Lyme borreliosis agents and the genetics and sex of their vector, Ixodes ricinus

The tick Ixodes ricinus is responsible for the transmission and maintenance of a wide variety of pathogenic organisms in the Northern Hemisphere, among which Lyme disease represents a major threat to humans. Despite numerous studies, the epidemiology of the different bacterial species responsible for this disease remains unclear. Recent evidence for a sex-biased genetic structure of its European vector leads us to analyse the consequences of this pattern on Borrelia transmission. Here we show that male and female ticks are not equivalently infected by Borrelia burgdorferi, that Borrelia afzelii affects tick migration capabilities, especially for the most vagile sex (i.e., male) and that Lyme borreliosis agents are consequently vectorised in a much more complex way than usually thought. Such results change the epidemiological perception of Lyme borreliosis and suggest new co-evolutionary pathways between the ticks and the borrelia.     

Migratory songbirds disperse ticks across Canada, and first isolation of the Lyme disease spirochete, Borrelia burgdorferi, from the avian tick, Ixodes auritulus

During a 3-yr comprehensive study, 196 ixodid ticks (9 species) were collected from 89 passerine birds (32 species) from 25 localities across Canada to determine the distribution of avian-associated tick species and endogenous Lyme disease spirochetes, Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt, and Brenner. We report the following first records of tick parasitism on avian hosts: the rabbit-associated tick, Ixodes dentatus Marx, from Manitoba and Ontario; the mouse tick, Ixodes muris Bishopp and Smith, from British Columbia; and the blacklegged tick, Ixodes scapularis Say, from New Brunswick. Moreover, we provide the first record of the Neotropical tick, Amblyomma humerale Koch (1 nymph), in Canada and its parasitism of any bird. This tick was compared morphologically with nymphs of other Neotropical Amblyomma spp., and genetically, using a 344-bp fragment of the 12S rDNA sequence of 41 New World Amblyomma species. The first collections of the western blacklegged tick, Ixodes pacificus Cooley and Kohls, from passerine species in Alberta and British Columbia, are also reported. Notably, we further report the first isolation of B. burgdorferi from the bird tick, Ixodes auritulus Neumann, collected from an American robin, Turdus migratorius L., on Vancouver Island. Furthermore, B. burgdorferi-positive I. auritulus larvae were collected from a reservoir-competent fox sparrow, Passerella iliaca (Merrem). Our findings indicate that ground-dwelling passerines, in particular, are parasitized by certain ixodid ticks and play an important role across Canada in the wide dispersal of B. burgdorferi-infected ticks and increased risk of Lyme disease exposure.     

Characterization of spirochetes isolated from ticks (Ixodes tanuki, Ixodes turdus, and Ixodes columnae) and comparison of the sequences with those of Borrelia burgdorferi sensu lato strains.

Ixodes persulcatus serves as a tick vector for Borrelia garinii and Borrelia afzelii in Japan; however, unidentified spirochetes have been isolated from other species of ticks. In this study, 13 isolates from ticks (6 from Ixodes tanuki, 6 from Ixodes turdus, and 1 from Ixodes columnae) and 3 isolates from voles (Clethrionomys rufocanus) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rRNA gene restriction fragment length polymorphism, partial sequencing of the outer surface protein C (OspC) gene, whole DNA-DNA hybridization, and 16S rRNA gene sequence comparison. All of the results revealed that these Borrelia strains clearly represent at least two new species. A third is also likely, although additional strains have to be isolated and characterized before a separate species is designated. We designated all isolates of I. tanuki and C. rufocanus as group Hk501 and all isolates of I. turdus as group Ya501. Phylogenetic analysis based on 16S rRNA gene sequences distinguished these Borrelia strains from those belonging to hitherto known Borrelia species. Furthermore, the genomic groups, each with its own tick vectors with enzootic cycles, were quite different from each other and also from those of Lyme disease Borrelia species known to occur in Japan. The results of 16S rRNA gene sequence comparison suggest that the strain Am501 from I. columnae is related to group Hk501, although its level of DNA relatedness is less than 70%.     

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.     

