A 2-pyridone amide inhibitor of transcriptional activity in Chlamydia trachomatis.

Chlamydia trachomatis is a strict intracellular bacterium that causes sexually transmitted infections and eye infections that can lead to life-long sequelae. Treatment options are limited to broad-spectrum antibiotics that disturb the commensal flora and contribute to selection of antibiotic-resistant bacteria. Hence, development of novel drugs that specifically target C. trachomatis would be beneficial. 2-pyridone amides are potent and specific inhibitors of Chlamydia infectivity. The first generation compound KSK120, inhibits the developmental cycle of Chlamydia resulting in reduced infectivity of progeny bacteria. Here, we show that the improved, highly potent second-generation 2-pyridone amide KSK213 allowed normal growth and development of C. trachomatis and the effect was only observable upon re-infection of new cells. Progeny elementary bodies (EBs) produced in the presence of KSK213 were unable to activate transcription of essential genes in early development and did not differentiate into the replicative form, the reticulate body (RB). The effect was specific to C. trachomatis since KSK213 was inactive in the closely related animal pathogen C. muridarum and in C. caviae The molecular target of KSK213 may thus be different in C. trachomatis or non-essential in C. muridarum and C. caviae Resistance to KSK213 was mediated by a combination of amino acid substitutions in both DEAD/DEAH RNA helicase and RNAse III, which may indicate inhibition of the transcriptional machinery as the mode of action. 2-pyridone amides provide a novel antibacterial strategy and starting points for development of highly specific drugs for C. trachomatis infections.

Circumpolar diversification of the Ixodes uriae tick virome.

Ticks (order: Ixodida) are a highly diverse and ecologically important group of ectoparasitic blood-feeding organisms. One such species, the seabird tick (Ixodes uriae), is widely distributed around the circumpolar regions of the northern and southern hemispheres. It has been suggested that Ix. uriae spread from the southern to the northern circumpolar region millions of years ago and has remained isolated in these regions ever since. Such a profound biographic subdivision provides a unique opportunity to determine whether viruses associated with ticks exhibit the same evolutionary patterns as their hosts. To test this, we collected Ix. uriae specimens near a Gentoo penguin (Pygoscelis papua) colony at Neko harbour, Antarctica, and from migratory birds-the Razorbill (Alca torda) and the Common murre (Uria aalge)-on Bonden island, northern Sweden. Through meta-transcriptomic next-generation sequencing we identified 16 RNA viruses, seven of which were novel. Notably, we detected the same species, Ronne virus, and two closely related species, Bonden virus and Piguzov virus, in both hemispheres indicating that there have been at least two cross-circumpolar dispersal events. Similarly, we identified viruses discovered previously in other locations several decades ago, including Gadgets Gully virus, Taggert virus and Okhotskiy virus. By identifying the same or closely related viruses in geographically disjunct sampling locations we provide evidence for virus dispersal within and between the circumpolar regions. In marked contrast, our phylogenetic analysis revealed no movement of the Ix. uriae tick hosts between the same locations. Combined, these data suggest that migratory birds are responsible for the movement of viruses at both local and global scales.

Pathogenesis of Relapsing Fever.

Relapsing fever (RF) is caused by several species of Borrelia; all, except two species, are transmitted to humans by soft (argasid) ticks. The species B. recurrentis is transmitted from one human to another by the body louse, while B. miyamotoi is vectored by hard-bodied ixodid tick species. RF Borrelia have several pathogenic features that facilitate invasion and dissemination in the infected host. In this article we discuss the dynamics of vector acquisition and subsequent transmission of RF Borrelia to their vertebrate hosts. We also review taxonomic challenges for RF Borrelia as new species have been isolated throughout the globe. Moreover, aspects of pathogenesis including symptomology, neurotropism, erythrocyte and platelet adhesion are discussed. We expound on RF Borrelia evasion strategies for innate and adaptive immunity, focusing on the most fundamental pathogenetic attributes, multiphasic antigenic variation. Lastly, we review new and emerging species of RF Borrelia and discuss future directions for this global disease.

K2S2O8-mediated coupling of 6-amino-7-aminomethyl-thiazolino-pyridones with aldehydes to construct amyloid affecting pyrimidine-fused thiazolino-2-pyridones.

