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Analysis of a VMP-like sequence (vls) locus in Borrelia garinii and Vls homologues among four Borrelia burgdorferi sensu lato species

Guiqing Wang, Alje P. van Dam, Jacob Dankert
DOI: http://dx.doi.org/10.1111/j.1574-6968.2001.tb10648.x 39-45 First published online: 1 May 2001

Abstract

The VMP-like sequence (vls) locus that consists of one expressed vlsE gene and 15 silent vls cassettes has been described in Borrelia burgdorferi sensu stricto B31. In the present study, the vls locus from a Borrelia garinii isolate A87SA was analyzed. DNA fragments that contained three complete and five partial vls cassettes were cloned and sequenced. Pulsed-field gel electrophoresis (PFGE) analysis and Southern hybridization of the PFGE blot indicated that the vls locus of B. garinii A87SA, consisting of at least eight vls cassettes, was located on a 21-kb linear plasmid. The size of the three complete vls cassettes varied from 573 to 612 bp. They had 93.8–94.3% identity at the nucleotide level and 84.9–87.3% amino acid identity. The amino acid sequences of the three vls cassettes of B. garinii A87SA exhibited 45.9–50.8% identity to the VlsE sequence of B. burgdorferi B31, and 30.0–33.8% identity to the VMP17 sequence of B. hermsii HS1. Homologues of the vls locus of B. garinii were detected by dot blot hybridization among 24 of the 30 (80.0%) isolates representing four B. burgdorferi sensu lato species distributed widely in Europe. Our findings indicate that B. garinii might possess a similar vls structure to that described in B. burgdorferi sensu stricto. The highly conserved nature of the vls locus among various B. burgdorferi sensu lato species suggests that it may be important in the physiology and pathogenesis of Lyme disease spirochetes.

Keywords
  • Lyme disease
  • VMP-like sequence
  • Antigenic variation
  • Borrelia burgdorferi
  • Borrelia garinii

1 Introduction

Infection with Borrelia burgdorferi sensu lato, the etiological agent of Lyme borreliosis (LB), may result in persistent clinical disease in humans and experimentally infected mice [1]. Several strategies that include differential gene expression, limited exposure and hindrance of access to outer membrane lipoproteins, immune mimicry to host tissue components and antigenic variation might be utilized by the spirochetes to evade host immunity and adapt to diverse environments (reviewed in [2]). Like other pathogenic bacteria and parasites such as Borrelia hermsii, Neisseria gonorrhoeae and African trypanosomes [3], sequence variations of various surface-exposed proteins, notably the outer surface proteins OspA, OspB, and OspC, and the decorin binding protein DbpA, were reported in B. burgdorferi sensu lato [46]. Lateral gene transfer and recombination among Borrelia isolates between and within species has been suggested to account for such sequence variations [4,6]. A variable major protein (VMP)-like sequence (vls) system that consists of one expressed vlsE gene and 15 silent vls cassettes on a 28-kb linear plasmid (lp28-1) was described in B. burgdorferi sensu stricto (ss) isolate B31 [7]. The Vls proteins of B. burgdorferi ss are variable and are expressed both in experimentally infected C3H/HeJ mice [7] and in patients with LB [8,9]. A cassette-specific, segmental gene conversion mechanism may be involved in yielding the extensive antigenic variations of these VlsE proteins [10]. More recently, VMP-like sequences were also reported to be present in more than 20 B. burgdorferi ss isolates cultured from patients with LB in North America [11]. Comparison of the vls sequences from low-passage clinical isolates indicates that both highly conserved and heterogeneous subgroups exist among the North American B. burgdorferi ss isolates [11].

In Europe, three Borrelia species, B. burgdorferi ss, Borrelia garinii and Borrelia afzelii, are responsible for causing human LB [12,13]. Although B. burgdorferi ss has been cultured or detected by PCR from specimens of some patients with LB, several studies in different countries show clearly that B. garinii and B. afzelii are the predominant pathogens of human LB in Europe [13,14]. The presence of a vls homologue of B. burgdorferi ss in B. garinii isolates 20047 and Ip90, and B. afzelii isolates ACA1 and P/Gau has been demonstrated by Southern hybridization [7,8]. However, no data are available to date on sequences of the vls locus of B. garinii and distribution of the vls homologues among different European B. burgdorferi sensu lato species. In the present study, we cloned and sequenced DNA fragments containing three complete and five partial vls cassettes of the vls locus from a cloned B. garinii isolate, A87SA. Furthermore, homologues of the vls locus were evaluated among 30 B. burgdorferi sensu lato isolates representing four Borrelia species distributed widely in Europe.

