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Anaerobic gut fungi (AGF) were the last phylum to be identified within the rumen microbiome and account for 7-9% of microbial biomass. They produce potent lignocellulases that degrade recalcitrant plant cell walls, and rhizoids that can penetrate the cuticle of plant cells, exposing internal components to other microbiota. Interspecies H 2 transfer between AGF and rumen methanogenic archaea is an essential metabolic process in the rumen that occurs during the reduction of CO 2 to CH 4 by methanogens. This symbiotic relationship is bolstered by hydrogensomes, fungal organelles that generate H 2 and formate. Interspecies H 2 transfer prevents the accumulation of reducing equivalents that would otherwise impede fermentation. The extent to which hydrogenosomes serve as a conduit for H 2 flow to methanogens is unknown, but it is likely greater with low quality forages. Strategies that alter the production of CH 4 could also have implications for H 2 transfer by anaerobic fungi. Understanding the factors that drive these interactions and H 2 flow could provide insight into the effect of reducing CH 4 production on the activity of ruminal fungi and the digestion of low-quality feeds., (© 2025, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

High-grain diets promote polymicrobial liver infections in cattle, commonly involving the bacterium Trueperella pyogenes . We have isolated T. pyogenes from the purulent material of abscesses and sequenced their genomes. These data enhance our understanding of the mechanisms underlying liver abscess development in cattle., Competing Interests: The authors declare no conflict of interest.

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.

Glucuronoyl esterases (GEs) are carbohydrate active enzymes in carbohydrate esterase family 15 which are involved in the hydrolysis of lignin-carbohydrate complexes. They are encoded by a wide range of aerobic and anaerobic fungi and bacteria inhabiting diverse environments. The rumen microbiome is a complex microbial community with a wide array of enzymes that specialize in deconstructing plant cell wall carbohydrates. Enzymes from the rumen tend to show low similarity to homologues found in other environments, making the rumen microbiome a promising source for the discovery of novel enzymes. Using a combination of phylogenetic and structural analysis, we investigated the structure-function relationship of GEs from the rumen bacteria Fibrobacter succinogenes and Ruminococcus flavefaciens, and from the rumen fungus, Piromyces rhizinflata. All adopt a canonical α/β hydrolase fold and possess a structurally conserved Ser-His-Glu/Asp catalytic triad. Structural variations in the enzymes are localized to loops surrounding the active site. Analysis of the active site structures in these enzymes emphasized the importance of structural plasticity in GEs with non-canonical active site conformations. We hypothesize that interkingdom HGT events may have contributed to the diversity of GEs in the rumen, and this is demonstrated by the phylogenetic and structural similarity observed between rumen bacterial and fungal GEs. This study advances our understanding of the structure-function relationship in glucuronoyl esterases and illuminates the evolutionary dynamics that contribute to enzyme diversity in the rumen microbiome., (© 2024. His Majesty the King in Right of Canada as represented by the Minister of Agriculture and Agri-Food, and Liam Worrel.)

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.

Liver abscesses (LA) resulting from bacterial infection in cattle pose a significant global challenge to the beef and dairy industries. Economic losses from liver discounts at slaughter and reduced animal performance drive the need for effective mitigation strategies. Tylosin phosphate supplementation is widely used to reduce LA occurrence, but concerns over antimicrobial overuse emphasize the urgency to explore alternative approaches. Understanding the microbial ecology of LA is crucial to this, and we hypothesized that a reduced timeframe of tylosin delivery would alter LA microbiomes. We conducted 16S rRNA sequencing to assess severe liver abscess bacteriomes in beef cattle supplemented with in-feed tylosin. Our findings revealed that shortening tylosin supplementation did not notably alter microbial communities. Additionally, our findings highlighted the significance of sample processing methods, showing differing communities in bulk purulent material and the capsule-adhered material. Fusobacterium or Bacteroides ASVs dominated LA, alongside probable opportunistic gut pathogens and other microbes. Moreover, we suggest that liver abscess size correlates with microbial community composition. 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 LA while reducing reliance on in-feed antibiotics., (© His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada, 2024.)

Although circular RNAs (circRNAs) play important roles in regulating gene expression, the understanding of circRNAs in livestock animals is scarce due to the significant challenge to characterize them from a biological sample. In this study, we assessed the outcomes of bovine circRNA identification using six enrichment approaches with the combination of ribosomal RNAs removal ( Ribo ); linear RNAs degradation ( R ); linear RNAs and RNAs with structured 3' ends degradation ( RTP ); ribosomal RNAs coupled with linear RNAs elimination ( Ribo-R ); ribosomal RNA, linear RNAs and RNAs with poly (A) tailing elimination ( Ribo-RP ); and ribosomal RNA, linear RNAs and RNAs with structured 3' ends elimination ( Ribo-RTP ), respectively. RNA-sequencing analysis revealed that different approaches led to varied ratio of uniquely mapped reads, false-positive rate of identifying circRNAs, and the number of circRNAs per million clean reads ( P adj <0.05). Out of 2,285 and 2,939 highly confident circRNAs identified in liver and rumen tissues, respectively, 308 and 260 were commonly identified from five methods, with Ribo-RTP method identified the highest number of circRNAs. Besides, 507 of 4,051 identified bovine highly confident circRNAs had shared splicing sites with human circRNAs. The findings from this work provide optimized methods to identify bovine circRNAs from cattle tissues for downstream research of their biological roles in cattle.

Several red seaweeds have been shown to inhibit enteric CH4 production; however, the adaptation of fermentation parameters to their presence is not well understood. The objective of this study was to examine the effect of three red seaweeds ( Asparargopsis taxiformis , Mazzaella japonica , and Palmaria mollis ) on in vitro fermentation, CH4 production, and adaptation using the rumen simulation technique (RUSITEC). The experiment was conducted as a completely randomized design with four treatments, duplicated in two identical RUSITEC apparatus equipped with eight fermenter vessels each. The four treatments included the control and the three red seaweeds added to the control diet at 2% diet DM. The experimental period was divided into four phases including a baseline phase (d 0-7; no seaweed included), an adaptation phase (d 8-11; seaweed included in treatment vessels), an intermediate phase (d 12-16), and a stable phase (d 17-21). The degradability of organic matter ( p = 0.04) and neutral detergent fibre ( p = 0.05) was decreased by A. taxiformis during the adaptation phase, but returned to control levels in the stable phase. A. taxiformis supplementation resulted in a decrease ( p < 0.001) in the molar proportions of acetate, propionate, and total volatile fatty acid (VFA) production, with an increase in the molar proportions of butyrate, caproate, and valerate; the other seaweeds had no effect ( p > 0.05) on the molar proportions or production of individual VFA. A. taxiformis was the only seaweed to suppress CH4 production ( p < 0.001), with the suppressive effect increasing ( p < 0.001) across phases. Similarly, A. taxiformis increased ( p < 0.001) the production of hydrogen (H2, %, mL/d) across the adaptation, intermediate, and stable phases, with the intermediate and stable phases having greater H2 production than the adaptation phase. In conclusion, M. japonica and P. mollis did not impact rumen fermentation or inhibit CH4 production within the RUSITEC. In contrast, we conclude that A. taxiformis is an effective CH4 inhibitor and its introduction to the ruminal environment requires a period of adaptation; however, the large magnitude of CH4 suppression by A. taxiformis inhibits VFA synthesis, which may restrict the production performance in vivo.

