Browsing by Author "Buechner-Maxwell, Virginia A."

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    Adaptation of Three Different Apoptotic Methods in Equine Bronchoalveolar Cells and Comparison of Bronchoalveolar Lavage Cell Apoptosis in Normal and COPD Affected Horses Before and After Dexamethasone Administration
    Leichner, Teri Lynn (Virginia Tech, 2001-06-21)
    Recent studies suggest that lymphocyte apoptosis serves to regulate pulmonary inflammation. Equine COPD, an allergic disease of the lower airway, is likely due to dysregulation of the pulmonary immune response. In this study, the hypothesis tested was COPD affected horses would have less apoptotic airway lymphocytes than control horses during clinical disease. To achieve this, 3 methods of measuring apoptosis, Vindelov's propidium iodide with Triton-X (PI/Triton-X), 7-aminoactinomycin D (7-AAD), and Annexin V with propidium iodide (Annexin/PI) were evaluated in equine airway lymphocytes. A significant linear relationship was found for equine bronchoalveolar lavage (BAL) lymphocytes stained with 7-AAD and Annexin/PI . No relationship was identified with cells stained with PI/Triton-X and Annexin/PI, and 7-AAD and PI/Triton-X indicating that methods which preserve cell membrane characteristics are more comparable when measuring BAL lymphocytes apoptosis in a heterogeneous population of cells. Additionally, all stains appear to perform the same in COPD and normal horses in remission and disease. Comparison of predominately BAL lymphocyte apoptosis using the above methods were performed at baseline, after natural challenge, and after dexamethasone administration in nine horses, five of which were affected with COPD. No differences in bronchoalveolar lavage lymphocyte apoptosis between COPD and control horses were detected either before or after dexamethasone administration, although numerical trends in COPD horses identified less apoptosis after natural challenge indicating that defective apoptosis may play a role in equine COPD pathogenesis. Dexamethasone administration was associated with trends of improvement in the pulmonary gas exchange and increased apoptosis toward baseline in the COPD horses.
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    Automatic Dynamic Tracking of Horse Head Facial Features in Video Using Image Processing Techniques
    Doyle, Jason Emory (Virginia Tech, 2019-02-11)
    The wellbeing of horses is very important to their care takers, trainers, veterinarians, and owners. This thesis describes the development of a non-invasive image processing technique that allows for automatic detection and tracking of horse head and ear motion, respectively, in videos or camera feed, both of which may provide indications of horse pain, stress, or well-being. The algorithm developed here can automatically detect and track head motion and ear motion, respectively, in videos of a standing horse. Results demonstrating the technique for nine different horses are presented, where the data from the algorithm is utilized to plot absolute motion vs. time, velocity vs. time, and acceleration vs. time for the head and ear motion, respectively, of a variety of horses and ponies. Two-dimensional plotting of x and y motion over time is also presented. Additionally, results of pilot work in eye detection in light colored horses is also presented. Detection of pain in horses is particularly difficult because they are prey animals and have mechanisms to disguise their pain, and these instincts may be particularly strong in the presence of an unknown human, such as a veterinarian. Current state-of-the art for detecting pain in horses primarily involves invasive methods, such as heart rate monitors around the body, drawing blood for cortisol levels, and pressing on painful areas to elicit a response, although some work has been done for humans to sort and score photographs subjectively in terms of a "horse grimace scale." The algorithms developed in this thesis are the first that the author is aware for exploiting proven image processing approaches from other applications for development of an automatic tool for detection and tracking of horse facial indicators. The algorithms were done in common open source programs Python and OpenCV, and standard image processing approaches including Canny Edge detection Hue, Saturation, Value color filtering, and contour tracking were utilized in algorithm development. The work in this thesis provides the foundational development of a non -invasive and automatic detection and tracking program for horse head and ear motion, including demonstration of the viability of this approach using videos of standing horses. This approach lays the groundwork for robust tool development for monitoring horses non-invasively and without the required presence of humans in such applications as post-operative monitoring, foaling, evaluation of performance horses in competition and/or training, as well as for providing data for research on animal welfare, among other scenarios.
