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High-throughput RNA sequencing offers broad opportunities to explore the Earth RNA virome. Mining 5,150 diverse metatranscriptomes uncovered >2.5 million RNA virus contigs. Analysis of >330,000 RNA-dependent RNA polymerases (RdRPs) shows that this expansion corresponds to a 5-fold increase of the known RNA virus diversity. Gene content analysis revealed multiple protein domains previously not found in RNA viruses and implicated in virus-host interactions. Extended RdRP phylogeny supports the monophyly of the five established phyla and reveals two putative additional bacteriophage phyla and numerous putative additional classes and orders. The dramatically expanded phylum Lenarviricota, consisting of bacterial and related eukaryotic viruses, now accounts for a third of the RNA virome. Identification of CRISPR spacer matches and bacteriolytic proteins suggests that subsets of picobirnaviruses and partitiviruses, previously associated with eukaryotes, infect prokaryotic hosts.

Changes in the sequence of an organism's genome, i.e., mutations, are the raw material of evolution. The frequency and location of mutations can be constrained by specific molecular mechanisms, such as diversity-generating retroelements (DGRs). DGRs have been characterized from cultivated bacteria and bacteriophages, and perform error-prone reverse transcription leading to mutations being introduced in specific target genes. DGR loci were also identified in several metagenomes, but the ecological roles and evolutionary drivers of these DGRs remain poorly understood. Here, we analyze a dataset of >30,000 DGRs from public metagenomes, establish six major lineages of DGRs including three primarily encoded by phages and seemingly used to diversify host attachment proteins, and demonstrate that DGRs are broadly active and responsible for >10% of all amino acid changes in some organisms. Overall, these results highlight the constraints under which DGRs evolve, and elucidate several distinct roles these elements play in natural communities.

This study introduces a novel approach to analyze glycosidic linkages in unfractionated polysaccharides from alcohol-insoluble residues (AIRs) of five brown seaweed species. GC-MS analysis of partially methylated alditol acetates (PMAAs) enables monitoring and comparison of structural variations across different species, harvest years, and tissues with and without blanching treatments. The method detects a wide array of fucose linkages, highlighting the structural diversity in glycosidic linkages and sulfation position in fucose-containing sulfated polysaccharides. Additionally, this technique enhances cellulose quantitation, overcoming the limitations of traditional monosaccharide composition analysis that typically underestimates cellulose abundance due to incomplete hydrolysis of crystalline cellulose. The introduction of a weak methanolysis-sodium borodeuteride reduction pretreatment allows for the detection and quantitation of uronic acid linkages in alginates. [ABSTRACT FROM AUTHOR]

The rumen is a complex ecosystem composed of anaerobic bacteria, protozoa, fungi, methanogenic archaea and phages. These microbes interact closely to breakdown plant material that cannot be digested by humans, whilst providing metabolic energy to the host and, in the case of archaea, producing methane. Consequently, ruminants produce meat and milk, which are rich in high-quality protein, vitamins and minerals, and therefore contribute to food security. As the world population is predicted to reach approximately 9.7 billion by 2050, an increase in ruminant production to satisfy global protein demand is necessary, despite limited land availability, and whilst ensuring environmental impact is minimized. Although challenging, these goals can be met, but depend on our understanding of the rumen microbiome. Attempts to manipulate the rumen microbiome to benefit global agricultural challenges have been ongoing for decades with limited success, mostly due to the lack of a detailed understanding of this microbiome and our limited ability to culture most of these microbes outside the rumen. The potential to manipulate the rumen microbiome and meet global livestock challenges through animal breeding and introduction of dietary interventions during early life have recently emerged as promising new technologies. Our inability to phenotype ruminants in a high-throughput manner has also hampered progress, although the recent increase in "omic" data may allow further development of mathematical models and rumen microbial gene biomarkers as proxies. Advances in computational tools, high-throughput sequencing technologies and cultivation-independent "omics" approaches continue to revolutionize our understanding of the rumen microbiome. This will ultimately provide the knowledge framework needed to solve current and future ruminant livestock challenges.

