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Chemical modification studies on Ricinus communis (Castor Bean) agglutinin.

Ricinus communis agglutinin was subjected to various chemical treatments and the effect on its hemagglutinating and saccharide-binding properties was studied. Acetylation, succinylation and citraconylation led to a complete loss in the activity of the agglutinin, whereas reductive methylation had no effect on the activity, showing that charged amino groups were involved in the hemagglutinating and saccharide-binding activity of Ricinus agglutinin. Modification of tryptophyl, arginyl and carboxyl-group-containing residues did not lead to any loss in the activity of the agglutinin. Acetylation of tyrosyl groups with N-acetylimidazole strongly reduced the hemagglutinating and saccharide-binding property of Ricinus agglutinin. The loss in activity was restored on deacetylation of the tyrosyl groups. Modification of tyrosyl residues also led to a change in the immunological properties of the agglutinin. The initial rate of modification of tyrosyl and amino groups and the concomitant loss of activity was reduced in the presence of lactose.

2-Hydroxy-5-nitrobenzyl Bromide↗

Widespread distribution and high prevalence of an alpha-proteobacterial symbiont in the tick Ixodes ricinus.

The tick Ixodes ricinus is responsible for the transmission of a number of bacterial, protozoan and viral diseases to humans and animals in Europe and Northern Africa. Female I. ricinus from England, Switzerland and Italy have been found to harbour an intracellular alpha-proteobacterium, designated IricES1, within the cells of the ovary. IricES1 is the only prokaryote known to exist within the mitochondria of any animal or multicellular organism. To further examine the distribution, prevalence and mode of transmission of IricES1, we performed polymerase chain reaction screening of I. ricinus adults from 12 countries across its geographic distribution, including tick colonies that have been maintained in the laboratory for varying periods of time. IricES1 was detected in 100% of field-collected female ticks from all countries examined (n = 128), while 44% of males were found to be infected (n = 108). Those males that are infected appear to harbour fewer bacteria than females. Sequencing of fragments of the 16S rRNA and gyrB genes revealed very low nucleotide diversity among various populations of IricES1. Transmission of IricES1 from engorged adult females to eggs was found to be 100% (n = 31). In tick colonies that had been maintained in the laboratory for several years, a relatively low prevalence was found in females (32%; n = 25). To our knowledge, IricES1 is the most widespread and highly prevalent of any tick-associated symbiont.

Alphaproteobacteria↗

A molecular marker for the identification of the zoonotic reservoirs of Lyme borreliosis by analysis of the blood meal in its European vector Ixodes ricinus.

The efficacy of the mitochondrially encoded cytochrome b gene as a molecular marker for the discrimination of the reservoir host species of the Lyme borreliosis spirochete, Borrelia burgdorferi sensu lato (s.l.), in its European vector Ixodes ricinus (Acari: Ixodidae) was determined. Degenerate PCR primers were designed which amplified orthologous regions of the cytochrome b gene in several animal species which act as B. burgdorferi s.l. reservoirs and hosts for I. ricinus. PCR products were amplified and characterized by hybridization and restriction fragment length polymorphism analysis. Restriction fragment length polymorphism analysis of a 638-bp PCR product with HaeIII and DdeI revealed unique restriction fragment profiles, which allowed the taxonomic identification of animals to the genus level. A system was devised for the detection of the larval host blood meal from the remnants in unfed nymphal I. ricinus ticks by nested PCR amplification. An inverse correlation was demonstrated between amplicon size and successful PCR amplification of host DNA from the nymphal stage of the tick. The stability of the cytochrome b product as a marker for the identification of the larval host species in the nymphal instar was demonstrated up to 200 days after larval ingestion (approximately 165 days after molting) by reverse line blotting with a host-specific probe. This assay has the potential for the determination of the reservoir hosts of B. burgdorferi s.l. by using extracts from the same individual ticks for both the identification of the host species and the detection of the Lyme borreliosis spirochete.

Animals↗

Rickettsia monacensis sp. nov., a spotted fever group Rickettsia, from ticks (Ixodes ricinus) collected in a European city park.

