Biomedical subjects

C J Newbold

Publications and source records attributed to C J Newbold.

At least 19 recordsLinked to original sources

PubMed2006

Duodenal flow and digestibility in fauna-free sheep and in sheep monofaunated with Entodinium caudatum or Polyplastron multivesiculatum.

Three groups of five rumen and duodenum cannulated fauna-free sheep were used in a 28 d experiment. One group remained fauna-free, whereas the second (EN) and third (PP) groups, respectively, were inoculated intraruminally with the protozoan species Entodinium caudatum and Polyplastron multivesiculatum. Rumen fluid, duodenal digesta and faecal samples were collected during the last 12 d. The flow of digesta to the duodenum was determined using Yb and Co as dual-phase markers. (15)Nitrogen and phosphatidylcholine were used as markers to calculate the duodenal flow of bacterial and protozoal N, respectively. Results showed an increase (P < 0.1) in the rumen concentration of NH3-N and total volatile fatty acids, and a decrease (P < 0.05) in the duodenal flow of non-NH3-N and bacterial N in sheep with EN and PP monofaunas, compared with fauna-free sheep. There were no differences (P > 0.05) in these variables between the two monofauna groups. Protozoal N accounted for 8 % of the duodenal non-NH3-N flow in the EN-monofaunated sheep, whereas no such flow was detected in the PP-monofaunated sheep. Apparent rumen digestibility of organic matter, neutral detergent fibre and acid detergent fibre were similar (P > 0.05) in the monofaunated groups of sheep, but rumen acid detergent fibre digestibility was higher (P < 0.05) in the monofaunated than in the fauna-free groups. Experimental results suggested that, unlike EN, the PP monofauna might not contribute to the duodenal flow of microbial protein, whereas both monofaunas showed a virtually equal degree of predation on rumen bacteria.

Animal Feed↗

PubMed2005

Propionate precursors and other metabolic intermediates as possible alternative electron acceptors to methanogenesis in ruminal fermentation in vitro.

Fifteen potential precursors of propionate were tested for their ability to decrease CH4 production by ruminal fluid in vitro. Sodium acrylate and sodium fumarate produced the most consistent effects in batch cultures, with 50 % of the added precursors being fermented to propionate and CH4 production decreasing by between 8 and 17 %, respectively. Additives were more effective when added as free acids, but this also decreased the pH and may have inhibited fibre digestion. Changing the dietary substrate from predominantly grass hay to predominantly concentrate had no influence on the effectiveness of acrylate and fumarate. In an in vitro fermentor (the rumen simulating technique, Rusitec) with a grass hay-concentrate (50:50, w/w) diet as substrate, both compounds were again fermented to propionate (33 and 44 % conversion to propionate, respectively). However, fumarate appeared more effective as a H2 sink compound. It was calculated to capture 44 % of the H2 previously used for CH4 formation compared with a 22 % capture of H2 with acrylate. Fumarate also caused a stimulation in fibre digestion. Thus, sodium fumarate was the preferred propionate precursor for use as a feed ingredient to decrease CH4 emissions from ruminants.

Acids↗

PubMed2005

Rumen microbial population dynamics in response to photoperiod.

AIMS: This work was carried out to determine if there was a difference in the microbial population of the rumen associated with daylength at which sheep are housed. METHODS AND RESULTS: Denaturing gradient gel electrophoresis (DGGE) was used to study the ciliate and bacterial diversity in the rumen of Soay rams kept in long day (16 h light) or short day (8 h light) photoperiods. Bacterial diversity varied according to the daylength conditions where the host animal was housed, as did total volatile fatty acids (VFA) concentrations. No differences associated with daylength were detected in ciliate diversity, branched VFA concentrations or the ruminal ammonia concentrations. CONCLUSIONS: As diets had identical composition, yet voluntary intakes levels were higher during long days, it is proposed that the differences in bacterial populations arise because of the differences in amount of food consumed. SIGNIFICANCE AND IMPACT OF THE STUDY: The outcome of this study demonstrated that factors beyond dietary composition must be taken into account when trying to study microbial populations, even in what can be considered a fairly constant environment.

