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

Abstract

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 the 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.

Diversity, abundance and novel 16S rRNA gene sequences of methanogens in rumen liquid, solid and epithelium fractions of Jinnan cattle

“…The most predominant species of methanogens in the three groups were related to the genus Methanobrevibacter. This is in agreement with Pei, Mao, Cheng and Zhu previous studies indicating species belonging to the genus Methanobrevibacter as the major methanogens in the rumen of sheep (Wright et al, 2004 andSkillman et al, 2004;Nicholson et al, 2007) and cattle (Whitford et al, 2001;Skillman et al, 2006;Nicholson et al, 2007;Ouwerkerk et al, 2008), of which some were associated with the rumen protozoa (Tokura et al, 1999a;Sharp et al, 1998;Chargan and Ushida, 2004). The predominance of this genus has also been suggested before based on cultivation studies (e.g.…”

Rumen-like methanogens identified from the crop of the folivorous South American bird, the hoatzin (Opisthocomus hoazin)

“…This finding is consistent with other studies where Methanobrevibacter strains accounted for the vast majority of methanogens in chicken ceca (Saengkerdsub et al, 2007), the lower termite Reticulitermes speratus (Shinzato et al, 1999) and the rumen Sharp et al, 1998;Tokura et al, 1999;Whitford et al, 2001;Irbis and Ushida, 2004;Skillman et al, 2004Skillman et al, , 2006Wright et al, 2004Wright et al, , 2008. Moreover, this finding supports our hypothesis that the genus Methanobrevibacter is the most dominant constituent of foregut fermentation systems.…”

“…Moreover, this finding supports our hypothesis that the genus Methanobrevibacter is the most dominant constituent of foregut fermentation systems. The remaining 13.2% of clones (26 of 197) had 94.3-96.3% identity to Methanosphaera stadtmanae, which has been identified in the rumen by others (Whitford et al, 2001;Wright et al, 2004Wright et al, , 2006Wright et al, , 2007Skillman et al, 2006). Despite Garcia-Amado et al (2007) reporting only two partial methanogen 16S rRNA sequences (o625 bp), one with 100% identity to Methanobrevibacter sp.…”

Diversity of gut methanogens in herbivorous animals

“…Methanobrevibacter ruminantium and unassigned Methanosphaera species were found in equal abundance (33.3%), followed by unassigned Methanobrevibacter species (26.7%). The nature and representation of methanogens from the Skillman et al (2006) study show significant overlap with that of the Holsteins analyzed by Whitford et al (2001) presented above. In a recent report by King et al (2011), the frequency of M. ruminantium (33.3%) and unassigned St-Pierre and Wright Methanobrevibacter species (28.6%) in Jersey cows from a mixed herd were also found to be similar to the study by Skillman et al (2006), but in contrast M. millerae was identified as a prominent methanogen (34.6%) and unassigned species of Methanosphaera were observed at a much lower frequency.…”

“…Two reports on Jersey rumen methanogen populations by 16S rRNA gene clone libraries are currently available. In a study conducted by Skillman et al (2006), 15 rumen 16S rRNA gene clones from the rumen of a Jersey cow grazing on ryegrass/clover pasture were analyzed. Methanobrevibacter ruminantium and unassigned Methanosphaera species were found in equal abundance (33.3%), followed by unassigned Methanobrevibacter species (26.7%).…”