Borrelia burgdorferi ospC Heterogeneity among Human and Murine Isolates from a Defined Region of Northern Maryland and Southern Pennsylvania
http://www.100md.com
微生物临床杂志 2005年第4期
W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205
Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Divisions of General Internal Medicine and Infectious Diseases, The Johns Hopkins University School of Medicine, Lutherville, Maryland 21093
ABSTRACT
B. burgdorferi invasiveness correlates with ospC genotype. To test this hypothesis and whether identical genotypes infect humans and small mammals in specific sites, B. burgdorferi ospC heterogeneity was tested among isolates from northern Maryland and southern Pennsylvania. Six culture-positive patients allowed collection of small animals from their properties, and spirochetes from animals trapped within 300 yards of each patient's home were isolated. 3' ospC sequences were compared to reference sequences. Of the 7 human and 15 mouse DNA templates that produced reliable sequences, all clustered with references into only four and seven distinct clades, respectively. A human and a mouse isolate with the same ospC were seen in only one locality, and five of six sites contained two or more B. burgdorferi ospC clones. Four invasive patient isolates and six small mammal isolates clustered with "noninvasive" reference ospC genotypes. A high degree of ospC diversity exists among B. burgdorferi isolates in Maryland and Pennsylvania, even in narrowly defined geographic localities. Dissemination in mice and humans by noninvasive ospC types contradicts the ospC invasiveness hypothesis. Alternative genetic markers for B. burgdorferi disseminated disease should be investigated.
INTRODUCTION
Lyme borreliosis is an infectious disease caused by Borrelia burgdorferi sensu lato (4). These spirochetes are transmitted by Ixodes spp. ticks that maintain an infectious cycle by feeding on reservoir hosts such as Peromyscus leucopus and other small mammals; humans are accidental hosts (5, 9). Three Borrelia species that cause human disease include B. burgdorferi sensu stricto (North America), B. afzelii, and B. garinii (Europe and Asia) (2, 27, 33). In North America the geographic distribution is well documented, with B. burgdorferi and Lyme disease expanding and clearly endemic in the mid-Atlantic region including Maryland (1, 22).
B. burgdorferi plasmid-encoded outer surface protein C genes (ospC) are implicated in transmission and dissemination of Borrelia between ticks and mammals (12, 18, 20, 24, 32). Diversity of ospC has lead to the classification of B. burgdorferi clones into human invasive and "noninvasive" groups (14, 16, 25, 28), but these studies have not evaluated ospC as an invasive factor in small mammals. Because of ospC heterogeneity, it has been used as marker for clonality in epidemiological studies that suggest that human and small mammal isolates from the same region are restricted to only a single or a few clones (14, 16). Neither geographic nor ospC heterogeneity of B. burgdorferi infecting humans and mice has been extensively studied in the mid-Atlantic States. The present study was designed to test two hypotheses: (i) that only limited ospC diversity exists among B. burgdorferi infecting humans and mice from the same localities in northern Maryland, and (ii) that ospC genotypes associated with invasiveness in humans extends to Maryland and to small mammals.
MATERIALS AND METHODS
Patients and Borrelia isolates. From a study of clinical Lyme disease in Maryland during 2001 (J. S. Dumler, unpublished data), eight Borrelia burgdorferi isolates from six culture-confirmed patients were selected from those who also agreed to allow collection of small animals from their properties. Six isolates were from human plasma, and two were from erythema migrans lesions. Isolates were confirmed to be B. burgdorferi by PCR amplification of the ospA gene (data not shown), as previously described (7). Five patients resided in Baltimore County, Maryland, and one was from neighboring York County, Pennsylvania. All patients had early Lyme disease with erythema migrans lesions, and five had manifestations suggesting early disseminated disease, including one with multiple erythema migrans and four with influenza-like illnesses. Patients provided informed consent prior to the study as approved by the John Hopkins Medicine Institutional Review Board.
Growth of human Borrelia isolates. Low-passage (<2 passages) bacteria were propagated from dimethyl sulfoxide-preserved frozen patient isolates. Approximately 0.5 ml of each frozen isolate was transferred to 5 ml of modified BSK-II medium (31) and incubated at 34°C in 5% CO2 until sufficient growth was confirmed by fluorescent microscopy using acridine-orange staining. Two control cultures were used: B. burgdorferi strain 297 (courtesy of Dennis Grab, the Johns Hopkins University School of Medicine) and a randomly selected human isolate previously prepared in the lab and used for internal control purposes (lab strain).
