Theses and Dissertations

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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
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
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
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.
Open Access
Developing an integrated livestock-human infectious disease management framework for the dairy farm environment
(Colorado State University. Libraries, 2023) Fathke , Robert, author; Rao, Sangeeta, advisor; Pinedo, Pablo, committee member; Reynolds, Stephen, committee member; Duncan, Colleen, committee member
This research aimed to develop a framework integrating cattle and human infectious disease prevention in the dairy farm environment. Infectious disease dynamics on dairy farms can be complex, with various factors impacting cattle and human health. The advent of the COVID-19 pandemic reminded the world of the complexities of disease dynamics and prevention. Biosecurity is key to infectious disease prevention on dairy farm settings, but preventive efforts might not focus on both cattle and human health. Those trained in veterinary medicine may be especially suited to help bridge this animal-human gap on dairy farms, as these professionals understand disease dynamics and may be trusted to serve in this capacity. Infectious disease risk assessment tools for dairy farms might not fully integrate human health. Developing more integrated risk assessment tools first requires a greater understanding of existing tools and dairy farmer knowledge, attitudes, and practices regarding cattle and human infectious disease prevention. The research described biosecurity, biosafety, and identified potential areas of overlap to create a foundational integrated animal-human infectious disease prevention model. A systematic literature review was conducted on animal producer knowledge, attitudes, and practices regarding personal protective equipment for zoonotic disease prevention. Current biosecurity and biosafety assessment surveys and tools applicable to dairy farm environment were also assessed for structure, content, and degree of human health integration. Assessment of the survey questions and score report from one of these tools included obtaining feedback from a small sample of Front Range Colorado dairy producers. A knowledge, attitudes, and practices questionnaire including elements of cattle and human infectious disease prevention was developed, and data was collected from 50 personnel, including workers and supervisors, across six Front Range Colorado dairy farms. This work found that the word "biosecurity" has many definitions that can vary by profession setting. Many elements of efforts aimed at preventing animal diseases can also be effective in preventing human diseases. Personal protective equipment is an example of such an element. Systematically reviewing literature on personal protective equipment knowledge, attitudes, and practices revealed that animal producers often fail to use preventive measures and may not always perceive zoonoses as a threat. Assessment of existing infectious disease risk assessment tools revealed that none fully and directly integrated human infectious disease prevention. Producer feedback on one tool focusing on cattle health provided valuable feedback on tool design and helped shape recommendations for developing integrated tools. Construction of the integrated knowledge, attitudes, and practices questionnaire was a novel approach to creating a research tool that integrates animal and human infectious disease prevention. Results revealed strengths and weaknesses in knowledge, attitudes, and practices regarding zoonotic disease prevention and helped identify elements that can be addressed to develop a shared understanding between dairy farm supervisors and workers.
Open Access
Development of an ovine cardiac model for the modified Blalock-Thomas-Taussig shunt
(Colorado State University. Libraries, 2023) Chu, Margaret Lucinda, author; Monnet, Eric, advisor; Marvel, Sarah, committee member; Randall, Elissa, committee member
This pilot study aimed to better describe the ovine thoracic vascular anatomy, to develop a model for placement of a modified Blalock-Thomas-Taussig (BT) shunt in sheep, and to evaluate whether hyaluronan-treated grafts would have decreased thrombosis and adherence compared to traditional polytetrafluoroethylene (PTFE) grafts. Ovine thoracic vasculature follows the pattern of a bovine aortic arch and branching of arterial vascular does not occur until the thoracic inlet. A prominent left azygos vein and ligamentum arteriosum are also present. Placement of a modified BT-shunt was possible in sheep and did appear to lead to some long-term changes in cardiac values. Hyaluronan-treated shunts were subjectively less adherent with incomplete thrombosis occurring in the subjects. Further research is needed to better evaluate the latter two aims of this project.
