Hosts have two main strategies for coping with infections: resistance and tolerance. Resistance is aimed at preventing or eliminating parasites, whereas the goal of tolerance is to maintain performance regardless of parasite burden. The balance between resistance and tolerance within a host may mediate host competence, or the propensity of a host to infect other hosts or vectors. Hosts with high tolerance and low resistance to an infection, for instance, may be highly competent and possess the greatest potential to act as superspreaders. These superspreading hosts will contribute disproportionately to transmission, thus posing the greatest risk to other hosts within a population or community. Understanding the drivers of heterogeneity in host competence therefore has broad implications for the management of infectious diseases in nature. Host tolerance is typically quantified as the slope of the relationship between host performance and parasite burden. The majority of host tolerance studies have been conducted at the level of genotypes, populations and species. Individual hosts often exhibit variation in competence, with some individuals contributing more or less to transmission than the population/species average. Despite the clear importance of understanding tolerance at the individual-level, such studies are rare and may be particularly challenging in field contexts due to the need for repeated performance-burden measurements. I used the house sparrow (HOSP) – West Nile virus (WNV) system to investigate differences among two alternative approaches to estimating individual tolerance: the scope and position methods. The scope method estimates tolerance traditionally as the slope of multiple performance-burden measurements over time within an individual; alternatively, the position method required only one measurement for each individual, thus characterizing tolerance via among-individual variation in host defense. We found strong relationships between scope and position estimates of individual tolerance, suggesting that the position method may be an appropriate proxy to use in field studies. I also compared tolerance estimates derived from different metrics of performance. There were weak correlations among these estimates of tolerance, implying that tolerance estimated by measuring a single trait may not be indicative of tolerance at the level of the whole individual or their contribution to disease processes. Understanding the physiological mediators of host competence may help to pinpoint at-risk and risky individuals (or genotypes, populations and species) within natural communities, thus facilitating the development of more targeted disease management strategies. Cytokines and glucocorticoids have been identified as potent mediators of host defense. Pro-inflammatory cytokines may act to promote resistance, whereas anti-inflammatory cytokines and glucocorticoids tend to mediate host tolerance. I investigated the dynamics of pro-inflammatory cytokine IFN-γ, anti-inflammatory cytokine IL-10, and the major avian glucocorticoid, corticosterone (CORT), following WNV exposure in HOSP. I then assessed the influence of these three mediators on resistance and tolerance to WNV infection. I found unusual dynamics for the three mediators across the infection period: IFN-γ expression was not induced by WNV exposure, IL-10 expression was dampened by WNV exposure, and CORT levels were higher in unexposed individuals. Despite the unique response of HOSP to WNV exposure seen here, we did find that constitutive expression of IFN-γ and IL-10 mediate resistance and tolerance to WNV, respectively. Unexpectedly, we also found evidence for protective (pro-resistance) effects of CORT, which contrasts with previous evidence for the role of CORT in mediating WNV infections. Combined, the results of this study suggest that hosts with constitutively high IL-10 and low IFN-γ expression may have high potential to act as superspreaders of disease, thus becoming critical targets in designing WNV-control strategies in passerines. The methods by which we quantify host tolerance may greatly affect the conclusions we are able to draw from such studies. To date, a variety of definitions and techniques have been used to study tolerance in animals. In chapter three, I briefly summarize past plant and animal tolerance research, highlighting discrepancies among researchers in their motivations, definitions and techniques for studying tolerance. For instance, I discuss biases in the literature regarding the use of range versus point tolerance, vigor, and laboratory versus field studies. In particular, I expound upon the nature of the performance metrics used in the majority of tolerance estimations in the literature, and discuss the ecological implications of these metrics. To conclude, I offer suggestions for overcoming the challenges associated with studying tolerance and encourage a unified way forward in the field, emphasizing the selection of system-specific and ecologically relevant tolerance metrics. My thesis research has employed physiological and behavioral methods in an ecological context to better understand the heterogeneities that exist in host competence. By combining empirical data in the HOSP-WNV system with conceptual and methodological strategies for assessing host defenses, this research has broadened our knowledge of host responses in the WNV system in a manner that may be applicable to understanding and managing disease dynamics in diverse natural communities.

