Symposium review: Understanding diet-microbe interactions to enhance productivity of dairy cows.

Ruminants are dependent on the microbiota (bacteria, protozoa, archaea, and fungi) that inhabit the reticulo-rumen for digestion of feedstuffs. Nearly 70% of energy and 50% of protein requirements for dairy cows are met by microbial fermentation in the rumen, emphasizing the need to characterize the role of microbes in feed breakdown and nutrient utilization. Over the past 2 decades, next-generation sequencing technologies have allowed for rapid expansion of knowledge concerning microbial populations and alterations in response to forages, concentrates, supplements, and probiotics in the rumen. Advances in gene sequencing and emerging bioinformatic tools have allowed for increased throughput of data to aid in our understanding of the functional relevance of microbial genomes. In particular, metagenomics can identify specific genes involved in metabolic pathways, and metatranscriptomics can describe the transcriptional activity of microbial genes. These powerful approaches help untangle the complex interactions between microbes and dietary nutrients so that we can more fully understand the physiology of feed digestion in the rumen. Application of genomics-based approaches offers promise in unraveling microbial niches and respective gene repertoires to potentiate fiber and nonfiber carbohydrate digestion, microbial protein synthesis, and healthy biohydrogenation. New information on microbial genomics and interactions with dietary components will more clearly define pathways in the rumen to positively influence milk yield and components.

Comparison of rumen bacterial communities in dairy herds of different production.

BACKGROUND: The purpose of this study was to compare the rumen bacterial composition in high and low yielding dairy cows within and between two dairy herds. Eighty five Holstein dairy cows in mid-lactation (79-179 days in milk) were selected from two farms: Farm 12 (M305 = 12,300 kg; n = 47; 24 primiparous cows, 23 multiparous cows) and Farm 9 (M305 = 9700 kg; n = 38; 19 primiparous cows, 19 multiparous cows). Each study cow was sampled once using the stomach tube method and processed for 16S rRNA gene amplicon sequencing using the Ion Torrent (PGM) platform. RESULTS: Differences in bacterial communities between farms were greater (Adonis: R2 = 0.16; p < 0.001) than within farm. Five bacterial lineages, namely Prevotella (48-52%), unclassified Bacteroidales (10-12%), unclassified bacteria (5-8%), unclassified Succinivibrionaceae (1-7%) and unclassified Prevotellaceae (4-5%) were observed to differentiate the community clustering patterns among the two farms. A notable finding is the greater (p < 0.05) contribution of Succinivibrionaceae lineages in Farm 12 compared to Farm 9. Furthermore, in Farm 12, Succinivibrionaceae lineages were higher (p < 0.05) in the high yielding cows compared to the low yielding cows in both primiparous and multiparous groups. Prevotella, S24-7 and Succinivibrionaceae lineages were found in greater abundance on Farm 12 and were positively correlated with milk yield. CONCLUSIONS: Differences in rumen bacterial populations observed between the two farms can be attributed to dietary composition, particularly differences in forage type and proportion in the diets. A combination of corn silage and alfalfa silage may have contributed to the increased proportion of Proteobacteria in Farm 12. It was concluded that Farm 12 had a greater proportion of specialist bacteria that have the potential to enhance rumen fermentative digestion of feedstuffs to support higher milk yields.

Metagenomic Evidence of the Prevalence and Distribution Patterns of Antimicrobial Resistance Genes in Dairy Agroecosystems.

Antimicrobial resistance (AR) is a global problem with serious implications for public health. AR genes are frequently detected on animal farms, but little is known about their origin and distribution patterns. We hypothesized that AR genes can transfer from animal feces to the environment through manure, and to this end, we characterized and compared the resistomes (collections of AR genes) of animal feces, manure, and soil samples collected from five dairy farms using a metagenomics approach. Resistomes constituted only up to 1% of the total gene content, but were variable by sector and also farm. Broadly, the identified AR genes were associated with 18 antibiotic resistances classes across all samples; however, the most abundant genes were classified under multidrug transporters (44.75%), followed by resistance to vancomycin (12.48%), tetracycline (10.52%), bacitracin (10.43%), beta-lactam resistance (7.12%), and MLS efflux pump (6.86%) antimicrobials. The AR gene profiles were variable between farms. Farm 09 was categorized as a high risk farm, as a greater proportion of AR genes were common to at least three sectors, suggesting possible horizontal transfer of AR genes. Taxonomic characterization of AR genes revealed that a majority of AR genes were associated with the phylum Proteobacteria. Nonetheless, there were several members of Bacteroidetes, particularly Bacteroides genus and several lineages from Firmicutes that carried similar AR genes in different sectors, suggesting a strong potential for horizontal transfer of AR genes between unrelated bacterial hosts in different sectors of the farms. Further studies are required to affirm the horizontal gene transfer mechanisms between microbiomes of different sectors in animal agroecosystems.

