A metagenomic analysis of DNA viruses in rumens of domestic caprines and ovines using goats and sheep as model organisms

Abstract

The rumen is a complex ecosystem (a metagenome) that is important as it embodies microbes responsible for digestion, efficient use of feeds and energy production in ruminants. A variety of viruses have been known to exist in the bovine rumen, but similar data on caprines and ovines is poorly explored. This study aimed at identifying by high-throughput sequencing (HTS), DNA viruses in the rumens of domestic goats and sheep. Rumen material was collected from eight goats and eight sheep, obtained from the Dagoretti abattoir in Nairobi. The rumen contents were filtered for host cells, bacteria and protozoa by a series of membrane filtration steps. The filtrates were concentrated for viruses by ultra-centrifugation, overnight PEG treatment and nuclease treatment. Total DNA was extracted using a commercial virus DNA kit (Roche). PCR amplification using primers from Methanobrevibacter ruminantium M1 was carried out to ascertain the presence of viral sequences. Purified DNA was submitted for pyrosequencing on the 454 GS FLX platform (Roche). The sequences obtained were processed using 454_QC 2.3.2. after which homology and functional analysis using MG-RAST. The 454-pyrosequencing yielded 149,318 high quality (100-646 bp) reads, 86.9% of the total reads. Of the total hits, 70.3% were bacterial, unassigned were 19.3%, Eukaryota 1.3%, and Archaea 0.4%. Only 3.5% of the above were viral sequences. A phylogenetic analysis of the sequences was achieved using MEGA 5.2.2 for alignment and RAxML for tree construction. The viruses found were mainly phages belonging to the families Siphoviridae (93.5%), Myoviridae (3.1%) and Podoviridae (0.1%). The remaining sequences consisted of Mimiviridae, Microviridae, Poxviridae, Tectiviridae and Marseille virus. 20% of the reads remained unassigned. Specific viral sequences identified included Lactococcus phage 1706, Lactobacillus phage LP65, Xanthomonas phage Xp15, Bacillus phage Bam35 and Acanthamoeba polyphaga mimivirus. These results point to the fact that Phages may be involved in maintenance of the rumen microbial populations of domestic caprines and ovines under their carrying capacity. The fact that majority of these phages have been isolated in other environments, implies that the rumen ecology may be influenced by extraneous factors such as feed/water sources and other ecological factors. The presence of a lytic gene mru_0320_endoisopeptidase ReiP in M. ruminantium M1 was also identified in this study. This may indicate the presence of phages targeting methanogenic bacteria, whose exploitation may hold a promise for reduction of methane emission from caprine and ovines. Furthermore, the 20% unassigned reads may represent novel genomes that need further study. In conclusion, improved protocols for viral enrichment and DNA isolation protocols are required so that data obtained would be representative of the rumen virome. Such protocols should also aim to include study of RNA viruses, because they were not targeted in the present study. Finally, future projects should aim to ascertain if the viruses exits as prophages or free viruses, as this information may offer further insights into the activity of phage viruses in caprine and ovine rumens.