Theses and Dissertations

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Metabolic support of preimplantation embryo growth and viability
(Colorado State University. Libraries, 2024) Fresa, Kyle Joseph, author; Carnevale, Elaine, advisor; Chicco, Adam, advisor; Tesfaye, Dawit, committee member; Krisher, Rebecca, committee member; Gentile, Christopher, committee member
Early embryo metabolism involves essential and dynamic biological reactions that support viability, growth, and pregnancy establishment. Embryo metabolism not only serves to provide energy through ATP synthesis, but also facilitates the production of macromolecules such as proteins, nucleotides, and lipids. The ways in which embryos balance catabolic and anabolic activity during the preimplantation stage are not well understood; however, understanding these processes may lead to improved fertility treatments, embryo culture, and pregnancy outcomes. The studies described in this dissertation utilize innovative methods, such as stable isotope tracer analysis to track carbon and nitrogen flux through various pathways, oxygen microsensors to determine individual embryo respiration under various conditions, and proteomic analysis to determine the impacts of metabolic disturbances on embryo viability. The overarching hypothesis of this dissertation is that embryo viability is dependent on efficient and tightly regulated metabolic activity, and disturbances to metabolic function ultimately lead to reduced developmental potential. To test this hypothesis, a series of projects were conducted to 1) evaluate the importance of phosphoenolpyruvate carboxykinase (PEPCK) during early development, 2) uncover the function of PEPCK to support catabolic and anabolic activity in early embryos, and 3) determine the impacts of delayed embryo development on embryo metabolism and pathway regulation. These projects revealed important insights into the impact of embryo metabolism on development, including the discovery of a novel, PEPCK-mediated pathway that embryos utilize to balance energy production and biosynthesis. Furthermore, the impact of delayed embryo development was demonstrated to alter embryo metabolic activity and pathway regulation, including increased aerobic activity and altered protein expression. These findings improve our understanding of metabolic activity and regulation during preimplantation development, highlighting the impact of metabolic activity to promote ATP production, biosynthesis, developmental kinetics, and ultimately survival. The experimental outcomes presented in this dissertation provide a foundation for targeted approaches to improve embryo development and reproductive success.
Open Access
Using a precision-cut lung slice co-culture paradigm to increase T-cell populations and model infection ex vivo
(Colorado State University. Libraries, 2024) Ehrlich, Alexis T., author; Tobet, Stuart, advisor; Perera, Rushika, committee member; Magee, Christianne, committee member
Precision cut lung slices (PCLS) bridge a gap between in vivo and in vitro studies by maintaining anatomical organization with structural integrity and intercellular signaling pathways. Applications of PCLS have included the modeling of inflammatory lung diseases, metabolism studies, and drug development. In the lungs, immune responses are carried out by a network of T- and B- cells, the latter of which are resident. The limited resident T-cell population of the lung diminishes accurate representations of pathogen response capacity in PCLS. Addressing this, we set out to increase pulmonary T-cell populations ex vivo. We hypothesized that thymus and bone marrow-derived T-cells would work synergistically to populate the lung in co-culture experiments. A murine organotypic lung co-culture model was developed and characterized for tissue health and T-cell recruitment over 3 days ex vivo using adult neurobasal media with 4 mM glucose + 2% B27 supplement. Lung slices were cultured independently, with bone marrow, thymus, or both. Immune colonization of the lung was assessed using immunohistochemistry for CD3+ T-cells, CD19+ B-cells and ACK2+ cells. Cells were counted in alveolar and airway spaces after 3 days of culture. Our results demonstrate that lung co-cultured with thymus and bone significantly increased T-cell populations ex vivo, whereas lung co-cultured with thymus or bone alone did not significantly alter T-cell counts. Additionally, B-cells and C-Kit+ cell populations were not influenced by the culture paradigm. Using this paradigm, we went on to explore this lung co-culture paradigm when stimulated by an immune modulatory agent – LPS- and when an active lung infection is present using Pseudomonas aeruginosa. Lung and lung co-cultures had increases in T-cell counts after immune stimulation and infection. Additionally, the co-cultures further increased T-cell counts after treatment. More strikingly, the co-cultures influenced the degree of bacterial infection in the lung slices without altering the B-cell populations among cultures. Viral infections are also common pathogens that affect the lungs, so we examined then examined the ability of viral pathogens to infect precision cut lung slices. As we did not get an infection with live virus, we explored the effect of a viral mimic – Resiquimod- on lung co-cultures on the immune responses. Resiquimod and the co-cultures are both able to increase T-cell populations in lung slices ex vivo. These results suggest that the increased T-cell population corresponding with thymus and bone marrow co-culture could be a result of cell-cell interaction or the secretion of growth factors. Cell secretions or growth factor release could stimulate thymic secretion of T-cells or could stimulate T-cell proliferation in the lung, suggesting that co-culture with thymus and bone marrow can elicit a T-cell response ex vivo. T-cells are necessary for host-pathogen immune responses, most commonly by CD8+ T-cells but there are other populations of T-cells. Although there are limitations to the use of lung slices in infection studies, the results of the co-culture and increasing T-cells is a promising step to studying pathogen response capacity in the future ex vivo.
Open Access
Stress during pregnancy leads to long-term consequences in the offspring
(Colorado State University. Libraries, 2024) Sheng, Julietta Angelina, author; Tobet, Stuart, advisor; Smith, Bret, advisor; Myers, Brent, committee member; Hale, Taben, committee member; Chanda, Soham, committee member
Neuropsychiatric disorders encompass a wide range of conditions that affect neurological health and brain function and lead to disabilities worldwide. Such disorders include, but are not limited to, Major Depressive Disorder, schizophrenia, and anxiety disorders. Risk factors for developing neuropsychiatric disorders are multifaceted and can range from genetic predisposition, lifestyle, and environmental influences. Exposure to maternal stress is one type of environmental factor that can lead to changes in brain function and signaling pathways and increase susceptibility for related diseases. Maternal stress encompasses a diverse array of environmental stimuli, ranging from acute traumatic events to chronic or day-to-day life stressors. Maternal stressors, experienced by pregnant women, lead to overexposure of stress hormones in the developing fetus and impact short- and long-term neurological health the offspring. These studies evaluated developmental, neuroendocrine, and behavioral outcomes in offspring exposed to different models of maternal stress. Chapter 1 provided a brief history of stress, the development of the hypothalamic-pituitary-adrenal axis that regulates the stress response, and maternal-fetal interactions in stress regulatory systems and related behaviors. Chapter 2 evaluated several models of maternal stress, maternal high fat diet, maternal caloric restriction, maternal exposure to synthetic glucocorticoids. Although there were vast discrepancies between each type of maternal stress, one similarity was an activated immune response with elevated maternal cytokines. Therefore, Chapter 3 characterized a model of maternal immune activation using a toll-like receptor agonist, Resiquimod, that increased maternal and fetal cytokines, produced delayed developmental milestones and stress-related behavioral impairments in prepubertal (social-like) and adult (social-like, depressive-like, anxiety-like) offspring. Because these behavioral phenotypes are partially regulated by the paraventricular nucleus of the hypothalamus (PVN), Chapter 4 examined the neuroendocrine stress response and blood-brain barrier of the PVN. Data showed altered stress response accompanied by impaired blood-brain barrier integrity in the PVN of the adult offspring exposed to maternal injection of Resiquimod. Taken together, Chapters 2, 3, and 4 suggest maternal stress led to negative developmental, behavioral, and cellular pathologies indicative of neuropsychiatric-like disease. By teasing apart these specific programming mechanisms, we can better diagnose and treat progression of neuro-related disorders.
