abstract: Emerging infectious diseases (EIDs) in vulnerable populations are a proposed cause of reduced global biodiversity due to local and regional extinctions. Chytridiomycosis, a fungal disease caused by Batrachochytrium dendrobatidis (Bd), is affecting amphibian populations worldwide. Chapter 1 of this thesis reports using lab-raised larval tiger salamanders (Ambystoma tigrinum nebulosum), collected as eggs, to test if Bd infects them. Bd infects metamorphosed tiger salamanders; however, it is currently unknown if larvae can be infected by Bd. Adult frogs tend to host Bd on ventral surfaces and hind legs while tadpoles host Bd in keratinized mouthparts. No research has considered differences in infection between life stages of salamanders. It was hypothesized that Bd can colonize larvae in the same manner as metamorphosed animals. Larval salamanders were inoculated to test if Bd concentrations differ among body regions in larvae compared to metamorphosed salamanders. Larvae can carry Bd with the concentration of Bd varying between body region. Chapter 2 report using native tiger salamanders (Ambystoma tigrinum nebulosum), from northern Arizona and Bd as a study system to test if Bd is native or introduced to Arizona. It was hypothesized that Bd is not endemic to Arizona, but is introduced. There are multiple hypotheses regarding potential routes Bd may have traveled through Arizona and into Mexico. These hypotheses were tested using the Kaibab Plateau in Coconino County, Arizona, as a study site. The plateau is isolated from surrounding areas by the Grand Canyon to the south and the Vermillion Cliffs to the north serving as major biogeographical barriers. It is hypothesized that tiger salamanders are not dispersing into or out of the Kaibab Plateau due to geological restrictions. Bd, therefore, should not be present on salamanders on the Kaibab Plateau due to geological restriction. Tiger salamanders in stock tanks located on the Kaibab as well as preserved museum specimens housed in the Arizona State University Natural History Collection were sampled. The results indicate that Bd occurs at low levels on Kaibab Plateau tiger salamanders. / Dissertation/Thesis / Masters Thesis Biology 2019

The effects of an organophosphorus pesticide, methyl parathion, on cellular immune defense mechanisms of the giant black tiger shrimp (Penaeus monodon) were studied. Animals were exposed for 96 hours at concentrations equal to, above and below the LC&\sb{lcub}50{rcub}& (3 ppb). Phagocytes were obtained from the heart and circulating hemolymph. Cellular immune responses, including chemotaxis, phagocytosis and the respiratory burst were examined. Chemotactic activity was determined by means of a modified Boyden double chamber technique. There was a dose-dependent decrease in the chemotactic activities of both circulating hemocytes and cardiac phagocytes. Phagocytic activity (percent phagocytosis) was examined by the microscopic enumeration of phagocytes which had internalized yeast cells. For both circulating and cardiac phagocytes, there was a decreasing trend in the phagocytosis of yeast cells as the concentration of methyl parathion was increased. Exposure to methyl parathion at and above 2 ppb resulted in a significant reduction in phagocytic activity for both cell types as compared to the control group. The phagocytic index for circulating and cardiac cells showed a decreasing trend with increasing concentrations with significant differences at and above 6 ppb. Since the measurement of superoxide has been accepted as an accurate way to quantify the intensity of the respiratory burst, superoxide production by hemocytes was measured by the reduction of the redox dye nitroblue tetrazolium (NBT). Both circulating and cardiac phagocytes exhibited significant increases in superoxide production at 6 and 10 ppb, as well as at 3 ppb in the case of cardiac phagocytes. Histopathological changes of the cells were observed in the gill, hepatopancreas, heart, muscle and ventral nerve, with the hepatopancreas containing the most pronounced changes. Electron microscopy revealed granular damage of the circulating hemocytes at 3, 6 and 10 ppb. These experiments indicate that methyl parathion alters cellular immune responses of Penaeus monodon in a dose-dependent manner. Further investigation of these immunological mechanisms is needed to explain the phenomenon of shrimp survival in contaminated environments.