Investigation of genotypes of Borrelia burgdorferi in Ixodes scapularis ticks collected during surveillance in Canada

The genetic diversity of Borrelia burgdorferi sensu stricto, the agent of Lyme disease in North America, has consequences for the performance of serological diagnostic tests and disease severity. To investigate B. burgdorferi diversity in Canada, where Lyme disease is emerging, bacterial DNA in 309 infected adult Ixodes scapularis ticks collected in surveillance was characterized by multilocus sequence typing (MLST) and analysis of outer surface protein C gene (ospC) alleles. Six ticks carried Borrelia miyamotoi, and one tick carried the novel species Borrelia kurtenbachii. 142 ticks carried B. burgdorferi sequence types (STs) previously described from the United States. Fifty-eight ticks carried B. burgdorferi of 1 of 19 novel or undescribed STs, which were single-, double-, or triple-locus variants of STs first described in the United States. Clonal complexes with founder STs from the United States were identified. Seventeen ospC alleles were identified in 309 B. burgdorferi-infected ticks. Positive and negative associations in the occurrence of different alleles in the same tick supported a hypothesis of multiple-niche polymorphism for B. burgdorferi in North America. Geographic analysis of STs and ospC alleles were consistent with south-to-north dispersion of infected ticks from U.S. sources on migratory birds. These observations suggest that the genetic diversity of B. burgdorferi in eastern and central Canada corresponds to that in the United States, but there was evidence for founder events skewing the diversity in emerging tick populations. Further studies are needed to investigate the significance of these observations for the performance of diagnostic tests and clinical presentation of Lyme disease in Canada.     

Prevalence of Borrelia burgdorferi sensu lato and Anaplasmataceae members in Ixodes ricinus ticks in Alsace, a focus of Lyme borreliosis endemicity in France

Due to the high Lyme borreliosis incidence in Alsace, in northeastern France, we investigated in 2003-2004 three cantons in this region in order to determine the density of Ixodes ricinus ticks infected by Borrelia burgdorferi sensu lato and Anaplasmataceae. The peak density of nymphs infected by B. burgdorferi sensu lato at Munster and Guebwiller, where the disease incidence was high, was among the highest reported in Europe (105 and 114 per 100 m(2), respectively). In contrast, the peak density of infected nymphs was low in the canton of Dannemarie (5/100 m(2)), where the disease incidence was low. The two main species detected in ticks were Borrelia afzelii, more frequent in nymphs, and Borrelia garinii, more frequent in adult ticks. The rates of tick infection by Anaplasma phagocytophilum were 0.4% and 1.2% in nymphs and adults, respectively.     

A tick mannose-binding lectin inhibitor interferes with the vertebrate complement cascade to enhance transmission of the lyme disease agent

The Lyme disease agent Borrelia burgdorferi is primarily transmitted to vertebrates by Ixodes ticks. The classical and alternative complement pathways are important in Borrelia eradication by the vertebrate host. We recently identified a tick salivary protein, designated P8, which reduced complement-mediated killing of Borrelia. We now discover that P8 interferes with the human lectin complement cascade, resulting in impaired neutrophil phagocytosis and chemotaxis and diminished Borrelia lysis. Therefore, P8 was renamed the tick salivary lectin pathway inhibitor (TSLPI). TSLPI-silenced ticks, or ticks exposed to TSLPI-immune mice, were hampered in Borrelia transmission. Moreover, Borrelia acquisition and persistence in tick midguts was impaired in ticks?feeding on TSLPI-immunized, B.?burgdorferi-infected mice. Together, our findings suggest an essential role for the lectin complement cascade in Borrelia eradication and demonstrate how a vector-borne pathogen co-opts a vector protein to facilitate early mammalian infection and vector colonization.     

Widespread dispersal of Borrelia burgdorferi-infected ticks collected from songbirds across Canada

