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. 1998 Mar;36(3):768–776. doi: 10.1128/jcm.36.3.768-776.1998

Molecular Typing of Borrelia burgdorferi Sensu Lato by Randomly Amplified Polymorphic DNA Fingerprinting Analysis

Guiqing Wang 1, Alje P van Dam 1,*, Lodewijk Spanjaard 1, Jacob Dankert 1
PMCID: PMC104623  PMID: 9508310

Abstract

To study whether pathogenic clusters of Borrelia burgdorferi sensu lato strains occur, we typed 136 isolates, cultured from specimens from patients (n = 49) with various clinical entities and from ticks (n = 83) or dogs (n = 4) from different geographic regions, by randomly amplified polymorphic DNA (RAPD) fingerprinting with four arbitrary primers. The RAPD patterns were reproducible up to the 95% similarity level as shown in duplicate experiments. In these experiments the purified DNAs prepared on different days, from different colonies, and after various passages were used as templates. With an intergroup difference of 55%, the 136 strains could be divided into seven genetic clusters. Six clusters comprised and corresponded to the established species B. burgdorferi sensu stricto (n = 23), Borrelia garinii (n = 39), Borrelia afzelii (n = 59), Borrelia japonica (n = 1), Borrelia valaisiana (n = 12), and genomic group DN127 (n = 1). One strain from a patient with erythema migrans (EM) did not belong to any of the species or genomic groups known up to now. The RAPD types of B. burgdorferi sensu stricto, B. garinii, and B. afzelii isolates, which may give rise to human Lyme borreliosis (LB), were associated with their geographic origins. A high degree of genetic diversity was observed among the 39 B. garinii strains, and six subgroups could be recognized. One of these comprised eight isolates from patients with disseminated LB only and no tick isolates. B. afzelii strains from patients with EM or acrodermatitis chronica atrophicans were not clustered in particular branches. Our study showed that RAPD analysis is a powerful tool for discriminating different Borrelia species as well as Borrelia isolates within species.

Lyme borreliosis (LB) is a tick-borne spirochetal disease endemic to regions in temperate climates throughout the world (7). The clinical spectrum of LB varies from cutaneous erythema migrans (EM) to severe arthritis, acrodermatitis chronica atrophicans (ACA), and cardiac and neurological manifestations (37). Borrelia burgdorferi sensu lato (19), the etiologic agent of LB, is genetically divergent. On the basis of DNA-DNA reassociation and the MseI restriction enzyme patterns of the 5S-23S rRNA intergenic spacer, B. burgdorferi sensu lato can be divided into at least 10 different species or genomic groups: B. burgdorferi sensu stricto, B. garinii, B. afzelii (5, 11), B. japonica (20), B. valaisiana (43), B. lusitaniae (22), B. andersonii (25), B. tanukii, B. turdi (15, 26), and group DN127 (33). In North America, only B. burgdorferi sensu stricto, B. andersonii, and group DN127 have been identified (3, 5, 25), whereas B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, and B. lusitaniae have been found in Europe (5, 11, 22, 43). B. japonica, B. tanukii, and B. turdi are limited to Japan (15, 20).

Not all strains from these species or genomic groups are pathogenic for humans. Up to now, only B. burgdorferi sensu stricto, B. garinii, B. afzelii, and group DN127 strains have been cultured from patients with LB (5, 32). Therefore, it may be that B. japonica, B. valaisiana, B. lusitaniae, B. andersonii, B. tanukii, and B. turdi are not pathogenic for humans. In addition, different species have been associated with distinct clinical manifestations of LB (4, 5, 11, 41). Arthritis is associated with B. burgdorferi sensu stricto infection and neuroborreliosis is associated with B. burgdorferi sensu stricto and B. garinii infection (4, 41), whereas B. afzelii has more frequently been cultured from skin biopsy specimens from patients with EM and ACA (10, 11, 41). However, it is not clear whether within species certain pathogenic clusters of strains which are responsible for the particular clinical syndromes of human LB occur and whether genetically distinguishable clusters causing EM, ACA, or neuroborreliosis can be found.

Randomly amplified polymorphic DNA (RAPD) analysis (46) or arbitrarily primed PCR (AP-PCR) (44), both of which use low-stringency PCR amplification with a single primer with an arbitrary sequence to generate strain-specific arrays of anonymous DNA fragments, has been used for the molecular typing of various microorganisms (39). In two studies, this technique was also used to type B. burgdorferi, although the procedure, including the use of radiolabelled nucleotides and analysis of the fragments on large sodium dodecyl sulfate-urea gels, was complex. In the first study, 29 B. burgdorferi isolates were divided by AP-PCR into three genospecific groups (45). In the second study, reported recently, the genetic diversity of 65 B. burgdorferi sensu stricto strains from various geographic locations in North America and Europe was also evaluated by AP-PCR (14).

In the present study we simplified and optimized RAPD analysis for the typing of Borrelia strains. Subsequently, we analyzed 136 B. burgdorferi sensu lato strains by using this method. The objectives of our study were to evaluate the usefulness of RAPD analysis as a molecular typing method for Borrelia strains, to identify pathogenic clusters of Borrelia strains causing particular clinical syndromes in patients with LB, and to assess the geographic diversity within different Borrelia species.

MATERIALS AND METHODS

Bacterial strains and DNA preparation.

The 136 B. burgdorferi sensu lato strains used in this study are listed in Table 1. Forty-nine strains were isolated from human clinical specimens (of which 20 and 12 were cultured from skin biopsy specimens from patients with EM and ACA, respectively; 7 were cultured from skin biopsy specimens from patients with disseminated LB [21]; 9 were cultured from cerebrospinal fluid specimens; and 1 was from a blood specimen). Of the remaining 87 strains, 83 were isolated from Ixodes ticks and 4 were isolated from dogs. The origins of the isolates covered North America, Europe, and Asia. Most of these strains have previously been typed in our laboratory by reactivity with monoclonal antibodies and rRNA gene restriction analysis (41). Strains not included in previous analyses were identified as belonging to the species listed in Table 1 by PCR-restriction fragment length polymorphism (RFLP) analysis of the 5S-23S intergenic spacer (33, 43). Strains of B. hermsii and B. anserina were used as controls.

