Isolation of Borrelia burgdorferi sensu lato from blood of patients with erythema migrans.

BACKGROUND: We assessed the isolation rate of Borrelia burgdorferi sensu lato from blood in European patients with typical erythema migrans and evaluated the course and outcome of their illness. PATIENTS AND METHODS: Adult patients diagnosed with erythema migrans and from whom borreliae cultured from blood were included in this study. RESULTS: Borreliae were isolated from the blood of 35/2,828 (1.2%) patients, on average 7 days (range 1-47 days) after the appearance of erythema migrans. Only seven (20%) patients reported constitutional symptoms. 24/35 isolates were typed of which 20 were Borrelia afzelii and four were Borrelia garinii. 31 (88.6%) patients were treated with oral antibiotics while four (11.4%) received ceftriaxone iv. The course and outcome of the illness were favorable in all patients. CONCLUSION: In European patients with erythema migrans the yield of blood culturing was low, spirochetemia was often clinically silent and the course and outcome of the illness were favorable; the predominantly isolated strain was B. afzelii.

Molecular characterization of Borrelia spp. isolates from greater metropolitan Chicago reveals the presence of Borrelia bissettii. Preliminary report.

Lyme Disease in the US is concentrated in three endemic areas: the Northeast, the upper mid-West, and the Pacific coast. In the mid-West, the range of Lyme disease has expanded to include large parts of Wisconsin and Minnesota. Despite its proximity to the mid-Western focus, Illinois, so far, has not been considered an endemic area. However, more recent data suggest that this situation may be changing. Also, the extent of borrelial diversity in the mid-West remains largely unexplored. Here, we present preliminary results on the molecular characterization of Borrelia isolates from rodents captured in Cook and Lake Counties, both of which are parts of the greater metropolitan Chicago area in Illinois. We investigated the rodent reservoir present in forested areas of suburban Chicago in order to determine the frequency of infection with the Lyme disease agent(s) by culture isolation of Borrelia spirochetes (Picken et al., unpublished). Rodent isolates of Borrelia were identified to the species level by genetic characterization. In total, 19 isolates were obtained over 3 years from NW Cook Co. and Lake Co. Pulsed-field gel electrophoretic analysis of Mlul digested DNA from these isolates showed macrorestriction patterns similar to that of the Californian isolate, strain DN127 (PF type I), New York isolate strain 25015 (PF type II), or a variant of the latter (PF type III). Sequence data generated from the rrf(5S)-rrl(23S) intergenic spacer region of the ribosomal RNA gene cluster confirmed the identity of all the Chicago isolates studied to date as B. bissettii. These strains are unlike our previous Borrelia isolates from NW Illinois and Wisconsin. In addition, there was a predominant association of B. bissettii infection with pratal rodent species such as Microtus pennsylvanicus and Zapus hudsonius. The relationship of this novel enzootic focus to the established mid-Western endemic focus of Lyme disease remains to be elucidated. The geographic range and reservoir diversity of this organism may have hitherto been underestimated.

Comparison of culture-confirmed erythema migrans caused by Borrelia burgdorferi sensu stricto in New York State and by Borrelia afzelii in Slovenia.

BACKGROUND: The clinical manifestations of Lyme borreliosis in North America and Europe seem to differ, but a systematic comparison has never been done. OBJECTIVE: To compare European and U.S. patients with culture-confirmed erythema migrans. DESIGN: Prospective, clinical cohort study. SETTING: University medical centers in Westchester County, New York, and Ljubljana, Slovenia. PATIENTS: 119 U.S. patients with Borrelia burgdorferi sensu stricto infection and 85 Slovenian patients with B. afzelii infection. MEASUREMENTS: Interview, physical examination, and laboratory assays. RESULTS: Compared with Slovenian patients, U.S. patients had erythema migrans for a briefer duration (median duration, 4 days compared with 14 days; P < 0.001) but were more likely to have systemic symptoms (P = 0.01), abnormal findings on physical examination (P < 0.001), and seroreactivity (P < 0.001). Central clearing of erythema migrans lesions was more likely in Slovenian patients (P < 0.001). CONCLUSIONS: Erythema migrans caused by B. afzelii in Slovenia and erythema migrans caused by B. burgdorferi in New York have distinct clinical presentations. Caution should be used when clinical and laboratory experience from one side of the Atlantic is applied to patients on the other.

