Infestation with pathogen-free nymphs of the tick Ixodes scapularis induces host resistance to transmission of Borrelia burgdorferi by ticks

Abstract

Female BALB/c mice were infested four times with pathogen-free Ixodes scapularis nymphs prior to infestation with nymphs infected with Borrelia burgdorferi B31. Each infestation was separated by a 14-day tick-free period. Mean weights of fed ticks and percentage reaching repletion did not indicate development of acquired resistance. Only 16.7% of mice repeatedly infested with pathogen-free ticks prior to infected I. scapularis nymph challenge became positive for B. burgdorferi. One hundred percent of control mice infested only with infected ticks were culture positive for B. burgdorferi.

Full text

INFECTION AND IMMUNITY,
0019-9567/97/$04.0010Jan. 1997, p. 335–338 Vol. 65, No. 1
Copyright q1997, American Society for Microbiology
Infestation with Pathogen-Free Nymphs of the Tick Ixodes
scapularis Induces Host Resistance to Transmission of Borrelia
burgdorferi by Ticks
STEPHEN K. WIKEL,
1
* RANGAPPA N. RAMACHANDRA,
1
DOUGLAS K. BERGMAN,
1
THOMAS R. BURKOT,
2
AND JOSEPH PIESMAN
2
Department of Entomology, Oklahoma State University, Stillwater, Oklahoma 74078,
1
and Centers for Disease Control
and Prevention, Division of Vector-Borne Infectious Diseases, Medical Entomology-Ecology Branch,
Fort Collins, Colorado 80522
2
Received 11 July 1996/Returned for modification 12 August 1996/Accepted 7 October 1996
Female BALB/c mice were infested four times with pathogen-free Ixodes scapularis nymphs prior to infes-
tation with nymphs infected with Borrelia burgdorferi B31. Each infestation was separated by a 14-day tick-free
period. Mean weights of fed ticks and percentage reaching repletion did not indicate development of acquired
resistance. Only 16.7% of mice repeatedly infested with pathogen-free ticks prior to infected I. scapularis nymph
challenge became positive for B. burgdorferi. One hundred percent of control mice infested only with infected
ticks were culture positive for B. burgdorferi.
The tick-host-pathogen interface is characterized by dy-
namic interactions among all three elements of the relation-
ship. Tick feeding induces a complex array of host immune
regulatory and effector responses involving antigen-presenting
cells, cytokines, immunoglobulins, complement, and T lympho-
cytes (1, 6, 24, 27, 29). Immunologically based acquired resis-
tance to ticks reduces engorgement, egg production, and via-
bility (24, 25). In turn, ticks have developed countermeasures
to host immune defenses (18, 25–28). Tick-induced host im-
munosuppression reduces both T-lymphocyte in vitro respon-
siveness to mitogens (23, 25) and antibody production (26) and
impairs the elaboration of macrophage and Th1-lymphocyte
cytokines (18). Tick modulation of host immune defenses
likely facilitates tick feeding and transmission of vector-borne
disease-causing agents (27, 28).
Transmission of Borrelia burgdorferi by the tick vector results
in a different array of host immune responses than needle
inoculation of the spirochetes (13, 21). Antibodies to the outer
surface proteins (Osp) A and B did not develop in animals
infected by tick transmission of B. burgdorferi, while animals
infected by needle inoculation of cultured spirochetes devel-
oped high titers of antibodies to both proteins. The antibody
response to needle inoculation of B. burgdorferi is dependent
upon the number of spirochetes inoculated (3). Administration
of greater than 10
4
spirochetes to mice induces an anti-OspA
response, while needle inoculation of fewer viable spirochetes
did not stimulate the production of OspA-reactive immuno-
globulins for up to 2 weeks postinjection.
B. burgdorferi, causative agent of Lyme borreliosis, is trans-
mitted by ticks of the genus Ixodes, and rodents serve as res-
ervoir hosts (2, 14). Ixodes scapularis is the primary vector of
Lyme borreliosis in the eastern and central United States and
Ixodes pacificus is the primary vector in the western United
States (2, 14, 15).
