A Molecular Enrichment Strategy Based on cpn60 for Detection of Epsilon-Proteobacteria in the Dog Fecal Microbiome
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Members of the rare microbiome can be important components of complex microbial communities. For example, pet dog ownership is a known risk factor for human campylobacteriosis, and Campylobacter is commonly detected in dog feces by targeted assays. However, these organisms have not been detected by metagenomic methods. The goal of this study was to characterize fecal microbiota from healthy and diarrheic pet dogs using two different levels of molecular detection. PCR amplification and pyrosequencing of the universal cpn60 gene target was used to obtain microbial profiles from each dog. To investigate the relatively rare epsilon-proteobacteria component of the microbiome, a molecular enrichment was carried out using a PCR that first amplified the cpn10–cpn60 region from epsilon-proteobacteria, followed by universal cpn60 target amplification and pyrosequencing. From the non-enriched survey, the major finding was a significantly higher proportion of Bacteroidetes, notably Bacteroides vulgatus, in healthy dogs compared to diarrheic dogs. Epsilon-proteobacteria from the genera Helicobacter and Campylobacter were also detected at a low level in the non-enriched profiles of some dogs. Molecular enrichment increased the proportion of epsilon-proteobacteria sequences detected from each dog, as well as identified novel, presumably rare sequences not seen in the non-enriched profiles. Enriched profiles contained known species of Arcobacter, Campylobacter, Flexispira, and Helicobacter and identified two possibly novel species. These findings add to our understanding of the canine fecal microbiome in general, the epsilon-proteobacteria component specifically, and present a novel modification to traditional metagenomic approaches for study of the rare microbiome.
KeywordsBacteroidetes Terminal Restriction Fragment Length Polymorphism Fecal Microbiota Fecal Microbiome Enrich Profile
This study was funded by the WCVM Companion Animal Health Fund and the Saskatchewan Health Research Foundation. BC was supported by a postdoctoral fellowship from the Saskatchewan Health Research Foundation. The authors are grateful to Manuel Chirino for donating the genomic DNA used in epsilon-enrichment primer development.
- 3.Ipsos-Reid (2001) Paws and claws: a syndicated study on Canadian pet ownership. Ipsos-Reid, Toronto, pp 1–176Google Scholar
- 4.AVMA (2007) US Pet ownership and demographics sourcebook. American Veterinary Medical Association, SchaumburgGoogle Scholar
- 5.Fullerton KE, Ingram LA, Jones TF, Anderson BJ, McCarthy PV, Hurd S, Shiferaw B, Vugia D, Haubert N, Hayes T, Wedel S, Scallan E, Henao O, Angulo FJ (2007) Sporadic Campylobacter infection in infants: a population-based surveillance case-control study. Pediatr Infect Dis J 26:19–24PubMedCrossRefGoogle Scholar
- 17.Swanson KS, Dowd SE, Suchodolski JS, Middelbos IS, Vester BM, Barry KA, Nelson KE, Torralba M, Henrissat B, Coutinho PM, Cann IK, White BA, Fahey GC Jr (2011) Phylogenetic and gene-centric metagenomics of the canine intestinal microbiome reveals similarities with humans and mice. ISME J 5:639–649PubMedCrossRefGoogle Scholar
- 30.Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541PubMedCrossRefGoogle Scholar
- 33.Felsenstein J (1989) PHYLIP—Phylogeny Inference Package (Version 3.2). Cladistics 5:164–166Google Scholar
- 34.Sakamoto M, Suzuki N, Benno Y (2010) hsp60 and 16S rRNA gene sequence relationships among species of the genus Bacteroides with the finding that Bacteroides suis and Bacteroides tectus are heterotypic synonyms of Bacteroides pyogenes. Int J Syst Evol Microbiol 60:2984–2990PubMedCrossRefGoogle Scholar