Borrelia burgdorferi from Rodents

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1 JOURNAL OF CLINICAL MICROBIOLOGY. Aug p /89/ $02.00/0 Copyright American Society for Microbiology Vol. 27. No. 8 Ear Punch Biopsy Method for Detection and Isolation of Borrelia burgdorferi from Rodents RICHARD J. SINSKY* AND JOSEPH PIESMAN Departinent of Epideiniology. School 'f Publi/ Healtl, University of Alabama at Birininghain, Birininghain, Alabama Received 10 March 1989/Accepted 20 April 1989 An ear punch biopsy method for the detection and isolation of Borrelia burgdorferi from rodents was developed. The ear punch biopsy proved to be extremely sensitive, detecting spirochetes in 100% (11 of 11) of laboratory hamsters infected by tick bite and 95.8% (23 of 24) of hamsters infected by intraperitoneal inoculation. When cultured at 4 to 6 weeks postinfection, 92 to 100% of the ear punches taken from individual hamsters yielded viable spirochetes. B. burgdorferi was detected in sequential cultures from animals as early as 4 days postinfection and as late as 20 weeks postinfection. A total of 86% (6 of 7) of field-collected white-footed mice (Peromyscus leucopus) which were positive for B. burgdorferi as determined by xenodiagnosis were also positive by the ear punch method. The ear punch biopsy method allows individual rodents to be sampled for B. burgdorferi serially over a long period and thus should prove useful for both field and laboratory experiments. Rodents are an important reservoir of Borrelia burgdorfèri, the etiologic agent of Lyme disease. This spirochete has been isolated from naturally and experimentally infected white-footed mice (Peroinyscus leucopus) by culturing the spleen, kidney, liver, blood, urinary bladder, and eye (1, 7, 9, 21). It has also been isolated from the blood, cerebrospinal fluid, skin, and liver of human patients with Lyme disease (3, 4, 7, 15, 22). Laboratory rodents such as the golden Syrian hamster and the white rat have been experimentally infected with B. buirgdoifjri (6, 12-14). The internal organs which have been the most successful for isolation of the spirochete from rodents have been the spleen, kidney (1, 2), and urinary bladder (21). While the cultivation of internal organs has yielded spirochetes, the disadvantage of this system is that the animals must be killed. Although blood culture has occasionally been successful in isolating B. biurgdoijeèi, it has been found to be an inefficient source of the organism (7). Alternatively, serology has been used to determine infection status. Serology, however, may be unreliable because of cross-reactions and may reflect past rather than current infection (16, 17). Another problem is that detectable antibodies may not be present until well after infection has been established (17). An alternative method for determining whether an animal is infected with B. biîrgdoiferi entails allowing uninfected tick larvae to feed on the test animal and examining the replete larvae for spirochetes (11). A disadvantage of this xenodiagnostic method is the requirement for a large supply of uninfected larvae. Thus, an easy and sensitive method is needed to repeatedly obtain cultures from individual rodents for B. bitr-gdotjeri, both in the field and in the laboratory. In humans, the skin is an important component of the pathology of Lyme disease and may be involved in the various stages of the illness. In the primary stage, the pathognomonic sign, erythema (chronicum) migrans, is often seen at the site of the infecting tick bite. B. burgdojeéri has been isolated from skin taken from erythema migrans lesions (4, 19, 22), even as early as 10 days after the skin has been fed upon by an infected tick (19). During the subsequent * Corresponding author. stages, multiple lesions may develop elsewhere on the patient's body. A Borrelia lymphocytoma, a dense, dermal lymphoreticular proliferation, may also develop, either at the site of the original tick bite or elsewhere on the body, especially the ear lobes (3). The same situation may hold true in rodents infected with B. burgdoiferi. In rodents, the ear is an easily obtainable source of skin. Accordingly, we evaluated the possibility that the skin, particularly skin from