Successful nutritional control of scratching and clinical signs associated with adverse food reaction: A randomized controlled COSCAD'18 adherent clinical trial in dogs in the United Kingdom

Abstract Background Adverse reactions to food are a common dermatological condition in dogs, requiring nutritional intervention using novel or hydrolysate protein‐based foods. Objective To evaluate a therapeutic food containing egg and phytonutrients in dogs with food allergies using an activity monitor and core outcome set for canine atopic dermatitis (COSCAD'18) in a controlled double‐masked, multicenter, prospective clinical trial. Animals Adult dogs with a history of adverse food reaction as diagnosed by a food elimination trial were recruited from general practices. Methods After a 21‐day baseline period, dogs were randomized to test or positive control (hydrolyzed protein) food for 21 days. Owner (pruritus visual analog score [PVAS], coat quality, food acceptance, and satisfaction) and veterinarian (canine atopic dermatitis lesion index [CADLI], physical examination) assessments were completed on days 0, 21, and 42. Dogs wore a collar‐mounted activity monitor to record sleep, scratching, and shaking behavior throughout the study. Statistical analysis included within‐group comparison to baseline and between‐group comparison at study end using a significance threshold of alpha = 0.05. Results At the end of the treatment period, all results were similar between groups for CADLI, PVAS, owner satisfaction, activity, and questionnaire data. Scores for hair dullness, brittleness, amount of dandruff, feces quality, and food acceptance were positive and were not statistically different between groups. Conclusions and Clinical Importance The therapeutic test food was well‐accepted and efficacious in managing signs of adverse reactions to food compared to baseline as well as compared to the positive control food.

K E Y W O R D S activity monitor, canine adverse food reactions, egg, novel protein, pruritus

| INTRODUCTION
Canine adverse food reaction (CAFR) accounts for about 10% of dogs presenting for pruritus or allergic skin disease 1 and 15% of those may be allergic to chicken whereas only 4% seem to be allergic to egg. 2 Clinical signs of CAFR typically are dermatological, gastrointestinal (GI), or both and affected dogs can be successfully managed using either alternate or hydrolyzed protein sources. 3,4 Controlled studies of CAFR are uncommonly reported in the literature and are not standardized. Lack of consistency in the design of interventional dermatology studies in dogs led to the proposal of the core outcome set for canine atopic dermatitis (COSCAD'18) which includes 3 endpoints: a veterinary assessment of skin lesions such as the canine atopic dermatitis lesion index (CADLI) or canine atopic dermatitis extent and severity index (CADESI), an owner-assessment of scratching behavior such as the pruritus visual analog score (PVAS), and an owner global assessment of treatment efficacy (OGATE). 5 We used COSCAD'18 criteria to evaluate 2 foods because the criteria could readily be applied to dogs with CAFR as well as dogs with atopic dermatitis. The major advantage of COSCAD'18 is that it allows for comparison of results between studies by standardizing outcomes. A drawback is that the measures used are subject to biased reporting by the veterinarian or a pet owner or both. Therefore, we included a wearable device to provide objective data. We previously described behavior algorithms for dogs based on continuous, high-resolution accelerometer data collected by collar-worn activity monitors. 6 The algorithms are sensitive, specific, and >99% accurate in quantifying scratching and shaking in dogs based on validation against >500 video assessments of scratching and shaking in a population of >300 dogs. 6 Activity monitors are capable of continuously and objectively quantifying behaviors in dogs 24 hours per day, 7 days per week, and have been used successfully in a nutrition intervention study. 7 Our objective was to conduct a randomized controlled clinical trial using COSCAD'18 criteria and activity monitor data to evaluate the efficacy of a therapeutic food containing egg and sources of phytonutrients compared to a hydrolyzed protein control food in managing clinical and owner-assessed pruritic signs in dogs with CAFR in a general practice setting.

| Ethics
This study was approved by the sponsor's Institutional Animal Care and Use Committee (CP824a, 11/17/2018) and was performed in accordance with the sponsor's animal welfare policy. Written, informed owner consent was obtained before the commencement of any study activities.

