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.     

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.     

Interdependence of environmental factors influencing reciprocal patterns of gene expression in virulent Borrelia burgdorferi

The paradigm for differential antigen expression in Borrelia burgdorferi, the agent of Lyme disease, is the reciprocal expression of its outer surface (lipo)proteins (Osp) A and C; as B. burgdorferi transitions from its arthropod vector into mammalian tissue, ospC is upregulated, and ospA is downregulated. In the current study, using B. burgdorferi cultivated under varying conditions in BSK-H medium, we found that a decrease in pH, in conjunction with increases in temperature (e.g. 34 degrees C or 37 degrees C) and cell density, acted interdependently for the reciprocal expression of ospC and ospA. The lower pH (6.8), which induced the reciprocal expression of ospC and ospA in BSK-H medium, correlated with a drop in pH from 7.4 to 6.8 of tick midgut contents during tick feeding. In addition to ospC and ospA, other genes were found to be regulated in reciprocal fashion. Such genes were either ospC-like (e.g. ospF, mlp-8 and rpoS) (group I) or ospA-like (lp6.6 and p22) (group II); changes in expression occurred at the mRNA level. That the expression of rpoS, encoding a putative stress-related alternative sigma factor (sigma(s)), was ospC-like suggested that the expression of some of the group I genes may be controlled through sigma(s). The combined results prompt a model that allows for predicting the regulation of other B. burgdorferi genes that may be involved in spirochaete transmission, virulence or mammalian host immune responses.     

Analysis of promoters in Borrelia burgdorferi by use of a transiently expressed reporter gene.

A transient chloramphenicol acetyltransferase (CAT) expression system was developed for Borrelia burgdorferi. An Escherichia coli vector containing a promoterless Streptococcus agalactiae cat gene was constructed. Promoters for ospA, ospC, and flaB were placed upstream of this cat gene, and CAT assays were performed in E. coli from these stably maintained plasmids. The plasmids with putative promoters ospA and flaB were found to be approximately 20-fold more active than were the plasmids with ospC or no promoter. The level of activity correlated well with the resistance to chloramphenicol that each plasmid provided. Next, the nonreplicative plasmid constructs were transformed by electroporation into B. burgdorferi. CAT assays were performed by both thin-layer chromatography and the fluor diffusion method. Measurement of CAT activity demonstrated that the ospA promoter was again about 20-fold more active than the promoterless cat gene. The flaB and ospC promoters increased the activity seven- and threefold, respectively, over that with the promoterless construct. This simple transient-expression assay was shown to be an effective method to study promoter function in B. burgdorferi in the absence of a well-developed genetic system.     

ospC diversity in Borrelia burgdorferi: different hosts are different niches

The outer surface protein C (ospC) locus of the Lyme disease bacterium, Borrelia burgdorferi, is at least an order of magnitude more variable than other genes in the species. This variation is classified into 22 ospC major groups, 15 of which are found in the northeastern United States. The frequency distributions of ospC within populations suggest that this locus is under balancing selection. In multiple-niche polymorphism, a type of balancing selection, diversity within a population can be maintained when the environment is heterogeneous and no one genotype has the highest fitness in all environments. Genetically different individuals within vertebrate species and different vertebrate species constitute diverse environments for B. burgdorferi. We examined four important host species of B. burgdorferi and found that the strains that infected each species had different sets of ospC major groups. We found no variation among conspecific hosts in the ospC major groups of their infecting strains. These results suggest multiple niches create balancing selection at the ospC locus.     

Pervasive recombination and sympatric genome diversification driven by frequency-dependent selection in Borrelia burgdorferi, the Lyme disease bacterium

How genomic diversity within bacterial populations originates and is maintained in the presence of frequent recombination is a central problem in understanding bacterial evolution. Natural populations of Borrelia burgdorferi, the bacterial agent of Lyme disease, consist of diverse genomic groups co-infecting single individual vertebrate hosts and tick vectors. To understand mechanisms of sympatric genome differentiation in B. burgdorferi, we sequenced and compared 23 genomes representing major genomic groups in North America and Europe. Linkage analysis of >13,500 single-nucleotide polymorphisms revealed pervasive horizontal DNA exchanges. Although three times more frequent than point mutation, recombination is localized and weakly affects genome-wide linkage disequilibrium. We show by computer simulations that, while enhancing population fitness, recombination constrains neutral and adaptive divergence among sympatric genomes through periodic selective sweeps. In contrast, simulations of frequency-dependent selection with recombination produced the observed pattern of a large number of sympatric genomic groups associated with major sequence variations at the selected locus. We conclude that negative frequency-dependent selection targeting a small number of surface-antigen loci (ospC in particular) sufficiently explains the maintenance of sympatric genome diversity in B. burgdorferi without adaptive divergence. We suggest that pervasive recombination makes it less likely for local B. burgdorferi genomic groups to achieve host specialization. B. burgdorferi genomic groups in the northeastern United States are thus best viewed as constituting a single bacterial species, whose generalist nature is a key to its rapid spread and human virulence.     

