INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI: 10.1128/IAI.69.6.4094–4102.2001

June 2001, p. 4094–4102 Vol. 69, No. 6

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Immunohistochemical Analysis of Lyme Disease in the Skin of Naiveand Infection-Immune Rabbits following Challenge

CELESTE CHONG-CERRILLO,1† ELLEN S. SHANG,1 DAVID R. BLANCO,1,2

MICHAEL A. LOVETT,1,2 AND JAMES N. MILLER1*

Department of Microbiology and Immunology,1 and Division of Infectious Disease, Department of Medicine,2

University of California, School of Medicine, Los Angeles, California 90095

Received 20 December 2000/Returned for modification 1 February 2001/Accepted 2 March 2001

In this study, skin histopathology from naive and infection-derived immune rabbits was compared followingintradermal challenge using Borrelia burgdorferi B31 strain. The presence or absence of spirochetes in relation-ship to host cellular immune responses was determined from the time of intradermal inoculation to the timeof erythema migrans (EM) development (;7 days in naive rabbits) and through development of challenge im-munity (;5 months in naive rabbits). Skin biopsies were obtained and analyzed for the presence of spirochetes,B cells, T cells, polymorphonuclear cells (PMNs), and macrophages by immunohistochemical techniques. In in-fected naive animals, morphologically identifiable spirochetes were detected at 2 h and up to 3 weeks post-infection. At 12 and 24 h postinfection there was a marked PMN response that decreased by 36 to 48 h; by 72 hthe PMNs were replaced by a few infiltrating macrophages. At the time of EM development and 14 days postin-fection, the PMNs and macrophages were replaced by a lymphocytic infiltrate. There was a greater number ofspirochetes at 14 days, a time when EM had resolved, than at 7 days postinfection. By 3 weeks postinfectionthere were few organisms and lymphocytes detectable. In contrast to infected naive rabbits, intact spirocheteswere never visualized in skin biopsies from infection-immune rabbits; only spirochetal antigen was detected at2, 12, and 24 h in the presence of a numerous PMN infiltrate. By 36 h postchallenge, spirochetal antigen couldnot be detected and the PMN response was replaced by a few infiltrating macrophages. By 72 h postchallenge,PMNs and macrophages were absent from the skin; B and T cells were never detected at any time point in skinfrom infection-immune rabbits. The destruction of spirochetes in immune animals in the presence of PMNsand in the absence of a lymphocytic infiltrate suggests that infection-derived immunity is antibody mediated.

Lyme disease is the most common vector-borne disease inthe United States (28). The disease is transmitted to humansby the bite of an infected Ixodes tick harboring either Borreliaburgdorferi sensu stricto, B. afzelii, or B. garinii, related spiro-chetes collectively termed B. burgdorferi sensu lato. In the ma-jority of patients, Lyme disease is characterized by the initialappearance of a rash-like skin lesion termed erythema migrans(EM) (39, 42). Early and late clinical manifestations includearthritis, neurological manifestations, lymphadenopathy, andcarditis, which reflects dissemination to visceral targets (1, 16,24, 29, 30, 34, 38, 40–43). Although Lyme disease is rarely fatal,it can be quite debilitating.

Implicit in the development of an effective vaccine againstLyme disease is a thorough understanding of the pathogeneticmechanisms as well as the host immune response operative dur-ing host-spirochete interaction. In the murine model of Lymedisease, the histopathology is characterized by a lymphocyticand plasma cell infiltration, occasional perivascular cuffing,and few or no detectable macrophages (8, 13, 18, 27, 32, 37).BALB/c and C3H mice have been used in elucidating theimmune response of the host following infection with B. burg-dorferi. Humoral immunity has been shown to play a major role

in resistance to Lyme disease, although cellular immunity hasalso been demonstrated to influence the outcome of arthritisseverity and infection (4–6, 20–23, 25, 26, 33, 47, 48). However,a direct relationship between the spirochetes and host immunecells has not been demonstrated. In addition, the chronic na-ture of the disease in the murine model precludes determiningthe immune mechanisms involved in clearance of organismsand resolution of the disease.

