Development of a sequence-based in silico OspA typing method for Borrelia burgdorferi sensu lato.

Lyme disease (LD), caused by spirochete bacteria of the genus Borrelia burgdorferi sensu lato, remains the most common vector-borne disease in the northern hemisphere. Borrelia outer surface protein A (OspA) is an integral surface protein expressed during the tick cycle, and a validated vaccine target. There are at least 20 recognized Borrelia genospecies, that vary in OspA serotype. This study presents a new in silico sequence-based method for OspA typing using next-generation sequence data. Using a compiled database of over 400 Borrelia genomes encompassing the 4 most common disease-causing genospecies, we characterized OspA diversity in a manner that can accommodate existing and new OspA types and then defined boundaries for classification and assignment of OspA types based on the sequence similarity. To accommodate potential novel OspA types, we have developed a new nomenclature: OspA in silico type (IST). Beyond the ISTs that corresponded to existing OspA serotypes 1-8, we identified nine additional ISTs that cover new OspA variants in B. bavariensis (IST9-10), B. garinii (IST11-12), and other Borrelia genospecies (IST13-17). The IST typing scheme and associated OspA variants are available as part of the PubMLST Borrelia spp. database. Compared to traditional OspA serotyping methods, this new computational pipeline provides a more comprehensive and broadly applicable approach for characterization of OspA type and Borrelia genospecies to support vaccine development.

Development and validation of a multi-target TaqMan qPCR method for detection of Borrelia burgdorferi sensu lato.

Reliable detection of bacteria belonging to the Borrelia burgdorferi sensu lato species complex in vertebrate reservoirs, tick vectors, and patients is key to answer questions regarding Lyme borreliosis epidemiology. Nevertheless, the description of characteristics of qPCRs for the detection of B. burgdorferi s. l. are often limited. This study covers the development and validation of two duplex taqman qPCR assays used to target four markers on the chromosome of genospecies of B. burgdorferi s. l. Analytical specificity was determined with a panel of spirochete strains. qPCR characteristics were specified using water or tick DNA spiked with controlled quantities of the targeted DNA sequences of B. afzelii, B. burgdorferi sensu stricto or B. bavariensis. The effectiveness of detection results was finally evaluated using DNA extracted from ticks and biopsies from mammals whose infectious status had been determined by other detection assays. The developed qPCR assays allow exclusive detection of B. burgdorferi s. l. with the exception of the M16 marker which also detect relapsing fever Borreliae. The limit of detection is between 10 and 40 copies per qPCR reaction depending on the sample type, the B. burgdorferi genospecies and the targeted marker. Detection tests performed on various kind of samples illustrated the accuracy and robustness of our qPCR assays. Within the defined limits, this multi-target qPCR method allows a versatile detection of B. burgdorferi s. l., regardless of the genospecies and the sample material analyzed, with a sensitivity that would be compatible with most applications and a reproducibility of 100% under measurement conditions of limits of detection, thereby limiting result ambiguities.

Lyme arthritis in Western Europe: a multicentre retrospective study.

To characterize Lyme arthritis, with a focus on management, and outcome. Observational retrospective multicentre study in Western France, of all consecutive cases of Lyme arthritis, documented by Borrelia burgdorferi IgG on ELISA serological testing, confirmed by Western blot, with or without positive Borrelia PCR in synovial fluid, with no alternative diagnosis. We enrolled 52 patients (29 males), with a mean age of 43 ± 19.4 years. Most patients had monoarthritis (n = 43, 82.7%), involving the knee (n = 51, 98.1%), with a median delay between symptoms onset and Lyme arthritis diagnosis of 5 months (interquartile range, 1.5-8). Synovial fluid analysis yielded median white cell count of 16,000/mm3 (9230-40,500), and positive PCR in 16 cases (39%), for B. burgdorferi sensu stricto (n = 5), B. garinii (n = 5), B. afzelii (n = 3), and undetermined (n = 3). All patients received antibiotics, for a median duration of 28 days (21-30), with doxycycline (n = 44, 84.6%), ceftriaxone (n = 6, 11.5%), or amoxicillin (n = 2). Twelve patients (23.1%) also received intra-articular injection of glucocorticoids as first-line treatment. Of 47 patients with follow-up, 35 (74.5%) had complete resolution of Lyme arthritis. Lyme arthritis in Western Europe may be due to B. burgdorferi ss, B. afzelii, or B. garinii. Clinical presentation is similar to Lyme arthritis in North America (i.e. chronic knee monoarthritis), with low sensitivity of synovial fluid PCR (39%).

