The rumen is a complex ecosystem composed of anaerobic bacteria, protozoa, fungi, methanogenic archaea and phages. These microbes interact closely to breakdown plant material that cannot be digested by humans, whilst providing metabolic energy to the host and, in the case of archaea, producing methane. Consequently, ruminants produce meat and milk, which are rich in high-quality protein, vitamins and minerals, and therefore contribute to food security. As the world population is predicted to reach approximately 9.7 billion by 2050, an increase in ruminant production to satisfy global protein demand is necessary, despite limited land availability, and whilst ensuring environmental impact is minimized. Although challenging, these goals can be met, but depend on our understanding of the rumen microbiome. Attempts to manipulate the rumen microbiome to benefit global agricultural challenges have been ongoing for decades with limited success, mostly due to the lack of a detailed understanding of this microbiome and our limited ability to culture most of these microbes outside the rumen. The potential to manipulate the rumen microbiome and meet global livestock challenges through animal breeding and introduction of dietary interventions during early life have recently emerged as promising new technologies. Our inability to phenotype ruminants in a high-throughput manner has also hampered progress, although the recent increase in “omic” data may allow further development of mathematical models and rumen microbial gene biomarkers as proxies. Advances in computational tools, high-throughput sequencing technologies and cultivation-independent “omics” approaches continue to revolutionize our understanding of the rumen microbiome. This will ultimately provide the knowledge framework needed to solve current and future ruminant livestock challenges.

The purpose of this study was to examine the stability and host specificity of a cow's ruminal bacterial community following massive challenge with ruminal microflora from another cow. In each of 2 experiments, 1 pair of cows was selected on the basis of differences in ruminal bacterial community composition (BCC), determined by automated ribosomal intergenic spacer analysis (ARISA), a culture-independent "community fingerprinting" technique. Each pair of cows was then subjected to a 1-time exchange of >95% of ruminal contents without changing the composition of a corn silage/alfalfa haylage-based TMR. In experiment 1, the 2 cows differed (P<0.01) in prefeed ruminal pH (mean = 6.88 vs. 6.14) and prefeed total VFA concentration (mean = 57 vs. 77 mM), averaged over 3 d. Following exchange of ruminal contents, ruminal pH and total VFA concentration in both cows returned to their preexchange values within 24h. Ruminal BCC also returned to near its original profile, but this change required 14 d for 1 cow and 61 d for the other cow. In experiment 2, the 2 other cows differed in prefeed ruminal pH (mean = 6.69 vs. 6.20) and total VFA concentration (mean = 101 vs. 136 mM). Following exchange of ruminal contents, the first cow returned to its preexchange pH and VFA values within 24h; the second cow's rumen rapidly stabilized to a higher prefeed pH (mean = 6.47) and lower prefeed VFA concentration (mean = 120 mM) that was retained over the 62-d test period. Both cows reached somewhat different BCC than before the exchange. However, the BCC of both cows remained distinct and were ultimately more similar to that of the preexchange BCC than of the donor animal BCC. The data indicate that the host animal can quickly reestablish its characteristic ruminal pH and VFA concentration despite dramatic perturbation of its ruminal microbial community. The data also suggest that ruminal BCC displays substantial host specificity that can reestablish itself with varying success when challenged with a microbial community optimally adapted to ruminal conditions of a different host animal.

Previous work indicated that Streptococcus bovis HC5 had significant antibacterial activity, and even nisin-resistant S. bovis JB1 cells could be strongly inhibited. S. bovis HC5 inhibited a variety of Gram-positive bacteria and the spectrum of activity was similar to monensin, a commonly used feed additive. The crude extracts (ammonium sulfate precipitation) were inactivated by Pronase E and trypsin, but the activity was resistant to heat, proteinase K and α-chymotrypsin. Most of the antibacterial activity was cell associated, but it could be liberated by acidic NaCl (100 mM, pH 20) without significant cell lysis. When glycolysing S. bovis JB1 cells were treated with either crude or acidic NaCl extracts, intracellular potassium declined and this result indicated the antibacterial activity was mediated by a pore-forming peptide. The peptide could be purified by HPLC and matrix-assisted laser desorption ionization time-of-flight analysis indicated that it had a molecular mass of approximately 2440 Da. The terminal amino acid sequence was VGXRYASXPGXSWKYVXF. The unnamed amino acid residues (designated by X) had approximately the same position as dehydroalanines found in some lantibiotics, but samples that were reduced and alkylated prior to Edman degradation did not have cysteine residues. The only other bacteriocin that had significant similarity was the lantibiotic precursor of Streptococcus pyogenes SF370, but the identity was only 55 %. Based on these results, the bacteriocin of S. bovis HC5 appears to be novel and the authors now designate it as bovicin HC5.