Open Access
Sex-specific cardiometabolic responses to chronic stress and the impact of prefrontal-medullary regulation
(Colorado State University. Libraries, 2024) Dearing, Carley, author; Myers, Brent, advisor; Smith, Bret, committee member; Fails, Anna, committee member; Hoke, Kim, committee member
Globally, cardiovascular and metabolic disease are leading causes of death and years lived with disability. Chronic stress is an etiologic factor in both diseases and biologic sex plays an important role in the progression and prognosis of each. However, the neurobiological basis of how chronic stress exposure intersects with sex, cardiovascular, and metabolic function to impact systemic physiology is poorly understood. Prior studies from our group indicate that, in rats, the prefrontal infralimbic cortex (IL)-rostral ventrolateral medulla (RVLM) circuit inhibits sympathetic and endocrine responses to stress. Therefore, we aimed to address the overarching hypothesis that the IL-RVLM circuit is necessary for homeostatic function and mitigation of deleterious changes to metabolic, cardiac, and microvascular function following chronic stress. To this end, an intersectional genetic approach was used to induce Cre-dependent expression of tetanus toxin light chain and inhibit neurotransmitter release from RVLM-projecting IL neurons in male and female rats. Rats were then exposed to 2 weeks of chronic variable stress (CVS). Metabolic function was assessed with a fasted glucose tolerance test. Cardiovascular function was examined with echocardiography and non-invasive hemodynamics. Additionally, microvascular function was quantified via ex-vivo resistance arteriole pressure myography. Our results indicate that glucose tolerance, left ventricular structure, and vascular function are all impacted in a sex-dependent manner. Following chronic stress, circuit-intact females show glucodysregulation characterized by decreased glucose clearance, elevated corticosterone, and insulin insensitivity. Regardless of stress, circuit inhibition in females also impaired glucoregulation but was characterized by elevated glucagon with no compensatory insulin response. Circuit inhibition also increased relative heart size, increased endothelial-dependent vasodilation at both normotensive and hypertensive pressures, and increased myogenic tone and diastolic wall strain. These changes indicate that chronic stress in females leads to broad endocrine-autonomic dysregulation of glucose homeostasis and microvascular function that is exacerbated by IL-RVLM inhibition. While chronic stress in males resulted in an adaptive metabolic response and no changes in normotensive vasodilation, circuit inhibition in chronically-stressed males lead to glucodysregulation and increased endothelial-dependent vasodilation at hypertensive pressures. Additionally, these animals had reduced ventricular wall thickness in diastole. Broadly, these results support the hypothesis that the IL-RVLM circuit is necessary for appropriate glucose homeostasis and vascular function and that circuit inhibition and chronic stress lead to sex-specific responses that may differentially impact the progression of cardiovascular and metabolic disease.
Open Access
Development of a bison-specific embryo culture system through targeted supplementation of media with stage-specific growth factors
(Colorado State University. Libraries, 2024) Acevedo Barriga, Carolina, author; Barfield, Jennifer P., advisor; Dawit, Tesfaye, committee member; Pinedo, Pablo, committee member; McCue, Patrick, committee member; Yuan, Ye, committee member
In vitro embryo production (IVP) offers a practical genetic exchange method for bison herds, eliminating the need for live animal transport and reducing stress on the animals. While successful in cattle, IVP efficiency is lower in bison. This study aims to enhance bison embryo quantity and quality by supplementing IVP media with stage-specific growth factors. Thirteen growth factor receptors (GFRs) were screened in bison and bovine embryos, with six GFRs falling within acceptable ranges. Abattoir-sourced oocytes were used for IVP (4 replicates). GFR expression, notably IGFR2, BMPR2, FGFR1, and EGFR1, peaked in bison embryos at the zygote and 8-16 cell stages, with higher IL6 expression at the morula stage. Bovine embryos displayed highest expression of BMPR2, EGFR, and IGFR1 at zygote and 8-16 cell stages, and of FGFR1 and IGFR2 at zygote stages. Corresponding growth factors were incorporated into bison culture media based on GFR expression. Treatment evaluations, included EGF, IGF1, IGF2, IL6, BMP2, FGF1, and a combination of all GFs to bison culture media. Results revealed that the addition of BMP2 resulted in a decrease in cleavage rates. Notably, EGF, IGF1, and IGF2 enhanced blastocyst rates, with IGF1 significantly higher than the control. Subjective embryo qualitative evaluation showed an upward trend in the number of high quality expanded blastocysts, and lipid content decreased (IGF1) while cell count increased (IGF1, IL6, BMP2, FGF1) with growth factor supplementation. In conclusion, supplementing IVP media with GFs, particularly IGF1 at 50 ng/mL, significantly improved both quantity and quality of bison embryos. Application of IVP theology has advance the use of assisted reproductive technologies for bison, potentially benefiting other species.
