Journal of the American Association for Laboratory Science Vol 57, No 2 Copyright 2018 March 2018 by the American Association for Laboratory Animal Science Pages 173–185

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

Melissa A Nashat,1 Rodolfo J Ricart Arbona,2 Elyn R Riedel,3 Olga Francino,4 Lluis Ferrer,5 Kerith R Luchins,2,§ and Neil S Lipman1,2,*

Demodex are microscopic, cigar-shaped, follicular mites often regarded as commensal microfauna in . Although Demodex spp. can cause dermatologic disease in any immunocompromised , they are rarely reported in laboratory mice. Recent identification ofDemodex 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—super- ficial 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.

Abbreviations: CD, caudal dorsum; CV, caudal ventrum; DSS, deep skin scrape; FP, fur pluck; IS, interscapular region; SSS, superficial skin scrape; TI, tape impression test

Demodex spp. mites are mammalian microfauna that typi- were infested (100% prevalence), and the colony provided us cally inhabit the pilosebaceous unit of healthy , where with the opportunity to evaluate the capability of various of they feed and reproduce.22,53 They are found in low numbers diagnostic modalities to detect D. musculi. Our goal was to find in normal skin and adnexa and usually do not cause clinical an antemortem test that was sensitive, minimally invasive, easy manifestations; however, in immunocompromised hosts, they to perform, and economical in terms of materials, sample col- can initiate dermatologic disease.13,16,34 Demodecid mites tend to lection, labor, and assessment. A sensitive diagnostic test would be host-specific and have been best studied in dogs and . be useful to identify Demodex mites in mice during quarantine Wild Mus musculus can host 6 Demodex spp. which can be found and routine biosecurity practices and could be used to assess in the skin, clitoral and preputial glands, and oral cavity.7,22,25-28 for D. musculi in mice with skin disease. Relatively few instances of Demodex infestation have been Diagnosis of D. musculi is challenging because the mites are reported in laboratory mice, all in immunocompromised strains microscopic (approximately 200 μm), transparent, and live and, in all cases, only Demodex musculi was identified.21,38,52,73 within hair follicles.22,53 Molecular diagnostic assays were not A colony of immunocompromised transgenic mice with a commercially available for rodent Demodex species at the initia- recombination activation gene 1 (RAG1) deficiency presented tion of the study (2014); therefore, we focused the investigation with exophthalmia and pruritus.52 The mice were susceptible on traditional microscopic methods of parasite detection, using to opportunistic infections due to compromised adaptive im- a PCR assay with primers directed against mite 18S rRNA on munity.51,58 Investigation into the cause of clinical signs revealed a subset of cases. In dogs, deep skin scrape (DSS) is the ‘gold opportunistic bacterial infections as well as an infestation with standard’ for the detection of D. canis, but fur pluck (FP) with D. musculi, first identified on histopathology. All mice examined trichoscopy (microscopic evaluation of hairs placed in mineral oil on a microscope slide), although not as sensitive as DSS, can be used in heavy infestations.49 In addition, FP is useful for Received: 27 Oct 2017. Revision requested: 02 Dec 2017. Accepted: 05 Dec 2017. 1 anatomic sites such as the face and interdigital regions where the Tri-Institutional Training Program in Laboratory Animal Medicine and Science, 14,49 Memorial Sloan Kettering Cancer Center, Weill Cornell Medicine, and The Rockefeller collection of DSS samples is challenging. In humans, the gold University;2 Center for Comparative Medicine and Pathology, Memorial Sloan Kettering standard for the detection of Demodex spp. is the standardized Cancer Center and Weill Cornell Medicine; and 3Epidemiology and Biostatistics Depart- skin surface biopsy;15 this method involves using an adhesive ment, Memorial Sloan Kettering Cancer Center, New York, New York; and 4Molecular Genetics Veterinary Service, Department of Animal and Food Science and 5Department to pull the top layers of epidermis and hairs (with roots) off of Animal Medicine and Surgery, Autonomous University of Barcelona, Barcelona, Spain. the skin’s surface. In the standardized skin surface biopsy, the *Corresponding author. Email:[email protected] skin’s surface and hairs are removed from a small region en § Current affiliation: Animal Resources Center, The University of Chicago, Chicago, masse, a process that likely would cause discomfort in densely Illinois

