Porphyromonas gulae and PPAD antibodies are not related to citrullination in rheumatoid arthritis.

INTRODUCTION: Porphyromonas gulae have the enzyme PPAD, as P. gingivalis, which is responsible for citrullination related to the pathophysiology of rheumatoid arthritis and periodontitis; this implies the presence of two species of PPAD-producing bacteria in the mouth as well as the presence of citrullinated proteins. There are no previous reports or studies investigating an association between P. gulae PPAD in rheumatoid arthritis (RA). OBJECTIVE: To assess the presence of P. gulae and anti-citrullinated peptide antibodies of P. gulae PAD in patients with RA and their possible relationship with clinical activity markers. SUBJECTS AND METHODS: A total of 95 patients with RA and 95 controls were included. Erythrocyte sedimentation rate (ESR), C-reactive protein, anti-citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF) were measured. Activity index-28 (DAS28) and SCDAI. The periodontal diagnosis was established. Presence of P. gulae and P. gingivalis. An ELISA was used to determine antibodies against citrullinated peptides of P. gulae PAD. RESULTS: A P. gulae frequency of 15.8% was observed in the RA group and 9.5% in the control group. Higher levels of ACPA were found in the P. gulae-positive patients of the RA group, finding no significant difference, but if in patients positive for P. gingivalis with statistical significance (p = 0.0001). The frequency of anti-VDK-cit and anti-LPQ-cit9 antibodies to PPAD of P. gulae was higher in the RA group than in the control group without significant difference. No relationship was found with the clinical variables despite the presence of P. gulae and anti-citrullinated peptide antibodies of P. gulae PPAD in patients with RA CONCLUSIONS: It was not possible to establish a connection with clinical variables in RA and P. gulae; as a result, the presence of P. gingivalis continues to contribute significantly to the increase in antibodies against citrullinated proteins/peptides from exogenous sources of citrullination in RA and periodontitis.

Presence of zoonotic black-pigmented periodontal pathogens in the oral microbiota of pet and stray cats.

Black-pigmented bacteria are one of the neglected species to cause periodontal disease in cats, and they are also zoonotic agents that pose an infection risk to humans. In this study, we aimed to determine the presence of Porphyromonas gingivalis, Porphyromonas gulae and Prevotella nigrescens in the oral microbiota of pet and stray cats. Dental swab samples were taken from 25 pet cats and 25 stray cats with symptoms of periodontal disease and then investigated by multiplex polymerase chain reaction using 16S rRNA species-specific primers. As a result of the multiplex PCR analysis, P. gingivalis 3/25 (12%), P. nigrescens 1/25 (4%), P. gingivalis + P. gulae 7/25 (28%), P. gingivalis + P. nigrescens 1/25 (4%), P. gulae + P. nigrescens 1/25 (4%), and P. gingivalis + P. gulae + P. nigrescens 2/25 (8%) were molecularly typed in the pet cats. In addition, 1/25 (4%) of P. gulae and 21/25 (84%) of P. gingivalis + P. gulae were typed in the stray cats. In 10/25 (40%) pet and 3/25 (12%) stray cat samples, no bacteria were detected by molecular typing. In summary, the results provide strong evidence that black-pigmented zoonotic pathogens are associated with cat periodontal disease.

Porphyromonas gulae Activates Unprimed and Gamma Interferon-Primed Macrophages via the Pattern Recognition Receptors Toll-Like Receptor 2 (TLR2), TLR4, and NOD2.

Porphyromonas gulae is an anaerobic, Gram-negative coccobacillus that has been associated with periodontal disease in companion animals. The aims of this study were to analyze the ligation of pattern recognition receptors by P. gulae and the subsequent activation of macrophages. Exposure of HEK cells transfected with Toll-like receptors (TLRs) or NOD-like receptors to P. gulae resulted in the ligation of TLR2, TLR4, and NOD2. The effects of this engagement of receptors were investigated by measuring the synthesis of nitric oxide (NO), CD86 expression, and inflammatory cytokine production by wild-type, TLR2-/-, and TLR4-/- macrophages. The addition of P. gulae to unprimed and gamma interferon (IFN-γ)-primed (M1 phenotype) macrophages significantly increased the surface expression of CD86, but only M1 macrophages produced nitric oxide. P. gulae-induced expression of CD86 on unprimed macrophages was dependent on both TLR2 and TLR4, but CD86 expression and NO production in M1 macrophages were only TLR2 dependent. P. gulae induced an increase in secretion of interleukin-1α (IL-1α), IL-1ß, IL-6, IL-12p70, IL-13, tumor necrosis factor alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1α (MIP-1α) by M1 macrophages compared to that by unprimed controls. Among these cytokines, secretion of IL-6 and TNF-α by M1 macrophages was dependent on either TLR2 or TLR4. Our data indicate that TLR2 and TLR4 are important for P. gulae activation of unprimed macrophages and that activation and effector functions induced in M1 macrophages by P. gulae are mainly dependent on TLR2. In conclusion, P. gulae induces a strong TLR2-dependent inflammatory M1 macrophage response which may be important in establishing the chronic inflammation associated with periodontal disease in companion animals.

Bacterial DNA and serum IgG antibody titer assays for assessing infection of human-pathogenic and dog-pathogenic Porphyromonas species in dogs.

Periodontal disease is highly prevalent in both humans and dogs. Although there have been reports of cross-infection of periodontopathic bacteria, methods for assessing it have yet to be established. The actual status of cross-infection remains to be seen. The purpose of this study was to evaluate the utility of bacterial DNA and serum immunoglobulin G (IgG) antibody titer assays to assess infection of human-pathogenic and dog-pathogenic Porphyromonas species in dogs. Four experimental beagles were used for establishing methods. Sixty-six companion dogs at veterinary clinics visiting for treatment and prophylaxis of periodontal disease were used and divided into healthy, gingivitis, and periodontitis groups. Periodontal pathogens such as Porphyromonas gingivalis and Porphyromonas gulae were investigated as target bacteria. DNA levels of both bacteria were measured using species-specific primers designed for real-time polymerase chain reaction (PCR). Serum IgG titers of both bacteria were measured by enzyme-linked immunosorbent assay (ELISA). PCR primers were confirmed to have high sensitivity and specificity. However, there was no relationship between the amount of bacterial DNA and the severity of the periodontal disease. In addition, dogs with periodontitis had higher IgG titers against both bacteria compared to dogs in the healthy and gingivitis groups; there was cross-reactivity between the two bacteria. Receiver operating characteristic (ROC) analysis of IgG titers against both bacteria showed high sensitivity (>90 %) and specificity (>75 %). Since both bacteria were distinguished by DNA assays, the combination of these assays may be useful in the evaluation of cross-infection.

The Effect of Subacute Oral Folic Acid Treatment on Growth of Porphyromonas gulae in Dogs.

Periodontitis is one of the most prevalent infectious diseases in humans and animals. It is a multifactorial disease resulting in attachment loss and tooth loss. Therefore, preventive dentistry, such as daily teeth cleaning or providing dental chews from puppyhood is essential. This study aimed to find an alternative option for preventive dentistry by examining both in vitro and clinically, the antibacterial, antihalitosis, and anti-inflammatory effects of folic acid (FA) in dogs with periodontal disease. The antibacterial and antihalitosis responses of FA were evaluated in vitro using Porphyromonas gulae, a bacterium that plays a significant role in the development of periodontal disease in dogs. Anti-inflammatory responses, such as secretion of IL-1ß, IL-6, and IL-8 induced by P. gulae infection in human gingival epithelium have been studied. This study used dogs with P. gulae-associated periodontal diseases and was conducted by providing a dental chew containing 0.13% FA for 28 days. The viability and halitosis production (hydrogen sulfide and methyl mercaptan) of P. gulae was significantly inhibited by FA in a dose and time-dependent manner. IL-1ß, IL-6, and IL-8 secretion were also significantly suppressed by FA treatment in a dose-dependent manner. In vitro bactericidal, antihalitosis, and anti-inflammatory effects of FA were confirmed in dogs with P. gulae-associated periodontal disease. One month of oral treatment with 0.13% FA-containing dental chews significantly reduced halitosis as well as P. gulae activity. This study suggests that oral treatment with FA can be a preventive option for periodontal disease in dogs as well as humans.

Bestatin as a treatment modality in experimental periodontitis.

BACKGROUND: Chronic periodontitis (CP), the most prevalent dysbiotic bacteria-driven chronic inflammatory disease, is an underestimated global health problem in itself, and due to a causative relationship with other disorders such as cardiovascular diseases or Alzheimer disease. The CP pathogenesis is primarily driven by Porphyromonas gingivalis in humans, and Porphyromonas gulae in dogs. These microorganisms initiate a pathogenic shift in the composition of the tooth-surface microflora. Our objective was to evaluate antimicrobial effects of bestatin, a potential CP drug candidate. METHODS: We evaluated bestatin bacteriostatic efficiency against periodontopathogens in planktonic cultures via microplate assay, and mono- and multispecies oral biofilm models. Neutrophil bactericidal activities, such as phagocytosis, were investigated in vitro using granulocytes isolated from the peripheral blood. The therapeutic efficacy and the immunomodulatory function of bestatin was assessed in a murine model of CP. RESULTS: Bestatin exhibited bacteriostatic activity against both P. gingivalis and P. gulae, and controlled the formation and species composition of the biofilm. We demonstrated that bestatin promotes the phagocytosis of periodontopathogens by neutrophils. Finally, we found that providing bestatin in the animal feed prevented alveolar bone resorption. CONCLUSIONS: We show that in a murine model of CP bestatin not only shifted the biofilm species composition from pathogenic to a commensal one, but also promoted bacteria clearance by immune cells and alleviated inflammation. Taken together, these results suggest that bestatin is a promising drug choice for the treatment and/or prevention of periodontitis and clinical trials are required to fully evaluate its potency.

Possible association of fimA genotype of Porphyromonas gulae with the severity of periodontal disease and the number of permanent teeth in dogs.

Previous research has demonstrated that Porphyromonas gulae (P. gulae) significantly contributes to the development of periodontal disease in dogs. Porphyromonas gulae is divided into three subtypes according to the 41-kDa filamentous appendage (fimA), defined as types A, B, and C. This study aimed to elucidate the association between fimA type of P. gulae with the number of permanent teeth, reflecting the severity of periodontal disease. Two hundred twenty-five dogs were categorized by P. gulae fimA type as negative, type A dominant, type B dominant, and type C dominant. The stage of periodontal disease in P. gulae-positive dogs increased with age, particularly in type C dominant dogs. Correspondingly, the number of permanent teeth in P. gulae fimA type C-dominant dogs was significantly lower than that of P. gulae-negative dogs, suggesting there is a significant association between fimA type of P. gulae and the number of permanent teeth resulting from the development of periodontal disease.

Volatile Sulfur Compounds Produced by the Anaerobic Bacteria Porphyromonas spp. Isolated from the Oral Cavities of Dogs.

Porphyromonas spp. are oral anaerobic Gram-negative bacteria that form black-pigmented colonies on blood agar and produce volatile sulfur compounds (VSCs), such as hydrogen sulfide (H2S), methyl mercaptan (CH3SH), and dimethyl sulfide ((CH3)2S), which cause halitosis and the destruction of periodontal tissues. P. gulae is considered the main pathogen involved in periodontal disease in dogs. However, the characteristics of the VSCs produced by P. gulae are unknown. In the present study, VSCs were measured in 26 isolates of P. gulae and some isolates of the other Porphyromonas spp. obtained from the oral cavities of dogs with periodontal disease using an in vitro assay with an Oral ChromaTM gas chromatograph. The results demonstrated that P. gulae was able to produce large amounts of H2S and CH3SH, and the dominant product was CH3SH (CH3SH/H2S was approximately 2.2). Other Porphyromonas spp. that were also obtained from the oral cavities of dogs with periodontal disease indicated similar levels of production of H2S and CH3SH to those of P. gulae. It is strongly suggested that the high levels of H2S and CH3SH produced by P. gulae and other Porphyromonas spp. contribute to halitosis and the destruction of periodontal tissues during the progression of periodontal disease in dogs.

