The pet population in Korea continues to grow, and a survey in 2019 reported that dogs accounted for 6 million of the total 10 million pets [1]. Consequently, there has been an increase in the interest in dog healthcare and related markets. Periodontal disease is a common disease in dogs, and dental care ranked 12th among the 20 major reasons for dogs visiting private veterinary clinics in Jeollabuk-do [2].

Periodontal disease is an infectious and inflammatory disease caused by microorganisms, including bacteria, viruses, fungi, and protozoa, that colonize the tooth surfaces below the gingival margins [3,4]. The microorganisms accumulate on the surface of the teeth to form a biofilm called plaque, which persists on the surface of the teeth, thickens, and becomes tartar when mineralized [5,6]. Plaque accumulation is one of the major factors of periodontitis development in dogs, because it releases enzymes derived from a specific pathogenic bacterium–host interaction that leads to the destruction of the supporting periodontium and alveolar bone around the teeth. This finally causes loss of attachment of the teeth [7]. Moreover, plaque is extremely common in dogs because the temperature and humidity in their oral cavity is appropriate for the growth of the microorganisms, providing an ideal environment for microbial life [8].

Plaque is formed by a variety of microorganisms, but periodontal disease mainly develops due to the enzymes secreted during the metabolism of Gram-negative anaerobic bacteria [9,10]. Although most of the periodontal disease-causing bacteria in dogs are different from those in humans, some of the species are common. Additionally, several periodontopathic species can be transmitted between humans and dogs [11]. Of these, three bacteria, Treponema denticola, Tannerella forsythia, and Porphyromonas gingivallis, are mainly associated with periodontitis in both dogs and humans; they are usually referred to as the red complex bacteria (RCB) [12,13].

Periodontitis is the most prevalent disease in dogs, affecting approximately 80% of dogs aged 4 years [14]. However, since the oral care in dogs is more easily neglected than in humans, gingivitis can rapidly progress to periodontitis without proper prevention and therapy.

Traditional clinical methods, such as pocket probing depth and bleeding on probing, are widely used in veterinary dentistry for assessing periodontal disease [15]. However, they do not provide sufficient information regarding the presence and absence of bacteria. Therefore, there is an increased interest in developing diagnostic methods for the prevention and treatment of periodontitis in dogs, which would be equally important for the oral health in humans.

The microbial-enzymatic N-benzoyl-DL-arginine-2-naphthylamide (BANA) has a unique ability to hydrolyze the trypsin substrate, and it can be used to detect the presence of the three putative pathogens in subgingival plaque samples known as RCB [3]. Furthermore, a previous study reported a strong relationship between a BANA-positive result and a high level of plaque spirochetes [16,17]. Accordingly, a BANA-based periodontitis diagnostic test kit was developed for use in humans [3,18]. However, especially in Korea, there are not many diagnostic test kits for assessing periodontitis in dogs using BANA yet.

MiriChek^TM Oral Bacter (O-Bacter; Mirimedix Incorporation, Chuncheon-si, Gwangwon-do, Korea) was developed as a rapid diagnostic BANA test kit, but formal clinical research have not yet been conducted.

In this clinical and in vivo study, we aimed to determine the usefulness of the O-Bacter as compared to the traditional clinical methods in the periodontal assessment of dogs with and without periodontitis.

We hypothesized that the O-Bacter test kit can detect the presence of BANA in plaque and help in diagnosing periodontitis in dogs, thereby achieving better prevention and treatment.

This study was conducted after explaining the diagnostic test kit method to the owners of 61 dogs visiting the veterinary clinic at Pet in zoo animal medical center, Uijeongbu-si, Gyeonggi Province and obtaining their consent. Clinical diagnosis of periodontitis was determined by two veterinarians based on probing depth, bleeding on gentle probing, and visual inspection. Briefly, healthy tooth and gums (stage 0), the gingival or gum tissue is a pink or coral color. The tissue is firm and resilient, and has minimal pocket depth. Gingivitis (stage 1), the gingival or gum tissue will be inflamed at the neck of the teeth, as opposed to the pinkish color, and there will be pocket depth and gingival bleeding on probing. And, was observed variable amounts of plaque and mildly reddened gums. Early periodontitis (stage 2), inflammation of periodontal ligaments and minor loss of attachment and pocket development. But, no tooth mobility at this stage. And, was observed larger amounts of plaque and bleeding gums. Moderate periodontitis (stage 3), moderate loss of attachment and/or moderate to deep pocket formation, and slight tooth mobility. Advanced periodontitis (stage 4), advanced breakdown of supporting periodontal tissue and severe pocket depth or significant gingival recession, and severe loss of tooth attachment. And, was observed dead tissue with blood and pus [19].