Pneumocystis species are opportunistic fungal pathogens that cause severe pneumonia in immunocompromised hosts, including AIDS patients. Pneumocystis species have a biphasic life cycle consisting of single-nucleated trophic forms and ascus-like cysts. Both stages live within the host, and, thus, must contend with threats from the host immune system. The cyst cell wall β-glucans have been shown to stimulate immune responses in lung epithelial cells, dendritic cells and alveolar macrophages. Little is known about how the trophic life forms, which do not have a fungal cell wall, interact with immune cells. In this study, the immune response to the life cycle stages of Pneumocystis murina was evaluated. Here, we report differences in the immune response of immunocompetent mice to the trophic and cystic life cycle stages of P. murina. Upon infection with purified trophic forms, wild-type adult mice developed a delayed innate and adaptive immune response compared to inoculation with the normal mixture of trophic forms and cysts. Cysts, but not trophic forms, stimulated Th1-type responses in the lungs of infected mice. Surprisingly, trophic forms are sufficient to generate protective adaptive responses, leading to clearance in immunocompetent mice. We report that CD4+ T cells primed in the presence of trophic forms are sufficient to mediate clearance of trophic forms and cysts. In addition, primary infection with trophic forms is sufficient to prime B cell memory responses capable of clearing a secondary infection with Pneumocystis following CD4+ T cell depletion. While trophic forms are sufficient for initiation of adaptive immune responses in immunocompetent mice, infection of immunocompromised RAG2-/- mice with trophic forms in the absence of cysts does not lead to the severe weight loss and infiltration of innate immune cells associated with the development of Pneumocystis pneumonia. Dendritic cells screen the alveolar spaces for pathogens, and are in a prime position to initiate the immune response against lung pathogens, including Pneumocystis. Our data demonstrate that trophic forms broadly dampen the ability of dendritic cells to respond to pathogen-associated molecular patterns. Bone marrow-derived dendritic cells were stimulated with trophic forms, a mixture of trophic forms and cysts, and various other inflammatory materials, including β-glucan. Trophic forms inhibited multiple components involved in antigen presentation by dendritic cells, including secretion of inflammatory cytokines and expression of MHC class II and costimulatory molecules on the cell surface. Furthermore, trophic forms suppressed or failed to induce the expression of multiple genes related to activation and maturation in dendritic cells. Dendritic cells silenced by trophic forms are unable to induce CD4+ T cell responses. These data suggest that immune evasion by trophic forms is dependent on the suppression of innate responses, and the development of adaptive immunity represents a “point of no return” at which the trophic forms are no longer able to escape clearance.

Plasmodium falciparum is a protozoan parasite that causes a severe form of malaria, a mosquito-borne infectious disease in humans. P. falciparum encodes a number of proteins to facilitate its life-cycle, including a type II heat shock protein 40 (Hsp40), Pfj2. Pfj2 shows a degree of homology to human ERdj5, a resident protein of the endoplasmic reticulum (ER) that promotes protein quality control by facilitating the degradation of misfolded proteins. The overall aim of this study was to further understand the function of Pfj2 in the P. falciparum cell by characterising it biochemically. A bioinformatic analysis of Pfj2 was carried out to enable the identification of a potential ER signal sequence and cleavage site. Furthermore, an analysis of Pfj2 protein sequence was performed to compare domain similarities and identities with typical type II Hsp40s namely, human ERdj5, S. cerevisiae Sis1, human Hsj1a and human DnaJB4. The method used included the insertion of the codon-optimised coding sequence for the processed ER form of Pfj2 into the prokaryotic expression vector, pQE30, to enable overproduction of a histidine-tagged protein. A 62 kDa His₆-Pfj2 was successfully expressed in Escherichia coli and purified using denaturing nickel affinity chromatography. ATPase assays were performed to determine the ability of His₆- Pfj2 to stimulate the chaperone activity of the ER Hsp70, also called immunoglobulin binding protein (BiP). Initial studies were conducted on readily available mammalian His₆-BiP as a control, which was shown to have an intrinsic activity of 12.07±3.92 nmolPi/min/mg. His₆- Pfj2 did not stimulate the ATPase activity of mammalian His₆-BiP, suggesting that it either could not act as a co-chaperone of mammalian His₆-BiP (specificity), or it required a misfolded substrate in the system. Therefore, ongoing studies are addressing the interaction of Pfj2 and misfolded substrates with P. falciparum BiP. The results of these studies will further our understanding of a poorly-studied parasite chaperone that represents a potential drug target for development of novel strategies for the control of a serious human disease

