Evaluation of intestinal colonization of mice by <i>C. difficile</i>.

<p>Colonization was evaluated at days 1, 4, 8, 10 and 14 in the GroEL- immunized group and in the control group after intra-gastric administration of <i>C.difficile</i> to mice.</p

Course of colonization and death of hamsters challenged with <i>Clostridium difficile</i> after pre-treatment with clindamycin.

<p>Each circle represents the same animal on different days (D) of observation. White circles: uncolonized hamsters; grey circles: colonized hamsters, black circles: colonized hamsters that died.</p

2-DE map on 18 cm IPG strip of cell wall proteins extracts used for hamsters immunization.

<p>Spots circled correspond to immuno-reactive proteins, DnaK (1), GroEL (2), S-layer protein precursor (3).</p

Kaplan-Meier survival estimates demonstrating time between challenge with <i>Clostridium difficile</i> and death.

<p>Clindamycin (50 mg/Kg) was administered 5 days before spore challenge. Animals were observed for 11 days. Experiments were performed with hamsters receiving PBS as control (n = 16) and hamsters immunized intra-rectally with cell wall extracts (CWE) (n = 20). Cholera toxin was used as adjuvant, for the two groups.</p

Immunoblots of cell wall extracts revealed by sera of hamster from the control group (A) and cell wall extracts revealed by sera of hamster from the cell wall extracts immunized group (B).

<p>Spots circled correspond to immuno-reactive proteins, DnaK (1), GroEL (2), S-layer protein precursor (3).</p

Protein identification by MS and MS/MS analysis.

<p>Protein identification by MS and MS/MS analysis.</p

Number of antibody producing cells specific to OVA in the intestinal lamina propria of immunized mice expressed in spot forming units (SFUs).

<p>Immunization by intra-rectal route with ovalbumin as antigen (OVA), with cholera toxin (CT) (n = 5), <i>Salmonella</i> Typhimurium flagellin (FLA-ST) (n = 5), <i>C</i>. <i>difficile</i> flagellin (FliC) (n = 5) respectively as adjuvant and PBS administration for the control group (n = 5). Immunization by intra-peritoneal route with OVA as antigen and FliC as adjuvant (FliC IP) (n = 5). (A) Anti-OVA IgA producing cells count; (B) anti-OVA IgG producing cells count *: statistically significant difference <i>p</i> < 0.05 (Mann-Whitney U-test).</p

Anti-OVA IgG level in sera of immunized mice.

<p>Immunization by intra-rectal route with ovalbumin as antigen (OVA), with cholera toxin (CT) (n = 14), <i>Salmonella</i> Typhimurium flagellin (FLA-ST) (n = 14), <i>C</i>. <i>difficile</i> flagellin (FliC) (n = 14) respectively as adjuvant and PBS administration for the control group (n = 14). Immunization by intra-peritoneal route with OVA as antigen and FliC as adjuvant (FliC IP) (n = 8). D0: before immunization, D15: first immunization, D30: first boost, D45: second boost. *: statistically significant difference *: 0.01<<i>p</i> < 0.05; **: 0.001<<i>p</i><0.01; ***: <i>p</i><0.001 (student t-test). Dotted-line corresponds to the cut-off value.</p

Anti-OVA antibody levels in intestinal contents of immunized mice.

<p>Immunization by intra-rectal route with ovalbumin as antigen (OVA), with cholera toxin (CT) (n = 14), <i>Salmonella</i> Typhimurium flagellin (FLA-ST) (n = 14), <i>C</i>. <i>difficile</i> flagellin (FliC) (n = 14) respectively as adjuvant and PBS administration for the control group (n = 14). Immunization by intra-peritoneal route with OVA as antigen and FliC as adjuvant (FliC IP) (n = 8). (A) Anti-OVA IgA level; (B) anti-OVA IgG level. *: statistically significant difference *: 0.01<<i>p</i><0.05; **: 0.001<<i>p</i><0.01; ***: <i>p</i><0.001 (student t-test). Dotted-line corresponds to the cut-off value.</p

<i>C</i>. <i>difficile</i> colonization in vaccinated mice after challenge.

<p><i>C</i>. <i>difficile</i> count from day 1 to day 10 in feces of mice intra-rectally vaccinated with SlpA as antigen and FliC (n = 12) or CT (n = 12) as adjuvant compared to control group (n = 12). *: statistically significant difference between the control and vaccinated groups <i>p</i> < 0.05 (Mann-Whitney U-test).</p