1. Introduction
The oral microbiota is a complex ecosystem in both humans and dogs [
1,
2]. Oral-associated bacteria have been implicated in diseases of inflammation and senescence, including cardiovascular disease, diabetes mellitus, cancers, and Alzheimer’s disease (AD) [
3,
4,
5,
6,
7]. Companion dogs make an excellent model system for healthy and unhealthy human aging as they share the human environment, have diseases in common with humans, and yet, age at an accelerated rate [
8]. Defining the oral microbiota of senior and geriatric companion dogs is critical to our understanding of healthy canine aging and senescence, which, in turn, can be applied to a greater understanding of human aging.
The field of canine microbiome research is in its early stages but it is advancing rapidly. A number of studies have focused on the gastrointestinal bacterial microbiome, with fewer investigating the oral and nasal environments. Early studies of oral bacteria were limited by their reliance on culture-based methods. In contrast, recent studies have taken advantage of next-generation sequencing, primarily 16S rRNA amplicon sequencing analysis, to reveal a much greater diversity of organisms in the canine oral cavity. These studies have primarily focused on populations of young to middle-aged dogs and, in many cases, have been limited to purpose-bred, working, or kenneled dogs of single or similar breeds [
9,
10,
11,
12]. One recent study reported a diverse fungal population in the canine mouth using internal transcribed spacer analysis [
13]. Still, research into the canine oral mycobiome is even less advanced than the bacterial microbiome.
In humans, the progression of periodontal disease correlates with AD progression [
14]. Chronic inflammation has been suggested as an indirect cause of this relationship [
15], whereas the keystone periodontal pathogen
Porphyromonas gingivalis has been suggested as a direct cause, mediated by virulence factors called gingipains [
16].
Canine cognitive dysfunction syndrome (CCDS) bears remarkable similarity at both the cellular and behavioral levels to AD. Dogs with CCDS develop pathognomonic central nervous system features of AD, including cerebral atrophy, ventricular widening, deposition of plaques of misfolded Aβ in the prefrontal cortex and cerebral vasculature as well as neuronal loss affecting the cerebral cortex, hippocampus, and limbic system [
17]. CCDS prevalence has been estimated as high as 67% for dogs aged 16–17 years based on owner-reported symptoms in the domains of disorientation, changes in social interactions, house training and the sleep/wake cycle [
18]. Further, findings in our laboratory show a significant decline in performance on laboratory tests of cognition in geriatric companion dogs [
19]. This decline is accompanied by escalating serum neurofilament light chain concentrations, and elevations followed by reductions in serum concentrations of amyloid beta 42, mirroring changes seen in patients with Alzheimer’s Disease [
18]. Based on these studies of histopathology, MRI findings, blood biomarker alterations, behavioral changes and reduced performance on cognitive tests, companion dogs can serve as an excellent real-world model for AD.
The primary objective of this exploratory study was to define the oral microbiota of senior dogs using whole genome sequencing to enhance species-level accuracy over previous studies relying on 16S rRNA amplicon sequencing. A secondary study aim was to evaluate the correlations between the oral microbiome and naturally occurring age-related disease processes, including CCDS.
4. Discussion
To our knowledge this study represents the first use of WGS sequencing to define the oral microbiota of aging companion dogs, and the first to investigate the oral microbiota in this population longitudinally. We identified a bacterial community consistent with oral dysbiosis in the majority of our subjects over the span of the study. Two closely-related organisms,
P. gulae and
P. gingivalis, were predominant. These bacteria are considered keystone species in periodontal disease in dogs [
43] and humans [
14], respectively. Moreover,
P. gingivalis has been implicated in AD in humans, either indirectly through chronic inflammation or directly through virulence factors including small proteases called gingipains [
44].
P. gulae produces gingipains and other virulence factors similar to
P. gingivalis [
50]. A recent study showed a statistically significant correlation between periodontal disease and cognitive decline in dogs [
51]. Additionally, in a small study of aged beagles,
P. gulae DNA and gingipains antigen was isolated from brain tissue, though cognition was not measured, and a gingipains inhibitor reduced clinical signs associated with periodontal disease in that study [
52].
Longitudinal analysis of individual taxa allows changes from baseline to be interrogated with respect to age and cognitive state regardless of initial state. This aspect is essential given the potentially vast variation in canine oral microbiome at later stages of life. We identified one organism with a statistically significant positive correlation with CADES score,
Leptotrichia sp. oral taxon 212, and one species with a statistically significant positive correlation with FoL,
Lactobacillus gasseri.