Canines experimentally infested with adult I. scapularis de-
veloped acquired resistance, which reduced tick viability and
oviposition with each successive exposure (11). Acquired re-
sistance to the vector might reduce transmission of spirochetes.
Clethrionomys glareolus (bank vole), a natural host of Ixodes
ricinus, which is the most important European vector of Lyme
borreliosis, acquired resistance to I. ricinus with repeated in-
festations (10). Acquired antitick resistance of C. glareolus
reportedly interferes with I. ricinus transmission of B. burgdor-
feri (9).
Acquired resistance to tick infestation has been shown to
alter transmission of another tick-borne pathogen, Francisella
tularensis (4). Rabbits pre-exposed to pathogen-free Derma-
centor andersoni were partially protected when exposed to F.
tularensis-infected nymphs. Those rabbits infested with patho-
gen-free D. andersoni also had heightened delayed cutaneous
reactivity upon intradermal inoculation of homogenates of
whole tick larvae, nymphs, or adults, indicating cell-mediated
immune reactivity to tick antigens (4).
The objective of this study was to determine whether re-
peated infestation with pathogen-free I. scapularis nymphs al-
ters the subsequent transmission of B. burgdorferi by infected
nymphs. Toward this aim, laboratory-reared nymphal I. scapu-
laris ticks used in this study were obtained from a colony
established in the Department of Entomology, Oklahoma
State University, Stillwater. Ticks were maintained in cotton-
plugged vials held over water in a desiccator at 158C with a
14:10 (light to dark) photoperiod. Laboratory colonization in-
volved feeding larvae and nymphs on BALB/c mice and feed-
ing adults on guinea pigs. Host animals were fed a commercial
diet and water ad libitum while being housed at 228Cinthe
Laboratory Animal Resources facility, Oklahoma State Uni-
versity. I. scapularis ticks were infected with the B31 strain of B.
burgdorferi as previously described (16). Only feedings that
resulted in a $80% infection rate in unfed nymphal ticks on
darkfield examination were included in this colony.
Experiments were initiated with 10- to 12-week-old female
BALB/c mice weighing between 20 and 25 g (Jackson Labo-
ratories, Bar Harbor, Maine). Mice were maintained at 228C
and fed a commercial diet and water ad libitum.
This study was repeated twice. The first experiment con-
sisted of 6 mice each in both the experimental and control
groups, and the second experiment utilized 12 mice in each
* Corresponding author. Mailing address: Dr. Stephen K. Wikel,
Department of Entomology, 127 Noble Research Center, Oklahoma
State University, Stillwater, Oklahoma 74078. Phone: (405) 744-9946.
Fax: (405) 744-6954.
335

group. The experimental group was exposed to four infesta-
tions with pathogen-free I. scapularis nymphs, followed by a
fifth infestation with B. burgdorferi B31-infected I. scapularis
nymphs. The first three infestations were with 10 nymphs per
animal, while the fourth pathogen-free infestation consisted of
8 nymphs per mouse. The infected-tick challenge was with six
nymphs per mouse. Control mice received one infestation with
B. burgdorferi B31-infected I. scapularis nymphs. Both experi-
mental and control mice were infested with spirochete-infected
ticks at the same time. Each infestation was allowed to proceed
until the nymphs engorged and detached or for 10 days, at
which time ticks were removed. A 14-day tick-free period was
maintained between each infestation. Each tick-free period
began 10 days after initiation of the preceding infestation,
regardless of the time when ticks were removed from the host.
All pathogen-free or B. burgdorferi-infected ticks used in a
given experiment were derived from a common pool of ticks
which were handled in a similar manner.
