Dysbiosis and Predicted Functions of the Dental Biofilm of Dairy Goats with Periodontitis.

Periodontitis is a polymicrobial biofilm-induced inflammatory disease associated with a dysbiotic microbial community and severely affects the health and welfare of animals. However, little is known regarding the dental microbiota associated with this disease in goats. In this study, we used high-throughput sequencing, network analysis, and predicted functions to investigate the microbiota of clinically healthy goats and those with periodontitis and identify possible pathogens and proteins associated with the disease. Dental microbiomes of goats with periodontitis were richer, and network analyses showed that the number of negative interactions was higher in the networks of animals with periodontitis. Based on the interrelationships, Porphyromonas, Fusobacterium, and Prevotella were suggested to play an important role in the dental microbiota associated with goat periodontitis. Protein families linked to translation, cytoplasmatic translation, and rRNA processing were more abundant in the dental microbiota of goats with periodontitis. In conclusion, the dental biofilm microbiota associated with goat periodontitis seems to be dysbiotic and has significant antagonistic interactions, which discriminate healthy animals from diseased animals and highlight the importance of key bacteria. Thus, these novel findings contribute to the evolution of knowledge regarding the etiopathogenesis of goat periodontitis and possibly to the development of periodontitis control measures.

Expression of Toll-like receptor and cytokine mRNAs in feline odontoclastic resorptive lesion (FORL) and feline oral health.

Feline odontoclastic resorptive lesion (FORL) is a common chronic inflammatory condition whose aetiopathogenesis remains unclear. FORL affects 20-75% of cats and causes excruciating pain and tooth loss. The purpose of this study was to evaluate chronic inflammation in FORL by assessing differences in Toll-like receptor (TLR) and cytokine transcripts in gingival tissues between diseased and healthy cats. Gingival tissue samples were collected from 14 healthy cats with no known clinical signs of oral disease and 41 cats with FORL. Levels of mRNA encoding TLR2, TLR3, TLR4, TLR7, TLR9 and the cytokines interleukin-1ß (IL-1ß), IL-4, IL-6, IL-10, IL-12, interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) was evaluated using quantitative real-time PCR. Statistical significance of the results was assessed using non-parametric tests. Levels of TLR and cytokine transcripts were upregulated in gingival tissue from cats with FORL as compared with healthy gingival tissue: TLR2, TLR3 and TLR9, p ≤ 0.001; TLR4 and TLR7, p ≤ 0.01; IFN-γ, IL-4, IL-6, IL-10, IL-12, IL-1ß and TNF-α, p ≤ 0.001). In conclusion, expression of TLR and both pro- and anti-inflammatory cytokines were significantly increased, confirming an ongoing chronic inflammatory response to the microbiome in FORL. It is likely that dysbiosis of the oral microbiota in cats with FORL activates the innate immune response, leading to active inflammation that results in tooth resorption.

Dysbiosis and predicted function of dental and ruminal microbiome associated with bovine periodontitis.

Extensive cattle livestock is advancing in Amazonia and its low productivity, with consequent pressure to open new areas, is partly due to sanitary problems and, among them, the periodontal diseases, whose environmental triggers or modifying factors are unknown. In this study, we used high-throughput sequencing, network analysis and predicted functions to investigate the dental and ruminal microbiota of cattle raised in new livestock areas in the Amazon and identify possible keystone pathogens and proteins associated with the disease. Ninety-three genera were common in dental and ruminal fluid microbiomes and among them periodontal pathogens such as Fusobacterium, Prevotella, Porphyromonas and Actinomyces were recognized. Network analysis showed that dental microbiomes of clinically healthy animals tend to comprise a group of OTUs in homeostasis and when analyzed together, dental and ruminal fluid microbiomes of animals with periodontitis had almost twice the number of negative edges, indicating possible competition between bacteria and dysbiosis. The incisor dental and ruminal fluid microbiomes were dominated by a core community composed of members of the phyla Firmicutes and Bacteroidetes. Network results showed that members of the Prevotella genus stood out among the top five OTUs, with the largest number of hubs in the dental and ruminal microbiota of animals with periodontitis. Protein families linked to an inflammatory environment were predicted in the dental and ruminal microbiota of cattle with periodontitis. The dissimilarity between dental microbiomes, discriminating between healthy cattle and those with periodontitis and the identification of possible key pathogens, represent an important reference to elucidate the triggers involved in the etiopathogenesis of bovine periodontitis, and possibly in the development of measures to control the disease and reduce the pressures for deforestation.

