Treponema pallidum ssp. pallidum is the causative agent of syphilis, a multi-stage bacterial infection, transmitted sexually or from mother-to-child, with an unparalleled range of symptoms arising from the ability of treponemes to penetrate any tissue and cross immune privileged endothelial barriers to access the brain, the eye, and the fetus. Further, without treatment T. pallidum evades immune clearance and persists within the host to establish a chronic infection. These characteristics suggest that T. pallidum may have evolved unique mechanisms for immune escape and to mediate host-cell interactions. The findings presented in this dissertation contribute to our knowledge of T. pallidum pathogenesis by investigating a previously unexplored host-cell interaction, between T. pallidum and human platelets. These results validate the hypothesis that, as a pathogen which successfully utilizes vascular dissemination, T. pallidum would not only encounter, but interact with human platelets, complex cells now viewed as vascular sentinels that participate in many host-pathogen interactions. This is the first study to demonstrate that T. pallidum interacts with human platelets and to characterize and quantify these interactions using high resolution microscope imaging techniques (video and frame analysis). These interactions were shown to be complex, reversible and mediated by motile treponemes localizing to stationary, (slide-adhered) activated platelets, versus to free-floating, inactive platelets. In addition, it was found that T. pallidum discriminates between the level of platelet activation and preferentially localized to the most activated platelet. Treponema pallidum was also able to induce platelet activation following an extended lag period. Modified chemotaxis assays quantified by flow cytometry, were used to investigate the migration of T. pallidum in response to the plasma of platelets differentially activated with infection-relevant host components (thrombin, collagen). The results herein reveal that T. pallidum discriminates between different mechanisms of platelet activation, with a significant preference towards the secretions of collagen-activated platelets (under these experimental conditions), compared with that of inactive or thrombin-activated platelets. Previously, T. pallidum chemotaxis had been investigated through genomic characterization and molecular interaction studies with recombinant proteins. This investigation is the first time live T. pallidum was utilized for in vitro chemotaxis assays and is also the first study of pathogen chemotaxis in response to the secretions of differentially activated platelets. The body of work in this dissertation provides a foundation to further investigate the role of T. pallidum-platelet interactions during infection, adding a new host-cell interaction to our understanding of T. pallidum pathogenesis. The evidence that the molecular gradients of host components can affect T. pallidum migration suggests an important role for chemotaxis during T. pallidum infection. Together, the characterization of platelet-interactions and treponeme chemotaxis in response to host components, adds to our knowledge of T. pallidum-host interactions, and eludes to additional pathogenic strategies that may facilitate T. pallidum dissemination and immune evasion. / Graduate / 2022-01-14

Colombia covers an area of approximately 114 million hectares (ha) of which the potential forestly land has been estimated at 60.7 million ha, about 53% of the total area. Only 54 million ha are considered as natural forest, leaving approximately 29 million ha to be used for livestock and agriculture. In order to provide an alternative source of timber, Colombian groups have planted approximately 327 000 ha to different species of Pinus, Eucalyptus and native species. This clearly represents only a small proportion of the total area that might be used for forestry, which is set to grow in the future. In general, trees established in plantations have been grown as monocultures that allow for substantial productivity per management unit. But this is also a homogeneous genetic resource that is highly susceptible to damage caused by insect pests and diseases. It is therefore, surprising that relatively little work has been conducted on pests and pathogens negatively affecting plantation foresty in Colombia. In this regard, the studies presented in this thesis present the first comprehensive treatment of the topic for the country. It is consequently hoped that these will form a basis for the future management and sustainability of forestry in Colombia. In the first Chapter of this thesis, I provide a historical background and the current pest and disease situation for the forestry sector in Colombia. This includes an overview of the main pest and diseases affecting commercial nonnative trees, especially species of Pinus and Eucalyptus. A wide range of sites occurring in Colombia were considered and the document also includes the impact of different climatic conditions on the incidence and management of the various pests and diseases treated. Some of the most important defoliators in Colombia reside in the order Phasmatodea and one of these, Litosermyle ocanae, was treated in studies presented in chapter two. The overall aim was to contribute basic knowledge of L. ocanae including an understanding of the biology, egg population dynamics, and potential biological control assessments based on early detection of the insect. vi Chapter three includes the discovery of one of the members of the Geometridae that causes serious damage due to defoliation of Pinus and