AEM
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Anderson, J. M.
Right arrow Articles by Norris, D. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Anderson, J. M.
Right arrow Articles by Norris, D. E.
Agricola
Right arrow Articles by Anderson, J. M.
Right arrow Articles by Norris, D. E.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, August 2006, p. 5331-5341, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00014-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Genetic Diversity of Borrelia burgdorferi Sensu Stricto in Peromyscus leucopus, the Primary Reservoir of Lyme Disease in a Region of Endemicity in Southern Maryland

Jennifer M. Anderson* and Douglas E. Norris

The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, Maryland 21205

Received 3 January 2006/ Accepted 8 June 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the north central and northeastern United States, Borrelia burgdorferi sensu stricto, the etiologic agent of Lyme disease (LD), is maintained in an enzootic cycle between the vector, Ixodes scapularis, and the primary reservoir host, Peromyscus leucopus. Genetic diversity of the pathogen based on sequencing of two plasmid-located genes, those for outer surface protein A (ospA) and outer surface protein C (ospC), has been examined in both tick and human specimens at local, regional, and worldwide population scales. Additionally, previous studies have only been conducted with tick or human specimens at the local population level in areas with high LD transmission rates. This study examined the genetic diversity of circulating borreliae in the reservoir population from a large region of the western coastal plains of southern Maryland, where moderate numbers of human LD cases are reported. Six ospA mobility classes, including two that were not previously described, and eight ospC groups were found among the P. leucopus samples. Twenty-five percent of all specimens were infected with more than one ospA or ospC variant. The frequency distribution of variants was homogeneous, both locally and spatially. The spirochete diversity found in Maryland was not as high as that observed among northern tick populations, yet similar genotypes were observed in both populations. These results also show that mice are important for maintaining Borrelia variants, even rare variants, and that reservoir populations should therefore be considered when assessing the diversity of B. burgdorferi.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Borrelia burgdorferi sensu lato is a complex of 11 pathogenic and nonpathogenic Borrelia species found in North America, Europe, and Asia (50). Of these, only three species have been isolated from clinically ill humans, B. burgdorferi sensu stricto, B. afzelii, and B. garinii, each possessing unique phenotypic and genotypic characteristics. In the United States, three Borrelia species from this complex have been isolated and characterized, B. burgdorferi sensu stricto, "B. bissettii," and "B. andersonii," yet only one, B. burgdorferi sensu stricto, is the etiologic agent of Lyme disease (LD) (30, 32, 72). The phylogenetic relationships among and genetic heterogeneity within various species of Borrelia have been widely studied (28, 39). Stable gene coding regions such as ribosomal subunits 5S and 23S (36, 50, 59) and functional chromosomal genes such as that for flagellin (fla) (14, 26, 42) have proven useful for taxonomic identification and for assessing phylogenetic relationships among these taxa. Other genes that code for immunodominant outer surface proteins (osp genes) have been valuable for determining the heterogeneity between and within populations (73). Two of the most widely studied proteins, OspA and OspC, are encoded by genes found on two separate plasmids (2, 38, 56). Because of their alternating expression patterns (58), they are differentially implicated in the maintenance of borreliae within the tick gut (ospA) (62, 77), dissemination to the salivary glands, and subsequent transmission to the vertebrate host (ospC) (17, 46). Although they were once considered to be clonal (8), it is now understood that the plasmids of B. burgdorferi are subject to recombination, lateral gene transfer, and immunological selection by vertebrate hosts, leading to genetic polymorphism in the bacterial population (4, 7, 53). Consequently, ospC and ospA are highly polymorphic within a single population and between geographically disjunct populations (18, 52, 54, 72).

B. burgdorferi sensu stricto circulates in an enzootic cycle between the primary vertebrate reservoir, the white-footed mouse (Peromyscus leucopus), and the black-legged tick (Ixodes scapularis) (34, 35). In North America, most of the information obtained about genetic variation in B. burgdorferi sensu stricto has been based on spirochetes and gene fragments amplified from I. scapularis ticks collected within regions where LD is highly endemic in New York (primarily Long Island and Shelter Island) (18, 52, 73) and a few scattered collections of ticks from the Mid-Atlantic region (54). These studies established that five clonal mobility class variants (MCVs) based on ospA (18) and 21 genetic groups based on ospC (60, 72) are found ubiquitously at most of the locations studied. In contrast, the diversity of ospC within human samples appears to be limited and varies between acute and disseminated infections (29, 31).

Numerous studies have assessed the genetic variation of B. burgdorferi in tick populations and human clinical specimens, but few studies have investigated the genetic heterogeneity of this pathogen in the rodent reservoir population (3, 31). White-footed mice mount a specific immune response against B. burgdorferi, primarily against the immunodominant OspC protein (16, 47, 76), yet they remain systemically and persistently infected and display no adverse effects due to the bacteremia (5, 20, 47). Additionally, the majority of studies assessing the diversity of B. burgdorferi have been conducted in regions where LD is considered to be highly endemic and only a few scattered samples have been collected in regions with moderate Lyme borreliosis endemicity (54).

To address these knowledge gaps, we conducted a phylogenetic study of B. burgdorferi recovered from field populations of P. leucopus collected from 96 locations in southern Maryland along the western coastal plains by using three molecular markers, gene fragments of flaB, ospA, and ospC. Specifically, we aimed to determine (i) if particular variants are more common among Peromyscus samples than reported among tick and human samples, (ii) if geographic differences in variant frequency exist between areas where LD is hyperendemic and areas where it is moderately endemic, and (iii) if new, previously undescribed variants exist in southern Maryland.

