Abstract
Objective—To evaluate the effectiveness of a topically applied gel containing essential oils (menthol and thymol) and polyphenolic antioxidants (phloretin and ferulic acid) for reducing halitosis in dogs.
Animals—20 dogs.
Procedures—A blinded crossover clinical trial was conducted. Dogs received a dental cleaning and examination (periodontal examination including periodontal probing and assessments of plaque, calculus, and gingivitis). Owners then applied a gel (active or placebo) to oral soft tissues twice daily for a 4-week period. Teeth of the dogs were cleaned again, and owners applied the other gel for a 4-week period. Clinicians scored halitosis immediately after the initial cleaning and at 4 and 8 weeks, and owners scored halitosis weekly.
Results—Halitosis assessment by clinicians revealed that both groups had improvement in halitosis scores. Two dogs were removed because of owner noncompliance. In the active-to-placebo group (n = 9), halitosis was significantly reduced during application of the active gel but increased during application of the placebo. Seven of 9 owners reported increased halitosis when treatment was changed from the active gel to the placebo. In the placebo-to-active group (n = 9), halitosis decreased during application of the placebo and continued to decrease during application of the active gel. Seven of 9 owners reported a decrease in halitosis with the active gel.
Conclusions and Clinical Relevance—An oral topically applied gel with essential oils and polyphenolic antioxidants applied daily after an initial professional dental cleaning decreased oral malodor in dogs.
Veterinarians recommend basic oral care for their patients, but many owners do not routinely clean or care for the teeth of their pets. Typically, owners only attend to the oral care of their pets because of severe halitosis or other health concerns. Professional dental treatment for oral disease in dogs includes cleaning the teeth to remove plaque and calculus as well as scaling and root planing when necessary. General anesthesia is required for the delivery of professional dental treatment in dogs. Owners often have an unrealistic perception of the risk associated with anesthesia and also may be reluctant to proceed with treatment because of the expense.
Periodontal disease and halitosis are common in dogs. Approximately 40% of dogs 1 to 4 years of age and 89.4% of dogs 12 to 13 years of age have periodontal disease.1 There is an association between bacterial accumulation, malodor, and periodontitis.2
Periodontitis and malodor are the result of a 2-stage process beginning with the accumulation of microbial biofilms and ending with an inflammatory reaction. The VSCs from the breakdown of gram-negative anaerobic organisms, food, protein, and oral cells are considered a cause for halitosis in dogs. The primary VSCs are considered to be methyl mercaptan and hydrogen sulfide and are the result of proteolytic breakdown of peptides and amino acids in the oral cavity.3
Saliva contains natural antioxidants that neutralize free radicals resulting from bacterial-induced inflammation. The use of antioxidants to mitigate inflammation from oral disease in animals is an emerging area of research. Dogs with advanced periodontal disease have lower amounts of antioxidants in the gingival crevicular fluid than do dogs with mild cases of periodontal disease. Total antioxidant capacity in gingival crevicular fluid is related to the degree of severity of periodontal disease. The mechanism in dogs is postulated to be the same as in humans; namely, ROS are released by activated phagocytes and fibroblasts in the inflamed periodontal tissues. The inflammatory response to infection, including the influx of phagocytes, PMNs, and chemical compounds, increases the amount of ROS and decreases the amount of antioxidants, which are depleted as they neutralize the ROS.4
Medicaments that have an effect on both the bacteria and depleted antioxidants may have a role in reducing periodontal inflammation and decreasing malodor. The objective of the study reported here was to evaluate the effectiveness of a topically applied gel containing antibacterials (menthol and thymol) and specific polyphenols (phloretin and ferulic acid) for reducing halitosis in client-owned dogs.
Materials and Methods
Animals—Twenty client-owned dogs were enrolled in a blinded crossover clinical trial conducted to measure the effect of topically applied essential oils and polyphenols for controlling halitosis. The study participants were adult dogs that ranged from 3 to 15 years of age. Body weight ranged from 2.6 to 48.5 kg (mean, 13.6 kg; median, 11.4 kg). Written informed consent was obtained from each owner before participation of dogs in the trial. The study was performed in compliance with guidelines established by the US Public Health Service Policy on Humane Care and Use of Laboratory Animals.
