A comparison of the occurrence of borreliae in nymphal and adult Ixodes ricinus ticks

The prevalence of borreliae in 209 nymphal and 251 adult Ixodes ricinus was investigated in two areas of southern Moravia, Czechoslovakia, using the dark-field and the Giemsa stained-smear techniques. The proportions of infected ticks were 3.8% in nymphs and 10.6% in adults of area A, while they were 29.1% in nymphs and 35.9% in adults of area B. The mean number of borreliae per tick was about 3 to 10 times greater in adult than in nymphal I. ricinus. The results indicate a significant role of nymphal I. ricinus in the ecology of Borrelia burgdorferi and in the epidemiology and epizootiology of Lyme borreliosis.     

Diversity of Babesia Infecting European sheep ticks (Ixodes ricinus)

Questing Ixodes ricinus (Acari: Ixodidae) adult and nymphal ticks collected in various parts of Slovenia were tested for the presence of babesial parasites with a PCR assay based on the nuclear small subunit rRNA gene (nss-ribosomal DNA [rDNA]). Thirteen of 135 ticks were found to contain babesial DNA. Sequence determination and analysis of amplified portions of nss-rDNA revealed their identity with Babesia microti and a high degree of homology with Babesia odocoilei and Babesia divergens. The results of this study represent the first genetic evidence of B. microti and B. divergens-like parasites in I. ricinus ticks in Europe.     

Prevalence of Borrelia burgdorferi in Ixodes ricinus ticks in urban recreational areas of Helsinki

Lyme borreliosis, an infection caused by the tick-borne spirochete Borrelia burgdorferi, is a major health problem for populations in areas of endemicity in the Northern Hemisphere. In the present study we assessed the density of ticks and the prevalence of B. burgdorferi sensu lato among ticks in popular urban recreational areas of Helsinki, Finland. Altogether 1,688 Ixodes ricinus ticks were collected from five areas located within 5 km of the downtown section of Helsinki, and 726 of them (303 nymphs, 189 females, and 234 males) were randomly chosen for laboratory analysis. The midguts of the ticks were divided into three pieces, one for dark-field microscopy, one for cultivation in BSK-II medium, and one for PCR analysis. Ticks were found in all the study areas; their densities varied from 1 to 36 per 100 m along which a cloth was dragged. The rate of tick infection with B. burgdorferi sensu lato varied from 19 to 55%, with the average being 32%. Borellia afzelii was the most predominant genospecies in all the areas, and no B. burgdorferi sensu stricto isolates were detected. Only two ticks were concurrently infected with both B. afzelii and Borrelia garinii. Dark-field microscopy gave more positive results for B. burgdorferi than did cultivation or PCR analysis. However, the agreement between all three methods was fairly good. We conclude that Lyme borreliosis can be contracted even in urban environments not populated with large mammals like deer or elk. The disease should be taken into account in the differential diagnosis of certain symptoms of patients from these areas, and the use of measures to improve the awareness of the general population and health care officials of the risk of contracting the disease is warranted.     

Diversity of Babesia in Ixodes ricinus ticks in Poland

PurposeThe aims of this study were: (1) to estimate Babesia prevalence in the most common species of tick in Poland, Ixodes ricinus, in two recreational areas (Urwita?t in the Mazury Lake District and Bielański Forest in Warsaw), and (2) to evaluate the molecular diversity of Babesia isolates in questing I. ricinus in Poland.Material and MethodsQuesting ticks were collected from vegetation in forest areas in Urwita?t near Miko?ajki and in Bielański Forest (Warsaw). Purified genomic DNA was used with specific primers to amplify a fragment of the Babesia spp. 18S rRNA gene.ResultsTick-drag indices for I. ricinus were high in both study areas, reaching somewhat higher values in Urwita?t than in Bielański Forest. The overall prevalence of Babesia spp. in examined ticks was 1.6%. In Urwita?t, two strains of B. microti were identified using rRNA sequences: the enzootic Munich strain and an isolate close to the zoonotic Jena strain. The proportion of infections due to these two strains in questing ticks reversed over a six-year period. During 3 years of study in Bielański Forest, all Babesia isolates obtained from I. ricinus were identical to Babesia sp. EU1 (B. venatorum), previously recognized as an agent of human babesiosis.ConclusionsThis study has confirmed the presence of enzoonotic and zoonotic Babesia species/strains in the abundant human-biting tick I. ricinus in recreational areas in Poland. It has also shown that the distribution of different genotypes has changed over time, however the reasons for these fluctuations still remain to be investigated.     

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.     

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.     

