Comparative genome analysis: selection pressure on the Borrelia vls cassettes is essential for infectivity

BACKGROUND: At least three species of Borrelia burgdorferi sensu lato (Bbsl) cause tick-borne Lyme disease. Previous work including the genome analysis of B. burgdorferi B31 and B. garinii PBi suggested a highly variable plasmid part. The frequent occurrence of duplicated sequence stretches, the observed plasmid redundancy, as well as the mainly unknown function and variability of plasmid encoded genes rendered the relationships between plasmids within and between species largely unresolvable. RESULTS: To gain further insight into Borreliae genome properties we completed the plasmid sequences of B. garinii PBi, added the genome of a further species, B. afzelii PKo, to our analysis, and compared for both species the genomes of pathogenic and apathogenic strains. The core of all Bbsl genomes consists of the chromosome and two plasmids collinear between all species. We also found additional groups of plasmids, which share large parts of their sequences. This makes it very likely that these plasmids are relatively stable and share common ancestors before the diversification of Borrelia species. The analysis of the differences between B. garinii PBi and B. afzelii PKo genomes of low and high passages revealed that the loss of infectivity is accompanied in both species by a loss of similar genetic material. Whereas B. garinii PBi suffered only from the break-off of a plasmid end, B. afzelii PKo lost more material, probably an entire plasmid. In both cases the vls gene locus encoding for variable surface proteins is affected. CONCLUSION: The complete genome sequences of a B. garinii and a B. afzelii strain facilitate further comparative studies within the genus Borrellia. Our study shows that loss of infectivity can be traced back to only one single event in B. garinii PBi: the loss of the vls cassettes possibly due to error prone gene conversion. Similar albeit extended losses in B. afzelii PKo support the hypothesis that infectivity of Borrelia species depends heavily on the evasion from the host response

Population-based study of diagnostic assays for Borrelia infection: comparison of purified flagella antigen assay (Ideia™, Dako Cytomation) and recombinant antigen assay (Liaison®, DiaSorin)

<p>Abstract</p> <p>Background</p> <p>Testing for <it>Borrelia</it>-specific IgM and IgG-antibodies are often performed on a variety of poorly defined symptoms, and isolated IgM results are a frequent finding, which results in diagnostic uncertainty and further testing. We wanted to test the hypothesis that Borrelia-specific assays using recombinant antigens perform differently from assays based on purified flagella antigen.</p> <p>Methods</p> <p>We compared the use of recombinant antigens (LIAISON^®DiaSorin, Saluggia, Italy) and purified flagella antigen (IDEIA™ Borrelia, DakoCytomation, Glostrup, Denmark) in the assay for <it>Borrelia</it>-specific IgM and IgG-antibodies. The assays were tested on an unselected population of serum samples submitted from general practice. A total of 357 consecutive samples for analysis of <it>Borrelia </it>IgM and IgG antibodies. Furthermore, we analysed 540 samples for <it>Borrelia</it>-specific IgM or IgG antibodies first by the IDEIA™ and, if they were positive, the samples were further analysed using the LIAISON^®assay. To verify the correctness of the patient's serological status, discrepant samples were analysed by line blots (EcoLine, Virotech).</p> <p>Results</p> <p>In the consecutive series of 357 samples, the IgM assays detected 308 negative and 3 positive samples with concordant results. Compared with the line blot, the IDEIA™ system produced 21 false-positive IgM results, whereas the LIAISON^®system produced only one false-positive IgM result. The IgG assays showed 1 positive and 328 negative concordant results. The LIAISON^®system produced 9 true IgG-positive samples that were not detected by the IDEIA™ system, but the former produced 4 positive IgG results that were negative by line blot.</p> <p>Conclusion</p> <p>Diagnostic assays based on flagella antigen seem to show more false-positive IgM and false-negative IgG results than assays based on recombinant antigens. The latter may reduce the number of presumably false-positive IgM results and identify more IgG-positive subjects, but this system also produces more false-positive IgG results.</p

Single cell bioprinting with ultrashort laser pulses

Tissue engineering requires the precise positioning of mammalian cells and biomaterials on substrate surfaces or in preprocessed scaffolds. Although the development of 2D and 3D bioprinting technologies has made substantial progress in recent years, precise, cell‐friendly, easy to use, and fast technologies for selecting and positioning mammalian cells with single cell precision are still in need. A new laser‐based bioprinting approach is therefore presented, which allows the selection of individual cells from complex cell mixtures based on morphology or fluorescence and their transfer onto a 2D target substrate or a preprocessed 3D scaffold with single cell precision and high cell viability (93-99% cell survival, depending on cell type and substrate). In addition to precise cell positioning, this approach can also be used for the generation of 3D structures by transferring and depositing multiple hydrogel droplets. By further automating and combining this approach with other 3D printing technologies, such as two‐photon stereolithography, it has a high potential of becoming a fast and versatile technology for the 2D and 3D bioprinting of mammalian cells with single cell resolution.Bavarian State Ministry for Science and EducationHorizon 2020 Framework ProgrammeProjekt DEA

Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

A serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n = 36) were analyzed. Both of these parameters were found to have coefficients of variation of \u3c 3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n = 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n = 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n = 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies against B. burgdorferi

Genome Stability of Lyme Disease Spirochetes: Comparative Genomics of Borrelia burgdorferi Plasmids

Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33–40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi ∼900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short ≤20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant

Improvement of Lyme Borreliosis Serodiagnosis by a Newly Developed Recombinant Immunoglobulin G (IgG) and IgM Line Immunoblot Assay and Addition of VlsE and DbpA Homologues

We developed and evaluated a recombinant Borrelia line immunoblot assay based on 18 homologues of seven different antigens, i.e., p100, p58, p41i, BmpA, VlsE, OspC, and DbpA. Each recombinant antigen can be detected separately and is distinct even from homologues with identical molecular weights. This blot was compared to the recently described recombinant Borrelia Western immunoblot assay (U. Schulte-Spechtel, G. Lehnert, G. Liegl, V. Fingerle, C. Heimerl, B. J. Johnson, and B. Wilske, J. Clin. Microbiol. 41:1299-1303, 2003). To verify sensitivity and specificity, both blots were evaluated for reactivity with Borrelia-specific immunoglobulin G (IgG) and IgM antibodies with 85 sera from patients with different manifestations of Lyme borreliosis and 110 controls. According to European interpretation criteria for Borrelia Western blots, which define a serum as positive when it recognizes at least two bands, sensitivity increased significantly from 70.6% (Western blot) to 84.7% (line blot) for IgG (P = 0.042) and from 40.0% (Western blot) to 73.8% (line blot) for IgM (P < 0.005). The increased sensitivity for IgG detection is due to the new line blot technique, whereas the improvement in detection of IgM is mainly achieved through incorporation of the additional antigens. Notably, the recombinant VlsE of Borrelia garinii strain PBi displayed the highest sensitivity of all antigens tested for IgG detection and is also one of the most useful antigens for IgM. Due to its excellent sensitivity and specificity combined with ease of evaluation, this line immunoblot assay offers a useful improvement in serodiagnosis of Lyme borreliosis