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Feed intake is a major factor in maintaining the balance between ruminal fermentation and the microbial community of dairy cows. To explore the relationship among feed intake, microbial metabolism, and ruminal fermentation, we examined the combined signatures of the microbiome and metabolome in dairy cows with different feed intake levels. Eighteen dairy cows were allocated to high feed intake (HFI), medium feed intake (MFI), and low feed intake (LFI) groups according to their average daily feed intake. 16S rDNA sequencing results revealed that the relative abundance of Firmicutes in the HFI group was significantly higher than that in the MFI and LFI groups (P < 0.05). The ratio of Bacteroidetes to Firmicutes was significantly lower in the HFI group than in the MFI and LFI groups (P < 0.05). The relative abundance of Lachnospiraceae_unclassified, Veillonellaceae_unclassified, and Saccharofermentants was significantly higher in the HFI group than in the LFI and MFI groups (P < 0.05). The relative abundance of Erysipelotrichaceae_unclassified and Butyrivibrio was significantly higher in the HFI group than in the MFI and LFI groups (P < 0.05). Ultra high performance liquid chromatography-mass spectrometry revealed five key pathways, including the linoleic acid metabolism pathway, alpha-linolenic acid metabolism, arginine and proline metabolism, glutathione metabolism, and valine, leucine, and isoleucine biosynthesis, which are closely related to energy and amino acid metabolism. Linoleic acid, glutamate, alpha-linolenic acid, l-methionine, and l-valine levels were significantly lower in the HFI group than in the MFI and LFI groups (q < 0.05), while the relative content of glutamate was significantly lower in the MFI group than in the LFI group (q < 0.05). Stearic acid content was significantly higher in the HFI group than in the LFI group (q < 0.05). Our findings provide insight into the rumen microbiome of dairy cows with different feed intake and the metabolic pathways closely associated with feed intake in early-lactating cows. The candidates involved in these metabolic pathways may be useful for identifying variations in feed intake. The signatures of the rumen microbiome and metabolome in dairy cows may help make decisions regarding feeding.

Despite the body of research into Zn for human and animal health and productivity, very little work has been done to discern whether this benefit is exerted solely on the host organism, or whether there is some effect of dietary Zn upon the gastrointestinal microbiota, particularly in ruminants. We hypothesized that (i) supplementation with Zn would alter the rumen bacterial community in yearling rams, but that (ii) supplementation with either inorganically sourced ZnSO4, or a chelated Zn AA complex, which was more bioavailable, would affect the rumen bacterial community differently. Sixteen purebred Targhee yearling rams were utilized in an 84-d completely randomized design, and allocated to one of three pelleted dietary treatments: control diet without fortified Zn (~1 × NRC), a diet fortified with a Zn AA complex (~2 × NRC), and a diet fortified with ZnSO4 (~2 × NRC). Rumen bacterial community was assessed using Illumina MiSeq of the V4 to V6 region of the 16S rRNA gene. One hundred and eleven OTUs were found with > 1% abundance across all samples. The genera Prevotella, Solobacterium, Ruminococcus, Butyrivibrio, Olsenella, Atopobium, and the candidate genus Saccharimonas were abundant in all samples. Total rumen bacterial evenness and diversity in rams were reduced by supplementation with a Zn AA complex, but not in rams supplemented with an equal concentration of ZnSO4, likely due to differences in bioavailability between organic and inorganically sourced supplement formulations. A number of bacterial genera were altered by Zn supplementation, but only the phylum Tenericutes was significantly reduced by ZnSO4 supplementation, suggesting that either Zn supplementation formulation could be utilized without causing a high-level shift in the rumen bacterial community which could have negative consequences for digestion and animal health.

