Several DNA extraction methods have been reported for use with digesta or fecal samples, but problems are often encountered in terms of relatively low DNA yields and/or recovering DNA free of inhibitory substances. Here we report a modified method to extract PCR-quality microbial community DNA from these types of samples, which employs bead beating in the presence of high concentrations of sodium dodecyl sulfate (SDS), salt, and EDTA, and with subsequent DNA purification by QIA columns [referred to as repeated bead beating plus column (RBB+C) method]. The RBB+C method resulted in a 1.5- to 6-fold increase in DNA yield when compared to three other widely used methods. The community DNA prepared with the RBB+C method was also free of inhibitory substances and resulted in improved denaturing gradient gel electrophoresis (DGGE) profiles, which is indicative of a more complete lysis and representation of microbial diversity present in such samples.

The gastrointestinal (GI) tract of poultry is densely populated with microorganisms which closely and intensively interact with the host and ingested feed. The gut microbiome benefits the host by providing nutrients from otherwise poorly utilized dietary substrates and modulating the development and function of the digestive and immune system. In return, the host provides a permissive habitat and nutrients for bacterial colonization and growth. Gut microbiome can be affected by diet, and different dietary interventions are used by poultry producers to enhance bird growth and reduce risk of enteric infection by pathogens. There also exist extensive interactions among members of the gut microbiome. A comprehensive understanding of these interactions will help develop new dietary or managerial interventions that can enhance bird growth, maximize host feed utilization, and protect birds from enteric diseases caused by pathogenic bacteria.

The objective of this study was to generate a phylogenetic diversity census of bacteria identified in the intestinal tract of chickens and turkeys using a naïve analysis of all the curated 16S rRNA gene sequences archived in public databases. High-quality sequences of chicken and turkey gastrointestinal origin (3,184 and 1,345, respectively) were collected from the GenBank, Ribosomal Database Project, and Silva comprehensive ribosomal RNA database. Through phylogenetic and statistical analysis, 915 and 464 species-equivalent operational taxonomic units (defined at 0.03 phylogenetic distance) were found in the chicken and the turkey sequence collections, respectively. Of the 13 bacterial phyla identified in both bird species, Firmicutes, Bacteroidetes, and Proteobacteria were the largest phyla, accounting for >90% of all the sequences. The chicken sequences represent 117 established bacterial genera, and the turkey sequences represent 69 genera. The most predominant genera found in both the chicken and the turkey sequence data sets were Clostridium, Ruminococcus, Lactobacillus, and Bacteroides, but with different distribution between the 2 bird species. The estimated coverage of bacterial diversity of chicken and turkey reached 89 and 68% at species-equivalent and 93 and 73% at genus-equivalent levels, respectively. Less than 7,000 bacterial sequences from each bird species from various locations would be needed to reach 99% coverage for either bird species. Based on annotation of the sequence records, cecum was the most sampled gut segment. Chickens and turkeys were shown to have distinct intestinal microbiomes, sharing only 16% similarity at the species-equivalent level. Besides identifying gaps in knowledge on bacterial diversity in poultry gastrointestinal tract, the bacterial census generated in this study may serve as a framework for future studies and development of analytic tools.

In this study, the collective microbial diversity in the rumen was examined by performing a meta-analysis of all the curated 16S rRNA gene (rrn) sequences deposited in the RDP database. As of November 2010, 13,478 bacterial and 3516 archaeal rrn sequences were found. The bacterial sequences were assigned to 5271 operation taxonomic units (OTUs) at species level (0.03 phylogenetic distance) representing 19 existing phyla, of which the Firmicutes (2958 OTUs), Bacteroidetes (1610 OTUs) and Proteobacteria (226 OTUs) were the most predominant. These bacterial sequences were grouped into more than 3500 OTUs at genus level (0.05 distance), but only 180 existing genera were represented. Nearly all the archaeal sequences were assigned to 943 species-level OTUs in phylum Euryarchaeota. Although clustered into 670 genus-level OTUs, only 12 existing archaeal genera were represented. Based on rarefaction analysis, the current percent coverage at species level reached 71% for bacteria and 65% for archaea. At least 78,218 bacterial and 24,480 archaeal sequences would be needed to reach 99.9% coverage. The results of this study may serve as a framework to assess the significance of individual populations to rumen functions and to guide future studies to identify the alpha and global diversity of ruminal microbiomes.

