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.

Integrated meta-omics analyses reveal a role of ruminal microorganisms in ketone body accumulation and ketosis in lactating dairy cows.

The extent to which a nutrition-related disorder such as ketosis alters the ruminal microbiota or whether microbiota composition is related to ketosis and potential associations with host metabolism is unknown. We aimed to evaluate variations occurring in the ruminal microbiota of ketotic and nonketotic cows in the early postpartum period, and how those changes may affect the risk of developing the disease. Data on milk yield, dry matter intake (DMI), body condition score, and blood ß-hydroxybutyrate (BHB) concentrations at 21 d postpartum were used to select 27 cows, which were assigned (n = 9 per group) to a clinical ketotic (CK, 4.10 ± 0.72 mmol BHB/L, DMI 11.61 ± 0.49 kg/d, ruminal pH 7.55 ± 0.07), subclinical ketotic (SK, 1.36 ± 0.12 mmol BHB/L, DMI 15.24 ± 0.34 kg/d, ruminal pH 7.58 ± 0.08), or control (NK, 0.88 ± 0.14 mmol BHB/L, DMI 16.74 ± 0.67/d, ruminal pH 7.61 ± 0.03) group. Cows averaged 3.6 ± 0.5 lactations and a body condition score of 3.11 ± 0.34 at the time of sampling. After blood serum collection for metabolomics analysis (1H nuclear magnetic resonance spectra), 150 mL of ruminal digesta was collected from each cow using an esophageal tube, paired-end (2 × 300 bp) sequencing of isolated DNA from ruminal digesta was performed via Illumina MiSeq, and sequencing data were analyzed using QIIME2 (v 2020.6) to measure the ruminal microbiota composition and relative abundance. Spearman correlation coefficients were used to evaluate relationships between relative abundance of bacterial genera and concentrations of serum metabolites. There were more than 200 genera, with approximately 30 being significant between NK and CK cows. Succinivibrionaceae UCG 1 taxa decreased in CK compared with NK cows. Christensenellaceae (Spearman correlation coefficient = 0.6), Ruminococcaceae (Spearman correlation coefficient = 0.6), Lachnospiraceae (Spearman correlation coefficient = 0.5), and Prevotellaceae (Spearman correlation coefficient = 0.6) genera were more abundant in the CK group and were highly positively correlated with plasma BHB. Metagenomic analysis indicated a high abundance of predicted functions related to metabolism (37.7%), genetic information processing (33.4%), and Brite hierarchies (16.3%) in the CK group. The 2 most important metabolic pathways for butyrate and propionate production were enriched in CK cows, suggesting increased production of acetyl coenzyme A and butyrate and decreased production of propionate. Overall, the combined data suggested that microbial populations may be related to ketosis by affecting short-chain fatty acid metabolism and BHB accumulation even in cows with adequate feed intake in the early postpartum period.

Symposium review: Understanding the role of the rumen microbiome in enteric methane mitigation and productivity in dairy cows.

Ruminants are one of the largest sources of global CH4 emissions. This enteric CH4 is exclusively produced by methanogenic archaea as a natural product during microbial fermentation in the reticulorumen. As CH4 formation leads to a gross energy loss for the ruminant host and is also an environmental issue, several CH4 mitigation approaches have been investigated, but results have been inconsistent, which may be partially attributed to a lack of understanding of the mechanistic basis of methanogenesis and the effect of inhibitors on individual methanogenic lineages and other fermenting microbes in the rumen. Methanogenic archaea are obligatory anaerobes that can reduce CO2, methanol, or methylamines or cleave acetate to form CH4. Although methanogens work toward a common goal of generating energy through the formation of CH4, individual methanogenic lineages differ in their physiological and metabolic capabilities, which can differentially affect H2 transactions and CH4 formation. Using advanced omic approaches, recent research has revealed that less abundant methanol-utilizing Methanosphaera and methylamine- and methanol-utilizing Methanomassiliicoccales lineages are positively correlated with CH4 emissions and may have a greater share in overall CH4 production compared with more abundant CO2-reducing methanogens than previously thought. These data imply that the diversity as well as the abundance of methanogens is important in CH4 formation, and that this diversity is influenced by H2 availability and interactions within and between H2-producing microbes in the rumen. These complex interactions between microbes and H2 are further influenced by variations in dietary, host, and environmental conditions. This review discusses critical knowledge gaps underlying methanogen diversity and its link to CH4 formation, formation of specific bacteria-archaeal cohorts, and how H2 production and utilization are regulated between these cohorts during normal and inhibited methanogenesis. Addressing these knowledge gaps has the potential to lead to the development of novel strategies or to complement existing strategies to effectively reduce CH4 formation while also improving productivity in dairy cows.

