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. / Master of Science

Introduced cool-season grasses are dominant in Virginia's grasslands, but their high digestible energy and non-structural carbohydrate (NSC) levels pose a risk for horses prone to obesity and laminitis. Native warm-season grasses (NWSG) have lower digestible energy and NSC levels that may be more suitable for horses susceptible to laminitis. The overall objectives of this research were to 1) assess voluntary intake, toxicological response, and apparent digestibility of NWSG hays fed to horses; 2) evaluate the characteristics of three NWSG species under equine grazing; and 3) evaluate establishment strategies for NWSG and wildflowers in Virginia. For the first objective, a hay feeding trial was conducted with 9 Thoroughbred geldings in a 3 x 3 Latin square design. Voluntary dry matter intake of indiangrass (Sorghastrum nutans) and big bluestem (Andropogon gerardii) hays by horses were 1.3% and 1.1% of BW/d, significantly lower than orchardgrass (Dactylis glomerata), an introduced cool-season grass, at 1.7% of BW/d. Biomarkers for toxicity remained within acceptable ranges for all treatments. Apparent DMD did not differ among hays, ranging from 39 to 43%. Non-structural carbohydrate levels were below the maximum recommended concentration for horses susceptible to laminitis. For the second objective, a grazing trial was conducted comparing indiangrass (IG), big bluestem (BB), and eastern gamagrass (Tripsacum dactyloides) (EG) yields, forage losses, changes in vegetative composition, and effects on equine bodyweight. Nine, 0.1-hectare plots were seeded with one of the three native grass treatments, and each plot was grazed by one Thoroughbred gelding in two grazing bouts, one in July and another in September 2019. Indiangrass had the highest available forage, at 4340 kg/ha, compared with 3590 kg/ha from BB (P < 0.0001). Eastern gamagrass plots established poorly, and had only 650 kg/ha available forage during the experiment. Grazing reduced standing cover of native grasses in IG and BB treatments by about 30%, and trampled forage constituted 36-68% of groundcover in those plots after each grazing bout. Horses lost weight on all treatments, but tended (P=0.09) greater weight loss on the indiangrass treatment at 1.5 kg/d compared to 0.5 kg/d in the BB and EG treatments. For the third objective, three experiments were conducted to evaluate different strategies for establishing NWSG and wildflowers. The first experiment compared large grazed plots with or without a 2 oz/acre rate of the herbicide imazapic. Imazapic led to higher biomass and percent cover in plots seeded only with NWSG. For plots seeded with a mix of NWSG and wildflowers, imazapic reduced wildflower establishment and resulted in higher biomass and percent cover of weeds over the course of the experiment. The second experiment examined four rates of imazapic application for NWSG and wildflower establishment in small plots seeded with either NWSG or a NWSG and wildflower mix, and found biomass and percent cover of weeds was lowest at a 6 oz/acre rate, while NWSG biomass and cover did not differ between treatments. Wildflower establishment was again reduced by imazapic. The third establishment experiment compared four site preparation strategies for wildflower establishment and found tillage resulted in the most cover and biomass of wildflowers. / Doctor of Philosophy / Introduced cool-season grasses are dominant in Virginia's grasslands, but their high digestible energy and non-structural carbohydrate (NSC) levels pose a risk for horses prone to obesity and laminitis. Native warm-season grasses (NWSG) have lower digestible energy and NSC levels that may be more suitable for horses susceptible to laminitis. The overall objectives of this research were to 1) assess voluntary intake, toxicological response, and apparent digestibility of NWSG hays fed to horses; 2) evaluate the characteristics of three NWSG species under equine grazing; and 3) evaluate establishment strategies for NWSG and wildflowers in Virginia. For the first objective, a hay feeding trial was conducted with 9 Thoroughbred geldings in a 3 x 3 Latin square design. Voluntary dry matter intake of indiangrass (Sorghastrum nutans) and big bluestem (Andropogon gerardii) hays by horses were 1.3% and 1.1% of BW/d, significantly lower than orchardgrass (Dactylis glomerata), an introduced cool-season grass, at 1.7% of BW/d. Biomarkers for toxicity remained within acceptable ranges for all treatments. Apparent DMD did not differ among hays, ranging from 39 to 43%. Non-structural carbohydrate levels were below the maximum recommended concentration for horses susceptible to laminitis. For the second objective, a grazing trial was conducted comparing indiangrass (IG), big bluestem (BB), and eastern gamagrass (Tripsacum dactyloides) (EG) yields, forage losses, changes in vegetative composition, and effects on equine bodyweight. Nine, 0.1-hectare plots were seeded with one of the three native grass treatments, and each plot was grazed by one Thoroughbred gelding in two grazing bouts, one in July and another in September 2019. Indiangrass had the highest available forage, at 4340 kg/ha, compared with 3590 kg/ha from BB (P < 0.0001). Eastern gamagrass plots established poorly, and had only 650 kg/ha available forage during the experiment. Grazing reduced standing cover of native grasses in IG and BB treatments by about 30%, and trampled forage constituted 36-68% of groundcover in those plots after each grazing bout. Horses lost weight on all treatments, but tended (P=0.09) greater weight loss on the indiangrass treatment at 1.5 kg/d compared to 0.5 kg/d in the BB and EG treatments. For the third objective, three experiments were conducted to evaluate different strategies for establishing NWSG and wildflowers. The first experiment compared large grazed plots with or without a 2 oz/acre rate of the herbicide imazapic. Imazapic led to higher biomass and percent cover in plots seeded only with NWSG. For plots seeded with a mix of NWSG and wildflowers, imazapic reduced wildflower establishment and resulted in higher biomass and percent cover of weeds over the course of the experiment. The second experiment examined four rates of imazapic application for NWSG and wildflower establishment in small plots seeded with either NWSG or a NWSG and wildflower mix, and found biomass and percent cover of weeds was lowest at a 6 oz/acre rate, while NWSG biomass and cover did not differ between treatments. Wildflower establishment was again reduced by imazapic. The third establishment experiment compared four site preparation strategies for wildflower establishment and found tillage resulted in the most cover and biomass of wildflowers.

