Changes in the Rumen Epimural Bacterial Diversity of Beef Cattle as Affected by Diet and Induced Ruminal Acidosis

Little is known about the nature of the rumen epithelial adherent (epimural) microbiome in cattle fed different diets. Using denaturing gradient gel electrophoresis (DGGE), quantitative real-time PCR (qPCR), and pyrosequencing of the V3 hypervariable coding region of 16S rRNA, epimural bacterial communities of 8 cattle were profiled during the transition from a forage to a high-concentrate diet, during acidosis, and after recovery. A total of 153,621 high-quality gene sequences were obtained, with populations exhibiting less taxonomic variability among individuals than across diets. The bacterial community composition exhibited clustering (P < 0.03) by diet, with only 14 genera, representing >1% of the rumen epimural population, differing (P ≤ 0.05) among diets. During acidosis, levels of Atopobium, Desulfocurvus, Fervidicola, Lactobacillus, and Olsenella increased, while during the recovery, Desulfocurvus, Lactobacillus, and Olsenella reverted to levels similar to those with the high-grain diet and Sharpea and Succinivibrio reverted to levels similar to those with the forage diet. The relative abundances of bacterial populations changed during diet transition for all qPCR targets except Streptococcus spp. Less than 5% of total operational taxonomic units (OTUs) identified exhibited significant variability across diets. Based on DGGE, the community structures of epithelial populations differed (P ≤ 0.10); segregation was most prominent for the mixed forage diet versus the grain, acidotic challenge, and recovery diets. Atopobium, cc142, Lactobacillus, Olsenella, RC39, Sharpea, Solobacterium, Succiniclasticum, and Syntrophococcus were particularly prevalent during acidosis. Determining the metabolic roles of these key genera in the rumens of cattle fed high-grain diets could define a clinical microbial profile associated with ruminal acidosis.     

Comparative Analysis of the Microbiota Between Rumen and Duodenum of Twin Lambs Based on Diets of Ceratoides or Alfalfa

In our previous study, diet directly impacted the microbiota of the rumen in twin lambs. The duodenum is the first part of the small intestine, so we seek to determine whether there is a difference in the digesta between the two feed groups HFLP (high fiber, low protein) and LFHP (low fiber, high protein), and its impact on the biodiversity and metabolism of the duodenum. Results showed that the number of Operational Taxonomic Units (OTUs) in the duodenum (2,373 OTUs) was more than those in the rumen (1,230 OTUs), and 143 OTUs were significantly different in the duodenum between the two groups. The two most predominant phyla were Bacteriodetes and Firmicutes, but this ratio was reversed between the rumen and duodenum of lambs fed different feedstuffs. The difference in the digesta that greatly changed the biodiversity of the rumen and duodenum could affect the microbial community in the gastrointestinal tract (GIT). Sixteen metabolites were significantly different in the duodenum between the two groups based on the metabolome analysis. The relationships were built between the microbiome and the metabolome based on the correlation analysis. Some metabolites have a potential role in influencing meat quality, which indicated that the diet could affect the microbiota community and finally change meat quality. This study could explain how the diet affects the rumen and duodenum’s microbiota, lay a theoretical basis for controlling feed intake, and determine the relationship between the duodenum’s microbiota and metabolism.     

Use of Pyrosequencing to Characterize the Microbiota in the Ileum of Goats Fed with Increasing Proportion of Dietary Grain

This study evaluated the effects of an increasing proportion of dietary grain on changes in bacterial populations in the goat ileum. Nine ruminally fistulated, castrated male goats were assigned to three diets in a completely randomized design. Goats were fed three different dietary treatments containing different proportions of corn grain (0, 25, and 50 %). The pH of the ileal contents and rumen fluid (P = 0.015) linearly decreased (P < 0.001), and the acetate, propionate, butyrate, and total volatile fatty acid in ileal contents increased (P < 0.05) with increases in dietary corn, and similar results were also observed in rumen fluid. The barcoded DNA pyrosequencing method was used to reveal 8 phyla, 70 genera, and 1,693 16S operational taxonomic units (OTUs). At the genus level, the proportions of Acetitomaculum, Enterococcus, Atopobium, unclassified Coriobacteriaceae, and unclassified Planctomycetaceae were linearly decreased (P < 0.05) with increases in corn grain. At the species level, high grain feeding linearly decreased the percentage of OTU8686 (unclassified Bacteria) (P = 0.004). To the best of our knowledge, this is the first study using barcoded DNA pyrosequencing method to survey the ileal microbiome of goats and the results suggest that increasing levels of dietary corn change the composition of the ileal bacterial community. These findings provide previously unknown information about the ileal microbiota of goats and a new understanding of the ileal microbial ecology, which may be useful in modulating the gut microbiome.     