Eucalyptus plantations. This pest, known as Chrysomima semiluteraria, has been known in Colombia for many years and this study included a comprehensive evaluation of its biology and field monitoring contributing to an Integrated Pest Management strategy for it. The insect was studied under field and laboratory conditions and a special emphasis was placed on its biological control using the egg parasitoid Telenomus alsophilae. Pinus plantations in Colombia have been affected by numerous pests, including recently, the adelgid Pineus sp. To establish management strategies to assist commercial forestry operations, the life cycle of this insect and the susceptibility of different Pinus species were determined in Chapter four. In addition, the possible role of a Ceraeochrysa sp. as a biological control agent was investigated. In chapter five, Fusarium circinatum is recorded for the first time on Pinus spp. in Colombia. The discovery of this fungus, known to cause the disease Pitch Canker has had an important impact on forestry, especially with regard to management strategies aimed at reducing its impact. Studies in this chapter included those to evaluate the susceptibility of families and provenances of Pinus spp., which are currently used in forestry planting programs in Colombia. The first emergence of the foliage pathogen Dothistroma septosporum as an important constraint to pine forestry in Colombia is treated in chapter six. This fungus is a serious pathogen of many species of Pinus around the world. It was, however, not recognised as a serious threat to forestry in Colombia until it emerged as a serious source of damage to Pinus tecunumanii. In this chapter, the aim was firstly to confirm the identity of the pathogen based on DNA sequence data. Subsequently, the host range and distribution of the pathogen was established in different Colombian forestry areas. The impact of the disease and the susceptibility of different provenances of P. tecunumanii was also assessed. In chapter seven, the main objective was to described the susceptibility of E. grandis clones to a new species of Ceratocystis and to identify clones resistant to it. The fungus was described as Ceratocystis neglecta and management strategies are suggested for it. This thesis includes two Appendices treating new reports of insect pests and an important Eucalyptus pathogen. In Appendix 1, I report on the importance of three different pest insects. Appendix 2 includes a description of a new disease, as well as the potential risks that this disease poses for the Colombian forestry sector. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Forestry and Agricultural Biotechnology Institute (FABI) / Unrestricted

Human alteration of the landscape and environment is favoring the expansion of ticks and the pathogens they vector throughout the United States. The changes in these distributions are documented through literature and the deposition of specimens in scientific collections. In southern Illinois, tick species records are sporadic and inconsistent with what is actually found in the region. This information is necessary for public health officials to develop prevention strategies against tick-borne illnesses. I conducted research from 2018-2020 to accomplish the following: (1) conduct a ticks species and tick-borne disease survey of southern Illinois, (2) create habitat maps for Amblyomma americanum and Dermacentor variabilis in the region, and (3) attempt to identify microhabitat variables influencing tick abundance. To accomplish my first objective, I sampled 26 sites from 11 counties in southern Illinois utilizing the tick drag method. Ticks were then tested for their associated pathogens using quantitative PCR. Four species of tick, Amblyomma americanum (the lone star tick), Dermacentor variabilis (the American dog tick), Ixodes scapularis (the black-legged deer tick), and Amblyomma maculatum (the Gulf Coast tick) were identified. Rickettsia rickettsii and Rickettsia parkeri causative agents of human rickettsiosis, Ehrilichia ewingii and Ehrlichia chaffeensis, causative agents of human granulocytic ehrlichiosis, and Rickettsia amblyommii whose effects are not known, were all found in the study region. The second objective, lone star and American dog tick species distribution models (SDMs), was accomplished using the popular SDM program, Maxent. Maxent uses species presence data and their associated environmental characteristics to create predictions of habitat suitability. The resulting models indicate a correlation for both species with the Shawnee National Forest, likely due to the fragmented habitat structure as indicated by the strong impact of cultivated and managed vegetation on both species models. Lastly, I attempted to identify microhabitat characteristics that influence tick species abundance. Temperature, humidity, canopy closure, and wind speed were measured and recorded and vegetation density estimates and soil moisture were recorded categorically at each site, each visit. Using a chi-squared test I proved that tick abundance is not static, and varies throughout the summer months. I then used a principal component analysis (PCA) to assess correlation between habitat variables and tick abundance, but no signal was detected. This study established a baseline for tick species and tick-borne disease presence in southern Illinois. The information resulting from this study can be used to inform public health officials and inform future tick-borne disease prevention strategies. I recommend continued surveillance of the area to monitor species distributions and resulting pathogen risk to residents.