Metagenomic assessment of the functional potential of the rumen microbiome in Holstein dairy cows.

The microbial ecology of the rumen microbiome is influenced by the diet and the physiological status of the dairy cow and can have tremendous influence on the yield and components of milk. There are significant differences in milk yields between first and subsequent lactations of dairy cows, but information on how the rumen microbiome changes as the dairy cow gets older has received little attention. We characterized the rumen microbiome of the dairy cow for phylogeny and functional pathways by lactation group and stage of lactation using a metagenomics approach. Our findings revealed that the rumen microbiome was dominated by Bacteroidetes (70%), Firmicutes (15-20%) and Proteobacteria (7%). The abundance of Firmicutes and Proteobacteria were independently influenced by diet and lactation. Bacteroidetes contributed to a majority of the metabolic functions in first lactation dairy cows while the contribution from Firmicutes and Proteobacteria increased incrementally in second and third lactation dairy cows. We found that nearly 70% of the CAZymes were oligosaccharide breaking enzymes which reflect the higher starch and fermentable sugars in the diet. The results of this study suggest that the rumen microbiome continues to evolve as the dairy cow advances in lactations and these changes may have a significant role in milk production.

Organic acid blend with pure botanical product treatment reduces Escherichia coli and Salmonella populations in pure culture and in in vitro mixed ruminal microorganism fermentations.

Foodborne pathogenic bacteria can live in the intestinal tract of food animals and can be transmitted to humans via food or indirectly through animal or fecal contact. Organic acid blend products have been used as nonantibiotic modifiers of the gastrointestinal fermentation of food animals to improve growth performance efficiency. However, the impact of these organic acid products on the microbial population, including foodborne pathogens, remains unknown. Therefore, this study was designed to examine the effects of a commercial organic acid and botanical blend product (OABP) on populations of the foodborne pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium. Pure cultures (2×10(6) colony-forming units [CFU]/mL) of each pathogen were added to tubes that contained water-solubilized OABP added at concentrations of 0, 0.1, 0.5, 1, 2, 5, and 10% (vol/vol; n=3). Water-solubilized OABP reduced (p<0.05) the growth rate and final populations of E. coli O157:H7 and Salmonella Typhimurium in pure culture at concentrations >2%. E. coli O157:H7 and Salmonella Typhimurium were added (2×10(5) and 3×10(6) CFU/mL, respectively) to in vitro mixed ruminal microorganism fermentations that contained water-solubilized OABP at concentrations of 0, 1, 2, 5, and 10% (vol/vol; n=3) that were incubated for 24 h. OABP addition reduced (p<0.05) final populations of E. coli O157:H7 and Salmonella Typhimurium in the ruminal fluid at concentrations ≥5%. The acetate-to-propionate ratios from the in vitro fermentations were reduced (p<0.05) by OABP treatment ≥5%. Treatments to reduce foodborne pathogens must be economically feasible to implement, and results indicate that organic acid products, such as OABP, can enhance animal growth efficiency and can be used to reduce populations of pathogenic bacteria.

Microbiome-informed study of the mechanistic basis of methane inhibition by Asparagopsis taxiformis in dairy cattle.