Taxonomic Identification of Commensal Bacteria Associated with the Mucosa and Digesta throughout the Gastrointestinal Tracts of Preweaned Calves

Bacterial colonization in the gastrointestinal tracts (GIT) of preweaned calves is very important, since it can influence early development and postweaning performance and health. This study investigated the composition of the bacteria along the GIT (rumen, jejunum, ileum, cecum, and colon) of preweaned bull calves (3 weeks old) using pyrosequencing to understand the segregation of bacteria between the mucosal surface and digesta. Phylogenetic analysis revealed that a total of 83 genera belonging to 13 phyla were distributed throughout the GIT of preweaned calves, with the Firmicutes, Bacteroidetes, and Proteobacteria predominating. Quantitative PCR (qPCR) analysis of selected abundant bacterial genera (Prevotella, Bacteroides, Lactobacillus, and Faecalibacterium) revealed that their prevalence was significantly different among the GIT regions and between mucosa- and digesta-associated communities. Rumens contained the most diverse bacterial population, consisting of 47 genera, including 16 rumen-specific genera, followed by the large intestine and then the small intestine. Bacterial species richness was higher at the mucosal surface than in the local digesta, with the exception of the rumen. The majority of bacteria found on the rumen epithelial surface and within the small intestine could not be identified due to a lack of known genus-level information. Thus, future studies will be required to fully characterize the microbiome during the development of the rumens and the mucosal immune systems of newborn calves. This is the first study to analyze in depth the bacterial composition of the GIT microbiome in preweaned calves, which extends previous findings regarding early rumen colonization and bacterial segregation between mucosa- and digesta-associated microbial communities.     

Characterization of rumen bacterial diversity and fermentation parameters in concentrate fed cattle with and without forage

Aims: To determine the effects of the removal of forage in high‐concentrate diets on rumen fermentation conditions and rumen bacterial populations using culture‐independent methods. Methods and Results: Detectable bacteria and fermentation parameters were measured in the solid and liquid fractions of digesta from cattle fed two dietary treatments, high concentrate (HC) and high concentrate without forage (HCNF). Comparison of rumen fermentation conditions showed that duration of time spent below pH 5·2 and rumen osmolality were higher in the HCNF treatment. Simpson’s index of 16S PCR‐DGGE images showed a greater diversity of dominant species in the HCNF treatment. Real‐time qPCR showed populations of Fibrobacter succinogenes (P = 0·01) were lower in HCNF than HC diets. Ruminococcus spp., F. succinogenes and Selenomonas ruminantium were at higher (P ≤ 0·05) concentrations in the solid vs the liquid fraction of digesta regardless of diet. Conclusions: The detectable bacterial community structure in the rumen is highly diverse. Reducing diet complexity by removing forage increased bacterial diversity despite the associated reduction in ruminal pH being less conducive for fibrolytic bacterial populations. Quantitative PCR showed that removal of forage from the diet resulted in a decline in the density of some, but not all fibrolytic bacterial species examined. Significance and Impact of the Study: Molecular techniques such as DGGE and qPCR provide an increased understanding of the impacts of dietary changes on the nature of rumen bacterial populations, and conclusions derived using these techniques may not match those previously derived using traditional laboratory culturing techniques.     

Next Generation Sequencing to Define Prokaryotic and Fungal Diversity in the Bovine Rumen