TABLE 1.

B. burgdorferi sensu lato strains used in this study

Species and strain Biological sourcea Geographic location RAPD typeb RAPD clusterc RFLP patternd Reference(s) or provider
B. burgdorferi sensu stricto (n = 23)
 M20 I. ricinus The Netherlands 1 I A 29
 M26 I. ricinus The Netherlands 1 I A 29
 M16 I. ricinus The Netherlands 2 I A 29
 VS293 I. ricinus Switzerland 3 I A 30
 M11 I. ricinus The Netherlands 4 I A 29
 M24 I. ricinus The Netherlands 4 I A 29
 M12 I. ricinus The Netherlands 5 I A 29
 VS219 I. ricinus Switzerland 6 I A 30
 M29 I. ricinus The Netherlands 7 I A 29
 VS215 I. ricinus Switzerland 8 I A 30
 M2 I. ricinus The Netherlands 9 I A 29
 M48 I. ricinus The Netherlands 10 I A 29
 VS130 I. ricinus Switzerland 11 I A 30
 VS134 I. ricinus Switzerland 11 I A 30
 A44S Human skin (EM) The Netherlands 11 I A 41
 A91-15 Dog United States 12 I A S. Rijpkema
 Hum3336 I. pacificus United States 13 I A 33
 A92-1-2 Dog United States 14 I A 2
 N40 I. scapularis United States 15 I A 13, 33
 A91-10 Dog United States 16 I A 2
 HB19 Human (blood) United States 17 I A 6
 A91-9 Dog United States 18 I A S. Rijpkema
 B31 I. scapularis United States 19 I A ATCC 35210
B. garinii (n = 39)
 NT29 I. persulcatus Japan 20 IIa C 33
 Ip89 I. persulcatus Russia 21 IIa C 33
 HT7 I. persulcatus Japan 22 IIa C 16
 A91S Human skin (NB) The Netherlands 23 IIb B 21, 41
 A91C Human (CSF) The Netherlands 23 IIb B 41
 A76S Human skin (CA) The Netherlands 23 IIb B 21, 41
 A104S Human skin (NB) The Netherlands 23 IIb B This study
 A94S Human skin (MY) The Netherlands 23 IIb B 21, 41
 A19S Human skin (NB) The Netherlands 24 IIb B 21, 41
 PBi Human (CSF) Germany 25 IIb B 47
 A01C Human (CSF) The Netherlands 26 IIb B 41
 HT55 I. persulcatus Japan 27 IIc B 16
 JP5 I. persulcatus China 28 IId B 23
 JP12 I. persulcatus China 28 IId B Z. F. Zhang
 JP2 I. persulcatus China 28 IId B 23
 HT19 I. persulcatus Japan 29 IId B 16
 M63 I. ricinus The Netherlands 30 IIe B 29
 M45 I. ricinus The Netherlands 31 IIf B 29
 PHei Human (CSF) Germany 32 IIf B 47
 A87S Human skin (NB) The Netherlands 33 IIf B 21, 41
 M3 I. ricinus The Netherlands 34 IIf B 29
 TN I. ricinus Germany 35 IIf B 47
 M4 I. ricinus The Netherlands 36 IIf B 29
 M64 I. ricinus The Netherlands 36 IIf B 29
 M6 I. ricinus The Netherlands 37 IIf B 29
 M44 I. ricinus The Netherlands 38 IIf B 29
 A77S Human skin (NB) The Netherlands 39 IIf B 21, 41
 A77C Human (CSF) The Netherlands 39 IIf B 41
 VSBM Human (CSF) Switzerland 39 IIf B 31
 M41 I. ricinus The Netherlands 40 IIf B 29
 VSBP Human (CSF) Switzerland 41 IIf B 30, 31
 20047 I. ricinus France 42 IIf B 33
 VS102 I. ricinus Switzerland 43 IIf B 30
 M8 I. ricinus The Netherlands 44 IIf B 29
 T25 I. ricinus Germany 45 IIf B 47
 M42 I. ricinus The Netherlands 46 IIf B 29
 M59 I. ricinus The Netherlands 47 IIf B 29
 PBr Human (CSF) Germany 48 IIf B 47
 VSDA Human (CSF) Switzerland 49 IIf B 31
B. afzelii (n = 59)
 A51T I. ricinus The Netherlands 50 III D 41
 A48T I. ricinus The Netherlands 51 III D 41
 A49T I. ricinus The Netherlands 52 III D 41
 A57T I. ricinus The Netherlands 53 III D 41
 A50T I. ricinus The Netherlands 54 III D 41
 A53T I. ricinus The Netherlands 55 III D 41
 A105S Human skin (EM) The Netherlands 56 III D This study
 A106S Human skin (EM) The Netherlands 57 III D This study
 A40S Human skin (EM) The Netherlands 58 III D 41
 A60T I. ricinus The Netherlands 59 III D 41
 A58T I. ricinus The Netherlands 60 III D 41
 A59T I. ricinus The Netherlands 61 III D 41
 A16S Human skin (ACA) The Netherlands 62 III D 41
 A95S Human skin (ACA) The Netherlands 62 III D This study
 A116S Human skin (EM) The Netherlands 63 III D This study
 A43S Human skin (EM) The Netherlands 64 III D 41
 A56T I. ricinus The Netherlands 65 III D 41
 PKo Human skin (EM) Germany 66 III D 33, 47
 A88S Human skin (ACA) The Netherlands 67 III D 41
 M10 I. ricinus The Netherlands 68 III D 29
 A11S Human skin (ACA) The Netherlands 69 III D 41
 A86S Human skin (ACA) The Netherlands 69 III D 41
 A10S Human skin (EM) The Netherlands 70 III D 41
 A13S Human skin (EM) The Netherlands 71 III D 41
 A72T I. ricinus The Netherlands 72 III D 41
 A61T I. ricinus The Netherlands 72 III D 41
 A71T I. ricinus The Netherlands 72 III D 41
 A63T I. ricinus The Netherlands 72 III D 41
 A67T I. ricinus The Netherlands 73 III D 41
 A108S Human skin (EM) The Netherlands 74 III D This study
 A64T I. ricinus The Netherlands 75 III D 41
 A68T I. ricinus The Netherlands 76 III D 41
 A65T I. ricinus The Netherlands 77 III D 41
 A20S Human skin (EM) The Netherlands 78 III D 41
 M55 I. ricinus The Netherlands 79 III D 29
 A09S Human skin (ACA) The Netherlands 80 III D 41
 A02S Human skin (EM) The Netherlands 80 III D 41
 A84S Human skin (EM) The Netherlands 81 III D 41
 A03S Human skin (EM) The Netherlands 82 III D 41
 A21S Human skin (EM) The Netherlands 82 III D 41
 A69T I. ricinus The Netherlands 83 III D 41
 A70T I. ricinus The Netherlands 84 III D 41
 A62T I. ricinus The Netherlands 85 III D 41
 A110S Human skin (ACA) The Netherlands 86 III D This study
 A114S Human skin (ACA) The Netherlands 86 III D This study
 A117S Human skin (EM) The Netherlands 87 III D This study
 A111aS Human skin (ACA) The Netherlands 88 III D This study
 A111bS Human skin (ACA) The Netherlands 88 III D This study
 A33S Human skin (EM) The Netherlands 89 III D 41
 A38S Human skin (EM) The Netherlands 90 III D 41
 VS461 I. ricinus Switzerland 91 III D 6, 33
 A73T I. ricinus The Netherlands 92 III D 41
 A54T I. ricinus The Netherlands 93 III D 41
 A27S Human skin (EM) The Netherlands 94 III D 41
 A52T I. ricinus The Netherlands 95 III D 41
 A17S Human skin (ACA) The Netherlands 96 III D 41
 M7Ce I. persulcatus China 97 III D 23, 33
 A66T I. ricinus The Netherlands 98 III D 41
 A26S Human skin (ACA) The Netherlands 99 III D 41
B. japonica HO14 (n = 1) I. persulcatus Japan 100 IV E 20, 33
B. valaisiana (n = 12)
 UK I. ricinus England 101 V F 33, 43
 M53 I. ricinus The Netherlands 102 V F 29, 43
 M57 I. ricinus The Netherlands 103 V F 29, 43
 AR-2 I. ricinus The Netherlands 104 V F 33, 43
 M50 I. ricinus The Netherlands 105 V F 29, 43
 M7 I. ricinus The Netherlands 106 V F 29, 43
 VS116 I. ricinus Switzerland 107 V F 30, 43
 M19 I. ricinus The Netherlands 107 V F 29, 43
 M38 I. ricinus The Netherlands 107 V F 29, 43
 M47 I. ricinus The Netherlands 107 V F 29, 43
 M49 I. ricinus The Netherlands 108 V F 29, 43
 M52 I. ricinus The Netherlands 109 V F 29, 43
Group DN127 25015 (n = 1) I. scapularis United States 110 VI K 3, 33
Borrelia sp. strain A14S (n = 1) Human skin (EM) The Netherlands 111 VII R 41
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a

