Maternal versus artificial rearing shapes the rumen microbiome having minor long-term physiological implications.

Increasing productivity is a key target in ruminant science which requires better understanding of the rumen microbiota. This study investigated how maternal versus artificial rearing shapes the rumen microbiota using 24 sets of triplet lambs. Lambs within each sibling set were randomly assigned to natural rearing on the ewe (NN); ewe colostrum for 24 h followed by artificial milk feeding (NA); and colostrum alternative and artificial milk feeding (AA). Maternal colostrum feeding enhanced VFA production at weaning but not thereafter. At weaning, lambs reared on milk replacer had no rumen protozoa and lower microbial diversity, whereas natural rearing accelerated the rumen microbial development and facilitated the transition to solid diet. Differences in the rumen prokaryotic communities disappear later in life when all lambs were grouped on the same pasture up to 23 weeks of age. However, NN animals retained higher fungal diversity and abundances of Piromyces, Feramyces and Diplodiniinae protozoa as well as higher feed digestibility (+4%) and animal growth (+6.5%) during the grazing period. Nevertheless, no correlations were found between rumen microbiota and productive outcomes. These findings suggest that the early life nutritional intervention determine the initial rumen microbial community, but the persistence of these effects later in life is weak.

A metabolomic study in oats (Avena sativa) highlights a drought tolerance mechanism based upon salicylate signalling pathways and the modulation of carbon, antioxidant and photo-oxidative metabolism.

Although a wealth of information is available on the induction of one or several drought-related responses in different species, little is known of how their timing, modulation and crucially integration influence drought tolerance. Based upon metabolomic changes in oat (Avena sativa L.), we have defined key processes involved in drought tolerance. During a time course of increasing water deficit, metabolites from leaf samples were profiled using direct infusion-electrospray mass spectroscopy (DI-ESI-MS) and high-performance liquid chromatography (HPLC) ESI-MS/MS and analysed using principal component analysis (PCA) and discriminant function analysis (DFA). The involvement of metabolite pathways was confirmed through targeted assays of key metabolites and physiological experiments. We demonstrate an early accumulation of salicylic acid (SA) influencing stomatal opening, photorespiration and antioxidant defences before any change in the relative water content. These changes are likely to maintain plant water status, with any photoinhibitory effect being counteracted by an efficient antioxidant capacity, thereby representing an integrated mechanism of drought tolerance in oats. We also discuss these changes in relation to those engaged at later points, consequence of the different water status in susceptible and resistant genotypes.

Polyphenol oxidase in leaves: is there any significance to the chloroplastic localization?

Polyphenol oxidase (PPO) catalyses the oxidation of monophenols and/or o-diphenols to o-quinones with the concomitant reduction of oxygen to water which results in protein complexing and the formation of brown melanin pigments. The most frequently suggested role for PPO in plants has been in defence against herbivores and pathogens, based on the physical separation of the chloroplast-localized enzyme from the vacuole-localized substrates. The o-quinone-protein complexes, formed as a consequence of cell damage, may reduce the nutritional value of the tissue and thereby reduce predation but can also participate in the formation of structural barriers against invading pathogens. However, since a sufficient level of compartmentation-based regulation could be accomplished if PPO was targeted to the cytosol, the benefit derived by some plant species in having PPO present in the chloroplast lumen remains an intriguing question. So is there more to the chloroplastic location of PPO? An interaction between PPO activity and photosynthesis has been proposed on more than one occasion but, to date, evidence either for or against direct involvement has been equivocal, and the lack of identified chloroplastic substrates remains an issue. Similarly, PPO has been suggested to have both pro- and anti-oxidant functions. Nevertheless, several independent lines of evidence suggest that PPO responds to environmental conditions and could be involved in the response of plants to abiotic stress. This review highlights our current understanding of the in vivo functions of PPO and considers the potential opportunities it presents for exploitation to increase stress tolerance in food crops.

Polyphenol oxidase-mediated protection against oxidative stress is not associated with enhanced photosynthetic efficiency.