A combination of Sanger and 454 sequences of small subunit rRNA loci were used to interrogate microbial diversity in the bovine rumen of 12 cows consuming a forage diet. Observed bacterial species richness, based on the V1–V3 region of the 16S rRNA gene, was between 1,903 to 2,432 species-level operational taxonomic units (OTUs) when 5,520 reads were sampled per animal. Eighty percent of species-level OTUs were dominated by members of the order Clostridiales, Bacteroidales, Erysipelotrichales and unclassified TM7. Abundance of Prevotella species varied widely among the 12 animals. Archaeal species richness, also based on 16S rRNA, was between 8 and 13 OTUs, representing 5 genera. The majority of archaeal OTUs (84%) found in this study were previously observed in public databases with only two new OTUs discovered. Observed rumen fungal species richness, based on the 18S rRNA gene, was between 21 and 40 OTUs with 98.4–99.9% of OTUs represented by more than one read, using Good’s coverage. Examination of the fungal community identified numerous novel groups. Prevotella and Tannerella were overrepresented in the liquid fraction of the rumen while Butyrivibrio and Blautia were significantly overrepresented in the solid fraction of the rumen. No statistical difference was observed between the liquid and solid fractions in biodiversity of archaea and fungi. The survey of microbial communities and analysis of cross-domain correlations suggested there is a far greater extent of microbial diversity in the bovine rumen than previously appreciated, and that next generation sequencing technologies promise to reveal novel species, interactions and pathways that can be studied further in order to better understand how rumen microbial community structure and function affects ruminant feed efficiency, biofuel production, and environmental impact.     

Rumen Microbial Population Dynamics during Adaptation to a High-Grain Diet

High-grain adaptation programs are widely used with feedlot cattle to balance enhanced growth performance against the risk of acidosis. This adaptation to a high-grain diet from a high-forage diet is known to change the rumen microbial population structure and help establish a stable microbial population within the rumen. Therefore, to evaluate bacterial population dynamics during adaptation to a high-grain diet, 4 ruminally cannulated beef steers were adapted to a high-grain diet using a step-up diet regimen containing grain and hay at ratios of 20:80, 40:60, 60:40, and 80:20. The rumen bacterial populations were evaluated at each stage of the step-up diet after 1 week of adaptation, before the steers were transitioned to the next stage of the diet, using terminal restriction fragment length polymorphism (T-RFLP) analysis, 16S rRNA gene libraries, and quantitative real-time PCR. The T-RFLP analysis displayed a shift in the rumen microbial population structure during the final two stages of the step-up diet. The 16S rRNA gene libraries demonstrated two distinct rumen microbial populations in hay-fed and high-grain-fed animals and detected only 24 common operational taxonomic units out of 398 and 315, respectively. The 16S rRNA gene libraries of hay-fed animals contained a significantly higher number of bacteria belonging to the phylum Fibrobacteres, whereas the 16S rRNA gene libraries of grain-fed animals contained a significantly higher number of bacteria belonging to the phylum Bacteroidetes. Real-time PCR analysis detected significant fold increases in the Megasphaera elsdenii, Streptococcus bovis, Selenomonas ruminantium, and Prevotella bryantii populations during adaptation to the high-concentrate (high-grain) diet, whereas the Butyrivibrio fibrisolvens and Fibrobacter succinogenes populations gradually decreased as the animals were adapted to the high-concentrate diet. This study evaluates the rumen microbial population using several molecular approaches and presents a broader picture of the rumen microbial population structure during adaptation to a high-grain diet from a forage diet.The rumen is a complex microbial ecosystem that is composed of an immense variety of bacteria, protozoa, fungi, and viruses (5). Among these microorganisms, bacteria are the most investigated population and have a significant effect on the animal''s performance. However, our understanding of how rumen bacteria change and adapt to different ruminal environments is in its infancy.In the feedlot cattle industry, when animals on a forage diet are directly put on a high-grain diet, a decrease in ruminal pH due to lactate production has been observed (23, 31, 42), which leads to the possibility of digestive disorders, which can cause a decrease in the animal''s performance (23, 45). Therefore, feeding programs have been implemented to adapt feedlot cattle from a high-forage diet to a high-concentrate diet by gradually increasing the concentration of grain in the diet and decreasing the fiber content (2, 35). During this adaptation to high-grain diets, significant changes in the ruminal environment and rumen bacterial population structure have been reported (17, 46, 48). However, the microbial changes that occur during this transition phase are poorly understood (17, 21, 26, 46). Studies performed to date have utilized culture-based techniques or have looked at the fluctuation of a few indicator bacteria (48, 47) to evaluate bacterial population changes. Due to limitations in culturing rumen bacteria, the use of culture-based techniques to evaluate bacterial populations substantially underestimates the diversity of microorganisms within the rumen. In this study, we have utilized culture-independent approaches to evaluate bacterial population structure and diversity using terminal restriction fragment length polymorphisms (T-RFLPs) and sequence analysis of 16S rRNA gene libraries to compare the rumen bacterial population structure in animals on prairie hay against that in animals adapting to a high-concentrate (high-grain) diet. We have also quantified the fluctuations in the populations of previously reported indicator bacterial species using quantitative real-time PCR (qRT-PCR) to assess the role of these organisms during adaptation to a high-concentrate diet.     