173 Vol 57, No 2 Journal of the American Association for Laboratory Animal Science March 2018 haired mammals such as mice. Therefore, we did not consider Citrobacter rodentium, Salmonella spp., ciliary-associated respira- including the standardized skin surface biopsy for comparison tory bacillus, and Clostridium piliforme; and fur mites (Myobia in mice. Instead we assessed other diagnostic methods, includ- musculi, Myocoptes musculinis, and Radfordia affinis), pinworms ing the superficial skin scrape (SSS) and tape impression (TI) (Syphacia spp. and Aspiculuris spp.), and Encephalitozoon cuniculi. tests, which have proven useful for detection of other parasites Experimental design. D. musculi-infested, TRP-1/TCR mice in animals.14,43,62,64,78 (n = 46 [22 male and 24 female mice], experimental and breeders; Although some fur mite species have preferred sites on hosts, age, 49 to 854 d [mean ± SEM, 172.5 ± 25.9 d]) were included studies in wild mice and our own experience with D. musculi in this study. Testing was performed immediately after carbon in laboratory mice indicate that these follicular mites can in- dioxide asphyxiation (n = 40) or under manual restraint (n = fest most areas of densely haired skin.18,26,52,62 We previously 6). Manually restrained mice were observed for adverse events reported that the sites with the highest number of mites per for 5 min after sample collection and 24 h later. We collected millimeter of skin were the middorsum, interscapular region samples by SSS, TI, FP, and DSS from the face, IS, CV, and CD (IS), caudal dorsum (CD), caudal ventrum (CV), and head.52 In of each mouse, unless otherwise noted. Each mouse was tested the current study, in addition to evaluating different diagnostic with a full complement or subset of tests once at each location methods, mite detection was compared at various anatomic and at a single time point. The same person (MN) collected and sites. We hypothesized that the DSS would be the best diagnostic evaluated all samples. test and that IS would be the best anatomic site for Demodex SSS procedure. A no. 10 or 20 scalpel blade (Bard-Parker; mite detection. The findings herein give insight into the most Aspen Surgical Products; Caledonia, MI) was run against the sensitive antemortem diagnostic modalities and sampling sites grain of the hairs rapidly for a distance of approximately 10 mm, for the detection of Demodex mites in laboratory mice. with the blade applying minimal pressure to the skin’s surface. Hair and skin debris were collected on a piece cellophane tape Materials and Methods (12.7 × 25 mm; Highland Brand 5190 transparent tape, 3M Sta- Animals. B6.Cg-Rag1tm1Mom Tyrp1B-W Tg(Tcra,Tcrb)9Rest tionary Products Division, St Paul, MN), which was adhered to (that is, TRP1/TCR) mice were imported to the Memorial Sloan a standard microscope slide and evaluated. SSS (n = 112) was Kettering Cancer Center from the National Cancer Institute in performed on 46 mice. 2006. The TRP1/TCR colony was expanded by using in vitro TI procedure. Using the thumb, the researcher pressed a piece fertilization and was subsequently found to be infested with of cellophane tape (12.7 × 25 mm) to the fur and removed it D. musculi.52 At the Memorial Sloan Kettering Cancer Center, rapidly by pulling perpendicularly to the skin. The tape was the line had been backcrossed onto the C57BL/6J strain and reapplied to and removed from the same region 3 or 4 times. then crossed to several other lines.52 TRP1/TCR mice contain 3 Hairs and surface debris were adhered to the tape, which was transgenes: the tyrosinase-related protein 1 transgene (Tcra,Tcrb) mounted on a glass microscope slide and evaluated. TI (n = 138) 9Rest, the white-based brown radiation-induced mutation of was performed on 36 animals. tyrosinase-related protein (Tyrp1B-W), and a Rag1 mutation FP procedure. A 4-in., curved Halstead mosquito hemostat (Rag1tm1Mom).51 The Rag1-null mutation inactivates an enzyme was used to grasp a 1- to 3-mm clump of hair from the region critical to normal lymphocyte development, which results in to be tested. The hairs were removed from the root by rapidly defective adaptive immunity due to the absence of functional pulling perpendicular to the skin’s surface. This process was mature T and B lymphocytes.46 repeated 3 or 4 times in each location. Hairs were placed in a Mice were housed in an AAALAC-accredited barrier facility drop of mineral oil on a microscope slide, a coverslip was ap- in accordance with the Guide for the Care and Use of Laboratory plied, and samples were evaluated. The hemostat tip was rinsed Animals (8th edition).24 Mice were maintained in IVC (no. 19, in 70% alcohol and clamped to a paper towel to remove hair and Thoren Caging Systems, Hazelton, PA) as breeding trios or were visible debris before sampling subsequent sites or animals. At housed by sex (n = 5 males or females per cage). Animals were the end of a sample harvest session, the hemostat was cleaned housed on autoclaved aspen-chip bedding (PWI Industries, by using a scrub brush and mild detergent and rinsed with tap Quebec, Canada), and fed a closed-formula, natural-ingredient, water and air-dried. FP (n = 158) was performed on 46 animals. γ-irradiated diet (PicoLab Mouse Diet 5053, Purina LabDiet, St DSS procedure. The researcher squeezed the region of skin 2 Louis, MO), and received acidified reverse-osmosis–purified to be sampled (1×1 to 1.5×1.5 cm ) between the thumb and water (pH 2.5 to 2.8, with hydrochloric acid) in polysulfone forefinger of one hand as a no. 10 scalpel blade, at an angle bottles with stainless steel caps and sipper tubes (Techniplast, of approximately 30 to 45° with regard to and with moderate West Chester, PA). As necessary, select mice were fed an irradi- pressure on the skin, was run against the grain of the hairs 3 or ated medicated feed containing 0.12% amoxicillin (TestDiet, 4 times. The collected debris was placed in a drop of mineral oil St Louis, MO) to prevent opportunistic infections.52 The cage on a microscope slide, a coverslip was applied, and the sample bottom was changed weekly, whereas the wire bar lid, water was evaluated. A new blade was used at each site for each DSS bottle, and filter top were changed biweekly within a vertical on each animal. DSS (n = 112) was performed on 46 mice. flow, HEPA-filtered, mass-air–displacement unit (model NU- Multiple samples from a single animal were mounted on S619-400, NuAire, Plymouth, MN). The room was maintained microscope slides, with 2 FP, 2 DSS, 4 TI, or 3 SSS samples on a 12:12-h light:dark cycle, relative humidity of 30% to 70%, mounted per slide. and room temperature of 72 ± 2 °F (22.2 ± 1.1 °C). Anatomic regions evaluated. Anatomic sites were selected Mice were free of mouse hepatitis virus, Sendai virus, mouse according to the literature and a recent study evaluating the 26,52 parvovirus, minute virus of mice, pneumonia virus of mice, topographic distribution of Demodex spp. A maximum of Theiler meningoencephalitis virus, mouse rotavirus (epizootic 14 tests were performed on each animal: face (nasal bridge and diarrhea of infant mice virus), ectromelia virus, reovirus 3, interocular region) by TI and FP; IS by SSS, TI, FP, and DSS; CV lymphocytic choriomeningitis virus, K virus, mouse adeno- by SSS, TI, FP, and DSS; and CD by SSS, TI, FP, and DSS). We virus types 1 and 2, polyoma virus, mouse cytomegalovirus, tested fewer animals (n = 20) on the CD because preliminary mouse thymic virus, and Hantaan virus; Mycoplasma pulmonis, results from FP and DSS indicated that mite yields were low at

