Assessing the Efficacy of Topical Fluralaner for the Treatment of Demodex musculi Infestations in NSG Mice (Mus musculus)
Demodex musculi is a microscopic mite that inhabits the hair follicles of mice. It is the most frequently diagnosed mite in laboratory colonies and infests up to 13% of mice shipped from noncommercial vendors. Depending on the mouse infested, D. musculi may cause immunomodulation or debilitating clinical signs that can impact experimental reproducibility. Current treatment options are limited, and many infested mice are ultimately euthanized. Fluralaner, an isoxazoline-class ectoparasiticide, successfully treats demodicosis in other species. However, to our knowledge, its efficacy in treating demodicosis in mice has not been reported. This study aimed to explore the effects of 2 different doses of topical fluralaner for the treatment of D. musculi in NSG mice. We hypothesized that both doses of topical fluralaner would eliminate infestations in all animals. Female NSG mice infested with D. musculi were treated with topical fluralaner at 2 different doses (100 mg/kg or 250 mg/kg) every 2 wk for 4 treatments. Efficacy was determined by routine deep skin scrapes, fur plucks, quantitative PCR, and a pelt scroll histologic analysis. All diagnostic modalities indicated that no cage in either treatment group was successfully cleared of mites. However, quantitative PCR indicated a significant reduction in mite burden following treatments. After treatments ceased, mite burden remained similar between time points and then increased. There were no significant differences in mite burden between treatment groups at any time point. These data suggest that topical fluralaner could be useful in treating D. musculi infestations in NSG mice. However, additional research is needed to determine if higher and/or more frequent doses will improve efficacy.
Introduction
The Demodex genus encompasses at least 65 species of microscopic, “cigar-shaped,” prostigmatid mites that inhabit the hair follicles of mammals.1–8 Demodex mites are becoming increasingly recognized for their potential impact on animal research, especially in mice.5,9 When infested, immunodeficient mice—notably, those with downregulated T-helper 2 cell responses—may exhibit blepharitis, pruritus, dermatitis, and potentially lethal comorbidities.3,4,8 Immunocompetent mice do not exhibit clinical signs but still have immunomodulation that may confound research.4,5,10 They may also serve as an asymptomatic reservoir and propagate infestations within vivaria.
Of the 6 species of Demodex known to infest wild Mus musculus, only Demodex musculi has been documented on laboratory mice.4,5 It is the most common mite in contemporary mouse colonies.4 Between 2003 and 2020 at one commercial diagnostic laboratory, D. musculi was present in 3.52% of submitted mouse samples.9 Other academic facilities estimate the prevalence of Demodex mites to be as high as 13% for animals coming from noncommercial vendors.10 As more transgenic mouse strains are developed, the extramural transfer of mice from noncommercial vendors will likely increase. Many programs do not exclude Demodex in their SPF programs, which further increases the risk of transmission.
Current therapies for demodicosis (Demodex infestation) in mice are limited. Outside of strain rederivation, the most effective treatment is weekly topical application of moxidectin and imidacloprid (Advantage Multi; Bayer Healthcare LLC, Shawnee Mission, KS) for 8 to 12 wk.4,11 Both options can be time and/or cost prohibitive, especially when large colonies are involved. In addition, moxidectin and imidacloprid treatments may cause high mortality in nursing pups—among other posttreatment effects that have not been fully evaluated.10–12 Investigating new compounds to treat demodicosis may improve the current standard of treatment and reduce the need to euthanize infested animals.
