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 (
The studies were conducted in dogs which were patients of the Department of Animal Surgery, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Poland. All procedures performed on the animals were accepted by the 2nd Local Ethics Committee for Animal Experiments in Lublin on 26 May 2015 (Resolution no. 29/2015). The owners of dogs qualified for the studies were informed about the purpose of the undertaken activities and gave their informed consent to the procedures explained to them. The studies were carried out on 105 dogs, comprising 65 males and 40 females of various breeds. The patients were aged 3–16 years and had body weights of 3–10 kg. The breeds qualified for the studies were mixed (n = 28), Yorkshire terrier (n = 28), poodle (n = 8), English cocker spaniel (n = 8), short-haired dachshund (n = 8), miniature schnauzer (n = 7), fox terrier (n = 3), pug (n = 3), Pekingese (n = 3), beagle (n = 2), papillon (n = 2), miniature pinscher (n = 2), Italian greyhound (n = 2) and Welsh terrier (n = 1). The animals were divided into two groups: experimental and control. The control group (C) comprised 18 clinically healthy dogs which came to the Department of Animal Surgery for periodic or prophylactic examinations. The dogs comprised 9 males and 9 females of various breeds. They were aged 5–13 years and had body weights of 5–10 kg. The breeds qualified for the studies in the control group were mixed (n = 8), Yorkshire terrier (n = 8) and poodle (n = 2). A physical examination and other examinations confirmed the good general condition of the animals.
The experimental group (E) comprised 87 dogs which were diagnosed with periodontal disease. They were examined for dental disorders because of the following symptoms: food intake disorders, foetor
Physical dental examination, periodontal examination and swabbing for microbiological tests were performed on the dogs under general anaesthesia before performing cavity cleaning, as part of the treatment of the periodontal disease. The anaesthesia regimen presented below was used for each patient. The premedication was xylazine (Sedazin; Biowet Puławy, Poland) at a dose of 2 mg/kg body weight (b.w.). After 15 min, the patient had an intravenous line inserted and received ketamine (Vetaketam; VetAgro, Lublin, Poland) at a dose of 8 mg/kg b.w., combined at a 1:1 ratio with diazepam (Relanium; Polfa, Warsaw, Poland) at a dose of 0.5 mg/kg b.w. (administered proportionately to the effect seen). Next, the patient was intubated, and a device monitoring vital signs (SurgiVet Advisor, Smiths Medical, Dublin, OH, USA) was connected. In order to maintain anaesthesia, inhalation (Matrx VMS Plus; Midmark Animal Health, Versailles, OH, USA) was used of a mixture of oxygen and isoflurane at a concentration of 2% (Aerrane; Baxter, Deerfield, IL, USA).
Swabs were collected with the use of sterile paper points (Pet Test; MIP Pharma, Berlin, Germany) from patients with gingival pockets deeper than 5 mm. Five swabs were collected from each patient from several sites in the periodontium, which were used as a pooled sample according to the procedure recommended by the manufacturer. Prior to insertion of a paper point into the gingival pocket, supragingival plaque was gently removed with the use of sterile gauze pads, paying attention not to cause bleeding. Then, the sites were dried with the use of compressed air. Each sterile point was inserted with tweezers into the bottom of the gingival pocket and left there for 15 s. Next, the points were combined into a pooled sample, which was placed in the separate transport containers supplied with the Pet Test kit. The samples were collected from 36 dogs in total (Fig. 1).
The samples were sent to the MIP Pharma GmbH analytical laboratory, where the material was analysed. The Pet Test uses a molecular-biological polymerase chain reaction – a real-time RT-PCR with the use of the TaqMan probe. Reference strains were cultured in accordance with the recommendations of the American Type Culture Collection. Sets of primers and probes of specific species were designed from the 16S rRNA gene sequence variable regions and used for a qualitative determination of the total proportion of bacterial cells in the samples.
Free DNA fragments were obtained from bacterial cells by lysis, which were then amplified and hybridised with fluorescence-labelled primers specific to particular periopathogens. Quantification was performed with the use of a reader measuring the fluorescence intensity versus reference samples.
The occurrence of the following bacteria was analysed:
All calculations were performed with computer methods using the Microsoft Excel 2010 spreadsheet and the Statistica 10.0 statistical package (StatSoft, now Tibco, Tulsa, OK, USA). Statistical significance was calculated using the Mann–Whitney U test. Relationships between the selected variables were calculated using Spearman’s correlation.
Based on the analysis of the oral cavity clinical assessment results from patients of the experimental group (n = 87), four stages of periodontal disease advancement were identified and dogs were accordingly allocated to subgroups:
Stage I (E1) – 19 dogs
Stage II (E2) – 24 dogs
Stage III (E3) – 20 dogs
Stage IV (E4) – 24 dogs.
The criterion of inclusion in a particular stage group was the degree of periodontal tissue destruction.
In none of the patients was
Mean number of bacteria isolated from gingival pockets presented in three subgroups
E2, E3, E4 – stage II, stage III and stage IV of the periodontal disease, respectively; x – mean bacterial count (× 104 CFU/mL); SD – standard deviation; * – statistically significant differences between particular bacteria in the analysed subgroups for P ≤ 0.05; Aa –
In 1998, Socransky
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
In the authors’ studies, the presence of
Bacteria of the
Such bacteria as
In their studies conducted on dogs with periodontal disease, Di Bello
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
Periodontal pocket microbiological analysis shows that microorganisms, namely
Mean number of bacteria isolated from gingival pockets presented in three subgroups