Adhesion of bacteria to epithelial cell surfaces within the reticulo-rumen of cattle.

Blocks of tissue were removed from various locations in the bovine digestive tract and fixed and processed for transmission and scanning electron microscopy by techniques that retained adherent bacteria. The distribution of bacteria on the surface of epithelial cells was examined by scanning electron microscopy. This showed intermittent colonization of the epithelia with the formation of occasional microcolonies of morphologically similar bacterial cells. Transmission electron microscopy of ruthenium red-stained material showed the presence of both the glycocalyx of the bovine epithelial cells and fibrous carbohydrate coats surrounding adherent bacteria. The carbohydrate coats appeared to mediate the attachment of bacteria to the epithelium, to food particles, and to each other so that microcolonies were formed. Careful examination of the bacterial colonization of keratinized cells in the process of being sloughed from the surface of the stratified squamous epithelium of the rumen showed that these dead cells were digested by adherent bacteria of a limited number of morphological types. The spatial relationship of this mixed, adherent, microbial population to living and dead epithelial cells and to food particles indicates that digestive processes of some importance may be accomplished by this stationary component of the microbial flora of the digestive tract.     

Adhesion of bacteria to epithelial cell surfaces within the reticulo-rumen of cattle

Blocks of tissue were removed from various locations in the bovine digestive tract and fixed and processed for transmission and scanning electron microscopy by techniques that retained adherent bacteria. The distribution of bacteria on the surface of epithelial cells was examined by scanning electron microscopy. This showed intermittent colonization of the epithelia with the formation of occasional microcolonies of morphologically similar bacterial cells. Transmission electron microscopy of ruthenium red-stained material showed the presence of both the glycocalyx of the bovine epithelial cells and fibrous carbohydrate coats surrounding adherent bacteria. The carbohydrate coats appeared to mediate the attachment of bacteria to the epithelium, to food particles, and to each other so that microcolonies were formed. Careful examination of the bacterial colonization of keratinized cells in the process of being sloughed from the surface of the stratified squamous epithelium of the rumen showed that these dead cells were digested by adherent bacteria of a limited number of morphological types. The spatial relationship of this mixed, adherent, microbial population to living and dead epithelial cells and to food particles indicates that digestive processes of some importance may be accomplished by this stationary component of the microbial flora of the digestive tract.     

Production of alkaline phosphatase by epithelial cells and adherent bacteria of the bovine rumen and abomasum

Three distinct bacterial populations have been defined in the bovine rumen: the rumen fluid population; the population associated with food particles; and the population adherent to the rumen epithelium. Alkaline phosphatase activity has been reported in cells of the first two populations and here we report that assays of rumen epithelial samples containing both tissue and bacteria also contain the enzyme. The reaction product technique has localized the enzyme both in adherent bacteria and in cell of the stratified squamous rumen epithelium. The epithelium of the abomasum shows much lower levels of alkaline phosphatase activity.     

Urease activity of adherent bacteria in the sheep rumen

In experiments on six sheep fed on a low protein diet (6.2 g N/day), it was found that the urease activity of the rumen fluid did not change significantly in the first 6 hours after feeding and that it ranged from 45 to 75 nkat.ml-1. The major portion was bound to the bacterial fraction and formed about 70% of total rumen fluid activity. Urease activity determined in food particles with adherent bacteria removed from the rumen before and 3 and 6 hours after feeding ranged from 20 to 26 nkat.g-1 food (wet weight), and on rumen wall samples with adherent bacteria from 30 to 800 nkat per 2.5 cm2 tissue. Again, no significant changes correlated to the time after feeding were found. The results show that urease activity in the sheep rumen is localized on food particles and on rumen wall epithelium with adherent bacteria, as well as in the rumen fluid.     

Novel microbial diversity adherent to plant biomass in the herbivore gastrointestinal tract, as revealed by ribosomal intergenic spacer analysis and rrs gene sequencing

