Molecular microbiological characteristics of gingival pockets in the periodontal diseases of dogs

The oral microbiota in dogs is composed of microorganisms belonging to several thousand species (19, 23). The composition of the bacteria forming dental plaque varies depending on the degree of plaque accumulation. In the balanced ecosystem of the gingival fissure, the microorganisms colonising the oral cavity do not have a pathogenic effect on the periodontium – they are a commensal microbiota. Disturbances in the living conditions of the bacteria lead to changes in the composition of the gingival pocket bacterial population (10, 18, 20). Microorganisms become pathogenic only if the host is susceptible to pocket colonisation. Host susceptibility enables the periopathogens to propagate and reach the spectrum of virulence. The normal bacterial flora is primarily conditioned by Gram-positive aerobic bacteria (4, 6, 18, 28). Under conducive conditions, a bacterial flora favoured by them adheres to the surfaces of the teeth, tongue, lips, cheeks and gums permanently and irremovably, breaks down the defence system of the host and penetrates its tissues. As inflammation of the periodontal tissues deepens and periodontal disease develops, the surface of the teeth becomes colonised with Gram-negative bacteria (19, 28, 29). Restricted oxygen diffusion and low oxidation reduction potential facilitates the development of obligate anaerobes, and the bacterial flora of the gingival pockets changes from a composition of aerobic Gram-positive species to one of anaerobic Gram-negative species (14, 16, 23). As a result of gingival swelling and deepening of the pocket, a subgingival biofilm is developed, where colonies of anaerobic Gram-negative (Bacterioides spp., Capnocytophaga spp., Fusobacterium spp., Porphyromonas spp., Prevotella spp., Tannerella spp. and Treponema spp.) and Gram-positive (Actinomyces spp., Corynebacterium spp., Eubacterium spp., Peptostreptococcus spp. and Streptococcus spp.) bacteria exist (10, 18, 22, 30). Microorganisms may affect periodontal tissues directly or indirectly. Direct tissue damage results from the activity of toxins, enzymes and the bacterial products of metabolism. An indirect effect involves the impairment of the defence response of the host by activation of the inflammatory cells, which produce and release mediators with high pro-inflammatory and catabolic activity (4). This activity plays a key role in the destruction of periodontal tissues. Moreover, certain bacteria may affect the host defence mechanism by inactivation of specific antibodies or inhibition of cellular phagocytosis (21). They can also penetrate periodontal tissues and consequently create specific subgingival reservoirs (16, 22).

In 1998, Socransky et al. (25) isolated bacterial complexes of 40 species by identifying the constituents of the subgingival biofilms with a molecular method and grouping them into five major microbial complexes (red, orange, yellow, green and purple). They assigned the highest importance to red complex bacteria, which in humans represent the marker bacteria for periodontitis. Bacteria belonging to red complexes are Gram-negative, non-spore-forming, anaerobic organisms carrying several virulence factors responsible for developing the inflammatory process, such as fimbriae, proteinases, exopolysaccharides and haemin-binding proteins (10). These include Treponema denticola, Tannerella forsythia and the main pathogen causing periodontitis in humans, Porphyromonas gingivalis (2). In dogs, various species of Porphyromonas are also considered important pathogens causing periodontitis, but their true significance in the pathogenesis is not fully known (19). The species of Porphyromonas isolated from the oral cavity of dogs include Porphyromonas gulae, P. macacae, P. cangingivalis, P. gingivicanis and P. circumdentaria (23). The second important complex in the course of periodontal diseases in humans is the green complex, which includes Capnocytophaga gingivalis, Campylobacter concisus and Eikenella corrodens. The orange complex, consisting of Fusobacterium nucleatum, Prevotella intermedia, P. nigrescens, Peptostreptococcus micros, Campylobacter rectus, C. gracilis, C. showae and Streptococcus constellatus is important because the presence of bacteria belonging to this complex determines formation of the red complex (9). The colonisation of specific bacterial species in the biofilm is an aetiological factor of periodontitis, and knowledge of the occurring pathogens makes it easier to select an adequate local and systemic therapy (10). However, while the presence of bacterial microflora causing periodontal diseases is a necessary condition for the development of the disease, by itself it is not sufficient for this (11, 17, 21, 24).