2 Materials and methods

2.1 Borrelia isolates

B. garinii isolate A87S was originally cultured from a skin biopsy of a patient with Lyme neuroborreliosis in The Netherlands [14]. Two cloned variants of this isolate, designated A87SA and A87SB, were obtained after plating spirochetes onto BSK II agar [15]. Thirty additional B. burgdorferi sensu lato isolates belonging to four different species and B. hermsii isolate HS1 were included to determine distribution of the vls homologues (Table 1). All spirochetes were cultured in modified BSK II medium at 33°C and Borrelia genomic DNA was extracted as described previously [14].

View this table:
Table 1

B. burgdorferi sensu lato isolates used in this study and detection of vls homologues by dot hybridization

Species and isolateSourcePassageaOspA serotypebDot hybridizationcProvider or reference(s)
B. burgdorferi ss
B31I. scapularis, USAHp1+/−ATCC35210
297Human CSF, USAHp1+/−[12]
N40I. scapularis, USAP41++E. Fikrig
A44SHuman skin (EM), The NetherlandsP41++[14]
VS293I. ricinus, SwitzerlandP91+O. Peter
VS130I. ricinus, SwitzerlandP91++O. Peter
VS215I. ricinus, SwitzerlandP91+O. Peter
B. garinii
20047I. ricinus, FranceHp4+/−[12]
A19SHuman skin (EM), The NetherlandsP74++[14]
A76SHuman skin (EM), The NetherlandsP144+[14]
A91CHuman CSF, The NetherlandsP64+[14]
A94CHuman CSF, The NetherlandsP64+[14]
T25I. ricinus, Germanyx7+B. Wilske
TNI. ricinus, Germanyx6++B. Wilske
A87SAHuman skin (EM), The NetherlandsP95++This study
A87SBHuman skin (EM), The NetherlandsP95This study
A77CHuman CSF, The NetherlandsP86++[14]
VSDAHuman CSF, SwitzerlandP95++O. Peter
VSBPHuman CSF, SwitzerlandP96++O. Peter
VSBMHuman CSF, SwitzerlandP96++O. Peter
PBiHuman CSF, Germanyx4+/−B. Wilske
B. afzelii
VS461I. ricinus, SwitzerlandHp2+/−[12], O. Peter
A67TI. ricinus, The NetherlandsP62++[14]
A48TI. ricinus, The NetherlandsP122++[14]
A17SHuman skin (EM), The NetherlandsP42++[14]
A20SHuman skin (EM), The NetherlandsP82++[14]
A26SHuman skin (EM), The NetherlandsP52++[14]
M7I. persulcatus, Chinaxnt+Z. Zhang
B. valaisiana
VS116I. ricinus, SwitzerlandHpnt+/−[18], O. Peter
M19I. ricinus, The Netherlandsxnt+[18], R. de Boer
M52I. ricinus, The Netherlandsxnt++[18], R. de Boer
M53I. ricinus, The Netherlandsxnt+[18], R. de Boer
B. hermsii
HS1Hp+
  • aHp: high passage (>P30); x: passage unknown.

  • bOspA serotypes were determined previously [15].

  • cBased on dot hybridization with a vls-containing probe pA693 as shown in Fig. 4. − no hybridization signal; +/−: average density value of the sample was over the mean density value plus 2 S.D. of the background; +: >mean plus 5 S.D.; ++: >mean plus 20 S.D.

