Borrelia burgdorferi Outer Surface Protein C Is Not the Sole Determinant of Dissemination in Mammals.

Lyme disease in the United States is most often caused by Borrelia burgdorferi sensu stricto. After a tick bite, the patient may develop erythema migrans at that site. If hematogenous dissemination occurs, the patient may then develop neurologic manifestations, carditis, or arthritis. Host-pathogen interactions include factors that contribute to hematogenous dissemination to other body sites. Outer surface protein C (OspC), a surface-exposed lipoprotein of B. burgdorferi, is essential during the early stages of mammalian infection. There is a high degree of genetic variation at the ospC locus, and certain ospC types are more frequently associated with hematogenous dissemination in patients, suggesting that OspC may be a major contributing factor to the clinical outcome of B. burgdorferi infection. In order to evaluate the role of OspC in B. burgdorferi dissemination, ospC was exchanged between B. burgdorferi isolates with different capacities to disseminate in laboratory mice, and these strains were then tested for their ability to disseminate in mice. The results indicated that the ability of B. burgdorferi to disseminate in mammalian hosts does not depend on OspC alone. The complete genome sequences of two closely related strains of B. burgdorferi with differing dissemination phenotypes were determined, but a specific genetic locus that could explain the differences in the phenotypes could not be definitively identified. The animal studies performed clearly demonstrated that OspC is not the sole determinant of dissemination. Future studies of the type described here with additional borrelial strains will hopefully clarify the genetic elements associated with hematogenous dissemination.

Association between body size and reservoir competence of mammals bearing Borrelia burgdorferi at an endemic site in the northeastern United States.

BACKGROUND: The reservoirs for the Lyme disease agent, Borrelia burgdorferi, are dominated by several different small to medium sized mammals in eastern North America. FINDINGS: To experimentally assess the competence of different mammalian species to transmit this pathogen to ticks, we carried out quantitative species-specific PCR of individual nymphal Ixodes scapularis ticks, which had been collected as replete larvae from animals captured at a field site in eastern Connecticut and then allowed to molt in the laboratory. The mammals, in order of increasing body mass, were the white-footed mouse, pine vole, eastern chipmunk, gray squirrel, Virginia opossum, striped skunk, and common raccoon. The prevalence of infection in the nymphs and the counts of spirochetes in infected ticks allometrically scaled with body mass with exponents of -0.28 and -0.29, respectively. By species, the captured animals from the site differed significantly in the mean counts of spirochetes in the ticks recovered from them, but these associations could not be distinguished from an effect of body size per se. CONCLUSIONS: These empirical findings as well as inferences from modeling suggest that small mammals on the basis of their sizes are more competent as reservoirs of B. burgdorferi in this environment than medium-to large-sized mammals.

Multilocus sequence typing of Borrelia burgdorferi suggests existence of lineages with differential pathogenic properties in humans.

The clinical manifestations of Lyme disease, caused by Borrelia burgdorferi, vary considerably in different patients, possibly due to infection by strains with varying pathogenicity. Both rRNA intergenic spacer and ospC typing methods have proven to be useful tools for categorizing B. burgdorferi strains that vary in their tendency to disseminate in humans. Neither method, however, is suitable for inferring intraspecific relationships among strains that are important for understanding the evolution of pathogenicity and the geographic spread of disease. In this study, multilocus sequence typing (MLST) was employed to investigate the population structure of B. burgdorferi recovered from human Lyme disease patients. A total of 146 clinical isolates from patients in New York and Wisconsin were divided into 53 sequence types (STs). A goeBURST analysis, that also included previously published STs from the northeastern and upper Midwestern US and adjoining areas of Canada, identified 11 major and 3 minor clonal complexes, as well as 14 singletons. The data revealed that patients from New York and Wisconsin were infected with two distinct, but genetically and phylogenetically closely related, populations of B. burgdorferi. Importantly, the data suggest the existence of B. burgdorferi lineages with differential capabilities for dissemination in humans. Interestingly, the data also indicate that MLST is better able to predict the outcome of localized or disseminated infection than is ospC typing.

Differentiation of reinfection from relapse in recurrent Lyme disease.