The mechanisms by which the facultative intracellular pathogen Francisella tularensis is recognized by the innate immune system and the strategies that F. tularensis uses to avoid this recognition are not well understood. We have identified the basic components of the inflammasome that assemble in response to F. tularensis Live Vaccine Strain (LVS) challenge as containing the cysteine protease caspase-1, the adaptor protein ASC and the PYHIN molecule AIM2. We have also shown here that the nucleotide-binding domain leucine-rich repeat containing receptors (NLRs), NLRC4, NLRP3, NLRP6, NLRP10, and NLRP12 were not necessary for activation of caspase-1 and subsequent IL-1β secretion in response to challenge with F. tularensis LVS in vitro. In vivo, NLRC4, NLRP3, NLRP6, NLRP10, and NLRP12 did not appear to enhance survival. However, caspase-1- and ASC-deficient mice succumbed more rapidly to infection, indicating that the inflammasome played a role in defense against F. tularensis LVS. Additionally, we identified a gene with homology to Escherichia coli mviN, a putative lipid II flippase, that functions as a F. tularensis virulence factor. In vivo infection of mice with a F. tularensis LVS mviN transposon mutant (mviN::Tn5) resulted in improved host survival and decreased bacterial burdens compared to infection with wild-type F. tularensis LVS. Wild-type F. tularensis LVS and the mviN::Tn5 mutant replicated at a similar rate in both macrophages and liquid broth culture. Additionally, the ability to induce the production of TNF-α or IL-6 was also similar between WT F. tularensis and the mviN::Tn5 mutant. In contrast to the similar levels of production of IL-6 and TNF-α, the mviN mutant induced increased AIM2 inflammasome-dependent IL-1β secretion and cytotoxicity in macrophages compared to wild-type F. tularensis. The compromised in vivo virulence associated with the mutation of mviN was dependent upon inflammasome activation, as caspase-1- and ASC-deficient mice did not exhibit preferential survival following infection. These data show that F. tularensis LVS activation of the inflammasome is caspase-1-, ASC-, and AIM2-dependent. These data also identify mviN as a novel F. tularensis virulence factor that enables F. tularensis LVS to evade some AIM2 inflammasome activation.

Endotoxins (E) are a unique and abundant family of glycolipids located in the outer leaflet of the outer membrane of Gram-negative bacteria. Host immune responses to endotoxin depend on ordered endotoxin-host protein interactions, resulting in delivery of an endotoxin monomer to MD-2 which acts as a potent agonist of Toll-Like Receptor (TLR) 4. Activated TLR4 is unique among TLRs in its ability to mobilize two distinct intracellular signaling pathways: the MyD88- and TRIF-dependent pathways. The regulated action of both pathways is likely important for optimal host immune responses to Gram-negative bacterial infection, but how this is achieved is not well understood Recent studies have indicated an essential role for host CD14 in TRIF-dependent signaling by activated TLR4 but the extent to which these observations reflect a general role of CD14 in endotoxin-triggered TRIF signaling or one more narrowly restricted to the specific endotoxins and/or cell types used is uncertain. We have addressed this question by identifying a novel CD14-independent mechanism for efficient delivery of E monomer to MD-2 and TLR4 activation, that is mediated by endotoxin.albumin complexes. We have used these complexes to demonstrate CD14-independent activation of MD-2⋅TLR4 by a wider range of endotoxin species than previously thought possible and activation of both MyD88- and TRIF-dependent pathways. Taken together, the findings in this thesis indicate that the molecular structure and physical presentation of endotoxin as well as CD14-independent properties of the host cell help determine the extent to which CD14 is required for TRIF-dependent signaling by activated TLR4.

