The current study characterized the salivary proteome profile of dogs with and without dental calculus. In total, we identified 1662 proteins through the SEQUEST filter criteria applied to MS/MS spectra. Among these salivary proteins, there were 658 (39.6%) described for the first time in saliva of dogs. Besides, it is important enphazises that 225 specific proteins were identified in dogs without clinical signs of dental calculus and 300 specific proteins of dogs with dental calculus, which demonstrated that saliva could be a valuable medium to get biomarkers of the dental calculus formation on dogs.
The whole saliva collection was performed by mechanical stimulation with aid of a device formulated for such purpose. The device used was Micro•SAL™ saliva collection device (Oasis Diagnostics® Corporation - Vancouver, WA, USA). The saliva collection device has no cellulose in its composition  and allows immediate visual confirmation of the volume of saliva obtained; the plunger compresses the absorbent pad and the saliva is collected in the Eppendorf tube portion of the device. Foremost, differently from previous studies where the collection of saliva in dogs was performed under anesthesia with stimulation with acid [28, 29], we have opted for a non-invasive collection without any other stimulation mechanism or general anesthesia. However, there were difficulties regarding the sample collection. The sample volumes obrained varied significantly due to the dog’s defense movements, such as head shaking or trying to move away from the owner’s containment position. These reports were also described by dog’s owners in the study of Wenger-Riggenbach et al. . Moreover, some dogs refused to open their mouths and became aggressive, consequently resulting in them being excluded from the study.
The visual clinical examination of the 20 analyzed dogs identified that 8 (40%) did not present any signs of dental calculus, and 12 (60%) presented some degree of dental calculus. The mean dental calculus scores of dogs with dental calculus was 1.8, indicating a moderate calculus scoring in these dogs. Since our main goal is to study dental calculus, we selected the breeds Shih Tzu and Lhasa Apso that possesses brachycephalic skulls and tend to show malocclusion, dental crowding and rotation [31, 32], which facilitates accumulation of dental calculus. It is known that dental calculus is a predisposing factor to periodontal disease, which is more frequent in middle-aged (above 7 years old) and small size animals (below 10 kg) . In addition, the breeds studied here are similar in appearance and the genetic investigation presented a close relationship between them . As our study only sampled these genetically similar breeds, this may have influenced the number of proteins identified. Sousa-Pereira et al.  identified 249 proteins on a mixed breed group and Torres et al.  identified 2.491 proteins in healthy dogs among 19 breeds. Notwithstanding, there is no description of oral health on the animals evaluated in the study of Sousa-Pereira et al.  nor the proteins functions. In this present study were identified 1.662 proteins, of which 658 (39.6%) were unique. The biological functions of the most abundant proteins of each group identified were: transportation, immune response, structural, enzymatic regulation and metabolism, and some proteins perform still unknown functions. Besides that, in our study, there was no difference in total protein concentration between males and females. The same result was found in the study by Lucena et al. , in which there was no major differences between genders.
In the last years, several studies have been carried out on proteomic analysis in different mammals. Sousa-Pereira et al. , analyzed the salivary proteome of distinct species, including dogs, and identified, the alpha amylase (among other) was not found in the present study. There are, differences between that study and the present one that make a direct comparison of results difficult. In Sousa Pereira study , the samples were collected in mixed breed while in our study two specific breeds were selected. Sanguansermsri et al. compared the salivary proteome between dogs and humans, and observed that alpha amylase was present in dogs saliva, but in lower levels than in humans saliva; however, the presence of this protein in dogs in that study may be due the fact that in Thailand these animals are often fed with rice and starch . Most likely due to a diet adaption, the dogs used in this study do not have this protein. This lack of amylase in dog’s saliva was also observed by other studies [4, 36, 37].