This is the first extensive report of the population structure of B. burgdorferi sensu stricto in the mid-Atlantic region of North America, as well as one of few extensive genetic surveys of spirochetes found in rodent reservoir populations over a large geographic region. Maryland reports moderate levels of LD transmission (12) and is at the geographic juxtaposition between regions with high and low LD transmission rates. Examining the genetic polymorphisms of B. burgdorferi found in the reservoir population in this region may help to elucidate the disparate LD transmission patterns observed along the eastern United States.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Amplification of ospA and ospC gene fragments from B. burgdorferi-positive mice.
P. leucopus mice were collected with small baited live traps (Sherman Traps, Tallahassee, FL) at 96 sites located within a five-county region of the western coastal plains of southern Maryland between June and September of 2001. A 2-mm ear punch biopsy was removed from each captured mouse (n = 548), and whole genomic DNA was extracted from surface-sterilized tissue with the QIAamp DNA Mini Kit (QIAGEN Corp.) by following the manufacturer's recommendations, with slight modification. Purified DNA was eluted in 50 µl of high-performance liquid chromatography grade water and stored at –80°C. DNA specimens positive for B. burgdorferi (n = 173) on the basis of PCR amplification of a 390-bp fragment of the flagellin B gene (flaB) (26) were used to investigate the phylogenetic structure of B. burgdorferi strains in southern Maryland. Two polymorphic markers, ospA and ospC, were employed for molecular typing by nested and seminested PCR amplification of gene fragments as previously described (18, 72). Only the 3' end of the ospC gene was amplified to produce a 314-bp fragment (1).

Cold single-stranded conformation polymorphism (SSCP) analysis.
B. burgdorferi flaB, ospA, and ospC gene fragments were analyzed for the presence of point mutations, insertions, and deletions by cold SSCP analysis (23, 44). A total of 5 µl of PCR product was added to 5 µl of a denaturing loading mixture (90% deionized formamide, 0.05% bromophenol blue, 0.05% xylene cyanol, 20% NaOH). The mixture was heated at 95°C for 2 min and then plunged into an ice bath to force single-strand annealing. The entire mixture was electrophoresed at 4 to 6°C on medium-format nondenaturing polyacrylamide gels (38.5%) run in 1x Tris-borate-EDTA buffer. Electrophoresis was conducted at a constant 20 mA (~400 V) for 4 to 5 h for best resolution. Electrophoretic products were visualized with SYBR green I (Cambrex Bio-Science, Rockland, ME) and photodocumented. Samples were grouped by banding pattern similarity, with each unique pattern designated an SSCP type. To identify and confirm the polymorphisms associated with each SSCP type, at least two templates representing each variant were purified with a QIAGEN QIAquick PCR purification kit (QIAGEN, Valencia, CA) and the products were sequenced directly in both directions.

Cloning of mixed alleles.
To investigate the possibility of a mixed-variant infection in a single mouse specimen, ospA and ospC PCR products representing suspected multiple SSCP profiles were cloned with a TOPO TA cloning kit (Invitrogen Corp., Carlsbad, CA). Ten to 20 transformed colonies were picked and used directly for PCR amplification with primers corresponding to the internal ospA or ospC nested product. PCR products from the cloned inserts were analyzed by SSCP to determine allele complexity and identification. Clones representing new banding patterns were then purified and sequenced as previously described.

Sequence and phylogenetic analysis.
Nucleic acid sequences for each of the gene fragments were assembled with SeqMan II (DNAstar Inc., Madison, WI), aligned with ClustalX (69), and manually realigned after translation to amino acid sequences with BioEdit software (19). B. burgdorferi sequences derived from rodent samples collected in Maryland were compared to published ospA and ospC sequences representing a variety of host sources and geographic regions. For sequence comparisons, PCR primer regions were removed and sequences were trimmed to equal length within each gene fragment under comparison. Alignment files were converted to Nexus files with ClustalX and then imported into PAUP* version Beta 10 (65).

Phylogenetic analysis was performed for each gene fragment separately. Before phylogenetic tree construction, each alignment was checked for the existence of phylogenetic structure (nonrandomness of the hierarchical structure) with a permutation test (9, 65) calculated with PAUP* (Sinauer Associates, Inc., Sunderland, MA). Alignments reporting a P value of <0.05 were considered acceptable for phylogenetic analysis. Phylogenetic trees based on maximum parsimony, maximum evolution (neighbor joining), and maximum likelihood were constructed by heuristic search methods, adding sequences to the initial tree randomly and with a tree bisection-reconnection branch-swapping algorithm. The GTR+ {Gamma} model of substitution, base frequencies, gamma distribution, invariant sites, transition/transversion ratios, and rate matrix values were determined with Modeltest 3.5 (49) for maximum-likelihood tree construction. Start branch lengths were calculated by the Rogers-Swofford approximation (55, 66). Each analysis was conducted on nucleotide and translated amino acid alignments, as well as gapped (representing insertions) and gap-stripped alignments. The reliability of individual branches was assessed by bootstrap replication (10) conducted under a maximum-likelihood criterion with 1,000 replicates.

Statistical analysis.
Genetic distance was calculated between pairs of sequences by the Tamura and Nei method (67) with the program Mega 2.1 (27). The mean pairwise distance, based on transitions and transversions, and the standard error (SE) were calculated for clades containing Maryland samples. The frequencies of ospA and ospC types were determined from quantification of identified unique SSCP types with Arlequin 2.0 (57).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Borrelia marker amplification.
One hundred seventy-three B. burgdorferi flaB and 168 ospA products were amplified from naturally infected P. leucopus mice collected during a cross-sectional study of the western coastal plains of southern Maryland. One hundred thirty-five of the flaB and ospA positive white-footed mice (80%) were positive for ospC with the seminested primer set.

Genetic diversity of PCR amplicons.
On the basis of SSCP analysis, no polymorphisms were detected in flaB PCR amplicons. This was confirmed by sequencing where all fragments were identical to the published sequence of the flaB gene from B. burgdorferi strains B31and 297 (GenBank accession numbers AE001126 and AB035616). Analysis of the ospA PCR products by SSCP analysis and subsequent sequencing revealed three dominant (MC1a, MC3, and MC4) and three rare (MC2, MC6, and MC7) mobility class variants among the mouse samples, and all variants appeared to be evenly distributed throughout the western coastal plains of southern Maryland (Fig. 1 and Table 1). Mobility class MC1a was the predominant type among P. leucopus mice (n = 103, frequency = 0.531). Sequence alignment of representative MCVs found in this and other studies (18, 54) verified that only five point mutations accounted for all of the variation observed (Table 2). Two novel MCVs were identified among the specimens collected in this study and were designated MDPlMC6 and MDPlMC7 (GenBank accession no. AY796112 and AY796113, respectively).


