diarrhea between groups (1-β=0.8; α=0.05). A total of 187 calves were enrolled and assigned to a treatment (IGY; n=91) or control group (CON; n=96). Health indicators included fecal score (FS), body weight, fecal shedding of pathogens (Salmonella culture and qPCR for BCoV, BRV, and Cryptosporidium parvum) were collected. Descriptive statistics were performed to summarize data. Chi-square was applied to establish statistical significance between groups for different outcomes (BRD prevalence, pathogen shedding for Salmonella, BCoV, BRV, and C. parvum. Multivariable models assessed the effect of IgY supplementation on diarrhea (FS ≥2, days 1-14), pathogen shedding, and average daily gain (ADG). A total of 187 calves were enrolled in the study, 91 animals assigned to the treatment group (IGY) and 96 to the control group (CON). Farms staff administered the supplement within 12 hours of birth to 66% of the calves in the IGY group. The mean ADG between groups was not statistically significant (p = 0.698, 0.81 kg +- 0.05;88). The IGY group was not significantly associated with the odds of having BRD (OR = 0.76, 95% CI [0.53-1.08], P = 0.1240). The treatment group was not associated with the likelihood of having at least one day with an FS ≥2 (OR = 1.05, 95% CI [0.76-1.45]) or FS ≥3 (OR = 0.67, 95% CI [0.36-1.2], p = 0.176). No statistically significant difference in Salmonella (p = 0.25) or Cryptosporidium parvum (p = 0.14) shedding. There were also no significant differences detected in the odds of pathogen shedding (P> 0.2), BCoV (OR: 2.43, CI: 0.7-8.88), and BRV (OR:1.08, CI: 0.30-3.85). Mortality remained low across groups, and farm treatment for diarrheic patients showed no statistically significant difference between groups for both parameters. In conclusion, a single dose of a mixture supplement with IgY administered within the first 12 hours of birth was not associated with BRD presentation, fecal score, prevalence of diarrhea, pathogen shedding, improvement of ADG, decreased mortality or treatments for diarrhea at the farm either during the first 14 days or during the first 60 days of life.
Embargo
Advancing disease surveillance and biosecurity in North American bison (Bison bison): multidisciplinary approaches to bison health monitoring
(Colorado State University. Libraries, 2025) Krus, Catherine Bridget, author; Mayo, Christie, advisor; Raabis, Sarah, advisor; Buttke, Danielle, committee member; Titcomb, Georgia, committee member
North American bison (Bison bison) play a critical ecological, cultural, and economic role, yet their long-term conservation and management face challenges from disease threats, anthropologic changes, and translocation-associated risks. This dissertation integrates a One Health approach to assess key aspects of bison health, disease surveillance, and biosecurity policy through a multidisciplinary framework. Specifically, this research (1) establishes a stakeholder-driven definition of bison health, (2) evaluates the epidemiology of Bluetongue Virus (BTV) and Epizootic Hemorrhagic Disease Virus (EHDV) in bison, and (3) assesses the diagnostic performance of serological assays for Mycoplasma bovis, a pathogen of increasing concern. To define bison health from a multisectoral perspective, a two-round Delphi survey was conducted with experts from public, tribal, nonprofit, and private sectors. Experts defined bison health as the ability of populations to express natural behaviors, demonstrate resilience to external stressors, and sustain high reproductive output with minimal intervention. Mycoplasma bovis was identified as a high-priority pathogen, with participants highlighting the urgent need for improved diagnostics, biosecurity measures, and cross-sectoral disease management strategies. In response to the research and biosecurity gaps in bison health identified in the Delphi manuscript, as well as the expanding geographical range of Orbiviruses in the United States, we conducted the first cross-sectional serosurvey of BTV and EHDV in North American bison. This study analyzed samples from 287 animals across nine herds in seven U.S. states. Competitive ELISA assays revealed a seroprevalence of 56.5% for BTV and 57.5% for EHDV, with logistic regression identifying age as a significant predictor of seropositivity (p < 0.01). PCR-based detection of circulating BTV serotypes (6, 11, 13, 17) was noted, indicating exposure to endemic serotypes. Additionally, a significant decline in viremia with increasing age suggesting age-related immune dynamics. These findings provide foundational data for incorporating bison into vector-borne disease surveillance and highlight the need for further investigation into their role as incidental hosts. Given the increasing impact of M. bovis on bison health along with the economic impacts highlighted in the Delphi manuscript, this dissertation also evaluates the diagnostic performance of a newly designed P48 ELISA compared to a commercially available ELISA for M. bovis detection in bison. This chapter assesses sensitivity, specificity, and cross-reactivity, identifying key limitations of current assays and providing recommendations for improving serological surveillance in bison populations. Findings from this study emphasize the need for species-specific diagnostic validation to enhance disease monitoring and outbreak prevention. By integrating Delphi consensus-building, epidemiological surveillance, and diagnostic evaluation, this dissertation provides critical insights into bison disease ecology, biosecurity needs, and translocation risks. The findings support evidence-based policies for disease mitigation and contribute to the sustainable management of bison populations under a One Health framework.