Procedures—The study was conducted at a veterinary clinic owned by one of the authors (RMP). Dogs were anesthetized and intubated, and full-mouth dental radiography, periodontal examination, and oral examination were performed, which was followed by dental cleaning (scaling and polishing). The periodontal examination included periodontal probing, scoring of plaque and calculus (scored in accordance with the modified Logan and Boyce index whereby anatomic landmarks are used for horizontal division; scale, 0 to 3), and gingivitis scoring (scored in accordance with the Loe gingivitis index; scale, 0 to 3). Dogs with periodontitis (probing depth > 3 mm; attachment loss > 1 mm measured from the cementoenamel junction to base of the sulcus; and alveolar bone loss measured on radiographs) were excluded.
A blinded crossover clinical trial was conducted. Each owner was provided gels to apply to the oral cavity of the dog at home. Owners were assigned by a randomization procedure via a coin toss to initially apply an active gela or a placebo. The active gel contained the antioxidants phloretin and ferulic acid and the antibacterials menthol, thymol, and chlorophyllin. The placebo consisted of a gel without the antioxidants and natural antibacterial agents. The active and placebo gels were identical in color, odor, and consistency. After the initial treatment was applied for 4 weeks, the dogs were anesthetized and their teeth were again cleaned and polished. The owners then applied the other gel for the subsequent 4-week period.
Owners were instructed to place a strip of gel (approx 0.75 mm in width and 12 mm in length) on their index finger and to apply the gel to the buccal surfaces of the attached gingiva of the maxillary and mandibular arches on 1 side of the mouth. The gel was to be applied from the incisor gingiva to as far back on the molar gingiva as possible. The owners were then to repeat this process for the other side of the mouth (ie, 12 mm of gel/side). Gel was applied twice daily for 4 weeks.
During the study, dogs were fed a dry Association of American Feed Control Officials–approved diet. All dogs were not fed identical diets, but each dog was fed the same diet throughout the study. Owners were not allowed to perform other homecare for the dog, such as brushing of teeth, rinsing of the oral cavity, or provision of treats, that could alter results. The owners were asked to record gel application daily and to assess and record the degree of halitosis weekly. Owners submitted all data at the end of each 4-week test period.
Assessment of halitosis—Prior to the start of the study, the clinicians performed calibration exercises to determine the degree of detectable odor and score assignment among the clinicians. These exercises were repeated with nonsubject dogs over a period of 1 week to ensure consistency in assessments among the clinicians.
Halitosis was scored on a scale of 0 to 10, with 0 being nonexistent and 10 being putrid and foul. Halitosis was scored by 3 clinicians immediately after the initial teeth cleaning (time 0) and at weeks 4 and 8 (ie, after each 4-week period of gel application). A halitosis score for each dog was obtained in the morning. All 3 clinicians scored each dog at the same time. Clinicians smelled the premolar and molar areas and separately recorded the halitosis score. The mean score was calculated for each dog.
Owners scored halitosis of their dog on a weekly basis. Owners used the same scale as that used by the clinicians. Owners did not perform calibration exercises. Owners received instructions on determining the halitosis score, which included a detailed explanation of the visual analogue scale and malodor categories.
Statistical analysis—Statistical analysis was conducted with the aid of statistical software.b A univariate ANOVA was used to test the effects of group (placebo to active vs active to placebo), clinician (3 clinicians), time of treatment (start of the study, 4 weeks, and 8 weeks), and their interactions on halitosis. The Shapiro-Wilk test was used to test for normality of the data. A Levene test was used to confirm equality of variances between the groups.
Results
Of the 20 dogs enrolled, 18 completed the study, with all data points collected over the 8 weeks of the study. Two dogs (both assigned to receive the active gel first followed by the placebo gel) were withdrawn from the study because of owner noncompliance. No adverse events were reported by the owners, and there was no clinical evidence of adverse effects detected by the clinicians.
Initial periodontal assessment—The mean gingivitis index score was 0.38 (526/540 [97%] sites had a score of 0 or 1), which indicated a population with mild inflammation. The plaque and calculus indices were 2.74 and 0.84, respectively.
Owner assessment of halitosis—Owner halitosis assessments of the placebo-to-active group and active-to-placebo group were summarized (Table 1). When treatment changed from the placebo gel to the active gel, improvement (percentage decrease in halitosis) was detected (range, 0% to 100%) during the second 4-week period. Seven of 9 dogs within the placebo-to-active group had improvement in halitosis, whereas only 2 dogs had no improvement (0% change) in halitosis for the owner assessment. In the active-to-placebo group, treatment was changed from the active gel to the placebo gel. The percentage increase in halitosis ranged from 0% to 60% during the second 4-week period. Seven of 9 dogs had an increase in halitosis for the owner assessment, whereas there was no effect in 2 dogs (0% change in halitosis).