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 fiber-degrading 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 His Majesty the King in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada for the contribution of O’Hara, Moote, Terry, Beauchemin, McAllister, Abbott, Gruninger.)

Supplementation of ruminant diets with the methane (CH4) inhibitor 3-nitrooxypropanol (3-NOP; DSM Nutritional Products, Switzerland) is a promising greenhouse gas mitigation strategy. However, most studies have used high grain or mixed forage-concentrate diets. The objective of this study was to evaluate the effects of supplementing a high-forage diet (90% forage DM basis) with 3-NOP on dry matter (DM) intake, rumen fermentation and microbial community, salivary secretion, enteric gas emissions, and apparent total-tract nutrient digestibility. Eight ruminally cannulated beef heifers (average initial body weight (BW) ± SD, 515 ± 40.5 kg) were randomly allocated to two treatments in a crossover design with 49-d periods. Dietary treatments were: 1) control (no 3-NOP supplementation); and 2) 3-NOP (control + 150 mg 3-NOP/kg DM). After a 16-d diet adaption, DM intake was recorded daily. Rumen contents were collected on days 17 and 28 for volatile fatty acid (VFA) analysis, whereas ruminal pH was continuously monitored from days 20 to 28. Eating and resting saliva production were measured on days 20 and 31, respectively. Diet digestibility was measured on days 38-42 by the total collection of feces, while enteric gas emissions were measured in chambers on days 46-49. Data were analyzed using the mixed procedure of SAS. Dry matter intake and apparent total-tract digestibility of nutrients (DM, neutral and acid detergent fiber, starch, and crude protein) were similar between treatments (P ≥ 0.15). No effect was observed on eating and resting saliva production. Relative abundance of the predominant bacterial taxa and rumen methanogen community was not affected by 3-NOP supplementation but rather by rumen digesta phase and sampling hour (P ≤ 0.01). Total VFA concentration was lower (P = 0.004) following 3-NOP supplementation. Furthermore, the reduction in acetate and increase in propionate molar proportions for 3-NOP lowered (P < 0.001) the acetate to propionate ratio by 18.9% as compared with control (4.1). Mean pH was 0.21 units lower (P < 0.001) for control than 3-NOP (6.43). Furthermore, CH4 emission (g/d) and yield (g/kg DMI) were 22.4 and 22.0% smaller (P < 0.001), respectively, for 3-NOP relative to control. Overall, the results indicate that enteric CH4 emissions were decreased by more than 20% with 3-NOP supplementation of a forage diet without affecting DM intake, predominant rumen microbial community, and apparent total-tract nutrients digestibility., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Lignocellulosic biomass represents an abundant, renewable resource that can be used to produce biofuels, low-cost livestock feed, and high-value chemicals. The potential of this bioresource has led to intensive research efforts to develop cost-effective methods to break down lignocellulose. The efficiency with which the anaerobic fungi (phylum Neocallimastigomycota) degrade plant biomass is well recognized and in recent years has received renewed interest. Transcriptomics has been used to identify enzymes that are expressed by these fungi and are involved in the degradation of a range of lignocellulose feedstocks. The transcriptome is the entire complement of coding and non-coding RNA transcripts that are expressed by a cell under a particular set of conditions. Monitoring changes in gene expression can provide fundamental information about the biology of an organism. Here we outline a general methodology that will enable researchers to conduct comparative transcriptomic studies with the goal of identifying enzymes involved in the degradation of the plant cell wall. The method described will include growth of fungal cultures, isolation and sequencing of RNA, and a basic description of data analysis for bioinformatic identification of differentially expressed transcripts., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Fungi utilize a unique mechanism of nutrient acquisition involving extracellular digestion. To understand the biology of these microbes, it is important to identify and characterize the function of proteins that are secreted and involved in nutrient acquisition. Mass spectrometry-based proteomics is a powerful tool to study complex mixtures of proteins and understand how the proteins produced by an organism change in response to different conditions. Many fungi are efficient decomposers of plant cell walls, and anaerobic fungi are well recognized for their ability to digest lignocellulose. Here we outline a protocol for the enrichment and isolation of proteins secreted by anaerobic fungi after growth on simple (glucose) and complex (straw and alfalfa hay) carbon sources. We provide detailed instruction on generating protein fragments and preparing these for proteomic analysis using reversed-phase chromatography and mass spectrometry. The interpretation of results and their relevance to a particular biological system is study-dependent and beyond the scope of this protocol., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The rumen simulation technique (RUSITEC) was used to investigate the effect of ergot alkaloids (EA) and a mycotoxin deactivating product (Biomin AA; MDP) on nutrient digestion, ruminal fermentation parameters, total gas, methane, and microbial nitrogen production. Ruminal fermentation vessels received a feedlot finishing diet of 90:10 concentrate:barley silage (DM basis). Using a randomized complete block design, treatments were assigned (n = 4 vessels/treatment) within two RUSITEC apparatuses in a 2 × 2 factorial arrangement. Treatments included: (1) control (CON) diet (no EA and no MDP); (2) CON diet + 1 g/d MDP; (3) CON diet + 20 mg/kg EA; and (4) CON diet + 20 mg/kg EA + 1 g/d MDP. The study was conducted over 14 d with 7 d of adaptation and 7 d of sample collection. Data were analyzed in SAS using PROC MIXED including fixed effects of EA, MDP, and the EA×MDP interaction. Random effects included RUSITEC apparatus and cow rumen inoculum (n = 4). Ergot alkaloids decreased dry matter (DMD) (P = 0.01; 87.9 vs. 87.2%) and organic matter disappearance (OMD) (P = 0.02; 88.8 vs. 88.4%). Inclusion of MDP increased OMD (P = 0.01; 88.3 vs. 88.9%). Neutral detergent fiber disappearance (NDFD) was improved with MDP; however, an EA×MDP interaction was observed with MDP increasing (P < 0.001) NDFD more with EA diet compared to CON. Acetate proportion decreased (P = 0.01) and isovalerate increased (P = 0.03) with EA. Consequently, acetate:propionate was reduced (P = 0.03) with EA. Inclusion of MDP increased total volatile fatty acid (VFA) production (P < 0.001), and proportions of acetate (P = 0.03) and propionate (P = 0.03), and decreased valerate (P < 0.001), isovalerate (P = 0.04), and caproate (P = 0.002). Treatments did not affect (P ≥ 0.17) ammonia, total gas, or methane production (mg/d or mg/g of organic matter fermented). The inclusion of MDP reduced (P < 0.001) microbial nitrogen (MN) production in the effluent and increased (P = 0.01) feed particle-bound MN. Consequently, total MN decreased (P = 0.001) with MDP. In all treatments, the dominant microbial phyla were Firmicutes, Bacteroidota, and Proteobacteria, and the major microbial genus was Prevotella. Inclusion of MDP further increased the abundance of Bacteroidota (P = 0.04) as it increased both Prevotella (P = 0.04) and Prevotellaceae&#95;UCG-003 (P = 0.001). In conclusion, EA reduced OMD and acetate production due to impaired rumen function, these responses were successfully reversed by the addition of MDP., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: The major greenhouse gas from ruminants is enteric methane (CH 4 ) which in 2010, was estimated at 2.1 Gt of CO 2 equivalent, accounting for 4.3% of global anthropogenic greenhouse gas emissions. There are extensive efforts being made around the world to develop CH 4 mitigating inhibitors that specifically target rumen methanogens with the ultimate goal of reducing the environmental footprint of ruminant livestock production. This study examined the individual and combined effects of supplementing a high-forage diet (90% barley silage) fed to beef cattle with the investigational CH 4 inhibitor 3-nitrooxypropanol (3-NOP) and canola oil (OIL) on the rumen microbial community in relation to enteric CH 4 emissions and ruminal fermentation., Results: 3-NOP and OIL individually reduced enteric CH 4 yield (g/kg dry matter intake) by 28.2% and 24.0%, respectively, and the effects were additive when used in combination (51.3% reduction). 3-NOP increased H 2 emissions 37-fold, while co-administering 3-NOP and OIL increased H 2 in the rumen 20-fold relative to the control diet. The inclusion of 3-NOP or OIL significantly reduced the diversity of the rumen microbiome. 3-NOP resulted in targeted changes in the microbiome decreasing the relative abundance of Methanobrevibacter and increasing the relative abundance of Bacteroidetes. The inclusion of OIL resulted in substantial changes to the microbial community that were associated with changes in ruminal volatile fatty acid concentration and gas production. OIL significantly reduced the abundance of protozoa and fiber-degrading microbes in the rumen but it did not selectively alter the abundance of rumen methanogens., Conclusions: Our data provide a mechanistic understanding of CH 4 inhibition by 3-NOP and OIL when offered alone and in combination to cattle fed a high forage diet. 3-NOP specifically targeted rumen methanogens and partly inhibited the hydrogenotrophic methanogenesis pathway, which increased H 2 emissions and propionate molar proportion in rumen fluid. In contrast, OIL caused substantial changes in the rumen microbial community by indiscriminately altering the abundance of a range of rumen microbes, reducing the abundance of fibrolytic bacteria and protozoa, resulting in altered rumen fermentation. Importantly, our data suggest that co-administering CH 4 inhibitors with distinct mechanisms of action can both enhance CH 4 inhibition and provide alternative sinks to prevent excessive accumulation of ruminal H 2 ., (© 2022. Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food.)