One promising treatment option is fluralaner, an isoxazoline-class ectoparasiticide.13 To the best of our knowledge, there is currently only one study documenting the use of fluralaner in laboratory mice.14 Fluralaner is commercially available (Bravecto; Merck, Rahway, NJ) as a flea/tick preventative for both dogs and cats and is used off-label to treat demodectic mange.15–17 In dogs and cats, fluralaner remains efficacious for 3 mo after a single dose and has a wide margin of safety.13,18 Once applied or ingested, fluralaner distributes in the blood, continuously eluting into the skin and adjacent fluids (for example, sebum). After being consumed by an arthropod like Demodex, fluralaner inhibits the GABA and L-glutamate-gated chloride channels, causing paralysis and death of the parasite.13,19 For the treatment of generalized demodicosis in dogs, a single topical dose of fluralaner has been found to be more efficacious than moxidectin and imidacloprid administered at multiple time points.15 In addition, fluralaner was used to successfully treat a mixed Demodex infestation in a Syrian hamster (Mesocricetus auratus) and was used to reduce the tick burden in wild Peromyscus mice.1,20,21 Together, these findings suggest that fluralaner may be useful for treating Demodex infestations in mice.
The goal of this study was to determine if topical fluralaner would be efficacious in treating D. musculi infestations in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. NSG strain mice are highly valuable in research because of their immunodeficiency, but this also makes them susceptible to D. musculi.4 We selected the topical route of administration over oral for ease of administration and more practical applicability. Here, we test 2 different biweekly doses of topical fluralaner in D. musculi infested, NSG mice. Deep skin scrapes (DSS), fur plucks (FP), and quantitative polymerase chain reaction (qPCR) of pelt swabs (PS) were used to characterize the infestation levels at monthly time points, and a histologic pelt scrolling analysis was performed at the end point. We hypothesized that both topical fluralaner doses would eliminate mite burden in the NSG mice.
Ethical review.
All procedures performed in this study were preapproved by Stanford University’s Administrative Panel on Laboratory Animal Care (IACUC). Stanford University is AAALAC accredited. All mice were treated in accordance with The Guide for the Care and Use of Laboratory Animals.22
Materials and Methods
Experimental animals.
In April 2022, a shipment of transgenic NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (Mus musculus) was imported from another academic institution. The transgene encoded for diphtheria toxin receptor under the CD169 promoter. During the quarantine period, one died. Rodentibacter pneumotropicus was identified as the cause of death, and D. musculi was found incidentally. The mice were bred in the quarantine room to maintain the strain as it was rederived. In July 2023, individuals (aged 9 to 11 mo) from this colony were cohoused with 5- to 10-wk-old, naïve, female, NSG mice bred within an inhouse barrier facility. Before being cohoused, the infested mice received 2 wk of enrofloxacin in their drinking water (approximately 85 mg/kg/day) to eliminate the Rodentibacter infection.23 Animals were left together for 5 mo to ensure the naïve NSG mice were infested with D. musculi. In addition, 2 cages of 17-wk-old NSG mice used in this study were infested with Demodex through a different, novel method that was developed in-house. A single, Demodex-positive animal was humanely euthanized and rubbed to the naïve animals for approximately 10 seconds, after which multiple FP were taken from the infested animal and left in the naïve cages. This occurred on the same day as cohousing, meaning that mites in these cages were also allowed to populate for 5 mo before the study start.
By study start, these methods yielded an infested population of 26 female NSG background mice. Twenty-two mice were 6 to 9 mo old, and the originally infested 4 were between 14 to 16 mo old. The older animals were included to optimize animal use. All animals infested with D. musculi for this study were confirmed positive on DSS and/or qPCR at least one week before start.
Animals were housed within individually ventilated cages (Innovive, San Diego, CA) with ALPHA-Dri bedding, a chlorinated water bottle (Innovive, San Diego, CA), and Enviro-dri (Lab Supply, Fort Worth, TX) for enrichment. Animals were fed an irradiated rodent chow (Teklad Global 18% Protein Rodent Diet 2018; Harlan Laboratories, Madison, WI) and maintained on a 12 h:12 h dark:light cycle at 68 to 79 °F (20 to 26 °C) between 30% and 70% relative humidity. Infested animals from the original shipment were maintained within the rodent quarantine room on their own half-rack while the other side was solely occupied by the animals in this study. Cages in the study were changed every 2 wk by experienced animal caretakers except during fluralaner dosing, when an author (CM) performed cage changes immediately after dosing.