2.2 Cloning and sequencing of the vls locus of B. garinii A87SA

A 10-kb HindIII fragment containing DNA homologous to the vls locus of B. burgdorferi B31 was identified in B. garinii A87SA by genome subtractive hybridization [16]. Briefly, DNA from strain A87SA (tester) was partially digested with Sau3A and hybridized to DNA from strain A87SB (driver) which had been sheared by ultrasonication [17]. The reassociated DNA mixture was ligated into a vector and resulting clones were tested for their reactivity with strains A87SA and A87SB. Among the clones reactive with A87SA but not with A87SB were clones pA213, pA248, pA501, pA503, pA529, and pA693. All these clones hybridized with the 10 kb fragment mentioned above. This fragment was purified from agarose gel with the QiaX II kit (Qiagen, Leusden, The Netherlands), partially digested with PstI, and cloned into PstI-digested, and both HindIII- and PstI-digested pBluescript KS vectors (Promega), respectively. One clone (pA87A) with a 4.5-kb HindIII–PstI fragment insert and several clones (e.g., pA87B1 and pB43) containing 0.6–1.2-kb PstI fragments were obtained. Directional subcloning of plasmid pA87A resulted in a subclone (pA87A1) with an insert 2.1-kb SpeI–PstI fragment (Fig. 1). Subsequently, DNA sequences were determined from both termini of clones pA87A, pA87B1, and pB43, and subclone pA87A1 on an A373 DNA sequencer [18].

1

Schematic structure of the vls locus of B. garinii A87SA. The three complete cassettes vlsH1 to vlsH3 of the vls locus are flanked by an 18-bp direct repeat (DR) sequence. Five other hypothetical vls cassettes included were based on the unique sequences of clones pA248, pA501, pA503, pA529, and pB43. Each of these clones contained sequences of the central region of a distinct vls cassette. Their exact locations were not determined in this study. A hypothetical vlsE expression site and a telomere according to the vls structure of B. burgdorferi ss B31 are shown in the figure [7].

Based on the DNA sequences from clones pA87A1 and pA87B1, two primers, A87A1R1 (5′-AGTAA TGCAT ATTTT GAGAA C-3′, reverse primer) and A87B1F1 (5′-TTGGT GCTGA CAATG CTGAT C-3′, forward primer), were designed and used to amplify the vls locus of B. garinii A87SA. The PCR was performed in a 50-μl mixture containing 100 ng of purified plasmid DNA, 10 mM Tris–HCl (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 0.1 mg ml−1 of bovine serum albumin, 200 μM of each deoxynucleotide triphosphate (Pharmacia Biotech), 1.5 U of Taq polymerase (Qiagen), and 0.5 μM of each primer. The PCR was carried out with a Biometra thermal cycler as described previously [13]. A DNA fragment with a size of 2064 bp was amplified by PCR, cloned directly into the TOPO-pCR2.1 vector (Invitrogen, Leek, The Netherlands) and sequenced.

2.3 Pulsed-field gel electrophoresis (PFGE) and Southern hybridization

The plasmid DNA of B. garinii variants A87SA and A87SB was extracted as described by Barbour et al. [19] and separated by PFGE with a CHEF-DRII apparatus (Bio-Rad Laboratories, Veenendaal, The Netherlands) with 0.9–2.5-s pulses at 14°C in 0.5×Tris–boric–EDTA buffer for 30 h. The gel was stained with 1×Gelstar (FMC Bioproduct, Rockland, ME, USA) solution, illuminated over UV light, and photographed. The plasmid DNAs separated by PFGE were transferred to a nylon membrane (Zeta-probe, Bio-Rad Laboratories) and hybridized with a 466-bp digoxigenin-labeled probe pA693 (GenBank accession number AF274076) which had 85.7–94.4% nucleotide identity with the 5′ sequences of the three complete cassettes vlsH1 to vlsH3.

2.4 Dot blot hybridization

Dot blots were prepared by spotting 1 μg of Borrelia genomic DNA from each isolate onto a Zeta-probe membrane, and hybridized with the digoxigenin-labeled DNA probe pA693 of B. garinii A87SA. Dot blot hybridization was performed at 60°C for 16 h in standard hybridization buffer (5×SSC, 0.1%N-lauroylsarcosine, 0.02% SDS and 1% blocking reagent) and the membrane was washed twice at 55°C in 0.5×SSC buffer with 0.1% SDS, followed by development of color as described previously [18]. Hybridization signal was analyzed with the Spot Density tools of the AlphaEase™ software (Alpha Innotech Corp., San Leandro, CA, USA), in which the average density value of each sample spot was estimated by dividing the integrated density value by spot area. Background density of the filter was calculated by selection of multiple representative blank spots from different regions on the membrane. Isolates with hybridization signal over the mean density value plus 2–20 S.D. of the background were described as weak (+/−) to strong (++), respectively (Table 1).