To the best of our knowledge, there are no proteomic studies regarding dental calculus in the saliva of dogs, so this is a pioneering study. Among the unique proteins identified, the existence of serum albumin was noted on every dog’s sample due to, as described in humans, a small volume of gingival crevicular fluid that enters the oral cavity even in healthy subjects, free of gingivitis, resulting the measurement of levels of this component of serum in saliva . Other components of serum have also been identified, among these elements, are: Hemoglobin subunit beta, Apolipoprotein A-I, Hemoglobin subunit alpha and Haptoglobin.
Further, mucin and lysozyme C proteins were identified. Mucins are glycoproteins of high molecular weight with elongated structure that contribute significantly to saliva’s viscoelastic behavior . They also play an antibacterial function of modulating selectively the coherence of microorganisms to the oral tissue surface, which assist with the control of bacterial and fungal colonization . Lysozymes, on the other hand, are enzymes with hydrolytic activities. In other words, they promote cell lysis in bacteria , since its biochemically function hydrolyze beta-1,4 bond between N-acetyl glucosamine and N-acetyl muramic acid residues of bacterial peptidoglycan, which is an essential part of bacterial cell wall and also promotes structural stiffness and neutralize osmotic pressure of the cytoplasm . Therefore, the lysozyme is considered a natural antibiotic and an important part of the innate immune system [41,42,43]. In humans, C-type lysozyme is found in all biofluids, including saliva. The C-type lysozymes, as the ones identified, are the main lysozymes produced by vertebrates .
Concerning the analyses in the two different groups, the most abundant proteins in the group of dogs without dental calculus participated in metabolism functions, whereas in the group with dental calculus there were major proteins related to immune response, enzymatic regulation and uncharacterized proteins, potentially this may be due the presence of dental calculus near the gingiva, which has tendency of more pronounced inflammatory response .
Among the specific proteins of dogs without dental calculus, the presence of Sphingosine-1-Phosphate Receptor Protein 1 (S1P1) was observed. This protein is highly expressed in humans in endothelial cells, brain, heart and immune system cells [46, 47]; it is coupled to G protein and binds to sphingosine-1-phosphate, which is a bioactive sphingolipid that behaves as an intracellular messenger of some cytokines and also as an autocrine and paracrine extracellular mediator . In addition, sphingosine-1-phosphate stimulates events of intracellular signaling, such as activation of phospholipase C, increased cytoplasmic calcium concentration, regulation of adenylate cyclase, activation of the MAP kinase pathway and the Rho cascade . When bound, they participate in several cellular processes such as proliferation, differentiation, adhesion, motility, angiogenesis, apoptosis, migration, morphogenesis and changes in the cytoskeleton . S1P1 proteins were also described in memory T cells and a cell immunophenotyping revealed that humans secrete CD4(+) T cells in saliva . Besides, the Voltage-dependent T-type calcium channel subunit alpha protein encoded by CACNA1G gene was equally identified as a specific protein of dogs without dental calculus. Interestingly, a similar protein encoded by CACNA1I gene was only identified in the saliva of dogs with dental calculus. It is deemed that proteins T-type calcium channel play important roles in neuronal activity and have been described in studies with rats  and humans ; but there are no reports in the literature relating these proteins to the dogs’ saliva.
According to Zhang and collaborators  up to 40 proteins can be named as protein biomarkers for periodontal diseases in humans, these biological mediators are released from host defense cells due to the presence of periodontopathic bacteria in the oral enviromment. They include numerous cytokines, such as prostaglandin E2; tumor necrosis factor (TNF); interleukins IL-1 and IL-6, proteinases as matrixmetalloproteinases (MMPs); elastase-like enzymes; trypsin-like proteases; aminopeptidases and dipeptidylpeptidases, epidermal; platelet-derived and vascular growth factors, pyridino-line cross-linked carboxyterminal telopeptide, osteocalcin, among others. Still, there is a lack of proteomic researches regarding dogs with chronic periodontitis, which compromises comparisons within this species.
