Browsing by Author "Landolt, Gabriele, committee member"
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Item Open Access Circulating micro RNA in insulin resistant horses(Colorado State University. Libraries, 2018) da Costa Santos, Hugo F., author; Hess, Tanja, advisor; Landolt, Gabriele, committee member; Bruemmer, Jason, committee member; Coleman, Stephen, committee member; Engle, Terry, committee memberInsulin resistance is a prevalent pathophysiological disorder among domestic horses and is associated with many other conditions such as abnormal adiposity, chronic low-grade systemic inflammation, equine metabolic syndrome, and pituitary pars intermedia dysfunction. However, the processes leading to equine insulin resistance and associated conditions remain a subject of study. Studies in human and laboratory animals have shown that a class of small non-coding RNAs, known as microRNAs (miRNAs), are involved in the regulation of many biological processes and are associated with many diseases. MiRNAs have emerged as potential biomarkers in studies for the diagnosis and prognosis of many diseases, including type 2 diabetes and metabolic syndrome. Profiling circulating miRNAs allows researchers to understand further the mechanisms involved in many diseases, including type 2 diabetes mellitus and metabolic syndrome in humans, and it has excellent potential for equine medicine. In our pilot study, we hypothesized that insulin resistant horses would have a different circulating miRNA profile than those that are healthy. 6 mares were selected from an initial population to represent the most insulin-sensitive (n = 3) and insulin resistant (n = 3) states. Serum samples were collected for miRNA profiling of these animals, investigating the presence and relative amount of 340 equine miRNAs. Results showed different miRNA profiles between groups, with a total of 14 miRNAs differently expressed between insulin resistant and insulin sensitive animals. Furthermore, results of this preliminary study suggested circulating miRNA profiles as potential new tools for evaluating the mechanisms of insulin resistance in horses and the development of novel diagnosis and treatment methods for this condition in equines. The current study aimed to follow up the pilot study by increasing the number of animals undergoing miRNA profiling and including both horses and ponies for testing. It was hypothesized that insulin resistant animals (horses and ponies) would have a different circulating miRNA profile than those that are healthy. Additionally, circulating miRNA profiles of horses and ponies were also compared. Two initial populations, one of horses and one of ponies, were screened for their insulin sensitivity state using basal proxies for insulin and glucose. Selected animals, 12 non-pregnant Thoroughbred/ Thoroughbred-cross and 12 non-pregnant Welsh/Dartmoor mares were evaluated for insulin sensitivity with the frequent sampling intravenous glucose tolerance test (FSIGTT), and serum samples collected for miRNA profiling. The quantification of miRNAs was done through qRT-PCR analysis performed to investigate the presence and relative amount of 340 equine miRNAs. Confirmation by quantitative real-time polymerase chain reaction revealed that miRNA was present in the serum of all animals. After diagnosis based on the FSIGTT results, mares were divided into groups representing their insulin sensitivity state: insulin sensitive (IS, n = 13, ten horses and three ponies) or insulin resistant (IR, n = 11, two horses and nine ponies) animals, and their miRNA profile compared. Results demonstrated that from the 340 miRNAs analyzed, 13 miRNAs were differentially expressed between insulin resistant and insulin sensitive horses, 15 differently expressed between insulin resistant and insulin sensitive ponies, 17 differently expressed between horses and ponies, and 14 differently expressed between insulin resistant and insulin sensitive animals, horses and ponies combined (p<0.05), with 4 of these miRNAs already noted when comparing horses versus ponies. In the horse groups, three miRNAs were expressed in the insulin resistant group only. Finally, eight circulating miRNAs are proposed as potential regulators of equine insulin resistance. The results of this study, in addition to our preliminary investigation, suggest potential new tools that could be used to understand further the mechanisms involved in equine insulin resistance and associated conditions and for the development of new, practical and efficient diagnosis and prognosis methods for this condition in horses.Item Open Access Dietary intake in a group of old mares fed a supplement containing long chain 18:3 (n-3) fatty acid and chromium(Colorado State University. Libraries, 2012) Otabachian-Smith, Silvia, author; Hess, Tanja, advisor; Carnevale, Elaine, committee member; Engle, Terry, committee member; Landolt, Gabriele, committee memberTo view the abstract, please see the full text of the document.Item Open Access Gene expression analysis before and after the pelvic flexure in the equine hindgut(Colorado State University. Libraries, 2024) Moss, Cameron D., author; Coleman, Stephen J., advisor; Engle, Terry, committee member; Metcalf, Jessica, committee member; Landolt, Gabriele, committee memberThe equine hindgut is the primary site of the horse's nutrient breakdown, absorption, and energy production. More