174 Methods and sites for detection of D. musculi

this site, but histology showed sufficient mites for testing.52 In Roche Applied Science, Mannheim, Germany). After overnight addition, a subset of mice (n = 10) did not undergo TI collection. incubation, proteinase K was inactivated by heating at 95 °C for Each anatomic region tested was divided into left and right sec- 10 min, and the samples were centrifuged for 10 min at 16,000 × g. tions, and when 4 tests were performed at a single anatomic site, DNA was extracted from the samples by using a commercial regions were divided into quadrants to ensure that individual kit (DNeasy Blood and Tissue Kit, Qiagen, Hilden, Germany) tests were performed on naïve sites. Test sampling for SSS, TI, according to the manufacturer’s instructions. Extracted DNA FP, and DSS were alternated between left and right or rotated samples were stored at –80 °C prior to testing, were shipped between left cranial, left caudal, right cranial, and right caudal for PCR processing in a chilled shipment box, and were thawed areas, to avoid sampling site bias. The researcher donned dis- on ice before analysis. posable nitrile gloves; gloves were changed between animals, TI samples with individual mites were extracted as previously and a fresh paper towel was placed on the workstation beneath described for individual canine Demodex mites.69 D. canis served each mouse, to avoid cross-contamination. as the positive control for the PCR analysis. An individual Microscopic evaluation of samples. Microscope slides were mite was harvested from a canine DSS sample. Extraction and evaluated by using a biologic light microscope (model CX31, amplification of D. canis DNA was performed as previously Olympus, Center Valley, PA) at 100× and 200× magnification. described.69 A low light level and reduced iris diaphragm aperture for PCR amplification was performed in a final reaction volume enhanced contrast were used to improve visualization of the of 50 μL, containing 5 μL of DNA solution, PCR buffer (1.5 mM transparent mites. Each sample was reviewed twice by a single MgCl2, 0.2 mM of each dNTP), 0.3 μM of each primer, and 2 U observer (MN), who was aware of the anatomic site and test of EcoTaq polymerase (Ecogen, Barcelona, Spain). Primer pairs type. A sample was deemed positive when it contained at least were Mite 410 (5′ TCC AAG GAA GGC AGC AGG CA 3′) and one mite or egg. In all samples, mites and eggs were counted. Mite 941 (5′ CGC GGT AGT TCG TCT TGC GAC G 3′; sequences In addition, for SSS, TI, and FP, hairs were counted under 50× provided by Susan Compton [Yale University]). These primers magnification. A minimum of 100 hairs was selected as the amplify a nonspecies-specific sequence in the 18S rRNA gene lower limit for an adequate sample, to maximize detection and of both Demodex spp. and Myobia musculi. The thermocycling remain within the recommended range of hairs as described for profile was 95° C for 3 min; followed by 40 cycles at 94 °C for FP sample collection in dogs.49 Select mites were photographed 30 s, 60 °C for 30 s, and 72 °C for 30 s; and final extension at 72 and measured by using an infield micrometer to confirm the °C for 5 min. Water was included as a negative control in each identity of the Demodex spp. Mite length was measured from the PCR assay. PCR products were visualized in a 1.2% agarose gel tip of the gnathosoma to the end of the opisthosoma. with molecular markers (DNA Molecular Weight Marker IX, Comparisons between sample sites with regard to mite detec- Roche Applied Science). tion and number of mites per sample. When at least one mite or The primers amplified a 537-bp fragment. The PCR product egg was observed in any sample, the sample and animal were was sequenced by using a commercial kit (BigDye Terminator deemed positive for D. musculi. The Demodex mite detection Cycle Sequencing Ready Reaction Kit, version 3.1, Life Tech- rate was determined for each test method or anatomic site as nologies; Carlsbad, CA), by using the same primers as described the number of positive tests divided by the total number of tests earlier. Amplification products were purified (SEQ96 Montage performed multiplied by 100%. The combined number of mites Sequencing Reaction Cleanup Kit, Millipore, Billerica, MA) and and eggs in each sample is presented as a median number ac- sequenced on an automated sequencer (PRISM 3730, Applied cording to the diagnostic method and anatomic site. Biosystems, Foster City, CA) according to the manufacturer’s To determine which combinations of tests and sites yielded protocol. The sequence obtained was examined and compared the highest detection rate, we compared the possible positive with those in the GenBank database by using the Basic Local test result permutations with the actual results by analyzing Alignment Search Tool.3 The sequences with the strongest agree- possible 2- and 4-way test–site combinations. Initially we con- ment are reported in the Results section. sidered 4 methods and 4 sites for comparison, but we excluded Data and statistical analyses. Summary statistics were calcu- the face from this analysis because it had the lowest detection lated for the numbers of mites and hairs by site and diagnostic rate and mite yield, thus resulting in 33, that is 27, potential test (SSS, TI, and FP). The number of mites was compared 2-way combinations of tests and anatomic sites, including DSS among all test–site combinations in aggregate by using the at 3 anatomic sites (IS, CD, and CV) and SSS, TI, and FP each Kruskal–Wallis test, and mite numbers were rank-ordered from at 3 anatomic sites. After 2-way analysis, DSS was determined highest to lowest among test–sites. The Demodex detection rate to have a high detection rate, and we selected the IS and CV was calculated as the percentage of samples collected by using a sites for additional 4-way test–site comparisons. For the sixteen particular method or from a specific anatomic site that contained 4-way test–site combinations evaluated, the possible permuta- any D. musculi eggs or mites. For all 14 test–site combinations, tions of positive and negative results for FP–IS, FP–CV, DSS–IS, the Demodex detection rate was rank-ordered to indicate the and DSS–CV are presented in addition to the actual results from test–site combinations with the highest and lowest detection the 46 test mice. rates. Paired results of detection rates from different tests on the PCR analysis. Fresh–frozen (n = 2) and formalin-fixed (n = 5) same animal were compared by using the McNemar test, which skin and samples from TI tests (n = 2; each containing an in- compares 2 related groups and examines agreement between the dividual Demodex mite) from 8 different mice were analyzed. tests. The McNemar test was used because multiple tests were The clinical and pathologic findings of these mice have been performed on the same subject at the same time point. DSS was described previously; their case identification numbers can be used as the test for comparison, because it serves as the standard used to obtain this information.52 DNA extraction from samples, in dogs.49 Differences in the median number of mites counted in except individual mites, was performed as follows. Sample vi- each sample were compared between anatomic sites by using als were thawed and incubated overnight at 56 °C in 200 μL of the Wilcoxon signed rank test. The Spearman rank correlation digestion buffer (50 mmol/L Tris-HCl, pH 8.5; 1 mmol/L EDTA) coefficient between the number of mites and the number of hairs with 4 μL of recombinant proteinase K solution (10 mg/mL, per test–site combination was calculated for individual mice.