The rodent quarantine room where the mice were housed restricts access to veterinary personnel only. The vivarium is SPF for the following agents: minute virus of mice, mouse hepatitis virus, mouse rotavirus, Theiler murine encephalomyelitis virus, Sendai virus, murine adenovirus 1 and 2, ectromelia virus, lymphocytic choriomeningitis virus, pneumonia virus of mice, respiratory enteric virus 3 (Reovirus 3), Mycoplasma pulmonis, Aspiculuris tetraptera, Syphacia spp., Ornithonyssus bacoti, and other endo/ectoparasites. Our institution does not regularly screen for D. musculi. Routine sentinel serology, necropsy, and PCR testing were used to maintain health status throughout this study.
Study design.
Mice included in this study tested positive for D. musculi on DSS, FP, and/or PS qPCR at least 1 wk prior to start (Figure 1). Four cages (3 to 4 mice each) were randomly allocated to each treatment group (100 or 250 mg/kg). On day 0, each animal was weighed to calculate the appropriate dose of fluralaner to be administered that day. Next, PS, DSS, and FP samples were collected for diagnostics (see Antemortem sampling). Mice were then gently restrained on the food hopper by using a single hand to loosely grasp the tail base while simultaneously scruffing the skin of the dorsum. The other hand then used a 200;μL micropipette to apply a topical dose of fluralaner (Bravecto feline 2.6 to 6.2 lb, Merck, Madison, NJ; lot no. AT2677A) at either 100 mg/kg (n = 14) or 250 mg/kg (n = 12) to the interscapular area. After application, the micropipette tip was used to gently spread the compound over the skin. The drug was not diluted (280 mg/mL), and all doses were between 10 and 31 μL. The skin was not clipped or prepared in any way before the fluralaner application. Animals were reweighed and redosed based on new weights every 2 wk for a total of 4 doses (at either 100 mg/kg or 250 mg/kg). All diagnostics (DSS, FP, and PS for qPCR) were repeated every 4 wk from day 0 until week 16. When diagnostics and treatments fell on the same day, diagnostic samples were collected first. At week 16, diagnostics were performed and the mice were then euthanized for postmortem analyses.
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-135
Given current diagnostic modalities and ease of intracage mite transmission, it would be very difficult (if not impossible) to standardize the mite burden for each mouse in this study. In addition, no mouse from the original colony appeared to spontaneously clear D. musculi over the year preceding this study (nor is this phenomenon likely given our experience). These factors—in addition to our repeated measures analyses—essentially eliminate the need to use a saline/vehicle drug control group. Therefore, in an effort to minimize animal use, this group was omitted. However, known positive and negative control PS were submitted with every qPCR run to ensure validity.
The PREPARE and ARRIVE 2.0 Guidelines were consulted before and throughout this study.24,25 Randomizations to allocate cages to treatment groups were performed using an online randomizer (https://www.random.org/lists).
Sample size.
In this study, we employed complex factorial repeated measures analyses, for which a priori power formulas do not exist. To optimize sample sizes, we used Mead’s Rule because it does not need apriori estimates of effect size or variance.26
Experimental procedures.
Antemortem sampling (PS, DSS, and FP).
Each mouse received a PS, DSS, and FP every 4 wk after initial fluralaner treatment. All procedures were performed in a laminar flow hood, and gloves were changed between cages. For all procedures, mice were gently restrained by the tail base and placed over the food hopper of the cage. Two sticky swabs (Pigeon Corporation, Tokyo, Japan) were then rolled over the entire pelt, with repeated rolls over the interscapular and caudal abdominal areas. The swab tips were broken off and placed in the same microcentrifuge tube for qPCR analysis. Next, while lightly scruffing the mouse, a sterile #15 blade coated in mineral oil was angled at 45° to gently scrape the skin of the left or right dorsal interscapular area approximately 15 times (sides alternated at each time point). Finally, mosquito forceps were used to epilate an approximately 3- to 5-mm area of fur from the same dorsal interscapular area. The forceps were submerged in 70% EtOH between sample collections and wiped dry before use with a clean paper towel. DSS and FP samples were plated together on a glass slide with a cover slip for light microscopy. With the exception of the third time point (week 12), samples were submitted to a board-certified veterinary clinical pathologist (FHdA) for immediate analysis. Samples were then reviewed by another veterinarian (CM) to improve diagnostic sensitivity (4 to 8 h later in the same day). At the third time point, a different veterinarian (RC) reviewed the slides first. Identification of at least one mite at any life stage (dead or alive) was considered a positive result. At 16 wk, samples were collected before euthanasia to reduce the likelihood of false negative results. All reviewers were blinded to the treatment group until the end of the study.
qPCR testing of PS.