The objective of this experiment, part of a larger study, was to investigate changes in rumen bacterial, archaeal, and fungal diversity in cows fed medium-chain saturated fatty acids. In the main study, 6 lactating dairy cows were dosed intraruminally with 240 g/(cow · d) of stearic (SA, control), lauric (LA), or myristic (MA) acid in a replicated 3 × 3 Latin square design trial. Experimental periods were 28 d, and cows were transfaunated between periods. Lauric acid decreased protozoal counts in the rumen by 96% compared with SA and MA (compared with SA, MA had no effect on ruminal protozoa). Whole ruminal contents samples were collected 2, 4, 6, 8, 10, 14, 18, and 24 h after the morning feeding on d 23 of each experimental period, stored frozen, and later composited by cow and period for microbial profile analyses, which involved tag-encoded flexible (FLX) amplicon pyrosequencing to provide diversity analyses of gastrointestinal bacterial, archaeal, and fungal populations of the cattle. The LA treatment, either directly or through its effect on protozoa, had a profound effect on the microbial ecology of the rumen. Ruminal populations of Prevotella, Bacteroides, and Enterorhabdus were decreased (P = 0.04 to P < 0.001) by more than 2-fold in LA treatments compared with SA, and Clostridium populations were decreased (P = 0.01) in LA- compared with MA-treated cows. The proportion of Ruminococcus was not affected by treatment, although the LA treatment had the least proportion of Ruminococcus. Proportions of Eubacterium, Butyrivibrio, Olsenella, and Lactobacillus genera were increased (P = 0.03 to 0.01) by LA compared with MA or SA. The LA treatment, possibly through its effect on protozoa physically associated with archaea, resulted in an increase (P = 0.01) in the archaeal methanogenic genus Methanosphaera and a decrease (P = 0.01) in Methanobrevibacter. Few changes in fungal populations caused by treatment were detected. Collectively, results indicate that LA, either through antiprotozoal or direct antimicrobial effects, altered bacterial and archaeal populations in the rumen of dairy cows, but effects on fungal populations were not clear.

The rumen bacterium Butyrivibrio proteoclasticus B316(T) has a 4.4-Mb genome composed of four replicons (approximately 3.55 Mb, 361, 302 and 186 kb). Mutagenesis of B316(T) was performed with the broad host-range conjugative transposon Tn916 to screen for functionally important characteristics. The insertion sites of 123 mutants containing a single copy of Tn916 were identified and corresponded to 53 different insertion points, of which 18 (34.0%), representing 39 mutants (31.7%), were in ORFs and 12 were where transposition occurred in both directions (top and bottom DNA strand). Up to eight mutants from several independent conjugation experiments were found to have the same integration site. Although transposition occurred in all four replicons, the number of specific insertion sites, transposition frequency and the average intertransposon distance between insertions varied between the four replicons. In silico analysis of the 53 insertion sites was used to model a target consensus sequence for Tn916 integration into B316(T) . A search of the B316(T) genome using the modelled target consensus sequence (up to two mismatches) identified 39 theoretical Tn916 insertion sites (19 coding, 20 noncoding), of which nine corresponded to Tn916 insertions identified in B316(T) mutants during our conjugation experiments.

Aims: To verify the taxonomic affiliation of bacterium Butyrivibrio fibrisolvens strain A from our collection and to characterize its enzyme(s) responsible for digestion of sucrose. Methods and Results: Comparison of the 16S rRNA gene of the bacterium with GenBank showed over 99% sequence identity to the species Pseudobutyrivibrio ruminis. Molecular filtration, native electrophoresis on polyacrylamide gel, zymography and thin layer chromatography were used to identify and characterize the relevant enzyme. An intracellular sucrose phosphorylase with an approximate molecular mass of 52 kDa exhibiting maximum activity at pH 6·0 and temperature 45°C was identified. The enzyme was of inducible character and catalysed the reversible conversion of sucrose to fructose and glucose-1-P. The reaction required inorganic phosphate. The Km for glucose-1-P formation and fructose release were 3·88 × 10−3 and 5·56 × 10−3 mol l−1 sucrose, respectively – while the Vmax of the reactions were −0·579 and 0·9 μmol mg protein−1 min−1. The enzyme also released free glucose from glucose phosphate. Conclusion: Pseudobutyrivibrio ruminis strain A utilized sucrose by phosphorolytic cleavage. Significance and Impact of the Study: Bacterium P. ruminis strain A probably participates in the transfer of energy from dietetary sucrose to the host animal.