Effect of Forage or Grain Diets with or without Monensin on Ruminal Persistence and Fecal Escherichia coli O157:H7 in Cattle

Twelve ruminally cannulated cattle, adapted to forage or grain diet with or without monensin, were used to investigate the effects of diet and monensin on concentration and duration of ruminal persistence and fecal shedding of E. coli O157:H7. Cattle were ruminally inoculated with a strain of E. coli O157:H7 (10¹⁰ CFU/animal) made resistant to nalidixic acid (Nal^r). Ruminal and fecal samples were collected for 11 weeks, and then cattle were euthanized and necropsied and digesta from different gut locations were collected. Samples were cultured for detection and enumeration of Nal^r E. coli O157:H7. Cattle fed forage diets were culture positive for E. coli O157:H7 in the feces for longer duration (P < 0.05) than cattle fed a grain diet. In forage-fed cattle, the duration they remained culture positive for E. coli O157:H7 was shorter (P < 0.05) when the diet included monensin. Generally, ruminal persistence of Nal^r E. coli O157:H7 was not affected by diet or monensin. At necropsy, E. coli O157:H7 was detected in cecal and colonic digesta but not from the rumen. Our study showed that cattle fed a forage diet were culture positive longer and with higher numbers than cattle on a grain diet. Monensin supplementation decreased the duration of shedding with forage diet, and the cecum and colon were culture positive for E. coli O157:H7 more often than the rumen of cattle.     

Impact of subacute ruminal acidosis on the diversity of liquid and solid-associated bacteria in the rumen of goats

This study was aimed to investigate the impact of subacute ruminal acidosis (SARA) on the diversity of liquid (LAB) and solid-associated bacteria (SAB) following high-grain feeding. Six ruminally cannulated goats were divided into two groups: one group was fed a hay diet (COD), and the other group was fed a high grain diet (SAID). Rumen liquids and rumen solids were sampled after 2 weeks adaption. SARA was diagnosed with a pH below 5.8 for 8 h. SAID decreased ruminal pH (P < 0.001) and increased the acetate (P = 0.017), propionate (P = 0.001), butyrate (P < 0.001) and total volatile fatty acid (P < 0.001) concentration in rumen compared with the COD. Denaturing gradient gel electrophoresis fingerprints analysis revealed a clear separation between both the diet and the fraction of rumen digesta in bacterial communities. Pyrosequencing analysis showed that the proportion of phylum Bacteroidetes in the SAID-LAB and SAID-SAB communities was less than in the COD group, whereas the SAID group had a greater percentage of Firmicutes in both the LAB and SAB libraries. UniFrac analyses and a Venn diagram revealed a large difference between the two diets in the diversity of rumen bacterial communities. Overall, our findings revealed that SARA feeding did alter the community structure of rumen liquids and rumen solids. Thus, manipulation of dietary factors, such as ratio of forage to concentrate may have the potential to alter the microbial composition of rumen liquid and rumen solid.     

Composition, spatial distribution, and diversity of the bacterial communities in the rumen of cows fed different forages

The species composition, distribution, and biodiversity of the bacterial communities in the rumen of cows fed alfalfa or triticale were investigated using 16S rRNA gene clone library analyses. The rumen bacterial community was fractionated and analyzed as three separate fractions: populations in the planktonic, loosely attached to rumen digesta particles, and tightly attached to rumen digesta particles. Six hundred and thirteen operational taxonomic units (OTUs) belonging to 32 genera, 19 families, and nine phyla of the domain Bacteria were identified from 1014 sequenced clones. Four hundred and fifty one of the 613 OTUs were identified as new species. These bacterial sequences were distributed differently among the three fractions in the rumen digesta of cows fed alfalfa or triticale. Chao 1 estimation revealed that, in both communities, the populations tightly attached to particulates were more diverse than the planktonic and those loosely attached to particulates. S-Libshuff detected significant differences in the composition between any two fractions in the rumen of cows with the same diet and between the communities fed alfalfa and triticale diets. The species richness estimated for the communities fed alfalfa and triticale is 1027 and 662, respectively. The diversity of the rumen bacterial community examined in this study is greater than previous studies have demonstrated and the differences in the community composition between two high-fiber diets have implications for sample selection for downstream metagenomics applications.     