PS for qPCR were performed in duplicate, generating 2 swab tips, which were placed into a single, 2-mL microfuge tube. The tubes were immediately submitted for qPCR analysis at the Animal Diagnostic Laboratory, Department of Comparative Medicine, at Stanford University. Total nucleic acids were extracted from the swabs using a commercial kit (NucleoSpin VET; 740842; Takara Bio). The extracted DNA was analyzed using a commercial TaqMan assay targeting the D. musculi 18S ribosomal RNA genomic sequence (GenBank accession no. JF834894.1; assay ID: 02077683 CDRWFPH; cat no. 4400295; Life Technologies, Carlsbad, CA). In addition, a commercial qPCR assay targeting the mouse housekeeping gene 18S rRNA (assay ID: Mm04277571_s1; Life Technologies, Carlsbad, CA) was used concurrently. This served as an internal control to ensure DNA recovery and the absence of PCR inhibitors in nucleic acid extracted from PS. In addition, it allowed for direct comparison of mite:mouse DNA to quantify mite burden.
The qPCR reactions, using TaqMan™ probes, were performed on a QuantStudio 3 (Applied Biosystems; Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. Briefly, 50 ng of template cDNA in a volume 4 µL was added in duplicates to an optical PCR-grade 96-well plate along with 6 µL of master mix, consisting of 5 µL of 2× TaqMan Fast Advanced Master Mix (Applied Biosystems; cat. no. 4444557), 0.5 µL of custom TaqMan assay (containing 20× assay stocks of 5 µM probe and 18 µM of each primer), and 0.5 µL of sterile water.
The reaction conditions were as follows: initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 10 s, and annealing/extension at 60 °C for 30 s, with a final extension at 60 °C for 30 s. Negative controls (no template DNA) and positive controls (for Demodex amplicon sequence) were included in each qPCR plate.
Postmortem pelt analysis.
At 16 wk, mice were euthanized via CO2 inhalation in accordance with the AVMA Guidelines for the Euthanasia of Animals: 2020 Edition.27 After confirmation of death and cardiac exsanguination, a rectangular aspect of the pelt extending from the inguinal area to axilla was collected (the “ventral” pelt; Figure 2). The body walls were then opened and visceral tissues (heart, lung, liver, spleen, kidney, gonads) were collected and immersion-fixed in 10% neutral buffered formalin. The remaining skin was carefully undermined from the underlying tissues using Mayo scissors and dissected along the lateral aspects to create the “dorsal” pelts. The dorsal and ventral pelts were then placed flat with the fascial planes against bibulous paper and allowed to adhere. The pelts were then rolled into a “Swiss roll.” The caudal aspect was rolled first so that it would be in the center of the roll and the cranial aspect toward the periphery. After fixation at room temperature, both the dorsal and ventral scrolls were cut along the midline, and 2 sections for the dorsal and the ventral skin were generated. These sections were left in 10% neutral buffered formalin at room temperature for another 24 hours. All tissues (skin and visceral organs) were then transferred to 70% ethanol, processed, embedded in paraffin, sectioned at 5 µm, routinely stained with hematoxylin and eosin, and coverslipped. Tissues were blindly evaluated by a board-certified anatomic pathologist (JV-M) using an Olympus BX43 upright brightfield microscope.