Aims: All members of the ruminal Butyrivibrio group convert linoleic acid (cis-9,cis-12-18 : 2) via conjugated 18 : 2 metabolites (mainly cis-9,trans-11-18 : 2, conjugated linoleic acid) to vaccenic acid (trans-11-18 : 1), but only members of a small branch, which includes Clostridium proteoclasticum, of this heterogeneous group further reduce vaccenic acid to stearic acid (18 : 0, SA). The aims of this study were to develop a real-time polymerase chain reaction (PCR) assay that would detect and quantify these key SA producers and to use this method to detect diet-associated changes in their populations in ruminal digesta of lactating cows. Methods and Results: The use of primers targeting the 16S rRNA gene of Cl. proteoclasticum was not sufficiently specific when only binding dyes were used for detection in real-time PCR. Their sequences were too similar to some nonproducing strains. A molecular beacon probe was designed specifically to detect and quantify the 16S rRNA genes of the Cl. proteoclasticum subgroup. The probe was characterized by its melting curve and validated using five SA-producing and ten nonproducing Butyrivibrio-like strains and 13 other common ruminal bacteria. Analysis of ruminal digesta collected from dairy cows fed different proportions of starch and fibre indicated a Cl. proteoclasticum population of 2–9% of the eubacterial community. The influence of diet on numbers of these bacteria was less than variations between individual cows. Conclusions: A molecular beacon approach in qPCR enables the detection of Cl. proteoclasticum in ruminal digesta. Their numbers are highly variable between individual animals. Significance and Impact of the Study: SA producers are fundamental to the flow of polyunsaturated fatty acid and vaccenic acid from the rumen. The method described here enabled preliminary information to be obtained about the size of this population. Further application of the method to digesta samples from cows fed diets of more variable composition should enable us to understand how to control these bacteria in order to enhance the nutritional characteristics of ruminant-derived foods, including milk and beef.

Aims: To characterize the cause for the lack of conjugated linoleic acid (CLA) reductase (CLA-R) activity in the Butyrivibrio fibrisolvens MDT-5 strain that rapidly isomerizes linoleic acid (LA) to CLA without hydrogenation, the CLA-R was purified and its gene (cla-r) sequence was determined. Methods and Results: CLA-R was purified to near homogeneity as a 53-kDa monomeric protein from the high CLA-R activity-expressing strain MDT-10. The purified CLA-R recognized conjugated double bonds. Unsaturated fatty acids containing 18 carbons markedly increased the CLA-R expression at the transcriptional level. Complete sequencing of the cla-r gene revealed that the CLA-R is a novel protein. Sequence analysis of the cla-r gene from the MDT-5 strain revealed that the MDT-5 CLA-R protein sequence differed from that of the MDT-10 at four consecutive amino acids. Northern and Western blotting analyses confirmed that the cla-r mRNA and protein are expressed normally in MDT-5. Conclusions: Strain MDT-5 expresses the CLA-R protein that lacks enzyme activity because of mutation, which explains why MDT-5 exclusively produces CLA from LA. Significance and Impact of the Study: The cla-r gene was sequenced for the first time. Exogenous fatty acids affected the cla-r transcription. These results will provide additional knowledge on biohydrogenation, and may also augment the CLA production in the gastrointestinal tract.

Butyrivibrio fibrisolvens is the most active bacterial species in the biohydrogenation of polyunsaturated fatty acids (PUFA) in the rumen. It needs to remove the unsaturated bonds in order to detoxify the PUFA to enable the growth of the bacterium. Here, we investigated the response of cell membrane-associated proteins in B. fibrisolvens to growth in the presence of PUFA. Numerous changes were observed in the cell membrane-associated proteome. One of the main modifications occurring when the 18:2 fatty acids, linoleic acid and conjugated linoleic acid, were added, was an increased expression of the molecular chaperone GroEL.

