REPORTS musculi in the of Transgenic Mice

LORI R. HILL, DVM,1 PAM S. KILLE, LAT,1 DALE A. WEISS, LATG,1 THOMAS M. CRAIG, DVM, PHD,2 AND LEZLEE G. COGHLAN, DVM1

Abstract ͉ Although by a number of Demodex species have been described in mice, the occurrence of Demodex musculi was last reported by Hirst in 1917. This communication describes the occurrence of D. musculi infestation in two lines of transgenic mice and their F1-hybrid offspring. We first found the Demodex mite in mouse hair samples collected during efficacy screenings in an ongoing ectoparasite treatment trial for the fur mite Radfordia affinis. An investigation was undertaken to determine the extent of the Demodex infestation within the facility and the original source of the parasite. D. musculi was found in three of the four mouse genotypes present in the index room and in one of these genotypes in two other rooms. The mite was not found in sentinel mice, other strains, or stocks within the facility. The were more easily recovered from the immunodeficient B6,CBA-TgN(CD3E)26Cpt transgenic (Tg) and the hybrid double-Tg (B6,CBA-TgN(CD3E)26Cpt x B6,SENCARB- TgN(pk5prad1)7111Sprd)F1 mice than from the B6,SENCARB-TgN(pk5prad1)7111Sprd Tg mouse, which is believed to be immunocompetent despite its thymic abnormalities. Histopathologic examination showed D. musculi superficially in hair follicles but not in the preputial or clitoral gland or in serial sections of the head, , or ears, the locations favored by other mouse demodicids. Physical and microscopic examination revealed no . The immune deficiency in the B6,CBA-TgN(CD3E)26Cpt mouse probably provided the permissive host conditions that contributed to the proliferation and subsequent detection of the Demodex. Preliminary transmission experiments conducted with other immunologic mutant mice and our sentinel strain demon- strated variation in mite transfer and in either detection or maintenance of infestation when naïve mice were housed with those carrying D. musculi. The original source of D. musculi was not conclusively identified, but this parasite appears to be of low patho- genicity in the examined genotypes.

Demodex mites, a diverse group of permanent semiendopara- copulatory transfer (26–29). Copulatory transfer was also postu- sites of , are host- and microhabitat-specific. Demodex lated for D. flagellurus, a parasite residing in the preputial and have been isolated from , , cats, cattle, swine, horses, clitoral glands of feral Mus musculus (22). In contrast, several sheep, goats, and numerous other (1). Among labora- investigators have shown that Demodex mites found in tory animals, Demodex has been described in the gerbil (2, 3), extracutaneous areas are usually nonviable, probably resulting golden hamster (4–9), rat (10–12), and rabbit (13). In mice, at from lymphatic drainage of the affected region of skin (30, 31). least four (possibly five) species of Demodex have been reported, How Demodex organisms physically move between hosts and with no contemporary accounts in laboratory mice (10, 14–18). whether this movement involves adult or immature stages have The immunodeficient SCID-beige mouse, when engrafted with not yet been documented. Although most authors regard the skin and infected with D. canis, has been used to study ca- presence of small numbers of Demodex mites as harmless com- nine demodectic disease. The lack of mite translocation to mouse mensals (1, 20, 30, 32, 33), descriptions of cutaneous disease are tissues in this model further affirmed the remarkable host speci- common, especially in the dog, where generalized ficity of Demodex (19). can be fatal (1, 24, 30, 33–36). Historically, disease states have The various species of demodectic mites reside not only in been thought to be associated with excessive numbers of prolif- the general epidermis but also in the excretory ducts of seba- erating mites, especially the immature forms represented by egg, ceous glands associated with hair follicles and within the , and nymph (30); however, the actual correlation between secretory and excretory portions of specialized glands, such as mite numbers and clinically apparent disease remains unclear the ceruminous glands of the ear canal and the meibomian (23). Decreased host immunity is thought to allow for progres- glands of the eyelids (11, 14–16, 20, 21). In mice, specimens sion to disease states (36, 37). have been recovered from the stratified epithelia of the oral This communication describes our discovery