Open Access
Estrogen receptors alpha and beta: Opposing roles in hypothalamic-pituitary-adrenal axis function and stress-related behaviors
(Colorado State University. Libraries, 2008) Weiser, Michael James, author; Handa, Robert, advisor
Estradiol has reported effects on mood ranging from anxiogenic to anxiolytic and depressant to anti-depressant. These opposing actions of estradiol may be explained by the existence of two distinct estrogen receptor (ER) systems, ER alpha (ERα) and ER beta (ERβ). Furthermore, there exists a sex difference in stress-related psychiatric disorders such as anxiety and depression, for which women are more susceptible than men. Common to the pathology of these disorders is a dysfunctional hypothalamic-pituitary-adrenal (HPA) axis where glucocorticoid negative feedback is impaired leading to chronically high levels of circulating glucocorticoids. The HPA axis is the main neuroendocrine axis that governs physiological responses to stressors. In rodents, basal and stress-induced activity of the HPA axis is higher in females than in males. This suggests that, if transferable to humans, the sex difference observed in HPA axis function in animal models may help explain the female predisposition for certain psychiatric disorders. The studies described in this dissertation were aimed at characterizing the distinct roles for ERα and ERβ in HPA axis activity and stress-related behaviors. The studies in Chapter 3 examine the effect of estradiol signaling through ERα or ERβ on glucocorticoid negative feedback of the HPA axis. Results indicate that estradiol impairs glucocorticoid-dependent negative feedback by activating ERÎ± specifically at the level of the paraventricular nucleus (PVN). The studies in Chapter 4 examine the effect of estradiol signaling through ERα or ERβ on anxiety-like and depressive-like behaviors. Results indicate that selective activation of ERα is anxiogenic and depressant, whereas selective activation of ERβ is anxiolytic and antidepressant. Finally, the studies in Chapter 5 examine the effect of estradiol signaling through ERβ on behavior and HPA axis activity induced by glucocorticoid receptor (GR) activation in the central nucleus of the amygdala (CeA). Results indicate that delivery of a GR agonist to the CeA is anxiogenic and augments the HPA axis response to a stressor, and peripheral administration of an ERβ agonist blocks this effect. Collectively, these studies point to an antagonistic relationship between estradiol signaling through ERα and ERβ with respect to HPA axis activity and stress-related behaviors.
Open Access
Characterization of periattachment factor
(Colorado State University. Libraries, 2008) Purcell, Scott H., author; Anthony, Russell V., advisor; Seidel, George E., advisor
The purpose of these studies was to characterize expression of periattachment factor (PF) mRNA in the developing sheep conceptus, localize PF in the sheep conceptus, determine if degrading PF mRNA in the sheep conceptus was detrimental to development, and characterize PF in human tissues and cells. Sheep conceptuses were collected on d 11, 13, 15, 16, 17, 21, and 30 post-mating. oPF mRNA exhibited a quadratic expression pattern (P<0.05). No oPF mRNA was detected in d 11 conceptuses. From d 13, oPF mRNA increased to a peak at d 16 before declining. Peak expression of oPF mRNA in the conceptus occurs during rapid elongation and initial attachment of the conceptus to the endometrium. Immunolocalization of PF in a d 15 conceptus showed predominantly nuclear staining in trophectoderm and trophendoderm. Next, embryos collected from superovulated ewes on d 8 post-mating were infected with either a lentivirus expressing a short-hairpin (sh) RNA designed to target PF mRNA for degradation, a lentivirus expressing a shRNA containing 3 mismatched nucleotides, or a control lentivirus expressing no shRNA. Following infection, blastocysts were transferred into recipient ewes and collected back on d 15 of gestation. While 94 and 88% of the control and mismatched shRNA-treated conceptuses elongated by d 15, none of the embryos treated with the lentivirus expressing shRNA against PF mRNA elongated, and most died. This indicates that oPF is required for conceptus elongation and survival. Human PF mRNA was detected in human carcinomas and in the first trimester cytotrophoblast cell line, hTR8. Immunohistochemistry showed PF in the nuclei of carcinomas and in first and second trimester cytotrophoblasts. PF was also present in the invading cytotrophoblast columns. In an in vitro invasion assay of first trimester cytotrophoblasts, hPF mRNA increased from 0, 3, to 12 h as invasion occurred. To further characterize PF in human cells, a lentiviral construct expressing an shRNA targeting the hPF mRNA sequence was developed that resulted in 63% mRNA reduction in BeWo cells.
Open Access
Characterization of mediators of cardiac and renal development in response to increased prenatal testosterone
(Colorado State University. Libraries, 2008) Maresh, Ryan W., author; Anthony, Russell, advisor
Previously reported differences in cardiac and kidney weights of offspring from ewes prenatally treated with testosterone suggested the development of systemic hypertension. To determine if prenatal androgen excess influences the expression of key mediators, angiopoietin 1 (Ang1), angiopoietin 2 (Ang2), tunica interna endothelial cell kinase-2 (Tie2), angiotensin II receptor subtypes 1 and 2 (AT1 and AT2), endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGF R1), VEGF receptor 2 (VEGF R2), insulin receptor β (IRβ), glucose transporter 1 (GLUT1), and glucose transporter 4 (GLUT4) were evaluated from fetal hearts, fetal kidney, left ventricle (LV), right ventricle (RV), and kidney. We hypothesized that mRNA and protein concentrations were altered and that the changes persist into adulthood. At fetal Day 90 a significant increase (p = 0.021) in IRβ mRNA concentration of was seen in the heart, whereas VEGF mRNA concentration was significantly reduced (p = 0.044) in the kidney. In 9 month RV, eNOS mRNA concentration was significantly reduced in the treatment group (p = 0.019). In the 9 month kidney, significant increases in GLUT4 mRNA concentration (p = 0.047) and eNOS protein concentration (p = 0.027) were seen in the treatment group. At 21 months of age, Ang1 mRNA concentration in the treatment group was significantly reduced (p = 0.025) in the LV. AT1 mRNA concentration was significantly decreased (p = 0.004) in the RV of the treatment group. LV eNOS concentration was significantly reduced (p = 0.032), while RV eNOS mRNA concentration was significantly decreased (p < 0.001) in the treatment group. Significant decreases in GLUT1 mRNA concentration were detected in the LV (p = 0.013) and RV (p = 0.006), and RV GLUT4 mRNA concentration was significantly decreased (p = 0.003) in the treatment group. LV IRβ concentration was significantly reduced (p = 0.036) in the treatment group. In the kidney, VEGF R2 mRNA concentration was significantly reduced (p = 0.024) in the treatment group. Taken together, the data supports the development of systemic hypertension and insulin resistance with age following prenatal androgen exposure.