BACKGROUND: Erythema migrans is the most common manifestation of Lyme disease. Recurrences are not uncommon, and although they are usually attributed to reinfection rather than relapse of the original infection, this remains somewhat controversial. We used molecular typing of Borrelia burgdorferi isolates obtained from patients with culture-confirmed episodes of erythema migrans to distinguish between relapse and reinfection. METHODS: We determined the genotype of the gene encoding outer-surface protein C (ospC) of B. burgdorferi strains detected in cultures of skin or blood specimens obtained from patients with consecutive episodes of erythema migrans. After polymerase-chain-reaction amplification, ospC genotyping was performed by means of reverse line-blot analysis or DNA sequencing of the nearly full-length gene. Most strains were further analyzed by determining the genotype according to the 16S-23S ribosomal RNA intergenic spacer type, multilocus sequence typing, or both. Patients received standard courses of antibiotics for erythema migrans. RESULTS: B. burgdorferi isolates obtained from 17 patients who received a diagnosis of erythema migrans between 1991 and 2011 and who had 22 paired episodes of this lesion (initial and second episodes) were available for testing. The ospC genotype was found to be different at each initial and second episode. Apparently identical genotypes were identified on more than one occasion in only one patient, at the first and third episodes, 5 years apart, but different genotypes were identified at the second and fourth episodes. CONCLUSIONS: None of the 22 paired consecutive episodes of erythema migrans were associated with the same strain of B. burgdorferi on culture. Our data show that repeat episodes of erythema migrans in appropriately treated patients were due to reinfection and not relapse. (Funded by the National Institutes of Health and the William and Sylvia Silberstein Foundation.).

Borrelia burgdorferi genotype predicts the capacity for hematogenous dissemination during early Lyme disease.

BACKGROUND: Lyme disease, the most common tickborne disease in the United States, is caused exclusively by Borrelia burgdorferi sensu stricto in North America. The present study evaluated the genotypes of >400 clinical isolates of B. burgdorferi recovered from patients from suburban New York City with early Lyme disease associated with erythema migrans; it is the largest number of borrelial strains from North America ever to be investigated. METHODS: Genotyping was performed by restriction fragment-length polymorphism polymerase chain reaction analysis of the 16S-23S ribosomal RNA spacer and reverse line blot analysis of the outer surface protein C gene (ospC). For some isolates, DNA sequence analysis was also performed. RESULTS: The findings showed that the 16S-23S ribosomal spacer and ospC are in strong linkage disequilibrium. Most B. burgdorferi genotypes characterized by either typing method were capable of infecting and disseminating in patients. However, a distinct subset of just 4 of the 16 ospC genotypes identified were responsible for >80% of cases of early disseminated Lyme disease. CONCLUSIONS: This study identified the B. burgdorferi genotypes that pose the greatest risk of causing hematogenous dissemination in humans. This information should be considered in the future development of diagnostic assays and vaccine preparations.

Effect of Borrelia burgdorferi genotype on the sensitivity of C6 and 2-tier testing in North American patients with culture-confirmed Lyme disease.

BACKGROUND: A potential concern with any serologic test to detect antibodies to Borrelia burgdorferi is whether the epitopes incorporated in the test provide sufficient cross-reactivity to detect infection with all of the pathogenic strains of the species. This is a particular concern for the C6 test, which is based on reactivity to a single peptide. METHODS: C6 testing and 2-tier testing were performed on acute-phase serum samples obtained from >158 patients with erythema migrans for whom the genotype of the borrelial isolate was defined on the basis of an analysis of the 16S-23S ribosomal DNA spacer region and/or on the genetic variation of the outer surface protein C gene (ospC). The sonicated whole cell-based enzyme-linked immunosorbent assay, the immunoblots used in the 2-tier testing, and the C6 assay all used antigens from B. burgdorferi sensu stricto strain B31. RESULTS: The sensitivity of C6 testing (69.5%) was greater than that of 2-tier testing (38.9%) (P<.001); the difference in sensitivity, however, was statistically significant only for patients infected with 2 of the 3 ribosomal spacer type-defined genotypes. The lower sensitivity of 2-tier testing was attributable to the low sensitivity of the immunoblot tests, rather than the first-tier enzyme-linked immunosorbent assay. There was also a trend for the sensitivity of 2-tier testing to vary according to the ospC genotype for the 14 genotypes represented in the study (P=.07); this relationship was not observed with C6 testing. CONCLUSIONS: Lack of sensitivity of the C6 test because of strain diversity seems less likely to be a limitation of this serologic test, compared with 2-tier testing in North American patients with early Lyme disease.

Borrelia burgdorferi sensu stricto is clonal in patients with early Lyme borreliosis.

Lyme borreliosis, the most commonly reported vector-borne disease in North America, is caused by the spirochete Borrelia burgdorferi. Given the extensive genetic polymorphism of B. burgdorferi, elucidation of the population genetic structure of the bacterium in clinical samples may be relevant for understanding disease pathogenesis and may have applicability for the development of diagnostic tests and vaccine preparations. In this investigation, the genetic polymorphism of the 16S-23S rRNA (rrs-rrlA) intergenic spacer and ospC was investigated at the sequence level in 127 clinical isolates obtained from patients with early Lyme borreliosis evaluated in suburban New York City. Sixteen distinct rrs-rrlA and 16 distinct ospC alleles were identified, representing virtually all of the genotypes previously found in questing Ixodes scapularis nymphs in this region. In addition, a new ospC group was identified in a single patient. The strong linkage observed between the chromosome-located rrs-rrlA and plasmid-borne ospC genes suggests a clonal structure of B. burgdorferi in these isolates, despite evidence of recombination at ospC.

