Th2 polarization of the immune response of BALB/c mice to Ixodes ricinus instars, importance of several antigens in activation of specific Th2 subpopulations.

BALB/c mice were infested with Ixodes ricinus larvae, nymphs or adults. Expression of IL-4 and IFN-gamma 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-gamma 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-gamma mRNA expression was detected. Draining lymph node cells of infested BALB/c produced higher level of IL-4 than IFN-gamma 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 pluri-infested with I. ricinus nymphs, which confirmed the Th2 polarization of the immune response.

Cytokines (IL-4 and IFN-gamma) and antibodies (IgE and IgG2a) produced in mice infected with Borrelia burgdorferi sensu stricto via nymphs of Ixodes ricinus ticks or syringe inoculations.

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.

PCR detection of granulocytic ehrlichiae in Ixodes ricinus ticks and wild small mammals in western Switzerland.

The presence of granulocytic ehrlichiae was demonstrated by PCR in Ixodes 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 ticks [corrected] 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 that A. 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 and Borrelia burgdorferi were found in ticks and small mammals.

Scored antibody reactivity determined by immunoblotting shows an association between clinical manifestations and presence of Borrelia burgdorferi sensu stricto, B. garinii, B. afzelii, and B. Valaisiana in humans.

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 to B. 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.

[Detection of Borrelia DNA in synovial fluid for diagnosis of Lyme arthritis]. / Nachweis von Borrelien-DNA in der Synovialflüssigkeit zur Diagnose der Lyme-Arthritis.

AIM: To test sensitivity and specificity of a polymerase chain reaction (PCR) targeting the Borrelia specific outer surface protein (Osp) A gene in synovial fluid for the diagnosis of Lyme arthritis, and thus permit an earlier start to treatment. PATIENTS AND METHODS: Prospectively we examined the synovial fluid of 37 patients with the clinical diagnosis of Lyme arthritis or with other arthropathies of known or unknown origin, searching for the presence of detectable borrelial DNA in both arms of the study. Retrospectively we examined the stored synovial fluid from 50 patients of the Department of Rheumatology of the University Hospital, Berne, with the clinical diagnosis of monarthritis or oligoarthritis of unknown etiology, juvenile chronic arthritis or rheumatoid arthritis. The laboratory biologist was unaware of the clinical diagnosis. RESULTS: In the prospective study no true false positive results were found: of the 28 patients without strong clinical suspicion of Lyme arthritis 27 were PCR negative. In one case with positive PCR for borrelial DNA the diagnosis could not be clarified, Lyme arthritis remaining a possibility. Therefore the specificity in the prospective study was at least 96%. Borrelial DNA in the synovial fluid was found in 5 out of 9 patients with strong clinical suspicion of Lyme arthritis. All 7 patients in this group were new, untreated cases. All the 5 PCR positive results belonged to this group, thus the "sensitivity" of the tested method was 71% in untreated cases of Lyme arthritis. In the retrospective study we found borrelial DNA in the synovial fluid of 2 patients. These 2 patients had gonarthritis of unknown origin. Retrospectively these 2 cases could be diagnosed as Lyme arthritis. CONCLUSION: In cases with clinical suspicion of Lyme arthritis the PCR method targeting a borrelial Osp A gene fragment common to all 3 European genospecies shows very good specificity and in untreated cases acceptable sensitivity. Introduction of the method studied into clinical practice is justified.

Influence of the genetic background and parasite load of mice on the immune response developed against nymphs of Ixodes ricinus.

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.

Susceptibility of BALB/c mice to nymphs and larvae of Ixodes ricinus after modulation of IgE production with anti-interleukin-4 or anti-interferon-gamma monoclonal antibodies.

BALB/c mice infested three times with nymphs or larvae of Ixodes ricinus ticks do not acquire resistance as assessed by evaluation of both tick attachment and the weight of engorged nymphs or larvae. Tick challenge causes a gradual increase in total IgE antibody production from the first to the third infestation. Anti-tick IgG antibodies are never detected. When the mice are treated with anti-interleukin-4 (anti-IL-4) or anti-interferon-gamma (anti-IFN-gamma) monoclonal antibodies (mAbs) 1 day before each infestation, they produce fewer or more IgE antibodies, respectively. No effect is observed on IgG antibodies. In IL-4-deficient mice, no IgE or IgG antibody is produced. However, these treatments and the use of IL-4-deficient mice have no negative effect on either tick attachment or the weight of engorged nymphs or larvae. Treatment with anti-IL-4 mAb and the use of IL-4-deficient mice inhibits and abolishes the switching of IgE, respectively, but these are apparently not sufficient to shift the response toward Th1 cells.

Identification of an Ixodes ricinus salivary gland fraction through its ability to stimulate CD4 T cells present in BALB/c mice lymph nodes draining the tick fixation site.