175 Vol 57, No 2 Journal of the American Association for Laboratory Animal Science March 2018

A P value of less than 0.05 indicates a correlation coefficient is, SSS, TI, and FP) was 0.47 (P = 0.001; Figure 2). For individual significantly different from 0. All analyses were performed by test types, the Spearman correlation coefficient was significantly using SAS version 9.4 (SAS Institute; Cary, NC). different from 0 (P < 0.05 for SSS, TI, and FP), with the TI test having the strongest correlation between the number of mites Results and the number of hairs plucked per sample (Table 4) Demodex detection rate and mite yield compared by diag- In tape-based tests (SSS and TI), adult mites and hairs were nostic method and anatomic site. All 46 mice were confirmed well preserved (Figure 3 A), but significant amounts of debris mite-infested by testing positive by at least one method at and air bubbles were present, making identification of mites a single site. The Demodex detection rate of each diagnostic challenging. Mineral oil-based samples (FP and DSS) had method was compared with the detection rate of DSS and is considerably less debris, making diagnosis easier than for tape- provided in Table 1. The rank order for highest to lowest over- based tests (Figure 3 B). The quality of a DSS sample depended all detection rate by test type, according to the total number of on skin squeezing (to extract mites) and the ability to obtain skin 48 tests performed, was DSS > FP > SSS > TI. The detection rates surface debris by using the blade. Although DSS samples con- of SSS, TI, and FP were not significantly different from one an- tained considerable debris with broken hair shafts, indicating other, but they were all significantly (P < 0.05) lower than the that a qualitatively sufficient area was scraped, sample quality sensitivity for DSS, for which 91.1% of the tests were positive. did not depend on the number of hairs and, compared with When results from all tests of each type in the same mouse were other sample types, DSS samples had less debris. In FP samples, analyzed, the percentage of mice positive for at least one test of the opisthosoma (caudal region) of the mite is approximately each type ranged from 76% to 98%. Testing multiple sites on an the same width as a mouse hair bulb, making it challenging individual mouse increased the detection rate, such that FP and to detect mites present in hair clusters. The decreased amount DSS had equivalent detection rates of 98%. In the single animal of debris and lack of hair bulbs made DSS the easiest method that had negative DSS on both IS and CV (CD was not tested in to evaluate. Mites were easiest to identify in fresh, oil-based this animal), FP tests were positive at both sites. In addition, DSS samples because they were often mobile, and their internal detected significantly (P < 0.05) more mites (approximately 7fold organs were visible (Figure 3 C). Guanine concretions (Figure 3 higher counts) per sample compared with all other methods, for B and C, arrowheads) were useful for detecting live or recently which mite yields did not differ significantly from one another. dead mites. In addition, the coverslip could be adjusted in oil- A comparison of detection rate according to anatomic site is based samples to dissociate hair clusters and mobilize hairs to provided in Table 2. When the total number of tests performed ascertain whether hair strands had obscured mites. Mites were at each site was considered, IS, CV, and CD supported similar more likely to be alive (moving, opaque) when samples were detection rates, ranging from 72.5% to 76.4%. The detection rate viewed within 1 to 6 h of collection, although live mites were for IS was significantly (P < 0.05) higher than for CD, and the observed as long as 53 h after sampling in oil-based tests. Mites rate for the face was significantly lower (29.3%; P < 0.05) than became transparent after death, making them more challenging for any of the other sites. When sites were compared among to detect (Figure 3 B). animals, IS, CV, and CD each yielded at least one positive test Adult female mites (n = 15) ranged in size from 170 to 220 per animal; the face had a significantly (P < 0.05) lower detec- μm, whereas adult males (n = 11) were shorter, at 135 to 175 μm. tion rate, with only 43% of mice testing positive. In addition, Dimensions and morphologic features were consistent with D. 22,26 IS yielded significantly (P < 0.05) more mites per sample than musculi. Dead mites are shorter than live mites, presumably other sites whereas the face had the lowest mite yield. due to muscle contraction upon death, which alters the exo- When the test–site combinations were rank-ordered, DSS–IS skeleton. Although the exoskeleton of adult mites can last for (93.5%), DSS–CD (90.0%), and DSS–CV (89.1%) yielded the months in tape-based samples or for weeks in oil-based samples, highest detection rates (Table 3). The lowest detection rates eggs and larvae were labile and were not identified in samples were for TI–face and FP–face (19.6% and 39.1%, respectively). older than 6 to 7 d. Adult mites were found on all 46 mice, but Other test–site combinations yielded detection rates ranging eggs or larvae were detected in fewer than half of the animals from 60.0% to 83.3%. (n = 21). In particular, eggs and larvae were not detected in The mite yields for all test–site combinations differed signifi- tape-based tests (SSS or TI), but they were visible on oil-based cantly (P < 0.05) from one another when compared with each tests (DSS and FP). Of the 21 mice from which eggs and larvae other in aggregate (maximum, 53 mites per sample; median, were detected, 46 (20 FP, 16 DSS) of 128 (35.9%) tests contained 10.5 mites per sample; Figure 1), with the greatest numbers of eggs or larvae. When the proportions of FP and DSS tests with mites detected by DSS–IS, which also had the greatest range of eggs or larvae were compared, FP was better (P < 0.05) than DSS mites or eggs detected when compared with all other test–site for detecting eggs or larvae. Nymphs were not detected on any combinations. The lowest median number of mites was detected test from any mouse. on the face (both TI and FP had a median of 0 mites per sample). Comparison of detection rates for test–site combinations. Two- Diagnostic sample quality and analysis. To assess sample qual- and 4-way test–site combinations were evaluated to determine ity, we determined whether samples had an adequate number which of the combinations yielded the highest and lowest mite of hairs (n ≥ 100; Table 4). Despite multiple sample collections detection rates. The face was excluded because it yielded the per site by using forceps and tape, some samples had fewer lowest detection rate and the fewest mites, thus resulting in 3 than the desired number of hairs; specifically 16 of 46 TI–face 3 , or 27, potential 2-way test combinations comprising DSS samples, 4 of 46 FP–face samples, 5 of 46 SSS–IS samples, and 2 with SSS, TI, and FP, each at 3 anatomic sites (Table 5). Of the of 46 SSS–CV samples failed to meet the 100-hair criterion. The 2-way test–site combinations considered, 9 of 27 yielded 100% percentage of representative tests that met the criterion ranged detection and 6 of these included DSS–IS. When DSS–IS was from 89% to 100% for most sites, but only 65% of the TI samples included in the 2-way comparison, 93% to 100% detection oc- from the face met this criterion. The Spearman correlation coef- curred; when DSS–CV was included, 93% to 100% detection ficient between the number of mites per sample and the number was present; and when DSS–CD was included, 89% to 100% of hairs per sample for all tests where hairs were counted (that detection was obtained. Therefore, the IS and CV sites were

176 Methods and sites for detection of D. musculi

Table 1. Demodex detection rate and mite yield by test type Superficial skin Tape Deep skin scrape impression Fur pluck scrapea.b No. of mice tested 46 36 46 46 Total no. of tests performed 112 138 158 112 No. of positive tests / total no. tests performed (% detection rate)a 70/112 (62.5)e 80/138 (57.5)d 99/158 (62.7)d 102/112 (91.1) No. of mice with at least 1 site positive /total no. of mice tested (% positive)d 35/46 (76.0)c 32/36 (88.9)e 45/46 (98.0) 45/46 (98) Median (interquartile range) no. of mites and eggs per samplee 1.0 (0.3–2.3)e 1.3 (0–2.9)e 1.3 (0.7–2.7)e 8.8 (3.5–17.0) aThe McNemar test method was used to compare the number of mice with at least one site positive after deep skin scraping, which served as the reference, with the number of animals positive by other methods. A positive test was recorded when at least a single mite or mite egg was detected, and an animal was positive when it had at least one positive test. The McNemar test also was used to compare the detection rates for superficial skin scraping, tape impression, and fur pluck with that for deep skin scraping. bWilcoxon signed-rank testing was used to compare the median number of mites after superficial skin scraping, tape impression, and fur pluck with that from deep skin scraping. cP ≤ 0.01 dP ≤ 0.001 eP ≤ 0.0001

Table 2. Demodex detection rate and mite yield by anatomic site Facea,b Interscapular region Caudal ventrum Caudal dorsum No. of mice tested 46 46 46 20 Total no. of tests performed 92 174 174 80 No. of positive tests / total no. of tests performed 27/92 (29.3) 133/174 (76.4)f,g 133/174 (76.4)f 58/80 (72.5)c (% detection rate)a No. of mice with at least one positive test / 20/46 (43) 46/46 (100)f 46/46 (100)f 20/20 (100)d total no. of animals tested (% positive)a Median (interquartile range) no. of mites 0 (0–1) 4.4 (2–9.5)f,h,i 2.5 (1–4.5)f,h 2.3 (1.5–4.4)e and eggs per sampleb aThe McNemar test was used to compare the number of mice with at least one test type positive for face with the number of animals positive at other test sites. A positive test was recorded when at least a single mite or mite egg was detected, and an animal was positive when it had at least one positive test. In addition, the McNemar test was used to compare the Demodex detection rates of the interscapular region, caudal ventrum, and caudal dorsum with the detection rate of the face. bThe Wilcoxon signed-rank test was used to compare the number of mites between anatomic sites. cP ≤ 0.05; dP ≤ 0.01; eP ≤ 0.001; and fP ≤ 0.0001 compared with value for face. gP ≤ 0.01 and hP ≤ 0.0001 compared with value for caudal dorsum. iP ≤ 0.001 compared with value for caudal ventrum.

