Progesterone in the mare declines to almost undetectable concentrations in late gestation. It’s metabolized into several pregnanes, some circulating at very high concentrations. Although the function of many pregnanes remains unclear, 5α-dihydroprogesterone and allopregnanolone are bioactive. Measurements of pregnanes in late gestation are typically by immunoassay, although results are confounded by cross-reactivity with related pregnanes. Conversely, liquid chromatography tandem mass spectrometry (LC-MS/MS) allows differentiation of individual pregnanes. The purposes of these studies were: 1) to evaluate the ability of a 5α-reductase inhibitor, dutasteride, to alter pregnane metabolism and pregnancy outcome, 2) to evaluate changes in target pregnanes in late gestation by LC-MS/MS in mares with ascending placentitis, and 3) compare immunoassay and LC-MS/MS detection of pregnanes in late gestation. Our findings suggest that dutasteride significantly altered pregnane metabolism without effects on pregnancy outcome. Pregnane measurement by LC-MS/MS resulted in a significant (p<0.05) differences in assay results, while correlation was observed between immunoassay measurements and actual progesterone concentrations by LC-MS/MS. These studies demonstrate the complexity of pregnane metabolism in late gestation in the mare and the necessity of LC-MS/MS to detect specific changes that immunoassays cannot differentiate.

The mare experiences a transient innate immune response to breeding, the resolution of which is crucial for optimal fertility. The majority of mares are able to modulate this inflammation in a timely fashion, but a subpopulation exists which fail to do so and are considered susceptible to persistent breeding-induced endometritis (PBIE). Seminal plasma has been shown to modulate aspects of this inflammation. Recently, two seminal plasma proteins have garnered interest for their immune modulating properties: cysteine-rich secretory protein-3 (CRISP-3) and lactoferrin. These proteins have been found to alter the binding between sperm and neutrophils based on sperm viability in vitro, but minimal work has evaluated their effect on endometrial mRNA expression of cytokines and inflammation in response to breeding. Experiments were performed to analyze the expression of equine CRISP-3. Found to be primarily synthesized in the ampulla of the vas deferens and to a lesser extent in the vesicular gland, CRISP-3 expression was only seen in the postpubertal stallion. Due to the effect of sperm viability on protein function in vitro, varying sperm populations were analyzed for their effect on gene expression in the uterus. It was determined that viable sperm suppressed the gene expression of the inflammatory modulating cytokine interleukin-6 (IL-6) in comparison to dead sperm. Next, the effect of CRISP-3 and lactoferrin on endometrial gene expression in the normal and susceptible mare was investigated. Neither protein had a significant effect on the mRNA expression of inflammatory cytokines in the normal mares at six hours post-breeding. In contrast, lactoferrin was found to significantly suppress the expression of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α in susceptible mares. Due to this, lactoferrin was further analyzed as an immunomodulant for the treatment of PBIE. Susceptible mares were infused with varying doses of lactoferrin at six hours post-breeding. Although not in a dose-dependent fashion, lactoferrin was found to decrease both fluid retention and neutrophil migration, in addition to suppressing the expression of the pro-inflammatory cytokine interferon gamma (IFNγ) and increasing the gene expression of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1RN). In conclusion, CRISP-3 expression occurs in secretory aspects of the male reproductive tract, and appears to be up regulated after sexual maturation. Viability of spermatozoa affects the immune response to breeding and should be taken into consideration for experimental design and interpretation of data. The seminal plasma proteins CRISP-3 and lactoferrin have minimal effect on endometrial gene expression in normal mares, but lactoferrin suppresses the expression of TNF in susceptible mares. Finally, lactoferrin was found to function as a potent anti-inflammatory for the persistent inflammation seen in susceptible mares when administered post-breeding. This protein should be further investigated as a potential therapeutic for the treatment of persistent breeding-induced endometritis.