Mouse B. burgdorferi isolates. Mice were trapped using 60 Sherman live traps (H. B. Sherman, Tallahassee, FL) baited with bird seed and placed within 300 yards of each patient's home. After administration of a light anesthetic, a 2-mm-diameter ear tissue sample was taken using a biopsy tool (Accuderm Inc., Ft. Lauderdale, FL) and placed in BSK-II medium for transport. A blood sample was taken from each capture by retro-orbital sampling and placed directly on ice for transport. Captured rodents were released at the site of capture. Trapping and sample collection were performed according to Hofmeister et al. (9) as approved by the John Hopkins University Institutional Animal Care and Use Committee.
Mouse blood and skin cultures were incubated at 34°C in 5% CO2 and examined weekly for up to 8 weeks. Acridine orange-positive samples were subcultured in BSK-II medium supplemented with 50 μg/ml of ciprofloxacin and 20 μg/ml of rifampin to reduce contamination with other bacterial species. When appropriate, 0.45-μm-pore-diameter syringe-driven filter units were used (11) to remove contaminating bacteria. After 2 to 3 weeks of log phase growth, aliquots of each culture were cryopreserved at –80°C. Despite the use of antimicrobials and filtering, contamination persisted for many cultures, resulting in poor Borrelia recovery in 11 field samples later excluded from further study. Despite all attempts, many isolates grew poorly in the medium preparations used.
DNA extraction. During log phase growth, 1.5 ml of each Borrelia culture was centrifuged at 13,800 x g for 25 min. The bacterial pellet was resuspended in 200 μl of phosphate-buffered saline, and DNA was prepared using a QIAamp DNA Mini kit (QIAGEN, Inc.) and the manufacturer's protocols. DNA preparations were stored at –20°C until analyzed.
PCR methods and conditions. The 3' and 5' ends of ospC were PCR amplified using conserved primers as described previously (25). PCR was performed using an eLONGase kit (Invitrogen Inc.) per manufacturer's instructions with the addition of 100 pmol of each primer and using 2 μl of B. burgdorferi isolate DNA that was not otherwise quantitated. The reaction was completed as follows: initial denaturation at 96°C for 2 min, followed by 40 cycles of denaturation at 95°C for 40 s, annealing at 54°C for 35 s, and extension at 72°C for 1 min. The 5' ospC amplification did not produce a reliable product from all samples; therefore, all subsequent analyses were based on amplification of a 314-bp fragment from the 3' end. A negative control (deionized water) was included in each PCR. PCR amplification of a B. burgdorferi-specific fragment from the flaB gene was used to confirm that small mammal isolates from which ospC was amplified were B. burgdorferi (13).
Sequencing and phylogenetic analysis. B. burgdorferi ospC PCR products (25 μl) were electrophoresed in 2% agarose gels, stained with ethidium bromide, and excised for purification using a QIAquick gel extraction kit (QIAGEN Inc.). Sequencing was performed using the forward and reverse PCR primers (25) and a fluorescent automated method (ABI 3100 genetic analyzer). The resulting forward and reverse ospC sequences were initially assembled using the Contig Assembly Program in BioEdit (8) to generate consensus sequences. Discrepancies were resolved by inspection of the original chromatograms or by additional sequence analysis.
To validate the use of the 3' ospC sequences for phylogenetic comparisons, 21 B. burgdorferi sequences corresponding to the major ospC groups (25, 30) were retrieved from GenBank (Table 1) and aligned using ClustalX (10). Primer sequences flanking the region of the alignment corresponding to the 3' amplicon were removed, and the resulting sequences were gap stripped and realigned for phylogenetic analysis. Full-length and 3' ospC trees were generated using the neighbor-joining algorithm, and topologies of the trees were compared. The consensus sequence assemblies of the study isolates were aligned with the reference 3' ospC sequences. These combined sequences were then trimmed for optimal pairwise alignments and used to generate a neighbor-joining tree with bootstrapping 1,000 times. The tree was viewed with TreeView software (19).
RESULTS
Isolation and detection of Borrelia. Human and mouse specimens. With the broad assumption that patient exposure occurred on or around their properties, small mammals were collected from the properties of the six selected patients. From the six locations, a total of 61 small mammals (48 white-footed mice [Peromyscus leucopus], 9 chipmunks [Tamias striatus], 1 vole [Microtus pennsylvanicus], and 2 shrews [Sorex spp.]) were captured. Blood and ear biopsies for culture were obtained from each animal except for the shrews, from which blood was not obtained, and two chipmunks, from which ear biopsies were not obtained. From 118 small mammal tissue or blood cultures, spirochetes were observed in 71 (60.2%), including 27 blood and 44 ear biopsy cultures (Table 2). Contamination was a significant problem, as 54 (45.8%) cultures contained other bacteria. Contamination levels were minimal in all but 11 cultures. Bacterial contaminants could not be cleared from these 11 cultures, and they were subsequently excluded from further analysis. Growth of borreliae was also poor for most samples despite adequate growth of laboratory control cultures (B. burgdorferi strain 297).
ospC amplification. An attempt to amplify the complete 650-bp sequence of ospC was initiated, as previously described (25), but amplification was inconsistent. Therefore, the 3' and 5' ends of ospC were separately amplified. Although the 5' ospC amplifications often failed, the 3' reactions were more often successful and were used as the basis for phylogenetic analysis. To validate this approach, full-length sequences of 21 previously published ospC reference sequences and sequences corresponding to the predicted 3' amplicons were separately aligned and resulted in similar tree topologies, for which each reference group identified a unique clade.
