16S rDNA directed PCR primers and detection of methanogens in the bovine rumen

Aims: To assess the diversity of ruminal methanogens in a grazing cow, and develop PCR primers targeting the predominant methanogens. Methods and Results: DNA was extracted from rumen contents collected from a cow grazing pasture. Archaeal 16S rRNA genes were amplified by PCR using two pairs of archaea-specific primers, and clone libraries prepared. Selected clones were sequenced. Phylogenetic analysis revealed that for one primer pair, most sequences clustered with Methanobrevibacter spp. whereas with the other primer pair most clustered with Methanosphaera stadtmanae. One sequence belonged to the Crenarcheota. PCR primers were designed to detect Msp. stadtmanae and differentiate between Mbb. ruminantium and Mbb. smithii and successfully tested. Conclusions: The ruminal methanogens included Mbb. ruminantium, Mbb. smithii, Mbb. thaueri and methanogens similar to Msp. stadtmanae. The study showed that apparent methanogen diversity can be affected by selectivity from the archaea-specific primers used to create clone libraries. Significance and Impact of Study: This study revealed a greater diversity of ruminal methanogens in grazing cows than previously recognized. It also shows the need for care in interpreting methanogen diversity using PCR-based analyses. The new PCR primers will enable more information to be obtained on Msp. stadtmanae and Methanobrevibacter spp. in the rumen.