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-135
For mite counting, one dorsal and one ventral skin section was assessed per mouse. Hair follicle profiles were individually tabulated and the number of recognizable D. musculi mites per follicular profile recorded—all while maintaining the caudal to cranial orientation. This allowed for determining the ratio of follicles containing mite profiles, the total number of mite profiles in a given skin section, and where along the caudal-cranial axis the mite profiles were present. Photomicrographs were obtained using the Olympus DP27 digital camera and the Olympus cellSens software.
Outcome measures and statistical methods.
In this study, we examined the results obtained from monthly diagnostics (qPCR, DSS, and FP) as well as the pelt scroll histology at the end point. Statistical analyses were performed in JMP Pro 17 for Windows and SAS 9.4 for Windows. JMP excels at visualization, model design, and assumption checking; while SAS excels at dealing with additional complexities, ease of post hoc testing, and human-readable data sharing.
qPCR.
To quantify mite burden in this study, we performed qPCR on PS to simultaneously amplify both mouse specific- and D. musculi-specific S18. The S18 gene is constitutively expressed in both species such that differences in threshold cycle values (ΔCT) are directly equivalent to log2(Demodex DNA/mouse DNA) and thus an indicator of mite burden. These values, expressed routinely as −ΔCT (so a higher value indicates a higher mite burden), were compared at monthly time points after biweekly dosing of topical fluralaner.
PCR data were analyzed using a repeated measures residual maximum likelihood (REML) mixed model, the gold standard for a time series of this kind. Best practices for model design, including sensitivity analysis (stress testing) were applied here and all subsequent analyses.28–32 Full details are provided in Data S1.
Pelt scrolls.
Each subject yielded a dorsal and ventral scrolled section for histology. For each section, each follicle profile (“space” where a mite could occupy) was identified in order, caudal to cranial, and the number of D. musculi profiles was counted. Our primary outcome variable was simply the number of follicle profiles containing mites, although we did look at the number of mites per follicular profile as a secondary measure. For data analysis, we divided each section into 10 caudal-cranial bins by percentile and summarized the number of follicle profiles, the total number of mites, and the total number of infested follicle profiles. These data were analyzed with a repeated measures REML mixed model. Bin was treated as a continuous variable to test for progressive caudal-cranial changes along each section and a quadratic, rather than simple linear regression provided the best fit to the data. The secondary outcome data were analyzed identically. Full details are provided in Data S1.
Agreement between diagnostics.
To assess the validity of our assays, we would ideally ask how the D. musculi PCR −ΔCT compared with skin-scrape (the historical standard) in terms of sensitivity and specificity. However, the nature of the experimental design presented challenges to conventional approaches. Instead, we exploited the fact that each skin-scrape slide was scored by 2 different observers and thus coded each slide as a Negative agreement, a Disagreement, or a Positive agreement. We predicted that these categories would be associated with low, intermediate, and high −ΔCT for D. musculi. To test this, we performed a Repeated Measures REML Mixed Model. Full details are provided in Data S1.
Results
Effect of topical fluralaner on mite burden as determined by qPCR.
Fluralaner treatment significantly reduced D. musculi infestation as measured by qPCR over time (Figure 3; F4,86.67 = 99.80; P < 0.0001). Reduction between the 2 doses did not differ significantly overall (F1,23.1 = 0.0676; P = 0.7927) or as a function of time (F4,86.67 = 2.102; P = 0.0874).
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-135
Pelt scroll analysis.
Each dorsal pelt scroll yielded an average of 525 follicle profiles for analysis (range: 328 to 783), while each ventral pelt scroll yielded an average of 714 follicle profiles (range: 366 to 1,000)—meaning that 28,310 follicles were analyzed in this study. When examining the proportion of follicle profiles infested in the pelt scrolls, there was a significant “U-shaped” curve by Bin (Caudal-Cranial position), with a larger proportion of follicles infested at the ends of the section compared with the middle (Figure 4; F1,392 = 7.052; P = 0.0082). The Dose by Section (Dorsal compared with Ventral) by Bin interaction was significant (F1,392 = 4.640; P = 0.0318), indicating that the inflection point of the curve differed as a product of Dose and Section. Post hoc contrasts showed that this was due to a right-shift of the modeled curve in dorsal scrolls from 250-mg/kg animals, resulting in a progressive Caudal-Cranial decrease (P < 0.0001), whereas the other 3 treatment combinations did not differ from the average underlying “U”-shaped curve (all P > 0.05). Furthermore, Ventral Scrolls had an overall lower level of infestation (F1,392 = 32.68; P < 0.0001), and Dose did not have a significant effect overall (F1,19 < 0.0001; P = 0.9960). For our secondary outcome (mean mites per follicle profile) the Dose-by-Bin-by-Scroll interaction was not significant, but the other terms in the model combined to produce essentially the same pattern of results.