BACKGROUND AND AIMS: Polyphenol oxidases (PPOs) catalyse the oxidation of monophenols and/or o-diphenols to highly reactive o-quinones, which in turn interact with oxygen and proteins to form reactive oxygen species (ROS) and typical brown-pigmented complexes. Hence PPOs can affect local levels of oxygen and ROS. Although the currently known substrates are located in the vacuole, the enzyme is targeted to the thylakoid lumen, suggesting a role for PPOs in photosynthesis. The current study was designed to investigate the potential involvement of PPOs in the photosynthetic response to oxidative stress. METHODS: Photosynthesis (A, Fv/Fm, ΦPSII, qN, qP, NPQ) was measured in leaves of a wild-type and a low-PPO mutant of red clover (Trifolium pratense 'Milvus') under control conditions and under a stress treatment designed to induce photooxidative stress: cold/high light (2 °C/580 µmol m(2 )s(-1)) or 0-10 µm methyl viologen. Foliar protein content and oxidation state were also determined. KEY RESULTS: Photosynthetic performance, and chlorophyll and protein content during 4 d of cold/high light stress and 3 d of subsequent recovery under control growth conditions showed similar susceptibility to stress in both lines. However, more extensive oxidative damage to protein in mutants than wild-types was observed after treatment of attached leaves with methyl viologen. In addition, PPO activity could be associated with an increased capacity to dissipate excess energy, but only at relatively low methyl viologen doses. CONCLUSIONS: The presence of PPO activity in leaves did not correspond to a direct role for the enzyme in the regulation or protection of photosynthesis under cold stress. However, an indication that PPO could be involved in cellular protection against low-level oxidative stress requires further investigation.

Ruminant Salivary Microbes: Passenger or Player in the Rumen?

Sampling of ruminant saliva has gained interest as a non-invasive proxy for exploring the structure of the rumen microbiome. However, the subsequent data analysis assumes that bacteria originating from the oral cavity are merely passengers in the rumen and play no active role. In this study, it was hypothesised that metabolically active oral bacteria present in the salivary microbiome play a role in the ruminal degradation of plant material. In vitro cultivation-based enumeration confirmed that the ruminant oral cavity harbours a significant number of anaerobic and cellulolytic bacteria that are metabolically active under ruminal conditions. Bacterial 16S rRNA gene profiling of in vitro enrichments also confirmed that oral-derived bacteria were capable of colonising plant material. Preliminary analysis of the colonising bacteria indicated that bacteria belonging to the genus Streptococcus were of particular interest. In conclusion, the findings of the current study clearly indicate that bolus-associated bacteria have the potential to play a metabolically active role in terms of ruminal colonisation and the degradation of plant material. This evidence confirms the merit of the hypothesis that the metabolically active oral bacteria present in the salivary microbiome may play a role in the ruminal degradation of plant material.

Evidence of a role for foliar salicylic acid in regulating the rate of post-ingestive protein breakdown in ruminants and contributing to landscape pollution.

Ruminant farming is important to global food security, but excessive proteolysis in the rumen causes inefficient use of nitrogenous plant constituents and environmental pollution. While both plant and microbial proteases contribute to ruminal proteolysis, little is known about post-ingestion regulation of plant proteases except that activity in the first few hours after ingestion of fresh forage can result in significant degradation of foliar protein. As the signal salicylic acid (SA) influences cell death during both biotic and abiotic stresses, Arabidopsis wild-type and mutants were used to test the effect of SA on proteolysis induced by rumen conditions (39 °C and anaerobic in a neutral pH). In leaves of Col-0, SA accumulation was induced by exposure to a rumen microbial inoculum. Use of Arabidopsis mutants with altered endogenous SA concentrations revealed a clear correlation with the rate of stress-induced proteolysis; rapid proteolysis occurred in leaves of SA-accumulating mutants cpr5-1 and dnd1-1 whereas there was little or no proteolysis in sid2-1 which is unable to synthesize SA. Reduced proteolysis in npr1-1 (Non-expressor of Pathogenesis Related genes) demonstrated a dependence on SA signalling. Slowed proteolysis in sid2-1 and npr1-1 was associated with the absence of a 34.6 kDa cysteine protease. These data suggest that proteolysis in leaves ingested by ruminants is modulated by SA. It is therefore suggested that influencing SA effects in planta could enable the development of forage crops with lower environmental impact and increased production potential.

Forage grass growth under future climate change scenarios affects fermentation and ruminant efficiency.

