The research presented in this dissertation involves the study of two systems in order to analyze and develop resistance in plants to pathogens. The first study considered the interaction between lettuce (Lactuca sativa L.) and Plasmopara lactucae-radicis, a recently described casual agent of downy mildew. This unique fungus is the only known casual agent of downy mildew that is restricted to the roots of its host. Thirty-eight lettuce cultivars were screened for resistance to P. lactucae-radicis. Two-wk-old lettuce plants grown hydroponically were challenged with this fungus and evaluated 2 and 3 wk after inoculation for resistance. Root necrosis and production of sporangia on roots was considered a susceptible reaction. Five cultivars were determined to be resistant to this fungus. Resistant cultivars, however, were colonized by the fungus but did not support the production of sporangia on roots. Data from F2 and F3 progenies demonstrated that resistance was conferred by a single recessive allele (plr). Fungal infection of susceptible plants resulted in significant decreases in fresh root and shoot weights and leaf number compared to decreases associated with infection of resistant plants. RFLP probes have been identified for mapping the plr gene. The second topic of this dissertation dealt with the development of transgenic tobacco plants that express a chimeric gene that consisted of the 35-S promoter and the coat protein gene of Beet Curly Top Virus, a member of the geminivirus group. Expression of viral coat protein genes in plants has resulted in resistance to the virus from which the coat protein gene was obtained. This type of "coat protein-mediated" resistance has not been demonstrated for any geminivirus. Tobacco leaf discs were inoculated with an Agrobacterium tumefaciens line that contained the chimeric gene. Three transgenic lines were determined by Southern and Northern analysis, and ELISA, to express the chimeric gene and produce coat protein. Trials to determine the resistance of these plant lines to BCTV are pending federal approval.

Campylobacter jejuni (C. jejuni) is a food-borne bacterial pathogen that is the leading cause of Traveller’s Diaharea and Guillian-Barré Syndrome. Previous research determined that bacterial surface carbohydrates are virulence factors. Specifically, the study of the capsular polysaccharide (CPS) structures has widespread applications to understanding serotype cross-reactivity and biosynthetic pathways, the function of bacterial surface carbohydrates, and glycoconjugate vaccine development. This thesis characterized the CPS of C. jejuni HS:10 and subsequently used the CPS for developing a glycoconjugate vaccine. This was accomplished through extraction and purification of sugar from the bacteria, followed by characterization using chemical degradation, GC-MS, NMR and conjugation techniques. These analyses determined that the CPS was composed of a 3-linked GalNAc backbone and 6d-Galhepf attached at the 4th position with non-stoichiometrically attached O-methyl phosphoramidate attached to the 3 position of the heptose. Using this structure, a successful glycoconjugate vaccine was prepared using periodate oxidation and reductive amination.

A luminescence-based detection system was developed to study changes in the survival and activity of cells following release from moribund and dead fish. <I>A.salmonicida</I> was chromosomally marked with the genes encoding bacterial luciferase, originally isolated from <I>Vibrio harveyi</I>. Characterisation of the growth and luminescence of the <I>lux</I>-marked strain demonstrated that light was directly proportional to cell biomass concentration during logarithmic growth. The survival of <I>lux</I>-marked and wild type <I>A.salmonicida</I> strains was investigated in sterile sea water at 4°C. The number of culturable cells declined rapidly, but the total number of cells remained relatively constant, suggesting <I>A. salmonicida</I> entered a nonculturable state. The survival of <I>lux</I>-marked <I>A. salmonicida</I> did not significantly differ from that of the wild type strain. A small number of cells remained culturable throughout starvation experiments and luminometry confirmed that the <I>lux</I>-marked cells were metabolically active, possibly surviving by cryptic growth. The viability of putative dormant cells could not be established since these cells could not be reactivated following the addition of a range of substrates. The <I>lux</I>-marked <I>A.salmonicida</I> strain was pathogenic only when injected at high doses. This poor virulence was probably due to loss of the proteinaceous A-layer which is responsible for hydrophobic cell interactions and cell defence against lytic agents. This prevented further studies aimed at determining the virulence of nonculturable cells using this strain. Preliminary experiments indicated the potential of the <I>lux</I>-marked system for studying vertical transmission of <I>A. salmonicida</I>. The main sites for attachment of the <I>lux</I>-marked strain were the gill and skin/mucus regions. Identical results were obtained using a wild type <I>virulent A. salmonicida</I> strain, but significantly higher numbers of cells were recovered from fish tissue.