B. burgdorferi ospC PCR and sequencing. All six B. burgdorferi DNA templates from human plasma cultures and one of the two human skin cultures produced an amplicon of approximately 314 bp with the 3' ospC primers. Only 18 of the 71 Borrelia cultures derived from small animals amplified with the ospC primers. From the 18 PCR positives, 2 were from blood and 16 were from ear biopsy cultures. Both controls (strain 297 and lab strain) amplified in all PCRs; negative controls (deionized water) did not produce detectable products. Unambiguous forward and reverse sequences were obtained from all except for three amplicons (one blood and two ear biopsies).
B. burgdorferi ospC heterogeneity among human and mouse isolates from the six properties. A total of 45 sequences, including 21 references from GenBank, 2 laboratory controls, and 22 obtained from human and small mammal cultures, were aligned to generate a phylogenetic tree. Sequences derived from human isolates were found in four distinct clusters, while sequences originating from animal reservoirs were found in seven clusters (Fig. 1). Table 3 summarizes the findings and shows the 7 human and 15 small mammal isolates and the sites from which they were collected. Only at site 2 were B. burg-dorferi isolates with the same ospC genotype observed in both human (isolate PL 2) and mouse (isolate EP2-12) isolates.
All B. burgdorferi sequences obtained from human isolates corresponded with ospC reference groups, including one each that clustered with groups K and B that are associated with invasive infections (Table 4) (25). The remaining blood isolates clustered with ospC groups not before identified to contain invasive strains, including PL6 in group H (Fig. 1) and PL3, PL4, and PL5 in group N (25). The one skin isolate (SK5) ex amined had an ospC sequence identical to that of a blood isolate from the same patient that clustered with group N as well.
From the 15 small mammal blood or ear biopsy isolate sequences, 10 clustered with invasive strains of major ospC groups (Fig. 1). Of those remaining, five clustered with noninvasive ospC groups (Fig. 1). Of those that matched invasive ospC groups, three each matched with group A (EP1-3 [site 1], EP3-1 [site 3], and EP3-3 [site 3]) and group K (EP2-1 [site 2], EP2-22 [site 2], and EP6-11 [site 6]). The remaining four matched with invasive ospC group B (EP2-12 [site 2], EP3-5 and BC3-4 [site 3], and EP6-6 [site 6]) (Table 4). The remaining four small-animal ear biopsy isolates EP2-14 (site 2; group G), EP4-3 and EP4-7 (site 4; group T), and EP6-12 (site 6; group H) clustered with ospC groups G, H, and T that are associated with noninvasive infections (Table 4) (25).
DISCUSSION
OspC of B. burgdorferi is a highly heterogeneous lipoprotein implicated as a potential virulence factor for tissue invasion and dissemination in humans (25). The diversity of ospC has been investigated extensively in tick and human isolates sampled from some regions (3, 9, 14, 23, 30) but has not been extensively explored among the isolates of small mammals that serve as critical reservoirs for maintaining the infectious cycle (6, 15). Moreover, few studies regarding the similarity of Borrelia isolates among small animals and humans from the same or related geographic regions have been conducted (16). The present study considers these factors in an examination of B. burgdorferi ospC heterogeneity in a Lyme disease-endemic region of northern Maryland and southern Pennsylvania.
PCR amplification of full-length ospC was unreliable for samples derived in this study; therefore, a modified method derived by comparing the sequences from 240 bp of the 3' end of ospC was used to yield a phylogenetic tree that allowed distinct identification of each reference group as demonstrated for the tree of the full-length gene (14). When applied to B. burgdorferi isolates from humans and mice collected within a limited geographic region in the Northern Maryland and Southern Pennsylvania, these data demonstrate that there is a high degree of ospC diversity. Matching human and animal isolates were only recovered from a single location/site, but this may be an artifact of small sample size. With the high degree of ospC polymorphism observed in this study, larger numbers of mammals may need to be captured to determine whether all or most mouse-infective clones are also infective for humans. Altogether, five of the six sites contained two or more B. burgdorferi ospC clones, including four at one site and three at another. Only a single clone was identified in site 5 (Pennsylvania). Unfortunately, ospC typing of animal isolates from that location was unsuccessful and would have likely revealed additional polymorphism.