Figure 1
View larger version (47K):
[in this window]
[in a new window]
  		FIG. 1. Representative ospA SSCP analysis of all MCVs observed. Numbers represent MCVs as defined by Guttman et al. (18). m = profile of a specimen infected with two or more Borrelia MCVs on the basis of ospA. L = 100-bp ladder.

 

View this table:
[in this window]
[in a new window]
  		TABLE 1. Outer surface protein A strains found in captured P. leucopus mice by county

 

View this table:
[in this window]
[in a new window]
  		TABLE 2. Nucleotide changes defining each ospA mobility class

 
Eight of the 22 previously described ospC groups were identified among collected P. leucopus mice by SSCP analysis and sequence analysis (Fig. 2). Groups A and K were predominant, representing 68% of the sample set, whereas groups C and D were rarely observed (n = 2 and 3, frequency = 0.013 and 0.018, respectively) (Table 3). Alignment of the ospC sequences revealed that the SSCP complexity observed was due to 127 point mutations and several major insertions (data not shown).


Figure 2
View larger version (80K):
[in this window]
[in a new window]
  		FIG. 2. SSCP analysis of predominant ospC groups observed among P. leucopus mice collected in southern Maryland. ospC group D is not shown. L = 100-bp ladder.

 

View this table:
[in this window]
[in a new window]
  		TABLE 3. Primary ospC groups found in P. leucopus specimens collected in southern Maryland

 
Multiple infections based on ospA and ospC.
SSCP analysis revealed multiple ospA MCVs in 24% (n = 39) of the amplification products from P. leucopus. Cloning studies indicated that these specimens were composed of a mixture of MC1a and MC4 (Table 2). Twenty-four percent of all ospC-positive P. leucopus samples (n = 32) were infected with more than one ospC group on the basis of the SSCP profile and subsequent cloning and sequence analysis. Complex infections were often composed of three different ospC groups (28% of mixed samples). The most common groups found in mixed infections were A and K, adding to the dominance of these two variants among mouse samples found in Maryland. Sequence analysis revealed that variation among clones in these complex infections was due to minor point mutations rather than having a sequence identical to one of the established ospC groups as seen in simply infected animals (GenBank accession numbers AY796075 through AY796111).

Phylogenetic analysis of ospA and ospC.
The ospA phylogenetic tree was constructed from 31 sequences that included reference mobility classes MC1 to MC5, additional non-B. burgdorferi sensu stricto ospA sequences and the two novel mobility classes (MC6 and MC7) described in this study (Table 4). The ospC phylogenetic tree was constructed by alignment of 104 ospC sequences, 43 of which were identified in this study (designated BbMDXXX). The remaining sequences were extracted from the GenBank database, including the reference sequence from each established ospC group (60, 72) (Table 5). All of the sequences obtained during this study that were identical in sequence to a reference strain (MC or OspC) were removed from the analysis to conserve processing speed. The phylogenetic analysis included 357 nucleotides (nt) from the ospA amplification product and 269 nt from the ospC amplification product. Among these, 156 ospA and 33 ospC characters were constant and 37 and 27 nt were parsimony uninformative, respectively, whereas 164 ospA characters were parsimony informative, the majority of which were contributed by non-B. burgdorferi taxa. In the ospC alignment, 170 characters were considered parsimony informative. Unlike ospA, the informative characters from ospC were widely dispersed among all of the taxa analyzed. There were no major topological differences between any of the derived trees for each gene, and only the maximum-likelihood tree is shown. Phylogenetic analysis of ospA and ospC gene fragments indicated that all of the amplified Borrelia products from samples collected in Maryland clustered with previously identified B. burgdorferi sensu stricto sequence fragments (Fig. 3 and 4).


View this table:
[in this window]
[in a new window]
  		TABLE 4. Sequenced ospA samples used in alignment and phylogenetic tree construction

 

View this table:
[in this window]
[in a new window]
  		TABLE 5. Origins, hosts, and accession numbers of Borrelia ospC gene sequences

 

Figure 3
View larger version (14K):
[in this window]
[in a new window]
  		FIG. 3. Maximum-likelihood tree based on ospA gene fragment alignments derived with PAUP. Maximum-likelihood bootstrap values (1,000 replicates) are listed for each supported node. Bb, B. burgdorferi; Bv, B. valaisiana; Ban, "B. andersonii"; Bbi, "B. bissettii"; Bg, B. garinii; Bj, B. japonica; Ba, B. afzelii; Bt, B. turdi; Bl, B. lusitaniae.

 

Figure 4
View larger version (36K):
[in this window]
[in a new window]
  		FIG. 4. Unrooted maximum-likelihood phylogenetic tree based on nucleotide sequence alignment of ospC fragments. Maximum-likelihood bootstrap values, based on 1,000 replicates, are listed at each supported node. Groups are based on the ospC allele nomenclature of Wang et al. (73). Maryland samples were found within encircled clades. All sample names are followed by a T, R, or H indicating the source of the material (tick, rodent, or human, respectively).

 
Genetic distance.
The mean average pairwise genetic distance analysis of ospA sequences analyzed in this study was 17% divergence. Within the B. burgdorferi sensu stricto clade, in which all of the Maryland samples clustered, the genetic divergence was only 1% while the divergence between B. burgdorferi sensu stricto and the nearest neighboring clade containing "B. bissettii" was 3.5% (Table 6). The overall genetic distance of the 108 ospC samples used to construct the maximum-likelihood tree presented in this study was 0.25, indicating a 25% mean average divergence in the ospC gene fragment (Table 6). To confirm the correct placement of Maryland samples among the previously described sequences, we calculated the genetic distance of the samples in each group into which our samples clustered. Our samples diverged less than 2% from published sequences. Several samples clustered in clades composed of few isolates. For this analysis, we calculated the genetic distance between the clades containing Maryland samples and the nearest neighboring clade with the hypothesis that the divergence would be greater than 8%. In each example where two or more distinct groups were compared, the genetic distance was always greater than 8% divergence (Table 6).


