<p>Unsafe food is responsible for causing more than 600 million cases of foodborne illnesses and 420,000 deaths each year. These foodborne illnesses have direct impact on growth and development in children, food and nutrition security, national economies, and sustainable development. Food manufactures, research institutions, governments, and consumers, together, play a pivotal role in establishing and implementing effective food safety systems. <em>Salmonella</em> spp. and <em>Listeria monocytogenes</em> are recognized as major threats to global food safety and security among other 31 known and unknown pathogens associated with foodborne illnesses and deaths. Since these pathogens can be transmitted through contaminated food and water, contaminated environmental surfaces, and subsequently from environment to food via cross-contamination, there is an urgent need for data-driven approaches to identify key points of contamination along the food systems to suggest interventions. While it is important to enhance food safety research in developed economies, developing capacity to enable conditions for food safety research translation and practice in developing economies is crucial for global food safety. In this dissertation, we presented three different research projects as summarized below-</p> <p>In Chapter 2 “Evaluating the efficacy of celery powder in ready-to-eat deli style turkey breast against <em>L. monocytogenes</em> under ideal and temperature abuse conditions”. In this study, we artificially inoculated “clean label” deli style turkey breast formulated with celery powder to evaluate the efficacy of this natural antimicrobial in inhibiting the growth of this pathogen. We stored the inoculated samples at ideal (4 °C) and abuse temperature conditions (7 °C, 10 °C, and 15 °C) for 21 d mimicking the possible temperature abuse along the cold chain, transportation, and at consumer refrigerator. Our findings indicated that although deli meat samples stored at 4 °C and 7 °C did not achieve significant growth of <em>L. monocytogenes;</em> increasing temperatures to 10 °C and 15 °C led to significant increase in the growth rate of this pathogen. This study evaluates the use and effectiveness of celery powder as an antimicrobial used by deli meat processors against <em>L. monocytogenes</em> in deli products. These data underscore the importance of maintaining refrigeration temperatures to complement the efficacy of antimicrobials. </p> <p>Chapter 3 “Investigating sanitary solutions to <em>L. monocytogenes, Salmonella enterica</em> ser. Typhimurium, and <em>Pseudomonas aeruginosa</em> dry surface biofilms”. In this study, we developed <em>in vitro</em> mono- and mix-culture dry surface biofilm (DSB) models of <em>L. monocytogenes, Salmonella enterica</em> ser. Typhimurium, and <em>Pseudomonas aeruginosa</em> leveraging the EPA MLB SOP MB-19 standard protocol using the CDC Biofilm Reactor and evaluated sanitation control strategies currently adopted by low-moisture food (LMF) food processors to determine their ability to inactive DSB, a decidedly difficult reservoir to eliminate. This study targets multiple biological hazards in a research area with very limited publicly available data and is the first of its kind to refine mono- and multi-species <em>in vitro</em> DSB models that mimic LMF facility conditions and combinations of relevant microorganisms for use cases (e.g., EPA adoption). The findings from this study indicated that these foodborne pathogens could form DSBs and serve as a source of pathogen reservoir and cross-contamination. Results from the efficacy testing of sanitizer and microfiber swabbing suggested that current sanitation practices may not be sufficient to remove or inactivate DSBs. This study will define future needs and new strategies to improve confidence in sanitation efficacy with private sector practitioners.