The seasonal effect of first cut (FC) and regrowth (RG) timothy (Phleum pratense) silage on the rumen fluid bacterial population and the ability of this microbial ecosystem to digest cellulose was compared to that of reindeer from natural pasture (NP). FC with 27% leaves contained 30.4% dry matter (DM) cellulose and 6.2% DM water soluble carbohydrates (WSC), while RG with 89% leaves contained 18.4% DM cellulose and 30.0% DM WSC. The number of culturable anaerobic bacteria per ml rumen fluid on a habitat simulating medium (M8V) ranged between 1.1×108 and 95.0×108 in the reindeer (n=18) investigated, and no significant (P>0.05) differences were found between animals from NP and animals from the experimental groups in summer or winter. Carboxymethyl cellulose hydrolysing bacterial strains (n=138) isolated from the rumen fluid of the animals (n=18) using an acid-swollen cellulose medium were characterised as Butyrivibrio spp. (69.6%), Eubacterium spp. (1.4%), Clostridium sp. (0.7%) and unknowns (28.3%). Cellulolytic strains of B. fibrisolvens were isolated from only one of the calves, which had been fed RG in summer. The rumen bacterial composition in reindeer on NP resembled that of the experimental groups. In vitro digestibility trials demonstrates that the ability of the rumen microbiota to digest cellulose is generally reduced in reindeer fed silage in summer compared to winter. Ruminal malfunction occurred in some of the animals, but no correlation between the composition of the bacterial population in the fluid fraction of the rumen and the ability of this microbiota to ferment cellulose was found.

R.J. FORSTER, R.M. TEATHER, J. GONG AND s.-J. DENG. 1996. Complete 16S rDNA sequences of six strains of Butyrivibrio fibrisolvens, including the type strain (ATCC 19171), were determined. The type strain was found to have less than 89% sequence similarity to the other isolates that were examined. The five plasmid-bearing strains formed a closely related cluster and three of these strains (OB156, OB157 and OB192) were very highly related (> 99%), indicating that they are isolates of the same genomic species. The phylogenetic position of Butyrivibrio was found to be within the subphylum Clostridzum, of Gram-positive bacteria. The closest relatives to the type strain were Eubacterium cellulosolvens and Cl. xylanolyticum and the closest relatives to the separately clustered strains were Roseburia cecicola, Lachnospira pectinoschiza and Eubacterium rectale.

A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79, bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved. The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM beta 1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and was stably maintained in both hosts. Both ORF1 and ORF2 were required for successful transformation of B. fibrisolvens.

Screening over 100 isolates from human faeces for cellulolytic activity led to the isolation of a weakly cellulolytic anaerobic, curved, motile bacterium which produced H 2 , lactate and butyrate from wheatbran. The mol% of G + C in the DNA was 39-42. These properties, together with the Gram-positive cell wall ultrastructure and SDS-PAGE profile, are consistent with the genus Butyrivibrio. The isolate is believed to be the most active wheatbran-degrading bacterium so far described

Despite evidence supporting improved incorporation of beneficial polyunsaturated fatty acids (PUFA) into ruminant products, such as meat and milk, following red clover and fish oil (FO) inclusion in the ruminant diet, little is known regarding the concomitant bacterial diversity. We evaluated the effects of feeding grass vs. red clover silage with incremental FO inclusion on known lipolytic, biohydrogenating, cellulolytic and proteolytic rumen bacterial communities of steers. Following 14 days of dietary adaptation, liquid-associated (LAB) and solid-associated (SAB) bacterial communities were harvested, DNA extracted and bacterial denaturing gradient gel electrophoresis (DGGE) and specific-bacterial quantitative PCR (QPCR) were undertaken. DGGE-derived dendrograms showed that diet caused the greatest change in LAB and SAB bacterial diversity, with FO inclusion at the 2% and 3% dry matter intake also causing some changes. QPCR revealed that diet resulted in changes in the DNA concentration of Anaerovibrio lipolytica, the Butyrivibrio proteoclasticus group, Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. FO inclusion caused changes in A. lipolytica, F. succinogenes and R. flavefaciens DNA concentration only. In the B. proteoclasticus group, which are the only known bacteria with the capacity to biohydrogenate PUFA to 18:0, DNA concentration did not correlate to 18:0 flow to the duodenum, however, suggesting that other bacteria may play a role in biohydrogenation. A greater understanding of microbial changes that accompany beneficial dietary changes will lead to novel strategies to improve ruminant product quality.