The percentage of ticks completing feeding and the mean
engorgement weights were determined for each infestation
group. An ear punch biopsy was obtained from each mouse, 4
weeks after termination of the infestation with infected ticks,
and cultured for spirochetes as described below. The surface of
the ear was swabbed with 70% ethanol and allowed to air dry
before a sterile punch biopsy was obtained. Tissue was placed
into sterile Barbour-Stoenner-Kelly medium according to the
method of Sinsky and Piesman (22). Cultures were maintained
at 348C and examined for spirochetes by darkfield microscopy
beginning on the 3rd day and ending on the 14th day of culture.
B. burgdorferi infections in I. scapularis ticks, which molted to
adults, were detected and densities were estimated by a two-
monoclonal antibody (MAb) OspA enzyme-linked immu-
nosorbent assay (ELISA), which was modified slightly from
one described previously (7). Briefly, wells of a polyvinylchlo-
ride ELISA plate were coated overnight at 48C with 0.17 mgof
MAb H5332 in 50 ml of phosphate-buffered saline (PBS). All
subsequent steps were performed at room temperature. The
next morning, wells were washed three times with a solution
containing PBS and 0.05% Tween 20 (PBS-T) before being
blocked with 200 ml of 2.5% nonfat dry milk blocker (Bio-Rad
Laboratories, Richmond, Calif.) in PBS-T for 1 h (blocking
solution). Nymphs were prepared for ELISA by homogeniza-
tion in 50 ml of 0.25% Nonidet P-40 in PBS after which PBS-T
was added to a final volume of 300 ml. After the wells were
washed as described above, 50 ml of tick homogenate was
added to each well for 90 min. Wells were again washed, and
8 ng of MAb 1-15 (a gift from Pfizer Animal Health) conju-
gated to horseradish peroxidase in 50 ml of blocking solution
was then added to each well for 1 h. MAb 1-15 reacts with a
nonprotective epitope of B. burgdorferi OspA. Wells were
washed again prior to addition of 100 mlof2,29-azino-bis(3-
ethylbenzthiazoline-sulfonic acid) per well. A
405
s were read
after 1 h. Spirochete numbers were estimated by comparing
absorbances generated from the culture samples with a stan-
dard curve of known low-passage B31 spirochetes.
Statistical significance was determined by analysis of vari-
ance followed by mean separation by the Newman-Keuls
method (12) or by chi-square.
The percentages of nymphs feeding to repletion (6standard
deviation) were determined for ticks collected from mice ei-
ther infested four times with pathogen-free ticks or uninfested
prior to challenge with B. burgdorferi-infected I. scapularis
nymphs. In the first experiment, the percentage of ticks feeding
to repletion recovered after each of the repeated infestations
did not differ significantly (P,0.05, Newman-Keuls method,
35 degrees of freedom) among each other or from ticks feeding
on control mice. Percentages (6standard deviation) of ticks
feeding to repletion for the first through fourth infestations
with pathogen-free ticks were 65.0 610.5, 50.0 66.3, 81.7 6
11.7, and 52.1% 620.0%, respectively. The percentages of
infected ticks feeding to repletion among the fifth infestation
of the experimental group and the first infestation of control
mice were 66.7 623.6 and 55.6% 632.8%, respectively. Dur-
ing the second experiment, the percentages of B. burgdorferi-
infected ticks completing feeding during the fifth infestation of
repeatedly infested mice and during infestation of the previ-
ously uninfested mice were significantly less than (P,0.05, 69
degrees of freedom) the percentages of pathogen-free ticks
feeding to repletion during each of the initial four exposures of
the previously infested group. Repletion percentages for the
first four infestations of the experimental group were 56.7 6
21.0, 60.0 623.0, 55.0 615.7, and 61.5% 621.0%, respec-
tively. The repletion percentage of infected ticks for the ex-
perimental group was 23.6% 626.1%, and for the control mice
it was 26.7% 621.1%.
Mean engorgement weights in milligrams (6standard devi-
ation) were determined for ticks obtained from each mouse at
the end of every infestation. Mean engorgement weights de-
creased significantly (P,0.05, 35 degrees of freedom) with
successive infestations during the first experiment but not dur-
ing the second experiment. Mean engorgement weights for the
first experiment for pathogen-free tick exposures one through
four were 3.0 60.3, 2.5 60.3, 2.5 60.3, and 1.9 mg 60.5 mg,
respectively, while for the infected-tick infestation (fifth expo-
sure) mean engorgement weight was 1.9 mg 60.5 mg. Mean
engorgement weight for the infected ticks infesting the control
mice was 1.9 mg 60.2 mg.
Ear punch biopsies from mice that became infected after
infestation with B. burgdorferi-infected ticks were positive for
spirochetes by the fourth day of culture in BSK-II medium.
During both experiments, repeated infestation with pathogen-
free I. scapularis nymphs resulted in resistance to subsequent
acquisition of B. burgdorferi infection when mice were fed upon
by infected ticks. During both the first and second experiments
16.7% of mice previously infested with pathogen-free ticks
became infected when given a challenge infestation with B.
burgdorferi-infected I. scapularis nymphs. Ear punch biopsies of
1 of 6 and 2 of 12 mice were BSK II culture positive at 4 weeks
postinfestation with infected ticks during the first and second
experiments, respectively. During both experiments, 100% of
control mice infested with only infected ticks became infected
with B. burgdorferi. Differences between these two treatment
groups were statistically significant for both the first (chi-
square 55.49, 1 degree of freedom, P50.0192) and second
(chi-square 513.89, one degree of freedom, P50.0002)
experiments.
Results of OspA antigen capture ELISA provided in Table
TABLE 1. Postinfestation B. burgdorferi infection status of I.
scapularis nymphs determined by OspA antigen capture ELISA
Group
No. of infected ticks/no. of ticks
tested
a
First expt Second expt
Pathogen-free ticks 0/7 0/22
B. burgdorferi-infected ticks
Previously infested hosts 5/7* 1/6**
Previously uninfested hosts 6/6* 9/9**
a
p, chi-square 50.43, 1 degree of freedom; P50.5142. pp, chi-square 57.81,
1 degree of freedom; P50.0052.
336 NOTES INFECT.IMMUN.

1 confirm the postchallenge presence of B. burgdorferi in the
infected I. scapularis nymphs. The sample population of patho-
gen-free ticks assayed postinfestation was negative for B. burg-
dorferi infection. All ticks collected from control mice which
received their only infestation with B. burgdorferi-infected ticks
were OspA positive (Table 1). Five of seven and one of six
infected ticks obtained at the termination of the challenge
infestation (fifth exposure) of mice repeatedly infested with
pathogen-free ticks were OspA antigen capture positive. Dif-
ferences between the number of OspA antigen-capture-posi-
tive challenge ticks infesting previously exposed versus initial
exposure animals were significantly different during the second
experiment (chi-square, P,0.05, 1 degree of freedom).
This study demonstrated that BALB/c mice repeatedly in-
fested with pathogen-free I. scapularis nymphs were resistant
to subsequent infection with tick-transmitted B. burgdorferi.
Transmission-blocking immunity was first described by Bell et
al. (4) for rabbits resistant to tick transmission of highly viru-
lent F. tularensis after repeated infestation with pathogen-free
adult D. andersoni. In another study, bank voles (C. glareolus)
expressing acquired resistance to I. ricinus did not become
infected with B. burgdorferi after infestation with infected ticks,
while bank voles not resistant to tick feeding became infected
with I. ricinus-transmitted spirochetes (9). The yellow-necked
mouse, Apodemus flavicollis, did not develop resistance to in-
festation with I. ricinus, indicating possible host species speci-
ficity of resistance to the vector and vector-borne pathogen (8,
9). This study describes a laboratory model for characterization
of vector-blocking immunity.