of D. musculi in cavity, tongue, and esophagus and from the ears, skin, eyelids, the skin of B6,CBA-TgN(CD3E)26Cpt (CD3E), a homozygous and preputial and clitoral glands (10, 14–18, 22). The speci- transgenic (Tg) mouse lacking mature T lymphocytes and natu- mens recovered appear to represent unique, anatomically ral killer cells (38); B6,SENCARB-TgN(pk5prad1)7111Sprd specific Demodex species. (Prad1), a Tg mouse overexpressing cyclin D1 and mani- Despite more than 150 years of collective investigation, very festing severe thymic hyperplasia (39); and the double-Tg F1 little is known about demodectic mite transmission and the fac- offspring of these two lines. Demodex was not recovered from the tors regulating subclinical carriage as opposed to clinically room sentinels—SSIN/UTSP//Vsp (SSIN; 40)—or from other apparent demodicosis (19, 20, 23). In the dog, cow, and golden genotypes within the facility. In addition, we present the pre- hamster, mites were transmitted to neonates by direct contact liminary results of transmission experiments with the sentinel with the dam during the first few days of life (7, 24, 25). The strain and several immunologically impaired mouse strains. finding of demodectic mites in the internal tissues and genitouri- nary systems of dogs and goats have suggested intrauterine and Case Report In May 1998, mice with a history of Radfordia affinis infesta- The University of Texas M. D. Anderson Cancer Center, Science Park, Depart- tion were being monitored for the presence of mites during the ment of Veterinary Sciences, Route 2, Box 151-B1, Bastrop, Texas, 786021, course of an experimental treatment. This treatment Department of Veterinary Pathobiology, The Texas Veterinary Medical Center, involved a novel use of an avermectin compound and was taking Texas A&M University, College Station, Texas, 77843-44672 place simultaneously in several rooms in the facility (41). An

Volume 38, No. 6 / November 1999 CONTEMPORARY TOPICS © 1999 by the American Association for Laboratory Science 13 FIG. 1. Photomicrograph of Demodex musculi located superficially in the hair follicle of a CD3E mouse. Note the lack of inflammatory response FIG.2. Demodex musculi adult female from the skin of a CD3E mouse. in the adjacent tissues. Hematoxylin and eosin; 500X magnification. Wet mount; 400X magnification. external parasite examination was performed by plucking hairs Wayne® Autoclavable Rodent Blox® #8656 (Harlan Teklad, Madi- from the dorsum of the neck, placing them in mineral oil on a son, WI) and autoclaved, reverse osmosis water were provided slide, and observing the slide under a dissecting microscope. ad libitum. Room temperature and humidity were 20 to 22ЊC Radfordia was present in samples collected from all four geno- and 55%, respectively, with a 12-h light/dark cycle and 15 air types of mice in the room. The samples collected from CD3E changes per hour. Incoming animals, including sentinels, un- mice contained several unidentified, cigar-shaped mites typical derwent a 3-week quarantine and were determined to be free of of Demodex. With repeated examinations, a few Demodex were even- major pathogens prior to release to investigation. Representa- tually recovered from two of the other genotypes, Prad1 and the tives from each shipment underwent a tape test of the anus, fecal double-Tg F1 hybrid offspring of CD3E and Prad1. examination (gross examination of cecal contents, fecal concen- Cellophane tape tests of the muzzles and rear limbs of pups tration, and wet mount for protozoa), pelt examination, gross that were 1 or 2 weeks of age and from the infected cages were necropsy, and serum collection for at least a six-pathogen en- negative for demodectic mites. Demodex was not recovered from zyme-linked immunosorbent assay panel (Sendai virus, the room sentinels, SSIN mice, despite their receiving soiled Pneumonia virus of mice, Mouse hepatitis virus, Mycoplasma bedding twice weekly from the cages of the three other geno- pulmonis, Theiler’s murine encephalomyelitis, and Minute virus types. When tested further in June 1998, these sentinels were of mice). negative for all assayed pathogens except Radfordia; Demodex was Shipments of the CD3E mice (The Jackson Laboratory, Bar not detected by examination of the pelt or by histopathologic Harbor, ME) had been received periodically beginning in June examination of the skin. 1995. After release from quarantine, CD3E mice were segregated Because the species of Demodex was not yet