The overall analysis is hampered by several potential biases. Although 60% of the 118 animal samples were culture positive, 18 were ospC PCR positive and only 15 were successfully sequenced despite repeated attempts. The inability to amplify ospC from field (mammal) isolates may be due to additional sequence variability at the priming sites resulting in failed PCR. Alternatively, the inability to amplify ospC and fla from small mammal blood cultures in which Borrelia were visualized could be the result of significant blood contamination and inhibition of PCR in spite of DNA purification methods. Another potential pitfall relates to the selection of B. burgdorferi clones in culture, possibly leading to a selection bias (17). For illustration, among the two human skin B. burgdorferi isolates examined, one failed to amplify with ospC primers, yet amplified with primers that target ospA, suggesting that even greater ospC heterogeneity exists among B. burgdorferi pathogenic for humans than is presently evaluated. Likewise, the inability to obtain unambiguous direct sequences from 17% of amplicons suggested that some cultures may be mixed. This proportion is similar to that described by Seinost et al. (26) and further illustrates the degree of potential heterogeneity in these regions.
Seinost et al. suggested that ospC typing could differentiate non-pathogenic from B. burgdorferi that are pathogenic for humans and may further differentiate strains that cause localized skin infection from those that cause systemic/invasive disease (25). However, four of the six human plasma isolates in the present study did not cluster with the major ospC groups previously implicated in invasion (25). In contrast, 10 of the 15 animal isolates clustered with the major ospC groups designated as invasive, potentially reflecting the innate ability of these groups to disseminate, even in the animal host. Unlike Seinost's observations, the major ospC groups A and K did not predominate in humans from this region (25, 29) but were found in 6 of 15 small mammal isolates (Table 4). Qiu et al. found group K to be one of the most common clones on the U.S. East Coast, including 14 of 22 B. burgdorferi identified in I. scapularis from Maryland (23), whereas it only accounted for 18% of our ospC-positive samples.
B. burgdorferi ospC groups G, H, J, N, and T are usually associated only with erythema migrans and not invasion in humans (25). However, in this study, their invasiveness in both animals and humans was demonstrated by identification in disseminated sites. Although not well investigated, animal invasiveness in groups G and T suggests the potential for invasion in humans.
The ospC groups D, E, F, and L, previously found only in ticks (25) were not identified in this study, confirming how infrequently they are observed in humans or reservoirs. Of more interest is the absence of these ospC groups in mice as well, despite their presence in ticks collected from this region (23). As B. burgdorferi exists in an obligate relationship that requires transmission between the tick and mammalian host (5), it is expected that these isolates should be identified in small animals. Alternatively, one may hypothesize that other mammalian hosts beside P. leucopus may harbor the ospC groups that have been characterized as predominantly tick associated. Another alternative could be that such ospC groups depend on transovarial transmission, a mechanism that is usually considered highly inefficient for B. burgdorferi (21). An expanded investigation may shed light on the extent of the host distribution of major ospC groups.
These Maryland and southern Pennsylvania studies confirm a high degree of B. burgdorferi genetic diversity and a lack of concordance between strains identified in animals and humans from the same locations. However, since 10 of the 15 animal isolates clustered with ospC types previously characterized as "invasive," it is reasonable to conclude that mouse invasiveness may be equivalent to human infectivity. Moreover, the identification of invasion in both mice and humans by noninvasive ospC types suggest that the grouping proposal of Seinost et al. (25) needs a more rigorous evaluation. Inclusion of additional isolates representing both a variety of geographic regions and infected human as well as reservoir hosts may help to determine whether all or some subpopulations of B. burgdorferi strains can cause disseminated human infection.
In accordance with other investigators using animal models (29), it is possible that ospC diversity is less important as a predictor of mammalian and human invasion than proposed previously (3, 25). Thus, given the extensive diversity of B. burgdorferi ospC types that may infect humans, alternative genetic markers should be investigated that may better correlate with and explain B. burgdorferi disseminated disease.
ACKNOWLEDGMENTS
The present study was supported in part by RO1A141213 from the National Institute of Allergy and Infectious Diseases to J.S.D. and a Cooperative Agreement Award to D.E.N. (U50/CCU319554) from the Centers for Disease Control and Prevention. J.P. was supported in part by a grant from KOSEF (Korean Science and Engineering Foundation). J.M.A. was supported in part by an National Institute of Environmental Health Sciences training award (T32ES07141).
This work was submitted in partial fulfillment of the Masters degree of M.Y.A. The isolation of B. burgdorferi from human patients was conducted with approval of The Johns Hopkins University School of Medicine (JHM IRB-2 RPN 01-01-12-05).
We thank Clara Lema, Amy Linhardt, and Peggy Coulter for their assistance with medium preparation and isolation and growth of B. burgdorferi from human patients.
Present address: Box 73265, Abu Dhabi, United Arab Emirates.