Copious amounts of methane, a major constituent of greenhouse gases currently driving climate change, are emitted by livestock, and efficient methods that curb such emissions are urgently needed to reduce global warming. When fed to cows, the red seaweed Asparagopsis taxiformis (AT) can reduce enteric methane emissions by up to 80%, but the achieved results can vary widely. Livestock produce methane as a byproduct of methanogenesis, which occurs during the breakdown of feed by microbes in the rumen. The ruminant microbiome is a diverse ecosystem comprising bacteria, protozoa, fungi, and archaea, and methanogenic archaea work synergistically with bacteria to produce methane. Here, we find that an effective reduction in methane emission by high-dose AT (0.5% dry matter intake) was associated with a reduction in methanol-utilizing Methanosphaera within the rumen, suggesting that they may play a greater role in methane formation than previously thought. However, a later spike in Methanosphaera suggested an acquired resistance, possibly via the reductive dehalogenation of bromoform. While we found that AT inhibition of methanogenesis indirectly impacted ruminal bacteria and fermentation pathways due to an increase in spared H2, we also found that an increase in butyrate synthesis was due to a direct effect of AT on butyrate-producing bacteria such as Butyrivibrio, Moryella, and Eubacterium. Together, our findings provide several novel insights into the impact of AT on both methane emissions and the microbiome, thereby elucidating additional pathways that may need to be targeted to maintain its inhibitory effects while preserving microbiome health and animal productivity. IMPORTANCE: Livestock emits copious quantities of methane, a major constituent of the greenhouse gases currently driving climate change. Methanogens within the bovine rumen produce methane during the breakdown of feed. While the red seaweed Asparagopsis taxiformis (AT) can significantly reduce methane emissions when fed to cows, its effects appear short-lived. This study revealed that the effective reduction of methane emissions by AT was accompanied by the near-total elimination of methane-generating Methanosphaera. However, Methanosphaera populations subsequently rebounded due to their ability to inactivate bromoform, a major inhibitor of methane formation found in AT. This study presents novel findings on the contribution of Methanosphaera to ruminal methanogenesis, the mode of action of AT, and the possibility for complementing different strategies to effectively curb methane emissions.

The effect of 3-nitrooxypropanol, a potent methane inhibitor, on ruminal microbial gene expression profiles in dairy cows.

BACKGROUND: Enteric methane emissions from dairy cows are an environmental problem as well as a gross feed energy loss to the animal. Methane is generated in the rumen by methanogenic archaea from hydrogen (H2) + carbon dioxide and from H2 + methanol or methylamines. The methanogenic substrates are provided by non-methanogens during feed fermentation. Methane mitigation approaches have yielded variable results, partially due to an incomplete understanding of the contribution of hydrogenotrophic and methylotrophic archaea to methanogenesis. Research indicates that 3-nitrooxypropanol (3-NOP) reduces enteric methane formation in dairy cows by inhibiting methyl-coenzyme M reductase (MCR), the enzyme responsible for methane formation. The purpose of this study was to utilize metagenomic and metatranscriptomic approaches to investigate the effect of 3-NOP on the rumen microbiome and to determine the fate of H2 that accumulates less than expected under inhibited methanogenesis. RESULTS: The inhibitor 3-NOP was more inhibitory on Methanobrevibacter species than methanol-utilizing Methanosphaera and tended to reduce the gene expression of MCR. Under inhibited methanogenesis by 3-NOP, fluctuations in H2 concentrations were accompanied by changes in the expression of [FeFe] hydrogenases in H2-producing bacteria to regulate the amount of H2 production. No previously reported alternative H2 sinks increased under inhibited methanogenesis except for a significant increase in gene expression of enzymes involved in the butyrate pathway. CONCLUSION: By taking a metatranscriptomic approach, this study provides novel insights on the contribution of methylotrophic methanogens to total methanogenesis and regulation of H2 metabolism under normal and inhibited methanogenesis by 3-NOP in the rumen. Video Abstract.

Proof of concept for developing novel feeds for cattle from wasted food and crop biomass to enhance agri-food system efficiency.

Modern agri-food systems generate large amounts of crop-based biomass that are unfit for direct human consumption but potentially suitable for livestock feeding in production of meats, milk, and eggs. This study aims to develop novel feeds for cattle from some of those biomass materials through the natural microbial-driven processes of ensiling. Fruit and vegetables resembling supermarket discards were ensiled alone or co-ensiled with corn crop residues, mushroom wastes, etc. via laboratory experiments. Longitudinal sample analyses showed that (co-)ensiling was successful, with pH and fermentation acids changing rapidly into desirable ranges (pH < 4.5, the acids 5-13% DM with lactic acid dominating). The (co-)ensiled products had key nutritional parameters comparable to those of good quality forages commonly used on dairy farms. Additionally, in vitro incubation experiments indicated that the ensiled products could substitute certain conventional feeds while maintaining diet digestibility. Findings from this pilot study provide a proof of principle that quality novel feeds for cattle can be generated by co-ensiling food discards and low-value crop residues. Future research and animal feeding trials to demonstrate the utility of this approach can help societies more effectively utilize untapped biomass resources, strengthening the regenerative capacity of agri-food systems towards a more sustainable food future.

Rumen bacterial diversity dynamics associated with changing from bermudagrass hay to grazed winter wheat diets.