2.5 Nucleotide sequence accession numbers

The sequences determined in this study have been assigned GenBank accession numbers AF274070AF274076. Sequences used for comparison in the study are U76405 (B. burgdorferi B31 vlsE) and L04788 (vmp17 gene of B. hermsii HS1).

3 Results and discussion

3.1 Vls locus of B. garinii A87SA

In an attempt to identify the genetic differences between two clonal B. garinii variants with distinct protein profiles and distinct serum sensitivity, 11 clones containing DNA fragments present only in the serum-sensitive variant A87SA were identified using genome subtractive hybridization [16]. Interestingly, the DNA sequence from one of these clones (clone pA693), which hybridized to a 10-kb HindIII plasmid-derived fragment of B. garinii A87SA, showed homology to the sequence of the vlsE gene of B. burgdorferi ss B31. DNA sequences from five other clones (pA213, pA248, pA501, pA503, and pA529) contained a Sau3A restriction fragment with sizes of 145–157 bp each, and showed more than 90% identity to each other, but no homology to other sequences in GenBank or to the sequence of pA693. However, probes containing DNA sequences from these five clones hybridized to the same 10-kb HindIII fragment of isolate A87SA as clone pA693 in Southern hybridization (data not shown). We assumed that a vls locus similar to that described in B. burgdorferi ss B31 might be present on this 10-kb plasmid fragment of B. garinii A87SA and that these clones represented cassettes of this locus. Cloning of the entire 10-kb HindIII fragment from B. garinii A87SA was not successful, possibly due to the telomeric localization of this fragment [20]. Alternatively, a HindIII–PstI partial digest of the purified 10-kb band and several digests of this fragment were subcloned as described in Section 2. As a result, three clones containing vls sequence were obtained and partially sequenced: clone pA87A contained a 4.5-kb HindIII–PstI fragment, while two clones, pA87B1 and pB43, possessed a 0.6- and a 1.2-kb PstI fragment, respectively. A clone, pA87A1, with an insert 2.1-kb SpeI–PstI fragment was also obtained by subcloning of pA87A (Fig. 1). Analysis of partial DNA sequences (about 550 bp each) of clones pA87A and pA87A1 revealed no vls-related sequence at the 5′ end (HindIII and SpeI restriction sites) of the inserts. Of the 550 bp of common DNA sequence of the 3′ terminus at the PstI restriction site of clones A87A and pA87A1, the first 272 bp had a G+C content of 50.5%, and showed more than 92% identity to the sequences from two other clones, pA87B1 and pB43. In contrast, the upstream 278 bp of DNA sequence had only 23.1% G+C content and no homology to any reported vls sequences. This finding suggested that the 3′ sequence of clones pA87A and pA87A1 contained part of the last vls recombination cassette in B. garinii A87SA. Thus, we designed two primers based on the DNA sequences from clones pA87A1 and pA87B1. A DNA fragment with a size of 2064 bp was amplified by PCR, cloned and sequenced. Three consecutive, complete cassettes of the vls locus, which were designated vlsH1, vlsH2 and vlsH3, were identified from the resulting clone pVSC3 (Fig. 1). In addition, partial DNA sequence of another vls cassette upstream of these three complete cassettes was recognized. Like in B. burgdorferi ss B31, each of the cassettes in B. garinii A87SA was flanked by an 18-bp direct repeat sequence, although the direct repeat nucleotides in B. garinii A87SA were clearly different from those of B. burgdorferi ss B31. The size of the three complete vls cassettes varied from 573 bp to 612 bp, showing 93.8–94.3% nucleotide identity and 84.9–87.3% amino acid identity to each other. The amino acid sequences of the three vls cassettes exhibited 45.9–50.8% identity to the VlsE sequence of B. burgdorferi ss B31, and 30.0–33.8% identity to the VMP17 sequence of B. hermsii isolate HS1 (Table 2).

View this table:
Table 2

Sequence similarity of the vls cassettes of B. garinii A87SA, B. burgdorferi ss B31 and the vmp17 of B. hermsiia

B. garinii A87S vlsH1B. garinii A87S vlsH2B. garinii A87S vlsH3B. burgdorferi ss B31 vlsE1B. hermsii HS1 vmp17
B. garinii A87SA vlsH184.987.350.830.2
B. garinii A87SA vlsH293.885.345.933.8
B. garinii A87SA vlsH394.194.349.830.0
B. burgdorferi ss B31 vlsE167.866.668.236.8
B. hermsii HS1 vmp1760.973.262.372.2
  • aNumbers indicate percentages of identity of the nucleotides and amino acids (shown in bold).