determined and into two rooms. One room remained a closed breeding colony, the involved mouse genotypes had immune and/or thymic ab- and Demodex has not been detected by gross or microscopic ex- normalities, we considered that the mice could be serving as amination in these mice or in subsequent shipments from the accidental hosts or that the mites could have been aberrantly same vendor. The CD3E mice in the other room were used for located in these transgenic lines. Therefore we examined the outcrossing and subsequent experimentation. Radfordia was be- anatomic locations in which Demodex mites had been previously lieved to have been introduced by the Prad1 mice, whose reported in the mouse (10, 14–18, 22). The examination was Radfordia infestation went undetected at the time of their trans- performed on two animals that were positive for Demodex and fer into the index room in January 1996. The quarantine period included the preputial or clitoral gland and serial sections of and screening of the Prad1 founders (B6,SJL-TgN the decalcified head including the oral cavity, tongue, esopha- (pK5prad1)7111) were as described. In addition to these pre- gus, ear canal, eyelids, and sections of skin. One animal was a cautions, none of the founders were released to investigation. 4-month-old male CD3E mouse that was negative for mites in all Only the offspring of these founders were used, and they had tissue sections. However, only 1 cm2 of skin was examined mi- been tested and were believed to be negative for external para- croscopically, because most of the pelt was submitted to one of sites on pelt examination prior to release from quarantine. the authors (T. M. C.) for collection and determination of the Previously, several cages of post quarantine mice with Rag1- species of Demodex involved. Both adult and nymphal stages were and Rag2-targeted mutations obtained from non-commercial recovered from this mouse. The other CD3E mouse was a fe- sources had been present in the index room, but they were male retired breeder. The same tissues were examined, as were killed and removed from the room prior to the introduction of more extensive sections of the skin taken from the dorsal trun- the Prad1 mice. cal area. Again, Demodex was found only in the skin, being located A retrospective examination of formalin-preserved skin superficially in the hair follicles (Figure 1). No inflammatory reac- samples collected from a CD3E mouse from the index room tion to the Demodex mites was noted on histologic examination of that was killed in August 1997 confirmed the presence of both the skin. The mites were identified as D. musculi (Figure 2). R. affinis and Demodex. Mice from the index room (Building C, All of the mice were housed in an AAALAC-accredited facility room A8) had been distributed only to one other room in the in microisolation cages (Micro-isolator Housing Unit; Lab Prod- facility (Building A, room 110), a room in which mice were ucts, Maywood, NJ) sterilized together with aspen chip bedding housed in suspended polycarbonate cages. Here, the detection (Aspen Sani-chips; P.J. Murphy Forest Products Corporation, of Demodex was limited to Prad1 mice that had been transferred Montville, NJ); the bedding and the cages were changed twice out of the index room, and only one D. musculi was recovered weekly in a biological safety cabinet (NuAire, Plymouth, MN). after several surveys. Because all mice in this room had recently

14 CONTEMPORARY TOPICS © 1999 by the American Association for Laboratory Animal Science Volume 38, No. 6 / November 1999 The SCID female was found to be positive for Demodex after 7 days; the male was removed 2 days later. Interestingly, the SCID female was negative on rechecks at days 27, 41, and 55, recon- verting to positive on day 70 and remaining so through day 82. Because the nu/nu female was negative on days 5, 20, 34, and 48, the male was not removed. Although this female was found to be positive on day 57, poor condition necessitated euthanasia at this time, and no mites were found through histologic exami- nation of the skin. The Rag1 female was negative at day 14 and positive at day 26, and the male was removed 4 days later. Demodex were found on this female at day 42 but not on days 52 or 70. After an initial negative test on day 21, the SSIN female was posi- tive for both Demodex and Radfordia on day 35; the male was removed 6 days later. Subsequent tests for Demodex on days 45 and 62 were negative in the SSIN female.