Present address: Department of Veterinary Internal Medicine, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561-756, Korea.
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Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
Divisions of General Internal Medicine and Infectious Diseases, The Johns Hopkins University School of Medicine, Lutherville, Maryland 21093
ABSTRACT
B. burgdorferi invasiveness correlates with ospC genotype. To test this hypothesis and whether identical genotypes infect humans and small mammals in specific sites, B. burgdorferi ospC heterogeneity was tested among isolates from northern Maryland and southern Pennsylvania. Six culture-positive patients allowed collection of small animals from their properties, and spirochetes from animals trapped within 300 yards of each patient's home were isolated. 3' ospC sequences were compared to reference sequences. Of the 7 human and 15 mouse DNA templates that produced reliable sequences, all clustered with references into only four and seven distinct clades, respectively. A human and a mouse isolate with the same ospC were seen in only one locality, and five of six sites contained two or more B. burgdorferi ospC clones. Four invasive patient isolates and six small mammal isolates clustered with "noninvasive" reference ospC genotypes. A high degree of ospC diversity exists among B. burgdorferi isolates in Maryland and Pennsylvania, even in narrowly defined geographic localities. Dissemination in mice and humans by noninvasive ospC types contradicts the ospC invasiveness hypothesis. Alternative genetic markers for B. burgdorferi disseminated disease should be investigated.
INTRODUCTION
Lyme borreliosis is an infectious disease caused by Borrelia burgdorferi sensu lato (4). These spirochetes are transmitted by Ixodes spp. ticks that maintain an infectious cycle by feeding on reservoir hosts such as Peromyscus leucopus and other small mammals; humans are accidental hosts (5, 9). Three Borrelia species that cause human disease include B. burgdorferi sensu stricto (North America), B. afzelii, and B. garinii (Europe and Asia) (2, 27, 33). In North America the geographic distribution is well documented, with B. burgdorferi and Lyme disease expanding and clearly endemic in the mid-Atlantic region including Maryland (1, 22).
B. burgdorferi plasmid-encoded outer surface protein C genes (ospC) are implicated in transmission and dissemination of Borrelia between ticks and mammals (12, 18, 20, 24, 32). Diversity of ospC has lead to the classification of B. burgdorferi clones into human invasive and "noninvasive" groups (14, 16, 25, 28), but these studies have not evaluated ospC as an invasive factor in small mammals. Because of ospC heterogeneity, it has been used as marker for clonality in epidemiological studies that suggest that human and small mammal isolates from the same region are restricted to only a single or a few clones (14, 16). Neither geographic nor ospC heterogeneity of B. burgdorferi infecting humans and mice has been extensively studied in the mid-Atlantic States. The present study was designed to test two hypotheses: (i) that only limited ospC diversity exists among B. burgdorferi infecting humans and mice from the same localities in northern Maryland, and (ii) that ospC genotypes associated with invasiveness in humans extends to Maryland and to small mammals.
MATERIALS AND METHODS
Patients and Borrelia isolates. From a study of clinical Lyme disease in Maryland during 2001 (J. S. Dumler, unpublished data), eight Borrelia burgdorferi isolates from six culture-confirmed patients were selected from those who also agreed to allow collection of small animals from their properties. Six isolates were from human plasma, and two were from erythema migrans lesions. Isolates were confirmed to be B. burgdorferi by PCR amplification of the ospA gene (data not shown), as previously described (7). Five patients resided in Baltimore County, Maryland, and one was from neighboring York County, Pennsylvania. All patients had early Lyme disease with erythema migrans lesions, and five had manifestations suggesting early disseminated disease, including one with multiple erythema migrans and four with influenza-like illnesses. Patients provided informed consent prior to the study as approved by the John Hopkins Medicine Institutional Review Board.
Growth of human Borrelia isolates. Low-passage (<2 passages) bacteria were propagated from dimethyl sulfoxide-preserved frozen patient isolates. Approximately 0.5 ml of each frozen isolate was transferred to 5 ml of modified BSK-II medium (31) and incubated at 34°C in 5% CO2 until sufficient growth was confirmed by fluorescent microscopy using acridine-orange staining. Two control cultures were used: B. burgdorferi strain 297 (courtesy of Dennis Grab, the Johns Hopkins University School of Medicine) and a randomly selected human isolate previously prepared in the lab and used for internal control purposes (lab strain).
Mouse B. burgdorferi isolates. Mice were trapped using 60 Sherman live traps (H. B. Sherman, Tallahassee, FL) baited with bird seed and placed within 300 yards of each patient's home. After administration of a light anesthetic, a 2-mm-diameter ear tissue sample was taken using a biopsy tool (Accuderm Inc., Ft. Lauderdale, FL) and placed in BSK-II medium for transport. A blood sample was taken from each capture by retro-orbital sampling and placed directly on ice for transport. Captured rodents were released at the site of capture. Trapping and sample collection were performed according to Hofmeister et al. (9) as approved by the John Hopkins University Institutional Animal Care and Use Committee.