We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/Munich(T)) from an Ixodes ricinus tick collected in a city park, the English Garden in Munich, Germany. Rickettsiae were propagated in vitro with Ixodes scapularis cell line ISE6. BLAST analysis of the 16S rRNA, the citrate synthase, and the partial 190-kDa rickettsial outer membrane protein A (rOmpA) gene sequences demonstrated that the isolate was a spotted fever group (SFG) rickettsia closely related to several yet-to-be-cultivated rickettsiae associated with I. ricinus. Phylogenetic analysis of partial rompA sequences demonstrated that the isolate was genotypically different from other validated species of SFG rickettsiae. R. monacensis also replicated in cell lines derived from the ticks I. ricinus (IRE11) and Dermacentor andersoni (DAE100) and in the mammalian cell lines L-929 and Vero, causing cell lysis. Transmission electron microscopy of infected ISE6 and Vero cells showed rickettsiae within the cytoplasm, pseudopodia, nuclei, and vacuoles. Hamsters inoculated with R. monacensis had immunoglobulin G antibody titers as high as 1:16,384, as determined by indirect immunofluorescence assay. Western blot analyses demonstrated that the hamster sera cross-reacted with peptides from other phylogenetically distinct rickettsiae, including rOmpA. R. monacensis induced actin tails in both tick and mammalian cells similar to those reported for R. rickettsii. R. monacensis joins a growing list of SFG rickettsiae that colonize ticks but whose infectivity and pathogenicity for vertebrates are unknown.

Actins↗

Prevalence of Borrelia burgdorferi and granulocytic and monocytic ehrlichiae in Ixodes ricinus ticks from southern Germany.

A total of 287 adult Ixodes ricinus ticks, collected in two regions of southern Germany (Frankonia and Baden-Württemberg) where Borrelia burgdorferi infections are known to be endemic, were examined for the presence of 16S ribosomal DNA specific for the Ehrlichia phagocytophila genogroup, E. chaffeensis, E. canis, and B. burgdorferi by nested PCR. Totals of 2.2% (6 of 275) and 21.8% (65 of 275) of the ticks were positive for the E. phagocytophila genogroup and B. burgdorferi, respectively. Two ticks (0.7%) were coinfected with both bacteria. Of 12 engorged I. ricinus ticks collected from two deer, 8 (67%) were positive for the E. phagocytophila genogroup and one (8%) was positive for B. burgdorferi. There was no evidence of infection with E. canis or E. chaffeensis in the investigated tick population. The nucleotide sequences of the 546-bp Ehrlichia PCR products differed at one or two positions from the original sequence of the human granulocytic ehrlichiosis (HGE) agent (S.-M. Chen, J. S. Dumler, J. S. Bakken, and D. H. Walker, J. Clin. Microbiol. 32:589-595, 1994). Three groups of sequence variants were detected; two of these were known to occur in other areas in Europe or the United States, whereas one has not been reported before. Thus, in the German I. ricinus tick population closely related granulocytic ehrlichiae are prevalent, which might represent variants of E. phagocytophila or the HGE agent.

Animals↗

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.

Animals↗

Transmission of Borrelia garinii OspA serotype 4 to BALB/c mice by Ixodes ricinus ticks collected in the field.

In Europe, Borrelia garinii OspA serotype 4 has been isolated from the cerebrospinal fluid of patients but, up to now, has never been identified among culture isolates from Ixodes ricinus ticks. This information raises the question of whether OspA serotype 4 is transmitted by I. ricinus in nature. In the present study, I. ricinus nymphs collected in an area of endemicity in southern Germany were allowed to feed on mice. Cultivation of ear biopsy specimens showed that six of seven B. garinii-infected mice were infected by OspA serotype 4. In contrast, very few B. garinii OspA serotype 4 organisms were isolated directly from the ticks which infected the mice; most isolates were B. afzelii. The infected mice transmitted mainly OspA serotype 4 to xenodiagnostic ticks, preferentially in combination with B. afzelii.

Animals↗

Determination of novel Borrelia genospecies in Swedish Ixodes ricinus ticks.

A total of 301 adult questing Ixodes ricinus ticks were collected at 15 different locations along the south and east coasts of Sweden to determine the Borrelia genospecies diversity. Thirty-two ticks (11%) were found to be positive by nested PCR with Borrelia burgdorferi sensu lato-specific primers. Species determination was based on partial sequencing of the 16S rRNA gene and the flagellin gene. Five different Borrelia species were found. The nucleotide sequence of the Borrelia DNA found in two ticks differed extensively from the nucleotide sequences of the Borrelia DNA found in the other ticks, and analysis revealed that they were closely related to the relapsing fever borrelia species Borrelia miyamotoi. This is the first report of a B. miyamotoi-like borrelia in I. ricinus and in Europe. Moreover, the Borrelia DNA of two ticks (6%) clustered within the B. valaisiana complex. B. valaisiana has not previously been reported in Sweden. B. afzelii DNA was found in 14 ticks (44%), and B. garinii DNA was found in 10 ticks (31%). B. burgdorferi sensu stricto DNA was found in four ticks (13%). We conclude that all of the known human-pathogenic species (B. garinii, B. afzelii, and B. burgdorferi sensu stricto) and B. valaisiana found elsewhere in Europe are also present in the Swedish host-seeking tick population and that a B. miyamotoi-like Borrelia species seems to be present in I. ricinus ticks in Europe.