Of the five clones obtained by genome subtractive hybridization, the DNA and amino acid sequence of clone pA213 (not shown in Fig. 1) were identical to that of the central region of cassette VlsH2. The amino acid sequences from four other subtractive clones (pA248, pA501, pA503, and pA529) and clone pB43, obtained in this study, possessed a high degree of similarity to sequences of the central region of the three complete vls cassettes. Each of these clones contained a unique Sau3A fragment. They might originate from five different vls cassettes of B. garinii A87SA since each of the three complete vls cassettes (VlsH1 to VlsH3) contained only one Sau3A fragment. The sequence of pA693 had a high homology to the 5′ part of the cassettes VlsH1 to VlsH3 and should therefore be mapped upstream one of the five Sau3A fragments to constitute an unsequenced cassette (Fig. 2). Given that almost 4.2 kb of the total 10-kb HindIII fragment contained non-vls sequence, the vls locus in B. garinii A87SA should be carried on an approximately 6-kb fragment. Assuming that each cassette had a size similar to the three identified cassettes, it is speculated that the vls locus of B. garinii A87SA would consist of 8–10 vls cassettes. This was supported by the identification of three complete and five partial vls cassettes as described in this study (Figs. 1 and 2). It is not clear whether an expression site, as described in B. burgdorferi ss B31, was also present in B. garinii A87SA, due to the fact that the entire vls locus has not yet been sequenced.

2

Comparison of Vls sequences of B. garinii (Bg), B. burgdorferi ss (Bbss) with Vmp of B. hermsii (Bh). Deduced amino acid sequences of three complete and six partial vls cassettes including the probe pA693 from B. garinii isolate A87SA, the VlsE sequence of B. burgdorferi ss B31 and the Vmp17 of B. hermsii HS1 are included. These sequences were aligned with ClustalW and formatted with the MEGA program. Amino acid residues are numbered to the left of the alignments according to the full-length sequences of VlsE and Vmp17 proteins or to the deduced sequences from individual cassettes or clones of isolate A87SA. Dots indicate amino acid residues that were identical to the VlsH1 sequences of B. garinii A87SA. Deletions are shown as dashes. The eight Sau3A fragments of B. garinii A87SA, each from a unique vls cassette, are boxed. The six variable regions (VR-I to VR-VI) were defined on sequences of B. burgdorferi ss B31 [7] and are shadowed in gray.

In B. burgdorferi ss, a high degree of sequence identity of the Vls locus was observed among clinical and tick isolates from North America [11]. Nearly all of the differences were restricted to the six variable regions (VR-I through VR-VI) which contain most of the sequence differences among the vls silent cassettes of B. burgdorferi ss B31 [7]. In B. garinii A87SA, sequence differences among the three Vls cassettes were also observed mainly in sequences corresponding to the six variable regions of B. burgdorferi ss B31 (Fig. 2). This finding implies that a vls locus showing comparable variable regions to that of B. burgdorferi ss was present in B. garinii.

3.2 Location of the vls locus in B. garinii A87SA

The vls locus in B. burgdorferi ss B31 was located on a 28-kb linear plasmid (lp28-1) [7]. However, the sizes of plasmids carrying the vls locus vary from 21 to 38 kb among the clinical isolates from North America [11]. To locate the vls locus in B. garinii, plasmids of B. garinii variants A87SA and A87SB were separated by PFGE (Fig. 3A). Subsequent hybridization of a PFGE blot with probe pA693 showed that the vls locus of B. garinii variant A87SA was carried on a 21-kb linear plasmid (Fig. 3B). This plasmid was absent in variant A87SB.

3

Plasmid profiles (A) of B. garinii variants A87SA (lane a) and A87SB (lane b) and Southern hybridization with a vls probe from clone pA693 (B). The plasmid carrying the vls locus in variant A87SA is indicated by the arrow at the left. Lane m: a 4.9-kb DNA ladder used as marker of molecular sizes (Bio-Rad Laboratories).