Two Boundaries Separate Borrelia burgdorferi Populations in North America

Understanding the spread of infectious diseases is crucial for implementing effective control measures. For this, it is important to obtain information on the contemporary population structure of a disease agent and to infer the evolutionary processes that may have shaped it. Here, we investigate on a continental scale the population structure of Borrelia burgdorferi, the causative agent of Lyme borreliosis (LB), a tick-borne disease, in North America. We test the hypothesis that the observed population structure is congruent with recent population expansions and that these were preceded by bottlenecks mostly likely caused by the near extirpation in the 1900s of hosts required for sustaining tick populations. Multilocus sequence typing and complementary population analytical tools were used to evaluate B. burgdorferi samples collected in the Northeastern, Upper Midwestern, and Far-Western United States and Canada. The spatial distribution of sequence types (STs) and inferred population boundaries suggest that the current populations are geographically separated. One major population boundary separated western B. burgdorferi populations transmitted by Ixodes pacificus in California from Eastern populations transmitted by I. scapularis; the other divided Midwestern and Northeastern populations. However, populations from all three regions were genetically closely related. Together, our findings suggest that although the contemporary populations of North American B. burgdorferi now comprise three geographically separated subpopulations with no or limited gene flow among them, they arose from a common ancestral population. A comparative analysis of the B. burgdorferi outer surface protein C (ospC) gene revealed novel linkages and provides additional insights into the genetic characteristics of strains.     

The role of lizards in the ecology of Lyme disease in two endemic zones of the northeastern United States

We examined the role of lizards in the ecology of Lyme disease in New York and Maryland. We collected data on vector tick infestations, measured lizard "realized" reservoir competence for the Lyme disease spirochete Borrelia burgdorferi, and estimated lizard population density. These data were incorporated into a model that predicts a host's ability to influence the prevalence of B. burgdorferi in the tick population, a primary risk factor in the epidemiology of Lyme disease. Published data on other northeastern hosts were included in the model to provide a reference for interpreting the importance of lizards as hosts. The model results indicate that 5-lined skinks (Eumeces fasciatus) are dilution hosts, capable of reducing infection prevalence in the tick population by 10.7-51.5 percentage points, whereas eastern fence lizards (Sceloporus undulatus) are not dilution hosts in the areas studied. Owing to moderate burdens of larval ticks, relatively high population densities, and reservoir incompetence, E. fasciatus may play an important role in the ecology of Lyme disease by reducing vector infection prevalence and associated human risk of infection.     

Borrelia burgdorferi and Babesia microti: efficiency of transmission from reservoirs to vector ticks (Ixodes dammini)

In endemic regions, Peromyscus leucopus, the mouse reservoir of the Lyme disease spirochete (Borrelia burgdorferi) and the piroplasm causing human babesiosis (Babesia microti), is nearly universally infected with both agents. Paradoxically, spirochetal infection is nearly twice as prevalent as is babesial infection in populations of field-collected nymphal Ixodes dammini, the tick vector. In the laboratory, a similarly disproportionate rate of infection was observed among nymphal ticks, feeding as larvae, on either B. burgdorferi- or B. microti-infected mice. Ticks which fed on mice with concurrent spirochetal and babesial infections also exhibited twice the incidence of spirochetal infection over that of the piroplasm. These data suggest that the efficiency of acquisition and transstadial passage of B. burgdorferi and B. microti infection differ by a factor of two. This discrepancy may explain differences observed both in the prevalence of infection in ticks collected in the field, as well as the apparently greater risk of spirochetal infection to humans in endemic areas.     