Animals↗

PubMed2005

Evaluation of a real-time PCR assay quantifying the ruminal pool size and duodenal flow of protozoal nitrogen.

We have recently developed a real-time polymerase chain reaction (PCR) assay to quantify copies of the genes encoding protozoal 18S rRNA. The assay includes procedures for isolating and concentrating protozoal cells from the rumen for use as a standard to convert 18S rRNA gene copies to a biomass basis. The current objectives were to 1) determine the degree of reduction of bacterial contamination in the protozoal standard, 2) determine if protozoal standards derived from ruminal fluid are appropriate for predicting duodenal flows, and 3) evaluate the assay's determined values for protozoal N in the rumen and flowing to the duodenum compared with independent measurements. Our protozoal collection method reduced non-associated bacterial contamination by 33-fold, the contamination of which could otherwise significantly bias RNA (microbial marker) and N percentages of concentrated protozoal fractions. Based on denaturing gradient gel electrophoresis, the use of protozoal cells isolated from ruminal fluid appears appropriate for use in quantitative assays determining protozoal N flow postruminally. Using real-time PCR, protozoal N was determined to be 4.8 and 12.7% of the rumen microbial N pool and 5.9 and 11.9% of the duodenal flow of microbial N on diets containing low (16%) or high (21%) forage neutral detergent fiber, respectively, which were comparable with independent measures and expectations.

Animals↗

PubMed2004

A re-appraisal of the diversity of the methanogens associated with the rumen ciliates.

The diversity of methanogenic archaea associated with different species of ciliated protozoa in the rumen was analysed. Partial fragments of archaeal SSU rRNA genes were amplified from DNA isolated from single cells from the rumen protozoal species Metadinium medium, Entodinium furca, Ophryoscolex caudatus and Diplodinium dentatum. Sequence analysis of these fragments indicated that although all of the new isolates clustered with sequences previously described for methanogens, there was a difference in the relative distribution of sequences detected here as compared to that of previous work. In addition, many of the novel sequences, although clearly of archaeal origin have relatively low identity to the sequences in database which are most closely related to them.

Animals↗

PubMed2004

Assessment of ciliates in the sheep rumen by DGGE.

AIMS: This work was carried out to develop a rapid molecular profiling technique to screen ciliate populations in the rumen of sheep. METHODS AND RESULTS: DGGE was used to study the ciliate diversity in the rumen of sheep. There was considerable variation between sheep which were co-housed, and fed the same diet. However, no difference in the major banding patterns was detected, when samples were collected from a single sheep sampled at different points. Following dietary changes, use of a pair-wise comparison of lanes, demonstrated that although there was still diversity between the ciliate population of sheep, the effects as a result of dietary changes were greater. CONCLUSIONS: The technique generated molecular profiles which are sufficiently different to allow comparison between samples, and to permit molecular ecological studies on the rumen ciliate population. SIGNIFICANCE AND IMPACT OF THE STUDY: The outcome of this study means that ciliate diversity in the rumen may now be studied by those unfamiliar with morphological identification of these organisms.

Animal Feed↗

PubMed2003

Methane oxidation and its coupled electron-sink reactions in ruminal fluid.

AIMS: This study was conducted to investigate the occurrence of methane oxidation in the rumen, and to identify the electron-sink reaction coupled to the oxidation if it occurred. METHODS AND RESULTS: Mixed ruminal microbes taken from sheep were incubated with 13CH4. Oxidation of methane, estimated from the flux of 13C to CO2 and microbial cells, occurred, but represented only 0.2-0.5% of the methane produced. Methane oxidation was suppressed by the presence of oxygen, and was also inhibited by 2-bromoethane-sulphonate, and molybdate, but not by tungstate. CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: Methane could be oxidized anaerobically in the rumen by reverse methanogenesis in consort with sulphate reduction.

Animals↗

PubMed2003

Eubacterium pyruvativorans sp. nov., a novel non-saccharolytic anaerobe from the rumen that ferments pyruvate and amino acids, forms caproate and utilizes acetate and propionate.

