Ehsan Khafipour

Rumen microbiome composition determined using two nutritional models of subacute ruminal acidosis.

ABSTRACT Subacute ruminal acidosis (SARA) is a metabolic disease in dairy cattle that occurs during early and mid-lactation and has traditionally been characterized by low rumen pH, but lactic acid does not accumulate as in acute lactic acid acidosis. It is hypothesized that factors such as increased gut permeability, bacterial lipopolysaccharides, and inflammatory responses may have a role in the etiology of SARA. However, little is known about the nature of the rumen microbiome during SARA. In this study, we analyzed the microbiome of 64 rumen samples taken from eight lactating Holstein dairy cattle using terminal restriction fragment length polymorphisms (TRFLP) of 16S rRNA genes and real-time PCR. We used rumen samples from two published experiments in which SARA had been induced with either grain or alfalfa pellets. The results of TRFLP analysis indicated that the most predominant shift during SARA was a decline in gram-negative Bacteroidetes organisms. However, the proportion of Bacteroidetes organisms was greater in alfalfa pellet-induced SARA than in mild or severe grain-induced SARA (35.4% versus 26.0% and 16.6%, respectively). This shift was also evident from the real-time PCR data for Prevotella albensis, Prevotella brevis, and Prevotella ruminicola, which are members of the Bacteroidetes. The real-time PCR data also indicated that severe grain-induced SARA was dominated by Streptococcus bovis and Escherichia coli, whereas mild grain-induced SARA was dominated by Megasphaera elsdenii and alfalfa pellet-induced SARA was dominated by P. albensis. Using discriminant analysis, the severity of SARA and degree of inflammation were highly correlated with the abundance of E. coli and not with lipopolysaccharide in the rumen. We thus suspect that E. coli may be a contributing factor in disease onset.

A grain-based subacute ruminal acidosis challenge causes translocation of lipopolysaccharide and triggers inflammation.

The effects of a grain-based subacute ruminal acidosis (SARA) challenge on translocation of lipopolysaccharide (LPS) into the peripheral circulation, acute phase proteins in blood and milk, feed intake, milk production and composition, and blood metabolites were determined in 8 lactating Holstein cows. Between wk 1 and 5 of 2 successive 6-wk periods, cows received a total mixed ration ad libitum with a forage to concentrate (F:C) ratio of 50:50. In wk 6 of both periods, the SARA challenge was conducted by replacing 21% of the dry matter of the total mixed ration with pellets containing 50% wheat and 50% barley. Rumen pH was monitored continuously using indwelling pH probes in 4 rumen cannulated cows. Rumen fluid samples were collected 15 min before feed delivery and at 2, 4, 6, 12, 14, 16, 18, and 24 h after feed delivery for 2 d during wk 5 (control) and wk 6 (SARA). Peripheral blood samples were collected using jugular catheters 15 min before feeding and at 6 and 12 h after feeding at the same days of the rumen fluid collections. The SARA challenge significantly reduced average daily pH from 6.17 to 5.97 and increased the duration of rumen pH below pH 5.6 from 118 to 279 min/d. The challenge reduced dry matter intake (16.5 vs. 19 kg/d), milk yield (28.3 vs. 31.6 kg/d), and milk fat (2.93 vs. 3.30%, 0.85 vs. 0.97 kg/d), and tended to increase milk protein percentage (3.42 vs. 3.29%), without affecting milk protein yield (1.00 vs. 0.98 kg/d). The challenge also increased the concentration of free LPS in rumen fluid from 28,184 to 107,152 endotoxin units (EU)/mL. This was accompanied by an increase in LPS in peripheral blood plasma (0.52 vs. <0.05 EU/mL) with a peak at 12 h after feeding (0.81 EU/mL). Concentrations of the acute phase proteins serum amyloid A, haptoglobin, and LPS-binding protein (LBP) in peripheral blood as well as LBP concentration in milk increased (438.5 vs. 167.4, 475.6 vs. 0, 53.1 vs. 18.2, and 6.94 vs. 3.02 microg/mL, respectively) during SARA. The increase in LBP in combination with the increase in LPS in peripheral blood provides additional evidence of translocation of LPS. Results suggest that the grain-based SARA challenge resulted in translocation of LPS into the peripheral circulation, and that this translocation triggered a systemic inflammatory response.

