INFECTION AND IMMUNITY, Oct. 1984, p. 220-2230019-9567/84/100220-04$02.00/0Copyright © 1984, American Society for Microbiology

Vol. 46, No. 1

Experimental Lyme Disease in Rabbits: Spirochetes Found inErythema Migrans and Blood

ARNOLD N. KORNBLATT,'t ALLEN C. STEERE,2* AND DAVID G. BROWNSTEIN'

Section of Comparative Medicine' and Department of Internal Medicine,2 Yale University School of Medicine, NewHaven, Connecticut 06510

Received 2 May 1984/Accepted 3 July 1984

In attempts to produce experimental Lyme disease, 33 rabbits were inoculated with Lyme spirochetes by tickfeeding or from tick organ homogenates or cultures. Two rabbits developed erythema chronicum migrans atthe site of inoculation, in one instance 2 days after injection of a tick organ homogenate and in the otherinstance, 17 days after feeding of infected Ixodes dammini ticks. Spirochetes were seen in skin biopsy specimens

of the second lesion with Warthin-Starry and immunoperoxidase stains. Spirochetes were also recovered fromblood cultures of two additional rabbits 2 weeks post-inoculation. These findings are characteristic of earlyLyme disease in humans and give additional support for the spirochetal etiology of Lyme disease.

Lyme disease, a complex, multisystem human illness,usually begins with a skin lesion, erythema chronicummigrans (ECM), that may be followed by neurological,cardiac, orjoint abnormalities (14). The illness is caused by anewly recognized spirochete (3, 5, 12) that is transmitted byIxodes dammini ticks and other species of the Ixodes ricinuscomplex (10, 13). The unique clinical feature of the disorderis the initial skin lesion, which occurs at the site of the tickbite after an incubation period of 3 to 32 days (9, 13). Thelesion begins as a red macule that gradually expands, oftenwith partial central clearing (9, 14). Spirochetes have beenseen in the lesion (4) and cultured from it (12).

In this study, we attempted to develop an animal model ofLyme disease. We chose rabbits as our first experimentalanimal because they are susceptible to Treponema pallidum,the agent of syphilis (1), and Treponema cuniculi causesvenereal spirochetosis in rabbits (6). More importantly, twoprevious investigations have described the development oferythematous skin lesions in rabbits fed upon by I. dammini(5, 8). However, unlike human ECM, the lesions noted byBurgdorfer and his colleagues did not occur at the site of thetick bite and did not develop until 10 to 12 weeks after tickfeeding (5). In addition, they stated that limited attempts toisolate spirochetes from skin biopsy specimens in Kelly'smedium were negative. The skin lesions described byKrinsky et al. were small macules (.5 mm in diameter) thatdeveloped within 2 to 3 days after initial tick exposure (8).The authors suggested that the lesions might have resultedfrom either a normal tick component or a microbe or toxinthat they were unable to detect. More recently, W. Burg-dorfer (Yale J. Biol. Med., in press) has shown that rabbitsare suitable as experimental hosts for infecting ticks withLyme disease spirochetes. In this study, we produced ex-perimental erythema migrans in rabbits and found spiro-chetes in the skin lesion and blood.

MATERIALS AND METHODSNew Zealand White rabbits were obtained from Junelove

Rabbitry, Missoula, Mont., and from Plummers Rabbitry,Grafton, Mass. Both male and female rabbits (3 to 5 kg) wereused. Before inoculation, physical examinations were con-

* Corresponding author.t Present address: Weizmann Institute of Science, Experimental

Animals Center, 76100 Rehovot, Israel.

220

ducted; oronasal and genital orifices and skin, after clippingthe back fur, were examined for lesions. Rectal temperatureswere taken daily, and activity and appetite were noted.Finally, blood was drawn for serological testing.

