Quantification of Demodex load in canine skin samples by real time qPCR

MARTÍNEZ S.; SASTRE N.; VELARDE R.; FRANCINO O.; FERRER L.

Copenhagen

Conferencia; 8th Conference of the Scandinavian-Baltic Society for Parasitology (SBSP) and the Annual Meeting of the European Veterinary Parasitology College (EVPC); 2019

European Veterinary Parasitology College

Demodex mites are considered normal inhabitants of the skin of the dog and of most mammals. Canine demodicosis is the result of an overpopulation of Demodex mites in the skin. The diagnosis is based on the detection of a high number of mites in skin scrapings. However, there is a lack of an objective and reliable method of quantifying the number of mites in a sample. The aim of this study was to develop a technique for quantifying the Demodex load in skin samples. For this purpose, we extracted and quantified the DNA of isolated Demodex mites, and of 10 and 100 mite pools. A qPCR real time was performed using primers that amplified a fragment of the 18S rRNA gene. Ten-fold dilutions of the DNA extracted from the isolated mites were used to elaborate the standard curve. The mite pools were used as controls of the standard curve. Moreover, forty-four 4-mm skin biopsies obtained post-mortem from nine dogs that died due to different causes but without skin disease, were used to determine the number of mites in the skin of healthy dogs. The total DNA of each skin biopsy was extracted and the qPCR was performed. Only six of the 44 sites sampling were positive (more than 1 mite/4 mm skin). This simple technique can be a useful tool for the diagnosis of canine demodicosis and for the follow-up of the dogs after the treatment.