Percentage change in halitosis assessed by owners of 18 dogs receiving an active gel containing the antioxidants phloretin and ferulic acid and the antibacterials menthol, thymol, and chlorophyllin and a placebo gel applied topically to oral soft tissues twice daily.
| Variable | Mean ± SD | Median (range) | Interquartile range |
|---|---|---|---|
| Placebo-to-active group (n = 9) | −47 ± 47 | −36 (0 to −100) | 5.5 to −100 |
| Active-to-placebo group (n = 9) | 26 ± 21 | 25 (0 to 60) | 5.5 to 45 |
In accordance with a crossover design, the teeth of each dog were cleaned, and dogs received a treatment for 4 weeks; the teeth were again cleaned and dogs then received the other treatment for the subsequent 4 weeks. Halitosis was scored immediately after the initial dental cleaning (time 0) and at the end of each 4-week treatment period. Halitosis was scored on a scale of 0 to 10, with 0 being nonexistent and 10 being putrid and foul. Negative numbers indicate a decrease in halitosis during the second 4-week treatment period, whereas positive numbers indicate an increase in halitosis during the second 4-week treatment period.
Clinician assessment of halitosis—Halitosis scores for the 3 clinicians were summarized (Table 2). Mean ± SD halitosis scores at time 0 did not differ significantly (P = 0.651) between the placebo-to-active group (3.11 ± 1.53) and active-to-placebo group (3.33 ± 2.13). At week 4, mean halitosis scores differed significantly (P = 0.017) between the placebo-to-active group (2.22 ± 1.22) and active-to-placebo group (1.44 ± 1.09). At week 8, mean halitosis scores did not differ significantly (P = 0.334) between the placebo-to-active group (1.93 ± 1.27) and active-to-placebo group (2.31 ± 1.57).
Mean ± SD halitosis score as assessed by 3 clinicians for dogs receiving an active gel and a placebo gel applied to the oral soft tissues.
| Placebo-to-active group | Active-to-placebo group | |||
|---|---|---|---|---|
| Time | No. of dogs | Mean ± SD | No. of dogs | Mean ± SD |
| 0 | 9 | 3.11 ± 1.53 | 11* | 3.33 ± 2.13 |
| Week 4 | 9 | 2.22 ± 1.22a | 9 | 1.44 ± 1.09b |
| Week 8 | 9 | 1.93 ± 1.27 | 9† | 2.31 ± 1.57 |
Two dogs were removed from the study because of issues with owner noncompliance. †Represents results of all 3 clinicians for 8 dogs but only 2 clinicians for 1 dog because one of the clinicians could not be present at the time of the evaluation.
Values with different superscript letters differ significantly (P = 0.017). See Table 1 for remainder of key.
Results of a univariate ANOVA revealed that there was not a significant effect of group (P = 0.833) or clinician (P = 0.123) or the group-by-clinician interaction (P = 0.347), group-by-time interaction (P = 0.107), clinician-by-time interaction (P = 0.987), or group-by-clinician-by-time interaction (P = 0.944). However, there was a significant (P ≤ 0.001) effect of time.
Mean scores of all dogs for all 3 clinicians were plotted on a graph (Figure 1). For the placebo-to-active group, the halitosis score at time 0 was not significantly (P = 0.052) different from the score at week 4 but was significantly (P = 0.005) higher than the score at week 8. For the active-to-placebo group, the halitosis score at time 0 was significantly (P < 0.001) higher than the score at week 4 but not significantly (P = 0.068) different from the score at week 8. Therefore, the amount of improvement was greater during the 4-week period of use of the active gel for both groups.
Mean ± SD halitosis score as assessed by 3 clinicians for 2 groups of dogs receiving an active gel containing the antioxidants phloretin and ferulic acid and the antibacterials menthol, thymol, and chlorophyllin and a placebo gel applied topically to oral soft tissues twice daily. In accordance with a crossover design, the teeth of each dog were cleaned, and dogs received a treatment for 4 weeks; the teeth were again cleaned, and dogs then received the other treatment for the subsequent 4 weeks. Halitosis was scored immediately after the initial dental cleaning (time 0) and at the end of each 4-week treatment period. Halitosis was scored on a scale of 0 to 10, with 0 being nonexistent and 10 being putrid and foul. Notice that halitosis score decreased during the initial 4-week period for dogs receiving either treatment, but halitosis score decreased during the second 4-week period when the placebo gel was applied first and was followed by the active gel (light gray bars), whereas halitosis score increased during the second 4-week period when the active gel was applied first and was followed by the placebo gel (dark gray bars). Values reported represent results for 9 dogs in the placebo-to-active group at all times and for 11 dogs at time 0 and 9 dogs at 4 and 8 weeks in the active-to-placebo group because 2 dogs were removed from that group owing to owner noncompliance.*, †Within a group, value differs significantly (*P < 0.001; †P = 0.005) from the value at time 0.