Table 3. Demodex detection rate (%) for each test–site combination. Face Interscapular region Caudal ventrum Caudal dorsum Superficial skin scrape NP 63.0 61.0 65.0 Tape impression 19.6 72.2 83.3 75.0 Fur pluck 39.1 76.1 74.0 60.0 Deep skin scrape NP 93.5 89.1 90.0 NP, not performed selected for 4-way test–site combination analysis due to their mammary gland sensitivity. Sampled animals began grooming higher range of detection rates in the 2-way analysis. Of the 16 to remove oil and debris and burrowing in the bedding immedi- possible permutations of results with 4-way diagnostic test–site ately after being returned to their home cages, perhaps to assist combinations (Table 6), when including FP–IS, FP–CV, DSS-IS, in removal of the mineral oil residue. Bleeding was not observed and DSS-CV, 10 combinations were reflected in the actual results. during DSS in mice, as occurs in other veterinary species.49 The resulting frequency in a 4-way test–site combination analy- PCR analysis for Demodex mite detection. Using nonspecies- sis of FP-IS, FP-CV, DSS-IS, and DSS-CV yielded 100% detection. specific primers that recognize a conserved region of 18S Among these test results, all mice had at least 2 positive tests, rRNA gene for both Demodex spp. and Myobia musculi detected but most had either 3 (22 of 46) or 4 (18 of 46) positive tests. Demodex mites in frozen skin (lanes 1 and 2), formalin-fixed Effects of skin testing. Samples from 6 of 46 (13%) of the test skin (lanes 3 through 5), and as individual D. musculi mites animals were collected antemortem. Momentary discomfort, preserved on tape (lanes 6 and 7; Figure 4). Samples were run primarily related to manual restraint, occurred occasionally twice, and a representative gel is shown. DNA from formalin- in these mice. In addition, some breeding females vocalized fixed skin consistently had a weaker band than fresh-frozen when fur was plucked from the ventrum, presumably due to skin and individual mites. A single 537-bp product present in

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a combination of PCR analysis and FP or DSS, we found that approximately 13% of the imported colonies were infested.63 A reliable detection method is therefore warranted, but com- parison of available detection methods for Demodex mites has not, to date, been conducted in rodents. Our current results shed light on how best to detect these ectoparasites by using traditional methodologies and are informative for laboratory animal personnel, diagnostic laboratories, and veterinary staff responsible for rodent health monitoring programs. There is debate as to whether Demodex mites are commensal, mutualistic, or parasitic in mammals.34,60 A beneficial role to the mammalian host has not been clearly defined and although almost 100% of certain host species (for example, humans and dogs) have been found to harbor Demodex mites, disease can develop as a result of mite overgrowth.13,16,60 Mice obtained from commercial vendors are produced from parental strains that have been rederived through cesarean or embryo transfer and are therefore presumably free of all ectoparasites. To our knowledge and in our experience, Demodex mite infestations do not cause significant morbidity in the majority of infested mouse strains and, until recently, the presence of Demodex mites went unrecognized or was perhaps considered by some to be inconsequential for SPF rodent colonies. Evidence suggests 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 that immunocompetent mice in laboratory colonies can be 21,52,63,73 the upper and lower quartiles, and the whiskers represent the lowest subclinical carriers of Demodex mites. Most imported and highest numbers of mites and eggs for each test–site. The face was Demodex-infested lines we evaluated were immunocompetent sampled by fur pluck and tape impression only. All values were signif- and were free of skin disease;63 however, in addition to previ- icantly different from one another (‡, P ≤ 0.0001, Kruskal–Wallis test). ously published reports, we have observed that some presumed immunocompetent mouse strains can have dermatologic all lanes was isolated and sequenced. When this sequence was manifestations. Importantly, subclinical infestations may have used as the query sequence for a homology search, 40 of the top a biologic impact and can potentially confound specific research 50 hits were from various Demodex rRNA gene sequences. Many studies. For example, infestations with other mite species can results were duplicates of mite sequences entered under cause elevated immunoglobulin production and immune different accession numbers. There was 100% homology with activation leading to amyloidosis in select strains.18,47,57,65 A the 18S rRNA gene from D. musculi and 96% to 99% sequence colony’s Demodex status is important in facilities that house im- homology with other Demodex spp., indicating that the region munocompromised animals, because they may be susceptible was highly conserved. The remaining sequences identified to clinical disease.21,38,52,73 were from rRNA genes of other mite species in both Trombid- Demodecid mites typically are located deep within hair fol- iformes and orders, some of which are parasitic, licles or sebaceous glands, and diagnosis requires a method in including Myobia musculi, with which D. musculi shares a 91% which the mites can be extracted from or observed within the sequence similarity. The mite species with the highest sequence follicle or gland. Trichoscopy of FP and DSS samples and his- homologies are listed (Figure 5). topathology have been used for diagnosis of Demodex mites in A summary of PCR results compared with 4 test–site combina- wild, laboratory, pet, and zoo-maintained rodents; however, de- tions is provided in Table 7 . All 8 animals were PCR-positive. tection methods had not been compared previously.8,29,42,54,73,75 In this subset, FP–IS and FP–CV had detection rates of 75% and In addition to the detection of ectoparasites in imported rodents, 62.5%, respectively, but the combined FP results at both sites a sensitive diagnostic test is needed to evaluate treatment effi- increased the detection rate to 100%. DSS from either site was cacy when treatment or eradication of Demodex infestation with equivalent to PCR analysis in detecting infested mice (100%) in parasiticides is elected. Although a sensitive detection method, this subset of test subjects. Despite the lower detection rates for the examination of histologic skin sections has the disadvantage FP in this subset, when compared with DSS and PCR analysis, that skin biopsies are invasive and are typically performed the differences were not statistically significant. postmortem, multiple biopsies may be necessary, analysis can be labor-intensive, and results may be delayed.38,52,73 An alternative Discussion sensitive antemortem diagnostic method would be ideal. Our Demodex mites have been reported in several immunocom- current results indicate that DSS, when combined with FP, at 2 promised laboratory mouse strains,21,38,52,73 but we and others different anatomic sites (IS and CV) yields 100% detection for have suggested that, although reported infestations are rare, the D. musculi in a moderately infested mouse strain. parasite is likely underrecognized and underreported.4,52,73 Just The diagnostic methods used and test type recommenda- as a close relationship between Demodex populations in humans tions in humans and veterinary species vary depending on the has been attributed to population movement, we propose that anatomic site being assessed. DSS is the most commonly used the frequent sharing of mice from noncommercial sources has method in dogs, although FP is recommended for diagnosis in contributed to the global spread of subclinical murine Demodex specific body regions, such as the periorbital and interdigital carriers.55 Although the true prevalence of Demodex mites in areas.50 In some cases, biopsy with histopathology is used when laboratory mouse colonies is unknown, testing of mice from negative findings are obtained by using traditional methods noncommercial vendors imported into our institution, by using or when the skin is thickened or scarred.50 In other veterinary

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Table 4. Test adequacy for the face, interscapular region, caudal ventrum, and caudal dorsum based on the number of hairs per sample for each test type. Interscapular Parametera Face region Caudal ventrum Caudal Dorsum Superficial skin scrape No. of tests performed NP 46 46 20 Median (IQR) no. of hairsb,c NP 294 (190–344) 311 (230–467) 367 (306–458) % of tests with ≥100 hairs NP 89 96 100

Tape impression # of tests performed 46 36 36 20 Median (IQR) no. of hairsb,d 132 (73–251) 381 (317–502) 370 (292–499) 512 (377–699) % of tests with ≥100 hairs 65 100 100 100