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    Biomarkers of oxidative stress and inflammation in biological samples collected from recurrent airway obstruction (RAO)-affected horses and their controls
    Tan, Rachel Hsing Hsing (Virginia Tech, 2008-03-12)
    Multiple biomarkers of oxidative stress have been measured and used in human medicine to diagnose and monitor airway disease. The purpose of the study was to determine if similar relationships existed between inflammatory and oxidative stress biomarkers in exhaled breath condensate (EBC), bronchoalveolar lavage fluid (BALF), red blood cells, white blood cells, and plasma; and cytokine expression in airway inflammatory cells and mucosal biopsies of RAO-affected horses and their controls. Sixteen horses in pairs were used: 8 non-RAO-affected (controls) and 8 RAO-affected horses. Samples from all horses were collected at remission (S1), during environmental challenge (S2) and at recovery (S3). RAO-affected horses had significant alterations in cellular glutathione peroxidase (cGPx) activity, ascorbic acid and pH in a number of biological samples. Concentrations of 8-isoprostanes, isofurans, amino acids and mRNA expression of interleukin 4 (IL4), gamma interferon (INFγ), inducible nitric oxide synthase (iNOS), extracellular glutathione peroxidase (GPx-3), and cytosolic superoxide dismutase (SOD-1) were not significantly different or were at the limits of detection. Conductivity was measured and assessed as a potential correctional factor for respiratory fluid dilution. The alterations in biomarker concentrations demonstrate that oxidative stress is an important component of airway inflammation in RAO-affected horses. Further research is warranted in the use of biomarkers and the effects of dietary interventions.
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    Can levamisole upregulate the equine cell mediated immune response in vitro?
    Santonastaso, Amy Marie (Virginia Tech, 2016-07-19)
    Equine Protozoal Myeloencephalitis (EPM) is arguably the most common and costly equine neurologic diseases nationwide. The national seroprevalence is >50%, but only 0.5-1% of all horses develops disease during their lifetimes. Some EPM affected horses have decreased immune response. A cell-mediated immune response has been shown to be protective for development of EPM after infection with Sarcocystis neurona in mouse models. Levamisole has been proposed as an adjunctive therapy for EPM to upregulate the cell-mediated immune response based on positive results in other species, but there are very limited studies in equids. We hypothesized that levamisole will upregulate the equine cell-mediated macrophage (M1) dendritic cell (DC1) CD4 T-helper 1 (Th1) CD8 Tc1 immune response in vitro. The first aim was to determine optimal conditions and effects of levamisole on cellular proliferation. Equine PBMCs were harvested from ten horses seronegative for S. neurona. The cells were cultured alone, or with one of the mitogens: concanavalin A (ConA) or phorbol 12-myristate 13-acetate and ionomycin (PMA/I), or with a combination of the above mitogens and levamisole at several conditions. Cellular proliferation was assessed using a colorimetric bromodeoxyuridine ELISA assay. The second aim was to determine the ability of levamisole, under optimized conditions, to upregulate the M1 DC1 CD4Th1 CD8 Tc1 response in vitro based on activation and function. PBMCs from the same 10 horses were cultured with each of the following: no stimulation, conA, and levamisole with and without ConA. To determine proliferation of each specific subset, cells were labeled with a fluorescent dye, CellTrace. Proliferation was determined based on dye dilution using flow cytometry. To determine the effects of levamisole on the specific immune response, cell subsets were labeled with fluorescent antibodies for cell surface markers (CD4, CD8, CD21, CD172a, CD14) and dendritic and macrophage activations markers (MHC Class II, CD86). Induction of T-regs was based on FoxP3 expression. Immune phenotypes were determined based on intracellular cytokine expression (IFNɣ, IL4, IL10). Study results indicate that levamisole alone did not significantly alter PBMC proliferation compared to the response of unstimulated cells. Cells cultured with either ConA or PMA/I resulted in a statistically significant increase (P<0.05) in proliferation compared to unstimulated cells. Cells cultured with ConA and levamisole at 1µg/mL resulted in a significant decrease (P<0.05) in proliferation compared with cells cultured with ConA alone. Flow cytometry data failed to elucidate the specific immune phenotype that is affected by levamisole. Subjectively, there appeared to be a trend for inceased IFNɣ production by CD14 and CD172a positive cells (macrophages and dendritic cells) and a decrease in IFNɣ production by CD4 and CD8 positive cells (T-lymphocytes). These results demonstrate that levamisole downregulates ConA stimulated PBMC proliferation. Based on these in vitro results, further studies to determine the effectiveness of levamisole on modulating the equine immune system in vivo and to more specifically evaluate the immune cell subets affected by levamisole are warranted.
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    Can levamisole upregulate the equine cell-mediated macrophage (M1) dendritic cell (DC1) T-helper 1 (CD4 Th1) T-cytotoxic (CD8) immune response in vitro?