Infections in the central nervous system pose a considerable challenge to the host. On one hand, a quick and rapid immune response is important to control the infection, while on the other hand, too robust a response can damage the CNS, which has poor regenerative properties. Therefore, nowhere else in the body is such a balance between pro- and anti-inflammatory mediators as important. Mice infected with the coronavirus, mouse hepatitis virus strain J2.2-V-1, are a useful model for understanding the two sides of the immune system. In this neurotropic viral infection, demyelination occurs secondary to the immune response's control of the viral infection. Thus, J2.2-V-1 infection also functions as an infectious animal model for multiple sclerosis (MS). Many arms of the pro-inflammatory immune system have been studied during J2.2-V-1 infection but the anti-inflammatory immune response has not been thoroughly investigated prior to this study. The data demonstrated here represent an in-depth look into the role of regulatory T cells and IL-10 during J2.2-V-1 infection. Specifically, by adoptive transfer of Tregs, I show that there is a relative paucity of Tregs during J2.2-V-1 infection in C57BL/6 mice and their addition decreases clinical scores, demyelination and the T cell response during infection without affecting viral clearance. A RAG1-/- adoptive transfer model demonstrates clinical results consistent with results obtained in B6 mice, while further demonstrating that Tregs function in the draining cervical lymph node by dampening dendritic cell activation and pro-inflammatory chemokine and cytokine release. There is also a relative decrease in T cell proliferation. Thus, Tregs are protective in J2.2-V-1-induced encephalomyelitis and their enhancement is a potential therapy for MS. Additionally, IL-10 is an important anti-inflammatory component of the immune response, as its absence causes increased immunopathology with increased demyelination in J2.2-V-1-infected B6 mice. Through the development of a recombinant J2.2-V-1 virus that produces IL-10, I also demonstrate that increasing the level of IL-10 at the site of infection is protective early in the immune response. Antigen-specific IFN-γ+ CD4 and CD8 T cells produce IL-10 at the height of the inflammation. CD8 T cells require a high level of antigen stimulation and the most recently activated CD69+CD8 T cells express high levels of IL-10. Additionally, this IL-10 expression is transient in both CD4 and CD8 T cells, presumably only by the recently stimulated cells. Through microarray analysis, protein expression and cytolytic assay, I show that IL-10+CD8 T cells are more activated than IL-10-CD8 T cells. Nonetheless, the IL-10 produced is anti-inflammatory and its production in CD8 T cells is protective in J2.2-V-1-infected mice. Thus, the most activated and cytotoxic CD8 T cells self-regulate the immune response through the production of IL-10. Overall, these studies show that the anti-inflammatory component of the immune system is vital to protecting the host from the immunopathology that occurs during J2.2-V-1 virus clearance. Specifically, the addition of Tregs and IL-10 helps ameliorate clinical disease and demyelination. These studies suggest that increasing Tregs and/or increasing the cytokine IL-10 in patients with MS may have therapeutic potential.

Pediatric acute respiratory tract infections (ARTIs) are a leading cause of morbidity and mortality. The objectives of this study were to describe the epidemiology of polymicrobial ARTI in a hospital-based pediatric population and to investigate the association of polymicrobial infection and severity of illness. We conducted a retrospective study of 559 archived respiratory specimens from 421 children under the age of 10 years collected from March 28, 2008 through June 30, 2009 and stored by the University of Iowa Hospital and Clinics Clinical Microbiology Laboratory. Specimens were tested by immunofluorescence assay and/or viral culture at the time of collection (influenza A and B, parainfluenza [PIV] 1-3, respiratory syncytial virus [RSV], adenovirus [Ad]) and uniformly by RT-PCR (human metapneumovirus [hMPV], rhinovirus [HRV], human bocavirus [HBoV]) and PCR (Ad) for the current study. Demographic and clinical data were abstracted from electronic medical records. Results from this study suggest that polymicrobial respiratory tract infections are common in this population. A virus was identified in 61.3% of 349 respiratory specimens from children with confirmed or suspected ARTI. HRV (27.5%), RSV (18.9%), HBoV (8.3%), hMPV (7.7%), and PIV (6.6%) were the most common viruses detected. A viral coinfection was identified in 21.5% of the 214 virus-positive specimens and was most often detected for Ad (53.3% of 15 Ad-positive specimens), HBoV (51.7% of 29 HBoV-positive specimens), PIV (43.5% of 23 PIV-positive specimens), HRV (35.4% of 96 HRV-positive specimens), and RSV (34.8% of 66 RSV-positive specimens). Among the 46 specimens with dual or triple viral coinfections detected, the most frequent virus-virus combination was HRV-RSV (n=12). We hypothesized that certain host-specific risk factors were associated with the likelihood of viral coinfection. While none of the covariates in the final model were significant, the results were suggestive. Male gender (OR 1.70, 95% CI 0.83-3.46), age between 6 months to 1 year (as compared to children less than 6 months old, OR 2.15, 95% CI 0.75-6.19), and history of any chronic condition that may result in immunosuppression (OR 2.05, 95% CI 0.99-4.23) were each associated with increased odds of viral coinfection (p > 0.05). We also hypothesized that children with coinfections would be more likely to have severe ARTI. Children with viral-bacterial coinfection, as compared to children with viral mono-infection, were more likely to be admitted to an intensive care unit (OR 6.00, 95% CI 2.51-14.33) even after controlling for age, history of prematurity, urban/rural residence, and leukocytosis. This study will inform medical and public health professionals with regard to the epidemiology of polymicrobial infections and their potential importance as a cause of severe acute respiratory tract infection in children. Furthermore, results of this study may contribute to the ongoing discussion of the importance of diagnostic ability to reliably detect multiple concurrent pathogens in a single individual.

