Megasphaera elsdenii Lactate Degradation Pattern Shifts in Rumen Acidosis Models.
ABSTRACT: Background:Megasphaera elsdenii is an ecologically important rumen bacterium that metabolizes lactate and relieves rumen acidosis (RA) induced by a high-grain-diet. Understanding the regulatory mechanisms of the lactate metabolism of this species in RA conditions might contribute to developing dietary strategies to alleviate RA. Methods:Megasphaera elsdenii was co-cultured with four lactate producers (Streptococcus bovis, Lactobacilli fermentum, Butyrivibrio fibrisolvens, and Selenomonas ruminantium) and a series of substrate starch doses (1, 3, and 9 g/L) were used to induce one normal and two RA models (subacute rumen acidosis, SARA and acute rumen acidosis, ARA) under batch conditions. The associations between bacterial competition and the shift of organic acids' (OA) accumulation patterns in both statics and dynamics manners were investigated in RA models. Furthermore, we examined the effects of substrate lactate concentration and pH on Megasphaera elsdenii's lactate degradation pattern and genes related to the lactate utilizing pathways in the continuous culture. Results and Conclusion: The positive growth of M. elsdenii and B. fibrisolvens caused OA accumulation in the SARA model to shift from lactate to butyrate and resulted in pH recovery. Furthermore, both the quantities of substrate lactate and pH had remarkable effects on M. elsdenii lactate utilization due to the transcriptional regulation of metabolic genes, and the lactate utilization in M. elsdenii was more sensitive to pH changes than to the substrate lactate level. In addition, compared with associations based on statics data, associations discovered from dynamics data showed greater significance and gave additional explanations regarding the relationships between bacterial competition and OA accumulation.
Project description:The transition period is a severe challenge to dairy cows. Glucose supply cannot meet demand and body fat is mobilized, potentially leading to negative energy balance (NEB), ketosis, or fatty liver. Propionate produces glucose by gluconeogenesis, which depends heavily on the number and species of microbes. In the present study, we analyzed the rumen microbiome composition of cows in the transition period, cows with ketosis, and nonperinatal cows by terminal restriction fragment length polymorphism (TRFLP) analysis of 16S rRNA genes and quantitative PCR. TRFLP analysis indicated that the quantity of Veillonellaceae organisms was reduced and that of Streptococcaceae organisms was increased in rumen samples from the transition period and ketosis groups, with the number of Lactobacillaceae organisms increased after calving. Quantitative PCR data suggested that the numbers of the main propionate-producing microbes, Megasphaera elsdenii and Selenomonas ruminantium, were decreased, while numbers of the main lactate-producing bacterium, Streptococcus bovis, were increased in the rumen of cows from the transition period and ketosis groups, with the number of Lactobacillus sp. organisms increased after calving. Volatile fatty acid (VFA) and glucose concentrations were decreased, but the lactic acid concentration was increased, in rumen samples from the transition period and ketosis groups. Our results indicate that the VFA concentration is significantly related to the numbers of Selenomonas ruminantium and Megasphaera elsdenii organisms in the rumen.