A mouse model was used to study the pathogenesis of systemic infection by the opportunistic pathogen <i>Candida albicans</i>. Using this model it was demonstrated that <i>C. albicans</i> yeast cells were more pathogenic for mice than hyphal forms. Polymorphonuclear leukocytes were shown to be important in resistance to systemic infection by <i>C. albicans</i>. Studies on conditions which promote germination of <i>C. albicans</i> yeasts showed that maximum numbers of yeast cells produced germ tubes when incubated in tissue culture media at 37°C, by 2 hours. A comparative ultrastructural examination of yeasts, germ tubes and hyphal forms demonstrated marked differences in the thickness and organisation of the cell walls between these forms. Furthermore, germination of <i>C. albicans</i> yeasts was shown to be accompanied by significant release of cell wall antigens. <i>In vitro</i> interactions between mouse polymorphonuclear leukocytes and <i>C. albicans</i> yeasts, germ tubes and hyphae in the absence of serum were examined. Mouse neutrophils were found to adhere readily to the surface of germ tubes and hyphae but not yeasts. This adherence resulted in damage of the fungus. Studies on the degradation of killed <i>C. albicans</i> yeasts following phagocytosis by murine macrophages <i>in vitro</i>, showed that progressive removal of yeast cell wall layers occurred. This was followed by dissolution of the cytoplasmic contents. During this process, cell wall and cytoplasmic antigens were released into the surrounding medium. An enzyme linked immunosorbent assay was developed to measure IgM, IgA and IgG class antibodies to <i>C. albicans</i> mannan and cytoplasmic antigens in patient's sera, and was shown to have diagnostic potential for candida infection. In particular, use of this assay to monitor the kinetics of antibody levels to these antigens was found to be of diagnostic value for immunocompromised patients at risk of candida infection. Finally a number of monoclonal antibodies were produced to <i>C. albicans</i> cytoplasmic proteins and have been partially characterised.

Risk-based inspection provides a framework whereby inspection resources can be prioritized and targeted towards foods that pose the highest risk to human health. To provide a risk assessment of the initial biological hazards associated with foods consumed, criteria related to hazard identification, hazard characterization and exposure assessment were developed for all foods inspected by the Canadian Food Inspection Agency.Using Canadian scientific data, food-pathogen pairs most responsible for foodborne illness were developed and ranked. To characterize the overall population burden of these food-pathogen pairs, a model adapted from the European Food Safety Authority (EFSA) was developed which incorporated criteria related to pathogen characteristics and probability of exposure of humans by food.The top risk-ranked food-pathogen pairs were Campylobacter spp. and poultry, pathogenic Escherichia coli and beef, Salmonella spp. and poultry, Salmonella spp. and produce, and Campylobacter spp. and dairy.