Glucans are (1→3)-β-linked linear and branched polymers containing anhydroglucose repeat units. They comprise a major portion of the cell wall of saprophytic and pathogenic fungi. Glucans activate a wide range of innate immune responses. They are also released from the fungal cell wall as exopolymers into the blood of patients with fungal infections. Extensive studies have been done on glucans isolated from saprophytic fungi, such as Saccharomyces cerevisiae; however, much less is known about the glucans produced by the polymorphic fungal pathogen Candida albicans. We have undertaken an extensive structural characterization and comparison of glucans isolated from C. albicans blastospores and hyphae using high-resolution, solution-state proton nuclear magnetic resonance spectroscopy (NMR). In addition, we developed a simple and straightforward method for the production of Candida hyphae that resulted in gram quantities of hyphal mass. Also, we compared and contrasted the Candida glucans isolated by two different protocols with those isolated from S. cerevisiae. Isolation protocols provide high purity glucans with source-based structural differences. Structural details provided by this NMR analysis included the degree of polymerization, molecular weight, degree and type of branching, and structural composition. We observed that Candida glucans, derived from blastospores or hyphae, are different compared to those isolated from S. cerevisiae with regard to side-chain branching along the backbone and at the reducing terminus. These structural details are an important prerequisite for biomedical studies on the interaction of isolated fungal cell wall glucans with the innate immune system.

Les protéines à domaine FIC (Filamentation induced by cAMP) sont très répandues chez les bactéries où elles catalysent l’ajout d’une modification post-traductionnelle contenant un phosphate à une protéine cible, en utilisant différents co-substrats comme l’ATP. Certaines de ces protéines sont des toxines sécrétées par des pathogènes humains, mais la fonction de la plupart d’entre elles reste mystérieuse. Plus d’une dizaine de structures de protéines FIC ont été déterminées récemment, qui ont permis d’élucider leur mécanisme catalytique. L’une des sous-familles de protéines FIC possède un glutamate dans leur site catalytique, dont il a été proposé qu’il aurait une fonction auto-inhibitrice pour la fixation de l’ATP. Durant ma thèse, j’ai étudié la structure et les mécanismes de régulation de deux familles de protéines FIC : les protéines FIC à glutamate inhibiteur, et la toxine AnkX de la bactérie pathogène Legionella pneumophila.La première étude s’est intéressée à la protéine FIC de la bactérie pathogène Enterococcus faecalis (EfFIC), qui fait partie de la sous-famille des protéines FIC possédant un glutamate inhibiteur. J’ai résolu plusieurs structures cristallographique d’EfFIC, qui ont permis de caractériser son site catalytique et comment elle fixe l’AMP et l’ADP. En utilisant une propriété fréquemment observée d’auto-AMPylation (modification par l’AMP), j’ai montré au moyen d’ATP radioactif qu’EfFIC possède une activité basale d’auto-AMPylation, et j’ai identifié une nouvelle activité de dé-AMPylation. En m’inspirant des métaux observés dans mes structures cristallographiques, j’ai montré que l’alternance entre les activités d’AMPylation et de de-AMPylation dépend de la nature du métal fixé dans le site actif et de la présence du glutamate. Ce glutamate régulateur est également présent chez une protéine humaine, HYPE, qui possède une double activité d’AMPylation et dé-AMPylation d’ une chaperone du réticulum endoplasmique. Par un test de fluorescence, j’ai enfin montré que l’activité de HYPE était elle aussi régulée par les métaux comme celle de EfFIC. Ces résultats suggèrent un nouveau modèle de régulation partagé par des protéines FIC de la bactérie à l’homme.La seconde étude a porté sur la toxine AnkX de Legionella