Project description:High-grain adaptation programs are widely used with feedlot cattle to balance enhanced growth performance against the risk of acidosis. This adaptation to a high-grain diet from a high-forage diet is known to change the rumen microbial population structure and help establish a stable microbial population within the rumen. Therefore, to evaluate bacterial population dynamics during adaptation to a high-grain diet, 4 ruminally cannulated beef steers were adapted to a high-grain diet using a step-up diet regimen containing grain and hay at ratios of 20:80, 40:60, 60:40, and 80:20. The rumen bacterial populations were evaluated at each stage of the step-up diet after 1 week of adaptation, before the steers were transitioned to the next stage of the diet, using terminal restriction fragment length polymorphism (T-RFLP) analysis, 16S rRNA gene libraries, and quantitative real-time PCR. The T-RFLP analysis displayed a shift in the rumen microbial population structure during the final two stages of the step-up diet. The 16S rRNA gene libraries demonstrated two distinct rumen microbial populations in hay-fed and high-grain-fed animals and detected only 24 common operational taxonomic units out of 398 and 315, respectively. The 16S rRNA gene libraries of hay-fed animals contained a significantly higher number of bacteria belonging to the phylum Fibrobacteres, whereas the 16S rRNA gene libraries of grain-fed animals contained a significantly higher number of bacteria belonging to the phylum Bacteroidetes. Real-time PCR analysis detected significant fold increases in the Megasphaera elsdenii, Streptococcus bovis, Selenomonas ruminantium, and Prevotella bryantii populations during adaptation to the high-concentrate (high-grain) diet, whereas the Butyrivibrio fibrisolvens and Fibrobacter succinogenes populations gradually decreased as the animals were adapted to the high-concentrate diet. This study evaluates the rumen microbial population using several molecular approaches and presents a broader picture of the rumen microbial population structure during adaptation to a high-grain diet from a forage diet.
Project description:Effects of subacute ruminal acidosis (SARA) challenges on the bacteria in rumen fluid, cecal digesta, and feces of dairy cows were determined using 16S rRNA gene pyrosequencing and real-time quantitative PCR. Six non-lactating Holstein cows with cannulas in the rumen and cecum were used in a 3 × 3 Latin square arrangement of treatments. During the first 3 wk of each experimental period, cows received a control diet containing 70% forages on a dry matter (DM) basis. In wk 4 of each period, cows received one of three diets: (1) the control diet; (2) a diet in which 34% of the dietary DM was replaced with pellets of ground wheat and barley (GBSC); or (3) a diet in which 37% of dietary DM was replaced with pellets of ground alfalfa (APSC). Rumen fluid, cecal digesta and feces were collected on d 5 of wk 4 of each period and the composition of the bacterial community was studied. Rumen fermentation responses were reported in a companion study. Both SARA-inducing challenges resulted in similar digesta pH depressions (as shown by the companion study), and reduced bacterial richness and diversity in rumen fluid, but GBSC had the larger effect. None of the challenges affected these measures in cecal digesta, and only GBSC reduced bacterial richness and diversity in feces. Only GBSC reduced the abundance of Bacteroidetes in rumen fluid. Abundances of limited number of bacterial genera identified by 16S rRNA gene sequencing in the rumen, cecum and feces were affected by the GBSC. The APSC did not affect any of these abundances. Both challenges increased the abundances of several starch, pectin, xylan, dextrin, lactate, succinate, and sugar fermenting bacterial species in the rumen, cecum, and feces as determined by qPCR. Only GBSC increased that of <i>Megasphaera elsdenii</i> in the rumen. Both challenges decreased the abundance of <i>Streptococcus bovis</i>, and increased that of <i>Escherichia coli</i>, in cecal digesta and feces, with GBSC having the larger effect. These results showed that the SARA challenges caused moderate and reversible changes of the composition of the bacteria in the foregut and hindgut, with the greater changes observed during GBSC.
Project description:Anaerobic bacteria insensitive to chlortetracycline (64 to 256 microg/ml) were isolated from cecal contents and cecal tissues of swine fed or not fed chlortetracycline. A nutritionally complex, rumen fluid-based medium was used for culturing the bacteria. Eight of 84 isolates from seven different animals were identified as Megasphaera elsdenii strains based on their large-coccus morphology, rapid growth on lactate, and 16S ribosomal DNA sequence similarities with M. elsdenii LC-1(T). All eight strains had tetracycline MICs of between 128 and 256 microg/ml. Based on PCR assays differentiating 14 tet classes, the strains gave a positive reaction for the tet(O) gene. By contrast, three ruminant M. elsdenii strains recovered from 30-year-old culture stocks had tetracycline MICs of 4 microg/ml and did not contain tet genes. The tet genes of two tetracycline-resistant M. elsdenii strains were amplified and cloned. Both genes bestowed tetracycline resistance (MIC = 32 to 64 microg/ml) on recombinant Escherichia coli strains. Sequence analysis revealed that the M. elsdenii genes represent two different mosaic genes formed by interclass (double-crossover) recombination events involving tet(O) and tet(W). One or the other genotype was present in each of the eight tetracycline-resistant M. elsdenii strains isolated in these studies. These findings suggest a role for commensal bacteria not only in the preservation and dissemination of antibiotic resistance in the intestinal tract but also in the evolution of resistance.