View this table:
[in this window]
[in a new window]
  		TABLE 6. Genetic distance calculated with the Tamura-Nei model within each OspA and OspC clade that contained Maryland samples

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Three molecular markers were used to determine the genetic heterogeneity of B. burgdorferi sensu stricto among P. leucopus mice collected at the southern edge of a region of the eastern United States where LD is highly endemic. The flaB gene proved to be inadequate to evaluate our objectives because of the absence of polymorphism even though this gene fragment had proven useful in other studies with even more restrictive geographic sampling (24, 42). A number of investigators have identified sufficient genetic diversity within the ospA gene for population level analysis and have found four mobility classes in the northeastern United States (18, 43, 52, 54). With the exception of MC2, which was never observed among our specimens or of those collected in Maryland by Qiu et al. (54), all other previously identified classes were observed. Qiu et al. (54) reported a new mobility class, MC5, that, to date, has only been reported from ticks collected in North Carolina and was described as a recombination event of MC1a and MC4 rather than a point mutation. Among the Maryland samples, a mixture of MC1a and MC4 represented 24% of the Peromyscus samples that were multiply infected. Cloning followed by SSCP analysis of samples suspected of representing mixed infections revealed a combination of the two previously described mobility classes. Sequencing verified that the mobility classes were differentiated by a single substitution that appeared as a mixed signal at nt 446. This finding illustrates that MC5 is likely to be not the result of a recombination event but rather a coinfection of one individual with two genetically similar variants.

In addition to recovering the four previously described mobility classes, we have identified two new ospA mobility classes among the P. leucopus samples and designated these MC6 and MC7. Unlike MC5, MC6 and MC7 represent novel combinations of mutations (Table 2 and Fig. 1). Mobility class 6 represents a new combination of previously reported mutations. Mobility class 7 introduces a new synonymous mutation at nt 474 (T->C) (Fig. 5). Both of the new mobility classes were rarely observed in our study. Even with the addition of two new mobility classes, our analysis confirms that ospA is a highly conserved gene and the predominant mobility classes have a homogeneous distribution in the reservoir population at both the local and regional levels. The scarcity of some MCVs like MC2 and the recovery of two new MCVs from Maryland suggest that local or regional variants may be abundant but circulate at low frequencies.


Figure 5
View larger version (6K):
[in this window]
[in a new window]
  		FIG. 5. Relationship of the B. burgdorferi strains based on ospA combining the findings of Guttman et al. (18) and Qiu et al. (54) and mobility classes defined in this study (italicized). Dashed lines indicate recombination events rather than mutation events (solid lines).

 
Of the three gene fragments examined in this study, the ospC gene was found to be the most informative for evaluating the phylogenetic relationships among B. burgdorferi sensu stricto samples recovered from specimens collected in the field. Genetic variation in the ospC gene has been used to define 22 ospC groups worldwide, of which 4 are found exclusively in Europe (61), 1 is found exclusively in the southeastern United States (31), and the remainder have been found relatively evenly distributed in the northeastern United States (54). Prior to this study, only four ospC sequences derived from sylvatic P. leucopus mice were listed in the GenBank database. Of those four, two were recovered from mice collected in Baltimore County, Maryland, by Hofmeister et al. (21) and were representative of groups A (strain 225e7) and K (strain 2253b6), the two most common groups found among our samples (Table 5 and Fig. 4). The other two Peromyscus-derived ospC sequences were from Wisconsin and Connecticut and represent groups L and M, neither of which was observed in our data set. It is difficult to assess whether this analysis suggests that regional variance exists in the reservoir population. However, the congruence between the isolates described by Hofmeister et al. (21) and data derived from P. leucopus collected from 80% of our sites suggests that ospC diversity is relatively stable in the reservoir population in this region.

The amount of genetic divergence between ospC variants is large (10 to 20%) compared with the polymorphism observed within a defined variant group (<2%) (60). All of the ospC sequences recovered in this study fall within the prescribed limits of inter- and intragroup relationships as shown by pairwise distance and phylogenetic analysis (Table 6). We did not identify unique ospC groups, yet we did observe minor point mutations in cloned products from animals infected with multiple ospC groups. These point mutations did not result in more than 2% sequence divergence from previously defined ospC groups. In addition, and consistent with all other reports, no variants were found to have >2% or <8% identity. Borrelia fragments from PCR-amplified material from infected P. leucopus (R) did not appear to cluster together in related clades or in particular branches on the phylogenetic tree separate from Borrelia fragments from humans (H) or ticks (T) (Fig. 4).

The phylogenetic tree obtained by analysis with a maximum-likelihood criterion of aligned ospC fragments yielded an unrooted tree in which most of the internal nodes were not supported by bootstrap analysis (Fig. 4). One explanation for the lack of internal node support, thus a lack of hierarchical structure, is the observation that ospC has undergone recombination events that have led to the 22 variant groups observed in nature. Divergence greater than 8% between different ospC groups suggests that small DNA fragments (1 kb or less) are recombining between different ospC variants (7) and that this recombination most likely occurs in the variable regions of the gene (72), as evidenced by a conserved sequence specific motif (33). Support for recombination events is also evident in the topology of the phylogenetic tree derived with ospC fragments. The star shape of the tree with long terminal branches and unsupported internal nodes reflects these events. Despite significant polymorphism, this characteristic indicates that ospC is not an ideal candidate for phylogenetic analysis or evolutionary inferences (7, 48). Recently, Bunikis et al. (4) suggested that the rrs-rrlA intergenic spacer region may be a more viable option for phylogenetic analysis because of its moderate polymorphism yet apparent lack of recombination.