Prevalence of Periodontal Pathogens in Slovak Patients with Periodontitis and Their Possible Aspect of Transmission from Companion Animals to Humans.

Oral health and diseases are greatly influenced by oral bacteria. During dysbiosis, bacterial composition changes, which can lead to periodontitis. Periodontitis in humans is associated with periodontal pathogens such as Treponema denticola, Porphyromonas gingivalis, Tannerella forsythia and Aggregatibacter actinomycetemcomitans. Animal-to-human transmission of some of these pathogens has also been reported. The aim of this study was to evaluate the prevalence of periodontal pathogens in Slovak patients and to assess the possible risk of transmission of these pathogens from animals to their owners. The presence of periodontal pathogens in dental plaque was monitored by PCR. Amplified products were analysed using Sanger sequencing. T. forsythia isolates were assessed for the susceptibility to different antibiotics using the disk diffusion method. In humans, T. denticola, P. gingivalis, T. forsythia and A. actinomycetemcomitans were present in 69.23%, 69.23%, 100% and 84.62%, respectively. Most isolates of T. forsythia were susceptible to amoxicillin-clavulanic acid, clindamycin and moxifloxacin, but they were resistant to metronidazole. The transmission of T. forsythia from animals to their owners was not proven based on sequence analysing. On the other hand, transmission of Porphyromonas gulae was confirmed, but the risk of its involvement in the pathogenesis of periodontitis in humans must be further investigated.

Erythritol inhibits the growth of periodontal-disease-associated bacteria isolated from canine oral cavity.

Periodontal disease (PD) is the most common oral disease that is caused by infection with periodontal-disease-associated bacteria (PDAB) such as Porphyromonas gulae and Porphyromonas macacae in dogs as well as in humans. Unlike humans, most dogs do not follow daily oral hygiene routine, and this results in many dogs being affected by PD. Thus, to prevent PD, it is important to control PDAB. Xylitol is a sugar alcohol that inhibits the growth of oral bacteria in humans. However, xylitol is poisonous to dogs and can lead to hypoglycemia and hepatic failure. Herein, we show the inhibitory effect of erythritol, a sugar alcohol that can be used safely in dogs, on the growth of PDAB isolated from dogs with PD. Oral bacteria were isolated from the oral cavities of dogs with PD, and the distribution of PDAB was evaluated. Interestingly, Porphyromonas gingivalis, a bacterium typically responsible for PD in humans, was not isolated from dog samples. The bacteriostatic effect of erythritol supplementation was investigated on isolated PDAB in vitro. Our results show that erythritol exert bacteriostatic effects on PDAB comparable to xylitol. Thus, application of erythritol can be suggested to control PDAB in dogs in the future.

Green tea catechins inhibit Porphyromonas gulae LPS-induced inflammatory responses in human gingival epithelial cells.

OBJECTIVES: To determine the anti-inflammatory effects of green tea catechins in immortalized human gingival epithelial cells (Ca9-22) stimulated with Porphyromonas gulae lipopolysaccharide (LPS). METHODS: Ca9-22 cells were incubated with P. gulae LPS (10 µg/ml) with or without green tea catechins, epigallocatechin-3-gallate (EGCg), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epicatechin (EC) (each at 50 µM), for 6 or 24 h. Real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay were used to determine the induction of cyclooxygenase 2 (COX2), tumor necrosis factor alpha (TNF-ɑ), interleukin 6 (IL-6), and IL-8. Furthermore, the expression of toll-like receptors (TLRs) 2 and 4 was examined using real-time PCR and western blotting analysis, and phosphorylation of the p38 and ERK1/2 was examined using western blotting analysis. RESULTS: At the mRNA and protein levels, EGCg, EGC, ECG, and EC were found to significantly inhibit COX2, TNF-ɑ, IL-6, and IL-8. Furthermore, the levels of ERK1/2 and p38 phosphorylation induced by P. gulae LPS were decreased following the addition of each of the catechins, as well as TLR2 and 4 mRNA and protein. CONCLUSIONS: These findings indicate that green tea catechins are potent inhibitors of inflammatory responses induced by P. gulae LPS, and may also be useful for prevention and/or attenuation of periodontitis.