Discussion In mice, demodectic mites have been reported only occa- FIG.3. Flow chart indicating history of backcrosses and movement of sionally. To the best of our knowledge, the only incident in the mice within the facility. Boxes with heavy borders indicate where D. United States was that of a Demodex sp. recovered from the oral musculi was found. cavity, tongue, and esophagus of grasshopper mice captured in Richland, Washington (17). Other accounts include finding been treated with an avermectin compound being used as an Demodex sp. in the tongue of a 3-month-old “white experimen- acaricide, the Prad1 mice were negative for R. affinis, as were tal” mouse in Europe (18) and D. flagellurus in the preputial the other genotypes. and clitoral glands from wild populations of the In an attempt to determine the source of the Demodex, seven in Czechoslovakia (14, 22). D. agrarii has been identified in the other rooms housing mice of the same strains as the index room cerumen and sebaceous glands of the ear of the striped field and other mice owned by investigators having mice in the index mouse in Eastern Slovakia (15), and D. lacrimalis has been re- room were surveyed monthly beginning in May 1998. These mice ported in the meibomian glands of the European wood mouse were not necessarily housed in microisolation caging, and in in the Netherlands and Italy (16). D. musculi, the species we some cases, mice in these rooms were known, or had recently identified, has been reported only by a single author in Great been found, to be positive for Radfordia. All genotypes in these Britain (10, 42). In his 1917 communication, Hirst stated that rooms, including the room sentinels, were evaluated by exami- four “tame” mice were examined and that D. musculi was found nation of plucked dorsal hairs. All hair-pluck tests were negative in three of them. The anatomic location of the mites was not for Demodex until September 1998, when one demodectic mite mentioned, although other species of Demodex discussed in this was found. Again, the host was a Prad1 mouse; neither Demodex report were located in the skin of the hosts. One other refer- nor Radfordia were found in other strains present in its room ence indicates that the D. musculi in Hirst’s report was located (Building B, room 129), in which all animals were held in in the skin (43). The results of our histologic surveys support microisolators. This mouse had been moved into this room from these findings. Because detection of skin-inhabiting demodectic the main Prad1 breeding room (Building A, room 125), the room mites had not been reported in plucked hairs, as opposed to that also supplied Prad1 mice to the index room (Figure 3). skin scrapings, we suspect that our finding of the mite by this An additional 13 mice representing the index room’s four method is due to the very superficial location of D. musculi in genotypes were Demodex-negative on scrapings of the skin and the hair follicles. Another superficially located Demodex is ex- tongue. Histopathologic examination of the skin conducted in emplified by an unnamed species found in the stratum corneum 3 (an SSIN sentinel, a B6-Tg(pK5prad1)7111Sprd, and a of cat skin (44, 45). B6,SENCARB-TgN (pK5prad1)7111Sprd) of these 13 animals, D. musculi is considerably shorter than other reported mu- also was negative except for the finding of rare mites on the B6- rine Demodex species. Females range in length from 113 to 170 Tg (pK5prad1)7111Sprd mouse (Building A, room 110). This ␮m and males, which have a distinct penile structure, from 126 mouse was housed in the one room to which mice from the in- to 154 ␮m (10, 42). The mean length for the D. flagellurus fe- dex room (Building C, room A8) had been distributed and in male is 430 ␮m and for the male is 787 ␮m (14). D. lacrimalis which a single mite had been found previously on a Prad1 mouse. females average 271 ␮m and males 225 ␮m (16). D. agrarii fe- Monthly pelt exams throughout the facility did not reveal the males average 610 ␮m and males 536 ␮m (15). The Demodex presence of Demodex in other rooms or mouse strains. mite that resides in the tongue measures greater than 400 ␮m To determine the communicability of D. musculi to other in length (18). strains of mice, we set up four microisolation cages for transmis- The original source of Demodex in our facility is unproven. sion studies. Each cage housed a male hybrid transgenic mouse However, the Prad1 Tg mice appear to have introduced R. affinis that was positive by hair examination for Demodex and Radfordia. to the CD3E Tg mice, and they may have simultaneously intro- These males were each paired with a single female of one of the duced D. musculi. This theory is supported by the negative mite following strains: C.B-17/IcrTac-scid (Taconic, Germantown, status of both the resident CD3E mice that had not been out- NY); nu/nu (CD-1; Charles River Laboratories, Wilmington, crossed and subsequent CD3E shipments as well as by the MA); B6,129-Rag1tm1Mom (Rag1; The Jackson Laboratory); SSIN population and mite infestation histories established for the in- (Dept. of Veterinary Sciences, Bastrop, TX). None of the mice dex room. Furthermore, the finding of a single Demodex mite on in the transmission study were treated with any acaricidal agent each of two Prad1 mice housed elsewhere in the facility, the find- either before or during the study. Transmission of mites was as- ing of rare mites on skin sections from a Prad1 mouse in one of sessed by hair plucking at approximately 7- to 10-day intervals; these rooms, and our inability to find the mite in other geno- skin scrapings were substituted in the case of the nu/nu female. types lend support to this hypothesis.