Fur pluck # of tests performed 46 46 46 20 Median (IQR) no. of hairsb,c 364 (203–501) 455 (351–610) 459 (302–590) 733 (594–1116) % of tests with ≥100 hairs 91 100 100 100 IQR, interquartile range; NP, not performed aThe hairs in each SSS, TI, and FP sample were counted. A test was considered adequate when it contained a minimum of 100 hairs per sample. bSpearman correlation coefficients between the number of mites (see Table 1) and the number of hairs counted per test were calculated: SSS, 0.3; TI, 0.53; and FP, 0.33. cP ≤ 0.05 dP ≤ 0.001 species, when DSS are performed correctly, the epidermis is scraped down to the dermis, and bleeding occurs with sufficient sampling depth.50 In contrast, when DSS samples are collected in mice, the skin does not bleed, likely because the murine dermis is poorly vascularized.77 Recently, alternatives to DSS have been proposed because it is mildly invasive. In dogs, FP or TI used with skin compres- sion have variable sensitivity but are less costly and invasive compared with DSS.41,56,66 In domestic cats, Demodex mites have been identified through coproscopy (fecal flotation), pre- sumably because cats are avid groomers.40,44,72 Because mice are also fastidious groomers, we have occasionally detected Demodex mites in fecal flotations, but, as in cats, fecal floatation does not appear to be as sensitive as DSS.44 Fecal PCR analysis for Demodex potentially could be useful when pooled samples are analyzed. Additional studies are necessary to ascertain the sensitivity of fecal PCR assays as compared with conventional sampling methods. In humans, Demodex mites are associated with several ocular and dermatologic conditions and are most 16 commonly found on the face and in eyelash follicles. Although Figure 2. Graphical representation of the Spearman rank correlation the standardized skin surface biopsy is the gold standard for between the number of mites per test site and the number of hairs testing lightly haired face and body areas in humans, lash counted at each test site for SSS, TI, and FP samples from each mouse. epilation is routinely used for cases of Demodex-associated The Spearman correlation coefficient was 0.47 (P = 0.001). .37 More recently, handheld confocal laser scanning microscopy has been proposed for surface mite detection in more frequently? We speculate that they are not typically an humans, but neither standardized skin surface biopsy nor confo- excluded pathogen, and routine careful assessments for Demo- cal laser microscopy is practical for testing rodent colonies for dex mites are not performed. Fur mites or eggs and pinworm Demodex mites.33,70 eggs are much larger and more opaque than Demodex mites, Our results indicate that, of the traditional microscopy making detection easier. Samples for fur mite diagnostics are methods, DSS had the highest detection rate by far, although typically examined at 50× magnification, which would likely any of the other methods tested can be used to detect Demodex preclude identification ofDemodex mites because of their small mites in mice. The enhanced sensitivity of DSS most likely size (approximately 200 μm) and transparency. For comparison, results from the expulsion of mites from the follicle as the skin a Demodex adult female is approximately the size of an adult fur is squeezed during sample collection. In addition, compared mite appendage. In facilities where SSS and TI are performed with other methods, DSS samples contain fewer hairs that can routinely to detect other parasites, increasing the magnification conceal mites, making sample analysis easier. Because SSS to 100× and adjusting contrast during microscopic assessment and TI are often used to detect other parasites in mice, such as may facilitate the detection of Demodex mites. fur mites and select pinworm species (for example, Syphacia We found that oil-based tests (FP and DSS) were superior to obvelata),20,30,62,78 and given that Demodex mites are likely more tape-based tests (SSS and TI) for Demodex mite detection. They prevalent than previously considered, why aren’t they detected are better at detecting both adult mites as well as eggs and

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Table 5. Comparison of 2-way test–site combinations Total no. of tests No. of positive tests Test A Test B performed (detection rate) DSS–IS SSS–IS 46 43 (93%) DSS–IS SSS–CV 46 43 (93%) DSS–IS SSS–CD 20 20 (100%) DSS–IS TI–IS 34 34 (100%) DSS–IS TI–CV 34 34 (100%) DSS–IS TI–CD 18 18 (100%) DSS–IS FP–IS 46 46 (100%) DSS–IS FP–CV 46 45 (98%) DSS–IS FP–CD 20 20 (100%) DSS–CV SSS–IS 46 43 (93%) DSS–CV SSS–CV 46 44 (96%) DSS–CV SSS–CD 20 19 (95%) DSS–CV TI–IS 34 33 (97%) DSS–CV TI–CV 34 34 (100%) DSS–CV TI–CD 18 18 (100%) DSS–CV FP–IS 46 45 (98%) DSS–CV FP–CV 46 45 (98%) DSS–CV FP–CD 20 19 (95%) DSS–CD SSS–IS 20 19 (95%) DSS–CD SSS–CV 20 19 (95%) DSS–CD SSS–CD 20 19 (95%) DSS–CD TI–IS 18 17 (94%) DSS–CD TI–CV 18 17 (94%) DSS–CD TI–CD 18 16 (89%) DSS–CD FP–IS 20 20 (100%) DSS–CD FP–CV 20 19 (95%) DSS–CD FP–CD 20 19 (95%) CD, caudal dorsum; CV, caudal ventrum; DSS, deep skin scrape; FP, fur pluck; IS, interscapular region; SSS, superficial skin scrape; TI, tape impression

the adhesive has a similar refractive index to eggs and larvae. In oil-based tests, mite visualization is easier because there is less debris. Furthermore, movement of adult mites is visible in fresh oil-based samples, enhancing detection. In addition, the coverslip can be manipulated, allowing repositioning of hairs and debris and revealing concealed mites. Importantly, some of our samples were positive for only a single mite and techni- cal staff training was required to identify individual mites in such samples. One structure that was helpful in identification of mites in oil-based tests was a small, opaque structure called the guanine concretion, which is the deposition of a pigmented nitrogenous waste product in the opisthosoma.74 The guanine concretion was refractive, making it identifiable with subtle Figure 3. Appearance of tape-based and oil-based samples. (A) Tape changes in light or depth of focus and was observed in most impression test under low magnification (scale bar, 300μ m). The mite, live or recently dead mites. There are caveats to using oil-based highlighted in the box, is shown at higher power in the inset (scale tests, because they are more invasive than tape-based tests and bar, 30 μm). (B) High-power magnification (scale bar, 100μ m) of FP. ideally should be analyzed in a timely manner to detect live Arrowheads indicate guanine concretions in transparent, dead mites. mites. Compared with other samples, oil-based test samples (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 are more difficult to store because they must remain flat and male, and adult female. Nymphs were not observed on ectoparasite cannot be stacked. tests. Arrowheads indicate guanine concretions. Interestingly, FP was better than DSS for detecting eggs and larvae. It is plausible that eggs and larvae are not well-anchored in the follicle and they are released more readily than mites larvae. Tape-based tests may not be useful for visualizing eggs when hairs are plucked. According to our observations, eggs and larvae because debris and air bubbles can obscure these and larvae are more labile than adult mites and, for easier detec- smaller mite stages. The adhesive tape may be suboptimal when tion of living mites, we recommend oil-based samples be read

180 Methods and sites for detection of D. musculi

Table 6. Assessment of 4-way test–site combinations Possible combinations Permutation DSS–IS DSS–CV FP–IS FP–CV Number of positive mice (frequency, %) 1 − − − − 0 (0) 2 − + − − 0 (0) 3 + − − − 0 (0) 4 − − + − 0 (0) 5 − − − + 0 (0) 6 − + − + 0 (0) 7 − − + + 1 (2) 8 − + + − 1 (2) 9 − + + + 1 (2) 10 + − − + 1 (2) 11 + − + − 1 (2) 12 + − + + 2 (4) 13 + + − − 2 (4) 14 + + − + 8 (17) 15 + + + − 11 (24) 16 + + + + 18 (39)