Open Access
Extracellular vesicles from the equine uterus: uptake by stallion spermatozoa and effect on capacitation parameters
(Colorado State University. Libraries, 2023) Granier, Shelby K., author; McCue, Patrick, advisor; Graham, James K., advisor; Hatzel, Jennifer, committee member; Tesfaye, Dawit, committee member
Fertilization in mammalian species relies on the activation of spermatozoa in the female reproductive tract by a consecutive series of events termed 'capacitation'. In vivo, ejaculated equine spermatozoa are deposited directly into the uterus and eventually arrive in the ampulla of the oviduct, which is the site of fertilization. However, the roles of the uterus, oviduct, and their secretions have on equine sperm capacitation is largely unknown. Extracellular Vesicles (EVs), including microvesicles and exosomes, are membrane enclosed nanoparticles released from most cell types that carry cargos of biologically active molecules that can affect nearby or distant recipient cells. EVs have recently been identified as playing a role in reproductive functions including sperm capacitation. The aims of the present study were: first characterize EVs collected from the uterine lumen of mares in both the estrus and diestrus phases of their reproductive cycles; and second investigate the effect these uterine EVs have on stallion sperm function. Uterine fluid from 6 mares was collected during both estrus and diestrus using a low volume uterine lavage then EVs were isolated from the fluid by ultracentrifugation, and EV concentration determined by nano-tracker analysis. The concentration of EVs obtained from estrus fluids (EEV) was 235 ± 164.029 billion EVs/mL and tended to be higher (p=0.07) than those obtained in diestrus fluids (DEV) (83.67 ± 89.328 billion EVs/mL). The average size of EVs were similar (p > 0.05) with values of 148.633 ± 11.35 nm for EEV and 146.183 ± 11.89 nm for DEV. Transmission electron microscopy delivered images of vesicles with characteristic cup-shape morphology and size consistent with NTA results. Immunoblotting confirmed the particles contained exosome markers TSG-101 and CD-63, and were negative for cytochrome C, a mitochondrial organelle marker, indicating these vesicles were indeed EVs. To determine the effect EVs have on sperm, semen from 3 Quarter Horse stallions were cryopreserved, and EVs added to samples after thawing. In the first experiment, EVs or PBS void of EVs were fluorescently labeled and incubated with frozen-thawed stallion spermatozoa for one hour and uptake was evaluated by fluorescent microscopy. Fluorescence was observed only in sperm incubated with EVs, and a greater fluorescent intensity detected in EEV treated sperm. In a second experiment, spermatozoa from each stallion were co-cultured with EEV, DEV, and PBS void of EVs (control) for 90 minutes and sperm functions associated with capacitation, including hyperactivated motility, and acrosome reactions, were evaluated using a computer assisted semen analysis unit (CASA) and flow cytometry. The percentages of hyperactively motile sperm were higher (p < 0.05) for EEV treated sperm compared to control and DEV. In addition, the percentage of acrosome reacted sperm was higher (p < 0.05) for sperm treated with EEV and DEV when compared to control. In summary, these results confirm that: 1) EVs can be isolated from uterine fluid of mares, 2) uterine derived EVs can be taken up by stallion spermatozoa, and 3) uterine derived EVs have a biological effect on stallion spermatozoa function in vitro. Consequently, it is hypothesized that EVs from the mare reproductive tract will have similar biological effects on stallion sperm function in vivo.
Open Access
The animal health components of a biosurveillance system
(Colorado State University. Libraries, 2023) Tan, Alwyn, author; Salman, Mo, advisor; McCluskey, Brian, committee member; VandeWoude, Susan, committee member
Biosurveillance defines a One Health approach of gathering, integrating, interpreting, and communicating information related to health hazards or diseases affecting human, animal, or plant health and their environment to achieve early detection and warning, situational awareness, and better decision making. Animal health surveillance is an important component within biosurveillance systems comprising a continuum of activities from detecting biological threats, to analyzing relevant data, to managing identified threats, and embracing a One Health concept. Despite ongoing health surveillance activities conducted by various stakeholders in different One Health sectors, numerous health crises continue to occur, affecting the health of humans and animals, the livelihoods of people, the economy, their environment, and social harmony. Zoonotic agents caused a large proportion of these health crises, and nations spent large amounts of resources on disease detection and control measures to safeguard the health of their citizens against these agents. This thesis will explore how the animal health community can strengthen biosurveillance in the following sections: 1) the components of a biosurveillance system from an animal health perspective and opportunities for the animal health surveillance community to enhance biosurveillance; 2) a pilot study on the use of air-sampling as a novel method for animal health surveillance; 3) a scoping review on behavioral barriers, enablers, and interventions for animal owners and producers reporting animal diseases to veterinary authorities; and 4) theoretical demonstration of a biosurveillance system.
