Developing diagnostic tools for canine periodontitis: combining molecular techniques and machine learning models.

BACKGROUND: Dental plaque microbes play a key role in the development of periodontal disease. Numerous high-throughput sequencing studies have generated understanding of the bacterial species associated with both canine periodontal health and disease. Opportunities therefore exist to utilise these bacterial biomarkers to improve disease diagnosis in conscious-based veterinary oral health checks. Here, we demonstrate that molecular techniques, specifically quantitative polymerase chain reaction (qPCR) can be utilised for the detection of microbial biomarkers associated with canine periodontal health and disease. RESULTS: Over 40 qPCR assays targeting single microbial species associated with canine periodontal health, gingivitis and early periodontitis were developed and validated. These were used to quantify levels of the respective taxa in canine subgingival plaque samples collected across periodontal health (PD0), gingivitis (PD1) and early periodontitis (PD2). When qPCR outputs were compared to the corresponding high-throughput sequencing data there were strong correlations, including a periodontal health associated taxa, Capnocytophaga sp. COT-339 (rs =0.805), and two periodontal disease associated taxa, Peptostreptococcaceae XI [G-4] sp. COT-019 (rs=0.902) and Clostridiales sp. COT-028 (rs=0.802). The best performing models, from five machine learning approaches applied to the qPCR data for these taxa, estimated 85.7% sensitivity and 27.5% specificity for Capnocytophaga sp. COT-339, 74.3% sensitivity and 67.5% specificity for Peptostreptococcaceae XI [G-4] sp. COT-019, and 60.0% sensitivity and 80.0% specificity for Clostridiales sp. COT-028. CONCLUSIONS: A qPCR-based approach is an accurate, sensitive, and cost-effective method for detection of microbial biomarkers associated with periodontal health and disease. Taken together, the correlation between qPCR and high-throughput sequencing outputs, and early accuracy insights, indicate the strategy offers a prospective route to the development of diagnostic tools for canine periodontal disease.

Development of Yorkshire Terrier Dentition.

The development of dentition in dogs has been associated with several problems including tooth over-crowding, missing permanent dentition, and persistent deciduous teeth (PDT). Information on dentition development in different breeds is lacking. This study of 61 Yorkshire terriers aimed to determine the (i) average age at deciduous tooth exfoliation, (ii) average age at permanent tooth eruption, (iii) PDT incidence, and influencing factors such as body weight. The ages of exfoliation of deciduous teeth and eruption of permanent dentition were influenced by body weight and tooth type. These dentition changes tended to occur later in dogs ≤ 3 kg versus dogs > 5 kg. Generally, incisors were exfoliated first, followed by premolars and then canines. At a body weight of 4.5 kg, the middle of the data range, the estimated age at loss of deciduous teeth (with 95% confidence intervals) was 21.9 (21.1, 22.9) weeks for incisors, 26.1 (24.9, 27.4) weeks for canines, and 23.9 (22.9, 24.9) weeks for premolar. The estimated age at eruption of permanent dentition was 22.3 (21.6, 23.0) weeks for incisors, 23.8 (23.0, 24.6) weeks for canines, 24.7 (24.0, 25.5) weeks for premolars, and 26.4 (25.5, 27.3) for molar teeth. However, this sequence was disrupted in dogs ≤ 3 kg. Yorkshire terriers had a high incidence of PDT. At a body weight of 4.5 kg, the estimated proportion of PDT was: incisors 0.86% (0.32, 2.31), canines 15.62% (7.62, 29.37) and premolars 3.57% (1.62, 7.66). Canines constituted the most frequently retained tooth type, with 89.1% retained in dogs ≤ 3 kg compared to 12.0% in dogs > 5 kg. This information will enable veterinarians to provide personalised advice regarding the oral care requirements for Yorkshire terriers and highlights the need to regularly monitor this breed between the ages of two and seven months, during the active phases of tooth development.

Comparison of subgingival and gingival margin plaque microbiota from dogs with healthy gingiva and early periodontal disease.