It is well recognized that a dynamic biofilm develops upon plant biomass in the herbivore gastrointestinal tract, but this component of the microbiome has not previously been specifically sampled, or directly compared with the biodiversity present in the planktonic fraction of digesta. In this study, the digesta collected from four sheep fed two different diets was separated into three fractions: the planktonic phase, and the microbial populations either weakly or tightly adherent to plant biomass. The community DNA prepared from each fraction was then subjected to both ribosomal intergenic spacer analysis (RISA) and denaturing gradient gel electrophoresis (DGGE). Both types of analysis showed that dietary factors influence community structure, and that the adherent fractions produced more complex profiles. The RIS-clone libraries prepared from the planktonic and adherent populations were then subjected to restriction fragment length polymorphism (RFLP) and DNA sequence analyses, which resulted in a far greater degree of discrimination among the fractions. Although many of the sequenced clones from the adherent populations were assigned to various clusters within the low G+C Gram-positive bacteria, the clone libraries from animals consuming an all-grass diet were largely comprised of novel lineages of Clostridium, while in animals consuming the starch-containing diet, Selenomonas and Ruminococcus spp. were the dominant low G+C Gram-positive bacteria. Additionally, the libraries from hay-fed animals also contained clones most similar to asaccharolytic Clostridia, and other Gram-positive bacteria that specialize in the transformation of plant phenolic compounds and the formation of cinnamic, phenylacetic and phenylpropionic acids. These results reveal, for the first time, the phylogeny of adherent subpopulations that specialize in the transformation of plant lignins and other secondary compounds, which potentiate polysaccharide hydrolysis by other members of the biofilm.     

Patterns of gastric evacuation, digesta characteristics and pH changes along the gastrointestinal tract of gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.)

A comparative study of gastric evacuation rates (GERs) and digesta content, moisture and pH values along the gastrointestinal tract was performed between gilthead sea bream and European sea bass. In order to distinguish species-specific differences from diet-elicited effects, all parameters were determined under either a fishmeal diet or a carob seed germ meal diet that contained high levels of total and soluble non-starch polysaccharides. GERs were significantly different between species and they were not affected by diet. Similarly, species-specific patterns were revealed in the distribution of digesta and water content along the gastrointestinal tract. In sea bream, stomach digesta and water content decreased with time, whereas in sea bass stomach retained the highest digesta and water content throughout the sampling period. The anterior and distal intestine exhibited the lowest accommodating capacities of digesta and water in either species. Overall, sea bream performed stomach digestion at lower hydration levels and higher pH compared with sea bass. Diet affected stomach moisture in both species and pH of stomach digesta in sea bass and of all intestinal sections in sea bream. The results obtained indicated that water and inorganic ion exchanges through the gut may differentiate between the species and warrant further investigation.     

Colonization of a portion of the bovine tongue by unusual filamentous bacteria.

Tongue samples from cattle on varied diets and ranging in age from 2 months to adult were studied by transmission and scanning electron microscopy to observe the in situ distribution and adhesion patterns of two readily identifiable genera of filamentous bacterial. The two, both members of the Simonsiellaceae, adhere to the epithelium by means of fibers which are produced on one side of the bacterial filaments and subsequently display a sidedness in their manner of adhesion to epithelial surfaces. Other bacterial populations found on the tongue were normally members of chains and seldom present as single cells. This suggests that filamentous or chain-forming bacteria may have a selective advantage over single bacteria in their ability to colonize and remain attached to the epithelium of the tongue.     

Colonization of a portion of the bovine tongue by unusual filamentous bacteria.

Tongue samples from cattle on varied diets and ranging in age from 2 months to adult were studied by transmission and scanning electron microscopy to observe the in situ distribution and adhesion patterns of two readily identifiable genera of filamentous bacterial. The two, both members of the Simonsiellaceae, adhere to the epithelium by means of fibers which are produced on one side of the bacterial filaments and subsequently display a sidedness in their manner of adhesion to epithelial surfaces. Other bacterial populations found on the tongue were normally members of chains and seldom present as single cells. This suggests that filamentous or chain-forming bacteria may have a selective advantage over single bacteria in their ability to colonize and remain attached to the epithelium of the tongue.     

Quantitative analysis of ruminal bacterial populations involved in lipid metabolism in dairy cows fed different vegetable oils

Vegetable oils are used to increase energy density of dairy cow diets, although they can provoke changes in rumen bacteria populations and have repercussions on the biohydrogenation process. The aim of this study was to evaluate the effect of two sources of dietary lipids: soybean oil (SO, an unsaturated source) and hydrogenated palm oil (HPO, a saturated source) on bacterial populations and the fatty acid profile of ruminal digesta. Three non-lactating Holstein cows fitted with ruminal cannulae were used in a 3×3 Latin square design with three periods consisting of 21 days. Dietary treatments consisted of a basal diet (Control, no fat supplement) and the basal diet supplemented with SO (2.7% of dry matter (DM)) or HPO (2.7% of DM). Ruminal digesta pH, NH₃–N and volatile fatty acids were not affected by dietary treatments. Compared with control and HPO, total bacteria measured as copies of 16S ribosomal DNA/ml by quantitative PCR was decreased (P<0.05) by SO. Fibrobacter succinogenes, Butyrivibrio proteoclasticus and Anaerovibrio lipolytica loads were not affected by dietary treatments. In contrast, compared with control, load of Prevotella bryantii was increased (P<0.05) with HPO diet. Compared with control and SO, HPO decreased (P<0.05) C18:2 cis n-6 in ruminal digesta. Contents of C15:0 iso, C18:11 trans-11 and C18:2 cis-9, trans-11 were increased (P<0.05) in ruminal digesta by SO compared with control and HPO. In conclusion, supplementation of SO or HPO do not affect ruminal fermentation parameters, whereas HPO can increase load of ruminal P. bryantii. Also, results observed in our targeted bacteria may have depended on the saturation degree of dietary oils.     