</p> <p>Chapter 4 “Enhancing research for development opportunities for Minority Serving Institutions: a case study in food safety”. Most developing economies have limited viable food safety systems due to underdeveloped research capabilities, competing resource demands, and insufficient enabling conditions, which undermines food security. United States Minority Serving Institution (MSIs) researchers and outreach specialists are familiar with and arguably best positioned to address global food safety and security challenges and needs, but MSIs implement limited research for development programs (e.g., U.S. university-led Feed the Future (FTF) Innovation Labs (ILs) funded by the United States Agency for International Development (USAID)) aimed to solve these challenges. Recognizing this opportunity, the Feed the Future Innovation Lab for Food Safety (FSIL) led by Purdue University in partnership with Cornell University, created and implemented an MSI-led research partnerships for global food safety research programs. In this chapter, we put together the process of a three-stage Request for Applications (RFA) process, which included non-competitive and competitive stages to encourage partnership and to refine ideas.  At the end of this process, seven individuals were invited to submit full proposals; two were funded. Intentional research opportunities and partnerships are essential to strengthen MSI competitiveness for research for development programs that develop and scale technologies to address urgent global agriculture, food security, and safety challenges.</p>

D. Tech. (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Salmonella enterica serovar Typhimurium and Campylobacter jejuni are the leading causes of Salmonellosis and Campylobacteriosis that is characterised by gastroenteritis. These waterborne diseases can be easily prevented by home water treatment methods such as solar disinfection (SODIS). The SODIS process involves placing microbiologically unsafe water in clear plastic or glass bottles and exposing them to direct sunlight for approximately six to eight hours. SODIS kills microbes through a combination of DNA-damaging effects of ultraviolet (UV) radiation and thermal inactivation from solar heating. The result is microbiologically safe water. Continuous drinking of SODIS treated water may confer some immunological effects on the consumer. These immunological effects have not been thoroughly explored. Therefore, the objectives of this study were to firstly, characterise the effects of solar irradiation on the viability of S. Typhimurium and C. jejuni; secondly, to determine the cytotoxicity and modulation of cell death of solar irradiated S. Typhimurium and C. jejuni on macrophages. Thirdly, to analyse the chemokine and cytokine profiles of macrophages infected with solar irradiated S. Typhimurium and C. jejuni. Lastly, to analyse the host-cell interactions of macrophages infected with solar-irradiated and non-solar irradiated S. Typhimurium and C. jejuni using a proteomic approach. In all the experiments, S. Typhimurium and C. jejuni were (i) heat/chemically treated, (ii) solar and non-solar irradiated for 4 and 8 hours. A murine macrophage cell line RAW264.7 was co-cultured with the differentially treated bacteria species for 3 and 24 hours. Appropriate controls were included. The impact of solar irradiated S. Typhimurium and C. jejuni on intracellular growth, proliferation, cytotoxicity, and apoptosis on macrophages was assessed. Intracellular growth of the both bacterial species was assessed with the gentamicin protection assay, and cytotoxicity was determined by Lactate Dehydrogenase Assay (LDH). The macrophages treated with solar irradiated S. Typhimurium and C. jejuni showed no intracellular growth after 48 hours post-infection. However, the non-irradiated S. Typhimurium survived within the macrophages and were highly toxic to the macrophages (average cytotoxicity of 91%±32). The non-solar irradiated C. jejuni were metabolically active but non-culturable, whereas the solar-irradiated C. jejuni was metabolically inactive. Thus, solar irradiated C. jejuni showed a lower percentage cytotoxicity (2.57% ± 0.32%) in comparison to non-solar irradiated C. jejuni at 24 hours post-infection (p.i.) (30.28% ± 0.05%). Flow cytometric analysis showed that the non-irradiated S. Typhimurium brought about a statistically significant increase in the percentage of necrotic cells (48% ± 2.99%), whereas bacteria irradiated for 8 hours produced a lower percentage of necrotic cells (25% ± 5.87%). The heat/chemical attenuated samples had the lowest percentage of necrotic cells (21.15% ± 5.36%) at 24 h p.i. Macrophages treated with solar irradiated and non-solar irradiated C. jejuni did not induce necrosis, but apoptotic cell death. At 24 h p.i., the highest proportion of apoptotic cell death was observed in macrophages treated with non-solar irradiated C. jejuni whereas the solar irradiated C. jejuni showed a lower percentage of apoptotic cell death. Therefore, there is great