The oral microbiota of dogs in the present study was highly rich and diverse, consistent with previous studies [9,10,11,12, 24, 25]. However, it has previously been described that the Shannon diversity index was significantly larger for SUP and significantly smaller for SAL samples compared to all other niches . In contrast to earlier findings in dogs, however, in the present study, SAL and SUB had similar Shannon index values that were higher than that of SUP. The lower diversity index value observed in the previous study for the canine saliva population may be due to the fact that the saliva was stimulated before collection, thus diluting the salivary microbiota. In the present study, the saliva was not stimulated, and therefore the samples in the present study were not diluted. Similar to the present study, in humans, studies focusing on multiple oral habitats described diversity parameters to be highest for both SUP and SAL samples [16, 26,27,28].
Davis et al.  reported that SUB samples from dogs of varying oral health statuses (72 with healthy gingiva; 77 with gingivitis; 74 with mild periodontitis) were colonized largely by Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Fusobacteria, and Spirochaetes regardless of disease stage. Additionally, in the healthy cohort, Proteobacteria and Bacteroidetes were the most abundant phyla; Porphyromonas, Moraxella, and Bergeyella were the most abundant genera in all dogs, and particularly higher in healthy animals . In another study evaluating a composite oral sample of healthy dogs (n = 6), whereby samples were collected by brushing the gums, tongue, teeth, and cheeks, the phyla Bacteroidetes, Proteobacteria, and Firmicutes predominated; the most commonly identified genera were Porphyromonas, Fusobacterium, Capnocytophaga, Derxia, and Moraxella . Additionally, in a healthy cohort (14 dogs), SUP samples were collected using plastic microbiological loops, buccal and tongue dorsum mucosa were collected using a CytoSoft cytology brush, and stimulated whole mouth saliva was collected using cotton wool swabs. In that study, Proteobacteria, Firmicutes, and Bacteroidetes were the most abundant phyla across all niches, although the ranking of these varied among niche . Similarly, in the present study, Bacteroidetes, Proteobacteria, and Firmicutes were the predominant phyla in the SUB and SAL samples. Porphyromonas was the most abundant genus, followed by Fusobacterium, Treponema, Enhydrobacter, and Moraxella. However, Capnocytophaga and Bergeyella had a low abundance. In SUP samples, Proteobacteria, Bacteroidetes, and Firmicutes were the predominant phyla, with Porphyromonas, Moraxella, Enhydrobacter, Fusobacterium, Corynebacterium, and Actinomyces being the predominant genera. The high variability of the oral microbiota among studies may be due to differences between animals, facilities (water, food, products used for cleaning), dental prophylaxis, type of swabs, extraction protocols, and/or the amplified 16S rRNA gene hypervariable region used in the microbiota analysis. Differences may also be due to interactions between the saliva, nutrient sources, host cell type, immunological factors, and exogenous factors such as oxygen availability and oral intake [9, 16].
In the present study, the microbiota populations were quite different among oral habitats (SAL, SUB, and SUP). The variation between SUP and SAL is consistent with data from a previous canine study whereby microbiota of SUP and oral swabs were shown to be distinct . These data are also similar to data from a human study that demonstrated that the buccal mucosa, gingivae, and hard palate microbiota populations were similar to one another and different than the populations present in saliva, tongue, tonsils and throat, and SUP and SUB that were similar to one another . The distinct microbiota communities of these microenvironments within the oral cavity are likely due to the differences in oxygen tension, pH, and mucosal surface characteristics [29, 30].