Citation: American Journal of Veterinary Research 75, 7; 10.2460/ajvr.75.7.653
Discussion
Halitosis is associated with periodontitis and a microbial cause. The organisms identified are primarily gram-negative anaerobes of the genera Porphyromonas, Bacteroides, and Prevotella.5 The malodor hypothesis centers around metabolic activity of the organisms on proteins that are food substrates or generated from endogenous cellular sources within an animal. The resultant malodor is in the form of VSCs such as hydrogen sulfide and methyl mercaptan.6 In turn, VSCs can induce additional inflammation.
Multiple methods for antimicrobial delivery, including dental hygiene chews, medications including antimicrobials such as clindamycin, and compounds such as chlorhexidine, cetylpyridinium chloride, and essential oils, have been used in studies of dogs.7,8 Similar to results of studies in humans, professional dental cleaning to decrease biofilms and remove calculus has also been found to decrease halitosis in dogs.9
The challenge is to maintain an acceptable amount of malodor for animals that do not have the ability to practice oral hygiene and that consume diets that could include a wide range of substances. Commercial antimicrobial products can be alcohol-based antiseptic mouth rinses that kill gram-negative microorganisms and hence reduce oral odor.8 However, these can have harsh overall effects. Although chlorhexidine appears to be clinically effective, it is not an agent that should be used routinely or for long periods, especially considering that it can stain teeth. Mouth rinses that contain essential oils are considered safer and are devoid of the long-term adverse effects of mouth rinses that contain chlorhexidine.10
When periodontal inflammation is initiated by a biofilm, ROS are increased. These oxygen-derived free radicals become toxic and are a major contributor to tissue destruction in the epithelial cells lining the periodontal pocket and to the release of VSCs. The more periodontal inflammation in a dog, the less total antioxidant capacity in the gingival crevicular fluid, and vice versa. It has been suggested4 that local delivery of antioxidants could help resolve periodontitis in dogs. Medicaments that have only an antibacterial effect may play a role in the destruction of bacteria but have no impact on decreasing the inflammatory reaction and cell death that also contribute to the release of VSCs.
An emerging treatment for periodontitis and other oral disorders in humans is topically administered polyphenols, with delivery systems such as oral gels, mouth rinses, and toothpastes. Considerable evidence supports the efficacy of polyphenols in managing oral disease in humans.11 Antioxidants are large, complex molecules found naturally in various foods and plants, and they also are produced by the body, including in saliva. In healthy individuals, the metabolic process results in a balance of free-radical ROS and antioxidants. If the oxidant-antioxidant balance is disturbed, it can result in a prooxidant burden (ie, proliferation of ROS and free radicals). An overabundance of oxidants is called oxidative stress. Oxidative stress can lead to cell injury through cell membrane microdamage, protein deactivation, and DNA damage.12
An important factor that increases the amount of free radicals and ROS is infection. Infection is the body's response to pathogens (eg, bacteria). Bacterial invasion triggers the release of chemicals, including cytokines (eg, interleukin-8), chemokines, prostaglandins, and destructive enzymes such as matrix metalloproteinases. These mediators cause an increase in the number and activity of macrophages and PMNs. As the PMNs attack and ingest the bacteria, they release additional cytokines, which creates large quantities of ROS.13
The amount of antioxidants in saliva is abnormally low in patients with periodontal disease. Most likely, antioxidant concentrations are depleted as they neutralize the overabundance of free radicals and ROS that result from infection and inflammation.14 Evidence suggests that green tea, which contains antioxidants such as polyphenols and catechins, can decrease malodor.15 A possible rationale is that polyphenols reduce halitosis by modulating VSCs.15
Dental professionals are evaluating protocols that fight inflammation by increasing the amount of antioxidants in the oral cavity. Polyphenols that are topically applied directly to the oral tissues are an emerging possibility with great potential. Specific combinations of the antioxidants phloretin, resveratrol, tetracurcuminoid, and ferulic acid (including formulations applied topically) can neutralize cell-damaging free radicals, particularly those caused by UV light, nicotine, alcohol, and hydrogen peroxide.16 A recent study17 has further confirmed that specific antioxidants, including phloretin, silymarin, hesperetin, and resveratrol, significantly inhibit the inflammatory response associated with Actinobacillus actinomycetemcomitans, one of the pathogens that causes periodontal disease. However, this organism has not been cultured from dogs. Plant-derived polyphenols can also adhere to epithelial surfaces over a prolonged period, which provides a time-release ability to neutralize tissue-damaging free radicals.17
In the present study, the active gel could have been responsible for a 2-part process for decreasing malodor: essential oils provided a direct antibacterial effect, and antioxidants neutralized free radicals resulting from the inflammation. Antioxidants in the gel were pure forms of the polyphenols phloretin and ferulic acid. Phloretin is found naturally in apples and other fruits; ferulic acid is derived from the bran of grasses such as wheat or oats. Both of these antioxidants have been rigorously tested to determine their efficacy for neutralizing ROS and free radicals in oral cells.16 Comparison of the effects of combinations of these 2 antioxidants with other polyphenols (ie, resveratrol and tetracurcuminoid) revealed that a combination of phloretin and ferulic acid exerts a synergistic effect that is greater than that for other combinations. Furthermore, the optimal concentration and pH for their efficacy have been determined.