Glycosidic linkage analysis was conducted on the unfractionated polysaccharides in alcohol-insoluble residues (AIRs) prepared from six red seaweeds (Gracilariopsis sp., Prionitis sp., Mastocarpus papillatus, Callophyllis sp., Mazzaella splendens, and Palmaria palmata) using GC-MS/FID analysis of partially methylated alditol acetates (PMAAs). The cell walls of P. palmata primarily contained mixed-linkage xylans and small amounts of sulfated galactans and cellulose. In contrast, the unfractionated polysaccharides of the other five species were rich in galactans displaying diverse 3,6-anhydro-galactose and galactose linkages with varied sulfation patterns. Different levels of cellulose were also observed. This glycosidic linkage method offers advantages for cellulose analysis over traditional monosaccharide analysis that is known for underrepresenting glucose in crystalline cellulose. Relative linkage compositions calculated from GC-MS and GC-FID measurements showed that anhydro sugar linkages generated more responses in the latter detection method. This improved linkage workflow presents a useful tool for studying polysaccharide structural variations across red seaweed species. Furthermore, for the first time, relative linkage compositions from GC-MS and GC-FID measurements, along with normalized FID and total ion current (TIC) chromatograms without peak assignments, were analyzed using principal component analysis (PCA) as a proof-of-concept demonstration of the technique's potential to differentiate various red seaweed species. [ABSTRACT FROM AUTHOR]

Seaweeds have received a great deal of attention recently for their potential as methane-suppressing feed additives in ruminants. To date, Asparagopsis taxiformis has proven a potent enteric methane inhibitor, but it is a priority to identify local seaweed varieties that hold similar properties. It is essential that any methane inhibitor does not compromise the function of the rumen microbiome. In this study, we conducted an in vitro experiment using the RUSITEC system to evaluate the impact of three red seaweeds, A. taxiformis, Palmaria mollis, and Mazzaella japonica, on rumen prokaryotic communities. 16S rRNA sequencing showed that A. taxiformis had a profound effect on the microbiome, particularly on methanogens. Weighted Unifrac distances showed significant separation of A. taxiformis samples from the control and other seaweeds (p < 0.05). Neither P. mollis nor M. japonica had a substantial effect on the microbiome (p > 0.05). A. taxiformis reduced the abundance of all major archaeal species (p < 0.05), leading to an almost total disappearance of the methanogens. Prominent fiberdegrading and volatile fatty acid (VFA)-producing bacteria including Fibrobacter and Ruminococcus were also inhibited by A. taxiformis (p < 0.05), as were other genera involved in propionate production. The relative abundance of several other bacteria including Prevotella, Bifidobacterium, Succinivibrio, Ruminobacter, and unclassified Lachnospiraceae were increased by A. taxiformis suggesting that the rumen microbiome adapted to an initial perturbation. Our study provides baseline knowledge of microbial dynamics in response to seaweed feeding over an extended period and suggests that feeding A. taxiformis to cattle to reduce methane may directly, or indirectly, inhibit important fiber-degrading and VFA-producing bacteria. [ABSTRACT FROM AUTHOR]

Liver abscesses (LA) pose a significant challenge to the Canadian beef industry as they are estimated to cost the industry ~ $61.2 million annually. This is likely an underestimate as it does not account for losses in animal productivity. Tylosin phosphate is widely used to reduce LA, but concerns over antimicrobial use selecting for antimicrobial resistance has created an urgency to explore alternative approaches. Understanding the impact of tylosin on the microbial ecology of LA is crucial to this. We hypothesized that altering the duration of in-feed administration of tylosin to feedlot cattle would alter the microbial community of LA. To investigate this, we collected abscessed livers from cattle fed a diet containing tylosin 1) throughout finishing, 2) during the first 78% of the feeding or 3) during the last 75% of the feeding period. We examined LA microbial ecology of purulent material, abscess capsule tissue, originating from abscesses of different sizes using a metataxonomic approach. Our findings revealed that shortening tylosin administration did not notably alter the alpha (P > 0.05) or beta-diversity (P > 0.05) of LA microbial communities. There was a significant difference in microbial richness associated with abscess capsule (P < 0.05) compared with bulk purulent material. Fusobacterium or Bacteroides ASVs dominated LA microbiomes, alongside probable opportunistic gut pathogens and other bacteria. Interestingly, classifying samples based on whether they originated from a liver with a single abscess, multiple abscesses or a very large abscess tended to differ in microbiome composition (P = 0.06). These insights contribute to our understanding of factors impacting liver abscess microbial ecology and will be valuable in identifying antibiotic alternatives. They underscore the importance of exploring varied approaches to address liver abscesses while reducing reliance on in-feed antibiotics. [ABSTRACT FROM AUTHOR]