In vitro effectiveness of pomegranate extract present in pet oral hygiene products against canine oral bacterial species.

Background and Aim: Pomegranate is known to possess antibacterial properties, partly because of its punicalagin content. However, its effect on canine oral bacterial species has not yet been elucidated. In this study, we evaluated the effect of pomegranate extract present in pet dental products on the growth and survival of five canine oral bacterial species in biofilms. Materials and Methods: Five bacterial species, Neisseria shayeganii, Neisseria canis, Porphyromonas gulae, Porphyromonas macacae, and Porphyromonas crevioricanis, were individually cultured for biofilm formation and exposed to pomegranate extract (or control) for 15 min. Cell survival was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and was compared between different conditions using a student's t-test. In addition, the individual strains were grown in planktonic suspensions and exposed to serial dilutions of the extract to determine the minimum inhibitory concentration. Results: At a concentration of 0.035% w/v, the extract significantly reduced the survival of P. gulae (-39%, p < 0.001) and N. canis (-28%, p = 0.08) in biofilms. At similar concentrations, the extract also completely or partially inhibited the growth of N. canis and Porphyromonas spp. in planktonic suspensions, respectively. Conclusion: The pomegranate extract found in some pet dental products can limit bacterial growth and survival in the biofilms formed by N. canis and P. gulae in vitro. As P. gulae is involved in periodontal disease progression, limiting its proliferation using products containing pomegranate extract could contribute to disease prevention. Further studies on dogs receiving such products are necessary to confirm these effects.

The bacteriostatic effect of erythritol on canine periodontal disease-related bacteria.

Erythritol helps both prevent and improve periodontal disease and is therefore widely used for dental care in humans. However, only a few studies have investigated the effects of erythritol on periodontal disease in animals. We hypothesized that erythritol could be used to prevent and improve periodontal disease also in canines and investigated the effects of erythritol on canine periodontal disease-related pathogenic bacteria using both in vitro and in vivo methods. The effect of erythritol on the proliferation of Porphyromonas gulae, which is reportedly associated with canine periodontal disease, was investigated in vitro. In addition, a 4-week intervention trial using an external gel preparation containing 5% erythritol was performed in canines with mild periodontal disease; changes in the microbiota around periodontal lesions were investigated using next-generation sequencing and bioinformatics analysis. The growth of P. gulae was significantly suppressed by erythritol in vitro. In the intervention study, the Shannon index, an indicator of the species distribution α-diversity, and the occupancy of several canine periodontal disease - related bacteria ( P. gulae, P. cangingivalis) were significantly decreased in periodontal lesions. Based on the results of in vitro and in vivo studies, we conclude that, as in humans, erythritol has bacteriostatic effects against periodontal disease - related bacteria in canines.

Detection of Periodontal Pathogens from Dental Plaques of Dogs with and without Periodontal Disease.

Dental plaque bacteria are one of the main factors responsible for the development of a periodontal disease, which is the most common infectious disease in dogs. The aim of this study was to identify the presence of periodontal disease-related bacteria in the dental plaque of dogs. Plaque samples were taken from dogs with and without periodontal disease. Samples were analyzed for the presence of Porphyromonas gulae, Tannerella forsythia and Treponema denticola using a PCR technique amplifying 16S rRNA genes of P. gulae and T. forsythia and flaB2 genes of Treponema species, including T. denticola. The presence of T. forsythia was confirmed in all samples. P. gulae was detected in all dogs with periodontal disease and in 71.43% of dogs without periodontal disease. Treponema spp. were detected in 64.29% of the samples. Based on Sanger sequencing and Basic Local Alignment Search Tool algorithm, Treponema spp. were identified as T. denticola and Treponema putidum. T. denticola was present in 28.57% of dogs with periodontal disease, while T. putidum was present in 42.86% of dogs with periodontal disease and in 57.14% of dogs without periodontal disease. T. putidum was positively correlated with both P. gulae and T. forsythia, suggesting that it may be involved in the development of periodontal disease.

Efficacy of FimA antibody and clindamycin in silkworm larvae stimulated with Porphyromonas gulae.