the ears, of infected hamsters and mice could provide suitable samples for culturing B. buirgdorjferi. In addition, the efficiency of the ear punch method was compared with xenodiagnosis and culturing internal organs as a means of detecting Borrelia infections in rodents. MATERIALS AND METHODS Spirochetes. Two strains of B. birgdo,:feri were utilized in these experiments, JD-1 and DW-18M. The JD-1 strain of B. blrgdoijeri was isolated and maintained in ticks by methods previously described (20). In vitro cultures of the JD-1 strain were isolates from the skin or ears of hamsters infected by tick feedings. These isolates were passaged in modified Barbour-Stoenner-Kelly (BSK) medium and frozen at -85 C in 30% sterile glycerol. The DW-18M strain was kindly provided by Russell Johnson (University of Minnesota, Minneapolis) as a third-passage isolate from an Ixodes daînrnini nymph removed from a bird in Minnesota. This culture was inoculated intraperitoneally into a hamster and reisolated from the skin and then maintained in culture in the same manner as the JD-1 strain. Cultures. Cultures were maintained in BSK medium as described previously (5), with the exception of the following additions: 0.023% L-cysteine hydrochloride, 0.015% DLdithiothreitol (Sigma Chemical Co., St. Louis, Mo.), 1,ug of L-glutamine (GIBCO Laboratories, Fairfield, N.J.) per ml, 0.15% soft agarose (SeaPrep; FMC BioProducts, Rockland, Maine), 50 pg of rifampin (Sigma) per ml, 20 pg of phosphomycin (Sigma) per ml, and 2.5 ptg of amphotericin B (Fungizone; GIBCO) per ml. Cultures were isolated and maintained either in 8-ml-capacity tubes, tightly sealed, containing 7 ml of medium, or in microdilution plates with 0.25 ml of medium per well stored in a glass desiccator used 1723

2 1724 SINSKY AND PIESMAN as a candle jar. Because of evaporation, fresh medium was added to the microdilution wells as needed. All cultures were maintained at 34 C and were examined for spirochetes by dark-field microscopy for 6 weeks. Hamsters. Female golden Syrian hamsters weighing approximately 80 to 100 g were infected with B. burgdorferi either by tick feeding or by intraperitoneal (i.p.) inoculation. Naturally infected hamsters were exposed to 5 to 15 I. dammini nymphs infected with the JD-1 strain of B. bzîrgdorferi. Animals infected i.p. were inoculated with 1 x 109 to 7.9 x 109 viable spirochetes of a passage 1 to 10 culture of either the JD-1 or DW-18 strain or with a whole-tick homogenate in phosphate-buffered saline containing 1,300 to 28,000 spirochetes of the JD-1 strain. Each animal was housed individually after infection. After the hamsters were infected, samples were obtained from them according to the following schedules: group A, animals that had been infected by tick feeding were euthanized and cultures were done at 21 to 32 days after the last day of tick feeding; group B, two animals had their ears punched every other day for 12 days prior to euthanasia and culturing of skin and bladders; group C, one animal had its ear punched every 2 weeks from week 6 to week 12 and then monthly thereafter; group D, animals that were inoculated i.p. with tick homogenate or cultured spirochetes were euthanized and cultures were obtained at 21 to 28 days postinoculation. The xenodiagnostic procedure was performed on a subset of animals from groups A and D, using larval I. dammini as described elsewhere (11, 20). White-footed mice. Ears from experimentally infected white-footed mice (P. leucopus) were supplied by Tom Schwan (Rocky Mountain Laboratory, Hamilton, Mont.). These mice had been inoculated i.p. with 5 x 106 cells of a second-passage culture of B. burgdorferi (strain ECM-NY- 86), and the mice were euthanized 56 days later. Ears from mice trapped in areas endemic for Lyme disease in Massachusetts were supplied by Sam Telford (Harvard School of Public Health, Boston, Mass.). The ears were removed from euthanized animals and frozen without cryoprotectant at -70 C, shipped on dry ice, and stored at -85 C until culturing. The ears from the experimentally infected P. leucopus were received in a partially thawed condition. Skin cultures. Skin samples were obtained from euthanized rodents by wetting down the skin of the back with 