E. coli O157:H7 is the most important serotype of enterohaemorrhagic E. coli (EHEC) which is of concern to public health worldwide. As a common cause of haemorrhagic colitis, EHEC infection can progress to life threatening sequelae including haemolytic uraemic syndrome (HUS) in humans. Human infection rates are higher in Scotland than found in the rest of the UK. Cattle are asymptomatic carriers of EHEC and are an important reservoir from which disease outbreaks can spread. The terminal rectum has been indicated as a site of E. coli O157:H7 colonisation in the bovine intestinal tract. This is the location of numerous lymphoid follicles which contain dendritic cells (DCs) which are professional antigen presenting cells and important directors of immune responses. DCs are likely to come into contact with EHEC and therefore could be key in this location for enabling EHEC to colonise the bovine host. The first aim of this project was to characterise dendritic cells within the bovine intestinal tract at various anatomical locations, including the terminal rectum, using immunohistochemistry techniques. Following this, work to extract and further phenotype dendritic cells from terminal rectal tissues was undertaken. Finally, a widely-used bovine dendritic cell model was employed to generate dendritic cells from circulating blood monocytes. This model was utilised to investigate the interactions of dendritic cells with EHEC strains compared with responses to bovine enterotoxigenic (ETEC) and bovine commensal E. coli strains. Early work identified that there are potentially numerous DCs within the bovine intestinal tissues and these cells were found in greater numbers at the terminal rectum. Protocols to extract and further characterise these cells were developed but proved inconsistent, with large variation between animals. Using the monocyte derived dendritic cells (moDCs), differences were observed between immunological responses to challenge with E. coli O157:H7 strains and bovine pathogenic or commensal E. coli strains. Cytokine production, cell surface molecule expression, cell phenotype and viability as well as intracellular bacterial counts were compared. The data presented here shows that the bovine moDCs respond differently to EHEC strains when compared with commensal or pathogenic E. coli in several key areas. This has important implications for the responses of the bovine host to various E. coli strains. This work also indicates that dendritic cells could be central to these responses and if studied further still, may hold the key to reducing the colonization and persistence of E. coli O157:H7 in cattle, and subsequent human disease outbreaks.

The performance of a novel, visible light-driven photoelectrocatalytic (PEC) batch reactor employing tungsten trioxide (WO₃) as a photocatalyst was assessed by studying the degradation of selected model pollutants (2,4-DCP, chloroform) and the disinfection of a human bacterial pathogen (<i>E. coli </i>O157:H7). Overall efficacy of the batch reactor was assessed by combining biological toxicity assessment (biosensing) with conventional analytical chemistry. Photoelectrocatalytic degradation of the organoxenobiotics (2,4-DCP, chloroform) was monitored toxicologically by applying bacterial <i>lux</i>-marked biosensors and analytically by HPLC. The bacterial biosensor traced the removal of the target, model pollutants during degradation experiments, and also monitored changes in toxicity in the analyte of the PEC batch reactor caused by the possible appearance/disappearance of toxic transient intermediates derived from the breakdown of the parent molecule. Chromosomally <i>lux</i>-marked, non-toxigenic <i>E. coli</i> O157:H7 was selected as a model human pathogenic bacterium to demonstrate the disinfection potential of the batch reactor. Results of disinfection experiments indicated that a substantial decline in the population density of culturable <i>E. coli </i> O157:H7 cells was achieved. Accurate differentiation between the effects of photoelectrocatalysis and photolysis on the cells of <i>E. coli</i> O157:H7 was not achieved. The observed rate of the degradation of the model chemical compounds and the disinfection of the model human pathogen, demonstrated that visible light-driven photoelectrocatalysis offers considerable potential for remediation of contaminated water. Furthermore, toxicological biosensing can bridge the gap between traditional chemical analysis and ecologically relevant sample evaluation and address suitability of reintroduction of treated solution back into mainstream wastewater treatment.