For human skin isolates, the clinical syndromes of the patient are indicated in parentheses, as follows: NB, neuroborreliosis; CA, carditis; MY, myalgia. CSF, cerebrospinal fluid. 

b

RAPD types were defined at the 95% similarity level. 

c

RAPD clusters were defined at the 45% similarity level between genospecies, and the subclusters of B. garinii were defined at the 60% similarity level. 

d

All RFLP patterns except pattern R for strain A14S were described by Postic et al. (33). 

e

In earlier studies, this strain was designated M7. 

All spirochetal strains were grown in modified Kelly’s medium (34) at 33°C. Three B. burgdorferi strains (strains A87S, A48T, and UK) were also incubated in solid culture medium to obtain pure colonies (12). DNA was extracted as described by Wilson (49), and the concentration of DNA was determined by spectrophotometry.

RAPD-PCR amplification.

Initially, 16 arbitrary oligonucleotide primers, synthesized by Perkin-Elmer Applied Biosystems (Perkin-Elmer, Cheshire, United Kingdom) and available in our laboratory, were tested for their usefulness for the typing of Borrelia species. On the basis of the initial experiments, four primers, primers 1254 (5′-CCGCA GCCAA-3′), 1283 (5′-GCGAT CCCCA-3′), 1247 (5′-AAGAG CCCGT-3′) (1), and AP13 (5′-TTGTC TAGTG GCAAG GCT-3′), were selected and used for the typing of the Borrelia isolates collected.

RAPD-PCR was performed under a layer of mineral oil in a 25-μl reaction mixture containing 20 ng of purified DNA, 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 4.0 mM MgCl2, 0.1 mg of bovine serum albumin per ml, a 200 μM concentration of each deoxynucleotide triphosphate (Pharmacia Biotech), 1 U of AmpliTaq polymerase (Perkin-Elmer, Gouda, The Netherlands), and a 0.4 μM concentration of a single primer (0.8 μM for primer AP13). The PCR was carried out with a Biometra thermocycler (Westburg B.V., Leusden, The Netherlands) by using the following steps: initial denaturation at 94°C for 2 min, followed by 3 cycles of 94°C for 5 min, 36°C for 5 min, and 72°C for 5 min and then 30 cycles of 94°C for 1 min, 36°C for 1 min, and 72°C for 2 min and a final incubation at 72°C for 10 min. Duplicate experiments with either the same DNA template or DNA extracted on different days or from different colonies or passages were performed to assess reproducibility.

RAPD fingerprinting analysis.

The amplified DNA fragments were separated on 1% (wt/vol) agarose gels (Boehringer, Mannheim, Germany) containing 10 μg of ethidium bromide per ml in the gel and in 1× Tris-acetate-EDTA buffer. For each experimental run, bacteriophage λ DNA digested with BstEII and Neisseria meningitidis ET80 DNA amplified with primer 1254 by the same protocol were included and were used as a size marker for the amplified fragments and as a reference for the normalization of different gels, respectively. All pictures were digitized with an Iris video digitizer and were analyzed with computer software (GelCompar; Applied Math, Kortrijk, Belgium). The bands with a faint intensity which were not reproducible in duplicate experiments were excluded in the final analysis.