In a previous study of 30 healthy adult Beagle dogs, SUP plaque and swabs from gums, tongue, and cheeks were sampled, reporting that Firmicutes and Spirochaetes were predominant in the plaque environment, and Proteobacteria and Firmicutes were predominant in the oral swabs . Similar data were reported in the present study, with the microbiota of SAL having lower Firmicutes than the tooth plaque sites. In contrast to the previous dog study  where the relative abundance of Actinobacteria was higher in SUP compared to an oral swab, the relative abundance of Actinobacteria was higher in SAL compared to tooth plaque site samples (SUB and SUP) in the present study. In a previous dog study, Treponema relative abundance was greater in SUP samples than oral swab samples [composite oral swabs - flocked nylon-tipped BD Liquid Amies Elution swabs (Becton, Dickinson and Company, USA) - collected by swabbing the gums, tongue, and cheeks for 10–15 s], Actinomyces and Pasteurella relative abundances were greater in swab samples than SUP samples, and there was no difference in Porphyromonas relative abundance among habitats . Contrary, in the present study, relative abundance of Actinomyces was higher in the SUP than SAL samples, relative abundance of Treponema and Porphyromonas were lower in the SUP than SAL samples, and relative abundance of Pasteurella was similar between SUP and SAL samples.
Furthermore, in a dog study whereby a checkerboard DNA–DNA hybridization of human probes was used, five intra-oral habitats (SUB, SUP, the tongue, tonsils and cheek mucosa) were evaluated in seven Beagle dogs . In that study, the prevalence of 26 species were different between SUP and SUB plaque samples, with 20 of them being higher in SUB plaque . SUP plaque contained higher proportions of P. gingivalis, F. periodonticum, F. nucleatum ss. vincentii, A. actinomycetemcomitans, Prevotella acnes and A. naeslundii genospecies 2-like species . In the present study, SUP contained higher proportions of Actinomyces, Corynebacterium, Leucobacter, Capnocytophaga, Bergeyella, Oscillospira, p-75-a5, Lautropia, Lampropedia, Desulfobulbus, Arcobacter, Pasteurella, Enhydrobacter, and Moraxella. In the previous dog study, the microbial profiles of the soft habitats (i.e., cheek and tongue mucosa, tonsils) and tooth plaque sites were markedly different, with 19 of 40 species differing among sample locations . P. gingivalis, T. denticola, Tannerella forsythia, S. constellatus, C. rectus and C. showae-like species were present in higher proportions on tooth plaque habitats . In the present study, the relative abundances of 7 genera were different among saliva and tooth plaque habitats. Tannerella, Peptostreptococcus, Schwartzia, and Neisseria were present in higher proportions on tooth plaque sites.
Similar to the previous study, Tannerella was present in a higher proportion on tooth plaque sites. One of the reasons could be that Tannerella are capable of producing proteolytic enzymes that can degrade host periodontal tissues and compromise the host immune system. Tannerella also possesses a surface-associated putative adhesin that serves as ligands to other bacteria (Fusobacterium), which provide this bacterial group with the ability to facilitate the development of complex communities and plaque formation [31,32,33,34,35,36,37,38,39,40]. Peptostreptococcus are capable of inducing a potent inflammatory reaction in macrophages, producing proteases that permit it to penetrate to the basement membrane, and creating a carbohydrate-mediated coaggregation with Fusobacterium and Porphyromonas [41,42,43], which also enable this bacterial genera to facilitate plaque development. In cats, Schwartzia was reported to be associated with gingivitis . In humans, periodontal patients not only had higher relative abundances of periopathogens, but also of other taxa (Anaeroglobus, Bulleidia, Desulfobulbus, Filifactor, Mogibacterium, Phocaeicola, Schwartzia, or TM7) whose role in oral health are not well-established but may be targeted in future research . In dogs, the primary colonizers of the tooth surface appear to be Neisseria and Moraxella [10, 46, 47]. Therefore, it was expected that a higher proportion of these bacterial groups would be measured in plaque habitats.
