Metagenomic analysis of bacterial populations associated with canine oral health through pyrosequencing

The oral bacterial flora is a large and complex community. It has long since been known that shifts in the members and proportions of bacterial species occurs with the onset of various diseases that either result in specific oral manifestations linked with systemic disease or that actually occur in the oral environment. Periodontal disease is the most significant oral disease in dogs with signs of this disease observed in over 70% of dogs over the age of 3 years. Furthermore multiple breeds characteristically suffer from an aggressive form of early-onset periodontitis which may progress to severe disease by the age of 2-3 years. Human periodontal disease is associated with the loss of so called 'beneficial' species and the appearance or overgrowth of 'disease-associated' species. Studies of the canine oral flora have also resulted in the identification of various organisms associated with the periodontally diseased state. In fact the development of oral disease has been described as involving three independent factors: 1) a change in the immunocompetency of the host; 2) a change in the numbers of beneficial bacteria in the mouth and 3) the succession of specific bacteria associated with disease from the normal 'health-associated' flora. It is unclear which or what interplay of these factors are responsible for the progression of breed-associated early onset canine periodontitis. Screening of amplified 16S rDNA libraries has identified that this same scenario exists in the canine oral flora with respect to periodontal disease. In this project we propose the use of 454 pyrosequencing using a metagenomic approach based upon analysis of 16S rRNA sequences from pooled plaque samples obtained from clinically healthy animals and from five breed-specific pooled plaque samples acquired from dogs with severe periodontal disease, where those breeds are associated with the early onset of disease (~2 years of age). This study will differ from those already done in two ways. First there are reports that DNA from some groups of microorganisms in complex communities is not amplified well by DNA polymerase, but can be analysed in a quantitative manner if amplified from a cDNA template, so rRNA species will be directly converted to cDNA, and this cDNA will be subjected to 454 pyrosequencing. Second, the sequencing will be done at a level to facilitate the analysis of over 333,000 sequences per pooled sample which allows a much greater depth of analysis of species and their represented numbers than can be easily done through a standard 16S rDNA plasmid-based library. The aims of this study will be to identify bacterial species that are associated with health and to determine whether any bacterial species are associated with predisposition of breeds to periodontitis. Following the identification of such species molecular tools will be developed that will allow the presence and numbers of these beneficial bacteria to be assessed from individual dogs in a quantitave and repeatable manner using qPCR. These protocols will support the industrial partner in studying the relative metabolic activity of the beneficial or disease associated species in response to nutritional formulations. Through comparison of such qPCR analyses on rRNA sequences and rDNA sequences the longitudinal monitoring of the effects of modifications in canine dietary formulation will be possible.