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-135
Diagnostic comparisons and assessment.
A total of 93 skin scrapes were analyzed sequentially by 2 blinded reviewers over the course of this study. Identification of just one live or dead mite at any life cycle stage was considered a “positive” result by either reviewer. The −ΔCT differed between skin scrape categories (F2,88.12 = 6.661; P = 0.0020) in the predicted order (Figure 5). Tukey post hoc tests showed that although the “Disagreement” category was intermediate it did not differ significantly from the “Negative agreement” category, only from the “Positive agreement” category.
Citation: Journal of the American Association for Laboratory Animal Science 64, 2; 10.30802/AALAS-JAALAS-24-135
Discussion
Effect of fluralaner dosing on mite burden as determined by qPCR.
Both topical fluralaner dosing regimens caused a significant and continuous decrease in mite DNA amplified by qPCR when compared with baseline (Figure 3). This reduction persisted for 2 wk after the last topical application and then began to increase—a trend that can be explained by the surviving mites continuing to reproduce. Mite DNA was amplified from all mouse samples at all time points. Overall, these data suggest that both fluralaner doses successfully reduced—but did not clear—mite burden. This did not fully support our hypothesis.
Mite burden between treatment groups was not significantly different at any time point. Because an overall reduction in mite burden was observed in both treatment groups, the most likely explanation is that both fluralaner doses were high enough to achieve a therapeutic plasma level that did not persist long enough to clear mite burden. In dogs and cats, fluralaner has a long half-life (greater than 3 mo) after absorption into the bloodstream so that it may continuously elute into perifollicular fluids to kill ectoparasites.13,19 However, due to metabolic scaling, smaller animals like mice typically require higher relative doses because drug clearance times are usually accelerated. It was recently demonstrated that CD1 and Peromyscus mice metabolize fluralaner much faster than dogs.14 Despite the doses in this study being 4 or 10 times higher than what is recommended in dogs (25 mg/kg), it is possible that NSG mice metabolized fluralaner too rapidly to allow for full mite clearance. If this hypothesis is correct, then increasing dose frequency rather than the dose itself would successfully clear D. musculi infestation. This may be why the current standard requires more frequent (weekly) topical application of moxidectin and imidacloprid for 8 to 12 wk.4,11,33 In this study, we attempted to take advantage of the longer half-life of fluralaner and extend the interval between treatments. However, this may not be practical given the higher metabolic rate of mice. In addition, oral dosing, as successfully performed in dogs (Canis lupus familiaris), deer mice (Peromyscus spp.), and a Syrian hamster (Mesocricetus auratus), may also be a more efficacious route of administration because it likely prolongs the bioavailability of fluralaner.1,13,17,18,34
It is worth noting that NSG mice are severely immunocompromised, which may impair mite clearance after fluralaner application. DNA from live or dead mites at any life stage would still contribute to qPCR results, so it would likely take longer for them to test negative due to the high sensitivity of PCR. While our other diagnostic modalities denoted that all infestations were continuous despite treatment, the hair cycle in mice is around 6 wk.10,35,36 It can take up to 8 mo for the hair coat to be shed entirely, so truly declaring mice free of D. musculi after treatment may take notable time after any successful therapy.10,35,36
Analysis of mouse pelt scrolls.
During microtome sectioning of the pelt for histology, hair follicles will inevitably be cut at a variety of angles. On subsequent microscopic examination, hair follicles can therefore have a variety of visible structures that mites like D. musculi can inhabit. We therefore favor the term “follicle profiles” over “follicles” to denote this.
