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.


Introduction
In veterinary medicine, periodontal diseases are the most common diseases of domestic carnivores and occur in approximately 80% of dogs older than 2 years [1,2]. The incidence of the disease increases significantly with age and weight of the animals. Epidemiological studies indicate higher occurrence in small breeds [3,4]. Based on the clinical signs, periodontal diseases are usually divided into two groups, gingivitis and periodontitis. The most common clinical signs of periodontal disease include halitosis, ptyalism, altered gingival color, gingival bleeding, tooth mobility, anorexia and behavioral changes. Periodontal diseases are the result of the accumulation of dental bacterial plaque on the surface of the teeth, which is exacerbated by the accumulation of mineralized dental calculus [5,6].
Periodontitis is a dysbiotic disease rather than an infection caused by only a few selected species of microorganisms. Dysbiosis of the periodontal microbiota represents a change in the relative number of individual components of the bacterial community, Pathogens 2022, 11, 480 2 of 8 namely, the shift of subgingival Gram-positive bacteria to Gram-negative bacteria [7,8]. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia (formerly Bacteroides forsythus) are considered to be the most important pathogens in periodontal disease in humans [9]. Porphyromonas gulae, previously known as the animal biotype of the human periodontal pathogen P. gingivalis, has virulence properties that are associated with P. gingivalis and may play a key role in the pathology of periodontitis in companion animals [10]. Several different species of Treponema are found in the oral cavity of healthy dogs, which suggests that they are a part of normal microbiota [11]. However, T. denticola is most commonly associated with periodontal disease [12,13]. The meaning of Treponema spp. in the etiology of periodontal disease is still not entirely clear and requires further investigation [11]. The presence of T. forsythia in human dental plaque is associated with an increased risk of periodontitis [14], while in dogs, studies show conflicting results in the presence of T. forsythia in dental plaque [15,16]. The aim of present study was to determine the presence of P. gulae, T. denticola and T. forsythia in supragingival dental plaque samples of dogs with and without periodontal disease by PCR (Figure 1).
Pathogens 2022, 11, x FOR PEER REVIEW 2 of 8 change in the relative number of individual components of the bacterial community, namely, the shift of subgingival Gram-positive bacteria to Gram-negative bacteria [7,8].
Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia (formerly Bacteroides forsythus) are considered to be the most important pathogens in periodontal disease in humans [9]. Porphyromonas gulae, previously known as the animal biotype of the human periodontal pathogen P. gingivalis, has virulence properties that are associated with P. gingivalis and may play a key role in the pathology of periodontitis in companion animals [10]. Several different species of Treponema are found in the oral cavity of healthy dogs, which suggests that they are a part of normal microbiota [11]. However, T. denticola is most commonly associated with periodontal disease [12,13]. The meaning of Treponema spp. in the etiology of periodontal disease is still not entirely clear and requires further investigation [11]. The presence of T. forsythia in human dental plaque is associated with an increased risk of periodontitis [14], while in dogs, studies show conflicting results in the presence of T. forsythia in dental plaque [15,16]. The aim of present study was to determine the presence of P. gulae, T. denticola and T. forsythia in supragingival dental plaque samples of dogs with and without periodontal disease by PCR ( Figure 1).

Study Population
Nine female and five male dogs of different breeds aged between 1-14 years were sampled. Based on the clinical signs, such as the amount of plaque, presence of dental calculus, inflammation of the gums or tooth loss, they were divided into two groupshealthy and periodontal disease (Table 1).

Study Population
Nine female and five male dogs of different breeds aged between 1-14 years were sampled. Based on the clinical signs, such as the amount of plaque, presence of dental calculus, inflammation of the gums or tooth loss, they were divided into two groups-healthy and periodontal disease (Table 1).