Although Rhodococcus equi (R. equi), previously known as Corynebacterium equi, was first isolated from pneumonic foals almost a century ago, it remains the most common cause of subacute or chronic granulomatous bronchopneumonia in foals. While the majority of foals exposed to R. equi develop a protective immune response (regressors), others exhibit a unique susceptibility to infection (progressors). The determinants for either outcome are not completely understood. Therefore, current diagnostic and preventive measures are suboptimal and require betterment. In light of this current need, we hypothesized that immunoglobulin G subisotype T [IgG(T)] against the virulence-associated protein A (VapA) of R. equi, and whole blood cytokine expression profile of foals predict the outcome of infection and can be used as diagnostic markers of clinical disease. Further, we hypothesized that the use of R. equi hyperimmune plasma (HIP) decreases severity of disease in naturally infected foals, playing an important role in disease prevention in the field. Lastly, we hypothesized that specific anti-Rhodococcus equi pili antibodies passively acquired by foals via colostrum after immunization of pregnant mares with a Rhodococcus equi pili-based candidate vaccine will confer protection against induced disease, and therefore have an immediate impact on R. equi pneumonia prophylaxis. The objectives of this study were: (1) to describe the humoral immune response of progressor and regressor foals to R. equi following experimental challenge and natural infection, (2) to compare the cytokine and cell-marker expression profile in whole blood of progressor and regressor foals after challenge, (3) to evaluate the Vap-A specific IgG profile of a commercially available HIP product and its value as a prophylactic tool on an endemic farm, and (4) to evaluate the efficacy of a vaccine based on the Rhodococcus equi pili (Rpl). Although the IgG(T) response of progressor foals after challenge or following natural infection tended to be more pronounced than that observed in regressor foals, its performance as a diagnostic test for predicting disease outcome was poor. Likewise, whole blood cell-marker and cytokine expression profiles of progressor and regressor foals were not significantly different, undermining its reliability as a diagnostic tool. Evaluation of the association of HIP VapA specific IgG profile and rhodococcal disease outcome in the field resulted in the conclusion that progressor foals received significantly less VapA specific IgG, suggesting that HIP may have provided some protection to regressor foals. Although HIP appeared to have provided some protection against clinical pneumonia, Rpl maternally-derived IgG failed to confer any advantage to foals born from vaccinated mares. The Rpl candidate vaccine failed to confer protection to foals after challenge, and did not decrease disease severity in comparison to a control group. In summary, the results of this study do not support the use of VapA specific IgG(T) or whole blood cytokine expression profile as predictors of disease outcome. Further, our results suggest a positive effect of HIP on disease outcome. Lastly, the presence of systemic and local Rpl antibodies was not protective in foals.

In horses, a painful and often debilitating disease known as laminitis can result in impaired function and, in severe cases, euthanasia. Equine Metabolic Syndrome (EMS) is a syndrome in horses that results in development of laminitis and is characterized by the presence of general and/or regional adiposity (“cresty neck”), aberrations in blood lipid concentrations, insulin resistance (IR) and/ or hyperinsulinemia. Therapies have focused on improving the state of obesity and insulin resistance with the goal of diminishing the likelihood of laminitis development. A definitive cause for laminitis has not been established, but hyperinsulinemia and IR are likely candidates as experimental states of hyperinsulinemia have been shown to induce laminitis and improvements in insulin sensitivity and obesity have been associated with a decreased risk of laminitis development. This dissertation discusses associations between obesity and IR, as well as potential therapies for alleviating insulin resistance with the ultimate goal of decreasing the risk of developing laminitis. Therapies evaluated included chromium and magnesium, levothyroxine sodium, and metformin hydrochloride. Horses were treated with each supplement for 10 to 36 weeks, depending on the supplement tested, and physical measurements such as body weight, neck circumference, and body condition score were obtained. Throughout each study, blood concentrations of glucose, insulin, and plasma lipids were analyzed. Chromium and magnesium currently do not appear to have any effect on insulin sensitivity, whereas results of levothyroxine administration indicate therapeutic responses, as does metformin, though results indicate further work are required. Research contained in this dissertation focuses on the potential of identifying animals at risk of developing IR and laminitis through measurement of blood biomarkers such as adiponectin and glucagon-like peptide 1. Assays to measure markers included enzyme-linked immunosorbent assays, western blots, and radioimmunoassays. Glucagon-like peptide 1 currently does not appear to differ between healthy and IR animals, but protein band density of high-molecular weight adiponectin does appear to be lower in horses with IR when compared to healthy animals. There is still much to learn about IR in horses, and therapy appears to be dependent on a case by case basis.

