16S ribosomal DNA probes for the detection and enumeration of proteolytic rumen bacteria :|ba thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Molecular Biology at Massey University

Abstract

Bacterial degradation of protein causes inefficient nitrogen retention in New Zealand ruminants. The 16S rRNA genes of a Butyrivibrio fibrisolvens-like strain and three Streptococcus bovis strains, isolated from New Zealand cattle were sequenced to further characterise these isolates. Based on 16S rDNA analysis the B. fibrisolvens-like isolate was classified as Clostridium proteoclasticum, while the three S. bovis isolates were confirmed as S. bovis strains. In the absence of selective media for enumeration of these bacteria, a competitive PCR (cPCR) approach was developed for enumeration of these bacteria from rumen samples. PCR primers were designed to variable regions within the 16S ribosomal RNA genes of both S. bovis and C. proteoclasticum. These primers were used in conjunction with the universal forward primer fD1*, to allow amplification of 16S rDNA fragments from these organisms. DNA database searches revealed that the B316 830 primer sequence was present in four B. fibrisolvens strains. Analysis of 16S rDNA sequences indicated that these B. fibrisolvens strains are closely related to C. proteoclasticum and that the B316 830 primer circumscribes these five strains.. The B315 454 primer sequence was found in the 16S rDNA of 10 Streptococcus species. Primer specificity was tested in amplification reactions with DNA extracted from 85 bacterial isolates, mainly of rumen origin. The C. proteoclasticum primer B316 830 and fD1* produced a specific PCR product from C. proteoclasticum DNA only, while the S. bovis primer B315 454 and fD1* gave specific PCR product from DNA of all strains of S. bovis tested but from no other rumen bacterium. An internal control was developed for both S. bovis and C. proteoclasticum to use in cPCR reactions for quantitation. Standard curves were constructed relating the PCR product intensity of target DNA extracted from a known number of cells and the intensity of internal control DNA PCR product. The standard curves were used to quantitate populations of S. bovis and C. proteoclasticum in rumen samples collected from eight dairy cows fed a rotation of four diets. Populations detected ranged from 2 x 106 to 2.8 x 107 for C. proteoclasticum and 1.7 x 107 to 1.3 x 108 for S. bovis. Diet had no significant effect on the populations of either of these proteolytic bacteria.