Otitis externa is one of the most common diseases in dogs. It is associated with bacteria and yeast, which are regarded as secondary causes. Cerumen is a biological substance playing an important role in the protection of ear skin. The involvement of cerumen in immune defense is poorly understood. MicroRNAs can modulate the host immune response and can provide promising biomarkers for several inflammatory and infectious disorder diagnosis. The aims of this study were to profile the cerumen miRNA signature associated with otitis externa in dogs, integrate miRNAs to their target genes related to immune functions, and investigate their potential use as biomarkers. Cerumen was collected from healthy and otitis affected dogs and the expression of miRNAs was profiled by Next Generation Sequencing; the validation of the altered miRNAs was performed using RT-qPCR. The potential ability of miRNAs to modulate immune-related genes was investigated using bioinformatics tools. The results pointed out that 32 miRNAs, of which 14 were up- and 18 down-regulated, were differentially expressed in healthy vs. otitis-affected dogs. These results were verified by RT-qPCR. To assess the diagnostic value of miRNAs, ROC analysis was carried out, highlighting that 4 miRNAs are potential biomarkers to discriminate otitis-affected dogs. Bioinformatics showed that cerumen miRNAs may be involved in the modulation of host immune response. In conclusion, we have demonstrated for the first time that miRNAs can be efficiently extracted and quantified from cerumen, that their profile changes between healthy and otitis affected dogs, and that they may serve as potential biomarkers. Further studies are necessary to confirm their diagnostic value and to investigate their interaction with immune-related genes.

Otitis externa is a common multifactorial disease in dogs, characterized by broad and complex modifications of the ear microbiota. The goal of our study was to describe the ear cerumen microbiota of healthy dogs, within the same animal and between different animals, and to compare the cerumen microbiota of otitis affected dogs with that of healthy animals. The present study included 26 healthy dogs, 16 animals affected by bilateral otitis externa and 4 animals affected by monolateral otitis externa. For each animal cerumen samples from the right and left ear were separately collected with sterile swabs, and processed for DNA extraction and PCR amplification of the 16S rRNA gene. Amplicon libraries were sequenced using an Ion Torrent Personal Genome Machine (PGM), and taxonomical assignment and clustering were performed using QIIME 2 software. Our results indicate that the bacterial community of the cerumen in healthy dogs was characterized by extensive variability, with the most abundant phyla represented by Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Fusobacteria. The analysis of both alpha and beta diversity between pairs of left and right ear samples from the same dog within the group of affected animals displayed higher differences than between paired samples across healthy dogs. Moreover we observed reduced bacterial richness in the affected group as compared with controls and increased variability in population structure within otitis affected animals, often associated with the proliferation of a single bacterial taxon over the others. Moreover, Staphylococcus and Pseudomonas resulted to be the bacterial genera responsible for most distances between the two groups, in association with differences in the bacterial community structure. The cerumen microbiota in healthy dogs exhibits a complex bacterial population which undergoes significant modifications in otitis affected animals.