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.

Assessment of fecal bacterial viability and diversity in fresh and frozen fecal microbiota transplant (FMT) product in horses.

BACKGROUND: Currently, lack of standardization for fecal microbiota transplantation (FMT) in equine practice has resulted in highly variable techniques, and there is no data on the bacterial metabolic activity or viability of the administered product. The objectives of this study were to compare the total and potentially metabolically active bacterial populations in equine FMT, and assess the effect of different frozen storage times, buffers, and temperatures on an equine FMT product. Fresh feces collected from three healthy adult horses was subjected to different storage methods. This included different preservation solutions (saline plus glycerol or saline only), temperature (-20 °C or -80 °C), and time (fresh, 30, 60, or 90 days). Samples underwent DNA extraction to assess total bacterial populations (both live and dead combined) and RNA extraction followed by reverse transcription to cDNA as a proxy to assess viable bacteria, then 16s rRNA gene amplicon sequencing using the V1-V2 region. RESULTS: The largest difference in population indices and taxonomic composition at the genus level was seen when evaluating the results of DNA-based (total) and cDNA-based (potentially metabolically active) extraction method. At the community level, alpha diversity (observed species, Shannon diversity) was significantly decreased in frozen samples for DNA-based analysis (P < 0.05), with less difference seen for cDNA-based sequencing. Using DNA-based analysis, length of storage had a significant impact (P < 0.05) on the bacterial community profiles. For potentially metabolically active populations, storage overall had less of an effect on the bacterial community composition, with a significant effect of buffer (P < 0.05). Individual horse had the most significant effect within both DNA and cDNA bacterial communities. CONCLUSIONS: Frozen storage of equine FMT material can preserve potentially metabolically active bacteria of the equine fecal microbiome, with saline plus glycerol preservation more effective than saline alone. Larger studies are needed to determine if these findings apply to other individual horses. The ability to freeze FMT material for use in equine patients could allow for easier clinical use of fecal transplant in horses with disturbances in their intestinal microbiome.

Acidification of colostrum affects the fecal microbiota of preweaning dairy calves.

Calf diarrhea is a leading cause of death in preweaning calves and it causes major economic losses to producers. Acidified milk has been shown to have beneficial effects on health and growth parameters in calves but there is little research into its effects on the microbiota, and few studies on the use of acidified colostrum. The purpose of this study was to compare how feeding acidified colostrum to calves at birth affects fecal microbiota from birth through 8 wk of age compared with calves fed nonacidified colostrum. In this study, 5 calves received acidified colostrum (treated group) and 5 calves received nonacidified colostrum (control group) at birth and at 12 h of age. All calves were subsequently fed acidified whole milk until weaning at 8 wk of age and had access to starter grain starting at d 3 and throughout the study. Fecal samples were collected at 24 h, 48 h, and at 1, 2, 3, 4, 5, 6, 7, and 8 wk of age. Samples were extracted for genomic DNA, PCR-amplified for the V1-V2 region of the 16S rRNA bacteria gene, sequenced, and analyzed using QIIME2. Bacterial richness (estimated by number of observed species) and bacterial diversity (estimated by Shannon diversity index) differed between time points but not between treatment groups, and both increased over time. Weighted and unweighted UniFrac analysis showed differences between bacterial communities across time points and treatments. Across all time points (lmer test), 6 bacterial genera were different between treatments: Faecalibacterium and unclassified Clostridiaceae were more abundant, whereas Atopobium, Collinsella, CF231, and unclassified Veillonellaceae were less abundant in treated versus control calves. Faecalibacterium is a butyrate-producing bacterium that has been linked to decreased prevalence of diarrhea in calves. Our results indicate that there is considerable flux in the calf microbiome through the neonatal period and weaning transition but that feeding acidified colostrum followed by acidified whole milk allowed early colonization of Faecalibacterium. Further studies are needed to verify the positive benefits of promoting Faecalibacterium on improving the health of preweaning calves.

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.

Using Structural Equation Modeling to Understand Interactions Between Bacterial and Archaeal Populations and Volatile Fatty Acid Proportions in the Rumen.