The rabbit model of Lyme disease has unique features rel-evant to the immunobiology of B. burgdorferi infection. It isthe only animal model besides the rhesus monkey (31) thatreproducibly results in EM that is indistinguishable from thatof human disease after intradermal inoculation of B. burgdor-feri sensu stricto (15). Most importantly, untreated skin andvisceral infection is ultimately cleared, resulting in completeimmunity against reinfection with up to 2 3 107 challengeorganisms (15). Thus, in the rabbit model of Lyme disease, it ispossible to examine the effective host immune factors, humoraland cellular, associated with acquired resistance against infec-tion with B. burgdorferi.

In the current study, we directed our efforts toward eluci-dating the outcome of host-spirochete interaction followingthe intradermal inoculation of naive and infection-derived im-mune rabbits with B. burgdorferi B31 from the time of in-tradermal inoculation to the time of EM development andthrough development of challenge immunity. We specificallyaddressed the location and persistence of spirochetes in rela-tionship to the presence and distribution of polymorphonu-clear cells (PMNs), macrophages, T cells, and B cells in an

* Corresponding author. Mailing address: Department of Microbi-ology, Immunology, and Molecular Genetics, CHS 43-239, UCLASchool of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095.Phone: (310) 825-1979. Fax: (310) 206-3865. E-mail: jmiller@mednet.ucla.edu.

† Present address: Department of Pathology, University of Califor-nia, Irvine, College of Medicine, Irvine, CA 92797.

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effort to determine the potential cellular mechanisms respon-sible for pathogenesis and host immunity.

MATERIALS AND METHODS

Animals. Adult, male, New Zealand White rabbits 6 to 9 months of age (IrishFarms, Norco, Calif.) were housed individually in a temperature-controlled en-vironment ranging from 18 to 21°C. Prior to intradermal (i.d.) inoculation withB. burgdorferi, the backs of the rabbits were clipped closely with an electricclipper fitted with a size 40 blade to expose the skin (Oster Professional Products,McMinnville, Tenn.).

Male C3H/HeJ mice 6 weeks of age were purchased from Jackson Laborato-ries (Bar Harbor, Maine) and housed in cages containing no more than fiveanimals.

Bacterial strains and preparation of inocula for infection and challenge.Virulent B. burgdorferi sensu stricto, strain B31, was isolated from infected rabbittissue, grown in BSK II medium (7) to maximum density, and then passagedtwice more in fresh BSK II medium. After the final passage (passaged two times),the organisms were centrifuged gently at 8,000 3 g for 10 min and washed threetimes in heat-inactivated (56°C for 30 min) normal rabbit serum (NRS), diluted1:1 with phosphate-buffered saline (PBS; pH 7.2) (NRS-PBS), in order to removeforeign protein components responsible for nonspecific reactions in the rabbit(15, 36). Inocula for infection and challenge were resuspended in NRS-PBS afterthe final wash to a final concentration containing 107 or 108 B. burgdorferi per mldepending on the experiment. As in previously published experiments (15, 36),the spirochetes in the final suspension were motile and virulent.

Generation and challenge of infection-derived immune rabbits. Based onprevious studies in which a high degree of infection-derived immunity to chal-lenge was established (15), rabbits were infected i.d. with doses ranging from 6 3106 to 8 3 107 B. burgdorferi depending on the experiment. Inocula were admin-istered in 0.1-ml doses at several sites. Six months after infection, at a time whenEM lesions had healed, dermal infection was absent and, on the basis of ourpreviously described studies, visceral dissemination was no longer demonstrable(15), the rabbits were challenged i.d. with doses ranging from 107 to 108 B.burgdorferi depending on the experiment. In addition, naive rabbits were inocu-lated in the same manner to serve as controls.