pneumophila, qui modifie les petites protéines G de la famille de Rab Rab1 et Rab35 (régulatrices du trafic cellulaire) par une molécule de phosphocholine (PC). En utilisant des liposomes de composition contrôlée, j’ai montré qu’AnkX interagit avec les membranes, et j’ai identifié par mutagenèse son domaine d’interaction avec les membranes. Au moyen de petites GTPases Rab ancrées artificiellement à la surface de liposomes par une queue 6his remplaçant le lipide naturel, j’ai montré que l’activité d’AnkX est stimulée par la présence de membranes. Des résultats préliminaires suggérent que Rab35 est un meilleur substrat que Rab1a, ce qui pourrait renseigner sur la fonction et le compartiment cellulaire où se trouve la toxine. J’ai ensuite mené une étude structurale d’AnkX par diffusion des rayons X aux petits angles (SAXS), qui permet d’obtenir des informations structurales en solution. AnkX est constitué d’un domaine FIC, de répétitions ankyrine et d’un domaine C-terminal. L’analyse en SAXS montre que ces domaines s’organisent en forme de fer à cheval, suggérant un modèle d’association bi-partite du complexe AnkX/Rab aux membranes. L’ensemble de ces résultats conduit à un modèle dans lequel l’activité d’AnkX est régulée spatialement par les membranes, ce qui pourrait lui permettre de cibler à la fois les petites GTPases Rab cellulaires et le compartiment membranaire. / FIC (Filamentation induced by cAMP ) domain containing proteins are widespread in bacteria where they use different substrate such ATP to modify a target protein with a phosphate containing post-translational modification. Some of those proteins are secreted toxins from pathogens but the function of the majority stay unknown. Some recently resolved structures explain the catalytic mechanism. A subfamily of FIC proteins was proposed to be auto-inhibited for ATP binding by a glutamate in their active site. In my thesis, I lead a structural and biochemical study of two FIC proteins family: the auto-inhibited by a glutamate FIC proteins and the Legionella pneumophila toxin AnkX.For the first study, I focused on the pathogenic bacteria Enterococcus faecalis protein EfFIC that is an auto-inhibited FIC protein. I solved several crystallographic structures to characterize the active site and the AMP and ADP binding. Using the classic auto-AMPylation (modification with an AMP molecule) mechanism and radioactive ATP, I showed that EfFIC is active and I identified a new de-AMPylation activity. Using metals found in my crystallographic structures, I showed that the AMPylation and de-AMPylation switch is controlled by the nature of the metal bound in the active site and that this switch is inhibitory glutamate-dependent. This glutamate is found in human HYPE that shows a double AMPyaltion and de-AMPylation activity of the ER chaperone BIP. Using fluorescence assays, I showed that those two activities are alors regulated by metals as in EfFIC. Those results point on a new regulation model shared between FIC proteins from bacteria to human.The second study focused on Legionella pneumophila toxin AnkX that modifies small GTPases Rab1 and Rab35 with a phosphocholine (PC) molecule. Using controlled composition liposomes, I showed that AnkX interact with membranes and mapped the interaction domain by mutagenesis. With artificially anchored to nickel containing liposomes surface Rab GTPases, I demonstrated the stimulation of AnkX activity by the membranes. Preliminary results also suggest that Rab35 is a better substrate than Rab1a, giving information on AnkX function and localization during infection. I lead a small angle X-ray scattering (SAXS) study on AnkX that gave low-resolution structural information on AnkX in solution. The analyses of SAXS results show that AnkX is horseshoe shaped, suggesting an association with the membrane and Rab of AnkX model. In this model, membranes spatially regulate AnkX, allowing a targeting of Rab and cellular compartment targeting.