Total 46 (100) CV, caudal ventrum DSS, deep skin scrape; FP, fur pluck; IS, interscapular region ideally within 12 h, but no later than 48 h, of collection. Dead mites are observable at later times, but the detection rate will likely decrease as mites become transparent and become more challenging to identify. Based on experience with fur mite species, which vary in their site preferences on hosts, selection of the anatomic site for Demodex collection affects test sensitivity.18,43,62 Even though Demodex mite distribution is generalized in immunocompro- mised TRP1/TCR mice, the face is not an ideal location for mite detection.52 We speculate that the hair on the face is shorter and more difficult to grasp with forceps or extract with tape, resulting in nonrepresentative samples. In addition, sampling from the face would be more irritating to the subject, given that the face is highly innervated. We found that the IS region is the best single location for detecting mites: it yielded the highest Figure 4. Representative PCR samples separated on an agarose gel. detection rate with DSS (93.5%) and the most mites per sample Lane sequence: positive control (D. canis), Lane 1, whole skin (frozen); (median, 4.4 mites). In fact, 100% of the mice we examined were 2, whole skin (frozen); 3, whole skin (formalin-fixed); 4, whole skin positive in IS with DSS or FP or both, indicating that it may be (formalin-fixed); 5, whole skin (formalin-fixed); 6, tape impression, possible to detect Demodex mites in moderately infested strains individual mite; 7, tape impression, individual mite; negative control by sampling only IS. The density of mites in IS may reflect that (water), and DNA ladder. Size markers are indicated. The PCR ampli- con was 537 bp. this site is difficult to groom or that the hair turnover rate in this region is slower.52,62 Although CD had a lower detection rate than the IS and CV regions when paired with FP, CD yielded and DSS should be performed at multiple sites, minimally the a 90% detection rate with DSS. It, therefore, remains a viable IS and CV regions, for optimal detection. anatomic site for testing; however, it is a more difficult region One limitation of the study was that a single person collected to sample during manual restraint, and the pelvis made it more and evaluated all samples. Another limitation was that a single difficult to scrape the skin evenly. This site would be easier to moderately infested, immunocompromised mouse strain was sample postmortem or after chemical restraint. Although we used. Therefore, systematically determining the effectiveness did not assess the middorsum in the current study, this site of DSS and FP in diagnosing Demodex mite infestation in im- might be useful, given that topographic analysis revealed high munocompetent mice commonly used as sentinels, such as numbers of mites in this region, and there is abundant loose Swiss Webster or CD1 stocks, is important. We surmise that a skin in this region.52 4 test–site combination strategy will be useful in strains with lower mite burdens, but the overall detection rate will likely When multiple tests were performed on individual mice, the 41,60 overall mite detection rate for a specific test increased in our be lower in healthy, immunocompetent animals. We used sample. This phenomenon has also been observed in dogs.60 the FP–DSS combination to determine the extent of Demodex As in other species, false negatives are possible in healthy, infestation in the animals housed in the same holding rooms as immunocompetent mice.14,60 We therefore recommend that FP TRP1/TCR mice. Although the majority of the 40 strains tested were Demodex-negative, we found mites in 3 of 5 mice (60%) in

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Figure 5. Mite species with highly homologous 18S rRNA sequences.

Table 7. Demodex detection rate of PCR and FP analyses compared with the detection rate of DSS in a subset of subjects Gel lanea Case IDb Sample type PCRa FP–IS FP–CV DSS–IS DSS–CV 1 17 Frozen, whole skin + + + + + 2 18 Frozen, whole skin + + − + + 3 42 Formalin-fixed, whole skin + + + + + 4 43 Formalin-fixed, whole skin + − + + + DNSa 44a Formalin-fixed, whole skin + + − + + DNS 45a Formalin-fixed, whole skin + + − + + 5 46 Formalin-fixed, whole skin + + + + + 6 46 Individual mite 7 20 Individual mite + − + + +

Detection rate (%)c 100% 75% 62.5% 100% 100% CD, caudal dorsum; CV, caudal ventrum; DNS: data not shown; DSS, deep skin scrape; FP, fur pluck; IS, interscapular region; SSS, superficial skin scrape; TI, tape impression aGel lane indicated corresponds to the gel shown in Figure 3. Results shown in the table are combined from multiple PCR assays. Samples 44 and 45 were run on another gel (not shown). bCase ID, for cross-referencing, refers to the animal identification number used in a previous publication.52 cDetection rates were compared between tests by using McNemar’s test. None of the values, including those for FP–IS and FP–CV, differed significantly from the DSS–IS or DSS–CV detection rate. each of 2 presumed immunocompetent strains.52 In addition, like eggs and larva, are more labile than mites and may not be we used FP with DSS, as well as a commercially available PCR detectable unless samples are evaluated immediately. assay (Charles River Laboratories) specific to Demodex spp., Given recommendations for sample collection in dogs, it was to assess whether mice imported from other institutions were important to obtain a representative sample for each method infested with Demodex mites. Positive results were obtained in evaluated. We determined that the number of hairs in a sample subclinical animals by using all methods.63 Therefore, DSS and is similarly important in the mouse, because there was a signifi- FP can be useful as well as cost-effective for testing mice for cant correlation between the number of hairs and the number of biosecurity and quarantine, when trained personnel are avail- mites detected in the sample. Whether a sample is representative able to collect and assess samples. is based on the number of hairs contained in the sample when In terms of life stage, we observed that adult females were using SSS, TI, and FP. SSS of IS and CV, TI of the face, and FP more numerous than adult male mites in the samples evaluated. of the face had fewer than the desired number of hairs in the This pattern is likely related to their tendency for arrhenotoky, sample, but all other test–site combinations met our criterion of a form of parthenogenesis.23,36,53 We also note that the maximal at least 100 hairs per sample. We speculate that as the number length we measured for adult males and females was longer of hairs increased, the number of follicles with potential mites than previously published.22,26 This outcome is not unexpected sampled increased; however, more densely haired samples be- because variability in length has been described in various De- came progressively more challenging to assess accurately. The modex spp., including D. musculi.5,10,26,39 Interestingly, nymphs narrow width of Demodex mites allows them to be concealed were not present in any of the samples examined, although beneath hair shafts or hair bulbs. Densely haired samples are they were identified previously when the skin of TRP1/TCR more time-consuming to analyze, and large numbers of hairs mice was assessed by using skin fragmentation digestion.52 This may hamper mite detection, potentially leading to false nega- finding may reflect that nymphs represent a small proportion tive results. In one report, false-negative results in detecting of the overall mite population. Nymphs appear deeply embed- murine fur mites was attributed to both sampling strategy and ded within sebaceous glands (data not shown) histologically, human error.62 Time-consuming, duplicative review of each perhaps making them more difficult to retrieve by using the sample (as in the current study) or independent evaluations by methods we evaluated. Another possibility is that nymphs, multiple technicians may reduce false-negative results. Some authors have suggested applying additives to samples as ways