70% ethanol and reflecting back the skin by making a transverse incision from leg to leg at the base of the tail followed by vertical incisions on either side of the animal. Adherent fat and the transparent connective tissue layer were than removed, and the skin was pulled taut over the fingers of one hand. Approximately 1 to 2 cm2 of the dermis was then gently shaved off with a pair of small sharp scissors which had been dipped in alcohol and flamed, taking care not to break through the epidermis. The sample was then inoculated into a 7-ml tube of modified BSK medium or divided and inoculated into 0.25 ml of medium in microdilution wells. Ear cultures. Initially, ear samples were obtained by cleaning the ear with 70% ethanol prior to removing it from euthanized animal and finely mincing it into separate 8-ml tubes. Subsequently, individual punches were obtained from euthanized animals by removing the ear as described above prior to taking samples from each ear with a standard 2-mm rodent ear-notching punch (MICH-CROWN, Bay City, Mich.), or a rodent ear tag punch (BAXTER, Atlanta, Ga.). The punch was dipped in alcohol between uses, and either single or multiple punches were taken from each ear; gener- J. CLIN. MICROBIOL. TABLE 1. Comparison of hamster ear, skin, and urinary bladder as sources of B. biur-gdoifè,ri in BSK cultures Source of Route of No. % of organs positive inoculum inoculation examined Ear Skin Bladder Tick feeding' Skin il Tick homogenate' I.p Culture' 1. p Totals B. burgdoiferi JD-1. "Five animals inoculated with B. buirgdoiféri DW-18, six animals inoculated with B. buirgdorferi JD-1. ally, three punches were taken per ear. Each punch was placed into an individual well of a microdilution plate with 0.25 ml of medium. Live animals from which cultures were repeatedly obtained were restrained, and each ear was soaked with 70% ethanol before the biopsy punches were taken. The punch was dipped in alcohol between samples. Frozen ears were quickly thawed immediately prior to sampling and dipped in 70% ethanol. Excess alcohol was drained off the ear, and the rest of the alcohol on the ear was allowed to evaporate before six punches were taken from each ear. Bladder cultures. The urinary bladders were removed aseptically, and the urine was expressed from the bladder. The tissues were then minced and ground in 3 ml of BSK medium, using a 7-ml-capacity tissue grinder. After the tissue was ground and particulate matter settled out, 1.0 ml of bladder homogenate was inoculated into 6 ml of modified BSK medium (21). RESULTS Among hamsters that acquired their infections by tick feeding, 11 of 11 were both skin and ear culture positive for B. burgdorferi, while only 5 were bladder culture positive (Table 1). Nine of these animals were tested by the xenodiagnostic method, and al were positive. Of the 13 animals that were inoculated i.p. with a tick homogenate, all were positive by ear culture, 12 were positive by skin culture, and 5 were positive by bladder culture. Two of these animals were tested by xenodiagnosis; both were positive. Eleven animals were inoculated i.p. with cultured spirochetes; 10 were positive by the ear culture method, 5 animals were skin culture positive, and 6 were bladder culture positive. Three of these animals were tested by xenodiagnosis; two of these were positive. Overall, 8.6% (3 of 35) of the infected hamsters were positive by ears only; none were positive by bladder or skin only. Both ears of a hamster that had acquired its infection by tick feeding and one ear from an animal inoculated i.p. with tick homogenate were completely examined by taking multiple ear punch samples. The punches were taken in an ordered sequence to map out their positions on the ear. Samples were obtained 30 days postinfection. It was possible to obtain as many as 71 individual punches per ear. The proportion of samples positive was 92.2% (left ear) and 100% (right ear) for the tick-fed animal and 95.8% (one ear) for the other (Table 2). The few negative samples were from the periphery of the ear. In general, the density of spirochete growth seen in the wells for individual samples increased as the position of the sample moved closer to the base and center of the ear.