Comparison of peripheral and central biopsy sites for the isolation of Borrelia burgdorferi sensu lato from erythema migrans skin lesions.

To compare peripheral and central biopsy sites for the isolation of Borrelia burgdorferi sensu lato from typical erythema migrans lesions, two biopsy specimens (one from the margin and the other from the center of the lesion) were taken from each of 53 adult patients. Thirty-four (32.1%) of the 106 biopsy specimens and 23 (43.4%) of the 53 patients were culture-positive. Spirochetes were isolated in 19 (35.9%) of the 53 central and 15 (28.3%) of the 53 peripheral biopsies (nonsignificant difference). In 8 cases only the central specimen, in 4 cases only the peripheral specimen, and in 11 cases both specimens were positive. In a comparison of 36 annular and 17 homogeneous lesions, borreliae were isolated from 15 (41.7%) of the former and 8 (47.1%) of the latter. In the annular-lesion biopsies, 11 central and 12 peripheral specimens were culture-positive. In the homogeneous-lesion biopsies, eight central and three peripheral specimens were culture-positive (nonsignificant difference); there were no culture-positive results solely for the margin of homogeneous lesions. This study demonstrates that B. burgdorferi sensu lato may be isolated from the central aspect of erythema migrans lesions as efficiently as from the peripheral aspect.

Concomitant infection with tick-borne encephalitis virus and Borrelia burgdorferi sensu lato in patients with acute meningitis or meningoencephalitis.

From September 1992 to August 1993, 338 patients over the age of 15 years presented to the Department of Infectious Diseases, University Medical Centre Ljubljana, with acute lymphocytic meningitis. In 89 of these patients (26.3%) serum IgM and IgG antibodies against tick-borne encephalitis (TBE) virus were detected, and in 59 patients (17.5%) a borrelial etiology of disease was demonstrated by one or more of the following presence of intrathecal antibody production, seroconversion to borrelial antigens, presence of erythema migrans, and/or isolation of Borrelia burgdorferi sensu lato from skin or cerebrospinal fluid. Of the 148 patients who fulfilled criteria for TBE or borrelial infection, concomitant infection with TBE virus and B. burgdorferi sensu lato was demonstrated in 12 patients (3.6% of all patients presenting with acute lymphocytic meningitis). In the majority of patients with concomitant infection the clinical features at presentation were characteristic of, or consistent with, TBE. In addition, during follow-up studies, eight of the 12 patients subsequently developed signs and symptoms compatible with minor and/or major manifestations of Lyme borreliosis. Six patients were diagnoses with neuroborreliosis based on signs or symptoms and/or laboratory tests. These findings show that in patients with acute lymphocytic meningitis or meningoencephalitis, originating in TBE and Lyme borreliosis endemic regions, the possibility of concomitant infection should be considered.

Identification of three species of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto, B. garinii, and B. afzelii) among isolates from acrodermatitis chronica atrophicans lesions.