Skin biopsy and culture. Skin punch biopsies were obtained from all experi-mental and control rabbits at various time points postinfection (p.i.) and post-challenge (p.c.). Rabbits were anesthetized by intramuscular injection with 45 mgof Ketaset (Fort Dodge Laboratories, Fort Dodge, Iowa) and 8.8 mg of Xylazine(Lloyd Laboratories, Shenandoah, Iowa) per kg of body weight. A 4- to 5-mmsterile punch biopsy (Baker and Cummings, Lakewood, N.J.) was taken adjacentto the inoculation site from the clipped back of each rabbit. Each biopsy speci-men was immediately minced and cultured in 5 ml of BSK II medium containing100 mg of phosphomycin per ml and 50 mg of rifampin per ml (Sigma, St. Louis,Mo.).

Cultures were incubated aerobically at 34°C for a period of 7 weeks, and thepresence or absence of B. burgdorferi was determined periodically by dark-fieldmicroscopy. Cultures were considered negative when no sprochetes were ob-served during the 7-week observation period.

Preparation of polyclonal mouse anti-B. burgdorferi immunoglobulin G (IgG)for immunohistochemical detection of B. burgdorferi in skin sections. VirulentB. burgdorferi was grown as described above to passage 3. Organisms werewashed three times by suspension in PBS followed by centrifugation at 8,000 3g for 10 min. After the final wash, organisms were resuspended in PBS to a finalvolume containing 1010 B. burgdorferi per ml. The organisms were then disruptedby ultrasonic treatment and stored at 280°C until ready for use.

Twenty C3H/HeJ mice were hyperimmunized with the above-prepared sus-pensions of B. burgdorferi. For the initial immunization, an equal volume oforganisms (ca. 100 ml) was mixed 1:1 with complete Freund adjuvant (Calbio-chem Corp., La Jolla, Calif.) and administered subcutaneously (s.c.). Twobooster immunizations at 4-week intervals were also given s.c. using 100 ml of theB. burgdorferi suspension mixed 1:1 with incomplete Freund adjuvant (Calbio-chem Corp.). Four weeks after the final boost immunization, all mice wereeuthanized and blood obtained by cardiac puncture. The sera were then pooledand the IgG fraction was isolated by fast-performance liquid chromatographyusing a protein A-Superose column (HR 10/2; Pharmacia Biotech, Uppsala,Sweden). As determined by enzyme-linked immunosorbent assay, the isolatedIgG fraction was found to have an anti-B. burgdorferi titer of 1:16,000 and wasreactive to all Amido black-stained B. burgdorferi antigens as determined bysodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immuno-blot analysis (data not shown).

Immunohistochemical staining of skin biopsies. Skin punch biopsies of 4 to 5mm were obtained from all experimental and control rabbits at various timepoints p.i. and p.c. The punch biopsies were embedded in O.C.T. compound(Sakura Finetek, U.S.A., Inc., Torrance, Calif.) and quick-frozen in a dry-ice–2-methylbutane bath. Skin punch biopsies from naive rabbits were also obtainedand frozen in O.C.T. compound to serve as negative controls for immunohisto-chemical staining. Frozen O.C.T.-embedded skin specimens were kept at 280°Cuntil ready for use.