Cervical stenotic myelopathy (CSM) is an important musculoskeletal and neurologic disease of the horse. Clinical disease occurs due to malformations of the vertebrae in the neck causing stenosis of the cervical vertebral canal and subsequent spinal cord compression. The disease is multifactorial in nature, therefore a clearer understanding of the etiology and pathogenesis of CSM will allow for improved management and therapeutic practices. This thesis examines issues of equine CSM diagnosis, skeletal tissue pathology, and inherited genetic determinants utilizing advances in biomedical imaging technologies and equine genomics. Magnetic resonance imaging (MRI) data provided a more complete assessment of the cervical column through image acquisition in multiple planes. First, MRI was compared to standing cervical radiographs for detection of stenosis. Using canal area or the cord canal area ratio, MRI more accurately predicted sites of compression in CSM cases. Secondly, articular process skeletal pathology localized on MRI was found to be more frequent and severe in CSM horses compared to controls. In addition, lesions were generalized throughout the cervical column and not limited to the spinal cord compression sites. A subset of lesions identified on MRI was evaluated using micro-CT and histopathology. Osteochondrosis, osseous cyst-like structures, fibrous tissue replacement of bone, and osteosclerosis were observed. These lesions support likely developmental aberrations of vertebral bone and cartilage maturation with secondary biomechanical influences. Bone cyst-like structures are a novel finding in this disease. Finally, the long-standing question of the contribution of genetic determinants to CSM was investigated using a genome wide association study (GWAS). Multiple significant loci were identified supporting the influence of a complex genetic trait in clinical disease. A simple Mendelian trait controlled by one gene is unlikely given the detection of variants across multiple chromosomes. Major contributions from this research include documentation of articular process bone and cartilage pathology in horses with CSM, support for abnormal cervical vertebrae development being an important contributing factor in the etiology and/or pathogenesis of equine CSM, and evidence that multiple genetic loci contribute to the CSM disease phenotype.

Lawsonia intracellularis and the disease it causes in horses, equine proliferative enteropathy (EPE), is an emerging pathogen of increasing importance to the horse industry from both an economic and welfare standpoint. Long recognized as an economically important disease of swine, the hallmark of EPE is a protein-losing enteropathy, where affected horses suffer weight loss and some ultimately succumb to the disease despite aggressive treatment. There are currently no known EPE preventative measures and the epidemiology of the disease remains poorly defined. While EPE is a sporadic disease affecting less than 25% of exposed horses, some farms experience clinical cases year after year. Further, weanlings are uniquely susceptible to this disease, although no conclusive reason for this predisposition has been identified. The overall hypothesis is that the host immune response plays a significant role in the susceptibility of weanlings to L. intracellularis infection and the occurrence of clinical equine proliferative enteropathy. To test this hypothesis, four individual hypotheses were proposed: (H1) previous farm history of EPE does not have an effect on weanling seroprevalence, (H2) passively-acquired antibodies do not have an effect on susceptibility to L. intracellularis and the occurrence of EPE, (H3) the serological status of mares can be used to determine the role they play in the epidemiology of EPE on endemic farms, and (H4) L. intracellularis-specific IFN-g expression is not associated with increased resistance to EPE.

Equine protozoal myeloencephalitis (EPM), caused by the protozoan parasite Sarcocystis neurona, is one of the most important neurological diseases of horses in the Americas. While seroprevalence of S. neurona in horses is high, clinical manifestation of EPM occurs in less than 1% of infected horses. Factors governing the occurrence and severity of EPM are largely unknown, although horse immunity might play an important role in clinical outcome. We hypothesize that EPM occurs due to an aberrant immune response, which will be discernable in the equine IgG subisotypes a, b, and (T) that recognize S. neurona in infected diseased horses versus infected but clinically healthy horses. Based on previously-established serum antibody concentrations for IgG subisotypes in healthy horses, standard curves were generated and served to establish the concentration of antigen-specific IgG subisotypes in equine serum and CSF in infected diseased and infected normal horses. The subisotype concentrations and ratios between subisotypes were analyzed to assess whether neurological disease is associated with detectable differences in the antibody response elicited by infection. Results indicate a type I biased immune response in infected diseased horses, implicating the role of immunity in the development of EPM.