Open Access
The use of chronic models of temporal lobe epilepsy in antiepileptic drug development
(Colorado State University. Libraries, 2007) Grabenstatter, Heidi, author; Dudek, F. Edward, advisor
A chronic animal model with altered ion channels, transmitter receptors and/or neural circuitry similar to temporal lobe epilepsy (TLE) may be useful in the discovery of new antiepileptic drugs (AEDs). The hypothesis was that rats with kainate-induced epilepsy are pharmacosensitive to AEDs, but high doses do not block all spontaneous seizures (i.e., these rats are "pharmacoresistant"). A repeated-measures cross-over protocol was used to show single intraperitoneal injections of topiramate, RWJ-333369, and carbamazepine reduced the frequency of spontaneous motor seizures. The same protocol with administration of 30 mg/kg and 100 mg/kg carbamazepine in specially-formulated food pellets was as effective as intraperitoneal injections, and 100 mg/kg carbamazepine administered in food three times per day completely suppressed motor seizures in 50% of the animals for a prolonged time period (i.e., 24 h) while reducing any stress to the animals. Video-EEG showed carbamazepine preferentially reduced spontaneous convulsive seizures compared to nonconvulsive seizures at 100 mg/kg, reduced seizure duration in some animals at 100 mg/kg, and caused a subtle decrease in the maximum frequency of population spikes during seizures at 30 mg/kg. These data suggest that animal models of TLE with spontaneous seizures can be used efficiently to test AEDs, and that this repeated-measures cross-over protocol is amenable to both dose-effect and time-course-of-recovery studies for the direct comparison of AEDs. This approach can provide statistical power to compensate for seizure clusters and variability across animals. These experiments also show that rats with kainate-induced epilepsy are pharmacosensitive to standard and experimental AEDs; additional studies are required to determine if this model is also pharmacoresistant.
Open Access
Status epilepticus, recurrent seizures, hippocampal damage and the estrous cycle in a model of temporal lobe epilepsy
(Colorado State University. Libraries, 2008) Fawley, Jessica, author; Dudek, F. Edward, advisor
Temporal lobe epilepsy is the most common form of epilepsy and is associated with hippocampal sclerosis and spontaneous recurrent seizures. These pathologies generally develop after a latent period from a precipitating brain injury, which often results in status epilepticus (SE). Sex and hormones have been reported to influence SE and mortality in both clinical and experimental settings. Temporal lobe epilepsy is also associated with an increase in reproductive disorders, which are often the result of altered pulsatile release of luteinizing hormone (LH). Gonadotropin-releasing hormone (GnRH) controls LH release; therefore, reproductive abnormalities associated with epilepsy could hypothetically involve hypothalamic disturbances, particularly to the GnRH network, resulting in altered secretion of GnRH. The aim of this dissertation was to (1) to examine the effects of SE and/or temporal lobe epilepsy on the GnRH neuronal network and (2) utilize recordings of electroencephalogram (EEG) activity to systematically quantify sex and hormone influences on SE and the subsequent recurrent seizures. I report that pilocarpine-induced SE resulted in reproductive alterations in two rodent models of temporal lobe epilepsy, which were not due to a detectable reduction in GnRH-positive neurons. There were no significant differences between the EEG parameters of SE or recurrent seizures between groups. Sex and the stage of the estrous cycle may influence pyramidal cell loss in the hippocampus at 24 h but the stage of the estrous cycle and/or sex do not seem to be predictors of long-term hippocampal damage. In summary, these data do not support the hypothesis that SE and/or temporal lobe epilepsy results in a reduction in the number of GnRH neurons or the hypothesis that sex/cycle stage influences SE, or the progression to temporal lobe epilepsy. However, these models of SE/temporal lobe epilepsy will be useful to further study temporal lobe epilepsy-associated reproductive alterations.
Open Access
The effect of aging on gene expression and mitochondrial DNA in the equine oocyte and follicle
(Colorado State University. Libraries, 2009) Campos-Chillon, Lino Fernando, author; Carnevale, Elaine, advisor
The decline in fertility of aged mares is linked to declining oocyte quality. Oocyte viability is dependant on the ability of oocytes to remain in meiotic arrest until the initiation of maturation and adequate cumulus communication. We hypothesize that aging is associated with quantitative and temporal differences in meiotic arrest and resumption in oocytes, decreased oocyte secretion of paracrine factors and lower mitochondrial numbers, ultimately resulting in a dissociation of oocyte and follicular maturation. The objectives of this study were to clone and determine quantitative and temporal differences in mRNA content of the LH receptor (LHR), amphiregulin (AREG) and epiregulin (EREG) in granulosa cells; PDE4 in cumulus cells; and PDE3A, GPR3, GDF9, BMP15, and mitochondrial DNA (mtDNA) in oocytes during in vivo maturation in young (3-12 yr) and old (>20 yr) mares. Oocytes and follicular cells were collected by transvaginal follicular aspiration. Follicle maturation was induced in estrous mares with a follicle >30 mm by injection of 750 µg of recombinant equine LH. Aspirations were conducted at 0, 6, 9, and 12 h after LH administration. Total RNA was isolated from single denuded oocytes and associated lysed cumulus and granulosa cells. For each gene, mean mRNA copy number for each time point and age group were compared by ANOVA and Fisher's LSD. Regression coefficients were generated to compare oocyte mitochondrial numbers and correlations between gene expression within age groups. Expression of LHR mRNA in granulosa cells was different (p<0.05) between age groups. Young mares displayed a significant drop in LHR mRNA between 0 h and 6, 9, and 12 h; while the pattern of expression in old mares was similar (p>0.05) among times and higher (p<0.05) at 6 h than in young mares. Expression of AREG mRNA in granulosa cells peaked (p<0.05) at 9 h; however, the magnitude of expression at 6 and 9 h was higher (p<0.05) in old than young mares. Similarly, EREG expression peaked (p<0.05) at 9 h in young and old mares but was higher (p<0.05) for old mares. Expression of PDE4D peaked (p<0.05) at 6 and 12 h in old and young mares, respectively. The patterns of expression of GPR3 for oocytes of young and old mares were different and peaked (p<0.05) at 9 and 12 h, respectively. Magnitude of expression of PDE3A for oocytes of old mares at 6 and 9 h was higher (p<0.05) than in young mares. Expression of GDF9 and BMP15 was different (p<0.05) between ages. Mean expression of both genes in the old group was similar over time; however, in young mare oocytes maximum expression was at 6 h (p<0.05). Correlation coefficients between GDF9 and BMP15 for old and young mares were 0.94 and 0.99, respectively. Numbers of copies of oocyte mtDNA did not vary in young mares; however, there was a temporal decrease (p<0.05) of oocyte mitochondrial copy numbers in old mares. The main effect for age for mtDNA was similar for old and young mares.