3 VOL. 27, 1989 EAR PUNCH METHOD FOR DETECTING SPIROCHETES 1725 Hamster TABLE 2. Proportions of whole-ear samples positive for B. bh",gdot"èri in hamsters No. of biopsy 157' of biopsy examined positive Source of Socurceof Ear samples samples 1 Tick feeding Left Right Tick homogenate Right Short-term sequential cultures were taken from individual hamsters that had been infected by tick feedings to determine how soon after infection spirochetes could be recovered. The earliest that a positive sample could be detected was 4 days after the last day of tick feeding (Table 3). In one animal, both ears became positive on day 4; in the other animal, one ear was positive on day 4, while the remaining ear did not yield positive samples until day 8. When cultured at day 12, the skin and bladder were positive in both animals. To determine whether spirochetes could be repeatedly isolated from the same animal, which had acquired its infection by tick feeding, sequential biopsy samples were taken over the course of 20 weeks. We went from sampling every 2 weeks to sampling every 4 weeks to maximize the amount of time that the animal could be sampled. Spirochetes were isolated at each sampling time through week 20 postinfection (Table 4). At 16 weeks, the proportion of punches which became positive appeared to decrease noticeably. Frozen ears from five experimentally infected P. leiic opus mice were sampled to determine whether they would be a suitable source for isolating spirochetes. All five animals were positive as determined by culturing bladder and spleen prior to freezing and shipping of the ears (T. Schwan, personal communication). Spirochetes were detected in three of these five experimentally infected animals by the ear punch method. White-footed mice from areas in Massachusetts endemic for Lyme disease were trapped and tested by xenodiagnosis prior to removal of the ears for shipping and culturing (Table 5). Both ears were available from seven animals, but only a single ear was available for culturing for two of these animals. Of nine animals tested, seven animals were positive by xenodiagnosis (S. Telford, personal communication). A total of six of the seven (85.7%) xenodiagnosis-positive mice were found to be infected by using the ear punch biopsy method. The animal which was xenodiagnosis positive and ear punch negative was also one of the animals which had only one ear cultured. Thus, the ear punch method proved to be sensitive for the detection of spirochetes in P. leiw sopius. TABLE 3. Short-term sequential ear punch culturing of B. burgdofiferi from hamsters infected by tick feeding No. of positive biopsies at the following Hamster Ear day postinfection": no. sampled o' () 12 1 Left O O Right O O Left O Right O O O In ail cases, a total of three biopsies wais examined. " Day 0 = last day of tick feeding. Ear TABLE 4. Long-term sequential ear punch culturing of B. hurgdoiferi from a hamster infected by tick feeding No. of positive biopsies at the following wk postinfection": ) Left Right " In ail cases, a total of three biopsies was examined. despite the fact that the ears were shipped on dry ice for more than 24 h. Positive skin and ear samples could generally be detected 48 to 72 h after tissues were placed into culture, although one sample was detected as early as 20 h postsampling. Cultures of frozen samples generally required 5 to 7 days of incubation before spirochetes could be detected. DISCUSSION The current method for culturing B. buirgdoiferi from rodents entails emulsifying the kidney, spleen, and bladder in BSK medium and then inoculating this material into 7-ml tubes of medium (14, 21). Unfortunately, this method of obtaining culture material necessitates killing the animal and does not allow follow-up studies of individual animals. Internal organ culture, if used in a long-term surveillance program, could remove animals from a wild population which might otherwise be monitored for the incidence or prevalence of infection with B. biurgdoiferi in an endemic area. Organ culture is also impractical for treatment trials in laboratory animals, since the infection status of an animal before treatment cannot be ascertained. Xenodiagnosis does allow for repeated testing; hamsters, however, may become resistant to repeated tick attachment (11). Therefore, xenodiagnosis might not be an effective method for sequentially testing individual hamsters. Another disadvantage is the time and effort required in establishing and maintaining a tick colony of adequate size for large-scale studies. It has been shown that Treponeina pallidum and other spirochetes are capable of growing better at temperatures 3 to 5 C lower than normal body temperature (8, 18). Apparently, B. bliogdo,:feri follows a similar pattern since its optimal growth is at 31 to 35 C (5). The ear of a rodent, by virtue of its vascular nature, large surface area, and small mass, would seem to provide an ideal environment for producing the lower temperature favorable for spirochetal growth. Additionally, given the predilection of ticks for feeding on the heads and ears of hosts, the ear could be the first site from which the spirochetes become recoverable. TABLE 5. Comparison of ear culture with xenodiagnosis for detection of B. burgdo, feri in P. leuctopus from endemic areas Area" Animal no. Xenodiagnosis Ear culture Nant i _ 1, 5 + _b Gl Nant. Nantucket Island. Mass.. Gl. Great Island. Mass. Only one ear sampled.