Animals↗

High diversity of ankA sequences of Anaplasma phagocytophilum among Ixodes ricinus ticks in Germany.

In Germany humans with acute granulocytic ehrlichiosis have not yet been described. Here, we characterized three different genes of Anaplasma phagocytophilum strains infecting German Ixodes ricinus ticks in order to test whether they differ from strains in other European countries and the United States. A total of 1,022 I. ricinus ticks were investigated for infection with A. phagocytophilum by nested PCR and sequence analysis. Forty-two (4.1%) ticks were infected. For all positive ticks, parts of the 16S rRNA and groESL genes were sequenced. The complete coding sequence of the ankA gene could be determined in 24 samples. The 16S rRNA and groESL gene sequences were as much as 100% identical to known sequences. Fifteen ankA sequences were >/=99.37% identical to sequences derived from humans with granulocytic ehrlichiosis in Europe and from a horse with granulocytic ehrlichiosis in Germany. Thus, German I. ricinus ticks most likely harbor A. phagocytophilum strains that can cause disease in humans. Nine additional sequences were clearly different from known ankA sequences. Because these newly described sequences have never been obtained from diseased humans or animals, their biological significance is currently unknown. Based on this unexpected sequence heterogeneity, we propose to use the ankA gene for further phylogenetic analyses of A. phagocytophilum and to investigate the biology and pathogenicity of strains that differ in the ankA gene.

Anaplasma phagocytophilum↗

Rickettsia spp. in Ixodes ricinus ticks in Bavaria, Germany.

This study aims to provide information on the occurrence of spotted fever rickettsiae in Ixodes ricinus ticks in southern Germany. A total of 2,141 I. ricinus ticks was collected in Bavaria. Pools of 5-10 ticks were studied by a PCR targeting the rickettsial citrate synthase gene gltA. The average prevalence rate was 12% (257 of 2,141). Sequencing data exclusively identified Rickettsia helvetica DNA. Results and other data demonstrate the possible role of R. helvetica in I. ricinus as a source of human infections in southern Germany.

Animals↗

Distribution, abundance, and habitat preferences of Ixodes ricinus (Acari: Ixodidae) in northern Spain.

Ixodes ricinus (L.) was collected by standard dragging in 2,082 different sites in 18 broad vegetation categories in northern Spain to explore the influence of vegetation on its abundance. Of these, 785 sites were surveyed in 1995, 636 in 1996, and 661 in 1997. The impact of habitat features on differences in tick numbers is addressed. The tick was present in low numbers in areas of old, heterogeneous coniferous forests. Ticks appeared to prefer sites that had substantial secondary plant growth such as river canopies, heterogeneous Pinus uncinata forests, mixed forests, and deciduous heterogeneous woods. Highest tick abundance was recorded for sites that contained Quercus spp., as well as for mixed old forests that had many ecotones. I. ricinus was absent in open habitats, homogeneous young coniferous forests, and open hillsides. These differences were attributed to greater shrub cover and litter depth, which created more favorable microclimatic conditions for tick survival. The abundance of I. ricinus nymphs was not homogeneous in sites within the same habitat category and vegetation physiognomy at these sites did not appear to cause differences in tick abundance. Analysis of variance (ANOVA) indicated that variation in tick abundance could be explained by the exposure of the sampled site, at least for some zones within deciduous forest categories. However, this factor did not explain the variation observed in other habitats. Temperature and vegetation (normalized derived vegetation index) features were recorded daily by remotely sensed imagery throughout the study period and the data were used to obtain long-term mean and maximum values of the physical parameters considered. Multiple regression analysis performed between these long-term abiotic factors and nymphal abundance in positive sites showed high relationship (R2 coefficients) for every habitat category and explained >50% of the variation in tick abundance.

Animals↗

Distribution of Borrelia burgdorferi sensu lato in Ixodes ricinus (Acari: Ixodidae) ticks from the Basque Country, Spain.

Borrelia burgdorferi was found widespread in ixodid ticks from the Basque Country (Spain) during a two-step study. In the first part, a total of 7,835 ixodids of eight different species was collected from vegetation, classified, and processed using polymerase chain reaction (PCR) for detection of B. burgdorferi ospA DNA. B. burgdorferi DNA wasdetectedin < or = 12.5% of adults and > or = 0.6% of Ixodes ricinus (L., 1758) nymphs (mean 1.5 and 0.05%, respectively), and in < or = 14.3% of adult Hemaphysalis punctata (Canestrini & Fanzago, 1877) analyzed (mean 1.2%). The second part of the study was undertaken 2 yr later to characterize B. burgdorferi distribution by focusing on the areas where L. ricinus was the predominant species. Ten areas were selected from which 1,535 nymphs and adults of I. ricinus were collected and processed by PCR and culture techniques. Infected ticks were found in all zones. B. burgdorferi DNA was detected in a mean of 9.3 and 1.5% of adults and nymphs, respectively. Nine isolates of B. burgdorferi were obtained, belonging to four different genospecies (B. burgdorferi sensu stricto, B. garinii, B. valaisiana, and B. lusitaniae). The results indicate that some areas of Spain have a potential risk for Lyme disease agent exposure and that B. borgdorferi appears to have an increasing occurrence in ticks in the Basque Country.

Animals↗

Distribution of the common tick, Ixodes ricinus (Acari: Ixodidae), in different vegetation types in southern Sweden.

The aim of this study was to investigate whether differences in Ixodes ricinus (L.) nymphal relative density exist among different vegetation types in southern Sweden. Nymphal I. ricinus were sampled in southeastern Scania in southern Sweden during June-August 1997. A total of 110-180 25-m2 samples were taken by blanket-dragging from each of nine different vegetation types. There was a highly significant difference in nymphal abundance between the open areas as one group and the forested areas as another group (P < 0.0001). Vegetation types that differed significantly in median nymphal abundance from all other vegetation types were pine forest (16 nymphs/100 m2) and beech forest (40 nymphs/100 m2). No significant differences in median nymphal density were revealed among mixed deciduous forest, alder forest, oak forest, and hazel forest (28-32 nymphs/100 m2), or among dry meadow, meadow, and heath (0 nymphs/100 m2). Forestation of open areas is likely to lead to increased I. ricinus abundance and disease risk.

Animals↗

Borrelia burgdorferi s.l. in Ixodes ricinus (Acari: Ixodidae) ticks collected from vegetation and small rodents in recreational areas of the city of Poznań.

During 1998-1999, Ixodes ricinus (L.) populations were investigated in three different biotopes (deciduous, mixed, coniferous forest) situated in popular recreational areas in Poznań, Poland. In total, 1,123 questing ticks (1,002 nymphs, 69 males, 52 females) were collected by flagging vegetation. Additionally, in 1998 between May and September small rodents were trapped and inspected for feeding ticks. Altogether, 213 rodents of three species: Apodemus agrarius Pall., A. flavicollis Melchior, Clethrionomys glareolus Schreber were captured. Of 323 engorged ticks, 304 were larvae and 19 nymphs. All ticks collected from vegetation, as well as from rodents were examined for the presence of Borrelia burgdorferi Johnson, Schmid, Hyde, Steigenwalt & Brenner s.l. spirochetes by indirect immunofluorescence assay (IFA) using PAB 1B29. The seasonal pattern of activity of questing I. ricinus was always bimodal (May/June and August/September). The most abundant tick population occurred in the deciduous forest. The total infection rate in questing ticks was 16.2%. Differences in mean infection prevalence of host-seeking ticks between three biotopes each year were not significant. On average more larvae parasitized on the genus of Apodemus than on C. glareolus. 17.8% of larvae and 31.6% of nymphs fed on rodents harbored spirochetes. The three rodent species contributed to a different degree in to transmission of the pathogen to subadult stages. Approximately 27% of larvae infested on A. agrarius, 22% on C. glareolus, and only 4.2% on A. flavicollis contained spirochetes. The results suggest that the prevalence of A. agrarius and C. glareolus in disturbed urban forests used for leisure activities seems to be crucial for the maintenance of B. burgdorferi s.l. in I. ricinus populations.

Animals↗

Ixodes ricinus density, and distribution and prevalence of Borrelia burgdorferi sensu lato infection along an altitudinal gradient.

In this study, we measured the phenology of Ixodes ricinus ticks and their infection with Borrelia burgdorferi sensu lato (sl) simultaneously along an altitudinal gradient to assess the impact of climate on the phenology of ticks and on their infection with B. burgdorferi sl. From 1999 to 2001, free-living I. ricinus ticks were collected monthly by flagging vegetation at three different altitudes (620, 740, and 900 m above sea level) on the slope of a mountain in Chaumont (Neuchâtel, Switzerland). I. ricinus ticks were examined for the presence of B. burgdorferi sl by using direct fluorescent antibody assay and isolation of spirochetes. Borrelia species were characterized by polymerase chain reaction followed by restriction fragment-length polymorphism. Tick density and tick phenology varied with altitude. Although the peak tick density decreased and the onset of ticks was delayed with altitude, the phenology was much more stable among years at the highest altitudes than at the lowest. The prevalence of B. burgdorferi infection in nymphs and adults decreased with altitude. The prevalence of infection differed significantly among years, and it was significantly higher in adults (30%) than in nymphs (21%). B. burgdorferi infection in adults was positively related with adult density, but this was not observed for nymphs. Five B. burgdorferi sl genospecies were successfully isolated: B. garinii, B. burgdorferi sensu stricto, B. afzelii, B. valaisiana, and B. lusitaniae. Mixed infections were obtained from five of 140 infected ticks. The greatest diversity in Borrelia species was observed at the lowest altitude where all five Borrelia species were present, whereas at the two highest altitudes, B. lusitaniae was not observed.

Altitude↗

Molecular phylogenetic analyses indicate that the Ixodes ricinus complex is a paraphyletic group.

The Ixodes ricinus species complex is a group of ticks distributed in almost all geographic regions of the world. Lyme borreliosis spirochetes are primarily transmitted by tick species within this complex. It has been hypothesized that the Lyme vector ticks around the world are closely related and represent a monophyletic group. This implies that vector competence in ixodid ticks for Lyme agents might have evolved only once. To test this hypothesis, we used a molecular phylogenetic approach. Two fragments of mitochondrial 16S ribosomal deoxyribonucleic acid were sequenced from 11 species in the I. ricinus complex and from 16 other species of Ixodes. Phylogenetic analysis using Bayesian methodology indicated that the I. ricinus complex is not a monophyletic group unless 3 additional Ixodes species are included in it. The known major vectors of Lyme disease agents in different areas of the world are not sister taxa. This suggests that acquisition of the ability to transmit borreliosis agents in species of Ixodes may have multiple origins.

Animals↗

Molecular identification of Babesia parasites isolated from Ixodes ricinus ticks collected in northwestern Poland.

In the present study, PCR has been applied to detect and analyze DNA of Babesia spp. extracted from Ixodes ricinus ticks. Collection of I. ricinus was made in 6 forested areas of Zachodniopomorskie Voivodship, Poland, during 2 seasonal peaks of tick activity, i.e., spring and autumn, 2001. In total, 1,328 I. ricinus were collected and processed for PCR with F34 and R323 primers. Babesia spp. was detected in 28 (2% of 1,328 tested) ticks; 26 were identified as B. divergens. The other 2 were identified as B. microti. PCR was conducted with 18S rRNA specific primers and sequencing was processed to precisely identify and compare these isolates with B. microti and B. divergens sequences from Europe, North America, and Asia obtained from the GenBank. Analysis revealed that sequences of B. microti from northwestern Poland are almost identical (99.94%) with those referred to as "Munich strain"; both form a clade different from other European strains, as well as those from Asia and North America (called B. microti, sensu stricto). An investigation performed with B. divergens sequences showed that the sequence from northwestern Poland is 99.94% homologous to an isolate from Ireland ("Purnel"), and differs in just a few nucleotides from other European sequences. Phylogenetic analysis revealed that the sequence of B divergens isolated from Polish ticks form a group that comprise 4 European sequences from Great Britain and Ireland and is, therefore, closely related to other European and North American B. divergnens sequences.

Animals↗

[Influence of temperature and humidity on the reproductive efficiency of Ixodes ricinus (Linnaeus, 1758) and Hyalomma marginatum Koch, 1844 (Ixodoidea, Ixodidae)].

Laboratory studies have been carried out on two species of ticks, I. ricinus and H. marginatum, showing different seasonal activity and different geographical distribution in nature. The purpose of the present study has been to investigate the effect of different combinations of temperature and humidity on the oviposition of I. ricinus and H. marginatum. An index of reproduction efficiency (no. eggs/wt female), established to relate the capacity of the females to produce eggs to their body weight, has been calculated; for I. ricinus 5 temperatures (18-20-25-27 and 30 degrees C) have been studied (Fig. 1), for H. marginatum 3 (25-30 and 32 degrees C) (Fig. 2) and for both species 2 R.H. (75-95%). An optimal range of temperature, within which the value of such an index doesn't change, has been demonstrated for both species by statistical testing (Table 1). A critical weight value as the minimum engorged female weight required for egg deposition has been identified with the weight of first female which has laid eggs. This weight, different in each species, is influenced by temperature and humidity.

Animals↗