Characterization of the rumen microbiome of Indian Kankrej cattle (Bos indicus) adapted to different forage diet

Present study described rumen microbiome of Indian cattle (Kankrej breed) to better understand the microbial diversity and largely unknown functional capacity of the rumen microbiome under different dietary treatments. Kankrej cattle were gradually adapted to a high-forage diet (four animals with dry forage and four with green forage) containing 50 % (K1), 75 % (K2) to 100 % (K3) forage, and remaining concentrate diet, each for 6 weeks followed by analysis of rumen fiber adherent and fiber-free metagenomic community by shotgun sequencing using ion torrent PGM platform and EBI-metagenomics annotation pipeline. Taxonomic analysis indicated that rumen microbiome was dominated by Bacteroidetes followed by Firmicutes, Fibrobacter, Proteobacteria, and Tenericutes. Functional analysis based on gene ontology classified all reads in total 157 categories based on their functional role in biological, molecular, and cellular component with abundance of genes associated with hydrolase activity, membrane, transport, transferase, and different metabolism (such as carbohydrate and protein). Statistical analysis using STAMP revealed significant differences (P?相似文献   

Biphasic assembly of the murine intestinal microbiota during early development

The birth canal provides mammals with a primary maternal inoculum, which develops into distinctive body site-specific microbial communities post-natally. We characterized the distal gut microbiota from birth to weaning in mice. One-day-old mice had colonic microbiota that resembled maternal vaginal communities, but at days 3 and 9 of age there was a substantial loss of intestinal bacterial diversity and dominance of Lactobacillus. By weaning (21 days), diverse intestinal bacteria had established, including strict anaerobes. Our results are consistent with vertical transmission of maternal microbiota and demonstrate a nonlinear ecological succession involving an early drop in bacterial diversity and shift in dominance from Streptococcus to Lactobacillus, followed by an increase in diversity of anaerobes, after the introduction of solid food. Mammalian newborns are born highly susceptible to colonization, and lactation may control microbiome assembly during early development.     

Rumen Microbiome from Steers Differing in Feed Efficiency

The cattle rumen has a diverse microbial ecosystem that is essential for the host to digest plant material. Extremes in body weight (BW) gain in mice and humans have been associated with different intestinal microbial populations. The objective of this study was to characterize the microbiome of the cattle rumen among steers differing in feed efficiency. Two contemporary groups of steers (n=148 and n=197) were fed a ration (dry matter basis) of 57.35% dry-rolled corn, 30% wet distillers grain with solubles, 8% alfalfa hay, 4.25% supplement, and 0.4% urea for 63 days. Individual feed intake (FI) and BW gain were determined. Within contemporary group, the four steers within each Cartesian quadrant were sampled (n=16/group) from the bivariate distribution of average daily BW gain and average daily FI. Bacterial 16S rRNA gene amplicons were sequenced from the harvested bovine rumen fluid samples using next-generation sequencing technology. No significant changes in diversity or richness were indicated, and UniFrac principal coordinate analysis did not show any separation of microbial communities within the rumen. However, the abundances of relative microbial populations and operational taxonomic units did reveal significant differences with reference to feed efficiency groups. Bacteroidetes and Firmicutes were the dominant phyla in all ruminal groups, with significant population shifts in relevant ruminal taxa, including phyla Firmicutes and Lentisphaerae, as well as genera Succiniclasticum, Lactobacillus, Ruminococcus, and Prevotella. This study suggests the involvement of the rumen microbiome as a component influencing the efficiency of weight gain at the 16S level, which can be utilized to better understand variations in microbial ecology as well as host factors that will improve feed efficiency.     

Intestinal microbiota of gibel carp (Carassius auratus gibelio) and its origin as revealed by 454 pyrosequencing