Dental biofilm and its ecological interrelationships in ovine periodontitis.

Introduction. Periodontitis, one of the most common oral disorders in sheep, is caused by a mixed and opportunistic microbiota that severely affects the health and welfare of animals. However, little is known about the ecological processes involved and the composition of the microbiota associated with the development of the disease.Hypothesis/Gap Statement. Using high-throughput sequencing of the 16S ribosomal RNA gene and network analysis it would be possible to discriminate the microbiomes of clinically healthy sheep and those with periodontitis and possibly identify the key microorganisms associated with the disease.Aim. The present study aimed to characterise the composition of dental microbiomes and bacterial co-occurrence networks in clinically healthy sheep and animals with periodontitis.Methodology. Dental biofilm samples were collected from ten sheep with periodontitis and ten clinically healthy animals. Bacteria were identified using high-throughput sequencing of the 16S ribosomal RNA gene.Results. The most prevalent genera in the dental microbiota of sheep with periodontitis were Petrimonas, Acinetobacter, Porphyromonas and Aerococcus. In clinically healthy animals, the most significant genera were unclassified Pasteurellaceae, Pseudomonas, and Neisseria. Fusobacterium was found at high prevalence in the microbiomes of both groups. The dental microbiota of sheep in the two clinical conditions presented different profiles and the diversity and richness of bacteria was greater in the diseased animals. Network analyses showed the presence of a large number of antagonistic interactions between bacteria in the dental microbiota of animals with periodontitis, indicating the occurrence of a dysbiotic community. Through the interrelationships, members of the Prevotella genus are likely to be key pathogens, both in the dental microbiota of healthy animals and those with periodontitis. Porphyromonas stood out among the top three nodes with more centrality and the largest number of hubs in the networks of animals with periodontitis.Conclusion. The dental biofilm microbiota associated with ovine periodontitis is dysbiotic and with significant antagonistic interactions, which discriminates healthy animals from diseased animals and highlights the importance of key bacteria, such as Petrimonas, Porphyromonas, Prevotella and Fusobacterium species.

Microbiome analysis of feline odontoclastic resorptive lesion (FORL) and feline oral health.

Introduction. Feline odontoclastic resorptive lesion (FORL) is one of the most common and painful oral diseases of the cat. It is characterised by tooth resorption due to destructive activity of odontoclasts. FORL can result in tooth loss. While the aetiology of FORL is not clearly understood, it is thought to be multifactorial and bacteria are likely to play a major role.Hypothesis. Dysbiosis of the normal feline oral microbiota leads to an alteration in commensal bacteria populations, which results in the development of FORL.Aim. The purpose of the current study was to determine the composition of the microbiomes associated with feline oral health and FORL.Methodology. Supragingival plaque was collected from 25 cats with a healthy oral cavity and 40 cats with FORL. DNA was extracted from each sample, the V4 region of the 16S rRNA gene amplified by polymerase chain reaction and amplicons sequenced. Diversity and species richness analyses were performed, principal component analysis was used to explore differences between the oral microbiomes of healthy cats and those with FORL, and linear discriminant analysis effect size was used to assess differences between the groups.Results. The six most abundant bacterial genera identified were Bergeyella, Capnocytophaga, Lampropedia, Morexella, Porphyromonas and Treponema. Two-step cluster analysis of the data identified two FORL sub-groups (FORL-1, FORL-2). The FORL-2 sub-group was very similar to the healthy group, whilst the FORL-1 sub-group was clearly different from both the FORL-2 sub-group and the healthy groups. In this analysis, Capnocytophaga (P <0.001) and Lampropedia (P <0.01) were found at significantly lower levels and Porphyromonas at a slightly higher level in the FORL-1 sub-group compared to the healthy and FORL-2 sub-groups. Microbial diversity was found to be less in the FORL-1 sub-group than in the healthy group. Lampropedia sp., a phosphate-accumulating oral commensal species, was significantly lower in the FORL-1 sub-group.Conclusion. The oral microbiota associated with the FORL-1 sub-group is distinct from that found in the healthy group and FORL-2 sub-group. Lampropedia species may influence the local calcium-phosphate ratio, which could be a factor in tooth and bone resorption observed in FORL.

Understanding the microbial components of periodontal diseases and periodontal treatment-induced microbiological shifts.