4 1726 SINSKY AND PIESMAN We obtained samples from animals that had acquired their infections by various routes and at differing doses and found both the skin and ear, either as a whole minced ear or as a biopsy punch, to be reliable for detecting infections of B. burgdorferi in hamsters and white-footed mice. For all three modes of infection, the ear was the most successful source for culturing spirochetes. The skin proved to be as good as or nearly as good as the ear for detecting infection when the animal was naturally infected or infected by inoculation with a fresh tick homogenate. Skin was the least effective of the culture sources for detecting infections acquired from inoculations with cultured spirochetes. The ear punch method compares favorably with xenodiagnosis in terms of how soon after exposure infected animals become test positive. In our laboratory, positive ear punches could be obtained as early as 4 days after infection, and xenodiagnosis can detect spirochetes as early as 5 days after infection (11). Ears that were shipped to us had not been placed in any type of cryoprotectant prior to freezing. This fact, coupled with the partial thawing of a portion of the samples in transit, may account for the reduction in the recovery rate compared with that of fresh samples. Subsequently, we found that placing the ears in a solution of 30% glycerol in phosphatebuffered saline (vol/vol) and allowing a 20- to 30-min refrigerated equilibrium period prior to freezing improved the recovery rates to approximately those of the fresh samples. When comparing samples taken from an animal immediately after euthanasia against those frozen with and without cryoprotectant, 66.6% (4 of 6) of the fresh samples were positive, while 58.3% (7 of 12) of the samples frozen with cryoprotectant and 16.6% (2 of 12) of the samples frozen without cryoprotectant were positive. There is also a greater problem with contamination of samples taken from frozen tissue versus those taken from fresh tissue. Although in some experiments as many as 65% of the samples taken were contaminated with either bacteria or fungus, viable spirochetes could still be detected in the majority of these cultures. Meticulous cleansing of the ears with either filter paper or a cotton swab soaked in 70 to 90% ethanol appears to reduce contamination. The microdilution plate method of culturing ear punch biopsies is much less labor intensive than currently used methods. A single live animal can be processed in approximately 5 min, which includes taking three punches from each ear and depositing them into individual wells. Additionally, if each punch is cultured separately, only 1.5 ml of medium per animal is needed, as opposed to the 40 to 120 ml per animal that would ordinarily be used (14, 21). The microdilution plates, however, must be topped off with fresh medium once or twice a week to compensate for evaporation. Depending on the density of the spirochetes in the tissue sample, positive ear punch cultures can generally be detected within 2 to 6 days postsampling. The culturing of ear punch biopsies could be useful in several areas of investigation. One possibility would be in setting up surveillance programs in wild rodents. By using a capture-release-recapture program, incidence and prevalence studies could be conducted to monitor rodent populations in areas enzootic for Lyme disease. The short time lag between an animal becoming infected and positive cultures being obtainable would make this method superior to serologic methods, with the added benefits that the ear punch would be less stressful than taking a blood sample, with less chance of accidentally killing the animal. This method could also be used to evaluate control programs in endemic areas. Surveillance programs could also be set up in nonendemic areas to check for the movement of B. hurgdorferi into naive rodent populations. Testing of treatment protocols would be feasible with ear punch biopsies, although the clearance of the organism from the skin and ear may not be entirely indicative of the status of the visceral organs. The possible role of antigenic variation as a mechanism for persistent spirochetal infection could be addressed through the ear punch biopsy method. An additional question concerns the possible sequestering of the spirochetes in the brain despite treatment (23) and whether the negative status of the ear biopsies would actually indicate that of the brain tissue. Further studies are needed to clarify this issue. Nevertheless, the ability to repeatedly sample naturally and experimentally infected animals provided by the ear punch biopsy method should prove useful in a variety of long-term studies investigating the ecology, immunology, and treatment of Lyme disease. ACKNOWLEDGMENTS J. CLIN. MICROBIOL. We thank Russell Johnson for providing the DW-18 strain of B. burgdoréferi, Tom Schwan for providing ears from experimentally infected mice, and Sam Telford for providing ears from naturally infected mice. We thank Jose Ribeiro for suggesting the use of the candle jar as a means of improving the culture environment. We also thank Willy Burgdorfer, Tom Schwan, Jose Ribeiro, and Martin Roop for their critical comments on the manuscript. 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