MLST of housekeeping genes captures geographic population structure and suggests a European origin of Borrelia burgdorferi.

Lyme borreliosis, caused by the tick-borne bacterium Borrelia burgdorferi, has become the most common vector-borne disease in North America over the last three decades. To understand the dynamics of the epizootic spread and to predict the evolutionary trajectories of B. burgdorferi, accurate information on the population structure and the evolutionary relationships of the pathogen is crucial. We, therefore, developed a multilocus sequence typing (MLST) scheme for B. burgdorferi based on eight chromosomal housekeeping genes. We validated the MLST scheme on B. burgdorferi specimens from North America and Europe, comprising both cultured isolates and infected ticks. These data were compared with sequences for the commonly used genetic markers rrs-rrlA intergenic spacer (IGS) and the gene encoding the outer surface protein C (ospC). The study demonstrates that the concatenated sequences of the housekeeping genes of B. burgdorferi provide highly resolved phylogenetic signals and that the housekeeping genes evolve differently compared with the IGS locus and ospC. Using sequence data, the study reveals that North American and European populations of B. burgdorferi correspond to genetically distinct populations. Importantly, the MLST data suggest that B. burgdorferi originated in Europe rather than in North America as proposed previously.

Blackbirds and song thrushes constitute a key reservoir of Borrelia garinii, the causative agent of borreliosis in Central Europe.

Blackbirds (Turdus merula) and song thrushes (Turdus philomelos) were found to carry 95% of all spirochete-infected tick larvae among 40 bird species captured in Central Europe. More than 90% of the infections were typed as Borrelia garinii and Borrelia valaisiana. We conclude that thrushes are key players in the maintenance of these spirochete species in this region of Central Europe.

Fitness variation of Borrelia burgdorferi sensu stricto strains in mice.

Lyme borreliosis in North America is caused by the tick-borne spirochete Borrelia burgdorferi, a zoonotic bacterium that is able to persistently infect a wide range of vertebrate species. Given the pronounced strain structure of B. burgdorferi in the northeastern United States, we asked whether the fitness of the different genotypes varies among susceptible vertebrate hosts. The transmission dynamics of two genetically divergent human isolates of B. burgdorferi, BL206 and B348, were analyzed experimentally in white-footed mice and in C3H/HeNCrl mice over a time period of almost 3 months. We found that the initially high transmission efficiency from white-footed mice to ticks declined sharply for isolate B348 but remained considerably high for isolate BL206. In contrast, in C3H/HeNCrl mice, high transmission efficiency persisted for both isolates. Our findings provide proof-of-principle evidence for intrinsic fitness variation of B. burgdorferi strains in vertebrate host species, perhaps indicating the beginnings of adaptive radiation.

Fundamental processes in the evolutionary ecology of Lyme borreliosis.

The evolutionary ecology of many emerging infectious diseases, particularly vector-borne zoonoses, is poorly understood. Here, we aim to develop a biological, process-based framework for vector-borne zoonoses, using Borrelia burgdorferi sensu lato (s.l.), the causative agent of Lyme borreliosis in humans, as an example. We explore the fundamental biological processes that operate in this zoonosis and put forward hypotheses on how extrinsic cues and intrinsic dynamics shape B. burgdorferi s.l. populations. Additionally, we highlight possible epidemiological parallels between B. burgdorferi s.l. and other vector-borne zoonotic pathogens, including West Nile virus.

Epidemic spread of Lyme borreliosis, northeastern United States.

We examined the degree of host specialization of different strains of Borrelia burgdorferi, the tickborne pathogen that causes Lyme borreliosis in the northeastern United States. We first assessed the genetic population structures of B. burgdorferi in ticks obtained from different mammalian host species and in questing ticks sampled in a woodland ecosystem in Connecticut. By comparing the patterns found in our study with data from another cross-sectional study, we demonstrate that B. burgdorferi is a generalist microparasite and conclude that efficient cross-species transmission of B. burgdorferi is a key feature that has allowed the rapid spread of Lyme borreliosis across the northeastern United States.

Association of Borrelia garinii and B. valaisiana with songbirds in Slovakia.

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.

The key roles of selection and migration in the ecology of Lyme borreliosis.

The roles of selection and migration of B. burgdorferi s. l. were studied. Questing adult Ixodes ricinus ticks were collected across Europe and analysed for infection with B. burgdorferi s. l. Analysis of the genospecies in individual ticks showed that B. garinii and B. valaisiana segregate from B. afzelii. Segregation of bird- and rodent-associated Borrelia genotypes can be explained by the operation of complement-mediated selection in the midgut of the feeding tick. Phylogenetic analyses of B. burgdorferi s. l. indicate high rates of migration for bird-associated genotypes. Altogether, it is emerging that the ecology of Lyme borreliosis is largely host-driven and that selection and migration are major forces shaping the population structures of B. burgdorferi s. l.