As the majority of energy and protein supplied to cattle arises as a result of ruminal fermentation, the rumen microbiome has an integral role in determining host feed efficiency. Counterintuitively, current evidence suggests that a less diverse rumen microbiome is associated with improved feed efficiency, possibly as a result of greater metabolic precision and avoidance of energy spilling fermentative pathways. The composition of the rumen microbiome is mainly determined by diet, but host traits such as rumen volume, rate of passage, rumination and immunity also have influence. Although less microbial diversity may improve feed efficiency in cattle fed a specific diet, reduced diversity may impair the ability of cattle to adapt to frequent changes in diet and the environment. Hydrogen exchange and capture is the energetic foundation of the rumen microbiome and considerable capital has been invested to develop additives that redirect hydrogen flow away from the reduction of CO2 to CH4 towards alternative sinks. These additives have been shown to reduce enteric CH4 emissions by 30 to 80%, but improvements in feed efficiency have been less than stoichiometric predictions. Approaches to improve the feed efficiency of cattle need to be multifaceted with consideration for host genetics, functional efficiency of the rumen microbiome, and the structure and composition of feed. Likewise, reductions in carbon emissions need to be broader than just CH4, with an appreciation of the role that cattle have within a circular bioeconomy to promote upcycling of nutrients and reductions in emissions from farming systems. Strategies to improve the efficiency of cattle production are a prerequisite for the sustainable intensification needed to ensure that the social license for milk and meat production from cattle is retained. [ABSTRACT FROM AUTHOR]

Ruminants are unique amongst livestock, having the ability to convert low cost and low-quality feedstuffs that cannot be consumed by humans, or non-ruminant animals, into high quality protein. Unfortunately, the low digestibility of these feeds also promotes reduced intake and performance, which limits their inclusion in ruminant diets. Variation in the ability of cattle to digest feed has also been considered one of the main factors affecting feed efficiency. In an extensive grazing system, greater feed intake and digestibility of forages are directly related to the performance of cattle fed. Development of technologies to enhance feed intake and digestibility of forage-based diets within the rumen is essential to increase their utilization in ruminant diets. Data will be presented from past, and current, efforts to understand inter-animal variability in feed digestibility, the linkages between the rumen microbiome and feed digestion efficiency, and to develop enzymes, and chemical pretreatment technologies that will enhance the digestion of lignocellulose in the rumen. [ABSTRACT FROM AUTHOR]

