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PubMedDate not supplied

Tick factors and in vitro cultivation influence the protein profile, antigenicity and pathogenicity of a cloned Borrelia garinii isolate from Ixodes ricinus hemolymph.

A Borrelia garinii isolate (NE11H) was obtained from the hemolymph of infed Ixodes ricinus. NE11H expressed four major proteins of 33 kDa, 32 kDa, 23 kDa and 22 kDa. During in vitro culture, NE11H successively lost the expression of the 22 kDa and 23 kDa proteins and the NE11H variant (NE11Hp15) was not recognized by an immune serum specific for the OspC protein (anti-OspC IS). However, when reintroduced into tick midguts, NE11Hp15 spirochetes present in the midgut again reacted with anti-OspC IS. A clone derived from the wild type line, cNE11H, lacked the 22 kDa but not the 23 kDa protein. The 23 kDa protein of cNE11H was recognized by anti-OspC IS. In addition, the two descendant lines (NE11Hp15 and cNE11H) lost their capacity to induce clinical arthritis in SCID mice. When cNE11H was reintroduced into ticks and reisolated from various tick organs, most reisolates presented the same reaction with anti-OspC IS as cNE11H. Interestingly, two reisolates obtained from the tick midgut reexpressed large amounts of the 22 kDa protein which was recognized by anti-OspC IS and these two reisolates induced clinical arthritis in SCID mice. The results confirm that proteins of 22/23 kDa are differentially expressed during in vitro subcultures and in ticks, and show that proteins which are not detectable after in vitro culture may be reexpressed after reexposure of B. burgdorferi to its former environment in the tick. The data suggest that the pathogenicity of B. burgdorferi for mice might be influenced by environmental factors via differential expression of 22/23 kDa proteins.

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PubMed1996

Spirochetal non-Borrelia-microorganism isolated from Ixodes ricinus.

Spirochetal microorganisms were isolated from female Ixodes ricinus in Slovakia. Morphological, immunochemical and molecular biological analysis showed that the microorganism shared several common antigens with Borrelia species while other genetic traits were distinct and not related to Borrelia burgdorferi sensu lato. Lyme disease patient's serum contained antibodies reacting with antigens of this microorganism. On the one hand the cross-reacting antigens represent a risk of false positive results in laboratory diagnostics, while on the other hand they have a certain potential for vaccine development against Lyme disease.

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PubMed2004

Combined infection of Ixodes ricinus with three Borrelia burgdorferi sensu lato genotypes.

Ixodes ricinus ticks were collected by random collections from western and central Slovakia during the years 1996-98. The midgut content of 240 ticks was examined by dark-field microscopy and submitted for cultivation for the presence of borrelias. Spirochetes were found in 21 unfed and host-seeking adults and nymphs (8.8%). By the analysis of restriction fragment length polymorphism (RFLP) one sample from unfed I. ricinus male from western Slovakia was identified as a triple infection of Borrelia burgdorferi sensu stricto, B. garinii and B. afzelii. The simultaneous presence of different B. burgdorferi genospecies in one midgut sample (triple infection in the tick) could be observed only after the multipart amplification of denaturated DNA and subsequent pooling of the products for further analysis.

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PubMed2006

In vitro cultivation and antibiotic susceptibility of a Cytophaga-like intracellular symbiote isolated from the tick Ixodes scapularis.

A Cytophaga-like organism (CLO), isolated from the tick Ixodes scapularis (IsCLO), was adapted to growth in insect cell lines and its antibiotic sensitivity was tested. IsCLO were introduced to four insect cell lines, and their growth was measured by quantitative polymerase chain reaction. IsCLO propagated well in a mosquito cell line, AeAl-2, and caused cytopathic effects in host cells. A lepidopteran cell line, HZ-AM1, was also suitable for propagation of IsCLO and kept a steady state with bacterial growth. Using IsCLO-infected AeAl-2, antibiotics effective against the bacteria included ampicillin, chloramphenicol, penicillin-G, rifampicin, and tetracycline. These antibiotics will be useful for eliminating CLO from host arthropods, which is necessary for in vivo studies of the intracellular facultative symbiotes.