Open Access
Cardiac computed tomography in dogs: insights into structural imaging and therapeutics
(Colorado State University. Libraries, 2025) Chi, I-Jung Bernard, author; Scansen, Brian, advisor; Orton, E. Christopher, advisor; Visser, Lance, committee member; Bark, David, committee member
Visualization of intracardiac structures with medical imaging has always been a formidable task due to the inherently complex structure of mammalian hearts. Traditional two-dimensional (2D) imaging such as fluoroscopy and 2D echocardiography present challenges in reconstructing the three-dimensional (3D) morphology of cardiac structures and alterations in intracardiac architecture caused by cardiovascular disease. Challenges in accurately visualizing complex cardiac geometry can lead to incomplete or inaccurate assessments during surgical and transcatheter interventions. To overcome this issue, cross-sectional imaging such as cardiac computed tomography (CCT) may be utilized to allow a comprehensive, dynamic, and 3D evaluation of cardiac valves, chambers and major vascular structures. One potential application of 3D imaging is preprocedural prediction of optimal fluoroscopic projections that can be used for intraprocedural guidance of cardiac interventions. For any given cardiac structure, there is an infinite number of 2D projections orthogonal to the straight-on (en face) projection forming a 360-degree circle of perpendicularity. The circle of perpendicularity becomes an arch of perpendicularity when considering projections above the fluoroscopic table only. In Chapter 1, this concept was explored by identifying the en face projections of various cardiac structures on CCT. The arch of perpendicularity (also known as the S curve) can be derived by using a trigonometric formula to solve the spatial relationship between the en face and perpendicular projections. Once the S curves are established, the optimal fluoroscopic projections (OFPs) can then be characterized and used to guide cardiac interventions. Cardiac computed tomography studies from eighteen healthy dogs were used to generate S-curves and OFPs for selected cardiac structures. The projections were defined by the cranial-caudal angulation and left-right rotation angles of the C-arm assuming the dog is in dorsal recumbency. Mean OFP S-curves and 95% confidence curve areas were determined for the aortic, mitral, and pulmonary valve along with interatrial septum. The impact of thoracic conformation on OFPs was found to be non-significant. By analyzing the CCTs from 18 dogs with severe pulmonary stenosis, the S-curves and OFPs of the pulmonary valve were described in Chapter 2. The coordinates of those optimized projections were found to be different from those of healthy dogs. Cardiac computed tomography from dogs with pulmonary stenosis were also used as a reference to examine the accuracy of pulmonary annular diameters measured on transthoracic echocardiography and angiocardiography. It was found that transthoracic echocardiography underestimates pulmonary annular diameter. Angiographic pulmonary annular diameters on standard lateral projection were also shown to be less precise when compared to the measurements made on CCT-derived OFPs. The inodilator pimobendan has been shown to delay the onset of congestive heart failure and decrease cardiac mortality in dogs with degenerative mitral valve disease. The benefit of pimobendan may go beyond its effect on cardiac contractility and vasodilation. In Chapter 3, CCTs from 20 dogs with subclinical degenerative mitral valve disease were used to investigate the effects of pimobendan on mitral annular dynamics. By comparing the mitral annular dynamics before and after the administration of pimobendan, it was shown that pimobendan augmented the systolic contraction of the mitral annulus which likely reduced the regurgitant orifice area and severity of mitral regurgitation. In conclusion, integration of CCT into clinical practice enhances our ability to evaluate and treat structural heart disease. Cardiac computed tomography can also be used as a reference to refine other imaging techniques and investigate the mechanism of action underlying various cardiovascular therapies.
Embargo
Developing preclinical models for intervertebral disc degeneration: analyzing mechanical, molecular, and immunological interventions
(Colorado State University. Libraries, 2024) Bonilla, Andres, author; Easley, Jeremiah, advisor; Dow, Steve, committee member; Puttlitz, Christian, committee member; Johnstone, Brian, committee member
Low back pain is a prevalent global health issue, significantly affecting quality of life and contributing to economic burdens through reduced productivity and healthcare costs. Intervertebral disc degeneration (IVDD) is recognized as a major underlying cause of chronic low back pain. Despite advances in therapeutic strategies for IVDD, the translation of these treatments from preclinical research to clinical application remains challenging due to the lack of appropriate animal models that accurately mimic the complex pathophysiology of human IVDD. This document aims to address these limitations by developing and validating novel ovine and immune-induced animal models of IVDD, and to provide insights into the molecular, biomechanical, and immunological aspects of the disease. Chapter 1 introduces the clinical significance of low back pain and the central role of IVDD in its development. The chapter highlights the unmet need for robust preclinical models to facilitate the evaluation of potential therapeutic interventions. Chapter 2 reviews the existing literature on ovine models of IVDD, emphasizing their relevance to human spinal disorders due to the anatomical, physiological, and histological similarities between sheep and humans. Ovine models are particularly valuable for studying both spontaneous and induced IVDD, providing a critical platform for translational research. Chapter 3 presents a groundbreaking study that conducts the first comparative analysis of surgical, imaging, histological, and proteomic characteristics between cervical and lumbar intervertebral discs (IVDs) in an ovine model of IVDD. The results demonstrate a comparable progression of IVDD in both regions, challenging the longstanding emphasis on lumbar IVDs in research and underscoring the importance of cervical models in advancing our understanding of the disease. These findings have substantial clinical and research implications, indicating that treatments traditionally developed and evaluated for lumbar IVDD may also be relevant for cervical pathology. Furthermore, the identification of specific biomarkers, could significantly enhance early diagnosis and inform the development of tailored therapeutic interventions. Chapter 4 introduces a novel model utilizing extracorporeal shock wave therapy (ESWT) applied to ovine IVDs. While no significant evidence of IVDD was observed during the 12-week study period, localized bone formation at the treatment sites was identified. This finding provides important insight into the effects of ESWT, suggesting that while it is conventionally used as a therapeutic modality, it may also have unintended consequences, such as promoting bone formation, which could potentially lead to tissue damage. These results highlight the need for further refinement of shock parameters to reliably induce progressive IVDD, offering valuable data for future research into both the therapeutic and adverse effects of ESWT in spinal treatments. In Chapter 5, a mechanical compression model utilizing MRI-compatible materials was developed to induce disc degeneration in ovine lumbar discs, marking the first report of its kind to our knowledge. This innovation allows for the longitudinal tracking of degeneration with a measurable rate of compression, leveraging MRI as the most critical tool for diagnosing IVDD. While the model successfully induced biomechanical changes, including reduced disc height and altered neutral zone dynamics, no significant histological or biochemical degeneration was observed. However, these findings provide valuable insights for future researchers using mechanically induced models, offering a foundation to refine and optimize the model for tracking the progression of degeneration more accurately. Chapter 6 explores the role of immune system in IVDD by developing an immune-induced model using a nucleus pulposus (NP) antigen vaccine. Rabbits were vaccinated against NP following IVD injury, leading to accelerated degeneration in comparison to non-vaccinated animals. This study highlights the potential of immune responses in accelerating disc degeneration, offering a novel avenue for understanding the interplay between immunity and IVDD progression. This document contributes to the advancement of IVDD research by establishing and validating novel animal models, including ovine, mechanical compression, and immune-induced models. The proteomic findings and biomechanical evaluations presented in this document offer critical insights into the molecular pathways involved in IVDD and lay the foundation for the development of customized therapeutic strategies. Future research should focus on refining these models to better replicate the complexities of human IVDD and explore long-term therapeutic interventions that can mitigate degeneration and restore disc function.