Howsoever, amidst the specific proteins identified in dogs with dental calculus in this study, some were found to function as immune response to the current peridodontitis. Between then it can be pointed out the presence of fragments of the Polymeric immunoglobulin receptor protein, which has been described in human saliva having the function of transporter of IgA, the main salivary antibody . Transforming growth factor beta (TGFB1) and complement C8 beta chain (C8B), were equally present in high abundance. TGFB1 was previously correlated to chronic periodontitis , its importance lies in the fact that this growth factor stimulate tissue remodeling and wound healing through increasing fibroblast proliferation, angiogenesis, and extracellular matrix production , and by inhibiting MMPs . Paradoxically, TGFB1 was also found to promote inflammation-associated tissue degradation by enhanced production of mediators that raise recruitment of eosinophils, lympocytes and monocytes, cells known to participate in chronic inflammation and tissue destruction . These facts could explain why TGFB1 is upregulated in cases of chronic periodontal pathologie.
Furthermore, another abundant protein detected by this research was the Calcium-sensing receptor (CaSR), whose function is calcium (Ca2+) transportation and regulation . In humans, CaSR is a G protein-coupled receptor that detects extracellular levels of Ca2+, which is expressed on plasma membranes of a broad variety of epithelial tissues including parathyroid, kidney, gastrointestinal tract and salivary glands [59, 60]. In salivary glands, it was showed that functional CaSR proteins can be stimulated by Ca2+ concentration, that is, CaSR can serve as a Ca2+ sensor in the luminal membrane of salivary gland ducts and regulate reabsorption of Ca2+ from the saliva via transient receptor potential canonical 3 (TRPC3), thus contributing to maintenance of salivary Ca2+ levels and representing a possible important protective mechanism against formation of salivary gland stones . Hence, as the CaSR stimulation in salivary glands can be related to an imbalance in the salivary Ca2+ concentration, it could as well contribute to the dental calculus accumulation that consist essentially of calcium phosphate. It is also important to note that in the kidney, the formation of stones is associated with changes in calcium reabsorption, causing hypercalcemia . Remarkably in the current investigation, CaSR was identified only in the saliva of dogs with dental calculus, which could suggest, together with its high abundance, an association with dental calculi presence. Further research is needed to better understand the relevance of CaSR protein in saliva and dental calculi formation, as it was demonstrated mainly in other human tissues .
An additional layer of information on possible pathways and processes involved in periodontal disease was granted by enrichment analysis. Overall, four of the five biological process that were encountered in dental calculus cases related to mitotic pathways that is the most common eukaryotic cell cycle. However, one critical finding merit being highlighted, which is the presence of the Rho GTPases signaling pathway. Rho GTPases can modulate the effects on human periodontal ligament cells of TNFB1, an important cytokine already known by its role in periodontal pathologies . Rho is a notable coordinator of the cytoskeleton , it was suggested in a previous study that the small Rho GTPase and its downstream effector Rho kinase (ROCK) regulate TGFB1-induced remodelling of mammary epithelial cell-to-cell contact . In accordance to the stated, Wang et al. showed that TGFB1 can induce proliferation and cytoskeletal rearrangement in periodontal ligament cells via Rho GTPase-dependent pathways . The high abundance of TGFB1 in dogs with dental calculus associated with the presence of Rho GTPase pathway is a relevant finding that may suggest, for futher investigations, TGFB1 as a biomarker candidate of periodontal disease in this species.
Future studies are needed for the evaluation of parameters such as pH values and determination of the buffer activity of each saliva sample collected. Quantification of electrolytes such as calcium and phosphate, which participate in the formation of the dental calculus and processes of demineralization and remineralization of dental enamel, should also be performed, not forgetting to mention sodium, potassium, zinc and magnesium, that are important in the metabolism of the salivary glands [65, 66]. The final task would be to combine the genomic, proteomic and other omic profiles together in an attempt to obtain a broader vision of how dental calculus accumulation impact dog salivary proteomic profile.