Host association of Borrelia burgdorferi sensu lato: A review.

Borrelia burgdorferi sensu lato (Bbsl) is a bacterial species complex that includes the etiological agents of the most frequently reported vector-borne disease in the Northern hemisphere, Lyme borreliosis. It currently comprises > 20 named and proposed genospecies that use vertebrate hosts and tick vectors for transmission in the Americas and Eurasia. Host (and vector) associations influence geographic distribution and speciation in Bbsl, which is of particular relevance to human health. To target gaps in knowledge for future efforts to understand broad patterns of the Bbsl-tick-host system and how they relate to human health, the present review aims to give a comprehensive summary of the literature on host association in Bbsl. Of 465 papers consulted (404 after exclusion criteria were applied), 96 sought to experimentally establish reservoir competence of 143 vertebrate host species for Bbsl. We recognize xenodiagnosis as the strongest method used, however it is infrequent (20% of studies) probably due to difficulties in maintaining tick vectors and/or wild host species in the lab. Some well-established associations were not experimentally confirmed according to our definition (ex: Borrelia garinii, Ixodes uriae and sea birds). We conclude that our current knowledge on host association in Bbsl is mostly derived from a subset of host, vector and bacterial species involved, providing an incomplete knowledge of the physiology, ecology and evolutionary history of these interactions. More studies are needed on all host, vector and bacterial species globally involved with a focus on non-rodent hosts and Asian Bbsl complex species, especially with experimental research that uses xenodiagnosis and genomics to analyze existing host associations in different ecosystems.

Spatial variability in prevalence and genospecies distributions of Borrelia burgdorferi sensu lato from ixodid ticks collected in southern Germany.

Lyme borreliosis (LB) is the most common arthropod-borne disease in Europe and North America and is caused by members of the Borrelia burgdorferi sensu lato (Bbsl) species complex. These bacteria are transmitted by ixodid tick vectors and therefore human LB risk is influenced by the prevalence and distribution of Bbsl genospecies within tick vectors throughout the wild. These distributions can easily change over spatiotemporal scales and, to understand LB risk fully, up to date information on prevalence and distribution of Bbsl is required. The last survey of Bbsl in southern Germany, including parts of the Munich metropolitan area, was completed in 2006 and new data is needed. Ixodid ticks were collected in seven plots located in and around Munich, Germany, from March to July 2019 and were screened for Bbsl. Borrelia burgdorferi s. l. positive ticks (52 adults, 158 nymphs) were found in all plots and adults (0-61.5 % Bbsl positive/plot) and nymphs (17.4-59.5 % Bbsl positive/plot) did not differ significantly in their overall Bbsl prevalence. The number of Bbsl positive nymphs did vary significantly between plots but the number of positive adults did not. In total, six Bbsl genospecies were located with B. afzelii and B. garinii dominating. Additionally, the relapsing-fever species B. miyamotoi was found in two sampling plots. Our results highlight the variability in Bbsl prevalence and genospecies distribution over short geographic distances and aid in understanding LB risk in and around the Munich metropolitan area.

Borrelia prevalence and species distribution in ticks removed from humans in Germany, 2013-2017.