Open Access
Presale diagnostic imaging in Thoroughbred horses: the prevalence and progression of radiological and ultrasonographical findings and their associations with racing performance
(Colorado State University. Libraries, 2024) Peat, Frances J., author; Kawcak, Christopher E., advisor; McIlwraith, C. Wayne, advisor; King, Melissa R., committee member; Selberg, Kurt T., committee member; Barrett, Myra F., committee member
Background: The sale of young horses at bloodstock auctions plays an important role in the success of the Thoroughbred industry worldwide. The Keeneland September Yearling Sale in Lexington, Kentucky, is the largest sale of Thoroughbred yearlings in the world. Conducted over 12 days and presenting between 2500 and 3000 horses through the auction ring, the sale now records gross receipts exceeding 400 million United States Dollars (USD) per year. Veterinarians at the sale perform presale inspections on future racing prospects and consult with prospective purchasers regarding a horse's suitability for its intended use. Radiography and ultrasonography are used in presale examinations to identify orthopaedic issues that may affect soundness during athletic training and racing. Modern diagnostic imaging technology produces high quality images that have enabled the detection of a number of presale findings of unknown significance in young horses. Variable interpretation of these findings and conflicting assignments of potential clinical importance have become a source of contention in the Thoroughbred industry and this requires resolution. Specific imaging findings for which further scientific evidence is needed include radiological changes in the equine medial femoral condyles (MFCs) and proximal sesamoid bones (sesamoids) and ultrasonographic findings in the medial and lateral branches of the suspensory ligament (branches). Changes in the sesamoid and the insertional region of the adjacent suspensory branch are of particular importance in young Thoroughbreds, due to the potential for catastrophic injury to the suspensory apparatus in which they are an integral structure. Little is known about the prevalence of concurrent ultrasonographic branch change relative to the various grades of radiological sesamoid appearance seen in horses. Objective scientific data would enable prepurchase and training management decisions to be made that are in the best interests of the horse and its connections and the wider industry. Objectives: In the equine stifle (femorotibial joint) and fetlock (metacarpo/metatarsophalangeal joint), the objectives of this doctoral research were firstly to identify the prevalence of subchondral lucencies (SCLs) in the distal aspect of the MFC and the prevalence of various sesamoid changes on sales repository radiographs in yearling and 2-year-old Thoroughbreds, and to identify the prevalence of ultrasonographic suspensory branch changes in the same population of horses. Secondly, the studies aimed to monitor changes in MFC, sesamoid and suspensory branch grades between yearling and 2-year-old sales in horses that presented for sale at both ages. Thirdly, the research aimed to determine any associations between grade of MFC, sesamoid or suspensory branch findings and future racing performance. Lastly, the research was designed to examine the existence of concurrent radiological and ultrasonographic findings in individual sesamoid-branch units in sales horses; to determine whether there are any radiological findings that are consistently accompanied by a particular degree of insertional branch change and to provide practical recommendations as to when suspensory branch ultrasonography may be warranted in the sales environment. The overriding objective was to provide an evidence-based determination of which presale imaging findings should be regarded as an acceptable appearance at a given age in sales horses and which findings constitute a risk to future performance. Methods: The research was performed via prospective cohort studies using enrolled samples. Sales repository radiographs were obtained with consignor permission from the 2016 Keeneland September Yearling Sale and the five major North American 2-year-old sales in 2017 run by Fasig-Tipton Company and Ocala Breeders' Sales Company. Ultrasonography was performed immediately prior to the sales on the forelimb suspensory branches of horses with consignor permission. Stifle and fetlock radiographs were evaluated for MFC and sesamoid changes, respectively. MFC SCL were graded on a scale of 0-3 according to radiological size and axial MFC lucencies were recorded separately. Sesamoid findings relating to vascular channel appearance (0-3), abaxial contour changes and apical and abaxial fragments were graded according to a grading system established for the purposes of this study. Ultrasonographic findings relating to suspensory branch size, fibrillar pattern, the presence of hyperechoic foci, periligamentar tissue thickness and the adjacent proximal sesamoid bone surface were recorded during post-sale image evaluation. Racing performance was assessed for all study horses until the end of their 4-year-old racing season and performance data was obtained from Equibase Company LLC. Racing performance was measured via eight outcome variables: whether the horse started at least one race by the end of their 4-year-old year, age at first race start, total number of race starts, total prizemoney earned, earnings per start, class of career best start achieved, weighted Listed and Group race starts and Class Performance Index. Clinical follow-up was sought to ascertain why horses that did not race never started. Distributions of imaging findings were examined using descriptive statistics at the individual bone and branch level and at the horse level. Associations between imaging findings and racing performance from 2 to 4 years of age were examined using multivariate regression analyses, controlling for horse sex. Analysis was via logistic, negative binomial or linear regression as appropriate, with the threshold for significance set at a=0.05. Results: Sales radiographs from 2,508 yearlings and 436 2-year-olds were included. This sample represented 11% of the annual US Thoroughbred foal crop. It comprised 36% of all yearlings sold at auction in North America in 2016 and 20% of all 2-year-olds sold at auction in North America in 2017. Radiographs of 5,016 yearling stifles and 872 2-year-old stifles were evaluated. MFC SCLs of Grades 1-3 were observed in 242 yearlings (9.7%) and 49 2-year-olds (11.2%). Bilateral MFC SCLs of Grades 1-3 were observed in 54 yearlings (2.2%) and 12 2-year-olds (2.8%). Yearling Grade 1 MFC SCLs had either resolved (11/31), remained unchanged (14/31) or progressed to a Grade 2 (6/31) by 2-year-old sales. Yearling Grade 2 MFC SCLs had either improved to a Grade 1 (2/10), remained unchanged (6/10) or progressed to a Grade 3 (2/10) by 2-year-old sales. Yearlings with a Grade 3 MFC SCL had a 78% probability of starting a race (95% Confidence Interval (CI): 58.2-89.6%), compared to 84% for MFC Grade 0 yearlings (95% CI: 82.7-85.8%). Six of the seven yearlings with axial MFC lucencies raced. Radiographs of 20,064 yearling sesamoids and 3,488 2-year-old sesamoids were evaluated. Interobserver agreement using the new radiological grading system was substantial. Yearling findings associated with a significantly reduced probability of starting a race were: Grade 3 vascular channels in forelimb sesamoids (0.52, P<0.001, 95% CI: 0.37-0.67), abaxial new bone in forelimb sesamoids (0.62, P=0.01, 95% CI: 0.49-0.73), apical or abaxial fragments in forelimb sesamoids (0.55, P=0.005, 95% CI: 0.37-0.72). For affected horses that did race, Grade 3 vascular channels in forelimb sesamoids were associated with fewer race starts (9.9 starts, P=0.03, 95% CI: 8.0-12.2) and Grade 3 vascular channels in hindlimb sesamoids were associated with a delayed start to racing careers (54 days, P=0.01, 95% CI: 20-89). Abaxial new bone in forelimb sesamoids was associated with a 54% reduction in total earnings (P=0.003, 95% CI: 24-72) and a 46% reduction in earnings per start (P=0.002, 95% CI: 21-64). Abaxial concavity occurred predominantly in yearling medial forelimb sesamoids, had no impact on racing performance and mostly resolved by two-year-old sale. A total of 593 sales yearlings and 367 2-year-olds had ultrasonography performed on all four forelimb suspensory branches per horse. Grade ≥2 fibrillar branch change was present in 8.9% of yearlings and 14.4% of 2-year-olds. A 0.25cm increase in branch width was associated with a 49-day delayed start to racing careers (P<0.001, 95% CI: 21-77 days). The