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- Materials and methods
Twenty-three strains of Ruminococcus isolated from ruminants were assessed for digestive ability on different plants and purified cellulose. Genetic diversity was assessed by ERIC, REP and 16–23S rDNA spacer polymorphisms. All ruminococci could be typed by ERIC, REP or 16–23S rDNA spacer, but all three typing methods had to be used in concert to differentiate closely related strains. Digestibility of lucerne (Medicago sativa), rhodes grass (Chloris gayana) and spear grass (Heteropogon contortus) were assessed. Dry matter (DM) digestibility was highly correlated (> 0·93) with neutral detergent fibre (NDF) digestibility, but cellulose disc digestibility was a poor indicator of DM and NDF digestibility. Studies demonstrate the wide variation in ability of ruminococci to digest forages, and some recently isolated strains (Y1, LP-9155, AR67, AR71 and AR72) were superior to reference strains (FD-1 and Ra8). Multivariate analysis showed that groupings derived from genotyping data closely resembled those determined by digestibility data. This study indicated that ruminococci are diverse in digestive ability and genotype, and this diversity suggests that there may be highly fibrolytic strains in nature that could be utilized for animal production.
Microbial diversity in the rumen is well recognized ( Krause & Russell 1996; Hespell et al. 1997 ) but there are few studies that have assessed genotypic diversity of a single genus in conjunction with a phenotype that is regarded as fundamental to the ecological fitness of the organism. Bacterial fibre digestion in the rumen is carried out primarily by Fibrobacter succinogenes, Ruminococcus (R. albus and R. flavefaciens) and Butyrivibrio fibrisolvens, but F. succinogenes and the Ruminococcus species are usually the most fibrolytic ( Hespell et al. 1997 ). Fibre fermenting ability by Ruminococcus is arguably central to its ability to compete in the rumen but it is not known if this important phenotype is related to its genotype.
Digestibility studies of different plants by a range of bacteria are few ( Dehority & Scott 1967; Morris & van Gylswyke 1980) and there are no studies which have investigated a range of ruminococci for digestive and genetic diversity. True assessments of diversity of digestive ability should be obtained from experiments using a variety of plants, and bacteria able to digest different plants efficiently have the best chance of surviving in the rumen. Cellulose digestibility has traditionally been used to evaluate activity of cellulolytic bacteria ( Halliwell & Bryant 1963; Stewart et al. 1990 ) but purified cellulose does not contain cell wall components (e.g. phenolics and lignin) that limit digestibility ( Akin et al. 1974 ; Akin 1982; McSweeney & Mackie 1997).
Several investigators ( Versalovic et al. 1991 ; van Belkum et al. 1998 ) have shown that repetitive extragenic palindromic sequences (REP) and enterobacterial repetitive intergenic consensus (ERIC) sequences can be used as bacterial typing methods. In addition, amplification of prokaryotic rDNA spacer regions with conserved 16S and 23S primers can be used to type bacteria ( Jensen & Hubner 1996). Cladistic analyses of genotype data (generated with ERIC, REP, and 16–23S rDNA spacers) and digestibility data have been used to investigate the relationship between genotype and phenotype (fibre digestibility).
- Top of page
- Materials and methods
An optimally functioning rumen is critical to animal productivity and superior performance in pasture fed animals is partly determined by efficiency of plant cell wall digestion ( Beveridge & Richards 1973; Broderick 1989; Ørskov 1991). As Ruminococcus species are one of the most fibrolytic groups of ruminal bacteria ( Weimer 1996), a study was undertaken to investigate their digestive potency and genetic diversity. These studies provide an appreciation of inherent phenotypic and genotypic variation within Ruminococcus that can be harnessed to improve ruminal function. 16S rRNA analysis differentiates strains at the species level ( Pace 1997) but to obtain an understanding of diversity within a species, subspecies genotyping methods are needed.
Rapid amplification of polymorphic DNA (RAPD) is frequently used as a typing method for bacteria and relies on the amplification of DNA with PCR primers at low stringency. RAPDs are not based on specific targets and do not require prior knowledge of sequence, but results are often variable ( Lawrence et al. 1993 ; Brikun et al. 1994 ). As ERICs, REPs and 16–23S spacers are based on conserved sites, they can be a more reliable way of typing bacteria ( Versalovic et al. 1991 ; De Bruijn 1992). REP elements may be regulatory in nature because of their ability to form stem-loop structures in transcribed RNA ( Versalovic et al. 1991 ), whereas ERIC elements contain inverted repeats located in extragenic regions ( Versalovic et al. 1991 ). Bacteria have several ribosomal operons and the spacer region between the 16S and 23S varies in sequence and length, providing an additional means of determining bacterial genotype ( Jensen & Hubner 1996).
Versalovic et al. (1991) found that the ERIC and REP elements were present in most Gram-negatives but not to the same extent in Gram-positives. They assessed more than 30 Gram-negatives and only 11 Gram-positives, but over 70% of the Gram-positives showed at least some hybridization with ERIC and REP consensus sequences. Ruminococcus are low G + C Gram-positives, related phylogenetically (by 16S rDNA analysis) to the clostridia, but no studies using ERICs, REPs and 16–23S spacers have assessed bacteria affiliated with the clostridia ( Rainey & Janssen 1995). We have surveyed (data not shown) a diverse group of Gram-positive and Gram-negative ruminal bacteria and ERIC, REP and 16–23S spacer polymorphisms can be extended to most of these bacteria.