In this study, all follicle profiles were documented for a single section through the dorsal and ventral pelt scrolls of each mouse. Demodex musculi were more frequently found on the cranial or caudal follicle profiles of either scroll, with the exception of the 250-mg/kg dorsal pelt (Figure 4). Relatively more mites were identified on the dorsal scroll sections than the ventral ones. Both findings may be indicative of mite location preference (due to local body temperature variations, hair follicle density, grooming accessibility, etc.) or an effect of fluralaner. Regardless, these results complement a previous study denoting how the interscapular and/or caudoventral pelt should be sampled when performing skin scrapes to maximize diagnostic sensitivity.5 Our findings further support this and suggest that sampling the dorsum may detect mites at a higher frequency than when sampling the ventrum.
Despite pelt analysis being 10 wk after the last topical treatment, there was still a significant decrease in relative mite burden at the cranial aspect of dorsal pelts when mice were dosed with topical fluralaner at 250 mg/kg (as compared with 100 mg/kg). Postmortem tissue harvesting and fixation methods were identical between groups. We can postulate 3 plausible reasons why this trend was observed. The first reason is that the topical doses of the 250-mg/kg group were more likely to encompass the skin section analyzed. It is possible that fluralaner exhibited local acaricidal effects because the estimated therapeutic concentration is much lower (on the nanogram level).13,37 In addition, the larger volume may have impacted the grooming behavior of the area to further reduce local mite density. Second, the reduction at the cranial aspect of the pelt may reflect D. musculi behaviors. The stimuli that instigate mite colonization of new follicles are not well understood, but other Demodex spp. can move 8 to 16 mm/h.6 It is possible that fluralaner doses impacted their pelt location even 10 wk after the last dose. Finally, anatomic differences may have impacted fluralaner’s distribution or efficacy. For example, the mouse dermis does not readily bleed during DSS, as compared with dogs and other species. If this reflects a reduction in relative vascularity, drug absorption or distribution to the perifollicular space would be impacted. Anatomic differences such as this may be at least partially responsible for the observed reduction in cranial mite burden for the 250-mg/kg dose group.
Agreement between diagnostic modalities.
Both reviewers considered a slide to be positive for D. musculi if one live or dead mite at any life stage was appreciable. This was to accommodate for the time interval between reviewers and how mites become increasingly translucent after death, making it difficult to adequately compare mite number and live/dead status. By doing so, judgment bias was eliminated.
There was no qPCR −ΔCT cutoff value that predicted positive or negative skin scrape results. However, as the qPCR −ΔCT increased (which is indicative of mite burden), the 2 raters were more likely to agree (Figure 5). This may be due to how we administered fluralaner to the same site that was sampled for DSS and FP. Our pelt scroll analysis suggested that fluralaner doses at 250 mg/kg caused a reduction in mite burden at the cranial aspect of the dorsal pelt. As such, the area sampled may have been less likely to contain a mite on DSS and FP regardless of the overall mite burden on the mouse detected by qPCR. As the overall mite burden increases, the likelihood of mites migrating to this area would also increase.6 It is also worth noting that the lack of association may also be due to how both live and dead mite DNA are not distinguishable on qPCR, but dead mites are more difficult to identify microscopically. D. musculi mites become increasingly more translucent after death and the longer they sit on a microscope slide.5 As such, interpretation becomes more difficult if the treatment is more efficacious.
These results further support the suspicion that DSS and FP suffer low diagnostic sensitivity. Like other diagnostic modalities, this can be increased by repeat testing. The test should be considered positive when any observer considers a slide positive. Furthermore, disagreements in observations are likely to occur when the mite burden is lower. Adjunct diagnostics like PCR are required to truly declare mice free of demodicosis.
Pathologic findings.