In the mid-1960s the microbial aetiology of periodontal diseases was introduced based on classical experimental gingivitis studies . Since then, numerous studies have addressed the fundamental role that oral microbiota plays in the initiation and progression of periodontal diseases. Recent advances in laboratory identification techniques have contributed to a better understanding of the complexity of the oral microbiome in both health and disease. Modern culture-independent methods such as human oral microbial identification microarray and next-generation sequencing have been used to identify a wide variety of microbial taxa residing in the gingival sulcus and the periodontal pocket. The first theory of the 'non-specific plaque' hypothesis gave rise to the 'ecological plaque' hypothesis and more recently to the 'polymicrobial synergy and dysbiosis hypothesis'. Periodontitis is now considered to be a multimicrobial inflammatory disease in which the various bacterial species within the dental biofilm are in a dysbiotic state and this imbalance favours the establishment of chronic inflammatory conditions and ultimately the destruction of tooth-supporting tissues. Apart from the known putative periodontal pathogens, the whole biofilm community is now considered to play a role in the establishment of inflammation and the initiation and progression of periodontitis in a susceptible host. Treatment is unlikely to eliminate putative pathogens but, when it is thoroughly performed it has the potential to establish a healthy ecosystem by altering the microbial community in numbers and composition and also contribute to the maturation of the host immune response.

Evaluation of tissue levels of Toll-like receptors and cytokine mRNAs associated with bovine periodontitis and oral health.

Bovine periodontitis is a progressive and purulent infection associated with an anaerobic subgingival biofilm, which induces irreversible damage to the dentition of affected animals. The aetiopathogenesis of the disease is unclear and treatment and control of the disease process in cattle are almost unknown. The aim of this study was to investigate the innate immune response by quantifying expression of Toll-like receptor (TLR) and cytokine genes in gingival tissue samples from cattle with and without periodontitis. Postmortem biopsies of gingival tissues were collected from 20 cattle with periodontitis and 20 cattle with no clinical signs of periodontal lesions. Tissue expression of TLR2, TLR4, TNF-α, IFN-γ, IL-1ß and IL-4 genes were determined using quantitative real-time PCR. Statistically significant increases in mRNA levels encoding TLR2 (p = 0.025), TLR4 (p = 0.037), TNF-α (p = 0.025), IFN-γ (p = 0.014), IL-1ß (p < 0.001) and IL-4 (p = 0.014) were observed in animals with periodontitis when compared to periodontally healthy animals. Increased levels of TLRs and inflammatory cytokines in periodontal tissue indicate an induction of the innate immune response of cattle and suggest that a substantial microbial challenge may be involved in the aetiopathogenesis of bovine periodontitis.

Microbiomes associated with bovine periodontitis and oral health.

Periodontitis is an infectious polymicrobial, immuno-inflammatory disease of multifactorial aetiology that has an impact on the health, production and welfare of ruminants. The objective of the present study was to determine the microbial profiles present in the gingival sulcus of cattle considered periodontally healthy and in the periodontal pocket of animals with periodontitis lesions using high-throughput bacterial 16S rRNA gene sequencing. Subgingival biofilm samples were collected from 40 cattle with periodontitis and 38 periodontally healthy animals. In total, 1923 OTUs were identified and classified into 395 genera or higher taxa. Microbial profiles in health differed significantly from periodontitis in their composition (p < 0.0001, F = 5.30; PERMANOVA) but no statistically significant differences were observed in the diversity of healthy and periodontitis microbiomes. The most prevalent taxa in health were Pseudomonas, Burkholderia and Actinobacteria, whereas in disease these were Prevotella, Fusobacterium and Porphyromonas. The most discriminative taxa in health were Gastranaerophilales, Planifilum and Burkholderia, and in disease these were Elusimicrobia, Synergistes and Propionivibrio. In conclusion, statistically significant difference exists between the microbiome in bovine oral health and periodontitis, with populations showing 72.6% dissimilarity. The diversity of the bacteria found in health and periodontitis were similar and bacteria recognised as periodontal pathogens showed increased abundance in disease. In this context, the main components of bacterial homeostasis in the biofilm of healthy sites and of dysbiosis in periodontal lesions provide unprecedented indicators for the evolution of knowledge about bovine periodontitis.

Microbiome of peri-implantitis affected and healthy dental sites in patients with a history of chronic periodontitis.