For each Borrelia strain, the DNA fingerprinting patterns obtained with the four primers were combined. These combined patterns were used for the similarity estimation and cluster analysis, in which the similarity among strains was estimated by means of the Dice comparison, and the clustering of strains was determined by the unweighted average pair group method (36) to facilitate the plotting of the dendrogram. Isolates with a level of similarity of more than 0.95 were assigned to the same RAPD type.

PCR amplification and sequencing of the 16S rRNA gene.

PCR amplification and DNA sequencing of the 5′ end of the 16S rRNA gene were performed as described previously (43).

Nucleotide sequence accession numbers.

The partial 16S rRNA gene sequences which we determined in this study have been assigned the following GenBank accession numbers: AF010163 (strain A01C), AF010164 (strain A76S), AF010165 (strain A87S), and AF010166 (strain M63). The accession numbers of the B. burgdorferi sensu lato strains which we used for comparison are as follows: U03396 (B. burgdorferi B31), D67018 (B. garinii 20047), X85199 (B. garinii PBi), and U78151 (B. afzelii VS461).

RESULTS

Reproducibility.

In order to assess the reproducibility and the day-to-day variation of RAPD fingerprinting, we performed PCR reamplification with 54 of the 136 Borrelia strains. In these duplicate experiments we used either the same DNA template (n = 36) or DNA extracted on different occasions (n = 18). The DNA fingerprints of these strains were very reproducible. Occasionally, a discrepancy in the appearance of faint bands was seen. Usually, the intensities of these faint bands were below the limit for inclusion in the analysis. In addition, second DNA samples from 10 strains were tested by one of the investigators who had no knowledge of the origins of these samples. In these analyses, DNA samples from the same strain showed at least 95% similarity. No differences were seen between the RAPD patterns for two Borrelia strains (A38S and A87S) at low passage numbers (5 and 3) and high passage numbers (25 and 27) (data not shown). The RAPD patterns of four different colonies of strain A48T were identical to each other, as were the RAPD patterns of 10 different colonies of strain UK (data not shown). Four colonies of strain A87S were also highly similar to each other; however, two of these colonies lacked one band after amplification with primer 1247, leading to a similarity level of 95.8% (Fig. 1).

FIG. 1.

FIG. 1

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Reproducibility of RAPD fingerprinting. The DNA fingerprints from different passages and different colonies of B. garinii A87S were obtained by PCR amplification with primers 1254 (A), 1283 (B), 1247 (C), and AP13 (D). Lanes 1 and 6, passages 3 and 27, respectively; lanes 2 to 5, four different colonies from passage 9, respectively; lane M, BstEII-digested bacteriophage lambda DNA. The molecular sizes (in kilobases) are indicated on the left.

RAPD fingerprinting among species.

In the combined gels obtained after four individual amplifications with different primers, the DNA fingerprints of Borrelia strains comprised 20 to 35 fragments with sizes ranging from 0.3 to 4.5 kb (Fig. 2). A total of 111 RAPD types, designated types 1 to 111, were identified for the 136 B. burgdorferi sensu lato strains (Table 1). The discrimination index of the RAPD technique calculated by application of the Simpson numerical index of diversity (18) was 0.996. Phylogenetic analysis showed that the 136 B. burgdorferi sensu lato strains used in this study could be divided into seven genetic clusters with an intergroup difference at least of 55% (Fig. 3). The RAPD fingerprints for representative strains of these seven clusters are included in Fig. 2. Clusters I, II, III, IV, and V corresponded to the well-known species B. burgdorferi sensu stricto (n = 23), B. garinii (n = 39), B. afzelii (n = 59), B. japonica (n = 1), and B. valaisiana (n = 12), respectively (5, 11, 20, 43). Cluster VI included one isolate from Borrelia genomic group DN127. The seventh cluster, with only one Borrelia isolate cultured from a sample from a patient in The Netherlands with EM who developed LB, has not been described previously.

FIG. 2.

FIG. 2

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RAPD fingerprints of the representative B. burgdorferi sensu lato strains from different species or genomic groups (RAPD clusters I to VII) obtained by using four primers as indicated in the legend to Fig. 1. Lanes 1 to 4, B. burgdorferi sensu stricto strains B31, A91-9, VS215, and A44S, respectively; lanes 5 to 10, B. garinii subgroup IIa to IIf strains NT29, PBi, HT55, HT19, M63, and 20047, respectively; lanes 11 to 15, B. afzelii VS461, A38S, A95S, A67T, and A71T, respectively; lane 16, B. japonica HO14; lanes 17 and 18, B. valaisiana VS116 and M19, respectively; lane 19, Borrelia group DN127 strain 25015; lane 20, Borrelia sp. strain A14S; lanes 21 and 22, B. hermsii and B. anserina, respectively; lane R, reference for normalization of different gels; lane M, BstEII-digested bacteriophage lambda DNA. The molecular sizes (in kilobases) are indicated on the right.

FIG. 3.

FIG. 3

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Simplified dendrogram of B. burgdorferi sensu lato strains. The 136 LB-associated Borrelia strains used in this study could be divided into seven clusters with an intergroup difference of 55%. Six of them (clusters I to VI) corresponded to the indicated established species or genomic groups. One strain (branch VII) could not be classified into one of the described LB-related Borrelia species. Isolates from patients are in boldface. The numbers in parentheses indicate the corresponding RAPD type of each strain.

All Borrelia strains from the three pathogenic species B. burgdorferi sensu stricto, B. garinii, and B. afzelii presented a 1.84-kb predominant band after amplification with primer 1254. This band was absent from Borrelia strains from species which were cultured only from nonhuman sources.

RAPD fingerprinting within species.

