Comprehensive analyses of transcriptomes induced by Lyme spirochete infection to CNS model system.

BACKGROUND: Lyme disease is a zoonotic disease caused by infection with Borrelia burgdorferi (Bb), the involvement of the nervous system in Lyme disease is usually referred to as Lyme neuroborreliosis (LNB). LNB has diverse clinical manifestations, most commonly including meningitis, Bell's palsy, and encephalitis. However, the molecular pathogenesis of neuroborreliosis is still poorly understood. Comprehensive transcriptomic analysis following Bb infection could provide new insights into the pathogenesis of LNB and may identify novel biomarkers or therapeutic targets for LNB diagnosis and treatment. METHODS: In the present study, we pooled transcriptomic dataset of Macaca mulatta (rhesus) from our laboratory and the human astrocyte dataset GSE85143 from the Gene Expression Omnibus database to screen common differentially expressed genes (DEGs) in the Bb infection group and the control group. Functional and enrichment analyses were applied for the DEGs. Protein-Protein Interaction network, and hub genes were identified using the Search Tool for the Retrieval of Interaction Genes database and the CytoHubba plugin. Finally, mRNA expression of hub genes was validated in vitro and ex vivo from Bb infected models and normal controls by quantitative reverse transcription PCR (qRT-PCR). RESULTS: A total of 80 upregulated DEGs and 32 downregulated DEGs were identified. Among them, 11 hub genes were selected. The pathway enrichment analyses on 11 hub genes revealed that the PI3K-Akt signaling pathway was significantly enriched. The mRNA levels of ANGPT1, TLR6, SREBF1, LDLR, TNC, and ITGA2 in U251 cells and/or rhesus brain explants by exposure to Bb were validated by qRT-PCR. CONCLUSION: Our study suggested that TLR6, ANGPT1, LDLR, SREBF1, TNC, and ITGA may be candidate mammal biomarkers for LNB, and the TLR6/PI3K-Akt signaling pathway may play an important role in LNB pathogenesis.

An unexpected case of Borrelia garinii liver infection.

BACKGROUND: Lyme borreliosis is the most prevalent arthropod-borne infection in the Northern Hemisphere. In Europe, Borrelia afzelii is predominantly involved in cutaneous manifestations, Borrelia garinii and Borrelia bavariensis in neurological manifestations, and Borrelia burgdorferi sensu stricto in articular ones. Liver impairement is not classical in Lyme borreliosis. Diagnosis is currently mainly based on serological testing, and is challenging in immunocompromised patients. CASE PRESENTATION: We report the first case of B. garinii infection revealed by liver involvement in an immunocompromised man. A 73-year-old man with marginal zone lymphoma, treated with bendamustine and rituximab, developed intermittent fever and inflammatory syndrome. Microbial investigations were all negative and FDG-PET showed complete remission of the lymphoma. Three months later, liver biopsy was performed and histology revealed spirochetes-like bacteria. Microbial diagnosis was performed by 16S rDNA sequencing, flagellin (flaB) gene sequencing and multi-locus sequence typing and identified B. garinii. The patient recovered successfully after a three weeks course of antibiotics. Diagnosis was challenging because Borrelia hepatic involvement is unusual and no erythema migrans nor tick bite were notified. CONCLUSION: This case highlights that unexplained fever and inflammatory syndrome in immunocompromised patients warrants specific investigations to identify bacteria such as spirochetes.

First isolation of Borrelia lusitaniae DNA from green lizards (Lacerta viridis) and Ixodes ricinus ticks in the Czech Republic.

This study aimed to reveal whether green lizards (Lacerta viridis), common hosts of tick larvae and nymphs, might be involved in the transmission cycle of Borrelia burgdorferi sensu lato in the Czech Republic. Green lizards were sampled in two areas at the Tiché Údolí Nature Reserve (site A: 50.1482 N, 14.3669E; site B: 50.1476 N, 14.3745 E), Central Bohemian Region, Czech Republic. The skin biopsy specimens and attached ticks (if any) were collected from 52 captured lizards. Also, questing ticks from both areas were collected by flagging. The touchdown polymerase chain reaction and gel electrophoresis revealed Borrelia lusitaniae in three lizard tissue samples. Most lizards (19/30, 63%) had at least one Borrelia positive tick. Borrelia lusitaniae formed 92% (34/37) and 59% (17/29) of all borreliae detected in larvae and nymphs, respectively. Borrelia lusitaniae (6/10, 60%) was also the major pathogen in questing nymphs from site B. At site A, 13% (2/16) of questing nymphs were positive for B. lusitaniae. Based on our data, it can be assumed that B. lusitaniae is a common pathogen at lizard sites in the Czech Republic, and further research to prove this hypothesis is therefore highly recommended. As lizards often inhabit urban areas, the data presented may also contribute to raising awareness of the possible spread and risk of Borrelia infection.