possibility that S. Typhimurium and C. jejuni could become avirulent after SODIS treatment and this could prevent gastroenteritis in consumers of SODIS-treated water. The activation of macrophages infected with solar irradiated S. Typhimurium and C. jejuni was also assessed in this study. The production of nitric oxide (NO) was determined using the Greiss Reagent Assay, whereas the production of chemokines, cytokines, and growth stimulating factors by the RAW264.7 cells in vitro was measured using the Luminex 200. The results showed that both solar and non-solar irradiated S. Typhimurium inhibited the production of nitric oxide in the RAW264.7 cells. The heat/chemically attenuated S. Typhimurium induced a significant increase (p<0.0.5) in the production of NO2− in the macrophages when compared to the unstimulated RAW264.7. The chemokine and cytokine levels produced by the macrophages were similar in the solar inactivated S. Typhimurium and the live untreated S. Typhimurium. However, macrophages treated with heat/chemically attenuated S. Typhimurium showed an anti-inflammatory response by inhibiting the production of pro-inflammatory cytokines such as IL-1, IL-1, IL-2, IL-6, and IL-17 in macrophages. The macrophages treated with solar and non-solar irradiated C. jejuni possibly produced an anti-inflammatory effect since the amount of pro-inflammatory cytokines in the samples was significantly reduced during the late infection period (24 h p.i.). This study also analysed the proteomic profiles of macrophages treated with LPS, non-solar irradiated, solar irradiated, heat/ chemical inactivated S. Typhimurium, and C. jejuni. This was carried out using SWATH-mass spectrophotometry-based proteomics. Proteins were extracted from infected macrophages after 24 hours p.i. HILIC-based sample clean-up and digestion, DDA LCMS-MS (spectral library), SWATH LCMS-MS, and data processing were carried out. A total of 15,077 peptides matching to 2,778 proteins were identified at 1% FDR with numerous differentially expressed proteins (DEPs) detected in macrophages treated with lipopolysaccharide (LPS), non-solar irradiated C. jejuni (NS), heat-attenuated C. jejuni (HA) and 4h-solar irradiated (SI4) and 8h-solar irradiated (SI8) C. jejuni, respectively. Pathway analysis revealed that most of the upregulated proteins in macrophages treated with solar irradiated C. jejuni were involved in oxidation-reduction processes, endoplasmic reticulum stress, transport, antigen processing and presentation of exogenous peptide antigens via MHC class I (TAP-dependant) and ATP-biosynthetic processes. The KEGG-pathways also revealed the roles of some upregulated proteins in lysosomal and phagosome pathways. In conclusion, our results revealed that there is coordinated up-regulation of MHC-I processing pathways occurred at 24 h p.i. It is likely that proteins from solar irradiated C. jejuni may undergo proteasomal degradation, and the peptides are transported to the endoplasmic reticulum (ER) and loaded onto MHC-I molecules. Peptide loading results in class I complexes consolidation and transit to the cell surface where antigens can be presented to circulating CD8 + T cells. Additionally, solar irradiated C. jejuni also undergoes degradation in the phagosome. The phagosome has the potential to create antigens that can be expressed on the cell surface of macrophages to stimulate different lymphocytes and induce appropriate immune responses, thus, connecting the innate to adaptive immunity, and this could also have health benefits via the consumption of SODIS treated water. However, proteomic analysis of S. Typhimurium showed no significant differentially expressed proteins in macrophages treated with LPS, non-solar irradiated, and solar irradiated S. Typhimurium. This may be due to an overestimation of the extracted protein. However, DEPs in macrophages treated with heat-attenuated S. Typhimurium showed that macrophages may have adapted an anti-inflammatory M2 phenotype because the IFN-γ signalling pathway was downregulated. This may have contributed to non-expression of the chemokine IFN-γ in RAW264.7 cells. Moreover, proteins such as Hmox1 and Sqstm1 were upregulated, and this is also characteristic of M2 macrophages. This study provided new insights on the effect of solar irradiated Salmonella Typhimurium and Campylobacter jejuni on the proliferation and activation of macrophages in vitro.

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