This work aimed to parameterize the ruminal degradation of neutral detergent fibre (NDF) from low-quality tropical forage using Michaelis-Menten kinetics. The intake, rumen outflow (L), fractional degradation rate (kd), discrete lag (LAG) and effective degradability (ED) of NDF, and the microbial flow of nitrogenous compounds into the small intestine (Nmic) were assessed in two 5 × 5 Latin square experiments by using five Holstein × Zebu heifers cannulated in the rumen. The experiments were carried out sequentially and the treatments were formed by increasing the level of supplementation with nitrogenous compounds. A low-quality signal grass (Brachiaria decumbens) hay was used as roughage. The nitrogen supplement was a mixture of urea, ammonium sulfate and albumin, at the ratios of 4.5:0.5:1.0, respectively. The crude protein contents in the diets ranged from 51.9 to 136.3 g/kg of dry matter. The rumen ammonia nitrogen (RAN) concentration was used as an independent variable. The NDF intake, L and Nmic showed a quadratic pattern (P b 0.05) as a function of RAN concentration, and the critical points (maximum responses) were observed with 15.17, 16.28, and 14.52 mg of RAN/dL of rumen fluid, respectively. On the other hand, ED and LAG presented a linear-response-plateau (P b 0.05) according to the RAN concentration, with break points close to 8 mg/dL for ED (maximum estimate) and LAG (minimum estimate). The RAN concentrations to optimize NDF degradation and intake were defined as 8 and 15 mg/dL, respectively. This difference between estimates appears to be due to a better adequacy of the metabolizable protein:metabolizable energy ratio in the animal metabolism, which increases the animal intake even after the rumen NDF degradation has been optimized. This observation was supported by Nmic pattern. An adapted Michaelis-Menten model was applied to the data, where RAN was the independent variable and kd the dependent variable. The relationship between these variables was found to be significant by using the Hanes-Woolf plot (P b 0.01). Based on this model, the rate of NDF degradation as a function of RAN concentration indicates that fibre degradation in the rumen could be considered a second order process. In this context, the RAN concentration of 8 mg/dL was assumed as the limit where zero order (below limit) and first order (above limit) reactions become predominant for NDF degradation in the rumen.Abbreviations: ADFom(n), Acid detergent fibre corrected for ash and nitrogenous compounds;ADIP, Acid detergent insoluble protein;BCVFA, Branched-chain volatile fatty acids;CP, Crude protein;DM, Dry matter;ED, Effective degradability of neutral detergent fibre;EE, Ether extract;kd, Fractional degradation rate of NDF; km, The Michaelis-Menten constant;L, Time-dependent rate parameter associated with rumen flow of fibrous particles;LAG, Discrete lag for fibre degradation; Lignin (sa), Lignin determined by solubilization of cellulose with sulphuric acid;aNDFom(n), Neutral detergent fibre assayed with a heat stable amylase and c...

At birth, calves display an underdeveloped rumen that eventually matures into a fully functional rumen as a result of solid food intake and microbial activity. However, little is known regarding the gradual impact of pre-weaning diet on the establishment of the rumen microbiota. Here, we employed next-generation sequencing to investigate the effects of the inclusion of starter concentrate (M: milk-fed vs. MC: milk plus starter concentrate fed) on archaeal, bacterial and anaerobic fungal communities in the rumens of 45 crossbred dairy calves across pre-weaning development (7, 28, 49, and 63 days). Our results show that archaeal, bacterial, and fungal taxa commonly found in the mature rumen were already established in the rumens of calves at 7 days old, regardless of diet. This confirms that microbiota colonization occurs in the absence of solid substrate. However, diet did significantly impact some microbial taxa. In the bacterial community, feeding starter concentrate promoted greater diversity of bacterial taxa known to degrade readily fermentable carbohydrates in the rumen (e.g., Megasphaera, Sharpea, and Succinivribrio). Shifts in the ruminal bacterial community also correlated to changes in fermentation patterns that favored the colonization of Methanosphaera sp. A4 in the rumen of MC calves. In contrast, M calves displayed a bacterial community dominated by taxa able to utilize milk nutrients (e.g., Lactobacillus, Bacteroides, and Parabacteroides). In both diet groups, the dominance of these milk-associated taxa decreased with age, suggesting that diet and age simultaneously drive changes in the structure and abundance of bacterial communities in the developing rumen. Changes in the composition and abundance of archaeal communities were attributed exclusively to diet, with more highly abundant Methanosphaera and less abundant Methanobrevibacter in MC calves. Finally, the fungal community was dominated by members of the genus SK3 and Caecomyces. Relative anaerobic fungal abundances did not change significantly in response to diet or age, likely due to high inter-animal variation and the low fiber content of starter concentrate. This study provides new insights into the colonization of archaea, bacteria, and anaerobic fungi communities in pre-ruminant calves that may be useful in designing strategies to promote colonization of target communities to improve functional development.