| Patients
Recruitment occurred between September 2019 and March 2020 and included dogs ≥1 year of age if they were free of concurrent disease, had a history of GI signs (tenesmus, diarrhea, or soft feces) with or without dermatological signs (erythema, scratching) related to an adverse reaction to food diagnosed by resolution of signs after a feeding trial and were currently stable for these clinical signs as determined by the attending veterinarian; patients with known allergy to egg were excluded.
Recruitment focused on dogs with cutaneous adverse reaction to food but those with concurrent GI manifestations were permitted provided the patient's GI signs were not consistent with inflammatory bowel disease (eg, undetermined weight loss, hypoproteinemia). Sample size calculations were made assuming a 40% change from baseline for the response variables. Monoclonal antibody-based medications and intermittently-dosed immunosuppressive agents were not allowed; other prior medications were allowed if owners were willing to keep the treatment regimen constant throughout the study. Pet owners were recruited by veterinarians from 11 general practices throughout the United Kingdom.

| Study foods
Details of the macronutrient content and ingredient list of the study foods are provided as supplemental information (Tables S1 and S2); the positive control food (PCF) was a protein hydrolysate-based food (Royal Canin Anallergenic, Lot #122027, Batch Codes 15/11/20, Purchased 06/2019) and the therapeutic test food (TTF) was a novel protein food with egg and rice as the major protein sources with additional sources of phytonutrients (Hill's Pet Nutrition Prescription Diet Derm Complete, Lot #122173, Produced 7/2019). Dogs were limited to their typical food during baseline and blocked based on CADLI scores (3 point increments). Patients were randomly assigned to either the TTF or PCF in a 1 : 1 allocation ratio using an electronic data capture system (Vision V9P3, Prelude Dynamics, Austin, Texas).
Study foods were provided in color-coded bags to mask food identity to all study participants. Dogs included in the analysis entered the study on a variety of foods, but all foods were novel or hydrolyzed protein foods or other limited ingredient or therapeutic foods. Supplements or treats were not allowed and dog owners were instructed to maintain the dog's feeding routine. The volume of food offered was based on the dog's current weight, caloric content of the study foods, and input from the veterinarian.

| Activity monitor
All dogs enrolled in the study were provided with a collar-mounted activity monitor which used triaxial accelerometer technology to generate a continuous record of the dog's motion throughout the baseline and treatment periods of the study. After collection, the data were processed using validated algorithms to quantify dermatologic related behaviors including scratching and shaking; sleeping and sleep quality (based on sleep disruptions) also were measured. 6 Study days in which a minimum of 20 hours of data from the activity monitor was obtained were considered "qualifying days" and included in the statistical analysis. Behavior data were summarized as the total duration of each behavior in seconds per day whereas sleep quality was assessed on a scale of 0 (frequently interrupted) to 100 (uninterrupted).

| Data collection
Study entry was considered day 0; questionnaire data from day 21 was considered baseline and compared to day 42 (end of study). Activity data were recorded on a continuous basis, the baseline period spanning days 0-21, and test period days 22-42. At each visit, veterinarians completed the CADLI and other questionnaires. For the CADLI, grades were assigned reflecting the degree of alopecia/lichenification/hyperpigmentation and erythema/excoriation/erosion for each of 5 body regions on a scale of 0 (none) to 5 (severe) yielding 2 subtotals that were added together for a maximum score of 50. 8 Scores <8/50 were considered clinically normal. 5 At the same time points, owners completed the PVAS using the scale of 0 (none) to 10 (severe). 9,10 Scores <3.6/10 were considered clinically normal. 5 Owners were asked to score overall skin and fecal quality and answered a series of questions regarding their dog's quality of life based on a questionnaire developed and validated for dogs with atopic dermatitis. 11 Quality of life responses consisted of a 5-point Likert scale ranging from strongly agree to strongly disagree. Other questions were based on a score of 1-100. Fecal quality was graded 1 (watery) to 5 (firm) using a scale that included both pictures and word descriptions. A successful food transition was defined as a transition to the study food without experiencing an adverse event or reporting poor palatability within 7 days of starting the study food, as reported by either the veterinarian or the owner.