Resistance of mice repeatedly infested with pathogen-free I.
scapularis nymphs prior to infected-tick challenge might be due
to an alteration of the ability of the tick to transmit spirochetes
and/or a modified host environment into which the spirochetes
are introduced. Initiation of feeding stimulates spirochetes to
disseminate from the tick gut to the hemocoel and salivary
glands for transmission to the host (20). Nymphal I. scapularis
ticks need to be attached to the host for approximately 48 h for
efficient transmission of B. burgdorferi (17). Alteration of the
events involved in spirochete activation could possibly alter
spirochete dissemination within the tick and transmission dur-
ing feeding. Host antibodies to B. burgdorferi OspA in the tick
blood meal blocked spirochete dissemination to the tick sali-
vary glands (8).
Using an infestation regimen similar to the one described
above, sera were obtained from BALB/c mice which had been
repeatedly infested with pathogen-free I. scapularis nymphs
derived from the colony maintained in this laboratory. BALB/c
mice infested with pathogen-free I. scapularis nymphs did not
develop antibodies reactive on immunoblots with an extract of
whole B. burgdorferi B31.
Other factors that might affect pathogen transmission and
establishment in hosts repeatedly infested with pathogen-free
ticks include alterations in the cutaneous environment at the
tick attachment site, which would interrupt feeding and/or be
deleterious to introduced spirochetes; reduction of duration of
attachment to the host; and host modification of pharmaco-
logically active components of the tick saliva.
Tick feeding suppresses innate and acquired immunity of the
host (19, 25, 26, 28). Tick-induced suppression of host immune
function reduces antibody responses, T-lymphocyte prolifera-
tion to mitogens, and elaboration of cytokines by macrophages
and Th1 lymphocytes. Greater suppression of host T-lympho-
cyte in vitro responsiveness was observed at the end of a pri-
mary infestation than at the termination of a second exposure
(25). Immunosuppressant proteins have recently been de-
scribed in tick salivary glands (5, 23). Repeated infestation with
pathogen-free ticks may induce a host immune response that
neutralizes the tick immunosuppressant(s) introduced into the
host during feeding and thus enhance resistance to infection
with B. burgdorferi. More complete expression of host innate
and acquired immune effector mechanisms might be a factor in
the resistance of repeatedly infested mice to tick transmission
of B. burgdorferi.
The OspA antigen capture ELISA results indicate that re-
peated infestation with pathogen-free ticks might impact B.
burgdorferi in infected challenge ticks. The B. burgdorferi-in-
fected ticks recovered after the fifth infestation of repeatedly
exposed mice had the lowest incidence of postinfestation in-
fection, as detected by OspA antigen capture. The differences
in infection status of ticks collected from control mice receiv-
ing a first exposure and repeatedly infested mice are not due to
differences in the ability to detect OspA, since the assays were
performed on ticks from a common pool at identical physio-
logical ages. The B. burgdorferi infection rate of ticks prior to
challenge infestation was 100%, as determined by darkfield
examination, compared with a 17% infection rate in ticks feed-
ing on mice previously infested with pathogen-free I. scapu-
laris. Ticks obtaining a blood meal from hosts repeatedly ex-
posed to pathogen-free tick bites might acquire factors that
affect B. burgdorferi development, multiplication, dissemina-
tion, and/or expression of OspA.
Prior infestation with pathogen-free ticks induces a host
transmission-blocking response which provides protection
against infection. Tick biology parameters evaluated could not
be used to definitively link acquired resistance to protection
against tick-transmitted B. burgdorferi infection.
Vector-blocking immunity, impairing pathogen transmission
and/or establishment within the host, is possibly a manifesta-
tion of acquired resistance to tick feeding. Expression of ac-
quired resistance in this study might be masked by serendipi-
tous variations in tick feeding. The host immune response to
repeated tick feeding could possibly neutralize immunosup-
pressive molecules introduced by the tick, allowing for greater
expression of host innate and acquired immune defenses
against tick-borne pathogens. The magnitude of tick-induced
suppression of host T-lymphocyte in vitro proliferative re-
sponses to mitogens and elaboration of macrophage and T-
lymphocyte cytokines progressively becomes less intense dur-
ing the course of four repeated tick exposures. A similar
phenomenon might have occurred during the repeated infes-
tations with pathogen-free I. scapularis nymphs in this study.