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PubMed2006

Habitat requirements of the seabird tick, Ixodes uriae (Acari: Ixodidae), from the Antarctic Peninsula in relation to water balance characteristics of eggs, nonfed and engorged stages.

The seabird tick Ixodes uriae is exposed to extreme environmental conditions during the off-host phase of its life cycle on the Antarctic Peninsula. To investigate how this tick resists desiccation, water requirements of each developmental stage were determined. Features of I. uriae water balance include a high percentage body water content, low dehydration tolerance limit, and a high water loss rate, which are characteristics that classify this tick as hydrophilic. Like other ticks, I. uriae relies on water vapor uptake as an unfed larva and enhanced water retention in the adult, while nymphs are intermediate and exploit both strategies. Stages that do not absorb water vapor, eggs, fed larvae and fed nymphs, rely on water conservation. Other noteworthy features include heat sensitivity that promotes water loss in eggs and unfed larvae, an inability to drink free water from droplets, and behavioral regulation of water loss by formation of clusters. We conclude that I. uriae is adapted for life in a moisture-rich environment, and this requirement is met by clustering in moist, hydrating, microhabitats under rocks and debris that contain moisture levels that are higher than the tick's critical equilibrium activity.

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PubMed2003

Experimental infection of dogs with Borrelia burgdorferi sensu stricto using Ixodes scapularis ticks artificially infected by capillary feeding.

Specific pathogen-free dogs were experimentally infected with Borrelia burgdorferi sensu stricto using nymphal or adult female Ixodes scapularis ticks artificially infected with spirochetes by capillary feeding. The ticks were capillary fed B. burgdorferi isolate 610, previously isolated from a dog with Lyme disease and grown in BSK medium. This isolate induced clinical signs in the dogs similar to those for dogs infested with ticks naturally infected with B. burgdorferi. Adult ticks were more efficient than nymphs in transmitting spirochetes to the dogs. One of five dogs infested with nymphal ticks capillary fed B. burgdorferi was skin biopsy culture and serologically positive, and demonstrated lameness. In contrast, all five dogs infested with adult female ticks that had been capillary fed with B. burgdorferi were culture and serologically positive, with one dog developing lameness. The immunoblot profiles of dogs challenged with female ticks infected by capillary feeding (8 weeks post challenge) were similar to immunoblots (4 weeks post challenge) from dogs challenged with naturally infected females collected in the field. These studies demonstrated that B. burgdorferi cultured in BSK medium can be capillary fed to either nymphal or adult female ticks under laboratory controlled conditions for the purpose of transmitting the spirochete to dogs during the tick's blood meal. This tick infection system would be useful for a controlled and defined challenge of vaccinated and non-vaccinated dogs for proper evaluation of vaccine efficacy, which is difficult to achieve using field-collected ticks. Furthermore, this system may also be useful for investigation of the pathogenesis of Lyme disease, evaluation of the pathogenicity of new isolates of B. burgdorferi, or evaluation of antibiotic therapy.

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PubMed1998

Expression of outer surface proteins A and C of Borrelia burgdorferi in Ixodes ricinus ticks removed from humans.

A total of 131 Ixodes ricinus (51 females, 1 male and 79 nymphs) removed from persons living in Southern Germany were investigated by immunofluorescence assay for the presence of Borrelia burgdorferi with a polyvalent rabbit immune serum and monoclonal antibodies specific for outer surface proteins (Osp) A or C. Borreliae were detectable in 48 (36.6%) of the ticks. Infection rates of these adults and nymphs were significantly higher than infection rates of unfed ticks from Southern Germany. Borreliae in 31.3% (n=15) of the infected ticks expressed solely OspA, solely OspC in 12.5% (n=6), and both OspA and OspC in 39.6% (n=19) of ticks, while in 16.7% (n=8) of ticks neither were expressed. Presentation of OspC by B. burgdorferi in I. ricinus was correlated with tick weight: in females, OspC was detectable only in ticks with a minimum weight of about 3.5 mg, and in nymphs weighing at least 1 mg. These results indicate that in I. ricinus removed from humans OspC is up-regulated during the blood meal of the tick, but in most ticks OspA is still detectable and might even be present in the absence of OspC expression in the midgut and salivary glands of nearly fully engorged nymphal ticks. Furthermore, we found strong evidence that borreliae expressing solely OspA while in the salivary glands can cause Lyme borreliosis. Our findings indicate that during tick feeding, humans are exposed to borreliae that may express either OspA or OspC or both, or lack both OspA and C. These findings suggests that, at the minimum, both OspA and C should be considered as vaccine candidates for prophylaxis of Lyme borreliosis in Europe.

