Abstract
The gingival contour plaque index (GCPI) is a method for measuring gingival margin plaque in dogs. The scoring approach usually characterizes accumulation of dental plaque via a “clean” baseline attained by toothbrushing of conscious animals. However, toothbrushing is a rare pet owner-led practice. Instead, the introduction and/or use of dental interventions is far more likely when a dog's teeth are in a somewhat “dirty” state. In line with such an assumption, 2 studies were conducted to gauge the efficacy of a single feeding instance of several different dental chews following either 7 or 5 days of intentional plaque accumulation. Each singly fed dental intervention led to a statistically significant removal of plaque compared to the no chew control (P ≤ 0.007). This adapted approach to employing GCPI offers a new opportunity to assess the plaque removal efficacy of oral care products, a novel clinical measure of principal value and relevance to the oral health of dogs in the real world.
Introduction
Over recent decades, a significant number of dietary oral care products have been developed for dogs, including specially designed dry diets 1 and a variety of dental chew types.2–7 Clinical studies have been and continue to be pivotal to evaluating the effectiveness of such dental interventions in vivo. In 1994, 2 seminal papers8,9 established a methodology characterized by a “clean tooth” model together with scoring systems for dental plaque and calculus. By referencing the baseline reset undertaken at the beginning of each experimental trial phase, in which the teeth of the dogs in a trial are scaled and polished, the approach ensures plaque and calculus levels are reduced to zero.
With universal adoption of the “clean tooth” model, it has become relatively standard practice to compare the new dietary dental innovation (test diet/treatment) to a control diet. Typically, in instances where the test treatment is a dental chew, all participating dogs will receive a complete and balanced dry main meal. This provides the sole source of nutrition to dogs during a control phase, while for a test phase, the amount fed to dogs will be reduced to compensate for the additional caloric contribution from the test product. When tested in a multiphase crossover study design, this enables a statistically robust approach by ensuring all participating dogs are exposed to all dietary regimes under investigation. In such trials, each phase is several days or weeks in duration, such that plaque and/or calculus can accumulate sufficiently and differences in effects of the diets can be detected. Notably, the Veterinary Oral Health Council (VOHC) stipulates a minimum of 28 days per trial phase if a protocol is being conducted as part of an application for their Seal of Acceptance. 10
An alternative approach to measuring the efficacy of dental interventions has been developed.11,12 Known as the “gingival contour plaque index” (GCPI), the method focuses only on plaque, specifically at the gum line. While this presents as a key limitation when compared with the Logan and Boyce model, the GCPI methodology offers a distinct advantage in not requiring general anesthesia or sedation of the participating dogs to perform a scale and polish procedure. With the sole focus on plaque as the only clinical index of interest, study designs employing the approach can be completed in much shorter timeframes.
The published methodologies discussed offer opportunities to assess a given intervention's ability to prevent plaque accumulation or build-up. Another way to assess the effectiveness of an intervention could be to determine the removal of existing plaque from a single feeding occasion, which is of particular interest with products such as a dental chew. Such a paradigm would enable an assessment of the immediate effect of a single exposure to the product. This could be achieved by measuring levels of accumulated plaque, feeding a chew, and scoring plaque levels again shortly after feeding. While anesthesia-based methods for scoring clinical indices would not be practical with the proposed sequence of events, the GCPI approach offers a more appropriate and viable means of achieving this. To elucidate this, the authors report 2 studies to determine the amount of plaque removed by a single feeding of several dental chews using the GCPI method.
Materials and Methods
Animals
Adult beagle dogs were housed at 2 locations. All procedures were conducted in accordance with the MARS Animal Research policy and approved by the WALTHAM Animal Welfare and Ethical Review Body. Routine housing, husbandry, and exercise regimes were maintained throughout the course of each study. Dogs were pair or group housed in comfortable temperature-controlled conditions with outdoor access throughout the day except when they were individually housed for feeding. The amount of the main meal diet was reduced to compensate for dogs receiving dental chew products. Water was provided ad libitum. The general health and overall condition of each animal were monitored daily by the animal care staff at each location.