The purpose of the investigation was to determine whether canine gingival margin (GM) plaque is a reliable surrogate for subgingival (SG) plaque from a microbial community (microbiota) perspective. SG and GM plaque samples were collected from 381 dogs visiting pet hospitals in the USA, China and Thailand. Dogs with clinically healthy gingivae through to early periodontitis were included in the study. The samples were subject to next generation Illumina sequence analysis to allow microbiota comparisons to be made between the two plaque sources. Overall, the SG and GM samples indicated commonality via the majority community that were shared between them; health associations led to the identification of some significant taxa-specific differences. GM microbiota exhibited lower variability and diversity and were shown to reflect a sub-population of those associated with SG plaque. Both plaque niches, however, demonstrated similar changes in microbial signatures with health and early periodontal disease and did not indicate divergent trends. The key, most abundant microbiota of GM plaque strongly reflect those observed with SG plaque across health and early periodontitis. Microbiota in plaque from above the gum line may therefore be employed as a biomarker of oral health. This opens up the potential to use plaque, sampled from conscious dogs, to define oral health status and improve the diagnosis, treatments and interventions for periodontal disease.

Effect of feeding a daily oral care chew on the composition of plaque microbiota in dogs.

The objective of this study was to investigate the influence of daily feeding of an oral care chew on the composition of canine supragingival plaque microbiota. Twelve beagle dogs were recruited to the randomized cross-over study. The dogs were fed one of two dietary regimes, both consisting of a commercially available wet and dry diet mix, either with or without daily supplementation with an oral care chew. After each 28-day test phase, supragingival plaque samples were collected and processed via Illumina sequencing to determine the microbiota composition. A comparative analysis of bacterial species associated with health and periodontal disease, identified from prior clinical studies, revealed differences between the dietary regimes. Consumption of the daily oral care chew, resulted in a significant increase in proportion of 6 health associated taxa but only 3 disease associated taxa compared to no chew. In contrast, 8 disease and 1 health associated taxa showed increased proportions for no chew versus the oral care chew. Daily feeding of the oral care chew tested in this study has therefore been shown to increase the proportion of health associated bacteria, over bacteria associated with periodontal disease, in supragingival plaque compared to no chew. By influencing plaque microbiota towards a bias for health associated bacteria, feeding of the oral care chew provides a means to reduce the prevalence of bacterial species shown to be associated with periodontal disease in dogs.

The canine oral microbiome: variation in bacterial populations across different niches.

BACKGROUND: Microbiota from different niches within the canine oral cavity were profiled and compared. Supragingival plaque and stimulated saliva, were collected alongside samples from the buccal and tongue dorsum mucosa, from 14 Labrador retrievers at three timepoints within a 1 month timeframe. The V3-V4 region of the 16S rRNA gene was sequenced via Illumina MiSeq. RESULTS: Supragingival plaque microbiota had the highest bacterial diversity and the largest number of significant differences in individual taxa when compared to the other oral niches. Stimulated saliva exhibited the highest variability in microbial composition between dogs, yet the lowest bacterial diversity amongst all the niches. Overall, the bacteria of the buccal and tongue dorsum mucosa were most similar. CONCLUSIONS: The bacterial community profiles indicated three discrete oral niches: soft tissue surfaces (buccal and tongue dorsum mucosa), hard tissue surface (supragingival plaque) and saliva. The ability to distinguish the niches by their microbiota signature offers the potential for microbial biomarkers to be identified in each unique niche for diagnostic use.

A longitudinal assessment of periodontal disease in Yorkshire terriers.

BACKGROUND: Periodontal disease is the most common oral disease of dogs and has been associated with systemic disease. The purpose of the present study was to determine the extent of periodontal disease in a population of Yorkshire terrier dogs with and without a tooth brushing regimen. Each dog was assessed under general anaesthesia two to five times between 37 and 78 weeks of age. The extent of gingivitis and periodontitis was ascertained for every tooth in the mouth. Gingivitis was measured using time to bleeding on probing, and periodontitis was based on extent of clinical attachment loss (probing depth, gingival recession and furcation exposure). RESULTS: Of the 49 dogs assessed at 37 weeks of age, 98% had at least one tooth or aspect with early periodontitis (PD2, < 25% attachment loss). The average percentage of teeth with periodontitis in the mouth was 29.6% with 95% confidence interval (23.6, 36.4). The odds of early periodontitis was 2.74 (2.23, 3.37) times higher at 78 weeks of age compared to 37 weeks of age. The canine teeth had a significantly higher probability of periodontitis compared to all other tooth types at both 37 and 78 weeks of age (p < 0.001). In addition, at the same time points, the incisors had a significantly higher probability of periodontitis compared to the molars and premolars (p < 0.001). CONCLUSIONS: Breeds of dog that are susceptible to developing periodontitis, such as Yorkshire terriers, require effective treatments for the prevention of periodontal disease from a young age. Although tooth brushing is one of the most effective methods when it comes to preventative homecare, this is not always realistic, as was found in this study. Therefore alternative ways to retard or prevent plaque accumulation that are practical for both dogs and their owners are required.

Genomes from bacteria associated with the canine oral cavity: A test case for automated genome-based taxonomic assignment.

Taxonomy for bacterial isolates is commonly assigned via sequence analysis. However, the most common sequence-based approaches (e.g. 16S rRNA gene-based phylogeny or whole genome comparisons) are still labor intensive and subjective to varying degrees. Here we present a set of 33 bacterial genomes, isolated from the canine oral cavity. Taxonomy of these isolates was first assigned by PCR amplification of the 16S rRNA gene, Sanger sequencing, and taxonomy assignment using BLAST. After genome sequencing, taxonomy was revisited through a manual process using a combination of average nucleotide identity (ANI), concatenated marker gene phylogenies, and 16S rRNA gene phylogenies. This taxonomy was then compared to the automated taxonomic assignment given by the recently proposed Genome Taxonomy Database (GTDB). We found the results of all three methods to be similar (25 out of the 33 had matching genera), but the GTDB approach required fewer subjective decisions, and required far less labor. The primary differences in the non-identical taxonomic assignments involved cases where GTDB has proposed taxonomic revisions.

Validation of Quantitative Light-Induced Fluorescence for Quantifying Calculus on Dogs' Teeth.

Periodontal disease is a common disease of dogs and is initiated by the buildup of plaque on the tooth surface. As plaque matures, it becomes mineralized to form calculus, which although not directly involved in the etiology of periodontal disease, provides an irregular surface to which plaque can adhere. Evaluation of the quantity of plaque and calculus on dogs' teeth is therefore essential to enable the efficacy of products, designed to prevent or retard plaque and calculus accumulation, to be evaluated. The objective of this study was to determine whether quantitative light-induced fluorescence (QLFTM) is a suitable tool to quantify the amount of calculus on the buccal surface of dogs' teeth following the removal of disclosed plaque by tooth brushing. The amount of calculus on the teeth of 26 miniature schnauzers was measured, using QLF and a calculus index method (Warrick-Gorrel), during a 28-day phase crossover study comparing feeding a daily dental chew versus providing no daily chew. Quantification of calculus using the Warrick-Gorrel method showed a 43.8% reduction in calculus buildup, with 95% confidence interval of 27.3 to 60.3 ( P < .001). With QLF, the percentage reduction in calculus accumulation was 65.8% (58.1-73.4, P < .001). A retrospective sample size analysis showed that fewer dogs were required for QLF analysis compared to the Warrick-Gorrel method. This study demonstrated that QLF is a sensitive and precise method for quantification of calculus on dogs' teeth. It removes the subjective element of human examiners and has greater accuracy and reduced variability through the continuous nature of the data.

Distinct fermentation and antibiotic sensitivity profiles exist in salmonellae of canine and human origin.

BACKGROUND: Salmonella enterica is a recognised cause of diarrhoea in dogs and humans, yet the potential for transfer of salmonellosis between dogs and their owners is unclear, with reported evidence both for and against Salmonella as a zoonotic pathogen. A collection of 174 S. enterica isolates from clinical infections in humans and dogs were analysed for serotype distribution, carbon source utilisation, chemical and antimicrobial sensitivity profiles. The aim of the study was to understand the degree of conservation in phenotypic characteristics of isolates across host species. RESULTS: Serovar distribution across human and canine isolates demonstrated nine serovars common to both host species, 24 serovars present in only the canine collection and 39 solely represented within the human collection. Significant differences in carbon source utilisation profiles and ampicillin, amoxicillin and chloramphenicol sensitivity profiles were detected in isolates of human and canine origin. Differences between the human and canine Salmonella collections were suggestive of evolutionary separation, with canine isolates better able to utilise several simple sugars than their human counterparts. Generally higher minimum inhibitory concentrations of three broad-spectrum antimicrobials, commonly used in veterinary medicine, were also observed in canine S. enterica isolates. CONCLUSIONS: Differential carbon source utilisation and antimicrobial sensitivity profiles in pathogenic Salmonella isolated from humans and dogs are suggestive of distinct reservoirs of infection for these hosts. Although these findings do not preclude zoonotic or anthroponotic potential in salmonellae, the separation of carbon utilisation and antibiotic profiles with isolate source is indicative that infectious isolates are not part of a common reservoir shared frequently between these host species.

Assessment of dental plaque coverage by Quantitative Light-induced Fluorescence (QLF) in domestic short-haired cats.