We herein present the synthesis of diversely functionalized pyrimidine fused thiazolino-2-pyridones via K2S2O8-mediated oxidative coupling of 6-amino-7-(aminomethyl)-thiazolino-2-pyridones with aldehydes. The developed protocol is mild, has wide substrate scope, and does not require transition metal catalyst or base. Some of the synthesized compounds have an ability to inhibit the formation of Amyloid-ß fibrils associated with Alzheimer's disease, while others bind to mature amyloid-ß and α-synuclein fibrils.

Joint and unique multiblock analysis of biological data - multiomics malaria study.

Modern profiling technologies enable us to obtain large amounts of data which can be used later for a comprehensive understanding of the studied system. Proper evaluation of such data is challenging, and cannot be carried out by bare analysis of separate data sets. Integrated approaches are necessary, because only data integration allows us to find correlation trends common for all studied data sets and reveal hidden structures not known a priori. This improves the understanding and interpretation of complex systems. Joint and Unique MultiBlock Analysis (JUMBA) is an analysis method based on the OnPLS-algorithm that decomposes a set of matrices into joint parts containing variations shared with other connected matrices and variations that are unique for each single matrix. Mapping unique variations is important from a data integration perspective, since it certainly cannot be expected that all variation co-varies. In this work we used JUMBA for the integrated analysis of lipidomic, metabolomic and oxylipins data sets obtained from profiling of plasma samples from children infected with P. falciparum malaria. P. falciparum is one of the primary contributors to childhood mortality and obstetric complications in the developing world, which makes the development of new diagnostic and prognostic tools, as well as a better understanding of the disease, of utmost importance. In the presented work, JUMBA made it possible to detect already known trends related to the disease progression, but also to discover new structures in the data connected to food intake and personal differences in metabolism. By separating the variation in each data set into joint and unique, JUMBA reduced the complexity of the analysis and facilitated the detection of samples and variables corresponding to specific structures across multiple data sets, and by doing this enabled fast interpretation of the studied system. All of this makes JUMBA a perfect choice for multiblock analysis of systems biology data.

Levels of human proteins in plasma associated with acute paediatric malaria.

BACKGROUND: The intimate interaction between the pathophysiology of the human host and the biology of the Plasmodium falciparum parasite results in a wide spectrum of disease outcomes in malaria. Development of severe disease is associated with a progressively augmented imbalance in pro- and anti-inflammatory responses to high parasite loads and sequestration of parasitized erythrocytes. Although these phenomena collectively constitute common denominators for the wide variety of discrete severe malaria manifestations, the mechanistic rationales behind discrepancies in outcome are poorly understood. Exploration of the human pathophysiological response by variations in protein profiles in plasma presents an excellent opportunity to increase the understanding. This is ultimately required for better prediction, prevention and treatment of malaria, which is essential for ongoing elimination and eradication efforts. RESULTS: An affinity proteomics approach was used to analyse 541 paediatric plasma samples collected from community controls and patients with mild or severe malaria in Rwanda. Protein profiles were generated with an antibody-based suspension bead array containing 255 antibodies targetting 115 human proteins. Here, 57 proteins were identified with significantly altered levels (adjusted p-values < 0.001) in patients with malaria compared to controls. From these, the 27 most significant proteins (adjusted p-values < 10-14) were selected for a stringent analysis approach. Here, 24 proteins showed elevated levels in malaria patients and included proteins involved in acute inflammatory response as well as cell adhesion. The remaining three proteins, also implicated in immune regulation and cellular adhesivity, displayed lower abundance in malaria patients. In addition, 37 proteins (adjusted p-values < 0.05) were identified with increased levels in patients with severe compared to mild malaria. This set includes, proteins involved in tissue remodelling and erythrocyte membrane proteins. Collectively, this approach has been successfully used to identify proteins both with known and unknown association with different stages of malaria. CONCLUSION: In this study, a high-throughput affinity proteomics approach was used to find protein profiles in plasma linked to P. falciparum infection and malaria disease progression. The proteins presented herein are mainly involved in inflammatory response, cellular adhesion and as constituents of erythrocyte membrane. These findings have a great potential to provide increased conceptual understanding of host-parasite interaction and malaria pathogenesis.

The oxylipin and endocannabidome responses in acute phase Plasmodium falciparum malaria in children.