Salmonella and Cronobacter sakazakii are two leading causes of foodborne illness associated with low-moisture foods, including infant formula. Both causative organisms can persist in food manufacturing processing environments and contaminate finished product if programs are not in place to limit their introduction and control their spread. An environmental sampling and monitoring program is an important tool that food manufacturers use to determine the effectiveness of their sanitation practices and pathogen control efforts. Guidance for initiating an environmental sampling plan and evaluating the plan is lacking. The objective of this study was to develop microbiological environmental sampling plans based on the answers to a series of questions related to product hazards, processing risks and controls, and knowledge of appropriate microbiological sampling and testing protocols. Furthermore, these initial sampling plans were related to the volume of product and size of the processing facility.  An interactive spreadsheet tool for designing sampling monitoring plans for an infant formula process was developed using Microsoft Excel. Additionally, the tool can be used to record qualitative and quantitative sample test results, and to alert the user how the upcoming sampling plan will be changed, if necessary, based on monthly test summaries. The sampling tool provides a simple method for selecting an appropriate environmental sampling plan (samples per zone per month) and provides a rationale and guidance for creating and modifying these plans.  Effective sampling plans and trend analysis of sample test results support the food processors decisions for implementing controls to enhance food safety. / Master of Science in Life Sciences

Though the past two decades have seen a dramatic increase in research toward self-cleaning repellent surfaces, multiple challenges exist in the creation of biorepellent surfaces for everyday use. Environmental concerns persist with many of the chemicals utilized in this field and the need for scalable, low-cost alternatives remains. Spread of pathogens including bacteria and viruses in healthcare and public settings also presents a need for stable surfaces. In the work presented here, we report on the current status of antimicrobial nanomaterials and coatings toward virus repellency, followed by an investigation into the application of polysiloxane nanostructures in creation of flexible hierarchical surfaces. Using n-propyltrichlorosilane (n-PTCS) coated on activated polyolefin (PO) we were able to demonstrate superhydrophobicity, reporting water contact angles above 153° paired with <1° sliding angles on hierarchical surfaces. A transfer assay, that closely mimics contact with high-touch surfaces, using Escherichia coli K-12 transfected with green fluorescent protein (GFP) reported a 1.6-log (97.5%) reduction in fluorescence on surfaces compared to planar PO controls, paired with a 1.2-log (93%) reduction in CFU/mL in comparison to control groups. Additionally, surfaces demonstrated a contact angle of 140.8° with citrated whole blood. Droplets of blood incubated on our surfaces for 15 min showed a 93% reduction in visible staining, while submersion in citrated whole blood for 20 minutes revealed an 87% reduction in blood adhered to the surfaces. The applications for these biorepellent surfaces have widespread potential, including the demonstrated need for prevention of surface contamination to minimize spread of hospital acquired infections (HAIs) within the healthcare system. / Thesis / Master of Applied Science (MASc) / The goal of creating a surface capable of repelling biological samples continues to present challenges due to surface stability, scalability, and cost of manufacturing techniques. Beyond this, many of the existing solutions use fluorine-based chemicals that present a risk to the environment due to the difficulty in breaking down these molecules. This thesis aims to understand the current state of repellent surfaces used for biological applications, including prevention of surface contamination by bacteria and viruses, then investigates the use of more environmentally friendly methods to produce repellent surfaces. Using a silicone-based coating combined with heat induced shrinking of shape memory polymers (SMPs), we have created a flexible surface with multiscale roughness that demonstrates repellency to bacteria and whole blood.

Palaeogenetic research on human pathogenic and microbiomic bacteria has been largely restricted to bloodborne pathogens from skeletal tissue and, due to short lengths of degraded ancient DNA, small-scale single loci studies. My thesis has expanded the breadth and depth of palaeomicrobial knowledge via the study of novel specimen types with next-generation technologies. Presented in sandwich thesis format, I discuss genome-scale studies of three previously-unstudied historical pathogens: 19th century Vibrio cholerae (cholera) from an alcohol-preserved intestine from Philadelphia, and medieval Staphylococcus saprophyticus (urinary tract infections) and Gardnerella vaginalis (bacterial vaginosis) from calcified urogenital infections of a Trojan woman. Cholera persists as a dangerous modern disease that was also responsible for severe historic epidemics. My research confirms that 19th century pandemics were caused by an O1 classical strain that may have possessed genomic features that contributed increased virulence. S. saprophyticus and G. vaginalis are opportunistic pathogens of the urogenital microbiome, especially in reproductive-age females. Using very high endogenous DNA content of the calcified infections, I have reconstructed one of the most complete ancient bacterial genomes for S. saprophyticus and coding genome for G. vaginalis. Both ancient pathogens possess most of the virulence and urogenital adaptive genes of modern strains, indicating similar ecological roles for these species in past female health. Finally, I successfully use LLMDA microarray technology (never before utilized for ancient DNA research) to detect ancient pathogens. LLMDA provides an inexpensive and informative alternative to high-throughput sequencing for assessing the metagenomic content of ancient samples. Together, my findings provide a framework emphasizing the need to broadly study past microbiomes in conjunction with specific pathogens. Using molecular data, this work supports anthropological views of infectious disease ecology related to the first epidemiological transition and historical narratives. Taken together with the recent literature on ancient pathogen genomes, my findings indicate that palaeogenome sequences may not necessarily reveal any specific signatures of greater virulence, and interpretations of past diseases must necessarily take into account additional host, environmental, and cultural factors. / Thesis / Doctor of Philosophy (PhD)

No description available.