Lyme borreliosis caused by spirochaetes of the Borrelia burgdorferi sensu lato (s.l.) complex is the most common tick-borne disease in Europe. In addition, the relapsing-fever spirochaete Borrelia miyamotoi, which has been associated with febrile illness and meningoencephalitis in immunocompromised persons, is present in Europe. This study investigated Borrelia prevalence and species distribution in ticks removed from humans and sent as diagnostic material to the Institute for Parasitology, University of Veterinary Medicine Hannover, in 2013-2017. A probe-based real-time PCR was carried out and Borrelia-positive samples were subjected to species determination by reverse line blot (RLB), including a B. miyamotoi-specific probe. The overall Borrelia-infection rate as determined by real-time PCR was 20.02 % (510/2547, 95 % CI: 18.48-21.63 %), with annual prevalences ranging from 17.17 % (90/524, 95 % CI: 14.04-20.68 %) in 2014 to 24.12 % (96/398, 95 % CI: 19.99-28.63 %) in 2015. In total, 271/475 (57.1 %) positive samples available for RLB were successfully differentiated. Borrelia afzelii was detected in 30.53 % of cases (145/475, 95 % CI: 26.41-34.89), followed by B. garinii/B. bavariensis (13.26 % [63/475], 95 % CI: 10.34-16.65). Borrelia valaisiana occurred in 5.89 % (28/475, 95 % CI: 3.95-8.41), B. spielmanii in 4.63 % (22/475, 95 % CI: 2.93-6.93), B. burgdorferi sensu stricto (s.s.)/B. carolinensis in 2.32 % (11/475, 95 % CI: 1.16-4.11), B. lusitaniae in 0.63 % (3/475, 95 % CI: 0.13-1.83) and B. bisettiae in 0.42 % (2/475, 95 % CI: 0.05-1.51) of positive ticks. Borrelia kurtenbachii was not detected, while B. miyamotoi was identified in 7.37 % (35/475, 95 % CI: 5.19-10.10) of real-time PCR-positive samples. Sanger sequencing of B. garinii/B. bavariensis-positive ticks revealed that the majority were B. garinii-infections (50/52 successfully amplified samples), while only 2 ticks were infected with B. bavariensis. Furthermore, 6/12 B. burgdorferi s.s./B. carolinensis-positive samples could be differentiated; all of them were identified as B. burgdorferi sensu stricto. Thirty-nine ticks (8.21 %, 95 % CI: 5.90-11.05) were coinfected with two different species. Comparison of the species distribution between ticks removed from humans in 2015 and questing ticks collected in the same year and the same area revealed a significantly higher B. afzelii-prevalence in diagnostic tick samples than in questing ticks, confirming previous observations. The obtained data indicate that Borrelia prevalence fluctuated in the same range as observed in a previous study, analysing the period from 2006 to 2012. Detection of B. miyamotoi in 7.37 % of Borrelia-positive samples points to the fact that clinicians should be aware of this pathogen as a differential diagnosis in cases of febrile illness.

Borrelia maritima sp. nov., a novel species of the Borrelia burgdorferi sensu lato complex, occupying a basal position to North American species.

Borrelia species are vector-borne parasitic bacteria with unusual, highly fragmented genomes that include a linear chromosome and linear as well as circular plasmids that differ numerically between and within various species. Strain CA690T, which was cultivated from a questing Ixodes spinipalpis nymph in the San Francisco Bay area, CA, was determined to be genetically distinct from all other described species belonging to the Borrelia burgdorferi sensu lato complex. The genome, including plasmids, was assembled using a hybrid assembly of short Illumina reads and long reads obtained via Oxford Nanopore Technology. We found that strain CA690T has a main linear chromosome containing 902176 bp with a blast identity ≤91 % compared with other Borrelia species chromosomes and five linear and two circular plasmids. A phylogeny based on 37 single-copy genes of the main linear chromosome and rooted with the relapsing fever species Borrelia duttonii strain Ly revealed that strain CA690T had a sister-group relationship with, and occupied a basal position to, species occurring in North America. We propose to name this species Borrelia maritima sp. nov. The type strain, CA690T, has been deposited in two national culture collections, DSMZ (=107169) and ATCC (=TSD-160).

Diversity of Borrelia burgdorferi sensu lato species in Ixodes ticks (Acari: Ixodidae) associated with cave-dwelling bats from Poland and Romania.