Open Access
Multi-modal investigation of tendon healing: tendinopathic injury models to novel rehabilitative strategies
(Colorado State University. Libraries, 2022) Johnson, Sherry, author; Frisbie, David, advisor; King, Melissa, advisor; Selberg, Kurt, committee member; Chicco, Adam, committee member
To view the abstract, please see the full text of the document.
Open Access
Perianesthesia analgesia, recovery efficacy, and financial impact of ultrasound-guided lumbar plexus and sciatic nerve analgesia in dogs undergoing tibial plateau leveling osteotomy
(Colorado State University. Libraries, 2022) Warrit, Kanawee, author; Boscan, Pedro, advisor; Monnet, Eric, advisor; Steffey, Eugene, committee member; Twedt, David, committee member; Rao, Sangeeta, committee member; Fails, Anna Dee, committee member
Perioperative analgesia is critical for patients undergoing surgery because uncontrolled pain can result in deleterious consequences and predispose chronic pain. Therefore, developing an appropriate analgesia technique is crucial, and in this study, an analgesia protocol was investigated in dogs undergoing tibial plateau leveling osteotomy (TPLO). The TPLO is a surgical technique used to stabilize the stifle joint for treating cranial cruciate ligament disease. This surgical procedure is invasive and painful. Therefore, multimodal analgesia is often required for controlling pain associated with TPLO surgery. This study used ultrasound-guided regional anesthesia of the lumbar plexus and sciatic nerve as a component of multimodal analgesia to control perioperative pain compared to patients only receiving a standard systemic analgesia. The study was designed to evaluate the efficacy of regional anesthesia and to determine the financial impact of this additional multimodal procedure. We hypothesized that ultrasound-guided lumbar plexus and sciatic nerve blocks would provide a better analgesic effect intraoperative and postoperatively. The second part of the study we hypothesized that ultrasound-guided lumbar plexus and sciatic nerve blocks would increase the anesthesia cost. It would then be possible to determine a cost – benefit of the procedure. The study was designed as a prospective, randomized, blinded clinical trial. Twenty dogs underwent TPLO surgery were enrolled in the study and randomly assigned to regional analgesia (RA) or control (CON) group. Dogs in the RA group received 0.5% ropivacaine for ultrasound-guided lumbar plexus and sciatic nerve blocks. The total dose of ropivacaine for both blocks was 0.15 mg/kg. Dogs in the CON group received sterile 0.9% saline for the blocks. All dogs received 0.2 mg/kg of hydromorphone and 0.02 mg/kg of atropine for anesthesia premedication. Propofol was administered for anesthesia induction to perform endotracheal tube intubation. Isoflurane in oxygen was delivered using a circle rebreathing system to maintain anesthesia. The ultrasound-guided lumbar plexus and sciatic nerve blocks were performed prior to TPLO surgery. Fentanyl was used for intraoperative as rescue analgesia to reduce the response from surgical stimulation or with an increase in heart rate, respiratory rate, or mean arterial blood pressure. Isoflurane was adjusted to maintain the appropriate anesthesia plane. Hypotension was treated following a stepwise protocol, in a sequencing fashion. The treatment protocol was added until the complications was resolved. The treatment protocol started from decreased isoflurane vaporizer setting, lactate Ringer's solution bolus, hetastarch bolus, and dopamine administration. At extubation, the recovery quality and pain were evaluated. Dexmedetomidine was used to treat poor recovery quality and pain. During the postoperative 12 hours period, pain and recovery quality were assessed by Colorado State University acute pain scale, visual analog pain scale, and modified University of Melbourne pain scale. Fentanyl or methadone was used for postoperative rescue analgesia. Dexmedetomidine or acepromazine was administered to calm the patient. The amount of every drug used, complications management, and extra nursing care were recorded to evaluate the blocks' efficacy. The micro-costing technique was used to collect the financial data and was analyzed to determine the financial impact. In the clinical study, there was a statistically significant difference in the amount of intraoperative fentanyl administered for rescue analgesia between the groups (p = 0.02), with lesser doses given to the RA group. Hypotension was found in 40% of dogs in the RA group and 80% of the dogs in the CON group (p = 0.16). Dogs in the RA group required less intensive treatment than in the CON group. There was a statistically significant difference in the recovery scores between the groups, with those in the RA group having lower recovery scores (p = 0.04). In the postoperative period, the time to receive the first dose