Discovery of the Lyme disease spirochete and its relation to tick vectors.

The various hypotheses concerning the etiologic agent of erythema chronicum migrans of Europe and of Lyme disease in the United States are reviewed, and an account of events that led to the discovery of the causative spirochetal agent in Ixodes dammini is presented. Spirochetes morphologically and antigenically similar, if not identical to, the organism detected in I. dammini were also found for the first time in Ixodes pacificus and Ixodes ricinus, the vectors hitherto incriminated, respectively, in western United States and Europe. In most infected ticks, spirochetal development was found to be limited to the midgut. Ticks with generalized infections were shown to transmit spirochetes via eggs, but infections decreased in intensity and became restricted to the central ganglion as filial ticks developed to adults. Although the mechanisms of transmission to a host are still under investigation, the spirochetes may be transmitted by saliva of ticks with generalized infectious and possibly also by regurgitation of infected gut contents, or even by means of infected fecal material.     

Impact of the experimental removal of lizards on Lyme disease risk

The distribution of vector meals in the host community is an important element of understanding and predicting vector-borne disease risk. Lizards (such as the western fence lizard; Sceloporus occidentalis) play a unique role in Lyme disease ecology in the far-western United States. Lizards rather than mammals serve as the blood meal hosts for a large fraction of larval and nymphal western black-legged ticks (Ixodes pacificus--the vector for Lyme disease in that region) but are not competent reservoirs for the pathogen, Borrelia burgdorferi. Prior studies have suggested that the net effect of lizards is to reduce risk of human exposure to Lyme disease, a hypothesis that we tested experimentally. Following experimental removal of lizards, we documented incomplete host switching by larval ticks (5.19%) from lizards to other hosts. Larval tick burdens increased on woodrats, a competent reservoir, but not on deer mice, a less competent pathogen reservoir. However, most larvae failed to find an alternate host. This resulted in significantly lower densities of nymphal ticks the following year. Unexpectedly, the removal of reservoir-incompetent lizards did not cause an increase in nymphal tick infection prevalence. The net result of lizard removal was a decrease in the density of infected nymphal ticks, and therefore a decreased risk to humans of Lyme disease. Our results indicate that an incompetent reservoir for a pathogen may, in fact, increase disease risk through the maintenance of higher vector density and therefore, higher density of infected vectors.     

Why Lyme disease is common in the northern US,but rare in the south: The roles of host choice,host-seeking behavior,and tick density

Lyme disease is common in the northeastern United States, but rare in the southeast, even though the tick vector is found in both regions. Infection prevalence of Lyme spirochetes in host-seeking ticks, an important component to the risk of Lyme disease, is also high in the northeast and northern midwest, but declines sharply in the south. As ticks must acquire Lyme spirochetes from infected vertebrate hosts, the role of wildlife species composition on Lyme disease risk has been a topic of lively academic discussion. We compared tick–vertebrate host interactions using standardized sampling methods among 8 sites scattered throughout the eastern US. Geographical trends in diversity of tick hosts are gradual and do not match the sharp decline in prevalence at southern sites, but tick–host associations show a clear shift from mammals in the north to reptiles in the south. Tick infection prevalence declines north to south largely because of high tick infestation of efficient spirochete reservoir hosts (rodents and shrews) in the north but not in the south. Minimal infestation of small mammals in the south results from strong selective attachment to lizards such as skinks (which are inefficient reservoirs for Lyme spirochetes) in the southern states. Selective host choice, along with latitudinal differences in tick host-seeking behavior and variations in tick densities, explains the geographic pattern of Lyme disease in the eastern US.

Lyme disease is common in the northeastern United States, but rare in the southeast, even though the tick vector is found in both regions. This study shows that this is largely because the tick vectors attach abundantly to rodents (which are good hosts for the Lyme bacteria) in the north, and to lizards (which are relatively poor hosts for Lyme bacteria) in the south.     

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.     