BACKGROUND: Oxylipins and endocannabinoids are low molecular weight bioactive lipids that are crucial for initiation and resolution of inflammation during microbial infections. Metabolic complications in malaria are recognized contributors to severe and fatal malaria, but the impact of malaria infection on the production of small lipid derived signalling molecules is unknown. Knowledge of immunoregulatory patterns of these molecules in malaria is of great value for better understanding of the disease and improvement of treatment regimes, since the action of these classes of molecules is directly connected to the inflammatory response of the organism. METHODS: Detection of oxylipins and endocannabinoids from plasma samples from forty children with uncomplicated and severe malaria as well as twenty controls was done after solid phase extraction followed by chromatography mass spectrometry analysis. The stable isotope dilution method was used for compound quantification. Data analysis was done with multivariate (principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA®) and univariate approaches (receiver operating characteristic (ROC) curves, t tests, correlation analysis). RESULTS: Forty different oxylipin and thirteen endocannabinoid metabolites were detected in the studied samples, with one oxylipin (thromboxane B2, TXB2) in significantly lower levels and four endocannabinoids (OEA, PEA, DEA and EPEA) at significantly higher levels in infected individuals as compared to controls according to t test analysis with Bonferroni correction. Three oxylipins (13-HODE, 9-HODE and 13-oxo-ODE) were higher in severe compared to uncomplicated malaria cases according to the results from multivariate analysis. Observed changes in oxylipin levels can be connected to activation of cytochrome P450 (CYP) and 5-lipoxygenase (5-LOX) metabolic pathways in malaria infected individuals compared to controls, and related to increased levels of all linoleic acid oxylipins in severe patients compared to uncomplicated ones. The endocannabinoids were extremely responsive to malaria infection with majority of this class of molecules found at higher levels in infected individuals compared to controls. CONCLUSIONS: It was possible to detect oxylipin and endocannabinoid molecules that can be potential biomarkers for differentiation between malaria infected individuals and controls and between different classes of malaria. Metabolic pathways that could be targeted towards an adjunctive therapy in the treatment of malaria were also pinpointed.

Integrin binding by Borrelia burgdorferi†P66 facilitates dissemination but is not required for infectivity.

P66, a Borrelia burgdorferi surface protein with porin and integrin-binding activities, is essential for murine infection. The role of P66 integrin-binding activity in B. burgdorferi infection was investigated and found to affect transendothelial migration. The role of integrin binding, specifically, was tested by mutation of two amino acids (D205A,D207A) or deletion of seven amino acids (Del202-208). Neither change affected surface localization or channel-forming activity of P66, but both significantly reduced binding to αv ß3 . Integrin-binding deficient B. burgdorferi strains caused disseminated infection in mice at 4 weeks post-subcutaneous inoculation, but bacterial burdens were significantly reduced in some tissues. Following intravenous inoculation, the Del202-208 bacteria were below the limit of detection in all tissues assessed at 2 weeks post-inoculation, but bacterial burdens recovered to wild-type levels at 4 weeks post-inoculation. The delay in tissue colonization correlated with reduced migration of the Del202-208 strains across microvascular endothelial cells, similar to Δp66 bacteria. These results indicate that integrin binding by P66 is important to efficient dissemination of B. burgdorferi, which is critical to its ability to cause disease manifestations in incidental hosts and to its maintenance in the enzootic cycle.

Study of the protein complex, pore diameter, and pore-forming activity of the Borrelia burgdorferi P13 porin.

P13 is one of the major outer membrane proteins of Borrelia burgdorferi. Previous studies described P13 as a porin. In the present study some structure and function aspects of P13 were studied. P13 showed according to lipid bilayer studies a channel-forming activity of 0.6 nanosiemens in 1 m KCl. Single channel and selectivity measurements demonstrated that P13 had no preference for either cations or anions and showed no voltage-gating up to ±100 mV. Blue native polyacrylamide gel electrophoresis was used to isolate and characterize the P13 protein complex in its native state. The complex had a high molecular mass of about 300 kDa and was only composed of P13 monomers. The channel size was investigated using non-electrolytes revealing an apparent diameter of about 1.4 nm with a 400-Da molecular mass cut-off. Multichannel titrations with different substrates reinforced the idea that P13 forms a general diffusion channel. The identity of P13 within the complex was confirmed by second dimension SDS-PAGE, Western blotting, mass spectrometry, and the use of a p13 deletion mutant strain. The results suggested that P13 is the protein responsible for the 0.6-nanosiemens pore-forming activity in the outer membrane of B. burgdorferi.

Expansion of the Chlamydia trachomatis inclusion does not require bacterial replication.