Millions of Lyme disease vector ticks are dispersed annually by songbirds across Canada, but often overlooked as the source of infection. For clarity on vector distribution, we sampled 481 ticks (12 species and 3 undetermined ticks) from 211 songbirds (42 species/subspecies) nationwide. Using PCR, 52 (29.5%) of 176 Ixodes ticks tested were positive for the Lyme disease spirochete, Borrelia burgdorferi s.l. Immature blacklegged ticks, Ixodes scapularis , collected from infested songbirds had a B. burgdorferi infection prevalence of 36% (larvae, 48%; nymphs, 31%). Notably, Ixodes affinis is reported in Canada for the first time and, similarly, Ixodes auritulus for the initial time in the Yukon. Firsts for bird-parasitizing ticks include I. scapularis in Quebec and Saskatchewan. We provide the first records of 3 tick species cofeeding on passerines (song sparrow, Swainson's thrush). New host records reveal I. scapularis on the blackpoll warbler and Nashville warbler. We furnish the following first Canadian reports of B. burgdorferi-positive ticks: I. scapularis on chipping sparrow, house wren, indigo bunting; I. auritulus on Bewick's wren; and I. spinipalpis on a Bewick's wren and song sparrow. First records of B. burgdorferi-infected ticks on songbirds include the following: the rabbit-associated tick, Ixodes dentatus, in western Canada; I. scapularis in Quebec, Saskatchewan, northern New Brunswick, northern Ontario; and Ixodes spinipalpis (collected in British Columbia). The presence of B. burgdorferi in Ixodes larvae suggests reservoir competency in 9 passerines (Bewick's wren, common yellowthroat, dark-eyed junco, Oregon junco, red-winged blackbird, song sparrow, Swainson's thrush, swamp sparrow, and white-throated sparrow). We report transstadial transmission (larva to nymph) of B. burgdorferi in I. auritulus. Data suggest a possible 4-tick, i.e., I. angustus, I. auritulus, I. pacificus, and I. spinipalpis, enzootic cycle of B. burgdorferi on Vancouver Island, British Columbia. Our results suggest that songbirds infested with B. burgdorferi-infected ticks have the potential to start new tick populations endemic for Lyme disease. Because songbirds disperse B. burgdorferi-infected ticks outside their anticipated range, health-care providers are advised that people can contract Lyme disease locally without any history of travel.     

Geographic uniformity of the Lyme disease spirochete (Borrelia burgdorferi) and its shared history with tick vector (Ixodes scapularis) in the Northeastern United States

Over 80% of reported cases of Lyme disease in the United States occur in coastal regions of northeastern and mid-Atlantic states. The genetic structure of the Lyme disease spirochete (Borrelia burgdorferi) and its main tick vector (Ixodes scapularis) was studied concurrently and comparatively by sampling natural populations of I. scapularis ticks along the East Coast from 1996 to 1998. Borrelia is genetically highly diverse at the outer surface protein ospC. Since Borrelia is highly clonal, the ospC alleles can be used to define clones. A newly designed reverse line blotting (RLB) assay shows that up to 10 Borrelia clones can infect a single tick. The clone frequencies in Borrelia populations are the same across the Northeast. On the other hand, I. scapularis populations show strong regional divergence (among northeastern, mid-Atlantic, and southern states) as well as local differentiation. The high genetic diversity within Borrelia populations and the disparity in the genetic structure between Borrelia and its tick vector are likely consequences of strong balancing selection on local Borrelia clones. Demographically, both Borrelia and I. scapularis populations in the Northeast show the characteristics of a species that has recently expanded from a population bottleneck. Major geological and ecological events, such as the last glacial maximum (18,000 years ago) and the modern-day expansion of tick habitats, are likely causes of the observed "founder effects" for the two organisms in the Northeast. We therefore conclude that the genetic structure of B. burgdorferi has been intimately shaped by the natural history of its main vector, the northern lineage of I. scapularis ticks.     

Variable exposure and immunological response to Lyme disease Borrelia among North Atlantic seabird species

Colonial seabirds often breed in large aggregations. These individuals can be exposed to parasitism by the tick Ixodes uriae, but little is known about the circulation of pathogens carried by this ectoparasite, including Lyme disease Borrelia. Here we investigated the prevalence of antibodies (Ab) against Borrelia burgdorferi sensu lato in seabird species sampled at eight locations across the North Atlantic. Using enzyme-linked immunosorbent assay tests, we found that the prevalence of anti-Borrelia Ab in adult seabirds was 39.6% on average (over 444 individuals), but that it varied among colonies and species. Common guillemots showed higher seroprevalence (77.1%+/-5.9) than black-legged kittiwakes (18.6%+/-6.7) and Atlantic puffins (22.6%+/-6.3). Immunoblot-banding patterns of positive individuals, reflecting the variability of Borrelia antigens against which Ab were produced, also differed among locations and species, and did not tightly match the prevalence of Borrelia phylogroups previously identified in ticks collected from the same host individuals. These results represent the first report of the widespread prevalence of Ab against Borrelia within an assemblage of seabird species and demonstrate that Borrelia is an integrated aspect in the interaction between seabirds and ticks. More detailed studies on the dynamics of Borrelia within and among seabird species at different spatial scales will now be required to better understand the implications of this interaction for seabird ecology and the epidemiology of Lyme disease.     