Background: Antibiotic-resistant Acinetobacter species are a growing public health threat, yet are not nationally notifiable, and most states do not mandate reporting. Additionally, there are no standardized methods to detect Acinetobacter species colonization., Methods: An outbreak of carbapenem-resistant Acinetobacter baumannii (CRAB) was identified at a Utah ventilator unit in a skilled nursing facility. An investigation was conducted to identify transmission modes in order to control spread of CRAB. Culture-based methods were used to identify patient colonization and environmental contamination in the facility., Results: Of the 47 patients screened, OXA-23-producing CRAB were detected in 10 patients (21%), with 7 patients (15%) having been transferred from out-of-state facilities. Of patients who screened positive, 60% did not exhibit any signs or symptoms of active infection by chart review. A total of 38 environmental samples were collected and CRAB was recovered from 37% of those samples. Whole genome sequencing analyses of patient and environmental isolates suggested repeated CRAB introduction into the facility and highlighted the role of shared equipment in transmission., Conclusions: The investigation demonstrated this ventilated skilled nursing facility was an important reservoir for CRAB in the community and highlights the need for improved surveillance, strengthened infection control and inter-facility communication within and across states., (Copyright © 2020. Published by Elsevier Inc.)

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.

The individual and combined effects of 3-nitrooxypropanol (3-NOP) and canola oil (OIL) supplementation on enteric methane (CH4) and hydrogen (H2) emissions, rumen fermentation and biohydrogenation, and total tract nutrient digestibility were investigated in beef cattle. Eight beef heifers (mean body weight ± SD, 732 ± 43 kg) with ruminal fistulas were used in a replicated 4 × 4 Latin square with a 2 (with and without 3-NOP) × 2 (with and without OIL) arrangement of treatments and 28-d periods (13 d adaption and 15 d measurements). The four treatments were: control (no 3-NOP, no OIL), 3-NOP (200 mg/kg dry matter [DM]), OIL (50 g/kg DM), and 3-NOP (200 mg/kg DM) plus OIL (50 g/kg DM). Animals were fed restrictively (7.6 kg DM/d) a basal diet of 900 g/kg DM barley silage and 100 g/kg DM supplement. 3-NOP and OIL decreased (P < 0.01) CH4 yield (g/kg DM intake) by 31.6% and 27.4%, respectively, with no 3-NOP × OIL interaction (P = 0.85). Feeding 3-NOP plus OIL decreased CH4 yield by 51% compared with control. There was a 3-NOP × OIL interaction (P = 0.02) for H2 yield (g/kg DM intake); the increase in H2 yield (P < 0.01) due to 3-NOP was less when it was combined with OIL. There were 3-NOP × OIL interactions for molar percentages of acetate and propionate (P < 0.01); individually, 3-NOP and OIL decreased acetate and increased propionate percentages with no further effect when supplemented together. 3-NOP slightly increased crude protein (P = 0.02) and starch (P = 0.01) digestibilities, while OIL decreased the digestibilities of DM (P < 0.01) and neutral detergent fiber (P < 0.01) with no interactions (P = 0.15 and 0.10, respectively). 3-NOP and OIL increased (P = 0.04 and P < 0.01, respectively) saturated fatty acid concentration in rumen fluid, with no interaction effect. Interactions for ruminal trans-monounsaturated fatty acids (t-MUFA) concentration and percentage were observed (P = 0.02 and P < 0.01); 3-NOP had no effect on t-MUFA concentration and percentage, while OIL increased the concentration (P < 0.01) and percentage (P < 0.01) of t-MUFA but to a lesser extent when combined with 3-NOP. In conclusion, the CH4-mitigating effects of 3-NOP and OIL were independent and incremental. Supplementing ruminant diets with a combination of 3-NOP and OIL may help mitigate CH4 emissions, but the decrease in total tract digestibility due to OIL may decrease animal performance and needs further investigation., (© Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada, 2021.)