Mouse blood and skin cultures were incubated at 34°C in 5% CO2 and examined weekly for up to 8 weeks. Acridine orange-positive samples were subcultured in BSK-II medium supplemented with 50 μg/ml of ciprofloxacin and 20 μg/ml of rifampin to reduce contamination with other bacterial species. When appropriate, 0.45-μm-pore-diameter syringe-driven filter units were used (11) to remove contaminating bacteria. After 2 to 3 weeks of log phase growth, aliquots of each culture were cryopreserved at –80°C. Despite the use of antimicrobials and filtering, contamination persisted for many cultures, resulting in poor Borrelia recovery in 11 field samples later excluded from further study. Despite all attempts, many isolates grew poorly in the medium preparations used.
DNA extraction. During log phase growth, 1.5 ml of each Borrelia culture was centrifuged at 13,800 x g for 25 min. The bacterial pellet was resuspended in 200 μl of phosphate-buffered saline, and DNA was prepared using a QIAamp DNA Mini kit (QIAGEN, Inc.) and the manufacturer's protocols. DNA preparations were stored at –20°C until analyzed.
PCR methods and conditions. The 3' and 5' ends of ospC were PCR amplified using conserved primers as described previously (25). PCR was performed using an eLONGase kit (Invitrogen Inc.) per manufacturer's instructions with the addition of 100 pmol of each primer and using 2 μl of B. burgdorferi isolate DNA that was not otherwise quantitated. The reaction was completed as follows: initial denaturation at 96°C for 2 min, followed by 40 cycles of denaturation at 95°C for 40 s, annealing at 54°C for 35 s, and extension at 72°C for 1 min. The 5' ospC amplification did not produce a reliable product from all samples; therefore, all subsequent analyses were based on amplification of a 314-bp fragment from the 3' end. A negative control (deionized water) was included in each PCR. PCR amplification of a B. burgdorferi-specific fragment from the flaB gene was used to confirm that small mammal isolates from which ospC was amplified were B. burgdorferi (13).
Sequencing and phylogenetic analysis. B. burgdorferi ospC PCR products (25 μl) were electrophoresed in 2% agarose gels, stained with ethidium bromide, and excised for purification using a QIAquick gel extraction kit (QIAGEN Inc.). Sequencing was performed using the forward and reverse PCR primers (25) and a fluorescent automated method (ABI 3100 genetic analyzer). The resulting forward and reverse ospC sequences were initially assembled using the Contig Assembly Program in BioEdit (8) to generate consensus sequences. Discrepancies were resolved by inspection of the original chromatograms or by additional sequence analysis.
To validate the use of the 3' ospC sequences for phylogenetic comparisons, 21 B. burgdorferi sequences corresponding to the major ospC groups (25, 30) were retrieved from GenBank (Table 1) and aligned using ClustalX (10). Primer sequences flanking the region of the alignment corresponding to the 3' amplicon were removed, and the resulting sequences were gap stripped and realigned for phylogenetic analysis. Full-length and 3' ospC trees were generated using the neighbor-joining algorithm, and topologies of the trees were compared. The consensus sequence assemblies of the study isolates were aligned with the reference 3' ospC sequences. These combined sequences were then trimmed for optimal pairwise alignments and used to generate a neighbor-joining tree with bootstrapping 1,000 times. The tree was viewed with TreeView software (19).
RESULTS
Isolation and detection of Borrelia. Human and mouse specimens. With the broad assumption that patient exposure occurred on or around their properties, small mammals were collected from the properties of the six selected patients. From the six locations, a total of 61 small mammals (48 white-footed mice [Peromyscus leucopus], 9 chipmunks [Tamias striatus], 1 vole [Microtus pennsylvanicus], and 2 shrews [Sorex spp.]) were captured. Blood and ear biopsies for culture were obtained from each animal except for the shrews, from which blood was not obtained, and two chipmunks, from which ear biopsies were not obtained. From 118 small mammal tissue or blood cultures, spirochetes were observed in 71 (60.2%), including 27 blood and 44 ear biopsy cultures (Table 2). Contamination was a significant problem, as 54 (45.8%) cultures contained other bacteria. Contamination levels were minimal in all but 11 cultures. Bacterial contaminants could not be cleared from these 11 cultures, and they were subsequently excluded from further analysis. Growth of borreliae was also poor for most samples despite adequate growth of laboratory control cultures (B. burgdorferi strain 297).
ospC amplification. An attempt to amplify the complete 650-bp sequence of ospC was initiated, as previously described (25), but amplification was inconsistent. Therefore, the 3' and 5' ends of ospC were separately amplified. Although the 5' ospC amplifications often failed, the 3' reactions were more often successful and were used as the basis for phylogenetic analysis. To validate this approach, full-length sequences of 21 previously published ospC reference sequences and sequences corresponding to the predicted 3' amplicons were separately aligned and resulted in similar tree topologies, for which each reference group identified a unique clade.
