Clinical evaluation of an antiinflammatory and antioxidant diet effect in 30 dogs affected by chronic otitis externa: preliminary results

The aim of this evaluation study was to assess the possible role of a specific nutraceutical diet in relieving main clinical symptoms of chronic bilateral otitis externa (occlusion of ear canal, erythema, discharge quantity, and odor) in 30 adult dogs. Thirty dogs of different breeds (mean age ± SEM; 6.03 ± 0.15 years and mean weight ± SEM; 32.01 ± 1.17 Kg; 53.3 % males, 46.6 % females) with evident chronic clinical otitis symptoms were equally divided and randomly assigned to receive either the nutraceutical diet (ND group) or a standard diet (SD group) over a period of 90 days. In all cases a topical pharmacological treatment was given. The nutraceutical diet, also endowed with anti-inflammatory and antioxidant activities, significantly decreased the mean score intensity of all symptoms after 90 days of intervention (P < 0.0001) with the exception of Malassezia pachydermatis infection which was only slightly reduced. Our investigation is one of the few evidence-based results where a commercial nutraceutical diet has been proven effective, in combination with drugs, in relieving otitis externa-related symptoms. This study opens new insights into otitis externa clinical management providing evidence of efficacy of a combined therapy with drugs and a specific nutraceutical diet.

Abstract The aim of this evaluation study was to assess the possible role of a specific nutraceutical diet in relieving main clinical symptoms of chronic bilateral otitis externa (occlusion of ear canal, erythema, discharge quantity, and odor) in 30 adult dogs. Thirty dogs of different breeds (mean age ± SEM; 6.03 ± 0.15 years and mean weight ± SEM; 32.01 ± 1.17 Kg; 53.3 % males, 46.6 % females) with evident chronic clinical otitis symptoms were equally divided and randomly assigned to receive either the nutraceutical diet (ND group) or a standard diet (SD group) over a period of 90 days. In all cases a topical pharmacological treatment was given. The nutraceutical diet, also endowed with anti-inflammatory and antioxidant activities, significantly decreased the mean score in-tensity of all symptoms after 90 days of intervention (P < 0.0001) with the exception of Malassezia pachydermatis infection which was only slightly reduced. Our investigation is one of the few evidence-based results where a commercial nutraceutical diet has been proven effective, in combination with drugs, in relieving otitis externa-related symptoms. This study opens new insights into otitis externa clinical management providing evidence of efficacy of a combined therapy with drugs and a specific nutraceutical diet.
Keywords Nutraceutical diet . Otitis externa . Symptoms intensity decrease . Antiinflammatory and antioxidant activities Alessandro Di Cerbo and Sara Centenaro contributed equally to this work.
The aim of this clinical evaluation was to observe the effect of a commercial nutraceutical diet, also endowed with antiinflammatory and antioxidant activities, as an adjuvant in pharmacological treatment of dogs affected by chronic otitis externa in order to improve the intensity of its clinical signs as well as the presence of Malassezia pachydermatis. The antiinflammatory and antioxidant activities of the proposed diet have to ascribed to the presence of the pool of fish hydrolyzed proteins, rice carbohydrates, Melaleuca alternifolia, Tilia cordata, Allium sativum L, Rosa canina L., Zinc and a well balanced Omega3:6 ratio (1:0.8).
Immunomodulatory activities have also been ascribed to zinc, whose deficiency affects innate and adaptive immunity, exacerbates inflammation (Bonaventura et al. 2014) and is closely related to skin desease and wound healing (Colombini 1999) since its absolute or relative deficiency can cause the onset of canine zinc-responsive dermatosis (Hensel 2010).
An optimal balance of the omega 3:6 fatty acids ratio in the food is considered a fundamental requirement for tissue to improve homeostasis and contrast the inflammatory processes. More in details, n-3 polyunsaturated fatty acids, usually found in fish oil, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are known to decrease the production of proinflammatory mediators and inhibit natural killer cell activity (Kelley et al. 1999). In addition, the n-6 polyunsaturated fatty acid gamma-linolenic acid (GLA) and EPA are endowed with specific antiinflammatory activity (DeLuca et al. 1999).
Based on such considerations, we performed a randomized placebo-controlled clinical evaluation on 30 dogs with evident chronic clinical otitis symptoms such as occlusion of ear canal, erythema, discharge quantity, and odor.

The animals
Thirty adult dogs of different breeds (mean age ± SEM; 6.03 ± 0.15 years and mean weight ± SEM; 32.01 ± 1.17 Kg; 53.3 % males, 46.6 % females) with evident chronic clinical otitis symptoms were randomly divided and assigned to receive either the specific diet (treatment group, n = 15) or the placebo (control group, n = 15) once a day for 90 days, accordingly with the following manifacture's table (Table 1). In addition, all dogs were also pharmacologically treated with a topic product (OTOMAX, Schering-Plough, Kenilworth, NJ, USA) 8 drops a day for 7 days.