Emerging or re-emerging tick-borne pathogens are expected to increase in prevalence and become more geographically widespread in Canada. Borrelia burgdorferi, the bacterium causing Lyme disease, is the most common vector-borne pathogen in North America, but additional tick-borne pathogens have started to be detected more frequently through surveillance efforts in Canada. The spread and transmission of these tick-borne pathogens are modulated by changes in the abundance and distribution of tick and host populations. Abiotic factors, such as temperature, precipitation, and snow, may affect tick and host abundances as well as host dispersal. Furthermore, biotic factors, such as the abundance and diversity of hosts, may alter tick abundance and consequent tick-borne disease risk. In this dissertation, I assess the historical associations and spatiotemporal changes of the tick vectors, hosts, and pathogens in Canada as well as the impact of abiotic and biotic factors on these key players. In Chapter 1, I present the first systematic assessment of the literature that identifies historical associations and spatiotemporal changes in the tick-host-pathogen disease systems in Canada over broad spatial and temporal scales. Borrelia burgdorferi was the most detected tick-borne pathogen and Ixodes scapularis harboured the greatest number of tick-borne pathogens. Several spatial outliers of high pathogen presence in ticks in addition to five spatiotemporal clusters were identified, which were located in areas of southern Canada with long-established tick populations. In addition, six spatiotemporal clusters of high pathogen presence were also identified, with four clusters associated with passive surveillance and two clusters related to active surveillance. In chapter 2, I concurrently evaluated high-resolution environmental and host-related factors to determine the relative impacts of abiotic and biotic factors on questing I. scapularis abundance in Ontario and Quebec. High-resolution abiotic factors were derived from remote sensing satellite imagery and meteorological towers, while biotic factors related to mammal hosts were derived from active surveillance data that I collected in the field. Important abiotic and biotic drivers of questing I. scapularis abundance were identified, which included monthly mean precipitation, accumulated snow, and mammal species richness. These results demonstrate the need to incorporate host active surveillance data with high-resolution environmental factors when trying to determine the key drivers impacting the abundance and distribution of tick populations and tick-borne pathogens. In Chapter 3, I analyzed the presence and prevalence of multiple tick-borne pathogens extracted from tick and small mammal specimens collected during field surveys in Ontario and Quebec. Three pathogen species were detected in ticks, which included Babesia odocoilei and B. burgdorferi in I. scapularis as well as Rickettsia rickettsii in Haemaphysalis leporispalustris. In small mammal hosts, three pathogen species were detected including B. odocoilei in one shrew, B. microti in one deer mouse, and Hepatozoon in one deer mouse and one white-footed mouse. My findings provide evidence that emerging or re-emerging tick-borne pathogens may be present outside currently defined risk areas identified by surveillance efforts in Canada. Finally, in chapter 4, I examined the effect of biotic factors related to I. scapularis and mammal hosts on the presence, prevalence, and diversity of pathogens in Ontario and Quebec using data from field surveys. Local infection prevalence ranged from 0% to 25.4% in questing ticks and from 0% to 16.7% in small mammal hosts. Local pathogen presence and prevalence were not impacted by I. scapularis abundance nor the abundance and diversity of mammal hosts. However, mammal species richness was a key predictor of the number of pathogen species. Collectively, my dissertation provides insight into the historical and contemporary relationships between ticks, hosts, and pathogens in Canada. My results demonstrate that additional tick species such as H. leporispalustris may be of public health importance due to their ability to maintain pathogens within the environment without needing a host. In addition, certain emerging or re-emerging tick-borne pathogens, such as B. odocoilei and R. rickettsii, were detected outside of currently defined risk areas in southeastern Quebec, which may impact future surveillance efforts in these regions. Furthermore, this work highlights the need for proactive and comprehensive surveillance efforts that test questing and feeding ticks of all life stages and species, as well as their hosts in areas outside currently defined risk areas or those targeted by sentinel surveillance to better determine the spread, transmission, and co-occurrence of tick-borne pathogens in Canada.