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  • 1
    ISSN: 1432-1831
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The ospC gene coding for the outer surface protein OspC and the fla gene coding for the flagellin have been investigated in three different Borrelia burgdorferi sensu lato strains. These strains (the North American strain B31 and the European strains PKo and PBi) derive from various biological sources (lxodes dammini, human skin and human CSF) and belong to three different B. burgdorferi OspA serotypes and genospecies (OspA serotype 1, B. burgdorferi sensu stricto; OspA serotype 2, group VS461 and OspA serotype 4, B. garinii, respectively). The ospC and fla genes of the respective strains have been amplified by polymerase chain reaction, cloned in pUC8 and sequenced. The fla as well as the ospC genes were different among the three strains investigated. In general the fla genes are more conserved than the ospC genes. The fla genes have the same length of 1008 nucleotides coding for proteins of 336 amino acids, whereas the ospC genes differ in length. The ospC genes of strains B31, PKo and PBi have 630, 636 and 621 nucleotides encoding proteins of 210, 212 and 207 amino acids, respecctively. The ospC genes exhibit sequence identities between 70% and 74% among each other, sequence identities of the fla genes are in the range 96–97%. The ospC genes could be expressed in Escherichia coli to obtain proteins with and without leader peptides. The expression of the fla gene and an internal gene fragment resulted in the complete flagellin protein and a truncated protein (amino acids 129–251). The different ospC and fla gene products were immunoreactive with monoclonal antibodies and human sera and, thus, enlarge the spectrum of recombinant antigens to improve antibody detection in patients with Lyme borreliosis.
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  • 2
    ISSN: 1432-1831
    Keywords: Borrelia burgdorferi, afzelii, garinii ; Outer surface protein A ; ospA genes ; ospA sequence analysis ; Outer surface protein A serotypes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract The genes coding for the outer surface protein A (OspA) of 19 different Borrelia burgdorferi strains belonging to the seven OspA-serotypes 1–7, previously described [Wilske et al. (1993) J Clin Microbiol, 31: 340–350], have been investigated. B. burgdorferi sensu lato strains were chosen from various biological sources (ticks, human skin and cerebrospinal fluid) as well as different geographical origins (Germany, Slovenia, Austria, United States). The open reading frames of all ospA genes consist of 819–825 nucleotides corresponding to proteins of approximately 30 kDa. The ospA sequences obtained in this study and previous published studies were compared with the results from OspA serotyping with monoclonal antibodies. The classification into the seven OspA serotypes could be confirmed on a genetic basis (ospA genotypes 1–7) for all strains analyzed so far (n=29). In addition, one strain without OspA expression could be assigned to ospA genotype 2. Genetic stability could be proven for the ospA gene of B. burgdorferi strain PWudI after inocculation and reisolation from a gerbil. However, we found evidence for intragenic recombination by cluster analysis of ospA sequence data. Accordance of ospA genotype 1 strains with B. burgdorferi sensu stricto and ospA genotype 2 strains with B. afzelii, as well as the ospA genotype strains 3–7 with B. garinii was confirmed by pulsed-field gel electrophoresis of MluI-digested genomic DNA. B. garinii is not only more heterogenous in respect to the OspA-encoding genes, but shows moreover major subgroups formed by genotypes 4, 5 and 6 and genotypes 3 and 7, respectively. The latter group has not been described previously and is specifically recognized by an OspA-specific monoclonal antibody L32 1F7.
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  • 3
    ISSN: 1439-0973
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Zusammenfassung Borrelia burgdorferi sensu lato, der Erreger der Lyme-Borreliose, ist in Europa ausgesprochen heterogen. Da die äußeren Membranproteine OspA und OspC die Hauptkandidaten für eine Borrelien-Vakzine sind, wurde deren immunologische und molekulare Heterogenität untersucht. Aufgrund von Westernblotanalysen mit monoklonalen Antikörpern und Sequenzanalysen von PCR-amplifiziertem OspA und OspC fanden wir sieben verschiedene OspA- und 16 verschiedene OspC-Typen. Während Hautisolate (n=68) ganz vorwiegend (84%) OspA-Serotyp 2 (oderBorrelia afzelii) angehörten, waren Isolate aus Liquor cerebrospinalis und von Zecken (n=43 bzw. n=90) ausgesprochen heterogen im OspA-Typ mit Prävalenz der mitBorrelia garinii assoziierten OspA-Typen. OspA-Typ 4 fand sich oft bei Liquorisolaten (28%). Von Zecken wurde OspA-Typ 4 bisher nicht isoliert. Wie in einer anderen Studie berichtet, konnte jedoch Typ 4-OspA in Zecken mit der hochsensitiven PCR nachgewiesen werden.