Enterohaemorrhagic Escherichia coli O157:H7 causes haemorrhagic diarrhoea and potentially fatal renal failure in humans. Ruminants are considered to be the primary reservoir for human infection. Vaccines that reduce shedding in cattle are only partially protective and their underlying protective mechanisms are unknown. Studies investigating the response of cattle to colonisation generally focus on humoral immunity, leaving the role of cellular immunity unclear. To inform future vaccine development, the cellular immune response of cattle during EHEC O157:H7 colonisation was examined. Calves were challenged with either a phage type (PT) 21/28 strain possessing the Shiga toxin (Stx) 2a and Stx2c genes or a PT32 strain possessing the Stx2c gene only. T-helper cell associated transcripts at the terminal rectum were analysed by reverse transcriptase quantitative PCR (RT-qPCR). Induction of interferon (IFN)γ and T-bet was observed, with peak expression of both genes at 7 days in PT32 challenged calves, whilst up regulation was delayed, peaking at 21 days in PT21/28 challenged calves. Cells isolated from gastro-intestinal lymph nodes demonstrated antigen-specific proliferation and IFNγ release in response to type III secreted proteins (T3SPs); however responsiveness was suppressed in cells isolated from PT32 challenged calves. Lymph node cells showed increased expression of the proliferation marker Ki67 in CD4+ T cells from PT21/28, NK cells from PT32 and CD8+ and γδ T cells from both PT21/28 and PT32 challenged calves following ex vivo stimulation with T3SPs. Epitope mapping of rectal lymph node CD4+ T cell responses to 16 EHEC O157:H7 proteins, identified 20 CD4+ T cell epitopes specific to E. coli. The highly conserved N-terminal region of Intimin, including the signal peptide, was consistently recognised by mucosal CD4+ T cell populations from multiple animals of different major histocompatibility complex (MHC) class II haplotypes. Studies investigating the impact of secreted bacterial proteins on bovine peripheral blood mononuclear cells (PBMC) identified the ability of these proteins to cleave the surface molecule CD8 and that this phenotype was dependent on the ler virulence regulator but not the type III secretory system (T3SS) machinery. This effect was also observed in murine and ovine, but not human lymphocytes. Preliminary in vitro experiments suggest that this activity may reduce the efficiency of CD8+ T cell killing. This study demonstrates that cattle mount cellular immune responses during colonisation with EHEC O157:H7, the temporality of which is strain dependent, with further evidence of strain-specific immunomodulation.

Shiga toxin (Stx) is a bacteriophage (phage)-encoded virulence factor of the Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 implicated in the pathogenesis of renal tissue damage and bloody diarrhoea in human. Cattle are the main asymptomatic reservoir for EHEC O157:H7 with the lymphoid-follicle rich areas of the terminal rectum identified as the primary colonisation site. However, the significance of Stx during bovine intestinal colonisation by EHEC O157:H7 remains unclear with mixed findings described in published studies. The objective of this study was to investigate if Stx and the Stx-encoding phage significantly contribute to EHEC O157:H7 colonisation particularly at the bovine terminal rectum. The expression of Stx receptor, Globotrioasylceramide (Gb3) at the bovine terminal rectum was analysed by fluorescence microscopy, revealing a similar pattern of Gb3 detection in the bovine colon with scattered positive detections limited to sub-epithelial, mesenchymal-associated cells. Purified Stx2 treatment of Gb3+ and Gb3- epithelial cell lines for 6 to 18 hours produced no effect on the cell cycle and proliferation. CD3+/CD8+, CD3+/γδ+ and CD21+ cells were significantly different between calves infected with EHEC O157:H7 Strain 9000 (Stx2a+/Stx2c+) and the uninfected calves, but not in calves with Strain 10671 (Stx2c+). Stx did not interfere with IFN-gamma (IFN-γ)-activation of the JAK/STAT1 pathway in epithelial cells. Bovine EHEC O157:H7 strains isolated from Scottish cattle farms in the IPRAVE study (Phage type 21/28 and 32) were used for a series of bacterial phenotypic characterisation assays. Total Stx production, Verocytotoxicity, growth in a competitive environment, epithelial cell adherence and Galleria mellonella virulence assay were performed to compare the IPRAVE EHEC O157:H7 strains (PT21/28 and PT32) and the isogenic Stx-phage mutants. Stx levels produced by the bovine-originated EHEC O157:H7 strains were significantly lower than that of the human isolated strains. The absence of Gb3 on the bovine terminal rectal epithelium, the non-significant changes in the cell cycle along with the uninterrupted IFN-γ activation of the JAK/STAT1 pathway in intestinal epithelial cells and the minute quantities of Stx generated by EHEC O157:H7 bovine strains suggest that the toxin is not involved in colonisation directly, at least at the intestinal epithelial level. Although future work is required to explain the mechanisms underlying the observed EHEC O157:H7 phenotypic changes particularly in the Stx-phage mutant strains, the work done has proven that the Stx-encoding phage indeed has the ability to exert changes in the bacterial cell leading to changes in bacterial phenotypes, which in turn, might affect the colonisation of the bovine intestine.

<p>The purpose of this study was to investigate the occurrence<strong> </strong>of Verotoxin-encoding bacteriophages in mussels, cultured downstream the sewage treatment plant<strong> </strong>in Lysekil.</p><p>Mussels were collected in three growing areas from April 2008 to March 2009. Real-time PCR was performed for detection of <em>vtx1</em> and <em>vtx2</em> genes and enrichment of bacteriophages on non Verotoxin-producing <em>Escherichia coli</em> O157: H7 was carried out. All samples in real-time PCR analysis were negative; no presence of Verotoxin-encoding phages was shown. No plaque was formed on blood agar base plates, indicating that no bacteriophages had been taken up by <em>E. coli</em> bacteria</p><p>The levels of Verotoxin-encoding phages and <em>E.coli</em> outside the sewage treatment plant in Lysekil were not high enough to be able to form VTEC in mussels, indicating that the faecal contamination was low. This does not exclude the presence of other more common pathogens such as norovirus and campylobacter.</p><p> </p><p> </p>

