D.O. Krause

Microbial interactions with tannins: nutritional consequences for ruminants

Polyphenolics are widely distributed in the plant kingdom and are often present in the diet of herbivores. The two major groups of plant polyphenolic compounds other than lignin are condensed and hydrolysable tannins. These compounds can have toxic and/or antinutritional effects on the animal. It is well established that tannins complex with dietary proteins can reduce nitrogen supply to the animal, but the ability of gastrointestinal microorganisms to metabolise these compounds and their effects on microbial populations have received little attention. In this paper, we review recent literature on the topic as well as present research from our laboratories on the effect of condensed tannins on rumen microbial ecology and rumen metabolism. Interactions of tannins with dietary components and endogenous protein in the rumen and post-ruminally, and their impact on the nutrition of the animal are considered

Molecular characterization of the microbial species that colonize human ileal and colonic mucosa by using 16S rDNA sequence analysis

The aim of this study was to characterize the bacterial community adhering to the mucosa of the terminal ileum, and proximal and distal colon of the human digestive tract.

Effect of the tropical forage calliandra on microbial protein synthesis and ecology in the rumen

C.S. MCSWEENEY, B. PALMER, R. BUNCH AND D.O. KRAUSE. 2001.

16S rDNA sequencing of Ruminococcus albus and Ruminococcus flavefaciens : design of a signature probe and its application in adult sheep

The ruminococci are an important group of fibrolytic bacteria inhabiting the rumen. Seventeen strains of presumptively identified Ruminococcus were evaluated by a combination of nearly complete and partial 16S rDNA sequence that identified all strains as either Ruminococcus albus or Ruminococcus flavefaciens. All sequences fell into cluster IV of the clostridia, while other species of ruminococci (e.g. Ruminococcus obeum, Ruminococcus gnavus, Ruminococcus lactaris) fall into cluster XIVa of the clostridia. Ruminococcus cluster IV sequences were used to design a 16S rRNA oligonucleotide probe to assess the relative abundance of target populations in a stable ruminal environment. A stable population (animals fed eight times per day) was established in sheep so that statistically robust comparisons could be made in the absence of variation due to diurnal rumen fluctuations. The steady state populations were sampled six times over a 24 d period and direct microscopic counts (DC), total culturable counts (TCC), and total cellulolytic counts (CEL) were determined. DC and culturable data (TCC and CEL) were compared with relative abundance estimates of Ruminococcus IV and Fibrobacter succinogenes. A combination of the Ruminococcus and F. succinogenes probes accounted for 4.0% of the bacterial population and cellulolytic bacteria (measured by most-probable numbers) were 5.2% of the total culturable count. These data suggest that a major portion of the Ruminococcus and Fibrobacter diversity has been cultured and is represented by available sequences. Steady state populations were measured over several days in three sheep and an estimate of variation in DC, TCC, CEL and 16S-based data were obtained. These variance estimates could be used to determine the theoretical sample sizes required to obtain statistically significant differences under different experimental conditions.

In vitro quality assessment of tannin-containing tropical shrub legumes: protein and fibre digestion

In vitro techniques were evaluated to determine the nutritive value of a selection of tanniniferous tree and shrub legumes (Calliandra calothyrsus, Leucaena leucocephala L. diversifolia and L. pallida) compared with lucerne (Medicago sativa). Polyethylene glycol (PEG) was also added to some in vitro fermentations (10 mg PEG/50 mg plant substrate) to assess the effects of tannins on digestion of dry matter (DM), neutral detergent fibre (NDF) and nitrogen (N). Total tannin content was poorly correlated with digestibility of dry matter and nitrogen. Apparent digestibility of dry matter and N were significantly different between plants and ranked in the following order; lucerne > L. leucocephala > L. diversifolia > L. pallida > C. calothyrsus. Ammonia was not produced (net accumulation) during 72 h fermentation of C. calothyrsus, L. diversifolia and L. pallida although apparent nitrogen digestion in these plants ranged from 36.9 to 44.3%. Acid-pepsin digestion resulted in a further 17‐22% of dry matter digestion in the shrub legumes compared with 8% in lucerne following 72 h fermentation. The amount of acid-pepsin digestible N available was lowest for lucerne (8.4%) and highest for L. pallida (38.9%) with the other legumes ranging from 26.5 to 36.8%. The PEG addition caused a significant increase in rate and extent of DM and NDF digestibility and ammonia production for all the tannin containing shrub legumes but not for lucerne. However, DM loss and fermentability of these plants appeared to be poorly correlated because PEG addition resulted in an increase in volatile fatty acid production ranging from 3.7 to 202% compared with an increase in apparent DM digestibility of 9.1‐30%. It is concluded that in vitro evaluation of apparent DM and N digestibility of tannin containing plants provides a poor indication of true digestion (fermentability) and thus measurements of

The diversity of Escherichia coli serotypes and biotypes in cattle faeces

Aim:  To study the diversity of commensal Escherichia coli populations shed in faeces of cattle fed on different diets.