Septoria nodorum blotch is a disease of wheat caused by the necrotrophic fungus Parastagonospora nodorum. In the wheat-P. nodorum pathosystem, recognition of pathogen-produced necrotrophic effectors (NEs) by dominant host genes leads to host cell death, which allows the pathogen to gain nutrients and proliferate. To date, nine host gene-NE interactions have been reported in this pathosystem. Among them, the Snn2-SnTox2 interaction has shown to be important in both seedling and adult plant susceptibility. A saturated genetic linkage map was developed using a segregating population of recombinant inbred lines and a high-resolution map was then developed using F2 plants derived from a cross between the SnTox2-insensitive wheat line BR34 and the SnTox2-sensitive line BG301. Over 10,000 gametes were screened for high-resolution mapping and the Snn2 gene was delineated to a genetic interval of 0.10 cM that corresponds to a physical segment of approximately 0.53 Mb on the short arm of wheat chromosome 2D. A total of 27 predicted genes present in this region and thirteen of them were identified as strong candidates. Seven EMS-induced Snn2-insensitive mutants were generated for gene validation. Results of this study provide the foundation for cloning of Snn2. The host sensitivity gene Snn1, which confers sensitivity to SnTox1, was previously cloned. Here, allelic diversity of Snn1 was studied to identify causal polymorphisms, and to develop markers useful for marker assisted selection (MAS). Twenty-seven coding sequence haplotypes that correspond to 21 amino acid haplotypes were identified. Three SNPs were identified as the possible mutations that caused the insensitive allele in wild emmer to become the sensitive allele in domesticated wheat. In addition, four SNPs that changed the sensitive allele into insensitive alleles were identified. SNP-based markers that could detect three of those SNPs were developed. Results of this study help to increase our knowledge in wheat-NE interactions and host sensitivity gene evolution. / USDA – Agricultural Research Service / National Institute of Food and Agriculture

Zoonotic pathogens shared with wild or domesticated animals are the cause of more than 60% of human infectious diseases. These pathogens are responsible for millions of deaths annually and have resulted in costs of over a hundred billion U.S. dollars in the past three decades. Investigating different aspects of zoonotic pathogens can help inform policy decisions on public health, agriculture, and conservation of biodiversity. Because pathogens play essential roles in natural communities, studying the variables that influence pathogen richness is important in determining the biological principles governing biodiversity. Gaining a better understanding of the factors that influence these pathogens can allow for the development of effective and targeted action plans to deal with zoonotic disease outbreaks. The aims of this work were twofold: (1) to review the current literature and identify statistically significant predictors of pathogen richness, and (2) to analyze responses by public health agencies to recent zoonotic outbreaks. This work also discussed current gaps in the literature and suggested future areas of proposed funding and research.

Clostridia are a class of gram-positive, anaerobic bacteria best known for their powerful toxins. These bacteria cause many diseases that are difficult to treat and often deadly, including colitis, botulism, tetanus and gas gangrene. These diseases are caused by the secretion of specific toxins, though current treatments do little to nullify these toxins and better therapeutics are urgently needed. The development of such treatments is hindered by our poor understanding of clostridial toxin secretion, which is itself hindered by the innate characteristics of these bacteria that make them difficult to study. Of the pathogenic clostridia, Clostridium perfringens is relatively easy to culture and straddles the line between pathogen and commensal, making it an attractive model organism for studying clostridial toxin secretion. C. perfringens is a bacterium found naturally in soils and in the gastrointestinal tracts of humans and animals that can also cause disease. C. perfringens produces more toxins than any other bacterium, and these toxins generally function as a means to lyse host cells so the bacteria may scavenge their intracellular nutrients. The primary focus of the research in this dissertation is the secretion of the toxin TpeL by a small membrane protein, TpeE. Preceding the study of TpeL secretion were two other projects, which are discussed in Chapters 2 and 3. Chapter 2 describes an experimental plan to characterize the genes involved in muscle cell adherence as a very basic model to mimic skeletal muscle attachment in gas gangrene. Like many other bacteria, C. perfringens can produce T4P, extracellular filaments that are synthesized, extended and retracted from the cell by the concerted effort of many proteins. Results from initial, proof-of-concept adherence assays are presented and demonstrate that statistical significance was lost when data were compiled. Despite efforts to troubleshoot this, robust test output was not achieved and the project was discontinued November 2016. Chapter 3 describes the experimental plan and initial findings of a project where a link