Rumen bacterial communities in forage-fed and grazing cattle continually adapt to a wide range of changing dietary composition, nutrient density, and environmental conditions. We hypothesized that very distinct community assemblages would develop between the fiber and liquid fractions of rumen contents in animals transitioned from bermudagrass hay diet to a grazed wheat diet. To address this hypothesis, we designed an experiment utilizing a 16S-based bTEFAP pyrosequencing technique to characterize and elucidate changes in bacterial diversity among the fiber and liquid rumen fractions and whole rumen contents of 14 (Angus x Hereford) ruminally cannulated steers sequentially fed bermudagrass hay (Cynodon dactylon; 34 days) and grazing wheat forage (28 days). Bermudagrass hay was a conserved C4 perennial grass lower in protein and higher in fiber (11% and 67%, respectively) content than grazed winter wheat (Triticum aestivum), a C3 annual grass with higher protein (20%) and a large (66%) soluble fraction.Significant differences in the OTU estimates (Chao1, Ace,and Rarefaction) were detected between fractions of both diets, with bermudagrass hay supporting greater diversity than wheat forage. Sequences were compared with a 16S database using BLASTn and assigned sequences to respective genera and genera-like units based on the similarity value to known sequences in the database. Predominant genera were Prevotella (up to 33%) and Rikenella-like (upto 28%) genera on the bermudagrass diet and Prevotella (upto 56%) genus on the wheat diet irrespective of the fractions. Principle component analyses accounted for over 95% of variation in 16S estimated bacterial community composition in all three fractions and clearly differentiated communities associated with each diet. Overall, bermudagrass hay diets clustered more clearly than wheat diets.These data are the first to explore bacterial diversity dynamics in a common population of animals in response to contrasting grass forage diets.

The distribution of microbiomes and resistomes across farm environments in conventional and organic dairy herds in Pennsylvania.

BACKGROUND: Antimicrobial resistance is a serious concern. Although the widespread use of antimicrobials in livestock has exacerbated the emergence and dissemination of antimicrobial resistance genes (ARG) in farm environments, little is known about whether antimicrobial use affects distribution of ARG in livestock systems. This study compared the distribution of microbiomes and resistomes (collections of ARG) across different farm sectors in dairy herds that differed in their use of antimicrobials. Feces from heifers, non-lactating, and lactating cows, manure storage, and soil from three conventional (antimicrobials used to treat cows) and three organic (no antimicrobials used for at least four years) farms in Pennsylvania were sampled. Samples were extracted for genomic DNA, processed, sequenced on the Illumina NextSeq platform, and analyzed for microbial community and resistome profiles using established procedures. RESULTS: Microbial communities and resistome profiles clustered by sample type across all farms. Overall, abundance and diversity of ARG in feces was significantly higher in conventional herds compared to organic herds. The ARG conferring resistance to betalactams, macrolide-lincosamide-streptogramin (MLS), and tetracyclines were significantly higher in fecal samples of dairy cows from conventional herds compared to organic herds. Regardless of farm type, all manure storage samples had greater diversity (albeit low abundance) of ARG conferring resistance to aminoglycosides, tetracyclines, MLS, multidrug resistance, and phenicol. All soil samples had lower abundance of ARG compared to feces, manure, and lagoon samples and were comprised of ARG conferring resistance to aminoglycosides, glycopeptides, and multi-drug resistance. The distribution of ARG is likely driven by the composition of microbiota in the respective sample types. CONCLUSIONS: Antimicrobial use on farms significantly influenced specific groups of ARG in feces but not in manure storage or soil samples.

Dysbiosis of the Fecal Microbiota in Cattle Infected with Mycobacterium avium subsp. paratuberculosis.

Johne's disease (JD) is a chronic, intestinal infection of cattle, caused by Mycobacterium avium subsp. paratuberculosis (MAP). It results in granulomatous inflammation of the intestinal lining, leading to malabsorption, diarrhea, and weight loss. Crohn's disease (CD), a chronic, inflammatory gastrointestinal disease of humans, has many clinical and pathologic similarities to JD. Dysbiosis of the enteric microbiota has been demonstrated in CD patients. It is speculated that this dysbiosis may contribute to the intestinal inflammation observed in those patients. The purpose of this study was to investigate the diversity patterns of fecal bacterial populations in cattle infected with MAP, compared to those of uninfected control cattle, using phylogenomic analysis. Fecal samples were selected to include samples from 20 MAP-positive cows; 25 MAP-negative herdmates; and 25 MAP-negative cows from a MAP-free herd. The genomic DNA was extracted; PCR amplified sequenced on a 454 Roche platform, and analyzed using QIIME. Approximately 199,077 reads were analyzed from 70 bacterial communities (average of 2,843 reads/sample). The composition of bacterial communities differed between the 3 treatment groups (P < 0.001; Permanova test). Taxonomic assignment of the operational taxonomic units (OTUs) identified 17 bacterial phyla across all samples. Bacteroidetes and Firmicutes constituted more than 95% of the bacterial population in the negative and exposed groups. In the positive group, lineages of Actinobacteria and Proteobacteria increased and those of Bacteroidetes and Firmicutes decreased (P < 0.001). Actinobacteria was highly abundant (30% of the total bacteria) in the positive group compared to exposed and negative groups (0.1-0.2%). Notably, the genus Arthrobacter was found to predominate Actinobacteria in the positive group. This study indicates that MAP-infected cattle have a different composition of their fecal microbiota than MAP-negative cattle.