3-Nitrooxypropanol (3-NOP) is an investigational compound that acts as an enzyme inhibitor to decrease ruminal methanogenesis. We hypothesized that when feeding 3-NOP to cattle fed a high-forage diet, H 2 would accumulate in the rumen, which could suppress microbial colonization of feed particles and fiber degradation. Therefore, the study investigated the effects of supplementing a high-forage diet with 3-NOP on ruminal fiber degradability and microbial colonization of feed particles using the in situ technique. Eight ruminally cannulated beef cattle were allocated to 2 groups (4 cattle/group) in a crossover design with 2 periods and 2 dietary treatments. The treatments were control (basal diet) and 3-NOP (basal diet supplemented with 3-NOP, 150 mg/kg of dry matter). The basal diet consisted of 45% barley silage, 45% chopped grass hay, and 10% concentrate (dry matter basis). Samples of dried, ground barley silage and grass hay were incubated in the rumen of each animal for 0, 4, 12, 24, 36, 48, 96, 120, 216, and 288 h to determine neutral detergent fiber (NDF) degradation kinetics. An additional 2 bags were incubated for 4 and 48 h to evaluate the bacterial community attached to the incubated forages. Dietary supplementation of 3-NOP decreased (-53%) the dissolved methane concentration and increased (+780%) the dissolved H 2 concentration in ruminal fluid, but did not substantially alter in situ NDF degradation. The addition of 3-NOP resulted in a decrease in the α-diversity of the microbial community with colonizing communities showing reduced numbers of amplicon sequence variants and phylogenetic diversity compared with control diets. Principal coordinate analysis plots indicated that forages incubated in animals fed 3-NOP resulted in highly specific changes to targeted microbes compared with control diets based on unweighted analysis (considering only absence and presence of taxa), but did not alter the overall composition of the colonizing community based on weighted UniFrac distances; unchanged relative abundances of major taxa included phyla Bacteroidetes, Firmicutes, and Fibrobacteres. The effect of 3-NOP on colonizing methanogenic microbes differed depending upon the forage incubated, as abundance of genus Methanobrevibacter was decreased for barley silage but not for grass hay. In conclusion, 3-NOP supplementation of a high-forage diet decreased ruminal methanogenesis and increased dissolved H 2 concentration, but had no negative effects on ruminal fiber degradation and only minor effects on relative abundances of the major taxa of bacteria adhered to forage substrates incubated in the rumen., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

This study examines the colonization of barley straw (BS) and corn stover (CS) by rumen bacteria and how this is impacted by ammonia fiber expansion (AFEX) pre-treatment. A total of four ruminally cannulated beef heifers were used to investigate in situ microbial colonization in a factorial design with two crop residues, pre-treated with or without AFEX. Crop residues were incubated in the rumen for 0, 2, 4, 8 and 48 h and the colonizing profile was determined using 16 s rRNA gene sequencing. The surface colonizing community clustered based on incubation time and pre-treatment. Fibrobacter, unclassified Bacteroidales, and unclassified Ruminococcaceae were enriched during late stages of colonization. Prevotella and unclassified Lachnospiraceae were enriched in the early stages of colonization. The microbial community colonizing BS-AFEX and CS was less diverse than the community colonizing BS and CS-AFEX. Prevotella, Coprococcus and Clostridium were enriched in both AFEX crop residues, while untreated crop residues were enriched with Methanobrevibacter. Several pathways associated with simple carbohydrate metabolism were enriched in the primary colonizing community of AFEX crop residues. This study suggests that AFEX improves the degradability of crop residues by increasing the accessibility of polysaccharides that can be metabolized by the dominant taxa responsible for primary colonization., (© Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-food Canada, 2020.)

The objective of this study was to evaluate the effect of ammonia fiber expansion (AFEX)-treated wheat straw pellets and a recombinant fibrolytic enzyme on the rumen microbiome, rumen fermentation parameters, total tract diet digestibility, and performance of lambs. Eight rumen cannulated wethers and 60 lambs (n = 15 per diet, 8 rams and 7 ewes) were used in a replicated 4 × 4 Latin square design digestibility study and a complete randomized growth performance study, respectively. Four treatment diets were arranged in a 2 × 2 factorial structure with AFEX wheat straw (0% or 30% AFEX straw pellets on a dietary DM basis replacing alfalfa hay pellets) and fibrolytic enzyme (with or without XYL10C, a β-1,4-xylanase, from Aspergillus niger) as main factors. Enzyme was applied at 100 mg/kg of diet DM, 22 h before feeding. Rumen bacteria diversity Pielou evenness decreased (P = 0.05) with AFEX compared with the control diet and increased (P < 0.01) with enzyme. Enzyme increased (P ≤ 0.02) the relative abundancies of Prevotellaceae UCG-004, Christensenellaceae R-7 group, Saccharofermentans, and uncultured Kiritimatiellaeota. Total protozoa counts were greater (P ≤ 0.04) in the rumen of lambs fed AFEX compared with control, with enzyme reducing (P ≤ 0.05) protozoa counts for both diets. Digestibility of DM did not differ (P > 0.10) among diets, but digestibility of CP was reduced (P = 0.001), and digestibility of NDF and ADF increased (P < 0.05) as AFEX replaced alfalfa. Compared with control, AFEX promoted greater DMI (P = 0.003) and improved ADG up to 42 d on feed (P = 0.03), but not (P = 0.51) over the full ~94-d experiment. Consequently, overall G:F was reduced (P = 0.04) for AFEX when compared with control (0.188 vs. 0.199), but days on feed were lower (P = 0.04) for AFEX (97 vs. 91 d). Enzyme improved DMI of AFEX up to day 70 (P = 0.01), but did not affect DMI of the control diet. Enzyme addition improved ADG of lambs fed both diets in the first 28 d (P = 0.02), but not over the entire feeding period (P ≥ 10). As a result, G:F was improved with enzyme for the first 28 d (P = 0.04), but not overall (P = 0.45). This study shows that AFEX-treated wheat straw can replace alfalfa hay with no loss in lamb growth performance. Additionally, the enzyme XYL10C altered the rumen microbiome and improved G:F in the first month of the feeding., (© Crown copyright 2020.)

The objective of this study was to examine the effect of a pine enhanced biochar (EB) on rumen fermentation, apparent total tract digestibility, methane (CH 4 ) emissions, and the rumen and fecal microbiome of Angus × Hereford heifers fed a barley silage-based diet. The experiment was a replicated 4 × 4 Latin square using 8 ruminally cannulated heifers (565 ± 35 kg initial BW). The basal diet contained 60% barley silage, 35% barley grain and 5% mineral supplement with EB added at 0% (control), 0.5, 1.0, or 2.0% (DM basis). Each period lasted 28 days, consisting of 14 days adaptation and 14 days of measurements. Samples for profiling of the microbiome in rumen liquid, solids and feces were collected on d 15 before feeding. Rumen samples for fermentation characterization were taken at 0, 3, 6, and 12 h post feeding. Total collection of urine and feces was conducted from days 18 to 22. Heifers were housed in open-circuit respiratory chambers on days 26-28 to estimate CH 4 emissions. Ruminal pH was recorded at 1-min intervals during CH 4 measurements using indwelling pH loggers. Data were analyzed with the fixed effects of dietary treatment and random effects of square, heifer within square and period. Dry matter intake was similar across treatments ( P = 0.21). Ammonia N concentration and protozoa counts responded quadratically ( P = 0.01) to EB in which both were decreased by EB included at 0.5 and 1.0%, compared to the control and 2.0% EB. Minimum pH was increased ( P = 0.04), and variation of pH was decreased ( P = 0.03) by 2.0% EB. Total tract digestibility, N balance and CH 4 production were not affected ( P ≥ 0.17) by EB. Enhanced biochar decreased the relative abundance of Fibrobacter ( P = 0.05) and Tenericutes ( P = 0.01), and increased the relative abundance of Spirochaetaes ( P = 0.01), Verrucomicrobia ( P = 0.02), and Elusimicrobia ( P = 0.02). Results suggest that at the examined concentrations, EB was ineffective at decreasing enteric CH 4 emissions, but did alter specific rumen microbiota., (Copyright © 2019 Terry, Ribeiro, Gruninger, Chaves, Beauchemin, Okine and McAllister.)