Project description:The study evaluated the potential of blends of eucalyptus oil and aqueous extract of mulethi (root of Glycyrrhiza glabra) to reduce rate of ruminal ammonia production without affecting feed digestion to improve nitrogen utilization efficiency and performance of Murrah buffalo (Bubalus bubalis). Based on preliminary independent studies with graded doses of eucalyptus oil and mulethi root aqueous extract in modulating in vitro rumen fermentation, four blends of feed additive comprising graded doses (5, 10, 15, and 25 ?L) of eucalyptus oil and a fixed quantity (15 ?L) of aqueous extract of mulethi roots were prepared and examined for their effects on in vitro rumen fermentation and on methane and gas production in 100-mL calibrated glass syringes by standard IVGP protocol. Rumen liquor was collected from four rumen fistulated Murrah buffaloes fed a total mixed ration. Out of four blends, blend-1 comprising 5 ?L of eucalyptus oil and 15 ?L of aqueous extract (233.6 g/L DW) of mulethi per 40 mL of in vitro medium was found to reduce ammonia production significantly (p < 0.001) without affecting feed digestibility. An equivalent dose of blend-1 (10.5 mL of eucalyptus oil and 7.35 g of mulethi root powder/h/day) fed to four rumen fistulated buffaloes for 24 days resulted in 50% reduction (p < 0.05) in rumen ammonia level with no inhibition in feed fermentation or short-chain fatty acid production. The total bacterial population including Ruminococcus albus, Fibrobacter succinogenes, Butyrivibrio fibrisolvens, and Megasphaera elsdenii as well as anaerobic fungi and methanogenic archaea remained unaffected (p > 0.05). Twelve buffalo calves (avg. BW 137.5 ± 9.2 kg, 8–12 months old) divided into two groups of six each and fed a total mixed ration (concentrate: roughage; 60:40) with or without supplementation of blend-1 for about 3 months demonstrated 14% increase (p < 0.05) in average daily gain in BW with a trend (p < 0.10) in improvement of feed or protein utilization efficiency (1.4 vs. 1.1 g crude protein/g average daily gain; 21.4% increase). Thus, supplementation of eucalyptus oil–mulethi root blend could reduce ruminal ammonia production and improve feed utilization efficiency in ruminants.
Project description:The objective of this study was to characterize oligofructose-induced acute rumen lactic acidosis and its consequences in zebu cattle. We used 29 Nellore heifers which were submitted to experimental induction of laminitis by oligofructose excess. During the induction period, the animals underwent clinical examination, including laminitis diagnosis (hoof pressure testing and locomotion score) and blood and ruminal fluid sampling every six hours (over the initial 24 h) and every 12 h (up to 72 h), after the highest dose. Almost half of the animals (48.1%) required treatment with bicarbonate and saline to correct metabolic acidosis and dehydration. Due to this treatment, the animals were analyzed in treated (n = 13) and non-treated (n = 14) groups. The induction model promoted marked reduction in rumen pH, rumen anaerobiosis, carbon dioxide pressure, and increase in rumen lactate, blood osmolarity, and cortisol concentration. The animals treated had lower values of rumen pH and marked dehydration, evidenced by the increase in globular volume and serum urea. The clinical condition caused by excess oligofructose is severe, with the differential of the appearance of ephemeral fever and respiratory compensation against systemic acidosis, in addition to the frequent appearance of laminitis.