presence of Grade 2 hyperechoic foci was associated with significantly lower total earnings (P=0.01, 95% CI: $2,000-$16,022) and lower earnings per start (P=0.003, 95% CI: $349-$1,718) in USD. Grade 3 fibrillar branch change had clinically important reductions in the probability of racing, calibre of racing performance and earnings. Grade 1 fibrillar pattern was associated with significantly higher earnings per start (P=0.004, 95% CI: $2,641-$5,759). A total of 2,204 yearling forelimb sesamoid-branch units and 1,336 2-year-old forelimb sesamoid-branch units were available for evaluation of concurrent imaging findings. The proportion of yearling sesamoids with Grade ≤1 vascular channels that had adjacent Grade ≥2 fibrillar branch change was 1.2%. The same proportion for 2-year-olds was 3.8%, with medial forelimb sesamoids with Grade 1 vascular channels overrepresented in 2-year-olds. In yearlings, 31% of sesamoids with Grade 2 vascular channels had adjacent Grade ≥2 fibrillar branch change and 59% of sesamoids with Grade 3 vascular channels had adjacent Grade ≥2 fibrillar branch change. In 2-year-olds, 47% of sesamoids with Grade 2 vascular channels had adjacent Grade ≥2 fibrillar branch change and 67% of sesamoids with Grade 3 vascular channels had the same. Only 1 yearling and 1 2-year-old sesamoid with radiological abaxial concavity had Grade 2 fibrillar branch change. Limitations: The samples used in this research are representative of the population of interest at Thoroughbred sales but may underestimate the prevalence of severe lesions in non-sale horses. The study design could not address exclusions prior to sale. The findings are applicable to horses prepared for public auction and deemed fit to be entered for sale by consignors and their veterinarians. Clinical examinations were not performed for the purposes of this research. Conclusions: Regarding stifle lucencies, Grade 1 MFC SCLs were the most common type seen in yearling and 2-year-old sales horses. The majority of yearling Grade 1 MFC SCLs resolved or remained unchanged by 2-year-old sales. It was also possible for Grade 2 and 3 MFC SCLs to improve one grade between sales. Fewer sales yearlings with a Grade 3 MFC SCL raced, but in those that did race there was no evidence of worse performance compared to unaffected peers. Axial MFC lucencies did not affect racing performance. For sesamoids, Grade 3 vascular channels, forelimb sesamoid abaxial new bone and forelimb sesamoid fragments are important findings in sales repository radiology. The new grading scale assigns a numerical grade for vascular channel appearance that matches the number of enlarged vascular channels evident in a given sesamoid. Abaxial contour changes, when present in sesamoids that are Grade 0 for vascular channels, are noted separately as either abaxial new bone or abaxial concavity. Fragments are also noted and interpreted separately. Reference values specific to young Thoroughbreds have been established for suspensory branch ultrasonography. Grade 1 fibrillar suspensory branch change should be regarded as an acceptable appearance in sales yearlings and 2-year-olds. Approximately one third of Grade 2 yearling branches progressed to a Grade 3 lesion. Evidence of enlarged branch width and Grade 2 hyperechoic foci at 2-year-old sales constitute a risk to racing performance. The existence and prevalence of concurrent radiological and ultrasonographic findings in the proximal sesamoid bones and adjacent suspensory ligament branches has been established in yearling and 2-year-old Thoroughbred sales horses. General recommendations have been made for selective branch ultrasonography on the basis of sesamoid radiological appearance. The results support a separate aetiology for radiological sesamoid abaxial concavity that does not primarily involve the suspensory branch insertion. This research provides veterinarians and the wider Thoroughbred industry with evidence-based determinations of the importance or otherwise of the various presale imaging findings seen in the MFCs, sesamoids and suspensory branches of yearlings and 2-year-olds. Many findings can be regarded as an acceptable appearance in yearlings and 2-year-olds. For those findings that are associated with reduced performance, sale and management decisions can be made based on quantitative evaluations of risk that are in the best interests of the horse.
Open Access
Investigation into the mechanisms of bone loss in a sheep model of osteoporosis
(Colorado State University. Libraries, 2024) Bisazza, Katherine T., author; Easley, Jeremiah T., advisor; Anthony, Russell V., committee member; McGilvray, Kirk, committee member; Goodrich, Laurie R., committee member; Nelson, Bradley B., committee member
Osteoporosis is the most common metabolic bone disease in humans and the leading cause of fragility fractures in the aging population. Given the invasiveness of researching bone diseases in people, appropriate animal models are essential to both build our understanding of the disease as well as examine novel therapeutics. While small animals and rodents are more commonly used as models in bone research, large animal models offer the ability to perform robust, long-term studies on bone quality with higher translational impact. Ovariectomized sheep are a well-established large animal model for osteoporosis because of the comparable bone size and microarchitecture that is shared with humans. While the ovariectomized sheep has been utilized for decades as a model for the study of bone, many gaps in the model characterization remain. Based on results from a preliminary literature search, we developed study objectives and hypotheses to expand upon current knowledge gaps in the characterization of the sheep model of osteoporosis. In order to test these aims, we performed a single 12-month in vivo study utilizing sixteen ewes. Osteoporosis was induced experimentally in ten of these ewes via ovariectomy followed by a 24-week regimen of high dose corticosteroids, while six ewes were used as healthy seasonal controls. In our first aim, we compared the bone density and microarchitectural changes in the osteoporotic animals as compared to healthy controls over the course of a year. Dual-energy x-ray absorptiometry (DXA) scans revealed significant bone density loss in the osteoporotic animals in both the lumbar spine and tibia as compared to control animals. We also noted significant microarchitectural changes in iliac crest bone biopsies of osteoporotic animals as indicated by micro-computed tomography (microCT), including decreased bone volume fraction, trabecular thickness, and trabecular number, as well as increased trabecular spacing. Additionally, we compared the use of quantitative computed tomography (QCT) and DXA to measure bone mineral density and correlated those findings with microarchitectural parameters in the osteoporotic animals. We demonstrated superior QCT sensitivity and specificity to subtle bone changes in the lumbar spine as compared to DXA, as well as demonstrated a higher correlation of QCT with iliac crest biopsy microarchitectural changes. The second aim of our study was to explore the systemic and clinical impacts of osteoporosis model development in our ten sheep compared to the healthy control animals. To test this aim, we collected blood, bone marrow, and body weights throughout the course of the year-long study. Osteoporotic animals demonstrated significant impacts to hematology and serology blood levels over the course of model development, primarily at 3 and 6-months when corticosteroids were at peak use. In particular, we note significant reductions in monocytes, lymphocytes, and eosinophils at 3-months with accompanying neutrophilia, as well as an increase in platelet count and volume. We also observed an increase in serum phosphorus and electrolytes, decrease in kidney enzymes and total protein, and an increase in select liver enzymes at 3 and 6-months in the same animals. Serum cortisol and estradiol were significantly depleted at 3 and 4-months, respectively, in the osteoporotic animals. However, estradiol levels were maintained to control levels for the remainder of the study. All these changes indicate disruptions to multiple physiologic systems over the course of osteoporosis induction in sheep which may highlight the acute effects of administering high-dose glucocorticoids. In the third and final aim of this study, we investigated the morphometrical and proteomic changes in the bone of sheep following osteoporosis induction over the course of a year. Histomorphometry of iliac crest bone biopsies revealed decreases in trabecular bone area in osteoporotic model