In Europe, at least three species of Borrelia are known to be causative agents of Lyme borreliosis: B. burgdorferi sensu stricto, B. garinii, and B. afzelii. Observable differences in the molecular characteristics of the three species have led to speculation that they may also differ in their pathogenic potential and/or tissue tropisms. Several studies have found an association between the chronic skin manifestation of Lyme borreliosis, acrodermatitis chronica atrophicans, and infection by B. afzelii. We sought to find further evidence for such a correlation by studying the genetic profiles of 22 strains of B. burgdorferi sensu lato derived from 21 patients who presented to the University Medical Center, Ljubljana, Slovenia between 1992 and 1995. Strains were isolated in culture from skin biopsies of acrodermatitis chronica atrophicans lesions; in the case of one patient two separate acrodermatitis chronica atrophicans lesions were cultured. All 21 patients had clinically typical lesions with "classic" histopathology and high IgG antibody titers to B. burgdorferi sensu lato. Strains were characterized and typed by 16S ribosomal RNA-specific polymerase chain reaction and determination of their large restriction fragment patterns using pulsed-field gel electrophoresis of MluI-digested genomic DNA. Of the 22 isolates studied, 17 possessed the highly conserved MLa1 pattern characteristic of B. afzelii. The remaining five isolates possessed large restriction fragment patterns that were typical of B. garinii (MLg2, four isolates from three patients) and B. burgdorferi sensu stricto (MLb2, one isolate). The results of 16S ribosomal RNA-specific polymerase chain reaction were concordant with these species designations. These data show that B. afzelii is the predominant, but not the exclusive, etiologic agent of acrodermatitis chronica atrophicans.

A two year prospective study to compare culture and polymerase chain reaction amplification for the detection and diagnosis of Lyme borreliosis.

AIM: To compare polymerase chain reaction (PCR) amplification of borrelial DNA and culture isolation of spirochaetes for the diagnosis of Lyme borreliosis by direct detection of Borrelia burgdorferi sensu lato in patients with erythema migrans and acrodermatitis chronica atrophicans lesions. METHODS: Skin biopsy specimens from erythema migrans and acrodermatitis chronica atrophicans lesions were subdivided and tested by PCR amplification assay and culture using two artificial growth media, Barbour-Stoenner-Kelly II (BSK II) and modified Kelly-Pettenkofer (MKP). Five classes of lesions were studied: typical erythema migrans, spontaneously resolved erythema migrans, atypical/partially treated erythema migrans, typical acrodermatitis chronica atrophicans, and atypical/partially treated acrodermatitis chronica atrophicans. RESULTS: For both erythema migrans and acrodermatitis chronica atrophicans lesions, the most sensitive detection method was MKP culture. PCR was less sensitive than MKP culture, but more sensitive than BSK II culture. Results for 758 typical erythema migrans specimens showed positivity rates of 36% for MKP, 25% for PCR, and 24% for BSK II. Differences were statistically significant. The overall positivity rate for all three methods combined was 54%, but few specimens (6%) were positive by all three methods. Examination of multiple erythema migrans lesions from the same patient increased the diagnostic yield. These findings, and similar results for acrodermatitis chronica atrophicans lesions, suggest that the distribution of spirochaetes in skin biopsies is not homogeneous. CONCLUSIONS: Although possessing the potential to provide a rapid diagnosis, PCR is not more sensitive than culture for the direct detection of borrelia. Spirochaetes appear to be unevenly distributed throughout biopsy specimens, suggesting that diagnosis of Lyme borreliosis by direct detection of the causative agent in skin lesions in vulnerable to sample bias.

Clinical findings for patients with Lyme borreliosis caused by Borrelia burgdorferi sensu lato with genotypic and phenotypic similarities to strain 25015.

In the course of performing culture isolation of Borrelia burgdorferi sensu lato for the diagnosis of Lyme borreliosis in Slovenia, we encountered nine patients who were infected with atypical strains. Molecular analyses of these strains suggested that they were more closely related to the North American tick isolate, strain 25015 (which belongs to the DN127 genomic group of B. burgdorferi sensu lato), than they were to the three species (B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii) hitherto found to be associated with European Lyme borreliosis. Review of the case histories of these patients revealed some atypical clinical features and variability in clinical presentation. In this study, we present the clinical findings for these patients and discuss their significance for the diagnosis of Lyme borreliosis. The DN127 genomic group shares with B. burgdorferi sensu stricto the distinction of being present in both the Old and New Worlds.

