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Salmonella enterica serovar Enteritidis (Salmonella Enteritidis), as the most relevant serovar associated to shell eggs, has caused numerous salmonellosis outbreaks. Salmonella Enteritidis exhibits great fitness in colonizing oviduct track of laying hen, leading to its predominance in shell eggs through vertical transmission route. Salmonella Enteritidis grown in egg yolk showed pronounced disease symptoms in a mouse model. However, the cause of the increasing virulence, attributed to the presence of the pathogen in egg environment, remains unrevealed. The high risk of S. enterica contamination prompted the food industry to develop egg pasteurization processes aiming to produce safer shell egg while maintaining the product’s fresh qualities. Water-immersion heating is often used to pasteurize shell eggs. Recently, ozone in combination with moderate heat treatment was introduced as an alternative to the purely thermal pasteurization technology. Although such processing technologies are promising, it is not known how the physiology of Salmonella changes during the treatment or what safety risks these pasteurization technologies would cause. To full fill these research gaps, the goals of the current study are: (I) to understand the contribution of shell eggs, as a food vehicle, to the increased risk of salmonellosis; and (II) to reveal the role of egg pasteurization parameters in increasing or mitigating the salmonellosis risk. The hypotheses related to the first goal of this research are: (1) egg environment can serve as an evolutionary drive for Salmonella Enteritidis to acquire genetic advantages, contributing to the virulence of the pathogen; and (2) growth of Salmonella Enteritidis in egg yolk allows changes in the transcription of pathogen’s virulence genes leading to increased virulence in a mouse model. To address these two hypotheses, the objectives under the first goal are: (1) using comparative genomic to explore whether egg environment provided Salmonella Enteritidis an evolutionary advantage favoring the pathogen’s ability to cause disease, and (2) using transcriptional analysis and the mouse model to measure the changes in virulence of Salmonella Enteritidis due to its growth in egg yolk, compared to growth in microbiological medium, tryptic soy broth (TSB), which serves as a control. The hypothesis related to the second goal is that subjection of Salmonella Enteritidis to improperly implemented heat or ozone during shell egg pasteurization induces an expression of stress responses leading to increased resistance to processing and elevated virulence. To test this hypothesis, the research objective was to measure phenotypical and transcriptional stress responses and virulence expression of Salmonella Enteritidis in egg yolk when treated with heat, ozone, and their combination. To reveal the effect of egg yolk as a food vehicle on the virulence of Salmonella Enteritidis (Goal I), the genomes of four Salmonella Enteritidis strains of eggs origin (three were sequenced and one was selected from NCBI database), along with eight genomes of Salmonella Enteritidis strains of clinical- and organ-origin, were compared to form a phylogenetic tree. The phylogenetic tree grouped egg–derived strains into one genomic cluster away from the rest of the strains. Unique genetic traits present in Salmonella Enteritidis of egg origin involved tyrosine metabolism, fatty acid degradation, and gluconolactone oxidation, which are related to the survival of Salmonella in egg environment. However, the association between these metabolic pathways and Salmonella virulence is yet to be investigated. To further fulfill the big picture of goal I, mice were gavaged with Salmonella Enteritidis that was (a) grown in egg yolk, (b) grown in TSB, or (c) grown in TSB with subsequent administration of sterile egg yolk to mice. The three treatment groups may help to identify whether the difference in contribution of these two media to Salmonella virulence was due to Salmonella pre-growth in egg yolk or to the mere presence of the pathogen in egg yolk during infection. Mice received Salmonella Enteritidis pre-grown in egg yolk showed significantly lower LD50, faster disease onset, and higher death rate, whereas the difference in disease manifestation between TSB group with or with administration of egg yolk was not statistically different. These findings suggest that growth of Salmonella in egg yolk was required for the increased virulence in the mouse model. Transcriptional analysis revealed that the enhanced virulence of Salmonella Enteritidis pre-grown in yolk to be a transcriptomic event. The upregulated virulence genes included genes encoded by Salmonella pathogenicity islands 1, 2, 4 and 5. Some of the upregulated genes are involved in type III secretion systems (i.e., spaR and sipA), and cell invasion and infection processes (i.e., invF). A relatively wide range of temperatures were suggested for egg pasteurization. The variation in applied temperatures leads to various heating rates during pasteurization temperature come-up stage. To determine if heating rate alters pathogen’s resistance during processing or virulence expression (Goal II), eggs inoculated with Salmonella Enteritidis were submerged in water baths held at 53°C and 59°C to mimic slow and fast heating rate, respectively. Slow heating during the thermal come-up time significantly upregulated a heat stress response-related gene, grpE; this also protected Salmonella from the lethal temperature at the holding stage and the remaining bactericidal heat at the beginning of the cooling stage. Due to the co-regulation of stress response and virulence, slow heating rate also significantly upregulated the expression of Salmonella virulence genes, sopB and sseA, which was confirmed through the in vivo model. However, ozone treatment following heat treatment significantly diminished Salmonella virulence. In conclusion, this research proved that both egg environment and egg processing could affect the safety of shell eggs. The study highlighted the importance of accurately evaluating the risk of salmonellosis as a function of food types. Our findings also informed food processors about the safety risks to consumers resulting from improper use of processing parameters during shell egg pasteurization. The study proved the value of treating shell eggs with ozone after heat treatment; the combined pasteurization process could protect consumer from natural Salmonella Enteritidis contaminants in shell eggs.