Within the clusters of B. burgdorferi sensu stricto, B. garinii, and B. afzelii, a correlation between the geographic origins of the strains and their RAPD patterns was observed. Among the B. burgdorferi sensu stricto isolates, the European and North American isolates were in different clusters (Fig. 4). B. garinii strains from various sources exhibited high degrees of genetic diversity. At the 60% similarity level, the 39 B. garinii strains could be divided into six subgroups. The eight B. garinii isolates from Ixodes persulcatus from the far eastern area of Russia, Japan, and China clustered into subgroups IIa, IIc, and IId, respectively, whereas the 30 European isolates from Ixodes ricinus as well as from patients were clustered into subgroups IIb and IIf (Fig. 5). Subgroup IIe contained only one strain, strain M63, which had previously been designated as an independent genomic group because of its unique rRNA restriction pattern (29). The majority (95%; 56 of 59) of the B. afzelii strains showed more than 70% identity in their RAPD fingerprints. However, strain M7C, originating from China (RAPD type 97), was rather different from the other strains, which were isolated from The Netherlands and Germany. Two other isolates from The Netherlands (RAPD types 98 and 99) were also distinct from the other strains (Fig. 6). Some of the B. afzelii tick isolates that had been collected from the same sampling site showed a tendency to cluster. Of the 12 tick isolates from Santpoort, in the dunes in the western part of The Netherlands, 9 clustered together into two groups (RAPD types 50 to 55 and 59 to 61, respectively), but 3 strains were divergent. A similar result was obtained for 13 tick isolates from a forest in Drenthe, in the northern part of The Netherlands: 11 isolates grouped into two subclusters (RAPD types 72 to 77 and 83 to 85, respectively) and 2 were unrelated (Fig. 6).

FIG. 4.

FIG. 4

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Dendrogram of B. burgdorferi sensu stricto strains (n = 23). The Borrelia isolates derived from North America and Europe clustered into separate branches.

FIG. 5.

FIG. 5

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Dendrogram of B. garinii strains (n = 39). The asterisks indicate strains from the cerebrospinal fluid of patients. See the legend to Fig. 3 for more information.

FIG. 6.

FIG. 6

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Dendrogram of B. afzelii strains from different geographic and biological sources (n = 59). Tick isolates from two different regions in The Netherlands are indicated. Isolates from patients with ACA are underlined. Sa (Santpoort) and Dr (Drenthe) are two geographic regions in the western and northern parts of The Netherlands, respectively. See the legend to Fig. 3 for more information.

RAPD types among isolates from patients.

Of the 49 Borrelia strains from human specimens, 48 were typed by RAPD analysis as B. burgdorferi sensu stricto (n = 2), B. garinii (n = 16), or B. afzelii (n = 30). These results were consistent with the classification of these strains as designated in Table 1. Only one strain, strain A14S, could not be classified by RAPD analysis into any of the six LB-related species or genomic groups included in this study. This strain also had a quite remarkable PCR-RFLP pattern: instead of a 246- to 255-bp fragment, a fragment of 225 bp was amplified. Digestion of this fragment with MseI resulted in a unique pattern, which was designated pattern R (Table 1). This pattern was not consistent with any pattern produced by the 10 LB-related Borrelia species or genomic groups identified up to now (unpublished data). The 16 B. garinii strains, which were all isolated from patients with extracutaneous syndromes, were found in subgroups IIb and IIf. Isolates from human skin biopsy specimens and cerebrospinal fluid were evenly distributed among both subgroups. Interestingly, subgroup IIb included only eight isolates from patients with disseminated LB and showed little heterogeneity. Isolates from both humans and ticks were found in subgroup IIf, with no particular clustering for the isolates from humans. For B. afzelii, isolates from patients (n = 30) did not differ from those from ticks (n = 29). Furthermore, no significant subcluster was found among isolates recovered from patients with EM (n = 18) and ACA (n = 12).

Further characterization of B. garinii subgroup IIb.

Since B. garinii subgroup IIb only contained isolates from humans, further characterization of this subgroup was performed by sequence analysis of the 16S rRNA gene. Partial 16S rRNA gene analysis of three isolates in this subgroup (isolates PBi, A01C, and A76S) showed the two conserved nucleotide substitutions at positions 469 and 635 (B. burgdorferi B31 numbering) (17) in comparison to the B. garinii consensus sequences (24) and to sequences from B. burgdorferi sensu stricto and B. afzelii, confirming that strains in this subgroup are genetically distinct from other B. garinii strains.

DISCUSSION

RAPD analysis has been used with increasing frequency as a method for the molecular typing and genetic characterization of various microorganisms (38, 39). However, considerable attention is directed to its reliability and reproducibility, since many studies have indicated that various factors can affect the results of RAPD fingerprinting (8, 28, 38, 40). In our experiments, optimal RAPD fingerprinting was found with the four primers that we selected and purified DNA. With these four different primers, the reproducibility of the RAPD fingerprints for a random subset of 54 of the 136 isolates was up to at least a 95% level of similarity. The number of passages did not affect the results of RAPD analysis. However, PCR amplification of different colonies of one strain resulted in a difference of one band, amplified by one of the primers. This band was absent from two colonies and was present in two other colonies. Whether this difference is caused by the presence of a mixed population of two related strains or by colonial variation within one strain must be elucidated. Since these RAPD fingerprints from different colonies still showed more than 95% similarity to each other and belonged to the same RAPD type according to our definition, we concluded that RAPD analysis is an appropriate method for the typing of uncloned Borrelia strains.

By RAPD fingerprinting 135 of the 136 B. burgdorferi sensu lato strains used in this study could be grouped into six different species or genomic groups. This result was consistent with our classification of these strains on the basis of rRNA gene restriction analysis (41) or PCR-RFLP. One strain, strain A14S, could not be classified into one of the six species or genomic groups included in this study and also had a highly divergent PCR-RFLP pattern. This strain has pathogenic potential, since it was originally isolated from a patient with EM.

In our study, strains belonging to the same species but originating from different geographical regions clustered into separate branches by RAPD analysis. Regarding B. burgdorferi sensu stricto, North American and European strains fell into separate subgroups. With the exception of one B. garinii strain, B. garinii strains from Europe clustered into two major subbranches. All eight B. garinii strains from far east Asia were clearly different from the European strains included in these two major subbranches. Among the B. afzelii strains, the heterogeneity was limited. Only 2 of the 56 Dutch strains and the one Chinese strain tested were more divergent than the majority of the Dutch strains. Since only one Chinese strain was studied, more strains from Asia should be typed by RAPD fingerprinting before a conclusion can be drawn as to whether B. afzelii strains from European and Asian sources differ. Regional differences among North American B. burgdorferi sensu stricto strains were also found with the use of pulsed-field gel electrophoresis and sequence analysis (27). Another recent study in which pulsed-field gel electrophoresis as well as the AP-PCR technique was used showed that B. burgdorferi sensu stricto strains could be subdivided into a number of subgroups, generally consisting of only North American or only European strains (14); however, a few North American strains, including strain B31, clustered with the European strains and vice versa. In contrast, in our study B31 clustered only with North American strains. Since Foretz et al. (14) studied different European and North American isolates, this may partly account for differences between the studies.