of rescue analgesia for dogs in the RA group was longer than dogs in the CON group (p=0.04). Micro-costing method was used for collecting the monetary information. The cost analysis was performed for evaluating the costs of dogs that received ultrasound-guided regional anesthesia with 0.5% ropivacaine and 0.5% sterile saline. The anesthesia fixed cost for the surgery was US$354. There was a statistically significant difference between the variable costs, with the RA group (US$82.65 (69.15-94.56); median (min-max)) having less anesthesia variable costs than dogs in the CON group (US$125.8 (55.23 to 156.35); p = 0.02). The additional cost for a charge per service of the use of ultrasound and electro-nerve stimulator machines (US$26.62) affects the total anesthesia cost for the RA group into both direction, it can enhance and save the total anesthesia cost. It can increases the total anesthesia cost by $US40.54 per dog and it can save the total anesthesia cost by $US35.17 per dog. From the clinical perspective, the number of dogs receiving TPLO surgery at the study hospital is approximately 160 cases per year. This number was used for estimating cost benefit per year performing nerve blocks for TPLO surgery and found that the nerve blocks would potentially increase the total cost for 160 dogs to US$6,486.40 per year but would decrease the total anesthesia cost by US$5,627.20 per year. Ultrasound-guided lumbar plexus and sciatic nerve regional analgesia was found to be an effective multimodal analgesia for TPLO surgery. The technique provided effective intraoperative analgesia that decreased rescue analgesia during surgery and led to better recovery from anesthesia for the dogs in this study. The ultrasound-guided analgesia technique would increase anesthesia costs but better analgesia, anesthesia, and decreased complications provided significant cost-saving benefits when performing regional analgesia for TPLO surgery.
Embargo
Culture-expanded articular chondrocytes: a potential cellular therapeutic for osteoarthritis with MSC-like properties
(Colorado State University. Libraries, 2022) Liebig, Bethany Ellen, author; Goodrich, Laurie, advisor; Kisiday, John, advisor; Regan, Daniel, committee member; Santangelo, Kelly, committee member; McGilvray, Kirk, committee member; Bahney, Chelsea, committee member
Osteoarthritis (OA) is a highly prevalent and debilitating joint disease in horses, dogs, and humans. OA affects more than 303 million people globally with an annual economic loss to Americans approaching $200 billion. It has a considerable impact on the patient, resulting in pain and disability and more than 1 million people undergo knee arthroscopy or joint replacement surgery each year due to end-stage OA in the United States. Therefore, OA therapies that produce lasting symptom- and disease-modifying effects are a medical priority. Mesenchymal stromal cells (MSCs) are considered 'medicinal signaling cells' that have been postulated to treat OA by reducing inflammation and restoring joint function. However, IA injection of MSCs into diseased human or companion animal joints has demonstrated only a modest benefit to date, as symptom-modifying effects are often temporary, and evidence of disease-modification has been minimal. It has been reported that culture-expanded chondrocytes (CECs) can assume many of the hallmark properties of MSCs, such as immunomodulation and immunophenotype. However, unlike MSCs, chondrocytes are known to thrive in suspension, which is important as IA injections release cells into synovial fluid. The goal of this research aims to characterize the growth, immunomodulatory properties, and gene expression of equine CECs as a function of expansion in vitro as well as CEC persistence in the joint after intra-articular injection using a validated model of OA in rats. Additional goals of this research are to 1) determine how CECs may (persistence) or may not (immunomodulation and molecular fingerprint) differ from bone marrow derived MSCs, and 2) compare cellular properties of CECs across age to determine an ideal donor for generating allogeneic therapies. The results shown in chapters 2 and 3 indicate that chondrocytes retain a strong propensity for immunomodulation, that increases with expansion and dedifferentiation does not coincide with other temporal changes in gene expression. Further, these data do not indicate a benefit of neonatal donors. Future in vitro studies should further characterize the immunomodulatory, redifferentiation (chondrogenic) and angiogenic potential of CECs. The preliminary results described in chapter 4 indicate that CECs may have greater persistence than MSCs in the first 3 days post IA injection. Future in vivo studies should focus on determining the symptom- and disease-modifying effects following IA injection of CECs in relevant preclinical models, such as the rodent, horse, and dog.
