| Summary: | The overall aim of this work was to develop molecular based techniques that would allow the identification and quantification of Prevotella spp. in samples of rumen fluid. This was achieved by developing two methods, both based on PCR amplification of 16S rDNA extracted from rumen and faecal samples. The first method involved the amplification of microbial DNA with universal primers, followed by probing with either a Bacteroides Prevotella specific oligonucleotide (Bac/Pre) or a universal oligonucleotide. Comparison with control DNA extracted from pure cultures allowed the relative abundances of Bacteroides and Prevotella spp. to be calculated for samples of rumen fluid and faecal material. In rumen fluid samples the abundance of Bacteroides and Prevotella spp. ranged from 12 to 22% in samples from sheep and up to 36% in a sample of cow rumen fluid. In the faecal samples only 2 to 6% of the total bacteria population belonged to Bacteroides and Prevotella spp. A second method based on PCR-RFLP was developed for the identification of different Bacteroides/Prevotella ribotypes in rumen fluid and faecal samples. The combination of the two techniques allows the Bacteroides and Prevotella spp. present in the rumen to be comprehensively studied and were used to follow changes occurring in the rumen of two sheep whose diets were changed from a high barley diet to a high hay diet. Considerable variation in the Bacteroides and Prevotella population was found between the two animals during the experiment. For example, when both animals were being fed the high hay diet, one animal had 43% of the Bacteroides/Prevotella population belonging to ribotype 7, whereas the other animal contained no DNA belonging to ribotype 7. This work clearly demonstrates the use of the molecular techniques to study changes occurring within the complex ecosystem, such as the rumen. The development of additional 16S rDNA directed oligonucleotides specific for other rumen bacteria could allow all the major rumen bacteria to be investigated using the techniques presented here.
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