The experimental protocols used in this study were approved by the internal animal care and use ethics committee of the Seojeong university (IACUC Approval Number: SJ2021-03), Korea.

Dogs of all breeds are at a risk of developing periodontal disease, which is caused by infection and inflammation due to the toxic enzymes secreted from the bacteria in the plaque [7,20]. Periodontal disease is divided into the following four stages based on the severity of the clinical manifestations: stage 0 (normal), stage 1 (gingivitis), stage 2 (early periodontitis), stage 3 (moderate periodontitis), and stage 4 (advanced periodontitis) [21]. Most veterinarians still use clinical experience and probes to determine the periodontal disease stage in dogs with clinical manifestations. However, these clinical assessment methods may provide insufficient information for the prevention and treatment of periodontal disease in dogs.

Currently, research and development of periodontitis diagnostic test kits for human plaque has seen a growing interest in the use of BANA [3,18]. However, we have limited knowledge about its application in dogs. O-Bacter is a BANA-based periodontitis diagnostic test kit for dogs. The objective of this study was to evaluate the usefulness of O-Bacter as compared to the traditional clinical methods of periodontal disease assessment in dogs.

In this study, periodontitis was found to be more common in the older dogs than in the younger ones, but there were no clinical manifestations observed due to differences in weight <Table 1>. Periodontal disease is a common disease affecting adult dogs, and it has been reported in 80% of dogs aged 4 years, and the incidence increases with age [14]. In this study, periodontal disease showed an association with the dog breed, but not body weight; dog breeds with brachycephaly and overcrowding of teeth are especially vulnerable [22]. Unfortunately, our data comparisons between groups did not reflect age as a control variable, and could not confirm the breed-related differences. As such, the above limited of data requires future study. These results suggest that as age increases in dogs, dental care becomes more important.

The positive and negative sensitivity rates of the O-Bacter test kit were 73.3% and 80.7%, respectively, when compared with the clinically determined periodontitis stages <Table 4>. The sensitivity of the O-Bacter test kit was slightly lower than that of the in vivo BANA-Zyme^TM test kit for humans as reported by Dhalla et al.[3]. BANA has shown potential as a periodontal disease marker by assessing the probing depth and the proportion of spirochetes in plaque [16,17]. Moreover, a previous study showed that BANA-Zyme^TM was useful in detecting the RCB of periodontal pathogens [3]. However, the result for periodontitis stage 2 (sensitivity: 53.9% positive and 46.2% negative) was unclear. This low sensitivity in determining periodontitis stage 2 might be related to the low intensity of RCB; hence, there is a need for measures to compensate for the low sensitivity.

The O-Bacter test kit determines the results based on the color change of the pad test line. Our results showed that there was no color change in periodontitis clinical stage 0, the color faded in periodontitis clinical stage 1, the color changed to light blue in periodontitis clinical stage 2, the color changed to complete blue in periodontitis clinical stage 3, and the color of the first line changed to complete blue and that of the second line changed to light blue in periodontitis clinical stage 4 <Fig. 3>. The readability and ability to detect the degree of periodontitis of the O-Bacter were similar to those of BANA-Zyme^TM [3]. Furthermore, we quantified the change in the color intensity of the first line of the pad using Image J <Fig. 4>. Based on these results, we identified the potential for improving the method of diagnosing the periodontitis stage using assist devices that can measure the intensity of color change in the future. Overall, these results are useful for the diagnosis of periodontitis in dogs and may pave the way for further research for using the O-Bacter test kit in humans.

This study aimed to provide alternative methods for the diagnosis of periodontal diseases in dogs using the O-Bacter test kit.

1. The periodontitis was more common in the older dogs than in the younger ones.

2. The average positive and negative sensitivity rates of the O-Bacter test kit were 73.3% and 80.7%, respectively, with high readability.

3. We identified the potential for improvement in the stepwise diagnosis method for periodontitis based on the color intensity.

4. In conclusion, the O-Bacter test kit can assist in the diagnosis of periodontal disease in dogs.