Molecular Microbiological and Immune Characterization of a Cohort of Patients Diagnosed with Early Lyme Disease.

Lyme disease is a tick-borne infection caused by the bacteria Borrelia burgdorferi Current diagnosis of early Lyme disease relies heavily on clinical criteria, including the presence of an erythema migrans rash. The sensitivity of current gold-standard diagnostic tests relies upon antibody formation, which is typically delayed and thus of limited utility in early infection. We conducted a study of blood and skin biopsy specimens from 57 patients with a clinical diagnosis of erythema migrans. Samples collected at the time of diagnosis were analyzed using an ultrasensitive, PCR-based assay employing an isothermal amplification step and multiple primers. In 75.4% of patients, we directly detected one or more B. burgdorferi genotypes in the skin. Two-tier testing showed that 20 (46.5%) of those found to be PCR positive remained serologically negative at both acute and convalescent time points. Multiple genotypes were found in three (8%) of those where a specific genotype could be identified. The 13 participants who lacked PCR and serologic evidence for exposure to B. burgdorferi could be differentiated as a group from PCR-positive participants by their levels of several immune markers as well as by clinical descriptors such as the number of acute symptoms and the pattern of their erythema migrans rash. These results suggest that within a Mid-Atlantic cohort, patient subgroups can be identified using PCR-based direct detection approaches. This may be particularly useful in future research such as vaccine trials and public health surveillance of tick-borne disease patterns.

Functional and Genomic Architecture of Borrelia burgdorferi-Induced Cytokine Responses in Humans.

Despite the importance of immune variation for the symptoms and outcome of Lyme disease, the factors influencing cytokine production during infection with the causal pathogen Borrelia burgdorferi remain poorly understood. Borrelia infection-induced monocyte- and T cell-derived cytokines were profiled in peripheral blood from two healthy human cohorts of Western Europeans from the Human Functional Genomics Project. Both non-genetic and genetic host factors were found to influence Borrelia-induced cytokine responses. Age strongly impaired IL-22 responses, and genetic studies identified several independent QTLs that impact Borrelia-induced cytokine production. Genetic, transcriptomic, and functional validation studies revealed an important role for HIF-1α-mediated glycolysis in the cytokine response to Borrelia. HIF-1α pathway activation and increase in glycolysis-derived lactate was confirmed in Lyme disease patients. In conclusion, functional genomics approaches reveal the architecture of cytokine production induced by Borrelia infection of human primary leukocytes and suggest a connection between cellular glucose metabolism and Borrelia-induced cytokine production.

Detection of Borrelia burgdorferi sensu stricto and Borrelia garinii DNAs in patient with Hyperkeratosis lenticularis perstans (Flegel disease).

Determination of the causative agent of erythema-like skin lesions in case of nonspecific superficial perivascular dermatitis was supported by histological examination and led to the latter diagnosis of Hyperkeratosis lenticularis perstans (Flegel disease) in patient. The presence of antibodies against Borrelia burgdorferi in patient serum was confirmed by a routine ELISA method and verified by Western blot technique. Skin biopsy and blood specimens were analyzed by PCR and multilocus sequence analysis (MLSA). Western blot method revealed IgG antibody response against two specific antigens, 17 and 83 kDa proteins. The recombinant test detected IgG antibody response against p100 and p41 antigens. The sequence analysis of amplicons from the selected genomic loci obtained from skin biopsy and serum samples revealed the presence of two species from B. burgdorferi sensu lato complex as a co-infection in this patient-B. burgdorferi sensu stricto (s.s.) and Borrelia garinii.

[Evaluation of 10 cases of Lyme disease presenting with erythema migrans in Istanbul, Turkey]. / Istanbul'da eritema migrans ile basvuran 10 Lyme olgusunun degerlendirilmesi.