With an increasing human population access to ruminant products is an important factor in global food supply. While ruminants contribute to climate change, climate change could also affect ruminant production. Here we investigated how the plant response to climate change affects forage quality and subsequent rumen fermentation. Models of near future climate change (2050) predict increases in temperature, CO2, precipitation and altered weather systems which will produce stress responses in field crops. We hypothesised that pre-exposure to altered climate conditions causes compositional changes and also primes plant cells such that their post-ingestion metabolic response to the rumen is altered. This "stress memory" effect was investigated by screening ten forage grass varieties in five differing climate scenarios, including current climate (2020), future climate (2050), or future climate plus flooding, drought or heat shock. While varietal differences in fermentation were detected in terms of gas production, there was little effect of elevated temperature or CO2 compared with controls (2020). All varieties consistently showed decreased digestibility linked to decreased methane production as a result of drought or an acute flood treatment. These results indicate that efforts to breed future forage varieties should target tolerance of acute stress rather than long term climate.

Using the forces of hydrodynamic countercurrent chromatography for the study of bacteriophages.

Bacteriophages (phages) are viruses that target bacteria, with the ability to lyse and kill host bacterial cells. Due to this, they have been of some interest as a therapeutic since their discovery in the early 1900s, but with the recent increase in antibiotic resistance, phages have seen a resurgence in attention. Current methods of isolation and purification of phages can be long and tedious, with caesium chloride concentration gradients the gold standard for purifying a phage fraction. Isolation of novel phages requires centrifugation and ultrafiltration of mixed samples, such as water sources, effluent or faecal samples etc, to prepare phage filtrates for further testing. We propose countercurrent chromatography as a novel and alternative approach to use when studying phages, as a scalable and high-yield method for obtaining phage fractions. However, the full extent of the usefulness and resolution of separation with this technique has not been researched; it requires optimization and ample testing before this can be revealed. Here we present an initial study to determine survivability of two phages, T4 and ϕX174, using only water as a mobile phase in a Spectrum Series 20 HPCCC. Both phages were found to remain active once eluted from the column. Phages do not fully elute from the column and sodium hydroxide is necessary to flush the column between runs to deactivate remaining phages.

Microbiomes attached to fresh perennial ryegrass are temporally resilient and adapt to changing ecological niches.

BACKGROUND: Gut microbiomes, such as the rumen, greatly influence host nutrition due to their feed energy-harvesting capacity. We investigated temporal ecological interactions facilitating energy harvesting at the fresh perennial ryegrass (PRG)-biofilm interface in the rumen using an in sacco approach and prokaryotic metatranscriptomic profiling. RESULTS: Network analysis identified two distinct sub-microbiomes primarily representing primary (≤ 4 h) and secondary (≥ 4 h) colonisation phases and the most transcriptionally active bacterial families (i.e Fibrobacteriaceae, Selemondaceae and Methanobacteriaceae) did not interact with either sub-microbiome, indicating non-cooperative behaviour. Conversely, Prevotellaceae had most transcriptional activity within the primary sub-microbiome (focussed on protein metabolism) and Lachnospiraceae within the secondary sub-microbiome (focussed on carbohydrate degradation). Putative keystone taxa, with low transcriptional activity, were identified within both sub-microbiomes, highlighting the important synergistic role of minor bacterial families; however, we hypothesise that they may be 'cheating' in order to capitalise on the energy-harvesting capacity of other microbes. In terms of chemical cues underlying transition from primary to secondary colonisation phases, we suggest that AI-2-based quorum sensing plays a role, based on LuxS gene expression data, coupled with changes in PRG chemistry. CONCLUSIONS: In summary, we show that fresh PRG-attached prokaryotes are resilient and adapt quickly to changing niches. This study provides the first major insight into the complex temporal ecological interactions occurring at the plant-biofilm interface within the rumen. The study also provides valuable insights into potential plant breeding strategies for development of the utopian plant, allowing optimal sustainable production of ruminants. Video Abstract.

Accumulation of chlorophyll catabolites photosensitizes the hypersensitive response elicited by Pseudomonas syringae in Arabidopsis.