| Statistical methods
All data were analyzed by a biostatistician (J. Brejda) who was blinded to the identity of the test and control foods and using Statistical Analysis Software (SAS, version 9.4). Differences in mean animal age and weight among dogs assigned to PCF and TTF were analyzed using a 2-sample t test. Distribution of body fat index (BFI) and sex between dogs enrolled into the PCF and TTF groups were analyzed using Fisher's exact test. Questionnaires that provided a numerical score ranging from 0-100 were assumed to be continuous and normally distributed and were analyzed using a linear mixed model with diet, day, and diet Â day as fixed effects in the model. The correlation between the repeated measures (day) was modeled by fitting an appropriate covariance structure selected using the corrected Akaike information criterion (AICC) and Bayesian information criterion (BIC) fit statistics. A simulation-based adjustment was used to control for inflation of the Type I error rate resulting from multiple comparisons. Trends over days were tested using orthogonal polynomial contrasts for linear and quadratic trends. The pet quality of life questionnaire had nominal ordinal responses such as strongly agree, agree, neutral, disagree, or strongly disagree and was analyzed using the Cochran-Mantel-Haenszel test with modified ridit scores. Separate analyses were performed for each day the questionnaire was completed. The PCFB values were analyzed using a general linear mixed-model with diet, week, and the diet Â week interaction as fixed effects. To account for the correlation between repeated measurements made at weekly intervals, 6 common covariance structures were fit to the data, and the AICC fit statistic was used to select the best covariance structure for each activity. To control for multiplicity, a simulation-based adjustment (ADJUST = SIMULATE) was used to control for inflation of the Type I error rate. To test for trends over time, orthogonal polynomial contrasts for linear, quadratic, and cubic effects were performed.
Differences were considered significant when P ≤ .05.

| Dogs
Patient signalment and characteristics are summarized and provided as supplemental information (Table S3). No significant differences were found between groups with respect to sex, weight, age, or breed. Thirtytwo dogs were screened, 29 were enrolled and 28 dogs completed the study and were included in the analysis (13 PCF and 15 TTF).
One dog (PCF) failed to complete the study due to deterioration of its dermatologic condition. Thirteen adverse events were reported for 8 dogs (4 PCF, 4 TTF) and were assessed by the attending investigator to be possibly or probably related to a food and ranged from mild to moderate and all resolved; 1 of these dogs ultimately was withdrawn from the study by its owner (1 PCF). The most common deviations reported were study visits conducted outside of the study design windows. Additional deviations included 14 instances in 9 dogs (4 PCF, 5 TTF) in which medications that may have had an effect on clinical signs were changed during the study period. These dogs still were included in the analysis because the timing or brevity or both of medication administration would not be expected to affect the response to the questions at subsequent visits. The details of these medications are provided in Table S4.

| Canine atopic dermatitis lesion index
No significant differences in CADLI scores were found between and within the PCF and TTF groups at end of baseline and end of study, and results are shown in Figure 1A. Most dogs entered and completed the study with a CADLI <8 (24/28; 86%), indicating a good level of control. 5 Dogs that entered the study with a CADLI ≥8 (2 PCF, 2 TTF), ended the study with scores ≤8 except 1 TTF dog (start, 9/50; end, 10/50).
Note: Values are represented as mean ± SD where appropriate. Scores were 0-100. Several questions were only assessed at study end. Abbreviations: PCF, positive control food; TTF, therapeutic test food. *P < .05 vs baseline. **P < .05 vs positive control. food (baseline). All other measures were not different between or within groups.