In the authors’ studies, clinical evaluation of the periodontal tissue condition was based on cross-referencing the depth of the gingival pockets to the Russell clinical index of periodontal diseases. An indicator and other indices used to assess the oral cavity in dogs used in studies include the presence of bleeding during probing, plaque index, calculus index modified by Logan and Boyce, gingivitis index, tooth mobility index or furcation index (17). However, authors have most frequently assessed the depth of the gingival pockets and the loss of connective tissue attachment. It should be noted that the results of the periodontal disease evaluation in dogs may be affected by the selection of dogs with regard to age and breed.

According to the literature data, most anaerobic bacteria isolated from gingival pockets in dogs belong to the Fusobacterium, Porphyromonas and Prevotella genera, members of which have for many years been considered key microorganisms that cause periodontal diseases, while Porphyromonas gingivalis in humans is considered a bacterium initiating periodontal disease and highly associated therewith (14, 15, 21). However, several differences have been documented between Porphyromonas in humans and their companion animals (23). Porphyromonas gingivalis isolated from dog and cat samples is catalase-positive and shows high phenotypic similarity to P. gulae, while the one isolated from human samples is catalase-negative (14). Moreover, both Porphyromonas species show similar virulence traits. The literature states that the high incidence of periodontal diseases in dogs may also be related to the occurrence of such Porphyromonas species as: P. macacae, P. cangingivalis, P. gingivicanis and P. circumdentaria (23). In the authors’ studies, Aggregatibacter actinomycetemcomitans was not isolated in any of the dogs undergoing microbiological analysis. This is probably due to the fact that this bacterium is highly correlated with aggressive periodontitis when identified in human cases. This form of inflammation is characterised by rapid loss of soft tissue attachment and destruction of the alveolar bones, while in dogs the disease is of a chronic nature whereby the development of periodontitis may last years (11, 12, 14).

In the authors’ studies, the presence of P. gingivalis was positively correlated with all the indices assessed in the dental examination, which may indicate the significant influence of this bacterium on periodontal tissue damage. Porphyromonas gingivalis was also strongly correlated with T. denticola as indicated by the calculated r coefficient. This may indicate a synergistic effect between bacteria and one between them and related occurrences. Analysing the rising trend of the counts of P. gingivalis and T. denticola with the stage of advancement of periodontal disease, one may suppose that they play an important role in progressive tissue destruction in the course of periodontitis. At the early stage of periodontitis in the analysed dogs, both bacteria were isolated in lower numbers than during more advanced stages, where the loss of periodontal tissues was much greater.

Bacteria of the Porphyromonas genus and some species of the Prevotella genus are called black-pigmented bacteria (12). Members of the latter genus were also frequently detected in the studied dogs. For many years, some bacteria of these genera have been identified as key factors involved in the pathogenesis of periodontitis in dogs. Black-pigmented bacteria have also been confirmed to be good markers of periodontal disease in dogs and cats. In the present studies, an upward tendency of the P. gingivalis and P. intermedia counts was observed with higher inflammation severity, which may classify them as indicator bacteria. Khazandi et al. (14) isolated Gram-negative bacteria producing black-pigmented colonies in 95% of the collected samples from canine and feline gingival pockets after four days of incubation, finding the majority of isolates to be Porphyromonas gulae. Charalampakis et al. (4) also used human strains of P. intermedia and T. denticola in their studies, recording an increased count of these bacteria in dogs with experimentally induced periodontitis.

Fusobacterium species show synergism with other bacteria. Existing in the gingival pocket, they have the ability to aggregate with other microorganisms. Fusobacterium nucleatum is one of the most frequently occurring bacterium in the gingival fissure of humans and dogs associated with periodontal diseases (26). The results of the analysis conducted by Conrads et al. (5) showed the existence of new strains of Fusobacterium belonging to species that are closely related to F. nucleatum. In the present studies, F. nucleatum was isolated in 80.5% of the experimental dogs. With regard to the total count of isolated pathogens, this bacterium made up 8%. A lower count of F. nucleatum was obtained in dogs with less severe inflammation of the periodontal tissues (4.62 × 10⁴ CFU/mL) than in dogs with severe periodontitis (9.69 × 10 ^bn CFU/mL). Similar results were obtained by Senhorinho et al. (23), who showed the presence of Fusobacterium nucleatum in the subgingival region in dogs with periodontitis. These researchers suggest that Fusobacterium plays an important role in periodontal diseases in dogs. Numerous examples of isolates from samples taken in the present research resolved to F. nucleatum, which raises the question of this bacterium possibly supporting other bacteria (e.g. Porphyromonas spp.) and facilitating their binding (2).