Dietary means of reducing plaque and calculus deposits are frequently sought for the maintenance of oral health in cats and dogs. In the development of such products sensitive, reliable, reproducible methods of measuring plaque and calculus are key. The aim of this study was to assess Quantitative Light-induced Fluorescence (QLF™) for the detection of dental plaque coverage in cats compared to the modified Logan and Boyce technique. The techniques were utilised in a crossover study, which compared two diets for their effect on plaque deposition in a cohort of 24 adult cats. Analysis of the effect of diet on plaque coverage by both the modified Logan and Boyce technique and QLF showed a significant effect of feeding regime (p=0.024 and p≤0.0001, respectively) with good agreement between the techniques in the percentage reduction of plaque accumulation. A within study assessment of QLF demonstrated excellent intra-operator repeatability (coefficient of variation 2.2%). Similarly, inter-operator reproducibility was also good (coefficient of variation 2.3%). A retrospective analysis, using the data to estimate the sample size required for at least 90% power to detect a 15% difference between treatments in a two-way crossover study, established that 10 cats would be sufficient for plaque measurement by QLF, while assessment by the modified Logan and Boyce method required over 30 cats. QLF was determined to be a reliable, reproducible method for the assessment of plaque deposition in cats and requires fewer subjects for the detection of differences between treatment effects compared to the modified Logan and Boyce method.

Quantification of Canine Dental Plaque Using Quantitative Light-Induced Fluorescence.

The aim of this work was to evaluate Quantitative Light-induced Fluorescence (QLF) as an alternative to the established Logan and Boyce method for determining plaque coverage of dogs' teeth. In a series of studies in conscious and anesthetized dogs, QLF showed good intra-photographer repeatability (coefficient of variation [CV] of 7.5% for undisclosed teeth) and inter-photographer reproducibility (CV of 3.2% for undisclosed teeth and 8.5% for disclosed teeth). The QLF software accurately identifies areas of plaque as demonstrated by comparison to the variability of 5 human scorers, manually marking plaque on QLF-acquired images (P = 0.1). There was good agreement with the modified Logan and Boyce method in the percentage reduction in plaque accumulation measured when dogs were fed an oral care chew versus no chew. To see a 15% difference in plaque accumulation, which is considered sufficient by the Veterinary Oral Health Council to differentiate between 2 treatments, a retrospective power analysis (90%) of the data established that only 7 dogs would be required, compared to 19 dogs for the modified Logan and Boyce method. QLF is a reliable method for measuring dental plaque in dogs with the added advantage that it is not subjective and requires fewer animals.

Characterising the Canine Oral Microbiome by Direct Sequencing of Reverse-Transcribed rRNA Molecules.

PCR amplification and sequencing of phylogenetic markers, primarily Small Sub-Unit ribosomal RNA (SSU rRNA) genes, has been the paradigm for defining the taxonomic composition of microbiomes. However, 'universal' SSU rRNA gene PCR primer sets are likely to miss much of the diversity therein. We sequenced a library comprising purified and reverse-transcribed SSU rRNA (RT-SSU rRNA) molecules from the canine oral microbiome and compared it to a general bacterial 16S rRNA gene PCR amplicon library generated from the same biological sample. In addition, we have developed BIONmeta, a novel, open-source, computer package for the processing and taxonomic classification of the randomly fragmented RT-SSU rRNA reads produced. Direct RT-SSU rRNA sequencing revealed that 16S rRNA molecules belonging to the bacterial phyla Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria and Spirochaetes, were most abundant in the canine oral microbiome (92.5% of total bacterial SSU rRNA). The direct rRNA sequencing approach detected greater taxonomic diversity (1 additional phylum, 2 classes, 1 order, 10 families and 61 genera) when compared with general bacterial 16S rRNA amplicons from the same sample, simultaneously provided SSU rRNA gene inventories of Bacteria, Archaea and Eukarya, and detected significant numbers of sequences not recognised by 'universal' primer sets. Proteobacteria and Spirochaetes were found to be under-represented by PCR-based analysis of the microbiome, and this was due to primer mismatches and taxon-specific variations in amplification efficiency, validated by qPCR analysis of 16S rRNA amplicons from a mock community. This demonstrated the veracity of direct RT-SSU rRNA sequencing for molecular microbial ecology.

Comparative Genomics of the Genus Porphyromonas Identifies Adaptations for Heme Synthesis within the Prevalent Canine Oral Species Porphyromonas cangingivalis.

