The presence of granulocytic ehrlichiae was demonstrated by PCR inIxodes ricinus ticks and wild small mammals in Switzerland in two areas of endemicity for bovine ehrlichiosis. Six ticks (three females and three nymphs) (1.4%) of 417 I. ricinus ticks collected by flagging vegetation contained ehrlichial DNA. A total of 201 small mammals from five species, wood mouse (Apodemus sylvaticus), yellow-necked mouse (Apodemus flavicollis), earth vole (Pitymys subterraneus), bank vole (Clethrionomys glareolus), and common shrew (Sorex araneus), were trapped. The analysis of I. ricinus mammals collected on 116 small mammals showed that nine C. glareolus voles and two A. sylvaticus mice hosted infected tick larvae. In these rodents, granulocytic ehrlichia infection was also detected in blood, spleen, liver, and ear samples. Further examinations of 190 small mammals without ticks or with noninfected ticks showed the presence of ehrlichial DNA in spleen and other tissues from six additional C. glareolus, three A. flavicollis, and one S. araneus mammals. This study suggests thatA. sylvaticus, A. flavicollis, S. araneus, and particularly C. glareolus are likely to be natural reservoirs for granulocytic ehrlichiae. Partial 16S rRNA gene sequences of granulocytic ehrlichiae from ticks and rodents showed a high degree of homology (99 to 100%) with granulocytic ehrlichiae isolated from humans. In contrast, groESL heat shock operon sequence analysis showed a strong divergence (approximately 5%) between the sequences in samples derived from rodents and those derived from samples from questing ticks or from other published ehrlichia sequences. Dual infections with granulocytic ehrlichia andBorrelia burgdorferi were found in ticks and small mammals.

SUMMARYT cells from BALB/c mice infested 9 days before with Ixodes ricinus nymphs had a suppressed response to in vitro concanavalin A (Con A) stimulation compared to cells from uninfested mice. When laminin (the main component of the extracellular matrix) was used as a coating agent, the Con A response of naive mice was characterized by a decrease in cell proliferation, whereas there was no significant effect on the mitogen response of cells from infested mice. In contrast, an increased response to lipopolysaccharide (LPS) was observed when assaying lymph node cells of infested mice, probably reflecting an increase in B-lymphocyte number or activity. LPS cell stimulation was not modified by laminin. Supernatants of lymph node cells, taken 9 days after the first infestation of mice, stimulated with Con A in vitro, contained interleukin-10 (IL-10) but no significant levels of IL-5 as tested by enzyme-linked immunosorbent assay. At this stage of the infestation all T cells reactive with tick antigens generated in lymph nodes that drain the tick fixation site, were CD4 cells, as determined by CD4 depletion. With cells taken 9 days after the third infestation an increase of IL-5 and IL-10 was observed. The IL-10 levels were higher than the IL-5. According to these observations, we conclude that the reduction of T-cell proliferation in response to Con A observed in lymph node cells from infested mice, may be due to the combined effect of laminin interaction with T lymphocytes during migration and IL-10 production by these lymphocytes.

BALB/c mice underwent 3 successive infestations with 15 Ixodes ricinus nymphs. No resistance was acquired as assessed by evaluating tick attachment, duration of blood meal, weights of engorged nymphs, and molting success. However, the hosts developed cutaneous immediate- and delayed-type hypersensitivity reactions when reinfested. Histological examination of tick attachment sites showed that inflammatory cells consisting of neutrophils, eosinophils, and mononuclear cells (lymphocytes and monocytes) infiltrated the skin more intensively during reinfestations. The number of intact mast cells did not vary between successive infestations, whereas the number of degranulated mast cells increased in the early stages of reinfestations. Basophils, which represent 12% of total infiltrating cells, were only observed and quantified in the skin of reinfested mice using transmission electron microscopy (TEM). Degranulating eosinophils were also observed by use of TEM.

SUMMARYBALB/c mice were infested with Ixodes ricinus larvae, nymphs or adults. Expression of IL-4 and IFN-g mRNA in axillary and brachial draining lymph node cells were measured by competitive quantitative reverse transcription-polymerase chain reaction 9 days after the beginning of primary-infestation. IL-4 mRNA was always higher than that of IFN-g mRNA for all tick instars. Moreover, IL-4 mRNA expression progressively increased during nymphal primary-infestation with a high burst of expression 7 days after the beginning of infestation. No evolution of IFN-g mRNA expression was detected. Draining lymph node cells of infested BALB/c produced higher level of IL-4 than IFN-g following in vitro restimulation with adult tick saliva, salivary gland extract (SGE) or with five selected different chromatographic fractions of SGE. Anti-tick IgG1 antibodies but no IgG2a were detected in BALB/c pluriinfested with IX ricinus nymphs, which confirmed the Th2 polarization of the immune response.