The emergence of Lyme disease in a changing environment in North America and Central Europe

Lyme disease has recently begun to emerge as a significant threat to human health, both in Europe and the United States. Late sequellae, resembling those of neurosyphilis and multiple sclerosis, may occur many years after initial infection. Spontaneous abortion accompanies arthritis, carditis and neuritis as burdensome short-term sequellae. Thousands of new infections are recognized each year on each side of the Atlantic, although reporting may be incomplete. The disease was described in Europe nearly a century ago and named erythema chronicum migrans, but its etiology has only recently been defined. The name Lyme disease was coined to describe a particularly intense American focus of disease, but the term has gained wide acceptance on both continents. The identity of the American and European etiological agents involved has yet to be determined.In America, a deer-associated, often bird-transported tick transmits this mouse-reservoired spirochete. The European situation seems more complex because the vector tick feeds on a greater variety of vertebrates. The reservoir hosts of the spirochete have yet to be determined. The role ofIxodes ricinus and possible other vectors in perpetuating transmission of the European infection remains to be defined. WhetherI. ricinus as well asI. dammini merely serve as a bridge to the human population or are important for the maintenance of the feral cycle remains to be seen.The capacity of a tick to maintain transmission of Lyme disease spirochetes depends upon a complex set of properties, including competence as a host for the spirochete, a pattern of feeding that focuses on a particular reservoir favored by a pattern of tick activity, during each transmission season, in which nymphs feed before larvae. Transmission would be favored by an environment, such as that of islands, in which the variety of potential reservoir hosts is restricted. Hosts, for example reptiles, that might fail to support growth of the spirochete would serve to dilute effective transmission in nature.Similarly, the capacity of a vertebrate to maintain the infection requires long-term support of the spirochete in a tissue site accessible to vector ticks, tolerance of repeated feeding by vector ticks and a pattern of host activity that exposes the host to numerous bites.The intensity of infection depends upon a continuous pattern of transmission in which each generation is infected anew. The rate event in which the vector inherits infection would serve mainly to transport the spirochete to a new site, most effectively by migrating birds.Due to the dispersed nature of Lyme disease and its recent emergence as an important hazard to health, measures for prophylaxis have only recently been devised. Lyme disease can be treated with antibiotics. But the effectiveness of such therapy depends upon correct and prompt diagnosis; delayed treatment is less effective, presumably because the spirochete becomes sequestered in immune-privileged sites.The present review lays stress on selected papers focusing on the epidemiological aspects and the experimental approach to the newly emerged disease rather than reviewing the complex literature concerning Lyme disease.     

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.     

The epidemiology of lyme borreliosis

The tick-transmitted bacterial infection known as Lyme disease, or Lyme borreliosis, has recently emerged as the leading arthropod-borne disease in Europe and North America. Several thousand new cases of human Lyme disease are reported each year from Europe and the USA. The causative agent, Borrelia burgdorferi, has been isolated from several species of mammals, birds, ticks and insects and, in this article, Thomas Jaenson discusses the geographical differences in the epidemiology of the infection.     

Vector competence of the Australian paralysis tick, Ixodes holocyclus, for the Lyme disease spirochete Borrelia burgdorferi

Clinical and serologic evidence of Lyme disease in Australia, including the typical rash, erythema migrans, has been reported. The vector tick transmitting Borrelia burgdorferi in Australia, however, has not been determined. The Australian paralysis tick, Ixodes holocyclus, is a logical candidate vector of the Lyme disease spirochete in Australia; therefore, we tested the ability of I. holocyclus to acquire and maintain a North American isolate of B. burgdorferi. Larval I. holocyclus ingested spirochetes, but none of 84 derived nymphs were infected. These experiments should be repeated with Australian strains of spirochetes.     

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.     

Detection of Borrelia burgdorferi Sensu Stricto ospC Alleles Associated with Human Lyme Borreliosis Worldwide in Non-Human-Biting Tick Ixodes affinis and Rodent Hosts in Southeastern United States

Comparative analysis of ospC genes from 127 Borrelia burgdorferi sensu stricto strains collected in European and North American regions where Lyme disease is endemic and where it is not endemic revealed a close relatedness of geographically distinct populations. ospC alleles A, B, and L were detected on both continents in vectors and hosts, including humans. Six ospC alleles, A, B, L, Q, R, and V, were prevalent in Europe; 4 of them were detected in samples of human origin. Ten ospC alleles, A, B, D, E3, F, G, H, H3, I3, and M, were identified in the far-western United States. Four ospC alleles, B, G, H, and L, were abundant in the southeastern United States. Here we present the first expanded analysis of ospC alleles of B. burgdorferi strains from the southeastern United States with respect to their relatedness to strains from other North American and European localities. We demonstrate that ospC genotypes commonly associated with human Lyme disease in European and North American regions where the disease is endemic were detected in B. burgdorferi strains isolated from the non-human-biting tick Ixodes affinis and rodent hosts in the southeastern United States. We discovered that some ospC alleles previously known only from Europe are widely distributed in the southeastern United States, a finding that confirms the hypothesis of transoceanic migration of Borrelia species.     