Frozen test and control skin biopsy specimens were cut into 4-mm-thick serialsections in a cryostat, collected on Superfrost Plus glass slides (Fisher Scientific,Pittsburgh, Penn.), and fixed with cold acetone for 20 min. Sections were allowedto air dry then incubated in Tris-buffered saline (TBS) for 5 min. Endogenousperoxidase activity was blocked by incubation with 3% H2O2 for 5 min at roomtemperature. Sections were rinsed well with double-distilled water (ddH2O)followed by incubation in TBS for 5 min. Sections were then blocked with Blotto(5% nonfat dry milk, 1% normal horse serum, and 0.2% sodium azide in TBS)at room temperature for 30 min. After the blocking step, five serial sections wereincubated at 4°C overnight with primary antibodies as follows. For the detectionof B. burgdorferi, sections were incubated with a 1:5,000 dilution in Blotto ofpolyclonal mouse anti-B. burgdorferi IgG; for the detection of B cells, T cells, andPMNs or macrophages, sections were incubated with a 1:50 dilution in ReagentDiluent (0.5% bovine serum albumin in TBS) of purified mouse anti-rabbitCD79a monoclonal antibody (MAb), purified mouse anti-rabbit CD3 MAb, orpurified mouse anti-rabbit CD11b MAb (Spring Valley Laboratories, Inc.,Woodbine, Md.), respectively. After three washes of 5 min each with PBScontaining 0.02% Tween 20 (PBS-Tween), the sections were incubated withhorse anti-mouse IgG conjugated to biotin (Vector Laboratories, Burlingame,Calif.) diluted 1:200 in PBS-Tween containing 1.5% NHS at 37°C for 30 min.Sections were then washed three times with PBS-Tween followed by incubationwith streptavidin peroxidase (Vector ABC Elite kit; Vector Laboratories) at 37°Cfor 30 min. Sections were washed once with PBS-Tween and then twice withddH2O, and the color was developed with 3-amino-9-ethyl-carbazole (AECChromogen Kit; Biomeda Corp., Foster City, Calif.) at 37°C for 10 min. Thereaction was stopped by washing sections in ddH2O, and then the sections werecounterstained with a 1- to 2-min incubation in aqueous hematoxylin (BiomedaCorp.). After a counter staining, the sections were rinsed well in running tapwater and then mounted with Crystal/Mount (Biomeda Corp.), followed by theaddition of a coverslip.

Frozen serial sections of each skin biopsy specimen were also stained withhematoxylin and eosin (H&E) for histopathological examination.

RESULTS

Detection of B. burgdorferi and characterization of the cel-lular infiltrate during EM in the skin of naive rabbits follow-ing infection. To identify and characterize the cellular immuneresponse in the development and resolution of EM, rabbitswere infected i.d. with 107 B. burgdorferi, and representativeskin biopsies were obtained at the time of EM development(day 7 p.i) and EM resolution (day 14 p.i.). Frozen serialsections of skin biopsies were analyzed for the presence ofB. burgdorferi, B cell, T cells, and CD11b1 PMNs or macro-phages by immunohistochemical staining. In addition, skin bi-opsy samples were analyzed for the presence of B. burgdorferi

TABLE 1. Histopathology in B. burgdorferi-infected rabbit skinduring development and resolution of EM lesions

Daysp.i.

No. of positiverabbits/total no.

of animalsaImmunohistochemical stainingb

EM lesion Cultures B. burgdorferi B cells T cells CD11b1

cells

7 4/4 4/4 1 1 1, 1/2 1/2c

14 0/4 4/4 11 1 1, 1/2 1/2c

a Number of positive rabbits per total number of animals observed.b Observations were made for four rabbits: 1/2, few/some; 1, moderate; 11,

numerous.c Identified as PMNs by microscopic analysis of H&E-stained serial sections.

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by culture in BSK II medium. As presented in Table 1, allrabbits developed culture-positive EM lesions at day 7 p.i. Atday 14 p.i., EM lesions had resolved, but all animals remainedskin culture positive. Histological examination showed thepresence of intact spirochetes at day 7 which increased inrelative numbers by day 14 p.i. (Fig. 1). At the time of theappearance of EM lesions that contained intact spirochetes(day 7 p.i.), there was predominantly a lymphocytic B-cellinfiltration with fewer T cells (Fig. 2). By comparison, CD11b1

cells representing PMNs were only occasionally detected inthese sections (Table 1). The type and relative quantity of thecellular infiltrate did not differ at day 14 (data not shown) from

that of day 7 even though the number of B. burgdorferi wasgreater at day 14.