Rabbits were divided into four groups for inoculation. Inthe first group, 25 nymphal or adult I. dammini, collected byflagging in endemic areas of Guilford and Lyme, Conn.,where ca. 20% of ticks have been shown to be infected (12),were placed in a chamber upon each of four rabbits. Themajority of ticks did not feed. A total of 13 ticks engorged onone rabbit, and only 4 or 5 ticks attached to each of theremaining three rabbits. In group 2, rabbits received anintradermal inoculation of tick organ homogenate (0.1 ml) atthree sites, and in group 3, 15 rabbits were given inoculationsof homogenate intradermally and intravenously (0.5 ml). Forpreparation of the homogenate, the internal organs of a meanof 70 I. dammini ticks (range, 60 to 75) were extracted undera dissecting microscope and crushed with forceps in a dropof modified Kelly's medium (7, 12). The resulting homoge-nate was transferred by pipette to a microcentrifuge tube,and additional medium was added to make a total volume of1 to 2 ml. Gross particulate matter was allowed to settle, and1 drop of each supernatant inoculum was examined by dark-field microscopy to confirm the presence of one or morespirochetes. The supernatant was used for the inoculation oftwo rabbits within 1 h of preparation. In the fourth group,four rabbits were inoculated intradermally and intravenouslywith cultured spirochetes (strain G39/40) which had beenpassaged ca. 50 times before inoculation. Spirochetes in one8-ml tube containing 106 organisms per ml were centrifugedat 12,000 x g for 10 min, and the pelleted organisms weresuspended in 1 ml of medium for inoculation. Hundreds ofmotile spirochetes could be seen in 1 drop of the suspensionby dark-field microscopy.

All of the rabbits were observed daily for signs of illnesssuch as depression, inappetance, or lameness. Their backskin was checked, and rectal temperatures were taken dailyfor 1 month. In addition, blood samples were drawn weeklyfor 1 month for culturing and serological studies. Bloodsamples (2 to 3 ml), collected in citrated tubes, were centri-fuged at 2,000 rpm for 5 min, and 3 drops of plasma weretransferred to an 8-ml Pyrex screw-cap tube that contained 7ml of a modified Kelly's medium (7). The remaining pelletwas centrifuged at 7,000 x g in an Eppendorf 5412 microfuge

LYME DISEASE IN RABBITS 221

(Brinkman Instruments, Inc., Westbury, N.Y.), and theplasma pellet was suspended and transferred to another tubeof medium. The cultures were incubated at 33°C and exam-ined weekly by dark-field microscopy for 1 month. Eachculture was subjected to one blind passage after 1 week.Antibody titers to the Lyme spirochete were determined byindirect immunofluorescence as previously described (12),except that goat anti-rabbit immunoglobulin G (IgG)-fluores-cein isothiocyanate (1:50) (Antibodies, Inc., Davis, Calif.)was used as the conjugate.

In an effort to find additional sites of infection, the rabbitsin groups 1 and 2 that had a fourfold or greater rise inantibody titer to the Lyme disease spirochete were sacrificedtwo at a time at 2, 3, or 6 weeks post-inoculation. Onesample of lung, liver, spleen, kidney, brain, and popliteallymph node or testis was cultured, and another sample ofeach was fixed in 10% Formalin for light microscopicstudies. Tissue samples were ground with a mortar andpestle in 1 ml of modified Kelly's medium. The emulsion wasspun at 2,000 rpm for 5 min, and 3 drops of supernatant werecultured in one tube of medium. The remaining supernatantwas centrifuged for 1 min at 7,000 x g, and the resultingpellet was suspended and transferred to another tube ofmedium. The cultures were then processed as above. Tissuespecimens were stained with hematoxylin and eosin and withthe Warthin-Starry silver stain (15). Specimens that werepositive for spirochetes were also stained with the avidin-biotin peroxidase complex detection system (Vector Labora-tories, Burling, Calif.) (2). After deparaffinizing, trypsiniz-ing, and quenching with normal goat sera, the tissue wasincubated with high-titered serum (1:100) from a patient withLyme disease, followed by 20 ,ul of biotinylated goat anti-human IgG and then 20 pul of avidin-biotinylated peroxidasecomplex. The substrate was 5 mg of diaminobenzidinetetrahydrochloride in 10 ml of 0.5 M Tris-hydrochloridebuffer (pH 7.6) with 4 p.l of 30% H202.To determine whether arthritis might develop, the rabbits

in groups 3 and 4 were kept for 6 months. They wereobserved daily for lameness, but temperatures were nottaken and blood samples were not drawn.

RESULTSOf four rabbits exposed to I. dammini (group 1), only one,

which was fed upon by 13 ticks, had a fourfold rise in IgGantibody titer to the Lyme disease spirochete (Fig. 1). This

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FIG. 1. Antibody titers against the Lyme disease spirochete in

rabbits, determined by indirect immunofluorescence. The meanvalue + one standard deviation is shown. IV, Intravenously; ID,intradermally.