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.

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.

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.

Establishment of methanogen bacterial interactions during the preweaning period of dairy cattle.

Ruminant livestock are major contributors to anthropogenic methane emissions in the United States and worldwide. Enteric methane is generated by methanogenic archaea residing in ruminant digestive tracts. Information on when methanogens colonize the gut and when they begin to interact with bacteria during the early phases of the ruminant life cycle is less explored. The objectives of this study were (i) to investigate the composition of the methanogenic archaeal community at birth and through the weaning transition and (ii) to determine if and when the methanogenic archaea begin to interact with bacteria in the lower gut of neonatal dairy calves. Ten female Holstein calves (approximately 45kg birth weight) were enrolled in the study. Fecal samples were collected every two weeks (Wk 2, 4, 6, 8, 10, and 12) between birth and weaning and analyzed for methanogenic archaeal diversity via 16S rRNA amplicon sequencing and quantitative real-time PCR (RT-qPCR). Estimates of alpha diversity (Observed species, and Shannon diversity index) and beta diversity (weighted and unweighted UniFrac distances) showed significant differences (P < 0.05) between archaeal communities across timepoints. Both 16S rRNA amplicon sequencing and RT-qPCR analyses revealed Methanobrevibacter was the most prevalent genus at Wk2, Wk4, and Wk6, whereas Methanosphaera gradually increased with time and was most abundant at Wk10 and Wk12. Correlation analysis revealed that Methanobrevibacter and Methanosphaera were inversely correlated with each other and formed distinct cohorts with specific bacterial lineages similar to those reported in the mature rumen, thus revealing that these associations are established during the preweaning period. Therefore, the preweaning period presents a window of opportunity to interfere with early-life methanogenic colonization with the ultimate goal of reducing enteric methane emissions without perturbing ruminal function later in the life of dairy cattle.

Effects of dietary zinc on the gut microbiome and resistome of the gestating cow and neonatal calf.

Zinc is an essential trace element required in the diet of all species. While the effects of zinc have been studied in growing calves, little is known about the effect of zinc on the microbiota of the gestating cow or her neonatal calf. Understanding factors that shape the gut health of neonatal animals and evaluating the effect of dietary supplements in adult gestating animals is important in promoting animal health and informing feeding practices. The aims of this study were to determine the effect of dietary zinc on the microbiota and resistome of the gestating cow and calf. Gestating cows received standard (40 ppm) or high (205 ppm) dietary zinc levels from dry off to calving. Fecal samples were collected from cows upon enrollment and at calving and from neonatal calves. Fecal samples underwent 16S rRNA sequencing and a subset also underwent shotgun metagenomic sequencing. The effect of zinc supplementation on the diversity and composition of the cow and calf microbiome and resistome was assessed. Alpha and beta diversity and composition of the microbiota were significantly altered over time but not by treatment in the cows, with alpha diversity decreasing and 14 genera found at significantly higher relative abundances at calving compared to enrollment. Levels of 27 antimicrobial resistance genes significantly increased over time. Only a small number of taxa were differentially expressed at calving in treatment and control groups, including Faecalibacterium, Bacteroides, Turicibacter, and Bifidobacterium pseudolongum. No effect of the dam's treatment group was observed on the diversity or composition of the neonatal calf microbiota. The calf resistome, which was relatively rich and diverse compared to the cow, was also unaffected by the dam's treatment group. The impact of high levels of dietary zinc thus appeared to be minimal, with no observed changes in alpha or beta diversity, and few changes in the relative abundance of a small number of taxa and antimicrobial resistance genes.