Such bacteria as Peptostreptococcus micros, Eubacterium nodatum and Capnocytophaga gingivalis were detected in some of the examined dogs. Given the low number of these pathogens in the collected samples and their low value as compared to other pathogens, it may be concluded that these bacteria are not significant for the pathogenesis of periodontal diseases in dogs. However, it must be noted that in each case, low numbers of these bacteria in the analysed samples gave way to increased counts as the inflammatory process deepened, which is probably related to an increased propagation of bacteria during moderate and severe periodontitis. Dilegge et al. (8) isolated not only the Capnocytophaga species typical of dogs (C. ochracea and C. haemolytica) but also those typical of humans (C. gingivalis and C. granulosa) in the dental plaque of dogs.

Wallis et al. (27) did not identify red complex bacteria (P. gingivalis, T. denticola and T. forsythia) in the subgingival plaque. Allaker et al. (2) did not identify Treponema sp. in studied dogs, which is in contrast to the authors’ own studies, since T. denticola was isolated from all the dogs, and as compared to other bacteria, it represented a major part of the pool of pathogens. The most frequently isolated bacteria in the studies of Riggio et al. (20) were Pseudomonas spp. (30.9%) and P. cangingivalis (16.1%). Other bacteria isolated in lower amounts included Tannerella forsythia. In a metagenomic analysis of the bacteria colonising the oral cavity of dogs, Sturgeon et al. (26) showed that the dominant genus among the isolates was Porphyromonas (39.2%). They isolated Fusobacterium spp. from 4.5%, Capnocytophaga spp. from 3.8%, Tannerella spp. from 2.2%, and Treponema spp. from 1.6% of samples. Other researchers identified P. gingivalis in 68% of the examined animals, while in 91% of animals they found the presence of black-pigmented bacteria (2). Nishiyama et al. (16) also showed the presence of P. gingivalis in 64% of individuals. Moreover, they showed the presence of P. intermedia and T. forsythia in 20% of the studied dogs, and F. nucleatum in 16% of the dogs. They did not isolate T. denticola from any of the dogs. In addition, they isolated P. gingivalis from one dog without periodontitis. Di Bello et al. (7) analysed in their studies the presence of red complex bacteria in dogs in the course of periodontal diseases. In 49.3% of the dogs, they observed a mixed P. gingivalis and T. forsythia infection, which was statistically significant. In other studies where various methods were used to treat experimentally induced periodontal disease, high levels of Prevotella intermedia and T. forsythia were observed. Where red complex bacteria were detected, Hall et al. (11) observed an increased mean count of these bacteria even after a four-week treatment period. These results are in contrast to the present results, since T. forsythia was only isolated in low amounts from 19% of canine samples. On the other hand, T. forsythia was isolated from humans from dog and cat bite wounds, which suggests the presence of these bacteria in the oral cavities of the animals (12). Senhorinho et al. (23) also reported the Porphyromonas genus as the one most often isolated from dogs with periodontal diseases (92% of the studied dogs), but also as a genus isolated from 56% of healthy dogs without periodontitis. Out of 46 dogs from which they isolated Porphyromonas spp., from 38 animals they also isolated P. gulae. Among isolates from gingival pockets in dogs studied by Radice et al. (19), the majority was represented by P. gingivalis (30.8%), Peptostreptococcus spp. (30.8%) and Prevotella intermedia (23.1%). Kato et al. (13) studied the occurrence of P. gingivalis, T. denticola, T. forsythia, Prevotella intermedia and A. actinomycetemcomitans in the saliva of dogs with gingivitis. The main bacteria isolated by them was T. forsythia, and in this the present investigation’s outcome differs. Regarding A. actinomycetemcomitans, they did not detect it in any of the dogs, as was the case in the authors’ own studies. They isolated P. intermedia from only three dogs and Porphyromonas gingivalis and T. denticola from only one dog. The authors also studied the occurrence of P. gulae and successfully isolated it from almost all the studied dogs. Yamasaki et al. (30) studied the occurrence of 10 bacteria associated with periodontitis in humans. In their study, the most frequently isolated pathogen was T. forsythia (77.3%), with which our results do not concur. However, they rarely isolated P. gingivalis, T. denticola, Prevotella intermedia or A. actinomycetemcomitans. In addition, in those authors’ studies the occurrence of Porphyromonas gulae was observed, being isolated from 71.2 % of the studied dogs. Porphyromonas gulae seems to be highly associated with periodontal disease in dogs but not in humans.