Associative patterns among anaerobic fungi, methanogenic archaea, and bacterial communities in response to changes in diet and age in the rumen of dairy cows.

The rumen microbiome represents a complex microbial genetic web where bacteria, anaerobic rumen fungi (ARF), protozoa and archaea work in harmony contributing to the health and productivity of ruminants. We hypothesized that the rumen microbiome shifts as the dairy cow advances in lactations and these microbial changes may contribute to differences in productivity between primiparous (first lactation) and multiparous (≥second lactation) cows. To this end, we investigated shifts in the ruminal ARF and methanogenic communities in both primiparous (n = 5) and multiparous (n = 5) cows as they transitioned from a high forage to a high grain diet upon initiation of lactation. A total of 20 rumen samples were extracted for genomic DNA, amplified using archaeal and fungal specific primers, sequenced on a 454 platform and analyzed using QIIME. Community comparisons (Bray-Curtis index) revealed the effect of diet (P < 0.01) on ARF composition, while archaeal communities differed between primiparous and multiparous cows (P < 0.05). Among ARF, several lineages were unclassified, however, phylum Neocallimastigomycota showed the presence of three known genera. Abundance of Cyllamyces and Caecomyces shifted with diet, whereas Orpinomyces was influenced by both diet and age. Methanobrevibacter constituted the most dominant archaeal genus across all samples. Co-occurrence analysis incorporating taxa from bacteria, ARF and archaea revealed syntrophic interactions both within and between microbial domains in response to change in diet as well as age of dairy cows. Notably, these interactions were numerous and complex in multiparous cows, supporting our hypothesis that the rumen microbiome also matures with age to sustain the growing metabolic needs of the host. This study provides a broader picture of the ARF and methanogenic populations in the rumen of dairy cows and their co-occurrence implicates specific relationships between different microbial domains in response to diet and age.

Bacterial diversity dynamics associated with different diets and different primer pairs in the rumen of Kankrej cattle.

The ruminal microbiome in herbivores plays a dominant role in the digestion of lignocellulose and has potential to improve animal productivity. Kankrej cattle, a popular native breed of the Indian subcontinent, were used to investigate the effect of different dietary treatments on the bacterial diversity in ruminal fractions using different primer pairs. Two groups of four cows were assigned to two primary diets of either dry or green forages. Each group was fed one of three dietary treatments for six weeks each. Dietary treatments were; K1 (50% dry/green roughage: 50% concentrate), K2 (75% dry/green roughage: 25% concentrate) and K3 (100% dry/green roughage). Rumen samples were collected using stomach tube at the end of each dietary period and separated into solid and liquid fractions. The DNA was extracted and amplified for V1-V3, V4-V5 and V6-V8 hypervariable regions using P1, P2 and P3 primer pairs, sequenced on a 454 Roche platform and analyzed using QIIME. Community compositions and the abundance of most bacterial lineages were driven by interactions between primer pair, dietary treatment and fraction. The most abundant bacterial phyla identified were Bacteroidetes and Firmicutes however, the abundance of these phyla varied between different primer pairs; in each primer pair the abundance was dependent on the dietary treatment and fraction. The abundance of Bacteroidetes in cattle receiving K1 treatment indicate their diverse functional capabilities in the digestion of both carbohydrate and protein while the predominance of Firmicutes in the K2 and K3 treatments signifies their metabolic role in fibre digestion. It is apparent that both liquid and solid fractions had distinct bacterial community patterns (P<0.001) congruent to changes in the dietary treatments. It can be concluded that the P1 primer pair flanking the V1-V3 hyper-variable region provided greater species richness and diversity of bacterial populations in the rumen of Kankrej cattle.