Lyme disease (LD) is a tick-borne, multisystemic infection caused by Borrelia burgdorferi. Although variable rates of seropositivity for B.burgdorferi have been reported between 2% to 44% in Turkey, its actual prevalence is not well-understood. The aim of this retrospective study was to evaluate the characteristics of 10 cases of LD presenting as erythema migrans (EM) between 2009 and 2013 from Istanbul which is one of the metropolitan cities of Turkey. Of the patients, five were male and five were female, ages between 9-51 years (mean age: 34.5 years). Five of the patients were admitted in June, three in October, one in November and two in December and all have the history of tick bite in last 1-2 weeks. There were no clinical symptoms for systemic infection among the patients with normal level routine laboratory test (whole blood count and biochemical tests) results. Five of the cases had EM lesions in the trunk, three in the upper extremities, and two in the lower extremities. Four patients presented with annular, three with solitary macular, and three with target-like EM lesions. In all cases, the biopsy specimens were positive for B.burgdorferi sensu lato DNA with polymerase chain reaction and all were also positive in terms of B.burgdorferi IgM antibodies with ELISA. Nine patients were treated with oral doxycycline, 100 mg twice daily and one child patient was treated with oral amoxicillin 500 mg twice daily for 21 days. EM lesions disappeared within 2-4 weeks in all patients. There was no clinical evidence for systemic involvement in any of the patients like neurologic, cardiac, and joint involvement at the follow-ups on the third, sixth and 12(th) months. To our best knowledge, 10 patients in this study are the largest EM series reported from Turkey. The increase in the number of LD cases may be associated with increased tick bite and increased awareness due to the emergence of concurrent Crimean-Congo hemorrhagic fever epidemic in Turkey. As a result, when enlarged erythematous lesions on the skin were observed, LH must also be considered in differential diagnosis, history of tick bite should be questioned and etiological diagnostic test should be performed.

Histopathology and immunophenotype of acrodermatitis chronica atrophicans correlated with ospA and ospC genotypes of Borrelia species.

BACKGROUND: Chronic cutaneous borreliosis (acrodermatitis chronica atrophicans, ACA) is a relatively rare manifestation of borreliosis attributed mainly to Borrelia afzelii. Chronic borreliosis has been associated with ospA and ospC genotypes. Literature on molecular investigations of Borrelia in lesions of ACA is scant. METHODS: Histopathological and immmunohistochemical features in 22 biopsies of ACA (16 patients) were examined. Paraffin-embedded biopsies were analyzed with polymerase chain reaction (PCR) assays targeting ospA and ospC genes, sequencing and phylogenetic analysis. RESULTS: Genotyping of ospA identified B. afzelii, serotype 2, in 12 of 16 patients. ospC-PCR was positive in seven patients revealing genotypes Af5 (n = 4), Af2 (n = 2) and Af6 (n = 1). Histopathologically, interstitial granulomatous infiltrates (CD68 positive) were common, combined with thickened collagen bundles and band-like infiltrates of CD4 positive T lymphocytes. Plasma cells were sparse/absent in 9 of 22 specimens even on staining with CD138. On CD34-staining, interstitial fibroblasts were often reduced akin to the situation in morphea. CONCLUSIONS: With assays targeting ospA and ospC genes we confirmed from paraffin-embedded biopsies that B. afzelii, serotype 2, osp C groups Af5, Af2 and Af6 is the main cause of ACA. Specimens commonly showed a combination of band-like T-cell-rich infiltrates with interstitial granulomatous features, a pattern previously under-recognized in ACA. This finding was particularly typical for lesions infected with ospC genotype Af5.

Geographical and genospecies distribution of Borrelia burgdorferi sensu lato DNA detected in humans in the USA.