Alfalfa pellet-induced subacute ruminal acidosis in dairy cows increases bacterial endotoxin in the rumen without causing inflammation

A study was conducted to determine if subacute ruminal acidosis (SARA) induced by feeding alfalfa pellets results in increases in free bacterial lipopolysaccharide (LPS) in rumen fluid and peripheral blood, and acute phase proteins in plasma, and to determine the effect of alfalfa pellet-induced SARA on feed intake, rumen fermentation characteristics, milk production and composition, and blood metabolites. Eight lactating Holstein cows, 4 of which were ruminally cannulated, were used in a 6-wk experiment and were fed once daily at 0900 h. During wk 1, cows received a diet containing 50% of DM as concentrate and 50% of DM chopped alfalfa hay. Between wk 2 and wk 6, alfalfa hay was gradually replaced with alfalfa pellets at the rate of 8% per week to reduce rumen pH. Rumen pH was monitored continuously in the ruminally cannulated cows using indwelling pH probes. Rumen fluid and peripheral blood were sampled 15 min before feed delivery and at 6 h after feed delivery. Based on adopted threshold of SARA of at least 180 min/d below pH 5.6, SARA was induced from wk 3 onwards. Replacing 40% of alfalfa hay with alfalfa pellets quadratically increased the DMI from 18.1 kg/d in wk 1 to 23.4 kg/d in wk 6. This replacement linearly decreased milk yield (32.7 vs. 35.9 kg/d) and milk fat percentage and yield (2.32 vs. 3.22%, and 0.77 vs. 1.14 kg/d, respectively), but increased milk protein percentage and yield (3.80 vs. 3.04%, and 1.23 vs. 1.07 kg/d, respectively). This gradual replacement also linearly increased the daily averages of total volatile fatty acids (90 to 121.9 mM), acetate (53.9 to 66.8 mM), propionate (21.5 to 39.6 mM), and osmolality (277.7 to 293.8 mmol/kg) in the rumen and decreased the acetate to propionate ratio from 2.62 to 1.73. Replacing alfalfa hay with alfalfa pellets linearly increased blood lactate from 1.00 mM in wk 1 to a peak of 3.46 mM in wk 5. Induction of SARA in this study increased free rumen LPS concentration from 42,122 endotoxin unit (EU)/mL in wk 1 to 145,593 EU/mL in wk 6. However, this increase was not accompanied by an increase in LPS (<0.05 EU/mL) and in acute phase proteins serum amyloid-A, haptoglobin, and LPS-binding protein in peripheral circulation. Results suggest that SARA induced by alfalfa pellets increased LPS in the rumen without causing translocation of LPS and an immune response.

Effects of subacute ruminal acidosis challenges on fermentation and endotoxins in the rumen and hindgut of dairy cows

The effects of a grain-based subacute ruminal acidosis (SARA) challenge (GBSC) and an alfalfa-pellet SARA challenge (APSC) on fermentation and endotoxins in the rumen and in the cecum, as well as on endotoxins in peripheral blood, were determined. Six nonlactating Holstein cows with cannulas in the rumen and cecum were used in the study. A 3×3 Latin square arrangement of treatments with 4-wk experimental periods was adopted. During the first 3 wk of each experimental period, all cows received a diet containing 70% forages [dry matter (DM) basis]. In wk 4 of each period, cows received 1 of the following 3 diets: the 70% forage diet fed during wk 1 to 3 (control), a diet in which 34% of the dietary DM was replaced with grain pellets made of 50% ground wheat and 50% ground barely (GBSC), or a diet in which 37% of dietary DM was replaced with pellets of ground alfalfa (APSC). Rumen pH was monitored continuously using indwelling pH probes, and rumen fluid, blood, cecal digesta, and fecal grab samples were collected immediately before feed delivery at 0900 h and at 6 h after feed delivery on d 3 and 5 of wk 4. The time for which rumen pH was below 5.6 was 56.4, 225.2, and 298.8 min/d for the control, APSC, and GBSC treatments, respectively. Compared with the control, SARA challenges resulted in similar reductions in cecal digesta pH, which were 7.07, 6.86, and 6.79 for the control, APSC, and GBSC treatments, respectively. Compared with the control, only GBSC increased starch content in cecal digesta, which averaged 2.8, 2.6, and 7.4% of DM for the control, APSC, and GBSC, respectively. Free lipopolysaccharide endotoxin (LPS) concentration in rumen fluid increased from 10,405 endotoxin units (EU)/mL in the control treatment to 30,715 and 168,391 EU/mL in APSC and GBSC, respectively. Additionally, GBSC increased the LPS concentration from 16,508 to 118,522 EU/g in wet cecal digesta, and from 12,832 to 93,154 EU/g in wet feces. The APSC treatment did not affect LPS concentrations in cecal digesta and feces. All concentrations of LPS in blood plasma were below the detection limit of >0.05 EU/mL of the technique used. Despite the absence of LPS in blood, only GBSC increased the concentration of LPS-binding protein in blood plasma, which averaged, 8.9, 9.5, and 12.1mg/L for the control, APSC, and GBSC treatments, respectively. This suggests that GBSC caused translocation of LPS from the digestive tract but that LPS was detoxified before entering the peripheral blood circulation. The higher LPS concentration in cecal digesta in the GBSC compared with the APSC suggests a higher risk of LPS translocation in the large intestine in GBSC than in APSC.