Skin histopathology in rabbits during initial infection andthrough the development of immunity. Based on previous stud-ies, rabbits infected i.d. 5 months earlier with B. burgdoreri B31develop complete immunity to challenge reinfection (15). Toinvestigate the role of cellular immunity in the progression andresolution of disease, rabbits were infected i.d. with 6 3 106 to9 3 106 B. burgdorferi, and representative skin biopsies wereobtained at 3 and 13 weeks p.i. and again at 24 weeks p.i. whenimmunity was established. Frozen serial sections of skin biop-sies were analyzed by immunohistochemical staining, as de-

FIG. 1. Immunohistochemical staining for the detection of B. burgdorferi in skin biopsies from infected naive rabbits at the time of EMdevelopment (7 days p.i.) (a) and at the time of EM resolution (14 days p.i.) (b). Magnification, 378.

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scribed above, and skin biopsy samples were cultured in BSK IImedium for detection of viable B. burgdorferi. At 3 weeks p.i.,as shown in Table 2, 80% of rabbits had culture-positive skin.Immunohistochemical detection showed that, compared to day14 p.i. (Table 1), the quantity of spirochetes, B cells, and T cellshad decreased. By 13 weeks p.i. as well as by 24 weeks p.i.,spirochetes were no longer histochemically detectable norcould they be cultured from the skin (Table 2). In addition,

lymphocytes were also not detected from skin sections; onlyrarely were macrophages detected at week 13 p.i. These dataare consistent with our previous observations demonstratingthat by 5 to 6 months p.i., skin and visceral infection in un-treated rabbits is cleared (15).

Comparative histopathology in the skin immediately follow-ing challenge of naive and infection-immune rabbits. To ex-amine early cell mediated immune events following challengeof immune animals, infection-derived immune rabbits previ-ously infected with 8 3 107 B. burgdorferi were challenged i.d.with 8 3 107 organisms, and representative skin biopsies wereobtained at 2, 12, 24, 36, 48, 72, and 96 h p.c. To compare theseearly events to those which occur in the nonimmune animaland to more thoroughly investigate immune mechanisms im-mediately following infection, naive rabbits were inoculated inthe same manner, and representative skin biopsies obtained atthe same time points as above. Serial sections of skin biopsieswere analyzed by immunohistochemical staining and for thepresence of viable B. burgdorferi by culture in BSK II mediumas before.

In naive animals, B. burgdorferi could be cultured from skinand detected by immunohistochemical staining at each timepoint p.i. The detection of morphologically intact spirochetesin the skin by immunohistochemical staining at the 36 h p.i.time point, as shown in Fig. 3c and d, was typical of all timepoints in infected naive rabbits. At 12 and 24 h p.i. there was amarked PMN response which decreased slightly by 36 to 48 hp.i. (Table 3 and Fig. 3e and f). By 72 and 96 h the PMNs werereplaced by a few infiltrating macrophages. Overall, there wasa mild inflammatory response at these time points. No lym-phocytes were evident at any of the above time points (data notshown); however, as mentioned earlier, the appearance of lym-phocytes did occur at the time of EM development (day 7 p.i.).

By comparison to the naive animals, skin biopsies from in-fection-derived immune rabbits were culture positive only upto 36 h p.c. (Table 4), while at 48 h dermal infection was notdetected in the skin biopsies examined. Immunohistochemicalanalysis showed that while B. burgdorferi antigen could be de-tected at 2, 12, and 24 h p.c. (Table 4 and Fig. 4c and d),morphologically intact spirochetes were never observed inthese biopsy sections compared to the infected naive animals(compare Fig. 4d with Fig. 3d). In addition, there was a markedPMN response at 12 and 24 h p.c. in immune animals similarto that observed in the infected naive rabbits (Table 4 and Fig.