between T4P and virulence was investigated. Such a link had been demonstrated in the T4P model organism Pseudomonas aeruginosa, where PilT, the T4P retraction ATPase, was shown to sense surface attachment and initiate virulence. In C. perfringens, PilT demonstrates a number of characteristics that lead us to think it may also function as a sensor, coordinating host cell attachment and colonization by alternatively associating with PilM and FtsA. We developed an experimental plan to determine if PilT binds both PilM and FtsA by co-immunoprecipitation with live-cell fluorescence imaging. However, we were unable to demonstrate the functionality of a PilT-fluorescent protein fusion with an anti-pilin ELISA assay, nor were we able to detect PilT or FtsA overexpression by immunoblotting, and the project was discontinued in November 2017. In retrospect, these experiments likely failed because of an inactive promoter region in the overexpression plasmid. Though clostridial diseases require secreted toxins, their secretion mechanisms are largely uncharacterized, and Chapter 4 describes the investigation of a potentially conserved toxin secretion mechanism. TpeL is a recently discovered C. perfringens toxin that is associated with chicken necrotic enteritis, a disease that costs the poultry industry billions of dollars each year. TpeL belongs to a subset of clostridial toxins characterized by their large size and conserved structure, the large clostridial toxins. The gene for tpeL and nearly all other large clostridial toxins lies next to a gene encoding a small membrane protein. Since bacterial genes with a shared function are often found in close proximity, it is suspected that these small proteins share some function with these toxins, and another research group has shown the two large clostridial toxins in C. difficile need this small membrane protein for their secretion. We isolated the small membrane protein and toxin genes tpeE and tpeL from native regulatory elements and overexpressed them heterologously in a different strain of C. perfringens. By immunoblotting, we found rapid TpeL secretion requires TpeE, and secretion was abolished when C-terminal sections of either protein were mutated. By immunoblotting and growth curve analyses, we found that TpeE is maintained at low concentrations and is not lethal in C. perfringens, but was expressed to high levels and was lethal in Escherichia coli. Our results, in conjunction with those from other research groups strongly suggest a conserved secretion mechanism dependent on small, membrane proteins. Our findings further the understanding of toxin secretion, a key step toward novel and effective clostridial disease strategies. Chapter 5 describes the outcome of an experimental approach where tpeE and tpeL were expressed from two different expression system plasmids. A number of off-target effects materialized with this approach which confounded our experimental results. The predominantly confounding effect was off-target protein secretion, found by immunoblotting to be associated with one of the expression systems. Despite efforts to minimize these effects, it became clear results from this approach would be uninterpretable and the two-plasmid approach for TpeE and TpeL expression was abandoned. A cut-and-paste strategy using the historical, single inducible expression system was implemented in its place. The exact mechanism for TpeL secretion by the small membrane protein TpeE is unclear. Chapter 6 outlines some hypotheses towards this mechanism and a nascent plan to uncover it. An efficient starting point is to determine if the two proteins are in close enough proximity to one another to interact in vivo. We developed a strategy to determine this by crosslinking and immunoblotting, using the size differential between the proteins to our advantage. Though the results of this study were confounded by an inability of TpeL to solubilize in buffer, the groundwork is laid for future endeavors. / Doctor of Philosophy / Clostridium perfringens is a bacterium found naturally in soils and in the gastrointestinal tracts of humans and animals worldwide. C. perfringens is an important organism to study due to its roles as a decomposer in our ecosystem and its ability to cause a number of diseases. These diseases cause considerable harm to livestock and poultry industries, as well as to human and animal life. Though these diseases vary wildly, they share this in common: they are defined by specific toxins and what defines a harmful lineage of C. perfringens is its ability to produce and secrete these toxins. In fact, this is the common denominator to all clostridial diseases, including the notorious diseases C. difficile-associated colitis, tetanus, botulism, and gas gangrene. Of primary concern in diseases caused by C. perfringens and other clostridia is that effective, novel therapies are grossly lacking. The effects of these powerful toxins can outpace antibiotic therapy and this often leads to extended periods of suffering, even in favorable cases. Of all the pathogenic clostridia, C. perfringens is easy to test in the laboratory and may even be used in place of more dangerous and difficult to work with bacteria. This is useful in developing better treatments and for studying treatment applications for the toxins themselves! Indeed, bacterial toxins have beneficial