B. burgdorferi ospC PCR and sequencing. All six B. burgdorferi DNA templates from human plasma cultures and one of the two human skin cultures produced an amplicon of approximately 314 bp with the 3' ospC primers. Only 18 of the 71 Borrelia cultures derived from small animals amplified with the ospC primers. From the 18 PCR positives, 2 were from blood and 16 were from ear biopsy cultures. Both controls (strain 297 and lab strain) amplified in all PCRs; negative controls (deionized water) did not produce detectable products. Unambiguous forward and reverse sequences were obtained from all except for three amplicons (one blood and two ear biopsies).
B. burgdorferi ospC heterogeneity among human and mouse isolates from the six properties. A total of 45 sequences, including 21 references from GenBank, 2 laboratory controls, and 22 obtained from human and small mammal cultures, were aligned to generate a phylogenetic tree. Sequences derived from human isolates were found in four distinct clusters, while sequences originating from animal reservoirs were found in seven clusters (Fig. 1). Table 3 summarizes the findings and shows the 7 human and 15 small mammal isolates and the sites from which they were collected. Only at site 2 were B. burg-dorferi isolates with the same ospC genotype observed in both human (isolate PL 2) and mouse (isolate EP2-12) isolates.
All B. burgdorferi sequences obtained from human isolates corresponded with ospC reference groups, including one each that clustered with groups K and B that are associated with invasive infections (Table 4) (25). The remaining blood isolates clustered with ospC groups not before identified to contain invasive strains, including PL6 in group H (Fig. 1) and PL3, PL4, and PL5 in group N (25). The one skin isolate (SK5) ex amined had an ospC sequence identical to that of a blood isolate from the same patient that clustered with group N as well.
From the 15 small mammal blood or ear biopsy isolate sequences, 10 clustered with invasive strains of major ospC groups (Fig. 1). Of those remaining, five clustered with noninvasive ospC groups (Fig. 1). Of those that matched invasive ospC groups, three each matched with group A (EP1-3 [site 1], EP3-1 [site 3], and EP3-3 [site 3]) and group K (EP2-1 [site 2], EP2-22 [site 2], and EP6-11 [site 6]). The remaining four matched with invasive ospC group B (EP2-12 [site 2], EP3-5 and BC3-4 [site 3], and EP6-6 [site 6]) (Table 4). The remaining four small-animal ear biopsy isolates EP2-14 (site 2; group G), EP4-3 and EP4-7 (site 4; group T), and EP6-12 (site 6; group H) clustered with ospC groups G, H, and T that are associated with noninvasive infections (Table 4) (25).
DISCUSSION
OspC of B. burgdorferi is a highly heterogeneous lipoprotein implicated as a potential virulence factor for tissue invasion and dissemination in humans (25). The diversity of ospC has been investigated extensively in tick and human isolates sampled from some regions (3, 9, 14, 23, 30) but has not been extensively explored among the isolates of small mammals that serve as critical reservoirs for maintaining the infectious cycle (6, 15). Moreover, few studies regarding the similarity of Borrelia isolates among small animals and humans from the same or related geographic regions have been conducted (16). The present study considers these factors in an examination of B. burgdorferi ospC heterogeneity in a Lyme disease-endemic region of northern Maryland and southern Pennsylvania.
PCR amplification of full-length ospC was unreliable for samples derived in this study; therefore, a modified method derived by comparing the sequences from 240 bp of the 3' end of ospC was used to yield a phylogenetic tree that allowed distinct identification of each reference group as demonstrated for the tree of the full-length gene (14). When applied to B. burgdorferi isolates from humans and mice collected within a limited geographic region in the Northern Maryland and Southern Pennsylvania, these data demonstrate that there is a high degree of ospC diversity. Matching human and animal isolates were only recovered from a single location/site, but this may be an artifact of small sample size. With the high degree of ospC polymorphism observed in this study, larger numbers of mammals may need to be captured to determine whether all or most mouse-infective clones are also infective for humans. Altogether, five of the six sites contained two or more B. burgdorferi ospC clones, including four at one site and three at another. Only a single clone was identified in site 5 (Pennsylvania). Unfortunately, ospC typing of animal isolates from that location was unsuccessful and would have likely revealed additional polymorphism.