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PubMed1999

Coexistence of ehrlichiae of the phagocytophila group with Borrelia burgdorferi in Ixodes ricinus from Southern Germany.

Human granulocytic ehrlichiosis (HGE) is an emerging infectious disease recognized in the Western hemisphere. HGE is well known to occur in North America, but records from outside the United States are sparse. The great majority of data from Europe are restricted to seroprevalence studies and molecular biological detection of granulocytic ehrlichiae (GE) in ticks and mammals, but include defined cases from Slovenia. They argue for the existence of this disease in many parts of Europe. In the present study, 510 Ixodes ricinus ticks collected in five different regions of Southern Germany were investigated for the presence of GE and Borrelia burgdorferi sensu lato using polymerase chain reaction. In all, 8 (1.6%) of the 492 ticks that could be evaluated (193 females, 208 males, and 91 nymphs) contained GE and 178 (36.2%) B. burgdorferi s.l.. Four of these ticks were infected with both pathogens. Interestingly, all ehrlichia-infected ticks were adults and all were collected in the English Garden, a recreational park area located in the city of Munich. Sequencing of the 16S rDNA (bp 1 1101) of four of the GE showed 100% sequence identity to each other and greater than 99.9% identity with the published sequence of the HGE agent. The four GE differed in respect to other hitherto described GE by a nucleotide exchange at position 336. These results show that GE that are closely related to the HGE agent are present in Southern Germany, and that coinfection with B. burgdorferi is common in GE-infected ticks. However, in contrast to B. burgdorferi which is endemic everywhere in Southern Germany, the distribution of GE seems to be focal.

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PubMed2002

Immunosuppressive effects of ixodes ricinus tick saliva or salivary gland extracts on innate and acquired immune response of BALB/c mice.

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 dose-dependent 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.

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PubMed2002

Low intraspecific variation in the rRNA internal transcribed spacer 2 (ITS2) of the Australian paralysis tick, Ixodes holocyclus.

Ixodes holocyclus has a narrow, discontinuous distribution along the east coast of Australia. We studied ticks from 17 localities throughout the geographic range of this tick. The ITS2 of I. holocyclus is 793 bp long. We found nucleotide variation at eight of the 588 nucleotide positions (1.4%) that were compared for all ticks. There were eight different nucleotide sequences. Most sequences were not restricted to a particular geographic region. However, sequences F, G and H, which had an adenine at position 197, were found only in the far north of Queensland--all other ticks had a guanine at this position. The low level of intraspecific variation in this tick (0.7%) contrasts with the sequence divergence between I. holocyclus and its close relative, I. cornuatus (13.1%). These data indicate that I. holocyclus does not contain cryptic species despite possible geographic isolation of some populations. We conclude that variation in the ITS2 is likely to be informative about the phylogeny of the group.

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PubMed2002

Salivary gland extract from Ixodes ricinus tick modulates the host immune response towards the Th2 cytokine profile.

In our previous work, the salivary gland extract (SGE) from Ixodes ricinus ticks impaired T-lymphocyte proliferation and clearly modulated the immune response towards the Th2 pattern in human peripheral blood mononuclear cell culture. In the present work, the results obtained on mouse splenocytes are compared with those on human leukocytes. ELISA (protein level) and RNAse protection assay (mRNA level) showed that SGE enhanced interleukin (IL)-1alpha, IL-1beta, IL-1Ra, IL-6, and IL-12p40 cytokines, whereas production of IL-2, IL-5, IL-10, and IL-13 was decreased. The minute levels of IL-9, IL-15 and IL-12p70 were not changed after the addition of tick saliva. IL-4 was upregulated, whereas the production of gamma interferon and migratory inhibition factor was downregulated after the addition of SGE. Tick saliva decreased concanavalin A-stimulated spleen cell proliferation and the percentage of activated T-cells. We conclude that the Th2 polarization did not involve all of the cytokines tested. However, the Th2 subset-augmenting effect of tick saliva was confirmed.