than 60% of the horse's energy comes from hindgut fermentation. In this process, commensal microbes in the hindgut aid in the digestion of plant materials to create volatile fatty acids that can be used by host cells to make energy. Many severe health issues- such as colic, laminitis, or colonic impactions- often occur in the equine hindgut, making it an important site to study to provide better management, treatment, and prevention options for horses suffering from gastrointestinal disease. Although much research exists focusing on the microbiome and overall physiology of the equine hindgut, relatively little addresses the role of gene expression in maintaining a complex yet essential homeostatic balance within the gastrointestinal tract. Previous from our lab found major differences in the microbial content of gastrointestinal compartments of the equine hindgut, separated by the pelvic flexure. The pelvic flexure is a short, narrow, horseshoe-shaped loop in the equine large colon. It defines the ventral and dorsal segments of the colon and is a common site of colonic impaction in horses. Although the pelvic flexure cannot and should not act as a "barrier," something "barrier-like" may be occurring around this region as it pertains to the hindgut microbiome. The mechanism for this action is not defined. As a result, this thesis aims to investigate gene expression in the intestinal epithelial cells of the ventral colon, pelvic flexure, and dorsal colon regions of a healthy hindgut to determine what differences exist. The insight gained from this analysis will provide a baseline for comparison to understand how gene expression patterns in these tissues adapt to changes in the microbiome and external factors like diet. The results of this thesis are the first steps towards a better understanding of homeostasis in the equine hindgut.Item Open Access RNA interference as an alternative preventive measure for avian influenza in poultry(Colorado State University. Libraries, 2014) Linke, Lyndsey M., author; Salman, Mo, advisor; Landolt, Gabriele, committee member; Olea-Popelka, Francisco, committee member; Wilusz, Jeffrey, committee memberAvian influenza virus (AIV) is a viral pathogen that causes a wide range of disease in poultry, from subclinical to severe clinical illness and can often result in death. In 1878, AIV was first described as a disease affecting poultry. Nearly 80 years later this disease-causing agent was identified as influenza A virus and a member of the family Orthomyxoviridae. AIV was not considered a significant human pathogen until 1997, when high pathogenic AIV H5N1 emerged from the wildfowl reservoir and was directly transmitted from domestic poultry to humans. Despite a long history of outbreaks in animals, this incident propelled AIV into a globally recognized disease associated with socioeconomic and animal health consequences. Each AIV outbreak highlights ways to improve upon current control strategies and stimulates new ideas for developing novel approaches and technologies to better mitigate AIV outbreaks worldwide. AIV is a dynamic pathogen to study. Host range and adaptation, pathogenicity, pathology, molecular composition, and the epidemiology of AIV all make this virus particularly challenging to control in poultry. Vaccines against AIV are available but the protection they provide for poultry is limited, especially when administered at the onset or in the midst of an outbreak. The most efficacious vaccines must be administered subcutaneously or intramuscularly, an impediment to successfully immunizing large numbers of poultry in a short window of time. Frequently, improper storage and handling leads to vaccine failure. To elicit efficient protection the vaccine must be HA-subtype specific to the outbreak virus. Often stockpiles of vaccines become obsolete and new vaccines must be generated. This is a time consuming process and can take months to secure and additional time to disseminate and administer. In the naive animal, protective antibody production takes two to three weeks to acquire following vaccination. Even if the decision to vaccinate during an outbreak is rapid and an appropriate vaccine is available for immediate use in poultry, vaccination alone would do little to protect against the threat of infection and break the chain of transmission, especially in areas lacking appropriate biosecurity measures. These limitations convey a genuine need to develop a prophylactic that would offer universal protection against any subtype or strain of AIV and would provide rapid protection in the face of an outbreak. Using RNA interference (RNAi) methodologies, this dissertation focuses on developing an innovative antiviral prophylactic that works rapidly to protect poultry against AIV shedding and transmission. The innovation behind this prophylactic technology lies in combining RNAi with the transkingdom RNAi (tkRNAi) delivery platform. This anti-AIV technology specifically targets conserved viral gene segments using small interfering RNA (siRNA) generated and delivered to chicken mucosal respiratory tissues using the tkRNAi system. The work presented in this dissertation details the steps taken to show proof of concept for using this technology to prevent AIV replication and shedding in vitro using an avian cell model and in vivo using commercial chickens. The overarching vision for this anti-AIV technology