Microbial syntrophy (obligate metabolic mutualism) is the hallmark of energy-constrained anaerobic microbial ecosystems. For example, methanogenic archaea and fermenting bacteria coexist by interspecies hydrogen transfer in the complex microbial ecosystem in the foregut of ruminants; however, these synergistic interactions between different microbes in the rumen are seldom investigated. We hypothesized that certain bacteria and archaea interact and form specific microbial cohorts in the rumen. To this end, we examined the total (DNA-based) and potentially metabolically active (cDNA-based) bacterial and archaeal communities in rumen samples of dairy cows collected at different times in a 24 h period. Notably, we found the presence of distinct bacterial and archaeal networks showing potential metabolic interactions that were correlated with molar proportions of specific volatile fatty acids (VFAs). We employed hypothesis-driven structural equation modeling to test the significance of and to quantify the extent of these relationships between bacteria-archaea-VFAs in the rumen. Furthermore, we demonstrated that these distinct microbial networks were host-specific and differed between cows indicating a natural variation in specific microbial networks in the rumen of dairy cows. This study provides new insights on potential microbial metabolic interactions in anoxic environments that have broader applications in methane mitigation, energy conservation, and agricultural production.

Short communication: Comparison of the fecal bacterial communities in diarrheic and nondiarrheic dairy calves from multiple farms in southeastern Pennsylvania.

Diarrhea is a major cause of illness and death in preweaned calves and causes significant economic losses to producers. A better understanding of the fecal microbiota in diarrheic and nondiarrheic calves could lead to improved treatment and prevention strategies. The purpose of this study was to compare the fecal microbiota of diarrheic and nondiarrheic calves to improve our understanding of what constitutes a healthy fecal microbiota in preweaned calves. At each of 7 farms, fecal samples were obtained from 1 to 3 diarrheic Holstein dairy calves (2 to 17 d old at sampling time) and age-matched (within 5 d) nondiarrheic controls for a total of 20 samples. Calves were fed either acidified bulk milk, pasteurized or unpasteurized waste milk, or milk replacer depending on farm. Fecal samples were extracted for genomic DNA, PCR-amplified for the V1-V2 region of the 16S rRNA bacterial gene, sequenced on the Illumina MiSeq (Illumina Inc., San Diego, CA) platform, and analyzed using QIIME2. Firmicutes and Bacteroidetes were the most abundant phyla in both groups; Fusobacteria was numerically more abundant in the diarrheic group, whereas Proteobacteria and Actinobacteria were numerically more abundant in the nondiarrheic group. At the genus level, Bacteroides was the most abundant genus in both groups and was numerically more abundant in the nondiarrheic group. Results from the mixed-effects regression model showed that Faecalibacterium and Butyricimonas were more abundant in the nondiarrheic calves, whereas Clostridium and Peptostreptococcus were more abundant in the diarrheic calves. Our results indicate that commensal bacteria acquired in the neonatal period may have been replaced with potential pathogens in diarrheic calves, which may have contributed to the incidence of diarrhea either directly or indirectly.

Experimental crossover study on the effects of withholding feed for 24 h on the equine faecal bacterial microbiota in healthy mares.

BACKGROUND: An association between equine gastrointestinal disease causing colic signs and changes in faecal bacterial microbiota has been identified. The reasons for these changes and their clinical relevance has not been investigated. Withholding feed, which is an integral part of managing horses with colic, may contribute to the observed changes in the microbiota and impact interpretation of findings in horses with colic. Study objectives were, therefore, to determine the effect of withholding feed for 24 h on equine faecal bacterial microbiota in healthy mares to differentiate the effects of withholding feed from the changes potentially associated with the disease. RESULTS: Species richness and Shannon diversity (alpha diversity) were significantly lower at the late withheld (10-24 h post withholding feed) and early refed (2-12 h post re-feeding) time points compared to samples from fed horses (P < 0.01). Restoration of species richness and diversity began to occur at the late refed (18-24 h post re-feeding) time points. Horses having feed withheld had a distinct bacterial population compared to fed horses (beta diversity). Bacteroidetes BS11 and Firmicutes Christensenellaceae, Christensenella, and Dehalobacteriaceae were significantly increased in horses withheld from feed primarily during the late withheld and early refed time points. Bacteroidetes Marinilabiaceae and Prevotellaceae, Firmicutes Veillonellaceae, Anaerovibrio, and Bulleidia, and Proteobacteria GMD14H09 were significantly decreased in horses with feed withheld at late withheld, early refed, and late refed time periods (P < 0.01). Changes in commensal gut microbiota were not significant between groups. CONCLUSIONS: Withholding feed has a significant effect on faecal bacterial microbiota diversity and composition particularly following at least 10 h of withholding feed and should be taken into consideration when interpreting data on the equine faecal bacterial microbiota in horses.