Denaturing gradient gel electrophoresis (DGGE) has become a widely used tool to examine microbial diversity and community structure, but no systematic comparison has been made of the DGGE profiles obtained when different hypervariable (V) regions are amplified from the same community DNA samples. We report here a study to make such comparisons and establish a preferred choice of V region(s) to examine by DGGE, when community DNA extracted from samples of digesta is used. When the members of the phylogenetically representative set of 218 rrs genes archived in the RDP II database were compared, the V1 region was found to be the most variable, followed by the V9 and V3 regions. The temperature of the lowest-meltingtemperature (T m(L) ) domain for each V region was also calculated for these rrs genes, and the V1 to V4 region was found to be most heterogeneous with respect to T m(L) . The average T m(L) values and their standard deviations for each V region were then used to devise the denaturing gradients suitable for separating 95% of all the sequences, and the PCR-DGGE profiles produced from the same community DNA samples with these conditions were compared. The resulting DGGE profiles were substantially different in terms of the number, resolution, and relative intensity of the amplification products. The DGGE profiles of the V3 region were best, and the V3 to V5 and V6 to V8 regions produced better DGGE profiles than did other multiple V-region amplicons. Introduction of degenerate bases in the primers used to amplify the V1 or V3 region alone did not improve DGGE banding profiles. Our results show that DGGE analysis of gastrointestinal microbiomes is best accomplished by the amplification of either the V3 or V1 region of rrs genes, but if a longer amplification product is desired, then the V3 to V5 or V6 to V8 region should be targeted.The inherent limitations associated with cultivation-based approaches to characterizing microbial communities are widely recognized, and a number of cultivation-independent, molecular methods have emerged in recent years to improve our understanding of this aspect of microbial ecology. Techniques such as denaturing gradient gel electrophoresis (DGGE) (8,20,21,27), terminal restriction fragment length polymorphism (15,22,28,38), length heterogeneity PCR (29, 34), automated rRNA intergenic spacer analysis (13), and ribosomal intergenic spacer length polymorphism (9, 40) are now widely used and reported in the literature. In the in silico analyses of 41 completely sequenced bacterial genomes, DGGE appeared to be one of the best molecular community fingerprinting techniques in terms of predicting the actual Shannon-Wiener diversity index, richness, and evenness (3). Additionally, DGGE supports the identification of community members because the amplification products can be recovered and sequenced (4,6,20,31). This may explain why DGGE has become the most frequently used method of molecular community fingerprinting.In PCR-DGGE, either a single hypervariable (V) region or a combinatio...

ABSTRACTFive essential oils (EOs), namely, clove oil (CLO), eucalyptus oil (EUO), garlic oil (GAO), origanum oil (ORO), and peppermint oil (PEO), were testedin vitroat 3 different doses (0.25, 0.50, and 1.0 g/liter) for their effect on methane production, fermentation, and select groups of ruminal microbes, including total bacteria, cellulolytic bacteria, archaea, and protozoa. All the EOs significantly reduced methane production with increasing doses, with reductions by 34.4%, 17.6%, 42.3%, 87%, and 25.7% for CLO, EUO, GAO, ORO, and PEO, respectively, at 1.0 g/liter compared with the control. However, apparent degradability of dry matter and neutral detergent fiber also decreased linearly with increasing doses by all EOs except GAO. The concentrations of total volatile fatty acids were not affected by GAO, EUO, or PEO but altered linearly and quadratically by CLO and ORO, respectively. All the EOs also differed in altering the molar proportions of acetate, propionate, and butyrate. As determined by quantitative real-time PCR, all the EOs decreased the abundance of archaea, protozoa, and major cellulolytic bacteria (i.e.,Fibrobacter succinogenes,Ruminococcus flavefaciens, andR. albus) linearly with increasing EO doses. On the basis of denaturing gradient gel electrophoresis analysis, different EOs changed the composition of both archaeal and bacterial communities to different extents. The Shannon-Wiener diversity index (H′) was reduced for archaea by all EOs in a dose-dependent manner but increased for bacteria at low and medium doses (0.25 and 0.50 g/liter) for all EOs except ORO. Due to the adverse effects on feed digestion and fermentation at high doses, a single EO may not effectively and practically mitigate methane emission from ruminants unless used at low doses in combinations with other antimethanogenic compounds.

Methanogenic archaea reside primarily in the rumen and the lower segments of the intestines of ruminants, where they utilize the reducing equivalents derived from rumen fermentation to reduce carbon dioxide, formic acid, or methylamines to methane (CH4). Research on methanogens in the rumen has attracted great interest in the last decade because CH4 emission from ruminants contributes to global greenhouse gas emission and represents a loss of feed energy. Some DNA-based phylogenetic studies have depicted a diverse and dynamic community of methanogens in the rumen. In the past decade, researchers have focused on elucidating the underpinning that determines and affects the diversity, composition, structure, and dynamics of methanogen community of the rumen. Concurrently, many researchers have attempted to develop and evaluate interventions to mitigate enteric CH4 emission. Although much work has been done using plant secondary metabolites, other approaches such as using nitrate and 3-nitrooxy propanol have also yielded promising results. Most of these antimethanogenic compounds or substances often show inconsistent results among studies and also lead to adverse effects on feed intake and digestion and other aspects of rumen fermentation when fed at doses high enough to achieve effective mitigation. This review provides a brief overview of the rumen methanogens and then an appraisal of most of the antimethanogenic compounds and substances that have been evaluated both in vitro and in vivo. Knowledge gaps and future research needs are also discussed with a focus on methanogens and methane mitigation.

Kigerl et al. show that spinal cord injury causes profound changes in gut microbiota and that these changes in gut ecology are associated with activation of GALT immune cells. They show that feeding mice probiotics after SCI confers neuroprotection and improves functional recovery.