Bats comprise one quarter of the world's mammal species. In Europe, three nidicolous Ixodes tick species, I. vespertilionis, I. simplex and I. ariadnae are specifically associated with cave-dwelling bats, but their role as potential vectors of zoonotic agents is unknown. In this study, we used PCR-based methods to provide the first evidence of Borrelia burgdorferi sensu lato (s.l.) infections in the three bat-associated tick species collected from ten bat species sampled in Poland and Romania. B. burgdorferi s.l. was detected in 24% (64/266) of tick samples, and 40.3% (60/149) of the bats carried infected chiropterophilic ticks. In Poland, the B. burgdorferi s.l. infection prevelance of I. ariadnae ticks parasitizing Myotis species was four times higher compared to the I. vespertilionis ticks derived from Rhinolophus hipposideros bats (44.4% vs.10%, respectively). The observed differences in infection prevalence could be explained by differences in reservoir potential between bat species. Bats from the genus Myotis and Miniopterus schreibersii carried more infected ticks than R. hipposideros regardless of the tick species. Analysis of the flaB gene sequences revealed seven species from the B. burgdorferi s.l. complex (B. afzelii, B. carolinensis, B. garinii, B. lanei, B. spielmanii, B. burgdorferi s.s., and B. valaisiana), of which five are considered as human pathogens. This large diversity of Borrelia species may reflect differences in susceptibility of chiropteran hosts and/or the tick vectors. Generally, mammal-associated B. burgdorferi s.l. species were more common than bird-associated species. Our study provides evidence for new enzootic transmission cycles of B. burgdorferi s.l. spirochetes involving nidicolous Ixodes tick species and cave-dwelling bats.

Ixodes persulcatus/pavlovskyi natural hybrids in Siberia: Occurrence in sympatric areas and infection by a wide range of tick-transmitted agents.

Ixodes persulcatus and Ixodes pavlovskyi ticks, two closely related species of the I. ricinus - I. persulcatus group, are widely distributed in the southern part of Western Siberia. Recently, the existence of natural hybrids of I. persulcatus and I. pavlovskyi ticks has been demonstrated. The aim of this study was to evaluate the abundance of I. persulcatus/pavlovskyi hybrids in several locations with different ratios of parental tick species and to investigate the prevalence and genetic variability of a wide range of infectious agents in these hybrids compared to the parental tick species. Natural hybrids of I. persulcatus and I. pavlovskyi ticks were identified in all examined locations in Altai and Novosibirsk, Western Siberia, Russia. The abundance of hybrids varied from 7% to 40% in different locations and was maximal in a location with similar proportions of I. persulcatus and I. pavlovskyi ticks. For the first time, it was shown that hybrids can be infected with the same agents as their parental tick species: tick-borne encephalitis and Kemerovo viruses, Borrelia afzelii, Borrelia bavariensis, Borrelia garinii, Borrelia miyamotoi, Rickettsia helvetica, Rickettsia raoultii, Rickettsia sibirica, "Candidatus Rickettsia tarasevichiae", Anaplasma phagocytophilum, Ehrlichia muris, "Candidatus Neoehrlichia mikurensis", and Babesia microti. The prevalence of most bacterial agents in hybrids was intermediate compared to their parental tick species. Most genetic variants of the identified agents have been previously found in the parental tick species. Wide distribution of I. persulcatus/pavlovskyi natural hybrids implies that I. persulcatus, I. pavlovskyi and their hybrids coexist in all I. persulcatus - I. pavlovskyi sympatric areas.

Diversity of Lyme borreliosis spirochetes isolated from ticks in Serbia.

Spirochetes from the Borrelia burgdorferi sensu lato (s.l.). (Spirochaetales: Spirochaetaceae) species complex, including the causative agents of Lyme borreliosis, have been isolated from ticks, vertebrate reservoirs and humans. Previous analyses based on direct molecular detection in ticks indicated a considerable diversity of B. burgdorferi s.l. complex in Serbia. The present study aimed (a) to isolate borrelia strains from Serbia; (b) to determine their genotypic characteristics; and (c) to establish a collection of viable B. burgdorferi s.l. strains for further biological, ecological and genetic studies. For the present study, 231 adult Ixodes ricinus (Ixodida: Ixodidae) ticks from 16 ecologically different localities in Serbia were individually processed to cultivate B. burgdorferi s.l. This led to the isolation of 36 strains. A hbb gene quantitative real-time polymerase chain reaction (PCR) based on melting temperature determination and ospA gene sequencing were used to genotype the isolated spirochetes. The species identified based on the hbb gene real-time PCR were: Borrelia lusitaniae (44.4%), Borrelia afzelii (36.1%), Borrelia garinii (13.9%) and Borrelia valaisiana (5.6%), whereas the ospA sequence analysis revealed the occurrence of Borrelia bavariensis. This is the first report of the isolation of B. lusitaniae, B. garinii, B. bavariensis and B. valaisiana strains in Serbia.