Characterization of the Core Rumen Microbiome in Cattle during Transition from Forage to Concentrate as Well as during and after an Acidotic Challenge

This study investigated the effect of diet and host on the rumen bacterial microbiome and the impact of an acidotic challenge on its composition. Using parallel pyrosequencing of the V3 hypervariable region of 16S rRNA gene, solid and liquid associated bacterial communities of 8 heifers were profiled. Heifers were exclusively fed forage, before being transitioned to a concentrate diet, subjected to an acidotic challenge and allowed to recover. Samples of rumen digesta were collected when heifers were fed forage, mixed forage, high grain, during challenge (4 h and 12 h) and recovery. A total of 560,994 high-quality bacterial sequences were obtained from the solid and liquid digesta. Using cluster analysis, prominent bacterial populations differed (P≤0.10) in solid and liquid fractions between forage and grain diets. Differences among hosts and diets were not revealed by DGGE, but real time qPCR showed that several bacteria taxon were impacted by changes in diet, with the exception of Streptococcus bovis. Analysis of the core rumen microbiome identified 32 OTU''s representing 10 distinct bacterial taxa including Bacteroidetes (32.8%), Firmicutes (43.2%) and Proteobacteria (14.3%). Diversity of OTUs was highest with forage with 38 unique OTUs identified as compared to only 11 with the high grain diet. Comparison of the microbial profiles of clincial vs. subclinical acidotic heifers found a increases in the relative abundances of Acetitomaculum, Lactobacillus, Prevotella, and Streptococcus. Increases in Streptococcus and Lactobacillus likely reflect the tolerance of these species to low pH and their ability to proliferate on surplus fermentable carbohydrate. The acetogen, Acetitomaculum may thereforeplay a role in the conversion of lactate to acetate in acidotic animals. Further profiling of the bacterial populations associated with subclinical and clinical acidosis could establish a microbial fingerprint for these disorders and provide insight into whether there are causative microbial populations that could potentially be therapeutically manipulated.     

Effect of condensed tannins on bacterial diversity and metabolic activity in the rat gastrointestinal tract

The effect of dietary condensed tannins (proanthocyanidins) on rat fecal bacterial populations was ascertained in order to determine whether the proportion on tannin-resistant bacteria increased and if there was a change in the predominant bacterial populations. After 3 weeks of tannin diets the proportion of tannin-resistant bacteria increased significantly (P < 0.05) from 0.3% +/- 5.5% to 25.3% +/- 8.3% with a 0.7% tannin diet and to 47.2% +/- 5.1% with a 2% tannin diet. The proportion of tannin-resistant bacteria returned to preexposure levels in the absence of dietary tannins. A shift in bacterial populations was confirmed by molecular fingerprinting of fecal bacterial populations by denaturing gradient gel electrophoresis (DGGE). Posttreatment samples were generally still distinguishable from controls after 3.5 weeks. Sequence analysis of DGGE bands and characterization of tannin-resistant isolates indicated that tannins selected for Enterobacteriaceae and Bacteroides species. Dot blot quantification confirmed that these gram-negative bacterial groups predominated in the presence of dietary tannins and that there was a corresponding decrease in the gram-positive Clostridium leptum group and other groups. Metabolic fingerprint patterns revealed that functional activities of culturable fecal bacteria were affected by the presence of tannins. Condensed tannins of Acacia angustissima altered fecal bacterial populations in the rat gastrointestinal tract, resulting in a shift in the predominant bacteria towards tannin-resistant gram-negative Enterobacteriaceae and Bacteroides species.     