Objective: Porphyromonas gulae, a major periodontal pathogen in animals, possesses fimbriae that have been classified into three genotypes (A, B, C) based on the diversity of fimA genes encoding fimbrillin protein (FimA). P. gulae strains with type C fimbriae were previously shown to be more virulent than other types. In this study, we further examined the host toxicity mediated by P. gulae fimbriae by constructing recombinant FimA (rFimA) expression vectors for each genotype and raised antibodies to the purified proteins. Methods and Results: All larvae died within 204 h following infection with P. gulae type C at the low-dose infection, whereas type A and B did not. Among fimA types, the survival rates of the larvae injected with rFimA type C were remarkably decreased, while the survival rates of the larvae injected with rFimA type A and type B were greater than 50%. Clindamycin treatment inhibited the growth of type C strains in a dose-dependent manner, resulting in an increased rate of silkworm survival. Finally, type C rFimA-specific antiserum prolonged the survival of silkworm larvae stimulated by infection with P. gulae type C strain or injection of rFimA type C protein. Conclusion: These results suggested that type C fimbriae have high potential for enhancement of bacterial pathogenesis, and that both clindamycin and anti-type C rFimA-specific antibodies are potent inhibitors of type C fimbriae-induced toxicity. This is the first report to establish a silkworm infection model using P. gulae for toxicity assessment.

Phylogenetic diversity in fim and mfa gene clusters between Porphyromonas gingivalis and Porphyromonas gulae, as a potential cause of host specificity.

BACKGROUND: Periodontopathic bacteria Porphyromonas gingivalis in humans and Porphyromonas gulae in animals are phylogenetically close and commonly have FimA and Mfa1 fimbriae. However, little is known about how fimA and mfa1 are phylogenetically different between P. gingivalis and P. gulae. Here, we examined phylogenetic diversity in their fim and mfa gene clusters. METHODS: Twenty P. gulae strains were isolated from the periodontal pocket of 20 dogs. For their genomic information, along with 64 P. gingivalis and 11 P. gulae genomes, phylogenetic relationship between the genotypes of fimA and mfa1 was examined. Variability of amino acid sequences was examined in the three-dimensional structure of FimA. The distance between strains was calculated for fim and mfa genes. RESULTS: Some fimA genotypes in P. gulae were close to particular types in P. gingivalis. Two types of mfa1 were classified as 70-kDa and 53-kDa protein-coding mfa1. The variable amino acid positions were primarily at the outer part of FimA. The genes encoding the structural proteins and the main component were similarly distant from the reference strain in P. gingivalis, but not in P. gulae. CONCLUSIONS: The differences in the gene clusters between P. gingivalis and P. gulae may result in their host specificity.

Glycyrrhizic acid suppresses inflammation and reduces the increased glucose levels induced by the combination of Porphyromonas gulae and ligature placement in diabetic model mice.

Diabetic patients are at an increased risk of developing severe and progressive periodontitis. Periodontal disease also increases the severity of diabetes by enhancing insulin resistance. Therefore, the regulation of periodontal inflammation in diabetic patients may contribute to the control of both diseases. Glycyrrhizic acid exerts anti-inflammatory effects by inhibiting high mobility group box 1 (HMGB1). HMGB1, one of the ligands of the receptor for advanced glycation end products (RAGE), is a damage-associated molecular pattern and induces inflammatory cytokine production. In the present study, we examined the effects of glycyrrhizic acid on ligature- and Porphyromonas gulae infection-induced periodontitis as well as the involvement of the HMGB1-RAGE axis in diabetic model mice. The molars of diabetic model mice, established by feeding HFD32 to KK/TaJcl mice, were subjected to silk thread ligation and P. gulae was then intraorally applied in the presence or absence of glycyrrhizic acid given topically. The topical application of glycyrrhizic acid suppressed ligature/P. gulae-induced increases in interleukin (IL)-6 and tumor necrosis factor (TNF)-α at the mRNA level in the gingiva and at the protein level in serum. Furthermore, glycyrrhizic acid suppressed ligature/P. gulae-induced increases in serum amyloid A (SAA) in serum and fasting blood glucose levels. It also suppressed ligature/P. gulae-induced increases of HMGB1 and RAGE at the mRNA level in the gingiva and at the protein level in serum. A mouse anti-HMGB1-neutralizing antibody inhibited increases in serum glucose levels. In conclusion, topical treatments with glycyrrhizic acid may suppress periodontal and systemic inflammation and reduce blood glucose levels through the HMGB1-RAGE axis in diabetic mice.