    Witonsky, Sharon G.; Buechner-Maxwell, Virginia A.; Santonastasto, Amy; Pleasant, R. Scott; Werre, Stephen R.; Wagner, Bettina; Ellison, Siobhan; Lindsay, David S. (Wiley, 2019-03-01)
    Background: Equine protozoal myeloencephalitis (EPM) is a common and devastating neurologic disease of horses in the United States. Because some EPM-affected horses have decreased immune responses, immunomodulators such as levamisole have been proposed as supplemental treatments. However, little is known about levamisole's effects or its mechanism of action in horses. Objective: Levamisole in combination with another mitogen will stimulate a macrophage 1 (M1), dendritic cell 1 (DC1), T-helper 1 (CD4 Th1), and T-cytotoxic (CD8) immune response in equine peripheral blood mononuclear cells (PBMCs) in vitro as compared to mitogen alone. Animals: Ten neurologically normal adult horses serologically negative for Sarcocystis neurona. Methods: Prospective study. Optimal conditions for levamisole were determined based on cellular proliferation. Peripheral blood mononuclear cells were then cultured using optimal conditions of mitogen and levamisole to identify the immune phenotype, based on subset-specific activation markers, intracellular cytokine production, and cytokine concentrations in cell supernatants. Subset-specific proliferation was determined using a vital stain. Results: Concanavalin A (conA) with levamisole, but not levamisole alone, resulted in a significant decrease (P <.05) in PBMC proliferation compared to conA alone. Levamisole alone did not elicit a specific immune phenotype different than that induced by conA. Conclusion and Clinical Importance: Levamisole co-cultured with conA significantly attenuated the PBMC proliferative response as compared with conA. If the mechanisms by which levamisole modulates the immune phenotype can be further defined, levamisole may have potential use in the treatment of inflammatory diseases.
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    Characterization of Apoptotic Cells in Equine Proximal Suspensory Desmitis
    Hewes, Christina Andrea (Virginia Tech, 2006-05-30)
    Suspensory desmitis is a common problem and affects a broad cross section of equine athletes in various disciplines. For this study, the proximal portion of the suspensory ligament was collected from 6 horses without suspensory ligament injury (16 ligaments) and 4 horses with degeneration of the suspensory ligament (11 ligaments). Specimens were collected immediately after euthanasia and placed in neutral-buffered 10% formalin. The tissue was fixed, sectioned, and stained with hematoxylin and eosin (H&E), Masson's trichrome, and for apoptosis by the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technique. Histological changes in the abnormal ligaments included mineralization, fibroplasias, neovascularization, collagen degeneration, and significant architecture disruption in 2 ligaments. There was a trend for increased apoptosis in the injured ligaments compared to the normal ligaments.
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    Characterization of basal and lipopolysaccharide-induced microRNA expression in equine peripheral blood mononuclear cells using Next-Generation Sequencing
    Parkinson, Nicholas J.; Buechner-Maxwell, Virginia A.; Witonsky, Sharon G.; Pleasant, R. Scott; Werre, Stephen R.; Ahmed, Sattar Ansar (PLOS, 2017-05-26)
    The innate immune response to lipopolysaccharide contributes substantially to the morbidity and mortality of gram-negative sepsis. Horses and humans share an exquisite sensitivity to lipopolysaccharide and thus the horse may provide valuable comparative insights into this aspect of the inflammatory response. MicroRNAs, small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in toll-like receptor signaling regulation but have not been studied in this context in horses. The central hypothesis of this study was that lipopolysaccharide induces differential microRNA expression in equine peripheral blood mononuclear cells in a manner comparable to humans. Illumina Next Generation Sequencing was used to characterize the basal microRNA transcriptome in isolated peripheral blood mononuclear cells from healthy adult horses, and to evaluate LPS-induced changes in microRNA expression in cells cultured for up to four hours. Selected expression changes were validated using quantitative reverse-transcriptase PCR. Only miR-155 was significantly upregulated by LPS, changing in parallel with supernatant tumor necrosis factor-α concentration. Eight additional microRNAs, including miR-146a and miR-146b, showed significant expression change with time in culture without a clear LPS effect. Target predictions indicated a number of potential immunity-associated targets for miR-155 in the horse, including SOCS1, TAB2 and elements of the PI3K signaling pathway, suggesting that it is likely to influence the acute inflammatory response to LPS. Gene alignment showed extensive conservation of the miR-155 precursor gene and associated promoter regions between horses and humans. The basal and LPS-stimulated microRNA expression pattern characterized here were similar to those described in human leukocytes. As well as providing a resource for further research into the roles of microRNAs in immune responses in horses, this will facilitate inter-species comparative study of the role of microRNAs in the inflammatory cascade during endotoxemia and sepsis.