The present study investigated the cause of illness in human patients primarily in the southern USA with suspected Lyme disease based on erythema migrans-like skin lesions and/or symptoms consistent with early localized or late disseminated Lyme borreliosis. The study also included some patients from other states throughout the USA. Several PCR assays specific for either members of the genus Borrelia or only for Lyme group Borrelia spp. (Borrelia burgdorferi sensu lato), and DNA sequence analysis, were used to identify Borrelia spp. DNA in blood and skin biopsy samples from human patients. B. burgdorferi sensu lato DNA was found in both blood and skin biopsy samples from patients residing in the southern states and elsewhere in the USA, but no evidence of DNA from other Borrelia spp. was detected. Based on phylogenetic analysis of partial flagellin (flaB) gene sequences, strains that clustered separately with B. burgdorferi sensu stricto, Borrelia americana or Borrelia andersonii were associated with Lyme disease-like signs and symptoms in patients from the southern states, as well as from some other areas of the country. Strains most similar to B. burgdorferi sensu stricto and B. americana were found most commonly and appeared to be widely distributed among patients residing throughout the USA. The study findings suggest that human cases of Lyme disease in the southern USA may be more common than previously recognized and may also be caused by more than one species of B. burgdorferi sensu lato. This study provides further evidence that B. burgdorferi sensu stricto is not the only species associated with signs and/or symptoms consistent with Lyme borreliosis in the USA.

Inter- and intra-specific pan-genomes of Borrelia burgdorferi sensu lato: genome stability and adaptive radiation.

BACKGROUND: Lyme disease is caused by spirochete bacteria from the Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) species complex. To reconstruct the evolution of B. burgdorferi s.l. and identify the genomic basis of its human virulence, we compared the genomes of 23 B. burgdorferi s.l. isolates from Europe and the United States, including B. burgdorferi sensu stricto (B. burgdorferi s.s., 14 isolates), B. afzelii (2), B. garinii (2), B. "bavariensis" (1), B. spielmanii (1), B. valaisiana (1), B. bissettii (1), and B. "finlandensis" (1). RESULTS: Robust B. burgdorferi s.s. and B. burgdorferi s.l. phylogenies were obtained using genome-wide single-nucleotide polymorphisms, despite recombination. Phylogeny-based pan-genome analysis showed that the rate of gene acquisition was higher between species than within species, suggesting adaptive speciation. Strong positive natural selection drives the sequence evolution of lipoproteins, including chromosomally-encoded genes 0102 and 0404, cp26-encoded ospC and b08, and lp54-encoded dbpA, a07, a22, a33, a53, a65. Computer simulations predicted rapid adaptive radiation of genomic groups as population size increases. CONCLUSIONS: Intra- and inter-specific pan-genome sizes of B. burgdorferi s.l. expand linearly with phylogenetic diversity. Yet gene-acquisition rates in B. burgdorferi s.l. are among the lowest in bacterial pathogens, resulting in high genome stability and few lineage-specific genes. Genome adaptation of B. burgdorferi s.l. is driven predominantly by copy-number and sequence variations of lipoprotein genes. New genomic groups are likely to emerge if the current trend of B. burgdorferi s.l. population expansion continues.

Seronegative lyme neuroborreliosis in a patient using rituximab.

A 66-year-old woman presented with severe shooting pains throughout her back and legs, followed by progressive deafness, weight loss and headache. She had a history of marginal zone B-cell lymphoma stage IV-B, for which she was successfully treated with immunochemotherapy and rituximab maintenance therapy. A relapse was suspected, but chemotherapy was not administered, since, despite elaborate investigations, malignancy could not be proven. Because of a history of tick bites she was tested for antibodies against Borrelia burgdorferi in serum and cerebrospinal fluid (CSF), which were negative. However, a B burgdorferi PCR on CSF came back positive. The patient was treated for seronegative Lyme neuroborreliosis with ceftriaxone intravenously and dramatically improved. This case presentation demonstrates that, in immunocompromised patients, it is important not to solely rely on antibody testing and to use additional diagnostic tests to avoid missing or delaying the diagnosis.

Ability to cause erythema migrans differs between Borrelia burgdorferi sensu lato isolates.

BACKGROUND: Lyme borreliosis is a tick-borne disease caused by Borrelia burgdorferi sensu lato. The variety of characteristic and non-specific clinical manifestations is partially explained by its genetic diversity. We investigated the ability of B. burgdorferi sl isolates to cause erythema migrans. METHODS: The genetic constellation of isolates from ticks was compared to isolates found in erythema migrans. PCR and sequence analysis was performed on the plasmid-encoded ospC and the chromosomal 5S-23S rDNA spacer region (IGS). RESULTS: Seven different B. burgdorferi sl genospecies were identified in 152 borrelia isolates from ticks and erythema migrans biopsies. B afzelii (51%) and B. garinii (27%) were the most common in ticks. From the 44 sequences obtained from erythema migrans samples 42 were B. afzelii, one B. garinii and one B. bavariensis. Significant associations with erythema migrans formation were found for four IGS and two ospC types. Five from 45 ospC types were associated with more than one genospecies. CONCLUSIONS: B. burgdorferi sl isolates differ in their propensity to cause erythema migrans. These differences were also found within genospecies. In other words, although B. afzelii was mostly associated with erythema migrans, some B. afzelii isolates had a low ability to cause erythema migrans. Our data further support the occurrence of plasmid exchange between borrelia genospecies under natural conditions.