B. afzelii strains grown from ticks collected in a limited area were often quite similar to each other, although we also found some exceptions. This is in accordance with the assumption that the migration rate of the vectors of B. afzelii, presumably being ticks and rodents, is rather low. It would be interesting to study a large collection of B. garinii strains in the same way, since birds are thought to be involved in the transmission of these spirochete species (9), and much more diversity of the strains within one area may occur because of this route of transmission.

Among the B. garinii strains, one cluster of eight strains (subgroup IIb) consisted only of isolates from humans. The strains were closely related to each other and were markedly different from the other B. garinii strains tested. The three subgroup IIb strains for which 16S rRNA gene sequencing was performed differed from the other B. garinii strains. All eight isolates in subgroup IIb originated from patients with disseminated disease. In another study, we showed that these strains all belonged to OspA type 4, as defined by Wilske et al. (47), and that they were resistant to the activity of normal human serum, in contrast to other B. garinii strains (42). Interestingly, these type 4 B. garinii strains have only been recovered from human specimens until now (48), and therefore, they may have an increased pathogenic potential.

The RAPD patterns of B. afzelii strains from ticks and humans were randomly distributed. In addition, strains from patients with ACA were not different from strains from patients with EM. This is in accordance with a recent study in which pulsed-field gel electrophoresis analysis of strains from patients with EM or ACA did not reveal differences between these groups (10). Therefore, those B. afzelii strains causing ACA have minor differences in their genomes in comparison with other B. afzelii strains. Alternatively, the persistence of these spirochetes, resulting in ACA, may mainly be determined by host factors.

In conclusion, RAPD analysis is a reliable technique for identifying the different Borrelia species, as well as for discriminating between strains within Borrelia species. Further use of this technique may lead to more information about the pathways of the geographic spread of the spirochetes. In addition, if more pathogenic subgroups exist, these may also be identified by this technique, which can be easily and rapidly performed after the isolation of a Borrelia strain.

ACKNOWLEDGMENTS

We thank S. J. Cutler (London, England), R. de Boer (Amsterdam, The Netherlands), M. Fukunaga (Fukuyama, Japan), O. Péter (Sion, Switzerland), S. G. T. Rijpkema (Bilthoven, The Netherlands), B. Wilske (Munich, Germany), and Z. F. Zhang (Beijing, People’s Republic of China) for supplying Borrelia strains. We also thank A. Oei for technical assistance, A. van der Ende and I. Schuurman for help in the development of RAPD analysis, and W. van Est for photography.