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FIG. 2. ECM in a rabbit. The lesion, which is 15 cm in diameter,has a red outer border (arrows) and partial central clearing. Thecenter of the lesion, where hair has regrown rapidly, is the attach-ment site of the tick feeding chambers.

rabbit developed an erythematous skin lesion (Fig. 2) at thesite of tick attachment 17 days after initial feeding. Thelesion, which covered much of the rabbit's back, had a redborder and partial central clearing. It expanded slightly andfaded within 3 days. The lesion was subtle and could havebeen overlooked. The rabbit developed no other signs ofinfection. Skin biopsy specimens were obtained from thecenter and periphery of the lesion on the day it was firstnoted. Histologically, the lesion showed loose aggregates oflymphoid cells and occasional small aggregates of hetero-phils around small blood vessels in the deep papillary andsuperficial reticular dermis. By using Warthin-Starry andimmunoperoxidase stains, numerous spirochetes were seenin areas of dermal inflammation in both the center andperiphery of the lesion (Fig. 3). Cultures of these tissues inmodified Kelly's medium, however, were negative.Of 10 rabbits inoculated intradermally with tick organ

homogenate (group 2), 5 (50%) seroconverted. In one ofthese five rabbits, dark-red indurated lesions (5 by 7 cm)appeared at two of the three inoculation sites 2 days later.Both lesions expanded ca. 3 cm and faded after 6 days. Skinbiopsies, obtained on the day the lesions were first noted,looked similar to ECM described in the first rabbit, butspirochetes were not seen and cultures of the tissue werenegative. The rabbits developed no other signs of illness.Additional sites of spirochetal infection were not detected inthe six seropositive rabbits in groups 1 and 2 that weresacrificed at 2, 3, or 6 weeks post-inoculation. Cultures ofbrain, lung, liver, spleen, kidney, cerebrospinal fluid, lymphnode, and testis were negative, and the histology of thesetissues was unremarkable.

In group 3, 15 rabbits were injected both intradermally andintravenously with tick organ homogenate; 5 (33%) serocon-verted (Fig. 1). None of these rabbits developed ECM, butspirochetes were recovered from blood cultures from two ofthem taken 2 weeks post-inoculation. The rabbits that sero-

VOL. 46, 1984

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222 KORNBLATT, STEERE, AND BROWNSTEIN

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,,* * ~~~~~FIG. 3. Lyme disease spirochetes in skin biopsy specimens of ECM from a rabbit demonstrated by Warthin-Starry (A) and immunoperoxi-

dase stains (B). For comparison, a control sample of Lyme disease spirochetes, also stained by the immunoperoxidase technique, are shown(C). The organisms ranged from 10 to 30 ,um in length. x 1,545.

converted in this group were observed for 6 months, but theyremained well throughout this period.The four rabbits in the final group were inoculated intra-

dermally and intravenously with cultured spirochetes. Theyhad rapid serological responses and high peak antibody titers(Fig. 1) but developed no signs of infection. Spirocheteswere not recovered from their blood.

DISCUSSIONIn this study, we produced expanding skin lesions in two

rabbits that were very similar to ECM in humans (4, 9). Thelesions occurred at the site of tick bites or inoculation of tickorgan homogenate within the incubation period observed inhumans (9, 14), and for the first time in animals, spirocheteswere demonstrated in the lesion produced by tick feeding.Two additional rabbits had spirochetemia 2 weeks afterinoculation. It is during this same period that spirocheteshave been recovered from the blood of patients with Lymedisease (3, 12). Thus, these findings are characteristic ofearly Lyme disease in humans and give further support forthe spirochetal etiology of Lyme disease.We examined three methods for inoculating spirochetes.

Each method has advantages and disadvantages. Tick feed-ing reproduces the way the disease is transmitted in nature,and the skin lesion produced in this manner looked most likeECM in humans. However, the ticks must be collected in thefield by flagging; they can only be obtained at particulartimes of the year; not all ticks will feed on rabbits; and thereis no way to determine in advance whether the tick isinfected. Although they may be dissected after engorgementand spirochetes can be sought by direct immunofluorescence(12), the rabbit may not seroconvert even if spirochetes areseen in the tick. This lack of seroconversion may be becausetoo few spirochetes were injected or because the rabbit isresistant to this infection. However, we believe that theserological test, indirect immunofluorescence, is sensitiveenough to detect the antibody response.