Project description:Dairy heifers were subjected to a non-life-threatening challenge designed to induce ruminal acidosis by feeding grain and sugar. Large among animal variation in clinical signs of acidosis, rumen metabolite concentrations, and the rumen microbiome occurred. This exploratory study investigates sources of the variation by examining associations between the genome, metabolome, and microbiome, albeit with a limited population. The broader objective is to provide a rationale for a larger field study to identify markers for susceptibility to ruminal acidosis. Initially, heifers (n = 40) allocated to five feed additive groups were fed 20-days pre-challenge with a total mixed ration and additives. Fructose (0.1% of bodyweight/day) was added for the last 10 days pre-challenge. On day 21 heifers were challenged with 1.0% of bodyweight dry matter wheat + 0.2% of bodyweight fructose + additives. Rumen samples were collected via stomach tube weekly (day 0, 7, and 14) and at five times over 3.6 h after challenge and analyzed for pH and volatile fatty acid, ammonia, D-, and L-lactate concentrations. Relative abundance of bacteria and archaea were determined using Illumina MiSeq. Genotyping was undertaken using a 150K Illumina SNPchip. Genome-wide association was performed for metabolite and microbiome measures (n = 33). Few genome associations occurred with rumen pH, concentration of acetate, propionate, total volatile fatty acids, or ammonia, or the relative abundance of the Firmicutes, Bacteroidetes, and Spirochaetes phyla. Metabolites and microbial phyla that had markers associated and quantitative trait loci (QTL) were: acetate to propionate ratio (A:P), D-, L-, and total lactate, butyrate, acidosis eigenvalue, Actinobacteria, Chloroflexi, Euryarchaeota, Fibrobacteres, Planctomycetes, Proteobacteria, and Tenericutes. A putative genomic region overlapped for Actinobacteria, Euryarchaeota, and Fibrobacteres and covered the region that codes for matrix extracellular phosphoglycoprotein (MEPE). Other overlapping regions were: (1) Chloroflexi, Tenericutes, and A:P, (2) L- and total lactate and Actinobacteria, and (3) Actinobacteria, Euryarchaeota, Fibrobacteres, and A:P. Genome-wide associations with the metabolome and microbiome occurred despite the small population, suggesting that markers for ruminal acidosis susceptibility exist. The findings may explain some of the variation in metabolomic and microbial data and provide a rationale for a larger study with a population that has variation in acidosis.
Project description:Megasphaera elsdenii is a lactate-fermenting, obligately anaerobic bacterium commonly present in the gastrointestinal tracts of mammals, including humans. Swine M. elsdenii strains were previously shown to have high levels of tetracycline resistance (MIC=64 to >256 ?g/ml) and to carry mosaic (recombinant) tetracycline resistance genes. Baby pigs inherit intestinal microbiota from the mother sow. In these investigations we addressed two questions. When do M. elsdenii strains from the sow colonize baby pigs? Can five antibiotic-sensitive M. elsdenii strains administered intragastrically to newborn pigs affect natural colonization of the piglets by antibiotic-resistant (AR) M. elsdenii strains from the mother? M. elsdenii natural colonization of newborn pigs was undetectable (<10(4) CFU/g [wet weight] of feces) prior to weaning (20 days after birth). After weaning, all pigs became colonized (4 × 10(5) to 2 × 10(8) CFU/g feces). In a separate study, 61% (76/125) of M. elsdenii isolates from a gravid sow never exposed to antibiotics were resistant to chlortetracycline, ampicillin, or tylosin. The inoculation of the sow's offspring with mixtures of M. elsdenii antibiotic-sensitive strains prevented colonization of the offspring by maternal AR strains until at least 11 days postweaning. At 25 and 53 days postweaning, however, AR strains predominated. Antibiotic susceptibility phenotypes and single nucleotide polymorphism (SNP)-based identities of M. elsdenii isolated from sow and offspring were unexpectedly diverse. These results suggest that dosing newborn piglets with M. elsdenii antibiotic-sensitive strains delays but does not prevent colonization by maternal resistant strains. M. elsdenii subspecies diversity offers an explanation for the persistence of resistant strains in the absence of antibiotic selection.