Open Access
The role of interferon-tau (IFNT) in luteal gene expression, steroidogenesis, and luteal lifespan in the ewe
(Colorado State University. Libraries, 2009) Bott, Rebecca Clark, author; Bruemmer, Jason, advisor; Niswender, Gordon, advisor
Interferon-tau (IFNT) was evaluated for endocrine actions on the corpus luteum (CL). The hypothesis was that infusion of IFNT would increase luteal expression of interferon-stimulated gene (ISG)-15, and the length of time for ewes to return to estrus. Osmotic pumps containing 200 μg IFNT or BSA (n=12 each) were connected to the uterine vein of non-pregnant ewes 10 days post-estrus. Messenger RNA encoding ISG15 was elevated in CL from pregnant and IFNT-infused ewes (P<0.05) compared to nonpregnant and BSA-treated ewes, respectively. Luteal mRNA encoding ISG15 from ewes treated with IFNT was greater than in ewes treated with BSA (P<0.05). Serum concentrations of progesterone were not different in ewes that received infusions of BSA or IFNT. Progesterone decreased by six hours (P<0.05) in ewes that received BSA+PGF or IFNT+PGF, but did not differ in ewes that received infusions of IFNT +/- PGF at 8, 10, or 12 hours after PGF. There were no differences in prostaglandin E synthase (PGES) or prostaglandin F synthase (PGFS), or in prostaglandin dehydrogenase (PGDH), steroidogenic acute regulatory protein (StAR), peripheral type benzodiazepine receptor (PBR), cytochrome P450 side chain cleavage enzyme (CYP-11A), or 3μ-hydroxysteroid dehydrogenase (3μ-HSD). Seven day infusion of IFNT during the time frame of maternal recognition of pregnancy resulted in 20% of IFNT-treated ewes returning to estrus by d19 compared to 100% of BSA-treated ewes (P<0.01). In conclusion IFNT acts systemically, alters gene expression in the corpus luteum, and decreases the number of ewes returning to estrus by d19.
Open Access
Two types of melanopsin retinal ganglion cell in the mouse retina: the regulation of melanopsin expression
(Colorado State University. Libraries, 2009) Baver, Scott Benjamin, author; Tobet, Stuart, advisor
Rods, cones and a subset of retinal ganglion cells (RGCs) that express the photopigment melanopsin are the sensory photoreceptors of the mammalian retina. The light-driven signals that are initiated by the photoreceptors are relayed from the retina to the brain. In addition to the role of light information in regulating the perception of colors, objects and movement, it also controls pupil size and the synchronization of daily physiological rhythms to the day/night cycle. The melanopsin-expressing RGCs, which are intrinsically photosensitive (ipRGCs), contribute especially to these two latter processes. The focus of this dissertation is the ipRGCs of the mouse retina.
Open Access
Energy substrates, metabolic regulators, and lipid accumulation during culture of in vitro-produced bovine embryos
(Colorado State University. Libraries, 2007) Barceló-Fimbres, Moisés, author; Seidel, George E., Jr., advisor
The main objective of this experiment was to optimize in vitro culture conditions for bovine embryonic development, using alternative energy sources and metabolic regulators. Replacing glucose with fructose in culture medium consistently increased blastocyst production per oocyte and decreased lipid content in bovine embryos. The use of phenazine ethosulphate (PES) or fetal calf serum (FCS) supplementation did not affect embryonic development; however, PES consistently decreased, and FCS increased lipid content of embryos compared to the control. There was no effect of glucose or fructose on survival of embryos after cryopreservation by slow freezing or vitrification; however, embryos-treated with PES to reduce lipid content resulted in improved cryotolerance, and FCS decreased cryotolerance compared to the control. Transfer of embryos treated with PES during in vitro culture did not affect pregnancy rates, conceptus losses, or fetal or post-natal development in calves born normally. The sex ratio of calves born was skewed toward males. This effect likely was due to a toxic effect of glucose to female embryos cultured in vitro. Therefore, the more expanded day 7 blastocysts were mostly male embryos.
Open Access
Identification and characterization of two ovine membrane receptors for progesterone
(Colorado State University. Libraries, 2007) Ashley, Ryan L., author; Nett, Terry M., advisor
Classically, progesterone (P4) has been thought to act only through the well-known genomic pathway involving hormone binding to nuclear receptors (PR) and subsequent modulation of gene expression. Alternatively, there is increasing evidence for rapid, nongenomic effects of P4 in a variety of mammalian tissues; however the receptor responsible for these actions is yet to be characterized. The likelihood of a membrane P4 receptor (mPR) causing these events is quite plausible. Sheep are the experimental model often used in our laboratory, because regulation of reproductive function is very similar in ewes and women. Nongenomic actions of P4 have been described in sheep, but the receptor(s) responsible has not been identified. The objective of this dissertation was to isolate and characterize an ovine mPR distinct from the nuclear PR. A membrane protein found to bind P4 was first isolated from porcine liver and is now known as P4 receptor membrane component 1 (PGRMC1). Since PGRMC1 is expressed in a variety of species and tissues, I hypothesized that PGRMC1 was the protein responsible for the nongenomic effects of P4 in sheep. As such, the purpose of my initial studies was to determine if PGRMC1 was expressed in the sheep and further verify if PGRMC1 was present in the plasma membrane of the ovine corpus luteum (CL). A protein lacking homology to the nPRs but homologous to other reported PGRMC1 proteins was isolated from the sheep and contains a single transmembrane domain at the N-terminus. Despite the transmembrane domain, the ovine PGRMC1 displays predominant localization in the endoplasmic reticulum. During my initial studies, another putative mPR was cloned from the seatrout that displayed seven transmembrane domains, indicative of a G-protein-coupled receptor (GPCR) and upon ligand activation, also caused rapid induction of second messenger pathways. As such, I wanted to determine if this mPR was also present in the sheep. An ovine mPR distinct from the nuclear PR was isolated and characterized. The ovine mPR is a 350 amino acid protein that, based on predicted structural analysis, possesses seven transmembrane domains and is expressed in the hypothalamus, pituitary, uterus, ovary and CL. The first characterization of mPR expression throughout the ovine estrous cycle in the hypothalamus, pituitary, uterus, ovary, and CL is also reported. In CHO cells that overexpress a mPR-GFP fusion protein the ovine mPR was uniquely localized to the endoplasmic reticulum and not the plasma membrane. The ovine mPR specifically binds only progestins. Progesterone, 20α-hydroxyprogesterone and 17α-hydroxyprogesterone significantly (P < 0.001) displaced binding of 3H-P4 to membrane fractions from CHO cells expressing ovine mPR. Additionally, the ovine mPR directly induces Ca2+ release from the endoplasmic reticulum upon ligand activation. Further, the ovine mPR appears to activate the MAPK pathway, specifically by phosphorylation of JNK1/2 upon ligand activation. This novel method of signaling at the endoplasmic reticulum adds to the intricacy of signaling in cells and provides a mechanistically unique method for initiating actions of P4 that may alter classical dogma regarding the mechanisms by which steroid hormones act.