Changes in the faecal bacterial microbiota during hospitalisation of horses with colic and the effect of different causes of colic.

BACKGROUND: Previous studies have identified alterations in the faecal microbiota of horses with colic; however, further work is needed to interpret these findings. OBJECTIVES: To compare the faecal microbiota of horses presenting for colic at hospital admission, day 1 and day 3/discharge and with different colic duration and lesion locations. STUDY DESIGN: Prospective observational clinical study. METHODS: Faecal samples were collected from 17 colic cases at hospital admission, on day 1 and on day 3 post-admission or at the time of hospital discharge if prior to 72 hours. Faecal samples were extracted for genomic DNA, PCR-amplified, sequenced and analysed using QIIME. Species richness and Shannon diversity (alpha diversity) were estimated. The extent of the relationship between bacterial communities (beta diversity) was quantified using pairwise UniFrac distances, visualised using principal coordinate analysis (PCoA) and statistically analysed using permutational multivariate analysis of variance (PERMANOVA). The relative abundance of bacterial populations at the different time points and in different types of colic was compared using ANCOM. RESULTS: There was a decrease in species richness from admission to day 3/hospital discharge (P < .05), and a lower species richness (P = .005) and Shannon diversity (P = .02) in horses with colic ≥60 h compared to <60 h. Based on PCoA and PERMANOVA, there was a significant difference in bacterial community composition for horses with different colic duration (P = .001) and lesion location (P = .006). Several differences in bacterial phyla and genera were observed at different time points and with different types of colic. MAIN LIMITATIONS: Relatively low numbers and a diverse population of horses. CONCLUSIONS: The microbiota change from hospital admission to day 3/discharge in horses with colic and horses with colic ≥60 h and large colon lesions have a distinct bacterial population compared to horses with colic <60 h and small intestinal lesions.

Temporal changes in the fecal bacterial community in Holstein dairy calves from birth through the transition to a solid diet.

The development of a robust microbiome is critical to the health of dairy calves, but relatively little is known about the progression of the microbiome through the weaning transition. In this study, fecal samples were obtained from ten female Holstein calves at 6 timepoints between 2-13 weeks of age. Calves were fed acidified milk until weaning at 8 weeks old and had access to starter grain throughout the study. Fecal samples were extracted for genomic DNA, PCR-amplified for the V1-V2 region of the 16S rRNA bacterial gene, sequenced on the Illumina MiSeq platform, and analyzed using the QIIME2 pipeline. Bacterial richness, estimated by number of observed species, and bacterial diversity, estimated by Shannon diversity index, both differed significantly between timepoints and both increased over time (P <0.05), with the largest increases occurring during weaning. Weighted and unweighted UniFrac analysis showed significant differences (P <0.05) between bacterial communities across timepoints; betadisper analysis revealed that the microbiomes of individual calves became more similar with time. Throughout the study, Firmicutes was the dominant phylum, followed by Bacteroidetes. Thirteen bacterial genera were found to be significantly influenced by time, including Faecalibacterium, Clostridium, unclassified S24-7, Collinsella, Sharpea, and Treponema. Unclassified Ruminococcaceae was the most prevalent genus at timepoints 1, 3, 5, and 6, but different amplicon sequence variants were detected at each timepoint suggesting the presence of different species of Ruminococcaceae at different times. Bacteroides was the most prevalent genus at timepoint 2, and Prevotella was most prevalent at timepoint 4. Our results indicate that there is considerable variation in the calf microbiome pre-weaning, but the microbial community stabilizes and becomes similar to the adult microbiome at weaning. Further studies to describe the phylogeny and functionality of core microbiota through the weaning transition are needed to improve health and reduce diarrhea in the neonatal period.

Comparison of Two Sampling Techniques for Evaluating Ruminal Fermentation and Microbiota in the Planktonic Phase of Rumen Digesta in Dairy Cows.