Pyrosequencing Reveals the Influence of Organic and Conventional Farming Systems on Bacterial Communities

It has been debated how different farming systems influence the composition of soil bacterial communities, which are crucial for maintaining soil health. In this research, we applied high-throughput pyrosequencing of V1 to V3 regions of bacterial 16S rRNA genes to gain further insight into how organic and conventional farming systems and crop rotation influence bulk soil bacterial communities. A 2×2 factorial experiment consisted of two agriculture management systems (organic versus conventional) and two crop rotations (flax-oat-fababean-wheat versus flax-alfalfa-alfalfa-wheat) was conducted at the Glenlea Long-Term Crop Rotation and Management Station, which is Canada’s oldest organic-conventional management study field. Results revealed that there is a significant difference in the composition of bacterial genera between organic and conventional management systems but crop rotation was not a discriminator factor. Organic farming was associated with higher relative abundance of Proteobacteria, while Actinobacteria and Chloroflexi were more abundant in conventional farming. The dominant genera including Blastococcus, Microlunatus, Pseudonocardia, Solirubrobacter, Brevundimonas, Pseudomonas, and Stenotrophomonas exhibited significant variation between the organic and conventional farming systems. The relative abundance of bacterial communities at the phylum and class level was correlated to soil pH rather than other edaphic properties. In addition, it was found that Proteobacteria and Actinobacteria were more sensitive to pH variation.

External influence of early childhood establishment of gut microbiota and subsequent health implications.

Postnatal maturation of immune regulation is largely driven by exposure to microbes. The gastrointestinal tract is the largest source of microbial exposure, as the human gut microbiome contains up to 1014 bacteria, which is 10 times the number of cells in the human body. Several studies in recent years have shown differences in the composition of the gut microbiota in children who are exposed to different conditions before, during, and early after birth. A number of maternal factors are responsible for the establishment and colonization of gut microbiota in infants, such as the conditions surrounding the prenatal period, time and mode of delivery, diet, mother’s age, BMI, smoking status, household milieu, socioeconomic status, breastfeeding and antibiotic use, as well as other environmental factors that have profound effects on the microbiota and on immunoregulation during early life. Early exposures impacting the intestinal microbiota are associated with the development of childhood diseases that may persist to adulthood such as asthma, allergic disorders (atopic dermatitis, rhinitis), chronic immune-mediated inflammatory diseases, type 1 diabetes, obesity, and eczema. This overview highlights some of the exposures during the pre- and postnatal time periods that are key in the colonization and development of the gastrointestinal microbiota of infants as well as some of the diseases or disorders that occur due to the pattern of initial gut colonization.

Characterization of Escherichia coli isolated from gut biopsies of newly diagnosed patients with inflammatory bowel disease

Background: Mucosal‐associated Escherichia coli may play a role in the pathogenesis of inflammatory bowel diseases (IBDs). In this study we assessed mucosal‐associated E. coli in adults at the time of first diagnosis. Materials and Methods: E. coli were isolated from 59 right colon biopsies of 34 newly diagnosed adult IBD patients (Crohns disease [CD] = 23, ulcerative colitis [UC] = 11) and 25 healthy controls (HC). Strains were serotyped, phylotyped into A, B1, B2, or D, and tested for their ability to survive in macrophages. The presence of various virulence factors was also assessed. The fimH subunit of type 1 fimbriae was sequenced and phylogenetically analyzed. Results: A total of 65 E. coli were isolated from CD (29 isolates from 23 patients), UC (11 isolates from 11 patients), and HC (25 isolates from 25 subjects). All E. coli were positive for fimH, crl, and cgsA and negative for vt1, vt2, hlyA, cnf, and eae. Significant positive associations were between CD and in between CD and afae (P = 0.002), and between UC and ompA (P = 0.02), afae (P = 0.03), and USP (P = 0.04). The B2+D phylotype was significantly associated with inflammation (P = 0.04) as it was with serine protease autotransporters (SPATE), malX, ompA, and kpsMTII (P < 0.05). Macrophage survival was the highest in UC‐isolated E. coli (P = 0.04). FimH amino acid substitutions N91S, S99N, and A223V were associated with IBD (P < 0.05). Conclusions: Adherent invasive E. coli are present at first diagnosis, suggesting that they may have a role in the early stages of disease onset. (Inflamm Bowel Dis 2010;)