Molecular Analysis
Species-specific PCR using the primers targeting species P. gulae showed that P. gulae was detected in all dogs with periodontal disease and in 71.43% of healthy dogs. Using specific PCR for Treponema species, including T. denticola, the presence of Treponema spp.
Pathogens 2022, 11, 480 3 of 8 was detected in 71.43% of dogs with periodontal disease and in 57.14% dogs without periodontal disease. T. forsythia was detected in all dogs with and without periodontal disease by species-specific PCR. The presence of P. gulae and T. forsythia was confirmed by Sanger sequencing and Basic Local Alignment Search Tool algorithm. Based on these methods, Treponema spp. were identified as T. denticola and Treponema putidum. T. denticola was present in 28.57% of dogs with periodontal disease, T. putidum in 42.86% of dogs with periodontal disease and in 57.14% of healthy dogs. The results of detected periodontal pathogens in samples of dental plaques of healthy and periodontal disease groups are shown in Figure 2.
Pathogens 2022, 11, x FOR PEER REVIEW 3 of Species-specific PCR using the primers targeting species P. gulae showed that P. gula was detected in all dogs with periodontal disease and in 71.43% of healthy dogs. Usin specific PCR for Treponema species, including T. denticola, the presence of Treponema spp was detected in 71.43% of dogs with periodontal disease and in 57.14% dogs without per iodontal disease. T. forsythia was detected in all dogs with and without periodontal diseas by species-specific PCR. The presence of P. gulae and T. forsythia was confirmed by Sange sequencing and Basic Local Alignment Search Tool algorithm . Based on these methods Treponema spp. were identified as T. denticola and Treponema putidum. T. denticola was pre sent in 28.57% of dogs with periodontal disease, T. putidum in 42.86% of dogs with perio dontal disease and in 57.14% of healthy dogs. The results of detected periodontal patho gens in samples of dental plaques of healthy and periodontal disease groups are shown in Figure 2. T. denticola was not present in any healthy dog. All three pathogens, P. gulae, T. for sythia and T. denticola, were detected in only two dogs with periodontal disease (28.57%) P. gulae together with T. forsythia were present in all dogs with periodontal disease and in five healthy dogs (71.43%) ( Table 2).  T. denticola was not present in any healthy dog. All three pathogens, P. gulae, T. forsythia and T. denticola, were detected in only two dogs with periodontal disease (28.57%). P. gulae together with T. forsythia were present in all dogs with periodontal disease and in five healthy dogs (71.43%) ( Table 2).

Discussion
Periodontal diseases represent a serious diagnostic and therapeutic problem in human and veterinary medicine. Therapy of periodontal diseases is focused on suppressing the progression of inflammation and often involves a combination of different therapeutic approaches, such as scaling and root planing, the use of antibiotics and antimicrobial agents (e.g., chlorhexidine), antimicrobial photodynamic therapy or even the use of probiotics [17,18]. Moreover, dental chews can be used in dogs to reduce periodontal disease by beneficially shifting the microbiota of dental plaque [19]. Periodontal pathogens from the dental plaque, apart from irreversible damaging periodontium, can also cause some systemic diseases, which points to the importance of their identification [16].
Both in humans and animals, composition of bacterial plaque has been studied for many decades using culture methods. In recent times, methods of molecular biology have come to the foreground because of its high specificity, low time-consuming character and technical demand. In the present study, we used molecular methods for the detection of P. gulae, T. forsythia and T. denticola, which are associated with canine periodontal disease. However, various research studies reported their different prevalence in plaque samples of dogs. forsythia in almost all dogs analyzed; therefore, they consider it a common member of the canine oral microbiota [15]. In the present study, all dogs with and without periodontal disease were positive for T. forsythia.
Taxa belonging to the genus Treponema are common members of the microbial community of the human oral cavity. However, specific treponemes may be involved in the etiopathology of periodontal disease [24]. Valdez et al. (2000) confirmed the presence of T. denticola, T. socranskii ssp., T. vincentii, T. maltophilum, T. medium and T. pectinovorum in dental biofilm of dogs [25]. In the present study, consensus primers for the flaB2 gene for the detection of T. denticola, T. vincentii, T. medium ssp. bovis and T. phagedenis ssp. vaccae were used. The flaB2 gene has been previously used to determine Treponema species associated with bovine digital dermatitis [26,27].
Several studies report a prevalence of T. denticola in dogs of less than 7% [15,20,22,23]. In addition, Nishiyama et al. (2007) did not detect T. denticola in samples from dogs with periodontitis [28]. On the other hand, Gołyńska et al. (2017) observed T. denticola in all tested dogs [16]. In the present study, T. denticola was detected in 28.57% of dogs with periodontal disease but not in dogs without periodontal disease. Additionally, the presence of T. putidum was determined in the plaque samples taken from dogs with and without periodontal disease.
Oral treponemes are classified into 10 phylogroups. T. denticola and T. putidum belong to phylogroup 2 and share 98.5% of their 16S rRNA gene sequence homology [29]. Both T. putidum and T. denticola are associated with human periodontal disease. T. putidum was isolated for the first time from human periodontitis lesions and acute necrotizing ulcerative gingivitis sites [30]. T. putidum has homologues of virulence factors previously described within T. denticola, such as factor H binding protein implicated in evading complement-mediated killing, the major surface protein involved in cellular adhesion processes and cystalysin (hemolysin) involved in volatile sulfur compound production and erythrolysis [29]. In the present study, the presence of T. putidum was in positive correlation with both P. gulae and T. forsythia. Nises et al. (2018) detected T. putidum in samples from dogs with periodontal disease [11]. Based on these findings, we can assume that T. putidum also plays a role in the development of canine periodontal disease.