TABLE 2. Histopathology in B. burgdorferi-infected rabbit skinduring disease progession and development of immunity

Cell typeImmunohistochemical staining at (wk p.i.)a

3 (16/20) 13 (0/20) 24 (0/20)

B. burgdorferi 1/2 2 1/2b

B lymphocytes 1, 1/2 2 2T lymphocytes 1/2 2 2CD11b1 cells 1/2c 1/2c 2

a The number of skin-culture-positive rabbits per total number of animalsobserved is indicated in parentheses. Observations were made for two rabbits: 2,none; 1/2, few/some; 1, moderate; 11, numerous.

b Detection of only B. burgdorferi antigen.c Macrophages as determined by microscopic analysis of H&E-stained serial

sections.

FIG. 2. Immunohistochemical staining of skin biopsies from in-fected naive rabbits at the time of EM development (7 days p.i.). Serialskin sections were stained for B. burgdorferi (a), B-cell lymphocytes (b),and T-cell lymphocytes (c). Magnification, 3125.

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4e and f); the PMNs were replaced by a sparse infiltrate ofmacrophages at 36 and 48 h p.c. (data not shown). By 72 h p.c.no inflammatory cells could be detected in the skin (data notshown). In addition, no lymphocytes were detected at any ofthe above time points in the challenged infection-immuneanimals. Further, skin biopsies obtained from 15 additionalinfection-immune rabbits at weeks 1 and 2 weeks p.c. were,as expected, culture negative (data not shown). Immunohisto-chemical staining of these 1-week p.c. sections showed no in-

tact spirochetes, only sparse detectable B. burgdorferi antigenand sparse detectable PMNs (data not shown).

DISCUSSION

In this study we compared the cellular events, including themorphological detection of spirochetes, in the skin of naiveand infection-derived immune rabbits following i.d. challengewith B. burgdorferi B31. The observations made encompass the

FIG. 3. Immunohistochemical staining of skin biopsies from infected naive rabbits 36 h p.i. Serial skin sections were stained by H & E (a andb), for B. burgdorferi (c and d), and for the CD11b PMN/macrophage marker (e and f). Magnification is at 331 (a, c, and e) and 3125 (b, d, andf) from an outlined area of panels a, c, and e, respectively.

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time from inoculation to the development of EM and throughthe development of challenge immunity. In the early eventsfollowing the infection of naive animals, morphologically iden-tifiable spirochetes were readily detected at 2 h and through 3weeks p.i., which paralleled the demonstration of culture-pos-itive skin infection during this time. We have recently reported,using quantitative PCR, that over 108 borreliae are present percm2 of EM biopsied skin (36), which indicates that the rabbitis a very permissive host during the early course of infection. At13 weeks p.i., however, skin cultures were consistently negativeand no intact spirochetes could be detected by immunohisto-chemical staining. By comparison, skin samples from infection-immune animals following challenge never showed intact spi-rochetes; only B. burgdorferi antigen was detected at 2, 12, and24 h p.c., which could no longer be detected at 36 h. In addi-tion, culture-positive samples were no longer demonstrablefrom these immune rabbits after the 36-h time point. Thesefindings demonstrate that the infection-derived immunity thatdevelops in experimental rabbit Lyme disease rapidly destroysspirochetes at the site of challenge, as evidenced by the loss ofspirochetal morphology and viability.

In order to determine whether specific cell-mediated im-mune mechanisms contribute to the immunity against chal-lenge that develops in rabbits, immunohistochemical analysisto identify infiltrating PMNs, macrophages, T cells, and B cellswas performed. In parallel, the same analysis of naive rabbitsfollowing challenge was performed for comparison to that ofthe immune animals and to characterize the cellular responsethat occurs from the start of infection to the development ofimmunity. It was observed that immediately following chal-lenge of either naive or infection-immune rabbits an influx ofPMNs occurred and were most prominently present at 12 and24 h. This PMN infiltration was subsequently replaced by onlya sparse infiltrate of macrophages, which in the naive animalspersisted for up to 13 weeks p.i. In immune animals, the mac-rophages were absent by 72 h, a finding concordant with thedisappearance of spirochetal antigen. T and B lymphocyteswere detected during the time when EM lesions appeared ininfected naive animals (day 7 p.i.) but also following the timethat EM lesions had resolved (day 14 p.i.). After 13 weeks p.i.,T and B lymphoctytes were no longer detectable. By compar-ison, T and B lymphocytes were never detected in the skin ofchallenged infection-immune challenged animals. Taken to-gether, these findings show that in naive animals, the resolu-tion of local skin infection correlates with the infiltration of T

