Growth in Egg Yolk Enhances Salmonella Enteritidis Colonization and Virulence in a Mouse Model of Human Colitis

Abstract

Salmonella Enteritidis (SE) is one of the most common causes of bacterial food-borne illnesses in the world. Despite the SE's ability to colonize and infect a wide-range of host, the most common source of infection continues to be the consumption of contaminated shell eggs and egg-based products. To date, the role of the source of SE infection has not been studied as it relates to SE pathogenesis and resulting disease. Using a streptomycin-treated mouse model of human colitis, this study examined the virulence of SE grown in egg yolk and Luria Bertani (LB) broth, and mouse feces collected from mice experimentally infected with SEE1 (SEE1 passed through mice). Primary observations revealed that the mice infected with SE grown in egg yolk displayed greater illness and disease markers than those infected with SE passed through mice or grown in LB broth. Furthermore, the SE grown in egg yolk achieved higher rates of colonization in the mouse intestines and extra-intestinal organs of infected mice than the SE from LB broth or mouse feces. Our results here indicate that the source of SE infection may contribute to the overall pathogenesis of SE in a second host. These results also suggest that reservoir-pathogen dynamics may be critical for SE's ability to establish colonization and priming for virulence potential.

Fig 1. Bacterial burden in the intestinal tract and fecal shedding.

Above are the results of the bacterial burden in the mouse intestines after infecting with SEE1 from different sources. Bacterial counts of the small intestine (A), cecum (B), colon (C) as well as fecal shedding differences (D) were taken from three experiments, normalized with corresponding controls, and the results analyzed by one-way ANOVA with a Tukey’s post-test (A-C) or Kruskall-Wallis test (D) and * represents p<0.05. There were five mice per group in the first two experiments and four mice per group in the last experiment. Mice that received the same treatment in each of the three experiments were considered one group for final data analysis.

Fig 2. Extra-intestinal dissemination of SEE1.

Bacterial burden of the liver (A) and spleen (B) from three experiments was taken and normalized with corresponding controls, and analyzed by Tukey’s post-test of one-way ANOVA, and significance of p<0.05 is indicated by *. There were five mice per group in the first two experiments and four mice per group in the last experiment. Mice that received the same treatment in each of the three experiments were considered one group for final data analysis.

Fig 3. Percent weight changes in mice.

The overall weight of mice is an indicator of gastrointestinal distress of mice. Mice from the third experiment (four mice in each experimental group and three mice in each control group) were weighed at the beginning and end of the experiment with SEE1 from various sources and the difference was compared to respective controls. The weight changes were taken from at least two experiments, normalized with corresponding controls, expressed as percent weight changes, and analyzed by Tukey’s pairwise comparison test following a one-way ANOVA. Significance was considered for p<0.05 and shown as *.

Fig 4. Gross pathological changes in mice.

Overall changes in the mice internal organs from the third experiment were photographed and analyzed. Mice infected with SEE1 grown in egg yolk (B) appear to have greater outward pathology than the mice infected with SEE1 from LB broth (A) or from mouse feces (C). Major changes in gross pathology and organs of interest are indicated by the arrows and Roman numerals. I, The liver displaying marked paling, and prominent blood vessels. II, The small intestine showing signs of paling compared to the small intestine in A and C. III, The cecum that is emptied and shriveled. IV, The colon section of the large intestine that is extremely pale and empty. V, Fluid build-up in the visceral tissues. There were four mice in each experimental group and three mice in each control group.

Fig 5. Biometrics of the ceca.

The overall size (A), weight (B), and pathology-driven morphological changes (C1-C3) were determined for the mice infected with SEE1 from the three sources. There were four mice in each experimental group and three mice in each control group. Measurements of the ceca from the last experiment were taken and normalized with corresponding controls, yielding the changes in size and weight. SEE1 grown in egg yolk appears to cause greater pathological changes in the ceca as compared to SEE1 grown in LB broth (C1) or passed through mice (C3). For A and B, significance was determined by one-way ANOVA Tukey’s pairwise comparison test, and significance was set at p<0.05; indicated by *.

Fig 6. Cytokine analysis of ceca of mice infected with SEE1 from three sources.

Pro-inflammatory and anti-inflammatory cytokine profiles had been determined by ELISA for ceca of mice infected with SEE1 from egg yolk (four mice), LB broth (four mice), and passed through mice (four mice) as well as corresponding controls for each source used (three mice each). Results were normalized with corresponding controls and analyzed through one-way ANOVA and subsequent Tukey’s Pairwise Comparison Test. Significance of p<0.05 is indicated by *.

Fig 7. Overall histopathology scores of ceca.

The primary site of colonization for SE in mice is the cecum; so the histopathology of the ceca of three mice from each control group and four mice from each infection group was determined. Statistical significance was determined by one-way ANOVA Tukey’s pairwise comparison test, and significance was set at p<0.05; indicated by *.

Fig 8. Changes in the histopathology of ceca infected with SEE1 from various sources.

This Fig shows a representative micrographs of sections of a healthy ceca from an uninfected mouse (A), and cecal sections of mice infected with SEE1 grown in egg yolk (B), SEE1 passed through mice (C), and SEE1 grown in LB broth (D). Abbreviations: GC (Goblet Cells), EP (Epithelial Cells), LP (Lamina Propria), TM (Tunica Muscularis), MM (Muscularis Mucosa), PMN (Polymorphonuclear leukocytes), SME (Sub-mucosal Edema), UL (Ulceration).