Ruminants are unique among livestock due to their ability to efficiently convert plant cell wall carbohydrates into meat and milk. This ability is a result of the evolution of an essential symbiotic association with a complex microbial community in the rumen that includes vast numbers of bacteria, methanogenic archaea, anaerobic fungi and protozoa. These microbes produce a diverse array of enzymes that convert ingested feedstuffs into volatile fatty acids and microbial protein which are used by the animal for growth. Recent advances in high-throughput sequencing and bioinformatic analyses have helped to reveal how the composition of the rumen microbiome varies significantly during the development of the ruminant host, and with changes in diet. These sequencing efforts are also beginning to explain how shifts in the microbiome affect feed efficiency. In this review, we provide an overview of how meta-omics technologies have been applied to understanding the rumen microbiome, and the impact that diet has on the rumen microbial community.

Frothy bloat is an often fatal digestive disorder of cattle grazing alfalfa pastures. The aim of this study was to investigate ruminal and fecal microbiota dynamics associated with development of alfalfa-induced frothy bloat and to further explore how bloat prevention strategies influence the composition of these microbial communities. In a 3 × 3 crossover experiment, twelve rumen-cannulated steers were sequentially subjected to: (1) pure alfalfa pasture, (2) pure alfalfa pasture supplemented with the pluronic detergent ALFASURE, and (3) alfalfa - sainfoin mixed pasture. Eleven out of 12 steers in pure alfalfa pasture developed clinical bloat, whereas ALFASURE treatment prevented the development of bloat in all 12 steers and alfalfa - sainfoin prevented bloat in 5 out of 11 steers. Development of bloat was associated with considerable shifts in the microbiota profile of rumen contents. In particular, the microbiota of solid rumen contents from bloated steers contained higher species richness and diversity. Streptococcus, Succinivibrio and unclassified Myxococcales were enriched in the rumen microbiota of bloated steers, whereas Fibrobacter and Ruminococcus were overrepresented in the rumen contents of non-bloated steers. Our results provide novel insights into bloat-associated shifts in the composition and predicted functional properties of the rumen microbiota of cattle grazing alfalfa pasture.

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.

Ruminants play an important role in food security, but there is a growing concern about the impact of cattle on the environment, particularly regarding greenhouse gas emissions. The objective of this study was to examine the effect of humic substances (HS) on rumen fermentation, nutrient digestibility, methane (CH4) emissions, and the rumen microbiome of beef heifers fed a barley silage-based diet. The experiment was designed as a replicated 4 × 4 Latin square using 8 ruminally cannulated Angus × Hereford heifers (758 ± 40.7 kg initial BW). Heifers were offered a basal diet consisting of 60% barley silage and 40% concentrate (DM basis) with either 0- (control), 100-, 200- or 300-mg granulated HS/kg BW. Each period was 28 d with 14 d of adaptation. Rumen samples were taken on day 15 at 0, 3, 6, and 12 h postfeeding. Total urine and feces were collected from days 18 to 22. Blood samples were taken on day 22 at 0 and 6 h postfeeding. Between days 26 and 28, heifers were placed in open-circuit respiratory chambers to measure CH4. Ruminal pH was recorded continuously during the periods of CH4 measurement using indwelling pH loggers. Intake was similar (P = 0.47) across treatments. Concentration of ammonia-N and counts of rumen protozoa responded quadratically (P = 0.03), where both increased at H100 and then decreased for the H300 treatments. Apparent total tract digestibility of CP (P = 0.04) was linearly increased by HS and total N retention (g/d, % N intake, g/kg BW0.75) was improved (P = 0.04) for HS when compared with the control. There was no effect of HS on CH4 production (g/d; P = 0.83); however, HS decreased the relative abundance of Proteobacteria (P = 0.04) and increased the relative abundance of Synergistetes (P = 0.01) and Euryarchaeota (P = 0.04). Results suggest that HS included at up to 300 mg/kg BW may improve N retention and CP digestibility, but there was no impact on CH4 production., (© Crown copyright 2018.)

The efficiency with which the anaerobic fungi (phylum Neocallimastigomycota) degrade plant biomass is well-recognized and in recent years has received renewed interest. To further understand the biological mechanisms that are utilized by the rumen anaerobic fungi to break down lignocellulose, we have used a transcriptomic approach to examine carbohydrate digestion by Neocallimastix frontalis, Piromyces rhizinflata, Orpinomyces joyonii, and Anaeromyces mucronatus cultured on several carbon sources. The number of predicted unique transcripts ranged from 6,633 to 12,751. Pfam domains were identified in 62-70% of the fungal proteins and were linked to gene ontology terms to infer the biological function of the transcripts. Most of the predicted functions are consistent across species suggesting a similar overall strategy evolved for successful colonization of the rumen. However, the presence of differential profiles in enzyme classes suggests that there may be also be niche specialization. All fungal species were found to express an extensive array of transcripts encoding carbohydrate active enzymes (CAZymes) ranging from 8.3 to 11.3% of the transcriptome. CAZyme families involved in hemicellulose digestion were the most abundant across all four fungi. This study provides additional insight into how anaerobic fungi have evolved to become specialists at breaking down the plant cell wall in the complex and, strictly anaerobic rumen ecosystem.

Although coccidioidomycosis in Arizona and California has been well-characterized, much remains unknown about its epidemiology in states where it is not highly endemic. We conducted enhanced surveillance in 14 such states in 2016 by identifying cases according to the Council of State and Territorial Epidemiologists case definition and interviewing patients about their demographic characteristics, clinical features, and exposures. Among 186 patients, median time from seeking healthcare to diagnosis was 38 days (range 1-1,654 days); 70% had another condition diagnosed before coccidioidomycosis testing occurred (of whom 83% were prescribed antibacterial medications); 43% were hospitalized; and 29% had culture-positive coccidioidomycosis. Most (83%) patients from nonendemic states had traveled to a coccidioidomycosis-endemic area. Coccidioidomycosis can cause severe disease in residents of non-highly endemic states, a finding consistent with previous studies in Arizona, and less severe cases likely go undiagnosed or unreported. Improved coccidioidomycosis awareness in non-highly endemic areas is needed.

A novel variant of the AAD(3″) class of aminoglycoside-modifying enzymes was discovered in fatal bovine respiratory disease-associated pathogens Pasteurella multocida and Histophilus somni . The aadA31 gene encodes a spectinomycin/streptomycin adenylyltransferase and was located in a variant of the integrative and conjugative element ICE Mh1 , a mobile genetic element transmissible among members of the family Pasteurellaceae . The gene was also detected in Mannheimia haemolytica from a case of porcine pneumonia and in Moraxella bovoculi from a case of keratoconjunctivitis. IMPORTANCE Aminoglycosides are important antimicrobials used worldwide for prophylaxis and/or therapy in multiple production animal species. The emergence of new resistance genes jeopardizes current pathogen detection and treatment methods. The risk of resistance gene transfer to other animal and human pathogens is elevated when resistance genes are carried by mobile genetic elements. This study identified a new variant of a spectinomycin/streptomycin resistance gene harbored in a self-transmissible mobile element. The gene was also present in four different bovine pathogen species.