Heterogeneous expression of glycopeptide resistance in enterococci associated with transfer of vanB.

In mating experiments using a clinical strain that constitutively expresses vanB-encoded glycopeptide resistance, resistance transfer was detectable at a frequency of <10(-7) transconjugants/donor. Vancomycin MICs for transconjugants were 2- to 10-fold lower than those for the donor; both inducibly and constitutively resistant transconjugants were obtained. These findings demonstrate that the transfer of vanB among enterococci can be associated with substantial alterations in the level and control of glycopeptide resistance expression.

Molecular subtyping of Borrelia burgdorferi sensu lato isolates from five patients with solitary lymphocytoma.

Solitary lymphocytoma is a rare cutaneous manifestation of Lyme borreliosis that has been reported almost exclusively from Europe. This suggests that its etiologic agent may be absent or extremely rare on the North American continent. All three species of B. burgdorferi sensu lato known to be associated with human Lyme borreliosis (B. burgdorferi sensu stricto, B. garinii, and B. afzelii have been isolated in Europe, whereas only B. burgdorferi sensu stricto has been found in North America. This suggests that either B. garinii or B. afzelii might be the etiologic agent of borrelial lymphocytoma. To investigate this hypothesis we characterized five strains of B. burgdorferi sensu lato isolated from lymphocytoma lesions of patients residing in Slovenia. The methods used included: large restriction fragment pattern analysis of restriction enzyme MluI-digested genomic DNA, plasmid profiling, protein profiling, ribotyping using 5S, 16S, and 23S rDNA probes, and polymerase chain reaction amplification of the rrf (5S)-rrl (23S) intergenic spacer region. Molecular subtyping showed that four of the five isolates belonged to the species B. afzelii; however, this species is the predominant patient isolate in Slovenia and, therefore, may not represent a preferential association with lymphocytoma. The fifth isolate appeared to be most closely related to the DN127 genomic group of organisms. Further characterization of the isolate revealed that it possessed a unique molecular "fingerprint." The results not only show that borrelial lymphocytoma can be caused by B. afzelii but also demonstrate an association with another genomic group of B. burgdorferi sensu lato that is present in North America as well.

Patient isolates of Borrelia burgdorferi sensu lato with genotypic and phenotypic similarities of strain 25015.

Strain 25015 is an atypical tick isolate that belongs to a distinct genomic group (DN127) within the general taxon Borrelia burgdorferi sensu lato. Similarities between this strain and a white-footed mouse isolate from Illinois, strain CT39, have been reported. In the course of isolating B. burgdorferi sensu lato in culture from Slovenian patients, 9 isolates were identified with the same genetic profiles as strains 25015 and CT39, as evidenced by restriction enzyme MluI digestion patterns of genomic DNA. The aim of the present study was to molecularly characterize all 11 isolates to examine the extent of their genotypic and phenotypic similarity. The results of molecular studies suggest a close relationship between the patient isolates and strains 25015 and CT39. However, CT39 and several patient isolates possessed unique characteristics that reflect their discrete ontogeny.

European Lyme borreliosis: 231 culture-confirmed cases involving patients with erythema migrans.

In 1994, we isolated Borrelia burgdorferi sensu lato from 231 patients with erythema migrans who presented to the University Medical Center in Ljubljana, Slovenia. Samples of erythema migrans-affected skin were placed in media to support the growth of Borrelia species and evaluated in Ljubljana and Chicago. Patients whose cultures were positive included 132 women and 99 men; 136 of these 231 patients recalled a tick bite. Patients noted a rash an average of 24 days after a bite and presented a mean of 34 days after the bite with erythema migrans (mean diameter. 16 cm). Itching (44%) burning (18%), and pain (11%) were the most common local symptoms. Systemic complaints (40%) included headache, fatigue, malaise, and arthralgia. Other than erythema migrans, findings on physical examination were minimal (< 5% had fever, and in < 10% local lymph nodes were affected). Serial serological studies using indirect immunofluorescence assay, ELISA, and Western blot methods were performed, and antibodies to B, burgdorferi sensu lato were detected in < 50% of samples from patients. This is the largest series reported to date of patients with culture-confirmed Lyme borreliosis. It highlights the deficiencies of serological tests in early disease, demonstrates the sensitivity of direct detection methods for evaluation of patients with erythema migrans, and suggests that patients with early Lyme borreliosis in Slovenia may suffer a milder illness than those in the United States.