and B lymphocytes as well as macrophages, suggesting a pri-mary role for cell-mediated immune mechanisms at the time ofborrelial clearance. The early inflammatory response of PMNsand macrophages does not appear to affect the initial estab-lishment and persistence of spirochetal infection. By compar-ison, no correlation was seen between the disappearance ofviable spirochetes and a cellular infiltrate of any particularimmune cell type in the immune rabbits following challenge,suggesting that humoral immunity plays the primary role in thepersistence of acquired resistance. This is consistent with ourrecent study showing that passive immunization with serumfrom infection-immune rabbits can completely protect naiverabbits against challenge with large numbers of virulentB. burgdorferi (12). Thus, both cell-mediated and humoral im-mune mechanisms would appear to be important in the devel-opment and persistence of acquired resistance that results dur-ing the course of experimental rabbit Lyme disease.

In contrast to the rabbit model of Lyme disease, whereinfected animals ultimately clear the infection and developimmunity to challenge reinfection, infection in mice and hu-mans is chronic. Thus, a comparison of the immune responsethat occurs following infection in mice with those in rabbitsmay provide insights into potential differences that account forthe development of immunity versus that of chronic infection.In the mouse model, the host-immune response to B. burgdor-feri infection has been studied primarily in BALB/c and C3Hstrains, the latter noted to be more permissive to B. burgdorferiinfection. The humoral arm of the murine immune responsehas been thought to play a major role in the relative resistanceof strains to Lyme disease while the cellular arm has beendemonstrated to influence the outcome of disease, includingthe severity of arthritis (23). Following infection, BALB/cmice, the strain relatively more resistant to Lyme disease in-fection, are known to express an early predominant Th2 re-sponse (humoral immunity), whereas the more susceptibleC3H mouse strain has an early predominant Th1 response(cell-mediated immunity) (20, 21, 22). B. burgdorferi-infectedC3H mice, compared to infected BALB/c mice, harbor anincreased spirochete burden in their joints and concomitantlyshow a more severe level of arthritis. It has been suggested thatdifferential cytokine production may contribute to the mousestrain-related differences in susceptibility to Lyme disease (22).In this regard, it has been shown that the ablation of the

TABLE 4. Histopathology in rabbit skin following i.d. challengeof infection-immune rabbits with B. burgdorferi

Cell typeImmunohistochemical staining at (h p.i.)a:

2 (5/5) 12 (5/5) 24 (2/5) 36 (2/5) 48 (0/5) 72 (0/5) 96 (0/5)

B. burgdorferi 1b 1b 1, 1/2b 2 2 2 2B lymphocytes 2 2 2 2 2 2 2T lymphocytes 2 2 2 2 2 2 2CD11b1 cells 1c 11c,d 11c 1/2d 1/2d 2 2

a The number of skin-culture-positive rabbits per total number of animalsobserved is indicated in parentheses. Observations made for four rabbits: 2,none; 1/2, few/some; 1, moderate; 11, numerous.

b No intact organisms were detected, but only B. burgdorferi antigen (CompareFig. 4d to Fig. 3d).

c PMNs as determined by microscopic analysis of H&E-stained serial sections.d Macrophages as determined by microscopic analysis of H&E-stained serial

sections.