The intestinal microbiota has received increasing attention, as it influences growth, feed conversion, epithelial development, immunity as well as the intrusion of pathogenic microorganisms in the intestinal tract. In this study, pyrosequencing was used to explore the bacterial community of the intestine in gibel carp (Carassius auratus gibelio), and the origin of these microorganisms. The results disclosed great bacterial diversities in the carp intestines and cultured environments. The gibel carp harbored characteristic intestinal microbiota, where Proteobacteria were predominant, followed by Firmicutes. The analysis on the 10 most abundant bacterial operational taxonomic units (OTUs) revealed a majority of Firmicutes in the intestinal content (by decreasing order: Veilonella sp., Lachnospiraceae, Lactobacillales, Streptococcus sp., and Lactobacillus sp.). The second most abundant OTU was Rothia sp. (Actinobacteria). The most likely potential probiotics (Lactobacillus sp., and Bacillus sp.) and opportunists (Aeromonas sp., and Acinetobacter sp.) were not much abundant. Bacterial community comparisons showed that the intestinal community was closely related to that of the sediment, indicating the importance of sediment as source of gut bacteria in gibel carp. However, 37.95 % of the OTUs detected in feed were retrieved in the intestine, suggesting that food may influence markedly the microbiota of gibel carp, and therefore may be exploited for oral administration of probiotics.     

Protozoa populations are ecosystem engineers that shape prokaryotic community structure and function of the rumen microbial ecosystem

Unicellular eukaryotes are an integral part of many microbial ecosystems where they interact with their surrounding prokaryotic community—either as predators or as mutualists. Within the rumen, one of the most complex host-associated microbial habitats, ciliate protozoa represent the main micro-eukaryotes, accounting for up to 50% of the microbial biomass. Nonetheless, the extent of the ecological effect of protozoa on the microbial community and on the rumen metabolic output remains largely understudied. To assess the role of protozoa on the rumen ecosystem, we established an in-vitro system in which distinct protozoa sub-communities were introduced to the native rumen prokaryotic community. We show that the different protozoa communities exert a strong and differential impact on the composition of the prokaryotic community, as well as its function including methane production. Furthermore, the presence of protozoa increases prokaryotic diversity with a differential effect on specific bacterial populations such as Gammaproteobacteria, Prevotella and Treponema. Our results suggest that protozoa contribute to the maintenance of prokaryotic diversity in the rumen possibly by mitigating the effect of competitive exclusion between bacterial taxa. Our findings put forward the rumen protozoa populations as potentially important ecosystem engineers for future microbiome modulation strategies.Subject terms: Microbial ecology, Food webs     

Effect of forage or grain diets with or without monensin on ruminal persistence and fecal Escherichia coli O157:H7 in cattle

Twelve ruminally cannulated cattle, adapted to forage or grain diet with or without monensin, were used to investigate the effects of diet and monensin on concentration and duration of ruminal persistence and fecal shedding of E. coli O157:H7. Cattle were ruminally inoculated with a strain of E. coli O157:H7 (10(10) CFU/animal) made resistant to nalidixic acid (Nal(r)). Ruminal and fecal samples were collected for 11 weeks, and then cattle were euthanized and necropsied and digesta from different gut locations were collected. Samples were cultured for detection and enumeration of Nal(r) E. coli O157:H7. Cattle fed forage diets were culture positive for E. coli O157:H7 in the feces for longer duration (P < 0.05) than cattle fed a grain diet. In forage-fed cattle, the duration they remained culture positive for E. coli O157:H7 was shorter (P < 0.05) when the diet included monensin. Generally, ruminal persistence of Nal(r) E. coli O157:H7 was not affected by diet or monensin. At necropsy, E. coli O157:H7 was detected in cecal and colonic digesta but not from the rumen. Our study showed that cattle fed a forage diet were culture positive longer and with higher numbers than cattle on a grain diet. Monensin supplementation decreased the duration of shedding with forage diet, and the cecum and colon were culture positive for E. coli O157:H7 more often than the rumen of cattle.     

Vaginal Microbiome Characterization of Nellore Cattle Using Metagenomic Analysis

Understanding of microbial communities inhabiting cattle vaginal tract may lead to a better comprehension of bovine physiology and reproductive health being of great economic interest. Up to date, studies involving cattle microbiota are focused on the gastrointestinal tract, and little is known about the vaginal microbiota. This study aimed to investigate the vaginal microbiome in Nellore cattle, heifers and cows, pregnant and non-pregnant, using a culture independent approach. The main bacterial phyla found were Firmicutes (~40–50%), Bacteroidetes (~15–25%) and Proteobacteria (~5–25%), in addition to ~10–20% of non-classified bacteria. 45–55% of the samples were represented by only ten OTUs: Aeribacillus, Bacteroides, Clostridium, Ruminococcus, Rikenella, Alistipes, Bacillus, Eubacterium, Prevotella and non-classified bacteria. Interestingly, microbiota from all 20 animals could be grouped according to the respiratory metabolism of the main OTUs found, creating three groups of vaginal microbiota in cattle. Archaeal samples were dominated by the Methanobrevibacter genus (Euryarchaeota, ~55–70%). Ascomycota was the main fungal phylum (~80–95%) and Mycosphaerella the most abundant genus (~70–85%). Hormonal influence was not clear, but a tendency for the reduction of bacterial and increase of archaeal populations in pregnant animals was observed. Eukaryotes did not vary significantly between pregnant and non-pregnant animals, but tended to be more abundant on cows than on heifers. The present work describes a great microbial variability in the vaginal community among the evaluated animals and groups (heifers and cows, pregnant and non-pregnant), which is significantly different from the findings previously reported using culture dependent methods, pointing out the need for further studies on this issue. The microbiome found also indicates that the vaginal colonization appears to be influenced by the gastrointestinal community.     