Single-tube nested polymerase chain reaction assay based on Flagellin gene sequences for detection of Borrelia burgdorferi sensu lato.

An inherent drawback of nested PCR systems to increase sensitivity of PCR-based assays is that tubes must be opened after the first round of amplification in order to transfer template molecules to the second amplification reaction; this procedure introduces the risk of carry-over contamination of negative specimens. To obviate this disadvantage, a nested PCR assay for detection of Borrelia burgdorferi in which both amplifications are performed in a single tube that remains closed throughout the entire process was devised. The assay is based on flagellin gene sequences with previously determined species-wide and species-specific properties. The nested PCR system proved to be 1000 times more sensitive than the conventional assay. Using the nested PCR system, ten spirochaetes could be routinely detected by agarose gel electrophoresis alone, whereas the conventional PCR system could detect only 10(4) spirochaetes under these conditions. After Southern transfer on amplification products and hybridization with 320- or chemiluminescent-labeled probes, the nested PCR system could easily detect a single spirochaete by both means, whereas the sensitivity of the conventional PCR assay varied from 10(1) (32P) to 10(3) (chemiluminescence) spirochaetes. This single-tube nested PCR system should be a useful addition to the current range of diagnostic assays for Lyme borreliosis.

Molecular characterization of Borrelia burgdorferi sensu lato from Slovenia revealing significant differences between tick and human isolates.

One hundred twenty-nine Slovenian isolates of Borrelia burgdorferi sensu lato derived from patients (69 strains) or Ixodes ricinus ticks (60 strains) were characterized. All of the strains were first- or second-passage isolates obtained in 1992 and 1993 from the same endemic region. The techniques used for the molecular analysis of strains included species-specific polymerase chain reaction (PCR) typing, and pulsed-field gel electrophoretic separation of undigested and MluI-digested genomic DNA. Isolates were identified to the species level by large restriction fragment pattern (LRFP) analysis and the results compared with the species-specific PCR result. Fifty-two patient isolates (75%) were typed as Borrelia afzelii (LRFP MLa1), 6 (9%) as Borrelia garinii (LRFPs MLg1-4), and 11 (16%) as Borrelia burgdorferi sensu stricto. The latter included 9 isolates (13%) with a new LRFP that is not typical of Borrelia burgdorferi sensu stricto and for which the designation MLx is suggested. In contrast, only 32 of 60 (53%) tick isolates were typed as Borrelia afzelii, while 20 strains (33%) were typed as Borrelia garinii and 8 strains (13%) as Borrelia burgdorferi sensu stricto. Three new LRFPs were found among the Borrelia garinii (MLg5 and 6) and Borrelia burgdorferi sensu stricto (MLb15) tick isolates. Large restriction fragment pattern analysis identified new groups of Borrelia burgdorferi sensu lato and revealed an apparent difference in the isolation frequency of different species from patients and ticks in the same endemic region.

Infection rate of Ixodes ricinus ticks with Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi sensu stricto in Slovenia.