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    Comparative efficacy of three common treatments for equine recurrent airway obstruction
    Lee, Laura Caryn (Virginia Tech, 2009-04-17)
    Objective - evaluate horses with acute airway obstruction using three treatment regimens: tapering doses of dexamethasone (DEX), environmental modification (ENV), and a combination of both treatments (DEX + ENV) by analyzing clinical parameters, pulmonary function testing, bronchoalveolar lavage fluid (BALF) cytology and BALF cell expression of the cytokines IFN-? and IL-4 Animals - 6 horses with recurrent airway obstruction (RAO) Procedures - Clinical examination, pulmonary function test, and collection of BALF prior to treatment and during 22 day treatment period Hypothesis - Alterations in clinical parameters, pulmonary function and airway inflammation in acute equine RAO will return to remission values by treating with DEX, ENV or DEX + ENV Results - All horses demonstrated clinical disease, reduced pulmonary dynamic compliance (Cdyn) and an increased maximum change in pleural pressures (?Pplmax) when in a challenge environment. All treatments improved clinical parameters, ?Pplmax and Cdyn. BALF cytology during an RAO crisis demonstrated neutrophilic inflammation. ENV or DEX + ENV resulted in a significant decrease in airway neutrophilia that was maintained throughout the treatment period. In contrast, treatment with DEX caused a reduction in airway neutrophilia initially followed by a rebound neutrophilia as the period between administrations of dexamethasone (0.05mg/kg) was increased to 72 hours. The rebound neutrophilia was not accompanied by equivalent deterioration in clinical parameters or pulmonary function. Conclusions - Environmental modification is important in the management of RAO horses. Treatment of clinical RAO with a decreasing dosage protocol of corticosteroids in the absence of environmental modification results in the persistence of airway inflammation without recrudescence of clinical disease.
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    Comparison of Airway Response in Recurrent Airway Obstruction-Affected Horses Fed Steamed Versus Non-steamed Hay
    Blumerich, Celeste Ann (Virginia Tech, 2012-06-21)
    Recurrent Airway Obstruction (RAO)-affected horses experience bronchoconstriction and airway inflammation in response to inhalation of irritants including hay molds. Steaming hay reduces fungal content, but the effect on the antigenic potential has not been investigated. We tested the hypothesis that RAO-affected horses develop less severe clinical disease when fed steamed versus non-steamed hay and this reduction coincides with decreased hay fungal content. Six RAO-affected horses in clinical remission were divided in two groups and fed steamed or non-steamed hay for 10 days using a two-way cross-over design. Hay was steamed using a commercial hay-steamer. Clinical assessment was performed daily. Full assessment, including airway endoscopy, tracheal mucous scores and maximal change in pleural pressure, was performed on days 1, 5, and 10. Bronchial fluid sampling and cytology were performed on days 1 and 10. Hay core samples were collected pre- and post-steaming and cultured to determine fungal and bacterial concentrations. Statistical analysis was based on data distribution and quantity and performed using SAS®. P-value <0.05 was significant. Steaming significantly decreased the number of bacterial and fungal colony-forming-units in hay. Horses fed non-steamed hay experienced a significant increase in clinical score and a trend towards airway neutrophilia, while parameters were unchanged in horses fed steamed hay. Only horses fed non-steamed hay experienced a significant increase in tracheal mucous score. Horses fed steamed hay gained significantly more weight compared to horses fed non-steamed hay, even though the amount of hay consumed not greater on a dry matter basis. These results indicate that steaming reduces the RAO-affected horse's response to hay which coincides with a reduction in viable fungal content of hay.
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    The Comparison of Airway Responses of Normal Horses Fed Round Bale versus Square Bale Hay
    Larson, Jennifer Lynn (Virginia Tech, 2012-06-13)
    Background – Feeding horses round bale hay (RBH) has been associated with airway inflammation. The purpose of this study was to determine if horses fed RBH for a 6-week period demonstrated more evidence of airway inflammation than horses fed square bale hay (SBH) of comparable quality. Hypothesis - The respiratory health of horses fed RBH will not differ from horses fed SBH of comparable quality. Animals – Two feeding groups of 15 healthy horses (mixed ages, breeds) from the University riding program. Methods – This was a prospective study performed during fall of 2009. At the beginning and end of a 6- week feeding trial, horses were examined (physical, upper airway endoscopic) and samples (tracheal aspirate (TA), bronchoalveolar lavage (BAL)) collected for cytology and/or bacterial/fungal culture. Hay was analyzed for nutritional value and bacterial/fungal content. Results – Horses fed RBH demonstrated an increase in pharyngeal lymphoid hyperplasia (p=0.0143) and percentage neutrophils (p=0.0078) in the TA samples post-feeding as compared to pre-feeding values. Nutritional analysis of hay and measurements of bacterial/fungal load did not differ over time and/or between hay types. Conclusions and clinical importance – The identification of airway inflammation in the horses fed RBH indicates that factors associated with the manner in which the hay is fed and consumed contribute to the development of subclinical airway inflammation. RBH affords horses continuous daily exposure to hay and as horses bury their muzzles in the bale, exposure to particulate matter is likely increased. These factors may partially explain the response in horses fed RBH. Further studies are required to confirm these predictions.
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    Comparison of Two Aerosolized Bronchodilators in the Treatment of Severe Equine Chronic Obstructive Pulmonary Disease
    Friday, Philippa Anne (Virginia Tech, 1999-08-04)
    Aerosolized bronchodilator drugs are commonly used for treatment of horses with chronic obstructive pulmonary disease. Relative efficacy of sympathomimetic and parasympatholytic bronchodilators for relief of acute airway obstruction and improvement of pulmonary gas exchange was compared in 6 horses with COPD. Physical examination, arterial and venous blood gas analysis and measurement of end-tidal CO₂ tension were performed at time zero, 30 minutes, 1, 2, 3 and 4 hours after administration of aerosol ipratropium (0.35 μg/kg), albuterol (1 μg/kg) or placebo via an equine Aeromask and metered dose inhaler. Physiologic shunt fraction (QS/QT), alveolar dead space fraction (VD/VT) and alveolar to arterial oxygen tension difference (p(A-a)O₂) were calculated using standard formulas. At time zero, horses demonstrated severe respiratory compromise and marked alterations in pulmonary gas exchange, indicative of alveolar hypoventilation, VA/Q mismatching and diffusion impairment. Ipratropium treatment significantly (p< 0.05) reduced arterial CO₂ tension and end-tidal CO₂ tension toward normal, but significantly increased p(A-a)O₂ from baseline. The change in paCO₂ after ipratropium treatment was significantly different from albuterol and placebo treatment groups. There were no significant changes in response variables after albuterol and there were no treatment by time interactions. These results indicate that, under the conditions of this study, ipratropium (0.35 μg/kg) improved alveolar ventilation and had superior bronchodilator efficacy than albuterol (1 μg/kg) in horses with severe COPD. Marked impairment of pulmonary gas exchange persisted after bronchodilators, emphasizing that anti-inflammatory therapy and environmental control are also necessary for effective treatment of severe equine COPD.
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    Designing outdoor spaces to support older adult dog walkers: A multi-method approach to identify and prioritize features in the built environment
    Shealy, Elizabeth Carlisle (Virginia Tech, 2021-04-02)
    Associations between the built environment and walking are well understood among the general population, but far less is known about how features of the built environment influence walking in older adults. As compared to other age groups, older adults, defined as those 65 years of age and older, are more likely to experience declines in physical activity, social interaction, and loss of community connectivity. Animal companionship can provide older adults the motivation to stay physically active and help them mitigate the feelings of isolation. Built environments that align with the needs and abilities of older adults and their animal companions, like dogs, can encourage and help sustain walking habits. The aim of this study was to identify and prioritize features within the built environment pertinent to older adult dog walkers. Existing literature served as the basis for identifying neighborhood design features associated with general walking and dog walking. Through the use of a three round Delphi study, 25 experts from urban planning and design, management of outdoor spaces, public health, gerontology, and human-animal relationships modified and rated the importance of the identified features as it pertains to older adult dog walkers. Following the Delphi study, 12 older adult dog owners from the Warm Hearth Village participated in a guided walk and interview using the Photovoice technique. The goal was to gather their perceptions of the outdoor walking environment. Among expert panelists, safety from motorized traffic, crime, unleashed dogs, and personal injury was paramount (mean (M)= 93.20, standard deviation (SD) = 11.54). Experts also saw the value and agreed upon the importance of dog supportive features within the built environment, like dog waste stations dog waste stations (desirable; M = 87.95, SD = 11.37), and dog policy signage (desirable; M = 79.91, SD = 11.22). Older adults also believed safety was important. They saw their dog as a protective safety factor against walking deterrents like aggressive or unleashed dogs. However, the feature that resonated most with older adult dog walkers in this study was their interaction with nature. They described the pleasure of observing seasons change and the connection with nature that came from the tree canopy cocooning the walking path. Path design is also a necessary consideration. Older adults emphasized the importance of having options between paved and unpaved walking paths. The panelists stressed the need for creating lines of sight (desirable; M = 66.46, SD = 20.71) and lighting (desirable; M = 77.92, SD =19.77). Those who plan, develop, and maintain spaces that support older adults can prioritize the features I identified in my research. Incorporating these features into the design of spaces for older adults has the potential to translate into increased walking and opportunities to socialize, contributing to mental and physical health.
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    Detection of apoptotic cells in horses with and without gastrointestinal disease
    Rowe, Emma L. (Virginia Tech, 2003-04-25)
    A study was performed to identify apoptotic cells in the equine intestine and to determine if the occurrence of apoptosis is affected by gastrointestinal disease and tissue layer of intestine. Samples of intestine were collected from 38 horses that underwent surgery or were humanely destroyed for small or large bowel obstruction, strangulation or distension. Samples were also taken from 9 horses which were humanely euthanized for reasons other than gastrointestinal disease or systemic disease. Specimens were collected at surgery from intestine involved in the primary lesion, distant to the primary lesion, or at necropsy from several sites including the primary lesion. Tissues were fixed, serially sectioned and stained with hematoxylin and eosin (H&E) and for apoptosis by the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technique. The number of apoptotic cells per high power field were counted in the mucosa, circular muscle, longitudinal muscle and serosa for each sample of intestine. Apoptotic staining nuclei were seen in all layers of intestine. An increased number of apoptotic cells were found in the circular muscle of the intestine from horses with simple obstruction. Intestine distant from the primary strangulating lesion had higher numbers of apoptotic cells than intestine distant from a simple obstruction lesion or intestine taken at the site of a strangulating or simple obstructive lesion. Intestine from horses with obstructing or strangulating lesions in the small intestine and large colon has increased numbers of apoptotic cells. Further investigation is required to determine whether increased apoptosis affects intestinal function.
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    Development and Resolution of Pulmonary Arterial Hypertension in RAO Horses
    Martin, Robert Brian (Virginia Tech, 2003-08-12)
    Equine recurrent airway obstruction (RAO) is associated with airway inflammation and bronchoconstriction in clinically affected horses. Horses demonstrating severe pulmonary compromise develop concurrent secondary pulmonary hypertension. The development of pulmonary hypertension is well documented in RAO affected horses, however, it is not known how rapidly increases in pulmonary artery pressure occur after the onset of RAO. It is also not known if pulmonary hypertension resolves concurrently with resolution of RAO. The goal of this study was to measure pulmonary artery pressure in RAO affected horses during the development and resolution of RAO. To accomplish this, three RAO affected and three normal horses were placed in a challenge environment where clinical parameters, pulmonary function, right heart and pulmonary artery pressures were measured on day 1, 3 and 5. After evaluating horses on day five, their environment was modified to reduce exposure to respirable debris and anti-inflammatory medication (dexamethasone) was initiated. Identical clinical parameters were measured on days 7 and 9. In our study, the arterial oxygen content in RAO horses was significantly less that that of control horses from day 1 through day 9. A concurrent increase in pulmonary artery pressure also developed on day 3 in RAO affected horses, and persisted through day 5. While some trend towards a difference between groups was noted, no other significant differences were observed between RAO and normal horses. These findings suggest that horses with severe RAO also develop significant increase in pulmonary artery pressure, which rapidly resolves with appropriate management of RAO.
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    Discovery of Novel Strains of Animal Hepatitis E Viruses in the United States: Antigenic and Genetic Characterization, Cross-Species Infection, and Public Health Implications
    Cossaboom, Caitlin Marie (Virginia Tech, 2015-03-17)
    Hepatitis E virus (HEV) is an important human pathogen, with pigs and likely other animal species serving as natural reservoirs. There are currently four recognized HEV genotypes that infect humans within the genus Hepevirus of the family Hepeviridae. Genotypes 1 and 2 are human viruses that are associated with waterborne and fecal-oral transmission in developing countries, while genotypes 3 and 4 have been identified in humans and other animal species and are zoonotic and endemic in both industrialized and developing countries. In my dissertation research, we identified the first strain of HEV from rabbits in the United States. We subsequently determined the complete genome sequence of the virus. Phylogenetic analyses of the full-length sequence indicated that U.S. rabbit HEV is a distant member of the zoonotic genotype 3, thus raising a potential concern for zoonotic infection. In order to investigate the cross-species potential of rabbit HEV, we then determined its antigenic cross-reactivity with other animal strains of HEV. Additionally, we demonstrated that the novel rabbit HEV could cross species barriers and infect pigs under experimental conditions. Finally, we attempted to determine the risk factors and sources of foodborne HEV infection in the United States. We detected HEV for the first time from non-liver pork commercial products in the United States and demonstrated consumption of undercooked meat a risk factor for HEV infection. HEV sequences of genotype 3 origin were detected from pork products purchased from grocery stores in Southwest Virginia. Approximately 6.3% (21/335) of university students tested seropositive for HEV antibodies and, importantly, those with a history of consuming undercooked meats were 13 times more likely to be seropositive. These results further underscore the importance of cooking pork thoroughly and using proper hygiene when preparing meals.
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    The effect of dog-human interaction on cortisol and behavior in registered animal-assisted activity dogs
    Ng, Zenithson Ying (Virginia Tech, 2013-06-06)
    Background: The effect of animal-assisted activities (AAA) on the animal participants has been minimally investigated and the welfare of these animals has been questioned. Cortisol, in conjunction with stress-associated behavior, has been utilized as an objective assessment of animal welfare. Objective: Salivary cortisol and behavior in AAA dogs were measured to test the null hypothesis that salivary cortisol concentration and behavior are not different in an AAA environment compared to home or neutral environments.  Hair cortisol was measured in AAA dogs to test the null hypothesis that there is no relationship between hair cortisol and salivary cortisol. Methods:  Fifteen healthy adult dogs registered with an AAA organization were recruited.  A hair sample was collected from each dog upon enrollment.  Saliva samples were collected from each dog every 30 minutes, starting 30 minutes prior to and 30 minutes after a standardized 60 minute session across 3 settings: an AAA session (AS) for college students in the communal area of a residence hall; a neutral session (NS) located in a novel room without interaction with a stranger; and a home session (HS).  Each session was videotaped continuously and behaviors were coded at three separate 5-minute intervals while the dog was petted by a stranger in the AS or handler in the NS and HS. Results:  Salivary cortisol levels were not different in the AS compared to HS, but were significantly higher in the NS compared to AS and HS.  Dogs exhibited significantly more standing and ambulating behavior in the AS compared to HS.  Salivary cortisol level was negatively correlated with panting and standing at specific time points in the NS and AS, respectively.  Hair cortisol level did not correlate with salivary cortisol level at any time point in any of the settings. Conclusions:  During a 60 minute AAA session, salivary cortisol concentration and stress-associated behavior were not different compared to when dogs spent the same amount of time in the home setting, suggesting that they were not stressed when being used as AAA animals. The physical environment may be an important consideration when evaluating the effect of AAA on dogs.  Hair cortisol did not correlate with salivary cortisol, suggesting that hair may not be a representative predictor of cortisol in these environments.  Additional investigation is required to support cortisol and behavior as measures of stress and welfare in AAA animals.
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    Effects of Diethylstilbestrol on Murine Early Embryonic Stem Cell Differentiation Using an Embryoid Body Culture System
    Ladd, Sabine Margaret (Virginia Tech, 2005-04-14)
    Objectives: The effects of estrogens on immune system formation and function are well documented. Diethylstilbestrol (DES), a synthetic estrogen, has been linked to neoplasia and immune cell dysfunction in humans and animals exposed in-utero. In-vitro effects of DES exposure of murine embryonic stem (ES) cells on the early embryonic immune system development and the expression of cellular surface markers associated with common hemangioblastic and hematopoietic precursors of the endothelial, lymphoid & myeloid lineages were investigated. Hypothesis: Early ES cell expression of CD45 a marker common to lymphoid lineage hematopoietic stem cells and differentiation of lymphoid lineage precursors are affected by in-vitro exposure to DES. Methods: Murine ES cells were cultured using a variety of techniques: an OP9 co-culture system, and formation of embryoid bodies (EBs) in a liquid medium and hanging drop system. The OP9 co-culture system did not appear to give rise to well differentiated lymphoid lineage cells during 12 days of differentiation. The hanging drop EB culture system, previously shown to promote differentiation of endothelial and lymphoid precursor cells, was chosen for further studies of ES cell differentiation. ES cells were harvested at five time points: undifferentiated (day 0), and differentiated (days 3, 8, 12 and 16). Differentiating ES cells were treated with DES beginning on day 3. The synthetic estrogen, DES, was chosen as a treatment because of its similar potency to 17β estradiol and documented association with neoplasia in women exposed in-utero. Surface marker expression, measured by real-time RT-PCR amplification, was recorded using fluorogenic TaqMan(R) probes designed specifically for the surface proteins of interest: oct4, c-Kit, Flk1, ERα, ERβ, CD45, Flt1, & VE-cadherin. Analysis & Results: Changes in surface marker gene expression between day 0 and day 16 of differentiation were analyzed using the RT-PCR threshold counts (CT) and the comparative threshhold cycle method. The expression of each target mRNA was normalized internally to a housekeeping gene (18s rRNA) and calculated relative to day 0. ANOVA (Type 3 fixed-effects analysis, SAS) was performed using the unexponentiated ΔΔCT values. The effects of DES, time, and the interaction between DES and time were evaluated for days 8, 12 and 16. Additionally, the effects of DES on the expression of each marker were evaluated for day 16. Expression of estrogen receptor receptor α & β (ERα & β) in the EBs was established, and did not appear to be affected at any time by treatment with DES. ERα was expressed in significant levels on day 16, while ERβ was expressed in low levels throughout the period of differentiation. The expression of the cell surface marker, c-Kit was significantly (P<0.0001) altered by the presence of DES between the three time points sampled. The expression of the VEGF receptor, Flt1, and the adhesion molecule, VE-cadherin, markers of endothelial cells, were also significantly (P<0.026) altered by treatment with DES on day 16 of differentiation. Treatment with DES appeared to have no effect on the expression of CD45, a marker common to lymphoid precursor cells. Conclusions: These results indicate the presence of estrogen receptors in differentiating ES cells as early as day three in-vitro (ERβ) until day 16 (ERα). DES alters expression of common hemangioblastic and hematopoietic precursor, as well as endothelial lineage markers, but has no effect on expression of the marker of lymphoid lineage development before day 16. These effects coincided with the expression of ERα. The enduring effects of DES exposure in-utero may not be manifest in this ES model, or may occur at later stages of differentiation or in selected subpopulations of CD45+ cells.
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    Effects of Experimental Sarcocystis neurona-Induced Infection on Immunity in an Equine Model.