Volume 38, No. 6 / November 1999 CONTEMPORARY TOPICS © 1999 by the American Association for Laboratory Animal Science 15 Our retrospective investigation revealed that the Prad1 Demodex was transmitted most quickly to the SCID mouse, from transgene had been first transferred to a new inbred background which the mite was recovered at the earliest examination point SENCARB/Pt/Vsp (Line B) beginning in May 1995, shortly af- (day 7). In addition, the SCID mouse maintained the infection, ter receipt of the original Prad1 founder in March 1995 (39). as mites were still present 82 days after initial contact. Alterna- The founder had been acquired from a facility with a docu- tively, the SCID mouse might have had a higher burden of mented history of negative screenings for ectoparasites. Although Demodex than did the other strains, thus aiding detection. How- a number of pathogens had been detected and eliminated dur- ever, the number of mites that were recovered by plucking dorsal ing the quarantine of the Line B mice, carriage of Radfordia and hair was typical of that seen in the CD3E or CD3E/Prad1 hybrid Pasteurella pneumotropica unfortunately were not detected prior mice (i.e., recovery of 3 to 5 mites per time point). That we were to release from quarantine. The Line B mice were outcrossed unable to recover D. musculi from the nu/nu mouse until day 57 with the Prad1 transgenic mice, and offspring were then back- was unexpected, and our inability to find Demodex histologically crossed to the Line B strain for several generations. After in a mouse that was positive by skin scraping further shows the detection of R. affinis and P. pneumotropica in the Line B strain, it difficulty in diagnosing an infestation of this parasite. Consider- was sent for cesarean rederivation; however, the matings that ation should also be given to the theory that the abnormalities had already taken place with the Prad1 Tg were overlooked in of the hair follicles and hair shafts in the nu/nu mouse may the process. Our current assumption is that the inbred Line B have been influential in the apparent inability to establish a stable strain may have transmitted R. affinis and D. musculi to the Prad1 infestation in this strain (46). The Rag1 and SSIN mice appeared transgenic mice, which in turn transmitted both mite species to equivalent in that Demodex was detected at 26 and 35 days, re- the CD3E mice. Unfortunately, to the best of our knowledge, no spectively, and neither strain appeared to maintain the representatives of the non-rederived inbred strain (Line B) or infestation. We are continuing to observe these mice to evaluate the original Prad1 founder strain are available for evaluation for longer end points and to determine whether the Rag1 and SSIN D. musculi. We have examined time-appropriate archival tissues mice could transmit Demodex to the CD3E mice by way of a pos- of seven of these Line B mice and confirmed the presence of R. sible occult infestation. Interestingly, Radfordia was not detected affinis only. However, carriage of low numbers of D. musculi can- in the three immunosuppressed strains in this experiment, al- not be ruled out at this time, as only a small percentage of the though it was transmitted to the SSIN mice. pelt was available for histologic examination. Indeed, we occa- In mice harboring the various species of Demodex, only mini- sionally were unable to detect D. musculi histologically in mice mal tissue reaction has been seen in studies that included that were positive by pelt exam. histopathologic examination (14–17). Therefore, this group of Caution should be exercised in concluding that a particular parasites appears to be of low virulence in mice, and the lack of mouse is truly negative for D. musculi on the basis of either gross dermal response in our animals concurs with findings from pre- or histologic examination, particularly in immunocompetent vious studies. However, the immunodeficient status of the animals. This cautionary statement is based on the extremely infested CD3E and F1 Tg mice must be considered as a possible low numbers of mites found in the immunocompetent Prad1 explanation for the lack of tissue reaction to the mites. and our inability to recover additional Demodex mites on re-sam- Further studies are needed to assess the pathogenicity of D. pling of the Prad1 mice. In