REFERENCES

  • 1.Akopyanz N, Bukanov N O, Westblom T U, Kresovich S, Berg D E. A diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res. 1992;20:5137–5142. doi: 10.1093/nar/20.19.5137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Appel M J G, Allan S, Jacobson R H, Lauderdale T L, Chang Y F, Shin S J, Summers B A. Experimental Lyme disease in dogs produces arthritis and persistent infection. J Infect Dis. 1992;167:651–654. doi: 10.1093/infdis/167.3.651. [DOI] [PubMed] [Google Scholar]
  • 3.Assous M V, Postic D, Paul G, Nevot P, Baranton G. Individualisation of two new genomic groups among American Borrelia burgdorferi sensu lato strains. FEMS Microbiol Lett. 1994;121:93–98. doi: 10.1111/j.1574-6968.1994.tb07081.x. [DOI] [PubMed] [Google Scholar]
  • 4.Balmelli T, Piffaretti J-C. Association between different clinical manifestations of Lyme disease and different species of Borrelia burgdorferi sensu lato. Res Microbiol. 1995;146:329–340. doi: 10.1016/0923-2508(96)81056-4. [DOI] [PubMed] [Google Scholar]
  • 5.Baranton G, Postic D, Saint Girons I, Boerlin P, Piffaretti J-C, Assous M, Grimont P A D. Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis. Int J Syst Bacteriol. 1992;42:378–383. doi: 10.1099/00207713-42-3-378. [DOI] [PubMed] [Google Scholar]
  • 6.Barbour A G, Tessier S L, Todd W J. Lyme disease spirochetes and Ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody. Infect Immun. 1983;41:795–804. doi: 10.1128/iai.41.2.795-804.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Bennett B E. Tick and Lyme disease. Adv Parasitol. 1995;36:344–405. doi: 10.1016/s0065-308x(08)60494-7. [DOI] [PubMed] [Google Scholar]
  • 8.Benter T, Papadopoulos S, Manns M, Poliwoda H. Optimization and reproducibility of random amplified polymorphic DNA in human. Anal Biochem. 1995;230:92–100. doi: 10.1006/abio.1995.1442. [DOI] [PubMed] [Google Scholar]
  • 9.Bunikis J, Olsén B, Fingerle V, Bonnedahl J, Wilske B, Bergström S. Molecular polymorphism of the Lyme disease agent Borrelia garinii in northern Europe is influenced by a novel enzootic Borrelia focus in the North Atlantic. J Clin Microbiol. 1996;34:364–368. doi: 10.1128/jcm.34.2.364-368.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Busch U, Hizo-Teufel C, Böhmer R, Fingerle V, Rößler D, Wilske B, Preac-Mursic V. Borrelia burgdorferi sensu lato strains isolated from cutaneous Lyme borreliosis biopsies differentiated by pulsed-field gel electrophoresis. Scand J Infect Dis. 1996;28:583–589. doi: 10.3109/00365549609037965. [DOI] [PubMed] [Google Scholar]
  • 11.Canica M M, Nato F, du Merle L, Maizie J C, Baranton G, Postic D. Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestation of Lyme borreliosis. Scand J Infect Dis. 1993;25:441–448. doi: 10.3109/00365549309008525. [DOI] [PubMed] [Google Scholar]
  • 12.Dever L L, Jorgensen J H, Barbour A G. In vitro antimicrobial susceptibility testing of Borrelia burgdorferi: a microdilution MIC method and time-kill studies. J Clin Microbiol. 1992;30:2692–2697. doi: 10.1128/jcm.30.10.2692-2697.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Fikrig E, Barthold S W, Kantor F S, Flavell R A. Protection of mice against the Lyme disease agent by immunizing with recombinant OspA. Science. 1990;250:553–556. doi: 10.1126/science.2237407. [DOI] [PubMed] [Google Scholar]
  • 14.Foretz M, Postic D, Baranton G. Phylogenetic analysis of Borrelia burgdorferi sensu stricto by arbitrarily primed PCR and pulsed-field gel electrophoresis. Int J Syst Bacteriol. 1997;47:11–18. doi: 10.1099/00207713-47-1-11. [DOI] [PubMed] [Google Scholar]
  • 15.Fukunaga M, Hamase A, Okada K, Nakao M. Borrelia tanukii sp. nov. and Borrelia turdae sp. nov. found from Ixodes ticks in Japan: rapid species identification by 16S rRNA gene-targeted PCR analysis. Microbiol Immunol. 1996;40:877–881. doi: 10.1111/j.1348-0421.1996.tb01154.x. [DOI] [PubMed] [Google Scholar]
  • 16.Fukunaga M, Koreki Y. A phylogenetic analysis of Borrelia burgdorferi sensu lato isolates associated with Lyme disease in Japan by flagellin gene sequence determination. Int J Syst Bacteriol. 1996;46:416–421. doi: 10.1099/00207713-46-2-416. [DOI] [PubMed] [Google Scholar]
  • 17.Gazumyan A, Schwartz J J, Liveris D, Schwartz I. Sequence analysis of the ribosomal RNA operon of the Lyme disease spirochete, Borrelia burgdorferi. Gene. 1994;146:57–65. doi: 10.1016/0378-1119(94)90833-8. [DOI] [PubMed] [Google Scholar]
  • 18.Hunter P R, Gaston M A. Numerical index of the discriminatory ability of typing systems: an application of Simpson’s index of diversity. J Clin Microbiol. 1988;26:2465–2466. doi: 10.1128/jcm.26.11.2465-2466.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Johnson R C, Schmid G P, Hyde F W, Steigerwalt A G, Brenner D J. Borrelia burgdorferi sp. nov.: etiological agent of Lyme disease. Int J Syst Bacteriol. 1984;34:496–497. [Google Scholar]
  • 20.Karabata H, Masuzawa T, Yanagihara Y. Genomic analysis of Borrelia japonica sp. nov. isolated from Ixodes ovatus in Japan. Microbiol Immunol. 1993;37:843–848. doi: 10.1111/j.1348-0421.1993.tb01714.x. [DOI] [PubMed] [Google Scholar]
  • 21.Kuiper H, van Dam A P, Spanjaard L, de Jongh B M, Widjojokusumo A, Ramselaar A C P, Cairo I, Vos K, Dankert J. Isolation of Borrelia burgdorferi from skin biopsy specimens taken from healthy-looking skin of patients with Lyme borreliosis. J Clin Microbiol. 1994;32:715–720. doi: 10.1128/jcm.32.3.715-720.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Le Fleche A, Postic D, Girardet K, Peter O, Baranton G. Characterization of Borrelia lusitaniae sp. nov. by 16S ribosomal DNA sequence analysis. Int J Syst Bacteriol. 1997;47:921–925. doi: 10.1099/00207713-47-4-921. [DOI] [PubMed] [Google Scholar]
  • 23.Liang J G, Zhang Z F. Analysis of rRNA gene restriction fragment length polymorphism of Borrelia burgdorferi sensu lato isolated in China. Chin J Microbiol Immunol. 1996;16:359–362. [Google Scholar]
  • 24.Marconi R T, Caron C F. Development of polymerase chain reaction sets for diagnosis of Lyme disease and for species-specific identification of Lyme disease isolates by 16S rRNA signature nucleotide analysis. J Clin Microbiol. 1992;30:2830–2834. doi: 10.1128/jcm.30.11.2830-2834.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Marconi R T, Liveris D, Schwartz I. Identification of novel insertion elements, restriction fragment length polymorphism patterns, and discontinuous 23S rRNA in Lyme disease spirochetes: phylogenetic analyses of rRNA genes and their intergenic spacers in Borrelia japonica sp. nov. and genomic group 21038 (Borrelia andersonii sp. nov.) isolates. J Clin Microbiol. 1995;33:2427–2434. doi: 10.1128/jcm.33.9.2427-2434.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Masuzawa T, Komikado T, Iwaki A, Suzuki H, Kaneda K, Yanagihara Y. Characterization of Borrelia sp. isolated from Ixodes tanuki, I. turdus, and I. columnae in Japan by restriction fragment length polymorphism of rrf (5S)-rrl (23S) intergenic spacer amplicons. FEMS Microbiol Lett. 1996;142:77–83. doi: 10.1111/j.1574-6968.1996.tb08411.x. [DOI] [PubMed] [Google Scholar]