Study Design
Study 1 comprised a 2-phase, and Study 2 a 3-phase, crossover trial in which each phase lasted 7 and 5 days, respectively. Each phase differed only on the final day (Day 7 or Day 5) of the assigned dietary regime. Figure 1 depicts the design for Study 1. Study 2 followed a similar design with the exceptions of the number and length of phases and the number of dogs.
Design Schematic for Study 1.
At the beginning of the first phase (Day 0, T0), the dogs had the total lengths of the gingival margins of the 22 selected teeth measured, as per the “GCPI Plaque Scoring” section, and received thorough toothbrushing to ensure complete, or close to complete, removal of plaque. All dogs received daily feeding of a commercially available standard dry main meal dieta, which was fed according to individual energy requirements to maintain bodyweight. On Day 7 (Study 1) or Day 5 (Study 2) of each phase, each dog had plaque measurements obtained (T1) and were offered their dietary regime in the subsequent hour according to their group assignment. One-hour post the initial plaque measurement, each dog then had plaque remeasured (T2) and received toothbrushing to prepare them for the next phase. Veterinarians or technicians scoring the length of gingival margins and/or plaque were blinded to the dietary regime assignments of the dogs in each study.
Study 1 (7 Days Plaque Accumulation)
Eight beagle dogs comprising 2 females (all intact) and 6 males (all neutered) with a mean age of 3.2 years (range 2.8-3.5 years) and weight 15.2 kg (range 11.6-18.8 kg) were enrolled into this study at the Department of Veterinary Sciences, Ludwig-Maximilians-Universität (LMU) Munich, Germany. Dogs were deemed suitable for the study if all 22 selected teeth were present, and their teeth had been scaled and polished within 6 months prior to the start of the study. The study comprised a 2-phase crossover in which each phase lasted 7 days. The dietary regime on the final day (Day 7) of each phase was either (i) no chew (control) or (ii) Chew Ab (composed of a single piece).
Study 2 (5 Days Plaque Accumulation)
Twelve beagle dogs comprising 3 females (all spayed) and 9 males (6 neutered) with mean age 4.1 years (range 1.9-7.6 years) and weight 14.0 kg (range 10.9-21.0 kg) were enrolled in the study at the Ontario Nutri Lab Inc., Fergus, Ontario, Canada, an independent, registered animal research facility. Dogs were deemed suitable for the study if all 22 selected teeth were present and their teeth were substantially free from calculus, such that plaque measurements could be made without significant interference from calculus deposits. Dogs not fulfilling this were either excluded or underwent a dental scale and polish prior to the start of the study. A 3-phase crossover study design was followed. Each phase comprised 5 days ending with one of the 3 dietary regimes on the final day of the phase: (i) no chew (control); (ii) Chew Bc (comprised of 2 pieces), or (iii) Chew Cd (comprised of a single piece).
GCPI Plaque Scoring
Plaque was measured according to a previously described GCPI method,11,12 with 2 modifications. Administration of atropine to reduce salivation and ease plaque measurement was not conducted in order to prioritize animal welfare. Such modification was made based on previous experience, which indicated that satisfactory plaque measurements could be achieved without administration of atropine. Secondly, assessment of maxillary first molar teeth at the caudal region of the mouth was avoided to minimize stress to the conscious dogs. Alternatively, the mandibular third incisor teeth were substituted to enable scoring of the same number of teeth (22),11,12 and ensure more consistency between the number of maxillary and mandibular teeth scored. The teeth scored were therefore the maxillary third incisor, canine, and first to fourth premolar, and the mandibular third incisor, canine, and second to fourth premolar.
Total length of each tooth along the gumline was measured (“L”) on Day 0 of each trial. Plaque measurements (lengths, “x”) of the same teeth before (T1) and after (T2) the dietary regime on the final day of the phase were determined by application of disclosing solution for Study 1e and 2f. GCPI was calculated in 2 ways: (i) by calculating “x/L” × 100 for each tooth and then averaging to determine the mean of all the teeth measured (individual tooth score) and (ii) by dividing the sum of “x” for all teeth measured by the sum of “L” for all teeth measured (whole mouth score) and multiplying by 100.