Open Access
An evaluation of estrus suppression in the mare through the use of an altrenogest delivering intravaginal device
(Colorado State University. Libraries, 2022) Lederman, Jessica Danielle Ruth, author; Hatzel, Jennifer, advisor; Easley, Jeremiah, committee member; Hollinshead, Fiona, committee member; Bouma, Jerry, committee member
The ability to avoid adverse behavior in mares through manipulation of their reproductive cyclicity has been a desired technique for many years. The mare's sour reputation when in heat, is known by competitive professionals and hobby riders alike. As the breeding season approaches during long daylight days in the northern hemisphere, the mare will begin to transition into her regular twenty-one day interovulatory cycle. The estrus or "standing heat" phase of a mare's cycle occurs for approximately 5-7 days within a given estrous cycle. During this phase, the mare is primarily under the influence of estrogen, produced by the dominant follicle, driving signs of the sexual receptivity. Outward signs of estrus for the mare include: overt interest in other horses (especially stallions), leaning their hind end toward another horse (teasing), lifting their tail, posturing and urinating frequently. Dangerous behaviors associated with estrus such as biting, kicking, irritability, and distraction commonly persuade owners to look into estrus suppression options. Following ovulation, a corpus luteum (CL) is formed and the luteal cells begin to produce progesterone. Progesterone levels increase once again, overcoming the effects of decreasing estrogen levels for twelve to fourteen days, and often alleviating the undesirable behavior. Altrenogest, an oral and injectable synthetic progestin, is the most effective supplement for providing estrus suppression in the mare. The equine industry is in need of a reliable pharmaceutical device to suppress adverse behavior commonly associated with the estrus phase of the mare's cycle, yet allow the mare to resume normal cyclicity upon removal of the device for pursuant of reproductive procedures. A custom intravaginal ring specifically designed for the unique anatomy of the mare will provide a novel and effective method for sustained release of altrenogest administration while being safer to handle and administer. The first experiment focuses on the unique anatomy of the mare's caudal reproductive tract compared to intravaginal ring sizes in order to obtain a pilot device for experimentation. An unmedicated toroidal silicone intravaginal ring measuring 14.2 cm in diameter was selected during experiment one. The second experiment was to evaluate the vaginal ring delivering altrenogest in a trial along with a placebo intravaginal ring and control group to evaluate several parameters associated with estrus behavior suppression. Both oil-based and solid suspension-based intravaginal rings for drug administration was evaluated during experiment two. Finally, the third experiment examined the in vivo evaluation of solid suspension altrenogest IVR as well as the marketability of this product and what the future holds for novel medical devices in equine reproduction. Twelve total mares were used over the course of this study to determine the pharmacokinetics (PKs) and pharmacodynamics (PDs) of intravaginally administered altrenogest. Variables such as: teasing behavior when presented with a stallion, ultrasonographic examination of reproductive changes throughout several cycles, uterine and vaginal cultures, and blood collections for drug hormone bioanalysis were collected and monitored to evaluate the PK and PD of this novel drug delivery device. Throughout experiment three, each mare went through three cycles in a crossover design. An oral form of the drug was administered to serve as a control group, a placebo vaginal ring control group, and therapeutic vaginal ring treatment group were used for this study. This study aims to provide horse owners and trainers an alternative method for delivering behavior modulating hormones, through an effective, therapeutic, steady-state release from a vaginal ring, and importantly enable normal reproductive cyclicity to resume upon removal.