3.3 Homologues of the vls locus among four B. burgdorferi sensu lato species

To determine whether homologues of the vls sequence of B. garinii A87SA are conserved among different species of B. burgdorferi sensu lato, a digoxigenin-labeled DNA probe from subtractive clone pA693, known to contain DNA homologous to the vls of B. burgdorferi ss B31, was used for hybridization with the genomic DNAs from seven B. burgdorferi ss, 12 B. garinii, seven B. afzelii, and four B. valaisiana isolates, as well as B. hermsii HS1, in dot blot hybridization. Twenty-four of the 30 (80.0%) B. burgdorferi sensu lato isolates showed clear signals of hybridization, indicating that sequences homologous to the vlsE gene from B. garinii A87SA were present in these isolates (Fig. 4). Six B. burgdorferi sensu lato isolates (B31, 297, 20047, PBi, VS461, and VS116) showed only a weak signal on the blot. This is possibly the result of their sequence diversity in this region as compared to that of the probe from B. garinii A87SA. Alternatively, some strains may have lost the entire plasmid containing the Vls locus, since most of these isolates were cultured for more than 30 passages in vitro. In that case, the weak signals would be the result of non-specific hybridization. DNA homologous to the vls of B. garinii A87SA was also detected in B. hermsii HS1 (Fig. 4), but not in other bacterial species such as Haemophilus influenzae and Helicobacter pylori (data not shown), indicating that the signal in the dot blot hybridization was Borrelia-specific.

4

Dot blot hybridization of genomic DNAs from various B. burgdorferi sensu lato isolates with a vls probe of B. garinii A87SA. The Borrelia isolates used as indicated as follows: Lane A, samples 1–7: B. burgdorferi ss isolates B31, HB19, N40, A44S, VS293, VS130, and VS215, respectively; line B, samples 1–7: OspA serotype 4 and 7 B. garinii isolates 20047, A19S, A76S, A91C, A94C, T25, and TN, respectively; line C, samples 1–7: OspA serotypes 5 and 6 B. garinii isolates A87SA, A87SB, A77C, VSDA, VSBP, VSBM, and PBi, respectively; line D, samples 1–7: B. afzelii isolates VS461, A67T, A48T, A17S, A20S, A26S, and M7, respectively; line E, samples 1–7: B. valaisiana isolates VS116, M19, M52, and M53, B. hermsii HS1, positive control (isolate A87S) and negative control (dH2O), respectively. See Table 1 for detailed source, passage and OspA serotype of each isolate.

Previously, we have shown that OspA serotype 5 or 6 B. garinii and B. valaisiana strains were serum-sensitive, B. afzelii and most OspA serotype 4 B. garinii isolates were serum-resistant, whereas the serum sensitivity of B. burgdorferi ss isolates was intermediate [15]. Thus, no correlation between the presence or absence of vls locus and serum sensitivity among Borrelia isolates was noted. Therefore, it seems unlikely that the difference in serum sensitivity between strains A87SA and A87SB is related to the absence of vlsE in strain A87SB. Since the parent strain, A87S, was not infective in the C3H mouse model (E. Fikrig, unpublished observation), it is difficult to perform in vivo experiments to study the role of the vlsE genes using these mutants.

In the present study, we cloned DNA fragments containing three complete vls cassettes and partial sequences from five other cassettes of the vls locus from a clinical B. garinii isolate, A87SA. Moreover, homologues of the vls locus of B. garinii A87SA were identified among most of the B. burgdorferi sensu lato isolates representing four different species that are widely distributed in Europe. Since currently available knowledge of the vls locus is based only on B. burgdorferi ss isolates, this study provides a view on the structure of the vls locus in B. garinii and the distribution of the vls homologues among various B. burgdorferi sensu lato species. Our data, taken together with other previous reports [7,8,11], indicate that the vls system may be important for different B. burgdorferi sensu lato species. Further studies on the conservation and divergence of the vls genes among various B. burgdorferi sensu lato species, particularly the pathogenic B. garinii and B. afzelii strains in Europe, will provide more information on its potential importance in the physiology and pathogenesis of the LB spirochetes.

Acknowledgements

The authors thank Drs. R. de Boer, E. Fikrig, O. Peter, B. Wilske, and Z.F. Zhang for providing B. burgdorferi isolates, Drs A. van de Ende and S.A.J. Zaat for helpful discussions, Dr. I. Schwartz for critical reading of the manuscript and, Wim van Est for photography.

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