Dietary wheat and reduced methane yield are linked to rumen microbiome changes in dairy cows.

Fermentation of pasture grasses and grains in the rumen of dairy cows and other ruminants produces methane as a by-product, wasting energy and contributing to the atmospheric load of greenhouse gasses. Many feeding trials in farmed ruminants have tested the impact of dietary components on feed efficiency, productivity and methane yield (MeY). Such diets remodel the rumen microbiome, altering bacterial, archaeal, fungal and protozoan populations, with an altered fermentation outcome. In dairy cows, some dietary grains can reduce enteric methane production. This is especially true of wheat, in comparison to corn or barley. Using a feeding trial of cows fed rolled wheat, corn or barley grain, in combination with hay and canola, we identified wheat-associated changes in the ruminal microbiome. Ruminal methane production, pH and VFA concentration data together with 16S rRNA gene amplicon sequences were used to compare ruminal bacterial and archaeal populations across diets. Differential abundance analysis of clustered sequences (OTU) identified members of the bacterial families Lachnospiraceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae, Selenomonadaceae, Anaerovoracaceae and Fibrobacteraceae having a strong preference for growth in wheat-fed cows. Within the methanogenic archaea, (at >99% 16S rRNA sequence identity) the growth of Methanobrevibacter millerae was favoured by the non-wheat diets, while Methanobrevibacter olleyae was unaffected. From the wheat-preferring bacteria, correlation analysis found OTU strongly linked to reduced MeY, reduced pH and raised propionic acid levels. OTU from the genera Shuttleworthia and Prevotella_7 and especially Selenomonadaceae had high anti-methane correlations. An OTU likely representing (100% sequence identity) the fumarate-reducing, hydrogen-utilising, rumen bacterium Mitsuokella jalaludinii, had an especially high negative correlation coefficient (-0.83) versus MeY and moderate correlation (-0.6) with rumen pH, strongly suggesting much of the MeY suppression is due to reduced hydrogen availablity. Other OTU, representing as yet unknown species from the Selenomonadaceae family and the genera Prevotella_7, Fibrobacter and Syntrophococcus also had high to moderate negative MeY correlations, but low correlation with pH. These latter likely represent bacterial species able to reduce MeY without causing greater ruminal acidity, making them excellent candidates, provided they can be isolated, for development as anti-methane probiotics.

Optimised Method for Short-Chain Fatty Acid Profiling of Bovine Milk and Serum.

Short-chain fatty acids (SCFA, C2-C5) in milk and serum are derived from rumen bacterial fermentation and, thus, have the potential to be used as biomarkers for the health status of dairy cows. Currently, there is no comprehensive and validated method that can be used to analyse all SCFAs in both bovine serum and milk. This paper reports an optimised protocol, combining 3-nitrophenylhydrazine (3-NPH) derivatisation and liquid chromatography-mass spectrometry (LC-MS) analysis for quantification of SCFA and ß-hydroxybutyric acid (BHBA) in both bovine milk and bovine serum. This method is sensitive (limit of detection (LOD) ≤ 0.1 µmol/L of bovine milk and serum), accurate (recovery 84-115% for most analytes) and reproducible (relative standard deviation (RSD) for repeated analyses below 7% for most measurements) with a short sample preparation step. The application of this method to samples collected from a small cohort of animals allowed us to reveal a large variation in SCFA concentration between serum and milk and across different animals as well as the strong correlation of some SCFAs between milk and serum samples.

Further development of a reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of foot-and-mouth disease virus and validation in the field with use of an internal positive control.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hooved animals. Global outbreaks have highlighted the significant economic, trade, psychosocial and animal welfare impacts that can arise from the detection of disease in previously 'FMD-free' countries. Rapid and early diagnosis provides significant advantages in disease control and minimization of deleterious consequences. We describe the process of further development and validation of a reverse-transcription loop-mediated isothermal amplification foot-and-mouth disease virus (RT-LAMP-FMDV) test, using a published LAMP primer set, for use in the field. An internal positive control (IPC) was designed and introduced for use with the assay to mitigate any intrinsic interference from the unextracted field samples and avoid false negatives. Further modifications were included to improve the speed and operability of the test, for use by non-laboratory trained staff operating under field conditions, with shelf-stable reaction kits which require a minimum of liquid handling skills. Comparison of the assay performance with an established laboratory-based real-time reverse transcriptase PCR (rRT-PCR) test targeting the 3D region of FMD virus (Tetracore Inc) was investigated. LAMP has the potential to complement current laboratory diagnostics, such as rRT-PCR, as a preliminary tool in the investigation of FMD. We describe a strategic approach to validation of the test for use in the field using extracted RNA samples of various serotypes from Thailand and then finally unextracted field samples collected from FMD-suspected animals (primarily oral lesion swabs) from Bhutan and Australia. The statistical approach to validation was performed by Frequentist and Bayesian latent class methods, which both confirmed this new RT-LAMP-FMDV test as fit-for-purpose as a herd diagnostic tool with diagnostic specificity >99% and sensitivity 79% (95% Bayesian credible interval: 65, 90%) on unextracted field samples (oral swabs).

High throughput whole rumen metagenome profiling using untargeted massively parallel sequencing.