In recent years, Seinost et al. (60) and Lagal et al. (29) have suggested that some ospC variants may facilitate infection and subsequent dissemination in human hosts. Following a comprehensive survey of ospC, Seinost et al. (60) concluded that certain ospC groups were associated with particular outcomes of LD pathogenesis. In particular, they found that ospC groups A, B, I, and K were found in 47% of patients with erythema migrans and representatives of these four groups were predominantly recovered from patients with secondary-site (disseminated) infections (84%). However, the same study recovered the four clinically dominant groups with less frequency from ticks (23%). In contrast, few studies have been conducted to analyze the ospC variants circulating in the small-animal population, primarily in the P. leucopus population (3). The comprehensive collection effort reported here, of greater than 800 small mammals over a more extensive geographic range, suggests that a limited number of dominant ospC variants circulate in the rodent reservoir population while other variants may also circulate but at much lower levels.

While P. leucopus appears to be the dominant reservoir for the B. burgdorferi sensu stricto strain associated with secondary-site infections in humans (ospC groups A, B, and K), all of the variants recovered in this study appear to represent disseminated and therefore "invasive" infections of small mammals since rodents become systemically infected (21). This conclusion is supported by two recent studies by Alghaferi et al. (1) and Lagal et al. (29) that suggest that the "invasive group hypothesis" is oversimplified and a broader number of alleles or even other molecular components may be involved in the pathogenesis of borreliae in humans. The bottleneck effect that appears to occur in P. leucopus is inconsistent with the diversity of Borrelia ospC variants found in tick specimens and suggests either that ticks feed on other hosts that harbor different ospC variants (4) or that some variants are present within rodent specimens at levels below our detection ability. In either case, the work reported here suggests that information regarding B. burgdorferi variants found in rodent reservoirs, especially P. leucopus, is not only useful for understanding the complete enzootic cycle of LD but is critical for understanding the potential disease outcomes in humans.


   ACKNOWLEDGMENTS
 
This work was supported by CDC cooperative agreement U50/CCU319554 awarded to D.E.N. and National Institute of Environmental Health Sciences training grant T32ES07141 awarded to J.M.A. Further support was granted to J.M.A. by the Otis and Calista Causey Fund, the Frederick B. Bang Award, and the Lloyd Rozeboom Memorial Scholarship.

We are grateful for the editorial comments made by J. Stephen Dumler during the preparation of this report. We thank Stuart Ray for comments and suggestions regarding the phylogenetic analysis. Thanks to Katherine I. Swanson and Timothy R. Schwartz for assisting with field collections.