• The staygreen (SGR) gene encodes a chloroplast-targeted protein which promotes chlorophyll degradation via disruption of light-harvesting complexes (LHCs). • Over-expression of SGR in Arabidopsis (SGR-OX) in a Columbia-0 (Col-0) background caused spontaneous necrotic flecking. To relate this to the hypersensitive response (HR), Col-0, SGR-OX and RNAi SGR (SGRi) lines were challenged with Pseudomonas syringae pv tomato (Pst) encoding the avirulence gene avrRpm1. Increased and decreased SGR expression, respectively, accelerated and suppressed the kinetics of HR-cell death. In Col-0, SGR transcript increased at 6 h after inoculation (hai) when tissue electrolyte leakage indicated the initiation of cell death. • Excitation of the chlorophyll catabolite pheophorbide (Pheide) leads to the formation of toxic singlet oxygen ((1)O(2)). Pheide was first detected at 6 hai with Pst avrRpm1 and was linked to (1)O(2) generation and correlated with reduced Pheide a oxygenase (PaO) protein concentrations. The maximum quantum efficiency of photosystem II (F(v)/F(m)), quantum yield of electron transfer at photosystem II (φPSII), and photochemical quenching (qP) decreased at 6 hai in Col-0 but not in SGRi. Disruption of photosynthetic electron flow will cause light-dependent H(2)O(2) generation at 6 hai. • We conclude that disruption of LHCs, possibly influenced by SGR, and absence of PaO produce phototoxic chlorophyll catabolites and oxidative stress leading to the HR.

A route to decreasing N pollution from livestock: Use of Festulolium hybrids improves efficiency of N flows in rumen simulation fermenters.

Ruminant agriculture suffers from inefficient capture of forage protein and consequential release of N pollutants to land. This is due to proteolysis in the rumen catalyzed by both microbial but initially endogenous plant proteases. Plant breeding-based solutions are sought to minimize these negative environmental impacts. The aim of this study was to perform an integrated study of rumen N metabolism using semi-continuous rumen simulation fermenters (Rusitec) to explore the extent to which swards containing Festulolium populations (interspecific hybrids between Lolium and Festuca grass species) with decreased rates of endogenous protein degradation conferred advantageous protein utilization in comparison with a National Listed perennial ryegrass. An in vitro experiment was conducted using three Festulolium hybrids (Lolium perenne × Festuca arundinacea var. glaucescens, LpFg; Lolium perenne × Festuca mairei, LpFm; and Lolium multiflorum × Festuca arundinacea var. glaucescens, LmFg) and a Lolium perenne, Lp control. LpFm and LmFg demonstrated significantly lower plant-mediated proteolysis than the control. Fresh forage was incubated in Rusitec with rumen fluid from four donor cows. Feed disappearance and production of gas, methane, and volatile fatty acids were similar across cultivars. Whereas no differences in microbial protein synthesis were noted across treatments during early fermentation (0-6 hr after feeding), an increased microbial N flow in LpFm (+30%) and LmFg hybrids (+41%) was observed during late fermentation (6-24 hr after feeding), with higher overall microbial N flows (+13.5% and + 20.2%, respectively) compared with the control (Lp). We propose an underpinning mechanism involving the partitioning of amino acid catabolism toward branched-chain amino acids and microbial protein synthesis in grasses with slow plant-mediated proteolysis instead of accumulation of rumen ammonia in grasses with fast plant-mediated proteolysis. These observations indicate the potential of Festulolium hybrids with a slow plant-mediated proteolysis trait to improve the efficiency of capture of forage protein and decrease the release of N pollutants onto the land.

The Isolation and Genome Sequencing of Five Novel Bacteriophages From the Rumen Active Against Butyrivibrio fibrisolvens.

Although the prokaryotic communities of the rumen microbiome are being uncovered through genome sequencing, little is known about the resident viral populations. Whilst temperate phages can be predicted as integrated prophages when analyzing bacterial and archaeal genomes, the genetics underpinning lytic phages remain poorly characterized. To the five genomes of bacteriophages isolated from rumen-associated samples sequenced and analyzed previously, this study adds a further five novel genomes and predictions gleaned from them to further the understanding of the rumen phage population. Lytic bacteriophages isolated from fresh ovine and bovine fecal and rumen fluid samples were active against the predominant fibrolytic ruminal bacterium Butyrivibrio fibrisolvens. The double stranded DNA genomes were sequenced and reconstructed into single circular complete contigs. Based on sequence similarity and genome distances, the five phages represent four species from three separate genera, consisting of: (1) Butyrivibrio phages Arian and Bo-Finn; (2) Butyrivibrio phages Idris and Arawn; and (3) Butyrivibrio phage Ceridwen. They were predicted to all belong to the Siphoviridae family, based on evidence in the genomes such as size, the presence of the tail morphogenesis module, genes that share similarity to those in other siphovirus isolates and phylogenetic analysis using phage proteomes. Yet, phylogenomic analysis and sequence similarity of the entire phage genomes revealed that these five phages are unique and novel. These phages have only been observed undergoing the lytic lifecycle, but there is evidence in the genomes of phages Arawn and Idris for the potential to be temperate. However, there is no evidence in the genome of the bacterial host Butyrivibrio fibrisolvens of prophage genes or genes that share similarity with the phage genomes.