BACKGROUND: Variation of microorganism communities in the rumen of cattle (Bos taurus) is of great interest because of possible links to economically or environmentally important traits, such as feed conversion efficiency or methane emission levels. The resolution of studies investigating this variation may be improved by utilizing untargeted massively parallel sequencing (MPS), that is, sequencing without targeted amplification of genes. The objective of this study was to develop a method which used MPS to generate "rumen metagenome profiles", and to investigate if these profiles were repeatable among samples taken from the same cow. Given faecal samples are much easier to obtain than rumen fluid samples; we also investigated whether rumen metagenome profiles were predictive of faecal metagenome profiles. RESULTS: Rather than focusing on individual organisms within the rumen, our method used MPS data to generate quantitative rumen micro-biome profiles, regardless of taxonomic classifications. The method requires a previously assembled reference metagenome. A number of such reference metagenomes were considered, including two rumen derived metagenomes, a human faecal microflora metagenome and a reference metagenome made up of publically available prokaryote sequences. Sequence reads from each test sample were aligned to these references. The "rumen metagenome profile" was generated from the number of the reads that aligned to each contig in the database. We used this method to test the hypothesis that rumen fluid microbial community profiles vary more between cows than within multiple samples from the same cow. Rumen fluid samples were taken from three cows, at three locations within the rumen. DNA from the samples was sequenced on the Illumina GAIIx. When the reads were aligned to a rumen metagenome reference, the rumen metagenome profiles were repeatable (P < 0.00001) by cow regardless of location of sampling rumen fluid. The repeatability was estimated at 9%, albeit with a high standard error, reflecting the small number of animals in the study. Finally, we compared rumen microbial profiles to faecal microbial profiles. Our hypothesis, that there would be a stronger correlation between faeces and rumen fluid from the same cow than between faeces and rumen fluid from different cows, was not supported by our data (with much greater significance of rumen versus faeces effect than animal effect in mixed linear model). CONCLUSIONS: We have presented a simple and high throughput method of metagenome profiling to assess the similarity of whole metagenomes, and illustrated its use on two novel datasets. This method utilises widely used freeware. The method should be useful in the exploration and comparison of metagenomes.

His1 and His2 are distantly related, spindle-shaped haloviruses belonging to the novel virus group, Salterprovirus.

Spindle-shaped viruses are a dominant morphotype in hypersaline waters but their molecular characteristics and their relationship to other archaeal viruses have not been determined. Here, we describe the isolation, characteristics and genome sequence of His2, a spindle-shaped halovirus, and compare it to the previously reported halovirus His1. Their particle dimensions, host-ranges and buoyant densities were found to be similar but they differed in their stabilities to raised temperature, low salinity and chloroform. The genomes of both viruses were linear dsDNA, of similar size (His1, 14,464 bp; His2, 16,067 bp) and mol% G+C (approximately 40%), with long, inverted terminal repeat sequences. The genomic termini of both viruses are likely to possess bound proteins. They shared little nucleotide similarity and, except for their putative DNA polymerase ORFs, no significant similarity at the predicted protein level. A few of the 35 predicted ORFs of both viruses showed significant matches to sequences in GenBank, and these were always to proteins of haloarchaea. Their DNA polymerases showed 42% aa identity, and belonged to the type B group of replicases that use protein-priming. Purified His2 particles were composed of four main proteins (62, 36, 28 and 21 kDa) and the gene for the major capsid protein was identified. Hypothetical proteins similar to His2 VP1 are present in four haloarchaeal genomes but are not part of complete prophages. This, and other evidence, suggests a high frequency of recombination between haloviruses and their hosts. His1 and His2 are unlike fuselloviruses and have been placed in a new virus group, Salterprovirus.

SH1: A novel, spherical halovirus isolated from an Australian hypersaline lake.

A novel halovirus, SH1, with a spherical morphology is described. Isolated from a hypersaline lake, SH1 is divalent, producing clear plaques on Haloarcula hispanica and a natural Halorubrum isolate. Single-step growth curves gave a latent period of 5-6 h and a burst size of around 200 PFU/cell. The host can differentiate to form tight clusters of thick cell-walled forms, and these were shown to be resistant to infection. Purified virions had no visible tail, were about 70 nm in diameter, and displayed a fragile outer capsid layer, possibly with an underlying membrane component. The structural proteins of the virion were analyzed by SDS-PAGE and several were found to be cross-linked, forming protein complexes. The genome was linear, dsDNA, of approximately 30 kb in length. This morphology and linear genome are features not observed in any other euryarchaeal viruses, but have properties similar to the bacterial virus PRD1.

Haloarchaeal viruses: how diverse are they?

Hypersaline lakes are highly productive microbial environments that provide many advantages for microbial ecologists, including stable communities of relatively low diversity (mainly haloarchaea). An important component of these communities is comprised of their non-cellular parasites, i.e., their viruses. Few viruses of halobacteria (haloviruses) have been isolated and studied even though a wide selection of host species have been formally described (and easily cultured) for ten years. Hypersaline waters have been shown to contain very high concentrations of virus-like particles (at least 10(7) particles/ml), particularly fusiform particles, but laboratory isolations of new haloviruses have been very slow and the detailed study of selected examples even slower. Here we provide an outline of the reported haloviruses, including fusiform and unpublished isolates from this laboratory, and we discuss their diversity and the future directions for this research.