Porphyromonads play an important role in human periodontal disease and recently have been shown to be highly prevalent in canine mouths. Porphyromonas cangingivalis is the most prevalent canine oral bacterial species in both plaque from healthy gingiva and plaque from dogs with early periodontitis. The ability of P. cangingivalis to flourish in the different environmental conditions characterized by these two states suggests a degree of metabolic flexibility. To characterize the genes responsible for this, the genomes of 32 isolates (including 18 newly sequenced and assembled) from 18 Porphyromonad species from dogs, humans, and other mammals were compared. Phylogenetic trees inferred using core genes largely matched previous findings; however, comparative genomic analysis identified several genes and pathways relating to heme synthesis that were present in P. cangingivalis but not in other Porphyromonads. Porphyromonas cangingivalis has a complete protoporphyrin IX synthesis pathway potentially allowing it to synthesize its own heme unlike pathogenic Porphyromonads such as Porphyromonas gingivalis that acquire heme predominantly from blood. Other pathway differences such as the ability to synthesize siroheme and vitamin B12 point to enhanced metabolic flexibility for P. cangingivalis, which may underlie its prevalence in the canine oral cavity.

A longitudinal assessment of changes in bacterial community composition associated with the development of periodontal disease in dogs.

Periodontal disease is the most widespread oral disease in dogs. Whilst the involvement of bacteria in the aetiology of periodontitis is well established the role of individual species and their complex interactions with the host is not well understood. The objective of this research was therefore to perform a longitudinal study in dogs to identify the changes that occur in subgingival bacterial communities during the transition from mild gingivitis to the early stages of periodontitis (<25% attachment loss). Subgingival plaque samples were collected from individual teeth of 52 miniature schnauzer dogs every six weeks for up to 60 weeks. The microbial composition of plaque samples was determined using 454-pyrosequencing of the 16S rDNA. A group of aerobic Gram negative species, including Bergeyella zoohelcum COT-186, Moraxella sp. COT-017, Pasteurellaceae sp. COT-080, and Neisseria shayeganii COT-090 decreased in proportion as teeth progressed to mild periodontitis. In contrast, there was less evidence that increases in the proportion of individual species were associated with the onset of periodontitis, although a number of species (particularly members of the Firmicutes) became more abundant as gingivitis severity increased. There were small increases in Shannon diversity, suggesting that plaque community membership remains relatively stable but that bacterial proportions change during progression into periodontitis. This is the first study to demonstrate the temporal dynamics of the canine oral microbiota; it showed that periodontitis results from a microbial succession predominantly characterised by a reduction of previously abundant, health associated taxa.

Investigating the prevalence of Salmonella in dogs within the Midlands region of the United Kingdom.

BACKGROUND: The intimate relationship between dogs and their owners has the potential to increase the risk of human exposure to bacterial pathogens. Over the past 40 years, there have been several reports on transmission of salmonellae from dogs to humans. This study therefore aimed to determine the prevalence of Salmonella in the faeces of dogs from the Midlands region of the United Kingdom to assess exposure risk and potential for zoonotic transmission. RESULTS: A total of 436 apparently healthy dogs without diarrhoea from households (n = 126), rescue centres (n = 96), boarding kennels (n = 43), retired greyhound kennels (n = 39) and a pet nutrition facility (n = 132) were investigated for Salmonella shedding. Faecal samples were processed by an enrichment culture based method. The faeces from one dog (0.23 %; 95 % confidence limit 0.006 %, 1.27 %) was positive for Salmonella. The species was S. enterica subspecies arizonae. CONCLUSION: This study showed that the prevalence of Salmonella from faeces from apparently healthy dogs from a variety of housing conditions is low; however, Salmonella shedding was still identified.

Draft genome sequences of 26 porphyromonas strains isolated from the canine oral microbiome.

We present the draft genome sequences for 26 strains of Porphyromonas (P. canoris, P. gulae, P. cangingavalis, P. macacae, and 7 unidentified) and an unidentified member of the Porphyromonadaceae family. All of these strains were isolated from the canine oral cavity, from dogs with and without early periodontal disease.

A longitudinal assessment of periodontal disease in 52 Miniature Schnauzers.