Animals and Sampling
A total of 14 dogs of different breeds, age and periodontal status were sampled at the Small Animal Clinic, University of Veterinary Medicine and Pharmacy in Kosice. Informed consent was obtained from the owners of the dogs for the study. The study was approved by the Ethics Commission of the University of Veterinary Medicine and Pharmacy in Kosice. Prior to the collection of dental plaque, an intraoral examination was performed to assess the periodontal status of each non-anesthetized dog by a veterinarian. The stage of periodontal disease was assessed according to Bauer et al. (2018) [31]. The dental plaque samples were taken from the buccal surfaces of the right upper canines and premolars with a syringe needle into an Eppendorf tube containing 300 µL of phosphate-buffered saline. After sampling, samples were stored at −70 • C until DNA extraction and PCR analysis were performed.

DNA Extraction
Thawed samples were centrifuged at 10,000× g for 10 min at 4 • C and the supernatant was discarded. The protocol for DNA extraction according to Vesty et al. (2017) [32] with some modifications was performed. Briefly, the pellet was resuspended in 180 µL of 10% sodium dodecyl sulphate and 25 µL of proteinase K was added to the mixture. The tubes were incubated at 55 • C for 2 h, with shaking at 300 rpm. Proteinase K was inactivated by heating at 95 • C for 5 min [33]. The tubes were then centrifuged at 10,000× g for 5 min at 23 • C and the supernatant was transferred to the Eppendorf tubes. Phenol and chloroform were equally (1:1) added to the supernatant and centrifuged at 10,000× g for 5 min at 23 • C. The upper aqueous phase was transferred to the Eppendorf tube. Isopropanol (0.6 volume of supernatant) and 3 M sodium acetate solution (0.1 volume of supernatant) were added. The nucleic acids were precipitated overnight at 4 • C. The following day, DNA was pelleted at 10,000× g for 10 min at 4 • C, washed with 100 µL of cold 70% ethanol and dried at 35 • C for 10 min. The pellets were resuspended in 30 µL of TE buffer [50 mM Tris-HCl, 10 mM EDTA]. The concentration of DNA was measured (NanoDrop 1000, Thermo Fisher Scientific, Waltham, WA, USA) and samples were diluted to a concentration of 50 ng/µL of template DNA.

PCR Assay
The PCR was processed using 2 µL (100 ng) of the sample (DNA template) added to 50 µL of reaction mixture containing OneTaq 2× Master Mix with Standard Buffer (New England Biolabs, Foster City, CA, USA), molecular grade water and primers. The primers used for PCR amplification of P. gulae, T. forsythia and Treponema species, including T. denticola, and PCR cycling conditions are listed in Table 3. PCR amplifications were performed in a thermocycler (TProfessional Basic, Biometra GmbH, Göttingen, Germany). A negative control (RNAse free H 2 O) was included in each PCR run. The amplicons were separated by gel electrophoresis and visualized with GelRed (Biotium, Inc., Hayward, CA, USA) under UV light [34]. A 100 bp DNA ladder (New England Biolabs, Foster City, CA, USA) was used as a molecular size standard.

Sequencing and Data Analysis
The amplification products were sent for Sanger sequencing in both forward and reverse directions (Microsynth, Wien, Austria). The obtained chromatograms of sequences were edited and aligned using Geneious alignment in Geneious 8.0.5 (Biomatters, Auckland, New Zealand). Homology searches were performed using the Basic Local Alignment Search Tool (BLAST) algorithm at the National Center for Biotechnology Information (NCBI). The nucleotide sequences were deposited in GenBank with accession numbers from MW595983 to MW595989, MW604827, MW604828, MW604829, MZ215849 and MZ215850, from OL839920 to OL839933, from OL906423 to OL906426, and from OM196210 to OM196214.

Conclusions
Based on our results, it can be assumed that T. forsythia is a common member of the oral microbiota of dogs, whereas T. denticola was detected only in dogs with periodontal disease. The higher prevalence of P. gulae was observed in dogs suffering with periodontal disease. Moreover, T. putidum may also be involved in the development of this disease. Further investigation is needed to clarify the possible co-involvement of T. putidum in the development of canine periodontal disease, and a larger sample size of studied population of dogs is needed as well.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki. The animal study protocol was approved by the Ethics Committee of the University of Veterinary Medicine and Pharmacy in Kosice (protocol code EKVP/2022-01, 14 January 2022).

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest.