Contrasts in Tick Innate Immune Responses to \u003cI\u003eBorrelia burgdorferi\u003c/I\u003e Challenge: Immunotolerance in \u003cI\u003eIxodes scapularis\u003c/I\u003e Versus Immunocompetence

ARTICLE

Contrasts in Tick Innate Immune Responses to Borrelia burgdorferiChallenge: Immunotolerance in Ixodes scapularis Versus

Immunocompetence in Dermacentor variabilis (Acari: Ixodidae)

ROBERT JOHNS,1 JUN OHNISHI,1, 2 ANNE BROADWATER,2 DANIEL E. SONENSHINE,ARAVINDA M. DE SILVA,2 AND WAYNE L. HYNES

Department of Biological Sciences, Old Dominion University, Norfolk, VA 23529

J. Med. Entomol. 38(1): 99Ð107 (2001)

ABSTRACT The blacklegged tick, Ixodes scapularis Say, transmits the Lyme disease spirocheteBorrelia burgdorferi, whereas the American dog tick, Dermacentor variabilis (Say), is unable totransmit the bacterium. We compared the innate immune response of these ticks against spirochetesdirectly inoculated into the hemocoel cavity of ticks. In I. scapularis, some Borrelia were foundassociated with hemocytes, while numerous other spiral-shaped, intact bacteria remained free in thehemolymph. In contrast, in D. variabilis only remnants of the bacteria were evident in the hemo-lymph, indicating lysis; intact spirochetes were rare. Spirochetes were observed bound to or withinthe organs of both tick species, although many more spirochetes were found associated with the I.scapularis organs. The few spirochetes observed with the D. variabilis organs appeared to be deadbecause D. variabilis tissues rarely contained culturable bacteria, unlike I. scapularis tissues. Whenspirochetes were incubated with I. scapularis hemolymph plasma in vitro, bacterial survival andmotility were not reduced. In contrast, incubation of spirochetes with D. variabilis hemolymphplasma resulted in .50% of the spirochetes becoming nonmotile by 45 min. The differences in theresponses of the two different tick species indicate that I. scapularis is immunotolerant whenchallenged with B. burgdorferi and dependent on a slow phagocytic response to clear Borrelia fromthe hemolymph. In contrast, D. variabilis is highly immunocompetent (i.e., innate immunity), usingplasma borreliacidal factors and a rapid increase in phagocytic cells to clear the infection and limittissue invasion.

KEY WORDS Borrelia burgdorferi, Ixodes scapularis, Dermacentor variabilis, immunotolerance,immunocompetence

LYME BORRELIOSIS IS caused by the spirochete Borreliaburgdorferi s.l. (Johnson et al. 1984) transmitted byticks of the genus Ixodes. Lyme disease, caused bythese bacteria, is now recognized as the most impor-tant vector-borne disease in the United States (CDC1999), with .15,000 cases per year, and many thou-sands of cases in Eurasia.

The passage of spirochetes from the midgut to thesalivary glands and other internal organs during feed-ing of infected vector ticks has been documented innumerous studies (Lane et al. 1991). In nymphs, dis-persal to the salivary glands occurs over a period ofseveral days, with the maximum numbers of spiro-chetes in the saliva within 72 h after the commence-ment of tick feeding (Zung et al. 1989, Piesman 1995,deSilva et al. 1995).However, little is knownabout thesurvival of these bacteria during hemolymph passage,

wherein they are likely to encounter antimicrobialpeptides and phagocytic hemocytes. Phagocytosis ofinvading Borrelia has been reported (Coleman et al.1997) and the possible role of lysozyme in bacteriol-ysis has been suggested (Podboronov 1991, Kuhn andHaug 1994). How the bacteria survive in the presenceof these immune challenges, how long they survive,the percentage of surviving bacteria that invadenearby tick organs, and other aspects of the dynamicsof spirochete passage in the ticks have received littleattention.

Evidence of spirocheticidal activity by antimicro-bial hemolymph peptides was described in Dermacen-tor variabilis (Say) (Johns et al. 2000), a tick whichdoes not transmit B. burgdorferi; it is not knownwhether similarpeptides are active in Ixodes scapularisSay. By following the fate of spirochetes inoculatedinto the hemocoel of ticks, it may be possible to gaina better understanding of the role of the tickÕs immunesystem in determining vector competence. In thisstudy, we used direct inoculation of cultured spiro-chetes into the hemocoel of ticks to follow the fate ofthebacteria in thehemolymphof competent (I. scapu-laris) and incompetent (D. variabilis) vectors of B.

All use of animals in this research was done in accordance withprotocols approved by the Old Dominion University Animal Use andCare Committee Protocols on 17 March 1999.

1 Robert Johns and June Ohnishi contributed equally to the per-formance of these studies.

2 Department of Microbiology and Immunology, University ofNorth Carolina, Chapel Hill, NC 27599.

0022-2585/01/0099Ð0107$02.00/0 q 2001 Entomological Society of America

burgdorferi. We report that spirochetes survived andinvaded organs in I. scapularis, whereas they wererapidly cleared from the hemolymph of D. variabilisticks.

Materials and Methods

Ticks. Dermacentor variabilis ticks were colonizedas described by Sonenshine (1993) using rats (Rattusnorvegicus) and rabbits (Oryctolagus cunniculus). I.scapularis was colonized from ticks collected nearArmonk, NY, fed on rabbits, and the spirochete-freeprogeny were reared to adults. All life stages were fedon rabbits and the fed tickswere incubated at 266 18Cand 92 6 1% RH. All I. scapularis used in the exper-iments were from the F1 generation.

Bacteria. The B. burgdorferi used in this study werethe low passage B31 strain from Center for DiseaseControl, Fort Collins, CO. The spirochetes were cul-tured and prepared for inoculation as described byJohns et al. (2000).

Tick Inoculations and Tick Tissue Collections. Alltick inoculations with B. burgdorferi were done asdescribed by Johns et al. (2000). Brießy, bacteriaweresuspended in phosphate-buffered saline (PBS) (pH7.4). Next, Þve 3-ml aliquots containing 35,000 spiro-chetes were injected into the hemocoel cavity of par-tially fed virgin female D. variabilis or I. scapularis viathe foramen between the capitulum and the anteriorend of the scutum. Ticks were also sham inoculatedwith PBS alone. A 50-ml Hamilton syringe (Hamilton,Reno, NV) with a 30-gauge hypodermic needle wasused for the bacterial inoculations. Hemolymph col-lections were made at one and 24 h following theinoculations. Hemolymph collected as described pre-viously (Johnset al. 1998)wasdiluted1:1 inShenÕs ticksaline (Oliver et al. 1974) with 10 mM phenylmethyl-sulfonyl ßuoride) (PMSF) (Sigma, St. Louis, MO);samples contaminated with midgut, malpighian tu-bules or other tissues were discarded. After hemo-lymph collection, 3Ð5 ml aliquots were smeared ontoCSS-100 silylated slides (CEL Associates, Houston,TX). Immediately after hemolymph collection, thesame tick specimens were dissected at the same timeintervalswith the aidof a stereoscopicmicroscopeandsamples of salivary glands and ovary were transferredto microscope slides. The same procedures were alsorepeated with inoculations of 3,500 spirochetes pertick so as to assess the effects of low dose inoculationson spirochete survival within ticks.

Direct Immunofluorescence Assay. A direct immu-noßuorescence assay (DFA) was used to detect spi-rochetes in tick hemolymph, salivary glands and ova-ries. The collected hemolymph was spotted directlyon CSS slides and air-dried. The tissue samples weredissectedout in adropof ShenÕs tick salineona regularglass slide, transferred to aCSS slide andair-dried.Theslides were Þxed in acetone for 20 min, then blockedwith 5% FCS, PBS and 0.05% sodium azide for 30 minat room temperature. Next, the slides were incubatedwith ßuorescein isothiocyante (FITC) conjugatedgoat anti-Borrelia antibody (KPL,Gaithersburg,MD),

diluted 1:50 with blocking buffer and incubated atroom temperature for 1 h. After incubation, the slideswerewashed three timeswithPBS for5mineach, thenmounted with SlowFade Light Antifade kit (Molecu-lar Probes, Eugene, OR).

Digital Imaging and Confocal Microscopy. Theslides were observed by epißuorescence with anECLIPS E 600 microscope (Nikon, Tokyo). BothbrightÞeldandßuorescent imageswerecapturedwitha SPOT II digital camera and processed using SPOTsoftware V2.2 (Diagnostic Instruments, SterlingHeights, MI). Slides were also observed with a LeicaTCS-NT confocal microscope system (Leica Micro-systems, Wetzlar, Germany). To determine whetherthe FITC-labeled spirochetes were inside of cells ormerely bound to their membranes, serial optical sec-tions were observed at 1-mm intervals. Images of thecells at the same optical section thickness were cap-tured using Normarski differential interference con-trast (DIC).

Bacterial Survival in Tissues of Infected I. scapu-laris. To assess the ability of B. burgdorferi to survivein salivary glands and ovaries after hemocoelic inoc-ulation, samples of these organs were dissected at 1and 24 h from B. burgdorferi-inoculated ticks, washed3 times inPBSbuffer, pH7.5, andcultured individuallyin BSK-H media that contained an antibiotic mixturefor Borrelia (Sigma) at 338C. The culture tubes wereexamined by dark Þeld microscopy and DFA for ev-idence of spirochete growth at 9Ð16 d after inocula-tion.

Borrelia Motility Inhibition Assays. To determineantimicrobial activity of I. scapularis hemolymphplasma against B. burgdorferi, hemolymph was col-lected at 1 h postinoculation from B. burgdorferi in-fected ticks or sham inoculated (noninfected) ticksand saved overnight at 48C. On the next day, thehemolymph was centrifuged at 12,000 3 g for 10 minto removehemocytes andnoncellular particulates andthe plasma was collected. Antimicrobial activity wasassessed by adding 10 ml of tick hemolymph plasmasample to 50 ml of a 3Ð5 d B. burgdorferi culture inBSK-H (adjusted to 4.5 3 103 cells per ml). Subse-quently, at deÞned intervals, sampleswere transferredto a hemocytometer and evaluated for bacterial num-bers and motility by dark Þeld microscopy.

Hemocyte Counts. The hemocyte counts weredone as previously described (Johns et al. 1998) usinga Brightline hemocytometer (Hausser ScientiÞc Co.,Horsham, PA) and viewed with a Nikon Optiphotcompound microscope. Hemolymph samples were as-sayed at 1, 6, 18, 24, 48, and 72 h postinoculation.

Results

Observation of B. burgdorferi Inoculated into theHemocoel of Ticks. One hour after spirochetes wereinoculated into I. scapularis, examination of the he-molymph by DFA revealed numerous spirochetes.Most appeared as free, intact, elongated immunoßu-orescent organisms in the plasma but some could beseen adjacent to the surface of hemocytes and, occa-

100 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 38, no. 1

sionally, several even appeared to be within the he-mocytes (Fig. 1a) [Compare the location of the or-ganismswith the locationof the cells in thebright Þeldimages.] In other instances, individual Borrelia ap-peared to be attached at their tips to a single hemo-cyte. Many spirochetes were also evident in or on thesurfaces of the salivary gland acini (Fig. 3a) and in theovarian tissues (data not shown).

At 24 h after B. burgdorferi inoculation into I. scapu-laris, examination of tissues revealed a different pic-ture. In the hemolymph, DFA revealed few intactspirochetes in the plasma but numerous ßuorescentfragments, presumably from spirochetes were evidentadjacent to or within the hemocytes (Fig. 1b). How-

ever, intact spirochetes were still evident in or on thesurfaces of the salivary gland acini (Fig. 3b) and in theovarian tissue samples (data not shown).

At 1 h afterB. burgdorferi inoculation intoD. variablisticks, few intact bacteria were evident in the hemo-lymph. Most organisms appeared to consist of small,cell-associated fragments (Fig. 1c). Confocal micros-copy conÞrmed that these spirochetes were inside thehemocytes,notmerelyonthesurfaces(Fig.2).Onlyoneintact spiral form characteristic of live spirochetes wasfound in the hemolymph plasma among the many Þeldsexamined in contrast to the numerous apparently intactspiral forms seen in the hemolymph plasma of I. scapu-laris. Examination of the salivary glands at 1 h revealed

Fig. 1. Images of tick hemolymph from fed female ticks following hemocoelic inoculation of B. burgdorferi. A bright Þeldimage of the hemolymph sample (left panel) and an immunoßuorescent image of spirochetes (green) in the same Þeld (rightpanel) are displayed in the Þgure. (a) I. scapularis, 1 h. Note some spirochetes free in the plasma (white arrow) versus othersattached to (orpossibly internalized) in ahemocyte (white arrowhead). (b) I. scapularis, 24h.Note thenumerousßuorescentfragments (white arrowhead), some adjacent to or possibly within hemocytes, but no free Borrelia.

January 2001 JOHNS ET AL.: TICK IMMUNE RESPONSES TO B. burgdorferi CHALLENGE 101

several spirochetes apparentlywithina single acinus sur-rounded by other acini without Borrelia (Fig. 3c). Sim-ilarly, a few spirochetes were found in the ovarian tissuesamples (data not shown).

At 24 h following inoculation, no spirochetes wereobserved in the hemolymph of D. variablis (Fig. 1d).Spirochetes were still evident in several local regionsof the salivary gland after 24 h, but most Þelds exam-ined were free of Borrelia (Fig. 3d). A few spirocheteswere also observed in the ovarian tissue at 24 h (datanot shown).

Survival of Borrelia burgdorferi in Tick Tissues.Experiments were performed to determine if the spi-rochetes observed with DFA in tick tissues were vi-able. I. scapularis and D. variablis ticks were inocu-lated with high (35,000 per tick) and low (3,500 pertick) doses of bacteria and then tick tissues wereremoved at 1 and 24 h after inoculation, washed 3times in PBS, and incubated with BSK-H media in anattempt to culture bacteria from the tissues. Whentissues from I. scapularis treated with the higher doseof spirochetes for 1 h were cultured in BSK II media,

Fig. 1. (continued) Images of tick hemolymph from fed female ticks following hemocoelic inoculation of B. burgdorferi.A bright Þeld image of the hemolymph sample (left panel) and an immunoßuorescent image of spirochetes (green) in thesame Þeld (right panel) are displayed in the Þgure. (c) D. variabilis, 1 h. Note the numerous immunoßuorescent fragmentsfree in the plasma (white arrowhead) and associated with hemocytes and possibly a single intact Borrelia (white arrow). (d)D. variabilis, 24 h no spirochetes were observed. In the bright Þeld images, hemocytes are marked by black arrows.MagniÞcation 4003.

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spirochetes were found in all three salivary glandstested and two of the three ovary samples (Table 1).Spirochetes were also cultured from two of the three

salivary glands and two of the three ovary samplescollected from I. scapularis at 24 h postinoculation(Table 1). When the studies were repeated with tis-sues from the ticks inoculated with the lower dose ofBorrelia, the resultswere similar. Spirochetes survivedin salivary gland and ovary samples for at least 24 h.Furthermore, bacteriawere also readily cultured fromhemolymph collected from I. scapularis treated withthe low dose of bacteria for 1 h.

In D. variabilis, spirochetes were cultured fromticks at 1 h postinoculation (high dose), includingthree of six hemolymph samples, but only one of sixsalivary gland or ovary samples (Table 1). No spiro-chetes could be cultured from tick tissues collected at24 h postinoculation. When the study was repeatedwith the low dose of Borrelia, at 1 h postinoculationspirochetes were cultured from only one of threesalivary glands while all the other samples were neg-ative (Table 1). No spirochetes were cultured fromtissue samples collected at 24hpostinoculation (Table1). In summary, culturing spirochetes from tissuestaken from B. burgdorferi-inoculated ticks was suc-cessful in only three out of 36 attempts, and then onlywhen taken at 1 h after inoculation. In contrast, cul-tures from I. scapularis were successful in 21 out of 24attempts.

Anti-Borrelia Activity in Hemolymph Plasma. B.burgdorferi were incubated with hemolymph plasmain vitro to directly test the effect of tick plasma onspirochetes. When I. scapularis plasma was incubatedwith cultured spirochetes in vitro, no signiÞcant dif-ferences in spirochetenumbersormotilitywere found(Fig. 4). However, when the same experiment wasdone with plasma from D. variabilis, spirochete sur-vival (assessed by motility) declined rapidly (Fig. 4).

HemocyteCounts.Hemocytes in thehemolymphofB. burgdorferi-challenged I. scapularis females weresigniÞcantly elevated at 1 h after bacterial inoculation(t 5 5.24, P , 0.01), but declined thereafter to normallevels (Fig. 5A). Similarly, Johns et al. (2000) showedhemocyte counts in the hemolymph of B. burgdorferi-challenged D. variabilis females increased eight-foldwithin 1 h and remained signiÞcantly greater than thecontrols for 6 h postinoculation (Fig. 5B) (data fromJohns et al. 2000, with permission of the publisher).

Discussion

Borrelia burgdorferi infect the gut and then dissem-inate into diverse tissues of I. scapularis and othermembers of the I. ricinus complex of ticks (Burgdorferet al. 1989, Zhu 1998). D. variabilis is often sympatricwith I. scapularis (Sonenshine 1993). Although B.burgdorferi have been observed in D. variabilis guts,both laboratoryandÞeld studies indicate thatD.varia-bilis is not a competent vector of B. burgdorferi (Pies-manandSinsky1988,MatherandMather1990,Matheret al. 1996). The results we report here provide a basisfor understanding the differences in vector compe-tence between I. scapularis and D. variabilis.

The I. scapularis hemocoel was a relatively benignenvironment for B. burgdorferi in comparision to the

Fig. 2. Confocal microscopic images conÞrming inter-nalization of spirochetes in a D. variabilis hemocyte. Right-hand panel shows a laser image of a spirochete at differentlevels from the base of the hemocyte. Left-hand panel showsNormarski DIC images of the hemocyte at the same level(arrow). Sections are 2 mm apart. MagniÞcation 1,0003.

January 2001 JOHNS ET AL.: TICK IMMUNE RESPONSES TO B. burgdorferi CHALLENGE 103

Fig. 3. Digital images of tick salivary glands from fed female ticks following hemocoelic inoculation of B. burgdorferi.Bright Þeld images of the salivary gland (left panel) and immunoßuorescent images of spirochetes (green) in the same Þeld(right panel) are displayed in the Þgures (a) I. scapularis 1 h, (b) I. scapularis 24 h, (c) D. variabilis 1 h, and (d) D. variabilis24 h. Arrows show (apparently intact) spirochetes. MagniÞcation 4003.

104 JOURNAL OF MEDICAL ENTOMOLOGY Vol. 38, no. 1

D. variabilis hemocoel.Within I. scapularis, numerousspirochetes remained free and intact in the hemo-lymph plasma 1 h after direct inoculation into thehemocoel. Moreover, this period proved sufÞcient forviable spirochetes to penetrate the surrounding tis-sues, including the salivary glands. In contrast, thehemolymphofD. variabilis, rapidly destroyedB. burg-dorferi and viable spirochetes were rarely recoveredfrom other tissues (Johns et al. 2000; Figs 1 and 4).

The I. scapularis hemocoel was not a completelybenign environment for spirochetes because we ob-

served a decline in the number of intact spirochetes24 h after intrahemocoelic inoculation. The gradualclearance of spirochetes from the I. scapularis hemo-coel may explain why B. burgdorferi infection wasusually conÞned to the gut of I. scapularis ticks. Spi-rochetes only disseminate beyond the gut to thehemocoel and other tick organs for a brief periodduring transmission (Piesman, 1995, de Silva andFikrig 1995). Others have also reported that spiro-chetes do not persist in the hemolymph of I. scapularisticks, suggesting that the hemolymph represents a

Fig. 4. Differences in antimicrobial activity of hemolymph plasma collected from I. scapularis and D. variabilis 1 h afterchallengewithB.burgdorferior saline(sham-treated).TheassaywasdonewithB.burgdorferi incubated inamixtureofBSK-Hplus tick hemolymph plasma. Time (minutes) after introduction of tick hemolymph plasma.

Table 1. In vivo survival of Borrelia burgdorferi in salivary glands and ovaries of I. scapularis and D. variabilis females followinghemocoelic inoculation of cultured spirochetes

Treatment% tissue or organs sampled with live spirochetes

Hemolymph 1 hr Hemolymph 24 h Sal glands 1 h Sal glands 24 h Ovaries 1 h Ovaries 24 h

I. scapularis

High dosea ND3 NDc 3/3 2/3 2/3 2/3Low doseb 3/3 NDc 3/3 3/3 3/3 3/3

D. variabilis

High dosea 3/6 0/6 1/6 0/6 1/6 0/6Low doseb 0/3 0/3 1/3 0/3 0/3 0/3

a Fed females were inoculated with 3.5 3 104 spirochetes (high dose) from a laboratory culture by intrahemocoelic injection, dissected at1 and 24 h postinoculation and organs removed. Samples of hemolymph or organs were incubated in BSK-H at 338C for 9Ð16 d and examinedfor spirochetes using dark Þeld microscopy and DFA.

b Females inoculated with 3.5 3 103 spirochetes (low dose).c ND 5 not done.

January 2001 JOHNS ET AL.: TICK IMMUNE RESPONSES TO B. burgdorferi CHALLENGE 105

hostile environment for these bacteria (Munderlohand Kurtti 1995, Coleman et al. 1997).

ThemechanismofBorrelia clearance in I. scapularisappears to be primarily cell-mediated because hemo-lymphplasma frombothB. burgdorferi challengedandunchallenged I. scapularis ticks was not borreliacidalin vitro. Phagocytosis appeared to be the primarymeans ofB. burgdorferi control in I. scapularisbecauseat 1 h after inoculation some spirochetes were cell-associated and by 24 h the bacteria were presentmostly as cell-associated fragments. Under naturalconditions, phagocytosis may proceed more slowly,perhaps, because the bacterial challenge itself is moregradual as spirochetes emerge from the infected mid-gut over several days, rather than in a single burst(Ribeiro et al.1987). In the Coleman et al. (1997)study, hemocytes with bound or incorporated spiro-chetes increased gradually, from only 2% of the he-mocyte population on feeding day 3Ð13% by feedingday 5. Hemocyte abundance was distinctly higher inthe B. burgdorferi-challenged I. scapularis ticks for atleast 1 h after spirochete inoculation indicating that anincrease in the number of hemocytes may contributeto the eventual clearance of spirochetes. Spirochetesintroduced into the hemocoel of D. variabilis experi-

encedadifferent fate than thebacteriawithin I. scapu-laris. Virtually all of these bacteria were destroyedwithin 1 h. The presence of innumerable ßuorescentfragments scattered throughout the hemolymphplasma or attached to hemocytes suggests a dualmechanism of spirochete control, namely, bacteriol-ysis by plasma factors and phagocytosis. This is con-sistent with previous Þndings by Johns et al. (2000),who showed that hemolymph plasma from B. burg-dorferi-challenged ticks rapidly cleared most of themicroorganisms when added to cultures of the spiro-chetes growing in BSK-H and that hemocyte popula-tions increased greatly during bacterial challenge.

We recognize that the unusually large number ofspirochetes inoculated into the tickÕs hemolymph,perhaps hundreds of times the numbers reported innaturally acquired infections, was artiÞcial. However,the results were similar with the lower dose, which ismore representative of natural conditions. The abilityof most Borrelia to survive in the hemolymph of I.scapularis for at least 1 h was long enough for them tomigrate to the surrounding tissues and survive. Thus,tissue invasion can proceed very rapidly in I. scapu-laris. As noted by Kurtti et al. (1993), B. burgdorferiadhere readily to actively transporting cells such asthose of the salivary glands that havenumerous coatedand uncoated pits. In contrast, in the more hostileenvironment of the D. variabilis hemolymph, mostspirochetes are lysed within a few minutes by antimi-crobial peptides (Johns et al. 2000). A few spirochetesdid reach organs in D. variablis also but these weremostly nonviable since spirochetes were rarely cul-tured from D. variabilis organs. Thus, in view of therobust immune system of D. variabilis, it is unlikelythat naturally acquired spirochetes would survive he-molymph passage and tissue penetration, even inthose rare occasions where they might survive in themidgut of ticks (Burgdorfer et al. 1989). We proposethat the vector competence of tick species for B. burg-dorferi is inßuenced strongly by the innate immuneresponse of the tick vector.

Acknowledgments

Weare grateful toMartinE. Schriefer (DivisionofVector-Borne Diseases, Centers for Disease Control, Fort Collins,CO) for the supply of the low passage B31 strain of B.burgdorferi used in this study. We are most grateful forfunding support for the research reported in this article fromthe Thomas F. Jeffress and Kate Miller Jeffress MemorialTrust (toD.S.), theOldDominionUniversityEminent Schol-ars Fund (to D.S.), the NIH (AR02061) (to A.deS.) and theArthritis Foundation (to A.deS.).

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Received for publication 30 May 2000; accepted 5 October2000.

January 2001 JOHNS ET AL.: TICK IMMUNE RESPONSES TO B. burgdorferi CHALLENGE 107