OBJECTIVE: To determine the composition of the microbiome of peri-implantitis sites and corresponding dental sites in subjects with a history of chronic periodontitis. DESIGN: Clinical and radiographic examination assessed the periodontal/peri-implant disease status. Plaque samples were collected from one diseased implant with peri-implantitis, functional for at least two years and healthy sites in ten non-smokers who had received periodontal treatment prior to implant placement. Following DNA extraction, the bacteria present in each sample were determined by high-throughput sequencing of V3-V4 region of the 16S rRNA gene using the Illumina MiSeq platform. OTUs were picked using QIIME. Differences between dental and implant sites were determined using linear discriminant analysis, effect size and diversity analyses were conducted using PAST v3.02. RESULTS: The microbiomes of healthy samples were more diverse than those found in disease, although disease was associated with a higher abundance of taxa relative to health. The genera Actinobacillus and Streptococcus were most closely associated with health, whereas Prevotella and Porphyromonas were most discriminative for disease. Synergistetes were highly associated with peri-implantitis. CONCLUSION: In patients with a history of periodontitis, putative periodontal pathogens prevailed in the microbiome of diseased implants. Diseased implants and corresponding healthy sites appear to have distinct microbiological ecosystems.

Black-pigmented anaerobic bacteria associated with ovine periodontitis.

Periodontitis is a polymicrobial infectious disease that causes occlusion change, tooth loss, difficulty in rumination, and premature culling of animals. This study aimed to detect species of the genera Porphyromonas and Prevotella present in the periodontal pocket of sheep with lesions deeper than 5mm (n=14) and in the gingival sulcus of animals considered periodontally healthy (n=20). The presence of microorganisms was evaluated by polymerase chain reaction (PCR) using specific primers for Porphyromonas asaccharolytica, Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonas gulae, Prevotella buccae, Prevotella intermedia, Prevotella loescheii, Prevotella melaninogenica, Prevotella nigrescens, Prevotella oralis, and Prevotella tannerae. Prevalence and risk analysis were performed using Student's t-test and Spearman's correlation. Among the Prevotella and Porphyromonas species detected in the periodontal lesions of sheep, P. melaninogenica (85.7%), P. buccae (64.3%), P. gingivalis (50%), and P. endodontalis (50%) were most prevalent. P. gingivalis (15%) and P. oralis (10%) prevailed in the gingival sulcus. P. gulae and P. tannerae were not detected in the 34 samples studied. Data evaluation by t-test verified that occurrence of P. asaccharolytica, P. endodontalis, P. gingivalis, P. buccae, P. intermedia, P. melalinogenica, and P. nigrescens correlated with sheep periodontitis. The findings of this study will be an important contribution to research on pathogenesis of sheep periodontitis and development of its control measures.

Community analysis of dental plaque and endotracheal tube biofilms from mechanically ventilated patients.

PURPOSE: Mechanically ventilated patients are at risk for developing ventilator-associated pneumonia, and it has been reported that dental plaque provides a reservoir of respiratory pathogens that may aspirate to the lungs and endotracheal tube (ETT) biofilms. For the first time, metataxonomics was used to simultaneously characterize the microbiome of dental plaque, ETTs, and non-directed bronchial lavages (NBLs) in mechanically ventilated patients to determine similarities in respective microbial communities and therefore likely associations. MATERIAL AND METHODS: Bacterial 16S rRNA gene sequences from 34 samples of dental plaque, NBLs, and ETTs from 12 adult mechanically ventilated patients were analyzed. RESULTS: No significant differences in the microbial communities of these samples were evident. Detected bacteria were primarily oral species (e.g., Fusobacterium nucleatum, Streptococcus salivarius, Prevotella melaninogenica) with respiratory pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcuspneumoniae, and Haemophilus influenzae) also in high abundance. CONCLUSION: The high similarity between the microbiomes of dental plaque, NBLs, and ETTs suggests that the oral cavity is indeed an important site involved in microbial aspiration to the lower airway and ETT. As such, maintenance of good oral hygiene is likely to be highly important in limiting aspiration of bacteria in this vulnerable patient group.

Prevalence of feline calicivirus in cats with odontoclastic resorptive lesions and chronic gingivostomatitis.