BACKGROUND: Periodontal disease (PD) is the most widespread oral disease in dogs and has been associated with serious systemic diseases. The disease is more prevalent in small breeds compared to large breeds and incidence increases with advancing age. In prevalence studies 84% of Beagles over the age of 3 and 100% of Poodles over the age of 4 were diagnosed with PD. Current knowledge of the rate of progression of PD is limited. The objective of this study was to determine the rate of PD progression in Miniature Schnauzers, an at risk small breed of dog. Dogs (n = 52, age 1.3-6.9 years) who had received a regular oral care regime prior to this study were assessed for levels of gingivitis and periodontitis around the whole gingival margin in every tooth under general anaesthetic. Assessments were conducted approximately every six weeks for up to 60 weeks following the cessation of the oral care regime. RESULTS: All of the 2155 teeth assessed entered the study with some level of gingivitis. 23 teeth entered the study with periodontitis, observed across 12 dogs aged between 1.3 and 6.9 years. 35 dogs had at least 12 teeth progress to periodontitis within 60 weeks. Of the teeth that progressed to periodontitis, 54% were incisors. The lingual aspect of the incisors was significantly more likely to be affected (p < 0.001). The severity of gingivitis in periodontitis-affected teeth was variable with 24% of the aspects affected having very mild gingivitis, 36% mild gingivitis and 40% moderate gingivitis. Periodontitis progression rate was significantly faster in older dogs. Only one dog (age 3.5) did not have any teeth progress to periodontitis after 60 weeks. CONCLUSIONS: This is the first study to have assessed the progression rate of periodontitis in Miniature Schnauzers and highlights that with no oral care regime, the early stages of periodontitis develop rapidly in this breed. An oral care regime and twice yearly veterinary dental health checks should be provided from an early age for this breed and other breeds with similar periodontitis incidence rates.

A cross-sectional survey of bacterial species in plaque from client owned dogs with healthy gingiva, gingivitis or mild periodontitis.

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.

Pyrosequencing the canine faecal microbiota: breadth and depth of biodiversity.

Mammalian intestinal microbiota remain poorly understood despite decades of interest and investigation by culture-based and other long-established methodologies. Using high-throughput sequencing technology we now report a detailed analysis of canine faecal microbiota. The study group of animals comprised eleven healthy adult miniature Schnauzer dogs of mixed sex and age, some closely related and all housed in kennel and pen accommodation on the same premises with similar feeding and exercise regimes. DNA was extracted from faecal specimens and subjected to PCR amplification of 16S rDNA, followed by sequencing of the 5' region that included variable regions V1 and V2. Barcoded amplicons were sequenced by Roche-454 FLX high-throughput pyrosequencing. Sequences were assigned to taxa using the Ribosomal Database Project Bayesian classifier and revealed dominance of Fusobacterium and Bacteroidetes phyla. Differences between animals in the proportions of different taxa, among 10,000 reads per animal, were clear and not supportive of the concept of a "core microbiota". Despite this variability in prominent genera, littermates were shown to have a more similar faecal microbial composition than unrelated dogs. Diversity of the microbiota was also assessed by assignment of sequence reads into operational taxonomic units (OTUs) at the level of 97% sequence identity. The OTU data were then subjected to rarefaction analysis and determination of Chao1 richness estimates. The data indicated that faecal microbiota comprised possibly as many as 500 to 1500 OTUs.

Characterization of cellular senescence mechanisms in human corneal endothelial cells.

The human cornea is a tri-laminar structure composed of several cell types with substantial mitotic potential. Age-related changes in the cornea are associated with declining visual acuity and the onset of overt age-related corneal diseases. Corneal transplantation is commonly used to restore vision in patients with damaged or diseased corneas. However, the supply of donor tissue is limited, and thus there is considerable interest in the development of tissue-engineered alternatives. A major obstacle to these approaches is the short replicative lifespan of primary human corneal endothelial cells (HCEC). Accordingly, a comprehensive investigation of the signalling pathways and mechanisms underpinning proliferative lifespan and senescence in HCEC was undertaken. The effects of exogenous human telomerase reverse transcriptase expression, p53 knockdown, disruption of the pRb pathway by over-expression of CDK4 and reduced oxygen concentration on the lifespan of primary HCEC were evaluated. We provide proof-of-principle that forced expression of telomerase, when combined with either p53 knockdown or CDK4 over-expression, is sufficient to produce immortalized HCEC lines. The resultant cell lines express an HCEC-specific transcriptional fingerprint, and retain expression of the corneal endothelial temperature-sensitive potassium channel, suggesting that significant dedifferentiation does not occur as a result of these modes of immortalization. Exploiting these insights into proliferative lifespan barriers in HCEC will underpin the development of novel strategies for cell-based therapies in the human cornea.