The objective of this experiment was to compare ruminal fluid samples collected through rumen cannula (RC) or using an oral stomach tube (ST) for measurement of ruminal fermentation and microbiota variables. Six ruminally cannulated lactating Holstein cows fed a standard diet were used in the study. Rumen samples were collected at 0, 2, 4, 6, 8, and 12 h after the morning feeding on two consecutive days using both RC and ST techniques. Samples were filtered through two layers of cheesecloth and the filtered ruminal fluid was used for further analysis. Compared with RC, ST samples had 7% greater pH; however, the pattern in pH change after feeding was similar between sampling methods. Total volatile fatty acids (VFA), acetate and propionate concentrations in ruminal fluid were on average 23% lower for ST compared with RC. There were no differences between RC and ST in VFA molar proportions (except for isobutyrate), ammonia and dissolved hydrogen (dH2) concentrations, or total protozoa counts, and there were no interactions between sampling technique and time of sampling. Bacterial ASV richness was higher in ST compared with RC samples; however, no differences were observed for Shannon diversity. Based on Permanova analysis, bacterial community composition was influenced by sampling method and there was an interaction between sampling method and time of sampling. A core microbiota comprised of Prevotella, S24-7, unclassified Bacteroidales and unclassified Clostridiales, Butyrivibrio, unclassified Lachnospiraceae, unclassified Ruminococcaceae, Ruminococcus, and Sharpea was present in both ST and RC samples, although their relative abundance varied and was influenced by an interaction between sampling time and sampling method. Overall, our results suggest that ruminal fluid samples collected using ST (at 180 to 200 cm depth) are not representative of rumen pH, absolute values of VFA concentrations, or bacterial communities >2 h post-feeding when compared to samples of ruminal fluid collected using RC. However, ST can be a feasible sampling technique if the purpose is to study molar proportions of VFA, protozoa counts, dH2, and ammonia concentrations.

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.

Characterization of the fecal microbiota of healthy horses.

OBJECTIVE To characterize the fecal microbiota of horses and to investigate alterations in that microbiota on the basis of sample collection site (rectum vs stall floor), sample location within the fecal ball (center vs surface), and duration of environmental exposure (collection time). ANIMALS 6 healthy adult mixed-breed mares. PROCEDURES From each horse, feces were collected from the rectum and placed on a straw-bedded stall floor. A fecal ball was selected for analysis immediately after removal from the rectum and at 0 (immediately), 2, 6, 12, and 24 hours after placement on the stall floor. Approximately 250 mg of feces was extracted from the surface and center of each fecal ball, and genomic DNA was extracted, purified, amplified for the V1-V2 hypervariable region of the 16S rDNA gene, and analyzed with a bioinformatics pipeline. RESULTS The fecal microbiota was unique for each horse. Bacterial community composition varied significantly between center and surface fecal samples but was not affected by collection time. Bacterial community composition varied rapidly for surface fecal samples. Individual bacterial taxa were significantly associated with both sample location and collection time but remained fairly stable for up to 6 hours for center fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that, for horses, fecal samples for microbiota analysis should be extracted from the center of fecal balls collected within 6 hours after defecation. Samples obtained up to 24 hours after defecation can be analyzed with the realization that some bacterial populations may deviate from those immediately after defecation.

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.

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.

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.

A comparison of rumen microbial profiles in dairy cows as retrieved by 454 Roche and Ion Torrent (PGM) sequencing platforms.

Next generation sequencing (NGS) technology is a widely accepted tool used by microbial ecologists to explore complex microbial communities in different ecosystems. As new NGS platforms continue to become available, it becomes imperative to compare data obtained from different platforms and analyze their effect on microbial community structure. In the present study, we compared sequencing data from both the 454 and Ion Torrent (PGM) platforms on the same DNA samples obtained from the rumen of dairy cows during their transition period. Despite the substantial difference in the number of reads, error rate and length of reads among both platforms, we identified similar community composition between the two data sets. Procrustes analysis revealed similar correlations (M (2) = 0.319; P = 0.001) in the microbial community composition between the two platforms. Both platforms revealed the abundance of the same bacterial phyla which were Bacteroidetes and Firmicutes; however, PGM recovered an additional four phyla. Comparisons made at the genus level by each platforms revealed differences in only a few genera such as Prevotella, Ruminococcus, Succiniclasticum and Treponema (p < 0.05; chi square test). Collectively, we conclude that the output generated from PGM and 454 yielded concurrent results, provided stringent bioinformatics pipelines are employed.

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.

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.