animals compared to healthy controls, while negligible differences were observed in cortical bone morphometry. Initial global untargeted proteomic outputs identified a total of 4,765 proteins from the iliac bone biopsy samples, 909 of which were determined to be differentially expressed over the course of model development in our osteoporotic sheep. Pathway analysis of differentially expressed proteins (DEPs) revealed unique enriched pathways at all time points. Enrichment of biological processes such as monocyte differentiation, metabolic processes, regulation of chromosome condensation, and immune responses were noted throughout osteoporosis development. When comparing the 909 DEPs between time points, we identified seven downregulated proteins shared between all time points as compared to baseline in the osteoporotic animals (CTR9, INPP5D, CDK6, PPP2R5C, NUP133, ITPRIPL1, W5PH60_SHEEP). Pathway analysis of these shared proteins revealed enrichment of p53 signaling, mRNA surveillance, sphingolipid signaling, and P13K-Akt signaling pathways. This study was the first to report on the proteomic changes of bone in conjunction with morphometry assessments in a sheep model of osteoporosis. All three of our described experiments allowed us to successfully fill in some of the knowledge gaps in the characterization of a large animal model of osteoporosis by further assessing both macro and micro changes in ovariectomized and steroid-dosed sheep over the course of a year. Large animal preclinical models offer researchers the ability to compare bone changes in the same animals over time, allowing for a more comprehensive insight into the progression of postmenopausal and age-related bone loss. Understanding the mechanisms driving bone loss and systemic changes in osteoporosis disease progression could aid in future cellular therapy research and investigation of novel pathway targets for osteoporosis treatment in humans.
Embargo
Comprehensive investigation of chronic enteropathy in dogs through a prospective clinical trial, immunoassays, and RNA-sequencing
(Colorado State University. Libraries, 2024) Manchester, Alison C., author; Dow, Steven, advisor; Lappin, Michael R., advisor; Avery, Anne, committee member; Webb, Craig, committee member
Chronic enteropathy is a common condition in dogs causing recurrent or persistent gastrointestinal clinical signs. Pathogenesis is thought to involve intestinal mucosal inflammatory infiltrates, but histopathological evaluation does not predict treatment response, inform prognosis, or correlate with clinical remission. Many dogs may improve clinically with dietary intervention, but between 15 to 40% of dogs are refractory to all therapies. This negatively impacts quality of life for dogs and their families and can lead to euthanasia. Better understanding of the cellular and molecular differences between CE and health is necessary to improve outcomes for these dogs, and to enable use of the dog as a translational model for study of inflammatory intestinal conditions across species. The goal of this work was to critically evaluate the pathogenesis of CE in dogs through use of in vitro assays, a prospective clinical trial, and next-generation sequencing based approaches. Preliminary studies have highlighted an important role for intestinal bile acids in the pathogenesis of canine and human chronic enteropathies. Fecal bile acid populations differ between healthy dogs and dogs with CE. However, there has been little work to evaluate potential consequences of these metabolic shifts in dogs. We therefore investigated potential immunomodulatory roles of primary and secondary bile acids through in vitro experiments with canine macrophages. Both the primary bile acid cholic acid (CA) and the secondary bile acid lithocholic acid (LCA) influenced LPS-induced cytokine production via canine monocyte-derived macrophages similarly, with suppression of TNF-α secretion and enhancement of IL-10 secretion. Neither BA altered the expression of the BA receptor TGR5. Transcriptomic analysis revealed that CA activated inflammatory signaling pathways in macrophages involving type II interferon signaling and the aryl hydrocarbon receptor, whereas LCA activated pathways related to nitric oxide signaling and cell cycle regulation. Thus, we concluded that both primary and secondary BAs are active modulators of macrophage responses in dogs, with differential and shared effects evident with sequencing analysis. Diet is the most effective management strategy for dogs with CE, enabling two-thirds of patients to achieve clinical remission from their disease. Various dietary strategies may be beneficial. Nutritional formulae sourcing protein from amino acids have been used for the induction of remission in human Crohn's disease patients for decades. We conducted a prospective clinical trial involving exclusive feeding of the first diet sourcing protein from individual amino acids to 23 client-owned dogs with CE to determine its ability to induce clinical remission and begin to tease apart mechanisms of action. After 2 weeks of EL, 68% of dogs consuming the diet were classified as responders. At the conclusion of the 8 week feeding trial, 16/23 dogs (70%) were considered clinical responders. Feeding EL caused shifts in fecal bacterial communities, which differed between responders and non-responders, suggesting that diet's ability to modulate gut bacterial populations may predict its efficacy. Serum biomarker concentrations were unchanged throughout the study apart from serum alkaline phosphatase activity. Results of this study indicate that an amino acid based diet is another option to treat dogs with CE and implicates the intestinal microbiota in achievement of remission in these patients. Most studies comparing healthy and CE dogs completed to date have been limited in scope, evaluating individual or a small collection of biomarkers or cell types. This has hampered advancement of the understanding of CE pathogenesis in dogs. Ultimately, this results in generic treatment strategies for dogs and leaves a substantial proportion unable to achieve clinical remission from their disease. To this end, we applied next-generation transcriptomic sequencing to mRNA from duodenal biopsies from CE dogs and healthy beagle dogs. Results of this analysis highlighted important roles for epithelial cell gene signatures in differentiating CE tissues from healthy ones. Commonly implicated cytokines like TNF-α, IL-12, or IL-10 were not differentially expressed, but pathway analysis highlighted a potential role for upregulation of anti-viral pathways in CE dogs. This preliminary study underscores the power of RNA sequencing to provide a broad overview of cellular activities in tissues of interest, and question widely accepted theories regarding dysfunction present in the gut of dogs with CE. Single-cell RNA sequencing offers a high-resolution molecular technique enabling characterization of gene expression on an individual cell basis. This approach overcomes traditional barriers to disease investigation (e.g., species-specific reagents) and allows for definition of cell subtypes within heterogeneous samples. We thus employed single-cell RNA sequencing to catalog and compare the diversity of cells present in duodenal mucosal endoscopic biopsies from 3 healthy dogs and 4 dogs with CE. We identified populations of epithelial cells, T cells, myeloid cells, and plasma cells, with contributions from both the healthy and CIE samples. Neutrophils from CE samples exhibited a more inflammatory transcriptional program. T cells were broadly divided into non-resident and tissue resident subtypes, though minimal transcriptomic differences were appreciated within this class of cells. One subset of epithelial cells from CE dogs showed differential expression of a gene encoding a 2-pore potassium channel (KCNK16). Our results reveal a previously unappreciated cellular heterogeneity in canine duodenal mucosa and provides insights into molecular mechanisms underlying CE in dogs. The cell type gene signatures determined through this work will enable better understand the subtleties of canine intestinal physiology to allow more accessible interrogation of cellular activities in health and disease. The results of the studies described add further nuance and detail to understanding of the pathogenesis and management of canine CE. We have documented the power of transcriptomic analysis for differentiation of intestinal mucosal molecular programs in health and CE. Further investigation into intestinal bile acids, duodenal mucosal T cell subtypes and neutrophils, and intestinal epithelial cell activities are indicated.