Determinations of cardiac output (CO) by M-mode echocardio-graphy were compared with simultaneous determinations by thermodilution in 2 conscious and 5 anesthetized horses. A range of cardiac outputs was induced by use of a pharmacological protocol (dopamine, 4 ug/kg/min, dobutamine, 4 ug/kg/min, and 10 ug/kg detomidine plus 20 ug/kg butorphanol, in sequence). Changes from baseline CO in response to each drug were evaluated, and data was analyzed to determine whether there were any interactions between drug treatment and measurement method. The mathematical relationship between CO as determined by M-mode echocardio-graphy (COecho) and as determined by thermodilution (COTD) was described and used to predict COTD from COecho. The 2 methods were compared with respect to bias and variability in order to determine the suitability of COecho as a substitute for COTD . Sources of the variability for each method were determined. Determination of CO by either method in standing horses was prohibitively difficult due to patient movement. The pharmacologi-cal protocol was satisfactory for inducing a range of cardiac outputs for the purpose of method comparison; however, use of dopamine did not offer any additional benefit over the use of dobutamine and was generally less reliable for increasing CO. Inclusion of detomidine provided an additional change in CO but did not increase the overall range of CO over that produced by halothane and dobutamine. COecho and COTD were significantly related by the predictive equation COTD = (0.63 +/- 0.157) x COecho + (16.6 +/- 3.22). The relatively large standard errors associated with COecho measurements resulted in a broad 95% prediction interval such that COecho would have to change by more than 100% in order to be 95% confident that the determined value represents true hemodynamic change. COecho underestimated COTD by a mean of 10 +/- 6.3 l/min/450 kg. The large standard deviation of the bias resulted in broad limits of agreement (-22.3 to +2.3 l/min/450 kg). Measurement-to-measurement variability accounted for 28% of the total variation in COTD values and 64% of the total variation in COecho values. Results might be improved if the mean of 3-5 consecutive beats was used for each measurement, but as determined in this experiment, COecho is too variable to have confidence in its use for precise determinations of CO. / Master of Science

