This study was designed following good clinical practices, although no suffering or health risks to the dogs were anticipated. The study was approved by the ethical committee of the Department of Veterinary Medicine of authors university. Ethical review was not necessary since requirements for administration of a nutraceutical are covered in the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes, which exempt it from ethics approval. The study was designed as a prospective randomized double-blinded multicentric study.

To be included in the study dogs should have a diagnosis of FAD, be aged between 1 and 12 years, and weighing between 1 kg and 20 kg. Furthermore, other parasitic infestations, microbial infections, autoimmune or immune-mediated diseases, metabolic or hormonal disorders had to be previously excluded. No ectoparasitic treatment had to be administered at least for 30 days (in the case of long-acting products at least 90 days). No any kind of nutraceutical with antiinflammatory or antipruritic effect have to be administered during the last 30 days. A written consent had to be signed by the owner to include his/her dog in the study for 2 months. To be eligible for the study the modified CADLI score had to be lower than 10 (in order to exclude severe cases that may need stronger antipruritic treatments with drugs).

Dogs with a diagnosis of FAD treated with systemic or topical antibiotics, steroids, IL-31 inhibitors, or antihistaminics were excluded as well as those receiving nutraceuticals in the last 30  days before inclusion.

In case of worsening of the clinical signs, appearance of new skin or systemic diseases, changes of diet, or unwillingness of the owner to continue the administration of product A or B, the selected dogs were dropped out of the study.

Forty-three dogs with FAD were included and completed the study, of those 24 received product A and 19 product B. Dogs belonging to the study groups were matched for sex (p=0.45), age (mean group A 5.5 years ±3.9 vs mean group B 4.9 years ±3.1, p=0.57) and bodyweight (mean group A 8.9 kg ±5.0 vs mean group B 8.7 kg ±6.2, p=0.87).

The dogs were of different breeds and sex (Table 2, Table 3).

The average age of the dogs included was 4,9 years (1 to 11 years) and 5,2 years (1 to 12 years) respectively for group A and B.

In both groups, a significant decrease of mCADLI, opVAS and vpVAS at days 30 and 60, compared to day 0, was observed. No differences were recorded in these values between day 30 and day 60 (Table 4, Table 5, Table 6).

At baseline, the mCADLI scores of Group B were significantly higher than Group A (95% CI: -4.0 to 0.0, p < 0.05), no differences were present at D30 and D60.

No differences were observed in opVAS and vpVAS between the study groups at baseline. In Group B, lower values of vpVAS were observed at D30 (95% CI: -2.5 to 0.0, p < 0.05) but not at D60, while the median values of opVAS in Group B were lower compared to Group A at both D30 (95% CI: -2.5 to 0.4, p < 0.01) and D60 (95% CI: -3.0 to 0.3, p < 0.05).

Flea allergy dermatitis is a common pruritic disease in dogs. The affected animals can have skin lesions of different severity as papules, crusts, excoriations, self-induced alopecia and secondary skin infections. As for the other pruritic skin diseases the diagnosis is based on identification and/or exclusion of fleas based on effective flea control 123456789. Subjects included in this prospective study had a previous diagnosis of flea allergy dermatitis based on these criteria. Other parasitic or allergic diseases have been excluded before the inclusion. All the animals selected received the same antiparasitic therapy to decrease the BIAS for statistical analysis.

Itch create a great discomfort and the decrease and control of it can be obtained through different treatments, depending on the severity of skin lesions. In mild cases essential fatty acids and/or aliamides can be useful to control erythema and decrease symptoms. In more severe cases, steroids and IL-31 inhibitors are of choice, alone or associated with nutraceuticals. This study included only dogs with mild pruritus related to FAD 137 associated with mild skin lesions.

Supplementation with dietary essential fatty acids is commonly recommended in allergic skin diseases such as flea allergy dermatitis and atopic dermatitis. Nutraceuticals help to improve the skin barrier and decrease water loss as well as decrease the production of inflammatory cytokines and inflammation 721. Skin inflammation can be affected by omega 3 and 6 fatty acids (FAs): ω-6 FAs are transformed into prostaglandins (PG) E-2 and leukotriene-4, while ω-3 FAs are changed into PGE-3 and leukotriene-5 that are less pro-inflammatory than their corresponding ω-6 fatty acid isomers. Neutrophil activation is hindered by these newly synthesized ω-3 based mediators resulting in a reduction of allergic response 72122. In this study we used a nutraceutical enriched in blackcurrant oil. This vegetable oil is a natural source of polyunsaturated fatty acids (PUFAs), especially gamma-linolenic acid and stearidonic acid. Previous studies have demonstrated the efficacy of black currant oil in decreasing pruritus and erythema in atopic dogs 721.