The overall analysis is hampered by several potential biases. Although 60% of the 118 animal samples were culture positive, 18 were ospC PCR positive and only 15 were successfully sequenced despite repeated attempts. The inability to amplify ospC from field (mammal) isolates may be due to additional sequence variability at the priming sites resulting in failed PCR. Alternatively, the inability to amplify ospC and fla from small mammal blood cultures in which Borrelia were visualized could be the result of significant blood contamination and inhibition of PCR in spite of DNA purification methods. Another potential pitfall relates to the selection of B. burgdorferi clones in culture, possibly leading to a selection bias (17). For illustration, among the two human skin B. burgdorferi isolates examined, one failed to amplify with ospC primers, yet amplified with primers that target ospA, suggesting that even greater ospC heterogeneity exists among B. burgdorferi pathogenic for humans than is presently evaluated. Likewise, the inability to obtain unambiguous direct sequences from 17% of amplicons suggested that some cultures may be mixed. This proportion is similar to that described by Seinost et al. (26) and further illustrates the degree of potential heterogeneity in these regions.
Seinost et al. suggested that ospC typing could differentiate non-pathogenic from B. burgdorferi that are pathogenic for humans and may further differentiate strains that cause localized skin infection from those that cause systemic/invasive disease (25). However, four of the six human plasma isolates in the present study did not cluster with the major ospC groups previously implicated in invasion (25). In contrast, 10 of the 15 animal isolates clustered with the major ospC groups designated as invasive, potentially reflecting the innate ability of these groups to disseminate, even in the animal host. Unlike Seinost's observations, the major ospC groups A and K did not predominate in humans from this region (25, 29) but were found in 6 of 15 small mammal isolates (Table 4). Qiu et al. found group K to be one of the most common clones on the U.S. East Coast, including 14 of 22 B. burgdorferi identified in I. scapularis from Maryland (23), whereas it only accounted for 18% of our ospC-positive samples.
B. burgdorferi ospC groups G, H, J, N, and T are usually associated only with erythema migrans and not invasion in humans (25). However, in this study, their invasiveness in both animals and humans was demonstrated by identification in disseminated sites. Although not well investigated, animal invasiveness in groups G and T suggests the potential for invasion in humans.
The ospC groups D, E, F, and L, previously found only in ticks (25) were not identified in this study, confirming how infrequently they are observed in humans or reservoirs. Of more interest is the absence of these ospC groups in mice as well, despite their presence in ticks collected from this region (23). As B. burgdorferi exists in an obligate relationship that requires transmission between the tick and mammalian host (5), it is expected that these isolates should be identified in small animals. Alternatively, one may hypothesize that other mammalian hosts beside P. leucopus may harbor the ospC groups that have been characterized as predominantly tick associated. Another alternative could be that such ospC groups depend on transovarial transmission, a mechanism that is usually considered highly inefficient for B. burgdorferi (21). An expanded investigation may shed light on the extent of the host distribution of major ospC groups.
These Maryland and southern Pennsylvania studies confirm a high degree of B. burgdorferi genetic diversity and a lack of concordance between strains identified in animals and humans from the same locations. However, since 10 of the 15 animal isolates clustered with ospC types previously characterized as "invasive," it is reasonable to conclude that mouse invasiveness may be equivalent to human infectivity. Moreover, the identification of invasion in both mice and humans by noninvasive ospC types suggest that the grouping proposal of Seinost et al. (25) needs a more rigorous evaluation. Inclusion of additional isolates representing both a variety of geographic regions and infected human as well as reservoir hosts may help to determine whether all or some subpopulations of B. burgdorferi strains can cause disseminated human infection.
In accordance with other investigators using animal models (29), it is possible that ospC diversity is less important as a predictor of mammalian and human invasion than proposed previously (3, 25). Thus, given the extensive diversity of B. burgdorferi ospC types that may infect humans, alternative genetic markers should be investigated that may better correlate with and explain B. burgdorferi disseminated disease.
ACKNOWLEDGMENTS
The present study was supported in part by RO1A141213 from the National Institute of Allergy and Infectious Diseases to J.S.D. and a Cooperative Agreement Award to D.E.N. (U50/CCU319554) from the Centers for Disease Control and Prevention. J.P. was supported in part by a grant from KOSEF (Korean Science and Engineering Foundation). J.M.A. was supported in part by an National Institute of Environmental Health Sciences training award (T32ES07141).
This work was submitted in partial fulfillment of the Masters degree of M.Y.A. The isolation of B. burgdorferi from human patients was conducted with approval of The Johns Hopkins University School of Medicine (JHM IRB-2 RPN 01-01-12-05).
We thank Clara Lema, Amy Linhardt, and Peggy Coulter for their assistance with medium preparation and isolation and growth of B. burgdorferi from human patients.
Present address: Box 73265, Abu Dhabi, United Arab Emirates.
Present address: Department of Veterinary Internal Medicine, College of Veterinary Medicine, Chonbuk National University, Jeonju, Jeonbuk 561-756, Korea.
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