  • 27.Mathiesen D A, Oliver J H, Jr, Kolbert C P, Tullson E D, Johnson B J B, Campbell G L, Mitchell P D, Reed K D, Telford III S R, Anderson J F, Lane R S, Persing D H. Genetic heterogeneity of Borrelia burgdorferi in the United States. J Infect Dis. 1997;175:98–107. doi: 10.1093/infdis/175.1.98. [DOI] [PubMed] [Google Scholar]
  • 28.Meunier J R, Grimont P A D. Factors affecting reproducibility of random amplified polymorphic DNA fingerprinting. Res Microbiol. 1993;144:373–379. doi: 10.1016/0923-2508(93)90194-7. [DOI] [PubMed] [Google Scholar]
  • 29.Nohlmans L M K E, de Boer R, van den Bogaard A E J M, Boven C P A. Genotypic and phenotypic analysis of Borrelia burgdorferi isolates from The Netherlands. J Clin Microbiol. 1995;33:119–125. doi: 10.1128/jcm.33.1.119-125.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Péter O, Bretz A G. Polymorphism of outer surface proteins of Borrelia burgdorferi as a tool for classification. Zentralbl Bakteriol Parasitenkd Infektkrankh Hyg Abt 1 Orig Reihe A. 1992;277:28–33. doi: 10.1016/s0934-8840(11)80867-4. [DOI] [PubMed] [Google Scholar]
  • 31.Péter O, Bretz A G, Zenhäusern R, Roten H, Roulet E. Isolement de Borrelia burgdorferi du liquide céphalorachidien de trois enfants avec une atteinte neurologique. Schweiz Med Wochenschr. 1993;123:14–19. [PubMed] [Google Scholar]
  • 32.Picken R N, Cheng Y, Strle F, Picken M M. Patients isolates of Borrelia burgdorferi sensu lato with genotypic and phenotypic similarities to strains 25015. J Infect Dis. 1996;174:1112–1115. doi: 10.1093/infdis/174.5.1112. [DOI] [PubMed] [Google Scholar]
  • 33.Postic D, Assous M V, Grimont P A D, Baranton G. Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf (5S)-rrl (23S) intergenic spacer amplicons. Int J Syst Bacteriol. 1994;44:743–752. doi: 10.1099/00207713-44-4-743. [DOI] [PubMed] [Google Scholar]
  • 34.Preac-Mursic V, Wilske B, Schierz G. European Borrelia burgdorferi isolated from humans and ticks: culture conditions and antibiotic susceptibility. Zentralbl Bakteriol Parasitenkd Infektkrankh Hyg Abt 1 Orig Reihe A. 1986;263:112–118. doi: 10.1016/s0176-6724(86)80110-9. [DOI] [PubMed] [Google Scholar]
  • 35.Rijpkema S G T, Molkenboer M J C H, Schouls L M, Jongejan F, Schellekens J F P. Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterization of the amplified intergenic spacer region between 5S and 23S rRNA genes. J Clin Microbiol. 1995;33:3091–3095. doi: 10.1128/jcm.33.12.3091-3095.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Sneath P H A, Sokal R R. Numerical taxonomy: the principle and practices of numerical classification. W. H. San Francisco, Calif: Freeman & Co.; 1973. [Google Scholar]
  • 37.Steere A C. Lyme disease. N Engl J Med. 1989;321:586–596. doi: 10.1056/NEJM198908313210906. [DOI] [PubMed] [Google Scholar]
  • 38.Tyler K D, Wang G, Tyler S D, Johnson W M. Factors affecting reliability and reproducibility of amplification-based fingerprinting of representative bacterial pathogens. J Clin Microbiol. 1997;35:339–346. doi: 10.1128/jcm.35.2.339-346.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.van Belkum A. DNA fingerprinting of medically important microorganisms by PCR. Clin Microbiol Rev. 1994;7:174–184. doi: 10.1128/cmr.7.2.174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.van Belkum A, Kluytmans J, van Leeuwen W, Bax R, Quint W, Peters E, Fluit A, Vandenbboucke-Grauls C, van der Brule A, Koeleman H, Melchers W, Meis J, Elaichouni A, Vaneechoutte M, Moonens F, Maes N, Struelens M, Tenover F, Verbrugh H. Multicenter evaluation of arbitrarily primed PCR for typing of Staphylococcus aureus strains. J Clin Microbiol. 1995;33:1537–1547. doi: 10.1128/jcm.33.6.1537-1547.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Van Dam A P, Kuiper H, Vos K, Widjojokusumo A, de Jongh B M, Spanjaard L, Ramselaar A C P, Kramer M D, Dankert J. Different genospecies of Borrelia burgdorferi are associated with distinct clinical manifestations of Lyme borreliosis. Clin Infect Dis. 1993;17:708–717. doi: 10.1093/clinids/17.4.708. [DOI] [PubMed] [Google Scholar]
  • 42.van Dam A P, Oei A, Jaspars R, Fijen C, Wilske B, Spanjaard L, Dankert J. Complement-mediated serum sensitivity among spirochetes that cause Lyme disease. Infect Immun. 1997;65:1228–1236. doi: 10.1128/iai.65.4.1228-1236.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Wang G, van Dam A P, Le Fleche A, Postic D, Peter O, Baranton G, de Boer R, Spanjaard L, Dankert J. Genetic and phenotypic analysis of Borrelia valaisiana sp. nov. (Borrelia genomic groups VS116 and M19) Int J Syst Bacteriol. 1997;47:926–932. doi: 10.1099/00207713-47-4-926. [DOI] [PubMed] [Google Scholar]
  • 44.Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 1990;18:7213–7218. doi: 10.1093/nar/18.24.7213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Welsh J, Pertzman C, Postic D, Saint Girons I, Baranton G, McClelland M. Genomic fingerprinting by arbitrarily primed polymerase chain reaction resolves Borrelia burgdorferi into three distinct phyletic groups. Int J Syst Bacteriol. 1992;42:370–377. doi: 10.1099/00207713-42-3-370. [DOI] [PubMed] [Google Scholar]
  • 46.Williams J G K, Kubelik A R, Livak K J, Antoni Rafalski J, Tingey S V. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 1990;18:6531–6535. doi: 10.1093/nar/18.22.6531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Wilske B, Preac-Mursic V, Gobel U B, Graf B, Jauris S, Soutschek E, Schwab E, Zumstein G. An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis. J Clin Microbiol. 1993;31:340–350. doi: 10.1128/jcm.31.2.340-350.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Wilske B, Busch U, Eiffert H, Fingerle V, Pfister H-W, Rossler D, Preac-Mursic V. Diversity of OspA and OspC among cerebrospinal fluid isolates of Borrelia burgdorferi sensu lato from patients with neuroborreliosis in Germany. Med Microbiol Immunol. 1996;184:195–201. doi: 10.1007/BF02456135. [DOI] [PubMed] [Google Scholar]
  • 49.Wilson K. Preparation of genomic DNA from bacteria. In: Ausubel F M, editor. Current protocols in molecular biology. Vol. 1. New York, N.Y: John Wiley & Sons, Inc.; 1988. pp. 2.4.1–2.4.5. [DOI] [PubMed] [Google Scholar]

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