The inclusion of antibiotic growth promoters, such as virginiamycin, at subtherapeutic levels in poultry feeds has a positive effect on health and growth characteristics, possibly due to beneficial effects on the host gastrointestinal microbiota. To improve our understanding of the chicken gastrointestinal microbiota and the effect of virginiamycin on its composition, we characterized the bacteria found in five different gastrointestinal tract locations (duodenal loop, mid-jejunum, proximal ileum, ileocecal junction, and cecum) in 47-day-old chickens that were fed diets excluding or including virginiamycin throughout the production cycle. Ten libraries (five gastrointestinal tract locations from two groups of birds) of approximately 555-bp chaperonin 60 PCR products were prepared, and 10,932 cloned sequences were analyzed. A total of 370 distinctcpn60sequences were identified, which ranged in frequency of recovery from 1 to 2,872. The small intestinal libraries were dominated by sequences from theLactobacillales(90% of sequences), while the cecum libraries were more diverse and included members of theClostridiales(68%),Lactobacillales(25%), andBacteroidetes(6%). To assess the effects of virginiamycin on the gastrointestinal microbiota, 15 bacterial targets were enumerated using quantitative, real-time PCR. Virginiamycin was associated with increased abundance of many of the targets in the proximal gastrointestinal tract (duodenal loop to proximal ileum), with fewer targets affected in the distal regions (ileocecal junction and cecum). These findings provide improved profiling of the composition of the chicken intestinal microbiota and indicate that microbial responses to virginiamycin are most significant in the proximal small intestine.

We previously reported marked differences in small intestinal morphology, including changes in crypt depth and villous height, after inoculation of germ-free pigs with different bacterial species. In an attempt to identify the mechanisms governing changes in villous morphology associated with bacterial colonization, 2 gnotobiotic experiments were performed. In each experiment, 16 piglets were allocated to 4 treatment groups including germ-free (GF), monoassociation with Lactobacillus fermentum (LF) or Escherichia coli (EC), or conventionalized with sow feces (SF). Piglets were reared under gnotobiotic conditions until 14 d of age, at which time whole intestinal tissue and enterocytes were collected for histological, gene expression, and protein analysis. Proliferating cell nuclear antigen, tumor necrosis factor α (TNFα), Fas ligand (FasL), CD3ε, caspase 3 (casp3), and toll-like receptors (TLR)2, 4, and 9 expression were measured by quantitative PCR. Activated casp3 was measured by Western blot. Increased abundance of activated casp3 and transcripts encoding proliferating cell nuclear antigen, TNFα, CD3ε, and FasL was observed in SF and EC treatment groups compared with GF and LF. Expression of TLR2 was increased (P < 0.05) in the SF treatment and tended to be greater (P < 0.08) in EC relative to LF and GF. Results indicate that conventional bacteria and E. coli but not L. fermentum increase overall cell turnover by stimulating increased apoptosis through the expression of FasL and TNFα and by increasing cell proliferation. The differential regulation of TLR expression indicates that microbially induced changes may be mediated in part by these receptors. Induction of inflammatory responses and activation of apoptosis through death receptors appears to play a significant role in enterocyte turnover mediated by commensal bacteria. Copyright © 2007. . Copyright 2007 Journal of Animal Science

Background: To gain insight into host-microbe interactions in a piglet model, a functional genomics approach was used to address the working hypothesis that transcriptionally regulated genes associated with promoting epithelial barrier function are activated as a defensive response to the intestinal microbiota. Cesarean-derived germfree (GF) newborn piglets were colonized with adult swine feces, and villus and crypt epithelial cell transcriptomes from colonized and GF neonatal piglets were compared using laser-capture microdissection and high-density porcine oligonucleotide microarray technology. Results: Consistent with our hypothesis, resident microbiota induced the expression of genes contributing to intestinal epithelial cell turnover, mucus biosynthesis, and priming of the immune system. Furthermore, differential expression of genes associated with antigen presentation (pan SLA class I, B2M, TAP1 and TAPBP) demonstrated that microbiota induced immune responses using a distinct regulatory mechanism common for these genes. Specifically, gene network analysis revealed that microbial colonization activated both type I (IFNAR) and type II (IFNGR) interferon receptor mediated signaling cascades leading to enhanced expression of signal transducer and activator of transcription 1 (STAT1), STAT2 and IFN regulatory factor 7 (IRF7) transcription factors and the induction of IFN-inducible genes as a reflection of intestinal epithelial inflammation. In addition, activated RNA expression of NF-kappa-B inhibitor alpha (NFκBIA; a.k.a I-kappa-B-alpha, IKBα) and toll interacting protein (TOLLIP), both inhibitors of inflammation, along with downregulated expression of the immunoregulatory transcription factor GATA binding protein-1 (GATA1) is consistent with the maintenance of intestinal homeostasis. Conclusion: This study supports the concept that the intestinal epithelium has evolved to maintain a physiological state of inflammation with respect to continuous microbial exposure, which serves to sustain a tight intestinal barrier while preventing overt inflammatory responses that would compromise barrier function.

