Temporal stability of the rumen microbiome and its longitudinal associations with performance traits in beef cattle.

The rumen microbiome is the focus of a growing body of research, mostly based on investigation of rumen fluid samples collected once from each animal. Exploring the temporal stability of rumen microbiome profiles is imperative, as it enables evaluating the reliability of findings obtained through single-timepoint sampling. We explored the temporal stability of rumen microbiomes considering taxonomic and functional aspects across the 7-month growing-finishing phase spanning 6 timepoints. We identified a temporally stable core microbiome, encompassing 515 microbial genera (e.g., Methanobacterium) and 417 microbial KEGG genes (e.g., K00856-adenosine kinase). The temporally stable core microbiome profiles collected from all timepoints were strongly associated with production traits with substantial economic and environmental impact (e.g., average daily gain, daily feed intake, and methane emissions); 515 microbial genera explained 45-83%, and 417 microbial genes explained 44-83% of their phenotypic variation. Microbiome profiles influenced by the bovine genome explained 54-87% of the genetic variation of bovine traits. Overall, our results provide evidence that the temporally stable core microbiome identified can accurately predict host performance traits at phenotypic and genetic level based on a single timepoint sample taken as early as 7 months prior to slaughter.

Infectious bronchitis virus vaccination, but not the presence of XCR1, is correlated with large differences in chicken caecal microbiota.

The chicken immune system and microbiota play vital roles in maintaining gut homeostasis and protecting against pathogens. In mammals, XCR1+ conventional dendritic cells (cDCs) are located in the gut-draining lymph nodes and play a major role in gut homeostasis. These cDCs sample antigens in the gut luminal contents and limit the inflammatory response to gut commensal microbes by generating appropriate regulatory and effector T-cell responses. We hypothesized that these cells play similar roles in sustaining gut homeostasis in chickens, and that chickens lacking XCR1 were likely to contain a dysbiotic caecal microbiota. Here we compare the caecal microbiota of chickens that were either heterozygous or homozygous XCR1 knockouts, that had or had not been vaccinated for infectious bronchitis virus (IBV). We used short-read (Illumina) and long-read (PacBio HiFi) metagenomic sequencing to reconstruct 670 high-quality, strain-level metagenome assembled genomes. We found no significant differences between alpha diversity or the abundance of specific microbial taxa between genotypes. However, IBV vaccination was found to correlate with significant differences in the richness and beta diversity of the microbiota, and to the abundance of 40 bacterial genera. In conclusion, we found that a lack of XCR1 was not correlated with significant changes in the chicken microbiota, but IBV vaccination was.

Including microbiome information in a multi-trait genomic evaluation: a case study on longitudinal growth performance in beef cattle.

BACKGROUND: Growth rate is an important component of feed conversion efficiency in cattle and varies across the different stages of the finishing period. The metabolic effect of the rumen microbiome is essential for cattle growth, and investigating the genomic and microbial factors that underlie this temporal variation can help maximize feed conversion efficiency at each growth stage. RESULTS: By analysing longitudinal body weights during the finishing period and genomic and metagenomic data from 359 beef cattle, our study demonstrates that the influence of the host genome on the functional rumen microbiome contributes to the temporal variation in average daily gain (ADG) in different months (ADG1, ADG2, ADG3, ADG4). Five hundred and thirty-three additive log-ratio transformed microbial genes (alr-MG) had non-zero genomic correlations (rg) with at least one ADG-trait (ranging from |0.21| to |0.42|). Only a few alr-MG correlated with more than one ADG-trait, which suggests that a differential host-microbiome determinism underlies ADG at different stages. These alr-MG were involved in ribosomal biosynthesis, energy processes, sulphur and aminoacid metabolism and transport, or lipopolysaccharide signalling, among others. We selected two alternative subsets of 32 alr-MG that had a non-uniform or a uniform rg sign with all the ADG-traits, regardless of the rg magnitude, and used them to develop a microbiome-driven breeding strategy based on alr-MG only, or combined with ADG-traits, which was aimed at shaping the rumen microbiome towards increased ADG at all finishing stages. Combining alr-MG information with ADG records increased prediction accuracy of genomic estimated breeding values (GEBV) by 11 to 22% relative to the direct breeding strategy (using ADG-traits only), whereas using microbiome information, only, achieved lower accuracies (from 7 to 41%). Predicted selection responses varied consistently with accuracies. Restricting alr-MG based on their rg sign (uniform subset) did not yield a gain in the predicted response compared to the non-uniform subset, which is explained by the absence of alr-MG showing non-zero rg at least with more than one of the ADG-traits. CONCLUSIONS: Our work sheds light on the role of the microbial metabolism in the growth trajectory of beef cattle at the genomic level and provides insights into the potential benefits of using microbiome information in future genomic breeding programs to accurately estimate GEBV and increase ADG at each finishing stage in beef cattle.

KOunt: a reproducible KEGG orthologue abundance workflow.

SUMMARY: Accurate gene prediction is essential for successful metagenome analysis. We present KOunt, a Snakemake pipeline, that precisely quantifies KEGG orthologue abundance. AVAILABILITY AND IMPLEMENTATION: KOunt is available on GitHub: https://github.com/WatsonLab/KOunt. The KOunt reference database is available on figshare: https://doi.org/10.6084/m9.figshare.21269715. Test data are available at https://doi.org/10.6084/m9.figshare.22250152 and version 1.2.0 of KOunt at https://doi.org/10.6084/m9.figshare.23607834.

A comparison of single-coverage and multi-coverage metagenomic binning reveals extensive hidden contamination.

Metagenomic binning has revolutionized the study of uncultured microorganisms. Here we compare single- and multi-coverage binning on the same set of samples, and demonstrate that multi-coverage binning produces better results than single-coverage binning and identifies contaminant contigs and chimeric bins that other approaches miss. While resource expensive, multi-coverage binning is a superior approach and should always be performed over single-coverage binning.

nf-core/isoseq: simple gene and isoform annotation with PacBio Iso-Seq long-read sequencing.

MOTIVATION: Iso-Seq RNA long-read sequencing enables the identification of full-length transcripts and isoforms, removing the need for complex analysis such as transcriptome assembly. However, the raw sequencing data need to be processed in a series of steps before annotation is complete. Here, we present nf-core/isoseq, a pipeline for automatic read processing and genome annotation. Following nf-core guidelines, the pipeline has few dependencies and can be run on any of platforms. AVAILABILITY AND IMPLEMENTATION: The pipeline is freely available online on the nf-core website (https://nf-co.re/isoseq) and on GitHub (https://github.com/nf-core/isoseq) under MIT License (DOI: 10.5281/zenodo.7116979).

High throughput analysis of MHC class I and class II diversity of Zambian indigenous cattle populations.

The classical MHC class I and class II molecules play key roles in determining the antigenic-specificity of CD8+ and CD4+ T-cell responses-as such characterisation of the repertoire of MHCI and MHCII allelic diversity is fundamental to our ability to understand, and potentially, exploit how genetic diversity influences the outcome of immune responses. Cattle remain one of the most economically livestock species, with particular importance to many small-holder farmers in low-and-middle income countries (LMICs). However, our knowledge of MHC (BoLA) diversity in the indigenous breeds that form the mainstay of cattle populations in many LMICs remains very limited. In this study we develop a MiSeq-based platform to enable the rapid analysis of BoLA-DQA and BoLA-DQB, and combine this with similar platforms to analyse BoLA-I and BoLA-DRB repertoires, to study a large cohort of cattle (~800 animals) representing the 3 major indigenous breeds (Angoni, Barotse, Tonga) in Zambia. The data presented confirms the capacity of this high-throughput and high-resolution approach to provide a full characterisation of the MHCI-MHCII genotypes of cattle for which little previous MHC sequence data has been obtained. The cattle in Zambia were found to express a diverse range of MHCI, MHCII and extended MHCI-MHCII haplotypes. The combined MHCI-MHCII genotyping now possible opens new opportunities to rapidly expand our knowledge of MHC diversity in cattle that could find applications in a related translational disciplines such as vaccine development.

Investigating the impact of database choice on the accuracy of metagenomic read classification for the rumen microbiome.

Microbiome analysis is quickly moving towards high-throughput methods such as metagenomic sequencing. Accurate taxonomic classification of metagenomic data relies on reference sequence databases, and their associated taxonomy. However, for understudied environments such as the rumen microbiome many sequences will be derived from novel or uncultured microbes that are not present in reference databases. As a result, taxonomic classification of metagenomic data from understudied environments may be inaccurate. To assess the accuracy of taxonomic read classification, this study classified metagenomic data that had been simulated from cultured rumen microbial genomes from the Hungate collection. To assess the impact of reference databases on the accuracy of taxonomic classification, the data was classified with Kraken 2 using several reference databases. We found that the choice and composition of reference database significantly impacted on taxonomic classification results, and accuracy. In particular, NCBI RefSeq proved to be a poor choice of database. Our results indicate that inaccurate read classification is likely to be a significant problem, affecting all studies that use insufficient reference databases. We observed that adding cultured reference genomes from the rumen to the reference database greatly improved classification rate and accuracy. We also demonstrated that metagenome-assembled genomes (MAGs) have the potential to further enhance classification accuracy by representing uncultivated microbes, sequences of which would otherwise be unclassified or incorrectly classified. However, classification accuracy was strongly dependent on the taxonomic labels assigned to these MAGs. We therefore highlight the importance of accurate reference taxonomic information and suggest that, with formal taxonomic lineages, MAGs have the potential to improve classification rate and accuracy, particularly in environments such as the rumen that are understudied or contain many novel genomes.