Feline odontoclastic resorptive lesion (FORL) and feline chronic gingivostomatitis (FCGS) are two of the most common diseases of the feline oral cavity. While evidence is emerging that FCGS is caused by gingival inflammation initiated and perpetuated by the oral microbiota, little is known in this regard for FORL. Feline calicivirus (FCV) has been associated with the presence of FCGS and is thought to play a role in the initiation of this disease. In this study, the incidence of FCV was investigated in cats with FORL and FCGS, and compared to unaffected controls. FCV was detected by viral culture. The incidence of FCV was as follows: 6 (24.0%) of 24 control cats, 9 (22.5%) of 40 cats with FORL and 15 (60.0%) of 25 cats with FCGS were positive for FCV. There was a significant difference in FCV incidence between all the groups (p=0.003) but none between the control group and the FORL group. However, significant differences were observed in the incidence of FCV between control and FCGS (p=0.010) and between FORL and FCGS (p=0.006). It is concluded that although FCV may be associated with FCGS, it appears unlikely to play a role in FORL.

Brief Report: Proatherogenic Cytokine Microenvironment in the Aortic Adventitia of Patients With Rheumatoid Arthritis.

OBJECTIVE: Patients with rheumatoid arthritis (RA) are at increased risk of developing cardiovascular disease (CVD) via mechanisms that have not yet been defined. Inflammatory pathways, in particular within the vascular adventitia, are implicated in the pathogenesis of primary CVD but could be amplified in RA at the local tissue level. The aim of this study was to examine the aortic adventitia of coronary artery disease (CAD) patients with or without RA to determine the cytokine profile contained therein. METHODS: Aortic adventitia and internal thoracic artery biopsy specimens obtained from 19 RA patients and 20 non-RA patients undergoing coronary artery bypass graft surgery were examined by immunohistochemistry. RESULTS: Interleukin-18 (IL-18), IL-33, and tumor necrosis factor (TNF) were expressed in aortic adventitia biopsy specimens from both groups, and expression of these cytokines was significantly higher in RA patients. In RA patients, IL-33 expression in endothelial cells correlated positively with the number of swollen joints, suggesting a link between the systemic disease state and the local vascular tissue microlesion. CONCLUSION: The presence of the proinflammatory cytokines IL-18, IL-33, and TNF may play a role in the inflammatory process within the adventitia that contributes to plaque formation and destabilization. In theory, the amplified expression of these cytokines may contribute to the known increased occurrence and severity of CAD in patients with RA.

Bacteria and Toll-like receptor and cytokine mRNA expression profiles associated with canine arthritis.

The major forms of inflammatory canine arthritis are immune-mediated arthritis (IMA) and septic arthritis (SA), although some cases of cruciate disease (CD) are associated with significant levels of synovitis. In this study, the bacteria associated with canine arthritis were identified and mRNA expression levels of Toll-like receptors (TLRs) and pro-inflammatory cytokines determined. Of the 40 synovial fluid samples analysed, bacteria were isolated from 12 samples by culture (2 CD, 10 SA) and detected in 4 samples (3 CD, 1 SA) using culture-independent methods. Statistically significant increases in TLR2, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-12 mRNA expression were seen in all disease groups compared to normal controls. All disease groups had decreased mRNA expression of other TLRs compared to normal controls, but this did not reach statistical significance. Synovial fluid cell counts revealed that the highest number and proportion of mononuclear cells and neutrophils were found in the IMA and SA samples, respectively. Age had an effect on the TLR and cytokine mRNA expression profiles: TNF-α (p=0.043) and IL-12 (p=0.025) mRNA expression was increased and TLR4 mRNA expression was reduced (p=0.033) in dogs up to 4 years of age compared to older animals. In the 10 SA samples from which bacteria were isolated, statistically significant increases in TLR2, TLR7, TNF-α and IL-6 mRNA expression were observed. It is concluded that canine arthritis is associated with increased mRNA levels of pro-inflammatory cytokines, which could in some cases be mediated by bacteria through activation of TLR2.

Bacteria in the adventitia of cardiovascular disease patients with and without rheumatoid arthritis.