Cut-Lengths of Perennial Ryegrass Leaf-Blades Influences In Vitro Fermentation by the Anaerobic Fungus Neocallimastix frontalis.

Anaerobic fungi in the gut of domesticated and wild mammalian herbivores play a key role in the host's ability to utilize plant biomass. Due to their highly effective ability to enzymatically degrade lignocellulose, anaerobic fungi are biotechnologically interesting. Numerous factors have been shown to affect the ability of anaerobic fungi to break down plant biomass. However, methods to reduce the non-productive lag time in batch cultures and the effect of leaf-blade cut-length and condition on the fungal fermentation are not known. Therefore, experimentation using a novel gas production approach with pre-grown, axenic cultures of Neocallimastix frontalis was performed using both fresh and air-dried perennial ryegrass leaf-blades of different cut-lengths. The methodology adopted removed the lag-phase and demonstrated the digestion of un-autoclaved leaf-blades. Fermentation of leaf-blades of 4.0 cm cut-length produced 18.4% more gas yet retained 11.2% more apparent DM relative to 0.5 cm cut-length leaf-blades. Drying did not affect fermentation by N. frontalis, although an interaction between drying and leaf-blade cut-length was noted. Removal of the lag phase and the use of un-autoclaved substrates are important when considering the biotechnological potential of anaerobic fungi. A hypothesis based upon sporulation at cut surfaces is proposed to describe the experimental results.

A computer vision approach to improving cattle digestive health by the monitoring of faecal samples.

The digestive health of cows is one of the primary factors that determine their well-being and productivity. Under- and over-feeding are both commonplace in the beef and dairy industry; leading to welfare issues, negative environmental impacts, and economic losses. Unfortunately, digestive health is difficult for farmers to routinely monitor in large farms due to many factors including the need to transport faecal samples to a laboratory for compositional analysis. This paper describes a novel means for monitoring digestive health via a low-cost and easy to use imaging device based on computer vision. The method involves the rapid capture of multiple visible and near-infrared images of faecal samples. A novel three-dimensional analysis algorithm is then applied to objectively score the condition of the sample based on its geometrical features. While there is no universal ground truth for comparison of results, the order of scores matched a qualitative human prediction very closely. The algorithm is also able to detect the presence of undigested fibres and corn kernels using a deep learning approach. Detection rates for corn and fibre in image regions were of the order 90%. These results indicate the potential to develop this system for on-farm, real time monitoring of the digestive health of individual animals, allowing early intervention to effectively adjust feeding strategy.

A Multi-Kingdom Study Reveals the Plasticity of the Rumen Microbiota in Response to a Shift From Non-grazing to Grazing Diets in Sheep.

Increasing feed efficiency is a key target in ruminant science which requires a better understanding of rumen microbiota. This study investigated the effect of a shift from a non-grazing to a grazing diet on the rumen bacterial, methanogenic archaea, fungal, and protozoal communities. A systems biology approach based on a description of the community structure, core microbiota, network analysis, and taxon abundance linked to the rumen fermentation was used to explore the benefits of increasing depth of the community analysis. A total of 24 sheep were fed ryegrass hay supplemented with concentrate (CON) and subsequently ryegrass pasture (PAS) following a straight through experimental design. Results showed that concentrate supplementation in CON-fed animals (mainly starch) promoted a simplified rumen microbiota in terms of network density and bacterial, methanogen and fungal species richness which favored the proliferation of amylolytic microbes and VFA production (+48%), but led to a lower (ca. 4-fold) ammonia concentration making the N availability a limiting factor certain microbes. The adaptation process from the CON to the PAS diet consisted on an increase in the microbial concentration (biomass of bacteria, methanogens, and protozoa), diversity (+221, +3, and +21 OTUs for bacteria, methanogens, and fungi, respectively), microbial network complexity (+18 nodes and +86 edges) and in the abundance of key microbes involved in cellulolysis (Ruminococcus, Butyrivibrio, and Orpinomyces), proteolysis (Prevotella and Entodiniinae), lactate production (Streptococcus and Selenomonas), as well as methylotrophic archaea (Methanomassiliicoccaceae). This microbial adaptation indicated that pasture degradation is a complex process which requires a diverse consortium of microbes working together. The correlations between the abundance of microbial taxa and rumen fermentation parameters were not consistent across diets suggesting a metabolic plasticity which allowed microbes to adapt to different substrates and to shift their fermentation products. The core microbiota was composed of 34, 9, and 13 genera for bacteria, methanogens, and fungi, respectively, which were shared by all sheep, independent of diet. This systems biology approach adds a new dimension to our understanding of the rumen microbial interactions and may provide new clues to describe the mode of action of future nutritional interventions.