Reduction of tick immunosuppression of the host might be an
effective strategy for enhancing resistance to tick-transmitted
disease-causing agents.
Research was supported by the Oklahoma Center of Advancement
of Science and Technology and by Oklahoma Agricultural Experiment
Station Project Number OKL02174.
We thank Alan Barbour for the use of MAb H5332 and Pfizer
Animal Health Division for MAb 1-15. We also thank Christine Happ
and Angela Koscelney for technical assistance.
REFERENCES
1. Allen, J. R. 1989. Immunology of interactions between ticks and laboratory
animals. Exp. Appl. Acarol. 7:5–13.
2. Anderson, J. F. 1989. Epizootiology of Borrelia in Ixodes tick vectors and
reservoir hosts. Rev. Infect. Dis. 11:S1451–S1459.
3. Barthold, S. W., E. Fikrig, L. K. Bockenstedt, and D. H. Persing. 1995.
Circumvention of outer surface protein A immunity by host-adapted Borrelia
burgdorferi. Infect. Immun. 63:2255–2261.
4. Bell, J. F., S. J. Stewart, and S. K. Wikel. 1979. Resistance to tick-borne
Francisella tularensis by tick sensitized rabbits: allergic klendusity. Am. J.
Trop. Med. Hyg. 28:876–880.
5. Bergman, D. K., R. N. Ramachandra, and S. K. Wikel. 1995. Dermacentor
andersoni: salivary gland proteins suppressing T-lymphocyte responses to
VOL. 65, 1997 NOTES 337

concanavalin A in vitro. Exp. Parasitol. 81:262–271.
6. Brossard, M., B. Rutti, and T. Haug. 1991. Immunological relationships
between host and ixodid ticks, p. 177–200. In C. A. Toft, A. Aeschliman, and
L. Bolic (ed.), Parasite-host associations: coexistence or conflict. Oxford
University Press, Oxford, England.
7. Burkot, T. R., J. Piesman, and R. A. Wirtz. 1994. Quantitation of the Borrelia
burgdorferi outer surface protein A in Ixodes scapularis: fluctuations during
the tick life cycle, doubling times and loss while feeding. J. Infect. Dis.
170:883–889.
8. DeSilva, A. M., S. R. Telford III, L. R. Brunet, S. W. Barthold, and E. Fikrig.
1996. Borrelia burgdorferi OspA is an arthropod-specific transmission-block-
ing Lyme disease vaccine. J. Exp. Med. 183:271–275.
9. Dizij, A., S. Arndt, H. M. Seitz, and K. Kurtenbach. 1994. Clethrionomys
glareolus acquires resistance to Ixodes ricinus: a mechanism to prevent spi-
rochete inoculation?, p. 228–231. In R. Cevenini, V. Sambri, and M. LaPlaca
(ed.), Advances in lyme borreliosis research. Proceedings of the VI Interna-
tional Conference on Lyme Borreliosis, Bologna, Italy.
10. Dizij, A., and K. Kurtenbach. 1995. Clethrionomys glareolus, but not Apode-
mus flavicollis, acquires resistance to Ixodes ricinus L., the main European
vector of Borrelia burgdorferi. Parasite Immunol. 17:177–183.
11. Gebbia, J. A., E. M. Bosler, R. D. Evans, and E. M. Schneider. 1995.
Acquired resistance in dogs to repeated infestation with Ixodes scapularis
(Acari: Ixodidae) reduces tick viability and reproductive success. Exp. Appl.
Acarol. 19:593–605.
12. Kachigan, S. K. 1986. Statistical analysis: an interdisciplinary introduction to
univariate and multivariate methods. Radius Press, New York, N.Y.