Statistical Methods
Analysis of change in GCPI between T1 and T2 in each phase was undertaken by linear mixed effects models using dog as a random effect and sequence, phase, and diet as fixed effects. Tukey HSD pairwise comparisons were made between each diet. However, residuals from Study 1 analyses failed normality, and the data were reanalyzed using the nonparametric Friedman test. Data were analyzedg, and statistical significance was taken as P < 0.05.
Results
Study 1 (7 Days Plaque Accumulation)
Across both phases mean ± SD plaque accumulation at T1, as measured by GCPI, was 65.9 ± 10.6 (individual tooth) or 69.7 ± 10.7 (whole mouth).
The change in plaque score determined for the dogs assigned to the control (no chew) group was 0.6, and for dogs assigned to the chew group (Chew A) was 39.4, using the individual tooth average analysis methods (Table 1). With the whole mouth average approach, the change in plaque score for the control (no chew) group was 0.5 and 41.1 for the chew group dogs (Table 1). The differences between the control (no chew) and chew (Chew A) groups were −38.8 (P = 0.005) and −40.69 (P = 0.005) using the individual tooth and whole mouth analysis methods, respectively.
Comparison of Plaque Score (GCPI) Differences (T1–T2) Between the Dietary Regimes in Study 1 (7 Days Plaque Accumulation).
Positive values for mean change in GCPI indicate a reduction in plaque between T1 and T2. Negative values for the difference indicate a reduction in the chew relative to the control.
Based on the mean of 22 teeth.
Based on the ratio of total plaque to total gumline length of 22 teeth.
Study 2 (5 Days Plaque Accumulation)
Of the 12 dogs enrolled for the 3-phase cross-over study, one of the dogs did not consume one of the chews. All data from this dog were excluded from the statistical analysis of the study plaque scores.
Across all phases, mean ± SD plaque accumulation at T1 as measured by GCPI was 64.4 ± 10.2 (individual tooth) or 70.7 ± 10.3 (whole mouth).
In this study, changes in plaque scores evaluated as per the individual tooth analysis approach were 7.7 for the control (no chew) group, 27.7 for dogs assigned to the Chew B group, and 19.6 for dogs assigned to the Chew C group (Table 2). Using the averages determined via the whole mouth approach, changes in plaque score for the control (no chew) group, Chew B group, and Chew C group were 7.8, 28.4, and 21.1, respectively (Table 2). Differences determined between the groups for changes in average plaque scores calculated using the individual tooth method were control (no chew) and Chew B −20.0 (P < 0.001); control (no chew) and Chew C −11.9 (P = 0.007); and Chew B and Chew C −8.1 (P = 0.077) (Table 2). Between the groups, differences in change score for the whole mouth approach were as follows: control (no chew) and Chew B −20.6 (P < 0.001); control (no chew) and Chew C −13.38 (P = 0.006); and Chew B and Chew C 7.3 (P = 0.166) (Table 2).
Comparison of Plaque Score (GCPI) Differences (T1–T2) Between the Dietary Regimes in Study 2 (5 Days Plaque Accumulation).
Positive values for mean change in GCPI indicate a reduction in plaque between T1 and T2. Negative values for the difference indicate a reduction in the chew relative to the control.
Based on the mean of 22 teeth.
Based on the ratio of total plaque to total gumline length of 22 teeth.
Discussion
Plaque control is fundamental to the maintenance of good periodontal health. As such, plaque is one of the key clinical indices in the assessment of dental intervention efficacy. Current evaluation methodologies rely on the establishment of clean teeth, resetting the baseline measurement to zero. By moving away from this paradigm, the 2 studies reported here have shown that efficacy against accumulated plaque can be demonstrated using “dirty” teeth via a single feed of different dietary interventions.