TABLE 3. Histopathology in B. burgdorferi-infected rabbit skinfollowing i.d. infection of naive rabbits

Cell typeImmunohistochemical staining at (h p.i.)a:

2 (9/9) 12 (9/9) 24 (9/9) 36 (9/9) 48 (9/9) 72 (9/9) 96 (9/9)

B. burgdorferi 1 1 1 1 1 1 1B lymphocytes 2 2 2 2 2 2 2T lymphocytes 2 2 2 2 2 2 2CD11b1 cells 1b 11b 11b 1b 1b 1/2c 1/2c

a The number of skin-culture-positive rabbits per total number of animalsobserved is indicated in parentheses. Observations made for six rabbits: 2, none;1/2, few/some; 1, moderate; 11, numerous.

b PMNs as determined by microscopic analysis of H&E-stained serial sections.c Macrophages as determined by microscopic analysis of H&E-stained serial

sections.

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endogenous cytokine interleukin-12 (IL-12), which helps topromote the development of a Th1 response, results in a re-duced level of arthritis. Conversely, the ablation of endogenousIL-6, which helps to promote the Th2 response, results in anincreased level of arthritis (4). These findings suggest that, inthe murine model, humoral mechanisms are primarily respon-sible for the relative resistance to infection, while a predomi-nant cell-mediated immune response is associated with in-creased disease severity. In contrast to these studies, Zeidner

and colleagues (47) have shown that when susceptible mice areinfected via the natural tick vector (Ixodes scapularis), exog-enously administered Th1 cytokines reduce the severity of in-fection. Thus, the difference between chronic infection in miceand complete immunity in rabbits may lie with differences inthe specific B. burgdorferi molecules recognized by the humoraland cell-mediated arms of immune response during infection.

We have recently shown that immune rabbits, but not rabbitshyperimmunized with purified outer membrane from in vitro-

FIG. 4. Immunohistochemical staining of skin biopsies from infection-immune rabbits 24 h after i.d. challenge with B. burgdorferi. Serial skinsections were stained by H & E (a and b), for B. burgdorferi (c and d), and for the CD11b PMN/macrophage marker (e and f). Magnification isat 331 (a, c, and e) and 3125 (b, d, and f) from an outlined area of a, c, and e, respectively. Note that similar results were obtained from serialskin sections of biopsies taken at 2 and 12 h p.c. (data not shown).

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cultivated borreliae, are completely protected following chal-lenge using organisms acquired from infected rabbit skin (36).We demonstrated that these organisms no longer expressOspA and upregulate proteins that have been associated withhost adaptation, such as OspC. These findings suggest thatupregulated and/or host-adapted borrelia specific moleculesare primarily responsible for the high degree of immunity thatresults following rabbit infection (36). Several proteins havebeen identified previously in the murine model as being up-regulated during infection, including DbpA (10), OspC (35),and OspE and OspF (44). Proteins identified as uniquely ex-pressed during infection include EppA (11), p35 and p37 (14),the OspE-F homologue p21 (45), the OspF homologues bbk2.1(3), pG (46), and the proteins encoded on operon 2.9-7lpB (2).Many of these proteins, however, have not shown great poten-tial as protective immunogens in the mouse model as a resultof either incomplete or no protection (14, 17) or due to anti-genic heterogeneity between strains which does not result inheterologous cross-immunity (9, 19). Similarly, we have foundin the rabbit model no correlation between challenge immunityand antibody detected against some of these proteins, includ-ing DbpA and OspC (12, 36). However, the finding in thisstudy that rabbit skin infection results in large numbers ofstructurally intact borrelia, which we have found to be host-adapted (36), provides a rich source for identifying potentiallynew candidate protective immunogens. Indeed, recent anti-genic analysis of extracted infected rabbit skin tissue has re-vealed several novel and strongly antigenic molecules not de-tected using cultivated borreliae (J. N. Miller and M. A. Lovett,unpublished data). We are currently studying the role theseantigens may play in the clearance of spirochetes during skininfection and the development of protective immunity in therabbit model of Lyme disease.

ACKNOWLEDGMENTS

This study was supported by NIH grant AI-37312 to J. N. Miller andNIH grant AI-29733 to M. A. Lovett.

We thank Xiao Yang Wu for his expert technical assistance andJonathan Said and Peter Shintaku for their guidance in the prepara-tion and interpretation of the histopathological sections.

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