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PubMed2004

Borrelia burgdorferi infection prevalences in questing Ixodes ricinus ticks (Acari: Ixodidae) in urban and suburban Bonn, western Germany.

From March to October 2003, a total of 2,518 host-seeking Ixodes ricinus ticks (1,944 nymphs, 264 females, 310 males) were collected by blanket dragging at 45 sites all over the city area of Bonn, western Germany, to be checked for Borrelia burgdorferi infection. The collection sites included 20 private gardens, nine public recreational parks, the boundaries of 14 sylvatic suburban areas and two footpaths between suburban farmed fields. Generally, numbers of specimens collected along sylvatic suburban areas and at urban sites with dense tree populations were significantly higher than at the other collection sites. Out of 1,394 specimens (865 nymphs, 241 females, 288 males) that were randomly chosen for Borrelia analysis by a simple PCR, 250 (17.9 %) were found to be infected with B. burgdorferi sensu lato. While the infection prevalences varied significantly between females (26.6%), males (12.5%) and nymphs (17.3%), there were no striking differences between sylvatic and unwooded sites. A total of 92.8% of the ticks Borrelia-positive by the simple PCR were also positive in a diagnostic nested PCR. Using genospecies-specific oligonucleotide probes, single Borrelia genospecies infections (91.4%) could be assigned to B. afzelii (39.5%), B. garinii (27.9%), B. burgdorferi sensu stricto (15.6%) and B. valaisiana (8.6%) by DNA hybridization. Various combinations of double infections were observed in 4.3% of the infected ticks. Another 4.3% of the Borrelia infections were untypeable. The B. burgdorferi genospecies distribution in the city area was shown to be variable from site to site and, even more, it was distinct from rural collection sites near Bonn. This is ascribed to a different spectrum of reservoir hosts. Taking into account the infection prevalences of host-seeking ticks in the forested surroundings of Bonn, our study demonstrates that the risk of acquiring Lyme disease after a tick bite in urban/suburban areas is comparably as high as in woodlands outside of the city.

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PubMed2007

Symbiotic bacteria in oocyte and ovarian cell mitochondria of the tick Ixodes ricinus: biology and phylogenetic position.

Under natural conditions, eukaryote cells may contain bacteria. Arthropods such as ticks, insects or mites are a group particularly favoured by the obligate intracellular bacteria. While arthropods are vectors for some of them, other bacteria inhabit invertebrate host cells having entered mutualistic interactions. Such endosymbionts dwell usually in the host cell vacuoles or cytoplasm but have been also reported from tick oocyte mitochondria. The microorganisms contribute to mitochondria degradation, but their colonies are not eliminated from the tick cells affected. So far, such bacteria have been detected in three research centres. The Italian centre has reported on results of microscope and molecular analyses, while the Polish centre published molecular data. The Danish centre registered a 16S rRNA gene fragment in GenBank. Independent comparisons of the 16S rRNA gene sequences, carried out in the Italian and Polish centres, confirmed that the nucleotide sequences of the Ixodes ricinus endosymbionts formed a single clade with certain non-identified tick bacterium species isolated from the tick Haemaphysalis wellingtoni. On the other hand, pathogenic species of the genera Anaplasma, Ehrlichia and Rickettsia detected in I. ricinus as well as symbionts of the genus Wolbachia present in Culex pipiens and Drosophila simulans have been placed at a different site on the phylogenetic tree.

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PubMed2000

Influence of saturation deficit and temperature on Ixodes ricinus tick questing activity in a Lyme borreliosis-endemic area (Switzerland).