Chlamydia trachomatis replication takes place inside of a host cell, exclusively within a vacuole known as the inclusion. During an infection, the inclusion expands to accommodate the increasing numbers of C. trachomatis. However, whether inclusion expansion requires bacterial replication and/or de novo protein synthesis has not been previously investigated in detail. Therefore, using a chemical biology approach, we herein investigated C. trachomatis inclusion expansion under varying conditions in vitro. Under normal cell culture conditions, inclusion expansion correlated with C. trachomatis replication. When bacterial replication was inhibited using KSK120, an inhibitor that targets C. trachomatis glucose metabolism, inclusions expanded even in the absence of bacterial replication. In contrast, when bacterial protein synthesis was inhibited using chloramphenicol, expansion of inclusions was blocked. Together, these data suggest that de novo protein synthesis is necessary, whereas bacterial replication is dispensable for C. trachomatis inclusion expansion.

Mutations in hemG mediate resistance to salicylidene acylhydrazides, demonstrating a novel link between protoporphyrinogen oxidase (HemG) and Chlamydia trachomatis infectivity.

Salicylidene acylhydrazides (SAHs) inhibit the type III secretion system (T3S) of Yersinia and other Gram-negative bacteria. In addition, SAHs restrict the growth and development of Chlamydia species. However, since the inhibition of Chlamydia growth by SAH is suppressed by the addition of excess iron and since SAHs have an iron-chelating capacity, their role as specific T3S inhibitors is unclear. We investigated here whether SAHs exhibit a function on C. trachomatis that goes beyond iron chelation. We found that the iron-saturated SAH INP0341 (IS-INP0341) specifically affects C. trachomatis infectivity with reduced generation of infectious elementary body (EB) progeny. Selection and isolation of spontaneous SAH-resistant mutant strains revealed that mutations in hemG suppressed the reduced infectivity caused by IS-INP0341 treatment. Structural modeling of C. trachomatis HemG predicts that the acquired mutations are located in the active site of the enzyme, suggesting that IS-INP0341 inhibits this domain of HemG and that protoporphyrinogen oxidase (HemG) and heme metabolism are important for C. trachomatis infectivity.

Complete genome sequence of Borrelia crocidurae.

We announce the draft genome sequence of Borrelia crocidurae (strain Achema). The 1,557,560-bp genome (27% GC content) comprises one 919,477-bp linear chromosome and 638,083-bp plasmids that together carry 1,472 open reading frames, 32 tRNAs, and three complete rRNAs, with almost complete colinearity between B. crocidurae and Borrelia duttonii chromosomes.

Adaptive and innate immune responsiveness to Borrelia burgdorferi sensu lato in exposed asymptomatic children and children with previous clinical Lyme borreliosis.

Why some individuals develop clinical manifestations in Lyme borreliosis (LB) while others remain asymptomatic is largely unknown. Therefore, we wanted to investigate adaptive and innate immune responsiveness to Borrelia burgdorferi sensu lato in exposed Borrelia-antibody-positive asymptomatic children (n = 20), children with previous clinical LB (n = 24), and controls (n = 20). Blood samples were analyzed for Borrelia-specific interferon (IFN)-γ, interleukin (IL)-4, and IL-17 secretion by ELISPOT and Borrelia-induced IL-1ß, IL-6, IL-10, IL-12(p70), and tumor necrosis factor (TNF) secretion by Luminex. We found no significant differences in cytokine secretion between groups, but a tendency towards an increased spontaneous secretion of IL-6 was found among children with previous clinical LB. In conclusion, the adaptive or innate immune responsiveness to Borrelia burgdorferi sensu lato was similar in Borrelia-exposed asymptomatic children and children with previous clinical LB. Thus, the immunological mechanisms of importance for eradicating the spirochete effectively without developing clinical manifestations of LB remain unknown.

Relapsing fever Borrelia binds to neolacto glycans and mediates rosetting of human erythrocytes.

A hallmark of acute relapsing fever borreliosis is severe bacteremia. Some Borrelia species, such as B. duttonii and B. crocidurae, associate with erythrocytes and induce aggregation recognized as erythrocyte rosetting. Erythrocyte rosettes contribute to disease severity by increased tissue invasiveness (such as invasion of CNS and encephalitis), hemorrhaging, and reduced blood flow in affected microcapillaries. Here we report that relapsing fever Borrelia binds to neolacto (Galbeta4GlcNAcbeta3Galbeta4Glcbeta1)-carrying glycoconjugates that are present on human erythrocytes. This interaction is of low affinity but is compensated for by the multivalency of neo-lacto-oligosaccharides on the erythrocyte cell surface. Hence, the protein-carbohydrate interaction is dependent on multivalent neolacto-glycans to mediate binding.

Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia.

The Borrelia consists of three groups of species, those of the Lyme borreliosis (LB) group, also known as B. burgdorferi sensu lato (s.l.) and recently reclassified into Borreliella, the relapsing fever (RF) group Borrelia, and a third reptile-associated group of spirochetes. Culture-based methods remain the gold standard for the laboratory detection of bacterial infections for both research and clinical work, as the culture of pathogens from bodily fluids or tissues directly detects replicating pathogens and provides source material for research. Borrelia and Borreliella spirochetes are fastidious and slow growing, and thus are not commonly cultured for clinical purposes; however, culture is necessary for research. This protocol demonstrates the methodology and recipes required to successfully culture LB and RF spirochetes, including all recognized species from B. burgdorferi s.l. complex including B. afzelii, B. americana, B. andersonii, B. bavariensis, B. bissettii/bissettiae, B. burgdorferi sensu stricto (s.s.), B. californiensis, B. carolinensis, B. chilensis, B. finlandensis, B. garinii, B. japonica, B. kurtenbachii, B. lanei, B. lusitaniae, B. maritima, B. mayonii, B. spielmanii, B. tanukii, B. turdi, B. sinica, B. valaisiana, B. yangtzensis, and RFspirochetes, B. anserina, B. coriaceae, B. crocidurae, B. duttonii, B. hermsii, B. hispanica, B. persica, B. recurrentis, and B. miyamotoi. The basic medium for growing LB and RF spirochetes is the Barbour-Stoenner-Kelly (BSK-II or BSK-H) medium, which reliably supports the growth of spirochetes in established cultures. To be able to grow newly isolated Borrelia isolates from tick- or host-derived samples where the initial spirochete number is low in the inoculum, modified Kelly-Pettenkofer (MKP) medium is preferred. This medium also supports the growth of B. miyamotoi. The success of the cultivation of RF spirochetes also depends critically on the quality of ingredients.

Identification of amino acid domains of Borrelia burgdorferi P66 that are surface exposed and important for localization, oligomerization, and porin function of the protein.

P66, a bifunctional integral outer membrane protein, is necessary for Borrelia burgdorferi to establish initial infection and to disseminate in mice. The integrin binding function of P66 facilitates extravasation and dissemination, but the role of its porin function during murine infection has not been investigated. A limitation to studying P66 porin function during mammalian infection has been the lack of structural information for P66. In this study, we experimentally characterized specific domains of P66 with regard to structure and function. First, we aligned the amino acid sequences of P66 from Lyme disease-causing Borrelia and relapsing fever-causing Borrelia to identify conserved and unique domains between these disease-causing clades. Then, we examined whether specific domains of P66 are exposed on the surface of the bacteria by introducing c-Myc epitope tags into each domain of interest. The c-Myc epitope tag inserted C-terminally to E33 (highly conserved domain), to T187 (integrin binding region domain and a non-conserved domain), and to E334 (non-conserved domain) were all detected on the surface of Borrelia burgdorferi. The c-Myc epitope tag inserted C-terminally to E33 and D303 in conserved domains disrupted P66 oligomerization and porin function. In a murine model of infection, the E33 and D303 mutants exhibited decreased infectivity and dissemination. Taken together, these results suggest the importance of these conserved domains, and potentially P66 porin function, in vivo.

Specificity and role of the Borrelia burgdorferi CtpA protease in outer membrane protein processing.

To further characterize the function of the Borrelia burgdorferi C-terminal protease CtpA, we used site-directed mutagenesis to alter the putative CtpA cleavage site of one of its known substrates, the outer membrane (OM) porin P13. These mutations resulted in only partial blockage of P13 processing. Ectopic expression of a C-terminally truncated P13 in B. burgdorferi indicated that the C-terminal peptide functions as a safeguard against misfolding or mislocalization prior to its proteolytic removal by CtpA. In a parallel study of Borrelia burgdorferi lipoprotein sorting mechanisms, we observed a lower-molecular-weight variant of surface lipoprotein OspC that was particularly prominent with OspC mutants that mislocalized to the periplasm or contained C-terminal epitope tags. Further investigation revealed that the variant resulted from C-terminal proteolysis by CtpA. Together, these findings indicate that CtpA rather promiscuously targets polypeptides that lack structurally constrained C termini, as proteolysis appears to occur independently of a specific peptide recognition sequence. Low-level processing of surface lipoproteins such as OspC suggests the presence of a CtpA-dependent quality control mechanism that may sense proper translocation of integral outer membrane proteins and surface lipoproteins by detecting the release of C-terminal peptides.