applications, Botox being a good example, as well as in cancer treatments. Like many other bacteria, C. perfringens can produce strong, rope-like appendages called pili that are made and extended and retracted from the cell, similar to a lasso, by the concerted effort of many different proteins. These pili confer a number of advantages for bacteria, one being a means for attachment to host cells, an important first step in establishing an infection. With the overarching goal toward sowing future therapeutic developments, Chapter 2 describes an experimental plan to identify and understand the genes for the proteins that allow C. perfringens to attach to muscle cells. Preliminary results are presented for a proof-of-concept method, which was ultimately discontinued November 2016 because reliable, robust results were not obtained. In addition to host cell attachment, pili have been shown to function a sensor for cell. For example, in another bacterium, pili "sense" a suitable surface for attachment and interpret this signal so the bacterium can attach and "set up shop" by releasing toxins. Based on considerable evidence, we thought C. perfringens might also "sense" a surface with its pili and interpret this signal for attachment and cell growth by means of interactions between three specific proteins. We designed a series of experiments to test this hypothesis, but due to the failure of important, initial studies, this project was discontinued in November 2017. Even though all clostridial diseases are caused by toxins, which must be secreted outside the bacterium to do harm, how these toxins are secreted is poorly understood. In Chapter 4, we investigate a toxin secretion method where a certain type of toxin is thought to be secreted through a temporary hole formed by many copies of a small, partner. First, we forced C. perfringens bacteria to artificially produce both the small protein and the toxin and found that the toxin needs this small protein to be secreted. We then deleted parts of both the toxin and the small protein and determined which parts of each are essential for this secretion method by linking an absence of secretion in bacteria whose proteins are missing essential parts. Further, we determined that production of this small, partner protein was kept to low levels and was harmless in C. perfringens, but was lethal in a different, unrelated bacterium, Escherichia coli, implying that C. perfringens bacteria have an ability control this hypothetical hole in themselves that E. coli does not. Our results, in conjunction with those from previous groups, suggest a pattern for secretion of this type using these small proteins. This information is a key first step towards developing better therapies for clostridial diseases, since without toxin secretion, clostridial diseases cannot occur. Chapter 5 describes the surprising outcome of an experimental approach where the toxin and small partner protein are produced in the bacterium by two different mechanisms. We found a number of off-target effects associated with this approach, one of which was the strange facilitation of off-target protein secretion. These off-target effects confused our experimental results and since it was likely that future experiments would also be uninterpretable, we abandoned this approach and used a simpler one instead. The mechanisms for toxin (TpeL) secretion by its small, partner protein (TpeE) are unclear. A key, initial step towards understanding this mechanism is to determine if the two proteins are in close enough proximity to one another in the bacterium. We developed a strategy to determine if the proteins are close enough together in the cell that takes advantage of the considerable size difference between the two proteins. Presented in this chapter are several initial experiments that can enable this experiment in the future.

Extracts from medicinal mushrooms were prepared and tested for anti-microbial activity against food pathogens and food spoilage microorganisms. The inhibitory activity was measured using a disk diffusion assay and with optical density (OD). For OD, 7 fractions were collected using HPLC for 4 (A. blazei Murrill, G. lucidum, G. frondosa, I. obiquus) medicinal mushrooms and 6 fractions from L. edodes and 8 fractions from P. linteus. The results from disk diffusion assay showed that most mushrooms displayed significant inhibition compared to the ethanol. The exceptions were: A. blazei Murrill, I. obliquus, and L. edodes against E. coli O157:H7; I. obliquus against L. monocytogenes V7; I. obliquus against S. cerevisiae Y99; L. edodes against Z. bailii Y03; and I. obliquus against Z. bailii/bisporus Y108. Inhibition was more effective in yeasts than bacteria. The result from Bioscreen C showed that against L. monocytogenes V7, fraction 7 in A. blazei Murrill; fraction 1, 4 and 5 in G. lucidum; fraction 4 in G. frondosa; and fraction 4 and 5 in I. obliquus significantly inhibited the growth compared to ethanol. Against Z. bailii Y03, fraction 7 in A. blazei Murrill; all fractions from G. lucidum, G. frondosa, and P. linteus; fraction 1, 2, 3, and 6 from I. obliquus; and fraction 4 and 6 from L. edodes significantly inhibited growth compare to ethanol. The results indicated that there is significant antimicrobial activity against food pathogens and spoilage organisms in the medicinal mushrooms studied. / Ph. D.

