Phylogenetic relationships and new genetic tools for the detection and discrimination of the three feline Demodex mites.

Two feline Demodex mite species have been described as causative agents of feline demodicosis, until recently a third species was detected. We provide an updated analysis on the phylogenetic relationship of Demodex mites. In addition, we present the first qPCR assay for the detection and differentiation of all three feline mite species in a single reaction. Specimen of Demodex cati, Demodex gatoi, and the recently discovered third species were collected from skin scrapings and fecal flotation for DNA extraction, conventional PCR, sequencing, and alignment. A total of 24 sequences of the partial 16S rRNA gene were used to estimate the evolutionary divergence in a p-distance model and a maximum likelihood phylogenetic tree. For the qPCR assay, new primers and fluorescent probes for the simultaneous detection of all three feline Demodex mites were designed. A consensus fragment of 351 bp was phylogenetically analyzed. The third species sequence of our study shares 98.6 % similarity to the available sequence in GenBank®. It is most similar to D. gatoi (82.41 %) and most distant to the canine Demodex injai (78.28 %). In contrast, D. gatoi is most similar to human Demodex brevis (87.01 %). The multiplex qPCR detected and discriminated the three different mite species in one reaction. The detection limit is ≤1.4 ng of mite DNA. The three feline Demodex species have distinct genotypes and did not cluster in one genetic clade. The species differentiation and assessment of evolutionary relationships will ultimately support correct diagnostics and treatment approaches.