From plants to animals; the role of plant cell death in ruminant herbivores.

Plant cell death occurring as a result of adverse environmental conditions is known to limit crop production. It is less well recognized that plant cell death processes can also contribute to the poor environmental footprint of ruminant livestock production. Although the forage cells ingested by grazing ruminant herbivores will ultimately die, the lack of oxygen, elevated temperature, and challenge by microflora experienced in the rumen induce regulated plant stress responses resulting in DNA fragmentation and autolytic protein breakdown during the cell death process. Excessive ruminal proteolysis contributes to the inefficient conversion of plant to microbial and animal protein which results in up to 70% of the ingested nitrogen being returned to the land as the nitrogenous pollutants ammonia and urea. This constitutes a significant challenge for sustainable livestock production. As it is estimated that 25% of cultivated land worldwide is assigned to livestock production, it is clear that understanding the fundamental biology underlying cell death in ingested forage will have a highly significant role in minimizing the impact of human activities. This review examines our current understanding of plant metabolism in the rumen and explores opportunities for exploitation of plant genetics to advance sustainable land use.

Dynamics of initial colonization of nonconserved perennial ryegrass by anaerobic fungi in the bovine rumen.

Anaerobic fungi (Neocallimastigales) are active degraders of fibrous plant material in the rumen. However, only limited information is available relating to how quickly they colonize ingested feed particles. The aim of this study was to determine the dynamics of initial colonization of forage by anaerobic fungi in the rumen and the impact of different postsampling wash procedures used to remove loosely associated microorganisms. Neocallimastigales-specific molecular techniques were optimized to ensure maximal coverage before application to assess the population size (quantitative PCR) and composition (automated ribosomal intergenic spacer analysis) of the colonizing anaerobic fungi. Colonization of perennial ryegrass (PRG) was evident within 5 min, with no consistent effect of time or wash procedure on fungal population composition. Wash procedure had no effect on population size unlike time, which had a significant effect. Colonizing fungal population size continued to increase over the incubation period after an initial lag of c. 4 min. This dynamic differs from that reported previously for rumen bacteria, where substantial colonization of PRG occurred within 5 min. The observed delay in colonization of plant material by anaerobic fungi is suggested to be primarily mediated by the time taken for fungal zoospores to locate, attach and encyst on plant material.

Using 'Omic Approaches to Compare Temporal Bacterial Colonization of Lolium perenne, Lotus corniculatus, and Trifolium pratense in the Rumen.

Understanding rumen plant-microbe interactions is central for development of novel methodologies allowing improvements in ruminant nutrient use efficiency. This study investigated rumen bacterial colonization of fresh plant material and changes in plant chemistry over a period of 24 h period using three different fresh forages: Lolium perenne (perennial ryegrass; PRG), Lotus corniculatus (bird's foot trefoil; BFT) and Trifolium pratense (red clover; RC). We show using 16S rRNA gene ion torrent sequencing that plant epiphytic populations present pre-incubation (0 h) were substantially different to those attached post incubations in the presence of rumen fluid on all forages. Thereafter primary and secondary colonization events were evident as defined by changes in relative abundances of attached bacteria and changes in plant chemistry, as assessed using Fourier transform infrared (FTIR) spectroscopy. For PRG colonization, primary colonization occurred for up to 4 h and secondary colonization from 4 h onward. The changes from primary to secondary colonization occurred significantly later with BFT and RC, with primary colonization being up to 6 h and secondary colonization post 6 h of incubation. Across all 3 forages the main colonizing bacteria present at all time points post-incubation were Prevotella, Pseudobutyrivibrio, Ruminococcus, Olsenella, Butyrivibrio, and Anaeroplasma (14.2, 5.4, 1.9, 2.7, 1.8, and 2.0% on average respectively), with Pseudobutyrivibrio and Anaeroplasma having a higher relative abundance during secondary colonization. Using CowPI, we predict differences between bacterial metabolic function during primary and secondary colonization. Specifically, our results infer an increase in carbohydrate metabolism in the bacteria attached during secondary colonization, irrespective of forage type. The CowPI data coupled with the FTIR plant chemistry data suggest that attached bacterial function is similar irrespective of forage type, with the main changes occurring between primary and secondary colonization. These data suggest that the sward composition of pasture may have major implications for the temporal availability of nutrients for animal.