Two similar gram-positive rods were isolated from 10(-6) dilutions of ruminal fluid from a sheep receiving a mixed grass hay/concentrate diet, using a medium containing pancreatic casein hydrolysate as sole source of carbon and energy. The isolates did not ferment sugars, but grew on pyruvate or trypticase, forming caproate as the main fermentation product and valerate to a lesser extent. Acetate and propionate were utilized. One of these strains, I-6T, was selected for further study. Strain I-6T was a non-motile coccal rod, 1.2 x 0.4 microm, with a gram-positive cell wall ultrastructure and a G + C content of 56.8 mol%. No spores were visible, and strain I-6T did not survive heating at 80 degrees C for 10 min. Its rate of NH3 production was 375 nmol (mg protein)(-1) min(-1), placing it in the 'ammonia-hyperproducing' (or HAP) group of ruminal bacteria. 16S rDNA sequence analysis (1296 bases) indicated that it represents a novel species within the 'low-G + C' gram-positive group, for which the name Eubacterium pyruvativorans sp. nov. is proposed. Among cultivated bacteria, strain I-6T was most closely related (89% identity) to other asaccharolytic Eubacterium isolates from the mouth and the rumen. It was 98% identical to uncultured bacterial sequences amplified by others from ruminal digesta.

Acetic Acid↗

PubMed2003

Two-step freezing procedure for cryopreservation of rumen ciliates, an effective tool for creation of a frozen rumen protozoa bank.

The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25 degrees C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kisidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of -30 degrees C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2 degrees C/min and 2.5 degrees C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.

Animals↗

PubMed2003

Effects of essential oils on ruminal microorganisms and their protein metabolism.

A commercial blend of essential oil (EO) compounds was added to a grass, maize silage, and concentrate diet fed to dairy cattle in order to determine their influence on protein metabolism by ruminal microorganisms. EO inhibited (P < 0.05) the rate of deamination of amino acids. Pure-culture studies indicated that the species most sensitive to EO were ammonia-hyperproducing bacteria and anaerobic fungi.

Animals↗

PubMed2002

Ammonia production by ruminal microorganisms and enumeration, isolation, and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen.

Excessive NH(3) production in the rumen is a major nutritional inefficiency in ruminant animals. Experiments were undertaken to compare the rates of NH(3) production from different substrates in ruminal fluid in vitro and to assess the role of asaccharolytic bacteria in NH(3) production. Ruminal fluid was taken from four rumen-fistulated sheep receiving a mixed hay-concentrate diet. The calculated rate of NH(3) production from Trypticase varied from 1.8 to 19.7 nmol mg of protein(-1) min(-1) depending on the substrate, its concentration, and the method used. Monensin (5 micro M) inhibited NH(3) production from proteins, peptides, and amino acids by an average of 28% with substrate at 2 mg/ml, compared to 48% with substrate at 20 mg/ml (P = 0.011). Of the total bacterial population, 1.4% grew on Trypticase alone, of which 93% was eliminated by 5 micro M monensin. Many fewer bacteria (0.002% of the total) grew on amino acids alone. Nineteen isolates capable of growth on Trypticase were obtained from four sheep. 16S ribosomal DNA and traditional identification methods indicated the bacteria fell into six groups. All were sensitive to monensin, and all except one group (group III, similar to Atopobium minutum), produced NH(3) at >250 nmol min(-1) mg of protein(-1), depending on the medium, as determined by a batch culture method. All isolates had exopeptidase activity, but only group III had an apparent dipeptidyl peptidase I activity. Groups I, II, and IV were most closely related to asaccharolytic ruminal and oral Clostridium and Eubacterium spp. Group V comprised one isolate, similar to Desulfomonas piger (formerly Desulfovibrio pigra). Group VI was 95% similar to Acidaminococcus fermentans. Growth of the Atopobium- and Desulfomonas-like isolates was enhanced by sugars, while growth of groups I, II, and V was significantly depressed by sugars. This study therefore demonstrates that different methodologies and different substrate concentrations provide an explanation for different apparent rates of ruminal NH(3) production reported in different studies and identifies a diverse range of hyper-ammonia-producing bacteria in the rumen of sheep.