In their studies conducted on dogs with periodontal disease, Di Bello et al. (7) showed the dominance of Actinomyces, Peptostreptococcaceae and Porphyromonas bacteria. The authors also demonstrated a correlation between the presence of Treponema denticola and mild periodontitis. This contrasts with the findings of this research, because in dogs showing stage II periodontal disease (early periodontitis), a negative correlation between T. denticola and the clinical indicator of periodontal disease was observed. In dogs in stage III (moderate periodontitis), this correlation was positive and high. The periodontal tissue damage was proportional to the number of isolated T. denticola. Perhaps together with the development of the periodontal disease, there is greater colonisation of the plaque with T. denticola, which in turn results in a further progressive loss of tissues.

Charalampakis et al. (4) showed an increased total bacteria count after removing ligatures causing inflammation of the periodontium when conducting a microbiological analysis of experimentally induced periodontitis in dogs, and demonstrated the formation of anaerobic Gram-negative bacterial flora. These data are in contrast with those of the authors’ present studies, since no correlation was observed between the total bacterial count and the age of the examined dogs. The analysis of the age of animals qualified to particular subgroups showed that the mean age of the dogs was directly proportional to the advancement of the inflammatory process.

The results of available studies confirm that periodontal diseases are caused by bacterial complexes. In recent years, traditional methods of bacterial culture have been replaced with methods of molecular analysis of the gene coding ribosomal RNA (16S rRNA). Sequencing of the 16S rRNA gene enabled the identification of bacteria which, for various reasons, cannot be cultured in media (1, 20). Test Pet uses a molecular and biological reverse transcriptase polymerase chain reaction in real time. This method enables quick and easy identification of specific microorganisms. Test Pet copies selected DNA/RNA fragments rapidly and makes it possible to analyse the number of copies in particular samples. It is characterised by high sensitivity – one fragment may give 10⁶ copies. Moreover, it enables the analysis of material even at low baseline DNA levels. Real-time PCR enables the specific and sensitive identification and quantification of the pathogens causing periodontitis. Comparing the real-time PCR with cell culture of five periopathogens for the purpose of their identification and quantification, Boutaga et al. (3) showed the high level of conformity of these analyses. They defined the real-time PCR method as an effective alternative for the quantitative assessment of anaerobic bacteria isolated from the subgingival region. In a subsequent investigation, they demonstrated the possibility of using the real-time PCR method to detect very low amounts of periopathogens in collected samples and particularly amounts below the detection limit with the use of the cell culture technique.

Elliott et al. (9) compared the DNA sequences used in their studies with human microflora DNA sequences in GenBank and showed approximately 7% difference in the 16S rRNA gene. They showed that most isolates from dogs were not typical of those occurring in the human oral cavity, while the bacteria were similar to those isolated from humans overall. Other researchers (7, 22) also conducted an analysis of bacterial species isolated from the dental plaque of dogs. Using the method of 16S rRNA gene sequencing, they identified 353 taxons, wherein 80% were completely new and only 16.4% were common to the microflora of the human oral cavity. With reference to the studies by Dahlen et al. (6), there are controversies related to the detection of microorganisms in the gingival pocket of dogs using microbiological tests based on molecular probes of human strains. The authors suggested the occurrence of cross-reactions between the DNA probes of human strains and strains isolated from dogs. According to them, P. gingivalis showed a strong cross-reaction with P. gulae and other Porphyromonas species. “Human” T. forsythia cross-reacts with Tannerella spp., but not with “canine” T. forsythia. Fusobacterium nucleatum cross-reacts with F. canifelinum. However, Prevotella intermedia does not cross-react with other canine species. Patients with periodontitis, regardless of the varied microbiological profile of the oral cavity, show a good response to treatment with the use of cavity cleaning (30).

Periodontal pocket microbiological analysis shows that microorganisms, namely Porphyromonas gingivalis, Treponema denticola and Prevotella intermedia, are present in the oral cavity of dogs with periodontal disease. In therapeutic categories, it is crucial to assess the role of these microorganisms in the aetiology of periodontitis in domestic animals. As the inflammatory process progresses, the number of isolated bacteria increases, showing a greater colonisation of the subgingival area associated with deepening local inflammation and progressive damage to periodontal tissues.