| DISCUSSION
We documented similar control of signs associated with CAFR between foods in owner-dog pairs managed in general practices in the United Kingdom using a core outcome set of measurements developed and endorsed by veterinary dermatologists 5 in conjunction with an activity monitor. Similarly, we chose the CADLI, PVAS, and owner evaluation results as primary outcome measures but also used activity data and veterinary outcomes to supplement these findings. A shortcoming of our study was that the population of dogs was not described based on the specific source of their food allergy. A practical approach for general practitioners is to recommend avoidance of common food allergens by prescribing foods with novel or hydrolyzed protein or both, and the results of our study support that approach. To our knowledge, a comparison showing similar outcomes between a hydrolyzed and a novel protein food in a population of dogs with CAFR managed in clinical practice has not been published previously.
The TTF incorporated ingredients to avoid as well as modulate the immune response. For the test food, egg was selected as a primary protein source because documented cases of allergies to egg represent approximately 4% of all food allergies in dogs, lower than other protein sources such as chicken or soy. [12][13][14] Several components of egg have been shown to have immunomodulating. 15 We previously have reported that a food with polyphenol containing phytonutrients modulates clinical signs in dogs with environmental allergies. 16,17 Polyphenols have been shown to modulate the immune system directly by inhibiting degranulation of mast cells, as well as cytokine and immunoglobulin production and indirectly by binding antigens, thus rendering them less recognizable by the immune system. 18,19 Clinically, once pruritus is controlled, skin healing can commence and is supported by dietary antioxidants, vitamins, fatty acids, and minerals. [20][21][22][23] To our knowledge, ours is the first study using COSCAD'18 criteria to evaluate food in managing CAFR. The COSCAD'18 was intended to be used in the evaluation of pharmaceuticals in dogs with environmental allergies but its use for food allergy has worked well. A benefit of the COSCAD'18 is that it demands a broader assessment of the clinical condition than a study design using only veterinary or owner input. Furthermore, our study showed that COSCAD'18 could be employed in study designs examining the maintenance of clinical condition.
We interpreted the CADLI and PVAS with reference to values considered normal because some level of scratching behavior can be habit-

ual. The upper threshold between what would be considered normal and
what would be clinically relevant is a CADLI score of 8 and a PVAS score of 3.6. 5 To be eligible, dogs were considered controlled with respect to dermatological signs, but minimum criteria were not set nor were dogs blocked based on score. Different scores were observed among participants at study entry but were not different between groups. The range was likely because of the fact that controlled is a subjective assessment.
Dogs entering the study with higher scores improved and improvements were similar between groups.
To our knowledge, ours is the first study to combine COSCAD'18 criteria with activity data in a nutrition intervention study. Activity monitors provided continuous and objective quantification of each dog's pruritic behavior. One limitation is that minimum technological requirements must be met, which may prohibit its use by certain owners Dogs began the study with normal fecal quality which did not change, and foods were similarly accepted in both groups. When asked specifically about their opinion of the foods, veterinarians and owners reported high satisfaction scores (>50/100) and willingness to recommend foods to a friend or colleague (>50%).
One limitation of our study is that the elimination trial was performed previously and not as part of the trial. As such, we were not able to set the conditions of the food elimination trial. This was because a prospective trial to recruit newly diagnosed cases of CAFR would take a long period of time. We controlled for this issue by recruiting animals where the veterinarian, and not the owner, attested to the presence of the food allergy and history of a food elimination trial.
A second limitation is that enrolled dogs could have been concurrently was not associated with early study withdrawal or with worsening signs at study end. 24 A final potential limitation to our study is the possibility of Type II error. To minimize this possibility, sample size calculations were made assuming a 40% change from baseline for the response variables. This is the magnitude of response anticipated as a result of feeding the therapeutic food. The anticipated response was observed over time, which from a clinical standpoint was the desired outcome because it shows the therapeutic food was successful. However, it is possible that the magnitude of the difference between the 2 foods was not as large as the magnitude of change over time. As a result, the sample size needed to show a significant change over time was insufficient to identify significant differences between the 2 foods, resulting in Type II error.

| CONCLUSION
In summary, we successfully used COSCAD'18 criteria and activity monitors to provide evidence for similar management of clinical signs associated with CAFR by 2 foods: a commercially available hydrolyzed protein-based food and a therapeutic food with egg, rice, and sources of polyphenols.