Questing Ixodes ricinus ticks were sampled monthly in a periurban mixed forest at Neuchâtel, Switzerland, in 1996, 1997, and 1998. A total of 5,530 I. ricinus nymphs and 1,385 adults were collected. The emergence of questing tick populations in spring varied among years in relation to the air temperature. The monthly variation in questing nymph density was negatively correlated with the saturation deficit; there was a strong decrease in tick numbers in late spring and summer as soon as the saturation deficit increased. A variation in tick density in relation to the saturation deficit was also observed between years; the tick density was high when the saturation deficit was low during spring and summer (1997) and was low when the saturation deficit was high (1998). During the 3-year study period, marked climatic differences among years highlighted the influence of temperature and saturation deficit on the phenology of ticks.

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PubMed1996

Ultrastructural localization of a sialic acid-specific hemolymph lectin in the hemocytes and other tissues of the hard tick Ixodes ricinus (Acari; Chelicerata).

Lectins have been suggested to function as pattern-recognition molecules in invertebrate immune mechanisms. A lectin from the hemolymph of the tick Ixodes ricinus with main specificity for sialic acid was characterized and antibodies directed against this lectin were prepared. In this study, these antibodies were used to localize the lectin in the tissues of I. ricinus. Immunoreactivity with poly- and monoclonal antibodies was detected in the granules of both types of granular hemocytes, at the membrane of hemocytes, and at the basal laminae surrounding the hemocoel. Furthermore, cells attached to the midgut, invaginations of Géné's organ, and granular inclusions of nephrocytes were labeled. The immunoreactivity detected in hemocytes and the hemocoel lining supports the idea that the hemolymph lectin may function as a recognition molecule in the immune system of I. ricinus. Another function could be protection of eggs that are coated with secretions by Géné's organ. The lectin activity could also be involved in transmission of Borrelia burgdorferi, the causative agent of Lyme borreliosis, and the tick-borne encephalitis virus.

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PubMed1996

Mitotic activity of the hemocytes in the tick Ixodes ricinus (Acari; Ixodidae).

The blood cells, or hemocytes, of Ixodes ricinus have been shown to recognize, attack, and phagocytose microorganisms invading the body cavity, or hemocoel, of this tick. Regulated proliferation and differentiation of hemocytes, also referred to as immunocytes, is basic to an effective immune response to invading microorganisms. Therefore, this study dealt with hemopoiesis in I. ricinus, the vector tick of the Lyme disease spirochete Borrelia burgdorferi. Histological evidence for the presence of hemopoietic tissue, a preferential proliferation site of hemocytes, is presented. Mainly the mitotic activity of free-floating hemocytes was examined. By means of microscopical photometry and flow cytometry, all three types of hemocytes in engorging female I. ricinus were found in different stages of the cell cycle. In the engorging tick, up to 40% of the hemocytes counted were in the S phase or the G2/M phase. From this study we conclude that the differentiated hemocyte types do not differentiate from stem cells in the adult tick. Moreover, microorganisms entering the hemocoel of engorging ticks are confronted with high numbers of hemocytes and, therefore, with an effective cellular immune response.

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PubMed1998

Prevalence rates of Borrelia burgdorferi sensu lato in host-seeking Ixodes ricinus ticks in Europe.

Borrelia burgdorferi sensu lato spirochetes have been found in all examined Ixodes ricinus (L.) populations in Europe. The overall mean proportions of unfed I. ricinus infected with B. burgdorferi s.l. were 1.9% (range 0-11%), 10.8% (2-43%) and 17.4% (3-58%) for larvae (n = 5699), nymphs (n = 48,804) and adults (n = 41,666), respectively. However, the results varied according to the method used. Cultivation in BSK medium is the least sensitive technique (an average of 11% adult ticks found infected), whereas polymerase chain reaction detecting spirochetal DNA is probably the most sensitive method (29% adults found infected). Microscopic methods (dark field, phase contrast, direct or indirect fluorescence) are generally comparable to each other (17-20% adults found infected) and should be regarded as standard procedures because they also make possible a quantitative estimation of spirochetes in the vector. Some technical problems of these methods are discussed.

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PubMed1998

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.

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