Leptotrichia species are facultative anaerobic Gram-negative bacilli, and are common inhabitants of the human oral microbiota [
3] that have been associated with both oral health and disease states in humans [
53] and in gingivitis in dogs [
11].
Leptotrichia species have been associated with AD [
54,
55] and periodontal disease [
53]. In humans.
Leptotrichia wadei, in particular, has been observed to be significantly enriched in subjects with mild cognitive impairment [
54]. However, species of the genus
Leptotrichia have been associated with both periodontal health and disease suggesting that individual
Leptotrichia species have “distinct pathogenic potential” [
53]. The relationship between CCDS and
Leptotrichia,
P. gulae and
P. gingivalis merits further investigation.
Several Lactobacillus species,
Lactobacillus gasseri in particular, have been previously identified in dog faeces [
56,
57] and human breast milk derived
Lactobacillus gasseri has been studied as a probiotic treatment for obesity in dogs [
58]. The study of association of
Lactobacillus gasseri with aging or cognition outside of model organisms [
59], however, remains largely limited to the study of probiotic supplementation [
60].
The biological significance of the fungal species identified in this study is unclear, but it is interesting that 12 species were found in all 26 samples, including
A. oryzaeh, the most abundant species present, and the opportunistic pathogen,
C. dubliniensis. This is in contrast to published findings of the canine oral mycobiome in a younger study population where none of the observed species occurred in all samples [
13]. We report a statistically significant increase in both bacterial and fungal alpha diversity over the course of the study. In humans, oral alpha diversity has been shown to decrease with age [
61]. Our results may represent a difference between the oral ecosystem of aged humans and senior companion dogs, differences in behaviors between humans and dogs, the onset of age-related behaviors (such as an increase or decrease in self-grooming) or study-related factors such as sample size or duration of this study.
This study was an exploratory evaluation of the oral microbiota and the small cohort size limits the conclusions that can be drawn. The fact that only one dog progressed into the MdSv CADES group during the course of the study limited the study’s power to examine the relationship between cognitive change and changes in the oral microbiota. With a larger sample size and a longer timeline, we anticipate a larger number of dogs progressing through CADES groups. This would allow examination of differences in the oral microbiome pre-CCDS and after the diagnosis of CCDS within individuals, potentially elucidating a role for the oral microbiome in CCDS development or progression. Moreover, cognitive aging occurs at different rates in different dogs and comparison of the oral microbiota between successful agers, those that develop mild impairment and those that develop severe impairment could also be evaluated [
62].
Because these data were generated as part of a broader study of neuro-aging intended to investigate natural aging in companion dogs, subjects with antibiotic use and dental prophylaxis were not excluded. As our study size and duration continue to increase, so too will the power, potentially enabling further understanding of the relationship between dental care, medications, oral microbiome, and cognition. At present, we found no correlation between recent dental prophylaxis and either alpha diversity, relative abundance of GPPs, or relative abundance of red complex bacteria. However, it is important to note that comprehensive oral examinations under anesthesia were not performed in these dogs. In this longitudinal study of aging dogs’ health, the central tenet is to cause no harm to the dogs during data collection. As such, anesthesia and sedation are avoided at the routine 6-monthly assessments. The use of the calculus, gingival and plaque indices allowed the description of some aspects of periodontal disease but should not be considered a complete evaluation of dental and oral health.
The high prevalence of cognitive decline in aging dogs coupled with the abundance of
P. gulae and
P. gingivalis in the aging cohort in the present study, leads us to postulate that these species may play a role in CCDS. While it is well-established that periodontal disease is very common in aging dogs [
63], the extent to which oral dysbiosis contributes to senescence in dogs has not been fully elucidated. Defining causative organisms in CCDS and elucidating the mechanisms involved will provide potential preventative and therapeutic targets for CCDS.
A limitation and advantage of this study is that the intraoral sites sampled (supragingival and buccal mucosa) is a combination of two distinct niches. This limits our ability to compare findings to prior studies. However, we chose this method for its ability to represent a greater portion of the oral cavity than single-site sampling and thus is likely more representative of the overall health state of the mouth. Moreover, establishing the aging oral microbiome using a method that does not require general anesthesia provides a baseline for future studies of the aging canine oral microbiome that may wish to avoid the risk or expense of anesthesia. Understanding this site is also valuable considering the availability of commercially-available tests of the oral microbiome which rely on owner-administered oral swabs for sample collection.