    Lewis, S. Rochelle; Ellison, Siobhan; Dascanio, John J.; Lindsay, David S.; Gogal, Robert M.; Werre, Stephen R.; Surendran, Naveen; Breen, Meghan E.; Heid, Bettina; Andrews, Frank M.; Buechner-Maxwell, Virginia A.; Witonsky, Sharon G. (2014)
    Sarcocystis neurona is the most common cause of Equine Protozoal Myeloencephalitis (EPM), affecting 0.5-1% horses in the United States during their lifetimes. The objective of this study was to evaluate the equine immune responses in an experimentally induced Sarcocystis neurona infection model. Neurologic parameters were recorded prior to and throughout the 70-day study by blinded investigators. Recombinant SnSAG1 ELISA for serum and CSF were used to confirm and track disease progression. All experimentally infected horses displayed neurologic signs after infection. Neutrophils, monocytes, and lymphocytes from infected horses displayed significantly delayed apoptosis at some time points. Cell proliferation was significantly increased in S. neurona-infected horses when stimulated nonspecifically with PMA/I but significantly decreased when stimulated with S. neurona compared to controls. Collectively, our results suggest that horses experimentally infected with S. neurona manifest impaired antigen specific response to S. neurona, which could be a function of altered antigen presentation, lack of antigen recognition, or both.
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    Endotoxin-induced microRNA expression in equine peripheral blood mononuclear cells
    Parkinson, Nicholas J. (Virginia Tech, 2016-07-22)
    The innate immune response to lipopolysaccharide (LPS) mediated by toll-like receptor 4 (TLR4) contributes substantially to the morbidity of equine gastrointestinal disease, neonatal sepsis and other diseases. MicroRNAs (miRNAs), small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in TLR4 signaling regulation in other species. The central hypothesis of this study was that LPS induces differential expression of miRNAs in equine peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from healthy adult horses and cultured with LPS or medium only for 2, 4 and 8 hours. Concentrations of inflammatory cytokines were measured in supernatants by immunoassay. Illumina Next-Generation Sequencing of the miRNA transcriptome was performed in PBMCs at 0, 2 and 4 hours. Selected expression changes were verified by qRT-PCR. 327 mature miRNAs were detected in equine PBMCs. Only miR-155 was significantly upregulated by LPS. 9 miRNAs showed statistically significant expression changes with time. Tumor necrosis factor-α concentration was significantly higher in supernatants from LPS-treated cells than controls from 2 hours, while interleukin-10 and interferon-γ were increased at 8 hours. miR-155 expression was correlated to all three cytokines. These data provide a foundation for future research into miRNA involvement in equine inflammatory responses. miR-155 is the principal LPS-induced miRNA in horses. Bioinformatic target predictions support roles in regulation of innate and adaptive immune responses including TLR4 signaling, as in humans. It is thus likely to influence the acute inflammatory response to LPS. Further research will be necessary to establish its role in naturally occurring disease.
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    Equine Herpesvirus Type 1: Filling Gaps Toward Improved Outbreak Management
    Saklou, Nadia Talal (Virginia Tech, 2023-09-06)
    Equine herpesvirus type 1 (EHV-1) is a common pathogen of horses that typically causes upper respiratory disease, however is also associated with late-term abortion, neonatal foal death and neurologic disease. Once a horse is infected, the virus concentrates to local lymphoid tissue, where it becomes latent. The virus can recrudesce during times of stress, which can lead to the initiation of devastating outbreaks. Some variants of EHV-1 have been associated with more severe disease outcomes. Appropriate outbreak management focuses on minimizing the movement of potentially exposed horses. This approach lacks a strategy for prevention at the level of latency largely due to a knowledge paucity in regards to carriage rate of latent EHV-1. Biosecurity decisions are also dependent on awaiting currently-available diagnostic testing that often take several days for results. Thus, our work has been focused on understanding the carriage rate of the latent virus in different geographic regions as well as improving diagnostic efficiency, both of which are essential for improving the management of EHV-1 disease. Loop mediated isothermal amplification (LAMP) is a method that amplifies nucleic acid rapidly at a constant temperature and is minimally affected by inhibitors that are often found in clinical samples. This procedure can be followed by multiple detection methods. A new, efficient sequencing method, called nanopore sequencing, has been developed in a handheld device, called MinION, that provides thorough output in a timely manner. When combined with LAMP, it has been referred to as LAMPore. The first objective of our work was to estimate the prevalence of latent EHV-1 and compare the frequency of each variant in the submandibular lymph nodes from horses in Virginia. Our second objective was to perform direct DNA sequencing of EHV-1 using the mobile MinION sequencer in combination with LAMP viral enrichment. Our findings demonstrated a low apparent prevalence of latent EHV-1 DNA in submandibular lymph nodes in this population of horses in Virginia as well as successful detection and identification of EHV-1 in equine nasal swab samples using LAMPore sequencing.