Original Contributions A Large Community Outbreak of Salmonellosis Caused by Intentional Contamination of Restaurant Salad Bars

Context.—This large outbreak of foodborne disease highlights the challenge of investigating outbreaks caused by intentional contamination and demonstrates the vulnerability of self-service foods to intentional contamination. Objective.—To investigate a large community outbreak of Salmonella Typhimurium infections. Design.—Epidemiologic investigation of patients with Salmonella gastroenteritis and possible exposures in The Dalles, Oregon. Cohort and case-control investigations were conducted among groups of restaurant patrons and employees to identify exposures associated with illness. Setting.—A community in Oregon. Outbreak period was September and October 1984. Patients.—A total of 751 persons with Salmonella gastroenteritis associated with eating or working at area restaurants. Most patients were identified through passive surveillance; active surveillance was conducted for selected groups. A case was defined either by clinical criteria or by a stool culture yielding S Typhimurium

O-mannosylation of the Mycobacterium tuberculosis adhesin Apa is crucial for T cell antigenicity during infection but is expendable for protection.

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis

T cell responses in nApa or rApa vaccinated mice.

<p>(A–D) Mice were vaccinated with nApa or rApa in DDA-MPL adjuvant or with adjuvant alone (controls), and their spleen or lung cells were stimulated <i>in vitro</i> with no Ag, nApa or rApa 1 wk after last vaccination. (A) The percentages (%) of TNF-α and IFN-γ (top) or IL-2 (bottom) producing cells among splenic CD4 T cells of 1 representative mouse from nApa or rApa vaccinated group are shown, and (B) the frequency (%) of nApa- or rApa-specific individual cytokine producing cells among splenic CD4⁺ T cells from 4 mice/vaccinated group is plotted ( means ± s.e.m.). (C) The frequency (%) of nApa- or rApa-specific total cytokine producing cells among CD4⁺ (top) and CD8⁺ (bottom) T cells from the spleen (means ± s.e.m., n = 4 mice/group) and lung (pools) of nApa or rApa vaccinated (vac) mice. The corresponding pie charts present the mean frequencies of 1+, 2+ and 3+ cytokine producers. (D) The frequency of nApa- or rApa-specific IFN-γ, IL-17 or IL-4 SFU/10⁶ spleen or lung cells of vaccinated (vac) and control mice. The corresponding pie charts present the mean cytokine frequencies. Data are means ± s.e.m. of 3–4 wells using pooled cells. The responses of 2 Apa vaccinated groups were compared. *Significant by 1-way ANOVA followed by Bonferroni's test comparing respective immunogen-specific responses. (E) Synthetic Gp281-325- or NGp281-325-specific cytokine response of nApa vaccinated mice 4 wks post-vaccination. Stimulation with p231-250, p271-288 or p301-320 was evaluated for comparison.</p

Pathogen-specific responses in Apa vaccinated and control mice after challenge.

<p>(A) The proportions of STCF-specific 1+, 2+ and 3+ cytokine producers (of IFN-γ, TNF-α and IL-2) constituting STCF-specific total cytokine positive (+) CD4⁺ or CD8⁺ T cells are plotted as % of CD4⁺ T cells or CD8⁺ T cells in the lung and spleen of Apa vaccinated and control mice. Data are means ± s.e.m. of 4 mice/group. (B) The frequency of STCF-specific IFN-γ, IL-17 or IL-4 SFU/10⁶ pooled spleen or lung cells of vaccinated and control mice. Data are means ± s.e.m. of 3 wells. The corresponding pie charts present mean cytokine frequencies. *Significant by 1-way ANOVA followed by Bonferroni's test comparing responses of each vaccinated group with adjuvant control group post-challenge.</p

Characterization of nApa-specific T cell hybridoma clone (4C3).

<p>(A–B) The T cell hybridoma 4C3 was generated using draining LN cells of nApa-FIA vaccinated mice. The Ag and epitope specificity of 4C3 T cell clone was determined using IL-2 assay. (A) The 4C3 T cells (5×10⁴ cells/well) were cultured with APCs pulsed with indicated amounts of nApa, rApa or whole trypsin digests using T cell to APC ratio of 1∶1 for 24 h and culture supernatants were evaluated using IL-2 ELISA. (B) Identification of peptide/epitope specific for 4C3 T cell clone. The nApa was digested with trypsin and the peptide fragments were resolved by RP-HPLC, total 25 fractions were collected. Fractions 7–25 containing nApa peptides were used to screen the biological activity of 4C3 clone in IL-2 assay. The whole digest of nApa and fractions 19, 21, and 22 containing single N-terminal glycopeptide (residues p40-145) produced a positive IL-2 response when used for Ag pulsing of APCs. The fraction 19 is characterized by LC-MS (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003705#ppat.1003705.s005" target="_blank">Figure S5B</a>). The data (A and B) are means (O.D. 405 nm) of culture supernatant evaluations from two separate cultures in IL-2 ELISA. The experiment was repeated with similar results.</p

T cell reactivity to <i>in vitro</i>-mannosylated rApa C-terminal.