Animals↗

PubMed2001

Influence of nitrogen source on the fermentation of fibre from barley straw and sugarbeet pulp by ruminal micro-organisms in vitro.

Incubations were carried out with a batch culture system to study the effects of different N sources on the fermentation by ruminal micro-organisms from Merino sheep of two fibre substrates derived from feedstuffs that differed in their fermentation rate. The substrates were neutral-detergent fibre (NDF) from barley straw and sugarbeet pulp. N sources were ammonia (NH4Cl) and peptides (Trypticase). Three treatments were made by replacing ammonia-N with peptide-N at levels of 0 (AMMO), 33 (PEPLOW) and 66 % (PEPHIGH) of total N. There were no differences (P>0.05) between treatments in NDF degradation for both the barley straw and the sugarbeet pulp. Peptides increased (P<0.05) total volatile fatty acids daily production for both substrates, with greater values (P<0.001) for PEPHIGH than for PEPLOW for the sugarbeet pulp. The presence of peptides also increased (P<0.05) microbial N synthesis compared with AMMO, with PEPHIGH supporting more growth (P<0.001) than PEPLOW when the sugarbeet pulp NDF was fermented. The presence of peptides increased (P<0.01) the amount of solids-associated micro-organisms (SAM)-N for both the barley straw and the sugarbeet pulp fibres, values in the PEPHIGH treatment being higher (P<0.001) than those in PEPLOW. The proportion of SAM-N in the total microbial N was not affected (P>0.05) by the presence of peptides compared with the AMMO treatment, but values were greater for the PEPHIGH compared with the PEPLOW N source, reaching statistical significance (P<0.05) only for the sugarbeet pulp. For liquid-associated micro-organisms, the AMMO treatment resulted in the greatest (P<0.05) proportion of N derived from ammonia for both substrates, with a further decrease (P<0.01) for the PEPHIGH treatment compared with the PEPLOW for the sugarbeet pulp, indicating preferential uptake of peptides when they were available. Microbial growth efficiency (g microbial N/kg NDF degraded) was not affected (P>0.05) by N source. These results indicate that N forms other than ammonia are needed for maximal growth of fibre-digesting ruminal micro-organisms.

Animal Feed↗

PubMed2001

Incorporation of [(15)N] ammonia by the cellulolytic ruminal bacteria Fibrobacter succinogenes BL2, Ruminococcus albus SY3, and Ruminococcus flavefaciens 17.

The origin of cell nitrogen and amino acid nitrogen during growth of ruminal cellulolytic bacteria in different growth media was investigated by using (15)NH(3). At high concentrations of peptides (Trypticase, 10 g/liter) and amino acids (15.5 g/liter), significant amounts of cell nitrogen of Fibrobacter succinogenes BL2 (51%), Ruminococcus flavefaciens 17 (43%), and Ruminococcus albus SY3 (46%) were derived from non-NH(3)-N. With peptides at 1 g/liter, a mean of 80% of cell nitrogen was from NH(3). More cell nitrogen was formed from NH(3) during growth on cellobiose compared with growth on cellulose in all media. Phenylalanine was essential for F. succinogenes, and its (15)N enrichment declined more than that of other amino acids in all species when amino acids were added to the medium.

Amino Acids↗

PubMed2000

The 3' untranslated region of messages in the rumen protozoan Entodinium caudatum.

The 3' untranslated regions of a number of cDNAs from the rumen protozoal species Entodinium caudatum were studied with a view to characterising their preference for stop codons, general length, nucleotide composition and polyadenylation signals. Unlike a number of ciliates, Entodinium caudatum uses UAA as a stop codon, rather than as a codon for glutamine. In addition, the 3' untranslated region of the message is generally less than 100 nucleotides in length, extremely A+T rich, and does not appear to utilise any of the conventional polyadenylation signals described in other organisms.

3' Untranslated Regions↗

PubMed2000

Effects of protozoa on bacterial nitrogen recycling in the rumen.