Open Access
Epidemiology and veterinary public policy
(Colorado State University. Libraries, 2008) Zepeda Sein, Cristóbal Andrés, author; Salman, Mo, advisor
Official Veterinary Services are increasingly required to base veterinary public policy decisions on scientific grounds, epidemiology and risk analysis play an important role in shaping these decisions. A formal, in-depth analysis of the multiple interactions between epidemiology, risk analysis and veterinary public policy was conducted to enable decision-makers to direct resources more efficiently and facilitate compliance with international agreements, in particular the Agreement on the Application of Sanitary and Phytosanitary Measures (SPS) of the World Trade Organization. The SPS Agreement recognizes the World Organization for Animal Health (OIE) as the international organization responsible for developing animal health standards. The OIE's Terrestrial Animal Health Code contains scientifically based recommendations for international trade in animals and animal products. However, to date, these recommendations have not been assessed from a risk-based perspective. The study is divided in two major sections: (1) the role of epidemiology in veterinary public policy and (2) the application of risk-based approaches to the assessment of international animal health standards. The first section addresses the international framework, risk analysis and its use worldwide, and the development of international standards. The second section focuses on quantitative risk assessment approaches for the international movement of animals and products, as well as the application of compartmentalization to aquaculture production systems emphasizing the use of a HACCP approach to biosecurity.
Open Access
A holistic approach to veterinary public health in animal shelters and other sites
(Colorado State University. Libraries, 2009) Steneroden, Kay K., author; Salman, M. D., advisor; Hill, Ashley E., advisor
Animal health and human health are intimately linked. Directly, through contact with or exposure to animals and their environments, and indirectly by way of food production, food safety and antimicrobial drug residues, humans are dependent upon and vulnerable to the health of animals. Veterinary public health is concerned with the interface of human and animal health and addressing problems at that interface. The potential impact of such exploration is greater human and animal health. Epidemiological needs assessment, problem investigation and subsequent outreach programs are essential tools of veterinary public health practice. These tools are used to explore infection control, infectious and zoonotic disease awareness, environmental contamination with infectious/zoonotic agents and monitoring the consequences of treatment of infectious and zoonotic diseases with antimicrobial drugs (i.e. antimicrobial drug resistance). The specific venues for these explorations for this dissertation include animal shelters, a veterinary teaching hospital, a former Soviet country and a United States governmental program. A holistic approach is used with animal shelters to assess infection control and zoonotic disease awareness needs, investigate environmental contamination with a zoonotic disease, develop training tools and train animal shelter workers and volunteers. The needs assessment provided valuable information on characteristics of animal shelters, provided impetus for the problem investigation and the basis for outreach training. The problem investigation tool provided the first available information on the prevalence and extent of salmonella contamination in Colorado animal shelters. The outreach components provided a tool and reference for training; the training itself indicated gaps in knowledge in various aspects of infection control and zoonotic disease awareness that could be addressed with training. Further, problem investigation is explored through the success of active surveillance in discovery and control of a zoonotic disease outbreak in a veterinary teaching hospital. Results of a needs assessment survey in the Republic of Armenia provide the basis for development of outreach materials for veterinarians, farmers and school-age children on their national animal health program. And a system of antimicrobial drug resistance monitoring is examined and challenged for completeness. Taken together, these studies further the examination of veterinary public health issues and highlight a holistic approach to their exploration.
Open Access
Immunoproteomic identification of bovine pericardium xenoantigens
(Colorado State University. Libraries, 2008) Griffiths, Leigh G., author; Orton, E. Christopher, advisor; Reardon, Kenneth F., advisor
Bovine pericardium (BP) is an important biomaterial used in the production of gluteraldehyde-fixed heart valves and tissue engineering applications. The ability to perform proteomic analysis on BP is potentially useful for several reasons including investigation of immune rejection after implantation. The importance of humoral and cell mediated rejection responses towards such xenogeneic tissues are becoming increasingly apparent. I have applied a novel immunoproteomic approach to survey the antigenic determinants of BP. Proteomic analysis of fibrous tissues like BP is challenging due to their relative low cellularity and abundance of extracellular matrix. A variety of methods for tissue homogenization, protein extraction, and fractionation were investigated with the aim of producing high quality 2-DE gels for both water- and lipid-soluble BP proteins. MALDI-TOF/TOF MS protein identifications were performed to confirm bovine origin and appropriate subcellular fractionation of resolved proteins. Sixteen unique predominantly cytoplasmic bovine proteins were identified from the water-soluble gels. Twenty-two unique predominantly membrane bovine proteins were identified from the lipid-soluble gels. These results demonstrate that the final 2-DE protocol produced high quality proteomic data from BP for both cytoplasmic and membrane proteins. Duplicate 2-DE gels were used to generate western blots from both water- and lipid-soluble gels. Western blots were probed with pre- and post-exposure anti-BP rabbit serum, with detection of immune complexes limited to the IgG subtype. Western blots were compared to duplicate 2-DE gels and spots matched using Delta 2D image analysis software. Protein identifications of matched spots were performed using either MALDI-TOF/TOF MS or ESI MS/MS. This approach identified 31 putative antigens, capable of stimulating an IgG humoral rejection response. To the best of my knowledge, this study was the first to apply an immunoproteomic approach for identification of antigenic targets in xenotransplanted tissues. The results provide important information for understanding and possibly mitigating the immune response to fixed and unfixed BP xenografts.