<p>(A) Pooled splenocytes from BCG infected mice (n = 4) were stimulated <i>in vitro</i> with nApa, rApa or 32 individual non-glycosylated synthetic Apa peptides in ELISPOT assay. IFN-γ SFU/10⁶ cells at 12 and 32 wk time points are shown. (B–C) Synthetic C-terminal glycopeptide (Gp281-325)- or control non-glycopeptide (NGp281-325)-specific T cell response (indicated inside dotted box) in mice. Stimulation with proteins (nApa or rApa) or non-glycopeptides (p271-288 or p301-320) was also included as controls. (B) Ag- or peptide-specific IFN-γ, IL-17 or IL-4 SFU/10⁶ pooled lung or spleen cells of BCG or <i>Mtb</i> infected mice at 32 and 26 wks, respectively. (C) The proportions of peptide-specific 1+, 2+ and 3+ cytokine producing T cell subsets constituting total cytokine positive (+) CD4⁺ T cells in the spleen of BCG infected mice at 32 wks, expressed as % of CD4⁺ T cells. Data in A–C are means ± s.e.m. *Significant using 1-way ANOVA followed by Bonferroni's test.</p

T cell responses to nApa and rApa in BCG infected mice.

<p>(A–E) Mice were infected s.c. with 1×10⁶ CFU BCG. At different time points after infection, as indicated, mice were euthanized (n = 4/time point) and their spleen or lung cells (pools) were stimulated <i>in vitro</i> with no Ag, nApa, rApa, nAg85B or WCL. (A) The percentages (%) of TNF-α and IFN-γ (top) or IL-2 (bottom) producing cells among lung CD4⁺ T cells from 1 representative experiment at the 52 wk time point are shown, and (B) the frequency (%) of nApa- or rApa-specific total cytokine producing cells among CD4⁺ and CD8⁺ T cells from the spleen and lung at 7 different time points encompassing entire life span are plotted. (C–D) nApa or rApa-specific cytokine co-expression profiles in the lung and spleen were determined, and (C) the proportions of single (1+), double (2+), or triple (3+) cytokine producing T cell subsets constituting total cytokine positive (+) CD4⁺ or CD8⁺ T cells at the peak of response are plotted as % of CD4⁺ T cells (top; at 52 wk) or CD8⁺ T cells (bottom; at 32 wk), respectively. The pie charts present the mean frequencies of 1+, 2+ and 3+ cytokine producers. (D) The percentages of 7 possible combinations of cytokine secreting subsets in the lung are also shown. Data at 12, 32, 52 and 72 wks (in B–D) are means ± s.e.m. of 3–4 independent mice experiments, while data (means) at 3, 6 and 104 wks are from one experiment evaluated in duplicate. Total as well as individual cytokine subset responses are compared. (E) nApa- or rApa-specific IFN-γ, IL-17 or IL-4 SFU/10⁶ lung or spleen cells at 12, 32 and 72 wks. Data are means ± s.e.m. of 2 independent mice experiments evaluated in triplicate. The pie charts present the mean IFN-γ, IL-17 and IL-4 SFU/10⁶ cells at 32 wk. *Significant using 1-way ANOVA followed by Bonferroni's multiple comparison test (B–E).</p

Protective potential of Apa against <i>Mtb</i> challenge and recall of T and B cell responses.

<p>(A) Protective efficacy of nApa or rApa vaccinated mice using 1 or 10 µg/dose in DDA-MPL against <i>Mtb</i> challenge. Twelve wks after first vaccine injection, mice were challenged with virulent <i>Mtb</i> Erdman. The differences in bacterial load between vaccinated and nonvaccinated control groups 6 wks after challenge are shown. Data are using 5–10 mice per group. (B–C) T cell responses of Apa vaccinated and control mice pre- and post-challenge. Respective immunogen (i.e. Apa form used for vaccination)-specific responses in Apa vaccinated (vac) groups while nApa-specific responses in control groups are shown. (B) The proportions of 1+, 2+, or 3+ cytokine producers (of IFN-γ, TNF-α and IL-2) constituting Apa-specific total cytokine positive (+) CD4⁺ or CD8⁺ T cells are plotted as % of CD4⁺ T cells (top) or CD8⁺ (bottom) T cells in the lungs and spleen of Apa vaccinated and control mice. Data are means ± s.e.m. of 4 mice/group. (C) The frequency of Apa-specific IFN-γ, IL-17 or IL-4 SFU/10⁶ pooled spleen or lung cells of vaccinated and control mice. Data are means ± s.e.m. of triplicate cultures. (D) Immunogen-specific IgG1, IgG2a and IgG2b antibody titers in the sera of Apa vaccinated mice. Data are means ± s.e.m. of 4 mice/group. Data in A, B and D are representative of 2 independent experiments. *Significant by Kruskal-Wallis followed by Dunn's test comparing vaccinated group with naïve and adjuvant controls in protection experiment (A), by 1-way ANOVA followed by Bonferroni's test comparing pre- and post-challenge responses of respective vaccinated groups (B and C) or comparing indicated groups (D).</p