The effects of protozoa on ruminal NH3-N kinetics and bacterial N recycling were measured in five sheep (57.6+/-7.1 kg BW, x +/- SD) with ruminal and duodenal cannulas in naturally faunated, defaunated, and refaunated periods. The sheep were fed a diet of 239 g of alfalfa haylage and 814 g of barley concentrate per day (DM basis) divided into 12 equal portions and allocated at 2-h intervals. A pulse dose of 300 mg of 15N as [15N]NH4Cl was administered into the rumen (on d 1 and 15) and 300 mg of 15N as [15N]urea was administered intravenously to the blood (d 8). Enrichment of 15N was measured in ruminal NH3-N, bacterial N, and plasma urea N over a period of 35 h. Total collection of urine was made for 5 d and analyzed for purine derivatives to calculate the flow of microbial N. Ruminal parameters and nutrient digestibilities were also measured. Sheep were defaunated using a rumen washing procedure 50 d prior to measurements in the defaunated period. Sheep were refaunated with ruminal contents from a faunated sheep receiving the same diet. Measurements began 26 d following refaunation, at which time protozoal numbers had returned to those in the originally faunated sheep. Data reported in parentheses are for faunated, defaunated, and refaunated sheep, respectively. Total culturable and cellulolytic bacterial numbers were unaffected by defaunation, but there was an increase in flow of microbial N from the rumen (10.8, 17.3, and 11.1 g N/d; P < .05) in the defaunated period. Flux, irreversible loss, and intraruminal recycling of NH3-N and recycling of NH3-N from plasma urea N were not affected by defaunation. Defaunation had no effect on reducing the absolute amount (13.8, 10.0, and 11.3 g N/d; P > .20) of bacterial N recycling and the percentage of N flux through the bacterial N pool. Total-tract digestion was reduced in defaunated compared with faunated sheep by 8, 17, 15, and 32% for OM, N, NDF, and ADF, respectively. In conclusion, defaunation improved ruminal N metabolism through the enhancement of bacterial protein synthesis, and improvement in the flow of microbial protein to the host animal.

Animals↗

PubMed1999

A xylanase produced by the rumen anaerobic protozoan Polyplastron multivesiculatum shows close sequence similarity to family 11 xylanases from gram-positive bacteria.

We report for the first time the cloning and characterisation of a protozoal enzyme involved in plant cell wall polysaccharide degradation. A cDNA library was constructed from the ruminal protozoan Polyplastron multivesiculatum and a stable clone expressing xylanase activity was isolated. The encoded enzyme belongs to the glycoside hydrolase family 11, and phylogenetic analysis indicates a closer relationship with catalytic domains from Gram-positive bacteria than the other fibrolytic eukaryotes from the rumen, the anaerobic fungi.

Amino Acid Sequence↗

PubMed1999

Protozoal sequences may reveal additional isoforms of the 14-3-3 protein family.

The phylogenetic position of eleven 14-3-3 proteins from five protozoal species was tested relative to other eukaryotic 14-3-3 versions representing many of the previously described isoforms. The protozoal proteins, four from Entodinium caudatum, three from Entameoba histolytica and four from apicomplexan parasites formed clusters closer to the plant and animal epsilon isoforms than to the animal beta, gamma/eta, sigma/theta, and zeta isoforms. This extends the preliminary findings of Wang and Shakes (1996) but data from a wider range of genera are still required to strengthen our hypothesis that the protozoan isoforms may constitute novel isoforms of the 14-3-3 family.

14-3-3 Proteins↗

PubMed1999

Influence of 1-[(E)-2-(2-methyl-4-nitrophenyl)diaz-1-enyl]pyrrolidine-2-carboxylic acid and diphenyliodonium chloride on ruminal protein metabolism and ruminal microorganisms.

The effects of 1-[(E)-2-(2-methyl-4-nitrophenyl)diaz-1-enyl]pyrrolidine-2-carboxy lic acid (LY29) and diphenyliodonium chloride (DIC) on the degradation of protein to ammonia were determined in a mixed rumen microbial population taken from sheep on a grass hay-concentrate diet. Both compounds decreased NH3 production by inhibiting deamination of amino acids. LY29, but not DIC, inhibited growth of the high-activity ammonia-producing species, Clostridium aminophilum and Clostridium sticklandii.

Animal Feed↗