Open Access
Investigation into disease events at the wildlife/livestock interface: lessons learned from bovine viral diarrhea virus in Colorado cervids
(Colorado State University. Libraries, 2009) Duncan, Colleen, author; Salman, Mo, advisor; VanCampen, Hana, advisor
Infectious agents may be transmitted between wild and domestic animals; these so called 'interface diseases' can have significant economic consequences. As such, effective tools and techniques with which to study disease in free ranging, wild animals is essential. Principles of wildlife disease surveillance were reviewed and it was concluded that while wildlife disease research may require unique logistical adaptations; basic principles of surveillance remain the same. A review of wildlife data sources utilized for surveillance suggests that information collected, and shared, is dependent on the group involved and that there are opportunities to improve the type and quality of material available. Bovine viral diarrhea virus (BVDV) is an important virus of domestic cattle that has recently been identified in wild ruminants worldwide. To investigate the presence, prevalence, distribution and significance of BVDV in wild cervids of Colorado a series of projects were conducted. Persistently infected (PI) deer were studied post mortem; immunohistochemical and molecular techniques used to look for viral antigen in deer tissue were found to be effective supporting the use of these tests in further studies. The prevalence and distribution of PI cervids in the state was evaluated using an opportunistic sampling technique; the prevalence is extremely low, but naturally occurring infection is present within Colorado. The cost associated with testing animals for an uncommon disease may be very high; techniques like pooling samples can help to keep costs down during such investigations. The sensitivity and specificity of RT-PCR on pooled samples was investigated in an experimental study and revealed that supernatant from a single positive deer skin sample may be diluted up to 10,000 times and still be detected. Another technique to focus research efforts on high risk areas is the use of simulation modeling. A stochastic risk assessment model was developed to identify regions in Colorado where PI cattle were likely to be born following exposure to a PI deer. Results of the model were consistent with both the cross-sectional survey for PI cervids and other reports on BVDV in wildlife of Colorado.
Open Access
Gene expression in phenotypically homogeneous chondrocytes from different articular cartilage layers of equine osteoarthritic and control joints: method validation and gene array analysis
(Colorado State University. Libraries, 2007) Düsterdieck, Katja Friederike, author; Frisbie, David D., advisor
Osteoarthritis remains a common and debilitating disease in horses, despite advances in diagnosis and treatment. Cartilage is commonly considered to play a central role in the pathophysiology of osteoarthritis. The investigation of differences in gene expression in cells from osteoarthritic and control cartilage is expected to yield genes possibly playing a role in the pathophysiology of osteoarthritis, representing new targets for treatment of the disease. The goals of this investigation were to develop a methodology to isolate RNA from phenotypically homogeneous cells of various cartilage layers for gene array analysis and to determine differentially expressed genes in these cells in osteoarthritic and control cartilage. A methodology to isolate phenotypically homogeneous chondrocytes from frozen sections of adult equine articular cartilage was developed using laser capture microdissection, RNA isolation, amplification and qrt-PCR. Expression levels of candidate genes were compared to those in conventionally isolated RNA from paired cartilage samples. The methodology was adequate to produce sufficient amounts of RNA for gene array analysis. Gene expression was found to be altered, but in a consistent fashion. The validated methodology, followed by gene array analysis was utilized to compare expression patterns in chondrocytes from tangential and radial layers of experimentally induced osteoarthritic and control cartilage. 154 genes were differentially expressed between tangential and radial cartilage layers and 17 genes were differentially expressed between osteoarthritic and control cartilage. The gene expression pattern of the tangential layer reflected support of cell proliferation, suppression of apoptosis and several genes involved in cell-matrix interactions or inflammatory processes. In contrast, the gene expression pattern of the radial layer was dominated by genes supporting the synthesis of proteins and proteoglycans. The gene expression pattern from osteoarthritic cartilage suggested an active response to oxidative stress, activation of the NF-κB pathway, decreased anti-apoptotic ability and downregulation of proteoglycan synthesis and glycolysis. This study was the first to determine gene expression patterns between two different layers of articular cartilage, improving our understanding of cartilage homeostasis in health and disease.
Open Access
Peri-slaughter ecology of Escherichia coli O157 and Salmonella enterica in feedlot beef cattle
(Colorado State University. Libraries, 2008) Dewell, Grant Alan, author; Salman, Mo, advisor
Risk factors for prevalence of E. coli O157 prior to slaughter and the genotypic relationship between feedlot and slaughter isolates were investigated. The odds of E. coli O157 positive fecal samples from cattle fed brewers grains were 6 times that for cattle not fed brewers grains. The odds of E. coli O157 positive fecal samples from cattle from Central Nebraska was 9 times that for cattle from Eastern Colorado. Within the sampled pens, 64% of the hide samples at the abattoir corresponded to a feedlot isolate. For carcass samples, 59% of isolates had a corresponding feedlot isolate. Transportation of cattle from the feedlot to the slaughter plant could influence hide contamination of Escherichia coli O157 or Salmonella enterica. Cattle held in E. coli O157 positive lairage pens had eight times greater relative risk of having E. coli O157 positive hide samples compared to cattle held in culture-negative pens. Cattle that were held in lairage pens contaminated with feces had three times greater relative risk for E. coli O157 positive hide samples and twice the relative risk for S. enterica positive hide samples compared to cattle held in clean pens. Cattle that were transported for long distances (> 160.9 km) had twice the relative risk of having E. coli O157 positive hide samples and twice the relative risk of having S. enterica positive hide samples compared to cattle transported shorter distances. Cattle with positive Salmonella enterica hide samples at the feedlot had almost twice the relative risk of having S. enterica positive hide samples compared to cattle without S. enterica positive feedlot hide samples. Cattle transported in trailers with positive S. enterica samples had over twice the relative risk of having S. enterica positive hide samples compared to cattle transported in culture negative trailers. Cattle held off feed longer than 18 hours before loading had a greater relative risk of having S. enterica positive hide samples compared to cattle held off feed for shorter times. Cattle that were agitated during loading had twice the relative risk of having S. enterica positive hide samples compared to cattle that were calm.
Open Access
Valve interstitial cell phenotypes and signaling pathways involved with canine myxomatous degenerative mitral valve disease
(Colorado State University. Libraries, 2008) Disatian, Sirilak, author; Orton, E. Christopher, advisor
Myxomatous mitral valve disease is a common heart disease of dogs that is similar to myxomatous mitral valve disease in humans. The first hypothesis of this dissertation is that interstitial cell phenotype transformation described in human myxomatous valves also occurs in dogs with myxomatous mitral valves and correlates with disease severity. Normal and early-, intermediate-, and late-stage myxomatous canine mitral valves were examined by immunohistochemistry for cytoskeletal (vimentin, desmin, smooth muscle α-actin, smooth muscle myosin, and non-muscle myosin), collagenolytic (MMP-1, MMP-13), cell surface (CD-31, CD-45, CD-68) and proliferation (Ki-67) proteins. Normal canine mitral valve interstitial cells were positive for vimentin, but negative for α-actin, desmin, and non-muscle myosin (i.e. fibroblast phenotype). Interstitial cells from myxomatous valves showed increased positive staining for α-actin and desmin, but were negative for smooth muscle myosin (i.e. myofibroblast phenotype). Positive cells first appeared as clusters in the subendocardial region of the lamina atrialis and extended into deeper layers with increasing severity. Interstitial cells from myxomatous valves showed positive staining for non-muscle myosin (i.e. activated mesenchymal cell phenotype). Positive staining cells increased with disease severity and were dispersed throughout the valve layers. Expression of MMP-1 and MMP-13 correlated with disease severity. Interstitial cellularity increased in degenerative valves however Ki-67 staining was mildly increased. In conclusion, two patterns of interstitial cell phenotype transformation were identified in dogs with myxomatous mitral valve disease and both correlated with disease severity.