Zecken als Krankheitsüberträger: Was tun bei einem Stich?

Because of its wide host‐range and capacity for transmission of multiple pathogens, Ixodes icinus poses a constant threat of human infection. Borrelia burgdorferi is the most prevalent tick‐borne pathogen affecting humans (Lyme Borreliosis), tick‐borne‐encephalitis (TBE) the most important viral tick‐borne disease in Europe. In natural foci the pathogens circulate between infected small mammals and ticks. Knowing the lifecycle of I.ricinus, their multistrategies for host finding, attachment and blood ingestion, we may understand, what makes the tick such an excellent vector. Instructions for individual behaviour in tick areas to avoid tick contact are given. Since transmission is closely related to the feeding period it is helpful to remove an attached tick as soon as possible. Protection against tick‐borne encephalitis by vaccination is possible.     

Phylogeographic relationships of Ixodes uriae (Acari: Ixodidae) and their significance to transequatorial dispersal of Borrelia garinii

The seabird tick Ixodes uriae (Acari: Ixodidae) has a bi- and circumpolar distribution and is commonly infected with Lyme disease Borrelia. Identical Borrelia flagellin gene sequences have been detected in I. uriae from both the Northern and Southern Hemispheres, suggesting a transequatorial transport of Borrelia. Parsimony analysis of the internal transcribed spacer 2 (ITS2) and a part of 16S rDNA of I. uriae from the Northern and Southern Hemispheres indicated that northern and southern I. uriae might be reproductively separated. We hypothesize that Borrelia is probably not dispersed from one hemisphere to the other by ticks attached to seabirds.     

Severe tick infestation in a hare and potential risk for transmitting pathogens to humans

Severe tick infestation was found in a hare in a suburban area of Nanchang, Jiangxi Province, China. We sampled ticks and identified them based on their morphologic characteristics. Three species, Ixodes sinensis, which is commonly found in China and can experimentally transmit Borrelia burgdorferi, Rhipicephalus haemaphysaloides, and Haemaphysalis longicornis which can transmit Lyme disease were detected with an optical microscope and a stereomicroscope. Risk of spreading ticks from suburban to urban areas exists due to human transportation and travel between the infested and non-infested areas around Nanchang.     

Lyme disease and migrating birds in the Saint Croix River Valley

During a study of migrating land birds in 1987, we examined over 9,200 individual birds representing 99 species from the Saint Croix River Valley, a Lyme disease-endemic area of east central Minnesota and northwestern Wisconsin. We found that 250 deer tick (Ixodes dammini) larvae and nymphs infested 58 birds from 15 migrant species; 56 ticks (22.4%) were positive for the Lyme disease spirochete Borrelia burgdorferi. Five ground-foraging migrant bird species favoring mesic habitats, veery (Catharus fuscescens), ovenbird (Seiurus aurocapillus), northern waterthrush (S. novaboracensis), common yellowthroat (Geothlypis trichas), and swamp sparrow (Melospiza georgiana), accounted for nearly three-quarters of parasitized individuals. Nearly half of the spirochete-positive ticks were removed from migrating birds taken in a riparian floodplain forest. Recaptured migrants with infected ticks indicate that they transmit B. burgdorferi to hexapod larvae. We suggest that birds may be both an important local reservoir in the upper Mississippi Valley and long-distance dispersal agents for B. burgdorferi-infected ticks to other regions of the continent.     

Lyme disease and migrating birds in the Saint Croix River Valley.

During a study of migrating land birds in 1987, we examined over 9,200 individual birds representing 99 species from the Saint Croix River Valley, a Lyme disease-endemic area of east central Minnesota and northwestern Wisconsin. We found that 250 deer tick (Ixodes dammini) larvae and nymphs infested 58 birds from 15 migrant species; 56 ticks (22.4%) were positive for the Lyme disease spirochete Borrelia burgdorferi. Five ground-foraging migrant bird species favoring mesic habitats, veery (Catharus fuscescens), ovenbird (Seiurus aurocapillus), northern waterthrush (S. novaboracensis), common yellowthroat (Geothlypis trichas), and swamp sparrow (Melospiza georgiana), accounted for nearly three-quarters of parasitized individuals. Nearly half of the spirochete-positive ticks were removed from migrating birds taken in a riparian floodplain forest. Recaptured migrants with infected ticks indicate that they transmit B. burgdorferi to hexapod larvae. We suggest that birds may be both an important local reservoir in the upper Mississippi Valley and long-distance dispersal agents for B. burgdorferi-infected ticks to other regions of the continent.     

