free (SPF) conditions. C57BL/6 mice were used in the experiments. Age matched female mice 3 weeks (right after weaning; infant mice) and 3 months of age (adult mice) as well as 6 months old IL-102/2 mice (C57BL/6 background) with overt chronic colitis were used. Clinical Score To assess clinical/macroscopic signs of C. jejuni induced infection on a daily basis, a standardized cumulative clinical score (maximum 12 points; modified according to [47] addressing the occurrence of blood in feces (0 points: no blood; 2 points: microscopic detection of blood by the Guajac method using Haemoccult, Beckman Coulter/PCD, Krefeld, Germany; 4 points: macroscopic blood visible), diarrhea (0: formed feces; 2: pasty feces; 4: liquid feces), and the clinical aspect (0: normal; 2: ruffled fur, less locomotion; 4: isolation, severely compromized locomotion, pre-final aspect) was used. Induction of Acute Ileitis For induction of ileitis, C57BL/6 mice were infected orally with 100 Toxoplasma (T.) gondii cysts (ME49 strain) from homogenized brains of intraperitoneally infected NMRI mice in a volume of 0.3 ml phosphate-buffered saline (PBS, pH 7.4) by gavage, as described previously [22,23,48,49]. C. jejuni Infection of Mice Mice were infected with approximately 109 viable CFU of C. jejuni strains ATCC 43431 or B2 by gavage in a total volume of 0.3 mL PBS on two consecutive days as described earlier in detail [17]. C. jejuni strain B2 was kindly provided by Prof. Dr. Uwe Groß, University of Go¨ ttingen, Germany. Elevation of Intestinal E. coli Loads In order to raise intestinal E. coli loads in 3-months-old adult mice harboring a conventional gut microbiota to supra-physiologic levels, live E. coli were isolated from na¨ıve, healthy 3 months old C57BL/6 mice by culture on MacConkey media. This strain was shown to be a common commensal in healthy mice. PCR-based detection revealed that this strain does not contain known virulence factors of pathogenic E. coli such as stx 1 and 2, catA, hlyA, cspA, katP, and astA. This commensal E. coli strain was subcultivated on blood agar and added to the drinking water at a final concentration of 108 CFU per mL suspension starting at day 0 in na¨ıve animals. The E. coli suspension was renewed daily. For kinetic experiments, the E. coli suspension was withdrawn after 8 days and replaced by regular tap water thereafter. For C. jejuni infection studies, the application of E. coli in drinking water was started 8 days prior the first oral C. jejuni infection and continued until the end of the experiment (12 days following the latest C. jejuni gavage). Sampling Procedures, Determination of Colon Length and Histopathology Mice were sacrificed by isofluran treatment (Abbott, Germany). From each mouse samples derived from the GI tract (stomach, duodenum, ileum, colon) were collected in parallel for histological, microbiological, immunobiological and molecular analyses. Car- diac blood and tissue samples from mesenteric lymphnodes, spleen, liver and the GI tract were asserved under sterile conditions. Histopathological changes were determined in colon samples immediately fixed in 5% formalin and embedded in paraffin. Sections (5 mm) were stained with respective antibodies for immunohistochemistry. E. coli Facilitates Murine Campylobacter Infection Immunohistochemistry In situ immunohistochemical analysis of colonic paraffin sections were performed as described previously [17,24,50]. Primary antibodies against CD3 (#N1580, Dako, Denmark, dilution 1:10), FOXP-3 (FJK-16s, eBioscience, 1:100), B220 (eBioscience, San Diego, CA, USA, 1:200), myeloperoxidase-7 (MPO-7, # A0398, Dako, 1:400), F4/80 (#14–4801, clone BM8, eBioscience, 1:50), and cleaved caspase-3 (Asp175, Cell Signaling, USA, 1:200) were used. For each animal, the average number of positive stained cells within at least six independent high power fields (HPF, 4006magnification) were determined microscopically and subjected to statistical analysis as indicated. Cytokine Detection in Colon Culture Supernatants Colon biopsies were cut longitudinally, washed in PBS and strips of 1 cm2 placed in 24-flat-bottom well culture plates (Nunc, Wiesbaden, Germany) containing 500 ml serum-free RPMI 1640 medium supplemented with penicillin (100 U/ml) and streptomy- cin (100 ug/ml; PAA Laboratories). After 18 h at 37uC, culture supernatants were tested for TNF-a, IL-6, MCP-1 and IFN-c by the Mouse Inflammation Cytometric Bead Assay (CBA; BD Biosciences) on a BD FACSCanto II flow cytometer (BD Biosciences). Nitric oxide (NO) was determined by Griess-reaction as described earlier [22]. Analysis of the Intestinal Microflora Cultural analyses, biochemical identification and molecular detection of luminal bacterial communities from stomach, duodenum, ileum, and colon as well as feces were performed as previously described [17,22,23,24]. Assessment of Bacterial Translocation Frequencies For bacterial translocation experiments, mesenteric lymphnodes 2 (MLNs) draining the small intestine, spleen, liver (1 cm ) and cardiac blood (1 mL) were transferred into thioglycollate enrich- ment broths under sterile conditions and cultivated for 7 days. Turbid broths were streaked onto sheep blood, MacConkey as well as Karmali agar in order to detect translocated live E. coli and C. jejuni followed by biochemical species identification (API 20E and API Campy, respectively, Biome´rieux). Given that bacterial growth in enrichment broths cannot be quantitated, the relative rates of translocating C. jejuni or E. coli into the respective extra- intestinal compartment are expressed in % (number of broths culture-positive for either E. coli or C. jejuni multiplied with 100 and divided by the total number of broths inoculated with the respective organ or blood). Statistical Analysis Mean values, medians, standard deviations, and levels of significance were determined using appropriate tests as indicated (two-tailed Student’s t-Test, Mann-Whitney-U Test). Two-sided probability (P) values #0.05 were considered significant. All experiments were repeated at least twice. Acknowledgments We thank Michaela Wattrodt, Ursula Ru¨schendorf, Gernot Reifenberger, Uwe Lohmann, Silvia Boelke, and the staff of the animal research facility for excellent technical assistance and animal breeding. We are grateful to Simone Spieckermann for immunohistochemistry staining of colon sections. PLoS ONE | www.plosone.org 12 May 2012 | Volume 7 | Issue 5 | e35988