This study evaluated beef heifers selected for high (efficient) or low (inefficient) digestible fiber intake (DFI). Initial analysis showed that high DFI animals had reduced methane production versus lowDFI under a high forage diet. Using the same cohort of animals maintained on a further 4 diets of varying forage:concentrate ratios, we employed multi-kingdom amplicon sequencing and metagenome shotgun sequencing of rumen digesta and feces alongside RNA sequencing of rumen epimural samples to evaluate the compositional and functional interplay between different microbial groups, and their relationship with host gene expression in cattle divergent for DFI. Samples were collected from 16 cattle during 2 metabolism trials, comprising 5 diets. Amplicon sequencing analysis was conducted using QIIME2; 16S rRNA and 18S rRNA reads were analyzed using the SILVA database, while LSU (fungal) sequences were analyzed using a custom D1/D2 database. Additional analysis of archaeal 16S rRNA sequences was conducted using the Rumen and Intestinal Methanogens (RIM) database. Metagenome shotgun reads underwent a two-pass classification with Kraken2 using a database of prokaryotic genomes derived from the GTDB taxonomy, with the unclassified output undergoing classification using a custom database containing all NCBI protozoa, fungi, and phage genomes, enriched with selected rumen-specific ciliate and fungal genomes. Downstream analysis of taxonomic data from all microbiome work was conducted in R, and differentially abundant taxa were identified using ANCOM-BC and Aldex2. Functional analysis of metagenome contigs using the CAZY database implemented in dbCAN3 is ongoing. RNA-seq data were analyzed using the ARS-UCD reference genome, with identification of DE genes conducted using DeSeq2. Preliminary results indicate no major effect of DFI ranking on host gene expression, bacterial 16S rRNA, or metagenome compositional profiles. Several bacterial genera were differentially abundant between digestibility groups (P < 0.05), but these were all minor (< 0.01%) members of the microbiome. Fungal and methanogen communities different significantly (P < 0.05) according to DFI group, with efficient (high DFI) containing and more diverse communities under highgrain diet (P < 0.05). The same difference showed a tendency toward significance for the 18S rRNA protozoa data (P < 0.1). These preliminary data indicate that the microbial factors underpinning divergence in efficiency measured by DFI vary according to diet and may be more prominent in the non-bacterial fraction of the microbiome. Ongoing functional analysis of metagenome data as well as integration of multiomic data will provide deeper insight into these relationships and how they contribute to feed digestibility and efficiency in cattle. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the factors which influence intake and fiber digestion efficiency of beef cattle previously selected for high or low digestible neutral detergent fiber (NDF) intake (DFI; g/kg BW0.75). Angus × Hereford cross heifers [n = 16; initial body weight (BW) = 584 ± 32.2 kg] previously classified as either high or low DFI when fed a high-forage diet (n = 8/treatment) were individually housed and fed one of 4 diets for ad libitum intake using a sequential offering of diets over four consecutive 28-d period. Diets included: 1) 100% grass hay; 2) 100% mature alfalfa hay; 3) 90% dry rolled barley + 10% alfalfa hay; and 4) 90% dry rolled barley + 10% grass hay (DM basis). Individual animal feed intake was recorded daily in addition to weekly BW. Rumen digesta samples were collected prior to feeding and 6 h after for 2 d within each period and analyzed for SCFA and NH3 -N concentrations, and total rumen protozoa counts and identification. High DFI heifers had similar (P > 0.11) DMI compared with low DFI heifers in periods 1, 3, and 4, but had a tendency for greater DMI in period 2 and NDF intake during period 2 and 3 (7.84 vs. 7.07 kg, P = 0.07; 4.71 vs. 4.27 kg, P = 0.08; 2.56 vs. 2.28 kg, P = 0.10). ADG was greater for high DFI during the first two periods (0.61 vs. 0.13, P = 0.04; 0.44 vs. -0.09, P = 0.02); moreover, high DFI cattle finished the study with a heavier BW (708 vs. 661 kg, P = 0.04). Total SCFA concentrations before and 6 h after feeding did not differ (P ≥ 0.36) between treatments. NH3 -N concentrations were not different before feeding during period 1, 3, and 4, but were greater for high DFI heifers during period 2 (9.85 vs. 8.39, P = 0.04). Additionally, there were no differences in NH3 -N concentrations 6 h after feeding during any period. No differences were observed (P > 0.25) between treatment groups for total rumen protozoa counts for periods 1 and 2; however, there was a tendency for a greater proportion of Isotrichia before feeding during period 1 (3.67 vs. 2.41%, P = 0.07). In conclusion, differences in intake and the small changes in ruminal fermentation parameters only partially explain the differences in ADG observed between high and low DFI heifers. [ABSTRACT FROM AUTHOR]