Consumption of Acidic Water Alters the Gut Microbiome and Decreases the Risk of Diabetes in NOD Mice

Infant formula and breastfeeding are environmental factors that influence the incidence of Type 1 Diabetes (T1D) as well as the acidity of newborn diets. To determine if altering the intestinal microbiome is one mechanism through which an acidic liquid plays a role in T1D, we placed non-obese diabetic (NOD)/ShiLtJt mice on neutral (N) or acidified H2O and monitored the impact on microbial composition and diabetes incidence. NOD-N mice showed an increased development of diabetes, while exhibiting a decrease in Firmicutes and an increase in Bacteroidetes, Actinobacteria, and Proteobacteria from as early as 2 weeks of age. NOD-N mice had a decrease in the levels of Foxp3 expression in CD4+Foxp3+ cells, as well as decreased CD4+IL17+ cells, and a lower ratio of IL17/IFNγ CD4+ T-cells. Our data clearly indicates that a change in the acidity of liquids consumed dramatically alters the intestinal microbiome, the presence of protective Th17 and Treg cells, and the incidence of diabetes. This data suggests that early dietary manipulation of intestinal microbiota may be a novel mechanism to delay T1D onset in genetically pre-disposed individuals.

An extended single‐index multiplexed 16S rRNA sequencing for microbial community analysis on MiSeq illumina platforms

The primary 16S rRNA sequencing protocol for microbial community analysis using Illumina platforms includes a single‐indexing approach that allows pooling of hundreds of samples in each sequencing run. The protocol targets the V4 hypervariable region (HVR) of 16S rRNA using 150 bp paired‐end (PE) sequencing. However, the latest improvement in Illumina chemistry has increased the read length up to 600 bp using 300 bp PE sequencing. To take advantage of the longer read length, a dual‐indexing approach was previously developed for targeting different HVRs. However, due to simple working protocols, the single‐index 150 bp PE approach still continues to be attractive to many researchers. Here, we described an extended single‐indexing protocol for 300 bp PE illumina sequencing that targets the V3‐V4 HVRs of 16S rRNA. The new primer set led to increased read length and alignment resolution, as well as increased richness and diversity of resulting microbial profile compared to that obtained from150 bp PE protocol for V4 sequencing. The β‐diversity profile also differed qualitatively and quantitatively between the two approaches. Both primer sets had high coverage rates and specificity to detect dominant phyla; however, their coverage rate with regards to the rare biosphere varied. Our data further confirms that the choice of primer is the most deterministic factor in sequencing coverage and specificity.

High Molecular Weight Barley β-Glucan Alters Gut Microbiota Toward Reduced Cardiovascular Disease Risk

The physiological cholesterol-lowering benefits of β-glucan have been well documented, however, whether modulation of gut microbiota by β-glucan is associated with these physiological effects remains unknown. The objectives of this study were therefore to determine the impact of β-glucan on the composition of gut microbiota in mildly hypercholesterolemic individuals and to identify if the altered microbiota are associated with bioactivity of β-glucan in improving risk factors of cardiovascular disease (CVD). Using a randomized, controlled crossover study design, individuals received for 5-week either a treatment breakfast containing 3 g high molecular weight (HMW), 3 g low molecular weight (LMW), 5 g LMW barley β-glucan, or wheat and rice. The American Heart Association (AHA) diet served as the background diet for all treatment groups. Phases were separated by 4-week washout periods. Fecal samples were collected at the end of each intervention phase and subjected to Illumina sequencing of 16S rRNA genes. Results revealed that at the phylum level, supplementation of 3 g/d HMW β-glucan increased Bacteroidetes and decreased Firmicutes abundances compared to control (P < 0.001). At the genus level, consumption of 3 g/d HMW β-glucan increased Bacteroides (P < 0.003), tended to increase Prevotella (P < 0.1) but decreased Dorea (P < 0.1), whereas diets containing 5 g LMW β-glucan and 3 g LMW β-glucan failed to alter the gut microbiota composition. Bacteroides, Prevotella, and Dorea composition correlated (P < 0.05) with shifts of CVD risk factors, including body mass index, waist circumference, blood pressure, as well as triglyceride levels. Our data suggest that consumption of HMW β-glucan favorably alters the composition of gut microbiota and this altered microbiota profile associates with a reduction of CVD risk markers. Together, our study suggests that β-glucan induced shifts in gut microbiota in a MW-dependent manner and that might be one of the underlying mechanisms responsible for the physiological benefits of β-glucan.