CowPI: A Rumen Microbiome Focussed Version of the PICRUSt Functional Inference Software.

Metataxonomic 16S rDNA based studies are a commonplace and useful tool in the research of the microbiome, but they do not provide the full investigative power of metagenomics and metatranscriptomics for revealing the functional potential of microbial communities. However, the use of metagenomic and metatranscriptomic technologies is hindered by high costs and skills barrier necessary to generate and interpret the data. To address this, a tool for Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was developed for inferring the functional potential of an observed microbiome profile, based on 16S data. This allows functional inferences to be made from metataxonomic 16S rDNA studies with little extra work or cost, but its accuracy relies on the availability of completely sequenced genomes of representative organisms from the community being investigated. The rumen microbiome is an example of a community traditionally underrepresented in genome and sequence databases, but recent efforts by projects such as the Global Rumen Census and Hungate 1000 have resulted in a wide sampling of 16S rDNA profiles and almost 500 fully sequenced microbial genomes from this environment. Using this information, we have developed "CowPI," a focused version of the PICRUSt tool provided for use by the wider scientific community in the study of the rumen microbiome. We evaluated the accuracy of CowPI and PICRUSt using two 16S datasets from the rumen microbiome: one generated from rDNA and the other from rRNA where corresponding metagenomic and metatranscriptomic data was also available. We show that the functional profiles predicted by CowPI better match estimates for both the meta-genomic and transcriptomic datasets than PICRUSt, and capture the higher degree of genetic variation and larger pangenomes of rumen organisms. Nonetheless, whilst being closer in terms of predictive power for the rumen microbiome, there were differences when compared to both the metagenomic and metatranscriptome data and so we recommend, where possible, functional inferences from 16S data should not replace metagenomic and metatranscriptomic approaches. The tool can be accessed at http://www.cowpi.org and is provided to the wider scientific community for use in the study of the rumen microbiome.

Polyomic tools for an emerging livestock parasite, the rumen fluke Calicophoron daubneyi; identifying shifts in rumen functionality.

BACKGROUND: Diseases caused by parasitic flatworms of rumen tissues (paramphistomosis) are a significant threat to global food security as a cause of morbidity and mortality in ruminant livestock in subtropical and tropical climates. Calicophoron daubneyi is currently the only paramphistome species commonly infecting ruminant livestock in temperate European climates. However, recorded incidences of C. daubneyi infection in European livestock have been increasing over the last decade. Whilst clinical paramphistomosis caused by adult worms has not been confirmed in Europe, fatalities have been attributed to severe haemorrhagic enteritis of the small intestine resulting from the migration of immature paramphistomes. Large numbers of mature adults can reside in the rumen, yet to date, the impact on rumen fermentation, and consequently on productivity and economic management of infected livestock, have not been resolved. Limited publicly available nucleotide and protein sequences for C. daubneyi underpin this lack of biological and economic understanding. Here we present for the first time a de novo assembled transcriptome, with functional annotations, for adult C. daubneyi, which provides a reference database for protein and nucleotide sequence identification to facilitate fundamental biology, anthelmintic, vaccine and diagnostics discoveries. RESULTS: This dataset identifies a number of genes potentially unique to C. daubneyi and, by comparison to an existing transcriptome for the related Paramphistomum cervi, identifies novel genes which may be unique to the paramphistome group of platyhelminthes. Additionally, we present the first coverage of the excretory/secretory and soluble somatic proteome profiles for adult C. daubneyi and identify the release of extracellular vesicles from adult C. daubneyi parasites during in vitro, ex-host culture. Finally, we have performed the first analysis of rumen fluke impacting upon rumen fermentation parameters using an in vitro gas production study resulting in a significant increase in propionate production. CONCLUSIONS: The resulting data provide a discovery platform (transcriptome, proteomes, EV isolation pipeline and in vitro fermentation system) to further study C. daubneyi-host interaction. In addition, the acetate: propionate ratio has been demonstrated to decrease with rumen fluke infection suggesting that acidotic conditions in the rumen may occur.