A host can adopt two strategies when facing infection: resistance, where host immune responses prevent or reduce pathogen replication; or tolerance, which refers to all mechanisms that reduce the impact of the infection on host health or performance. Both strategies may be under host genetic control, and could thus be targeted for genetic improvement. Although there is ample evidence of genetic variation in resistance to infection, there is limited evidence to suggest that individuals also differ genetically in tolerance. Furthermore, although resistance and tolerance are typically considered as alternative host defense mechanisms, relatively little is known about the genetic relationship between them and how they change together over time and jointly determine infection outcome. In this thesis, two datasets from experimental challenge infection experiments were considered for investigating tolerance genetics: Porcine Reproductive & Respiratory Syndrome (PRRS), an endemic viral disease which causes loss of growth and mortality in growing pigs; and Listeria monoctyogenes (Lm), a bacterium which causes food-borne infections in mammals. The two datasets differed substantially in size and genetic structure; the PRRS dataset consists of thousands of records from outbred commercial pig populations, whereas the Listeria dataset comprises much fewer records from genetically diverse highly inbred strains of a mice as a model species. The aims of this thesis were to: 1) Identify if genetic variation in host tolerance to infection exists, with case studies in PRRS and listeria, using conventional reaction-norm methodology; 2) Identify if host tolerance, along with resistance, changes longitudinally as infection progresses; 3) Identify whether the WUR genotype is associated with tolerance slope; 4) Analyse the dynamic relationship between host performance and pathogen load over the time-course of infection by examining the relationship at different stages of infection using GWAS; 5) Develop novel trajectory methodology to offer insight into health-infection dynamics, and identify whether there is genetic variation in trajectories; 6) Develop novel trajectory-derived phenotypes that analyse changes in host performance with respect to changes in pathogen load, as an alternative to tolerance, and identify whether genetic variation exists. This study found that conventional reaction-norm methodology is limited to capture genetic variation in tolerance in outbred populations without measures of performance in the absence of infection. However, by utilising repeated longitudinal data on the same dataset, stages of infection (early, mid and late) were defined for each individual, based on host pathogen load. Using these stages of infection, genetic variation in tolerance was identified over all stages of infection and at mid to late stage of infection. Genetic correlation between resistance and tolerance was strong and positive over all stages of infection, and evidence suggested that resistance and tolerance may be under pleiotropic control. Furthermore, this research found that genetic correlations between resistance and growth changed considerably over time, and that individuals who expressed high genetic resistance early in infection tended to grow slower during that time-period, but were more likely to clear the virus by late stage, and thus recover in growth. However, at mid-late stage of infection, those with high virus load also had high growth, indicating potential epidemiological problems with genetic selection of host resilience to infection. Furthermore, genome wide association studies for pathogen load and growth associated with different stages of infection did not identify novel genetic loci associated with these traits than those previously reported for the whole infection period. By adopting conventional methodology, this study found genetic variation in tolerance of genetically diverse mouse strains to Lm and pigs to PRRS, despite statistical problems. The relationship between resistance and tolerance indicated that both traits should be considered in genetic selection programs. By adopting novel trajectory analysis, this study demonstrated that level of expression of resistance and tolerance changed throughout the experimental infection period and, furthermore, that expression of resistance, followed by tolerance, determined survival of infection. Survivors and non-survivors followed different infection trajectories, which were partially determined by genetics. By deriving novel phenotypes from trajectories that explained changes in growth in relation to change in pathogen load at specific time points, and applying these to the PRRS data, this study did not identify genetic variation in these phenotypes. The genetic signal in these phenotypes may have been masked by the fact that individuals were likely at different stages of infection. In summary, this study has shown that genetic improvement of tolerance, in addition to resistance may be desirable, but could be difficult to achieve in practice due to shortcomings in obtaining accurate and unbiased tolerance estimates based on conventional reaction-norms. Infection trajectories have proven to be a promising tool for achieving an optimally timed balance between resistance and tolerance, but further work is needed to incorporate them in genetic improvement programs.