Comparison of Diagnostic Methods and Sampling Sites for the Detection of Demodex musculi

Abstract

Demodex mites are microscopic, cigar-shaped, follicular mites often regarded as commensal microfauna in mammals. Although Demodex spp. can cause dermatologic disease in any immunocompromised mammal, they are rarely reported in laboratory mice. Recent identification of Demodex musculi in a colony of immunodeficient mice with dermatitis afforded us the opportunity to investigate the comparative sensitivity of 4 antemortem diagnostic techniques to detect D. musculi-superficial skin scrape (SSS), tape impression (TI), fur pluck (FP), and deep skin scrape (DSS)-which we performed on 4 anatomic sites (face, interscapular region [IS], caudal ventrum [CV], and caudal dorsum [CD]) in 46 mice. DSS had an overall detection rate of 91.1% (n = 112 tests), with the highest detection rates in IS (93.5%), CV (89.1%), and CD (90.0%). The detection rates for SSS (62.5%; n = 112 tests), TI (57.5%; n = 138 tests), and FP (62.7%; n = 158 tests) were all lower than for DSS. IS was the most reliable site. Results from combined FP and DSS samples collected from IS and CV yielded 100% detection, whereas the face was not a desirable sampling site due to inadequate sample quality and low detection rate. Demodex eggs and larvae were observed from FP more often than DSS (19.0% of 158 tests compared with 14.3% of 112 tests). In a subset of samples, an 18S rRNA PCR assay was equivalent to DSS for detection of mites (both 100%, n = 8). We recommend collecting samples from both IS and CV by both FP and DSS to assess for the presence of D. musculi and performing further studies to assess whether PCR analysis can be used as a diagnostic tool for the detection of Demodex mites in laboratory mice.

Figure 1.

Mite yield per sample by site, displayed as a box plot. The middle line represents the median, the bounds of the box represent the upper and lower quartiles, and the whiskers represent the lowest and highest numbers of mites and eggs for each test–site. The face was sampled by fur pluck and tape impression only. All values were significantly different from one another (‡, P ≤ 0.0001, Kruskal–Wallis test).

Figure 2.

Graphical representation of the Spearman rank correlation between the number of mites per test site and the number of hairs counted at each test site for SSS, TI, and FP samples from each mouse. The Spearman correlation coefficient was 0.47 (P = 0.001).

Figure 3.

Appearance of tape-based and oil-based samples. (A) Tape impression test under low magnification (scale bar, 300 μm). The mite, highlighted in the box, is shown at higher power in the inset (scale bar, 30 μm). (B) High-power magnification (scale bar, 100 μm) of FP. Arrowheads indicate guanine concretions in transparent, dead mites. (C) Life stages of D. musculi as observed in mineral oil from either DSS or FP (scale bar, 20 μm). From left to right, egg, hexapod larvae, adult male, and adult female. Nymphs were not observed on ectoparasite tests. Arrowheads indicate guanine concretions.

Figure 4.

Representative PCR samples separated on an agarose gel. Lane sequence: positive control (D. canis), Lane 1, whole skin (frozen); 2, whole skin (frozen); 3, whole skin (formalin-fixed); 4, whole skin (formalin-fixed); 5, whole skin (formalin-fixed); 6, tape impression, individual mite; 7, tape impression, individual mite; negative control (water), and DNA ladder. Size markers are indicated. The PCR amplicon was 537 bp.

Figure 5.

Mite species with highly homologous 18S rRNA sequences.