Transmission studies of Babesia microti in Ixodes ricinus ticks and gerbils

In order to investigate the possible role of Ixodes ricinus as a vector of zoonotic Babesia microti infection in Europe, a European rodent isolate (HK) and a zoonotic American isolate (GI) were studied in transmission experiments. PCR detected B. microti in the blood and spleens of infected gerbils (Meriones unguiculatus) and also in laboratory-induced infections of I. ricinus ticks. B. microti DNA was detected by PCR in all pooled samples of nymphs and the majority of adults that had fed as larvae and nymphs, respectively, on gerbils with acute infection of the European isolate, confirming that I. ricinus could serve as a vector in Europe. The American isolate, GI, proved to be equally infective for larval and nymphal I. ricinus as the HK strain, despite a very different appearance in gerbil erythrocytes. Nymphs infected with the HK and GI strains readily infected gerbils. In contrast to the finding in acute infections, ticks that fed on gerbils with chronic infections of HK and GI did not become infected. It was also found that the HK strain was not transmitted transovarially. The finding that a B. microti strain (GI) from a distant geographical region (United States) can infect and be transmitted by I. ricinus suggests that other European B. microti strains, in addition to the HK strain used here, are probably infective for I. ricinus, supporting the view that infection of humans with European B. microti may be a regular occurrence.     

Prevalence of tick-borne encephalitis virus in Ixodes ricinus ticks in Finland

Approximately 20 cases of tick‐borne encephalitis (TBE) occur annually in Finland. The known endemic areas are situated mainly in the archipelago and coastal regions of Finland, with highest incidence in Åland islands. Ixodes ricinus panels collected in 1996–1997 from two endemic areas were screened for the presence of RNA. Two distinct RT‐PCR methods were applied, and were shown to have an approximate detection limit of 10 focus forming doses (FFD)/100 μl. One out of 20 pools (a total of 139 ticks) from Helsinki Isosaari Island and one out of 48 pools (a total of 450 ticks) from Åland were positive with both methods, whereas the remaining pools were negative. The observed overall frequency (0.34%) in ticks in endemic areas of Finland, was similar to the low incidence found by virus isolation in mice in the 1960s (0.5%). Viral RNA was detectable in a diluted sample representing 0.005% of a positive pool of ten nymphs suggesting that the viral RNA load within an infected tick pool was approximately equivalent to 20,000–200,000 FFD. Sequence analysis did not show geographical clustering of the Finnish strains, suggesting an independent emergence of different TBE foci from the south. TBE virus RNA positive ticks were not found in I. ricinus panels consisting of 130 pools (726 ticks) from Helsinki city parks or 41 pools (197 ticks) from Võrmsi Island in Estonia. J. Med. Virol. 64:21–28, 2001. © 2001 Wiley‐Liss, Inc.     

Isolation of Tettnang virus from Ixodes ricinus ticks in Czechoslovakia.

Three virus strains closely related to Tettnang virus were isolated from nymphs and males of Ixodes ricinus tick in Moravia and Slovakia. The virus isolates were lethal for suckling mice on intracerebral (i.c.) inoculation but the virus titres in the brain were low. The incubation period of 5--12 days in the first mouse passage was shortened to 3 days in further passages. Infection of mice was accompanied by ultrastructural changes in the cytoplasm of neurons. Groups of spherical virus particles 75 to 80 nm in diameter occurred within simple vacuoles.     

Investigation of the mechanisms of anti-complement activity in Ixodes ricinus ticks

The feeding success of a tick upon a host depends on its ability to suppress host anti-tick responses which include activation of the complement system. We investigated the mechanism of inhibition of the alternative pathway of complement by salivary gland extract (SGE) of the ixodid tick species, Ixodes ricinus. SGE treatment strongly inhibited C3a generation and factor B cleavage in serum when rabbit erythrocytes were used as complement activator, but not when cobra venom factor (CVF) was used as an activator. SGE treatment strongly inhibited C3b deposition on rabbit erythrocytes, and the turnover of C3 (to C3b/iC3b) in serum. However, there was no significant effect upon the formation, stability or activity of C3 convertase (C3bBb) when formed from purified C3b, factor B and factor D. SGE treatment of isolated C3 resulted in a shift in mobility of the alpha-chain (by about 5 kDa). N-terminal sequencing of this species suggests that cleavage occurs at the C-terminus of the alpha-chain of C3. Consistent with this hypothesis, the modified alpha-chain was still a substrate for pre-formed convertase. The activity was specific for the alpha-chain of C3 but not of C3(H2O) nor the alpha'-chain of C3b. It is proposed that SGE-modified C3 does not participate in convertase formation, probably having a reduced affinity for factor B.     

Molecular epidemiology of tick-borne encephalitis virus in Ixodes ricinus ticks in Lithuania

In Lithuania, 171-645 serologically confirmed cases of tick-borne encephalitis occurred annually [Mickiene et al. (2001): Eur J Clin Microbiol Infect Dis 20:886-888] in 1993-1999, and the tick-borne encephalitis virus (TBEV) seroprevalence in the general population was found previously to be 3.0% [Juceviciene et al. (2002): J Clin Virol 25:23-27]. To assess the risk for TBEV virus infection in Lithuania and to characterize the agent a panel of 3,234 ticks combined into 436 pools [Juceviciene et al., 2005] were tested for presence of TBEV RNA by a nested RT-PCR targeting at the NS5 gene. Six pools were confirmed positive and the prevalence of the infected ticks was 0.2% (if one tick per pool [Juceviciene et al., 2005] was considered positive) and the proportion of positive tick pools was 1.4%. The prevalence of the infected ticks in the Panevezys, Siauliai, and Radviliskis regions (in central Lithuania) was 0.1%, 0.4%, and 1.7% corresponding with a higher TBE disease burden in these regions. The 252-nucleotide NS5-region amplicons, and a longer sequence (737 nucleotides) obtained from one sample from the PrM-E gene region, were sequenced. Phylogenetic analysis of the latter showed that all western type TBEV PrM-E sequences, including the Lithuanian strains, were monophyletic, showed no clustering and had very little variation. The NS5 sequences, although identical within one locality, did not show any mutations common to strains from the two Lithuanian regions, nor could any geographical clustering be found among western type TBEV strains from other areas.     

Borrelia burgdorferi sensu lato genospecies in questing Ixodes ricinus ticks in Austria

Unfed ticks of all instars (Ixodes ricinus, n=853; Haemaphysalis concinna, n=11) collected in all nine federal states of Austria were individually examined for the presence of Borrelia burgdorferi sensu lato (s.l.) using PCR. The mean overall infection rate was 14.4%. Infection rates were 24.5% in adult ticks, 16.1% in nymphs, and 1.6% in larvae. Four genospecies were detected, including B. valaisiana which was detected for the first time in Austria. The most common B. burgdorferi s.l. genospecies was B. garinii (66.9%), followed by B. valaisiana (13.7%), B. afzelii (11.3%), and B. burgdorferi sensu stricto (s.s.) (6.5%). Two specimens (1.6%) could not be identified to the genospecies level. Geographically, the highest infection rates were detected in the federal state of Vorarlberg (33.3%), B. garinii and B. afzelii being the most prevalent genospecies. B. valaisiana occurred most often in the federal state of Lower Austria, and B. burgdorferi s.s. was focally distributed in the Tyrol, in the surroundings of Imst.     

Marker stability of the Skalica strain (from the tick-borne encephalitis complex) propagated in Ixodes ricinus ticks

Ixodes ricinus larvae from the laboratory breed were infected on viraemic suckling mice inoculated with the Skalica strain from the tick-borne encephalitis complex. The strain recovered from individual nymphs on days 39 and 54 after metamorphosis had the markers (ic+, sc-, t-, v-) identical with the original Skalica strain.     

Evidence of the human granulocytic ehrlichiosis agent in Ixodes ricinus ticks in Switzerland

A total of 1,667 Ixodes ricinus ticks were collected from five regions in Switzerland where there have been sporadic occurrences of granulocytic ehrlichiosis in dogs and horses. The ticks were examined for rickettsiae of the Ehrlichia phagocytophila group via nested PCR. Twenty-one ticks (1.3%) were positive; 3 (0.5%) were nymphs, 6 (1.3%) were adult males, and 12 (1.9%) were adult females. The number of positive ticks varied with the stage of development and with the geographical origin. Nucleotide sequencing of the isolated PCR products identified these products as part of the 16S rRNA gene of Ehrlichia. In addition, these products had 100% homology with the agent of human granulocytic ehrlichiosis. The occurrence of this agent in I. ricinus in Switzerland presents a potential danger of transmission of granulocytic ehrlichiosis to dogs, horses, and humans.     

Identification of host bloodmeal source and Borrelia burgdorferi sensu lato in field-collected Ixodes ricinus ticks in Chaumont (Switzerland)

To evaluate the importance of vertebrate species as tick hosts and as reservoir hosts in two endemic areas for Lyme borreliosis in Switzerland, we applied molecular methods for the analysis of bloodmeal source and Borrelia infection in questing Ixodes ricinus L. ticks. In total, 1326 questing ticks were simultaneously analyzed for Borrelia and for blood meal remnants by using reverse line blot. An overall infection prevalence of 19.0% was recorded for Borrelia sp., with similar rates in both sites. Using a newly developed method for the analysis ofbloodmeal targeting the 12S rDNA mitochondrial gene, identification of host DNA from field-collected ticks was possible in 43.6% of cases. Success of host identification at the genus and species level reached 72%. In one site, host identification success reached its maximum in spring (93% in May), decreasing in summer (20% in July) and rising in autumn (73% in October). In the other site, identification rate in ticks remained low from April to July and increased in autumn reaching 68% in October and November. The most prevalent identified host DNA was artiodactyls in both sites. Red squirrel DNA was significantly more frequently detected in ticks collected in one site, whereas insectivore DNA was more frequent in ticks in the other site. DNA from more than one vertebrate host was detected in 19.5% of nymphs and 18.9% of adults. Host DNA was identified in 48.4% of the Borrelia infected ticks. Although DNA from all Borrelia species was found in at least some ticks with DNA from mammals and some ticks with DNA from birds, our results confirm a general association of B. afzelii and B. burgdorferi sensu stricto with rodents, and B. valaisiana and B. garinii with birds.     

Molecular evidence of Anaplasma phagocytophilum in Ixodes ricinus ticks and wild animals in Austria

Real-time PCR analysis of a groESL heat shock operon segment showed the presence of two genetic lineages of Anaplasma phagocytophilum in Ixodes ricinus ticks as well as one variant in wild red and roe deer, the latter supposedly representing the natural reservoir of one variant of A. phagocytophilum.