Open Access
Surveillance and diagnosis of transmissible spongiform encephalopathies in the United States
(Colorado State University. Libraries, 2007) Dennis, Michelle Marie, author; Salman, Mo, advisor
Since limited knowledge of transmissible spongiform encephalopathies (TSEs) restricts treatment and successful control interventions, and since some may cause fatal food-borne disease in humans, the United States (U.S.) has established TSE surveillance programs to support control efforts and to protect agriculture-based economy. The enhanced BSE surveillance system was conducted to characterize the extent of the presence of BSE in the U.S. cattle population in order to reassure consumers and trading partners of the U.S. BSE status. Given the level of importance and the cost of the enhanced BSE surveillance program, surveillance system evaluation was conducted to provide feedback for improving future surveillance and to determine the extent to which the system had met its objectives. Recommendations were made to improve efficiency and quality of future BSE surveillance systems. The enhanced BSE surveillance certainly met its stated objectives. Surveillance interests in the U.S. were subsequently re-directed towards efficiently assuring that BSE control measures remain effective, and to maintain assurance of trading partners of the U.S. BSE status. A plan for ongoing BSE surveillance was constructed using the standards and guidelines for animal health surveillance established by the National Surveillance Unit (NSU). Results derived from the enhanced BSE surveillance system and its evaluation prompted appriopriate adaptations for maintenance surveillance methods. Conditions which naturally degrade prions need to be elucidated to facilitate disposal of prion-contaminated biowastes. In order to determine whether long-term heating could destroy prions, the immunodetection of protease-resistant, disease-associated prion protein (PrPres) was evaluated in brain from chronic wasting disease (CWD)-affected elk. Using 3 diagnostic assays for CWD, progressive loss of PrPres immunodetectability, which increased with incubation temperature, was demonstrated when brain homogenates were incubated at 37, 55, and 80° C over a period of 200 days. Disposal systems which use heat over time may effectively degrade prions. Furthermore, the validity of test results derived from tissues which have been exposed to such conditions is questionable. In the U.S., scrapie surveillance uses PrPres immunohistochemistry (IHC) applied to tissues collected postmortem. The only live animal test available, PrPres IHC applied to third eyelid biopsy, is limited by comparatively lower sensitivity, high frequency of inconclusive test results, and the limited amount of tissue available for repeat testing. A study evaluated PrP res IHC applied to recto-anal mucosa associated lymphoid tissue (RAMALT) biopsy for scrapie diagnosis in live sheep. Biopsy-related complications were rare. The sensitivity of RAMALT biopsy PrPres IHC ranged from 87.5-89.3%, and approximated or exceeding that applied to third eyelid biopsy. The use of PrPres IHC applied to RAMALT biopsies for scrapie diagnosis in live high-risk sheep is expected to improve the surveillance activities that support the success of the U.S. National Scrapie Eradication Program.
Open Access
Tissue engineering of heart valves: antigen removal from xenogeneic tissue scaffolds
(Colorado State University. Libraries, 2009) Arai, Shiori, author; Orton, E. Christopher, advisor
Tissue-engineered heart valves hold the promise of an ideal heart valve substitute by using appropriate and functional cells and scaffolds. An ideal heart valve should be durable, non-immunogenic, non-thrombogenic, resistant to infection and capable of regeneration and growth. Xenogeneic tissues are potential candidates for scaffolding of tissue-engineered heart valves. Anionic detergent-based decellularization has been employed to eliminate xenogeneic tissue immunogenicity. The present studies were performed to develop a technique to detect antigenic proteins in xenogeneic tissue scaffolds, to evaluate the efficacy of antigen removal of current detergent-based decellularization of xenogeneic tissues, to develop novel techniques to enhance antigen removal, and to address issues related to the cytotoxic effects of sodium dodecyl sulfate (SDS).
Open Access
Epidemiology of reported scrapie in the United States: 1947-1991
(Colorado State University. Libraries, 1993) Wineland, Nora E., author; Salman, M., advisor; Kimberling, Cleon V., committee member; Gould, Daniel H., committee member; Weber, Stephen, committee member
Data collected in support of the United States Department of Agriculture (USDA) scrapie eradication program between 1947 and September 30, 1991 were evaluated to determine the presence of trends or patterns which might help further the understanding of natural sheep scrapie. The USDA records from 957 confirmed positive cases of natural scrapie in 581 flocks from 39 states were reviewed and compiled into a database. Possible host and management risk factors for scrapie such as age at death, within-flock mortality, breed, sex, sire and dam disease status, flock size, and location were examined. There were several significant findings from the study. The proportion of reported positive flocks in those states reporting positive cases showed a steady increase between 1965 and 1991. In addition, the average flock mortality showed a slight increase between 1947 and 1991. These increases did not seem to be directly related to any changes in the USDA eradication program. The average age at death for confirmed cases was 43.6 months. Rams died of scrapie an average of five months younger than did the ewes. This difference was statistically significant, but likely due to the small numbers of rams included in the study. There were insufficient numbers of twins (26 pairs) to allow any significant conclusions to be drawn. There were no statistically significant differences between age at death for the eight geographical regions or the various sheep breeds affected. The Suffolk breed comprised 88% of the reported cases, and Hampshire sheep accounted for 6% of the cases. Attempts were made to further define the role of vertical transmission in natural scrapie. The scrapie disease status of the sire had no appreciable effect on the age of death of positive offspring. The scrapie disease status of the dam had a detectable effect with positive offspring from positive dams diagnosed at a significantly younger age than positive offspring from other dams. Unfortunately it was not possible to determine when a positive dam might begin shedding the scrapie agent and consequently present a threat to her offspring. All of the positive dams in the study gave birth to their positive offspring in flocks where there were other active cases of scrapie which might have been the source of infection for the offspring. The source of infection could not be determined for over half of the reported cases. Several possible explanations for this situation were presented. Failure to detect the sources of infection may in part be responsible for the apparent increase in the magnitude of the scrapie problem in the United States. Data quality and consistency was a major issue for this study. The records available from the technical program staff of USDA contained varying amounts of information about each of the positive animals and flocks. In addition to variation in the records, the eradication program itself went through several phases during the study period. These different phases may have had multiple effects on the levels of disease reported to USDA. Unfortunately these effects could not be measured or corrected for in the analysis.

Browse

Recent Submissions