The incidence of atherosclerosis is significantly increased in rheumatoid arthritis (RA). Infection is one factor that may be involved in the pathogenesis of both diseases. The cause of RA and atherosclerosis is unknown, and infection is one of the factors that may be involved in the pathogenesis of both diseases. The aims of this study were to identify bacteria in the aortic adventitia of patients with cardiovascular disease (CVD) in the presence and absence of RA, and to determine the effect of identified candidate pathogens on Toll-like receptor (TLR)-dependent signalling and the proinflammatory response. The aortic adventitia of 11 CVD patients with RA (RA+CVD) and 11 CVD patients without RA (CVD) were collected during coronary artery bypass graft surgery. Bacteria were detected in four samples from CVD patients and three samples from RA+CVD patients and identified by 16S rRNA gene sequencing. Methylobacterium oryzae was identified in all three RA+CVD samples, representing 44.1% of the bacterial flora. The effect of M. oryzae on TLR-dependent signalling was determined by transfection of HEK-293 cells. Although mild TLR2 signalling was observed, TLR4 was insensitive to M. oryzae. Human primary macrophages were infected with M. oryzae, and a TLDA qPCR array targeting 90 genes involved in inflammation and immune regulation was used to profile the transcriptional response. A significant proinflammatory response was observed, with many of the up-regulated genes encoding proinflammatory cytokines (IL-1α, IL-1ß, IL-6, TNF-α) and chemokines (CCR7, IL-8). The aortic adventitia of CVD patients contains a wide range of bacterial species, and the bacterial flora is significantly less diverse in RA+CVD than CVD patients. M. oryzae may stimulate an proinflammatory response that may aggravate and perpetuate the pathological processes underlying atherosclerosis in RA patients.

Culture-independent identification of bacteria associated with ovine 'broken mouth' periodontitis.

'Broken mouth' periodontitis (BMP) is a painful condition of sheep grazed on rough pasture and involves periodontal infection of the incisor teeth and progressive tooth loss. This can reduce the efficiency of grazing of sheep, which contributes to malnutrition, weight loss, systemic health problems, poor quality of life and early culling from flocks. Consequently, this condition is a major economic problem to sheep farmers. However, there are no treatment or control methods available. The aim of this study was to identify the bacteria associated with BMP and oral health in sheep. Swabs were collected from the gingival pockets of three sheep with BMP and from the gingival margin of three orally healthy (normal) sheep. Bacteria were identified using culture-independent (16S rRNA gene sequencing) methods. In the normal samples, 26 phylotypes were identified. The most prevalent species were Enterobacter hormaechei (21.3% of analysed clones) and Hafnia alvei (21.3%), with uncultured (4.4%) and novel (5.0%) phylotypes also being identified. For the BMP samples, 24 phylotypes were identified. The most prevalent species were Mannheimia ruminalis (28.4%) and Moraxella caprae (13.5%), with uncultured (2.6%) and novel (24.5%) phylotypes also being identified. In conclusion, a distinct microflora is associated with BMP and oral health in sheep and M. ruminalis may be involved in the aetiology of BMP.

Influence of periodontal disease, Porphyromonas gingivalis and cigarette smoking on systemic anti-citrullinated peptide antibody titres.

BACKGROUND: Anti-citrullinated protein antibody (ACPA) responses may precede clinical onset of rheumatoid arthritis. Porphyromonas gingivalis peptidylarginine deiminase can citrullinate proteins possibly inducing autoimmunity in susceptible individuals. AIM: To determine whether periodontitis, carriage of P. gingivalis, smoking and periodontal therapy influence ACPA titres. METHODS: Serum and plaque samples were collected from 39 periodontitis patients before and after non-surgical periodontal treatment, and from 36 healthy subjects. Carriage of P. gingivalis was determined by PCR of plaque DNA. ACPA was determined by anti-cyclic citrullinated peptide (CCP) enzyme-linked immunosorbent assay (ELISA). Anti-P. gingivalis titres were determined by ELISA. RESULTS: Untreated periodontitis patients had higher anti-CCP antibody titres than healthy controls [three patients (8%) greater than manufacturer suggested assay diagnostic threshold (5 Assay Units/AU) versus none (0%); mean ± SEM: 1.37 ± 0.23 versus 0.40 ± 0.10 AU, p < 0.0001]. Periodontitis patients who smoked demonstrated lower anti-P. gingivalis (15956 ± 4385 versus 2512 ± 1290 Units/ml, p < 0.05), but similar anti-CCP than non-smoking periodontitis patients (smokers: 1.31 ± 0.35; non-smokers: 1.41 ± 0.32 AU). Healthy smokers demonstrated elevated anti-CCP titres (0.75 ± 0.19 AU), at levels between healthy non-smokers (0.15 ± 0.05 AU) and non-smoker periodontitis patients. Six months after periodontal treatment, there were significant reductions in anti-CCP (non-smokers p < 0.05) and anti-P. gingivalis (all participants p < 0.01). CONCLUSION: In subjects with periodontitis, P. gingivalis infection may be responsible for inducing autoimmune responses that characterize rheumatoid arthritis.

