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Glucocorticoids are widely used in horses for a variety of autoimmune and inflammatory conditions. Its potent antiinflammatory properties have been associated with the suppression of a number of different inflammatory cytokines. The purpose of the study was to evaluate the effect of dexamethasone treatment in horses on mRNA cytokine expression, including interleukin-1Î , interferon-gamma, interleukin-4 and interleukin-6, during a five day treatment period and a five day post treatment period. A randomized complete block design was performed on 16 healthy horses. Group I (8 horses) received 0.1 mg/kg of dexamethasone sodium phosphate by intravenous injection once daily for 5 days. Group II (8 horses) received an equivalent volume of sterile saline by intravenous injection daily for 5 days. A sample of 5x10 mililiters of blood in acid citrate dextrose was obtained prior to initial treatment. Thirty minutes after each treatment injection (placebo or dexamethasone) a sample of blood was obtained during the 5 day treatment period and 24, 48, 72, 96 and 120 hours after the last treatment injection was administered. Peripheral-blood mononuclear cells were isolated from the blood samples and stimulated with concavalin A. RNA was isolated using the QIAGEN RNeasy kit. cDNA first strand synthesis was achieved using QIAGEN's OMMISCRIPT RT KIT. cDNA was also constructed for the house keeping gene Î actin. Primer pairs specific for each cytokine were designed using equine cytokine sequences available on Genbank. cDNA for each cytokine and Î -actin was amplified using Real Time PCR technique. Interleukin-4, interleukin-6 and interferon-gamma mRNA expression was statistically significant suppressed in horses treated with dexamethasone when compared to control horses. Interleukin-1Î was only significantly suppressed on day 5. Interleukin-4, interleukin-6 and interferon-gamma mRNA expression suppression was initially observed on day 2 and lasted 24 hours after the last dose of dexamethasone was administered. Interleukin-6 mRNA expression was significantly higher when compared to control group on day 10. Our results suggest that dexamethasone treatment of healthy horses suppresses mRNA expression of several cytokines, including interleukin-4, interleukin-6 and interferon-gamma. This effect could explain part of corticosteroid's mechanism of action for controlling inflammation in a variety of disease conditions. The time-course effect of dexamethasone showed that the effect on mRNA cytokine expression suppression is only observed on day 2 of treatment and mRNA suppression is maintained for 24 hours after discontinuation of treatment. / Master of Science

Forage is an essential part of the equine diet for health and performance. Pasture nonstructural carbohydrates (NSC) have been shown to fluctuate diurnally and seasonally throughout the year due to various factors including environmental conditions and plant stress. The intake of elevated NSC content is linked with metabolic and digestive diseases, including colic and laminitis. A yearlong grazing study was conducted at the Virginia Tech Middleburg Agricultural Research and Extension Center from October 2016 through September 2017 to investigate fluctuations in pasture NSC concentrations as well as the metabolic and digestive response of grazing horses. Twelve sporthorse mares (15 ± 3.4 yrs) were maintained on an 8.5-ha cool season mixed grass pasture with water, mineral (Buckeye Nutrition, Dalton, OH), and white salt ad libitum. Weekly pasture samples (200 g wet weight) were clipped at random 2.5 cm from the plant base at 0800 (AM) and 1600 h (PM) on d 1 and 0800 (AM) and 1300 h (PM) on d 2. Samples were weighed and dried at 70º C and submitted to a commercial laboratory (Equianalytical, Ithaca, NY) to determine NSC content (water soluble carbohydrates [WSC] + starch). Environmental conditions were measured including ambient temperature, relative humidity, solar radiation, rainfall, soil and canopy temperature, and photosynthetically active radiation (PAR). Corresponding weekly blood samples were collected at 1300 h on d 2 via jugular venipuncture into 4 mL potassium oxalate, and 7 mL EDTA vacutainer tubes and analyzed for glucose (mg/dL), insulin (μIU/mL), and L-lactate (mg/dL). Each month, fecal grab samples were collected from the midrectum to measure pH and Dlactate (µM). Additionally, monthly glucose and insulin dynamics (% ∆) were assessed via a modified oral sugar test. Pasture nutrients including DE (2.35 ± 0.12 Mcal/kg), NSC (25.45 ± 4.02% DM), WSC (19.65 ± 3.47% DM), and starch (7.25 ± 1.29% DM) were higher in the afternoon hours (PM) compared to morning measurements (AM). Pasture CP and carbohydrate fractions were higher in the spring and fall months compared to summer and winter months with NSC concentrations being highest in May (wk 19) at 25.45% DM. Pasture NSC content was correlated (P ≤ 0.05) with relative humidity (r = 0.38), solar radiation (r = 0.32), and PAR (r = 0.51) and tended (P ≤ 0.1) to have a relationship with ambient temperature (r = 0.23) and rainfall (r = 0.23). There was seasonal variation in all morphometric measures in grazing horses. BW was highest in the spring (P < 0.0001), while BCS and CNS were highest in the fall (P = 0.0021 and P < 0.0001, respectively). Metabolic responses in grazing horses also fluctuated seasonally with glucose and insulin concentrations being most elevated in the spring (P < 0.0001). There was also seasonal variation in digestive measures in grazing horses. Plasma Llactate and fecal D-lactate means differed by month (P < 0.05) with the highest concentrations in April (11.8 ± 0.91 mg/dL and 4220.4 ± 185.5 µM, respectively). Fecal pH was most acidic in April (6.52 ± 0.08). Pasture NSC content was correlated with weight (r = 0.35), glucose (r = 0.21), and insulin (0.26) in grazing horses and tended to have a relationship with CNS (r = 0.14). There was also a relationship between NSC and plasma L-lactate (r = 0.33), fecal D-lactate (r = 0.48) and pH (r = -0.27). Lastly, glucose and insulin % ∆ (P < 0.0001) were greatest during spring months, but there was no effect of fasting insulin (P < 0.2787) or fasting glucose (P < 0.2055) on glucose % ∆. These data indicate a relationship between seasonal changes in pasture NSC content and the physiological response in grazing horses. Future aims include evaluating possible seasonal fluctuations in the hindgut microbiome of grazing horses to better understand the link between the equine microbiome and nutritionally-related disturbances. Improved grazing management strategies are needed to reduce the risk of metabolic and gastrointestinal disorders in horses, which may lead to subsequent colic and pasture associated laminitis. / Doctor of Philosophy / Lush pastures are an important part of the equine diet for overall health and performance. However, there are several nutrition-related diseases that can occur when environmental conditions favor starch and sugar (nonstructural carbohydrates, NSC) accumulation in pasture grasses. Environmental conditions such as air temperature, intensity of sunlight, frost, and drought can all lead to increased accumulation of NSC in pasture grasses, especially in spring and fall months. When horses graze pastures with elevated NSC concentrations they can develop several conditions such as obesity, insulin resistance, and gastrointestinal upset. One of the most common but least understood equine diseases is pasture-associated laminitis, in which inflammation causes pain and damage to the structure of the equine hoof. The objectives of our research were to measure seasonal changes in pasture NSC concentrations as well as the metabolic and digestive response in grazing horses to better understand how the intake of pasture NSC content may lead to disturbances or disease in the horse. A yearlong grazing study was conducted at the Virginia Tech Middleburg Agricultural Research and Extension Center from October 2016 through September 2017 to investigate the relationship between pasture NSC and grazing horses. Twelve sporthorse mares were maintained on a 21-acre mixed grass pasture with water, mineral (Buckeye Nutrition, Dalton, OH), and white salt ad libitum. Weekly pasture samples collected to determine NSC content of the grasses. Weekly blood samples were collected from the horses to measure glucose, insulin, and L- lactate concentrations. Each month, fecal samples were collected to measure pH and Dlactate. Additionally, monthly glucose and insulin dynamics (% ∆) were assessed via a modified oral sugar test. Pasture NSC content fluctuated throughout the year and was most elevated in the spring and fall months. There was seasonal variation in the metabolic response of grazing horses with glucose and insulin concentrations being highest in the spring months. There was also seasonal variation in digestive measures in grazing horses. Plasma L-lactate and fecal D-lactate were most elevated in the spring and fecal pH was most acidic in the spring. These results indicate a relationship between seasonal changes in pasture NSC content and the physiological response in grazing horses. Future aims include evaluating possible seasonal fluctuations in the hindgut microbiota of grazing horses to better understand the link between the equine gastrointestinal bacteria and nutritionally-related diseases. Improved grazing management strategies are needed to reduce the risk of metabolic and gastrointestinal disorders in horses, which may lead to diseases such as colic and pasture-associated laminitis.

Recurrent airway obstruction (RAO) affects middle-age horses, inducing bronchoconstriction and airway inflammation. β2-agonists like salbutamol are used as treatment, promoting airway smooth muscle (ASM) relaxation and bronchodilation. In addition to ASM, inflammatory cells express the β2-adrenoreceptors (β2-AR). In other species, β2-agonists promote peripheral blood lymphocyte (PBL) cytokine expression towards a pro-inflammatory phenotype. RAO horses are a good model for evaluating chronic changes in human asthma. However, little is known about the effect of β2-agonist stimulation on equine PBL inflammatory response. The aims of this study were to develop an indirect method to evaluate the response of equine PBLs to β2-agonist stimulation, and to compare it between cells from RAO and non-affected horses. Isolated PBLs were activated with ConA and stimulated with salbutamol. Response to agonist binding was indirectly determined using flow cytometric methodology and verified by Western blot. Activated PBLs from RAO horses demonstrated a significant response to β2-agonist binding whereas cells from non-affected horses did not. Response of PBLs from RAO horses was attenuated when pre-treated with a β2-antagonist but unaffected following pre-treatment with a β1-antagonist, indicating that the response of PBLs from these horses to salbutamol binding was mainly through the β2-AR. Preliminary investigation of bronchoalveolar lavage (BAL) lymphocytes from RAO horses demonstrated that they also responded to β2-agonist binding, while cells from non-affected horses did not. These findings represent a novel tool for further investigation of the role of β2-agonist binding in diseases like asthma and RAO, and support the use of this model for future studies. / Master of Science

Objectives - To evaluate the effects of equine sCD14 and monoclonal antibodies (mAbs) to equine CD14 on LPS-induced TNF° expression of equine peripheral blood mononuclear cells (PBMCs). To determine serum concentrations of soluble (sCD14) in a population of horses with gastrointestinal diseases or other illnesses likely to result in endotoxemia; and identify relationships with clinical data. Animals - Part 1; 10 healthy horses. Part 2; 55 clinical cases and 23 healthy control horses. Procedure - Part 1; PBMCs were incubated with Escherichia coli LPS, CD14 mAb, sCD14, CD14 mAb plus E coli LPS or sCD14 plus E coli LPS. Supernatants were collected at 6 hours and assayed for tumor necrosis factor ° (TNF°) activity. Part 2; Serum sCD14 was measured at admission and then at 24 and 48 hours after admission using a bead-based multiplex assay. Results - Part 1; Pre-incubation with CD14 mAb did not inhibit LPS-induced TNF° protein production in isolated equine monocytes. Use of sCD14 inhibited LPS-induced TNF° protein production in isolated monocytes in a concentration-dependent manner. Part 2; Serum concentration of sCD14 was positively related to duration of clinical signs (P = 0.007), respiratory rate (P=0.04) and band neutrophil count (P = 0.0002). There was no correlation between serum concentration of sCD14 and heart rate, temperature, hematocrit, lactate, white blood cell count, fibrinogen, creatinine, urea nitrogen, glucose and anion gap values. Serum sCD14 did not correlate with outcome at any time point for clinical cases. / Master of Science

Justification - Endotoxemia is an important contributor to mortality and loss of use in the horse and results in significant losses to the equine industry on an annual basis. Objective - To determine the effect of PGG-Glucan on the cytokine response to endotoxin in the horse. Animals - Part 1; 6 adult horses. Part 2; 12 adult horses. Procedure - Part 1; Whole blood was collected, aliquoted, and incubated in vitro in four groups; saline control, endotoxin (LPS) (100 ng/ml), PGG-Glucan (0.1, 1.0, 10 and 100 μg/ml) and LPS (100 ng/ml) plus PGG-Glucan (0.1, 1.0, 10 and 100 μgg/ml). Supernatants were collected at 0, 6 and 12 hours and assayed for tumor necrosis factor £\ (TNF£\) activity. Part 2; Horses received either PGG-Glucan (1 mg/kg) or an equal volume of isotonic saline (0.9% NaCl) IV over 15 minutes. Twenty four hours later blood was collected and mononuclear cells isolated for cell culture. Cells were treated with LPS (100 ng/ml) and RNA extractions were performed at 0, 6, 12, 24 and 48 hours. Relative mRNA expression of TNFα, interleukin-1β (IL-1β),, interleukin-10 (IL-10) and interferon-γ (IFN-γ) was determined by reverse transcription and real time polymerase chain reaction. Results - Using an in vitro endotoxin challenge method PGG-Glucan altered the production of TNFα in a dose-dependent manner. PGG-Glucan had no effect upon the ex vivo cytokine mRNA expression of TNFα, IL-1β, IL-10 or IFN-γ. Conclusions and Relevance - Although mild changes were observed in TNFα production in vitro, it is not likely that PGG-Glucan will have a significant effect upon clinical endotoxemia. / Master of Science