Distribution of Borrelia burgdorferi s.l. spirochaete DNA in British ticks (Argasidae and Ixodidae) since the 19th Century, assessed by PCR

The distribution of Borrelia burgdorferi sensu lato , the Lyme borreliosis agent, was surveyed in British ticks in the collection of the Natural History Museum, London. Alcohol-preserved specimens of eight species of ticks known to attack humans were studied: Ixodes ricinus , I. hexagonus , I. uriae , I. trianguliceps , Dermacentor reticulatus , Haemaphysalis punctata , Rhipicephalus sanguineus and Argas vespertilionis. The sample comprised all life stages and originated from a wide range of host species, collection dates (1896–1994) and geographical localities in England, Scotland and Wales.
Borrelia burgdorferi s. l. DNA, detected by a polymerase chain reaction that targeted the outer surface protein A gene, was found in all eight species. The overall proportion of PCR-positive specimens ranged from 7.8% for I. hexagonus (mostly from mustelids and hedgehogs) to 98.3% for I. uriae (mostly from seabirds). Borrelia burgdorferi s. l. DNA was found for the first time in the bat parasite A. vespertilionis (85.3%). The spirochaete is newly recorded in British populations of I. trianguliceps (97.4%, mostly from voles, mice and shrews), D. reticulatus (12.5% from dog and man) and R. sanguineus (30% from dogs and human dwellings). Of the four tick species with larvae available for testing, examples of I. ricinus, I. uriae and A. vespertilionis were PCR positive, as were significantly more nymphs than adults of I. ricinus, I. hexagonus and A. vespertilionis. Analyses showed that B. burgdorferi s. l. has been consistently present in British tick populations since at least 1897. Ticks positive for B. burgdorferi s. l. DNA were collected in all months of the year, throughout Britain, and were found on a wide range of mammal and bird species. PCR positivity does not prove vector or reservoir competence, but the use of archived material has demonstrated an extensive range of host–tick relationships involving B. burgdorferi s.l. in Britain for >100 years.     

Methods for estimating gene frequencies and detecting selection in bacterial populations

Recent breakthroughs in molecular technology, most significantly the polymerase chain reaction (PCR) and in situ hybridization, have allowed the detection of genetic variation in bacterial communities without prior cultivation. These methods often produce data in the form of the presence or absence of alleles or genotypes, however, rather than counts of alleles. Using relative allele frequencies from presence-absence data as estimates of population allele frequencies tends to underestimate the frequencies of common alleles and overestimate those of rare ones, potentially biasing the results of a test of neutrality in favor of balancing selection. In this study, a maximum-likelihood estimator (MLE) of bacterial allele frequencies designed for use with presence-absence data is derived using an explicit stochastic model of the host infection (or bacterial sampling) process. The performance of the MLE is evaluated using computer simulation and a method is presented for evaluating the fit of estimated allele frequencies to the neutral infinite alleles model (IAM). The methods are applied to estimate allele frequencies at two outer surface protein loci (ospA and ospC) of the Lyme disease spirochete, Borrelia burgdorferi, infecting local populations of deer ticks (Ixodes scapularis) and to test the fit to a neutral IAM.     

Molecular identification and analysis of Borrelia burgdorferi sensu lato in lizards in the southeastern United States

Lyme borreliosis (LB) group spirochetes, collectively known as Borrelia burgdorferi sensu lato, are distributed worldwide. Wild rodents are acknowledged as the most important reservoir hosts. Ixodes scapularis is the primary vector of B. burgdorferi sensu lato in the eastern United States, and in the southeastern United States, the larvae and nymphs mostly parasitize certain species of lizards. The primary aim of the present study was to determine whether wild lizards in the southeastern United States are naturally infected with Lyme borreliae. Blood samples obtained from lizards in Florida and South Carolina were tested for the presence of LB spirochetes primarily by using B. burgdorferi sensu lato-specific PCR assays that amplify portions of the flagellin (flaB), outer surface protein A (ospA), and 66-kDa protein (p66) genes. Attempts to isolate spirochetes from a small number of PCR-positive lizards failed. However, PCR amplification and sequence analysis of partial flaB, ospA, and p66 gene fragments confirmed numerous strains of B. burgdorferi sensu lato, including Borrelia andersonii, Borrelia bissettii, and B. burgdorferi sensu stricto, in blood from lizards from both states. B. burgdorferi sensu lato DNA was identified in 86 of 160 (54%) lizards representing nine species and six genera. The high infection prevalence and broad distribution of infection among different lizard species at different sites and at different times of the year suggest that LB spirochetes are established in lizards in the southeastern United States.     

Evolution of the Borrelia burgdorferi outer surface protein OspC.