   FOOTNOTES
 
* Corresponding author. Present address: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12735 Twinbrook Parkway, Rm. 2E-22, Rockville, MD 20852. Phone: (301) 435-3587. Fax: (301) 594-5373. E-mail: jenanderson{at}niaid.nih.gov. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Alghaferi, M. Y., J. M. Anderson, J. Park, P. G. Auwaerter, J. N. Aucott, D. E. Norris, and J. S. Dumler. 2005. Borrelia burgdorferi ospC heterogeneity among human and murine isolates from a defined region of northern Maryland and southern Pennsylvania: lack of correlation with invasive and noninvasive genotypes. J. Clin. Microbiol. 43:1879-1884.[Abstract/Free Full Text]
  2. Barbour, A. G., and C. F. Garon. 1988. The genes encoding major surface proteins of Borrelia burgdorferi are located on a plasmid. Ann. N. Y. Acad. Sci. 539:144-153.[Medline]
  3. Brisson, D., and D. E. Dykhuizen. 2004. ospC diversity in Borrelia burgdorferi: different hosts are different niches. Genetics 104:713-722.
  4. Bunikis, J., U. Garpmo, J. Tsao, J. Berglund, D. Fish, and A. G. Barbour. 2004. Sequence typing reveals extensive strain diversity of the Lyme borreliosis agents Borrelia burgdorferi in North America and Borrelia afzelii in Europe. Microbiology 150:1741-1755.[Abstract/Free Full Text]
  5. Bunikis, J., J. Tasao, C. Luke, M. Luna, D. Fish, and A. G. Barbour. 2004. Borrelia burgdorferi infection in natural populations of Peromyscus leucopus mice: a longitudinal study in an area where Lyme borreliosis is highly endemic. J. Infect. Dis. 189:1515-1523.[CrossRef][Medline]
  6. Caporale, D. A., and T. D. Kocher. 1994. Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi. Mol. Biol. Evol. 11:51-64.[Abstract]
  7. Dykhuizen, D. E., and G. Baranton. 2001. The implications of a low rate of horizontal transfer in Borrelia. Trends Microbiol. 9:344-350.[CrossRef][Medline]
  8. Dykhuizen, D. E., D. S. Polin, J. J. Dunn, B. Wilske, V. Preac-Mursic, R. Dattwyler, and B. J. Luft. 1993. Borrelia burgdorferi is clonal: implications for taxonomy and vaccine development. Proc. Natl. Acad. Sci. USA 90:10163-10167.[Abstract/Free Full Text]
  9. Faith, D. P. 1991. Cladistic permutation tests for monophyly and nonmonophyly. Syst. Zool. 40:366-375.[CrossRef]
  10. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783-791.[CrossRef]
  11. Fikrig, E., S. W. Barthold, N. Marcantonio, K. DePonte, F. Kantor, and R. A. Flavell. 1992. Roles of OspA, OspB, and flagellin in protective immunity to Lyme borreliosis in laboratory mice. Infect. Immun. 60:657-661.[Abstract/Free Full Text]
  12. Frank, C., A. D. Fix, C. A. Pena, and G. T. Strickland. 2002. Mapping Lyme disease incidence for diagnostic and preventive decisions, Maryland. Emerg. Infect. Dis. 8:427-429.[Medline]
  13. Fukunaga, M., and A. Hamase. 1995. Outer surface protein C gene sequence analysis of Borrelia burgdorferi sensu lato isolates from Japan. J. Clin. Microbiol. 33:2415-2420.[Abstract]
  14. Fukunaga, M., K. Okada, M. Nakao, T. Konishi, and Y. Sato. 1996. Phylogenetic analysis of Borrelia species based on flagellin gene sequences and its application of molecular typing of Lyme disease Borreliae. Int. J. Syst. Bacteriol. 46:898-905.[Abstract/Free Full Text]
  15. Fukunaga, M., M. Sohnaka, Y. Takahashi, M. Nakao, and K. Miyamoto. 1993. Antigenic and genetic characterization of Borrelia species isolated from Ixodes persulcatus in Hokkaido, Japan. J. Clin. Microbiol. 31:1388-1391.[Abstract/Free Full Text]
  16. Fung, B., G. McHugh, J. Leong, and A. Steere. 1994. Humoral immune response to outer surface protein C of Borrelia burgdorferi in Lyme disease: role of the immunoglobulin M response in the serodiagnosis of early infection. Infect. Immun. 62:3213-3221.[Abstract/Free Full Text]
  17. Grimm, D., K. Tilly, R. Byram, P. E. Stewart, J. G. Krum, D. M. Bueschel, T. G. Schwan, P. F. Policastro, A. F. Elias, and P. A. Rosa. 2004. Outer-surface protein C of the Lyme disease spirochete: a protein induced in ticks for infection of mammals. Proc. Natl. Acad. Sci. USA 101:3142-3147.[Abstract/Free Full Text]
  18. Guttman, D. S., P. W. Wang, I.-N. Wang, E. M. Bosler, B. J. Luft, and D. E. Dykhuizen. 1996. Multiple infections of Ixodes scapularis ticks by Borrelia burgdorferi as revealed by single-strand conformation polymorphism analysis. J. Clin. Microbiol. 34:652-656.[Abstract]
  19. Hall, T. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41:95-98.
  20. Hofmeister, E., and J. Childs. 1995. Analysis of Borrelia burgdorferi sequentially isolated from Peromyscus leucopus captured at a Lyme disease enzootic site. J. Infect. Dis. 172:462-469.[Medline]
  21. Hofmeister, E., B. A. Ellis, G. E. Glass, and J. E. Childs. 1999. Longitudinal study of infection with Borrelia burgdorferi in a population of Peromyscus leucopus at a Lyme disease-enzootic site in Maryland. Am. J. Trop. Med. Hyg. 60:598-609.[Abstract]
  22. Hofmeister, E., G. E. Glass, J. E. Childs, and D. H. Persing. 1999. Population dynamics of a naturally occurring heterogeneous mixture of Borrelia burgdorferi clones. Infect. Immun. 67:5709-5716.[Abstract/Free Full Text]
  23. Hongyo, T., G. Buzard, R. Calvert, and C. Weghorst. 1993. Cold SSCP: a simple, rapid and non-radioactive method for optimized single stranded conformation polymorphism analysis. Nucleic Acids Res. 21:3637-3642.[Abstract/Free Full Text]
  24. Jauris-Heipke, S., R. Fuchs, M. Motz, V. Preac-Mursic, E. Schwab, E. Soutschek, G. Will, and B. Wilske. 1993. Genetic heterogeneity of the genes coding for the outer surface protein C (ospC) and the flagellin of Borrelia burgdorferi. Med. Microbiol. Immunol. 182:37-50.[Medline]
  25. Jauris-Heipke, S., G. Liegl, V. Preac-Mursic, D. Rossler, E. Schwab, E. Soutschek, G. Will, and B. Wilske. 1995. Molecular analysis of genes encoding outer surface protein C (OspC) of Borrelia burgdorferi sensu lato: relationship to ospA genotype and evidence of lateral gene exchange of ospC. J. Clin. Microbiol. 33:1860-1866.[Abstract]
  26. Johnson, B. J., C. M. Happ, L. W. Mayer, and J. Piesman. 1992. Detection of Borrelia burgdorferi in ticks by species-specific amplification of the flagellin gene. Am. J. Trop. Med. Hyg. 47:730-741.[Abstract/Free Full Text]