Abstract Background Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the cellulosic biofuel industry. The rumen is a highly adapted environment for the degradation of cellulose and a promising source of enzymes for industrial use. To identify cellulase enzymes that may be of such use we have undertaken a functional metagenomic screen to identify cellulase enzymes from the bacterial community in the rumen of a grass-hay fed dairy cow. Results Twenty five clones specifying cellulose activity were identified. Subcloning and sequence analysis of a subset of these hydrolase-positive clones identified 10 endoglucanase genes. Preliminary characterization of the encoded cellulases was carried out using crude extracts of each of the subclones. Zymogram analysis using carboxymethylcellulose as a substrate showed a single positive band for each subclone, confirming that only one functional cellulase gene was present in each. One cellulase gene, designated Cel14b22, was expressed at a high level in Escherichia coli and purified for further characterization. The purified recombinant enzyme showed optimal activity at pH 6.0 and 50°C. It was stable over a broad pH range, from pH 4.0 to 10.0. The activity was significantly enhanced by Mn2+ and dramatically reduced by Fe3+ or Cu2+. The enzyme hydrolyzed a wide range of beta-1,3-, and beta-1,4-linked polysaccharides, with varying activities. Activities toward microcrystalline cellulose and filter paper were relatively high, while the highest activity was toward Oat Gum. Conclusion The present study shows that a functional metagenomic approach can be used to isolate previously uncharacterized cellulases from the rumen environment.

This study evaluated beef heifers selected for high or low digestible fiber intake (DFI) and investigated its relationship with methane production, residual feed intake (RFI), and total tract digestibility. Sixteen recently weaned black Angus beef heifers (n = 8/treatment) were selected from a group 64 heifers (224 ± 17.2 kg) fed a high forage-based diet [70% barley silage:30% pelleted concentrate (DM basis)]. The 64 heifers were fed in 6 outdoor pens equipped with GrowSafe bunks to monitor feed intake for 60 d (14 d adaptation + 46 d for data collection). Individual fecal samples and BW, and feed samples were taken once weekly. Fecal samples were pooled by animal. Feed and fecal samples were analyzed for dry matter (DM), neutral detergent fibre (NDF), and undigested neutral detergent fiber (uNDF) content. The internal marker uNDF was used to estimate total tract diet DM and NDF digestibility. The 8 heifers with the greatest and the 8 with the least digestible NDF intake (g/kg BW0.75) were selected and used for methane measurements using the GreenFeed system (42 d) and in a total-tract digestibility trial with total fecal and urine collection using the same high forage based diet. Results from the GrowSafe selection trial indicated no differences between the average BW of low and high DFI heifers (264 vs. 274 kg, P = 0.20). Heifers selected for high DFI had greater (P < 0.01) DM and NDF intake (kg/d or % of BW), and digestibility (49.6 vs. 42.1% NDF digestibility). The heifer groups differed in RFI (P < 0.01), with high DFI categorized as inefficient (+0.84 RFI) and low DFI as efficient (-0.34 RFI). No differences in ADG were observed between low and high DFI heifers during this short 46 d study period (0.614 vs 0.773, P = 0.16). High DFI heifers had lower methane production than low DFI heifers (15.9 vs. 19.0 g/kg of DMI, P = 0.02). The results of the total-tract digestibility trial showed that the high DFI heifers had a greater DMI compared with the low DFI heifers (10.9 vs. 10.2 kg/d; P = 0.04). The DMI intake was not different between groups when expressed as a % of BW (2.06 vs 2.04, P = 0.65). There was also no difference observed for DM digestibility (73.0 vs 73.1%) between the two groups. It is important to note that in the digestibility study, the DMI was less than in the GrowSafe selection trial (2.5 vs 3.2% BW) and this may have influenced results. Heifers with high DFI were heavier than low DFI heifers (533 vs. 505, P = 0.02) during the digestibility trial. Results suggest that selecting heifers for high DFI may increase growth rate and reduce methane production. [ABSTRACT FROM AUTHOR]