is to provide a cost effective means to protect commercial and backyard flocks against AIV outbreaks. The long-term goal is to promote this prophylactic as a complement to vaccination with the intention of developing a more effective and robust control plan against AIV in poultry. If this technology is successful, it could be applied in the face of an outbreak to reduce the shedding and transmission of virus within poultry, between farms, and across borders, thereby improving animal health and reducing the economic impact of outbreaks worldwide. Additionally, this work could provide the framework and valuable evidence for developing a similar anti-influenza prophylactic for humans.Item Open Access The effects of corn on microRNA expression within horses(Colorado State University. Libraries, 2021) Carver, Clarissa, author; Hess, Tanja, advisor; Bruemmer, Jason E., committee member; Coleman, Stephen, committee member; Landolt, Gabriele, committee memberNutrition has been shown to play a major role in the health of horses in all life stages and levels of work. In recent years the prevalence of equine obesity has increased as more horses are kept in stalls with lower workloads, while receiving high energy and calorically dense feeds like grain, many of which contain corn, in addition to forage. The increase in equine obesity has been accompanied by more cases of metabolic diseases developing, often linked to poor nutrition and diets high in non-structural carbohydrates (NSC). Although more cases of metabolic disorders are emerging there currently are no good biomarkers to diagnose these diseases or identify horses on diets providing them with poor nutrition. Diets high in NSCs have been linked to insulin resistance and laminitis within horses, two of the main components of Equine Metabolic Syndrome (EMS), however nutrigenomic studies looking at the interaction of diets high in NSCs on gene expression, specifically through the regulation of endogenous microRNAs (miRNA) are rare. Recent research on mice and human models has demonstrated the large impact diet has on levels of miRNAs within the body and mRNA targets for these miRNAs resulting in the regulation of gene expression, in addition to identifying miRNAs in circulation that can be used as biomarkers for obesity, type 2 diabetes, and metabolic syndrome. Research has also demonstrated the ability of diet-derived exogenous miRNAs to be absorbed from the digestive tract, appear within circulation, and be taken up by various tissues throughout the body. Diet-derived miRNAs specifically from plants have been detected in tissue and circulating within the blood suggesting the possibility of cross-species gene regulation, but the exact role these miRNAs play physiologically is still unknown. miRNAs are small non-coding molecules that affect post-transcriptional gene regulation and RNA silencing by translational repression or degradation. Previous research revealed that some plant miRNAs could be identified in equine serum exosomes and tissues but was not able to identify a corn specific miRNA within any equine samples. We first hypothesized that diet-derived corn miRNAs can be detected in equine serum and muscle after corn supplementation. For this study twelve mares were blocked by weight and BCS and assigned to one of two treatments (n=6/group): 1) control, (basal diet: 20 lbs./head/d of chopped mixed alfalfa-grass hay and ad libitum mixed grass hay), 2) basal diet supplemented with 1 lb./d steam flaked corn. Muscle biopsies of the Gluteus medius and serum samples were collected from all horses on d0 and d28. Samples were analyzed using real-time RT-qPCR for 3 plant miRNAs. Our results revealed the presence of plant miRNAs in equine total serum and skeletal muscle. Our results also revealed the level of plant miRNAs, including the corn specific miRNA, within circulation vary after ingestion, suggesting plant miRNAs are capable of being taken up by equine tissues. These results are important for understanding how physiological processes may be impacted by diet-derived plant miRNAs. Moreover, these results suggest plant miRNAs could potentially serve a therapeutic role in helping to regulate endogenous gene expression in addition to the nutrients being provided by ingestion. The large impact diet can have on equine health and the association between diets high in NSC and insulin resistance, caused us to be interested in the effects a diet supplemented with corn would have on endogenous miRNAs within the horse. We hypothesized that supplementing horses with corn would alter the endogenous miRNA profiles within both serum and skeletal muscle. For this objective, we utilized the same serum and muscle samples collected for the feed trial horses as the plant miRNAs. Samples were analyzed using real-time RT-qPCR for 277 endogenous equine miRNAs. Our results showed 13 differentially expressed (P<.05) miRNAs in equine serum after 28 days of corn supplementation. Six of these miRNAs (eca-mir16, -4863p, -4865p, -126-3p, -296, and -192), were linked to obesity and/or metabolic disease. Within skeletal muscle, our results showed three miRNAs differentially expressed (P<.05) and three miRNAs with a trend toward differential expression (.05Item Open Access Viral shedding and antibody response of mallard ducks to avian influenza viruses(Colorado State University. Libraries, 2012) Muth, Jack