Open Access
Mechanism of neuronal cell death in canine glaucoma
(Colorado State University. Libraries, 2009) Alyahya, Khaleel I., author; Madl, James E., advisor
Glaucoma is one of the most important causes of blindness in human and dogs. Glaucoma is characterized by a progressive loss of retinal ganglion cells that is often associated with increased intraocular pressure and decreased retinal blood flow. In previous investigations, we found that changes in glutamate distribution occur selectively in damaged areas of retinas of dogs with primary glaucoma. This glutamate redistribution is consistent with high levels of extracellular glutamate (â†‘GluE) contributing to excitotoxic damage to neurons. In this dissertation, we used immunohistochemical methods to test three mechanisms by which this glutamate redistribution may occur in retinas from clinical cases of canine glaucoma. First, we tested if ischemia due to microvessel loss causes the changes in glutamate distribution. We found significantly lower microvessel density in damaged regions, consistent with ischemia occurring in canine glaucoma. Second, we tested if loss of glutamine synthetase induces the glutamate redistribution. We have found significantly decreased amounts of glutamine synthetase in areas with neuronal damage and glutamate redistribution, consistence with decreased glutamate synthetase contributing to glutamate redistribution. Third, leakage of glutamate from blood vessels from inflamed areas may lead to glutamate redistribution. We found that there is albumin leakage from blood vessels in damaged regions with other inflammatory indicators in those areas. The smaller size of glutamate suggests that it should also diffuse out of blood into the extracellular fluid of the retina even more readily than albumin. Increase leakiness of blood vessels in canine glaucoma is consistent with glutamate leakage contributing to glutamate redistribution. In conclusion, our results are consistent with all three mechanisms contributing to glutamate redistribution in canine glaucoma. The dissertation includes further discussion of more refined hypotheses of the mechanisms by which glutamate redistribution and neuronal damage may occur.
Open Access
Functional organization of a cortical-medullary neural circuit mediating organismal adaptation to stress
(Colorado State University. Libraries, 2023) Pace, Sebastian A., author; Myers, Brent, advisor; Hentges, Shane, advisor; Tobet, Stuart, committee member; Foster, Michelle, committee member
Hindbrain regions responsible for epinephrine and norepinephrine production are critical for orchestrating stress responses, maintaining physiological equilibrium and integrating afferent information. The nuclei central to hindbrain epinephrine and norepinephrine production, create a neural network that interfaces with forebrain and spinal cord regions, facilitating the integration of neuroendocrine and autonomic functions. Despite significant strides in our comprehension of stress response systems, questions concerning the roles of sex, stress history, and circuit mechanisms endure. In this study, we unveil and characterize a prefrontal-medullary circuit crucial for the suppression of stress responses. First, anterograde and retrograde tract-tracing studies demonstrated a stress-reactive vmPFC-RVLM circuit. Activation of this vmPFC-RVLM circuit mitigates glucocorticoid stress reactivity in both males and females, by targeting non-catecholaminergic neurons. Therefore, vmPFC-RVLM circuit activation may utilize local inhibitory neurons to limit catecholaminergic activation. To better understand how chronic stress affects the medulla, we explored the impact of chronic stress on signaling machinery and revealed elevated tyrosine hydroxylase (TH) levels in both male and female rats following chronic variable stress (CVS). To understand how CVS interacts with the vmPFC-RVLM circuit, we used an intersectional TeLC (Tetanus toxin - light chain) approach to disrupt the circuit and evaluate multiple stress response systems. In males, circuit disruption and CVS largely left behavioral and cardiovascular stress reactivity unaltered, however, some neuroendocrine endpoints were affected. Conversely, females exposed to circuit disruption and chronic stress exhibited heightened stress reactivity in glycemic, corticosterone, and arterial pressure responses, coupled with avoidant-like behaviors. These findings underscore the sex-specific necessity of the vmPFC-RVLM circuit in countering chronic stress-related outcomes, emphasizing a greater protective role in females relative to males. To gain deeper insights into the role of vmPFC inputs to the RVLM in females, we once again utilized a circuit-based TeLC approach, employing in situ hybridization (ISH) coupled with immunohistochemistry (IHC) to assess TH and phenylethanolamine N-methyltransferase (PNMT) transcript density across various VLM subregions. Notably, the TeLC-induced elevation of PNMT expression in females suggests that disrupting this circuit could potentially enhance epinephrine production by RVLM neurons, potentially intensifying stress reactivity post-CVS. This comprehensive study demonstrated the critical role of the vmPFC-RVLM circuit in modulating stress responses and revealing female-specific effects in mitigating physiological, behavioral, and transcriptional outcomes after chronic stress. These findings emphasize the significance of the vmPFC-RVLM circuit in managing stress reactivity in the context of chronic stress and identify the circuit as a potential candidate for reducing stress responding.