  27. Kumar, S., K. Tamura, I. Jakobsen, and M. Nei. 1993. MEGA: molecular evolutionary genetics analysis. Bioinformatics 17:1244-1245.
  28. Lagal, V., D. Postic, and G. Baranton. 2002. Molecular diversity of the ospC gene in Borrelia. Impact on phylogeny, epidemiology and pathology. Wien. Klin. Wochenschr. 114:562-567.[Medline]
  29. Lagal, V., D. Postic, E. Ruzic-Sabljic, and G. Baranton. 2003. Genetic diversity among Borrelia strains determined by single-strand conformation polymorphism analysis of ospC gene and its association with invasiveness. J. Clin. Microbiol. 41:5059-5065.[Abstract/Free Full Text]
  30. Lane, R. S., J. Piesman, and W. Burgdorfer. 1991. Lyme borreliosis: relation of its causative agent to its vectors and hosts in North America and Europe. Annu. Rev. Entomol. 36:587-609.[CrossRef][Medline]
  31. Lin, T., J. H. Oliver, and L. Gao. 2002. Genetic diversity of the outer surface protein C gene of southern Borrelia isolates and its possible epidemiological, clinical, and pathogenetic implications. J. Clin. Microbiol. 40:2572-2583.[Abstract/Free Full Text]
  32. Lin, T., J. H. Oliver, Jr., L. Gao, T. M. Kollars, Jr., and K. L. Clark. 2001. Genetic heterogeneity of Borrelia burgdorferi sensu lato in the southern United States based on restriction fragment length polymorphism and sequence analysis. J. Clin. Microbiol. 39:2500-2507.[Abstract/Free Full Text]
  33. Livey, I., C. P. Gibbbs, R. Schuster, and F. Dorner. 1995. Evidence for lateral transfer and recombination in OspC variation in Lyme disease Borrelia. Mol. Microbiol. 18:257-269.[CrossRef][Medline]
  34. Magnarelli, L., J. Anderson, K. E. Hyland, D. Fish, and J. McAninch. 1988. Serologic analyses of Peromyscus leucopus, a rodent reservoir for Borrelia burgdorferi, in northeastern United States. J. Clin. Microbiol. 26:1138-1141.[Abstract/Free Full Text]
  35. Magnarelli, L., and J. F. Anderson. 1988. Ticks and biting insects infected with the etiologic agent of Lyme disease, Borrelia burgdorferi. J. Clin. Microbiol. 26:1482-1486.[Abstract/Free Full Text]
  36. Marconi, R. T. 1995. 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. 33:2427-2434.[Abstract]
  37. Marconi, R. T., S. Hohenberger, S. Jauris-Heipke, U. Schulte-Spechtel, C. P. LaVoie, D. Robler, and B. Wilske. 1999. Genetic analysis of Borrelia garinii OspA serotype 4 strains associated with neuroborreliosis: evidence for extensive genetic homogeneity. J. Clin. Microbiol. 37:3965-3970.[Abstract/Free Full Text]
  38. Marconi, R. T., M. E. Konkel, and C. F. Garon. 1993. Variability of osp genes and gene products among species of Lyme disease spirochetes. Infect. Immun. 61:2611-2617.[Abstract/Free Full Text]
  39. Marti Ras, N., D. Postic, M. Foretz, and G. Baranton. 1997. Borrelia burgdorferi sensu stricto, a bacterial species "made in the USA"? Int. J. Syst. Bacteriol. 47:1112-1117.[Abstract/Free Full Text]
  40. Masuzawa, T., T. Fukui, M. Miyake, H. B. Oh, M. K. Cho, W. H. Chang, Y. Imai, and Y. Yanagihara. 1999. Determination of members of a Borrelia afzelii-related group isolated from Ixodes nipponensis in Korea as Borrelia valaisiana. Int. J. Syst. Bacteriol. 49:1409-1415.[Abstract/Free Full Text]
  41. Masuzawa, T., T. Komikado, and Y. Yanagihara. 1997. PCR-restriction fragment length polymorphism analysis of the ospC gene for detection of mixed culture and for epidemiological typing of Borrelia burgdorferi sensu stricto Clin. Diagn. Lab. Immunol. 4:60-63.
  42. Norris, D. E., B. J. Johnson, J. Piesman, G. O. Maupin, J. L. Clark, and W. C. Black. 1997. Culturing selects for specific genotypes of Borrelia burgdorferi in an enzootic cycle in Colorado. J. Clin. Microbiol. 35:2359-2364.[Abstract]
  43. Norris, D. E., J. S. H. Klompen, J. E. Keirans, R. S. Lane, J. Piesman, and W. C. T. Black. 1997. Taxonomic status of Ixodes neotomae and I. spinipalpis (Acari: Ixodidae) based on mitochondrial DNA evidence. J. Med. Entomol. 34:696-703.[Medline]
  44. Orita, M., H. Iwahana, H. Kanazawa, K. Hayashi, and T. Sekiya. 1989. Detection of polymorphisms of human DNA by gel electrophoresis as single-stranded conformation polymorphisms. Proc. Natl. Acad. Sci. USA 86:2766-2770.[Abstract/Free Full Text]
  45. Padula, S. J., A. Sampieri, F. Dias, A. Szczepanski, and R. W. Ryan. 1993. Molecular characterization and expression of p23 (ospC) from a North American strain of Borrelia burgdorferi. Infect. Immun. 61:5097-5105.[Abstract/Free Full Text]
  46. Pal, U., X. Yang, M. Chen, L. K. Bockenstedt, J. F. Anderson, R. A. Flavell, M. V. Norgard, and E. Fikrig. 2004. OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands. J. Clin. Investig. 113:220-230.[CrossRef][Medline]
  47. Piesman, J., M. Dolan, C. M. Happ, B. J. Luft, S. Rooney, T. N. Mather, and W. T. Golde. 1997. Duration of immunity to reinfection with tick-transmitted Borrelia burgdorferi in naturally infected mice. Infect. Immun. 65:4043-4047.[Abstract]
  48. Posada, D., and K. Crandall. 2002. The effect of recombination on the accuracy of phylogeny estimation. J. Mol. Evol. 54:396-402.[Medline]
  49. Posada, D., and K. Crandall. 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14:817-818.[Abstract/Free Full Text]
  50. Postic, D., M. V. Assous, P. A. Grimont, and G. Baranton. 1994. Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf (5S)-rrl (23S) intergenic spacer amplicons. Int. J. Syst. Bacteriol. 44:743-752.[Abstract/Free Full Text]
  51. Probert, W., M. Crawford, R. Cadiz, and R. LeFebvre. 1997. Immunization with outer surface protein (Osp) A, but not OspC, provides cross-protection of mice challenged with North American isolates of Borrelia burgdorferi. J. Infect. Dis. 175:400-405.[Medline]
  52. Qiu, W.-G., E. M. Bosler, J. R. Campbell, G. Ugine, I.-N. Wang, B. J. Luft, and D. E. Dykhuizen. 1997. A population genetic study of Borrelia burgdorferi sensu stricto from eastern Long Island, New York, suggested frequency-dependent selection, gene flow and host adaptation. Hereditas 127:203-216.[CrossRef][Medline]