Pituitary pars intermedia dysfunction (PPID) is one of the most common endocrine diseases affecting senior horses. PPID causes abnormally high concentrations of adrenocorticotropic hormone (ACTH) in the plasma and a very distinct, long, shaggy haircoat (hypertrichosis). At present, the recommended treatment for PPID is daily oral administration of pergolide mesylate. Due to the increased ACTH levels associated with PPID, it is commonly thought that these horses are immunosuppressed and at increased risk of opportunistic infections, although current research in this area is sparse. Additionally, it is not well-understood how treatment with Prascend® (pergolide tablets) affects endocrine measures other than ACTH and if it also impacts the immune response. To better understand how PPID influences endocrine and immune function in the horse, Non-PPID horses (n=10), untreated PPID horses (n=9), and PRASCEND-treated PPID horses (n=9) were followed over 15 months. Endocrine measures assessed included basal ACTH, ACTH responses to thyrotropin-releasing hormone (TRH) stimulation tests, basal insulin, insulin responses to oral sugar tests (OST), total cortisol, and free cortisol. Systemic immune function measures included basal and stimulated whole blood and peripheral blood mononuclear cell (PBMCs) cytokine and receptor expression, plasma myeloperoxidase levels, and complete blood counts. Localized immune function measures within the lung included cytokine and receptor expression after stimulation of cells obtained via bronchoalveolar lavage (BAL), myeloperoxidase levels in BAL fluid, and BAL fluid cytology. We hypothesized that PPID would affect immune function, but that any alterations would be corrected by treatment with PRASCEND. Results for the endocrine analyses showed that basal ACTH was reduced in the PRASCEND-treated horses to the levels of the Non-PPID horses, but ACTH in response to TRH stimulation was only reduced in the PRASCEND-treated horses at non-fall timepoints. PPID did not affect basal insulin, insulin responses to OSTs, total cortisol, or free cortisol, and PRASCEND treatment did not appear to have an impact on these measures either. These results suggest that PPID and hyperinsulinemia/insulin dysregulation are distinct endocrine conditions, and that the excess ACTH in horses with PPID is inactive, as it is unable to stimulate a normal cortisol response. In the immune function analyses, PPID horses had decreased expression of interferon gamma (IFNγ) from PBMCs stimulated with Rhodococcus equi and Escherichia coli and increased transforming growth factor beta (TGFβ) expression from the E. coli-stimulated PBMCs. TGFβ was also increased in PPID horses in the unstimulated whole blood samples. These results suggest that PPID horses are unable to mount an appropriate Th1 response, and that the regulatory subset of T-lymphocytes may be contributing to this decreased Th1 response. Results for the localized immune function analyses may indicate altered Th2 responses within the lung of PPID horses, although these results were severely limited by the sample size available for analyses. PRASCEND did not appear to affect immune function as measured in this study. In summary, PRASCEND successfully reduces basal ACTH in PPID horses and remains the best choice for veterinarians in monitoring dosage and response to PRASCEND treatment. Insulin, total cortisol, and free cortisol were not affected by PPID status or PRASCEND treatment in this study. Immune function was altered in horses with PPID, and it is likely that these horses are indeed at increased risk of opportunistic infection. PRASCEND treatment did not correct the differences in immune function in this study. Additional research is needed to further understand which mechanisms are driving the alterations in immune function for horses with PPID.

Kentucky 31 tall fescue (KY31) infected with the common toxic endophyte strains of Epichloё coenophiala produces toxic alkaloids that improve plant vigor, but cause numerous adverse effects in grazing animals. Researchers developed a variety of KY31 containing an alternative strain of E. coenophiala, termed novel endophyte (NE). Adverse health effects in mares have not been evaluated. Experiments in this thesis tested the hypothesis that the NE pasture does not cause adverse effects typically associated with KY31. Specific aims were to: 1) compare forage ergovaline concentrations between KY31 vs NE pastures; 2) evaluate palmar artery diameters in mares grazing KY31, NE, or orchardgrass-bluegrass (OGBG) pastures; 3) determine mare serum prolactin, estradiol, and progesterone concentrations associated with ingesting each pasture type over time; and 4) measure foaling outcomes, including percentage of live foals, foal birth weights, and foal growth rates. In 2015, six nonpregnant mares grazed KY31, six pregnant mares grazed NE and six pregnant mares grazed OGBG pastures. In 2016, eighteen mares were used; six mares grazed each pasture type. Study results showed that ergovaline did not appear to be produced by NE. Novel endophyte pasture did not have negative effects on palmar artery diameter, reproductive hormones, or foaling outcomes.

The objectives of this thesis were: (1) to evaluate the downregulation of the stallion hypothalamic-pituitary-gonadal (HPG) axis by a GnRH antagonist (acyline) based upon endocrine, seminal, testicular and behavioral effects, and (2) to assess recovery after treatment. Stallions were treated for 50 days (n=4; 330µg/kg acyline q 5d) and controls (n=4) received vehicle alone. Stallions were assessed pre-treatment and for 72 days after last treatment. Treatment induced declines (p<0.05) in FSH, LH, testosterone (to castrate levels) and estrone sulfate. Gonadotropins and testosterone returned to control values within nine days and estrone sulfate by 14 days after treatment discontinuation. Acyline-treated stallions failed to respond with FSH, LH and testosterone increase after exogenous GnRH stimulation (25µg gonadorelin, IV) compared to pre-treatment and control stimulation. Total sperm numbers and motility were reduced in acyline-treated stallions, as well as total seminal plasma protein and testicular volume (p<0.05). Time to ejaculation was increased in acyline group (p<0.5). Testicular, sexual behavior and most seminal parameters regained normal levels within 72 days after treatment ceased. Sperm output of acyline-treated stallions was regained within seven months after ending treatment. Acyline reversibly suppressed the stallion HPG axis, thus has potential for treating the androgen-dependent Equine-Arteritis-Virus carrier state and as behavior modulator.