Spatial Dynamics of Lyme Disease: A Review

Lyme disease (LD), the most frequently reported vector-borne disease in the United States, requires that humans, infected vector ticks, and infected hosts all occur in close spatial proximity. Understanding the spatial dynamics of LD requires an understanding of the spatial determinants of each of these organisms. We review the literature on spatial patterns and environmental correlates of human cases of LD and the vector ticks, Ixodes scapularis in the northeastern and midwestern United States and Ixodes pacificus in the western United States. The results of this review highlight a need for a more standardized and comprehensive approach to studying the spatial dynamics of the LD system. Specifically, we found that the only environmental variable consistently associated with increased LD risk and incidence was the presence of forests. However, the reasons why some forests are associated with higher risk and incidence than others are still poorly understood. We suspect that the discordance among studies is due, in part, to the rapid developments in both conceptual and technological aspects of spatial ecology hastening the obsolescence of earlier approaches. Significant progress in identifying the determinants of spatial variation in LD risk and incidence requires that: (1) existing knowledge of the biology of the individual components of each LD system is utilized in the development of spatial models; (2) spatial data are collected over longer periods of time; (3) data collection and analysis among regions are more standardized; and (4) the effect of the same environmental variables is tested at multiple spatial scales.     

UNCOORDINATED PHYLOGEOGRAPHY OF BORRELIA BURGDORFERI AND ITS TICK VECTOR,IXODES SCAPULARIS

Vector‐borne microbes necessarily co‐occur with their hosts and vectors, but the degree to which they share common evolutionary or biogeographic histories remains unexplored. We examine the congruity of the evolutionary and biogeographic histories of the bacterium and vector of the Lyme disease system, the most prevalent vector‐borne disease in North America. In the eastern and midwestern US, Ixodes scapularis ticks are the primary vectors of Borrelia burgdorferi, the bacterium that causes Lyme disease. Our phylogeographic and demographic analyses of the 16S mitochondrial rDNA suggest that northern I. scapularis populations originated from very few migrants from the southeastern US that expanded rapidly in the Northeast and subsequently in the Midwest after the recession of the Pleistocene ice sheets. Despite this historical gene flow, current tick migration is restricted even between proximal sites within regions. In contrast, B. burgdorferi suffers no barriers to gene flow within the northeastern and midwestern regions but shows clear interregional migration barriers. Despite the intimate association of B. burgdorferi and I. scapularis, the population structure, evolutionary history, and historical biogeography of the pathogen are all contrary to its arthropod vector. In the case of Lyme disease, movements of infected vertebrate hosts may play a larger role in the contemporary expansion and homogenization of the pathogen than the movement of tick vectors whose populations continue to bear the historical signature of climate‐induced range shifts.     

Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis.

We studied 48 Borrelia isolates that were associated with Lyme borreliosis or were isolated from ticks and identified three DNA relatedness groups by using the S1 nuclease method. The three DNA groups (genospecies) were associated with specific rRNA gene restriction patterns, protein electrophoresis patterns, and patterns of reactivity with murine monoclonal antibodies. Genospecies I corresponded to Borrelia burgdorferi sensu stricto since it contained the type strain of this species (strain ATCC 35210); this genospecies included 28 isolates from Europe and the United States. Genospecies II was named Borrelia garinii sp. nov. and included 13 isolates from Europe and Japan. Genospecies III (group VS461) included seven isolates from Europe and Japan.     

Reduced variation among northern deer tick populations at an autosomal microsatellite locus.

To determine whether genes flow freely between populations of the Ixodes ricinus-like ticks of eastern North America, and to determine whether the abundant northerly populations of these vectors of Lyme disease and other zoonotic infections may have arisen recently from a small cohort of ancestral founders, we characterized the nuclear IR27 microsatellite alleles in ticks sampled from the geographic extremes of their ranges. These microsatellite alleles differentiated populations located in the southeastern, northcentral and northeastern United States, respectively. Although evident heterozygous genotypes are about as frequent as would be expected in randomly mating populations, particular microsatellite alleles and diploid genotypes occur more frequently in certain populations than in others. Ticks from the Northeast and upper Midwest are markedly more related to each other than to ticks from the Southeast. Patterns of diversity present in this nuclear microsatellite marker correspond to those evident at a mitochondrial locus and indicate that the deer ticks of the Northeast and upper Midwest are genetically isolated from those in the Southeast. The Ixodes ricinus-like ticks that impose a public health burden in the northeastern and northcentral United States originated recently in a common founder population.     