Use of 16S-rRNA based techniques to investigate the ecological succession of microbial populations in the immature lamb rumen: Tracking of a specific strain of inoculated Ruminococcus and interactions with other microbial populations in vivo

A bstractThe establishment of microorganisms in the rumen is a critical step if rumen manipulation is to be accomplished by use of microbial inoculants. Microbial populations in the maturing rumen undergo successional changes and, while in a state of flux, provide a possible opportunity for the introduction of specific strains of bacteria. While the rumen of the young lamb was maturing, we measured changes in several microbial populations with 16S-rRNA specific oligonucleotides: Rumincoccus, Fibrobacter, eukaryotes, Gram-positive bacteria, the Bacteroides–Porphromonas–Prevotella group, and anaerobic rumen fungi. In this study we repeatedly dosed 15 lambs with approximately 3.4 × 108 to 0.8 × 109Ruminococcus cells dose-1, twice a week, for 7 wk from 23 d to 63 d of age. Of the five Ruminococcus strains dosed (R. albus SY3 and AR67, and R. flavefaciens Y1, LP9155, and AR72) the most specific primers (based on 16S rDNA) were obtained for strain SY3. There was an increase in the eukaryotic population during dosing, and it was hypothesized that protozoal predation contributed to the disappearance of strain SY3. At the end of dosing PCR amplification showed that SY3 were approximately 109 cells ml-1, but decreased to below the detection limit of the PCR system (8.6 × 104 ml-1) within 28 d postdosing. These experiments showed that fibrolytic populations increased significantly (P < 0.1) above the controls during the dosing period and were elevated for several days postdosing. This suggests that dosing of highly fibrolytic bacteria makes more of the fiber available to other organisms able to degrade fiber, and in so doing increases the overall fibrolytic activity of the rumen. Examination of the succession of gram-positive bacteria and the Bacteroides–Porphromonas–Prevotella group showed a decline in relative abundance as the lambs matured.

Repeated ruminal dosing of Ruminococcus spp. does not result in persistence, but changes in other microbial populations occur that can be measured with quantitative 16S-rRNA-based probes.

Digestibility of fibre in ruminants may be improved by the introduction of highly fibrolytic strains of ruminal bacteria. This approach may be feasible if, for example, strains of Ruminococcus that are significantly more fibrolytic than the normal population of Ruminococcus are used for inoculation purposes. Introduced strains of bacteria, irrespective of ecosystem, often decline after inoculation, and in this study, highly fibrolytic strains of Ruminococcus were continuously dosed to ensure that measurements of fibre digestion were made in the presence of significant numbers of the introduced bacteria. During dosing the total culturable count increased significantly (P<0.05), but declined post-dosing. The level of dosed Ruminococcus, and total Ruminococcus, Fibrobacter succinogenes and eukaryotes measured by 16S rRNA probes increased significantly (P<0.05) during the dosing period, but also declined post-dosing. When in vitro nylon bag digestibility, feed intake or whole-tract digestibility was measured, no improvement could be measured.

Effect of finishing diets on Escherichia coli populations and prevalence of enterohaemorrhagic E. coli virulence genes in cattle faeces.

Aim:  To determine the effect of different carbohydrate‐based finishing diets on fermentation characteristics and the shedding of Escherichia coli and enterohaemorrhagic E. coli (EHEC) virulence genes in cattle faeces.

The application of rumen biotechnology to improve the nutritive value of fibrous feedstuffs: pre- and post-ingestion

Abstract Rumen biotechnology has the potential to improve the nutritive value of ruminant feedstuffs that are fibrous, low in nitrogen and of limited nutritional value for other animal species. For the purposes of this paper we define rumen biotechnology as the application of knowledge of forestomach fermentation and the use and management of both natural and recombinant rumen microorganisms to improve the efficiency of ruminant production. Knowledge of rumen digestion that is relevant to modifying both the nutritive value of feedstuffs and the rumen microbial ecosystem by biotechnology is reviewed. Examples of the use and potential of biotechnology to alter the amount and availability of carbohydrate and protein in plants as well as the rate and extent of fermentation and metabolism of these nutrients in the rumen are discussed. The potential applications of biotechnology to rumen microorganisms are manifold and have been reviewed extensively whereas this paper concentrates on the technical difficulties that are limiting its progress. Current limitations include: isolation and taxonomic identification of strains for inoculation and DNA recombination; isolation and characterization of candidate enzymes; level of production, localisation and efficiency of secretion of the recombinant enzyme; stability of the introduced gene; fitness, survival and functional contribution of introduced new strains. Ultimately, the success of rumen biotechnology will depend on the environmental and regulatory concerns of the public being addressed.