The genes coding for outer surface protein OspC from 22 Borrelia burgdorferi strains isolated from patients with Lyme borreliosis were cloned and sequenced. For reference purposes, the 16S rRNA genes from 17 of these strains were sequenced after being cloned. The deduced OspC amino acid sequences were aligned with 12 published OspC sequences and revealed the presence of 48 conserved amino acids. On the basis of the alignment, OspC could be divided into an amino-terminal relatively conserved region and a relatively variable region in the central portion. The distance tree obtained divided the ospC sequences into three groups. The first group contained ospC alleles from all (n = 13) sensu stricto strains, the second group contained ospC alleles from seven Borrelia afzelii strains, and the third group contained ospC alleles from five B. afzelii and all (n = 9) Borrelia garinii strains. The ratio of the mean number of synonymous (dS) and nonsynonymous (dN) nucleotide substitutions per site calculated for B. burgdorferi sensu stricto, B. garinii, and B. afzelii ospC alleles suggested that the polymorphism of OspC is due to positive selection favoring diversity at the amino acid level in the relatively variable region. On the basis of the comparison of 16S rRNA gene sequences, Borrelia hermsii is more closely related to B. afzelii than to B. burgdorferi sensu stricto and B. garinii. In contrast, the phylogenetic tree obtained for the B. hermsii variable major protein, Vmp33, and 18 OspC amino acid sequences suggested that Vmp33 and OspC from B. burgdorferi sensu stricto strains share a common evolutionary origin.     

Genetic structure of marine Borrelia garinii and population admixture with the terrestrial cycle of Lyme borreliosis

Despite the importance of population structure for the epidemiology of pathogenic bacteria, the spatial and ecological heterogeneity of these populations is often poorly characterized. Here, we investigated the genetic diversity and population structure of the Lyme borreliosis (LB) spirochaete Borrelia garinii in its marine cycle involving colonial seabirds and different host races of the seabird tick Ixodes uriae. Multilocus sequence analyses (MLSA) on eight chromosomal and two plasmid loci (ospA and ospC) indicate that B. garinii circulating in the marine system is highly diverse. Microevolution in marine B. garinii seems to be mainly clonal, but recombination and selection do occur. Sequence types were not evenly distributed among geographic regions, with substantial population subdivision between Atlantic and Pacific Ocean basins. However, no geographic structuring was evident within regions. Results of selection analyses and phylogenetic discordance between chromosomal and plasmid loci indicate adaptive evolution is likely occurring in this system, but no pattern of host or vector-associated divergence was found. Recombination analyses showed evidence for population admixture between terrestrial and marine strains, suggesting that LB spirochaetes are exchanged between these enzootic cycles. Importantly, our results highlight the need to explicitly consider the marine system for a complete understanding of the evolutionary ecology and global epidemiology of Lyme borreliosis.     

Diversity of Borrelia burgdorferi sensu lato in Russian Far East

Thirty strains of Borrelia burgdorferi sensu lato have been isolated from Ixodes persulcatus ticks and from skin lesions of Lyme disease patients in the Russian Far East from 1997 to 2003. We amplified full-length outer surface protein A (ospA) gene of all strains. BLAST search and following phylogenetic analysis showed that strains form four well-defined groups. Four strains belong to Borrelia afzelii species. Other strains distributed into tree major groups, identified as Borrelia garinii. Indeed, based on the ospA gene comparison, phylogenetic relationship of these groups among each other does not differ from relationship among other previously defined groups inside B. burgdorferi sensu lato genogroup, such as B. afzelii or Borrelia bissettii. Further investigations of genetic and serologic properties of the strains belonging to those groups are required in order to clarify their taxonomic status.     

Outer surface protein B is critical for Borrelia burgdorferi adherence and survival within Ixodes ticks

Survival of Borrelia burgdorferi in ticks and mammals is facilitated, at least in part, by the selective expression of lipoproteins. Outer surface protein (Osp) A participates in spirochete adherence to the tick gut. As ospB is expressed on a bicistronic operon with ospA, we have now investigated the role of OspB by generating an OspB-deficient B. burgdorferi and examining its phenotype throughout the spirochete life cycle. Similar to wild-type isolates, the OspB-deficient B. burgdorferi were able to readily infect and persist in mice. OspB-deficient B. burgdorferi were capable of migrating to the feeding ticks but had an impaired ability to adhere to the tick gut and survive within the vector. Furthermore, the OspB-deficient B. burgdorferi bound poorly to tick gut extracts. The complementation of the OspB-deficient spirochete in trans, with a wild-type copy of ospB gene, restored its ability to bind tick gut. Taken together, these data suggest that OspB has an important role within Ixodes scapularis and that B. burgdorferi relies upon multiple genes to efficiently persist in ticks.