Discriminating between Ixodes Ticks by Means of Mitochondrial DNA Sequences

Ticks of the genus Ixodes have recently assumed prominence because they frequently serve as vectors of important zoonoses, including Lyme disease and babesiosis. The morphological characteristics that have been used in their identification often are ambiguous and are useful solely at a particular stage of development. Here we report the DNA sequence of the mitochondrially encoded 16S rRNA gene of nine different Ixodes ticks and an outgroup from another genus, Dermacentor. The sequences readily discriminate between these ticks. Samples of I. dammini from the northeastern and upper midwestern United States differ from southeastern I. scapularis at about 2% of the nucleotides. This difference is about half that separating other members of the I. ricinus group of species, but exceeds typical levels of intraspecific variation. Two major clades exist within the I. ricinus complex. One includes I. cookei , I. hexagonus,and I. angustus. The other includes I. persulcatus, I. pacificus, I. muris, I. ricinus, I. scapularis, and I. dammini. We conclude that mtDNA sequences are useful for unravelling the systematics of these important vectors of human disease.     

Variation in the Microbiota of Ixodes Ticks with Regard to Geography,Species, and Sex

Ixodes scapularis is the principal vector of Lyme disease on the East Coast and in the upper Midwest regions of the United States, yet the tick is also present in the Southeast, where Lyme disease is absent or rare. A closely related species, I. affinis, also carries the pathogen in the South but does not seem to transmit it to humans. In order to better understand the geographic diversity of the tick, we analyzed the microbiota of 104 adult I. scapularis and 13 adult I. affinis ticks captured in 19 locations in South Carolina, North Carolina, Virginia, Connecticut, and New York. Initially, ticks from 4 sites were analyzed by 454 pyrosequencing. Subsequently, ticks from these sites plus 15 others were analyzed by sequencing with an Illumina MiSeq machine. By both analyses, the microbiomes of female ticks were significantly less diverse than those of male ticks. The dissimilarity between tick microbiomes increased with distance between sites, and the state in which a tick was collected could be inferred from its microbiota. The genus Rickettsia was prominent in all locations. Borrelia was also present in most locations and was present at especially high levels in one site in western Virginia. In contrast, members of the family Enterobacteriaceae were very common in North Carolina I. scapularis ticks but uncommon in I. scapularis ticks from other sites and in North Carolina I. affinis ticks. These data suggest substantial variations in the Ixodes microbiota in association with geography, species, and sex.     

Avian and mammalian hosts for spirochete-infected ticks and insects in a Lyme disease focus in Connecticut.

Spirochetes and their vectors and reservoirs were studied in a Lyme disease focus in East Haddam, Connecticut, from mid-May through September 1983. Ixodes dammini subadults were comparable in number on white-footed mice (Peromyscus leucopus) (means = 2.9 +/- 3.6 SD) to those on 27 different species of birds (means = 2.3 +/- 4.2 SD) representing 11 families within the order Passeriformes. Less commonly found ticks on birds (means less than or equal to 0.1) were immature Ixodes dentatus and Haemaphysalis leporispalustris. Although spirochete-infected I. dammini larvae and nymphs were taken off eight and nine different species of birds, respectively, significantly fewer positive larvae were removed from birds than from white-footed mice. Spirochetes were detected in the midguts of I. dammini, Dermacentor variabilis, and H. leporispalustris and two species of insects (Cuterebra fontinella and Orchopeas leucopus). Possibly, arthropods other than I. dammini vector these spirochetes in northeastern United States. Spirochetes grew in a cell-free medium inoculated with bloods from four white-footed mice, one woodland jumping mouse (Napaeozapus insignis), one northern mockingbird (Mimus polyglottos), one gray catbird (Dumetella carolinensis), two prairie warblers (Dendroica discolor), one orchard oriole (Icterus spurius), one common yellowthroat (Geothlypis trichas), and one American robin (Turdus migratorius). We suggest that avian hosts, like mammals, develop spirochetemias of the causative agent of Lyme disease. Erythematous tissues from a white-footed mouse were infected with spirochetes.     

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.     

<Emphasis Type="Italic">Babesia microti</Emphasis> Infection in Europe

The majority of babesia infections in Europe are life-threatening and caused by Babesia divergens and B. bovis. Although Babesia microti has been detected in ticks from Switzerland, few if any cases of babesiosis have been caused by B. microti. This first reported case, diagnosed by serology, DNA detection, and microscopy, is additionally interesting because there appears to be coinfection with the Lyme disease organism, Borrelia burgdorferi.