BALB/c mice infested with larvae or nymphs of Ixodes ricinus develop in their lymph nodes a T cell-specific immune response triggered by salivary gland soluble antigens (SGA). SGA are apparently conserved in the 3 biological stages of I. ricinus ticks and are species specific. SGA derived from partially fed females I. ricinus stimulate lymph node T cells from mice infested with I. ricinus larvae or nymphs. In contrast, lymph node cells from mice infested with Amblyomma hebraeum nymphs do not respond. A chromatographic fraction enriched with a 65 kDa protein (IrSG65) isolated from salivary glands of I. ricinus partially fed females induces in vitro a specific T cell proliferation of lymph node cells from mice infested with I. ricinus nymphs. The depletion of CD4+ T cells drastically reduces the ability of lymphocytes from infested mice to proliferate after IrSG65 stimulation.

Cytokine production by lymph node cells from mice infested with Ixodes ricinus ticks and the effect of tick salivary gland extracts on IL-2 production.

In BALB/c mice repeatedly infested with nymphal Ixodes ricinus ticks, lymphocytes from axillary and brachial lymph nodes which drain the tick attachment site produced significant levels of IL-2, TNF-alpha and GM-CSF when stimulated in vitro with Con A or anti-CD3 antibodies. Cytokine production by cells from lymph nodes of the opposite flank was equivalent to that of cells from uninfested mice. Nine days after the first infestation and IL-2, GM-CSF were produced primarily by the CD4+ T cells, while some other cell types contributed also to the TNF-alpha production. In mice repeatedly infested, a gradual increase of lymph node cell production of IL-2 was observed. The IL-2 levels regularly increased from the first to the third infestation compared to TNF-alpha levels which gradually decreased. The in vitro production of GM-CSF was not affected by successive infestations. Spleen lymphocytes from naive mice produced higher levels of IL-2 than lymphocytes from axillary and brachial lymph nodes. Both tick salivary gland extracts and D-mannose inhibited IL-2 production by these lymphocytes.

Immunosuppression and cytokine production in mice infested with Ixodes ricinus ticks: a possible role of laminin and interleukin-10 on the in vitro responsiveness of lymphocytes to mitogens.

T 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.

In vitro production of interleukin-4 and interferon-gamma by lymph node cells from BALB/c mice infested with nymphal Ixodes ricinus ticks.

In this study we compared the ability of lymphocytes taken from axillary and brachial lymph nodes of BALB/c mice that had been infested once three times with 15 nymphal Ixodes ricinus ticks, to produce interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) after in vitro stimulation with concanavalin A (Con A). They released high levels of IL-4 and low levels of IFN-gamma. An increase of IFN-gamma between the first and the third tick infestation was observed. Salivary gland extracts from female I. ricinus ticks induced specific in vitro proliferation of lymphocytes from infested mice. IL-4 production was correlated with the salivary gland extracts' ability to stimulate tick-specific lymphocyte proliferation. Its levels remained high from the first to the third infestation. IFN-gamma production was not necessarily associated with tick salivary gland antigen stimulation. In BALB/c mice, anti-tick immune response induction is regional and the contribution of other similar secondary lymphoid organs is negligible. Only cells from the lymph nodes which drained the tick-fixation site proliferated in vitro in the presence of tick antigens, and when stimulated with Con A produced IL-4 and IFN-gamma.

Infiltration of CD4+ CD8+ T cells, and expression of ICAM-1, Ia antigens, IL-1 alpha and TNF-alpha in the skin lesion of BALB/c mice undergoing repeated infestations with nymphal Ixodes ricinus ticks.

The skin cellular immune response of BALB/c mice was examined during three successive infestations with nymphal Ixodes ricinus ticks. An immunohistochemical analysis of skin cryostat sections 72 hr post-tick attachment revealed that CD4+ T cells outnumbered CD8+ T cells in all infestations. The CD4+:CD8+ T-cell ratio was 2.2:1 in the primary infestation, then increased to 3.2:1 and 4.7:1 in the secondary and tertiary infestations. No B lymphocytes (CD45R) were detected in the skin of control and infested mice. A positive staining of intercellular adhesion molecule-1 (ICAM-1) on vascular endothelial cells, dendritic cells and some other mononuclear cells was observed in the dermis. Also, a strong positive staining of Ia antigens on dendritic cells and infiltrated mononuclear cells was noted. The staining pattern was more intense and positive cells increased in number in the skin of re-infested mice compared to the primary infestation. In addition, cells such as epidermal keratinocytes, dermal dendritic cells and infiltrated mononuclear cells positive for the 'pro-inflammatory' cytokines interleukin-1 alpha (IL-1 alpha) and tumour necrosis factor-alpha (TNF-alpha) were localized in the skin of infested mice, as detected at the mRNA level by in situ hybridization and at protein level by immunostaining with antibodies. These results suggest that an antigen was presented to infiltrating T lymphocytes which then became activated. This event may explain the cutaneous delayed-type hypersensitivity previously described in tick-infected BALB/c mice. Importantly, this cutaneous reaction was not sufficient to protect the mouse against tick re-infestation. Furthermore, ICAM-1 could mediate, at least in part, the extravasation of inflammatory cells into the skin of infested mice.