In a human study, it was suggested that various oral habitats (buccal mucosa, keratinized gingiva, hard palate, throat, palatine tonsils, tongue dorsum, SAL, SUP, SUB) could be characterized and then easily sampled sites (e.g., SAL, tongue) could be used as surrogate markers for the others . Similarly in animals, SAL and SUP samples are relatively easy to collect and do not require sedation for the majority of the animals. The data from the present study and that of a previous study , however, shows that the use of the oral salivary swabs to assess the oral plaque microbiota is not recommended because their communities are distinct from those of the plaque populations and would most likely be misleading. In a previous study, higher relative abundance of Treponema and Clostridiales in plaque, and higher relative abundance of Psychrobacter, Mannheimia, and Pasteurella in swab samples (gums, tongue, and cheeks), demonstrated that plaque microbiota harbor greater populations of anaerobic and biofilm-associated taxa . Similarly in the present study, Paludibacter, Filifactor, Peptostreptococcus, Fusibacter, Anaerovorax, Fusobacterium, Leptotrichia, Desulfomicrobium, and TG5 (anaerobic bacteria) were enriched in SUB samples. Actinomyces, Corynebacterium, Leucobacter, Euzebya, Capnocytophaga, Bergeyella, Lautropia, Lampropedia, Desulfobulbus, Enhydrobacter, and Moraxella (aerobic and anaerobic bacteria) were enriched in SUP samples. Prevotella, SHD-231, Helcococcus, Treponema, and Acholeplasma (aerobic and anaerobic bacteria) were enriched in SAL samples.
Identifying the relationships between oral microbiota and periodontal disease is extremely important to understand the disease process and how to prevent or treat it. Past studies have focused on Porphyromonas, as it is well known to be one of the most important bacteria for the development and progression of periodontal disease in humans [48,49,50,51]. In past studies with dogs, Porphyromonas was the most abundant genus, being particularly higher in healthy dogs [9, 25]. In the present study, Porphyromonas was highly prevalent, providing strong evidence that they are part of the commensal oral microbiome. The data from the current and past studies suggest that instead of having a complete absence of pathogenic organisms in the normal microbiota, disease occurs when there is an imbalance [52, 53]. Nevertheless, other groups of bacteria seem to be key components of periodontal disease in dogs, including Peptostreptococcus, Actinomyces, and Peptostreptococcaceae that have been shown to be the most predominant taxa in dogs with mild periodontitis, with Corynebacterium canis being more abundant in dogs with mild periodontitis and gingivitis, and Leptotrichia sp., Neisseria canis, and an uncultured Capnocytophaga sp. being associated with gingivitis .
In the present study we correlated bacterial genera and oral scores, and in SUB samples, Actinomyces, Corynebacterium, and Leptotrichia were strongly and positively correlated with higher pocket score, gingivitis score, and OHS. We also observed a strong positive correlation between Capnocytophaga and pocket and gingivitis scores. In SUP samples, a strong positive correlation between Actinomyces and pocket and gingivitis scores, and strong positive correlations between Corynebacterium, Leptotrichia, and Neisseria and pocket score were observed. Actinomyces belong to the group of bacteria that can overcome the immune barrier, pass through endothelial gaps and pores, penetrate the bloodstream. Therefore, it plays a significant role in gingivitis and the progression of periodontal diseases because they are able to cause inflammation, periapical lesion, and induce soft and hard tissue destruction [54,55,56,57,58,59]. Capnocytophaga spp. possess a trypsin-like enzyme and are considered to be periodontopathic . Leptotrichia species typically colonize the oral cavity and have been reported to participate in oral disease in humans (gingivitis, necrotizing ulcerative gingivitis, adult/juvenile periodontitis, ‘‘refractory’’ periodontitis). L. buccalis is highly saccharolytic and produces lactic acid, a property that may implicate participation in tooth damage [61,62,63,64,65,66]. Additionally, non-plaque induced gingival lesions can result from specific bacterial pathogens such as Neisseria gonorrhea . Therefore the correlation of those bacterial genera with higher gingivitis and pocket scores was expected. Even though ease of access and the lack of anesthesia would reduce the cost and complication of saliva collection, bacteria related to the development of periodontal disease and gingivitis are present in greater concentrations in oral plaque. Therefore, plaque collection is suggested. Unreliable data coming from SAL samples would likely lead to inaccurate diagnosis and monitoring of oral health, potentially delaying proper care and tooth cleaning that would worsen periodontal disease.
Limitations of the present study are that the (1) animals with severe oral disease were not included; this information would help understand differences in the microbiota community due to periodontal disease and (2) samples were only taken from one point of collection, with longitudinal samples over time providing a better description of the changes in oral microbiota during the development of oral disease.