Due to the detrimental side effects of NSAID administration, such as gastrointestinal ulceration and renal papillary necrosis, there is a profound need for clinical pain relief in horses with long term orthopedic disease whereby gastrointestinal side effects are obviated. Acetaminophen is one of the most commonly used analgesic drugs in humans, and is readily available as an inexpensive generic over-the-counter preparation. Acetaminophen has a number of mechanisms of action that differ from NSAIDs, including actions on the serotonergic, opioid, endocannabinoid and lipoxygenase pathways. These alternate pathways may provide greater efficacy against chronic or neuropathic pain in equine patients. Acetaminophen was preferred by physicians over COX-2 and nonselective NSAIDs, even when those drugs were coupled with proton-pump inhibitors to reduce gastrointestinal side effects; due to cost considerations and the occurrence of adverse side effects from those drugs. In horses, acetaminophen has been reported to be efficacious as an adjunct treatment for laminitis in one pony, and was an effective analgesic agent when combined with NSAIDs in a model of inducible foot pain. However, no studies have been performed to validate a dose-response curve in horses. A study recently completed by our group demonstrated rapid absorption following oral administration of acetaminophen. Reported human therapeutic plasma concentrations were achieved within 30 minutes of administration, with no clinical or clinicopathologic evidence of adverse side effects after two weeks of repeated dosing. Dose simulation trials indicate that a change in dosage schedule may be required in order to provide adequate plasma concentrations. / Master of Science / The use of non-steroidal anti-inflammatory drugs (NSAIDs) such as phenylbutazone in horses is widespread, and can be associated with detrimental side effects such as gastrointestinal ulceration and kidney damage. The clinical need for pain relief in horses with long-term lameness that minimizes gastrointestinal side effects has led to the development of cyclooxygenase-2 (COX-2) selective NSAIDs, such as firocoxib, but the expense of this therapy is often a major consideration limiting its use and few alternatives are available. Acetaminophen is one of the most commonly used analgesic drugs in humans, and is readily available as an inexpensive generic over-the-counter preparation. Despite the lower efficacy of acetaminophen in trials of human patients with chronic osteoarthritis, acetaminophen remains the preferred analgesic in humans due to its increased tolerance and improved cost-benefit analysis when compared to nonselective and COX2 selective NSAIDs. Acetaminophen has a number of mechanisms of action that differ from the current mainstays of equine analgesic therapy, which may provide greater efficacy against chronic or neuropathic pain in equine patients. A recent study of acetaminophen in horses has shown rapid absorption and achievement of levels reported to be therapeutic in humans, with no adverse side effects after two weeks of repeated dosing. In horses, acetaminophen has demonstrated efficacy as an adjunct treatment for laminitis in one pony, and was an effective analgesic agent when combined with NSAIDs in a model of inducible foot pain.

Establishment and maintenance of a successful pregnancy requires signaling from the embryo to the mare, a process known as maternal recognition. Six days after fertilization, the trophectoderm (TE), a placenta precursor is formed. Signals emanating from the TE to the uterine environment are critical to maternal recognition of pregnancy. The identity of factors necessary for this process remain unknown. A novel equine induced trophoblast cell line (iTr) that closely mimics the genotype and phenotype of native equine TE was created. Transcriptome analysis of iTr revealed increased expression of growth factor (GF) receptors for Epidermal GF (EGF), Hepatocyte GF (HGF), Fibroblast GF-2 (FGF-2) and Insulin GF (IGF-1), suggesting these GF may be important targets during TE development in the early embryo. We hypothesized that treatment of iTr cells with these GF would induce changes in cell proliferation and expression of genes likely involved in maternal recognition. The objectives of this experiment were to evaluate the effect of these GFs on iTr mitotic response and regulation of genes involved in steroidogenesis. Equine iTr cells (n = 3) were cultured with 10 ng/mL EGF, HGF, FGF-2 or IGF-1 for 24 hr, with 5-ethynyl-2'-deoxyuridine (EdU) supplementation during the final 2 hr. Subsequently, cells were fixed and EdU positive and total nuclei were enumerated. A parallel plate of iTr cells was treated in a similar manner and lysed for total RNA isolation. Quantitative PCR using gene-specific primers for CYP11A1, PTGS2, PTGES2, and PTGES3 was performed. Data were analyzed by ANOVA with Tukey's post hoc adjustment using the GLM procedure of SAS. Treatment with EGF, FGF-2, HGF, and IGF-1 increased (P < 0.05) iTr proliferation from control levels of 25.33 ± 1.03% to 38.58 ± 1.61%, 45.50 ± 2.94%, and 38.23 ± 2.01% respectively. The 2-&#916;&#916;CT method was used to calculate the fold change (FC) using GAPDH as the reference gene for normalization. Expression of CYP11A2, PTGES2, and PTGES3 was not affected by GF, as measured by qPCR. By contrast, PTGS2 transcript abundance increased (P < 0.05) following FGF-2 (FC = 3.327 ± 0.8291) and HGF (FC = 11.88 ± 4.572) treatment. These results indicate that FGF-2 and HGF may simultaneously induce proliferation and prostaglandin production by TE cells. The combined results of these experiments will improve our understanding of TE morphogenesis and its response to uterine-derived growth factors. / Master of Science / Establishment and maintenance of a pregnancy requires that the mare uterus recognize the presence of the embryo, a process known as maternal recognition of pregnancy. The trophectoderm (TE) are cells on the outer layer of the embryo formed six days after fertilization, which later give origin to the placenta. The TE sends signals from the embryo to the uterus, that are very important for the mare’s recognition of the embryo’s presence. The specific nature of these signals are still unknown in the horse. A cell line (iTr) very similar in aspect and genes to the horse’s native TE has been created in our laboratory. A set of comparative assays have showed that, during the developmental stage of maternal recognition, both the horse TE and the iTr cells share significant identity, and have receptors for the same set of growth factors (GF), suggesting these GF are important for early embryo development and potentially involved in the signaling process of maternal recognition. We proposed that treatment with these GF would induce iTr cells to proliferate and express signals likely involved in maternal recognition in horses. The objectives of this experiments were to evaluate the effect of EGF, HGF, FGF-2 and IGF-1 on iTr cells by measuring proliferation and cellular mechanisms of maternal recognition already established in in other species. Equine iTr cells were cultured with different GF and right before analysis a fluorescent dye that stain dividing cells was added in order to measure the proliferation. Equivalent cell cultures were used to evaluate if the treatment affected the production of hormones involved in signaling maternal recognition. Treatment with all GF induced higher cell proliferation, but HGF also increased the production of one enzyme that participates in producing a very important hormone (prostaglandin E2). The combined results of these experiments add to our understanding of maternal recognition in horses.

Like any other agricultural industry, many different career paths in the equine field are becoming more technologically advanced. To adapt to technological progress, current standards need to be integrated into school-based agricultural education courses to properly train and teach students about current industry standards. In addition to equine science curriculum development, emphasis must also be focused on providing assistance to secondary-school-based agriculture teachers so they can effectively teach industry-based equine science objectives in their programs. The equine curriculum objectives were measured in terms of perceived importance by equine professionals and post-secondary equine educators as well as Arizona school-based agriculture teachers. Further, the Arizona school-based agriculture teachers indicated their self-perceived ability to teach the objectives. Demographics for each group (equine industry professionals and Arizona school-based agriculture teachers) were also collected. A Borich needs assessment was calculated to prioritize areas needed for professional development of school-based agriculture teachers. Recommendations include the importance of bringing secondary-school-based agricultural education equine science curricula in line with industry standards. Further, providing professional development to secondary-school-based agriculture teachers is key to increasing teachers’ competence and ability to teach industry-based equine science.