Identification and molecular characterization of Porphyromonas gulae fimA types among cat isolates.

Porphyromonas gulae, a Gram-negative black-pigmented anaerobe, is one of several major periodontal pathogens of animals. P. gulae isolates from dogs have been classified into three genotypes based on a 41-kDa filamentous appendage (FimA) on the cell surface, which is closely related to virulence in periodontal disease. However, other specific bacterial virulence factors contributing to the aggravation of periodontal disease in cats remain elusive. In the present study, we assessed FimA diversity in P. gulae isolates from cats and examined whether this diversity influenced periodontal condition. The putative amino acid sequences of FimA from 15 P. gulae isolates from 13 cats were classified into three genotypes (types A, B, and C), which showed 95-100% identity and similarity to the fimA types in dogs. The type C isolate showed greater adhesion and invasion properties in periodontal ligament fibroblasts as well as stronger inhibition of scratch closure of the cells compared with type A and B isolates. Next, a PCR-based method for identification of fimA genotype was developed and used to analyze 99 oral swab specimens from cats. High fimA type A detection rates were observed regardless of the periodontal condition, whereas types B and C were frequently detected from subjects with moderate and severe periodontitis, respectively. These results suggest that P. gulae isolates from cats can be classified into three types based on fimA genotype, which may be closely related to virulence in periodontitis.

Interspecies dynamics among bacteria associated with canine periodontal disease.

The etiology and pathogenic mechanisms associated with canine periodontal disease are less well understood than the disease in humans. In this study we have reconstructed defined consortia biofilms in vitro of microorganisms identified as prevalent in a same-breed cohort of dogs with or without periodontal disease. Frederiksenia canicola and Neisseria canis were selected as potential early colonizers of salivary pellicle, and Fusobacterium nucleatum and Porphyromonas gulae were included as high incidence canine oral bacteria. N. canis formed a biofilm substratum under aerobic conditions, but was unable to tolerate anaerobic conditions. Fr. canicola exhibited synergistic biofilm growth with Po. gulae under anaerobic conditions, but displayed an antagonistic relationship with Fu. nucleatum. However, strong co-adhesion between Fu. nucleatum and Po. gulae was able to overcome the inhibitory effects of Fr. canicola to facilitate three-species biofilm formation. Parvimonas micra, an anaerobic, asaccharolytic Gram-positive coccus found only under disease conditions in vivo, was able to form biofilms in conjunction with Fr. canicola and Po. gulae. Furthermore, the specific proteolytic activities of biofilms containing Fr. canicola and Po. gulae or Fu. nucleatum and Po. gulae were increased several-fold upon the addition of Pa. micra. This suggests that anaerobic cocci such as Pa. micra might provide a catalyst for progressive tissue destruction, inflammation and alveolar bone loss in canine periodontal disease, in keeping with the keystone-pathogen hypothesis.

Short communication: Distribution of Porphyromonas gulae fimA genotypes in oral specimens from dogs with mitral regurgitation.

Porphyromonas gulae, a suspected pathogen for periodontal disease in dogs, possesses approximately 41-kDa fimbriae (FimA) that are encoded by the fimA gene. In the present study, the association of fimA genotypes with mitral regurgitation (MR) was investigated. Twenty-five dogs diagnosed with MR (age range 6-13 years old, average 10.8 years) and 32 healthy dogs (8-15 years old, average 10.8 years) were selected at the participating clinics in a consecutive manner during the same time period. Oral swab specimens were collected from the dogs and bacterial DNA was extracted, then polymerase chain reaction analysis was performed using primers specific for each fimA genotype, with the dominant genotype determined. The rate for genotype C dominant specimens was 48.0% in the MR group, which was significantly higher than that in the control group (18.8%) (P <0.05). These results suggest that P. gulae fimA genotype C is associated with MR.