Microbial communities play critical roles in the gastrointestinal tracts (GIT) of preruminant calves by influencing performance and health. However, little is known about the establishment of microbial communities in the calf GIT or their dynamics during development. In this study, next-generation sequencing was used to assess changes in the bacterial communities of the rumen, jejunum, cecum, and colon in 26 crossbred calves at four developmental stages (7, 28, 49, and 63 days old). Alpha diversity differed among GIT regions with the lowest diversity and evenness in the jejunum, whereas no changes in alpha diversity were observed across developmental stage. Beta diversity analysis showed both region and age effects, with low numbers of operational taxonomic units (OTUs) shared between regions within a given age group or between ages in a given region. Taxonomic analysis revealed that several taxa coexisted in the rumen, jejunum, cecum, and colon but that their abundances differed considerably by GIT region and age. As calves aged, we observed lower abundances of taxa such as ,, and with higher abundances of and in the rumen. The jejunum also displayed taxonomic changes with increases in and taxa in older calves. In the lower gut, taxa such as, , and decreased and S24-7, , and increased as calves aged. These data support a model whereby early and successive colonization by bacteria occurs across the GIT of calves and provides insights into the temporal dynamics of the GIT microbiota of dairy calves during preweaning development. The gastrointestinal tracts (GIT) of ruminants, such as dairy cows, house complex microbial communities that contribute to their overall health and support their ability to produce milk. For example, the rumen microbiota converts feed into usable nutrients, while the jejunal microbiota provides access to protein. Thus, establishing a properly functioning GIT microbiota in dairy calves is critical to their productivity as adult cows. However, little is known about the establishment, maintenance, and dynamics of the calf GIT microbiota in early life. In this study, we evaluated the bacterial communities in the rumen, jejunum, cecum, and colon in dairy calves across preweaning development and show that they are highly variable early on in life before transitioning to a stable community. Understanding the dairy calf GIT microbiota has implications for ensuring proper health during early life and will aid in efforts to develop strategies for improving downstream production.

The growth of Streptococcus bovis JB1 was initially inhibited by nisin (1 M), and nisin caused a more than 3-log decrease in viability. However, some of the cells survived, and these nisin-resistant cells grew as rapidly as untreated ones. To see if the nisin resistance was merely a selection, nisin-sensitive cells were obtained from agar plates lacking nisin. Results indicated that virtually any nisin-sensitive cell could become nisin-resistant if the ratio of nisin to cells was not too high and the incubation period was long enough. Isolates obtained from the rumen were initially nisin sensitive, but they also developed nisin resistance. Nisin-resistant cultures remained nisin resistant even if nisin was not present, but competition studies indicated that nisin-sensitive cells could eventually displace the resistant ones if nisin was not present. Nisin-sensitive, glucose-energized cells lost virtually all of their intracellular potassium if 1 M nisin was added, but resistant cells retained potassium even after addition of 10 M nisin. Nisin-resistant cells were less hydrophobic and more lysozymeresistant than nisin-sensitive cells. Because the nisin-resistant cells bound less cytochrome c, it appeared that nisin was being excluded by a net positive (i.e., less negative) charge. Nisin-resistant cells had more lipoteichoic acid than nisin-sensitive cells, and deesterified lipoteichoic acids from nisin-resistant cells migrated more slowly through a polyacrylamide gel than those from nisin-sensitive cells. These results indicated that lipoteichoic acids could be modified to increase the resistance of S. bovis to nisin. S. bovis JB1 cultures were still sensitive to monensin, tetracycline, vancomycin, and bacitracin, but ampicillin resistance was 1,000-fold greater.Streptococcus bovis is a rapidly growing and opportunistic bacterium that is usually found at low numbers in the rumen, but its numbers can increase dramatically if cattle are switched abruptly from hay to grain diets. S. bovis produces lactate when carbohydrates are in excess, and lactate accumulation in the rumen can cause acidosis (31). Ruminal acidosis causes decreases in food intake and ruminal ulceration and founder, and it can even kill the animal (23, 31). S. bovis has also been implicated in the colon cancer of humans (9).Since the 1970s, the feed additive monensin has been used to modify ruminal fermentations, and this antibiotic is most effective against gram-positive bacteria (28). In vitro studies have indicated that monensin can inhibit S. bovis (21), but only higher than normal doses of monensin (approximately 350 mg/animal/day) could prevent S. bovis proliferation and acute ruminal acidosis (22). Recent work has indicated that nisin and monensin have similar effects on in vitro ruminal fermentations, but the effect of nisin on S. bovis has not been described (4).Nisin is a small peptide (34 amino acids) that forms pores in cell membranes, has "generally recognized as safe" (GRAS) status, and is approved for use as a food preservative (13,20...

Antimicrobial peptides (AMPs) are promising drug candidates to target multi-drug resistant bacteria. The rumen microbiome presents an underexplored resource for the discovery of novel microbial enzymes and metabolites, including AMPs. Using functional screening and computational approaches, we identified 181 potentially novel AMPs from a rumen bacterial metagenome. Here, we show that three of the selected AMPs (Lynronne-1, Lynronne-2 and Lynronne-3) were effective against numerous bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). No decrease in MRSA susceptibility was observed after 25 days of sub-lethal exposure to these AMPs. The AMPs bound preferentially to bacterial membrane lipids and induced membrane permeability leading to cytoplasmic leakage. Topical administration of Lynronne-1 (10% w/v) to a mouse model of MRSA wound infection elicited a significant reduction in bacterial counts, which was comparable to treatment with 2% mupirocin ointment. Our findings indicate that the rumen microbiome may provide viable alternative antimicrobials for future therapeutic application.