Metatranscriptomics can be used to examine both the composition of a microbial community as well as its metabolic activity under a particular set of conditions and complement metagenomic studies. The availability of low-cost, high-throughput next-generation sequencing has led to a rapid increase in the number of metatranscriptomic studies being undertaken. One of the primary difficulties when conducting transcriptomics is the ability to isolate high-quality RNA from samples of interest. The application of metatranscriptomics in rumen microbiology is still relatively novel but there is a significant push toward applying this technology in this field. In this protocol, we outline the method that is used routinely in our laboratory to purify high quality RNA from rumen contents that are suitable for metatranscriptomic sequencing using RNA-seq.

myo -Inositol phosphates (IPs) are important bioactive molecules that have multiple activities within eukaryotic cells, including well-known roles as second messengers and cofactors that help regulate diverse biochemical processes such as transcription and hormone receptor activity. Despite the typical absence of IPs in prokaryotes, many of these organisms express IPases (or phytases) that dephosphorylate IPs. Functionally, these enzymes participate in phosphate-scavenging pathways and in plant pathogenesis. Here, we determined the X-ray crystallographic structures of two catalytically inactive mutants of protein-tyrosine phosphatase-like myo -inositol phosphatases (PTPLPs) from the non-pathogenic bacteria Selenomonas ruminantium (PhyAsr) and Mitsuokella multacida (PhyAmm) in complex with the known eukaryotic second messengers Ins(1,3,4,5)P 4 and Ins(1,4,5)P 3 Both enzymes bound these less-phosphorylated IPs in a catalytically competent manner, suggesting that IP hydrolysis has a role in plant pathogenesis. The less-phosphorylated IP binding differed in both the myo -inositol ring position and orientation when compared with a previously determined complex structure in the presence of myo -inositol-1,2,3,4,5,6-hexa kis phosphate (InsP 6 or phytate). Further, we have demonstrated that PhyAsr and PhyAmm have different specificities for Ins(1,2,4,5,6)P 5 , have identified structural features that account for this difference, and have shown that the absence of these features results in a broad specificity toward Ins(1,2,4,5,6)P 5 These features are main-chain conformational differences in loops adjacent to the active site that include the extended loop prior to the penultimate helix, the extended Ω-loop, and a β-hairpin turn of the Phy-specific domain., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Enzyme activities that improve digestion of recalcitrant plant cell wall polysaccharides may offer solutions for sustainable industries. To this end, anaerobic fungi in the rumen have been identified as a promising source of novel carbohydrate active enzymes (CAZymes) that modify plant cell wall polysaccharides and other complex glycans. Many CAZymes share insufficient sequence identity to characterized proteins from other microbial ecosystems to infer their function; thus presenting challenges to their identification. In this study, four rumen fungal genes ( nf2152 , nf2215 , nf2523 , and pr2455 ) were identified that encode family 39 glycoside hydrolases (GH39s), and have conserved structural features with GH51s. Two recombinant proteins, NF2152 and NF2523, were characterized using a variety of biochemical and structural techniques, and were determined to have distinct catalytic activities. NF2152 releases a single product, β1,2-arabinobiose (Ara 2 ) from sugar beet arabinan (SBA), and β1,2-Ara 2 and α-1,2-galactoarabinose (Gal-Ara) from rye arabinoxylan (RAX). NF2523 exclusively releases α-1,2-Gal-Ara from RAX, which represents the first description of a galacto-(α-1,2)-arabinosidase. Both β-1,2-Ara 2 and α-1,2-Gal-Ara are disaccharides not previously described within SBA and RAX. In this regard, the enzymes studied here may represent valuable new biocatalytic tools for investigating the structures of rare arabinosyl-containing glycans, and potentially for facilitating their modification in industrial applications., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Future growth in demand for meat and milk, and the socioeconomic and environmental challenges that farmers face, represent a "grand challenge for humanity". Improving the digestibility of crop residues such as straw could enhance the sustainability of ruminant production systems. Here, we investigated if transfer of rumen contents from bison to cattle could alter the rumen microbiome and enhance total tract digestibility of a barley straw-based diet. Beef heifers were adapted to the diet for 28 days prior to the experiment. After 46 days, ~70 percent of rumen contents were removed from each heifer and replaced with mixed rumen contents collected immediately after slaughter from 32 bison. This procedure was repeated 14 days later. Intake, chewing activity, total tract digestibility, ruminal passage rate, ruminal fermentation, and the bacterial and protozoal communities were examined before the first and after the second transfer. Overall, inoculation with bison rumen contents successfully altered the cattle rumen microbiome and metabolism, and increased protein digestibility and nitrogen retention, but did not alter fiber digestibility.

Fungi utilize a unique mechanism of nutrient acquisition involving extracellular digestion. To understand the biology of these microbes, it is important to identify and characterize the function of proteins that are secreted and involved in this process. Mass spectrometry-based proteomics is a powerful tool to study complex mixtures of proteins and understand how the proteins produced by an organism change in response to different conditions. Many fungi are efficient decomposers of plant cell wall, and anaerobic fungi are well recognized for their ability to digest lignocellulose. Here, we outline a protocol for the enrichment and isolation of proteins secreted by anaerobic fungi after growth on simple (glucose) and complex (straw and alfalfa hay) carbon sources. We provide detailed instruction on generating protein fragments and preparing these for proteomic analysis using reversed phase chromatography and mass spectrometry.

Background: Shigella species (spp.) are a leading cause of moderate to severe diarrhea in children worldwide. The recent emergence of quinolone-resistant Shigella spp. gives cause for concern, and South Asia has been identified as a reservoir for global spread. The influence of socioeconomic status on antimicrobial resistance in developing countries, such as those in South Asia, remains unknown., Methods: We used data collected from 2009 to 2014 from a hospital specializing in the treatment of diarrhea in Dhaka, Bangladesh, to determine the relationship between Ciprofloxacin-resistant Shigella spp. isolates and measures of socioeconomic status in Bangladeshi children less than 5 years of age., Results: We found 2.7% (230/8,672) of children who presented with diarrhea had Shigella spp. isolated from their stool, and 50% (115/230) had resistance to Ciprofloxacin. Using multivariable logistic regression analysis, we found that children from families where the father's income was in the highest quintile had significantly higher odds of having Ciprofloxacin-resistant Shigella spp. compared to children in the lowest quintile (OR = 6.1, CI 1.9-19). Factors protective against the development of resistance included access to improved sanitation (OR = 0.27, CI 0.11-0.7), and improved water sources (OR = 0.48, CI 0.25-0.92). We did not find a relationship between Ciprofloxacin resistance and other proxies for socioeconomic status, including the presence of animals in the home, nutritional status, paternal education level, and the number of family members in the home., Conclusions: Although the associations between wealth and antimicrobial resistance are not fully understood, possible explanations include increased access and use of antibiotics, greater access to healthcare facilities and thus resistant pathogens, or greater consumption of commercially produced foods prepared with antibiotics., Competing Interests: Potential Conflicts Of Interest: The authors do not have a conflict to declare.