Next generation monoclonal antibodies (mAbs) are unique in that they are specifically designed to enhance their mechanisms of action, primarily complement fixation and antibody-dependent cellular cytotoxicity (ADCC). Recent studies suggest that complement-fixing properties of a mAb can counter its ability to activate NK cells and mediate ADCC. GA101, a third generation (type II anti-CD20) mAb, and rituximab-MAGE (glyco-engineered type I mAb) show enhanced ADCC and direct cell killing; while ofatumumab, a second generation anti-CD20 mAb, shows enhanced complement-mediated cytotoxicity (CMC). These studies set out to determine the primary mechanisms of actions of these various mAbs, and compare the effect of complement on their ability to activate NK cells and mediate ADCC or CMC. We also studied the efficiency of rituximab vs. rituximab-MAGE to deplete B-cells in vivo in mice expressing human transgenic CD20. In vitro, rituximab and ofatumumab fixed more complement and mediated a greater degree of CMC, than GA101 and rituximab-MAGE. Additionally, complement inhibited the ability of both rituximab and ofatumumab to bind to and activate NK cells, whereas, addition of complement to GA101 or rituximab-MAGE did not affect their NK cell activating ability. Complement also blocked rituximab-induced NK-cell mediated ADCC, but not GA101-induced NK-cell mediated ADCC. Finally, GA101 and rituximab-MAGE depleted a higher percentage of B cells in whole blood compared to rituximab and ofatumumab, whereas rituximab-MAGE depleted fewer B cells, in vivo, in a complement-dependent fashion. We conclude from these studies that there are significant differences among these antibodies and that the ability of a given antibody to mediate CMC and complement fixation correlates with the ability of complement to block the interaction between the antibody and NK cells.

The application of spatial methods to epidemic estimation and prediction problems is a vibrant and active area of research. In many cases, however, well thought out and laboratory supported models for epidemic patterns may be easy to specify but extremely difficult to fit efficiently. While this problem exists in many scientific disciplines, epidemic modeling is particularly prone to this challenge due to the rate at which the problem scope grows as a function of the size of the spatial and temporal domains involved. An additional barrier to widespread use of spatiotemporal epidemic models is the lack of user friendly software packages capable of fitting them. In particular, compartmental epidemic models are easy to understand, but in most cases difficult to fit. This class of epidemic models describes a set of states, or compartments, which captures the disease progression in a population. This dissertation attempts to expand the problem scope to which spatio-temporal compartmental epidemic models are applicable both computationally and practically. In particular, a general family of spatially heterogeneous SEIRS models is developed alongside a software library with the dual goals of high computational performance and ease of use in fitting models in this class. We emphasize the task of model specification, and develop a framework describing the components of epidemic behavior. In addition, we establish methods to estimate and interpret reproductive numbers, which are of fundamental importance to the study of infectious disease. Finally, we demonstrate the application of these techniques both under simulation, and in the context of a diverse set of real diseases, including Ebola Virus Disease, Smallpox, Methicillin-resistant Staphylococcus aureus, and Influenza.