Novel bacterial phylotypes associated with the healthy feline oral cavity and feline chronic gingivostomatitis.

Feline chronic gingivostomatitis (FCGS) is a painful inflammatory disease of the oral cavity. Treatment options for FCGS are very limited and little is known regarding its aetiology. The aim of this study was to investigate the presence of putative novel species in the oral cavity of cats with and without FCGS. Bacterial DNA was extracted from oral swabs and identified by 16S rRNA gene sequencing. The 16S rRNA genes of 54 clones representing distinct potentially novel species were sequenced (1202-1325 base pairs). Obtained sequences were compared to the BLAST database, aligned using the ClustalW2 alignment tool and a phylogenetic tree created. Twenty-two clones (18 from control and four from FCGS samples) had a similarity of less than 97% and were considered novel. The proportion of novel phylotypes in each group was 19.6% (control) and 2.3% (FCGS). In the derived phylogenetic tree, 15 novel phylotypes clustered together and branched away from known species and phyla. This suggests the presence of a group of novel, previously unidentified bacteria that are associated with the feline oral cavity in both health and disease.

Molecular identification of bacteria associated with canine periodontal disease.

Periodontal disease is one of the most common diseases of adult dogs, with up to 80% of animals affected. The aetiology of the disease is poorly studied, although bacteria are known to play a major role. The purpose of this study was to identify the bacteria associated with canine gingivitis and periodontitis and to compare this with the normal oral flora. Swabs were obtained from the gingival margin of three dogs with gingivitis and three orally healthy controls, and subgingival plaque was collected from three dogs with periodontitis. Samples were subjected to routine bacterial culture. The prevalent species identified in the normal, gingivitis and periodontitis groups were uncultured bacterium (12.5% of isolates), Bacteroides heparinolyticus/Pasteurella dagmatis (10.0%) and Actinomyces canis (19.4%), respectively. Bacteria were also identified using culture-independent methods (16S rRNA gene sequencing) and the predominant species identified were Pseudomonas sp. (30.9% of clones analysed), Porphyromonas cangingivalis (16.1%) and Desulfomicrobium orale (12.0%) in the normal, gingivitis and periodontitis groups, respectively. Uncultured species accounted for 13.2%, 2.0% and 10.5%, and potentially novel species for 38.2%, 38.3% and 35.3%, of clones in the normal, gingivitis and periodontitis groups, respectively. This is the first study to use utilise culture-independent methods for the identification of bacteria associated with this disease. It is concluded that the canine oral flora in health and disease is highly diverse and also contains a high proportion of uncultured and, in particular, potentially novel species.

Identification of bacteria associated with feline chronic gingivostomatitis using culture-dependent and culture-independent methods.

Feline chronic gingivostomatitis (FCGS) is a chronic inflammatory disease of the oral cavity that causes severe pain and distress. There are currently no specific treatment methods available and little is known regarding its aetiology, although bacteria are thought to play a major role. The purpose of this study was to identify the oral bacterial flora in normal and diseased cats. Oral swabs were obtained from the palatoglossal folds of eight cats (three normal and five FCGS) and were subjected to microbiological culture. Pasteurella pneumotropica and Pasteurella multocida subsp. multocida were the most prevalent species identified by culture methods in the normal and FCGS samples, respectively. Bacteria were also identified using culture-independent methods (bacterial 16S rRNA gene sequencing). For the normal samples, 158 clones were analysed and 85 clones were sequenced. Capnocytophaga canimorsus (10.8% of clones analysed) was the predominant species. Uncultured species accounted for 8.2% of clones analysed, and 43.7% of clones analysed represented potentially novel species. For the FCGS samples, 253 clones were analysed and 91 clones were sequenced. The predominant species was P. multocida subsp. multocida (51.8% of clones analysed). Uncultured species accounted for 8.7% of clones analysed, and 4.7% of clones analysed represented potentially novel species. It is concluded that the oral flora in cats with FCGS appears to be less diverse than that found in normal cats. However, P. multocida subsp. multocida is found to be significantly more prevalent in FCGS than in normal cats and consequently may be of aetiological significance in this disease.