Ixodes dammini: occurrence and prevalence of infection with Borrelia spp. in Minnesota

The distribution of Ixodes dammini in Minnesota was studied by collecting adult ticks from hunting dogs during the grouse seasons in September and October of 1985 and 1986. The tick was most frequently found in the east-central part of the state. Borrelia spp. were observed by immunofluorescence in 10% of the ticks. The locations where ticks were found coincide with the primary endemic areas for Lyme disease in the state.     

Borrelia burgdorferi in ticks and dogs in the province of Vojvodina, Serbia

Lyme disease is a tick borne zoonotic infection, caused by Borrelia burgdorferi s.l. bacteria. For the transmission of the disease, the presence of ticks is a prerequisite. Lyme borreliosis mostly occurs in people and dogs, but it may occur in other animals. Ticks which carry B. burgdorferi s.l. in Serbia are of the Ixodes ricinus specis. In Serbia, Lyme disease was detected for the first time in the late '80-es. In dogs, clinical symptoms may occur even months after a tick bite, and include weakness, lymphadenopathy, fever, lameness, arthritis, etc. In our survey, we have observed tick and dog populations in the province of Vojvodina (northern part of Serbia). I. ricinus ticks were collected and examined for the presence of B. burgdorferi s.l. in several chosen locations. In addition, blood samples were collected from house dogs and pets from the same locations, and analyzed for the presence of antibodies specific for B. burgdorferi s.l. The results showed a mean infection of ticks of 22.12%, and a mean seroprevalence of Lyme disease in dogs of 25.81%. We conclude that in Vojvodina there is an actual risk of Lyme borreliosis for other animals and humans, because of the persistence of B. burgdorferi s.l. in both tick and dog populations.     

Improving the specificity of 16S rDNA-based polymerase chain reaction for detecting Borrelia burgdorferi sensu lato-causative agents of human Lyme disease

AIMS: 16S rDNA sequences of Borrelia burgdorferi sensu lato were aligned with the 16S rDNA sequences of Borrelia hermsii, Borrelia turicatae, and Borrelia lonestari in order to identify primers that might be used to more specifically identify agents of human Lyme disease in ticks in human skin samples. METHODS AND RESULTS: Standard polymerase chain reaction (PCR), using an oligonucleotide sequence, designated TEC1, was shown, in combination with a previously developed primer (LD2) to amplify strains of B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii, but not the non-Lyme causing B. hermsii or B. turicatae. This primer pair, designated Bbsl, was successfully used to amplify B. burgdorferi sensu lato from skin biopsies of patients with Lyme disease symptoms as well as from Ixodes scapularis, Amblyomma americanum and Dermacentor variabilis ticks. CONCLUSIONS: The primer set Bbsl allows for the rapid detection and differentiation of B. burgdorferi sensu lato from non-Lyme disease-causing Borrelia species in ticks and human tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR primer set, Bbsl, will greatly facilitate detection of the causative agents of Lyme disease in infected ticks and human skin samples assisting in epidemiological studies, and potentially allowing for a more rapid diagnosis of the disease in patients.     

Absence of spirochaetes (Borrelia burgdorferi) and piroplasms (Babesia microti) in deer ticks (Ixodes dammini) parasitized by chalcid wasps (Hunterellus hookeri)

An entomophagous wasp (Hunterellus hookeri Howard) parasitizes about a third of the host-seeking nymphal Ixodes dammini Spielman et al. ticks on Naushon Island in Massachusetts (U.S.A.) where the agents of Lyme disease (Borrelia burgdorferi Johnson et al.) and human babesiosis (Babesia microti Franca) are enzootic. Following blood-feeding, wasp-parasitized ticks are destroyed by the developing wasp. The prevalence of either human pathogen in host-seeking ticks collected in wasp-infested sites is nearly 40% lower than that found in other sites. Nymphal ticks, collected early in their season of activity, are more frequently parasitized by the wasp and less frequently by the Lyme disease spirochaete than those collected later in the summer. Spirochaetes never infected wasp-infected ticks, and few wasp-infected ticks were concurrently infected by the Babesia piroplasm. Taken together, these correlations indicate that the wasp may render the tick inhospitable to both pathogens. The presence of the wasp may have reduced risk of human infection on the island by either pathogen by as much as a third.