Saliva and salivary gland extract (SGE) of Ixodes ricinus ticks have suppressive effects on the innate immune response of BALB/c mice. Tick saliva prevents hemolysis of sheep red blood cells (SRBC) by the human alternative pathway of complement. The adaptive immune response is also modulated by tick antigens (saliva or SGE). When stimulated in vitro with increasing doses of tick antigens, the proliferation and IL-4 production of draining lymph node T cells of mice infested with nymphal ticks increase, peak and then decrease. These results indicate that immunostimulative and immunosuppressive molecules have competing effects in tick saliva or in SGE. I. ricinus saliva inhibits, in a dosedependent manner, splenic T cell proliferation in response to concanavalin A (ConA). Tick SGE or saliva injected intraperitoneally to BALB/c mice simultaneously with SRBC systemically immunosuppress the anti-SRBC response as shown in vitro by the reduced responsiveness of sensitized splenic T cells to restimulation with SRBC. In brief some components of SGE or tick saliva reduce the responsiveness of draining lymph node T cells and of sensitized splenic T cells in vitro. The responsiveness of naive splenic T cells to ConA stimulation in vitro is also decreased by tick saliva. Modulation of host responses by tick antigens may facilitate tick feeding, transmission and the propagation of pathogens.

Mice were tolerant to tick bites during three infestations with nymphs of Ixodes ricinus infected with Borrelia burgdorferi sensu stricto. To determine whether tick bites influence the immune response against B. burgdorferi, we examined the production of cytokines IL-4 and IFN-gamma by lymph node cells of BALB/c mice and IL-4 deficient BALB/c mice after tick inoculation versus syringe inoculation of B. burgdorferi. We also measured IgG2a anti-borrelial antibodies and total IgE in these mice. Results showed that BALB/c mice developed a Th2 immune response against B. burgdorferi after tick inoculation and a mixed Th1/Th2 response after syringe inoculation of B. burgdorferi. IL-4 deficient mice produced a Th1 immune response in both cases. IL-4 produced following tick bites greatly decreased the production of anti-borrelial IgG2a antibodies by comparison with the production of anti-borrelial IgG2a antibodies produced following syringe injection of B. burgdorferi.

The immune response of BALB/c (H-2d), DBA (H-2d), C57BL/6 (H-2b), C3H (H-2k), CBA (H-2k), SJL (H-2s), and FVB (H-2q) mice infested once with 15 nymphs of Ixodes ricinus is polarized toward Th2 as suggested by cytokines produced by lymph node cells stimulated with concanavalin A. The parasite load does not influence the polarization of the immune response as observed in BALB/c mice, which developed a Th2 response when infested with 5 or 45 nymphs. As assessed by attachment and weights of engorged nymphs, no resistance was acquired by BALB/c, C57BL/6, or C3H mice undergoing three successive infestations. However, these mice produced a gradual increase in IgE.

An immunoglobulin G immunoblot was developed with antigenic extracts of Borrelia burgdorferi sensu stricto,B. garinii, B. afzelii, and B. valaisiana genospecies and was reacted with sera from patients with neuroborreliosis, acrodermatitis, and Lyme arthritis. A detailed analysis of the reactivities of the protein bands was performed, and a two-step scoring procedure was selected to determine the preferential reactivity of sera to one particular genospecies. The discriminative potential of 5 proteins (12-kDa, 16-kDa, 18-kDa, OspA, and 66-kDa proteins) was used as a rapid first-step scoring method, followed by scoring of 14 additional protein bands if necessary. The advantage of this procedure is the low percentage of serum samples with inconclusive results for one of the four species (10% for patients with neuroborreliosis, 6% for patients with acrodermatitis chronica atrophicans, and 6% for patients with Lyme arthritis). Among 31 serum samples from patients with neuroborreliosis, 16 were more reactive toB. garinii, 7 were more reactive to B. afzelii, 3 were more reactive to B. valaisiana, and 2 were more reactive to B. burgdorferi sensu stricto. Of 31 serum samples from patients with acrodermatitis, 26 showed a higher level of reactivity to B. afzelii. Of 34 serum samples from patients with Lyme arthritis, 21 were more reactive to B. burgdorferi sensu stricto, 10 were more reactive to B. afzelii, and 1 was more reactive to B. valaisiana. Our results suggest an organotropism of Borrelia species and provide some evidence of a pathogenic potential of B. valaisiana in humans.