Lignocellulosic biomass represents an abundant, renewable resource that can be used to produce biofuels, low-cost livestock feed, and high-value chemicals. The potential of this resource has led to intensive research efforts to develop cost effective methods to breakdown lignocellulose. The efficiency with which the anaerobic fungi (phylum Neocallimastigomycota) degrade plant biomass is well recognized and in recent years has received renewed interest. Transcriptomics has been used to identify enzymes that are expressed by these fungi and are involved in the degradation of a range of lignocellulose feedstocks. The transcriptome is the entire complement of coding and noncoding RNA transcripts that are expressed by a cell under a particular set of conditions. Monitoring changes in gene expression can provide fundamental information about the biology of an organism. Here we outline a general methodology that will enable researchers to conduct comparative transcriptomic studies with the goal of identifying enzymes involved in the degradation of the plant cell wall. The method described here includes growth of fungal cultures, isolation and sequencing of RNA, and a basic description of data analysis for bioinformatic identification of differentially expressed transcripts.

Despite recent large-scale cholera outbreaks, little is known about the immunogenicity of oral cholera vaccines (OCV) in African populations, particularly among those at highest cholera risk. During a 2015 preemptive OCV campaign among internally displaced persons in South Sudan, a year after a large cholera outbreak, we enrolled 37 young children (1-5 years old), 67 older children (6-17 years old) and 101 adults (≥18 years old), who received two doses of OCV (Shanchol) spaced approximately 3 weeks apart. Cholera-specific antibody responses were determined at days 0, 21 and 35 post-immunization. High baseline vibriocidal titers (>80) were observed in 21% of the participants, suggesting recent cholera exposure or vaccination. Among those with titers ≤80, 90% young children, 73% older children and 72% adults seroconverted (≥4 fold titer rise) after the 1 st OCV dose; with no additional seroconversion after the 2 nd dose. Post-vaccination immunological endpoints did not differ across age groups. Our results indicate Shanchol was immunogenic in this vulnerable population and that a single dose alone may be sufficient to achieve similar short-term immunological responses to the currently licensed two-dose regimen. While we found no evidence of differential response by age, further immunologic and epidemiologic studies are needed.

The North American Beaver (Castor canadensis) is the second largest living rodent and an iconic symbol of Canada. The beaver is a semi-aquatic browser whose diet consists of lignocellulose from a variety of plants. The beaver is a hindgut fermenter and has an enlarged ceacum that houses a complex microbiome. There have been few studies examining the microbial diversity in gastrointestinal tract of hindgut fermenting herbivores. To examine the bacterial and archaeal communities inhabiting the gastrointestinal tract of the beaver, the microbiome of the ceacum and feaces was examined using culture-independent methods. DNA from the microbial community of the ceacum and feaces of 4 adult beavers was extracted, and the16S rRNA gene was sequenced using either bacterial or archaeal specific primers. A total of 1447 and 1435 unique bacterial OTUs were sequenced from the ceacum and feaces, respectively. On average, the majority of OTUs within the ceacum were classified as Bacteroidetes (49.2%) and Firmicutes (47.6%). The feaces was also dominated by OTUs from Bacteroidetes (36.8%) and Firmicutes (58.9%). The composition of bacterial community was not significantly different among animals. The composition of the ceacal and feacal microbiome differed, but this difference is due to changes in the abundance of closely related OTUs, not because of major differences in the taxonomic composition of the communities. Within these communities, known degraders of lignocellulose were identified. In contrast, to the bacterial microbiome, the archaeal community was dominated by a single species of methanogen, Methanosphaera stadtmanae. The data presented here provide the first insight into the microbial community within the hindgut of the beaver.

Lignocellulosic biomass is a promising renewable resource; however, deconstruction of this material is still the rate-limiting step. Major obstacles in the biocatalytic turnover of lignocellulose are ester-linked decorations that prevent access to primary structural polysaccharides. Enzymes targeting these esters represent promising biotools for increasing bioconversion efficiency. Ruminant livestock are unique in their ability to degrade lignocellulose through the action of their gut microbiome. The anaerobic fungi (phylum Neocallimastigomycota) are key members of this ecosystem that express a large repertoire of carbohydrate-active enzymes (CAZymes) with little sequence identity with characterized CAZymes [Lombard, Golaconda, Drula, Coutinho and Henrissat (2014) Nucleic Acids Res. 42: , D490-D495]. We have identified a carbohydrate esterase family 1 (CE1) ferulic acid esterase (FAE) belonging to Anaeromyces mucronatus(AmCE1/Fae1a), and determined its X-ray structure in both the presence [1.55 Å (1 Å=0.1 nm)] and absence (1.60 Å) of ferulic acid. AmCE1 adopts an α/β-hydrolase fold that is structurally conserved with bacterial FAEs, and possesses a unique loop, termed the β-clamp, that encloses the ligand. Isothermal titration calorimetry reveals that substrate binding is driven by enthalpic contributions, which overcomes a large entropic penalty. A comparative analysis of AmCE1 with related enzymes has uncovered the apparent structural basis for differential FAE activities targeting cross-linking ferulic acid conjugates compared with terminal decorations. Based on comparisons to structurally characterized FAEs, we propose that the β-clamp may define the structural basis of exolytic activities in FAEs. This provides a structure-based tool for predicting exolysis and endolysis in CE1. These insights hold promise for rationally identifying enzymes tailored for bioconversion of biomass with variations in cell wall composition., (© 2016 Authors; published by Portland Press Limited.)

Unique to Gram-positive bacteria, wall teichoic acids are anionic glycopolymers cross-stitched to a thick layer of peptidoglycan. The polyol phosphate subunits of these glycopolymers are decorated with GlcNAc sugars that are involved in phage binding, genetic exchange, host antibody response, resistance, and virulence. The search for the enzymes responsible for GlcNAcylation in Staphylococcus aureus has recently identified TarM and TarS with respective α- and β-(1-4) glycosyltransferase activities. The stereochemistry of the GlcNAc attachment is important in balancing biological processes, such that the interplay of TarM and TarS is likely important for bacterial pathogenicity and survival. Here we present the crystal structure of TarM in an unusual ternary-like complex consisting of a polymeric acceptor substrate analog, UDP from a hydrolyzed donor, and an α-glyceryl-GlcNAc product formed in situ. These structures support an internal nucleophilic substitution-like mechanism, lend new mechanistic insight into the glycosylation of glycopolymers, and reveal a trimerization domain with a likely role in acceptor substrate scaffolding.

Anaerobic fungi (phylum Neocallimastigomycota) inhabit the gastrointestinal tract of mammalian herbivores, where they play an important role in the degradation of plant material. The Neocallimastigomycota represent the earliest diverging lineage of the zoosporic fungi; however, understanding of the relationships of the different taxa (both genera and species) within this phylum is in need of revision. Issues exist with the current approaches used for their identification and classification, and recent evidence suggests the presence of several novel taxa (potential candidate genera) that remain to be characterised. The life cycle and role of anaerobic fungi has been well characterised in the rumen, but not elsewhere in the ruminant alimentary tract. Greater understanding of the 'resistant' phase(s) of their life cycle is needed, as is study of their role and significance in other herbivores. Biotechnological application of anaerobic fungi, and their highly active cellulolytic and hemi-cellulolytic enzymes, has been a rapidly increasing area of research and development in the last decade. The move towards understanding of anaerobic fungi using -omics based (genomic, transcriptomic and proteomic) approaches is starting to yield valuable insights into the unique cellular processes, evolutionary history, metabolic capabilities and adaptations that exist within the Neocallimastigomycota., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Bdellovibrio bacteriovorus is an unusual δ-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases) are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP) class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.