ERICs ( Fig. 1a) and REPs ( Fig. 1b) both demonstrated the presence of highly polymorphic DNA, and primers amplified many bands. In contrast, amplification of the 16–23S rDNA spacer gave one to three bands ( Fig. 1c) but these banding patterns could be used to differentiate between bacteria. A combination of ERICs, REPs and 16–23S rDNA spacers was required to differentiate effectively between bacteria (see Results above). We found in the course of these studies that culture purity could be problematic and it was difficult to differentiate between ruminococci microscopically. These genotyping methods provide a benchmark for quality control and for differentiating among closely related strains.
Cellulose disc digestion was not the best way of evaluating organisms because of low correlations with forage NDF digestion ( Fig. 2). This observation is supported by work by Stewart et al. (1990) which showed that R. flavefaciens 007 lost its ability to degrade cotton fibres after repeated transfer on cellobiose medium. One of these colonies could be resuscitated and was slightly more active than the ‘mutated’ low cotton digesting form, but there was hardly any difference when wheat straw was the substrate. These authors concluded that the factors important in cotton digestion had little relevance to wheat straw digestion.
We considered that NDF digestion was the most important characteristic because ruminants consume a diet containing various plants and assessments of digestive ability of several plants give a more accurate understanding of variations in digestive potential. Dehority & Scott (1967) examined digestion of a wide range of forages by various species of ruminal bacteria. Digestive abilities among the fibrolytic organisms varied, but only four strains of R. flavefaciens (strains B1a, B34b, C1a and C-94) and one strain of R. albus (strain 7) were studied. Kock & Kistner (1969) and Morris & van Gylswyke (1980) studied digestibility of Eragrostis tef hay, and found similar results to Dehority & Scott (1967). However, assessments in all three studies used mostly the same strains.
As rate of passage and rate of fermentation compete in the rumen, only bacteria that ferment substrate at rates greater than the passage rate will be competitive ( Colucci et al. 1982 ). If extent of digestion does not reflect the rate of digestion, then this is an important consideration when bacteria are ranked for digestive potency. We compared the rate and extent of digestion of three ruminococci which differed in their ability to digest fibre ( Fig. 3) and found that rate and extent of digestion were highly correlated.
Lucerne has a greater soluble fraction than rhodes or spear grass and DM digestibilities were usually higher ( Table 2). These differences in the easily digestible fraction of a forage have implications for diet composition and competitive fitness of ruminococci. Fibrolytic bacteria such as Butyrivibrio fibrisolvens grow more rapidly on the soluble component of forages than ruminococci and produce bacteriocins that inhibit ruminococci growth ( Kalmokoff & Teather 1997). As B. fibrisolvens requires amino acids as a nitrogen source and ruminococci, only ammonia ( Hespell et al. 1997 ), a forage diet low in soluble nutrients (rhodes or spear) and with only a urea supplement might select for ruminococci. van Gylswyk (1970) found that a teff (Eragrostis tef) diet fed to sheep selected for ruminococci in preference to butyrivibrios, and that supplementation with urea and branched-chain volatile fatty acids increased their numbers.
Clustering based on genotype ( Fig. 4a) did not group bacteria according to their 16S rDNA determined species ( Table 2). This ‘mismatch’ is not particularly surprising because 16S rRNA phylogeny is based on highly conserved genes ( Pace 1997) while ERIC, REP and 16–23S rDNA spacers are based on chromosomal elements that are not necessarily stable and may be evolving at significantly different rates to the 16S rDNA genes ( van Belkum et al. 1998 ). There was, however, a very close relationship between phenotype ( Fig. 4b) and genotype ( Fig. 4a) based clustering, indicating that certain genetic types were associated with fibrolytic ability.
The above conclusions are supported by work done on Bradyrhizobium with ERIC, REP and 16–23S rDNA spacers ( Vinuesa et al. 1998 ). Root nodulating bacteria from a variety of geographical sources were analysed for 16S rDNA RFLP, ERIC, REP and 16–23S rDNA spacer polymorphisms and ability to nodulate legumes. There was not a good relationship between 16S rDNA grouping and grouping determined with ERIC, REP and 16–23S rDNA spacers. There was, however, a strong correlation between ERIC, REP and 16–23S rDNA spacers derived genotypes and nodulating ability, a phenotype fundamental to the competitive fitness of Bradyrhizobium.
The ruminococci are clearly a diverse group of bacteria and plant digesting ability and genotype differs widely between strains. Significant observations were that ranking’s of bacteria remained virtually the same on all forages but changed with cellulose discs ( Table 2), and that bacteria performing well on plant substrates have the best opportunity for success in the rumen. It is likely that ruminal communities are made up of a variety of ruminococci with digestive abilities not unlike those in Table 2. However, there are few studies which have indicated ruminal manipulations (dietary or otherwise) that would enable a different/introduced population to establish in the rumen. A significant research effort in this area is an essential element in furthering our understanding of the ruminal ecosystem.