  53. Qiu, W.-G., S. E. Schutzer, J. F. Bruno, O. Attie, Y. Xu, J. J. Dunn, C. M. Fraser, S. Casjens, and B. J. Luft. 2004. Genetic exchange and plasmid transfer in Borrelia burgdorferi sensu stricto revealed by three-way genome comparisons and multilocus sequence typing. Proc. Natl. Acad. Sci. USA 101:14150-14155.[Abstract/Free Full Text]
  54. Qiu, W., D. Dykhuizen, M. Acosta, and B. Luft. 2002. Geographic uniformity of the Lyme disease spirochete (Borrelia burgdorferi) and its shared history with tick vector (Ixodes scapularis) in the northeastern United States. Genetics 160:833-849.[Abstract/Free Full Text]
  55. Rogers, J. 1972. Measure of genetic similarity and genetic distance. Stud. Genet. 7213:145-153.
  56. Sadziene, A., B. Wilske, M. S. Ferdows, and A. G. Barbour. 1993. The cryptic ospC gene of Borrelia burgdorferi B31 is located on a circular plasmid. Infect. Immun. 61:2192-2195.[Abstract/Free Full Text]
  57. Schneider, S., D. Roessli, and L. Excoffier. 2000. Arlequin: a software for population genetics data analysis. Ver 2.000. Genetics and Biometry Lab, Department of Anthropology, University of Geneva, Geneva, Switzerland.
  58. Schwan, T. G., W. Burgdorfer, and P. Rosa. 1995. Borrelia, p. 625-635. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed. ASM Press, Washington, D.C.
  59. Schwartz, I., G. P. Wormser, J. J. Schwartz, D. Cooper, P. Weissensee, A. Gazumyan, E. Zimmermann, N. S. Goldberg, S. Bittker, G. L. Campbell, and C. S. Pavia. 1992. Diagnosis of early Lyme disease by polymerase chain reaction amplification and culture of skin biopsies from erythema migrans lesions. J. Clin. Microbiol. 30:3082-3088.[Abstract/Free Full Text]
  60. Seinost, G., D. E. Dykhuizen, R. Dattwyler, W. T. Golde, J. J. Dunn, I.-N. Wang, G. P. Wormser, M. E. Schriefer, and B. J. Luft. 1999. Four clones of Borrelia burgdorferi sensu stricto cause invasive infection in humans. Infect. Immun. 67:3518-3524.[Abstract/Free Full Text]
  61. Seinost, G., W. T. Golde, B. Berger, J. J. Dunn, D. Qiu, D. Dunkin, D. E. Dykhuizen, B. J. Luft, and R. Dattwyler. 1999. Infection with multiple strains of Borrelia burgdorferi sensu stricto in patients with Lyme disease. Arch. Dermatol. 135:1329-1333.[Abstract/Free Full Text]
  62. Seshu, J., and J. Skare. 2000. The many faces of Borrelia burgdorferi. J. Mol. Microbiol. Biotechnol. 2:463-472.[Medline]
  63. Shih, C. M., and L. L. Chao. 2002. An ospA-based genospecies identification of Lyme disease spirochetes (Borrelia burgdorferi) isolated in Taiwan. Am. J. Trop. Med. Hyg. 66:611-615.[Abstract]
  64. Stevenson, B., L. K. Bockenstedt, and S. W. Barthold. 1994. Expression and gene sequence of outer surface protein C of Borrelia burgdorferi reisolated from chronically infected mice. Infect. Immun. 62:3568-3571.[Abstract/Free Full Text]
  65. Swofford, D. 2003. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer Associates, Sunderland, Mass.
  66. Swofford, D., and S. Berlocher. 1987. Inferring evolutionary trees from gene frequency data under the principle of maximum parsimony. Syst. Zool. 36:293-325.[CrossRef]
  67. Tamura, K., and M. Nei. 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol. Biol. Evol. 10:512-526.[Abstract]
  68. Theisen, M., M. Borre, M. J. Mathiesen, B. Mikkelsen, A.-M. Lebech, and K. Hansen. 1995. Evolution of Borrelia burgdorferi outer surface protein OspC. J. Bacteriol. 177:3036-3044.[Abstract/Free Full Text]
  69. Thompson, J., T. Gibson, F. Plewniak, F. Jeanmougin, and D. Higgins. 1997. The ClustalX Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876-4882.[Abstract/Free Full Text]
  70. Wallich, R., C. Helmes, U. Schaible, Y. Lobet, S. Moter, M. Kramer, and M. Simon. 1992. Evaluation of genetic divergence among Borrelia burgdorferi isolates by use of OspA, fla, HSP60, and HSP70 gene probes. Infect. Immun. 60:4856-4866.[Abstract/Free Full Text]
  71. Wang, G., A. P. van Dam, and J. Dankert. 2000. Two distinct ospA genes among Borrelia valaisiana strains. Res. Microbiol. 151:325-331.[Medline]
  72. Wang, G., A. P. van Dam, I. Schwartz, and J. Dankert. 1999. Molecular typing of Borrelia burgdorferi sensu lato: taxonomic, epidemiological, and clinical implications. Clin. Microbiol. Rev. 12:633-653.[Abstract/Free Full Text]
  73. Wang, I.-N., D. E. Dykhuizen, W. Qiu, J. J. Dunn, E. M. Bosler, and B. J. Luft. 1999. Genetic diversity of ospC in a local population of Borrelia burgdorferi sensu stricto. Genetics 151:15-30.
  74. Will, G., S. Jauris-Heipke, E. Schwab, U. Busch, D. Rossler, E. Soutschek, B. Wilske, and V. Preac-Mursic. 1995. Sequence analysis of ospA genes shows homogeneity within Borrelia burgdorferi sensu stricto and Borrelia afzelii strains but reveals major subgroups within the Borrelia garinii species. Med. Microbiol. Immunol. 184:73-80.[Medline]
  75. Wilske, B., S. Jauris-Heipke, R. Lobentanzer, I. Pradel, V. Preac-Mursic, D. Rossler, E. Soutschek, and R. C. Johnson. 1995. Phenotypic analysis of outer surface protein C (OspC) of Borrelia burgdorferi sensu lato by monoclonal antibodies: relationship to genospecies and OspA serotype. J. Clin. Microbiol. 33:103-109.[Abstract]
  76. Wilske, B., V. Preac-Mursic, G. Schierz, and K. Busch. 1986. Immunochemical and immunological analysis of European Borrelia burgdorferi strains. Zentbl. Bakteriol. Mikrobiol. Hyg. A 263:92-102.
  77. Yang, X. F., U. Pal, S. M. Alani, E. Fikrig, and M. V. Norgard. 2004. Essential role for OspA/B in the life cycle of the Lyme disease spirochete. J. Exp. Med. 199:641-648.[Abstract/Free Full Text]
  78. Zumstein, G., R. Fuchs, A. Hofmann, V. Preac-Mursic, E. Soutschek, and B. Wilske. 1992. Genetic polymorphism of the gene encoding the outer surface protein A (ospA). Med. Microbiol. Immunol. 181:57-70.[Medline]

Applied and Environmental Microbiology, August 2006, p. 5331-5341, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00014-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Anderson, J. M.
Right arrow Articles by Norris, D. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Anderson, J. M.
Right arrow Articles by Norris, D. E.
Agricola
Right arrow Articles by Anderson, J. M.
Right arrow Articles by Norris, D. E.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
J. Bacteriol. Microbiol. Mol. Biol. Rev. Eukaryot. Cell All ASM Journals