PCR-DGGE analysis reveals a distinct diversity in the bacterial population attached to the rumen epithelium

Bacteria attached to the rumen epithelium (or epimural community) are not well characterised and their role in rumen functioning is not totally understood. There is just one published report of a clone library from one cow that suggests that this epimural community differs from the bacteria associated with the rumen digestive contents. However, this time-consuming approach is not adapted for examining microbial population changes in groups of animals. In in vivo studies, when samples from several animals have to be analysed simultaneously, a simpler technique has to be used. In this study, a genetic fingerprinting technique, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), was used to characterise the structure of the bacterial population attached to the rumen epithelium. This community was compared with that present in the solid and liquid phases of rumen content under two contrasting diets. Rumen samples were obtained from four forage-fed and four high-concentrate-fed (80 : 20, wheat grain : hay) 5-month-old lambs. After slaughter, samples from five epithelial sites and the solid and liquid digesta phases were taken for DNA extraction and analysis. Bacterial communities were profiled by PCR-DGGE using bacterial-specific 16S rDNA primers. Analysis of the fingerprint revealed that the epithelial community differed from those of rumen content in both diets. As expected, the nature of the feed influenced the bacterial communities from the solid and liquid rumen phases but no diet effect was observed in the rumen epithelial profiles suggesting a strong host effect on this bacterial population. Additionally, no differences were observed among the five epithelial sampling sites taken from each animal. The profile of the bacterial population attached to the rumen epithelium presented a high inter-animal variation, whether this difference has an influence in the function of this community remains to be determined.     

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.     

Apparent absorption of methionine and 2-hydroxy-4-methylthiobutanoic acid from gastrointestinal tract of conventional and gnotobiotic pigs

The effect of commensal microbiota and feeding corn or wheat/barley-based diets on the apparent gastrointestinal absorption of dl-methionine (MET) and 2-hydroxy-4-methylthiobutanoic acid (MHA-FA) was studied in conventional (n = 32) and gnotobiotic pigs (n = 24). Conventional pigs (CON) were vaginally delivered and sow-reared until weaning at 14 days of age. Gnotobiotic pigs were derived by caesarian section and reared in HEPA (high efficiency particulate air)-filtered isolator units with ad libitum access to a milk-based formula. Corn or wheat/barley-based diets were fed to all pigs from 14 to 24 days of age. At 24 days of age, after an overnight fast, pigs were fed 20 g/kg BW of experimental diet supplemented with 107 Bq of either 3H-l-MET or 3H-l-MHA-FA per kg of feed and chromic oxide (0.5% wt/wt). Pigs were killed for sample collection 3 h after consuming the meal. Residual 3H-MET and 3H-MHA-FA were estimated in gastrointestinal contents as the ratio of 3H : chromic oxide in digesta samples to the ratio of 3H : chromic oxide in feed. In CON pigs, feeding a wheat/barley-based diet increased (P < 0.05) total aerobes, whereas supplementation with MHA-FA increased (P < 0.05) total aerobes and lactobacilli populations in proximal small intestine (SI). Among the gnotobiotic pigs, bacterial contamination occurred such that eight pigs (two isolators) were monoassociated with a Gram-negative bacteria closely related to Providencia spp. and 16 pigs (four isolators) were monoassociated with Gram positive Enterococcus faecium. Species of monoassociated bacterial contaminant and diet composition did not affect residual methionine or MHA-FA in digesta. In both CON and monoassociated (MA) pigs, methionine and MHA-FA were retained in stomach (92%) but disappeared rapidly from proximal SI. Residual methionine and MHA-FA in digesta was not different in MA pigs; however, in CON pigs, less (P < 0.01) apparent residual methionine was found in digesta recovered at 25% (from cranial to caudal) and 75% of SI length compared with MHA-FA. Apparent residual methionine was 16% and 8% compared with 34% and 15% for MHA-FA, at the 25% and 75% locations, respectively. In proximal SI tissue, significantly (P < 0.05) higher radioactivity (cpm/mg wet tissue) was associated with MET pigs (8.56 ± 0.47) as compared to MHA-FA ones (5.45 ± 0.50). This study suggests that microbial metabolism of MHA-FA increases retention in small intestinal digesta relative to methionine and contributes, in part, to the lower bioefficacy of MHA-FA compared to methionine.     

Enrichment and Association of Bacteria and Particulates in Salt Marsh Surface Water

Elevated counts of bacteria were found during outgoing tides in surface microlayers (~300 μm) of Sippewissett salt marsh, Falmouth, Massachusetts, and Palo Alto salt marsh, Palo Alto, California. At both sampling sites, the degrees by which bacteria were concentrated into the surface microlayer were linearly dependent upon surface concentration of particulate material. A significant percentage of bacteria in the microlayer were found to be attached to particulate material, while bacterial populations in the subsurface water were largely planktonic. Proportions of the bacterial populations which could be grown on seawater nutrient agar were also greater in the microlayer than in the subsurface waters and were positively correlated with the fraction of bacteria attached to particulate matter. Data from these studies suggest that particulates in the microlayer waters of the salt marsh influenced the observed increase in both the readily grown and the total numbers of bacteria.     

