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Verocytotoxigenic Escherichia coli (VTEC) is a major cause of diarrheal infections. It is increasingly associated with the consumption of fresh produce and responsible for several international outbreaks with severe sequelae. Quantitative risk assessment (QRA) aim to evaluate the risk of contamination, quantify uncertainties and identify potential mitigation steps. Current published QRA focus on post-harvest process steps without consideration of pre-harvest factors. This study aimed to identify preharvest knowledge gaps and quantify the potential for growth of VTEC in plant tissue prior to harvest. Risk factors were used to develop a QRA for VTEC O157:H7 in ready-to-eat (RTE) salad from farm-to-fork. Plant host metabolites were analysed by mass spectrometry and chromatography. Further plant and bacteria derived factors involved in pre-harvest colonisation were determined and implemented into a QRA. The burden of disease was estimated as the probability of illness by consuming RTE salad in the United Kingdom (UK). VTEC was shown to frequently internalise into lettuce and spinach to high concentrations. There, it can persist, evade surface decontamination and crosscontaminate equipment and produce during processing. The probability of illness was estimated to be 2.66 * 10-5 for lettuce and 1.51 * 10-5 for spinach. Internalisation was determined as the driving factor for 1820 illnesses per year in the UK associated with the consumption of lettuce and 950 with spinach, respectively. Mitigation steps had limited impact on internalised VTEC O157:H7 and mostly reduced external bacteria. Outputs from the QRA indicate the insufficiency of current mitigation steps to reduce internal VTEC O157:H7 and highlights the need for advanced decontamination procedures. The QRA tool can be used for the development of industrial guidelines in order to improve food safety in the UK and Europe.

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Submitted in complete fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2012. / Immunomodulation using plants is of primary interest in scientific communities because it provides an alternative to conventional chemotherapy for a wide range of diseases. It is based on the ability of the plants to effectively modulate immune functions, thus being able to promote positive health and maintain the body’s resistance to infection. This research is aimed to evaluate the immunomodulatory potential of fourteen traditional leafy vegetables from Kwa-Zulu Natal, South Africa on human peripheral blood mononuclear cells (PBMC). In this study the methanolic and aqueous extracts were screened for lymphocyte proliferation using the MTT assay. The cytokine response was evaluated by measuring the secretion of interleukin 10 (IL-10) and interferon-gamma (IFN-γ) using the ELISA assay. The subpopulation of T cells viz., CD4+, CD8+, NK and B cells were measured by flow cytometry. Most of the methanolic extracts stimulated PBMC’s whilst a few suppressed lymphocyte proliferation. Most of the aqueous extracts were inactive. The methanolic extracts of Amaranthus hybridus and Centella asiatica stimulated PBMC’s and showed an increase in IFN-γ secretion and the CD8+ cytotoxic T cells and B cells. Thus, they induced the Tc-1 immune response and stimulated cell mediated immunity. The methanolic extracts of Asystasia gangetica, Bidens pilosa, Emex australis, Justicia flava Momordica balsamina, Oxygonum sinuatum, Senna occidentalis and Sonchus oleraceous and the aqueous extracts of Amaranthus spinosus and Asystasia gangetica, Ceratotheca triloba, Oxygonum sinuatum, Physalis viscosa and Sonchus oleaceous stimulated PBMC’s and showed an increase in IL-10 secretion and the CD8+ cytotoxic T cells and B cells. Thus, they induced the Tc-2 immune response and stimulated humoral immunity. Also, the methanolic extracts of Amaranthus spinosus and Ceratotheca triloba and the aqueous extracts of Bidens pilosa and Justicia flava increased both IL-10 and IFN-γ secretion and the CD8+ vii cytotoxic T cells indicating the stimulation of both the Tc1 and Tc2 cytokine profiles. The elevated secretion of IFN-γ and IL-10 caused by the extracts can be attributed to the CD8+ cytotoxic T cells and B cells. The findings of this study show that leafy vegetables hold promise as immunomodulatory candidates. They may enhance cell-mediated immune functions by a pro-inflammatory response whilst some can promote humoral immune functions by means of an anti-inflammatory response. Further investigation should be considered on the effect of the extracts on other immune parameters. / National Research Foundation

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