Microbiome-driven breeding strategy potentially improves beef fatty acid profile benefiting human health and reduces methane emissions.

BACKGROUND: Healthier ruminant products can be achieved by adequate manipulation of the rumen microbiota to increase the flux of beneficial fatty acids reaching host tissues. Genomic selection to modify the microbiome function provides a permanent and accumulative solution, which may have also favourable consequences in other traits of interest (e.g. methane emissions). Possibly due to a lack of data, this strategy has never been explored. RESULTS: This study provides a comprehensive identification of ruminal microbial mechanisms under host genomic influence that directly or indirectly affect the content of unsaturated fatty acids in beef associated with human dietary health benefits C18:3n-3, C20:5n-3, C22:5n-3, C22:6n-3 or cis-9, trans-11 C18:2 and trans-11 C18:1 in relation to hypercholesterolemic saturated fatty acids C12:0, C14:0 and C16:0, referred to as N3 and CLA indices. We first identified that ~27.6% (1002/3633) of the functional core additive log-ratio transformed microbial gene abundances (alr-MG) in the rumen were at least moderately host-genomically influenced (HGFC). Of these, 372 alr-MG were host-genomically correlated with the N3 index (n=290), CLA index (n=66) or with both (n=16), indicating that the HGFC influence on beef fatty acid composition is much more complex than the direct regulation of microbial lipolysis and biohydrogenation of dietary lipids and that N3 index variation is more strongly subjected to variations in the HGFC than CLA. Of these 372 alr-MG, 110 were correlated with the N3 and/or CLA index in the same direction, suggesting the opportunity for enhancement of both indices simultaneously through a microbiome-driven breeding strategy. These microbial genes were involved in microbial protein synthesis (aroF and serA), carbohydrate metabolism and transport (galT, msmX), lipopolysaccharide biosynthesis (kdsA, lpxD, lpxB), or flagellar synthesis (flgB, fliN) in certain genera within the Proteobacteria phyla (e.g. Serratia, Aeromonas). A microbiome-driven breeding strategy based on these microbial mechanisms as sole information criteria resulted in a positive selection response for both indices (1.36±0.24 and 0.79±0.21 sd of N3 and CLA indices, at 2.06 selection intensity). When evaluating the impact of our microbiome-driven breeding strategy to increase N3 and CLA indices on the environmental trait methane emissions (g/kg of dry matter intake), we obtained a correlated mitigation response of -0.41±0.12 sd. CONCLUSION: This research provides insight on the possibility of using the ruminal functional microbiome as information for host genomic selection, which could simultaneously improve several microbiome-driven traits of interest, in this study exemplified with meat quality traits and methane emissions. Video Abstract.

Bovine host genome acts on rumen microbiome function linked to methane emissions.

Our study provides substantial evidence that the host genome affects the comprehensive function of the microbiome in the rumen of bovines. Of 1,107/225/1,141 rumen microbial genera/metagenome assembled uncultured genomes (RUGs)/genes identified from whole metagenomics sequencing, 194/14/337 had significant host genomic effects (heritabilities ranging from 0.13 to 0.61), revealing that substantial variation of the microbiome is under host genomic control. We found 29/22/115 microbial genera/RUGs/genes host-genomically correlated (|0.59| to |0.93|) with emissions of the potent greenhouse gas methane (CH4), highlighting the strength of a common host genomic control of specific microbial processes and CH4. Only one of these microbial genes was directly involved in methanogenesis (cofG), whereas others were involved in providing substrates for archaea (e.g. bcd and pccB), important microbial interspecies communication mechanisms (ABC.PE.P), host-microbiome interaction (TSTA3) and genetic information processes (RP-L35). In our population, selection based on abundances of the 30 most informative microbial genes provided a mitigation potential of 17% of mean CH4 emissions per generation, which is higher than for selection based on measured CH4 using respiration chambers (13%), indicating the high potential of microbiome-driven breeding to cumulatively reduce CH4 emissions and mitigate climate change.

Effect of cecal microbiota transplantation between different broiler breeds on the chick flora in the first week of life.

The cecal microbiota plays numerous roles in chicken health and nutrition. Where such microbiota differs between lines exhibiting distinct phenotypes, microbiota transplantation offers scope to dissect the role of gut microbial communities in those traits. However, the composition and stability of transplants over time is relatively ill-defined and varying levels of success have been reported. In this study, we transplanted cecal contents from adult Roslin broilers into chicks from a different broiler line. Within <12 h posthatch, Ross 308 chicks received an oral gavage of cecal contents (n = 26) or a PBS control (n = 24). Cecal contents samples were collected postmortem from birds on d 1, 2, 3, 4, and 7 posthatch. DNA was extracted from these samples and the transplant inoculum and the V4 region of the 16S rRNA gene was amplified and sequenced. The cecal microbiota of chickens receiving the microbiota transplant was significantly different in composition and significantly richer and more diverse, in comparison to control birds. At the final timepoint (d 7), of the 150 Operational Taxonomic Units (OTUs) that were >0.1% abundant (average) in the donor sample, 137 were detected in the treated group (75 were >0.1% abundant (average)) while only 88 were detected in the control group (29 were >0.1% abundant (average)). Our data therefore suggests that stable transplantation of the cecal microbiota between lines is achievable using the methods described in this paper.

Extensive microbial diversity within the chicken gut microbiome revealed by metagenomics and culture.

BACKGROUND: The chicken is the most abundant food animal in the world. However, despite its importance, the chicken gut microbiome remains largely undefined. Here, we exploit culture-independent and culture-dependent approaches to reveal extensive taxonomic diversity within this complex microbial community. RESULTS: We performed metagenomic sequencing of fifty chicken faecal samples from two breeds and analysed these, alongside all (n = 582) relevant publicly available chicken metagenomes, to cluster over 20 million non-redundant genes and to construct over 5,500 metagenome-assembled bacterial genomes. In addition, we recovered nearly 600 bacteriophage genomes. This represents the most comprehensive view of taxonomic diversity within the chicken gut microbiome to date, encompassing hundreds of novel candidate bacterial genera and species. To provide a stable, clear and memorable nomenclature for novel species, we devised a scalable combinatorial system for the creation of hundreds of well-formed Latin binomials. We cultured and genome-sequenced bacterial isolates from chicken faeces, documenting over forty novel species, together with three species from the genus Escherichia, including the newly named species Escherichia whittamii. CONCLUSIONS: Our metagenomic and culture-based analyses provide new insights into the bacterial, archaeal and bacteriophage components of the chicken gut microbiome. The resulting datasets expand the known diversity of the chicken gut microbiome and provide a key resource for future high-resolution taxonomic and functional studies on the chicken gut microbiome.

Whole-Genome Sequence Data Suggest Environmental Adaptation of Ethiopian Sheep Populations.

Great progress has been made over recent years in the identification of selection signatures in the genomes of livestock species. This work has primarily been carried out in commercial breeds for which the dominant selection pressures are associated with artificial selection. As agriculture and food security are likely to be strongly affected by climate change, a better understanding of environment-imposed selection on agricultural species is warranted. Ethiopia is an ideal setting to investigate environmental adaptation in livestock due to its wide variation in geo-climatic characteristics and the extensive genetic and phenotypic variation of its livestock. Here, we identified over three million single nucleotide variants across 12 Ethiopian sheep populations and applied landscape genomics approaches to investigate the association between these variants and environmental variables. Our results suggest that environmental adaptation for precipitation-related variables is stronger than that related to altitude or temperature, consistent with large-scale meta-analyses of selection pressure across species. The set of genes showing association with environmental variables was enriched for genes highly expressed in human blood and nerve tissues. There was also evidence of enrichment for genes associated with high-altitude adaptation although no strong association was identified with hypoxia-inducible-factor (HIF) genes. One of the strongest altitude-related signals was for a collagen gene, consistent with previous studies of high-altitude adaptation. Several altitude-associated genes also showed evidence of adaptation with temperature, suggesting a relationship between responses to these environmental factors. These results provide a foundation to investigate further the effects of climatic variables on small ruminant populations.

Metagenomic analysis of the cow, sheep, reindeer and red deer rumen.

The rumen microbiota comprises a community of microorganisms which specialise in the degradation of complex carbohydrates from plant-based feed. These microbes play a highly important role in ruminant nutrition and could also act as sources of industrially useful enzymes. In this study, we performed a metagenomic analysis of samples taken from the ruminal contents of cow (Bos Taurus), sheep (Ovis aries), reindeer (Rangifer tarandus) and red deer (Cervus elaphus). We constructed 391 metagenome-assembled genomes originating from 16 microbial phyla. We compared our genomes to other publically available microbial genomes and found that they contained 279 novel species. We also found significant differences between the microbiota of different ruminant species in terms of the abundance of microbial taxonomies, carbohydrate-active enzyme genes and KEGG orthologs. We present a dataset of rumen-derived genomes which in combination with other publicly-available rumen genomes can be used as a reference dataset in future metagenomic studies.

