This study was designed as an open uncontrolled trial. Six dogs with chronic non-seasonal atopic dermatitis were selected. Dogs were enrolled based on itching intensity and on a history of allergic dermatitis. Inclusion to the study also was based on elimination of resembling non-immune-mediated pruritic dermatoses by clinical assessment only. Inclusion and exclusion criteria followed those of a previously published clinical trial [5]. Briefly, dogs were otherwise healthy, were not, and did not require, active treatment for other conditions; dogs were not receiving and had not received any new form of treatment for AD for 8 weeks (immunosuppressants, antibiotics, antihistamines).
The owners received explanation about the trial, and the study followed those who accepted the free and informed consent form. The study was performed under the licence of the Committee for the Use of Animals in Research of Imunova Análises Biológicas LTDA., protocol 003.2018. All dogs were treated with the experimental compound. Five animals received 10,000 IU/kg of rhIFNα-14 orally for 8 weeks and one dog received it for only five weeks due to a sprained spleen, which led to an emergency surgery.
Each dog was evaluated for the presence or absence of papule, macula, pustules, dandruff, skin scabs, lichenification, nodules, tumors, hyperkeratosis, vesicles, hyperpigmentation, erythema, and alopecia by the veterinarian once a week. Efficacy outcomes were based on a veterinary-conducted simplified version of the CADESI (canine atopic dermatitis extent and severity index) score [5, 6]. Owner assessment of the status of the dog was also collected during the clinical consultation. An analogical scoring table was used by owners for quantification of itching [5].
Blood samples were collected for haematological analysis. Samples were collected every fifteen days, totaling four samples from each animal. Haematological analyses consisted of determination of blood cell counts. For the determination of adverse events, deviations in blood parameters that occurred following drug administration were counted.
Skin microbiota was assessed by next-generation sequencing. Skin swabs were collected biweekly, always from the cranial face of the right hand. DNA was extracted from swabs using the ZR Fecal DNA MiniPrep® (Zymo Research). The variable V4 region of 16S rRNA was amplified using the universal primers 515F and 806R (Caporaso et al., 2011). PCR conditions were as follows: 94°C, 3 min; 18 cycles of 94°C, 45 s, 50°C, 30 s e 68°C, 60 s; followed by 72°C, 10 min. These amplicons were then sequenced (Illumina MiSeq). Sequencing reads were normalized at 8010 reads and analysed with the QIIME (Quantitative Insights into Microbial Ecology) platform (Caporaso et al., 2010, 2011). Sequences were classified into bacterial genera through the recognition of operational taxonomic units (OTUs) based on the homology at 97% of the sequences when compared to the SILVA 128 ribosomal sequence database [2017 release] (Yilmaz et al., 2013). Basic diversity was assessed using QIIME. Shannon’s homogeneity index was calculated as previously described [7].
Statistical analysis was conducted using GraphPad Prism 8. Tests are indicated with each figure.