Several rabbits injected with tick organ homogenate alsodeveloped either erythema migrans or spirochetemia. Withthis method, the problems of tick feeding are eliminated, andthe chances of having spirochetes in the inoculum areincreased, since the internal organs of ca. 70 ticks were usedto prepare each homogenate. Even so, only one or a fewspirochetes were seen in 1 drop of the homogenate by dark-field microscopy, and only one-third to one-half of inoculat-ed rabbits seroconverted. The likelihood of infection would

probably be greater if more ticks were used in preparing eachhomogenate.The easiest method is to obtain spirochetes from stock

cultures. The inoculum can be quantified, and millions oforganisms can be injected. Rabbits inoculated in this waydeveloped higher antibody titers sooner than rabbits inocu-lated with tick organ homogenate, but similar to the titer ofthe rabbit that was infected via tick bites. However, exceptfor seroconversion, the four rabbits inoculated with strainG39/40 did not develop signs of infection. This findingimplies that this isolate of Lyme disease spirochetes, whichhad already been passaged 50 times before inoculation, mayhave lost pathogenicity in culture. Alternatively, the numberof rabbits tested may have been too small to demonstrateinfection. Because of the obvious advantages of using cul-tured spirochetes, it will be important to determine whethersome isolates maintain pathogenicity for a certain period inculture or whether pathogenicity may be restored by passagethrough an appropriate animal.We are particularly interested in developing an animal

model ofLyme disease to help determine the pathogenesis ofLyme arthritis, which in humans is similar histologically torheumatoid arthritis (11, 14). Although several rabbits in thisstudy developed ECM or spirochetemia, the illness wasmild. None of them appeared ill, had fever, or manifestedany of the later signs of Lyme disease, which in humans isassociated with an increased frequency of the B-cell alloanti-gen DR2 (11). Thus, it seems that rabbits are not a suitableanimal for studies of pathogenesis of the later stages ofLymedisease.

ACKNOWLEDGMENTSThis work was supported by Public Health Service grants AM-

20358, AM-07107, AM-5639, RR-00125, and RR-00393 from theDivision of Research Resources, National Institutes of Health, andthe Arthritis Foundation and its Connecticut chapter.We thank residents of Guilford, East Haddam, and Lyme, Conn.,

for permission to collect ticks on their property; Marc Kornblatt,John Strathman, Mahesh Shrestha, and Judith Kornblatt for assist-ance in collecting ticks; and Stephen Barthold, Robert Grodzicki,Elizabeth Johnson, Frank Cappiello, and Rachel Ardito for labora-tory support.

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2. Bayer, E. A., and M. Wilchak. 1980. The use of avidin-biotin

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LYME DISEASE IN RABBITS 223

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3. Benach, J. L., E. M. Bosler, J. P. Hanrahan, J. L. Coleman,G. S. Habicht, T. F. Bast, D. J. Cameron, J. L. Ziegler, A. G.Barbour, W. Burgdorfer, R. Edelman, and R. A. Kaslow. 1983.Spirochetes isolated from the blood of two patients with Lymedisease. N. Engl. J. Med. 308:740-742.

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9. Steere, A. C., N. H. Bartenhagen, J. E. Craft, G. J. Hutchinson,J. H. Newman, D. W. Rahn, L. H. Sigal, P. N. Spieler, K. S.Stenn, and S. E. Malawista. 1983. The early clinical manifesta-

tions of Lyme disease. Ann. Intern. Med. 99:76-82.10. Steere, A. C., T. F. Broderick, and S. E. Malawista. 1978.

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11. Steere, A. C., A. Gibofsky, M. E. Patarroyo, R. J. Winchester,J. A. Hardin, and S. E. Malawista. 1979. Chronic Lyme arthritis:clinical and immunogenetic differentiation from rheumatoidarthritis. Ann. Intern. Med. 90:286-291.

12. Steere, A. C., R. L. Grodzicki, A. N. Kornblatt, J. E. Craft,A. G. Barbour, W. Burgdorfer, G. P. Schmid, E. Johnson, andS. E. Malawista. 1983. The spirochetal etiology of Lyme dis-ease. N. Engl. J. Med. 308:733-740.

13. Steere, A. C., and S. E. Malawista. 1979. Cases of Lyme diseasein the United States: locations correlated with distribution ofIxodes dammini. Ann. Intern. Med. 91:730-733.

14. Steere, A. C., S. E. Malawista, J. A. Hardin, S. Ruddy, P. W.Askenase, and W. A. Andiman. 1977. Erythema chronicummigrans and Lyme arthritis: the enlarging clinical spectrum.Ann. Intern. Med. 86:685-698.

15. Thompson, S. W. 1966. Modified Warthin-Starry method for thedemonstration of spirochetes, p. 1048-1049. In Selected histo-chemical and histopathological methods. Charles C Thomas,Springfield, Ill.

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