P., author; Bowen, Richard, advisor; Landolt, Gabriele, committee member; Mason, Gary, committee member; Zabel, Mark, committee memberWild ducks are a key reservoir for avian influenza (AI) viruses. Their long distance migrations, coupled to frequent contact with domestic poultry enhances risk for spread of highly pathogenic avian influenza (HPAI) viruses. Despite years of study, our understanding of how AI viruses are maintained and transmitted in nature remains poorly understood. The work described here examines several aspects of avian influenza virus infections that play a role in perpetuation and spread of this disease, including persistence of virus in duck feces, effect of prior exposure to AI viruses on subsequent infections and the passage of maternal antibodies between hen and duckling. In recent years, the emergence of H5N1 HPAI virus stimulated establishment of massive international surveillance programs to detect that virus in wild waterfowl. One deficit in these efforts was a lack of data on the stability of AI virus and AI virus RNA in bird feces under different environmental conditions. Consequently, an experiment was designed to address this knowledge gap. Feces were collected from mallards infected with a low pathogenic avian influenza (LPAI) virus (H5N2) on days 3 and 4 post infection and kept in environmental chambers for 21 days under the following conditions: 32°C/20% relative humidity (RH), 32°C/50%RH, 32°C/90%RH, 4.5°C/50%RH, 4.5°C/90%RH, and 0°C/50%RH. Sensitivity of detection of infectious virus in fresh fecal material was equivalent to that from cloacal swab samples, while time and environmental conditions did not significantly affect detection of AI virus RNA by PCR. Infectious virus was isolated from feces for considerably shorter intervals than RNA could be detected and was isolated for longer periods of time when feces were maintained under cold conditions. High relative humidity also had a negative effect on virus isolation at 4.5°C. Use of quantitative reverse transcriptase PCR to detect AI virus in fecal samples is as a valuable tool in limiting the labor involved in surveying wild ducks for AI virus. Few prior studies have examined virus shedding over the course of short interval, sequential infections of ducks with LPAI viruses, as likely occurs in natural settings such as breeding grounds. We characterized such infections by sequential inoculation of ducks with homosubtypic versus heterosubtypic with H5N2 and H3N8 LPAI viruses. We found that prior infection with either virus reduced the duration of viral shedding during a subsequent infection initiated 14 or 28 days later. Further, shedding was significantly shorter when the secondary infection occurred 28 days following the initial infection compared to 14 days. No difference in rate of shedding for the secondary infection were noted based on the viral subtype causing the initial infection, suggesting induction of some degree of heterosubtypic immunity. As reported from previous studies, some ducks shed virus but did not develop detectable antibody titers. There was no evidence of subtype cross-reactivity by antibodies as demonstrated by hemagglutination inhibition testing. The antibody response to a heterosubtypic virus was not improved by a prior infection while a second infection with the same virus was capable of boosting the antibody response to that virus. This information should be useful in parameterizing models examining the ecology of avian influenza infection. Another factor of significance in understanding transmission of AI viruses among wild ducks is the influence of passive immunity. A third study was performed to evaluate the magnitude of passive transfer of anti-influenza virus antibodies in mallard ducks and to determine their rate of decay in ducklings. Since not all ducks develop antibodies following natural infection with AI virus and the antibody titers are typically low, a vaccine was used to induce consistent seroconversion. Four, 11 month-old mallard hens were inoculated with a recombinant H5 protein in adjuvant. Specifically, hens received a single injection of 20 μg of hemagglutinin protein derived from A/Vietnam/1203/2004 emulsified in Freund's incomplete adjuvant. Beginning two weeks post-vaccination, eggs were collected daily. Yolk was harvested from eggs laid at one-week intervals and the remainder of the eggs incubated for hatching. All hens developed detectable antibody titers with an average log 2 hemagglutination inhibition titer (HI) of 6.4. Maternal-origin antibodies were detected in the yolk of eggs laid by all hens. Antibody titers peaked in yolks three weeks post vaccination for two hens and were still rising four weeks post vaccination for the other two hens. The highest yolk HI antibody titer was 32. Serum samples from the ducklings hatched from vaccinated hens were collected between days 0 and 22 post-hatch. The calculated mean half-life of maternal antibody in ducklings was 2.3 days with a range of 1.6 to 4.0 days. The short duration of passive immunity in ducks is similar to what has been reported for other species of birds and suggests that maternal antibodies may not play a major role in modulating protection against AI virus infection in natural populations. The strong immune response elicited by the H5 protein suggested that further evaluation should be performed to determine the viability of this vaccine for ducks.