    Notes: Summary Borrelia burgdorferi sensu lato, the etiological agent of Lyme borreliosis is considerably heterogeneous in Europe. Since the outer surface proteins OspA and OspC are the most promising candidates for aBorrelia vaccine the immunological heterogeneity of these proteins was investigated. By immunological analysis with monoclonal antibodies and sequence analysis of PCR amplified OspA and OspC at least seven and 16 different types, respectively, were found. Whereas skin isolates (n=68) were quite homogeneous (84% belonged to OspA-serotype 2 orBorrelia afzelii), isolates from human cerebrospinal fluid and from ticks (n=43 and n=90 respectively) were highly heterogeneous in their OspA-serotypes with prevalence of theBorrelia garinii associated types (about 70%). OspA-type 4 was often found among isolates from cerebrospinal fluid (28%). In ticks type 4 OspA has not been detected by culture so far. However, as reported in a previous study, type 4 OspA could be detected in ticks by the highly sensitive PCR technique.
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  • 4
    ISSN: 1432-1831
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Immunodominant proteins are variable in molecular and antigenic structure among different genospecies of Borrelia burgdorferi sensu lato. We have recently developed an immunoblot using five recombinant antigens: the chromosomal-encoded B. burgdorferi proteins p 100, the flagellin and an internal flagellin fragment thereof, and the plasmid-encoded outersurface proteins A (OspA) and C (OspC). In the present study the same antigens (derived from strain PKo, genospecies B. afzelii) were compared with the homologous recombinant proteins from strain B31 (genospecies B. burgdorferi sensu stricto) and with OspA, OspC and the internal flagellin fragment from strain PBi (genospecies B. garinii). Patients with neuroborreliosis (n=28) and patients with acrodermatitits chronica atrophicans (n=20) were investigated in the IgG immunoblot; the IgM immunoblot was performed only in patients with neuroborreliosis. There was a small increase in the detection rate of OspA-specific IgG or IgM antibodies using the different variants of recombinant OspA; however, OspA remained an insensitive antigen for antibody detection in Lyme borreliosis. The same was true to OspC-specific IgG antibodies. The sensitivity of OspC, which is the immunodominant antigen for IgM antibody detection, could not be increased using recombinant antigens derived from different strains. However, some sera which were negative in the recombinant immunoblot reacted with OspC in the conventional immunoblot using B. burgdorferi whole cell lysate as antigen. The most unexpected finding was the high degree of immunological heterogeneity of the internal flagellin fragments: IgG antibodies were detected in 18 of 48 patients using B31 fragments, in 25 of 48 using PKo fragments, in 23 of 48 using PBi fragments versus 33 of 48 when the three recombinant proteins were combined. PKo-derived fragments were more sensitive for antibody detection in patients with acrodermatitis chronica atrophicans, B31- and PBi-derived fragments for antibody detection in patients with neuroborreliosis. This is in agreement with the fact that isolates from patients with neuroborreliosis are predominantly belonging to the genospecies B. burgdorferi sensu stricto and B. garinii. For detection of IgM antibodies in sera from patients with neuroborreliosis, recombinant internal fragments derived from strains B31 and PBi were more sensitive than the PKo-derived fragment. The best discrimination between neuroborreliosis sera and control sera was achieved when the IgM blot was performed using recombinant internal flagellin fragments derived from strains PKo and PBi and OspC derived from B31 or PKo.
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  • 5
    ISSN: 1420-9071
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Conclusions As in OspA-serotyping experiments, theB garinii group (OspA-sterotype 3–7) showed highest diversity within this internal fragment of p83/100, whereas theB. afzelii group (OspA-type 2) and theB. burgdorferi sensu stricto group (OspA-type 1) were nearly identical. Determination of the size of the PCR products as well as restriction fragment length polymorphism analysis (DraI) can be used for classification into the three species ofB. burgdorferi sensu lato. Since p83/100 is chromosomally encoded, this protein might be a more stable marker for classification than the plasmid-encoded OspA. In contrast to the flagellin gene a subclassification of theB. garinii group is possible due to the diversity of the p83/100 internal fragment.
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