IFN-gamma IL-2, and IL-4 mRNA expression in the skin and draining lymph nodes of BALB/c mice repeatedly infested with nymphal Ixodes ricinus ticks.

The skin and draining lymph nodes of BALB/c mice were examined for IFN-gamma, IL-2, and IL-4 mRNA expression by in situ hybridization in three successive infestations with nymphal Ixodes ricinus ticks. IFN-gamma and IL-2 mRNA positive cells were readily detected in lymph node sections during primary antigenic stimulation (72 hr post-tick attachment), whereas hybridization with IL-4 probe yielded no or only a faint positive signal. No changes in the cytokine pattern were observed in lymph node sections from reinfested mice, with IL-4 mRNA always being expressed to a lesser extent than IFN-gamma and IL-2 mRNA. Seventy-two hours post-tick attachment in primary infestation, some infiltrating cells in the skin were positive for IFN-gamma and IL-4 mRNA, but not for IL-2 mRNA. In skin sections of reinfested mice, mRNA coding for IFN-gamma, IL-2, and IL-4 were detected in infiltrating cells. Cells positive for IL-4 mRNA were lower in number than those positive for IFN-gamma and IL-1 mRNA. A significant decrease in the number of IL-4 mRNA positive cells in the tertiary infestation was noted. All together, these results indicate that I. ricinus nymphal ticks antigens are able to elicit expression of IFN-gamma, IL-2 mRNA and to a lesser extent IL-4 mRNA in both skin and draining lymph nodes. In addition, repeated infestations with ticks led to strong expression of IFN-gamma and IL-2 mRNAs in the skin that may be correlated with previous observations showing the occurrence of cutaneous delayed-type hypersensitivity in tick-infested mice. Notably, the cytokine pattern observed in the skin and draining lymph nodes is not associated with a protective immune response in mice against I. ricinus nymphal ticks infestations.

Absence of acquired resistance to nymphal Ixodes ricinus ticks in BALB/c mice developing cutaneous reactions.

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.

Vaccination of cattle against Rhipicephalus appendiculatus with detergent solubilized tick tissue proteins and purified 20 kDa protein.

Three groups of 4 cattle have been vaccinated with either detergent solubilized tick tissue proteins (SMP) of male and female Rhipicephalus appendiculatus, a 20 kDa soluble integumental antigen, a mixture of both SMP and 20 kDa. Two weeks after one booster injection all cattle were challenged by infestation with adult ticks. Treatment had no influence on tick attachment but on cattle vaccinated by the 20 kDa 32.5% fed ticks died (p < 0.001). Moreover, the mean weight of ticks fed on 7 out of 12 vaccinated cattle was significantly lower (p < 0.05 to p < 0.001). Individual differences could be seen where the mean weight reduction was up to 30%. Moreover, ticks fed on 1 (group SMP) or 2 cattle (group 20 kDa) had some difficulties in converting their blood meal into eggs (p < 0.05 to p < 0.001).

Induction of host resistance to Rhipicephalus appendiculatus in rabbits: effects of immunizing with detergent-solubilized tick tissue proteins.

Resistance to the hard tick, Rhipicephalus appendiculatus, was induced in rabbits by immunizing them with tick tissue proteins extracted with a detergent, Triton X-100. There was 25% mortality in female ticks fed on immunized rabbits as compared with those fed on controls. Similarly, there was a 40% and 60% reduction in the engorged weight and the weight of egg batches, respectively, of ticks fed on immunized rabbits. Western blot analysis of detergent-solubilized tick tissue proteins, carried out using immune sera, recognized a complex pattern of proteins. A strong reaction was observed with proteins with apparent molecular weights of 94,000 and 40,000 daltons.

Repetitive detection by immunoblotting of an integumental 25-kDa antigen in Ixodes ricinus and a corresponding 20-kDa antigen in Rhipicephalus appendiculatus with sera of pluriinfested mice and rabbits.

Mice were pluriinfested with nymphs and rabbits, with adult Ixodes ricinus. As determined by immunoblotting, greater than 50% of sera from these animals reacted against a tick antigen with a molecular weight of 25 kDa, which was detected in total extracts of partially fed I. ricinus females and in tick integumental extract. It was also found in engorged nymphs but was absent from larvae. Sera of I. ricinus-infested rabbits and mice or of rabbits infested with Rhipicephalus appendiculatus adults reacted with a 20-kDa antigen in total extracts of partially fed R. appendiculatus females and the integument of this species.