Embargo
An investigation of synaptic vesicle docking and priming and a proposed method for quantitatively measuring both in Drosophila using electron tomography
(Colorado State University. Libraries, 2023) Twiggs, Jasmin A., author; Reist, Noreen, advisor; Hoerndli, Frederic, committee member; Hoke, Kim, committee member; Tamkun, Michael, committee member
The nervous system, as the body's command center, plays a crucial role in cellular communication within the brain and between the brain and other body systems. Neurons, the individual cellular units, transmit electrical information and communicate with other cells through neurotransmitter release in response to electrical stimuli. Chapter 1 introduces the foundational concepts of neuronal structure and function and delves into the mechanisms underlying neurotransmitter release. Special attention is given to the neuromuscular junction (NMJ), a well-studied chemical synapse crucial for muscle movement. The synaptic vesicle cycle is introduced, with particular emphasis on docking and priming. The significance of active zones, specialized sites for efficient signal transmission, and their associated structural components are underscored. Synaptotagmin, a pivotal protein in calcium-triggered vesicle fusion, is discussed with emphasis on its C2B polylysine motif. Throughout the chapter, the utility of Drosophila as a model system for studying synaptic processes, particularly at the NMJ, is emphasized. In sum, Chapter 1 provides the foundational knowledge essential for comprehending the intricate cellular and molecular facets of synaptic communication within the nervous system, serving as a precursor to subsequent chapters' investigations. Chapter 2 examines synaptotagmin's C2B polylysine motif and its role in synaptic vesicle docking at the Drosophila NMJ. It explores the polylysine motif's potential involvement in endocytosis, demonstrates an unaffected interaction with AP-2, and uses electron microscopy to find no significant changes in vesicle distribution. The findings suggest that the reduced neurotransmitter release in the polylysine mutant is likely due to an impairment in vesicle priming. Chapter 3 introduces a method for studying synaptic vesicle docking and priming in Drosophila, using electron tomography. I address the limitations of conventional electron microscopy and underscore the need for higher-resolution techniques to assess molecular structures that mediate physiological processes. Chapter 3 also emphasizes the significance of the contact area between docked vesicles and the presynaptic membrane as a correlate of vesicle priming. The protocol, expected results, and key considerations are discussed. The methods presented in Chapter 3 offer a promising approach for understanding synaptic processes. In Chapter 4, I discuss key considerations for when standard electron microscopy can be used for assessing vesicle docking. Then, I discuss how the electron tomography method presented in Chapter 3 could not only confirm the results found in Chapter 2, that the synaptotagmin C2B polylysine motif is not implicated in vesicle docking but could also be used to directly test the mutant's role in priming. Specific aims for future studies on the synaptotagmin polylysine mutation in Drosophila are presented, potential results and interpretations are discussed. Finally, I showcase interesting, unpublished findings from electron tomograms I have taken at the Drosophila NMJ and discuss their potential significance.
Embargo
IQGAP1 is a novel effector of gonadotropin-releasing hormone receptor signaling
(Colorado State University. Libraries, 2023) Alqahtani, Huda A., author; Amberg, Gregory, advisor; Clay, Colin, committee member; Tamkun, Michael, committee member; DeLuca, Jennifer, committee member
Stimulation of gonadotropin-releasing hormone (GnRH) receptors on the surface of anterior pituitary gonadotrope cells is a key signaling event for the hypothalamic-pituitary-gonadal axis. One important downstream component of GnRH receptor signaling is activation of the mitogen-activated protein kinase ERK (extracellular signal-regulated kinase), which is essential for the production of the gonadotropin luteinizing hormone. Evidence suggests that GnRH receptors reside in low-density plasma membrane domains where they participate in multiprotein signaling complexes. Here we used quantitative proteomics to identify proteins associated with low-density plasma membrane domains and to measure changes in their relative abundance in these domains in response to GnRH. Using αT3-1 gonadotropes, we identified 537 proteins in detergent-free subcellular fractions containing low-density plasma membranes. SILAC (stable isotope labeling by amino acids in cell culture) in combination with mass spectrometry demonstrated that GnRH, within 10 min, altered the association of 87 proteins with this plasma membrane fraction. Ontology analysis revealed that GnRH promoted an enrichment of actin cytoskeletal and adherens junction-related proteins including the molecular scaffold IQGAP1 and the small GTPase Rac1. Subsequent investigation revealed that the association between Rac1 and IQGAP1 increased with GnRH receptor stimulation and that GnRH increased Rac1 activity. Demonstrating functional relevance, inhibiting Rac1 reduced GnRH-dependent ERK activation. Our data reveals an upstream activation of signaling and structural molecules, including Ca2+, CDC42 and Rac1, E-cadherin, N-cadherin, and β-catenin. We also identified interactions between the scaffold protein IQGAP1 and these molecules, indicating that IQGAP1 is a fundamental regulator of GnRH-dependent signaling in gonadotropes. Furthermore, our data shows that IQGAP1 has a transcriptional regulatory role in gonadotropes treated with GnRH. In sum, these data indicate that IQGAP1 complexed with Rac1 modulates ERK activity and as such serves as an essential effector in modulating cell polarity and cell-cell contacts in gonadotropes. Altogether, our proteomics data show that acute stimulation of GnRH receptors (3 nM for 10 min) alters the PAM fraction abundance of proteins, such as IQGAP1, mechanistically linked to gonadotrope activation.
Embargo
Evaluation of feral swine as potential reservoirs, sentinels, and vectors of emerging and re-emerging zoonotic pathogens in the United States
(Colorado State University. Libraries, 2023) Maison, Rachel Marie, author; Bosco-Lauth, Angela, advisor; Bowen, Richard, advisor; Borlee, Bradley, committee member; Brown, Vienna, committee member; Han, Sushan, committee member
Feral swine are an extremely adaptable and prolific invasive species present in many regions of the United States. A generalist diet, high reproductive capacity, and opportunistic nature make them a significant threat to native flora and fauna, and their destructive foraging behaviors have been attributed to substantial crop loss and property damage throughout their range. Feral swine have also been demonstrated as vectors and reservoirs for many diseases, some of which are transmissible to humans. Despite the efforts of government agencies to impede range expansion of feral swine and monitor populations for disease, human-mediated movements as well as continued natural dispersal in some regions have made management difficult. Subsequently, this invasive species continues to threaten human and animal health throughout its current range. Reported herein are a series of laboratory and field-based investigations that seek to address the potential role(s) of feral swine in the epidemiology of a few zoonotic pathogens that are emerging or re-emerging in the United States. As pigs interact closely with soils through their natural rooting and wallowing behaviors, we were primarily interested in soil-dwelling zoonoses, and identified three of particular interest. The two bacterial species Bacillus anthracis and Burkholderia pseudomallei, as well as the fungus Coccidioides, are the causative agents of anthrax, melioidosis, and coccidioidomycosis respectively, and each is understudied as they relate to feral swine. While each of these organisms is unique in their biology and the relative threat(s) they've posed to humans and animals throughout their respective histories, they are similar in that much of their ecology remains undescribed. The often-complex ecological relationships exhibited by pathogenic organisms are vital to their continued survival and evolution and can play an important role in their dissemination to human populations. The presence of high-density populations of feral swine in many regions of the United States, as well as their ability to occupy a range