Assessing the Efficacy of Topical Fluralaner for the Treatment of Demodex musculi Infestations in NSG Mice (Mus musculus)

Study timeline. Starting at week 0, Demodex musculi-infested NSG mice received biweekly topical fluralaner at doses of either 100 mg/kg or 250 mg/kg (4 doses total). Also beginning at week 0, all animals received pelt swab, deep skin scrape, and fur pluck diagnostics monthly until end point (week 16). On weeks 0 and 4, all diagnostics were performed before treatments. At the end point, animals were humanely euthanized for postmortem analyses. Created in BioRender. Morrill, C. (2024) BioRender.com/k96r501.

A depiction of the step-by-step process for pelt scroll analysis. After humane euthanasia, Mayo scissors were used to trim a rectangular portion of the ventral pelt from the inguinal to axillary areas. The remaining pelt was also collected. Both pelt sections were placed flat on bibulous paper and then rolled in a caudal-to-cranial direction, creating a “Swiss roll.” The Swiss roll was immersion fixed in 10% neutral buffered formalin at room temperature for 48 h before collecting 2 smaller sections at midline. Both sections were placed flat in cassettes, fixed using the same method for 24 h, and then sent to a commercial laboratory for processing and staining with hematoxylin and eosin. Created in BioRender. Morrill, C. (2024) BioRender.com/h41e149.

Demodex musculi infestation in mice (as measured by qPCR) is reduced by topical fluralaner treatment. Average infestation levels between the 2 doses do not differ at any timepoint (including week 12). Data are plotted as least square means ± SE. Mice were treated at baseline, 2, 4, and 6 wk (black triangles). Swabs for qPCR at baseline and 4 wk were taken before treatment. Given that the doses did not differ, Tukey post hoc tests were performed on the mean of the 2 treatments at any given week. Thus, time points with the same letter do not differ in mean DNA levels. Accordingly, by 6 wk after the cessation of treatment, we saw that Demodex DNA levels began to increase.

Relative Demodex musculi density (percentage of follicle profiles infested) in mice, as a function of Caudal-Cranial position. For each section, follicle profiles were binned into 10 quantiles (that is, 0% to 10%, 10% to 20%, etc.). Within each bin, data are represented as the LSM+/-SE. For clarity, dorsal and ventral sections are presented on separate panels. However, all 4 combinations of dose compared with section were analyzed together. Overall, the data followed a “U” shaped curve (P = 0.0082). Only the 250-mg/kg dorsal sections had an inflection point that differed from the average curve (P < 0.0001), such that this treatment showed a progressive decrease in Demodex musculi density. The other 3 conditions did not differ from the overall “U”-shaped curve (all P > 0.05). Note that data were analyzed as a repeated measures mixed model such that each animal acted as its own control and the analysis inherently corrects for individual differences. Created in BioRender. Morrill, C. (2024) BioRender.com/s75e729.

Demodex musculi infestation burden (as measured by qPCR) in mice differs as a function of agreement between raters of the same skin scrape slides. Data are plotted as least square means ± SE. Given that the doses did not differ, the mean −ΔCT is plotted. Datapoints that did not differ after Tukey adjustment are given the same letter. Slides where raters either agreed that the slide was negative or disagreed did not differ in −ΔCT . However, −ΔCT was significantly lower than the level at which raters agreed that the slide was positive.