Project description:Several studies have evaluated the effects of live yeast supplementation on rumen microbial population; however, its effect on differential microbial genes and their functional potential has not been described. Thus, this study applied shotgun metagenomic sequencing to evaluate the effects of live yeast supplementation on genetic and functional potential of the rumen microbiota in beef cattle. Eight rumen-cannulated Holstein steers were randomly assigned to two treatments in a cross-over design with two 25-day experimental periods and a 10-day wash-out between the two periods. The steers were housed in individual pens and fed 50% concentrate-mix and 50% red clover/orchard hay ad libitum. Treatments were (1) control (CON; basal diet without additive) and (2) yeast (YEA; basal diet plus 15?g/d of live yeast product). Rumen fluid samples were collected at 3, 6, and 9?h after feeding on the last d of each period. Sequencing was done on an Illumina HiSeq 2500 platform. Dietary yeast supplementation increased the relative abundance of carbohydrate-fermenting bacteria (such as Ruminococcus albus, R. champanellensis, R. bromii, and R. obeum) and lactate-utilizing bacteria (such as Megasphaera elsdenii, Desulfovibrio desulfuricans, and D. vulgaris). A total of 154 differentially abundant genes (DEGs) were obtained (false discovery rate?<?0.01). Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analysis of the DEGs revealed that 10 pathways, including amino sugar and nucleotide sugar metabolism, oxidative phosphorylation, lipopolysaccharide biosynthesis, pantothenate and coenzyme A biosynthesis, glutathione metabolism, beta-alanine metabolism, polyketide sugar unit biosynthesis, protein export, ribosome, and bacterial secretory system, were enriched in steers fed YEA. Annotation analysis of the DEGs in the carbohydrate-active enzymes (CAZy) database revealed that the abundance of genes coding for enzymes belonging to glycoside hydrolases, glycosyltransferases, and carbohydrate binding modules were enriched in steers fed YEA. These results confirm the effectiveness of a live S. cerevisiae product for improving rumen function in beef steers by increasing the abundance of cellulolytic bacteria, lactic acid-utilizing bacteria, and carbohydrate-active enzymes in the rumen.
Project description:Thiamine is known to attenuate high-concentrate diet induced subacute ruminal acidosis (SARA) in dairy cows, however, the underlying mechanisms remain unclear.The major objective of this study was to investigate the metabolic mechanisms of thiamine supplementation on high-concentrate diet induced SARA.Six multiparous, rumen-fistulated Holstein cows were used in a replicated 3?×?3 Latin square design. The treatments included a control diet (CON; 20% starch, dry matter basis), a SARA-inducing diet (SAID; 33.2% starch, dry matter basis) and SARA-inducing diet supplemented with 180 mg of thiamine/kg of dry matter intake (SAID?+?T). On d21 of each period, ruminal fluid samples were collected at 3 h post feeding, and GC/MS was used to analyze rumen fluid samples.PCA and OPLS-DA analysis demonstrated that the ruminal metabolite profile were different in three treatments. Compared with CON treatment, SAID feeding significantly decreased rumen pH, acetate, succinic acid, increased propionate, pyruvate, lactate, glycine and biogenic amines including spermidine and putrescine. Thiamine supplementation significantly decreased rumen content of propionate, pyruvate, lactate, glycine and spermidine; increase rumen pH, acetate and some medium-chain fatty acids. The enrichment analysis of different metabolites indicated that thiamine supplementation mainly affected carbohydrates, amino acids, pyruvate and thiamine metabolism compared with SAID treatment.These findings revealed that thiamine supplementation could attenuate high-concentrate diet induced SARA by increasing pyruvate formate-lyase activity to promote pyruvate to generate acetyl-CoA and inhibit lactate generation. Besides, thiamine reduced biogenic amines to alleviate ruminal epithelial inflammatory response.