A Cross-Sectional Survey of Bacterial Species in Plaque from Client Owned Dogs with Healthy Gingiva, Gingivitis or Mild Periodontitis

Gingivitis or Mild Periodontitis. PLoS ONE 8(12): e83158. doi:10.1371/journal.pone.0083158 A Cross-Sectional Survey of Bacterial Species in Plaque from Client Owned Dogs with Healthy Gingiva, Gingivitis or Mild Periodontitis Ian J. Davis 0 Corrin Wallis 0 Oliver Deusch 0 Alison Colyer 0 Lisa Milella 0 Nick Loman 0 Stephen Harris 0 Malcolm Gracie Semple, University of Liverpool, United Kingdom 0 1 The Waltham Centre for Pet Nutrition, Mars Petcare UK , Leicestershire , United Kingdom , 2 The Veterinary Dental Surgery, Surrey , United Kingdom , 3 Centre for Systems Biology, University of Birmingham , Birmingham , United Kingdom Periodontal disease is the most widespread oral disease in dogs which if left untreated results in significant pain to the pet and loss of dentition. The objective of this study was to identify bacterial species in canine plaque that are significantly associated with health, gingivitis and mild periodontitis (<25% attachment loss). In this survey subgingival plaque samples were collected from 223 dogs with healthy gingiva, gingivitis and mild periodontitis with 72 to 77 samples per health status. DNA was extracted from the plaque samples and subjected to PCR amplification of the V1-V3 region of the 16S rDNA. Pyrosequencing of the PCR amplicons identified a total of 274 operational taxonomic units after bioinformatic and statistical analysis. Porphyromonas was the most abundant genus in all disease stages, particularly in health along with Moraxella and Bergeyella. Peptostreptococcus, Actinomyces, and Peptostreptococcaceae were the most abundant genera in mild periodontitis. Logistic regression analysis identified species from each of these genera that were significantly associated with health, gingivitis or mild periodontitis. Principal component analysis showed distinct community profiles in health and disease. The species identified show some similarities with health and periodontal disease in humans but also major differences. In contrast to human, healthy canine plaque was found to be dominated by Gram negative bacterial species whereas Gram positive anaerobic species predominate in disease. The scale of this study surpasses previously published research and enhances our understanding of the bacterial species present in canine subgingival plaque and their associations with health and early periodontal disease. - Funding: This work was funded by Mars Petcare UK. The WALTHAM Institute of Pet Nutrition is the fundamental research center for Mars Petcare. The WALTHAM Institute was involved at all levels of this research including study design, data collection and analysis, decision to publish, or preparation of the manuscript. However there are no conflicts of interest, this survey represents an unbiased view (in the commercial sense) of the bacterial species present in canine plaque. The oligonucleotide primers used and the parameters used in subsequent analysis were chosen on guidance from external academic colleagues. No current external funding sources for this study. Competing interests: The authors have the following interests. This work was funded by Mars Petcare UK, the employer of Ian. J. Davis, Corrin Wallis, Oliver Deusch, Alison Colyer and Stephen Harris. Lisa Milella is employed by The Veterinary Dental Surgery. A patent has been filed based on information from this work. There are no products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. Periodontal disease (PD) is the most widespread oral disease in dogs. Studies have demonstrated between 44% and 63.6% of dogs are affected [1-4]. Variation in prevalence estimates are likely to be due to the different age and breed compositions of the study groups and the criteria used to establish diagnosis of periodontal disease. It is accepted that bacteria present in human dental plaque are the aetiological agent of periodontal disease; though the specific organisms involved in the initiation of disease and the basis of the subsequent events thereafter are unclear [5]. A working hypothesis is that specific antigens or enzymes produced by bacteria in the plaque biofilm initiate activation of the host inflammatory response, the latter being the main pathological agent of periodontal disease. The initial stages of disease are observed clinically as red and inflamed gums, defined as gingivitis. Without treatment by removal of the plaque biofilm, gingivitis may progress to early periodontitis. The earliest stage of periodontitis (PD1) is characterised by initial tissue breakdown and loss of up to 25% attachment of the periodontal ligament surrounding the tooth root [6]. In humans this switch from gingivitis to periodontitis appears to be restricted to 10-15% of the population [7]. The onset of periodontitis is defined by irreversible tissue destruction and if left untreated will progress to extreme periodontitis (PD3-PD4). This is characterised by extensive (>50%) destruction of the periodontal ligament, gum recession and breakdown of supporting tissues eventually leading to the loss of the tooth. The periodontal disease process can be inhibited in the early stages (PD1) by dental scaling and polishing of the periodontal pocket to remove the source of inflammation (dental plaque) with subsequent regular plaque removal by tooth brushing. As such this work is focused on understanding the early stages of disease, (gingivitis through to PD1) in pet dogs where non-surgical interventions may be effective. The diversity of bacterial species found in the canine oral microbiome has been reported using culture independent molecular methods from 51 dogs [8]. Based on full length 16S rDNA Sanger sequencing 353 taxa were identified; of these 80% were novel and only 16.4% were shared with the human oral microbiome. Not only does this indicate clear differences between the bacterial populations in human versus canine mouths but also the under representation of 16S rDNA sequences from canine oral species in public DNA sequence databases. A survey of the canine oral microbiota of pooled samples from gums, tongue, teeth and cheeks, of 6 clinically healthy dogs has been completed using pyrosequencing methods [9]. This approach enabled the estimation of the relative abundance of genera with the most common being Porphyromonas (39.2% of sequences), Fusobacterium (4.5%) and Capnocytophaga (3.8%). Significant differences in bacterial populations by oral health status have also been reported in a study of dogs with healthy mouths, gingivitis or periodontal disease (n=3 per health state; [10]). The study used culture dependant and independent approaches with identification of taxa via Sanger sequencing of 16S rDNA. Significant associations of specific bacterial taxa with disease stages were not observed due to the relatively small population size and low sequence d (...truncated)