Mice were weaned onto the ~12% fat diet at three weeks of age and then either kept on that diet, gradually shifted to the ~6% fat diet at least two weeks prior to inoculation with C. jejuni at 8 to 12 weeks of age or shifted abruptly to the ~6% fat diet just prior to inoculation at 8 to 12 weeks of age. C. jejuni strains Details concerning the strains used appear in Table 1. Growth media and inoculum preparation were as previously described [40]. Genetic methods Total DNA was extracted from tissue and fecal samples using DNeasy Tissue Kit (Qiagen, Valencia, CA) and was assayed by species-specific MK-4827 clinical trial PCR for the C. jejuni
gyrA gene as previously described [40, 44]. Pathogenicity gene complements of the C. jejuni strains were determined using published PCR assays cited in Table 2; the 9.6 kbp LOS
fragment was generated using the Expand Long Template PCR System (Roche, Mannheim, Germany). Primers for luxS were generated using the web-based Primer3 program [68] http://jura.wi.mit.edu/rozen/papers/rozen-and-skaletsky-2000-primer3.pdf: GGTTGTCGCACGGGTTTTTA (forward) and GGCAATTTGTTTGGCTTCAT (reverse). Cycling conditions were 2.0 mM MgCl2, denaturation at 95°C for 1 min followed by 30 cycles of 94°C for 30 s, 49°C for 1 min, 72°C for 2 min, and final extension at 72°C, 10 min. RFLP analysis of virulence determinants was conducted as follows. PCR products for flaA, LOS, cdtABC, selleck inhibitor ceuE, pldA, ciaB, dnaJ, and cgtB were digested with DdeI, RsaI, or HhaI to generate restriction fragment length polymorphism (RFLP) patterns. DNA extraction from bacterial cultures, restriction enzyme digestion, agarose gel electrophoresis, and ethidium bromide staining were performed using standard methods [69].
Stained gels were visualized and photographed using an Alpha Bacterial neuraminidase Innotech UV transilluminator (Alpha Innotech, San Leandro, CA). Banding patterns were scored visually. Multilocus sequence typing (MLST) of strain NW (RAD001 GenBank accession numbers FJ361183 through FJ361189) was performed using genes, primer sets, and cycling conditions described at the Campylobacter jejuni Multi Locus Sequence Typing website http://pubmlst.org/campylobacter/ developed by Keith Jolley and Man-Suen Chan and sited at the University of Oxford [7]. DNA sequencing was performed at the MSU Genomics Technology Support Facility. Each PCR product was initially sequenced in both directions; additional sequencing was done as necessary to resolve discrepancies. DNA:DNA microarrray comparison of C. jejuni strains 11168 and NW An in-house whole-open-reading frame (ORF) microarray for C. jejuni 11168 (95% coverage) was developed using primers and clones described in Parrish et al. [51]. See NCBIGEO series number GSE13794 for a full description of chip manufacture. ORFs from pVir, C. jejuni strain 81–176 were also spotted on the chips.