Senior horses (≥20 years) exhibit inflamm-aging, or chronic, low-grade inflammation that occurs systemically with aging, similarly to humans. Inflamm-aging has previously been characterized in the horse in circulation as well as specifically being mediated by lymphocytes and monocytes. In humans, inflamm-aging has been associated with increased morbidity and mortality. However, in the horse, relatively little about inflamm-aging is known regarding clinical effects or factors influencing severity. The contribution of lymphocytes to inflamm-aging of senior horses was examined, specifically through determining the relationships of inflamm-aging with various other health parameters, effects of seasonality, and the extent to which inflamm-aging can be modulated by anti-inflammatory phytonutrient curcumin. The overall hypothesis of this research is that lymphocyte-mediated inflamm-aging of the senior horse is associated with various factors including season, endocrine function, body composition, and nutritional status, and may be modulated by polyphenol curcumin. The effect of season on lymphocyte-mediated inflamm-aging was examined, and senior horses exhibited elevated inflammation compared to adult horse as expected, while also exhibiting changes in inflammatory cytokine production and gene expression throughout the year. In addition to season, pituitary pars intermedia dysfunction (PPID), a common endocrinopathy in senior horses that is associated with immunosuppression, was examined in a group of senior horses to determine any effects on degree of inflamm-aging. Results indicated no significant differences between age-matched PPID and non-PPID horses for lymphocyte-mediated inflammatory cytokine production or gene expression. The immunosuppressive aspect of PPID does not appear to be associated with the degree of lymphocyte-mediated inflammation of the aged horse. Additionally, an expansive correlative study was undertaken to determine relationships between inflamm-aging and basal nutritional status, body composition, age, and PPID within a similarly-managed senior horse population. Results showed various relationships between inflammatory markers and nutritional status, particularly yielding positive associations with serum folate and with serum fatty acids C22:2n6c and C22:5n3c. Inflammation was also associated with age itself but was not associated with body composition parameters and showed mild association with PPID (and serum inflammatory C-reactive protein). As a whole, this study demonstrates that nutritional status can be associated with inflammatory markers. Similarly, many phytonutrients have exhibited anti-inflammatory properties, which may be beneficial to the senior horse exhibiting inflamm-aging. Specifically, the effects of polyphenols including curcuminoids, resveratrol, quercetin, pterostilbene, and hydroxypterostilbene on lymphocyte production of inflammatory cytokines by senior horses were examined in vitro and found to significantly reduce inflammation similarly to common non-steroidal anti-inflammatory drugs. This study led to the in vivo investigation of the effectiveness of curcumin in modulating chronic inflammation of the senior horse. No significant differences were seen between groups receiving curcumin and placebo for the various inflammatory parameters, which may be due to the dose or low bioavailability of curcumin. As a whole, this research provides further understanding of factors associated with inflamm-aging of the senior horse.

"Troubling Breath," the product of fourteen months of fieldwork, examines the experience of tuberculosis sufferers in rural Rajasthan, India. In it, I engage the Indian national tuberculosis control program, local health institutions, informal biomedical providers, non-biomedical healers and sufferers to consider how global tuberculosis control initiatives interact with social life and subjectivity among the rural poor. I ask how tuberculosis affliction and healing builds and reveals the diversity and limit of relationships between state and citizen, individual and kin, body and social, global and local, and formal and informal healthcare. / Anthropology

With over 35 million people currently infected, the World Health Organization considers HIV a global pandemic. HIV is characterized by a high mutation rate, which allows it to evade the host immune system and develop resistance to drugs. However, this extraordinary adaptive ability may also be the key to HIV’s demise. Through the field of phylodynamics, the evolutionary behavior of the virus is being studied in an attempt to control the epidemic. In this thesis, three papers are presented in which we analyze sequences generated through the Canadian HIV Strain and Drug Resistance Surveillance program. In chapter 2 we validate a classifier which distinguishes between recent and established infections based on the proportion of mixed bases observed in population-based pol sequences. Our results will help identify recent infections and improve incidence calculations. In chapter 3, we investigate immune-induced patterns in HIV that are shared by patients of the same ethnicity. An understanding of the forces shaping HIV evolution is instrumental to the development of a vaccine relevant to the Canadian epidemic. In chapter 4, we present preliminary results of a historical reconstruction of HIV across the provinces of Canada. This analysis will highlight strategies that have succeeded or failed in controlling the epidemic. Furthermore, our work will establish whether non-B subtypes of HIV are an increasing threat to Canadian public health. Overall, this thesis provides the first country-wide evolutionary and phylogenetic analysis of the HIV epidemic.

Listeria monocytogenes is a facultative intracellular bacterium that causes foodborne disease in humans. L. monocytogenes invade the gut mucosa and then disseminate, causing systemic infections associated with high mortality rates in immunocompromised individuals. It is unknown how L. monocytogenes traffic to the mesenteric lymph nodes, which represent an important bottleneck for systemic spread. In addition, little is known about the gastrointestinal stage of infection due to the general resistance of mice to oral infection with L. monocytogenes. Our laboratory developed a novel foodborne mouse model of listeriosis utilizing a murinized strain of L. monocytogenes to investigate the gastrointestinal stage of infection. First, we found that the majority of L. monocytogenes isolated from the intestinal tissue and MLN were extracellular; however, the minimal fraction of intracellular L. monocytogenes was vital for persistence in the gut and spread to the MLN. The vast majority of cell-associated L. monocytogenes in the MLN were adhered to inflammatory monocytes, but these cells did not support the intracellular growth of L. monocytogenes. A minor proportion of L. monocytogenes were associated with migratory dendritic cells in the intestinal lamina propria and MLN, but like monocytes, these cells did not appear to serve as an intracellular growth niche for L. monocytogenes. Lastly, extracellular L. monocytogenes were observed migrating in mesenteric lymphatic vessels that drain from the intestine to the MLN, suggesting that L. monocytogenes can spread beyond the intestinal mucosa independent of migratory immune cells. Overall, these studies are the first to characterize the interaction of L. monocytogenes with immune cells in the intestine and MLN following foodborne infection and suggest that extracellular, and not cytosolic L. monocytogenes, primarily drive innate immune responses in the gut.