Explosive radiation of a bacterial species group.

The current diversity of life on earth is the product of macroevolutionary processes that have shaped the dynamics of diversification. Although the tempo of diversification has been studied extensively in macroorganisms, much less is known about the rates of diversification in the exceedingly diverse and species-rich microbiota. Decreases in diversification rates over time, a signature of explosive radiations, are commonly observed in plant and animal lineages. However, the few existing analyses of microbial lineages suggest that the tempo of diversification in prokaryotes may be fundamentally different. Here, we use multilocus and genomic sequence data to test hypotheses about the rate of diversification in a well-studied pathogenic bacterial lineage, Borrelia burgdorferi sensu lato (sl). Our analyses support the hypothesis that an explosive radiation of lineages occurred near the origin of the clade, followed by a sharp decay in diversification rates. These results suggest that explosive radiations may be a general feature of evolutionary history across the tree of life.

Prevalence of Borrelia burgdorferi infection in a series of 98 primary cutaneous lymphomas.

Borrelia burgdorferi has been variably associated with different forms of primary cutaneous lymphoma. Differences in prevalence rates among reported studies could be a result of geographic variability or heterogeneity in the molecular approaches that have been employed. In the present study, we investigated the prevalence of Borrelia burgdorferi sensu lato DNA in diagnostic tissue samples from fresh cutaneous biopsies of 98 primary cutaneous lymphomas and 19 normal skin controls. Three different polymerase chain reaction (PCR) protocols targeting the hbb, flagellin, and Osp-A genes were used. Direct sequencing of both sense and antisense strands of purified PCR products confirmed the specificity of the amplified fragments. Sequence specificity was assessed using the Basic Local Alignment Search Tool, and MultAlin software was used to investigate the heterogeneity of target gene sequences across the different samples. Borrelia DNA was not detected in 19 controls, 23 cases of follicular lymphoma, 31 cases of extranodal marginal zone lymphoma, or 30 cases of mycosis fungoides. A single case of 14 diffuse large B-cell lymphoma cases was positive for B. burgdorferi. This study does not support a pathogenic role of B. burgdorferi in primary cutaneous B- and T-cell lymphomas from areas nonendemic for this microorganism and the consequent rationale for the adoption of antibiotic therapy in these patients.

Introduced Siberian chipmunks (Tamias sibiricus barberi) harbor more-diverse Borrelia burgdorferi sensu lato genospecies than native bank voles (Myodes glareolus).

Little attention has been given in scientific literature to how introduced species may act as a new host for native infectious agents and modify the epidemiology of a disease. In this study, we investigated whether an introduced species, the Siberian chipmunk (Tamias sibiricus barberi), was a potentially new reservoir host for Borrelia burgdorferi sensu lato, the causative agent of Lyme disease. First, we ascertained whether chipmunks were infected by all of the B. burgdorferi sensu lato genospecies associated with rodents and available in their source of infection, questing nymphs. Second, we determined whether the prevalence and diversity of B. burgdorferi sensu lato in chipmunks were similar to those of a native reservoir rodent, the bank vole (Myodes glareolus). Our research took place between 2006 and 2008 in a suburban French forest, where we trapped 335 chipmunks and 671 voles and collected 743 nymphs of ticks that were questing for hosts by dragging on the vegetation. We assayed for B. burgdorferi sensu lato with ear biopsy specimens taken from the rodents and in nymphs using PCR and restriction fragment length polymorphism (RFLP). Chipmunks were infected by the three Borrelia genospecies that were present in questing nymphs and that infect rodents (B. burgdorferi sensu stricto, B. afzelii, and B. garinii). In contrast, voles hosted only B. afzelii. Furthermore, chipmunks were more infected (35%) than voles (16%). These results may be explained by the higher exposure of chipmunks, because they harbor more ticks, or by their higher tolerance of other B. burgdorferi sensu lato genospecies than of B. afzelii. If chipmunks are competent reservoir hosts for B. burgdorferi sensu lato, they may spill back B. burgdorferi sensu lato to native communities and eventually may increase the risk of Lyme disease transmission to humans.