The population of dogs in the present study was recruited from patients at a private clinical practice; therefore, these dogs may have had frequent visits to the clinic. The amount of overall inflammation was mild; therefore, substantial changes in inflammatory variables would not be anticipated over the 8-week study period. A thorough dental cleaning (scaling and polishing) was performed prior to starting each 4-week application of the active or placebo gel. The rationale was to remove as many local factors (in the form of biofilms and calculus) as possible to minimize inflammation and decrease possible malodor. Application of an active gel containing phloretin, ferulic acid, and essential oils resulted in both the clinicians and owners detecting a decrease in halitosis. Similarly, during application of the placebo gel, halitosis increased.
Application of the active gel resulted in a wide range of improvement among dogs and a similar range of increasing halitosis with the use of the placebo gel. Several explanations could exist for these findings. The subjective organoleptic measurement of halitosis raises questions as to reliability and validity. Clinician assessments were aided by calibration exercises, whereas owners did not perform calibration exercises for the halitosis assessments. The scale had a range of 0 to 10, with the endpoints being no odor and putrid odor. A smaller scale of 0 to 5 and a smell identification test (ie, calibration exercise) for owners prior to assessments could have enhanced validity and reliability. An instrument to measure sulfide gas would have provided an objective method for the clinician assessment. The active and placebo gels were similar in color, but the active gel contained menthol and thymol; thus, clinicians and owners may have detected a difference as a result of a thermal sensation to the skin. Although all dogs were fed similar diets throughout the study, specific food intake for each dog during a 24-hour period could not be strictly controlled, so certain foodstuffs and debris may have contributed to malodor. Owners were instructed to place approximately 12 mm of the gel of an index finger and spread the gel over the gingiva from the incisors to the molars of both the maxillary and mandibular quadrants on one side of the mouth and to then repeat the procedure on the other side of the mouth. It is possible that the quantity of gel may not have been evenly distributed among quadrants of the mouth. In addition, all dogs received the same amount of gel independent of the weight of each dog. Finally, there are variations in the genetic susceptibility to inflammation among subjects.
One possibility for maintaining an acceptable amount of malodor in dogs following dental cleaning is that daily application of antioxidants can reduce the free-radical content and thus decrease the number of pathogens and cell death that cause malodor. Halitosis is caused by both the accumulation of gram-negative anaerobes and resulting periodontal inflammation with subsequent cellular death. A topical oral gel that contains essential oils and the polyphenolic antioxidants phloretin and ferulic acid applied daily may decrease bacterial biofilms, reduce inflammation, and neutralize VSCs. The subsequent result is fewer VSCs produced by the microbial flora or through cell death. Analysis of the results of the present study supported the contention that professional dental cleaning combined with daily topical administration of an active gel containing antimicrobials with specific antioxidant combinations can be used to reduce halitosis and maintain acceptable breath in dogs.
ABBREVIATIONS
| PMN | Polymorphonuclear neutrophilic leukocyte |
| ROS | Reactive oxygen species |
| VSC | Volatile sulfur compound |
PerioSciences LLC with exclusive technology licensing to Tooth To Tail Animal Inc, Dallas, Tex.
SPSS, version 18, SPSS Inc, Chicago, Ill.
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