In spring 1993, Ixodes ricinus ticks were collected from six regions of Slovenia to determine their overall rate of infection with Borrelia burgdorferi sensu lato and to assess the frequency of individual species in these tick populations. Ticks were dissected and midgut tissue inoculated into modified Barbour-Stoenner-Kelly (BSK II) medium. Borrelia isolates were differentiated into separate species using species-specific polymerase chain reaction (PCR) primers and by large restriction fragment pattern (LRFP) analysis. Infected ticks were found in all six regions surveyed. Spirochaetes were isolated from 69 of 363 ticks (19%): the isolation rate from adult female ticks was 35% (23/66 ticks cultured), from adult male ticks 22% (20/91), and from nymphal ticks 13% (26/206). Determination of the species of 60 isolates revealed that 32 were Borrelia afzelii (53%), 20 were Borrelia garinii (33%), and 8 were Borrelia burgdorferi sensu stricto (13%). In the Ljubljana region Borrelia afzelii and Borrelia garinii predominated (43% and 40%, respectively), whereas Borrelia burgdorferi sensu stricto constituted only 17% of isolates. In three other regions of the country Borrelia afzelii was isolated exclusively, although the number of isolates investigated was small. This study demonstrates the presence of all three European species of Borrelia burgdorferi sensu lato within the Slovenian tick population and also within a geographic area of less than 100 m2.

Genotypic and phenotypic characterization of Borrelia burgdorferi isolated from ticks and small animals in Illinois.

We have characterized 33 isolates of Borrelia burgdorferi from northern Illinois (32 isolates) and Wisconsin (1 isolate) representing the largest series of midwestern isolates investigated to date. The techniques used for molecular analysis of strains included (i) genospecies typing with species-specific PCR primers, (ii) plasmid profiling by pulsed-field gel electrophoresis of total genomic DNA, (iii) large-restriction-fragment pattern (LRFP) analysis by pulsed-field gel electrophoresis of MluI-digested genomic DNA (J. Belfaiza, D. Postic, E. Bellenger, G. Baranton, and I. Saint Girons, J. Clin. Microbiol. 31:2873-2877, 1993), (iv) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total proteins, (v) microsequencing of high-performance liquid chromatography-purified peptides derived from proteins showing high levels of expression, (vi) amino acid composition analysis of proteins, and (vii) immunological analysis of proteins with a polyclonal antiserum of human origin. Five reference strains as well as two atypical tick isolates from California (DN127) and New York (25015) were included for comparison. All of the Illinois and Wisconsin isolates were typed as B. burgdorferi sensu stricto with genospecies-specific PCR primers. The isolates were found to be heterogeneous with regard to their plasmid and protein profiles. One isolate from Illinois possessed two large-molecular-size plasmids instead of the usual 49-kb plasmid. Fragment patterns resulting from MluI digestion of genomic DNA from the 33 isolates and strains DN127 and 25015 were separable into six distinct LRFPs, five of which have not previously been described. Strain 25015 and an isolate from Illinois (CT39) shared an unusual LRFP that is not typical of other B. burgdorferi sensu stricto strains, suggesting that they may represent a fifth species of B. burgdorferi sensu lato. Five of the 33 isolates and strains DN127 and 25015 showed high-level expression of proteins with molecular masses of approximately 22 kDa. Investigation of these proteins by microsequencing of individual peptides and total amino acid composition analysis indicated that the 22-kDa proteins expressed by the seven strains were polymorphic OspC proteins. By using a polyclonal serum of human origin, expression of OspC could be detected in all 33 Illinois and Wisconsin isolates.

Persistence of Borrelia burgdorferi sensu lato in resolved erythema migrans lesions.

Erythema migrans skin lesions resulting from a tick bite and infection with Borrelia burgdorferi sensu lato eventually resolve, even without antibiotic therapy. The aim of the present study was to gauge the frequency of persistence of B. burgdorferi sensu lato in such lesions. Thus, the site of a previous lesion was biopsied and cultured in 48 patients: 39 with systemic or localized symptoms/signs and nine with no symptoms. B. burgdorferi sensu lato was isolated from biopsied skin from three symptomatic patients. Cultures of other tissues and fluids were negative. By genospecies-specific polymerase chain reaction (PCR) and pulsed-field gel electrophoretic (PFGE) analysis, two isolates were classified as Borrelia afzelii. The remaining isolate was a member of an unusual group of strains that type as B. burgdorferi sensu stricto by genospecies PCR but possess an atypical PFGE profile. All three patients had a dramatic clinical response to antibiotic treatment. These findings demonstrate the capacity of viable B. burgdorferi sensu lato organisms to persist in clinically normal-appearing skin at the site of a resolved erythema migrans rash for periods ranging from 2 months to 3.5 years. This observation may provide new insight into the organisms' ability to evade the host's immune response.