Open Access
Oropharyngeal bacteria, with respect to animal health classification, and viral serology of Montana bighorn sheep (Ovis canadensis) and domestic (Ovis aries) near to and distant from the wildlife/domestic animal interface
(Colorado State University. Libraries, 2010) Miller, David Steven, author; Campbell, Terry W., advisor; Garry, Franklyn, advisor; Chapman, Phillip L., committee member; Kimberling, Cleon V., committee member; Rhyan, Jack C., committee member
Respiratory disease outbreaks attributed to pasteurellosis have lead to conflict at the wildlife/domestic interface, where domestic sheep have been hypothesized to be a reservoir of Pasteuerellaceae strains that cause disease in bighorn sheep. This dissertation compares bighorn sheep ( Ovis canadensis) and domestic sheep ( O. aries) oropharyngeal Pasteurellaceae biovariants from animals classified as diseased and healthy. It also compares bacteriology and viral serology of populations of these species near to and distant from the wildlife/domestic livestock interface. A retrospective study of clinical submissions (1990 - 2004) indicated that 94 Pasteurellaceae biovariants have been associated with domestic sheep classified as diseased. A second retrospective study (1989 - 2004) indicated that 37 Pasteurellaceae biovariants have been associated with bighorn sheep classified as diseased. A prospective study of domestic and bighorn sheep near to and distant from the wildlife/domestic interface indicated that Pasteurellaceae biovariants commonly associated with disease in the retrospective studies were also common in healthy animals, and that there was extensive interspecific sharing of biovariants. This suggests that a simple agent/disease relationship may not exist for Pasteurellaceae in these host species. In addition, it is not clear that either species serves as a reservoir for Pasteurellaceae that are pathogenic for the sympatric species. However, unstated assumptions that single samples represent an animal's Pasteurellaceae microflora are questionable, based on the minimal concordance of biovariants of individual domestic livestock (n = 118) sampled six months apart. Based on the populations in the prospective study, bighorn sheep populations were naive to Mycoplasma, and both Ovis species were largely naive to infectious bovine rhinotracheitis and bovine virus diarrhea 1 and 2. This suggests that these agents may cause outbreaks if introduced into these populations. Cluster analysis of Pasteurellaceae and viral serology results identified four different clusters (P < 0.0001), but these did not closely correspond to species and location categories. The results from this study suggest that emphasis on single determinants for causes of respiratory disease outbreaks in domestic and bighorn sheep, rather than determination of risk factors for multiple determinants, may not provide results that are useful for managing disease in these species.
Open Access
Glucose transporter-1 expression and the antiproliferative effects of 2-deoxy-d-glucose in osteosarcoma models
(Colorado State University. Libraries, 2010) Lori, Janet Carolyn, author; Lana, Susan E., advisor; Thamm, Douglas, advisor; Biller, Barbara J., committee member; Ehrhart, Eugene J., committee member
Osteosarcoma (OSA) is the most common bone tumor in the dog, more common in large to giant breed dogs. 90% of dogs diagnosed with OSA will die of metastatic disease within one year of diagnosis. There have been no great advances in therapy for canine OSA over the last 20 years. Hypoxia in tumors has been associated with an increased resistance to radiation and chemotherapy, and increased metastatic potential. Hypoxia-inducible factor 1-a (HIF-la) is a transcription factor stabilized by hypoxia. Glucose transporter 1 (GLUT-1), a downstream product of HIF-la pathway activation, is over-expressed in a variety of human tumors. We sought to determine if GLUT-1 is expressed in canine OSA and if expression is related to tumor necrosis and outcome. Immunohistochemistry was performed on 44 histologically confirmed OSA tissue samples to assess expression of GLUT-1. Of 44 cases, 27 (61%) expressed GLUT-1. There was no statistical correlation between GLUT-1 and disease-free interval, survival time, or percent necrosis. As hypothesized, GLUT-1 is present in most canine appendicular OSA. A more objective evaluation of GLUT-1 and other proteins in the HIF-la pathway may be warranted. Some cells within a tumor may be poorly perfused, and therefore less susceptible to traditional chemotherapy. Cancer cells, especially those hypoxic cells that are distant from the stromal blood vessels, require more glucose than normal cells as they utilize anaerobic glycolysis, rather than oxidative phosphorylation, to survive. 2-deoxy-Dglucose (2-DG) is a glucose analog that is preferentially captured by cancer cells, _ blocking the first step of glycolysis. We evaluated the sensitivity of various OSA (canine and murine) cell lines to 2-DG, and attempted correlation to the protein GLUT-1 with western analysis. There was no statistical correlation between 2-DG and GLUT-1 or Akt expression, although it did correlate with total ERK expression. In a murine OSA model, 2-DG was shown to inhibit metastasis, possibly through the inhibition of invasion and migration, as assessed by Boyden chamber assays in vitro using the same OSA murine cell line.