Clinical spectrum of Lyme disease.

Lyme disease (borreliosis) is one of the most common vector-borne diseases worldwide. Its incidence and geographic expansion has been steadily increasing in the last decades. Lyme disease is caused by Borrelia burgdorferi sensu lato, a heterogeneous group of which three genospecies have been systematically associated to Lyme disease: B. burgdorferi sensu stricto Borrelia afzelii and Borrelia garinii. Geographical distribution and clinical manifestations vary according to the species involved. Lyme disease clinical manifestations may be divided into three stages. Early localized stage is characterized by erythema migrans in the tick bite site. Early disseminated stage may present multiple erythema migrans lesions, borrelial lymphocytoma, lyme neuroborreliosis, carditis, or arthritis. The late disseminated stage manifests with acordermatitis chronica atrophicans, lyme arthritis, and neurological symptoms. Diagnosis is challenging due to the varied clinical manifestations it may present and usually involves a two-step serological approach. In the current review, we present a thorough revision of the clinical manifestations Lyme disease may present. Additionally, history, microbiology, diagnosis, post-treatment Lyme disease syndrome, treatment, and prognosis are discussed.

Competition Between Strains of Borrelia afzelii in Immature Ixodes ricinus Ticks Is Not Affected by Season.

Vector-borne pathogens often consist of genetically distinct strains that can establish co-infections in the vertebrate host and the arthropod vector. Co-infections (or mixed infections) can result in competitive interactions between strains with important consequences for strain abundance and transmission. Here we used the spirochete bacterium, Borrelia afzelii, as a model system to investigate the interactions between strains inside its tick vector, Ixodes ricinus. Larvae were fed on mice infected with either one or two strains of B. afzelii. Engorged larvae were allowed to molt into nymphs that were subsequently exposed to three seasonal treatments (artificial summer, artificial winter, and natural winter), which differed in temperature and light conditions. We used strain-specific qPCRs to quantify the presence and abundance of each strain in the immature ticks. Co-infection in the mice reduced host-to-tick transmission to larval ticks and this effect was maintained in the resultant nymphs at 1 and 4 months after the larva-to-nymph molt. Competition between strains in co-infected ticks reduced the abundance of both strains. This inter-strain competition occurred in the three life stages that we investigated: engorged larvae, recently molted nymphs, and overwintered nymphs. The abundance of B. afzelii in the nymphs declined by 40.5% over a period of 3 months, but this phenomenon was not influenced by the seasonal treatment. Future studies should investigate whether inter-strain competition in the tick influences the subsequent strain-specific transmission success from the tick to the vertebrate host.

Competition between strains of Borrelia afzelii inside the rodent host and the tick vector.

Multiple-strain pathogens often establish mixed infections inside the host that result in competition between strains. In vector-borne pathogens, the competitive ability of strains must be measured in both the vertebrate host and the arthropod vector to understand the outcome of competition. Such studies could reveal the existence of trade-offs in competitive ability between different host types. We used the tick-borne bacterium Borrelia afzelii to test for competition between strains in the rodent host and the tick vector, and to test for a trade-off in competitive ability between these two host types. Mice were infected via tick bite with either one or two strains, and these mice were subsequently used to create ticks with single or mixed infections. Competition in the rodent host reduced strain-specific host-to-tick transmission and competition in the tick vector reduced the abundance of both strains. The strain that was competitively superior in host-to-tick transmission was competitively inferior with respect to bacterial abundance in the tick. This study suggests that in multiple-strain vector-borne pathogens there are trade-offs in competitive ability between the vertebrate host and the arthropod vector. Such trade-offs could play an important role in the coexistence of pathogen strains.

Multilocus sequence typing of clinical Borreliella afzelii strains: population structure and differential ability to disseminate in humans.