of habitats, may be playing a significant role in the persistence and dissemination of these organisms. Furthermore, the interactions of feral swine with contaminated substrates within their environment, as well as high levels of interspecies interactions throughout their current range could make them a likely source of additional emerging or novel pathogens. After a summary of the literature concerning feral swine in the United States, followed by that for the causative agents of anthrax, coccidioidomycosis, melioidosis, and a few select emerging disease threats, each respective chapter within this work details investigations largely by pathogen. Within Chapter 2, we present the results of a field serosurvey in feral swine residing across regions of known anthrax endemicity in Texas, and retrospectively document bacterial exposure via enzyme-linked immunosorbent assay (ELISA). This study was performed principally to evaluate the biosentinel utility of feral swine for anthrax contaminated environments, and the ELISA utilized was developed and optimized for detecting antibodies of swine origin to B. anthracis protective antigen. We additionally report on a laboratory-based experimental infection study where which a group of juvenile feral swine were either intranasally or subcutaneously exposed to varying amounts of B. anthracis strain Sterne 34F2 spores, and seroconversion as well as bacterial shedding through the nasal passages documented over time. Through both the field-based serosurvey, as well as the experimental inoculation study, we report that feral swine serology may be used as a management strategy to indirectly identify regions contaminated with anthrax bacteria. Moreover, we report that some feral swine intranasally exposed to high amounts of B. anthracis Sterne contained detectable and viable spores within their nasal passages up to 56 days past their initial exposure event as demonstrated by bacterial culture. The presence of viable B. anthracis within the nasal mucosa well after an exposure event suggests that feral swine may be capable of spreading infectious anthrax bacteria as mechanical vectors. Chapter 3 of this work details an experimental infection study similar to that reported in the previous chapter, however, instead intranasally exposes a group of juvenile feral swine to Coccidioides posadasii strain Silveira spores. This study was performed to evaluate the pathogenesis and immune response of feral swine to one of the causative agents of coccidioidomycosis. Fungal culture of tissues collected from inoculated individuals at necropsy was performed to assess whether animals might act as reservoirs for the fungus in addition to describing the characteristics of infection. Feral swine utilized for this study did not display overt clinical signs of disease, and despite being inoculated with a large dose of fungal spores, also did not seroconvert based on the results of porcine agar gel immunodiffusion assays. Fungal culture of lung and mediastinal lymph node tissues of a subset of pigs revealed the presence of viable C. posadasii, however, examination of additional sections of the same tissues did not reveal the presence of the granulomatous lesions often associated with coccidioidomycosis. Most individuals had significant comorbidities as illustrated via histology, most notably Metastrongylus nematodes in the lungs. Despite an absence of lesions and organisms histologically, isolation of the fungus from tissues by culture indicated active infection and imply that feral swine are mildly susceptible to acute Coccidioides infection. These results suggest that in some instances, feral swine may aid in fungal dispersal on the landscape due to the presence of spherules in tissues post-mortem. Chapter 4, concerning melioidosis, reports on laboratory-based experiments evaluating an indirect ELISA for measuring antibodies to B. pseudomallei in domestic and wild swine. Described previously to test human sera, this ELISA utilized whole-cell lysate derived from B. pseudomallei strain Bp82 and was optimized and preliminarily validated by testing sera from domestic goats and domestic swine experimentally inoculated with virulent and avirulent strains of B. pseudomallei, respectively. Further evaluation of assay specificity was performed by testing sera against similar antigen preparations of the closely related bacterial species Pseudomonas aeruginosa strain PAO1. Examination of sera from laboratory animals against Bp82 and PAO1 whole-cell lysate antigens revealed that most animals seroconverted and displayed B. pseudomallei antibodies, and that little cross-reactivity existed between antigens. After preliminary assay validation, serum from wild pig populations originating from Arizona, California, and Puerto Rico were assessed using both antigens to document B. pseudomallei exposure in wild pigs residing in regions where bacterial absence or endemicity can be confidently inferred at this time. In stark contrast to the serology displayed by laboratory animals, analysis of field sera collected from these regions demonstrated high levels of cross-reactivity between Bp82 and PAO1 antigens, suggesting the assay is not suitable for use in wild pig populations. Lastly, Chapter 5 describes a general survey of feral swine removed from the Aransas National Wildlife Refuge in Texas for each of the organisms investigated in the proceeding chapters, as well as additional bacterial and viral pathogens; added pathogens that were included within this survey included Francisella tularensis, and SARS-CoV-2, the causative agents of tularemia and COVID-19, respectively. A suite of samples, including external parasites (e.g., ticks), nasal swabs, blood, and a series of tissues were collected from each feral swine in the field and analyzed by bacterial and fungal culture, serology, and viral assays to determine if any pigs were actively infected or previously exposed to a range of pathogens. General and selective culture of tissue samples did not reveal active infection with B. anthracis, Coccidioides spp., B. pseudomallei, or other pathogenic bacteria, however, serology illustrated that a subset of pigs were previously exposed to B. anthracis. Further examination of tissues histologically illustrated a high degree of parasitic infection in the majority of pigs, particularly with Metastrongylus nematodes. Finally, three ixodid tick species, all adults, were collected off of the majority of feral swine sampled, none of which appeared to be carrying infectious virus based on the results of general cytopathic effects assays. Results generated for this study further confirm that feral swine are hosts for a range of ixodid ticks present in Texas and include species that are known to pose a risk to the health of livestock, wildlife, and humans. Moreover, serological results indicating previous exposure to anthrax-causing bacteria agree with past ecological modeling studies that have suggested environmental suitability for B. anthracis in the region of Aransas National Wildlife Refuge. Taken together, these investigations demonstrate that feral swine in the United States very likely are contributing to the ecology of anthrax and coccidioidomycosis by being mechanical vectors and possible reservoirs of B. anthracis and Coccidioides, respectively. Serological data demonstrated from field and laboratory studies additionally support the use of feral swine as biosentinels within landscapes undergoing invasive species management and that may be contaminated with B. anthracis. While the serological data generated through the B. pseudomallei investigations reported here currently do not support use of a crude whole-cell lysate-based ELISA to evaluate samples collected from the field, we hypothesize that feral swine may be reservoirs and/or vectors of melioidosis-causing bacteria. Future investigations should continue to examine additional antigens for serologic testing, as laboratory-infected swine demonstrated a measurable antibody response after intranasal exposure with Bp82. Additional follow-up studies should also be conducted to further describe the relative role(s) that feral swine may play in the ecology and epidemiology of B. anthracis and Coccidioides, particularly if regions with high incidence of animal or human disease correspond with high densities and activity of feral swine. Future research should continue to screen feral swine as well as the ectoparasites that feed upon them for emerging or re-emerging pathogens, especially in areas with high biodiversity or species of conservation concern, as well as in areas with high levels of human activity.