All mice were grossly evaluated at the end point during pelt harvest. Five mice (2 in the 100-mg/kg group and 3 in the 250-mg/kg group) exhibited mild dilatation of one uterine horn consistent with normal age-related changes in mice.38,39 One mouse from the 250-mg/kg group had multiple pale tan nodules along one aspect of the ovary. These findings were further examined histologically and attributed to age-related changes. In addition, histologic analysis was performed on the heart, lungs, liver, spleen, kidney, and gonads for all animals in the 250-mg/kg group. No evidence of toxicity was appreciable in the examined sections. These findings are complementary to those in a recent study where no signs of toxicity were observed in CD1 mice after oral administration of fluralaner at 1,000 mg/kg.14
Five animals did not make it to the end point for pelt scroll analysis. Three exhibited poor body condition scores (≤2/5) and one animal presented with a prolapsed uterus so they were all humanely euthanized. One mouse acutely died overnight with lesions over the rump consistent with fight wounds. The carcass was extensively cannibalized so a necropsy was not performed. Gross necropsies were performed on all other animals, and no overt lesions or signs of toxicity were appreciable. None of these animals were from the original naïve NSG group and so they were between 12 and 17 mo at the time of death. NSG mice housed in conventional facilities are at higher risk of morbidities and death so some level of attrition is not uncommon.40
Study limitations.
One potential limitation of this study was that the fluralaner dosing regimen was not concurrently verified by pharmacologic testing. Without plasma concentrations, we cannot confirm that fluralaner achieved a therapeutic level. However, our doses were based on those documented in other species.1,15,18,37,41–43 In addition, our dosing frequency was selected to coincide with the D. musculi life cycle (estimated at approximately 14.5 d), as the mite life stage possibly impacts drug susceptibility.4,6 The study duration encompassed 4 treatments over 3 mite life cycles while allowing for additional life cycles for the mites to repopulate. Our study design helped to mitigate this limitation but does not definitively establish how topical fluralaner is reducing mite burden.
The second potential limitation of this study is that no mice were singly housed throughout. Demodex mites are capable of transferring between animals within a cage, and if treatments were not efficacious for just a single animal, the whole cage will remain burdened. We deliberately chose not to singly house animals because we wanted to understand how one might use fluralaner in a real-world clinical setting. However, single housing might be appropriate in future studies attempting to understand fluralaner metabolism or efficacy on an individual scale.
A third potential limitation is that this study only used female mice. This was an ethical decision because to reliably achieve our study population, we needed to place a mouse from the original, infested colony in a cage with younger, naïve animals. Introducing males to each other would almost certainly induce fighting so it was not performed. We attempted to infest cages of naïve male littermates by manually rubbing the dorsa of infested animals to naïve ones and placing “infested” FP in the naïve cages, but infestation was not consistently successful (which was also the case with females). These findings may be indicative of Demodex transmission characteristics and warrant further study. Given that fluralaner doses in other species do not differ between sexes, we determined that a male cohort was not ethical or feasible.
Conclusions
Four biweekly topical fluralaner doses at 100 mg/kg and 250 mg/kg reduced—but did not clear—D. musculi mite burden in group housed, female NSG mice. No signs of toxicity were observed in this study. Our data suggest a potential for the use of topical fluralaner to treat D. musculi infestations in NSG mice. Developing a successful fluralaner treatment regimen would promote the 3Rs (Replacement, Reduction, and Refinement) by salvaging unusable animals, reducing pain/discomfort associated with the infestation, and preventing the spread of mites within/between vivaria. Here we also demonstrate how quantitative PCR and pelt scrolling histology techniques may be helpful for characterizing ectoparasite burdens in future studies.
Additional research is required to establish appropriate fluralaner dosing regimens and more thoroughly assess for toxicity. Given mite burden was similar between treatment groups in this study, more frequent dosing—as compared with higher doses—may be more efficacious. The oral route of administration may be more reliable and should be investigated further. Regardless of which route is deemed more appropriate, fluralaner may prove to be a more efficacious and economical treatment option for D. musculi infestations in mice.
Supplementary Materials
Data S1. The Supplementary Information provides statistical analysis notes and SAS data and code.
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.
Contributor Notes
This article contains supplemental materials online.