No abstract

Terminal restriction fragment length polymorphism (T-RFLP) analysis was conducted on the 16S rRNA genes of the bacterial communities colonizing the epithelial surfaces of the terminal ilea of open conventionally housed mice in an institutional small-animal facility. Polymeric-immunoglobulin-receptor-deficient (pIgR ؊/؊ ) mice that were unable to secrete antibodies across mucosal surfaces were cohoused with normal and otherwise genetically identical wild-type (C57BL/6) mice for 4 weeks. If secretory antibodies played a role in modeling the gastrointestinal microbiota, C57BL/6 mice would have had a more distinct and uniform microbiota than their pIgR ؊/؊ cage mates. The T-RFLP profiles of the bacterial communities were compared by using Sorensen's pairwise similarity coefficient, a newly developed weighted pairwise similarity coefficient, and on the basis of Shannon's and Simpson's diversity indices. No systematic differences were observed between the dominant components of the mucosa-associated bacterial communities of the terminal ileal walls of the two types of mice, indicating that secretory antibodies do not control the composition of this microbiota. Similar analyses of experiments conducted at two different times, between which the bacterial community composition of the mouse colony in the small-animal facility appeared to have changed, showed that differences could have been detected, had they existed.

The humoral response to the gastrointestinal (GI) flora was analyzed in secretory Ig (sIg)-deficient polymeric IgR (pIgR)(-/-) mice and otherwise congenic C57BL/6 mice. While both strains carried an ileal flora of similar size and composition, increased bacterial translocation to mesenteric lymph node was demonstrated in pIgR(-/-) mice. Serum IgA was greatly increased in pIgR(-/-) mice compared with C57BL/6 mice and reacted with commensal organisms and food. Serum IgG levels in pIgR(-/-) mice were increased to 6-fold above that of C57BL/6 mice and included specificities that bound to selected flora antigens. The enhanced recognition of flora antigens in pIgR(-/-) mice was explored using ovalbumin (OVA)-specific CD4(+) T cells and feeding of low concentrations of OVA. Increased proliferation of transgenic T cells was observed in pIgR(-/-) mice, relative to C57BL/6 mice, suggesting elevated net uptake of protein antigens from the GI tract in the absence of sIg. These studies suggest that there is increased recognition of GI flora antigens by systemic antibodies in pIgR(-/-) mice, most probably as a result of increased access of antigens from the GI flora to the systemic immune compartment, and support the hypothesis that a major function of the secretory immune system is to return environmental antigens to mucosal surfaces.

We examined the impact of Helicobacter pylori infection on the murine gastric microbiota by culture and terminal-restriction fragment length polymorphism and found that neither acute nor chronic H. pylori infection substantially affected the gastric microbial composition. Interestingly, the total H. pylori burden detected by real-time PCR was significantly higher than that revealed by viable counts, suggesting that the antigenic load sustaining H. pylori-induced gastritis could be considerably higher than previously believed.Helicobacter pylori colonizes the stomachs of an estimated 50% of the world's population and is classified as a group 1 carcinogen (4, 15). Various animal models are currently used to study the pathogenesis of H. pylori infection, but there is little information on the gastric bacterial composition of rodents after H. pylori infection (1, 2, 7, 12). We therefore compared the gastric flora compositions, over time, of uninfected mice and of mice infected with H. pylori using conventional culture methods and terminal-restriction fragment length polymorphism (T-RFLP) analysis.Six-to 8-week-old female C57BL/6 mice were reared under specific-pathogen-free conditions at the animal facility of the Department of Microbiology and Immunology, University of Melbourne. All animal experiments were approved by the University of Melbourne Animal Ethics and Experimental Committee and complied with relevant legislation. Statistical significance was assessed by the nonparametric Mann-Whitney U test, and two-tailed P values of less than 0.05 were considered statistically significant.Preliminary results from culture of stomach homogenates and generation of 16S rRNA gene clone libraries from naïve mice confirmed that the murine stomach was highly colonized by lactobacilli, with the dominant species being Lactobacillus reuteri and Lactobacillus murinus (supplementary data), which is consistent with other studies (1, 2, 10, 12). In addition, T-RFLP data from an initial short-term H. pylori infection experiment with the SS1 strain (6) indicated that the gastric microbiotas of H. pylori-infected mice were similar to those of age-matched naïve mice, implying that an acute H. pylori in-* Corresponding author. Mailing address:

Artefacts consisting of concatenated oligonucleotide primer sequences were generated during sub-optimally performing polymerase chain reaction amplification of bacterial 16S rRNA genes using a commonly employed primer pair. These artefacts were observed during amplification for terminal restriction fragment length polymorphism analyses of complex microbial communities, and after amplification from DNA from a microbial culture. Similar repetitive motifs were found in gene sequences deposited in GenBank. The artefact can be avoided by using different primers for the amplification reaction.