Cloning, sequencing, and expression of two class B endoflagellar genes of Treponema pallidum subsp. pallidum encoding the 34.5- and 31.0-kilodalton proteins

Champion, Cheryl I.; Miller, James N.; Lovett, Michael; Blanco, David R.

doi:10.1128/iai.58.6.1697-1704.1990

Cited by 54 publications

(30 citation statements)

References 34 publications

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“…The flagellin-encoding gene, on the other hand, is highly conserved among different B. burgdorferi strains, with there being only a few nucleotide differences even between U.S. and European strains (13,51). Being aware of the extensive DNA homology between flagellin genes of B. burgdorfen and even taxonomically remotely related bacteria such as Salmonella typhi and E. coli (10,41,51), we placed our oligonucleotide primers in areas that were nonhomologous when we compared them with the sequence data for the two closely related spirochetes B. hermsii and T. pallidum (9,41,45). Our PCR assay proved to be species wide and B. burgdorfen specific (Table 3; Fig.…”

Section: Discussionmentioning

confidence: 99%

Detection of Borrelia burgdorferi DNA in urine samples and cerebrospinal fluid samples from patients with early and late Lyme neuroborreliosis by polymerase chain reaction

1992

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A polymerase chain reaction (PCR) was developed for use in the identification of a 248-bp fragment of the Borrelia burgdorferi flagellin gene in urine and cerebrospinal fluid (CSF) from patients with Lyme neuroborreliosis. The specificities of the PCR products were confirmed by DNA-DNA hybridization with an internal probe. The assay had a detection limit of 10 in vitro-cultivated B. burgdorferi. The PCR assay seemed to be species wide as well as species specific, since DNA from all 21 B. burgdorferi isolates from humans tested but not from Borrelia hermsii or Treponema pallidum could be amplified. We tested 10 consecutively diagnosed patients with untreated neuroborreliosis. There was lymphocytic pleocytosis and intrathecal B. burgdorferispecific antibody synthesis in the CSF of all patients. Urine and CSF samples were investigated by PCR before, during, and up to 8.5 months after therapy. B. burgdorferi DNA was detected in urine samples from nine patients; five patients, including two patients with chronic neuroborreliosis, were PCR positive prior to treatment, whereas urine samples from the remaining four patients obtained 3 to 6 days after the onset of therapy became PCR positive. All urine samples obtained >4 weeks after therapy were negative by PCR. PCR of CSF was less sensitive, and samples from only four patients, including one with chronic neuroborreliosis, were positive. We conclude that urine is a more suitable sample source than CSF for use in B. burgdorferi DNA detection by PCR. Normalization of inflammatory CSF changes and the negative PCR results during follow-up even in patients with chronic neuroborreliosis do not point to a persistent infection. The future role of PCR as a diagnostic tool for Lyme neuroborreliosis is still uncertain.

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Section: Discussionmentioning

confidence: 99%

Detection of Borrelia burgdorferi DNA in urine samples and cerebrospinal fluid samples from patients with early and late Lyme neuroborreliosis by polymerase chain reaction

1992

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“…This concept is supported by the antiendoflagellar antibody present in NHS, which has been shown to contribute to NHS treponemicidal activity (3,4). The recent cloning and sequence analysis of genes which encode all of the T. pallidum endoflagellar filament proteins (7,11,16) can now allow determination of the epitopes which are the targets for this treponemicidal activity.…”

Section: While Complete Protection Against Experimental Syphilismentioning

confidence: 99%

Immunization with Treponema pallidum endoflagella alters the course of experimental rabbit syphilis

et al. 1990

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Rabbits were immunized over a 32-week period with a total of 450 ,ug of purified Treponema pallidum endoflagella. As measured by enzyme-linked immunosorbent assay, sera from immunized rabbits had antiendoflagellar antibody titers that were fivefold greater than titers of sera from infected immune rabbits and patients with secondary disease. Sera from all immunized animals possessed complement-dependent trepone-* Corresponding author. t Present address: National Institutes of Health, Bethesda, MD 20892.

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“…Oligonucleotide primers were based on the nucleotide sequence of the highly conserved flagellin-encoding gene of B. burgdorferi (14). To obtain a B. burgdorferi-specific amplification, the primers were placed in areas nonhomologous to the nucleotide sequences of the Borrelia hermsii (28) and Treponema pallidum (8,26) flagellin genes.…”

Section: Methodsmentioning

confidence: 99%

Comparison of in vitro culture, immunohistochemical staining, and PCR for detection of Borrelia burgdorferi in tissue from experimentally infected animals

1995

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An avidin-biotin-amplified immunophosphatase staining method with a purified polyclonal rabbit anti-Borrelia burgdorferi hyperimmune serum was developed for identification of B. burgdorferi in tissue specimens. The diagnostic efficacy was compared with those of in vitro culture and PCR with fresh and fixed, paraffinembedded tissues. A nested PCR assay was developed for identification of a 276-bp fragment of the B. burgdorferi flagellin gene. The diagnostic sensitivities of the different techniques were evaluated with spleen, renal, and urinary bladder tissues from eight experimentally infected gerbils. A systemic infection was verified by positivity of 23 of 24 (96%) organ cultures. B. burgdorferi was visualized immunohistochemically in 9 of 23 (39%) of the specimens. Among these nine specimens, an average of 33% of the 15 sections examined were positive. The spirochetes accumulated in discrete clusters and were associated with focal lymphocytic infiltration. The diagnostic sensitivity obtained by PCR with fixed, paraffin-embedded tissue was 21%, considerably lower than that with fresh tissue (71%). Thus, the reliable demonstration of B. burgdorferi by immunohistochemical staining is possible but extremely laborious, and considering the fact that the density of B. burgdorferi in human tissue is even lower than that in experimentally infected animals, the method is not useful in a clinical setting. It may, however, still be valuable in pathogenetic research. Detection of B. burgdorferi DNA by PCR should be performed with fresh tissue specimens and not with fixed, paraffinembedded specimens.

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Cloning, sequencing, and expression of two class B endoflagellar genes of Treponema pallidum subsp. pallidum encoding the 34.5- and 31.0-kilodalton proteins

Cited by 54 publications

References 34 publications

Detection of Borrelia burgdorferi DNA in urine samples and cerebrospinal fluid samples from patients with early and late Lyme neuroborreliosis by polymerase chain reaction

Detection of Borrelia burgdorferi DNA in urine samples and cerebrospinal fluid samples from patients with early and late Lyme neuroborreliosis by polymerase chain reaction

Immunization with Treponema pallidum endoflagella alters the course of experimental rabbit syphilis

Comparison of in vitro culture, immunohistochemical staining, and PCR for detection of Borrelia burgdorferi in tissue from experimentally infected animals

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