In parallel with the original GCPI method,11,12 the studies reported here were initiated, in the most part, by toothbrushing of the dogs’ teeth. Descale and polish procedures under general anesthesia were minimized, with the intention to maximize animal ethics and welfare standards during the studies. Between the 2 investigations conducted, the variation in study design aimed to assess different timeframes for plaque build-up before exposure to the respective test interventions. Initially, a 7-day accumulation was trialed (Study 1) that led to a statistically significant difference in the average plaque measurements between the control and chew groups. Subsequently, in Study 2, the accumulation phase was reduced to 5 days, yet it also yielded statistically significant differences between the respective control and chew groups. Moreover, these statistically significant findings were yielded in both the individual tooth and whole mouth analyses conducted. It is important to note the difference; the individual tooth approach gives equal weight (importance) to all teeth regardless of size, while the whole mouth method assigns greater weight to larger teeth. Overall, these findings suggest flexibility in the exact protocol used to enable sufficient plaque build-up for clinical assessment, similar to that used here. Furthermore, the statistically significant differences show that the effect of a single instance of a dental intervention can be determined. The authors believe this approach offers a first of this kind, reporting the impact on existing plaque accumulation of one dental treat.
The authors acknowledge the notable difference in the average plaque scores determined for the control (no chew) group between the 2 studies reported. As expected, the mean change in GCPI, evaluated by the 2 analysis routes, was negligible (close to zero) for Study 1, but larger for Study 2. The authors are uncertain of the reasons for this observation but speculate that it may be due to differences in dog behavior between the 2 trials. More specifically, the authors suspect the cohort comprising Study 2 had a greater tendency to drink water, being conducted at a time of year when temperatures were relatively warm. In contrast, Study 1 took place at a much cooler time of year. This is a key consideration given the seasonal influences at play in the deployment of each study design. Consequently, a temperature-associated drinking bias in Study 2 may have led to regions of accumulated plaque being dislodged after the T1 measurements, despite no test dietary intervention, which were subsequently detected during the T2 measurements. It is also important to note that this would likely have affected the average plaque scores determined for all Study 2 groups. Given that assumption and the confidence in the statistical approach, the authors are confident in the conclusions drawn.
The oral care products evaluated in the 2 studies were dietary, dental chew interventions. However, the demonstrated approach has applicability to other formats, including dental diets and water additives. In addition, while the methodology presented has assessed the different dietary interventions following consumption of each at a single frequency, the number of feeding occasions could be adjusted to explore multiples. By utilizing the approach employed here or adjusting to accommodate the variations discussed, the “dirty” mouth model enables the novel assessment of the plaque removal capability of oral care interventions. By such means, therefore, adaptation to employment of GCPI provides a truer reflection of the impact of oral care products by resembling natural plaque levels and the oral environment of the average pet dog.
Alongside the conventional method, the alternative approach to GCPI presented here offers an animal welfare-friendly means for assessing the impact of oral health interventions. Furthermore, the dual benefit offered by this adapted method is the ability to evaluate the effect of a single administration of a given intervention in a “dirty” mouth in which plaque biofilms have been established. As well as providing a different lens on efficacy evaluation, which is perhaps more relevant to the real world, the authors believe this offers an opportunity to more rapidly, cheaply, and ethically evaluate products for plaque removal.
Materials
Josera JosiDog Agilo Sport for Study 1; Royal Canin Medium Adult Dry Dog Food for Study 2, Royal Canin, Somerset, UK
Commercial Product (twice-weekly chew), Mars Petcare, Leicestershire, UK
Prototype Product (bite-sized chew), Mars Petcare, Leicestershire, UK
Commercial Product (daily chew), Mars Petcare, Leicestershire, UK
PlaqSearch™, TePe®, Malmö, Sweden
Garnet, DHARMA Research Inc., Miami, FL, USA
Minitab21, Minitab LLC, Coventry, UK
Footnotes
Acknowledgments
The authors would like to acknowledge members of Ludwig-Maximilians-Universität (LMU) Munich and Ontario Nutri Lab Inc., including Kelly Cameron, for conducting the studies.
Author Note
Avika Ruparell, Tim Sparks, and Phil McGenity contributed to the article as employees of Mars and therefore Mars has a copyright interest in that portion of the article.
Ethical Approval
All procedures were conducted in accordance with the MARS Animal Research policy and approved by the WALTHAM Animal Welfare and Ethical Review Body.
Funding
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was funded by Mars Petcare.
Declaration of Conflicting Interest
The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: All authors except Britta Dobenecker were employees of Mars Petcare at the time of the study. Data in this article have been used to support a patent application.
Data Availability Statement
The datasets generated and/or analyzed during the current study are not publicly available due to commercial reasons, but are available from the corresponding author on reasonable request.