The diets
The two diets were based on the same receipt and completely fulfil the recommendations for proteins, carbohydrates and fats content in order to obtain a complete food for a daily ration in dog (as reported in Nutritional Guidelines for complete and complementary pet food for cats and dogs by The European Pet Food Industry Federation). In particular, the two foods reported similar analytical composition in nutrients (24 % of crude protein, 12 % of crude oils and fats, 3.7 %, of crude fibre 5 % of crude ash, 9 % of moisture) and, as a consequence, similar Metabolised Energy (ME) of 3.477 kcal/ kg corresponding to 14.6 MJ/kg. Both foods are commercially available and in the form of kibbles industrially produced with extrusion technique. The specific nutraceutical diet was composed by two mixed components: kibbles, included in the ideal percentage of 93-94 % in weight, and cold-pressed microcapsules at the 6-7 % in weight of complete food (European patent n.EP 2526781). Overall nutrient profile of the product was obtained by the sum of a first nutrient profile of the kibbles, for feeding purpose, and a second nutrient profile of the microcapsules for both nutrient and therapeutic purposes. Microcapsules were composed of 60-80 % of hydrolyzed proteins (of fish and vegetable origin), 20-40 % of minerals, used as glidants, and therapeutical substances (Melaleuca alternifolia, 0.00343 %, Tilia platyphyllos scapoli et cordata, 0.0147 %, Allium sativum L., 0.0245 %, Rosa canina L., 0.098 %, and Zinc, 0.00479 %).

Malassezia pachydermatis determination
A small-tip cotton swab was inserted into the external ear canal removing some exudate. The swab was then rolled along a microscope slide with the sequence number. The slides were dried and stained with modified Wright's stain, and evaluated microscopically (Cole et al. 2007) with an Olympus 60BX polarized light microscope (New York Microscope Company Inc, Hicksville, NY, USA). Malassezia pachydermatis organisms were identified morphologically. The sample was considered pathological if the average of identified yeasts resulted more than 10 per high- power field (HPF) in several fields, (Fig. 1) (Cowell et al. 2008). The precedure was performed before intervention (time 0); after 30 days (time 30); after 60 days (time 60) and at the end of intervention (time 90).

Clinical evaluation and scoring system
Dogs received veterinary inspections, before intervention (time 0); after 30 days (time 30); after 60 days (time 60) and at the end of intervention (time 90). Operative procedures and animal care were performed in compliance with the national and international regulations (Italian regulation D.L.vo 116/1992 and European Union regulation 86/609/EC). The protocol was examined and approved prior to the beginning of the study by the Veterinary Ethical Review Committee. The recommendations of the ARRIVE guidelines in animal research were also consulted and considered (Kilkenny et al. 2012).

Statistical analysis
Data were analyzed using GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA). All data are presented as the means ± standard error of the mean and were first checked for normality test using the D'Agostino-Pearson normality test. Differences in occlusion of the ear, erythema, discharge quantity and odor score between the two supplements at the end of treatment versus baseline for each ear were analyzed using a two-way analysis of variance (ANOVA) followed by Sidak's multiple comparisons test. A p < 0.05 was considered significant.

Results
Following clinical and cytological evaluation 28 out of 30 dogs presented an excessive amount of ear wax related to Malassezia pachydermatis infection. Only 2 out of 30 dogs reported an additional bacterial presence (either cocci or bacilli), therefore we considered such condition as not worth of clinical monitoring.
No adverse effects, such as cutaneous atrophy, secondary infections (Muller et al. 2001), increased licking (Bensignor and Olivry 2005), occasional skin itching or burning (Caffier   (Mason et al. 2013) were reported by the owners or noted on otoscopic examinations with any treatment, and all dogs completed the 90-day evaluation period. In Fig. 2, the overall improvement of dogs hears before and at the end of the 90-days evaluation is shown (Fig. 2).
The nutraceutical diet significantly decreased dog's ear canal occlusion, erythema, odor and mucus discharge scores after 90 days of evaluation, if compared with baseline while the mean number of Malassezia pachydermatis organisms slightly decresed (Fig. 3). Fig. 3 Graphical representations of symptoms trend during the evaluation. (a) Mean Malassezia organisms in OIF before and after 90 days of evaluation for SD and ND group, organisms resulted slightly decreased in ND group; (b) mean occlusion of ear canal score before and after 90 days of evaluation for SD and ND group, a significant decrease (****P < 0.0001) was observed in ND group; (c) mean discharge quantity score before and after 90 days of evaluation for SD and ND group, a significant decrease (****P < 0.0001) was observed in ND group t; (d) mean odor score before and after 90 days of evaluation for SD and ND group, a significant decrease (****P < 0.0001) was observed in ND group; (e) mean erythema score before and after 90 days of evaluation for SD and ND group, a significant decrease (****P < 0.0001) was observed in ND group After 90-days of evaluation Malassezia pachydermatis organisms decreased from a baseline value of 5.32 ± 0.4 to 4.2 ± 0.3 in the ND group and from a baseline value of 5.4 ± 0.4 to 5.4 ± 0.3 in the SD group (Fig. 3a).
Dogs ear canal occlusion resulted decresead from a baseline value of 1.87 ± 0.1 to 1.84 ± 0.1 in the SD group and from a baseline value of 1.89 ± 0.1 to 0.78 ± 0.1 in the ND group (Fig. 3b, ****P < 0.0001).
As to discharge quantity, the scores decreased from a baseline value of 1.72 ± 0.1 to 1.74 ± 0.1 in the SD group and from a baseline value of 1.77 ± 0.1 to 0.31 ± 0.07 in the ND group (Fig. 3c, ****P < 0.0001). Also odor scores resulted decreased after 90-days of evaluation, with respect to the baseline.
More in details, the scores decreased from a baseline value of 1.89 ± 0.1 to 1.87 ± 0.1 in the SD group and from a baseline value of 1.91 ± 0.1 to 1.0 ± 0.1 in the ND group (Fig. 3d, ****P < 0.0001).
Finally, erythema dicreased from a baseline value of 1.28 ± 0.1 to 1.30 in the SD group and from a baseline value of 1.26 ± 0.1 to 0.58 ± 0.1 in the ND group (Fig. 3e, ****P < 0.0001).