Characterization of the Ruminal Microbiota in Sheep and Goats Fed Different Levels of Tannin-Rich Sericea Lespedeza Hay.

Understanding ruminal microbiota and diet-host breed interactions under forage feeding conditions is essential for optimizing rumen fermentation and improving feed efficiency in small ruminants. This study aimed to investigate the effects of different ratios of condensed tannin (CT)-rich Sericea lespedeza (Lespedeza cuneate) in the diets on changes and interactions of ruminal microbiota and host species (i.e., sheep and goats). Katahdin sheep (n = 12) and Alpine goats (n = 12) at approximately 10 to 12 months of age were blocked by body weight (BW = 30.3 kg and 25.5 kg, respectively) and randomly assigned to one of the three treatments. Diets contained 75% coarsely ground forage and 25% concentrate. The forages were (1) 100% alfalfa hay (AL), (2) 100% Sericea lespedeza hay (SL), and (3) 50 % AL + 50% SL (ASL). In the present study, the diversity and composition of ruminal microbiota differed between sheep and goats fed similar diets. Based on the taxonomic analysis, there was a distinct clustering pattern (P < 0.05) for sheep by diets, but such a pattern was not observed for goats (P > 0.1). The most predominant phyla were Firmicutes, Bacteroidetes, Ascomycota, and methanogen species of Methanobrevibactor sp. in the rumen of sheep and goats, regardless of diets. The Bacteroidetes and Ascomycota were enriched in sheep fed AL and ASL. In contrast, these microbial phyla were enhanced in goats fed tannin-rich SL diets, with the diet by host species interaction (P < 0.02) for the Bacteroidetes phylum. Sheep rumen fluid samples showed a higher degree of variability in microbial community composition compared to goat rumen fluid samples. The relative proportion of the Aspergillus fungi population was reduced to 90.7% in the SL group compared with the AL group, regardless of host species. The antimicrobial activity of tannins and greater sensitivities of selected microbiota species to these tannin compounds during SL feeding in sheep and goats perhaps caused this difference. The results from this study suggest that differences in the microbiota were associated with differences in diets and host species. Therefore, this study provides a better understanding of ruminal microbiota and diet-host species interactions under various tannin-rich diets, which could advance consolidative information on rumen microbiome community diversity changes and may improve sheep and goat production.

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.

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.

Distinct Gut Microbiota Signatures in Mice Treated with Commonly Used Food Preservatives.

Diet is one of the most important factors regulating and influencing the composition of our gut microbiome, but the specific effects of commonly used antimicrobial agents i.e., food preservatives present within foods, are not completely understood. In this study, we examined the effect of the three widely used food-grade preservatives i.e., benzoic acid, potassium sorbate, and sodium nitrite, in recommended levels, on the gut microbiota diversity and composition in a mouse model. The analysis of ß-diversity reveals distinct signatures of the gut microbiota between mice consuming different preservatives. Further analyses of α-diversity indices also show that the three preservatives induce specific patterns of microbial diversity, with diversity being lowest in mice consuming potassium sorbate. In terms of bacterial abundance, each of the three preservatives demonstrated unique microbial signatures, mainly affecting the proportions of bacterial taxa belonging to Bacteroidetes, Verrucomicrobia, and Proteobacteria. Specifically, we find the increased proportion of Bacteroides, Blautia, Ruminococcus, Oscillospira, and Dorea in mice fed with benzoate; increased abundance of Firmicutes, Turicibacter, and Alkaliphilus by sodium nitrate; and increased proportion of Parabacteroides and Adlercreutzia by potassium sorbate. The findings improve our understanding of how food-grade preservatives may influence the gut microbiota composition and diversity and should facilitate prospective studies investigating diet-microbiome interactions in relation to intestinal and metabolic health.