Surveying the Microbiome of Ants: Comparing 454 Pyrosequencing with Traditional Methods To Uncover Bacterial Diversity

We are only beginning to understand the depth and breadth of microbial associations across the eukaryotic tree of life. Reliably assessing bacterial diversity is a key challenge, and next-generation sequencing approaches are facilitating this endeavor. In this study, we used 16S rRNA amplicon pyrosequencing to survey microbial diversity in ants. We compared 454 libraries with Sanger-sequenced clone libraries as well as cultivation of live bacteria. Pyrosequencing yielded 95,656 bacterial 16S rRNA reads from 19 samples derived from four colonies of one ant species. The most dominant bacterial orders in the microbiome of the turtle ant Cephalotes varians were Rhizobiales, Burkholderiales, Opitutales, Xanthomonadales, and Campylobacterales, as revealed through both 454 sequencing and cloning. Even after stringent quality filtering, pyrosequencing recovered 445 microbe operational taxonomic units (OTUs) not detected with traditional techniques. In comparing bacterial communities associated with specific tissues, we found that gut tissues had significantly higher diversity than nongut tissues, and many of the OTUs identified from these groups clustered within ant-specific lineages, indicating a deep coevolutionary history of Cephalotes ants and their associated microbes. These lineages likely function as nutritional symbionts. One of four ant colonies investigated was infected with a Spiroplasma sp. (order Entomoplasmatales), a potential ant pathogen. Our work shows that the microbiome associated with Cephalotes varians is dominated by a few dozen bacterial lineages and that 454 sequencing is a cost-efficient tool to screen ant symbiont diversity.     

Effects of Propionibacterium strains on ruminal fermentation,nutrient digestibility and methane emissions in beef cattle fed a corn grain finishing diet

Twenty ruminally cannulated beef heifers were fed a high corn grain diet in a randomized block design to determine the effect of three direct fed microbial (DFM) strains of Propionibacterium on ruminal fermentation, nutrient digestibility and methane (CH₄) emissions. The heifers were blocked in five groups on the basis of BW and used in five 28-day periods. Dietary treatments included (1) Control and three strains of Propionibacterium (2) P169, (3) P5, and (4) P54. Strains were administered directly into the rumen at 5×10⁹ CFU with 10 g of a maltodextrin carrier in a gel capsule; Control heifers received carrier only. All heifers were fed the basal diet (10 : 90 forage to concentrate, dry matter basis). Rumen contents were collected on days 15 and 18, ruminal pH was measured continuously between days 15 and 22, enteric CH₄ emissions were measured between days 19 and 22 and diet digestibility was measured from days 25 to 28. Mean ruminal pH was 5.91 and was not affected by treatments. Similarly, duration of time that pH<5.8 and 5.6 was not affected by treatment. Likewise, total and major volatile fatty acid profiles were similar among all treatments. No effects were observed on dry matter intake and total tract digestibility of nutrients. Total enteric CH₄ production (g/day) was not affected by Propionibacterium strains and averaged 139 g/day. Similarly, mean CH₄ yield (g CH₄/kg of dry matter intake) was similar for all the treatments. The relative abundance of total Propionibacteria in the rumen increased with administration of DFM and were greater 3 h post-dosing relative to Control, but returned to baseline levels before feeding. Populations of Propionibacterium P169 were higher at 3 and 9 h as compared with the levels at 0 h. In conclusion, moderate persistency of the inoculated strains within the ruminal microbiome and pre-existing high propionate production due to elevated levels of starch fermentation might have reduced the efficacy of Propionibacterium strains to increase molar proportion of propionate and subsequently reduce CH₄ emissions.