Discussion
Dogs affected by chronic, recurrent otitis externa are considered one of the most frustrating pathologies of daily veterinary clinical practice (Rosser 2004).
In this study, we use a specific nutraceutical diet, based on a combination of fish hydrolyzed proteins, rice carbohydrates, Melaleuca alternifolia, Tilia cordata, Allium sativum L, Rosa canina L., Zinc and a Omega3/6 (1:0.8 ratio), as an adiuvant approach for the clinical management of canine otitis externa.
We observed a significant and encouraging reduction of the main symptoms of otitis externa -as the external ear canal occlusion, erythema, odor and mucus discharge -in enrolled dogs fed the nutraceutical diet if compared to those who received the standard diet. To this regard, we recently described the efficacy of a similar diet in relieving some otitis externa clinical symptoms, such as malodor, shaking, pus presence, earwax, itch, edema, blood presence, auricular function and auricular flush, in 107 dogs after 30 days of evaluation (Di Cerbo et al. 2014).
Our results appear in agreement with those observed by Sarrell et al. that compared the effectiveness of a naturopathic herbal extract, containing also Allium sativum, with anaesthetic ear drops in the management of ear pain associated with acute otitis media (Sarrell et al. 2001). Specifically, 61 out of 103 children treated with naturopathic herbal extract had an overall improvement in ear pain score due to analgesic, antiinflammatory, anti-occlusive effects and anti-infective properties of the naturopathic product.
Here, we evidenced that the specific nutraceutical diet was also highly tolerated throughout the whole evaluation period as no adverse effects were observed in all dogs completing the study. In addition, we observed that most of clinical symptoms were substantially halved. These intersting occurences strongly encourage the use of the nutraceutical diets, endowed with anti-inflammatory and antioxidant acitivities, as valid and safe support to the conventional pharmacological therapy for dogs affected by chronic otits externa.
With regard to comorbidity of Malassezia infection, the addition of zinc in our diet was based on previous in vitro and in vivo studies, which highlighted its role in reducing yeasts number (DeAngelis et al. 2005;Mendelsohn et al. 2005). However, clinically apparent yeast presence seemed unvaried in the SD group. Althought our study showed a slightly reduction in the number of Malassezia pachydermatis organisms in ND group, it is reasonable to hypothesize a possible synergistic action of this antiinflammatory and antioxidant diet with antibiotic therapy. In this regard, therapy usually is topically applied for a reduced time, in order to avoid an antibiotic resistance phenomenon. It is noteworthy that an increased risk of antibiotic resistance may occur after a routine topical antibiotic administration in the treatment of otitis externa (Voget et al. 2012). In this respect, the antiinflammatory and antioxidant effects of a diet could likely reduce the needing and the frequency of local antibiotic administration and contribute to avoid the emergence of drug resistence.
The results achieved in this study, concerning ear canal occlusion and erythema, are in agreement with those observed by Sarrell et al. that compared the effectiveness of a naturopathic herbal extract, containing also Allium sativum, with anaesthetic ear drops in the management of ear pain associated with acute otitis media (Sarrell et al. 2001). Specifically, the authors reported that 61 out of 103 children, belonging to the naturopathic herbal extract-treated group, had an overall improvement in ear pain score due to analgesic, antiinflammatory, anti-occlusive effects and anti-infective properties of the naturopathic product.
Many studies, regarding both dog and human, claim for nutraceutical administration benefits in otitis externa. Our investigation further outlines the quick symptoms relieving of otitis externa by means of a commercially available nutraceutical diet.
To the best of our knowledge this is the first report of a veterinary clinical evaluation concerning a anti-inflammatory and antioxidant diet effect on dogs affected by chronic otitis externa. Although further studies with a larger sample and time of observation are needed these results can be considered very promising in light of a possible traslation on the human side.

Compliance with ethical standards
Conflict of interest statement The authors declare that they have no conflict of interest. This research was performed in collaboration with some scientists from the Division of Research and Development, Sanypet SpA, Padova, Italy (as indicated in the Author's affiliation) according to scientific and ethical principles of the scientific community. No financial funding was obtained from Sanypet Industry for this research study.
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