Single-cell RNA-seq reveals CD16- monocytes as key regulators of human monocyte transcriptional response to Toxoplasma.

Monocytes are among the major myeloid cells that respond to Toxoplasma, a ubiquitous foodborne that infects ≥ 1 billion people worldwide, in human peripheral blood. As such, a molecular understanding of human monocyte-Toxoplasma interactions can expedite the development of novel human toxoplasmosis control strategies. Current molecular studies on monocyte-Toxoplasma interactions are based on average cell or parasite responses across bulk cell populations. Although informative, population-level averages of monocyte responses to Toxoplasma have sometimes produced contradictory results, such as whether CCL2 or IL12 define effective monocyte responses to the parasite. Here, we used single-cell dual RNA sequencing (scDual-Seq) to comprehensively define, for the first time, the monocyte and parasite transcriptional responses that underpin human monocyte-Toxoplasma encounters at the single cell level. We report extreme transcriptional variability between individual monocytes. Furthermore, we report that Toxoplasma-exposed and unexposed monocytes are transcriptionally distinguished by a reactive subset of CD14+CD16- monocytes. Functional cytokine assays on sorted monocyte populations show that the infection-distinguishing monocytes secrete high levels of chemokines, such as CCL2 and CXCL5. These findings uncover the Toxoplasma-induced monocyte transcriptional heterogeneity and shed new light on the cell populations that largely define cytokine and chemokine secretion in human monocytes exposed to Toxoplasma.

1200 high-quality metagenome-assembled genomes from the rumen of African cattle and their relevance in the context of sub-optimal feeding.

BACKGROUND: The Boran (Bos indicus), indigenous Zebu cattle breed from sub-Saharan Africa, is remarkably well adapted to harsh tropical environments. Due to financial constraints and low-quality forage, African livestock are rarely fed at 100% maintenance energy requirements (MER) and the effect of sub-optimal restricted feeding on the rumen microbiome of African Zebu cattle remains largely unexplored. We collected 24 rumen fluid samples from six Boran cattle fed at sub-optimal and optimal MER levels and characterised their rumen microbial composition by performing shotgun metagenomics and de novo assembly of metagenome-assembled genomes (MAGs). These MAGs were used as reference database to investigate the effect of diet restriction on the composition and functional potential of the rumen microbiome of African cattle. RESULTS: We report 1200 newly discovered MAGs from the rumen of Boran cattle. A total of 850 were dereplicated, and their uniqueness confirmed with pairwise comparisons (based on Mash distances) between African MAGs and other publicly available genomes from the rumen. A genome-centric investigation into sub-optimal diets highlighted a statistically significant effect on rumen microbial abundance profiles and a previously unobserved relationship between whole microbiome shifts in functional potential and taxon-level associations in metabolic pathways. CONCLUSIONS: This study is the first to identify 1200 high-quality African rumen-specific MAGs and provides further insight into the rumen function in harsh environments with food scarcity. The genomic information from the rumen microbiome of an indigenous African cattle breed sheds light on the microbiome contribution to rumen functionality and constitutes a vital resource in addressing food security in developing countries.

An improved pig reference genome sequence to enable pig genetics and genomics research.

BACKGROUND: The domestic pig (Sus scrofa) is important both as a food source and as a biomedical model given its similarity in size, anatomy, physiology, metabolism, pathology, and pharmacology to humans. The draft reference genome (Sscrofa10.2) of a purebred Duroc female pig established using older clone-based sequencing methods was incomplete, and unresolved redundancies, short-range order and orientation errors, and associated misassembled genes limited its utility. RESULTS: We present 2 annotated highly contiguous chromosome-level genome assemblies created with more recent long-read technologies and a whole-genome shotgun strategy, 1 for the same Duroc female (Sscrofa11.1) and 1 for an outbred, composite-breed male (USMARCv1.0). Both assemblies are of substantially higher (>90-fold) continuity and accuracy than Sscrofa10.2. CONCLUSIONS: These highly contiguous assemblies plus annotation of a further 11 short-read assemblies provide an unprecedented view of the genetic make-up of this important agricultural and biomedical model species. We propose that the improved Duroc assembly (Sscrofa11.1) become the reference genome for genomic research in pigs.