Extraction and characterization of DNA from Demodex canis.

Canine demodicosis occurs when Demodex mites colonize hair follicles in large numbers. Their characterization is based on morphologic differences. A molecular genetic analysis within Demodex mite species and between other mite species would better characterize their phylogenetic diversity and could assist in treatment strategies as well as improve our understanding of disease progression. Demodex canis mites were obtained during routine deep scrapings of client-owned dogs and collected in Tris EDTA buffer (TE). The mites were separated from debris with a microhematocrit suction apparatus. Their outer chitin layer was ruptured using chitinase, glass beads, and vortexing. Membranes and proteins were disrupted with Sodium Dodecyl Sulfate (SDS) and proteinase K. Phenol/chloroform extraction, ethanol precipitation, and resuspension in TE further purified the nucleic acids. One hundred ng of mite DNA was amplified by a Random Amplified Polymorphic DNA (RAPD) method as DNA yields were low. The amplified DNA was randomly cloned into plasmids and 10 clones were sequenced. The BLAST program in Macvector version 9.0TM was used to identify sequences that matched insect-type DNAs. Two oligonucleotide primer sets were designed to amplify the mite DNA by polymerase chain reaction (PCR). One sequence was homologous to ubiquitin and the other matched a bacterial species related to those commonly found as commensals in insects. The latter primer set also amplified a similar sequence from flea and mosquito DNA. Neither amplified dog DNA. This method was successful in isolating DNA from Demodex canis mites. Oligonucleotide primers were developed that amplify Demodex sequences and will be useful in analyzing phylogenetic relationships and may assist in understanding demodicosis progression. Extraction and Characterization of DNA from Demodex canis Elizabeth Toops, MS, DVM1 Byron Blagburn, MS, PhD2 Scott Lenaghan, PhD3 Robert. Kennis, MS, DVM, DACVD4 John MacDonald, MEd, DVM, DAVCD4 Christine Dykstra, MS, PhD2 1Pittsburgh Veterinary Specialty and Emergency Center, Pittsburgh Veterinary Dermatology, Pittsburgh, Pennsylvania, USA 2Department of Pathobiology, Auburn University College of Veterinary Medicine, Auburn, Alabama, USA 3Department of Mechanical, Aerospace and Biomedical Engineering, University of Tennessee, Knoxville, Tennessee, USA 4Department of Clinical Sciences, Auburn University College of Veterinary Medicine, Auburn, Alabama, USA