This study investigated successional colonization of fresh perennial ryegrass (PRG) by the rumen microbiota over time. Fresh PRG was incubated in sacco in the rumens of three Holstein × Friesian cows over a period of 8 h, with samples recovered at various times. The diversity of attached bacteria was assessed using 454 pyrosequencing of 16S rRNA (cDNA). Results showed that plant epiphytic communities either decreased to low relative abundances or disappeared following rumen incubation, and that temporal colonization of the PRG by the rumen bacteria was biphasic with primary (1 and 2 h) and secondary (4-8 h) events evident with the transition period being with 2-4 h. A decrease in sequence reads pertaining to Succinivibrio spp. and increases in Pseudobutyrivibrio, Roseburia and Ruminococcus spp. (the latter all order Clostridiales) were evident during secondary colonization. Irrespective of temporal changes, the continually high abundances of Butyrivibrio, Fibrobacter, Olsenella and Prevotella suggest that they play a major role in the degradation of the plant. It is clear that a temporal understanding of the functional roles of these microbiota within the rumen is now required to unravel the role of these bacteria in the ruminal degradation of fresh PRG.

Motile, anaerobic gram-negative bacilli belonging to the genera Butyrivibrio, Succinimonas, Succinivibrio, Anaerovibrio, Wolinella, Campylobacter, Desulfovibrio, Selenomonas, and Anaerobiospirillum are being recognized in clinical specimens with increasing frequency. Over a 12.5-year period at the VA Wadsworth Medical Center, 13 clinical specimens yielded one of these organisms. Six isolates were recovered from infected wounds, five from respiratory tract specimens obtained from patients with anaerobic pleuropulmonary infection, and two from peritoneal fluid of patients with intraabdominal infection. The distribution of isolates among the genera was as follows: Wolinella, five; Selenomonas, three; unidentifiable motile gram-negative bacilli, two; Desulfovibrio vulgaris, one; Campylobacter concisus, one; and Succinimonas, one. Our experience provides the first case reports of infection involving the last two organisms mentioned. Clinical features of the infections caused by these motile anaerobes were studied, and the current medical literature on such infections was reviewed.

The menaquinone (vitamin K2) composition of a wide range of obligately anaerobic bacteria was investigated using chromatographic and physicochemical techniques. Members of the genera Bifidobacterium, Butyrivibrio, Clostridium, Fusobacterium, Lachnospira, Megasphaera, Propionispira, Ruminococcus, Selenomonas, Succinovibrio and Succinomonas were found to lack menaquinones, In contrast, all species of the genera Actinomyces, Arachnia, Bacteroides, Propionibacterium, Veillonella and Wolinella examined possessed menaquinones. Considerable variation in length and degree of saturation of the C-3 isoprenyl side-chains of the menaquinones isolated from these taxa were evident. In addition to menaquinone, strains of Wolinella and Eubacterium lentum contained methylated menaquinones. The possible contribution of bacterial menaquinones to the total pool of vitamin K in the human gut is discussed.

SUMMARY Amino Acid (AA) degradation profiles of five major genera of rumen bacteria were determined using physiological levels of AA under in vitro conditions. The results indicated that (1) not all AA are degraded by all strains of rumen bacteria and (2) degradation occurs at different rates. The genera Megaspbaera, Eubacterium and Streptococcus isolate 19D degraded all 14 AA tested. Members of the genus Butyrivibrio totally degraded Ser, Asp and Glu, with Gly being the only AA not attacked. The two subspecies of Selenomonas tested, differed in their AA degradation patterns. Subspecies lactilytica degraded all AA tested except His and Tyr. Subspecies ruminantium Could not degrade Gly, Leu, lie, Thr, His, Arg, Lys, Trp nor Tyr. Met appeared unique in that it was produced by members of Megaspbaera, Eubacterium and isolate 19D of Streptococcus. Selenomonas, Butyrivibrio and Streptococcus isolated 12D, however, degraded Met. It thus appears that the total ruminal degradation of dietary AA occurs as a result of extensive bacterial interaction.