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 th e rumen. Seventeen strains of presumptively identified Ruminococcus were ev aluated by a combination of nearly complete and partial 165 rDNA sequence t hat identified all strains as either Ruminococcus albus or Ruminococcus fla vefaciens. All sequences fell into cluster IV of the clostridia, while othe r species of ruminococci (eg. Ruminococcus obeum, Ruminococcus gnavus, Rumi nococcus lactaris) fall into cluster XIVa of the clostridia. Ruminococcus c luster IV sequences were used to design a 16S rRNA oligonucleotide probe to assess the relative abundance of target populations in a stable ruminal en vironment. A stable population (animals fed eight times per day) was establ ished in sheep so that statistically robust comparisons could be made in th e absence of variation due to diurnal rumen fluctuations. The steady state populations were sampled six times over a 24 d period and direct microscopi c counts (DC), total culturable counts (TCC), and total cellulolytic counts (CEL) were determined. DC sind culturable data (TCC and GEL) were compared with relative abundance estimates of Ruminococcus IV sind Fibrobacter succ inogenes. A combination of the Ruminococcus and F. succinogenes probes acco unted for 4.0 % of the bacterial population and cellulolytic Bacteria (meas ured by most-probable numbers) were 5.2 % of the total culturable count. Th ese data suggest that a major portion of the Ruminococcus and Fibrobacter d iversity has been cultured and is represented by available sequences. Stead y state populations were measured over several days in three sheep and an e stimate of variation in DC, TCC, CEL and 16S-based data were obtained. Thes e variance estimates could be used to determine the theoretical sample size s required to obtain statistically significant differences under different experimental conditions.