13. Kurtenbach, K., A. Dizij, H. M. Seitz, G. Margos, S. E. Muter, M. D.
Kramer, R. Wallich, U. E. Schaible, and M. M. Simon. 1994. Differential
immune responses to Borrelia burgdorferi in European wild rodent species
influence spirochete transmission to Ixodes ricinus L. (Acari: Ixodidae). In-
fect. Immun. 62:5344–5352.
14. Lane, R. S., J. Piesman, and W. Burgdorfer. 1991. Lyme borreliosis: relation
of its causative agent to its vectors and hosts in North America and Europe.
Annu. Rev. Entomol. 36:587–609.
15. Oliver, J. H., Jr., M. R. Owsley, H. J. Hutcheson, A. M. James, C. Chen, W. S.
Irby, E. M. Dotson, and D. K. McClain. 1993. Conspecificity of the ticks
Ixodes scapularis and I. dammini (Acari: Ixodidae). J. Med. Entomol. 30:54–63.
16. Piesman, J. 1993. Standard system for infesting ticks (Acari: Ixodidae) with the
Lyme disease spirochete Borrelia burgdorferi. J. Med. Entomol. 30:199–203.
17. Piesman, J. 1993. Dynamics of Borrelia burgdorferi transmission by nymphal
Ixodes dammini ticks. J. Infect. Dis. 167:1082–1085.
18. Ramachandra, R. N., and S. K. Wikel. 1992. Modulation of host immune
responses by ticks (Acari: Ixodidae): effects of salivary gland extracts on host
macrophages and lymphocyte cytokine production. J. Med. Entomol. 29:
818–826.
19. Ribeiro, J. M. C. 1995. Blood-feeding arthropods: live syringes or inverte-
brate pharmacologists? Infect. Agents Dis. 4:143–152.
20. Ribeiro, J. M. C., T. N. Mather, J. Piesman, and A. Spielman. 1987. Dis-
semination and salivary delivery of Lyme disease spirochetes in vector ticks
(Acari: Ixodidae). J. Med. Entomol. 24:201–205.
21. Roehrig, J. T., J. Piesman, A. R. Hunt, M. G. Keen, C. M. Happ, and B. J. B.
Johnson. 1992. The hamster immune response to tick-transmitted Borrelia
burgdorferi differs from the response to needle-inoculated, cultured organ-
isms. J. Immunol. 149:3648–3653.
22. Sinsky, R. J., and J. Piesman. 1989. Ear punch biopsy method for detection
and isolation of Borrelia burgdorferi from rodents. J. Clin. Microbiol. 27:
1723–1727.
23. Urioste, S., L. R. Hall, S. R. Telford III, and R. G. Titus. 1994. Saliva of the
Lyme disease vector, Ixodes dammini, blocks cell activation by a nonpros-
taglandin E
2
-dependent mechanism. J. Exp. Med. 180:1077–1085.
24. Wikel, S. K. 1982. Immune responses to arthropods and their products.
Annu. Rev. Entomol. 27:21–48.
25. Wikel, S. K. 1982. Influence of Dermacentor andersoni infestation on lym-
phocyte responsiveness to mitogens. Ann. Trop. Med. Parasitol. 76:627–632.
26. Wikel, S. K. 1985. Effects of tick infestation on the plaque-forming cell
response to a thymic dependent antigen. Ann. Trop. Med. Parasitol. 79:195–
198.
27. Wikel, S. K. 1996. Host immunity to ticks. Annu. Rev. Entomol. 41:1–22.
28. Wikel, S. K., R. N. Ramachandra, and D. K. Bergman. 1994. Tick-induced
modulation of the host immune response. Int. J. Parasitol. 24:59–66.
29. Willadsen, P. 1980. Immunity to ticks. Adv. Parasitol. 18:293–313.
Editor: R. E. McCallum
338 NOTES INFECT.IMMUN.