Hemp (Cannabis sativa) is an angiosperm plant belonging to the Cannabaceae family. Hemp contains a large number of compounds, such as phytocannabinoids, flavonoids, amino acids, fatty acids, vitamins, etc. 23. The endocannabinoid system (ECS) is present in many species: from mammals to more primitive phyla (Cnidaria). The pervasive and early emergence of ECS in the evolution of life indicates its biological importance. ECS is composed of endogenous ligands, G-protein-coupled receptors (CBR) and a group of enzymes to degrade and recycle the ligands. Some of the receptors can modulate a healthy immune response by increasing anti-inflammatory and decreasing pro-inflammatory pathways. CBRs are able to induce apoptosis of T cells and induce inhibition of T cell proliferation while promoting the production of regulatory T cells. CBRs can be activated also by plant cannabinoids: phytocannabinoids can induce apoptosis of immune cells by activation of CD95, increase the production of the anti-inflammatory cytokine IL10 and reduce TNF-α and other pro-inflammatory cytokines 24. Cannabidiol (CBD) is the main non-psychotropic cannabinoid found in Cannabis sativa. In recent years CBD has been used as a nutraceutical for its high content of polyunsaturated fatty acids (especially ω-3 series) and phenolic compound as a support in inflammatory diseases in dogs and humans 23. Cannabidiol has been used in atopic dogs as an adjunct therapy to decrease pruritus with good results 25.

The association between blackcurrant oil and CBD oil tested in this prospective study was useful in decreasing the clinical signs of FAD. Even if control of the allergic response to flea bites is obtained by preventing parasite feeding with antiparasitic treatment, this treatment alone is not enough to decrease the erythema, pruritus and secondary self-induced skin lesion already present. Product A was able to decrease the opVAS (p < 0.001), vpVAS (p < 0.001) and mCADLI (p < 0.05) already during the first month of use.

Vitamin E is a fat-soluble nutrient that is essential for animal s body to develop strong and healthy muscles and healthy circulatory and immune systems. It s also an antioxidant, helping to protect cells from damage caused by free radicals. In dogs with excessive moulting, thin or balding patches on their coat, and dry or flaky skin, Vitamin E supplementation may improve the conditions 26. In more recent years it has been reported to contribute to decrease many other inflammatory diseases of dogs and cats 2728. The presence of vitamin E can contribute to the anti-inflammatory effects of the formulation.

Palmitoylethanolamide (PEA), belongs to aliamides, a family of fatty acid amides whose name comes from their mechanism of action, the Autacoid Local Injury Antagonism (ALIA) 23. PEA is a bioactive lipid compound and an endocannabinoid-like molecule. Endocannabinoids and their mediators, such as PEA, are produced in response to inflammation and tissue damage and play a key role in controlling cutaneous inflammation and immunity 13. PEA is generally used in allergic skin diseases with moderate clinical signs to decrease pruritus and skin lesions. In cats, this nutraceutical has been proven to decrease clinical signs of allergic dermatitis and reduce the use of systemic steroids 29. Even if there are no specific indications for its use in canine FAD, the anti-inflammatory effects are well known 30. In this study, all the dogs that received the product based on PEA had a significant decrease of opVAS (p < 0.05), v pVAS (p < 0.01) and mCADLI (p < 0.01) within the first month of treatment.

Active nutraceutical components of products A and B are commercialized respectively in pearls and capsules with inactive gel components. These forms of presentation are necessary to avoid oxidation of active components.

This study has some limitations. Forty-three dogs are a small group of dogs for a prospective study but FAD is an uncommon diagnosis nowadays in developed countries, thanks to extensive use of antiparasitic treatments. Animals selection was performed by a veterinary dermatologist as a guarantee of the correct diagnosis. It could have been useful to compare the efficacy of tested products in decreasing pruritus with a group of dogs treated only with antiparasitic drugs ( control group ), but this was avoided to diminish animal discomfort. Skin allergic reaction can decrease only by avoiding exposure to fleabite, but secondary lesions due to itch are responsible for major dog discomfort and can lead to skin barrier damage, alopecia, crusts and secondary skin infections. Despite extensive research and always new antiparasitic drugs flea infestation is still a major problem in lots of countries. 95% of the flea population is represented by immature stages present in the environment versus only 5% of adults on the host. Pupal stages can survive a long time without animal presence and are highly resistant to challenging environmental conditions, together with the high and fast rate of population growth provide excellent explanations for failure of parasite control 31. For all these reasons, even with complete systemic antiparasitic treatment is impossible to achieve a quick control of the environmental infestation and a recrudescence of itch, in allergic dogs can be expected during the third week of treatment. In study trials two to four months are necessary to achieve complete flea control and subsequent remission of clinical signs of FAD in animals housed in infested environments 10. Even with a complete antiparasitic treatment allergic reaction takes time to decrease. As a confirmation of this hypothesis, at day 30 some of the included dogs had still positive pVAS and mCADLI values. FAD affected dogs generally receive antiinflammatory drugs such as steroids or IL-31 inhibitors to decrease inflammatory reactions and avoid self-inflicted skin lesions 1712. Nutraceuticals with antiinflammatory action can avoid the use of systemic drugs in mild cases.

Another limitation of the study is related to the different age, sex and breed of the selected dogs. It was not possible to assess by statistical analysis if differences in these parameters had influenced the results, but the decrease of mCADLI, opVAS and vpVAS in both groups seems to confirm that age, sex and breed are not significant on the success of the nutraceutical anti-pruritic effects.