Gut microbiota features associated with Clostridioides difficile colonization in dairy calves.

Diarrheal disease, a major cause of morbidity and mortality in dairy calves, is strongly associated with the health and composition of the gut microbiota. Clostridioides difficile is an opportunistic pathogen that proliferates and can produce enterotoxins when the host experiences gut dysbiosis. However, even asymptomatic colonization with C. difficile can be associated with differing degrees of microbiota disruption in a range of species, including people, swine, and dogs. Little is known about the interaction between C. difficile and the gut microbiota in dairy calves. In this study, we sought to define microbial features associated with C. difficile colonization in pre-weaned dairy calves less than 2 weeks of age. We characterized the fecal microbiota of 80 calves from 23 different farms using 16S rRNA sequencing and compared the microbiota of C. difficile-positive (n = 24) and C. difficile-negative calves (n = 56). Farm appeared to be the greatest source of variability in the gut microbiota. When controlling for calf age, diet, and farm location, there was no significant difference in Shannon alpha diversity (P = 0.50) or in weighted UniFrac beta diversity (P = 0.19) between C. difficile-positive and-negative calves. However, there was a significant difference in beta diversity as assessed using Bray-Curtiss diversity (P = 0.0077), and C. difficile-positive calves had significantly increased levels of Ruminococcus (gnavus group) (Adj. P = 0.052), Lachnoclostridium (Adj. P = 0.060), Butyricicoccus (Adj. P = 0.060), and Clostridium sensu stricto 2 compared to C. difficile-negative calves. Additionally, C. difficile-positive calves had fewer microbial co-occurrences than C. difficile-negative calves, indicating reduced bacterial synergies. Thus, while C. difficile colonization alone is not associated with dysbiosis and is therefore unlikely to result in an increased likelihood of diarrhea in dairy calves, it may be associated with a more disrupted microbiota.

Antibiotic resistance, antimicrobial residues, and bacterial community diversity in pasture-raised poultry, swine, and beef cattle manures.

Animal manure can be a source of antibiotic-resistant genes (ARGs) and pharmaceutical residues; however, few studies have evaluated the presence of ARG in pasture-raised animal production systems. The objective of this study was to examine changes in microbiome diversity and the presence of antibiotic residues (ABRs) on three farms that contained a diverse range of animal species: pasture-raised poultry (broiler and layer), swine, and beef cattle. Total bacterial communities were determined using 16S rRNA microbiome analysis, while specific ARGs (sulfonamide [Sul; Sul1] and tetracycline [Tet; TetA]) were enumerated by qPCR (real-time PCR). Results indicated that the ARG abundances (Sul1 [P < 0.05] and TetA [P < 0.001]) were higher in layer hen manures (16.5 × 10-4 and 1.4 × 10-4 µg kg-1, respectively) followed by broiler chickens (2.9 × 10-4 and 1.7 × 10-4 µg kg-1, respectively), swine (0.22 × 10-4 and 0.20 × 10-4 µg kg-1, respectively) and beef cattle (0.19 × 10-4 and 0.02 × 10-4 µg kg-1, respectively). Average fecal TetA ABR tended to be greater (P = 0.09) for broiler chickens (11.4 µg kg-1) than for other animal species (1.8 to 0.06 µg kg-1), while chlortetracycline, lincomycin, and oxytetracycline ABRs were similar among animal species. Furthermore, fecal microbial richness and abundances differed significantly (P < 0.01) both among farms and specific species of animal. This study indicated that the microbial diversity, ABR, ARG concentrations, and types in feces varied from farm-to-farm and from animal species-to-animal species. Future studies are necessary to perform detailed investigations of the horizontal transfer mechanism of antibiotic-resistant microorganisms (ARMs) and ARG.

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.