Effect of environmental factors and influence of rumen and hindgut biogeography on bacterial communities in steers

Feces from cattle production are considered important sources of bacterial contamination of food and the environment. Little is known about the combined effects of arctic temperatures and fodder tannins on rumen and hindgut bacterial populations. Individual rumen liquor and rectal fecal samples from donor steers fed either alfalfa silage or sainfoin (Onobrychis viciifolia Scop.) silage and water ad libitum were collected weekly on the first three sampling days and fortnightly afterwards. The daily ambient temperatures were registered and averaged to weekly mean temperatures. Steers fed sainfoin silage had lower (P < 0.05) concentrations of branched-chain volatile fatty acids (VFA) than those fed alfalfa silage. All VFA concentrations were higher (P < 0.001) in rumen liquor samples than in fecal samples. The interaction of sample type and diet showed a significant effect (P < 0.05) on the proportions of the bacterial community that were from the phyla Proteobacteria and Verrucomicrobia. Ambient temperature had an indirect effect (P < 0.05) on the phylum Firmicutes, as it affected its proportional balance. The bacterial population diversity in samples appeared to decrease concurrently with the ambient temperature. The phylum Firmicutes explained the first principal component at 64.83 and 42.58% of the total variance in rumen liquor and fecal samples, respectively. The sample type had a larger effect on bacterial communities than diet and temperature. Certain bacterial populations seemed to be better adapted than others to environmentally adverse conditions, such as less access time to nutrients due to higher motility and rate of passage of digesta caused by extreme temperatures, or antimicrobials such as tannins, possibly due to an influence of their biogeographical location within the gut.     

Digestive performance and selective digesta retention in the long-nosed bandicoot,Perameles nasuta,a small omnivorous marsupial

Bandicoots are opportunistic omnivores that feed on invertebrates, fungi and both epigeal and hypogeal plant parts. We examined the performance of the digestive tract of the long-nosed bandicoot (Perameles nasuta) in terms of intake and total digestibility, patterns of excretion of inert digesta markers, and likely sites of digesta retention, on two diets designed to mimic part of their natural plant and insect diets. On the insect diet (mealworm larvae), bandicoots virtually maintained body mass at a digestible energy intake of 511 kJ · kg^-0.75 · day^-1 and were in strongly positive nitrogen balance. In contrast, on the plant diet (shredded sweet potato), bandicoots ate only one-third as much digestible energy, lost 7% body mass, and were in negative nitrogen balance. Mean retention times of two particle markers on the plant diet (27.5 and 27.0 h) were more than double those on the insect diet (12.4 and 11.2 h), and on both diets the mean retention time of the fluid digesta marker was greater than those of the particle markers, indicating consistent selective retention of fluid digesta in the gut. It was seen radiographically than in mealwormfed bandicoots major sites of digesta retention were the distal colon and rectum, whereas in the sweet potato-fed animals the caecum and proximal colon were principal sites. It was concluded that retention of plant material in the caecum and proximal colon (the main sites of microbial digestion) and the preferential retention of fluid digesta (together with bacteria and small feed particles) in the caecum were important factors in the ability of bandicoots to switch between insect and plant foods, depending on relative availabilities, and thus to exploit nutritionally unpredictable environments.Abbreviations ADF acid-detergent fibre - bm body mass - Co-ED-TA cobalt-ethylenediaminetetra-acetic acid - CWC cell wall constituents - DE digestable energy - dm dry matter - EUN endogenous urinary nitrogen - ICP inductively-coupled plasma atomic emission spectroscopy - MFN metabolic faecal nitrogen - MRT mean retention time - NDF neutral-detergent fibre - ww wet weight     

Scanning Electron Microscope Study of Bacteria Associated with the Rumen Epithelium of Sheep

Examination of the rumen epithelium of sheep by scanning electron microscopy revealed bacteria associated with the epithelial surface. Comparison of epithelial surfaces from 10 sheep revealed areas that were consistently densely covered with bacteria and other areas where the cover was consistently light. The bacterial populations were frequently of mixed morphological types, but areas populated with a single type were also observed. This finding, together with the discovery of bacterial forms not previously described in rumen contents, suggests that a specific flora may exist on the rumen epithelial surface. The functional significance of such a population is discussed.