Equine assisted psychotherapy (EAP) is an emerging experiential methodology<br />that has recently gained recognition as a method for addressing a range of presenting<br />problems for a wide variety of client populations. Couples therapy is one area that the<br />practice of equine assisted psychotherapy has recently gained traction. This study<br />describes the practice of equine assisted couples therapy in terms of practitioner<br />characteristics, approach to treatment, therapeutic goals and outcomes. Mental health<br />professionals currently using equine assisted psychotherapy to treat couples share their<br />experiences and perspectives to provide an overview of this emerging modality. / Master of Science

With overdose deaths and residential addiction treatment admittance increasing, more treatment options are necessary to meet the demand of services. Equine assisted psychotherapy and learning (EAPL) is a relatively new therapeutic tool in addiction treatment for individuals battling substance use disorder (SUD), and thus, lacks a solid evidence base to be implemented consistently and effectively. The purpose of this dissertation was to determine the effectiveness of EAPL in creating an emotionally safe environment for learning for college-age young adults with a particular focus on those struggling with SUD. This was accomplished in a series of projects focused on: 1) implementing evaluation methodology for tracking emotional safety and learning in young adults participating in equine interaction activities without therapeutic intentions, 2) determining the relationship between physiological responses and emotional safety and learning in young adults participating in outpatient EAPL programming for SUD, and 3) evaluating the treatment outcomes associated with emotional safety and learning for young adults with SUD participating in EAPL at a residential treatment facility. Each project utilized the same three evaluations for evaluating emotional safety and learning: 1) emotional safety evaluation, 2) equine knowledge exam, and 3) physical skills evaluation. In each of the studies, a correlation was found between emotional safety and equine knowledge and handling skills for college-age young adults, however, equine interaction with therapeutic intentions revealed a stronger correlation indicating it is not just the horse that provides the mental health benefits. An emotionally safe environment for learning, was established for college-age young adults within a residential treatment facility, this was not dependent on the length of stay or level of participation suggesting this is an efficient therapeutic option for SUD for this population. Emotional safety for the college-age young adults participating in EAPL was correlated to vital sign measurements, but vital sign measurements of young adults were independent of the vital signs of the therapy horse. Each of the three projects provided vital information filling the gap of knowledge surrounding EAPL as an effective clinical intervention strategy for SUD in college-age young adults and created a basis for further research using more objective measures.

The equine influenza virus is a significant cause of respiratory disease in horses. Even though horses generally recover from this virus, sometimes horses with equine influenza develop secondary bacterial infections which can cause severe pneumonia, thereby increasing recovery times. Owners and managers are faced with the decision of whether to delay preventative treatment in hopes of the horse avoids contracting a secondary bacterial infection (“wait and see”) or aggressively treat the horse with an antibiotic in hopes of avoiding a serious infection (“treat now”). From a decision making standpoint, the economic considerations include explicit treatment costs as well as nonmonetary costs the owner or manager bear when caring for an ill horse. This study investigating horse owner/manager preferences for treatment alternatives is approached in two parts. The first part of the study collects data from field practitioners to estimate the cost of treatment strategies under different scenarios. The second part consists of a questionnaire presented to horse owners and managers and includes four choices between alternative treatment strategies. Analyzing the data using a conjoint analysis approach, respondents’ willingness to pay for different elements of a treatment strategy are estimated. Based on treatment strategies and demographic interactions, a respondent was willing to pay to cover the cost of a horse who became ill with the equine influenza, but individual price sensitivities suggested horse owners and managers are willing to “treat now” versus “wait and see” in order to not see their horse feel poorly and miss training time.

Unwanted horses are a growing problem in the United States, and through the use of surveys, researchers can understand public opinion and experiences related to this problem. This study administered surveys specific to horse and non-horse owners through face-toace delivery and to rescue/retirement and auction facilities through the mail. Data was analyzed using a CRD, GLM and Fisher’s Protected LSD with á of 0.05. Comparisons were completed between horse and non-horse owners, horse owners and non-horse owners with equine experience, non-horse owners with and without equine experience, Quarter horse and other breeds owners, and auction and rescue/retirement facilities. Similarities were seen between groups for questions associated with participant location, understanding of processing/harvesting regulations and procedures, processing/harvesting facility restrictions preferences, and knowledge of management cutbacks and rescue facility locations. The only question that was significantly different for all comparisons was concerning the support towards the reopening of equine processing/harvesting facilities.