We have combined the culture-independent methods of high-throughput sequencing of chaperonin-60 PCR product libraries and quantitative PCR to profile and quantify the small-intestinal microflora of pigs fed diets based on corn, wheat, or barley. A total of 2,751 chaperonin-60 PCR product clones produced from samples of ileum digesta were examined. The majority (81%) of these clones contained sequences independently recovered from all three libraries; 372 different nucleotide sequences were identified, but only 14% of the 372 different sequences were recovered from all three libraries. Taxonomic assignments of the library sequences were made by comparison to a reference database of chaperonin-60 sequences combined with phylogenetic analysis. The taxa identified are consistent with previous reports of pig ileum microflora. Frequencies of each sequence in each library were calculated to identify taxa that varied in frequency between the corn, barley, and wheat libraries. The chaperonin-60 sequence inventory was used as a basis for designing PCR primer sets for taxon-specific quantitative PCR. Results of quantitative PCR analysis of ileum digesta confirmed the relative abundances of targeted taxa identified with the library sequencing approach. The results of this study indicate that chaperonin-60 clone libraries can be valid profiles of complex microbial communities and can be used as the basis for producing quantitative PCR assays to measure the abundance of taxa of interest during experimentally induced or natural changes in a community.

One mechanism through which S. cerevisiae may improve the performance of pigs is by altering the composition of the gut microbiota, a response that may be enhanced by early postnatal supplementation of probiotics. To test this hypothesis, newborn piglets (16 piglets/group) were treated with either S. cerevisiae yeast (5 x 10⁹ cfu/pig: Low) or (2.5 x 10¹⁰ cfu/piglet: High) or equivalent volume of sterile water (Control) by oral gavage every other day starting from day 1 of age until weaning (28±1 days of age). Piglet body weight was recorded on days 1, 3, 7, 10, 17, 24 and 28 and average daily gain (ADG) calculated for the total period. At weaning, piglets were euthanized to collect cecum content for microbial profiling by sequencing of the 16S rRNA gene. ADG was higher in both Low and High yeast groups than in Control group (P<0.05). Alpha diversity analyses indicated a more diverse microbiota in the Control group compared with Low yeast group; the High yeast being intermediate (P < 0.01). Similarly, Beta diversity analyses indicated differences among treatments (P = 0.03), mainly between Low yeast and Control groups (P = 0.02). The sparse Partial Least Squares Discriminant Analysis (sPLS-DA) indicated that Control group was discriminated by a higher abundance of Veillonella, Dorea, Oscillospira and Clostridium; Low yeast treated pigs by higher Blautia, Collinsella and Eubacterium; and High yeast treated pigs by higher Eubacterium, Anaerostipes, Parabacteroides, Mogibacterium and Phascolarctobacterium. Partial Least Squares (PLS) analysis showed that piglet ADG was positively correlated with genus Prevotella in High yeast group. Yeast supplementation significantly affected microbial diversity in cecal contents of suckling piglets associated with an improvement of short chain fatty acid producing bacteria in a dose-dependent manner. In conclusion, yeast treatment improved piglet performance and shaped the piglet cecum microbiota composition in a dose dependent way.

As an alternative to antibiotic growth promoters, live yeast supplementation has proven useful in reducing weaning stress and improving performance parameters of piglets. Here, we compared the performance and hindgut microbiota of weanling piglets subjected to different pre- and post-weaning yeast supplementation regimens using a live strain of Saccharomyces cerevisiae (Actisaf Sc 47). Average feed intake and average daily weight gain of piglets within Yeast-Control and Yeast-Yeast groups were higher than those in the Control-Control group. Yeast supplementation resulted in development of microbial communities that were phylogenetically more homogenous and less dispersed compared to the microbiota of control piglets. Key bacterial taxa overrepresented in the microbiota of yeast supplemented piglets included phylum Actinobacteria, specifically family Coriobacteriaceae, as well as Firmicutes families Ruminococcaceae, Clostridiaceae, Peptostreptococcaceae, and Peptococcaceae. Correlation network analysis revealed that yeast supplementation was associated with enrichment of positive correlations among proportions of different bacterial genera within the hindgut ecosystem. In particular, within the cecal microbiota of supplemented piglets, higher numbers of positive correlations were observed among potentially beneficial genera of the phyla Actinobacteria and Firmicutes, suggesting a mechanism by which yeast supplementation may contribute to regulation of intestinal homeostasis and improved performance of piglets.