Many college students exercise individually or participate in collegiate and intramural sports in addition to fulfilling their stressful academic requirements. The combination of accumulated stress and vigorous exercise could result in an impaired immune system, prompting the onset of disease and absences in class and sports practice. Twenty-six male and female participants aged 18 to 23 were recruited for this study. Over the course of an academic semester, participants completed weekly electronic surveys documenting stress levels, aerobic exercise, and symptoms related to upper respiratory tract infections. Participants were evaluated at four different time points (Baseline, Post-Midterm Exam, Baseline Reassessment, and Post-Final Exam) for body fat percentage, cardiovascular fitness, heart rate, blood pressure, and a 10mL blood draw. Blood samples were used to measure blood glucose, cortisol, IL-6, and CD11b levels. Analysis of cortisol and IL-6 concentrations required ELISA kits for protein quantification in plasma samples. CD11b levels in peripheral blood mononuclear cell samples were measured by Western Blot analysis. There was a significant increase in blood pressure during the final exam compared to rest for systolic (p=0.005) and diastolic (p=0.004) blood pressures. There was a significant decrease in anxiety during the final exam compared to anxiety during the mid-term exam (p=0.022). The acute stress of an exam was strong enough to illicit physiologic blood pressure change, but the chronic stress throughout the semester was not intense enough did not illicit physiologic or immune responses. The volume of aerobic exercise in the vigorous workout group was not great enough to influence immune responses nor disease incidence.

Mastitis represents one of the major economical and animal welfare concerns within the dairy industry. Animals affected with this disease can experience a range of clinical symptoms from mild discomfort and swelling of the udder to a severe systemic inflammatory response that could result in the death of the animal. This range of responses is due to differences in pathogen, environment, and inter-animal differences in their innate immune response. A dermal fibroblast model was used to predict the magnitude of an animal's innate immune response towards an intra-mammary S. aureus challenge. Animals whose fibroblasts exhibited a low response phenotype, characterized by lower levels of IL-8 following in vitro immune stimulation, suffered less mammary tissue damage and a less severe reduction in milk quality following the in vivo S. aureus challenge as compared to animals classified as high responders. Furthermore, the heightened inflammatory response of the high responders offered no advantage in bacterial clearance. For a S. aureus infection, the lower response phenotype is preferred. To further explore inter-animal variation in the innate immune response, fibroblast cultures were established and challenged with LPS from two breeds of cattle, Holsteins, a dairy breed and Angus, a beef breed. Cultures from Holstein animals exhibited a higher responding phenotype than cultures from Angus animals. As these two breeds undergo selection for different traits and are reared differently as calves, whole transcriptome analysis (RNA-Seq) and DNA methylation analysis (Methylated CpG Island Recovery Assay; MIRA-Seq) of their fibroblasts was completed to examine the genetic and epigenetic basis for the contrasting responses. RNA-Seq revealed several immune associated genes that were expressed at higher levels in Holstein cultures compared to Angus cultures, including TLR4, IL-8, CCL5, and TNF-α, both basally and following LPS exposure. Although MIRA-Seq analysis revealed 49 regions with differential methylation between the Holstein and Angus cultures, overall, the methylation of the fibroblast genome was similar between these breeds. A combination of genetic and epigenetic factors seems to contribute to the breed-dependent differences observed between Holstein and Angus fibroblasts. Early life exposure to bacterial compounds or inflammatory mediators can have long-term effects on the magnitude of an animal's innate immune response, and may contribute to inter-animal variation in this response. To determine if an early life exposure to LPS would modify the response to a subsequent LPS challenge in dairy animals, neonatal Holstein calves were treated with LPS or saline at 7 days of age and subsequently challenged with LPS 25 days later. Calves that received LPS at 7 days of age had greatly elevated levels of plasma IL-6 and TNF-α compared to calves that received saline, indicating a substantial inflammatory response. However, following the subsequent LPS challenge completed on all calves, there were no differences in plasma IL-6 and TNF-α between the LPS- and saline- treated calves. Alternative exposure strategies in calves may generate the long-term effects observed in other model systems. There is a wide range in the responses observed in the innate immune response of the bovine. Animals with a lower innate immune response effectively clear the infection, but avoid the collateral tissue damage from excessive inflammation. Therefore, it seems that a reduced innate immune response would be more beneficial to the dairy cow.