Functional screening of a metagenomic library constructed with DNA extracted from the rumen contents of a grass/hay-fed dairy cow identified a protein, β-glucosidase/β-xylosidase/α-arabinosidase gene (Bgxa1), with high levels of β-glucosidase activity. Purified Bgxa1 was highly active against p-nitrophenyl-β-D-glucopyranoside (pNPG), cellobiose, p-nitrophenyl-β-D-xylopyranoside (pNPX) and p-nitrophenyl-α-D-arabinofuranoside (pNPAf), suggesting it is a multifunctional β-glucosidase/β-xylosidase/α-arabinosidase. Kinetic analysis of the protein indicated that Bgxa1 has the greatest catalytic activity against pNPG followed by pNPAf and pNPX, respectively. The catalytic efficiency of β-glucosidase activity was 100× greater than β-xylosidase or α-arabinosidase. The pH and temperature optima for the hydrolysis of selected substrates also differed considerably with optima of pH 6.0/45 °C and pH 8.5/40 °C for pNPG and pNPX, respectively. The pH dependence of pNPAf hydrolysis displayed a bimodal distribution with maxima at both pH 6.5 and pH 8.5. The enzyme exhibited substrate-dependent responses to changes in ionic strength. Bgxa1 was highly stable over a broad pH range retaining at least 70 % of its relative catalytic activity from pH 5.0-10.0 with pNPG as a substrate. Homology modelling was employed to probe the structural basis of the unique specificity of Bgxa1 and revealed the deletion of the PA14 domain and insertions in loops adjacent to the active site. This domain has been found to be an important determinant in the substrate specificity of proteins related to Bgxa1. It is postulated that these indels are, in part, responsible for the multifunctional activity of Bgxa1. Bgxa1 acted synergistically with endoxylanase (Xyn10N18) when incubated with birchwood xylan, increasing the release of reducing sugars by 168 % as compared to Xyn10N18 alone. Examination of Bgxa1 and Xyn10N18 synergy with a cellulase for the saccharification of alkali-treated straw revealed that synergism among the three enzymes enhanced sugar release by 180 % as compared to cellulase alone. Our results suggest that Bgxa1 has a number of properties that make it an interesting candidate for the saccharification of lignocellulosic material.

Interest in the bacteria responsible for the breakdown of lignocellulosic feedstuffs within the rumen has increased due to their potential utility in industrial applications. To date, most studies have focused on bacteria from domesticated ruminants. We have expanded the knowledge of the microbial ecology of ruminants by examining the bacterial populations found in the rumen of non-domesticated ruminants found in Canada. Next-generation sequencing of 16S rDNA was employed to characterize the liquid and solid-associated bacterial communities in the rumen of elk (Cervus canadensis), and white tailed deer (Odocoileus virginianus). Despite variability in the microbial populations between animals, principle component and weighted UniFrac analysis indicated that bacterial communities in the rumen of elk and white tail deer are distinct. Populations clustered according to individual host animal and not the association with liquid or solid phase of the rumen contents. In all instances, Bacteroidetes and Firmicutes were the dominant bacterial phyla, although the relative abundance of these differed among ruminant species and between phases of rumen digesta, respectively. In the elk samples Bacteroidetes were more predominant in the liquid phase whereas Firmicutes was the most prevalent phyla in the solid digesta (P = 1×10(-5)). There were also statistically significant differences in the abundance of OTUs classified as Fibrobacteres (P = 5×10(-3)) and Spirochaetes (P = 3×10(-4)) in the solid digesta of the elk samples. We identified a number of OTUs that were classified as phylotypes not previously observed in the rumen environment. Our results suggest that although the bacterial diversity in wild North American ruminants shows overall similarities to domesticated ruminants, we observed a number of OTUs not previously described. Previous studies primarily focusing on domesticated ruminants do not fully represent the microbial diversity of the rumen and studies focusing on non-domesticated ruminants should be expanded.

Mycobacteria use specialized type VII (ESX) secretion systems to export proteins across their complex cell walls. Mycobacterium tuberculosis encodes five nonredundant ESX secretion systems, with ESX-1 being particularly important to disease progression. All ESX loci encode extracellular membrane-bound proteases called mycosins (MycP) that are essential to secretion and have been shown to be involved in processing of type VII-exported proteins. Here, we report the first x-ray crystallographic structure of MycP1(24-407) to 1.86 Å, defining a subtilisin-like fold with a unique N-terminal extension previously proposed to function as a propeptide for regulation of enzyme activity. The structure reveals that this N-terminal extension shows no structural similarity to previously characterized protease propeptides and instead wraps intimately around the catalytic domain where, tethered by a disulfide bond, it forms additional interactions with a unique extended loop that protrudes from the catalytic core. We also show MycP1 cleaves the ESX-1 secreted protein EspB from both M. tuberculosis and Mycobacterium smegmatis at a homologous cut site in vitro.

Background: Interest in cellulose degrading enzymes has increased in recent years due to the expansion of the ellulosic 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.

Protein-tyrosine phosphatase-like inositol polyphosphatases are microbial enzymes that catalyze the stepwise removal of one or more phosphates from highly phosphorylated myo-inositols via a relatively ordered pathway. To understand the substrate specificity and kinetic mechanism of these enzymes we have determined high resolution, single crystal, x-ray crystallographic structures of inactive Selenomonas ruminantium PhyA in complex with myo-inositol hexa- and pentakisphosphate. These structures provide the first glimpse of a myo-inositol polyphosphatase-ligand complex consistent with its known specificity and reveal novel features of the kinetic mechanism. To complement the structural studies, fluorescent binding assays have been developed and demonstrate that the K(d) for this enzyme is several orders of magnitude lower than the K(m). Together with rapid kinetics data, these results suggest that the protein tyrosine phosphatase-like inositol polyphosphatases have a two-step, substrate-binding mechanism that facilitates catalysis.

Mitsuokella multacida expresses a unique inositol polyphosphatase (PhyAmm) that is composed of tandem repeats (TRs). Each repeat possesses a protein tyrosine phosphatase (PTP) active-site signature sequence and fold. Using a combination of structural, mutational, and kinetic studies, we show that the N-terminal (D1) and C-terminal (D2) active sites of the TR have diverged and possess significantly different specificities for inositol polyphosphate. Structural analysis and molecular docking calculations identify steric and electrostatic differences within the substrate binding pocket of each TR that may be involved in the altered substrate specificity. The implications of our results for the biological function of related PTP-like phytases are discussed. Finally, the structures and activities of PhyAmm and tandemly repeated receptor PTPs are compared and discussed. To our knowledge, this is the first example of an inositol phosphatase with tandem PTP domains possessing substrate specificity for different inositol phosphates.