BACKGROUND: Lyme borreliosis in humans results in a range of clinical manifestations, thought to be partly due to differences in the pathogenicity of the infecting strain. This study compared European human clinical strains of Borreliella afzelii (previously named Borrelia afzelii) using multilocus sequence typing (MLST) to determine their spatial distribution across Europe and to establish whether there are associations between B. afzelii genotypes and specific clinical manifestations of Lyme borreliosis. For this purpose, typing was performed on 63 strains, and data on a further 245 strains were accessed from the literature. RESULTS: All 308 strains were categorized into 149 sequence types (STs), 27 of which are described here for the first time. Phylogenetic and goeBURST analyses showed short evolutionary distances between strains. Although the main STs differed among the countries with the largest number of strains of interest (Germany, the Netherlands, France and Slovenia), the B. afzelii clinical strains were less genetically structured than those previously observed in the European tick population. Two STs were found significantly more frequently in strains associated with clinical manifestations involving erythema migrans, whereas another ST was found significantly more frequently in strains associated with disseminated manifestations, especially neuroborreliosis. CONCLUSIONS: The MLST profiles showed low genetic differentiation between B. afzelii strains isolated from patients with Lyme borreliosis in Europe. Also, clinical data analysis suggests the existence of lineages with differential dissemination properties in humans.

Seroprevalence of Lyme disease in Eastern Slovakia.

OBJECTIVE: Lyme disease (LD) is chronic, multi-system zoonosis transmitted by ticks, and LD aetiological agents are spirochetes of the Borrelia burgdorferi sensu lato complex. The aim of the cross-sectional study was to analyze the LD incidence on the basis of the presence of specific antibodies in the serum of patients in Eastern Slovakia, and to compare the results of serological ELISA and immunoblot assays. METHODS: Venous blood with questionnaires was obtained by field sampling of respondents from Eastern Slovakia. Overall, we examined 537 human sera by the ELISA and for confirmation we tested all positive IgG antibodies against the Borrelia immunoblot assay. RESULTS: Our results confirmed the high serum prevalence of anti-Borrelia antibodies (17.9% for IgG), while the immunoblot seropositive test was confirmed in 69.8% of responders from ELISA IgG positive sera. Positive antibodies of the IgM class were found in 7.6% of the population under study. Most commonly found were antibodies against VlsE (80.2%), p41 (66.7%), p18 (56.3%), p100 (41.7%), p58 (31.3%), and p39 (30.2%). CONCLUSION: It should be noted that detection of antibodies against B. burgdorferi s.l. is only an indirect evidence of the presence of this bacterium in the development of clinical signs of LD in humans. Laboratory LD tests should be performed in accordance with valid standards, positive and uncertain results must be confirmed by the Western Blot/Immunoblot assay.

Species Identification and Phylogenetic Analysis of Borrelia burgdorferi Sensu Lato Using Molecular Biological Methods.

Bacterial species identification is required in different disciplines and-depending on the purpose-levels of specificity or resolution of typing may vary. Nowadays, molecular methods are the mainstay for bacterial identification and sequence-based analyses are of ever-growing importance. For diagnostics, immediate results are needed and often real-time PCR of one or two loci is the method of choice while for epidemiological or evolutionary studies sequence data of several loci improve phylogenetic resolution to required levels. Multilocus sequence typing (MLST) and multilocus sequence analyses (MLSA) utilize sequences information of several housekeeping loci (eight for Borrelia) to distinguish between species. This method has been widely used for bacterial species and strain identification and will be described in this chapter.As more and more diversity is being detected in the Borrelia burgdorferi sensu lato species complex, the importance of accurate species and strain typing has come to the fore. This is particularly significant with a view of differentiating human pathogenic and non-pathogenic strains or species and understanding the epidemiology, ecology, population structure, and evolution of species.

Surveillance for and Discovery of Borrelia Species in US Patients Suspected of Tickborne Illness.