The thesis traces the successive urban responses made by the Hong Kong government from 1997 to 2014, in an attempt to achieve “imagined immunity” for the city. The urban responses being analysed are efforts to regulate the ways in which “live poultry” (especially live chickens) is metabolized and circulating in the city. The efforts are made to re-order the human-birds-microbes relationships in Hong Kong - a process conceptualized as “re-urbanization of nature.” The consequence of these re-urbanization of nature processes, led to changes in the specific practice of consuming “live poultry” in the city. Four periods of re-urbanization of nature are identified in the analysis, and it is argued that in each wave of restructuring there were markedly different frames constructed to generate distinctive meanings of the “contagion condition,” imagined urban immunity, and practices of re-urbanization of nature. Their meanings and resultant practices were products of negotiations, within an entangled web of human and nonhuman features in particular periods. The context of these interventions and the biopolitical contestations are analyzed in the thesis. It is then argued that such contingencies and context-sensitive processes, call for further studies of post-epidemic urban changes. The thesis also explores the possibility of developing a theoretical framework of “urban biopolitical immunology” to accomplish the inquiry. By so doing, it seeks to contribute to studies of the politics of contemporary epidemics, and to research on the production of urban nature.

Buruli ulcer (BU), an infectious disease caused by Mycobacterium ulcerans, is the third most common mycobacterial disease after leprosy and tuberculosis and a WHO-defined neglected tropical disease. Despite years of research, the mode of transmission of BU remains unknown. This master’s thesis provides an integrated spatial analysis of disease dynamics in Ghana, West Africa, an area of comparatively high BU incidence. Within a case/matched control study design, environmental factors associated with BU infection and spatial behaviors are investigated to uncover possible links between individual daily activity spaces and terrains of risk across disturbed landscapes. This research relies upon archival and field-collected data and analyses conducted with geographical information systems (GIS).

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen. Several antibiotic resistant strains of P. aeruginosa are commonly found as secondary infection in immune-compromised patients leaving significant mortality and healthcare cost. Pseudomonas aeruginosa successfully avoids the process of phagocytosis, the first line of host defense, by secreting several toxic effectors. Effectors produced from P. aeruginosa Type III secretion system are critical molecules required to disrupt mammalian cell signaling and holds particular interest to the scientists studying host-pathogen interaction. Exoenzyme S (ExoS) is a bi-functional Type III effector that ADP-ribosylates several intracellular Ras (Rat sarcoma) and Rab (Response to abscisic acid) small GTPases in targeted host cells. The Rab5 protein acts as a rate limiting protein during phagocytosis by switching from a GDP- bound inactive form to a GTP-bound active form. Activation and inactivation of Rab5 protein is regulated by several Rab5-GAPs (GTPase Activating Proteins) and Rab5-GEFs (Rab5-Guanine nucleotide Exchange Factors). Some pathogenic bacteria have shown affinity for Rab proteins during infection and make their way inside the cell. This dissertation demonstrated that Rab5 plays a critical role during early steps of P. aeruginosa invasion in J774-Eclone macrophages. It was found that live, but not heat inactivated, P. aeruginosa inhibited phagocytosis that occurred in conjunction with down-regulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and more than one arginine sites in Rab5 are possible targets for ADP-ribosylation modification. However, the expression of Rin1, but not other Rab5GEFs (Rabex-5 and Rap6) reversed this down-regulation of Rab5 in vivo. Further studies revealed that the C-terminus of Rin1 carrying Rin1:Vps9 and Rin1:RA domains are required for optimal Rab5 activation in conjunction with active Ras. These observations demonstrate a novel mechanism of Rab5 targeting to phagosome via Rin1 during the phagocytosis of P. aeruginosa. The second part of this dissertation investigated antimicrobial activities of Dehydroleucodine (DhL), a secondary metabolite from Artemisia douglasiana, against P. aeruginosa growth and virulence. Populations of several P. aeruginosa strains were completely susceptible to DhL at a concentration between 0.48~0.96 mg/ml and treatment at a threshold concentration (0.12 mg/ml) inhibited growth and many virulent activities without damaging the integrity of the cell suggesting anti-Pseudomonas activity of DhL.