<p>Escherichia coli is a natural inhabitant of the intestines of both humans and animals, but there are also several pathogenic types of E. coli which cause disease in humans.</p><p>Strains of enterohemorrhagic E. coli (EHEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans. Most clinical signs of disease arise as a consequence of the production of shigatoxin 1 and 2 or combination of these toxins. Other major virulence factors include EHEC hemolysin and intimin, the product of the eae gene that is involved in attaching and effacing adherence phenotype. EHEC has also been associated with uncomplicated diarrhea.</p><p>The capacity to control EHEC disease and to limit the scale of outbreaks is dependent upon prompt diagnosis and identification of the source of infection.</p><p>The principal reservoirs of EHEC are cattle and food products, which presumably have come into contact with domestic animal manure and/or are inadequately pasteurised, these are important vehicles of infection.</p><p>In the present study, the PCR technique with primers detecting the verocytotoxin genes was shown to be a possible method to screen for and identify EHEC.</p><p>In summary stx genes were detected in 16 samples of 228 sampels and the eae gene was detected in 2 samples using PCR.</p>

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (p. 98-113).

The purpose of this study was to investigate the occurrence of Verotoxin-encoding bacteriophages in mussels, cultured downstream the sewage treatment plant in Lysekil. Mussels were collected in three growing areas from April 2008 to March 2009. Real-time PCR was performed for detection of vtx1 and vtx2 genes and enrichment of bacteriophages on non Verotoxin-producing Escherichia coli O157: H7 was carried out. All samples in real-time PCR analysis were negative; no presence of Verotoxin-encoding phages was shown. No plaque was formed on blood agar base plates, indicating that no bacteriophages had been taken up by E. coli bacteria The levels of Verotoxin-encoding phages and E.coli outside the sewage treatment plant in Lysekil were not high enough to be able to form VTEC in mussels, indicating that the faecal contamination was low. This does not exclude the presence of other more common pathogens such as norovirus and campylobacter.

<i>Escherichia coli</i> O157 is a food borne pathogen of increasing public health concern worldwide. Cattle have been implicated as the primary reservoir of <i>E. coli</i> O157. The fact that the livestock industry has rapidly expanded in Saskatchewan makes it imperative to have a clear scientific understanding of the prevalence of <i>E. coli</i> O157 in this province as well as its survival in soil under ambient conditions.<p>Longitudinal and point studies were employed to determine the prevalence of <i>E. coli</i> O157 among Saskatchewans cattle. During a 2-year period, 23 feedlot and cattle operations were examined and an overall prevalence of 15.6% was reported. The most important finding was that the prevalence rates were highly dependent on cattle density. All <i>E. coli</i> O157 isolates obtained from this study were characterized by using multiplex PCR, RAPD fingerprinting, a Vero cell cytotoxicity assay and antibiotic susceptibility tests. This characterization revealed a surprisingly highly virulent and heterogenous population of <i>E. coli</i> O157 isolates. <p>Subsequently, the survival characteristics of <i>E. coli</i> O157:H7 ATCC 43894 in sterile soil and manure-amended soil microcosms, as well as in situ under ambient environmental conditions were examined. Findings from this work indicated that desiccation had the most lethal effect on cell viability, whereas nutritionally-rich soils significantly increased survival times of the pathogen population. <p>A final study was designed to examine the survival strategy of hyper- and hypothermally adapted <i>E. coli</i> O157 cells exposed to high and low temperatures, with specific focus on the role of RpoS. Using wild type and its rpoS null allele <i>E. coli</i> O157 strains, in combination with 2D PAGE, It was found that both heat and cold post-acclimation stimulons consisted of two large sub-groups: (i) stress proteins, and (ii) housekeeping proteins. Comparative proteomic analyses revealed that the GroEL/S chaperonin complex and Pnp ribonuclease played a crucial role in growth resumption during high and low temperatures, respectively. Notably, RpoS had no control over key stress proteins in either stress stimulon. RpoS, however, showed a significantly more pronounced role during cold temperatures, where it was seen to regulate key proteins involved in homoeoviscous adaptation as well as various housekeeping proteins of both stress stimulons.

Escherichia coli is a natural inhabitant of the intestines of both humans and animals, but there are also several pathogenic types of E. coli which cause disease in humans. Strains of enterohemorrhagic E. coli (EHEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome in humans. Most clinical signs of disease arise as a consequence of the production of shigatoxin 1 and 2 or combination of these toxins. Other major virulence factors include EHEC hemolysin and intimin, the product of the eae gene that is involved in attaching and effacing adherence phenotype. EHEC has also been associated with uncomplicated diarrhea. The capacity to control EHEC disease and to limit the scale of outbreaks is dependent upon prompt diagnosis and identification of the source of infection. The principal reservoirs of EHEC are cattle and food products, which presumably have come into contact with domestic animal manure and/or are inadequately pasteurised, these are important vehicles of infection. In the present study, the PCR technique with primers detecting the verocytotoxin genes was shown to be a possible method to screen for and identify EHEC. In summary stx genes were detected in 16 samples of 228 sampels and the eae gene was detected in 2 samples using PCR.