P66 porins are present in both Lyme disease and relapsing fever spirochetes: a comparison of the biophysical properties of P66 porins from six Borrelia species.

The genus Borrelia is the cause of the two human diseases: Lyme disease (LD) and relapsing fever (RF). Both LD and RF Borrelia species are obligate parasites and are dependent on nutrients provided by their hosts. The first step of nutrient uptake across the outer membrane of these Gram-negative bacteria is accomplished by water-filled channels, so-called porins. The knowledge of the porin composition in the outer membranes of the different pathogenic Borrelia species is limited. Only one porin has been described in relapsing fever spirochetes to date, whereas four porins are known to be present in Lyme disease agents. From these, the Borrelia burgdorferi outer membrane channel P66 is known to act as an adhesin and was well studied as a porin. To investigate if P66 porins are expressed and similarly capable of pore formation in other Borrelia causing Lyme disease or relapsing fever three LD species (B. burgdorferi, B. afzelii, B. garinii) and three RF species (B. duttonii, B. recurrentis and B. hermsii) were investigated for outer membrane proteins homologous to P66. A search in current published RF genomes, comprising the ones of B. duttonii, B. recurrentis and B. hermsii, indicated that they all contained P66 homologues. The P66 homologues of the six Borrelia species were purified to homogeneity and their pore-forming abilities as well as the biophysical properties of the pores were analyzed using the black lipid bilayer assay.

Concomitant infection decreases the malaria burden but escalates relapsing fever borreliosis.

About 500 million cases of malaria occur annually. However, a substantial number of patients who actually have relapsing fever (RF) Borrelia infection can be misdiagnosed with malaria due to similar manifestations and geographic distributions of the two diseases. More alarmingly, a high prevalence of concomitant infections with malaria and RF Borrelia has been reported. Therefore, we used a mouse model to study the effects of such mixed infection. We observed a 21-fold increase in spirochete titers, whereas the numbers of parasitized erythrocytes were reduced 15-fold. This may be explained by polarization of the host immune response toward the intracellular malaria parasite, resulting in unaffected extracellular spirochetes and hosts that succumb to sepsis. Mixed infection also resulted in severe malaria anemia with low hemoglobin levels, even though the parasite counts were low. Overall, coinfected animals had a higher fatality rate and shorter time to death than those with either malaria or RF single infection. Furthermore, secondary malaria infection reactivated a quiescent RF brain infection, which is the first evidence of a clinically and biologically relevant cue for reactivation of RF Borrelia infection. Our study highlights the importance of investigating concomitant infections in vivo to elucidate the immune responses that are involved in the clinical outcome.

Prevalence and diversity of Borrelia species in ticks that have bitten humans in Sweden.

Members of the genus Borrelia are among the most common infectious agents causing tick-borne disease in humans worldwide. Here, we developed a Light Upon eXtension (LUX) real-time PCR assay that can detect and quantify Borrelia species in ticks that have fed on humans, and we applied the assay to 399 such ticks. Borrelia PCR-positive ticks were identified to species level by sequencing the products of conventional PCR performed using Borrelia group-specific primers. There was a 19% prevalence of Borrelia spp. in the detached ticks, and the number of spirochetes per Borrelia PCR-positive tick ranged from 2.0 × 10(2) to 4.9 × 10(5), with a median of 7.8 × 10(3) spirochetes. Adult ticks had a significantly larger number of spirochetes, with a median of 8.4 × 10(3) compared to the median of nymphs of 4.4 × 10(3). [corrected] Adult ticks also exhibited a higher prevalence of Borrelia (33%) than nymphs (14%). Among the identified species, Borrelia afzelii was found to predominate (61%) and was followed by B. garinii (23%), B. valaisiana (13%), B. burgdorferi sensu stricto (1%), B. lusitaniae (1%), and B. miyamotoi-like (1%). Also, 3% of the ticks were coinfected with multiple strains of B. afzelii. Notably, this is the first report of B. lusitaniae being detected in ticks in Sweden. Our LUX real-time PCR assay proved to be more sensitive than a corresponding TaqMan assay. In conclusion, the novel LUX real-time PCR method is a rapid and sensitive tool for detection and quantification of Borrelia spp. in ticks.