182 Methods and sites for detection of D. musculi

to enhance mite visibility in trichoscopy samples, but they have investigation is needed to determine whether immunologic the disadvantage of being lethal to parasites or hydrophilic and changes result from colonization of immunocompetent mouse thus incompatible with mineral oil.6,32 strains. In conclusion, FP and DSS are valuable diagnostic PCR analysis is increasingly used as a diagnostic tool in labo- methods for Demodex mite detection in laboratory mice. Future ratory rodent health surveillance programs. Specific genes, such studies will determine whether PCR analysis could be used as 18S rRNA and chitin synthase, are highly conserved among alone or as an adjunct to standard detection methods, as is the Demodex spp.11,81 Research in humans, domestic animal species, case with other parasites.20,78 and wildlife indicates that PCR assays of these genes may be used to detect Demodex mites.9,17,45,59,67,80 The DNA sequence Acknowledgments for D. musculi 18S rRNA was posted to GenBank in 2011 after We thank the members of Taha Merghoub’s laboratory, especially the isolation of mites from an immunocompromised laboratory Hong Zhong; the staff of the Center of Comparative Medicine and mouse, and it has been compared with the DNA of various mite Pathology’s (CCMP) Laboratory of Comparative Pathology, especially species.19,79,82 The primers we used in the current study were Jacqueline Candelier; and the CCMP’s Veterinary Services staff for their used to amplify the original D. musculi sequence submitted assistance with this project. We also thank Clifford Desch for his valu- to GenBank, which is 100% homologous with the D. musculi able advice, Michelle Lepherd for teaching and training the staff how to detect Demodex mites, and Susan Compton for her insights regarding rRNA sequence we isolated. In agreement with other studies, all PCR assays and for providing primer sequences. This work was sup- Demodex rRNA sequences in GenBank had strong homology ported in part by a grant from the National Cancer Institute (P30 CA 35,69 (96% to 99%) when compared with our amplicon. However, 008748) to the Memorial Sloan Kettering Cancer Center. the M. musculi rRNA sequence had less homology (91%). Our results agree with previous reports demonstrating that indi- vidual Demodex mites can be detected by using PCR analysis, References 69,82 1. Akilov OE, Kazanceva SV, Vlasova IA. 2001. Particular features indicating that this method is highly sensitive. Although of immune response after invasion of different species of human the primers we used were effective, their usefulness may be Demodex mites. Russ J Immunol 6:399–404. limited due to their lack of specificity. We were in the process 2. Akilov OE, Mumcuoglu KY. 2004. Immune response in demodi- of constructing more specific primers when PCR assays for cosis. J Eur Acad Dermatol Venereol 18:440–444. D. musculi became commercially available in 2015. 3. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. In dogs and cats, PCR analysis has been used to identify new Basic local alignment search tool. J Mol Biol 215:403–410. mite species and the etiologic mite species in clinical infesta- 4. Barthold SW, Griffey SM, Percy DH, editors. 2016. Pathology of laboratory rodents and rabbits, 4th ed. Ames (IA): Blackwell tions.12,17,59,69,71 PCR analysis is not routinely used to diagnose Publishing. Demodex in dogs and humans because 100% of subjects are in- 5. Bourdeau PJ. 2010. Variation of size in Demodex canis: from the fested (the majority subclinically), but recently, quantitative PCR shortest to the longest forms. The 24th Annual Congress of the analysis in humans has been proposed, because clinical mani- ECVD-ESVD, 17–19 September 2009, Bled, Slovenia. Vet Dermatol festations are associated with Demodex mite overgrowth.9,31,76 21:213. PCR assays may be more effective as a diagnostic tool in mice 6. Bruet V, Bourdeau P. 2011. Evolution over time of Demodex mite because vendor-sourced mice should be free of ectoparasites. detection in skin scrapings with Amann’s lactophenol in dogs. Vet Because a commercially available PCR assay was unavailable Dermatol 22:378–379. 7. Bukva V. 1985. Demodex flagellurus sp. n. (: Demodicidae) for D. musculi at the time this study was initiated, we evalu- from the preputial and clitoral glands of the , Mus ated traditional parasitology methods as well as an inhouse, musculus L. Folia Parasitol (Praha) 32:73–81. nonspecific assay. The detection rate of DSS was equivalent to 8. Bukva V. 1995. Demodex species (Acari:) parasitiz- that of PCR assays performed on skin biopsies or mites from ing the brown rat, Rattus norvegicus (Rodentia): redescription of TI, but FP was not as sensitive. In a separate study, we opted Demodex ratti and description of D. norvegicus sp. n. and D. ratticola to compare one commercially available PCR assay with FP and sp. n. Folia Parasitol (Praha) 42:149–160. DSS to determine parasiticide treatment efficacy. Our results 9. Casas C, Paul C, Lahfa M, Livideanu B, Lejeune O, Alvarez- (manuscript under review) indicate that PCR analysis of pelt Georges S, Saint-Martory C, Degouy A, Mengeaud V, Ginisty H, Durbise E, Schmitt AM, Redoulès D. 2012. Quantification of swabs can be used as a highly sensitive antemortem diagnostic by PCR in and its relationship to skin tool. Therefore, based on the Demodex detection rates observed innate immune activation. Exp Dermatol 21:906–910. in this study, we recommend that DSS of IS should always be 10. Chesney CJ. 1999. Short form of Demodex sp. mite in the dog: oc- included in a dermatologic work-up when ectoparasites are currence and measurements. J Small Anim Pract 40:58–61. suspected. In addition, when time and resources are available, 11. de Rojas M, Riazzo C, Callejon R, Guevara D, Cutillas C. 2012. the combination of 4 test–sites (DSS–IS, DSS–CV, FP–IS, FP–CV) Molecular study on 3 morphotypes of Demodex mites (Acarina: is ideal because of enhanced detection due to multiple site test- Demodicidae) from dogs. Parasitol Res 111:2165–2172. ing and the ability to detect eggs and larvae. PCR analysis and 12. Ferreira D, Sastre N, Ravera I, Altet L, Francino O, Bardagi M, Ferrer L. 2015. Identification of a 3rd feline Demodex species histopathology are both important tools that can be combined through partial sequencing of the 16S rDNA and frequency with antemortem tests to enhance mite detection. of Demodex species in 74 cats using a PCR assay. Vet Dermatol The intimate relationship between Demodex mites and mam- 26:239–e53. mals is ancient, potentially dating back to the radiation of 13. Ferrer L, Ravera I, Silbermayr K. 2014. Immunology and patho- mammals from synapsids more than 200 million years ago.67 genesis of canine . Vet Dermatol 25:427–e65. Although the mammalian host’s immune system is tolerant of 14. Fondati A, De Lucia M, Furiani N, Monaco M, Ordeix L, these ubiquitous mites, it has been demonstrated in both hu- Scarampella F. 2010. Prevalence of Demodex canis-positive healthy mans and dogs that significant immune system perturbations dogs at trichoscopic examination. Vet Dermatol 21:146–151. 15. Forton F, Seys B. 1993. Density of Demodex folliculorum in rosacea: can result in response to colonization with Demodex 1,2,9,13,16,61 a case-control study using standardized skin-surface biopsy. Br J mites. Immune responses are likely altered in Demodex- Dermatol 128:650–659. infested mice, especially those with dermatologic disease, po- tentially affecting research as well as animal welfare. Further

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