Background: Tick-transmitted Borrelia fall into 2 heterogeneous bacterial complexes comprised of multiple species, the relapsing fever (RF) group and the Borrelia burgdorferi sensu lato group, which are the causative agents of Lyme borreliosis (LB), the most common tickborne disease in the Northern Hemisphere. Geographic expansion of LB in the United States and discovery of emerging Borrelia pathogens underscores the importance of surveillance for disease-causing Borrelia. Methods: De-identified clinical specimens, submitted by providers throughout the United States, for patients suspected of LB, anaplasmosis, ehrlichiosis, or babesiosis were screened using a Borrelia genus-level TaqMan polymerase chain reaction (PCR). Borrelia species and sequence types (STs) were characterized by multilocus sequence typing (MLST) utilizing next-generation sequencing. Results: Among 7292 specimens tested, 5 Borrelia species were identified: 2 causing LB, B. burgdorferi (n = 25) and B. mayonii (n = 9), and 3 RF borreliae, B. hermsii (n = 1), B. miyamotoi (n = 8), and Candidatus B. johnsonii (n = 1), a species previously detected only in the bat tick, Carios kelleyi. ST diversity was greatest for B. burgdorferi-positive specimens, with new STs identified primarily among synovial fluids. Conclusions: These results demonstrate that broad PCR screening followed by MLST is a powerful surveillance tool for uncovering the spectrum of disease-causing Borrelia species, understanding their geographic distribution, and investigating the correlation between B. burgdorferi STs and joint involvement. Detection of Candidatus B. johnsonii in a patient with suspected tickborne disease suggests this species may be a previously undetected cause of illness in humans exposed to bat ticks.

Molecular identification of Borrelia spirochetes in questing Ixodes ricinus from northwestern Spain.

BACKGROUND: Ixodes ricinus, the predominant tick species in Europe, can transmit the causative agents of important human diseases such as Lyme borreliosis (LB), caused by Borrelia spirochetes. In northern Spain, LB is considered endemic; recently, a significant increase of the annual incidence of LB was reported in the northwestern (NW) region. METHODS: In order to provide information on the prevalence of Borrelia spp., pooled and individually free-living I. ricinus from NW Spain were molecularly analyzed. Positive samples were characterized at the fla and Glpq genes and the rrfA-rrlB intergenic spacer region to identify Borrelia species/genospecies. RESULTS: Borrelia burgdorferi (sensu lato) (s.l.) individual prevalence and MIR were significantly higher in adult females (32.3 and 16%) than in nymphs (18.8 and 6.2%) and adult males (15.6 and 8.4%). Five Borrelia genospecies belonging to the B. burgdorferi (s.l.) group were identified: B. garinii was predominant, followed by B. valaisiana, B. lusitaniae, B. afzelii and B. burgdorferi (sensu stricto) (s.s.). One species belonging to the tick-borne relapsing fever group (B. miyamotoi) was also found, showing low individual prevalence (1%), positive pool (0.7%) and MIR (0.1%) values. To our knowledge, this is the first citation of B. miyamotoi in free-living ticks from Spain. CONCLUSIONS: The significant prevalences of B. burgdorferi (s.l.) genospecies detected in questing ticks from NW Spain are similar to those detected in northern and central European countries and higher to those previously found in Spain. These results together with the high incidence of LB in humans and the high seroprevalence of B. burgdorferi (s.l.) in roe deer shown in other studies reveal that the northwest area is one of the most risky regions for acquiring LB in Spain.

Borrelia lanei sp. nov. extends the diversity of Borrelia species in California.

The diversity of Borrelia species discovered in California appears to be particularly high. A divergent group of Borrelia strains collected from Ixodes ticks in California was described by Postic and co-workers and designated 'genomospecies 2' (Postic D, Garnier M, Baranton G. Int J Med Microbiol 2007;297:263-271; Postic D, Ras NM, Lane RS, Hendson M, Baranton G. J Clin Microbiol 1998;36:3497-3504). We performed multilocus sequence analysis (MLSA) using eight housekeeping loci (clpA, clpX, nifS, pepX, pyrG, recG, rplB and uvrA) on 12 strains of this Borreliagenospecies to confirm that these strains form a distinct group within the Borreliaburgdorferi s. l. complex (Margos G, Hojgaard A, Lane RS, Cornet M, Fingerle V et al.Ticks Tick Borne Dis 2010;1:151-158). Phylogenetic and genetic distance analyses based on sequences of the MLSA housekeeping genes corroborated the distinctness of this group; genetic distances to all other members of the B. burgdorferi s.l. complex were 96 % or lower. We propose the name Borrelia lanei sp. nov. for this genospecies in honor of Professor Robert S. Lane, University of California Berkeley, for his contributions to Borrelia and tick research. The type strain for Borrelia lanei sp. nov., strain CA28-91T, has been deposited to two culture collections (=DSM 17992T=CIP 109135T).