Polymerase chain reaction primers and probes derived from flagellin gene sequences for specific detection of the agents of Lyme disease and North American relapsing fever.

By cloning and sequencing the flagellin gene of Borrelia hermsii and comparing this sequence with that of the corresponding gene from B. burgdorferi, I identified a central region within the two genes which showed a reduced level of sequence similarity. Oligonucleotide sequences selected from this region produced species-specific amplimers when used in polymerase chain reaction experiments. Thus, primers derived from the B. burgdorferi sequence amplified a 276-bp fragment from 22 strains of B. burgdorferi of diverse geographic origin but not from 5 strains of B. hermsii, 5 other Borrelia species, 16 Treponema, Leptospira, and Spirochaeta species, or representatives of 10 other bacterial genera. However, when the amplified fragments were tested for hybridization with an oligonucleotide probe derived from the nonhomologous region, seven strains from either Germany or Switzerland did not hybridize. Cloning and sequencing of the amplified fragments from these strains revealed that the 22 strains of B. burgdorferi tested could be divided into three groups based on the nucleic acid sequence of the central region of the flagellin gene. With this information, oligonucleotide probes that hybridized to the amplified fragments and were able to differentiate the three groups of B. burgdorferi were designed. The corresponding primers, derived from the B. hermsii gene sequence, were tested for their ability to amplify DNA from this collection of strains. Although no amplification was obtained with representatives of the three groups of B. burgdorferi or various Treponema, Leptospira, and Spirochaeta species, amplification was obtained with the five other Borrelia species (B. parkeri, B. turicatae, B. crocidurae, B. anserina, and B. coriaceae) in addition to the five strains of B. hermsii. Sequencing of the amplified fragments from one strain of B. hermsii as well as B. parkeri and B. turicatae allowed the design of oligonucleotide probes that were able to differentiate the three species of North American relapsing fever spirochetes into two separate groups. These studies suggest that there is sufficient diversity within the flagellin gene sequences of closely related Borrelia species to differentiate them into groups and to pursue taxonomic studies both within and between species.

Isolation of a species-specific DNA probe for Neisseria gonorrhoeae using a novel technique particularly suitable for use with closely related species displaying high levels of DNA homology.

The use of nucleic acid probes has become an increasingly common method for detecting pathogenic micro-organisms in clinical specimens. In the course of our efforts to isolate species-specific DNA probes for bacterial pathogens, we encountered a special problem with regard to Neisseria gonorrhoeae. As a consequence of the high degree of DNA homology that this organism displays with its nearest relative, Neisseria meningitidis, the isolation of such probes could not be readily achieved. We therefore developed a novel method of probe isolation which overcomes this problem. This methodology relies upon the application of a 'sandwich' hybridization assay to screen an M13 'shotgun' library derived from N. gonorrhoeae genomic DNA. For this, genomic DNA from N. gonorrhoeae and N. meningitidis was immobilized on nitrocellulose filters and probed with recombinant phage DNA from candidate clones. Those clones which had hybridized to target sequences were then detected using labelled vector sequences in a second hybridization step. By this means we obtained a numerical assessment of the degree of specificity of candidate clones for the target organism as compared to one or more related species. Using this technique we have isolated three DNA probes which are highly specific for N. gonorrhoeae and which display no cross-reactivity with N. meningitidis or other members of the Neisseriaceae. This paper presents the basis of the methodology and describes the isolation and characterization of three N. gonorrhoeae-like specific probes.