Disulfide-mediated oligomer formation in Borrelia burgdorferi outer surface protein C, a critical virulence factor and potential Lyme disease vaccine candidate.

Borrelia burgdorferi OspC is an outer membrane lipoprotein required for the establishment of infection in mammals. Due to its universal distribution among B. burgdorferi sensu lato strains and high antigenicity, it is being explored for the development of a next-generation Lyme disease vaccine. An understanding of the surface presentation of OspC will facilitate efforts to maximize its potential as a vaccine candidate. OspC forms homodimers at the cell surface, and it has been hypothesized that it may also form oligomeric arrays. Here, we employ site-directed mutagenesis to test the hypothesis that interdimeric disulfide bonds at cysteine 130 (C130) mediate oligomerization. B. burgdorferi B31 ospC was replaced with a C130A substitution mutant to yield strain B31::ospC(C130A). Recombinant protein was also generated. Disulfide-bond-dependent oligomer formation was demonstrated and determined to be dependent on C130. Oligomerization was not required for in vivo function, as B31::ospC(C130A) retained infectivity and disseminated normally. The total IgG response and the induced isotype pattern were similar between mice infected with untransformed B31 and those infected with the B31::ospC(C130A) strain. These data indicate that the immune response to OspC is not significantly altered by formation of OspC oligomers, a finding that has significant implications in Lyme disease vaccine design.

First isolation and molecular identification of Borrelia burgdorferi sensu stricto and Borrelia afzelii from skin biopsies of patients in Taiwan.

OBJECTIVES: In order to clarify the transmission cycle and genetic identity of Borrelia spirochetes in the non-endemic country of Taiwan, the causative agents responsible for human borreliosis were isolated from skin biopsies of patients and their genetic identities were determined. METHODS: Serum samples and skin biopsy specimens were collected from 95 patients: 85 with suspected Lyme disease and 10 controls. Infection with Borrelia burgdorferi was verified by Western immunoblot analysis and isolation of the Borrelia spirochetes from skin biopsy specimens. The genetic identities of these isolated spirochetes were determined by analyzing the gene sequences amplified by polymerase chain reaction assay based on the 5S (rrf)-23S (rrl) intergenic spacer amplicon gene of B. burgdorferi sensu lato. RESULTS: Serological evidence of B. burgdorferi infection was confirmed by elevated IgG and IgM antibodies against the major protein antigens of B. burgdorferi. Borrelia spirochetes were isolated from the skin biopsies of two patients. Phylogenetic analysis revealed that these detected spirochetes were genetically affiliated to the genospecies Borrelia burgdorferi sensu stricto and Borrelia afzelii, with a high sequence homology within the genospecies of B. burgdorferi sensu stricto (98.7-100%) and B. afzelii (100%), respectively. CONCLUSIONS: This study provides convincing evidence of B. burgdorferi sensu stricto and B. afzelii isolated and identified for the first time in Taiwan, and highlights the significance of genetic diversity of Borrelia spirochetes among patients in Taiwan.

Species of Borrelia burgdorferi complex that cause borrelial lymphocytoma in France.

BACKGROUND: Only about 30 cases of borrelial lymphocytoma (BL) with identification of the causative species of Borrelia have been published to date, mainly from Eastern or Central European countries. OBJECTIVES: To identify the species of B. burgdorferi complex responsible for BL in France. METHODS: Nine patients with BL acquired in France and for whom skin samples were sent to the national reference centre laboratory between 1994 and 2007 were included in this retrospective study. Direct detection of Borrelia in skin samples was made by polymerase chain reaction targeting the fla gene. Culture was performed when technically possible, and identification of each species was made by hybridization of a fragment of the fla gene with a panel of species-specific oligonucleotides. RESULTS: Borrelia afzelii was identified in three cases, B. garinii and B. burgdorferi sensu stricto in one case each. Culture was positive in only one case (B. garinii). CONCLUSIONS: Borrelia afzelii seems to be the predominant species of Borrelia responsible for BL in France, as already reported in other European countries.