fact, determining the extent of musculi and determine how the superimposed Radfordia or the examination sufficient to consider a particular mouse to be free acariasis treatment may have influenced the behavior and distri- of D. musculi may be difficult. Perhaps housing suspect mice with bution of the parasite. How Demodex interacts with the highly T-cell deficient transgenics (serving as sentinels) would be the complex series of responses of the immune system as a whole, method most likely to confirm the presence of Demodex mites. particularly in immune-compromised animals, is not yet known. We theorize that the immunodeficient status of the CD3E Tg Our preliminary findings of the enhanced susceptibility in the mice allowed the Demodex infestation to increase to a point that CD3E Tg mice and the variable transmissibility in the other this rare murine parasite was eventually detected. Although the mouse strains examined demonstrate the usefulness of a mouse infestation was subclinical, three to five demodectic mites were model for the study of skin-inhabiting demodectic mites. Fur- often observed from a tuft of plucked CD3E hair. In contrast, ther investigation of D. musculi in these and other immunologic the reportedly immunocompetent Prad1 mouse probably har- mutant and Tg mice should allow better understanding of the bors much lower numbers of Demodex, as exemplified by the factors involved in clinical demodicosis and provide insight into extreme difficulty in mite recovery from this strain. Whether the the biology of this curious family of parasites. In addition, mice Prad1 phenotype of epidermal proliferation predisposed this with severe immunodeficiency (e.g., CD3E) may be particularly strain to infestation with D. musculi is unknown. effective sentinels for D. musculi. In canine demodicosis, the long-accepted theory has been that mite proliferation results from failure of the host to mount an Acknowledgments adequate cell-mediated immune response, because of either spe- This work is supported in part by NIH-NCI CA 16672. The cific or non-specific suppression of T-cell function. (30, 36). authors thank Gerald Costello and Annie Limmer for their help According to some, a hereditary T-cell defect may allow for mas- with photography and Eric Simmonds and Drs. Pete Teel, Rob- sive mite proliferation, which in turn causes release of a humoral ert Dunstan, and Clifford Desch for providing further verification substance leading to generalized T-cell suppression (30, 34, 36). of mite identification. We also thank Donna Schutz for assistance Both the humoral factors and the cell-mediated immunosup- with record retrieval, C. J. Maliniemi for manuscript prepara- pression disappear when the mites are eradicated (30). In tion, and Dr. Wallace Baze for his assistance with histopathology contrast, there is no evidence of humoral immunodeficiency in and manuscript review. dogs with generalized demodicosis; in fact, the B-cell responses appear to be excessive (30, 36). Recently, immunohistochemi- cal studies have suggested that cytotoxic T lymphocytes are References important in the immune response to D. canis (23, 36). How- 1. Georgi, J. R., and M. E. Georgi. 1990. : mites, ever, whether these cells participate in pathologic self-injury or p. 57–76. In J. Dyson, D. Kilmer, and E. Dick (eds.), Parasi- act to limit mite proliferation is as yet unknown (23). tology for veterinarians, 5th ed. W. B. Saunders Company, Our preliminary transmission studies demonstrated that Philadelphia.

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Volume 38, No. 6 / November 1999 CONTEMPORARY TOPICS © 1999 by the American Association for Laboratory Animal Science 17 43. Flynn, R. J. 1973. Mites, p. 425–492. In Parasites of labora- 45. McDougal, B. J. and C. P. Novak. 1986. Feline demodicosis tory animals. The Iowa State University Press, Ames, IA. caused by unnamed Demodex mite. Compend. Continuing 44. Conroy, J. D., M. C. Healey, and A. G. Bane. 1982. New Ed. Pract. Vet. 11:820–822. Demodex sp. infesting a cat: a case report. J. Am. Anim. Hosp. 46. Sundberg, J. P. 1994. The nude (nu) and streaker (nustr) Assoc. 18:405. mutations, chromosome 11, p. 379–389. In J. P. Sundberg (ed.), Handbook of mouse mutations with skin and hair abnormalities. CRC Press, Inc., Boca Raton, FL.

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