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Pyrosequencing-based profiling of bacterial 16S rRNA genes identifies the unique Proteobacteria attached to the rumen epithelium of bovines
The objective of this research is to characterize the unique bacterial community attached to the rumen epithelium in both dairy cows and beef cattle. The rumen content and rumen epithelium samples were collected from four Chinese Holstein dairy cows and five Australian beef cattle when the animals were slaughtered. The microbial DNA was extracted from rumen content and epithelium using CTAB plus bead beating method. Community compositions of bacterial 16S rRNA genes from content and wall were examined using a tag-encoded amplicon pyrosequencing assay with bacterial-targeting universal primers 8F and 533R. The sequencing was performed with Roche 454 GS FLX instrument. The platform QIIME was used for reads trimming and OTU analysis. The reads sharing more than 97% identity were clustered into OTUs. The results showed that 158 729 trimmed sequences with the length of 300-560 bp were kept. After OTU picking, 2735 OTUs were formed, and the average reads counts per sample was 7280. The bacterial species richness from epithelium was significantly lower (P<0.01) than that from content according to Chao1 index, and bacterial alpha diversity from epithelium was also significantly lower (P<0.01) than that from content according to Shannon index. The PCoA analysis revealed that epimural bacterial community was distinctly different from that in content, which implied some unique bacterial community attached to the epithelium of bovine. To further find out what the unique community was, taxonomy and significance test were carried out. While the Fimicutes (~42%) and Bacteroidetes (~20%) were the predominant abundant bacteria on epithelium, significant higher (P<0.01) abundance of Proteobacteria (~26%) were present on epithelium than that in content (~3%). Hylemonella, Desulfobulbus, Campylobacter and unclassified Neisseriaceae were the predominant bacteria attached to epithelium compared to the content in both dairy cows and beef. These unique bacteria were microaerophilic (except for Desulfobulbus), and all of them were able to hydrolyze urea, which could further certify the function of oxygen scavenging and urea hydrolysis for bacteria community attached to epithelium.
Keywords: rumen epithelium, bacterial community, pyrosequencing